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. 2022 Dec 8;11:e74576. doi: 10.7554/eLife.74576

Inhibition of β1-AR/Gαs signaling promotes cardiomyocyte proliferation in juvenile mice through activation of RhoA-YAP axis

Masahide Sakabe 1,2, Michael Thompson 1,2, Nong Chen 1,2, Mark Verba 1,2, Aishlin Hassan 1,2, Richard Lu 1,2, Mei Xin 1,2,✉
Editors: Enzo R Porrello3, Didier YR Stainier4
PMCID: PMC9767473  PMID: 36479975

Abstract

The regeneration potential of the mammalian heart is incredibly limited, as cardiomyocyte proliferation ceases shortly after birth. β-adrenergic receptor (β-AR) blockade has been shown to improve heart functions in response to injury; however, the underlying mechanisms remain poorly understood. Here, we inhibited β-AR signaling in the heart using metoprolol, a cardio-selective β blocker for β1-adrenergic receptor (β1-AR) to examine its role in heart maturation and regeneration in postnatal mice. We found that metoprolol enhanced cardiomyocyte proliferation and promoted cardiac regeneration post myocardial infarction, resulting in reduced scar formation and improved cardiac function. Moreover, the increased cardiomyocyte proliferation was also induced by the genetic deletion of Gnas, the gene encoding G protein alpha subunit (Gαs), a downstream effector of β-AR. Genome wide transcriptome analysis revealed that the Hippo-effector YAP, which is associated with immature cardiomyocyte proliferation, was upregulated in the cardiomyocytes of β-blocker treated and Gnas cKO hearts. Moreover, the increased YAP activity is modulated by RhoA signaling. Our pharmacological and genetic studies reveal that β1-AR-Gαs-YAP signaling axis is involved in regulating postnatal cardiomyocyte proliferation. These results suggest that inhibiting β-AR-Gαs signaling promotes the regenerative capacity and extends the cardiac regenerative window in juvenile mice by activating YAP-mediated transcriptional programs.

Research organism: Mouse

Introduction

The capacity to regenerate and repair in response to cardiac injury in the adult mammalian heart is limited. Neonatal mouse hearts retain regenerative potential following cardiac injury up to 7 days after birth Porrello et al., 2011; Porrello et al., 2013; Xin et al., 2013b. Changes after birth such as metabolic state, oxygen level, cardiomyocyte structure and maturity, hormones, and polyploidy are among the factors contributing to the loss of the regenerative potential in the heart Hirose et al., 2019; Vivien et al., 2016; Nakada et al., 2017; Kimura et al., 2015; Siedner et al., 2003; Derks and Bergmann, 2020; Puente et al., 2014. For instance, the postnatal metabolic shift from glycolysis to fatty acid oxidation or aerobic respiration-mediated oxidative DNA damage can lead to cardiomyocyte cell cycle arrest postnatally Nakada et al., 2017; Kimura et al., 2015; Puente et al., 2014. In addition, signaling pathways such as Hippo, Neuregulin, ERBB2, Agrin, and thyroid hormone have been shown to regulate cardiac regeneration Hirose et al., 2019; D’Uva et al., 2015; Bassat et al., 2017; Mahmoud et al., 2015; Lin et al., 2014.

The evolutionarily conserved Hippo signaling pathway is known as a pivotal regulator of organ size and cell proliferation Zhao et al., 2011; Pan, 2010. It consists of a series of kinases, including MERLIN, MST1/2, and LATS1/2, that phosphorylate the downstream effectors YAP and TAZ, preventing their nuclear translocation and activation of target gene expression Varelas, 2014; Hansen et al., 2015. Activation of YAP in the embryonic heart, either through the loss of Mst1/2, Sav1, or forced expression of a constitutively active form of YAP, induces cardiomyocyte proliferation and increases heart size Xin et al., 2011; Heallen et al., 2011. Moreover, activation of YAP in adult hearts improves cardiac function and reduces scar formation after myocardial infarction (MI) Xin et al., 2013a; Heallen et al., 2013; Leach et al., 2017, suggesting that YAP activation promotes cardiomyocyte regeneration even in the adult mouse heart. However, it remains unknown what upstream signaling cues regulate the Hippo signaling pathway for cardiac regeneration.

The Hippo pathway can be activated by several molecular signals, including G-protein-coupled receptors (GPCRs), cell-cell interactions, and alterations in cytoskeletal dynamics Yu et al., 2012; Wada et al., 2011; Kim et al., 2011. Beta-adrenergic receptors (β-ARs), members of GPCRs that couple to a stimulatory G protein alpha-subunit (Gαs), are essential components of the sympathetic nervous system Lymperopoulos et al., 2013. Stimulation of the β-ARs activates Gαs activity leading to an increase in intracellular cAMP levels and the subsequent activation of protein kinase A (PKA), which can result in increased heart rate and contractility Rockman et al., 2002; Molkentin and Dorn, 2001. Overexpression of Gαs-protein leads to increased myocardial collagen content and fibrosis with variable hypertrophy in mice Iwase et al., 1997; Geng et al., 1999, suggesting an important role of Gαs in controlling cardiac contractility or hypertrophic response in the heart. Furthermore, activation of Gαs by epinephrine inactivates YAP and inhibits cell growth in various cell lines Yu et al., 2012. This presents an opportunity to manipulate cardiomyocyte proliferation through inhibition of the β-AR signaling pathway.

Inhibition of β-AR by β-adrenergic receptor blockade (β-blockers) has been shown to improve survival and symptoms in heart failure patients Patel and Shaddy, 2010; MERIT-HF Study group, 1999. Gene variants in GNAS, which encodes the Gαs protein, are associated with β-blocker-related survival or risk in patients after coronary artery bypass grafting Frey et al., 2014. A recent study suggested that a non-selective β-blocker (propranolol) increased the number of cardiomyocytes in neonatal mice Liu et al., 2019. Since propranolol inhibits both the β1-AR that is a predominant receptor in the heart muscle and β2-AR that is highly expressed in vascular and non-vascular smooth muscle cells and endothelial cells in non-heart tissues Cannavo et al., 2013; Cannavo and Koch, 2017; Flacco et al., 2013; Woo and Xiao, 2012; Zhu and Steinberg, 2021; Wang et al., 2018, it is not suitable for patients with diabetes or bronchospasm. The 2nd generation β1-blocker, metoprolol, selectively binds to the β1-AR receptor on cardiomyocytes Ladage et al., 2013; however, its effect on cardiomyocyte proliferation and heart regeneration have not been explored. Furthermore, the mechanisms by which β-AR-Gαs signaling modulates cardiomyocyte proliferation and heart regeneration remains to be defined.

In this study, we show that treatment with the β1-blocker metoprolol promotes cardiomyocyte proliferation, reduces scar formation, and improves cardiac function after myocardial injury in juvenile mice. Inhibition of the β1-AR downstream effector Gαs activity by genetic deletion of Gnas enhances cardiomyocyte proliferation by activating YAP activity through its nuclear localization. Thus, our study demonstrates that β-AR-Gαs signaling represses the regenerative capacity of postnatal cardiomyocytes by inhibiting YAP-activated transcriptional programs. Inhibition of β-AR-Gαs signaling extended the cardiac regenerative window, suggesting a potential therapeutic target for extending the cardiac regeneration window.

Results

β1-blocker treatment promotes cardiomyocyte proliferation

β1-AR is the most abundant β-AR subtype present in cardiomyocytes, comprising about 80% of total β-AR. β2-AR, comprising about 20% of β-AR in cardiomyocytes, is highly expressed in vascular and non-vascular smooth muscle cells Cannavo et al., 2013; Cannavo and Koch, 2017; Flacco et al., 2013; Woo and Xiao, 2012; Zhu and Steinberg, 2021; Wang et al., 2018. To investigate whether blockade β1-AR specifically has a significant effect on cardiomyocyte proliferation and cardiac regeneration, we injected the β1-AR blocker metoprolol (hereinafter, referred to as “β-blocker”) into mice for two weeks starting at postnatal day (P) 1 via daily intraperitoneal injection (IP) (Figure 1a). The heart rate of β-blocker treated mice was significantly reduced and thickening of the myocardial wall of β-blocker treated hearts was evident at P14 (Figure 1b and c). β-blocker treatment induced an increase in the heart weight-to-body weight ratio at P14, but not at P7 (Figure 1d and Figure 1—figure supplement 1a). Increased proliferating cardiomyocytes in β-blocker-treated hearts at P7 or P14 was confirmed by 5-ethynyl-2'-deoxyuridine (EdU) incorporation, Ki67, PH3, and aurora kinase B (AURKB) immunostaining (Figure 1e and f, Figure 1—figure supplement 1b–e). We also observed an increased total number of cardiomyocytes and mononucleated cardiomyocytes (a proliferative and regenerative subpopulation of the postnatal heart), in the β-blocker-treated hearts (Figure 1g and Figure 1—figure supplement 1f). To examine the possibility that the increased heart size is due to cardiac hypertrophy, cardiomyocyte size was measured. No significant difference in cardiomyocyte size was detected between control and β-blocker-treated hearts (Figure 1—figure supplement 1g and h), suggesting that the enlarged heart phenotype by the β-blocker treatment is not likely caused by cardiac hypertrophy. Moreover, β-blocker treatment promoted cardiomyocyte proliferation at later time points from P14 to P28, when the majority of cardiomyocytes are matured (Figure 1h), suggesting that β1-AR-selective blocker treatment reactivates cell proliferation even in relatively matured cardiomyocytes in vivo.

Figure 1. β-blocker (Metoprolol) treatment promotes neonatal-juvenile cardiomyocyte proliferation.

(a) Schematic of experimental timeline. (b) Heart rate of the saline (control) and β-blocker-treated mice at P7 and P14. Data are as mean ± SD, Student’s t-test, P7, n≥5; P14, n≥5. (c) H&E staining of control and β-blocker-treated hearts at P14. Scale bar: 500μm. (d) Heart weight (HW) to body weight (BW) ratio at P14. Data are as mean ± SD, Student’s t-test, n≥8. (e) Co-immunostaining of PH3 and PCM1 of heart sections from control and β-blocker-treated mice at P14 (left panel). Arrows indicate PH3-positive CMs. Inset shows high-magnification image of PH3-positive CM. Quantification of PH3 positive cardiomyocytes (right panel). Data are as mean ± SD, Student’s t-test, n=3. Scale bar: 50μm. (f) Aurora kinase B (AURKB) staining of P7 β-blocker treated heart. Scale bar: 10 μm (left panel). Quantification of AURKB positive cardiomyocytes at P7 (right panel). Data are as mean ± SD, Student’s t-test, n=3. (g) DAPI staining of isolated cardiomyocytes from P14 control and β-blocker treated hearts (right panel). Quantification of mono-nucleated, bi-nucleated and multi-nucleated cardiomyocytes in P14 control and β-blocker treated hearts (left panel). Data are as mean ± SD, Student’s t-test, n=5. N.S., not significant. Scale bar: 100μm. (h) PH3 and PCM1 co-immunostaining of P28 heart sections of control and mice with daily β-blocker treatment from P14 (left panel). Arrow indicates PH3-positive CMs. Inset shows high-magnification image of PH3-positive CM. Quantification of PH3 positive cardiomyocytes (right panel). Data are as mean ± SD, Student’s t-test, n=3. Scale bar: 50μm.

Figure 1.

Figure 1—figure supplement 1. β-blocker treatment promotes cardiomyocyte proliferation after the cardiac regeneration window.

Figure 1—figure supplement 1.

(a) Heart weight (HW) to body weight (BW) ratio of β-blocker treated heart at P7 (Student’s t-test, n≥3). (b) Ki67 and cardiac α-actinin staining of heart sections from control and β-blocker treated mice at P7 and P14. Insets show high-magnification image of Ki67-positive CM. Scale bar: 50μm. (c) Quantification of Ki67 positive cardiomyocytes at P7 and P14. Data are as mean ± SD, Student’s t-test, n=4. (d) Co-immunostaining of EdU and PCM1 of heart sections from control and β-blocker-treated mice at P14 (left panel). Arrows indicate EdU-positive CMs. Quantification of EdU positive cardiomyocytes (right panel). Data are as mean ± SD, Student’s t-test, n=3. Scale bar: 25μm. (e) Aurora kinase B (AURKB) staining of P14 β-blocker treated heart. Scale bar: 20 μm (left panel). Quantification of AURKB positive cardiomyocytes at P14 (right panel). (f) Images of isolated cardiomyocytes and quantification of number of isolated cardiomyocytes from control and β-blocker treated hearts at P14. Pellets of isolated cardiomyocytes from a whole heart are shown in 1.5 ml tubes. Data are as mean ± SD, Student’s t-test, n=3. (g) Wheat germ agglutinin (WGA)-stained sections of the LV compact zone in hearts of P14 control and β-blocker treated mice (n=3). Scale bar: 50μm. Quantification of relative pixel per area of WGA-positive cardiomyocytes (Student’s t-test, n=8) (right panel). N.S., not significant. (h) Images (left) and quantification (right) of isolated cardiomyocytes from control and β-blocker treated mice. Twenty cardiomyocytes from 10 individual hearts were measured. Student’s t-test was used. Scale bar: 100μm.
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β1-blocker treatment extends the cardiac regeneration window after myocardial infarction

To determine whether β-blocker treatment extends the regenerative window in mice, we induced myocardial infarction by permanent ligation of the left anterior descending coronary artery (LAD) at P7 and treated the mice with β-blocker from P8 until P28 (Figure 2a). Whereas the vehicle treated mice showed loss of heart tissue, extensive scarring, and dilation post-MI, β-blocker treated mice exhibited significantly reduced left ventricle fibrosis and increased myocardial tissue (Figure 2b and Figure 2—figure supplement 1). Cell proliferation of cardiomyocytes was upregulated by β-blocker treatment in both the infarct border zone and the remote zone, evidenced by an increase in the number of PH3 positive cardiomyocytes (Figure 2c–e), suggesting that β-blocker treatment extends the cardiac regenerative window and enhances cardiomyocyte proliferation in the injured hearts. Moreover, echocardiography analysis indicated that β-blocker treatment led to an improvement in cardiac function post-MI. Both ejection fraction (EF) and fractional shortening (FS) were decreased in all mice relative to sham control mice 1 week after MI, but cardiac function was dramatically enhanced in the β-blocker treated hearts by 3 weeks post-MI (Figure 2f and g). To investigate whether the improved cardiac function is due to an acute cardio-protective effect of β-blocker, we measured the infarcted area 1 day post-MI. No significant difference of ischemic area between control and β-blocker-treated hearts was observed (Figure 2—figure supplement 2), suggesting that β-blocker didn’t provide substantial cardio-protection immediately after MI. These data indicate that β1-selective blocker treatment can extend the cardiac regeneration window and sustain cardiac functions post-MI injury.

