Cardiomyocyte‐Specific Overexpression of Activated Yes‐Associated Protein Modified‐RNA Promotes Cardiomyocyte Proliferation and Myocardial Regeneration

Abstract

Background

The proliferative capacity of cardiomyocytes in adult mammalian hearts is far too low to replace the cells that are lost to myocardial infarction. Both cardiomyocyte proliferation and myocardial regeneration can be improved via the overexpression of a constitutively active variant of YAP5SA (Yes‐associated protein, 5SA [active] mutant), but persistent overexpression of proliferation‐inducing genes could lead to hypertrophy and arrhythmia, whereas off‐target expression in fibroblasts and macrophages could increase fibrosis and inflammation.

Methods and Results

Transient overexpression of YAP5SA or GFP (green fluorescent protein; control) was targeted to cardiomyocytes via our cardiomyocyte‐specific modified mRNA translation system (^YAP5SACM‐SMRTs or ^GFPCM‐SMRTs, respectively). YAP5SA‐cardiomyocyte specificity was confirmed via in vitro experiments in cardiomyocytes and cardiac fibroblasts that had been differentiated from human induced‐ pluripotent stem cells and in human umbilical‐vein endothelial cells, and the regenerative potency of ^YAP5SACM‐SMRTs was evaluated in a mouse myocardial infarction model. In cultured human induced‐pluripotent stem cells‐cardiomyocytes, YAP was abundantly expressed for 3 days after ^YAP5SACM‐SMRTs administration and was accompanied by increases in the expression of markers for proliferation, before declining to near‐background levels after day 7, whereas GFP fluorescence remained high from days 1 to 3 after ^GFPCM‐SMRTs treatment and then slowly declined. GFP fluorescence was also observed in human induced‐pluripotent stem cells‐cardiac fibroblasts and human umbilical‐vein endothelial cells on day 1 after ^GFPCM‐SMRTs administration but declined substantially by day 3. In the mouse myocardial infarction model, echocardiographic assessments of left‐ventricular ejection fraction and fractional shortening were significantly greater, whereas infarct sizes were significantly smaller in ^YAP5SACM‐SMRTs–treated mice than in vehicle‐treated control animals, and ^YAP5SACM‐SMRTs appeared to promote cardiomyocyte proliferation.

Conclusions

The CM‐SMRTs can be used to transiently and specifically overexpress YAP5SA in cardiomyocytes, and this treatment strategy significantly promoted cardiomyocyte proliferation and myocardial regeneration in a mouse myocardial infarction model.

Nonstandard Abbreviations and Acronyms

ATP2A2

ATPase Sarcoplasmic/Endoplasmic Reticulum Ca²⁺ Transporting 2

CM‐SMRTS

cardiomyocyte‐specific, modRNA translation system

COL‐I+

Type I collagen positive

CTGF

Connective Tissue Growth Factor

cTnT

cardiac troponin T

Cyr61

Cysteine‐rich angiogenic inducer 61

DDR2+

Discoidin Domain Receptor Tyrosine Kinase 2 positive

EBM‐2

Endothelial Basal Medium

EGM‐2

Endothelial Cell Growth Medium

FGF2

Fibroblast Growth Factor 2

GSK

Glycogen synthase kinase

hiPSCq

human induced‐pluripotent stem cell

HUVEC

human umbilical vein endothelial cell

L7Ae

archaeal RNA binding protein L7Ae

miR1/208

microRNA‐1 and microRNA‐208

miR1/208^RE

recognizing (seeding) sequence for microRNA‐1 and microRNA‐208

miR1/208^RE‐L7Ae

the first sequence in the modified RNA designed, miR1/208RE combined with L7Ae

modRNA

modified RNA

MYH6

Myosin Heavy Chain 6

MYL2

Myosin Light Chain 2

PH3

phosphorylated Histone H3

pRKAG

Protein Kinase AMP‐Activated Non‐Catalytic Subunit Gamma

RYR2

Ryanodine Receptor 2

TCF21+

Transcription Factor 21 positive

TNNT2

Cardiac Troponin Type 2

YAP

Yes‐associated protein

YAP5SA

Yes‐associated protein, 5SA (active) mutant

Clinical Perspective.

What Is New?

• This study demonstrates the use of a cardiomyocyte‐specific modified mRNA translation system for transiently overexpressing an activated variant of YAP5SA (Yes‐associated protein, 5SA [active] mutant) in infarcted mouse hearts.

• In vitro experiments confirmed that genes delivered via the cardiomyocyte‐specific modified mRNA translation system were highly expressed for approximately 1 week in cardiomyocytes but for <3 days in endothelial cells and cardiac fibroblasts.

• Cardiomyocyte‐specific modified mRNA translation system‐mediated YAP5SA overexpression significantly increased cardiomyocyte proliferation and myocardial regeneration in a mouse model of myocardial infarction.

What Are the Clinical Implications?

• Myocardial regeneration can be promoted by overexpressing proliferation‐inducing genes in cardiomyocytes; however, uncontrolled cardiomyocyte proliferation has been associated with arrhythmogenesis and hypertrophy, whereas off‐target overexpression in fibroblasts and macrophages could increase fibrosis and inflammation.

• The cardiomyocyte‐specific modified mRNA translation system minimizes these risks, because modified RNA is only transiently expressed after administration, and the administered gene is only expressed in cardiomyocytes.

