Mechano-matching hydrogel patch promotes myocardial infarction repair via YAP-Bcl-2 pathway activation

Abstract

Although elastic cardiac patches have demonstrated efficacy in alleviating ventricular wall stress and restoring cardiac function following myocardial infarction (MI), the mechanistic basis governing their therapeutic effects remains incompletely elucidated. In this study, three distinct acellular hydrogel patches with tailored elastic moduli were fabricated, namely soft (1.61 kPa), mechano-matching (16.82 kPa, corresponding to the moduli of native adult myocardium), and rigid (602.61 kPa). These patches were implanted in a rat model of MI to evaluate their therapeutic potential. Among the three groups, the mechano-matching hydrogel patch exhibited superior performance, significantly improving cardiac function (with left ventricular ejection fraction [LVEF] elevated by 15.89 %, p = 0.002), reducing infarct size by 14.49 % (p < 0.001), mitigating myocardial fibrosis, and attenuating cardiomyocyte apoptosis. To dissect the underlying mechanism, an in vitro cyclic stretch model mimicking the in vivo myocardial mechanical microenvironment was established. Results revealed that hydrogels with moderate stiffness (16.82 kPa) transduced mechanical cues to promote nuclear translocation of Yes-associated protein (YAP) in cardiomyocytes. This key mechanotransduction event upregulated the expression of anti-apoptotic protein Bcl-2, thereby suppressing cardiomyocyte apoptosis. Notably, this study uncovers a previously unelucidated mechanistic paradigm by which moderate mechanical stimuli, matching the intrinsic stiffness of native myocardium, confer cardioprotection specifically through activation of the YAP-Bcl-2 signaling axis. Furthermore, it establishes that acellular biomaterials can exclusively harness their intrinsic mechanical properties to reverse pathological myocardial remodeling post-MI, without relying on cellular components or bioactive molecules. This finding provides strategy guided by mechanobiology for cardiac regeneration, substantially enhancing the clinical translatability of acellular cardiac patches.

Graphical abstract

1. Introduction

Myocardial infarction (MI) is defined by the acute occlusion of a coronary artery, triggering irreversible cardiomyocyte loss due to ischemia-hypoxia. This pathological process makes MI one of the leading causes of mortality and morbidity in cardiovascular diseases worldwide, posing a severe burden to global public health [1,2]. Currently, standard therapeutic strategies for MI include pharmacological thrombolysis, percutaneous coronary intervention (PCI), coronary artery bypass grafting (CABG), and heart transplantation. While these approaches effectively restore blood perfusion to the ischemic myocardium in the early phase and limit infarct expansion, they fail to address the subsequent pathological ventricular remodeling, a cascade involving cardiomyocyte apoptosis, compensatory hypertrophy of residual cardiomyocytes, ventricular wall thinning, and excessive collagen deposition in the infarcted region [3]. This remodeling process progressively impairs cardiac structural integrity and contractile function, ultimately progressing to chronic heart failure (HF), a terminal stage with poor prognosis. Thus, there remains an urgent unmet clinical need for therapeutic strategies that can directly intervene in post-MI remodeling to prevent HF progression.

A growing body of evidence indicates that the disruption of the cardiac mechanical microenvironment is a pivotal driver of the transition from acute MI to HF [4,5]. Critically, these mechanical abnormalities regulate cell phenotypes and tissue function via mechano-biochemical transduction, further exacerbating cardiomyocyte apoptosis and cardiac dysfunction. Regrettably, current pharmacotherapies and device-based interventions (e.g., PCI) do not target the core regulatory axis between the mechanical microenvironment and cardiomyocyte apoptosis, leaving a critical gap in MI repair strategies.

Among emerging MI therapeutics, cardiac patches have garnered substantial attention due to their unique therapeutic potential. Traditional cardiac patches primarily exert effects by delivering exogenous factors including stem cells, drugs, cytokines, and exosomes to the infarcted region, relying on these bioactive components to mediate repair [6]. In contrast, acellular elastic patches, which are devoid of cellular or bioactive additives, function autonomously via their intrinsic mechanical and structural properties. This mechanical regulation strategy offers distinct advantages in that mechanical signals can directly modulate host cell behavior and material properties can be precisely tuned to match pathological demands. Previous studies have confirmed that elastic patches passively constrain the infarcted myocardium, reduce decompensatory ventricular dilatation, and inhibit left ventricular remodeling [7]. However, the therapeutic efficacy of these patches is largely attributed to general mechanical support and reduced wall stress, with their underlying biological mechanisms remaining incompletely elucidated. Furthermore, reported elastic moduli of cardiac patches span a wide range, from several kilopascals to tens of megapascals [8,9]. The optimal elastic modulus for targeting post-MI myocardium and how specific mechanical properties of biomaterials drive repair remain undefined, representing a major bottleneck in the field.