Figure 2. β-blocker treatment promotes cardiac regeneration and cardiomyocyte proliferation following injury in juvenile hearts.

(a) Schematic of experimental timeline. (b) Masson’s trichrome staining of heart sections from control and β-blocker-treated mice 3 weeks post-MI (left panel). Scale bar: 500μm. Quantification of the fibrotic areas (right panel). Data are as mean ± SD, Student’s t-test, n=4. (c–e) PH3 and cardiac α-actinin staining of injured hearts treated with saline or β-blocker at 3 weeks post-MI. Data are as mean ± SD, Student’s t-test, n≥3. Scale bar: 25μm. (f–g) Echocardiographic analysis of control and β-blocker-treated mice at 3 weeks post MI surgery. Serial echocardiographic measurements of EF and FS of injured hearts treated with saline (Control) or β-blocker (n=9). ANOVA test, *, p<0.01; N.S., not significant. (h) P7 mice were subjected to LAD ligation and treated with β-blocker from P8 to P28. 4 days after final β-blocker treatment, the heart function was assessed by echocardiography. Data are as mean ± SD, Student’s t-test, n=3.

Figure 2.

Figure 2—figure supplement 1. Scar area is reduced in β-blocker treated hearts 3 weeks post MI.

Figure 2—figure supplement 1.

Hearts were subjected to LAD ligation at P7 and treated with β-blocker from P8 for 3 weeks. Scar area was analyzed by sirius red staining of transverse sections at P28. Serial sections were cut at 200μm intervals from the site of the ligature to the apex. Red region indicates fibrotic scar area. Scale bar: 500μm.
Figure 2—figure supplement 2. β-blocker doesn’t have cardioprotective effect at juvenile period.

Figure 2—figure supplement 2.

2,3,5-Tripherylterazolium chloride (TTC) staining of control and β-blocker treated hearts 1 day post-MI. Right panel showed the % of ischemic area of each heart. Student t-test. N.S., not significant.
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The improved cardiac function in the β-blocker treated mice could be due to a larger volume of blood flow into the ventricle, leading to an increase in the force of contraction associated with a slower heart rate. To rule out this possibility, we assessed cardiac function at 4 days after the last β-blocker treatment. Since metoprolol has a short half-life of 3–7 hr, it should be metabolized during these 4 days. Although the heart rates were comparable between β-blocker-treated and saline-treated control mice, we found that cardiac function was still significantly improved in the β-blocker-treated heart (Figure 2h). Therefore, these results suggest that the β-blocker treatment improves cardiac function by promoting cardiac regeneration in the injured heart.

Deletion of Gnas promotes cardiomyocyte proliferation

β1-AR is associated with the stimulatory G protein (Gαs) and the activated Gα subunit then regulates the downstream effector molecules such as PKA and cAMP. We hypothesize that blockade of β1-AR promotes cardiomyocyte proliferation through the inhibition of Gαs activity. To test this hypothesis, we deleted Gnas, the gene encoding Gαs, in the heart by crossing Gnasflox/flox mice with Myh6Cre mouse line. The Gnasflox/flox; Myh6Cre (Gnas cKO) hearts did not show any abnormal phenotype compared with littermate controls at birth. However, from P7 onwards, Gnas cKO hearts were markedly enlarged and the heart weight-to-body weight ratio was significantly increased (Figure 3a–b, Figure 3—figure supplement 1a). Furthermore, enzyme-linked immunosorbent assay (ELISA) showed that cAMP levels were significantly reduced in Gnas cKO hearts (Figure 3c), suggesting that Gαs function was reduced in Gnas cKO hearts. Consistent with the downregulation of cAMP level, heart rate was also decreased in Gnas cKO (Figure 3d).

Figure 3. Deletion of Gnas promotes cardiomyocyte proliferation.

(a) Hematoxylin and eosin staining of P7 control and Gnas cKO heart sections. Scale bar: 500μm. (b) Heart weight (HW) to body weight (BW) ratio of P7 Gnas cKO mice. Data are as mean ± SD, Student’s t-test, n≥5. (c) Relative cAMP level in P7 control and Gnas cKO hearts. Data are as mean ± SD, Student’s t-test, n=3. (d) Heart rate at P14 in control and Gnas cKO mice. Data are as mean ± SD, Student’s t-test, n≥4. (e) PH3 and PCM1 staining of left ventricle sections of control and Gnas cKO mice at P14 (left panel). Arrow indicates PH3-positive CM. Inset shows high-magnification image of PH3-positive CM. Quantification of PH3 positive cardiomyocytes (right panel). Data are as mean ± SD, Student’s t-test, n=3. Scale bar: 50μm. (f) Aurora kinase B (AURKB) staining of P7 Gnas cKO heart (left panel). Quantification of AURKB-positive cardiomyocytes in the control and Gnas cKO mice (right panel). Data are as mean ± SD, Student’s t-test, n=3. Scale bar: 10 μm. (g) DAPI staining of isolated cardiomyocytes from P14 control and Gnas cKO hearts (left panel). Quantification of mono-nucleated, bi-nucleated and multi-nucleated cardiomyocytes in P14 Gnas cKO hearts (right panel). Data are as mean ± SD, Student’s t-test, n=3. Scale bar: 50μm.

Figure 3.

Figure 3—figure supplement 1. Gnas cKO hearts exhibit enlarged phenotype but do not show cardiac hypertrophy.

Figure 3—figure supplement 1.

(a) H&E staining of control and Gnas cKO hearts at P1 (upper panels) and P14 (lower panels) and quantification of heart weight-to-body weight ratio of control and Gnas cKO neonates at P1 and P14, respectively. Data are as mean ± SD, Student’s t-test, n≥3. Scale bar: 500μm. (b) Aurora kinase B (AURKB) staining of P14 Gnas cKO heart (left panel). Quantification of AURKB positive cardiomyocytes in the control and Gnas cKO mice (right panel). Scale bar: 10 μm. (c) Pellet of cardiomyocytes isolated from a WT and Gnas cKO heart at P14 (upper panel) and quantification of number of isolated cardiomyocytes (lower panel). Data are as mean ± SD, Student’s t-test, n=3. (d) Isolated cardiomyocytes from control and Gnas cKO mice at P14 (left panel). Scale bar: 100μm. Quantification of the area of isolated cardiomyocytes measured with Image J software (right panel). Data are as mean ± SD, 20 cardiomyocytes from 9 individual hearts were measured, Student’s t-test was used. (e) Wheat germ agglutinin (WGA) staining of P14 control and Gnas cKO heart sections (left panel). Quantification of relative pixel per area of WGA-positive cardiomyocytes (right panel). N.S., not significant. Data are as mean ± SD, Student’s t-test, n=8. Scale bar: 50μm.
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Immunobiological analysis revealed that the deletion of Gnas in the heart resulted in enhanced proliferation of cardiomyocytes as evidenced by the increased number of PH3-positive, and also AURKB-positive cardiomyocytes in Gnas cKO hearts (Figure 3e and f and Figure 3—figure supplement 1b). Further, we dissociated P14 hearts with collagenase and found that the number of cardiomyocytes was significantly increased in Gnas cKO hearts (Figure 3—figure supplement 1c). No significant difference in cardiomyocyte cell size was detected between the control and Gnas cKO hearts (Figure 3—figure supplement 1d, e), suggesting that the increased heart size was not due to cardiac hypertrophy. The percentage of mononucleated cardiomyocytes in Gnas cKO hearts was indeed higher than that in control hearts (Figure 3g). These data suggest that Gnas ablation leads to increased cardiomyocyte proliferation and heart size but not cardiac cell hypertrophy.

Inhibition of β1-AR-Gαs signaling promotes metabolic switch from fatty acid oxidation to glycolysis in cardiomyocytes

To determine the potential mechanisms by which inhibition of β-AR-Gαs promotes cardiomyocyte proliferation and cardiac regeneration, we performed RNA-sequencing analysis (RNA-Seq) using RNA isolated from P7 control, Gnas cKO, and β-blocker treated hearts. We identified approximately 2000 differentially regulated genes (fold change ≥1.2) between control vs. Gnas cKO and control vs. β-blocker treated hearts. We found that 1076 and 733 genes were down-regulated in Gnas cKO and β-blocker treated hearts, respectively, and that 109 genes overlapped between Gnas cKO and β-blocker treated hearts (Figure 4—figure supplement 1a). Gene ontology (GO) analysis using Enrichr Chen et al., 2013 indicated that the expression of genes related to fatty acid metabolism, a major source of energy for mature cardiomyocytes, was down-regulated in Gnas cKO and β-blocker treated hearts (Figure 4a). Gene set enrichment analysis (GSEA) also showed that fatty acid metabolism-related genes were down-regulated in Gnas cKO and β-blocker-treated hearts (Figure 4b, Figure 4—figure supplement 1c, d). In contrast, the expression of genes related to glycolysis, the metabolic pathway utilized by immature cardiomyocytes and associated with cardiomyocyte proliferation, was upregulated in Gnas cKO and β-blocker treated hearts (Figure 4—figure supplement 1c–f). Quantitative PCR (q-PCR) analysis of fatty acid metabolism-related genes confirmed the RNA-seq results (Figure 4c). Moreover, ultrastructural analysis of the heart by electron microscopy (EM) revealed elongated mitochondria in control hearts, whereas mitochondria of Gnas cKO hearts were small and round, which is a distinctive phenotype of immature mitochondria, suggesting down-regulation of total energy metabolism in Gnas cKO and β-blocker treated hearts. (Figure 4d). Consistent with EM images, the copy number of mitochondrial DNA was less in the Gnas cKO hearts (Figure 4e). These data suggest that cardiomyocytes in the Gnas cKO and β-blocker treated hearts exhibit a characteristic feature of immature cardiomyocytes.

Figure 4. Inhibition of βAR-Gαs signaling leads to elevation of YAP activity in the cardiomyocytes.

(a) Functional enrichment of GO terms for the common down-regulated genes in Gnas cKO and β-blocker treated hearts at P7 (fold change ≤0.8). (b) GSEA plot shows that fatty acid metabolic genes are down-regulated in Gnas cKO and β-blocker-treated hearts. (c) Q-PCR analysis of fatty acid metabolism related genes in control and Gnas cKO hearts at P14. Data are as mean ± SD, Student’s t-test, n=3. (d) Transmission electron microscopy images of mitochondria in ventricular cardiomyocytes of P14 control and Gnas cKO hearts. Scale bar: 600 nm. (e) Q-PCR analysis of mitochondrial DNA in control and Gnas cKO hearts at P14. Mitochondrial DNA copy number was normalized to nuclear DNA copy number (mtDN1 vs. H19, and mtDN2 vs. Mx1). Data are as mean ± SD, Student’s t-test, n=3. (f) Functional enrichment of GO term for the up-regulated genes in Gnas cKO and β-blocker treated hearts at P7 (fold change ≥1.2). (g) GSEA plot showed that YAP signature genes are up-regulated in Gnas cKO and β-blocker treated hearts. (h) Q-PCR analysis of YAP target gene expression, Ccn2 and Ankrd1, in control and β-blocker treated hearts at P14. Data are as mean ± SD, Student’s t-test, n=3. (i) YAP and cardiac α-actinin immunostaining of heart sections from control and β-blocker treated mice at P7 (n=3). Inset shows high-magnification image of nuclear YAP in CMs. Scale bar: 50μm. (j) Heart weight (HW) to body weight (BW) ratio of P14 control (n=15), control with β-blocker (n=13), Yap cKO (n=4), and Yap cKO with β-blocker (n=5) treated mice. Data are as mean ± SD, ANOVA test, N.S., not significant. (k) Quantification of the number of PH3 positive cardiomyocytes per view. Data are as mean ± SD, ANOVA test, n=4. N.S., not significant.

Figure 4.

Figure 4—figure supplement 1. Differential gene expression in Gnas cKO and β-blocker-treated hearts.

Figure 4—figure supplement 1.

(a) The Venn diagram shows the down-regulated genes in Gnas cKO and β-blocker treated hearts at P7 (fold change ≤0.8). The total and overlay numbers of down-regulated genes are 1700 and 109, respectively. The overlapped genes were used for GO analysis. (b) The Venn diagram shows the up-regulated genes in Gnas cKO and β-blocker-treated hearts at P7 (fold change ≥1.2). The total and overlay numbers of up-regulated genes are 2860 and 161, respectively. The overlapped genes were used for GO analysis. (c) Heat map of fatty acid metabolism, glycolysis, and YAP signature related gene expression from RNA-seq data, Control vs. Gnas cKO hearts. (d) Heat map of fatty acid metabolism, glycolysis, and YAP signature related gene expression from RNA-seq data, Control vs. β-blocker-treated hearts. (e) GSEA identified significant enrichment of glycolysis related gene expression in Gnas cKO hearts. (f) GSEA identified significant enrichment of glycolysis related gene expression in β-blocker treated hearts.
Figure 4—figure supplement 2. YAP activity is regulated by Gαs.

Figure 4—figure supplement 2.