• Thus, the cardiomyocyte‐specific modified mRNA translation system is a promising platform for promoting cardiomyocyte proliferation and myocardial regeneration in patients with cardiac disease.

Cardiovascular disease is the leading cause of death worldwide, with nearly half of cardiovascular mortality attributed to myocardial infarction (MI). ¹ The intrinsic mechanisms of myocardial repair are limited by the inability of postnatal cardiomyocytes to proliferate. Numerous strategies for promoting myocardial regeneration have been developed, ² but cell‐based approaches are limited by the small number of exogenous cells that are engrafted at the administration site, and genetic methods often raise long‐term safety concerns associated with persistent, uncontrolled overexpression of the targeted gene. Modified mRNA (modRNA) technology ³ circumvents these therapeutic obstacles, because the transcript is taken up by cells already present in the myocardium but is not incorporated into the cellular genome, ⁴ , ⁵ leading to transient gene expression. The clinical feasibility of modRNA administration is also well supported by the effectiveness and safety of modRNA‐based vaccines against SARS‐CoV‐2. ⁶

modRNA is typically generated by replacing uridine residues in the original sequence with pseudouridine, yielding a sequence with greater (though still limited) stability and low immunogenicity that can be quickly and efficiently translated by the cell's translation machinery. modRNA can be used to upregulate the activity of paracrine mechanisms that increase cell survival, promote angiogenesis, and impede fibrosis, or to deliver genes that directly induce cell‐cycle activity and proliferation in cardiomyocytes. ³ We have shown that the delivery of modRNA coding for the CCND2 (cell‐cycle regulatory molecule cyclin D2) promoted cardiomyocyte proliferation, reduced infarct size, and improved cardiac function in both mouse and pig models of MI ⁷ ; however, off‐target overexpression of cell‐cycle regulators could be detrimental (eg, upregulating the proliferation of cardiac fibroblasts could increase fibrosis). Thus, we used a novel, cardiomyocyte‐specific, modRNA translation system (CM‐SMRTs) ⁸ to ensure that the CCND2 transcript was only translated in cardiomyocytes. ⁷

YAP (Yes‐associated protein) is a key mediator of Hippo signaling, which plays a crucial role in cardiac development, ⁹ , ¹⁰ and previous studies have demonstrated that myocardial regeneration and contractility in infarcted hearts can be improved via the overexpression of YAP, ¹¹ whereas YAP deficiency impaired cardiac function by increasing apoptosis and reducing proliferation in cardiomyocytes. ¹² Thus, this report investigates whether the CM‐SMRTs can be used to drive the transient, cardiomyocyte‐specific, overexpression of a constitutively active YAP variant (YAP5SA), and whether this strategy can promote cardiomyocyte proliferation and improve cardiac regeneration in infarcted mouse hearts while limiting the detrimental effects of persistent or off‐target YAP upregulation.

METHODS

The data that support the findings of this study are available from the corresponding author upon request for purposes of reproducing the results or replicating the procedure. All experiments and procedures involving animals were approved by the Institutional Animal Care and Use Committee of the University of Alabama Birmingham School of Medicine and were consistent with the Guidelines for the Care and Use of Laboratory Animals published by the US National Institutes of Health (2011).

modRNA Production

Three modRNAs were generated for these studies, one that coded for L7Ae (archaeal RNA binding protein L7Ae) and contained recognition sites for the cardiomyocyte‐specific microRNAs miR‐1 and miR‐208 (miR1/208^RE‐L7Ae), a second that coded for GFP ((green fluorescent protein) and contained a kink‐turn motif (ktm) that served as a binding site for L7Ae (ktm‐GFP), and a third that coded for YAP5SA and also contained a ktm motif (ktm‐YAP5SA).

YAP5SA, the activated human form of YAP, contains a total of 8 serine (S) to alanine (A) mutations (S61A, S109A, S127A, S128A, S131A, S163A, S164A, and S381A) located in the 5 canonical Lats‐dependent phosphorylation motifs. YAP5SA was generated from plasmid PB‐TRE3‐YAP5SA‐P2A‐EGFP via polymerase chain reaction (PCR), ¹³ and a pRKAG plasmid vector was linearized with enzyme AleI/Afel. Then, both YAP5SA and pRKAG were purified, and the YAP5SA sequence was inserted into pRKAG with a NEBuilder HiFi DNA Assembly Cloning Kit (NEB; E5520S) to generate pRKAG‐YAP5SA, which was used as a template for synthesizing YAP5SA modRNA via in vitro transcription, as described previously. ¹⁴ Briefly, a tailed template was generated via PCR, purified with a Monarch PCR&DNA Cleanup Kit (NEB; T1030S), and then combined with a customized ribonucleotide mixture containing CleanCap Reagent AG, m7G(5′)ppp(5′)(2′OMeA)pG (TriLink Biotechnologies), guanosine triphosphate (Invitrogen), adenosine triphosphate (Invitrogen), cytidine triphosphate (Invitrogen), N1‐methylpseudouridine‐5′‐triphosphate (TriLink Biotechnologies), T7 enzyme, and 10× reaction buffer. The modRNA was purified with a Nonarch RNA cleanup kit (NEB), characterized with an Agilent 2100 bioanalyzer at the University of Alabama Birmingham Heflin Center for Genomic Science, and concentrated with Amicon Ultra‐4 10k Centrifugal Filters (Millipore; UFC801024).

