Apoptotic bodies derived from human umbilical cord mesenchymal stem cells improve recovery from myocardial infarction in swine

ABSTRACT

Apoptotic bodies (ABs) are a type of extracellular vesicles (EVs) that could contribute to the paracrine effect of stem cells. However, their potential in treating cardiovascular diseases is largely unexplored. This study investigated the therapeutic effects of ABs derived from human umbilical cord mesenchymal stem cells (MSCs) on cardiac recovery in a porcine model of myocardial infarction (MI). In vitro, ABs reduced apoptosis and cytotoxicity in cardiomyocytes under oxygen and glucose deprivation (OGD) conditions and enhanced the capacity of migration and tube formation in endothelial cells. In vivo, akin to MSCs, administration of ABs improved contractile function, reduced infarct size, and mitigated adverse remodeling in pig hearts with MI, concomitantly with increased cardiomyocyte survival and angiogenesis. These cardioprotective effects were mediated through the regulation of autophagy by activating the adenosine monophosphate – activated protein kinase (AMPK) and transcription factor EB (TFEB) signaling pathways. microRNAs contained in ABs were sequenced, revealing that let-7f-5p was the most abundant. let-7f-5p promoted AMPK phosphorylation by targeting protein phosphatase 2 regulatory subunit B alpha (PPP2R2A) and decreased TFEB phosphorylation by targeting MAP4K3 to regulate autophagy, thereby contributing to the effects of ABs. Overall, these findings indicate that MSC-derived ABs have the potential to be a promising and effective acellular therapeutic option for treating MI.

Introduction

Cardiovascular diseases, especially myocardial infarction (MI), are a major cause of mortality and morbidity worldwide [1]. Various types of stem cells and their derivatives have been applied in animal models and clinical trials to promote cardiac repair and combat MI [2,3]. Among them, human umbilical cord mesenchymal stem cells (MSCs) have attracted the attention of a wide range of researchers and clinicians [4] due to their ease of acquisition and expansion, favorable self-renewal and proliferation, plasticity, and low immunogenicity [5]. Transplantation of MSCs has been shown to have protective effects in several medical conditions, including MI [6–8]. However, the underlying mechanism is still largely unclear.

It has been widely demonstrated that stem cells have the ability to repair the heart with MI in a paracrine way, primarily mediated by extracellular vesicles (EVs) [9,10]. EVs are bubble-shaped membrane vesicles released from cells into the extracellular space [11], where they can be taken up by other cells via receptor-dependent endocytosis, micropinocytosis, plasma membrane fusion, or phagocytosis [12,13]. Depending on their origin, EVs are classified into three populations, namely exosomes, microvesicles, and apoptotic bodies (ABs). Current studies on the paracrine function of stem cells mainly focus on exosomes and microvesicles [12,14], while the effects and therapeutic application of ABs are largely unexplored.

ABs, a distinct type of EVs released by cells that undergo programmed cell death (apoptosis), possess biological activity in tissue regeneration and repair [15,16]. However, there have been limited reports on the effect of ABs on cardiovascular diseases. There is a paradoxical mismatch between the low engraftment of transplanted MSCs and their beneficial therapeutic effects [3]. Moreover, the transplanted cells quickly become apoptotic [17,18], resulting in a reduction of exosome- and microvesicle-mediated effects and production of ABs in the process of cell apoptosis. Thus, we speculate that ABs may play an essential role in the therapeutic effects of MSCs against MI. It is also worth investigating whether MSC-derived ABs could become a novel acellular option to promote myocardial healing after MI. Considering that microRNAs (miRNAs) are one of the most active components of EVs [19], there is a solid rationale to elucidate the types of miRNAs that are abundant in ABs and the primary factor responsible for safeguarding the heart with MI. Additionally, it is important to clarify the underlying mechanism.

Experimental data from small-animal models offer limited guidance for clinical translation due to significant disparities in cardiac anatomy, electrophysiology, and hemodynamics between rodents and humans [20]. Consequently, studies utilizing large-animal models are comparable to clinical trials and may have higher translational value for cardiovascular disease research [21]. Here, we mainly investigated whether MSC-derived ABs could restore heart function and reverse cardiac remodeling after MI in pigs. To the best of our knowledge, this study was the first to focus on the therapeutic effects of ABs in a preclinical porcine model of MI.

Therefore, this study aimed to (i) examine the effect of MSC-derived ABs on cardiomyocytes and endothelial cells (ECs) in vitro, (ii) explore the effects of ABs on promoting cardiac recovery in a porcine model of MI, (iii) identify which miRNA is the most abundant and contributes the most to AB-mediated beneficial effects, and (iv) investigate the underlying mechanism.

Materials and Methods

A detailed description of the experimental procedures used in this study is provided in the Supplemental Material.

Results

Characteristics of MSCs and MSC-derived ABs

First, MSCs were successfully isolated from human umbilical cords and cultured, as previously described [22]. Immunohistochemical staining showed that MSCs expressed CD73, CD90, and CD105 but did not express CD34 and CD45 (Figure 1A). Similarly, flow cytometry revealed that MSCs strongly expressed CD73, CD90, and CD105 but did not express CD34, CD45, human leukocyte antigen DR (HLA-DR), CD11, and CD19 (Figure S1A). After induction, MSCs could differentiate into osteocytes (Figure S1Bi), adipocytes (Figure S1Bii), and chondrocytes (Figure S1Biii). Next, ABs were obtained by treating MSCs with staurosporine and a sequential centrifugation procedure [23,24]. Nanoparticle tracking analysis (NTA) (Figure 1B) and scanning electron microscopy (SEM) (Figure 1C) revealed that the isolated ABs typically showed a round shape and had a diameter ranging from 400 to 800 nm; western blot showed that ABs expressed the specific apoptotic marker cleaved caspase-3 (Figure 1D); and immunofluorescence staining showed that the apoptotic marker annexin A5 (ANXA5) bound to phosphatidylserine was exposed on the membrane surface of ABs (Figure 1E). These results demonstrate that the generated MSCs and MSC-derived ABs possess typical characteristics and are suitable for further investigation.

Figure 1.