(a) Western blot analysis of lysate from P4, P7, and P9 hearts using phospho-YAP (P-YAP) or total YAP antibodies. (b) YAP and cardiac α-actinin immunostaining of P4 and P7 control and Gnas cKO heart sections (left panel). Inset shows high-magnification image of nuclear YAP in CMs. Scale bar: 25μm. Quantification of nuclear YAP localization in cardiomyocytes of control and Gnas cKO hearts (right panel). Data are as mean ± SD, Student’s t-test, n≥3. (c) Fractionation assay of P14 control and β-blocker-treated hearts. Histone H3 is a positive marker for the nuclear fraction and GAPDH is a positive marker for the cytoplasmic fraction. (d) Fractionation assay of P14 control and Gnas cKO hearts. Histone H3 is a positive marker for the nuclear fraction and GAPDH is a positive marker for the cytoplasmic fraction. (e) Schematic illustration of experimental design. Tamoxifen (TAM) was injected into Gnasflox/flox; Myh6MerCreMer (Gnas-MCM) mice at P14, and hearts were harvested at P17 (upper panel). YAP and cardiac α-actinin co-immunostaining of heart sections from control and Gnas cKO hearts at P17 (lower panel). Inset shows high-magnification image of nuclear YAP in CMs. Scale bar: 50μm. (f) YAP immunostaining of P4 WT and Gnas cKO hearts treated with saline or epinephrine (left panel). Scale bar: 25μm. Quantification of nuclear YAP localization in cardiomyocytes of P4 WT and Gnas cKO heart treated with saline or epinephrine (right panel). Data are as mean ± SD, Student’s t-test, n=3. N.S., not significant. The online version of this article includes the source data for Figure 4-figure supplement 2.
Figure 4—figure supplement 2—source data 1. Raw data of Western Blots.
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Inhibition of β1-AR-Gαs signaling promotes YAP transcriptional activity leading to proliferative immature cardiomyocytes

We found that 1176 and 1845 genes were up-regulated in Gnas cKO and β-blocker-treated hearts, respectively, and that 161 overlapping genes identified between Gnas cKO and β-blocker-treated hearts (Figure 4—figure supplement 1b). GO and GSEA analysis indicated that the expression of genes related to the Hippo signaling pathway was increased in Gnas cKO and β-blocker-treated hearts (Figure 4f and g, Figure 4—figure supplement 1c, d). q-PCR analysis confirmed the upregulation of YAP target genes Ccn2 and Ankrd1 in Gnas cKO and β-blocker treated hearts (Figure 4h). YAP cellular localization in cardiomyocytes correlates with heart regenerative capacity. Nuclear YAP is high at P4 when the heart remains regenerative, but low at P7 when the regeneration window is closed. These results are confirmed by western blotting analysis that there is an increase in phospho-YAP (inactive form of YAP) in cardiomyocytes from P4 to P9 mice (Figure 4—figure supplement 2a, b). Consistent with upregulated YAP target gene expression, blockade of β1-AR-Gαs signaling by either β-blocker treatment or deletion of Gnas promoted retention of YAP nuclear localization in the P7 hearts (Figure 4i, Figure 4—figure supplement 2b). Fractionation assay also showed that more YAP was detected in the nuclear fraction of P14 β-blocker-treated and Gnas cKO hearts compared with littermate controls (Figure 4—figure supplement 2c, d). Furthermore, nuclear YAP was detected when the Gnas gene was deleted later at P14 upon tamoxifen treatment in the Gnas-Myh6MerCreMer cKO mouse line (Figure 4—figure supplement 2e). This suggests that Gαs inhibits YAP nuclear localization not only in juvenile but also in young-adult cardiomyocytes. Conversely, when Gαs was activated by epinephrine, an agonist for β-AR, YAP was detected mainly in the cytoplasm at P4 in the control mice; however, YAP remained in the nucleus in the Gnas cKO cardiomyocytes when treated with epinephrine (Figure 4—figure supplement 2f), suggesting that β-AR signaling inhibits YAP nuclear localization through Gαs activation during cardiomyocyte maturation.

Ablation of YAP abolished the β-blocker induced cardiomyocyte proliferation

We next investigated whether the increased proliferation phenotype seen with β-blocker treatment was caused by an activation of YAP. We performed β-blocker treatment in the cardiac specific Yap1 knockout mice Yap1; Myh6Cre (Yap cKO), which did not show any morphological phenotype in hearts at P14, and found that β-blocker treatment did not increase cardiomyocyte proliferation in the Yap cKO hearts (Figure 4j and k), suggesting that β-blocker-induced cardiomyocyte proliferation is dependent on YAP functions. Together, our data suggest that Gαs mediates adrenergic signaling to inhibit cardiomyocyte proliferation via inhibition of YAP activity.

Gαs inhibits cardiomyocyte proliferation through inactivation of the Rho signaling pathway

To gain insight into the molecular mechanisms of how Gαs regulates YAP, we performed pathway analysis based on transcriptomic profiles and found that Rho signaling pathway was activated in Gnas cKO and β-blocker-treated hearts (Figure 5a). To examine whether Gαs regulates RhoA activity in vivo, we performed active-RhoA pull-down assay using P7 control and Gnas cKO hearts. As expected, RhoA activity was increased while the phospho-YAP, the inactive form of YAP (cytoplasmic localized) was dramatically decreased in the Gnas cKO heart, compared with littermate controls at P7 (Figure 5b).

Figure 5. GαS regulates cardiomyocyte proliferation through RhoA mediated YAP activation.

(a) Functional enrichment of GO terms for the common up-regulated genes. (b) Active-RhoA pull-down assay and western blot analysis of P7 control and Gnas cKO hearts. (c) Active-RhoA pull-down assay and western blot analysis of cultured cardiomyocytes with epinephrine treatment. (d) Immunostaining of YAP in cultured cardiomyocytes treated with C3 (Rho inhibitor). Scale bar: 50μm (left panel) and 25μm (right panel). (e) EdU incorporation assay on rat neonatal cardiomyocytes treated with Rho inhibitors (left panel). Quantification of EdU-labelled proliferating cardiomyocytes stained with cardiac α-actinin. Data are as mean ± SD, n=3 (right panel). Scale bar: 50μm. (f) Model of β1-AR-Gαs signaling regulation of cardiomyocyte proliferation. The online version of this article includes the source data for Figure 5.

Figure 5—source data 1. Raw data of Western Blots.

Figure 5.

Figure 5—figure supplement 1. RhoA activity is only detected in the embryonic and postnatal hearts.

Figure 5—figure supplement 1.

Active-RhoA pull-down assay of wild type hearts at several time points. The online version of this article includes the following source data for Figure 5—figure supplement 1.
Figure 5—figure supplement 1—source data 1. Raw data of Western Blots.
Figure 5—figure supplement 2. Comparison of heart rate at different ages.

Figure 5—figure supplement 2.

Heart rate at P0, P7, and P14 in C57BL6 mice. Data are as mean ± SD, n≥5.
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To confirm this inhibitory effect of Gαs against RhoA, we stimulated Gαs with epinephrine in cultured neonatal rat cardiomyocytes. Epinephrine treatment resulted in a decrease in RhoA activity and an increase in YAP phosphorylation (Figure 5c). Moreover, when cardiomyocytes were treated with C3 toxin, a Rho inhibitor, YAP was localized in the cytoplasm, while YAP remained in the nucleus in the saline-treated cardiomyocytes (Figure 5d). Treatment with the Rho inhibitors C3 and G04 greatly reduced cardiomyocyte proliferation, as demonstrated by EdU incorporation (Figure 5e). Thus, our data suggest that β1-AR-Gαs signaling negatively regulates cardiomyocyte proliferation through the inhibition of RhoA mediated YAP activity. Given that pharmacological inhibition of Gαs promotes cardiomyocyte proliferation through activation of YAP, β-blocker could be used as a potential therapeutic strategy to promote cardiomyocyte proliferation and heart regeneration (Figure 5f).

Discussion

Since the adult mammalian heart has limited potential for re-entry into the cell cycle, cardiomyocyte replenishment after the loss of cardiomyocytes due to myocardial infarction is insufficient to restore heart function Zhao et al., 2020; Cardoso et al., 2020; Broughton et al., 2018. To repair or improve heart function of the injured heart, several strategies using cellular therapies, such as direct cardiac reprogramming, and noncellular therapies have been published Wang et al., 2021; Hashimoto et al., 2018; Tzahor and Poss, 2017; He et al., 2020. In this study, we identify β1-adrenergic/Gαs-protein signaling as an inhibitory pathway that restricts the capacity of cardiomyocytes to return to immature proliferative states. When the heart was injured at P7, we show that both pharmacological and genetic inhibition of β1-adrenergic/Gαs-protein reactivate cardiomyocyte proliferation programs and heart regeneration by activating Hippo-YAP signaling, suggesting that β1-adrenergic/Gαs-YAP signaling contributes to extend the cardiac regeneration window at the juvenile stage. At the young-adult age, combination treatment with α/β-blocker and thyroid hormone inhibitor, but not β-blocker itself, is required to enhance cardiomyocyte regeneration after MI surgery Payumo et al., 2021, suggesting that additional molecules might be needed to promote adult cardiac regeneration. Further investigation is needed to elucidate the molecular mechanism of how different pathways are involved in promoting adult heart regeneration.

Although neonatal cardiomyocytes have the capacity to proliferate, this regenerative potential is lost in mice one week after birth. During this narrow window, cardiomyocytes undergo one round of DNA synthesis and nuclear division without cytokinesis, which leads to binucleated cardiomyocytes and cell cycle arrest Li et al., 1996. At the same time, metabolism in cardiomyocytes switches from glycolysis to fatty acid metabolism, and contractile proteins change from embryonic to neonatal isoforms Ng et al., 1991; Kolwicz et al., 2013; Lopaschuk and Jaswal, 2010. It has been suggested that cardiomyocyte maturation is conversely correlated with its proliferative ability with the increase of DNA content and well aligned contractile structures. Recent studies have also demonstrated that oxidative stress after birth is one driver for cell cycle arrest in neonatal cardiomyocytes Nakada et al., 2017; Kimura et al., 2015. Despite these findings, the molecular mechanisms of postnatal cardiomyocyte cell cycle withdrawal are not fully understood. In the present study, we found that Gαs signaling activity was correlated with loss of proliferative or regenerative ability of neonatal cardiomyocytes, and inhibition of Gαs activity promotes mono-nucleated cardiomyocyte division and cardiac regeneration through YAP transcriptional activity after the regenerative window. In the canonical Hippo signaling pathway, the major YAP regulators are the LATS1/2 kinases, which phosphorylate and inhibit YAP activity. A previous study reported a model that Rho GTPases inhibit LATS1/2 activity through actin cytoskeleton organization and activate YAP transcriptional activity. Therefore, we consider that Gαs-Rho-cytoskeleton-LATS pathway inhibits YAP and induces loss of proliferative or regenerative ability during cardiomyocyte maturation. Together, inhibition of Gαs induces the de-differentiation of mature cardiomyocytes to an immature state and reactivates YAP transcriptional activity to extend the regeneration window.

It has been shown that cardiac Gαs regulates heart rate and myocardial contractility Molkentin and Dorn, 2001; Tilley and Rockman, 2011. However, the role of Gαs in cardiomyocytes proliferation is still unclear. Our cardiomyocyte-specific Gnas KO mice showed an enlarged heart phenotype with an increase in cardiomyocyte proliferation at the juvenile stage. Conversely, transgenic mice overexpressing Gαs displayed myocardial hypertrophy with increased myocardial collagen content and fibrosis Iwase et al., 1997. Together, these results suggest that Gαs plays an important role in maintaining cardiomyocyte homeostasis.

Several signals such as mechanical and oxidative stress have been identified as regulators of the Hippo signaling pathway Wang et al., 2016b; Wang et al., 2016a; Lehtinen et al., 2006; Geng et al., 2015. YAP is known as a mechanical sensor due to its ability to alter its localization in response to various environmental stimuli, including alternation of cytoskeletal dynamics and blood flow Wang et al., 2016b; Wang et al., 2016a; Foster et al., 2017; Vite et al., 2018. We found that treatment of cultured cardiomyocytes with a Rho inhibitor hinders YAP activity. This is consistent with a previous study showing that the Rho-mediated pathway promotes nuclear localization of YAP in human embryonic stem cells Yu et al., 2012. At present, it is still unclear whether Rho directly regulates YAP activity in cardiomyocytes. A previous report using a cardiomyocyte-specific RhoA transgene revealed heart rate depression and decreased cardiomyocyte contraction Sah et al., 1999. We found that RhoA activity was gradually downregulated between P0 and P14 (Figure 5—figure supplement 1), and heart rate gradually increased from ~250 beats/min at P0 to ~550 beats/min at P14 (Sato, 2008; Figure 5—figure supplement 2). During this period, YAP translocates from the nucleus to the cytoplasm, suggesting that RhoA may regulate YAP activity by inhibiting cardiomyocyte contraction rate.

β-AR signaling has been shown to increase cardiac output by enhancing heart rate and contractility via activation of Gαs protein Salazar et al., 2007; Bers, 2002. β1-AR is expressed in all cardiomyocytes with equal distribution between the left and right ventricles. On the other hand, β2-AR is not only expressed in the cardiomyocytes, but also in vascular and non-vascular smooth muscle cells in multiple tissues. Both β1- and β2-AR are coupled to GαS, while β2-AR can be coupled to Gαi/o as well Tilley and Rockman, 2011. Stimulation of β1-AR results in adenylyl cyclase-mediated cAMP generation and activation of PKA, MAP kinase, and calmodulin-dependent protein kinase II (CAMKII) Bers, 2002; Oestreich et al., 2009; Mangmool et al., 2010. Clinical studies have indicated that β-blockers, especially the β1-AR-specific blocker, metoprolol, improve cardiac function and reduce mortality in patients with heart failure and MI MERIT-HF Study group, 1999. However, mechanisms underlying the therapeutic effects of β-blockers in heart failure patients are poorly understood. A recent study indicated that in heart failure patients, YAP activity is inactivated by phosphorylation, and that blocking the inhibitory kinase LATS1/2 can reverse heart failure post-MI in mice Leach et al., 2017. Similarly, we demonstrate that at the juvenile stage, β-blocker treatment could promote nuclear YAP translocation and cardiac regeneration after MI. Given that YAP activity enhances cardiac regeneration and promotes survival post-MI in our mouse models, activation of Hippo-YAP signaling might be one of the reasons why β-blocker treatment is able to improve heart function in patients.