Cell Differentiation, Culture, and Transfection

Human induced‐pluripotent stem cells were maintained in mTeSR Plus medium (Stemcell Technologies) and differentiated into human induced‐pluripotent stem cells‐cardiomyocytes (hiPSC‐CMs) ¹⁵ and human induced‐pluripotent stem cells‐cardiac fibroblasts (hiPSC‐CFs), ¹⁶ , ¹⁷ as described previously. Briefly, hiPSC‐CM differentiation was initiated by culturing hiPSCs until they were ≈80% confluent and then changing the culture medium to (1) basal medium (RPMI 1640 medium supplemented with 1× B27 without insulin) with 6 μmol/L CHIR‐99021 (a GSK‐3 inhibitor) for 24 hours; (2) basal medium with 3 μmol/L CHIR‐99021 for 48 hours; (3) basal medium with 10 μmol/L IWR‐1 (a Wnt inhibitor) for 48 hours; (4) fresh basal medium for 48 hours; and (5) cardiomyocyte maintenance medium (RPMI 1640 medium supplemented with 1× B27 with insulin). hiPSC‐CMs were purified for 1 week via metabolic selection in glucose‐free RPMI 1640 medium containing 1× B27 and 0.2% D‐lactate. Then, the hiPSC‐CMs were cultured for 60 days before use in subsequent experiments to ensure that their intrinsic proliferative capacity was minimal. hiPSC‐CF differentiation was followed, as previously reported. ¹⁷ Briefly, hiPSCs were about 80% confluence and induced into mesoderm cell by treating with 6 μmol/L of CHIR99021 for 2 days, then replaced with RPMI+B27(with insulin) for 24 hour. Applying with 5 μmol/L of IWR1 (Sigma) for 2 days to generate cardiac progenitor cells and replated at a density of 20 000 cells/cm² in advanced DMEM medium (Life Technologies, 12634028). iPSC cardiac progenitor cells were treated with 5 μmol/L of CHIR99021 and 2 μmol/L of retinoic acid (R2625; Sigma) for 3 days and recovered in advanced DMEM for another 4 days to generate epicardial cells. Human iPSC epicardial cells were replated and treated with 10 μmol/L of FGF2 (PeproTech; 100‐18B) and 10 μmol/L of SB431542 (Selleck chemicals; S1067) in a fibroblast growth medium for another 6 days. Usually >90% purity of iPSC‐CFs (COL‐I+/DDR2+/TCF21+) can be achieved, and hiPSC‐CFs were maintained in FIBGRO fibroblast growth medium (Lonza) with 10 μmol/L of SB431542. Human umbilical vein endothelial cells (HUVECs) were purchased from ATCC and maintained in EBM‐2 Basal Medium with EGM‐2 SingleQuots Supplements (Lonza); the culture medium was changed every other day and cells were passaged when reaching 90% confluency.

hiPSC‐CMs, hiPSC‐CFs, and HUVECs were transfected with modRNAs by using Lipofectamine MessengerMax Reagent (Invitrogen; LMRNA015) as directed by the manufacturer's instructions. Transfection was performed in 12‐well plates seeded with 2×10⁴ (hiPSC‐CMs) or 6‐well plates seeded with 2×10⁵ (hiPSC‐CFs and HUVECs) cells per well. hiPSC‐CMs were quantified with an automatic cell counter (Countess 3; Invitrogen).

Mouse MI Model and Treatment

Experiments were conducted in a total of 40 adult male C57BL/6J mice (Jackson Laboratory; stock number 000664;).

MI was induced, as described previously. ¹⁸ Briefly, 6‐ to 8‐week‐old mice were anesthetized with inhaled 2% isoflurane. Then, a 1.2‐cm cut was made over the left chest, the major and minor pectoral muscles were dissected and retracted, and a small opening was made in the fourth intercostal space. The pleural membrane and pericardium were opened, and a mosquito clamp was used to extract the heart. The left anterior descending coronary artery was permanently ligated with an 8‐0 nonabsorbable suture, and treatment was injected into 3 sites around the infarcted zone. Treatments consisted of 100 μg ktm‐GFP and 50 μg miR1/208^RE‐L7Ae in 30 μL sucrose‐citrate buffer (^GFPCM‐SMRTs, n=6), 100 μg ktm‐YAP5SA, and 50 μg miR1/208^RE‐L7Ae in 30 μL sucrose‐citrate buffer (^YAP5SACM‐SMRTs, n=14), or 30 μL sucrose‐citrate buffer (ie, the delivery vehicle, n=10). Doses were determined in previous studies of CM‐SMRTs administration in the same animal model, ⁷ and treatments were administered by researchers who were blinded to the experimental group. After treatment, the heart was immediately repositioned into the intrathoracic space, air was manually evacuated from the chest cavity, and the muscles and skin were closed. Mice were administered buprenorphine (0.1 mg/kg every 12 hours for 3 consecutive days) and carprofen (5 mg/kg every 12 hours for 1 day) after surgery for pain control during recovery, and left‐ventricular ejection fraction was measured via echocardiography 12 hours after surgery. Mice in the Sham group (n=6) underwent all surgical procedures for MI induction and recovery except for the ligation step.