Hippo-YAP signaling pathway is a central mediator linking the mechanical microenvironment to cardiomyocyte fate determination [10,11]. As an evolutionarily conserved pathway, Hippo maintains organ size and tissue homeostasis by regulating cell growth, proliferation, survival, and differentiation. Yes-associated protein (YAP), a key downstream effector of Hippo, exhibits inherent mechanosensitivity. In the active Hippo state, upstream kinases phosphorylate YAP, thereby sequestering it in the cytoplasm [12,13]. In the inactive state, YAP translocates to the nucleus, where it functions as a transcriptional co-activator to regulate target gene expression [14,15]. Accumulating evidence confirms that the Hippo-YAP pathway is critical for maintaining cardiac homeostasis, facilitating injury repair, and promoting regeneration, making it a promising therapeutic target for MI [16]. However, existing studies predominantly focus on YAP-mediated cardiomyocyte cycle re-entry and proliferation as the core mechanism for MI repair [17,18]. Research investigating how YAP regulates cardiomyocyte apoptosis remains scarce. Notably, cardiomyocyte apoptosis drives ventricular remodeling [19], and the disruption of the mechanical microenvironment exacerbates this apoptotic process [20]. Direct experimental evidence is lacking regarding whether YAP can regulate cardiomyocyte apoptosis following changes in the mechanical environment, as well as its specific downstream targets. Elucidating this mechanism fills the knowledge gap in the mechano-microenvironment-YAP-cardiomyocyte apoptosis axis, enable YAP-targeted precise interventions, and inhibit ventricular remodeling at its source to reduce the incidence of heart failure. It also represents a critical breakthrough for MI therapy and advances cardiovascular mechanobiology.

To address the aforementioned gaps, this study fabricated three acellular elastic hydrogel patches with distinct elastic moduli, namely soft hydrogel patches (SHP), mechano-matching hydrogel patches (MMHP, elastic modulus matched to that of native adult myocardium), and rigid hydrogel patches (RHP). These patches were implanted in a rat model of acute MI to systematically evaluate their therapeutic efficacy. In vivo results demonstrated that only MMHP exerted significant therapeutic effects, including reducing infarct size, mitigating cardiomyocyte apoptosis, and alleviating myocardial fibrosis (Fig. 1). Subsequent in vitro experiments, which utilized a cyclic stretch model to mimic in vivo cardiac mechanical cues and employed molecular intervention approaches, verified that MMHP promotes YAP nuclear translocation. This translocation, in turn, upregulates the anti-apoptotic protein Bcl-2, ultimately suppressing cardiomyocyte apoptosis and conferring cardioprotection.

Fig. 1.

Schematic illustration of the synthesis of the mechano-matching hydrogel patch and its effects in reducing infarct size, decreasing myocardial apoptosis, and alleviating myocardial fibrosis after MI (Created with Biorender.com).

This work holds notable scientific significance. Specifically, by establishing a direct link between the mechanical properties of acellular hydrogels and the YAP-Bcl-2 signaling axis, it uncovers a novel “mechanical cue-molecular signal” regulatory mechanism for MI repair, one that advances our understanding of cardiovascular mechanobiology. More importantly, this study defines the mechano-matching modulus as the optimal mechanical parameter for cardiac patches and clarifies a role of YAP in regulating cardiomyocyte apoptosis via Bcl-2. Collectively, these findings lay a theoretical and experimental foundation for the development of myocardial repair strategies based on mechanical regulation and their clinical translation.