Materials and methods

Mouse experiments

All animal experiments were performed with the approval of the Institutional Animal Care and Use Committee of Cincinnati Children’s Hospital Medical Center. Mouse lines harboring the Gnas and Yap floxed alleles have been described previously Xin et al., 2011; Chen et al., 2005. The α-Myosin heavy chain (Myh6)-Cre (Myh6Cre) and Myh6MerCreMer mice were obtained from Jeff Robbins and Jeffery D. Molkentin, respectively Sanbe et al., 2003; Sohal et al., 2001. Tamoxifen (Sigma) was dissolved in 90% sunflower oil (Sigma)/10% ethanol and stored at –20℃. The tamoxifen solution was injected by intraperitoneal (IP) injection once a day (50 mg/kg). For 5-ethynyl-2-deoxyuridine (EdU) studies, mice were administered an IP injection of EdU (5 μg/kg) once a day. The β-blocker (metoprolol) was dissolved in saline and injected by IP (2 mg/kg) once a day. C57BL/6 J mice were used for the β-blocker treatment studies.

Myocardial infarction

To induce myocardial infarction (MI) in juvenile mice, we permanently ligated the left anterior descending artery on P7 as previously described Blom et al., 2016; Mahmoud et al., 2014. Briefly, mice were anaesthetized with isoflurane and the heart was exposed via thoracotomy through the fourth or fifth intercostal space. An 8–0 nylon suture was tied around the left anterior descending coronary artery (LAD). Subsequently, the chest and skin were closed in layers using 6–0 nylon sutures. The mouse was allowed to recover from surgery on a heating pad. Sham-operated mice underwent the same procedure involving anesthesia and thoracotomy without LAD ligation.

Echocardiography

Assessment of cardiac function on conscious, non-sedated mice was performed with the Vivo 2100 micro-ultrasound system (VisualSonics). Cardiac function and heart rate were measured on M-mode and doppler images.

Neonatal rat cardiomyocyte isolation and culture

Neonatal rat cardiomyocyte culture was performed using the neonatal cardiomyocyte isolation kit (Cellutron). P2 neonatal cardiomyocytes were plated on tissue culture dishes pre-coated with SureCoat (Cellutron) at a density of 2x105/cm2. After 24 hr, cardiomyocytes were treated with epinephrine (100 μM, Sigma), C3 (1 mg/ml, Cytoskeleton), and GO4 (100 μM, provided by Dr. Yi Zheng).

Mouse cardiomyocyte isolation

The mouse cardiomyocyte isolation was performed as previously described Mahmoud et al., 2013. In brief, P14 hearts were harvested and immediately fixed with 4% paraformaldehyde (PFA) at 4°C overnight. Subsequently, samples were incubated with collagenase B (1.8 mg/ml, Roche) and D (2.4 mg/ml, Roche) for 12 hr at 37°C. The hearts were minced to smaller pieces and the procedure was repeated until no more cardiomyocytes were dissociated from the tissue. The digested cardiomyocytes were stained with 4',6-diamidino-2-phenylindole (DAPI) for nucleation counts. A hemocytometer was used for counting cardiomyocytes.

Assessment of cardiomyocyte size

The cross-sectional area of cardiomyocytes was assessed using wheat germ agglutinin (WGA) staining. Cryosections were rinsed in PBS and then incubated with WGA conjugated with Alexa Fluor 488 (1:100, Invitrogen). Slides were imaged by Eclipse Ti confocal microscopy with a C2 laser-scanning head (Nikon). ImageJ software (National Institutes of Health) was used to quantify the size of each cell. The area of the digested cardiomyocytes was quantified using ImageJ software based on phase contrast images.

Histological analysis

Hearts were fixed in 4% PFA at 4°C overnight, embedded in paraffin, and sectioned at 5 μm thickness. Hematoxylin and eosin (H&E) staining was performed following standard protocol. Masson’s trichrome and Picrosirius red staining was performed according to standard procedures at CCHMC’s pathology core. Fibrotic area was quantified using ImageJ software.

Immunofluorescence experiments

For immunostaining, hearts were fixed in 4% PFA at 4 °C overnight, embedded in OCT compound (Sakura), and sectioned at 8 μm thickness. For PCM1 staining, we used fresh-frozen (non-fixed) samples and sections were fixed in 10% formalin for 10 min. Sections were blocked with 1% bovine serum albumin (BSA), incubated with primary antibodies against PH3 (rabbit polyclonal, 1:200; Millipore), PCM1 (1:1000; Sigma), cardiac α-actinin (1:200; Sigma), cardiac Troponin T (1:200; Thermo), YAP (1:100; Cell Signaling), and smooth muscle α-actin conjugated with AlexaFluor594 (1:200; Sigma), and were further incubated with Alexa Fluor-conjugated secondary antibodies against mouse or rabbit IgG and with DAPI. For EdU staining, postnatal mice were administered an intraperitoneal (IP) injection of EdU (5 μg/g of mouse body weight) at P5, P6, P12, and P13, and we collected the hearts at P14. EdU incorporation was assessed using Click-IT EdU system (Invitrogen). Fluorescent images were captured using Eclipse Ti confocal microscopy with a C2 laser-scanning head (Nikon).

RNA sequencing

RNA was extracted from hearts using TRIzol (Invitrogen) followed by purification using RNeasy Mini kit (Qiagen). RNA-seq was performed using two individual animals for control and Gnas cKO hearts, or control and β-blocker treated hearts. RNA sequencing was performed by the Center for Medical Genomics, Indiana University School of Medicine. The RNA-seq data generated for this study have been made publicly available via NCBI’s GEO (GSE186099). Gene Ontology analysis of gene expression changes was performed using Enrichr Chen et al., 2013 and Gene Set Enrichment Analysis (GSEA) software.

Real-time qPCR

Total RNA was isolated using TRIzol according to the manufacture’s protocol. cDNA was synthesized from 500 ng of total RNA using PrimeScript RT Master Mix (Takara). Quantitative real-time PCR (qPCR) was performed using SYBR-Green Master Mix (KAPA) on a StepOnePlus Real-Time PCR system (Applied Biosystems). Values for specific genes were normalized to 18s ribosomal RNA.

Active RhoA assay

RhoA activity was examined by an effector domain, GST-fusion pull-down protocol, as previously described Zhu et al., 2000. Cultured cardiomyocytes or heart tissues were lysed in a lysis buffer containing 1% Triton X-100 and incubated with the glutathione bead-bound GST-Rhotekin. The bead-immobilized GTP-bound RhoA and total RhoA in the lysates were probed by immunoblotting with anti-RhoA antibody (Cell Signaling).

Western blot analysis

Cultured cardiomyocytes or heart tissues were lysed with 2x sample buffer (BioRad) containing 2-mercaptoethanol and heated for 5 min at 95°C. Equal amounts of protein were run on SDS-polyacrylamide gel and transferred to Immobilon-P membrane (Millipore). Membranes were incubated with anti-YAP (Novus), anti-phospho-YAP (Cell Signaling), and anti-GAPDH (Cell Signaling) antibodies at 4℃ overnight. Anti-rabbit horse-radish peroxidase (GE healthcare) was used as the secondary antibody, followed by detection with Super Signal West Pico chemiluminescent substrate (Thermo).

Fractionation assay

The fractionation assay was performed as previously described Chen et al., 2009. For the cytoplasmic extract, hearts were lysed in hypotonic buffer (10 mM HEPES (pH 8), 1.5 mM MgCl2, 10 mM KCl, and 1 mM DTT). Nuclei were then resuspended in hypotonic buffer with 420 mM NaCl, 0.2 mM EDTA, 25% glycerol, 1% NP-40, and 1 mM PMSF.

Statistical analysis

All datasets were taken from n≥3 biological replicates. Used animal numbers or group numbers are described in the respective figure legends. Animals were genotyped before the experiments and were caged together and treated in the same way. The experiments were not randomized. We calculated p values with unpaired Student’s t test or analysis of variance (ANOVA) followed by Tukey-Kramer test with Excel (Microsoft Office). P-value < 0.05 was considered to represent a statistically significant difference. Data are presented as mean ± SD.

Acknowledgements

The authors thank Dr. Jeff Molkentin for insightful discussions and suggestions; Dr. Masayuki Fujii for helpful discussions; Zhifei Xu, Bin Liu, and Hui Sun for technical support; Lingli Xu for Data analysis; Dr. Yi Zheng for the Rho inhibitor GO4; Dr. Eric N Olson for Yap floxed mice; Dr. Lee Weinstein for Gnas floxed mice. This work was supported by the National Institutes of Health (Grant HL-132211).

Appendix 1

Appendix 1—key resources table.

Reagent type (species) or resource Designation Source or reference Identifiers Additional information
gene (Mus musculus) Gnas NCBI Gene:14683
strain, strain
background
(Mus musculus)		 C57BL/6 J The Jackson
Laboratory		 IMSR_JAX:000664
genetic reagent
(Mus musculus)		 Gnas flox/flox The Jackson
Laboratory		 IMSR_JAX:035239
genetic reagent
(Mus musculus)		 Yap flox/flox Xin et al., 2011
genetic reagent
(Mus musculus)		 Myh6-Cre Sanbe et al., 2003
genetic reagent
(Mus musculus)		 Myh6-MerCreMer Sohal et al., 2001
biological sample
(Rattus norvegicus)		 Primary neonatal rat
cardiomyocytes		 In this paper Freshly isolated
from neonatal rats
antibody PCM1 (rabbit
polyclonal)		 Sigma Car#: HPA023370 IHC (1:1000)
antibody Phspho-histon H3 (PH3)
(mouse monoclonal)		 Millipore Car#: 05–806 IHC (1:100)
antibody Sarcomeric a-actinin
(mouse monoclonal)		 Sigma Car#: A7811 IHC (1:200)
antibody Cardiac Troponin T
(mouse monoclonal)		 Thermo Car#: MA295-P1 IHC (1:200)
antibody YAP (Rabbit
monoclonal)		 Cell Signaling Cat#: 14074 IHC (1:100)
antibody YAP (Rabbit
polyclonal)		 Novus Cat#: NB110-58358 WB (1:1000)
antibody Phospho-YAP
(Rabbit monoclonal)		 Cell Signaling Cat#: 13008 WB (1:1000)
antibody RhoA (Rabbit
monoclonal)		 Cell Signaling Cat#: 2117 WB (1:1000)
antibody GAPDH (Rabbit
monoclonal)		 Cell Signaling Cat#: 2118 WB (1:1000)
antibody Ki67 (Rabbit
monoclonal)		 Thermo Cat#: RM9106 IHC (1:200)
antibody Aurora B (AurkB)
(Rabbit polyclonal)		 Abcam Cat#: ab2254 IHC (1:100)
antibody Histon H3 (Rabbit polyclonal) Abcam Cat#: ab1791 WB (1:3000)
sequence-
based reagent		 Hmgcs2-F This paper qPCR primer GAAGAGAGCGA
TGCAGGAAAC
sequence-
based reagent		 Hmgcs2-R This paper qPCR primer GTCCACATATT
GGGCTGGAAA
sequence-
based reagent		 Nqo1-F This paper qPCR primer AGGATGGGAG
GTACTCGAATC
sequence-
based reagent		 Nqo1-R This paper qPCR primer TGCTAGAGATG
ACTCGGAAGG
sequence-
based reagent		 Pla2g4e-F This paper qPCR primer AGGTGGAGTTC
CTACTCGAAG
sequence-
based reagent		 Pla2g4e-R This paper qPCR primer TGTTCTCGAAGG
AGTCTGTCA
sequence-
based reagent		 Pla2g5-F This paper qPCR primer CCAGGGGGCT
TGCTAGAA
sequence-
based reagent		 Pla2g5-R This paper qPCR primer AGCACCAATC
AGTGCCATCC
sequence-
based reagent		 mtDN1-F This paper qPCR primer CTCTTATCCACG
CTTCCGTTACG
sequence-
based reagent		 mtDN1-R This paper qPCR primer GATGGTGGTAC
TCCCGCTGTA
sequence-
based reagent		 mtDN2-F This paper qPCR primer CCCATTCCACT
TCTGATTACC
sequence-
based reagent		 mtDN2-R This paper qPCR primer ATGATAGTAGAG
TTGAGTAGCG
sequence-
based reagent		 CTGF-F This paper qPCR primer GGGCCTCTT
CTGCGATTTC
sequence-
based reagent		 CTGF-R This paper qPCR primer ATCCAGGCAAG
TGCATTGGTA
sequence-
based reagent		 Ankrd1-F This paper qPCR primer GGATGTGCCGA
GGTTTCTGAA
sequence-
based reagent		 Ankrd1-R This paper qPCR primer GTCCGTTTATAC
TCATCGCAGAC
commercial
assay or kit		 Neonatal cardiomyocyte
isolation kit		 Cellutron NC-6031
commercial
assay or kit		 RNeasy mini kit Qiagen 74104
chemical
compound, drug		 Epinephrine Sigma E4375 100 mM
chemical
compound, drug		 C3 Cytoskeleton CT04 1 mg/ml
chemical
compound, drug		 GO4 Provided by
Dr. Yi Zheng		 100 mM
chemical
compound, drug		 Tamoxifen Sigma T5648 50 mg/kg, IP injection
chemical
compound, drug		 Metoprolol (b-blocker) Sigma M5391 2 mg/kg, IP injection
chemical
compound, drug		 5-ethynyl-2-
deoxyuridine (EdU)		 Thermo Cat#: C10337 5 mg/kg, IP injection
software,
algorithm		 ImageJ National Institutes of
Health (NIH)
other 4',6-diamidino-2-
phenylindole (DAPI)		 Invitrogen D1306 For nuclear staining
other Wheat germ
agglutinin (WGA)		 Invitrogen W11261 For plasma membrane staining
other Collagenase B Roche 11088815001 1.8 mg/ml For heart digestion
other Collagenase D Roche 11088866001 2.4 mg/ml For heart digestion
other 2,3,5-
Tripherylterazolium
chloride (TTC)		 Sigma T8877 For staining of ischemic region
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Funding Statement

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Contributor Information

Mei Xin, Email: mei.xin@cchmc.org.