Echocardiography

Echocardiographic assessments of left ventricular ejection fraction, left ventricular fractional shortening, and diastolic radial strain were performed, as described previously. ⁷ , ¹⁹ Mice were anesthetized with 1% to 2% inhaled isoflurane, and heart rates were maintained at 400 to 500 bpm. Then, 2‐dimensional B‐mode images of the heart were acquired from both the long‐axis and short‐axis views with a high‐resolution microultrasound system (Vevo F2; VisualSonics).

Histological Sectioning

Mice were anesthetized, and the hearts were quickly harvested, washed with cold PBS, fixed in 4% paraformaldehyde at 4 °C overnight, dehydrated in 30% sucrose buffer at 4 °C overnight, embedded in optimal cutting temperature compound (SAKURA), and frozen. Transverse sections (10 μm) were cut at 400‐μm intervals with a cryostat (Leica) and mounted on glass slides.

Infarct Size

Sections were stained with a Trichrome Stain Kit (Abcam; ab150686) as directed by the manufacturer's instructions, and infarct size was determined via midline length measurement, as reported previously. ²⁰ Briefly, the midline of the left ventricle wall was drawn equidistant from the epicardial and endocardial surfaces. Then, the ventricle circumference was determined by measuring the length of the midline, and the length of the infarct was determined by measuring the portion of the midline that was located in a region where the infarct occupied ≥50% of the thickness of the myocardial wall. Infarct size was calculated by dividing the sum of midline infarct lengths by the sum of midline circumferences for 9 to 12 sections per heart and presented as a percentage.

Immunofluorescence Analysis

Cells were seeded in chamber slides, treated with modRNA or the CM SMRTs, washed 3 times with PBS, and then fixed in 4% paraformaldehyde. The frozen sections were brought up to room temperature for 15 minutes and washed in Dulbecco's phosphate‐buffered saline for 5 minutes. Cells in chamber slides or tissue in frozen sections were permeabilized with 90% acetone for 3 minutes at −20 °C, blocked with 5% donkey serum for 30 minutes, incubated with primary antibodies (Table S1) at 4 °C overnight, incubated with Alexa 488‐, 594‐ or 647‐conjugated secondary antibodies (Table S1) for 60 minutes at room temperature in the dark, and then mounted on slides with Antifade Mounting Medium containing 4,6‐diamidino‐2‐phenylindole (Vector Laboratories). Slides were imaged with an inverted fluorescence microscope or a confocal microscope (Olympus, Japan). For in vivo studies, immunofluorescence staining was quantified in every tenth serial section from the region of interest for each heart; 5 randomly selected high‐resolution (×40 magnification) images were evaluated for each of 25 to 30 short‐axis sections (out of a total of 250–300) per heart, and the results were quantified with Image J software. For in vitro studies, quantification was performed in 5 randomly selected high‐resolution (×40 magnification) images were evaluated for each sample, 5 biological replications were performed, and the results were quantified with ImageJ software.

Western Blotting

Tissues were homogenized in RIPA lysis buffer (Pierce; 89 900) containing a protease inhibitor cocktail (Boster; AR1182), and cells were lysed directly in RIPA buffer; then, the samples were sonicated and centrifuged at 12 000g for 10 minutes. The supernatant was collected, and protein concentrations were quantified with a Pierce BCA Protein Assay Kit (Thermo Scientific). Then, 30‐ to 50‐μg protein was added to the 4× laemmli sample buffer (Bio‐Rad; 1610747) and boiled at 100 °C for 5 minutes. Samples were run on Mini‐PROTEANN TGX Stain‐Free Gels (Bio‐Rad) and transferred to polyvinylidene difluoride membranes via the semidry transfer method. The blots were blocked with 5% nonfat milk for 1 hour at room temperature and then incubated with primary antibodies (Table S1) at 4 °C overnight, with secondary antibodies (Table S1) for 1 hour at room temperature, and with SuperSignal West Pico PLUS Chemiluminescent Substrate (Thermo Scientific; 34 577). Blots were imaged with a Bio‐Rad ChemiDoc Imager, and the protein signal was digitized and quantified with ImageJ software.

Reverse Transcriptase‐Quantitative PCR

Total RNA was extracted from cells with Trizol (Invitrogen) and from tissues with a RNeasy Plus Universal Mini Kit (Qiagen; 73 404). cDNA was synthesized with the PrimeScript RT Reagent Kit (Takara; RR037A) and reverse transcriptase‐quantitative PCR was performed with the SYBR Green qPCR MasterMix (Bio‐Rad) and appropriate primers (Table S2) on a Step‐One real‐time PCR system (ABI).

Statistical Analysis

Mouse MI model and modRNA or vehicle treatments were administered by researchers who were blinded to treatment group, and data analysis was unblinded. Data normalcy was tested with the Shapiro‐Wilk test. For normally distributed data, statistical significance was determined with the unpaired t test for comparisons between 2 groups, via ANOVA and the Tukey test for comparisons among ≥3 groups, and via 2‐way ANOVA followed by the Tukey test or Šídák multiple comparisons test for multigroup comparisons involving >2 variables. Data that were not normally distributed were evaluated for significance via the Mann‐Whitney U test for 2‐group comparisons or the Kruskal‐Wallis H test for comparisons among ≥3 groups. A P value of <0.05 was considered statistically significant. Analyses were conducted with Graphpad Prism 9 software, and results were presented as mean±SEM.