2. Results and discussion

2.1. Synthesis and characterization of SilMA hydrogel

SilMA (Silk Fibroin Methacryloyl) solution was prepared following a previously established protocol [21] (Fig. 2A). After photocrosslinking, the resultant SilMA hydrogel exhibited excellent shape retention, maintaining its structural integrity even under inversion (Fig. 2B). The chemical modification of SF with glycidyl methacrylate (GMA) was driven by the reaction between the primary amino groups (-NH₂) of SF and the epoxide groups of GMA via epoxide ring opening, which proceeds under both basic and neutral conditions as previously described. The success of GMA modification on SF was verified using ¹H nuclear magnetic resonance (¹H NMR) spectroscopy (Fig. 2C). In the ¹H NMR spectrum of SilMA, characteristic peaks corresponding to the vinyl protons of methacrylate groups were observed at δ = 6.0–6.2 ppm and δ = 5.6–5.8 ppm, while an additional peak at δ = 1.8 ppm was attributed to the methyl protons (-CH₃) of GMA. These signals are absent in unmodified SF, directly confirming the conjugation of GMA to the SF backbone. The degree of methacrylation was calculated to be 24 % using the corresponding formula. Owing to its excellent biocompatibility and gradual degradation into non-toxic products, unmodified silk fibroin remains a safe material even when retained in tissues. For Fourier-transform infrared (FT-IR) spectroscopy analysis (Fig. S1), SilMA exhibited distinct characteristic peaks at wavenumbers of 947 cm⁻¹ and 1167 cm⁻¹. These peaks were either absent or significantly weaker in unmodified SF, further validating the successful chemical modification of SF into SilMA and supporting its suitability for subsequent cardiac patch fabrication.

Fig. 2.

Characterization of the SilMA hydrogel. (A) Schematic for preparing SilMA hydrogel. (B) Photographs of SilMA hydrogel before and after UV curing, and the stability of hydrogel was proved by placing in different orientations. (C) ¹H NMR spectra of SF and SilMA. (D) The stress-strain curves of three groups of hydrogel patches. (E) The Young’s modulus of three groups of hydrogel patches (n = 3 per group). (F) The conductivity in three groups of SilMA hydrogel (n = 4 per group). (G) Swelling property of three groups of SilMA hydrogels under the PBS (n = 5 per group). SF: Silk fibroin,GMA: Glycidyl methacrylate,AKG: α-Ketoglutaric acid. Data are means ± standard deviation. ∗p < 0.05, ∗∗p < 0.01.

Each tissue or organ possesses a specific modulus to meet its physiological requirements. The elastic modulus of native adult myocardial tissue is approximately 15 kPa at the end of a diastole [6]. Following myocardial infarction (MI), however, the damaged myocardium undergoes progressive fibrotic remodeling, with the infarcted region gradually replaced by fibrous tissue, leading to a marked increase in modulus [22,23]. The mechanical properties of biomaterials employed in MI therapy are of pivotal importance, as mismatched modulus can either fail to provide sufficient mechanical support, thereby exacerbating ventricular dilatation, or induce excessive stress, which aggravates myocardial injury. To investigate the effects of mechanical stimuli as a single variable on cardiac tissue and cardiac cells, we fabricated three groups of SilMA hydrogels with identical chemical compositions but distinct elastic moduli (Fig. S2). This was accomplished by adjusting the concentration of the SilMA solution and the duration of alcohol dehydration, a facile and reproducible approach that eliminates the introduction of confounding chemical variables. The elastic moduli of the hydrogels were quantified via compression tests, and the three groups were defined as soft hydrogel patch (SHP, 1.61 ± 0.29 kPa), mechano-matching hydrogel patch (MMHP, 16.82 ± 0.29 kPa, whose modulus is comparable to that of native myocardial tissue) and rigid hydrogel patch (RHP, 602.61 ± 87.36 kPa), respectively (Fig. 2D and E). Moreover, there is no significant change in the elastic modulus of RHP after rehydration (Fig. S3). RHP is highly stable and can retain its designed mechanical properties in vivo. Cardiac tissue relies on efficient transmission of bioelectrical signals to maintain synchronized contraction. The electrical conductivity of native myocardium was measured to range from 0.005 to 0.16 S/m [24,25]. To evaluate the electrochemical performance of our SilMA hydrogels, we measured the electrical conductivity of the three groups using an electrochemical workstation. The results showed that the electrical conductivities of SHP, MMHP, and RHP were not statistically different and much lower than the intrinsic electrical conductivity range of native myocardium (Fig. 2F). This result confirms that any post-implantation differences in cardiac function are not attributable to variations in electrical conductivity, thereby providing further validation of the single mechanical variable design principle employed in this study. The swelling behavior of hydrogels is a critical property, as it directly influences their mechanical stability, biocompatibility, and in vivo performance. Excessive swelling can alter modulus and disrupt the mechanical microenvironment, while insufficient swelling may lead to dehydration and structural collapse. All three groups of hydrogels exhibited a consistent swelling kinetic profile and absorbed water rapidly within the first 1 h, with this process accompanied by volume expansion and weight gain. The swelling rate slowed between 2 and 4 h, and swelling equilibrium was achieved after 24 h (Fig. 2G). Quantitatively, the swelling ratios were 378.76 ± 38.22 % (SHP), 182.66 ± 16.81 % (MMHP) and 41.46 ± 3.40 % (RHP), respectively. Notably, the swelling ratio decreased with increasing SilMA concentration, and none of the three groups exhibited over-swelling. Scanning electron microscopy (SEM) images revealed that all SilMA hydrogels possessed a porous structure with interconnected networks (Fig. S4). For MMHP, porous structure combined with its myocardium-matching modulus creates a microenvironment that mimics native myocardium, laying the foundation for regulating host cell behavior in subsequent in vivo experiments.