Enzo R Porrello, Murdoch Children's Research Institute, Australia.

Didier YR Stainier, Max Planck Institute for Heart and Lung Research, Germany.

Funding Information

This paper was supported by the following grant:

  • National Institutes of Health HL-132211 to Mei Xin.

Additional information

Competing interests

No competing interests declared.

No competing interests declared.

Author contributions

Conceptualization, Data curation, Formal analysis, Investigation, Visualization, Methodology, Writing – original draft.

Validation.

Validation.

Validation.

Validation.

Resources, Supervision, Validation, Writing - review and editing.

Conceptualization, Resources, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Writing – original draft, Project administration, Writing - review and editing.

Ethics

All animal experiments were performed with the approval of the Institutional Animal Care and Use Committee of Cincinnati Children's Hospital Medical Center (IACUC 2019-0086).

Additional files

Transparent reporting form

Data availability

RNA seq data have been deposited to GEO under accession code GSE186099.

The following dataset was generated:

Sakabe M, Xin M. 2021. Gene expression changes in beta-blocker treated neonatal hearts. NCBI Gene Expression Omnibus. GSE186099

References

  1. Bassat E, Mutlak YE, Genzelinakh A, Shadrin IY, Baruch Umansky K, Yifa O, Kain D, Rajchman D, Leach J, Riabov Bassat D, Udi Y, Sarig R, Sagi I, Martin JF, Bursac N, Cohen S, Tzahor E. The extracellular matrix protein agrin promotes heart regeneration in mice. Nature. 2017;547:179–184. doi: 10.1038/nature22978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bers DM. Cardiac excitation–contraction coupling. Nature. 2002;415:198–205. doi: 10.1038/415198a. [DOI] [PubMed] [Google Scholar]
  3. Blom JN, Lu X, Arnold P, Feng Q. Myocardial infarction in neonatal mice, a model of cardiac regeneration. Journal of Visualized Experiments. 2016;1:e100. doi: 10.3791/54100. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Broughton KM, Wang BJ, Firouzi F, Khalafalla F, Dimmeler S, Fernandez-Aviles F, Sussman MA. Mechanisms of cardiac repair and regeneration. Circulation Research. 2018;122:1151–1163. doi: 10.1161/CIRCRESAHA.117.312586. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Cannavo A, Liccardo D, Koch WJ. Targeting cardiac β-adrenergic signaling via GRK2 inhibition for heart failure therapy. Frontiers in Physiology. 2013;4:264. doi: 10.3389/fphys.2013.00264. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cannavo A, Koch WJ. Targeting β3-adrenergic receptors in the heart: selective agonism and β-blockade. Journal of Cardiovascular Pharmacology. 2017;69:71–78. doi: 10.1097/FJC.0000000000000444. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cardoso AC, Pereira AHM, Sadek HA. Mechanisms of neonatal heart regeneration. Current Cardiology Reports. 2020;22:33. doi: 10.1007/s11886-020-01282-5. [DOI] [PubMed] [Google Scholar]
  8. Chen M, Gavrilova O, Zhao WQ, Nguyen A, Lorenzo J, Shen L, Nackers L, Pack S, Jou W, Weinstein LS. Increased glucose tolerance and reduced adiposity in the absence of fasting hypoglycemia in mice with liver-specific Gsα deficiency. Journal of Clinical Investigation. 2005;115:3217–3227. doi: 10.1172/JCI24196. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Chen Y, Wu H, Wang S, Koito H, Li J, Ye F, Hoang J, Escobar SS, Gow A, Arnett HA, Trapp BD, Karandikar NJ, Hsieh J, Lu QR. The oligodendrocyte-specific G protein-coupled receptor GPR17 is a cell-intrinsic timer of myelination. Nature Neuroscience. 2009;12:1398–1406. doi: 10.1038/nn.2410. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Chen EY, Tan CM, Kou Y, Duan Q, Wang Z, Meirelles GV, Clark NR, Ma’ayan A. Enrichr: interactive and collaborative HTML5 gene list enrichment analysis tool. BMC Bioinformatics. 2013;14:128. doi: 10.1186/1471-2105-14-128. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Derks W, Bergmann O. Polyploidy in cardiomyocytes: roadblock to heart regeneration? Circulation Research. 2020;126:552–565. doi: 10.1161/CIRCRESAHA.119.315408. [DOI] [PubMed] [Google Scholar]
  12. D’Uva G, Aharonov A, Lauriola M, Kain D, Yahalom-Ronen Y, Carvalho S, Weisinger K, Bassat E, Rajchman D, Yifa O, Lysenko M, Konfino T, Hegesh J, Brenner O, Neeman M, Yarden Y, Leor J, Sarig R, Harvey RP, Tzahor E. Erbb2 triggers mammalian heart regeneration by promoting cardiomyocyte dedifferentiation and proliferation. Nature Cell Biology. 2015;17:627–638. doi: 10.1038/ncb3149. [DOI] [PubMed] [Google Scholar]
  13. Flacco N, Segura V, Perez-Aso M, Estrada S, Seller JF, Jiménez-Altayó F, Noguera MA, D’Ocon P, Vila E, Ivorra MD. Different β-adrenoceptor subtypes coupling to cAMP or NO/cGMP pathways: implications in the relaxant response of rat conductance and resistance vessels. British Journal of Pharmacology. 2013;169:413–425. doi: 10.1111/bph.12121. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Foster CT, Gualdrini F, Treisman R. Mutual dependence of the MRTF-SRF and YAP-TEAD pathways in cancer-associated fibroblasts is indirect and mediated by cytoskeletal dynamics. Genes & Development. 2017;31:2361–2375. doi: 10.1101/gad.304501.117. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Frey UH, Muehlschlegel JD, Ochterbeck C, Fox AA, Shernan SK, Collard CD, Lichtner P, Peters J, Body S. Gnas gene variants affect β-blocker-related survival after coronary artery bypass grafting. Anesthesiology. 2014;120:1109–1117. doi: 10.1097/ALN.0000000000000189. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Geng YJ, Ishikawa Y, Vatner DE, Wagner TE, Bishop SP, Vatner SF, Homcy CJ. Apoptosis of cardiac myocytes in Gsalpha transgenic mice. Circulation Research. 1999;84:34–42. doi: 10.1161/01.res.84.1.34. [DOI] [PubMed] [Google Scholar]
  17. Geng J, Sun X, Wang P, Zhang S, Wang X, Wu H, Hong L, Xie C, Li X, Zhao H, Liu Q, Jiang M, Chen Q, Zhang J, Li Y, Song S, Wang HR, Zhou R, Johnson RL, Chien KY, Lin SC, Han J, Avruch J, Chen L, Zhou D. Kinases MST1 and MST2 positively regulate phagocytic induction of reactive oxygen species and bactericidal activity. Nature Immunology. 2015;16:1142–1152. doi: 10.1038/ni.3268. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hansen CG, Moroishi T, Guan KL. YAP and TAZ: a nexus for hippo signaling and beyond. Trends in Cell Biology. 2015;25:499–513. doi: 10.1016/j.tcb.2015.05.002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Hashimoto H, Olson EN, Bassel-Duby R. Therapeutic approaches for cardiac regeneration and repair. Nature Reviews. Cardiology. 2018;15:585–600. doi: 10.1038/s41569-018-0036-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. He L, Nguyen NB, Ardehali R, Zhou B. Heart regeneration by endogenous stem cells and cardiomyocyte proliferation: controversy, fallacy, and progress. Circulation. 2020;142:275–291. doi: 10.1161/CIRCULATIONAHA.119.045566. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Heallen T, Zhang M, Wang J, Bonilla-Claudio M, Klysik E, Johnson RL, Martin JF. Hippo pathway inhibits wnt signaling to restrain cardiomyocyte proliferation and heart size. Science. 2011;332:458–461. doi: 10.1126/science.1199010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Heallen T, Morikawa Y, Leach J, Tao G, Willerson JT, Johnson RL, Martin JF. Hippo signaling impedes adult heart regeneration. Development. 2013;140:4683–4690. doi: 10.1242/dev.102798. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Hirose K, Payumo AY, Cutie S, Hoang A, Zhang H, Guyot R, Lunn D, Bigley RB, Yu H, Wang J, Smith M, Gillett E, Muroy SE, Schmid T, Wilson E, Field KA, Reeder DM, Maden M, Yartsev MM, Wolfgang MJ, Grützner F, Scanlan TS, Szweda LI, Buffenstein R, Hu G, Flamant F, Olgin JE, Huang GN. Evidence for hormonal control of heart regenerative capacity during endothermy acquisition. Science. 2019;364:184–188. doi: 10.1126/science.aar2038. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Iwase M, Uechi M, Vatner DE, Asai K, Shannon RP, Kudej RK, Wagner TE, Wight DC, Patrick TA, Ishikawa Y, Homcy CJ, Vatner SF. Cardiomyopathy induced by cardiac Gs alpha overexpression. American Journal of Physiology-Heart and Circulatory Physiology. 1997;272:H585–H589. doi: 10.1152/ajpheart.1997.272.1.H585. [DOI] [PubMed] [Google Scholar]
  25. Kim NG, Koh E, Chen X, Gumbiner BM. E-cadherin mediates contact inhibition of proliferation through hippo signaling-pathway components. PNAS. 2011;108:11930–11935. doi: 10.1073/pnas.1103345108. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Kimura W, Xiao F, Canseco DC, Muralidhar S, Thet S, Zhang HM, Abderrahman Y, Chen R, Garcia JA, Shelton JM, Richardson JA, Ashour AM, Asaithamby A, Liang H, Xing C, Lu Z, Zhang CC, Sadek HA. Hypoxia fate mapping identifies cycling cardiomyocytes in the adult heart. Nature. 2015;523:226–230. doi: 10.1038/nature14582. [DOI] [PubMed] [Google Scholar]
  27. Kolwicz SC, Purohit S, Tian R. Cardiac metabolism and its interactions with contraction, growth, and survival of cardiomyocytes. Circulation Research. 2013;113:603–616. doi: 10.1161/CIRCRESAHA.113.302095. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Ladage D, Schwinger RHG, Brixius K. Cardio-Selective beta-blocker: pharmacological evidence and their influence on exercise capacity. Cardiovascular Therapeutics. 2013;31:76–83. doi: 10.1111/j.1755-5922.2011.00306.x. [DOI] [PubMed] [Google Scholar]
  29. Leach JP, Heallen T, Zhang M, Rahmani M, Morikawa Y, Hill MC, Segura A, Willerson JT, Martin JF. Hippo pathway deficiency reverses systolic heart failure after infarction. Nature. 2017;550:260–264. doi: 10.1038/nature24045. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Lehtinen MK, Yuan Z, Boag PR, Yang Y, Villen J, Becker EB, DiBacco S, Iglesia N, Gygi S, Blackwell TK, Bonni A. A conserved MST-FOXO signaling pathway mediates oxidative-stress responses and extends life span. Cell. 2006;125:987–1001. doi: 10.1016/j.cell.2006.03.046. [DOI] [PubMed] [Google Scholar]
  31. Li F, Wang X, Capasso JM, Gerdes AM. Rapid transition of cardiac myocytes from hyperplasia to hypertrophy during postnatal development. Journal of Molecular and Cellular Cardiology. 1996;28:1737–1746. doi: 10.1006/jmcc.1996.0163. [DOI] [PubMed] [Google Scholar]
  32. Lin Z, von Gise A, Zhou P, Gu F, Ma Q, Jiang J, Yau AL, Buck JN, Gouin KA, van Gorp PRR, Zhou B, Chen J, Seidman JG, Wang D-Z, Pu WT. Cardiac-Specific YAP activation improves cardiac function and survival in an experimental murine MI model. Circulation Research. 2014;115:354–363. doi: 10.1161/CIRCRESAHA.115.303632. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Liu H, Zhang C-H, Ammanamanchi N, Suresh S, Lewarchik C, Rao K, Uys GM, Han L, Abrial M, Yimlamai D, Ganapathy B, Guillermier C, Chen N, Khaladkar M, Spaethling J, Eberwine JH, Kim J, Walsh S, Choudhury S, Little K, Francis K, Sharma M, Viegas M, Bais A, Kostka D, Ding J, Bar-Joseph Z, Wu Y, Yechoor V, Moulik M, Johnson J, Weinberg J, Reyes-Múgica M, Steinhauser ML, Kühn B. Control of cytokinesis by β-adrenergic receptors indicates an approach for regulating cardiomyocyte Endowment. Science Translational Medicine. 2019;11:eaaw6419. doi: 10.1126/scitranslmed.aaw6419. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Lopaschuk GD, Jaswal JS. Energy metabolic phenotype of the cardiomyocyte during development, differentiation, and postnatal maturation. Journal of Cardiovascular Pharmacology. 2010;56:130–140. doi: 10.1097/FJC.0b013e3181e74a14. [DOI] [PubMed] [Google Scholar]
  35. Lymperopoulos A, Rengo G, Koch WJ. Adrenergic nervous system in heart failure: pathophysiology and therapy. Circulation Research. 2013;113:739–753. doi: 10.1161/CIRCRESAHA.113.300308. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Mahmoud AI, Kocabas F, Muralidhar SA, Kimura W, Koura AS, Thet S, Porrello ER, Sadek HA. Meis1 regulates postnatal cardiomyocyte cell cycle arrest. Nature. 2013;497:249–253. doi: 10.1038/nature12054. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Mahmoud AI, Porrello ER, Kimura W, Olson EN, Sadek HA. Surgical models for cardiac regeneration in neonatal mice. Nature Protocols. 2014;9:305–311. doi: 10.1038/nprot.2014.021. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Mahmoud AI, O’Meara CC, Gemberling M, Zhao L, Bryant DM, Zheng R, Gannon JB, Cai L, Choi WY, Egnaczyk GF, Burns CE, Burns CG, MacRae CA, Poss KD, Lee RT. Nerves regulate cardiomyocyte proliferation and heart regeneration. Developmental Cell. 2015;34:387–399. doi: 10.1016/j.devcel.2015.06.017. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Mangmool S, Shukla AK, Rockman HA. β-arrestin–dependent activation of Ca2+/calmodulin kinase II after β1–adrenergic receptor stimulation. Journal of Cell Biology. 2010;189:573–587. doi: 10.1083/jcb.200911047. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. MERIT-HF Study group Effect of metoprolol CR/XL in chronic heart failure: metoprolol CR/XL randomised intervention trial in congestive heart failure (MERIT-HF) Lancet. 1999;353:2001–2007. [PubMed] [Google Scholar]