RESULTS

^YAP5SACM‐SMRTs Activated YAP5SA Expression in Cardiomyocytes for ~1 Week After Administration

The CM‐SMRTs consists of 2 modRNA constructs, one that codes for the archaea ribosome protein L7Ae and contains recognition elements for the cardiomyocyte‐specific microRNAs miR‐1 and miR‐208 (miR1/208^RE‐L7Ae), and another that codes for the gene of interest (YAP5SA) and contains a ktm that serves as the binding site for L7Ae (ktm‐YAP5SA) (Figure 1A). Thus, L7Ae is expressed in noncardiomyocytes, where it blocks YAP5SA modRNA translation, whereas endogenous miR‐1 and miR‐208 expression degrades the L7Ae construct and activates YAP5SA expression in cardiomyocytes. The ktm‐YAP5SA and miR1/208^RE‐L7Ae constructs, as well as a control modRNA sequence containing the ktm and a GFP‐coding sequence (ktm‐GFP), were verified via both plasmid sequencing and modRNA integrity bioanalysis (Figure S1), and the cardiomyocyte‐specificity of the ^YAP5SACM‐SMRTs (ktm‐YAP5SA and miR1/208^RE‐L7Ae cotransfection) and the ^GFPCM‐SMRTs (ktm‐GFP and miR1/208^RE‐L7Ae cotransfection) was evaluated in hiPSC‐CMs and hiPSC‐CFs, as well as HUVECs.

Figure 1. CM‐SMRTs drives cardiomyocyte‐specific YAP5SA and GFP expression.

A, The sequences of the modRNA constructs composing the ^YAP5SACM‐SMRTs and ^GFPCM‐SMRTs are displayed as a schematic. B, hiPSC‐CMs were transfected with ^YAP5SACM‐SMRTs at the indicated concentration of ktm‐YAP5SA, and the abundance of total YAP, p‐YAP, and GAPDH was evaluated via Western blot 48 hours later. C, YAP and p‐YAP measurements were normalized to GAPDH to control for unequal loading. D and E, hiPSC‐CMs were transfected with ^YAP5SACM‐SMRTs (2.5 μg ktm‐YAP5SA and 1.25 μg miR1/208^RE‐L7Ae) or ^GFPCM‐SMRTs (2.5 μg ktm‐GFP and 1.25 μg miR1/208^RE‐L7Ae), and the abundance of (D) total YAP, p‐YAP, and cTnT or (E) GFP was evaluated via Western blot at the indicated time points. GAPDH abundance was also evaluated to control for unequal loading. F, GFP fluorescence (green) was monitored in hiPSC‐CMs at the indicated time points after ^GFPCM‐SMRTs transfection. G, GFP fluorescence was monitored in HUVECs and hiPSC‐CFs 24 hours and 3 days after transfection with GFP modRNA or ^GFPCM‐SMRTs. Scale bar=200 μm. N=3 biological replicates per experimental condition. *P<0.05, **P<0.01, ***P<0.001. Statistical significance was tested via 1‐way ANOVA with t test. CM‐SMRTS indicates cardiomyocyte‐specific modRNA translation system; cTnT, cardiac troponin T; GAPDH, glyceraldehyde phosphate dehydrogenase; GFP, green fluorescent protein; hiPSC‐CMs, human induced‐pluripotent stem cells‐cardiomyocytes; HUVECs, human umbilical vein endothelial cells; L7Ae, Archaeal RNA binding protein L7Ae; ktm, kink‐turn motif; miR1/208, microRNA‐1 and microRNA‐208; modRNA, modified RNA; miR1/208^RE: recognizing (seeding) sequence for microRNA‐1 and microRNA‐208; miR1/208^RE‐L7Ae, the first sequence in the modified RNA designed, miR1/208RE combined with L7Ae, ns, not significant; p‐YAP, phosphorylated YAP; YAP, Yes‐associated protein; and YAP5SA, Yes‐associated protein, 5SA (active) mutant.

A dose‐finding study in hiPSC‐CMs determined that maximum YAP protein abundance was achieved when 1×10⁶ cells were transfected with 2.5 μg ktm‐YAP5SA and 1.25 μg miR1/208^RE‐L7Ae (Figure 1B and 1C). When monitored over time, YAP abundance increased from 6 hours to 1 day after transfection and was maintained through day 3 before gradually declining to near‐background levels after day 7 (Figure 1D); the abundance of phosphorylated YAP was largely unchanged, and far lower than total YAP levels, regardless of the ktm‐YAP5SA dose or time point. Unexpectedly, cardiac troponin T (cTnT) expression also declined over time, perhaps because YAP‐overexpressing cardiomyocytes acquired a more primitive phenotype that reduced sarcomeric protein expression, as has been previously reported. ²¹ GFP protein abundance in hiPSC‐CMs remained high for at least 1 week after ^GFPCM‐SMRTs transfection (Figure 1E) and then declined substantially during the second week, whereas GFP fluorescence remained high from day 1 through day 3 before slowly declining (Figure 1F). Substantial GFP fluorescence was also observed in HUVECs and hiPSC‐CFs on the first day after ^GFPCM‐SMRTs transfection, but by day 3, the fluorescence signal declined substantially and was far lower than that observed in HUVECs and hiPSC‐CFs that had been transfected with modRNA coding for GFP alone (Figure 1G).