Characterization and cytoprotection of apoptotic bodies (ABs) derived from human umbilical cord mesenchymal stem cells (MSCs). (A) MSCs were characterized by immunofluorescence expression of CD73, CD90, and CD105 but not of CD34 and CD45. Bar = 75 μm. (B) the size distribution of MSC-derived ABs was measured by using a zetasizer nano series instrument. (C) the morphology of ABs was examined by using a scanning electron microscope (SEM). Bar = 500 nm. (D) the protein expression of caspase-3 (casp-3) and cleaved caspase-3 (cleaved casp-3) in MSCs and MSC-derived ABs was detected by western blot. (E) ABs were immunofluorescently stained with annexin A5 (ANXA5) to show the exposed phosphatidylserine on the surface. Bar = 75 μm. (F) hiPSC-derived cardiomyocytes (hiPSC-CMs) were incubated with 1,1”-dioctadecyl-3,3,3,”3’-tetramethylindocarbocyanine perchlorate (Dil)-labeled ABs (Dil-ABs) for 12 h; then, the cardiomyocytes were fixed and immunofluorescently stained for cardiac troponin T (cTnT) expression, and nuclei were counterstained with DAPI. ABs that the cardiomyocytes had internalized were identified by Dil fluorescence (bar = 25 μm). (G – I) hiPSC-CMs were cultured under oxygen and glucose deprivation (OGD) conditions and treated with phosphate-buffered saline (PBS) or a concentration gradient of ABs (5, 10, 20, and 30 μg/mL) for 48 h. (G) the cardiomyocytes were immunofluorescently stained for cTnT expression and stained by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL); then, nuclei were counterstained with DAPI (bar = 50 μm). (H) quantification for TUNEL+ cardiomyocytes. (I) the cytotoxicity of hiPSC-CMs cultured under OGD conditions was assessed via a lactate dehydrogenase (LDH) release kit. n = 4 independent experiments. Significance was evaluated via one-way analysis of variance (ANOVA), followed by Tukey’s post-hoc test in (H and I). *p < 0.05 and **p < 0.01.

MSC-derived ABs protect cardiomyocytes from injury caused by oxygen and glucose deprivation (OGD) and promote the angiogenic potential of ECs in vitro

Then, we examined whether MSC-derived ABs have a protective effect on cardiomyocytes under injury conditions in vitro. Analysis of dye 1,1”-dioctadecyl-3,3,3,”3’-tetramethylindocarbocyanine perchlorate (Dil) fluorescence images indicated that when human induced pluripotent stem cell – derived cardiomyocytes (hiPSC-CMs) were co-cultured with Dil-labeled ABs for 12 h, a large number of ABs were internalized by the cardiomyocytes (Figure 1F), and most of them were co-localized with lysosomes (Figure S1C). Next, hiPSC-CMs were cultured under OGD conditions and treated with phosphate-buffered saline (PBS) or a concentration gradient of ABs for subsequent analyses. TUNEL staining revealed significantly less apoptosis of cardiomyocytes under OGD injury conditions after 10, 20, or 30 μg/mL AB treatment compared with PBS treatment (Figure 1G,H). In line with this, lactate dehydrogenase (LDH) release assay showed that ABs at concentrations greater than or equal to 10 μg/mL significantly mitigated the cytotoxicity of hiPSC-CMs (Figure 1I) in a dose-dependent manner.

Furthermore, we conducted tube formation and migration assay to evaluate the pro-angiogenic efficacy of ABs. The Dil and CD31 co-expression images confirmed that ABs could also be endocytosed by human umbilical vein ECs (HUVECs) (Figure S2A). The tube-forming activity of ECs was markedly improved following the treatment with ABs (10, 20, or 30 μg/mL), as evidenced by the augmented number and total length of branches (Figure S2B – D). Similarly, the wound healing assay showed that ABs at concentrations greater than or equal to 10 μg/mL significantly boosted HUVEC migration, as evidenced by a reduced wound area (Figure S2E and S2F). Collectively, these findings demonstrate that ABs have the potential to safeguard cardiomyocytes from OGD injury and promote the angiogenic capacity of ECs.

MSC-derived ABs improve cardiac function in a murine model of MI

To the best of our knowledge, no study has reported the benefits of transplantation of ABs in a large-animal model of MI. To determine the optimal dose of MSC-derived ABs for pigs in this study, ABs in different concentrations (5, 10, and 20 μg) were administered to assess the most effective therapeutic outcome in enhancing cardiac contractile function in a murine MI model. Immunofluorescence analyses revealed that Dil-labeled ABs were largely taken up by cardiomyocytes and ECs (Figure 2A,B) in the border zone (BZ) of the murine heart after injection. Furthermore, echocardiographic assessment showed that ABs significantly improved cardiac function in a dose-dependent manner, as evidenced by restoring the left-ventricular (LV) ejection fraction (LVEF) and LV fractional shortening (LVFS) at day 28 after MI (Figure 2C,D). Sirius Red and Fast Green staining also showed that ABs markedly reduced cardiac infarct size in a dose-dependent manner (Figure 2E,F). Consequently, given that 20 μg of ABs emerged as the optimal therapeutic dosage in the murine MI model without any abnormalities in mouse activities, we extrapolated and implemented a dosage of 2 mg of ABs to assess its effectiveness and explore the underlying mechanisms in the subsequent porcine MI study.

Figure 2.

Abs improve cardiac function recovery in a mouse model of myocardial infarction (MI). Mice were divided into five groups. One group underwent sham surgery (sham group), and the other four groups underwent MI surgery, followed by treatment with PBS (MI group) or ABs (5, 10, or 20 µg: MI+AB groups). (A – B) mice underwent MI surgery, after which Dil-labeled ABs (20 µg) were intramyocardially injected into the ischemic myocardium. Three days later, immunofluorescence staining was performed on frozen heart sections to observe the internalization of ABs (Dil⁺) into (A) cardiomyocytes (cTnT⁺) and (B) endothelial cells (CD31⁺) in vivo. Bar = 25 µm. (C – D) cardiac function was evaluated at day 28 after MI or sham surgery via echocardiographic assessments of (C) the left-ventricular ejection fraction (LVEF) and (D) fractional shortening (LVFS). (E – F) hearts were harvested 28 days after MI. Fibrosis was assessed in cryosections (E) stained with Sirius red (fibrotic tissue) and Fast Green (functional cardiac tissue) and then (F) quantified as the ratio of the fibrotic area to the total area. n = 7–9 animals per group. Significance was evaluated via one-way ANOVA, followed by Tukey’s post-hoc test in (C, D, and F). *p < 0.05 and **p < 0.01.