  41. Molkentin JD, Dorn GW. Cytoplasmic signaling pathways that regulate cardiac hypertrophy. Annual Review of Physiology. 2001;63:391–426. doi: 10.1146/annurev.physiol.63.1.391. [DOI] [PubMed] [Google Scholar]
  42. Nakada Y, Canseco DC, Thet S, Abdisalaam S, Asaithamby A, Santos CX, Shah AM, Zhang H, Faber JE, Kinter MT, Szweda LI, Xing C, Hu Z, Deberardinis RJ, Schiattarella G, Hill JA, Oz O, Lu Z, Zhang CC, Kimura W, Sadek HA. Hypoxia induces heart regeneration in adult mice. Nature. 2017;541:222–227. doi: 10.1038/nature20173. [DOI] [PubMed] [Google Scholar]
  43. Ng WA, Grupp IL, Subramaniam A, Robbins J. Cardiac myosin heavy chain mRNA expression and myocardial function in the mouse heart. Circulation Research. 1991;68:1742–1750. doi: 10.1161/01.res.68.6.1742. [DOI] [PubMed] [Google Scholar]
  44. Oestreich EA, Malik S, Goonasekera SA, Blaxall BC, Kelley GG, Dirksen RT, Smrcka AV. Epac and phospholipase Cϵ regulate Ca2+ release in the heart by activation of protein kinase Cϵ and calcium-calmodulin kinase II. Journal of Biological Chemistry. 2009;284:1514–1522. doi: 10.1074/jbc.M806994200. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Pan D. The Hippo signaling pathway in development and cancer. Developmental Cell. 2010;19:491–505. doi: 10.1016/j.devcel.2010.09.011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Patel AR, Shaddy RE. Role of β-blocker therapy in pediatric heart failure. Pediatric Health. 2010;4:45–58. doi: 10.2217/phe.09.65. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Payumo AY, Chen X, Hirose K, Chen X, Hoang A, Khyeam S, Yu H, Wang J, Chen Q, Powers N, Chen L, Bigley RB, Lovas J, Hu G, Huang GN. Adrenergic-thyroid hormone interactions drive postnatal thermogenesis and loss of mammalian heart regenerative capacity. Circulation. 2021;144:1000–1003. doi: 10.1161/CIRCULATIONAHA.121.054846. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Porrello ER, Mahmoud AI, Simpson E, Hill JA, Richardson JA, Olson EN, Sadek HA. Transient regenerative potential of the neonatal mouse heart. Science. 2011;331:1078–1080. doi: 10.1126/science.1200708. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Porrello ER, Mahmoud AI, Simpson E, Johnson BA, Grinsfelder D, Canseco D, Mammen PP, Rothermel BA, Olson EN, Sadek HA. Regulation of neonatal and adult mammalian heart regeneration by the mir-15 family. PNAS. 2013;110:187–192. doi: 10.1073/pnas.1208863110. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Puente BN, Kimura W, Muralidhar SA, Moon J, Amatruda JF, Phelps KL, Grinsfelder D, Rothermel BA, Chen R, Garcia JA, Santos CX, Thet S, Mori E, Kinter MT, Rindler PM, Zacchigna S, Mukherjee S, Chen DJ, Mahmoud AI, Giacca M, Rabinovitch PS, Aroumougame A, Shah AM, Szweda LI, Sadek HA. The oxygen-rich postnatal environment induces cardiomyocyte cell-cycle arrest through DNA damage response. Cell. 2014;157:565–579. doi: 10.1016/j.cell.2014.03.032. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Rockman HA, Koch WJ, Lefkowitz RJ. Seven-transmembrane-spanning receptors and heart function. Nature. 2002;415:206–212. doi: 10.1038/415206a. [DOI] [PubMed] [Google Scholar]
  52. Sah VP, Minamisawa S, Tam SP, Wu TH, Dorn GW, Ross J, Chien KR, Brown JH. Cardiac-specific overexpression of rhoa results in sinus and atrioventricular nodal dysfunction and contractile failure. The Journal of Clinical Investigation. 1999;103:1627–1634. doi: 10.1172/JCI6842. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Salazar NC, Chen J, Rockman HA. Cardiac GPCRs: GPCR signaling in healthy and failing hearts. Biochimica et Biophysica Acta. 2007;1768:1006–1018. doi: 10.1016/j.bbamem.2007.02.010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Sanbe A, Gulick J, Hanks MC, Liang Q, Osinska H, Robbins J. Reengineering inducible cardiac-specific transgenesis with an attenuated myosin heavy chain promoter. Circulation Research. 2003;92:609–616. doi: 10.1161/01.RES.0000065442.64694.9F. [DOI] [PubMed] [Google Scholar]
  55. Sato S. Quantitative evaluation of ontogenetic change in heart rate and its autonomic regulation in newborn mice with the use of a noninvasive piezoelectric sensor. American Journal of Physiology. Heart and Circulatory Physiology. 2008;294:H1708–H1715. doi: 10.1152/ajpheart.01122.2007. [DOI] [PubMed] [Google Scholar]
  56. Siedner S, Krüger M, Schroeter M, Metzler D, Roell W, Fleischmann BK, Hescheler J, Pfitzer G, Stehle R. Developmental changes in contractility and sarcomeric proteins from the early embryonic to the adult stage in the mouse heart. The Journal of Physiology. 2003;548:493–505. doi: 10.1113/jphysiol.2002.036509. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Sohal DS, Nghiem M, Crackower MA, Witt SA, Kimball TR, Tymitz KM, Penninger JM, Molkentin JD. Temporally regulated and tissue-specific gene manipulations in the adult and embryonic heart using a tamoxifen-inducible cre protein. Circulation Research. 2001;89:20–25. doi: 10.1161/hh1301.092687. [DOI] [PubMed] [Google Scholar]
  58. Tilley DG, Rockman H. G protein–dependent and G protein–independent signaling pathways and their impact on cardiac function. Circulation Research. 2011;109:217–230. doi: 10.1161/CIRCRESAHA.110.231225. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Tzahor E, Poss KD. Cardiac regeneration strategies: staying young at heart. Science. 2017;356:1035–1039. doi: 10.1126/science.aam5894. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Varelas X. The hippo pathway effectors TAZ and YAP in development, homeostasis and disease. Development. 2014;141:1614–1626. doi: 10.1242/dev.102376. [DOI] [PubMed] [Google Scholar]
  61. Vite A, Zhang C, Yi R, Emms S, Radice GL. α-catenin-dependent cytoskeletal tension controls YAP activity in the heart. Development. 2018;145:dev149823. doi: 10.1242/dev.149823. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. Vivien CJ, Hudson JE, Porrello ER. Evolution, comparative biology and ontogeny of vertebrate heart regeneration. NPJ Regenerative Medicine. 2016;1:16012. doi: 10.1038/npjregenmed.2016.12. [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. Wada KI, Itoga K, Okano T, Yonemura S, Sasaki H. Hippo pathway regulation by cell morphology and stress fibers. Development. 2011;138:3907–3914. doi: 10.1242/dev.070987. [DOI] [PubMed] [Google Scholar]
  64. Wang L, Luo JY, Li B, Tian XY, Chen LJ, Huang Y, Liu J, Deng D, Lau CW, Wan S, Ai D, Mak KLK, Tong KK, Kwan KM, Wang N, Chiu JJ, Zhu Y, Huang Y. Integrin-YAP/TAZ-JNK cascade mediates atheroprotective effect of unidirectional shear flow. Nature. 2016a;540:579–582. doi: 10.1038/nature20602. [DOI] [PubMed] [Google Scholar]
  65. Wang KC, Yeh YT, Nguyen P, Limqueco E, Lopez J, Thorossian S, Guan KL, Li YSJ, Chien S. Flow-dependent YAP/TAZ activities regulate endothelial phenotypes and atherosclerosis. PNAS. 2016b;113:11525–11530. doi: 10.1073/pnas.1613121113. [DOI] [PMC free article] [PubMed] [Google Scholar]
  66. Wang J, Gareri C, Rockman HA. G-Protein-Coupled receptors in heart disease. Circulation Research. 2018;123:716–735. doi: 10.1161/CIRCRESAHA.118.311403. [DOI] [PMC free article] [PubMed] [Google Scholar]
  67. Wang H, Yang Y, Liu J, Qian L. Direct cell reprogramming: approaches, mechanisms and progress. Nature Reviews. Molecular Cell Biology. 2021;22:410–424. doi: 10.1038/s41580-021-00335-z. [DOI] [PMC free article] [PubMed] [Google Scholar]
  68. Woo AYH, Xiao R. Β-Adrenergic receptor subtype signaling in heart: from bench to bedside. Acta Pharmacologica Sinica. 2012;33:335–341. doi: 10.1038/aps.2011.201. [DOI] [PMC free article] [PubMed] [Google Scholar]
  69. Xin M, Kim Y, Sutherland LB, Qi X, McAnally J, Schwartz RJ, Richardson JA, Bassel-Duby R, Olson EN. Regulation of insulin-like growth factor signaling by YAP governs cardiomyocyte proliferation and embryonic heart size. Science Signaling. 2011;4:2278. doi: 10.1126/scisignal.2002278. [DOI] [PMC free article] [PubMed] [Google Scholar]
  70. Xin M, Kim Y, Sutherland LB, Murakami M, Qi X, McAnally J, Porrello ER, Mahmoud AI, Tan W, Shelton JM, Richardson JA, Sadek HA, Bassel-Duby R, Olson EN. Hippo pathway effector YAP promotes cardiac regeneration. PNAS. 2013a;110:13839–13844. doi: 10.1073/pnas.1313192110. [DOI] [PMC free article] [PubMed] [Google Scholar]
  71. Xin M, Olson EN, Bassel-Duby R. Mending broken hearts: cardiac development as a basis for adult heart regeneration and repair. Nature Reviews. Molecular Cell Biology. 2013b;14:529–541. doi: 10.1038/nrm3619. [DOI] [PMC free article] [PubMed] [Google Scholar]
  72. Yu FX, Zhao B, Panupinthu N, Jewell JL, Lian I, Wang LH, Zhao J, Yuan H, Tumaneng K, Li H, Fu XD, Mills GB, Guan KL. Regulation of the Hippo-YAP pathway by G-protein-coupled receptor signaling. Cell. 2012;150:780–791. doi: 10.1016/j.cell.2012.06.037. [DOI] [PMC free article] [PubMed] [Google Scholar]
  73. Zhao B, Tumaneng K, Guan KL. The Hippo pathway in organ size control, tissue regeneration and stem cell self-renewal. Nature Cell Biology. 2011;13:877–883. doi: 10.1038/ncb2303. [DOI] [PMC free article] [PubMed] [Google Scholar]
  74. Zhao MT, Ye S, Su J, Garg V. Cardiomyocyte proliferation and maturation: two sides of the same coin for heart regeneration. Frontiers in Cell and Developmental Biology. 2020;8:594226. doi: 10.3389/fcell.2020.594226. [DOI] [PMC free article] [PubMed] [Google Scholar]
  75. Zhu K, Debreceni B, Li R, Zheng Y. Identification of Rho GTPase-dependent sites in the Dbl homology domain of oncogenic Dbl that are required for transformation. Journal of Biological Chemistry. 2000;275:25993–26001. doi: 10.1074/jbc.M003780200. [DOI] [PubMed] [Google Scholar]
  76. Zhu J, Steinberg SF. Β1-Adrenergic receptor N-terminal cleavage by ADAM17; the mechanism for redox-dependent downregulation of cardiomyocyte β1-adrenergic receptors. Journal of Molecular and Cellular Cardiology. 2021;154:70–79. doi: 10.1016/j.yjmcc.2021.01.012. [DOI] [PMC free article] [PubMed] [Google Scholar]

Editor's evaluation

Enzo R Porrello 1

This manuscript provides strong evidence that β adrenergic signaling regulates cardiomyocyte proliferation in the postnatal period. The authors provide compelling data that inhibition of β adrenergic signaling promotes cardiomyocyte proliferation in juvenile mice through activation of a RhoA-YAP signaling axis.

Decision letter

Editor: Enzo R Porrello1
Reviewed by: Enzo R Porrello2

Our editorial process produces two outputs: (i) public reviews designed to be posted alongside the preprint for the benefit of readers; (ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.

Decision letter after peer review:

Thank you for submitting your article "Inhibition of adrenergic b1-AR/Gas signaling promotes cardiomyocyte proliferation through activation of RhoA-YAP axis" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, including Enzo R. Porrello as the Reviewing Editor and Reviewer #1, and the evaluation has been overseen by a Reviewing Editor and Didier Stainier as the Senior Editor.

The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.

Essential revisions:

1) Cardio-protection vs regeneration: The authors conclude that their results "suggest that β blockade improves cardiac function by promoting cardiac regeneration in the injured heart". However, effects on CM proliferation are fairly modest (~0.3% pH3-positive CM following MI at P7) and it is unclear whether improvements in cardiac function/fibrosis post-MI are due to induction of CM proliferation. Moreover, the authors have not excluded an acute cardioprotective effect post-MI. Infarct size should be assessed (e.g. using tetrazolium chloride staining) and quantified acutely post-MI (e.g 24 hrs) to distinguish acute cardioprotective effects from bona fide regeneration. If the authors have access to cardiac MRI, this could be used to prove remuscularization has occurred post-MI.