^YAP5SACM‐SMRTs Promoted the Proliferation of Cultured hiPSC‐CMs

Both the abundance of YAP mRNA (Figure 2A) and the frequency of YAP protein expression (Figure 2B) were significantly greater when hiPSC‐CMs were transfected with ^YAP5SACM‐SMRTs rather than ^GFPCM‐SMRTs, and these increases were accompanied by the upregulation of 2 downstream targets of YAP, CTGF, and Cyr61 (Figure 2A), which confirmed that ^YAP5SACM‐SMRTs significantly increased YAP pathway activity in hiPSC‐CMs. Notably, the increase in YAP mRNA abundance (≈2500‐fold) far exceeded increases in YAP protein (25‐fold, Figure 1C) or CTGF and Cyr61 mRNA (~2‐fold), which suggests that although ^YAP5SACM‐SMRTs transfection was exceptionally efficient, only a fraction of the YAP5SA transcript was translated into YAP protein that subsequently entered the nucleus to activate the expression of YAP‐targeted genes. The abundance of mRNA transcripts (Figure 2C) and the frequency of protein expression (Figure 2D through 2F) for markers of cell proliferation (Ki67, PCNA [proliferating cell nuclear antigen], CCND2, PH3 [phosphorylated histone 3], and Aurora B) were also significantly greater in hiPSC‐CMs after ^YAP5SACM‐SMRTs transfection than in ^GFPCM‐SMRTs–transfected hiPSC‐CMs, and cell counts were greater in hiPSC‐ CMs 14 days after the cells were transfected with ^YAP5SACM‐SMRTs than at the same time point after transfection with CM‐SMRTs coding for luciferase (^LucCM‐SMRTs) (Figure 2G). However, ^YAP5SACM‐SMRTs–transfected hiPSC‐CMs expressed lower levels of genes associated with contractile activity (TNNT2, MYH6, MYL2) and calcium handling (ATP2A2, RYR2) (Figure 2H). Collectively, these observations indicate that the ^YAP5SACM‐SMRTs not only promoted proliferation but also induced a less functionally mature phenotype in cultured hiPSC‐CMs.

Figure 2. Treatment with ^YAP5SACM‐SMRTs promotes a more proliferative, less mature phenotype in cultured hiPSC‐CMs.

A through F, hiPSC‐CMs were transfected with ^YAP5SACM‐SMRTs or ^GFPCM‐SMRTs. A, Forty‐eight hours later, the abundance of mRNA transcripts for YAP and 2 downstream targets of YAP signaling, CTGF and CYR61, were measured via RT‐qPCR. B, Cells were stained for the expression of YAP and cTnT, nuclei were counterstained with DAPI, and the percentage of hiPSC‐CMs that were YAP‐positive was calculated. C, Abundance of mRNA transcripts for the proliferation/cell‐cycle–activity markers Ki67 (Ki67), PCNA, and CCND2 was measured via RT‐qPCR. D through F, Cells were stained for the expression of the proliferation markers (D) Ki67, (E) PH3, or (F) AuroraB, and for cTnT, nuclei were counterstained with DAPI, and the percentage of cells that were positive for each proliferation marker was calculated. G, hiPSC‐CMs cell counts were determined before (0 day) and 14 days after transfection with ^YAP5SACM‐SMRTs or CM‐SMRTs driving luciferase expression (^LucCM‐SMRTs). H, hiPSC‐CMs were transfected with ^YAP5SACM‐SMRTs or ^GFPCM‐SMRTs; 48 hours later, the abundance of mRNA transcripts for the calcium‐handling and contractile proteins troponin T2 (TNNT2, cTNT), MYH6, MYL2, sarcoplasmic/endoplasmic reticulum calcium ATP2A2, and RYR2 was measured via RT‐qPCR. Scale bar=100 μm. N=3 biological replicates per experimental condition. *P<0.05, **P<0.01, and ***P<0.001. Statistical significance was tested via unpaired t test in (A) through (F) and (H), and 2‐way ANOVA with Šídák multiple comparisons in (G). ATP2A2 indicates ATPase 2; AuroraB, Aurora kinase B; CCND2, cell‐cycle regulatory molecule cyclin D2; CM, cardiomyocyte; CM‐SMRTS, cardiomyocyte‐specific modRNA translation system; CTGF, connective tissue growth factor; cTnT, cardiac troponin T; CYR61, cysteine‐rich angiogenic inducer 61; DAPI, 4′,6‐diamidino‐2‐phenylindole; GFP, green fluorescent protein; hiPSC‐CMs, human induced‐pluripotent stem cells‐cardiomyocytes; Ki67, activity markers Ki67; Luc, luciferase; MYH6, myosin heavy chain 6; MYLT, myosin light chain 2; PCNA, proliferating cell nuclear antigen; PH3, phosphorylated histone 3; RT‐qPCR, reverse transcriptase‐quantitative polymerase chain reaction; RYR2, ryanodine receptor 2; YAP, Yes‐associated protein; TNNT2, Cardiac Troponin Type 2; and YAP5SA, Yes‐associated protein, 5SA (active) mutant.

^YAP5SACM‐SMRTs Improved Cardiac Function and Infarct Size When Delivered to Mouse Hearts After MI

To investigate whether the increases in cardiomyocyte proliferation associated with ^YAP5SACM‐ SMRTs transfection in vitro may promote recovery from cardiac injury in vivo, MI was experimentally induced in mice via ligation of the left anterior descending coronary artery, and the animals were then treated with ^YAP5SACM‐SMRTs (100 μg ktm‐YAP5SA and 50 μg miR1/208^RE‐L7Ae in 30% sucrose; the MI+ ^YAP5SACM‐SMRTs group) or the delivery vehicle (the MI+ vehicle group) (Figure 3A). The treatments were delivered to the border zone of the infarct via direct intramyocardial injection, and a third group of animals (the Sham group) underwent all surgical procedures for MI induction except the ligation step and recovered without ^YAP5SACM‐SMRTs or vehicle administration. No treatment‐associated adverse events were observed.