MSC-derived ABs improve heart performance and limit cardiac remodeling in a porcine MI model

MSCs and MSC-derived ABs were separately injected into the ischemic regions of pig myocardium to examine the cardioprotective effects of MSCs and ABs against MI. Twenty-eight days after MI and MSC transplantation, the existence of MSCs in pig hearts was identified using immunofluorescence staining for human-specific nuclear antigen expression. However, we found a minimal residual number of these cells. Furthermore, most of them were TUNEL-positive, indicating that these cells underwent apoptosis (Figure S3A). This implies that the therapeutic effect of MSCs might be, at least partly, mediated through the release of ABs. We also found that the transplanted Dil-labeled ABs could be internalized by porcine cardiomyocytes (Figure S3B) and ECs (Figure S3C). Cardiac function was evaluated 28 days after MI and treatments via echocardiographic and hemodynamic analyses before the animals were euthanized for the assessment of cardiac infarct size, fibrosis, and hypertrophy. Compared with the MI group, both MSC and MSC-derived AB treatments significantly enhanced LVEF and LVFS (Figure 3A–C); decreased LV end-diastolic volume (LVEDV), LV end-systolic volume (LVESV), and LV end-diastolic pressure (LVEDP) (Figure S3D and S3E, Figure 3D); conserved the maximum rising rate of LV pressure (+dp/dt_max) and maximum declining rate of LV pressure (−dp/dt_max) (Figure 3E,F); and had no effect on heart rate (Figure S3F), and there were no differences between MSC and AB treatments. Furthermore, analysis of fresh heart slides and Sirius Red and Fast Green staining showed that the treatment with MSCs or ABs significantly reduced the cardiac infarct size (Figure 3G,H), as well as the fibrosis in the BZ and infarct zone (IZ) of the hearts with MI (Figure 3I,J, Figure S3G and S3H). Consistent with this, the apparent cardiac hypertrophy was progressively reversed after MSC and AB treatments, as evidenced by a reduction in the ratio of heart weight to body weight (Figure S3I) and cardiomyocyte size detected by wheat germ agglutinin (WGA) staining (Figure 3K,L). Taken together, these results demonstrate that like MSCs, ABs derived from MSCs have the potential to enhance cardiac function, diminish infarct size, limit cardiac fibrosis, and reduce cardiac hypertrophy in the porcine model of MI.

Figure 3.

MSC-derived ABs ameliorate myocardial function and mitigate cardiac remodeling in a porcine model of MI. Pigs were divided into four groups; one group underwent sham surgery (sham group), and the other three groups underwent MI surgery, followed by treatment with PBS (MI group), MSCs (MI+MSC group), or ABs derived from MSCs (MI+AB group). (A – C) cardiac function was evaluated via (A) echocardiography at day 28 after MI or sham surgery. (B) the LVEF and (C) LVFS were examined. (D – F) hemodynamic measurements of (D) LV end-diastolic pressure (LVEDP), (E) maximum ascending rate of pressure (+dp/dt_max), and (F) maximum declining rate of pressure (−dp/dt_max) were performed 28 days after MI or sham surgery. (G – H) hearts were harvested on day 28 after MI or sham surgery, and cut into five equally spaced transverse slices (R1-R5) perpendicular to the long axis of the ventricles. The scarred (pale) area on the slices were identified as the infarct zones. (G) Representative third slices are shown. (H) infarct size was quantified as the ratio of the total LV scar surface area to the total LV surface area and presented as a percentage. (I – J) fibrosis in the infarcted zone was assessed by staining with (I) Sirius red (fibrotic tissue) and Fast Green (functional cardiac tissue) and (J) quantified as a ratio of the fibrotic area to the total area. (K) wheat germ agglutinin (WGA) staining was performed to visualize cardiomyocyte borders in the border zone (BZ) of the infarcted hearts (bar = 75 μm). (L) cardiomyocyte cross-sectional surface areas (CSAs) were quantified. n = 6–8 animals per group. Significance was evaluated via one-way ANOVA, followed by Tukey’s post-hoc test in (B – F, H, J, and L). *p < 0.05 and **p < 0.01.

MSC-derived ABs promote cardiomyocyte survival and angiogenesis in pig hearts with MI

Next, we investigated the critical roles of MSC-derived ABs in the key pathological processes related to cardiac remodeling after MI, including cardiomyocyte apoptosis and angiogenesis in pig hearts with MI. The immunofluorescence staining showed that MSC and AB treatments were almost equally effective in reducing the proportion of apoptotic (TUNEL-positive) cardiomyocytes (Figure 4A,B) in BZ. In line with this, western blot showed that the treatments with MSCs or ABs dramatically promoted the restoration of the expression of the anti-apoptotic protein B-cell lymphoma 2 (Bcl-2) (Figure 4C,D) and reduced the expression of pro-apoptotic proteins, including Fas and Bcl-2-associated X protein (Bax). Moreover, immunofluorescence analyses also indicated that measures of vascular density (structures expressing CD31) (Figure 4E,F) and arteriole density (structures expressing both CD31 and α-SMA) (Figure 4E,G) in the BZ of the infarcted hearts from the MI+MSC and MI+AB groups were similar and significantly greater than those from the animals in the MI group. Collectively, these results suggest that transplanted ABs can replicate the effect of MSCs to alleviate cardiomyocyte apoptosis and promote the growth of blood vessels and arterioles in porcine hearts with MI.

Figure 4.

MSC-derived ABs promote cardiomyocyte survival and angiogenesis in pig hearts after MI. Frozen pig heart tissue was collected from sham, MI, MI+MSC, and MI+AB animals. (A) sections obtained from the BZ were immunofluorescently stained for cTnT expression and stained by TUNEL; nuclei were counterstained with DAPI. Bar = 75 μm. (B) apoptosis was quantified as the percentage of cells that were TUNEL-positive. (C) western blot was performed to assess the protein expression of fas, B-cell lymphoma 2 (Bcl-2), and Bcl-2-associated X protein (bax) in the BZ of the infarcted hearts. (D) quantification of protein expression via densitometry analysis and normalization to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). n = 4 animals per group. (E) sections of the BZ at day 28 after MI or sham surgery were immunofluorescently stained for CD31, α-smooth muscle actin (α-SMA), and cTnT expression (bar = 75 μm). (F) vascular density was determined by quantifying the number of structures that expressed CD31. (G) arteriole density was determined by quantifying the number of structures co-expressing CD31 and α-SMA. n = 6 animals in each group. Significance was evaluated via one-way ANOVA, followed by Tukey’s post-hoc test in (B, D, F, and G). *p < 0.05 and **p < 0.01.

ABs activate adenosine monophosphate – activated protein kinase (AMPK)- and transcription factor EB (TFEB)-mediated autophagy to exert effects on hiPSC-CMs and HUVECs in vitro