2) Cardiomyocyte proliferation: Additional data are required to demonstrate bona fide cardiomyocyte division following β blockade and Gnas loss-of-function. AurkB staining should be performed to identify cardiomyocytes undergoing cytokinesis, which should be used as a marker for CM division as per other publications in the field. In addition, Supp Figure 1D (quantification of cardiomyocyte number following β blocker treatment) is a critical experiment and should be presented in the main data panels of Figure 1.

3) Given the potential clinical significance and therapeutic implications of the current findings, the authors should determine whether β blockers are sufficient to induce cardiomyocyte cell cycle re-entry and cardiac regeneration following myocardial infarction in adult mice.

4) Additional biochemical evidence is required to demonstrate Yap translocation to the nucleus in Supplemental Figure 5. Immunofluorescence images provided in Supplemental Figures 5 d&f are difficult to interpret. Nuclear translocation of the factor, which is essential for its activation, is not shown here biochemically. On the contrary, the authors show that there is nuclear YAP in basal conditions by immunofluorescence, while this drops to virtually null after epinephrine treatment (bar chart in Suppl. Figure 5f). Incidentally, the over 30% nuclear positivity shown in the graph in basal conditions is difficult to reconcile with the lack of nuclear staining in Suppl. Figure 5b.

5) Please clarify the connection of canonical Hippo signaling and the RhoA effect on Yap. In the current manuscript the mechanistic connection remains somewhat vague. In the original Yu Cell paper from 2021 there is a connection to cytoskeleton. Is that the case here?

6) The authors refer to effects of β blocker treatment on "neonatal heart regeneration". This terminology could be confusing as it is most commonly used to describe experiments in P1 (regenerative) neonatal mice not P7 (non-regenerative) mice. For clarity, it is suggested that the authors rephrase these statements to refer to "prolongation of the regenerative window" in neonatal mice.

7) On several occasions the authors refer to expression of b1-AR specifically in the heart. Is it known whether b1-AR expression is restricted to cardiomyocytes and/or whether it is developmentally regulated from neonatal to adult stages? These data are important with regards to interpretation of systemic metoprolol administration studies. These data should be provided or cited from relevant literature.

8) Β blocker treatment and Gas knockout were both associated with repression of fatty acid oxidation and induction of glycolysis. Are these metabolic transcriptional programs dependent on RhoA/Yap signaling?

9) It would be helpful if the authors showed the overlap between β blocker and Gas KO RNA-seq data sets as a Venn diagram in Figure 4 (or associated Supplementary Figure). Was an FDR cut-off applied to identify DEGs?

10) Figure 4K: data should be presented as %pH3-positive CM as per other figures.

11) Figure 5B: Please replace poor quality Western blot image for Active RhoA.

12) Figure 2f and 2g. Why do EF and FS progressively worsen in the sham animals without MI?

13) Typo: Agrin, not agarin.

14) Please provide dot plots rather than bar graphs.

15) Please clearly state the statistical test used in each figure panel.

Reviewer #1 (Recommendations for the authors):

1. Cardio-protection vs regeneration: The authors conclude that their results "suggest that β blockade improves cardiac function by promoting cardiac regeneration in the injured heart". However, effects on CM proliferation are fairly modest (~0.3% pH3-positive CM following MI at P7) and it is unclear whether improvements in cardiac function/fibrosis post-MI are due to induction of CM proliferation. Moreover, the authors have not excluded an acute cardioprotective effect post-MI. Infarct size should be assessed (e.g. using tetrazolium chloride staining) and quantified acutely post-MI (e.g 24 hrs) to distinguish acute cardioprotective effects from bona fide regeneration. In addition, the Discussion should be tempered to account for alternative physiological mechanisms mediating therapeutic effects observed post-MI in addition to regeneration (e.g. immunomodulation, inhibition of cell death, angiogenesis, reduced contractile loading, improved coronary flow, etc).

2. It is unclear why experiments were not performed in adult mice to determine whether β blockade or Gas loss-of-function are sufficient to induce adult cardiomyocyte cell cycle re-entry. Given the potential clinical significance and therapeutic implications of the authors findings, this experiment would significantly strengthen the paper.

3. The authors refer to effects of β blocker treatment on "neonatal heart regeneration". This terminology could be confusing as it is most commonly used to describe experiments in P1 (regenerative) neonatal mice not P7 (non-regenerative) mice. For clarity, it is suggested that the authors rephrase these statements to refer to "prolongation of the regenerative window" in neonatal mice.

4. On several occasions the authors refer to expression of b1-AR specifically in the heart. Is it known whether b1-AR expression is restricted to cardiomyocytes and/or whether it is developmentally regulated from neonatal to adult stages? These data are important with regards to interpretation of systemic metoprolol administration studies. These data should be provided or cited from relevant literature.

5. Β blocker treatment and Gas knockout were both associated with repression of fatty acid oxidation and induction of glycolysis. Are these metabolic transcriptional programs dependent on RhoA/Yap signaling?

Other:

6. Supp Figure 1D (quantification of cardiomyocyte number following β blocker treatment) is a critical experiment and should be presented in the main data panels of Figure 1.

7. It would be helpful if the authors showed the overlap between β blocker and Gas KO RNA-seq data sets as a Venn diagram in Figure 4 (or associated Supplementary Figure). Was an FDR cut-off applied to identify DEGs?

8. Figure 4K: data should be presented as %pH3-positive CM as per other figures.

9. Figure 5B: Please replace poor quality Western blot image for Active RhoA.

Reviewer #2 (Recommendations for the authors):

My recommendation to the authors is to strengthen the data on cardiomyocyte proliferation and cardiac regeneration. The use of cardiac MRI to prove remuscularization, should the authors have a microMRI available, the study of BrdU incorporation, the evidence for aurora B localization in midbodies would all strengthen the claim for regeneration.

An additional set of required experiments concerns YAP, as in my earlier text. Providing a mechanism linking betaAR to YAP activation would be also important in this context.

Specific issues.

Figure 2f and 2g. Why do EF and FS progressively worsen in the sham animals without MI?

Agrin, not agarin.

There is no established evidence that Yap translocates from the nucleus to the cytoplasm, quite the reverse. A decrease in the levels of nuclear YAP is the consequence of increased cytoplasmic phosphorylation and degradation.

Reviewer #3 (Recommendations for the authors):

The authors study mammalian heart regeneration and study the connection between Yap and β-adrenergic receptor (β-AR) blockade. Interestingly, metoprolol robustly enhanced cardiomyocyte proliferation and promoted cardiac regeneration post myocardial infarction, resulting in reduced scar formation and improved cardiac function. The conclusion was also supported by genetic deletion of Gnas. CMs had an immature cell state with enhanced activity of Hippo-effector YAP. They also find that increased YAP activity is modulated by RhoA.

Comments

1) AurkB should be used as a marker for CM division as per other publications in the field. PHH3 will also mark CMs that are undergoing Karyokinesis without division for example.

2) Please provide dot plots rather than bar graphs.

3) Please clearly state the statistical test used in each figure panel.

4) Figure 5 B Western blot needs to be improved and quantified.

5) Please clarify the connection of canonical Hippo signaling and the RhoA effect on Yap. In the current manuscript the mechanistic connection remains somewhat vague. In the original Yu Cell paper from 2021 there is a connection to cytoskeleton. Is that the case here? Please clarify.

[Editors' note: further revisions were suggested prior to acceptance, as described below.]

Thank you for resubmitting your work entitled "Inhibition of adrenergic β1-AR/Gαs signaling promotes cardiomyocyte proliferation through activation of RhoA-YAP axis" for further consideration by eLife. Your revised article has been evaluated by Didier Stainier (Senior Editor) and a Reviewing Editor.

The manuscript has been improved but there are some remaining issues that need to be addressed, as outlined below:

As you will see from the reviewer comments below, there continue to be some concerns about the lack of definitive evidence for cardiomyocyte proliferation and cardiac regeneration in this study. Additional data are required to unequivocally demonstrate that adrenergic β1-AR/Gαs signaling promotes cardiomyocyte proliferation including full progression through cytokinesis. Moreover, as metoprolol is not sufficient to induce adult cardiomyocyte proliferation, the authors' conclusions need to be substantially toned down to reflect the restricted effects on cardiac regeneration in the neonatal period. In addition, reviewer 2's remaining concerns about the biochemical validation of YAP activation should be directly addressed.

Reviewer #2 (Recommendations for the authors):

The authors have essentially NOT responded to my comments. First, there is no additional evidence of cardiomyocyte proliferation in this revised version of the manuscript. The images for histone H3 phosphorylation show few positive cells, the identity of which remains debatable. The authors themselves admit that there are so few Aurora B positive cells that not even statistical assessment can be attempted. These findings do not support the statement that "metoprolol robustly enhanced cardiomyocyte proliferation and promoted cardiac regeneration (abstract)". In addition, the new experiment in adult mice (asked by another reviewer) shows that the drug does NOT induce regeneration. Hence, the whole message of the manuscript is quite deceptive.

Second, as far as YAP activation is concerned, I asked for molecular or biochemical evidence of nuclear translocation (for example, by cytoplasmic and nuclear fractionation). This was not provided, while the IF images remain doubtful (cf. my original comments). YAP is known to also regulate cardiomyocyte hypertrophy, which could explain some of the findings presented in this manuscript.

eLife. 2022 Dec 8;11:e74576. doi: 10.7554/eLife.74576.sa2

Author response

Essential revisions:

1) Cardio-protection vs regeneration: The authors conclude that their results "suggest that β blockade improves cardiac function by promoting cardiac regeneration in the injured heart". However, effects on CM proliferation are fairly modest (~0.3% pH3-positive CM following MI at P7) and it is unclear whether improvements in cardiac function/fibrosis post-MI are due to induction of CM proliferation. Moreover, the authors have not excluded an acute cardioprotective effect post-MI. Infarct size should be assessed (e.g. using tetrazolium chloride staining) and quantified acutely post-MI (e.g 24 hrs) to distinguish acute cardioprotective effects from bona fide regeneration. If the authors have access to cardiac MRI, this could be used to prove remuscularization has occurred post-MI.

We thank the reviewer for pointing out the possibility of an acute cardioprotective effect of b blocker post-MI. As reviewer mentioned that some papers reported the cardioprotective effect of b-blocker, but it is still controversial. To address this question, we performed TTC staining to measure the infarct size 24 hrs post-MI (cardiac MRI is not available in our institute). We observed ischemic area in both saline and b-blocker treated hearts, and there is no significant difference of ischemic area between them, suggesting that the b-blocker (metoprolol) may not have any cardioprotective effects at this neonatal period. We added the data in Supplemental Figure3.

Previous studies regarding neonatal heart regeneration showed that promoting cardiomyocyte proliferation can reduce the infarct size and improve cardiac function post MI. The range of percentage of proliferating CM was 0.1-0.3% (Mahmoud et al., 2013; Nakada et al., 2017; Leach et al., 2017). This range is the same as what we observed in b-blocker-treated MI hearts, it is appropriate to say that β blockade improves cardiac function by promoting cardiac regeneration in the injured heart.

2) Cardiomyocyte proliferation: Additional data are required to demonstrate bona fide cardiomyocyte division following β blockade and Gnas loss-of-function. AurkB staining should be performed to identify cardiomyocytes undergoing cytokinesis, which should be used as a marker for CM division as per other publications in the field. In addition, Supp Figure 1D (quantification of cardiomyocyte number following β blocker treatment) is a critical experiment and should be presented in the main data panels of Figure 1.

We performed AurkB staining using P14 control, b-blocker-treated, and Gnas cKO hearts. We didn’t observe any AurkB-positive CMs in the control hearts, whereas b-blocker-treated or Gnas cKO hearts showed a few AurkB-positive CMs. Since AurkB-postive CM is rare (one or two AurkB-positive CMs per section), it is hard to perform the statistical analysis. Therefore, we included the AurkB staining images in Supplemental Figure1d and Supplemental Figure 4b. Also, we moved the quantification data of cardiomyocyte number from the original Supplemental Fig1D to Fig1F in the revision.

3) Given the potential clinical significance and therapeutic implications of the current findings, the authors should determine whether β blockers are sufficient to induce cardiomyocyte cell cycle re-entry and cardiac regeneration following myocardial infarction in adult mice.

As the reviewer mentioned, we performed MI surgery using adult mice to investigate whether b-blocker is sufficient to induce cardiac regeneration. We observed huge ischemic area in both the control and b-blocker-treated hearts as shown in Author response image 1. Therefore, in the adult stage, b-blocker treatment may not be sufficient to promote cardiac regeneration. Recent study showed that combination treatment with a/b-blocker and thyroid hormone inhibitor enhanced cardiomyocyte regeneration after MI surgery at P14 when the regeneration window has already closed, suggesting that it may need additional drug to promote cardiac regeneration at adult stage.

Author response image 1.

Author response image 1.

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4) Additional biochemical evidence is required to demonstrate Yap translocation to the nucleus in Supplemental Figure 5. Immunofluorescence images provided in Supplemental Figures 5 d&f are difficult to interpret. Nuclear translocation of the factor, which is essential for its activation, is not shown here biochemically. On the contrary, the authors show that there is nuclear YAP in basal conditions by immunofluorescence, while this drops to virtually null after epinephrine treatment (bar chart in Suppl. Figure 5f). Incidentally, the over 30% nuclear positivity shown in the graph in basal conditions is difficult to reconcile with the lack of nuclear staining in Suppl. Figure 5b.

In the canonical Hippo signaling pathway, the major YAP regulators are the LATS1/2 kinases, which phosphorylate and inhibit YAP activity. In this paper, we found that loss of Gas function up-regulates RhoA activity in cardiomyocytes. Previous paper (Yu et al., Cell, 2012) reported a model that Rho GTPases inhibit LATS1/2 activity through actin cytoskeleton organization and thereby promote YAP activation. From these results, we consider that Gas-Rho-cytoskeleton-LATS signaling pathway regulates YAP activity in neonatal cardiomyocytes. However, it is still unclear the mechanisms by which actin cytoskeleton controls LATS1/2 activity, therefore further investigation is required to solve the question. We added a Discussion section about the signaling pathway that regulates YAP activity in cardiomyocytes.