Figure 3. Treatment with ^YAP5SACM‐SMRTs improved recovery from MI in mice.

A, Experimental timeline is displayed as a schematic. B through F, MI was experimentally induced in mice, and then the animals were treated with (B) ^GFPCM‐SMRTs or (C through F) ^YAP5SACM‐SMRTs (MI+^YAP5SACM‐SMRTs) or the delivery vehicle (MI+vehicle). A third group of animals (the Sham group) underwent all surgical procedures for MI induction except the ligation step and recovered without ^YAP5SACM‐SMRTs or vehicle administration. B, Two days after MI and treatment administration, GFP fluorescence (green) was visualized in heart sections stained for the expression cTnT (red); nuclei were counterstained with DAPI (blue). Scale bar=50 μm. C through E, Echocardiographic images were collected before MI induction (day 0) and 1, 7, 14, 21, and 28 days afterward. C, Representative images are displayed for each animal group at the indicated time points. D and E, Left ventricular (D) ejection fraction, (E) fractional shortening, and (F) radial strain were determined from images collected at the indicated time points. Hearts were harvested 28 days after Sham surgery or MI induction and treatment. G, Representative images of trichrome‐stained left ventricle short‐axis sections are displayed for each group; the infarcted region appears blue. Scale bar=2 mm. H, Infarct sizes (as a percentage of the circumference of the left ventricle) and (I) heart weight:body weight ratios were calculated for each group. N=6 per group for (D) through (F), and n=7 per group for (G) and (H). Each point represents 1 animal in (H). *P<0.05, **P<0.01, and ***P<0.001. Statistical significance was tested via 2‐way ANOVA with Tukey test in (D) and (E), 1‐way ANOVA with Tukey test in (D) through (F) and (H) and (I). ATP2A2 indicates ATPase 2; AuroraB, Aurora kinase B; CM‐SMRTS, cardiomyocyte‐specific modRNA translation system; cTnT, cardiac troponin T; DAPI, 4′,6‐diamidino‐2‐phenylindole; GFP, green fluorescent protein; HW/BW, heart weight/body weight; LAD, left anterior descending; Luc, luciferase; MI, myocardial infarction; MYH6, myosin heavy chain 6; MYLT, myosin light chain 2; PCNA, proliferating cell nuclear antigen; PH3, phosphorylated histone 3; RYR2, ryanodine receptor 2; YAP, Yes‐associated protein; and YAP5SA, Yes‐associated protein, 5SA (active) mutant.

Preliminary experiments with ^GFPCM‐SMRTs (100 μg ktm‐GFP and 50 μg miR1/208^RE‐L7Ae) confirmed that the CM‐SMRTs was highly cardiomyocyte‐specific after delivery to infarcted mouse hearts: GFP fluorescence was observed exclusively in cells that expressed cTnT (Figure 3B). Echocardiographic assessments (Figure 3C) of left ventricular ejection fraction (Figure 3D) and fractional shortening (Figure 3E) were equivalent in all 3 groups before MI induction, and 1 day after MI, measurements in the MI+^YAP5SACM‐SMRTs and MI+ vehicle groups remained equivalent but were significantly lower than in the Sham group. However, although both left ventricular ejection fraction and fractional shortening remained stable (or declined slightly) in the MI+vehicle group at later time points, measurements in MI+^YAP5SACM‐SMRTs animals improved and were significantly greater than in MI+vehicle animals on days 14 to 28 after MI induction. Also, radian strain in MI+^YAP5SACM‐SMRTs mice was significantly higher than in MI+vehicle mice (Figure 3F). Analysis of trichrome‐stained heart sections (Figure 3G) obtained at day 28 indicated that infarcts were significantly smaller in the MI+^YAP5SACM‐SMRTs group than in MI+vehicle animals (Figure 3H), but heart weight to body weight ratios in the 2 groups did not differ significantly (Figure 3I). Immunofluorescence imaging assessments of Ki67 and PH3 expression suggested that ^YAP5SACM‐SMRTs treatment promoted cardiomyocyte proliferation (Figure 4A through 4C) and cardiomyocyte cross‐sectional surface areas were smaller in MI + ^YAP5SACM‐SMRTs hearts than in MI + vehicle hearts (Figure 4D). Thus, ^YAP5SACM‐SMRTs administration appeared to improve recovery from MI in mice by promoting cardiomyocyte proliferation.

Figure 4. Treatment with ^YAP5SACM‐SMRTs promoted cardiomyocyte proliferation in infarcted mouse hearts.