Biodegradability and degradation rate are critical for hydrogels applied in myocardial repair, as they determine the duration of mechanical support and compatibility with tissue integration. To evaluate these properties, the three hydrogel patches were implanted into the infarcted myocardial region of rats, isolated at 1, 2, 3, and 4 weeks post-implantation, and their degradation rates were calculated. Experimental results demonstrated that MMHP exhibited a degradation profile matching the demand of myocardial repair: it maintained structural integrity to rapidly provide suitable and sustained mechanical support for the injured myocardium within the first 2 weeks after myocardial infarction. After 2 weeks, MMHP gradually degraded while retaining sufficient mechanical strength, thus maintaining the supportive effect and simultaneously reserving space for myocardial tissue integration. In contrast, SHP degraded rapidly, resulting in insufficient mechanical support that failed to constrain ventricular dilatation. Although RHP exhibited good in vivo stability and slow degradation, its excessively high modulus was much higher than that of native myocardial tissue, exerting excessive continuous mechanical stimulation on the myocardium and potentially exacerbating pathological remodeling (Fig. S5).

2.2. Favorable in vivo and in vitro biosafety of the three SilMA hydrogel groups

Biosafety, which encompasses biocompatibility and non-toxicity, is a prerequisite for the clinical translation of biomaterials. To evaluate the in vivo biosafety of the SilMA hydrogels, we first implanted three hydrogel patches (SHP, MMHP, RHP) onto the healthy rat hearts and monitored the animals for 1 and 4 weeks (Fig. 3A). Over the entire observation period, the patches did not induce any detectable impairment of cardiac function. Echocardiographic analysis of healthy rats with or without hydrogel implantation showed that key cardiac function parameters, including left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS), remained within the normal physiological range and exhibited no significant differences between the implanted and non-implanted groups (Fig. 3B and C).

Fig. 3.

Biocompatibility of three hydrogel patches. (A) Experimental design. (B) The echocardiographic images of healthy rats with and without different cardiac patches for 1 week and 4 weeks. (C) Representative parameters of cardiac function of healthy rats with and without three groups of hydrogel cardiac patches for 1week and 4 weeks (n = 3 per group). (D) Live/dead staining of H9c2 cardiomyocytes after seeding on three groups of hydrogels or regular cell culture plate for 3 days (Scale bar, 100 μm). Data are means ± standard deviation. ∗p < 0.05, ∗∗p < 0.01.