We apologize for having an incorrect information regarding the epinephrin treated hearts in the figure legend of Supplemental Figure5. We used “P4 pups” for this experiment instead of “P7 pups” as indicated in the original figure legend. We have made corrections in the new figure legend. As it’s shown in Supplemental Figure6b, YAP localized in the nucleus at P4 when the regeneration window is still open; however, YAP translated to the cytoplasm at P7 when the regeneration window closed. We hypothesized that this cytoplasmic YAP translocation is promoted by b1-AR-Gas signaling pathway. To test our hypothesis, we injected epinephrin into “P4 pups” and we observed cytoplasmic YAP translocation in the WT hearts, but not in the Gnas cKO hearts. These data indicate that b1-AR stimulation promotes cytoplasmic YAP localization through Gas. The immunostaining results in Supplemental Figure6c using “P17 hearts” confirms our hypothesis. Gnas deletion leads to YAP nuclear localization when YAP has already translocated to the cytoplasm at P17 in the control mice.

Additionally, we performed western blotting analysis using hearts from P4 to P9 and found that phospho-YAP level increased as the heart regeneration window closed. This result correlates with the immunostaining data confirming that YAP nuclear localization correlates with heart regenerative capacity. This piece of data is in the new Supplemental Fig6a.

5) Please clarify the connection of canonical Hippo signaling and the RhoA effect on Yap. In the current manuscript the mechanistic connection remains somewhat vague. In the original Yu Cell paper from 2021 there is a connection to cytoskeleton. Is that the case here?

The canonical Hippo signaling pathway is mediated by MST kinases, LATS kinases, and the downstream transcriptional coactivators YAP and TAZ. As the reviewer mentioned, Yu et al. (Cell, 2012) reported that activation of GPCRs-Rho signaling by Epinephrine inactivates YAP through actin cytoskeleton organization which may controls LATS phosphorylation. In this study, we also found that YAP activity in the neonatal cardiomyocytes is affected by b1AR-Gas-Rho signaling pathway. Moreover, Yu et al. reported that YAP nuclear localization under LPA (Ga12/13 agonist) treatment correlated with levels of cellular actin filaments. As shown in Author response image 2, we also have a piece of data indicating that LPA (Ga12/13 agonist) treatment activates YAP in the wild type P7 hearts, as shown in Author response image 2 (data not included in the manuscript). Therefore, we argue that YAP activity in the cardiomyocyte appears to be regulated by Rho-cytoskeleton-LATS signaling pathway. Since the mechanisms by which actin cytoskeleton organization regulates LATS activity is still unclear, further investigation is required to elucidate it. We added the sentence to clarify the Hippo-RhoA-cytoskeleton-LATS signaling pathway in the discussion.

Author response image 2.

Author response image 2.

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6) The authors refer to effects of β blocker treatment on "neonatal heart regeneration". This terminology could be confusing as it is most commonly used to describe experiments in P1 (regenerative) neonatal mice not P7 (non-regenerative) mice. For clarity, it is suggested that the authors rephrase these statements to refer to "prolongation of the regenerative window" in neonatal mice.

We totally agreed the reviewer’s comment. We have edited it in the revision.

7) On several occasions the authors refer to expression of b1-AR specifically in the heart. Is it known whether b1-AR expression is restricted to cardiomyocytes and/or whether it is developmentally regulated from neonatal to adult stages? These data are important with regards to interpretation of systemic metoprolol administration studies. These data should be provided or cited from relevant literature.

It is well known that b1-AR is the predominant bAR subtype in cardiac muscle comprising 80% of bARs in cardiomyocytes. It is also expressed in sinoatrial node, and atrioventricular node, but juxtaglomerular cell and adipose tissue. Since b1-AR knockout mice die prenatally between E10.5 and E18.5, b1-AR may have a critical role for the adrenergic system during embryonic heart development. However, previous paper reported that b1-AR mRNA expression in adult hearts significantly increased by fivefold than E11.5 hearts. Moreover, basal cAMP level was significantly increased in the developed hearts (Feridooni et al., 2017, Am. J. Physiol. Heart Circ. Physiol.). Therefore, these results suggest that b1-AR expression and stimulation is developmentally regulated from neonatal to adult stage. These data also support our model that inhibition of b1-AR signaling by b-blocker extended the cardiac regenerative window. We discussed b1-AR expression and stimulation during heart development in the discussion part.

8) Β blocker treatment and Gas knockout were both associated with repression of fatty acid oxidation and induction of glycolysis. Are these metabolic transcriptional programs dependent on RhoA/Yap signaling?

There are pieces of evidence indicating that in several cancer cell lines, YAP is involved in metabolism regulation to promote glycolysis (Koo and Guan, 2018, Cell Metabolism). Other studies also reported that cells having constitutively active YAP promoted glucose utilization, indicating an increase in glycolysis. In fact, YAP promotes transcription of glycolysis related genes, such as GLUT3 or HK2, by interacting with TEAD. In the ischemic hearts, upregulation of YAP occurs one week after TAC, and then we can see increased glucose uptake in adult cardiomyocytes (Kashihara and Sadoshima, 2019, J. Cardiovasc. Pharmacol.). Our transcriptome RNA seq data also suggest that YAP may promotes glycolysis in cardiomyocytes in response to loss of Gas function.

9) It would be helpful if the authors showed the overlap between β blocker and Gas KO RNA-seq data sets as a Venn diagram in Figure 4 (or associated Supplementary Figure). Was an FDR cut-off applied to identify DEGs?

We added the Venn diagram of down-regulated and up-regulated genes in Gnas cKO and b-blocker treated hearts in Supplemental Figure 5a and 5b. We used FPKM≥0.5 and |fold change|≥1.2 (not FDR cut-off) to identify the differential gene expression. We used FDR for GSEA analysis.

10) Figure 4K: data should be presented as %pH3-positive CM as per other figures.

We revised the data, following the reviewer’s suggestion.

11) Figure 5B: Please replace poor quality Western blot image for Active RhoA.

We have replaced the Western blot image for active RhoA in Figure 5B.

12) Figure 2f and 2g. Why do EF and FS progressively worsen in the sham animals without MI?

As reviewer mentioned, we can see that EF and FS of sham animals appear to be getting worse when we compared 1 week (P14) and 3 weeks (P28) post-MI data. It has been previously reported that the neonatal EF and FS are slightly higher than juvenile mice (P10 vs. P35) (Wiesmann et al., Am.J.Physiol.Heart Circ.Physiol., 2000). Therefore, we consider that the reduction of EF and FS in the sham animals what we observed could be normal phenomenon as shown by other group for the control mice.

13) Typo: Agrin, not agarin.

We have corrected the typo (Page3, Line8).

14) Please provide dot plots rather than bar graphs.

We revised all graphs from bar graph to dot plot.

15) Please clearly state the statistical test used in each figure panel.

We added the types of statistical method in each figure legends.

[Editors' note: further revisions were suggested prior to acceptance, as described below.]

The manuscript has been improved but there are some remaining issues that need to be addressed, as outlined below:

As you will see from the reviewer comments below, there continue to be some concerns about the lack of definitive evidence for cardiomyocyte proliferation and cardiac regeneration in this study. Additional data are required to unequivocally demonstrate that adrenergic β1-AR/Gαs signaling promotes cardiomyocyte proliferation including full progression through cytokinesis. Moreover, as metoprolol is not sufficient to induce adult cardiomyocyte proliferation, the authors' conclusions need to be substantially toned down to reflect the restricted effects on cardiac regeneration in the neonatal period. In addition, reviewer 2's remaining concerns about the biochemical validation of YAP activation should be directly addressed.

We thank the reviewers of their positive feedback about our revised manuscript. We are sorry that some concerns regarding cardiomyocyte proliferation and YAP activation still remain not fully addressed. In this resubmission, we have provided EdU and Aurora B Kinase immunostaining data to demonstrate that β1-AR/Gαs signaling promotes cardiomyocyte proliferation from DNA synthesis (EdU) to nuclear division (Phospho- Histone H3) eventually to cytokinesis (Aurora B). We further validated YAP activation via nuclear translocation by Fractionation Assay. Please see the detailed responses to reviewer 2’s concerns below.

We also toned down the conclusions to reflect the restricted cardiac regeneration effect in the juvenile stage (in the revised abstract, text and discussion). The changes are marked in the text.

Reviewer #2 (Recommendations for the authors):

The authors have essentially NOT responded to my comments. First, there is no additional evidence of cardiomyocyte proliferation in this revised version of the manuscript. The images for histone H3 phosphorylation show few positive cells, the identity of which remains debatable. The authors themselves admit that there are so few Aurora B positive cells that not even statistical assessment can be attempted. These findings do not support the statement that "metoprolol robustly enhanced cardiomyocyte proliferation and promoted cardiac regeneration (abstract)". In addition, the new experiment in adult mice (asked by another reviewer) shows that the drug does NOT induce regeneration. Hence, the whole message of the manuscript is quite deceptive.

We appreciate the reviewer’s comments. To show the proliferating cardiomyocytes more clearly, in this new revision, we included insets of higher magnification in the Phospho-histone H3 staining images in New Figures (Figure 1e and h and Figure 3e).

To show the full spectrum of proliferating cardiomyocytes, we performed EdU incorporation assay in the β-blocker treated hearts. A significant increase in the number of EdU positive and PCM1 (cardiomyocyte nuclear marker) positive cells was detected in the β-blocker treated hearts at P14, suggesting an increase in DNA synthesis (New Figure 1—figure supplement 1d).

In the last revision, we did observe Aurora B positive cardiomyocytes in β-blocker treated and Gnas cKO hearts at P14, but did not detect any Aurora B positive cells in the control hearts confirming that cardiomyocytes ceased proliferation after the regeneration window in the control but ablation of Gnas indeed improved cytokinesis. In this new revision, we provided statistical analysis of Aurora B staining of P14 hearts (New Figure 1—figure supplement 1e) as well as new Aurora B data of β-blocker treated and Gnas cKO hearts at P7. As shown in the new Figure 1f, 3f, Figure 1—figure supplement 1e and Figure 3—figure supplement 1b the number of Aurora B positive cardiomyocytes was significantly increased in both β-blocker treated hearts and Gnas cKO hearts at P7 and P14. These new results suggest that inhibition of β1-AR/Gαs signaling promotes cardiomyocyte proliferation at the juvenile stage. The texts were edited to reflect these changes (New Page4; line22-23 and Page7; line14).

We have toned down our conclusions regarding the effect of b-blocker on cardiac regeneration strictly to juvenile stage as it’s not sufficiently to induce adult heart regeneration. Please see the new abstract (page1), text (Page4; line1) and discussion (Page12; line5-13, Page13; line16, and Page14; line22). Our study suggests that inhibition of β1-AR/Gαs contributes to extending cardiac regeneration at the juvenile stage. Other pathways may be needed to promote adult cardiomyocyte proliferation and cardiac regeneration, which will need further investigation.

Second, as far as YAP activation is concerned, I asked for molecular or biochemical evidence of nuclear translocation (for example, by cytoplasmic and nuclear fractionation). This was not provided, while the IF images remain doubtful (cf. my original comments). YAP is known to also regulate cardiomyocyte hypertrophy, which could explain some of the findings presented in this manuscript.

We appreciate the reviewer’s comments and have a better understanding of the experiment that the reviewer suggested. As requested, we performed fractionation assay using P14 β1- blocker treated and Gnas cKO hearts. YAP is present in the nuclear fraction in both β1- blocker treated and Gnas cKO, but barely detectable in the control hearts. Cytoplasmic YAP is comparable with the controls. The nuclear fraction and cytoplasmic fraction were confirmed by Histone H3 and GAPDH, respectively. These data support the immunostaining results of increased YAP nuclear localization in both β1- blocker treated and Gnas cKO hearts. We have included these new data in Figure 4—figure supplement 2c and 2d and clarified these points in the manuscript (Page9; line19-21). Also, we included an inset with higher magnification to show nuclear YAP in the cardiomyocytes (new Figure 4—figure supplement 2b and e).

We appreciate that the reviewer pointed out that YAP may regulate cardiomyocyte hypertrophy, which was also one of our concerns. We used two independent methods (WGA and isolated cardiomyocytes) and showed in our last revision that no significant difference in cardiomyocyte size was observed between control and b-blocker treated or Gnas cKO hearts. The data are now in Figure 1—figure supplement 1g and h and Figure 3—figure supplement 1d and e. Therefore, the enlarged heart phenotype is not likely due to cardiac hypertrophy (Page5 line5-10 and Page7; line 18-20).

Associated Data

    This section collects any data citations, data availability statements, or supplementary materials included in this article.

    Data Citations

    1. Sakabe M, Xin M. 2021. Gene expression changes in beta-blocker treated neonatal hearts. NCBI Gene Expression Omnibus. GSE186099

    Supplementary Materials

    Figure 4—figure supplement 2—source data 1. Raw data of Western Blots.
    elife-74576-fig4-figsupp2-data1.zip (8MB, zip)
    Figure 5—source data 1. Raw data of Western Blots.
    elife-74576-fig5-data1.zip (14.5MB, zip)
    Figure 5—figure supplement 1—source data 1. Raw data of Western Blots.
    elife-74576-fig5-figsupp1-data1.zip (1.4MB, zip)
    Transparent reporting form
    elife-74576-transrepform1.docx (246.3KB, docx)

    Data Availability Statement

    RNA seq data have been deposited to GEO under accession code GSE186099.

    The following dataset was generated:

    Sakabe M, Xin M. 2021. Gene expression changes in beta-blocker treated neonatal hearts. NCBI Gene Expression Omnibus. GSE186099

    Articles from eLife are provided here courtesy of eLife Sciences Publications, Ltd

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