Cumulative evidence suggests that the therapeutic effects of stem cell-derived EVs in heart injury are primarily mediated by specific miRNAs [25,26]. Therefore, we investigated miRNAs contained in MSC-derived ABs to determine the possible mechanism behind their function. Total RNA was extracted from MSC-derived ABs and subjected to miRNA sequencing (Figure 5A,B). Interestingly, the KEGG pathway enrichment analysis indicated that the AMPK signaling pathway may be the crucial pathway involved in the biological function of miRNAs contained in ABs (Figure 5C). Previous studies have suggested that the AMPK pathway plays a role in multiple heart diseases by promoting the survival of cardiomyocytes, enhancing angiogenesis, and reducing cardiac fibrosis through mammalian target of rapamycin (mTOR) and unc-51-like autophagy-activating kinase 1 (ULK1)-linked autophagy [27–29]. Thus, an in vitro investigation was conducted to examine whether ABs exert their effects through the AMPK-mediated mTOR/ULK1/autophagy pathway. Western blot performed on hiPSC-CMs showed that ABs were able to enhance AMPK phosphorylation, inhibit mTOR phosphorylation, and increase ULK1 phosphorylation (Figure 5D–I). TFEB is a member of the bHLH leucine-zipper family of transcription factors. Under elevated stress conditions, cytoplasmic TFEB is dephosphorylated and translocated into the nucleus, driving the expression of autophagy genes [30,31]. Here, we found that AB treatment decreased the phosphorylation level of TFEB (Figure 5J,K), increased the mRNA expression of genes involved in autophagy and lysosomal biogenesis (Figure S4A), and promoted the translocation of endogenous TFEB (Figure S4B – F). Similarly, ABs increased the expression of the pro-autophagy protein, microtubule-associated protein 1 light chain 3-II (LC3-II) (Figure 5L,M); decreased the expression of P62, an autophagic substrate protein that is degraded by autophagy (Figure 5L,M); enhanced the autophagic flux (Figure S4G – I); and augmented the number of autophagosome and autolysosome puncta within hiPSC-CMs (Figure 5N,O). These effects of ABs were abolished after administering the AMPK inhibitor Compound C, siRNA-TFEB (si-TFEB), or the autophagy inhibitor 3-methyladenine (Figure 5L–O). Similar western blot results were observed in HUVECs after AB treatment (Figure S5A – J). Furthermore, Compound C, si-TFEB, or 3-methyladenine reversed the AB-induced effects on reducing apoptosis and cytotoxicity in hiPSC-CMs under OGD conditions (Figure 5P–R) and on promoting tube formation and migration capability of HUVECs (Figure S5K – O). Taken together, these results demonstrate that the protective effects of ABs involve modulating autophagy through the activation of the AMPK/mTOR/ULK1 and TFEB signaling pathways in vitro.

Figure 5a.

Activation of adenosine monophosphate – activated protein kinase (AMPK)- and transcription factor EB (TFEB)-mediated autophagy is responsible for the cytoprotective effects of ABs on OGD-injured cardiomyocytes. (A) identification of miRNA content of ABs by a bulk miRNA-sequencing analysis. (B) heat map showing the relative abundance of miRnas in ABs from three individuals. (C) the miRNA-sequencing results underwent kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analysis to identify the critical signaling pathway associated with the biological function of the miRnas contained in ABs. (D – R) hiPSC-CMs were cultured under normal or OGD conditions and treated with PBS or ABs (20 μg/mL) in the presence or absence of AMPK inhibitor Compound C (CC, 10 μM), si-TFEB (20 nM), or autophagy inhibitor 3-methyladenine (3-MA, 5 mM). (D) the protein expression of AMPK and phosphorylated AMPK (p-AMPK) was detected via western blot. (E) the ratio of p-AMPK to total AMPK protein expression was calculated via densitometry analysis. (F) the protein expression of mammalian target of rapamycin (mTOR) and phosphorylated mTOR (p-mTOR) was detected via western blot. (G) the ratio of p-mTOR to total mTOR protein expression was analyzed. (H) the protein expression of UNC-51-like kinase 1 (ULK1) and phosphorylated ULK1 (p-ULK1) was detected via western blot. (I) the ratio of p-ULK1 to total ULK1 protein expression was analyzed. (J) the protein expression of TFEB and phosphorylated TFEB (p-TFEB) was detected via western blot. (K) the ratio of p-TFEB to total TFEB protein expression was analyzed. (L) the protein expression of autophagy-related proteins, including microtubule-associated protein 1 light chain 3-II (LC3-II) and P62, was detected via western blot. (M) protein amounts were quantified via densitometry analysis and normalized to GAPDH. (N) hiPSC-CMs were transfected with adenovirus containing fluorescent mRFP-GFP-LC3. Autophagosomes were shown as yellow puncta in green and red-merged images, and autolysosomes were shown as red puncta in immunofluorescent images. Bar = 25 μm. (O) the autophagosome and autolysosome puncta per cell were quantified. (P) the cardiomyocytes were immunofluorescently stained for cTnT expression and stained by TUNEL to detect the apoptosis of hiPSC-CMs; nuclei were counterstained with DAPI. (Q) quantification for TUNEL+ cardiomyocytes (bar = 50 μm). (R) the cytotoxicity of hiPSC-CMs cultured under OGD conditions was measured via an LDH release kit. n = 4 independent experiments. Significance was evaluated via one-way ANOVA, followed by Tukey’s post-hoc test in (E, G, I, K, M, O, Q, and R). *P < 0.05 and **P < 0.01 compared as indicated on the panels; ^&P < 0.05 compared with corresponding OGD+PBS values; and ^#P < 0.05 compared with corresponding OGD+AB values.

Figure 5.

(Continued).

ABs activate AMPK- and TFEB-mediated autophagy in porcine hearts after MI

We further investigated whether MSC-derived ABs confer cardioprotection by modulating autophagy through the activation of the AMPK/mTOR/ULK1 and TFEB signaling pathways in porcine hearts after MI. Western blot showed that both MSC and AB treatments obviously increased the phosphorylation levels of AMPK and ULK1 while repressing the phosphorylation levels of mTOR and TFEB (Figure 6A–F) in the BZ of the infarcted hearts compared with those in the MI group. Similarly, both AB and MSC treatments significantly enhanced the protein expression of LC3-II and decreased the protein expression of P62 (Figure 6G,H). Immunofluorescence staining was performed to detect the autophagy marker LC3 dot in the peri-infarct cardiac tissue (Figure 6I). Compared with the MI group, there was a higher number of LC3 dots per high-power field (HPF) in the MI+AB and MI+MSC groups (Figure 6J). Similar results were observed in the number of autophagosomes and autolysosomes in the heart tissue detected by transmission electron microscopy (TEM) (Figure 6K). Moreover, the mRNA and protein expression of autophagic regulators, including autophagy-related gene 5 (ATG5) and autophagy-related gene 7 (ATG7), was significantly increased in the BZ of the infarcted hearts from the MI+AB and MI+MSC groups compared with those from the MI group (Figure 6L, S6A and S6B). In addition, the protective effects of MSCs and ABs in terms of improving cardiac function and reducing infarct size in MI mice were largely abolished by applying the autophagy inhibitor 3-methyladenine (Figure S6C – F). Overall, these results show the AMPK/mTOR/ULK1- and TFEB-mediated autophagy activation following both MSC and AB treatments in porcine MI hearts. Thus, similar to MSCs, ABs may exhibit cardioprotective effects by regulating autophagy in the porcine MI model.

Figure 6.