A through C, Sections were collected from the border zone of the infarct in the hearts of mice from the MI+^YAP5SACM‐SMRTs and MI+vehicle groups on day 28 after MI induction and then stained for the expression of (A) YAP or the proliferation markers (B) Ki67 or (C) PH3 and cTnT; nuclei were counterstained with DAPI. Scale bar=50 μm. D, Sections were collected from the border zone of the infarct in the hearts of mice from the MI+^YAP5SACM‐SMRTs and MI+vehicle groups, and from the corresponding region of hearts from the Sham group on day 28 and then stained with WGA (light blue) to identify cell borders and for the expression of cTnT. nuclei were counterstained with DAPI. Scale bar=20 μm. N=5 biological replicates per experimental condition. **P<0.01 and ***P<0.001. Statistical significance was tested via unpaired t test in (A) through (C) and 1‐way ANOVA with Tukey test in (D). CM indcates cardiomyocyte; CM‐SMRTS, cardiomyocyte‐specific modRNA translation system; DAPI, 4′,6‐diamidino‐2‐phenylindole; cTnT, cardiac troponin T; Ki67, activity markers Ki67; GFP, green fluorescent protein; MI, myocardial infarction; PH3, phosphorylated histone 3; WGA, wheat‐germ agglutinin; YAP, Yes‐associated protein; and YAP5SA, Yes‐associated protein, 5SA (active) mutant.

DISCUSSION

YAP is one of many genes, microRNAs, and small molecules that appear to have a role in myocardial regeneration. ²² , ²³ , ²⁴ , ²⁵ , ²⁶ , ²⁷ , ²⁸ , ²⁹ Epicardial YAP expression contributes to the recruitment of T‐regulatory cells, which suppress the immune response and limit cardiac remodeling after myocardial injury, ³⁰ whereas treatment with 2 different activated YAP variants has been linked to improvements in cardiomyocyte necrosis, cardiac inflammation, and hypertrophy in models of myocardial ischemia–reperfusion injury, ³¹ as well as cardiomyocyte dedifferentiation and nuclear replication when combined with mutations in serum response factor. ³² However, YAP upregulation increased both the proliferation of cardiac fibroblasts and their transdifferentiation into myofibroblasts, leading to increases in fibrosis and inflammation, ³³ whereas macrophage YAP expression appears to have a role in cardiac fibrosis and hypertrophy. ³⁴ Furthermore, although studies in transgenic mice have shown that the persistent, cardiomyocyte‐specific overexpression of constitutively active YAP variants can promote myocardial regeneration and contractility in infarcted hearts, ¹¹ it also led to the development of heart failure. ²¹ Thus, the clinical usefulness of genetic approaches to YAP upregulation is crucially dependent on controlling both its duration and cell‐type specificity.

The results presented in this report conclusively demonstrate that our CM‐SMRTs can be used to transiently overexpress YAP5SA in cardiomyocytes, and that this treatment strategy significantly promoted myocardial regeneration in a mouse MI model. modRNAs are easily synthesized in vitro and less immunogenic than endogenous mRNA transcripts with no risk of genomic integration. They are also more stable than unmodified mRNAs, but whereas GFP fluorescence was detected in hiPSC‐CMs for 2 weeks after treatment with ^GFPCM‐SMRTs, YAP expression declined to near‐background levels just 1 week after the cells were treated with ^YAP5SACM‐SMRTs, which suggests that the duration of modRNA expression can vary substantially depending on the sequence of the encoded gene. GFP fluorescence was also observed in hiPSC‐CFs and HUVECs 1 day after ^GFPCM‐SMRTs administration but declined dramatically by day 3, which suggests that the miR1/208^RE‐L7Ae construct had to be translated for approximately 2 days before intracellular L7Ae protein levels were sufficiently high to block GFP expression.

The role of cardiomyocyte YAP activity in myocardial regeneration extends well beyond promoting cardiomyocyte proliferation. YAP also regulates the expression of genes that induce F‐actin polymerization and cytoskeletal remodeling, ³⁵ as well as noncanonical Wnt signaling, and the Wnt ligands produced by cardiomyocytes appear to activate Wnt receptors in fibroblasts that reduce fibrosis. ³⁶ Furthermore, the results from spatial transcriptomics and single‐cell RNA sequencing studies indicated that when YAP5SA expression was induced in the cardiomyocytes of adult mice, a subpopulation of cardiomyocytes with high YAP activity colocalized with C3‐expressing cardiac fibroblasts and macrophages that expressed the C3ar1 receptor to form a cellular triad that promoted cardiac regeneration. ³⁷

The number of animals in each group was calculated based on our previous experiences using modRNA and the mouse model, ⁷ and the Rosner's equation ³⁸ using values for power=0.80 and significance level=0.05. According to this calculation, animals are required for each group of this study were: buffer (GFP174 CMSMRTs, n=6), 100 μg ktm‐YAP5SA and 50 μg miR1/208RE175‐L7Ae in 30 μL sucrose‐citrate buffer (YAP5SA176 CM‐SMRTs, n=14), and 30 μL sucrose‐citrate buffer (ie, the delivery vehicle, n=10).

In conclusion, intramyocardial injections of ^YAP5SACM‐SMRTs significantly upregulated YAP activity in cardiomyocytes and promoted both cardiomyocyte proliferation and myocardial regeneration in a mouse MI model. Alternative delivery methods, such as lipid nanoparticles ³⁹ , ⁴⁰ , ⁴¹ may further improve the efficiency of cellular modRNA uptake, and future experiments in large mammals (eg, pigs) will be necessary to facilitate the development of this strategy for clinical applications.

Sources of Funding

This work was supported in part by the following funding sources: National Institutes of Health RO1s: HL114120, HL131017, HL 149137, NIH UO1 HL134764, NIH PO1 HL160476.

Disclosures

None.

Supporting information

Tables S1–S2

Figure S1