Activation of AMPK- and TFEB-mediated autophagy by ABs in pig hearts after MI. The pig hearts were harvested 28 days after MI or sham surgery, and the tissues in the BZ were examined. (A) the protein expression of AMPK and p-AMPK was detected via western blot. (B) the ratio of p-AMPK to total AMPK protein expression was calculated via densitometry analysis. (C) the protein expression of p-mTOR, mTOR, p-ULK1, and ULK1 was detected via western blot. (D) ratios of p-mTOR/mTOR and of p-ULK1/ULK1 protein expression were analyzed. (E) the protein expression of TFEB and p-TFEB was detected via western blot. (F) the ratio of p-TFEB to total TFEB protein expression was analyzed. (G) the protein expression of LC3-II and P62 was detected via western blot. (H) quantification of protein expression. (I) the heart tissue sections were stained with antibodies against cTnT and Lc3; nuclei were counterstained with DAPI. Bar = 25 μm. (J) the numbers of LC3 dots per high-power field (HPF) were calculated. (K) autophagosomes (red arrows) and autolysosomes (red arrowheads) in BZ myocardium were detected via transmission electron microscopy (TEM). (L) the mRNA expression of autophagy-related gene 5 (ATG5) and autophagy-related gene 7 (ATG7) was measured via quantitative real-time polymerase chain reaction (qRT-PCR) and normalized to GAPDH. n = 4–6 animals per group. Significance was evaluated via one-way ANOVA, followed by Tukey’s post-hoc test in (B, D, F, H, J, and L). *p < 0.05 and **p < 0.01.

miRNA let-7f-5p contained in ABs enhances AMPK phosphorylation by targeting protein phosphatase 2 (formerly 2A) regulatory subunit B alpha (PPP2R2A) and decreases TFEB phosphorylation by targeting MAP4K3

Given the pivotal role of miRNA as one of the most critical mediators in EV-mediated cardiac repair [25,26], it is worth noting that highly expressed miRNAs often exhibit functional similarities to EVs [32]. As shown in Figure 5B, the 10 most abundant miRNAs in ABs were identified, and they are shown in a heat map in Figure 7A. Given that miRNA let-7f-5p was found to be the most abundant miRNA in ABs obtained from MSCs and its expression was further validated by qRT-PCR (Figure 7B), the impact and mechanism of let-7f-5p were further investigated. Interestingly, it was observed that let-7f-5p in ABs was capable of evading lysosomal confinement and being released into the cytosol following the internalization of ABs by lysosomes within recipient cells (Figure S7A and S7B). Using databases such as TargetScan, microRNA.org, and miRbase, we predicted the potential target genes of let-7f-5p and selected PPP2R2A and MAP4K3 as candidate target genes. PPP2R2A is one of the major regulatory subunit B isoforms of PP2A, which is a central serine/threonine phosphatase in the heart that regulates multiple myocyte functions through several target molecules, including AMPK [33,34]. MAP4K3, also known as germinal-center kinase-like kinase, has been reported to inactivate TFEB by directly or indirectly phosphorylating it to regulate autophagy [31]. Dual-luciferase reporter (DLR) assays revealed a notable decrease in luciferase activity in the presence of let-7f-5p for PPP2R2A or MAP4K3 3”-UTR wild type (WT), while this effect was negated when PPP2R2A or MAP4K3 3”-UTR mutant was used (Figure 7C–F); this confirmed the interaction between let-7f-5p and PPP2R2A, as well as that between let-7f-5p and MAP4K3. Consistently, western blot revealed a significant downregulation of PPP2R2A and MAP4K3 protein expression in cardiomyocytes transfected with let-7f-5p agomir (Figure 7G,H), further validating PPP2R2A and MAP4K3 as the downstream targets of let-7f-5p. To establish the regulatory link between PPP2R2A and AMPK, as well as the link between MAP4K3 and TFEB, we utilized siRNA-mediated gene silencing in cardiomyocytes. Notably, a pronounced increase in AMPK phosphorylation or a marked decrease in TFEB phosphorylation was observed in the si-PPP2R2A or si-MAP4K3 group compared with the corresponding scramble group (Figure 7I–L), underscoring the pivotal function of PPP2R2A in regulating AMPK phosphorylation and the function of MAP4K3 in regulating TFEB phosphorylation in this model. Additionally, the si-PPP2R2A and si-MAP4K3 treatments enhanced the protein expression of LC3-II and decreased the protein expression of P62 (Figure S8A-D). These findings collectively illustrate that miRNA let-7f-5p, which is enriched in MSC-derived ABs, specifically targets PPP2R2A and MAP4K3. This targeting subsequently enhances AMPK phosphorylation and decreases TFEB phosphorylation, respectively, thereby activating autophagy.

Figure 7.

PPP2R2A and MAP4K3 are the direct targets of miRNA let-7f-5p. (A) the expression of the 10 most abundant miRnas in ABs is shown. (B) the expression level of let-7f-5p in human fibroblast-derived ABs (FB-ABs) and MSC-derived ABs (MSC-ABs) was measured by qRT-PCR and normalized to measurements in MSC-ABs. (C-F) luciferase activity analysis to investigate the let-7f-5p target genes, including protein phosphatase 2 (formerly 2A) regulatory subunit B alpha (PPP2R2A) and mitogen-activated protein kinase kinase kinase kinase 3 (MAP4K3). (C) wild-type or mutant dual-luciferase reporter plasmid was constructed according to the predicted binding sequence in the 3-UTR of PPP2R2A or mutant sequence; then, (D) the activities of Renilla and firefly luciferase were determined using the dual-luciferase reporter assay system in human embryonic kidney 293T (HEK293T) cells. (E) wild-type or mutant dual-luciferase reporter plasmid was constructed in line with the predicted binding sequence in the 3-UTR of MAP4K3 or the mutant sequence. (F) the activities of Renilla and firefly luciferase were determined using a dual-luciferase reporter assay system. (G-H) hiPSC-CMs were transfected with miRNA let-7f-5p agomir and agomir negative control (NC), and the protein expression of PPP2R2A and MAP4K3 in cells was detected via western blot. (G) Representative immunoblots and (H) quantitative protein expression. (I – J) si-PPP2R2A and scramble control were transfected into hiPSC-CMs, in which the protein expression of PPP2R2, AMPK, and p-AMPK was detected by western blot. (I) Representative immunoblots and (J) quantitative protein expression. (K-L) si-MAP4K3 and scramble control were transfected into hiPSC-CMs, in which the protein expression of MAP4K3, TFEB, and p-TFEB was detected by western blot. (K) Representative immunoblots and (L) quantitative protein expression. Significance was evaluated via two-tailed Student’s t tests in (B) or one-way ANOVA, followed by Tukey’s post-hoc test in (D, F, H, J, and L). *p < 0.05 and **p < 0.01.

Let-7f-5p plays an important role in AB-mediated cytoprotection in safeguarding OGD cardiomyocytes and promoting the angiogenic capacity of ECs

To determine the critical role of let-7f-5p in AB-mediated cardioprotection, we performed the let-7f-5p gain – loss function experiments on cardiomyocytes under OGD injury. The miRNA let-7f-5p expression remained stable in ABs^NC-anta but decreased significantly in ABs^let-7p-anta (Figure S9A). Western blot showed that the markedly enhanced phosphorylated AMPK (p-AMPK) protein level and decreased phosphorylated TFEB (p-TFEB) protein expression in the OGD+AB groups were abolished in the OGD+AB^let-7f-anta group and reproduced in the OGD+let-7f-5p group (Figure 8A–D). In addition, the notably increased protein expression of LC3-II and decreased P62 protein expression in the OGD+AB group were mostly attenuated in the OGD+AB^let-7f-anta group and maintained in the OGD+let-7f-5 group (Figure 8E,F). Furthermore, loss of let-7f-5p abrogated, whereas let-7f-5p agomir reproduced, the effects of ABs on increasing the number of autophagosome and autolysosome puncta in hiPSC-CMs (Figure 8G) and on reducing cardiomyocyte apoptosis (Figure 8H) and LDH leakage (Figure 8I).

Figure 8.

Let-7f-5p loss abolishes, while let-7f-5p agomir reproduces, the protective effect of ABs on cardiomyocytes against OGD injury. hiPSC-CMs were treated with PBS, AB (20 μg/mL), AB^NC-anta (20 μg/mL), AB^let-7p-anta (20 μg/mL), let-7f-5p agomir (100 nM), or agomir NC under OGD conditions. Protein samples were collected from these groups for subsequent experiments. (A) the protein expression of AMPK and p-AMPK was detected via western blot. (B) quantification of protein expression. (C) the protein expression of TFEB and p-TFEB was detected via western blot. (D) quantification of protein expression. (E) the protein expression of LC3-II and P62 was detected via western blot. (F) quantification of protein expression. (G) hiPSC-CMs were transfected with an adenovirus containing tandem fluorescent mRFP-GFP-LC3. The quantification for autophagosome and autolysosome puncta per cell was analyzed. (H) apoptosis of hiPSC-CMs was detected through TUNEL staining, and the percentage of TUNEL-positive cardiomyocytes was analyzed. (I) the cytotoxicity of hiPSC-CMs was evaluated using an LDH release kit. n = 4 independent experiments. Significance was evaluated via one-way ANOVA, followed by Tukey’s post-hoc test in (B, D, F, G, H, and I). *p < 0.05 and **p < 0.01 compared as indicated on the panels; ^#p < 0.05 compared with corresponding OGD+AB^NC-anta values; ^{^}p < 0.05 compared with corresponding OGD+AB values, and ^&p < 0.05 compared with corresponding OGD+agomir NC values.

Congruously, the loss of let-7f-5p abrogated the effects of ABs on the protein expression of p-AMPK, p-TFEB, LC3-II, and P62 in ECs, while the addition of let-7f-5p agomir reproduced these effects (Figure S9B – G). We also conducted tube formation and migration assay to evaluate the pro-angiogenic effectiveness of miRNA let-7f-5p. Loss of let-7f-5p abrogated the effects of ABs in promoting the tube-forming activity of ECs, as evidenced by the decreased number and total length of branches, whereas let-7f-5p agomir reproduced these effects (Figure S9H and S9I). Similarly, loss of let-7f-5p abrogated, whereas let-7f-5p agomir reproduced, the migration-promoting effects of ABs on HUVECs (Figure S9J). Taken together, these results demonstrate that the enriched miRNA let-7f-5p contributes to the cytoprotective effects of MSC-derived ABs in regulating autophagy through the activation of the AMPK and TFEB pathways to decrease apoptosis and cytotoxicity in OGD cardiomyocytes and enhance angiogenic ability in ECs.

Delivery of let-7f-5p activates autophagy and enhances cardiac repair in pigs after MI injury

We conducted in vivo experiments in pigs to investigate the effect of let-7f-5p in promoting cardiac repair after MI. Endogenous let-7f-5p expression was progressively decreased in the BZ of the infarcted hearts (Figure 9A), and the intramyocardial injection of let-7f-5p agomir resulted in a significant elevation of let-7f-5p levels that persisted for up to 28 days after MI (Figure 9B). Treatment with let-7f-5p agomir significantly improved cardiac function (enhanced LVEF and LVFS) (Figure 9C,D), reduced cardiomyocyte apoptosis (Figure 9E,F), and promoted angiogenesis (increased vascular and arteriole density) in the BZ myocardium (Figure 9G–I), compared with the agomir negative control (NC) treatment. In addition, let-7f-5p agomir obviously inhibited the protein expression of PP2R2A, MAP4K3, p-TFEB, and P62, and upregulated the protein expression of p-AMPK and LC3-II in the BZ myocardium (Figure 9J–O). Furthermore, let-7f-5p agomir treatment obviously augmented the production of autophagosomes and autolysosomes in the porcine myocardium relative to NC treatment (Figure 9P). Consistently, after transfection with miRNA let-7f-5p antagomir, MSC-derived ABs lost their autophagy regulation and cardioprotective effects (Figure 9C–P). In addition, the application of the autophagy inhibitor 3-methyladenine counteracted the improvement in cardiac function and reduction in infarct size in MI mice medicated by let-7f-5p agomir (Figure S10A – D). Collectively, these findings demonstrate that let-7f-5p plays an important role in AB-mediated cardioprotection in promoting porcine cardiac repair following MI injury via activation of AMPK- and TFEB-mediated autophagy.

Figure 9a.

Let-7f-5p agomir activates AMPK- and TEFB-medicated autophagy and enhances myocardial function in pigs with MI. The pigs were divided into four groups, and they underwent MI surgery, followed by the administration of let-7f-5p agomir (Mi+let-7f-5p group), agomir NC (MI+NC group), AB^NC-anta (MI+ AB^NC-anta group), or AB^let-7p-anta (MI+ AB^let-7p-anta group). (A) the expression of let-7f-5p in the sham hearts and BZ of the infarcted hearts 3 and 28 days after MI. (B) quantification of the let-7f-5p level in the sham, MI, MI+NC, and MI+let-7f-5p myocardium on day 28 after MI. n = 4 animals per group. (C – D) cardiac function was evaluated via echocardiography 28 days after MI, including (C) LVEF and (D) LVFS. n = 5–6 animals per group. (E) sections obtained from the BZ of the infarcted hearts were immunofluorescently stained for cTnT expression and stained by TUNEL; nuclei were counterstained with DAPI. Bar = 25 μm. (F) apoptosis was quantified as the percentage of TUNEL-positive cells. (G) sections obtained from the BZ myocardium were immunofluorescently stained with CD31, α-SMA, and cTnT (bar = 75 µm). (H) vascular density was determined by quantifying the number of structures that expressed CD31. (I) arteriole density was determined by quantifying the number of structures co-expressing CD31 and α-SMA. (J) western blot was performed to detect the protein expression of PPP2R2A, AMPK, and p-AMPK in the BZ of the infarcted heart. (K) quantification of protein expression. (L) the protein expression of MAP4K3, TFEB, and p-TFEB was detected via western blot. (M) quantification of protein expression. (N) the protein expression of LC3-II and P62 was detected via western blot. (O) quantification of protein expression. n = 4 animals per group. (P) autophagosomes (red arrows) and autolysosomes (red arrowheads) in pig cardiomyocytes from the BZ myocardium were detected via transmission electron microscopy. Significance was evaluated via one-way ANOVA, followed by Tukey’s post-hoc test in (A, B, C, D, F, H, I, K, M, and O). *P < 0.05 and **P < 0.01.

Figure 9b.

(Continued).

Discussion

In the present investigation, the therapeutic effects of MSC-derived ABs on cardiac recovery in a porcine MI model were evaluated initially. The main findings (Figure 10) are as follows: (i) in vitro, ABs exhibit the potential to safeguard cardiomyocytes from OGD injury, including impeding cell apoptosis and cytotoxicity, and to enhance the angiogenic capacity of ECs through the promotion of migration and tube formation; (ii) in vivo, ABs can reproduce the effects of MSCs to improve contractile function, reduce infarct size, and mitigate adverse remodeling in pig hearts with MI, concomitantly with enhanced cardiomyocyte survival and angiogenesis; (iii) mechanistically, ABs modulate autophagy through the activation of the AMPK/mTOR/ULK1 and TFEB signaling pathways, thereby exerting effects on cardiomyocytes and ECs and subsequently promoting porcine heart recovery after MI; and (iv) miRNA let-7f-5p is the predominant miRNA in ABs, and it has the potential to increase AMPK phosphorylation by targeting PPP2R2A and decrease TFEB phosphorylation by targeting MAP4K3 to regulate autophagy, thereby contributing to the effects of Abs. These findings extend the previous knowledge and provide insights into the mechanisms underlying the cardioprotection conferred by MSC-derived ABs, supporting the view that ABs may be a beneficial acellular therapeutic option for treating MI.

Figure 10.

Schematic representation of the effects of ABs derived from MSCs on cardiac function and remodeling in swine with MI.

It is increasingly evident that ABs originating from stem cells or differentiated cells contain signaling cues and recyclable materials that have distinct regulatory effects on recipient cells [15]. Among them, MSC-derived ABs have the potential to play a role comparable to that of maternal stem cells, aiding in tissue homeostasis and regeneration in several disease models, such as skin wounds, muscle injuries, and osteopenia [35–37]. Previous research has demonstrated the cardioprotective effect of ABs in a mouse MI model [38]. Herein, we further investigated the protective effect of ABs on human cardiomyocytes (hiPSC-CMs) in vitro and provided evidence that transplantation of MSC-derived ABs could replicate the effects of MSCs in enhancing cardiac recovery in pigs following MI. Specifically, after being taken up by recipient cells, ABs improved cardiac contractile function, attenuated infarct size, limited cardiac fibrosis, and suppressed cardiac hypertrophy, while also reducing cardiomyocyte apoptosis and improving angiogenesis, in hearts suffering from MI. The discovery of the AB-exerted protective effects presents a novel approach to investigating the cardiac repair capability of MSCs. Additionally, considering that ABs are durable, easy to preserve, and less prone to risks of immune rejection and tumor formation [36,39], they offer a promising and beneficial cell-free therapeutic option for MI. It is noteworthy that we demonstrated the role and mechanisms of ABs in a preclinical porcine model of MI for the first time. Pigs are frequently utilized as a model for cardiovascular diseases because of their similarity to human cardiovascular anatomy and physiology in terms of coronary artery distribution, ventricular performance, cardiac metabolism, electrophysiology, and collateralization following an acute MI [40]. By clarifying the cardioprotective effect of ABs in the porcine model of MI, we presented a more accurate preclinical foundation for the forthcoming clinical utilization of ABs.

Autophagy is a significant catabolic process that is crucial for eradicating protein aggregates and damaged or excess organelles to preserve intracellular homeostasis and keep the cell healthy [41]. In certain dangerous situations, autophagy is enhanced to maintain energy levels and protein synthesis, ultimately exerting a cardioprotective function [42,43]. In this study, we demonstrated that activation of autophagy was a crucial factor in AB-mediated cardioprotection. AB treatment activated autophagy in cardiomyocytes, as evidenced by the increased presence of autophagosomes and autolysosomes under OGD conditions, as detected by tandem GFP-RFP-LC3 assay, and under MI injury, as detected by TEM examination. In contrast, inhibition of autophagy in vitro abolished all the effects of ABs on increasing cardiomyocyte viability. Autophagy was also suggested to maintain EC homeostasis under stress conditions, promote the production of pro-angiogenic factors, and regulate the proliferation and migration of ECs [44,45]. Inhibition of autophagy abolished the angiogenic capacity – enhancing effects of ABs in vitro, suggesting that ABs also induce autophagy in ECs to promote angiogenesis. This finding is consistent with the observation that AB treatment markedly augmented the vascular density in the BZ of the infarcted myocardium. During the formation of autophagosomes, LC3-I transforms into the autophagic vesicle-bound LC3-II [42,43]. The selective autophagy receptor p62 is degraded via autophagy and can link ubiquitinated proteins to the autophagic machinery, which allows for their lysosomal degradation [43]. Consistent with these findings, treatment with ABs resulted in increased expression of LC3-II protein and decreased expression of P62 protein in ODG cardiomyocytes, ECs, and MI myocardium, and these effects were abolished by the addition of an autophagy inhibitor. Furthermore, in vivo, the application of the autophagy inhibitor largely abolished the protective effects of ABs in MI mice. In conclusion, treatment with ABs induces autophagy, which may lead to an increase in energy supply and the recycling of macromolecules to meet the cellular demand in the face of excessive wall stress and ischemia/hypoxia, subsequently improving post-infarction cardiac dysfunction and remodeling.

The AMPK signaling pathway is recognized as a crucial mechanism in the regulation of autophagy [43]. A reduction in ATP level triggers AMPK activation and subsequently inhibits mTOR, resulting in the formation of ULK1-mediated autophagosome to stimulate autophagy [43]. AMPK also negatively regulates mTOR via a pathway that includes the tuberous sclerosis complex (TSC1/2) and its substrate, a Ras-related small GTPase [42]. In the present study, we showed that MSC-derived ABs elevated the AMPK and ULK1 phosphorylation levels and suppressed the mTOR phosphorylation level in ODG cardiomyocytes, ECs, and MI myocardium, indicating the activation of the AMPK/mTOR/ULK1 signaling pathway following treatment with ABs. TFEB has been regarded as a master regulator of lysosome biosynthesis and autophagy-related gene transcription [38]. When its phosphorylation levels at Ser3, Ser142, or Ser211 reach a sufficiently low level, TFEB is activated and can translocate from the cytoplasm to the nucleus to drive the expression of autophagy and lysosomal genes, thereby promoting the repair of autophagic flux [30,31]. Interestingly, there is unique crosstalk between the TFEB, AMPKα, and mTOR signaling pathways [46–48]. AMPKα and mTOR can regulate the phosphorylation of TFEB to influence its activation [46,48]; TFEB, in turn, can activate AMPK/mTOR signaling to upregulate autophagy [47]. Herein, we also found that MSC-derived ABs decreased the TFEB phosphorylation level in both ODG cardiomyocytes and MI myocardium, as well as promoting TFEB nuclear translocation, indicating that the TFEB pathway was activated. Furthermore, the activation of this pathway and regulation of autophagy, as well as the enhancement of cardiomyocyte survival and angiogenic capacity of ECs induced by ABs, were abolished by both the AMPK inhibitor and TFEB silencing. These findings demonstrate that MSC-derived ABs exert their effects by modulating autophagy through the activation of the AMPK/mTOR/ULK1 and TFEB signaling pathways.

As a type of EVs, ABs also function by delivering a range of substances [23,24,49–51]. In this study, we sequenced the miRNA profile of MSC-derived ABs and found that let-7f-5p stood out as the most abundant miRNA in ABs. let-7f-5p belongs to the human let-7 family, which contains 13 members [52], and it has been reported that let-7f can promote angiogenesis, regulate the growth and development of the heart, and improve cardiac function after MI [53,54]. In this study, we proved that let-7f-5p contributed to the AB-exerted cardioprotective effects. The evidence was derived from the loss/gain-of-function experiments, which showed that treatment with let-7f-5p antagomir eliminated, while treatment with let-7f-5p agomir reproduced, the AB-mediated effects on activating autophagy and on improving the survival of OGD cardiomyocytes and the angiogenic capacity of ECs. Additionally, let-7f-5p administration had an apparent cardioprotective effect in the porcine MI model. It improved cardiac contractile function, reduced apoptosis of hiPSC-CMs, enhanced angiogenesis, and activated AMPK/mTOR/ULK1- and TFEB-mediated autophagy in infarcted hearts. In contrast, let-7f-5p antagomir abolished AB-mediated autophagy regulation and protection in pig MI hearts.

The protein phosphatase 2A (PP2A), a prominent member of the serine/threonine phosphatases family, acts as a crucial cardiac phosphatase for regulating a wide range of myocyte functions via multiple target molecules [55,56. PP2]A regulates the activity of AMPK, and inhibition of PP2A can promote the activation of AMPK [33,34. PP2]A comprises three subunits, namely catalytic (subunit C), scaffold (subunit A), and regulatory (subunit B) subunits. PPP2R2A is one of the main isoforms of regulatory subunit B ^55,56. In our study, PPP2R2A was identified as a downstream target gene of let-7f-5p via bioinformatics analysis, which was further confirmed by DLR assay. A series of gain-of-function experiments also verified that let-7f-5p inhibited PPP2R2A expression and promoted AMPK phosphorylation, thereby leading to enhanced autophagy. Furthermore, we established the regulatory link between PPP2R2A and AMPK by silencing the PPP2R2A gene in cardiomyocytes. This silencing increased the phosphorylation level of AMPK. MAP4K3 belongs to the Ste20 subfamily of mitogen-activated protein kinases (MAPKs), which control a wide range of cellular processes in all eukaryotes. Previous studies have shown that MAP4K3 can inactivate TFEB by directly or indirectly phosphorylating it through the stimulation of mTOR complex 1 (mTORC1) [31]. In this study, we also identified MAP4K3 as another downstream target gene of let-7f-5p. let-7f-5p inhibited MAP4K3 expression and decreased TFEB phosphorylation, thereby leading to enhanced autophagy. Meanwhile, MAP4K3 silencing in cardiomyocytes decreased the phosphorylation level of TFEB, which proved the regulatory link between MAP4K3 and TFEB. Collectively, this study demonstrates that enriched let-7f-5p in ABs could regulate AMPK- and TFEB-mediated autophagy via the inhibition of PPP2R2A and MAP4K3 activity, respectively.

Our research has some limitations. In this study, ABs were administered via in situ intramyocardial injections in pigs. This delivery method is primarily suitable for patients who require thoracotomy, such as those undergoing coronary artery bypass grafting or heart valve replacement surgery. Targeted delivery of ABs with efficacy, safety, and operational availability in treating MI requires exploration for clinical application in future studies. Moreover, considering the length limit, we only focused on the most abundant miRNA in ABs, let-7f-5p, and elucidated its role and mechanism. The function of other miRNAs and compounds in ABs should be investigated in future studies. Furthermore, while our findings indicate that autophagy activation plays a role in the effectiveness of ABs against MI, it remains possible that other mechanisms also contribute to the cardioprotective effects of ABs.

In summary, like MSCs, administration of MSC-derived ABs can alleviate ischemic myocardial injury in pigs after MI. This is achieved by modulating autophagy to improve cardiomyocyte survival and EC angiogenesis through the AMPK/mTOR/ULK1 and TFEB signaling pathways, which restores heart function, reduces infarct size, and suppresses LV remodeling. Highly enriched let-7f-5p contributes to the beneficial effects of ABs by targeting PPP2R2A and MAP4K3 to increase the activation of the AMPK and TFEB pathways. These findings suggest that administration of ABs is a noteworthy and efficient cell-free therapeutic option for treating MI.

Funding Statement

This work was supported by the National Key Research and Development Program of China [2022YFA1105100], the National Natural Sciences Foundation of China [82270261 and 82070260], the Eastern Talent Plan Leading Project in Shanghai [BJKJ2024014], the Academic Leaders Training Program of the Pudong Health Bureau of Shanghai [PWRd2024–02], and the Peak Disciplines (Type IV) of Institutions of Higher Learning in Shanghai.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Ethics approval statement

All animal procedures and protocols were performed in accordance with the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health and were approved by the Institutional Animal Care and Use Committee of Tongji University (Shanghai, China; Approval Number: TJLAC-019–135).

Author contributions

L.G. and Z.L. conceived and designed the research. W.L., H.L., P.Z., H.C., Y.D., Y.G., D.Z., and Y.X. contributed to the acquisition, analysis, and interpretation of the data. W.L., H.L., P.Z., L.G., and Z.L. wrote and revised the manuscript. All authors read and approved the final manuscript.

Supplementary material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/15548627.2025.2536449