In addition to in vivo biosafety, in vitro cytotoxicity assessment is essential for predicting the compatibility of the material with host cells, as cytotoxicity can directly negate therapeutic effects by inducing cell death. H9c2 cardiomyocytes were seeded onto the three SilMA hydrogel scaffolds, and live/dead staining was performed after 3 days of culture to visualize cell viability. Calcein-AM emits green fluorescence in live cells, while propidium iodide emits red fluorescence in dead cells. The staining results revealed no significant differences in cardiomyocyte viability between the three hydrogel groups and the tissue culture plate (TCP) control group (Fig. 3D). Specifically, the number of dead (red) cells was negligible in all groups, indicating that the hydrogels do not release cytotoxic factors and provide a cytocompatible microenvironment for cardiomyocyte adhesion and survival. Based on the above results, the three prepared hydrogels exhibit excellent biosafety both in vitro and in vivo.

2.3. The MMHP enhances cardiac function following MI

Mechanical cues are widely acknowledged regulators of cellular and tissue behavior, with their effects on post-MI myocardial repair closely tied to the mechanical properties of implanted biomaterials. To specifically investigate how modulus-dependent mechanical signals of elastic hydrogel patches modulate MI repair, we established a rat model of acute MI via left anterior descending (LAD) coronary artery ligation, followed by epicardial implantation of the three hydrogel patches and survival curves were plotted (Fig. S6). The results showed that most deaths occurred within 72 h after surgery, which is directly associated with acute pathophysiological disturbances caused by myocardial ischemic injury and surgery-related complications. Cardiac function was serially evaluated via echocardiography at 1 and 4 weeks post-MI. All animals were euthanized at 4 weeks after surgery and myocardial tissues were harvested for subsequent histological and molecular analyses (Fig. 4A). Representative echocardiography images (Fig. 4B) revealed distinct differences in left ventricular (LV) contractile behavior across groups. The sham group exhibited stable cardiac contraction with prominent LV wall motion amplitude, consistent with normal cardiac function. In contrast, the MI control group showed no apparent contraction of the LV anterior wall, reflecting extensive myocardial necrosis and loss of contractile function. Notably, the MMHP group displayed significantly enhanced contractile activity of the LV anterior wall compared to the MI control group, suggesting that mechanical properties of MMHP may mitigate contractile dysfunction. Quantitative analysis of echocardiographic parameters further validated these qualitative observations (Fig. 4C and D). At 4 weeks after MI, the MI group exhibited marked declines in key systolic function indicators. The values of LVEF decreased to 29.42 % ± 10.69 % and LVFS decreased to 16.26 % ± 7.48 %, which were significantly lower than those in the sham group (LVEF: 77.26 % ± 2.14 %, LVFS: 47.07 % ± 2.28 %) and thus confirmed severe cardiac dysfunction after MI (Fig. 4C and D).

Fig. 4.

The MMHP improved heart function after MI. (A) Experimental design. (B) The echocardiographic images of rats with MI after patching with or without three groups of hydrogels and sham-operated rats for 1 week and 4 weeks. (C) Echocardiographic assessment of cardiac function in five groups of rats (n = 3 for sham group, n = 9–10 for other four groups). (D) Echocardiographic assessment of cardiac function in five groups of rats at 4 weeks after surgery. Data are means ± standard deviation. ∗p < 0.05, ∗∗p < 0.01.

In stark contrast, the MMHP group showed significant recovery of systolic function. The values of LVEF increased to 45.31 % ± 5.71 % and LVFS increased to 29.36 % ± 7.28 %. Importantly, no such functional improvement was observed in the SHP or RHP groups. LVEF values were 28.99 % ± 9.21 % (SHP) and 33.95 % ± 12.64 % (RHP), while LVFS values were 15.98 % ± 6.11 % (SHP) and 17.75 % ± 6.98 % (RHP), all statistically indistinguishable from the MI control group (Fig. 4C and D). In addition, the left ventricular end-systolic diameter (LVIDs) and left ventricular end-diastolic diameter (LVIDd) were decreased in the MMHP group and were significantly lower than those in the MI group, the SHP group, and the RHP group. The above results demonstrate that only the MMHP group improved cardiac function after MI.

2.4. The MMHP attenuates pathological cardiac remodeling following MI

Pathological cardiac remodeling after MI is characterized by myocardial fibrosis, infarct expansion, ventricular wall thinning, and cardiomyocyte hypertrophy, and represents the core pathological process driving progression to heart failure. To evaluate the effect of elastic hydrogel patches on this process, we analyzed cardiac morphology and pathological changes in the infarct area of rats across five groups through histological and immunofluorescence approaches. Masson’s Trichrome staining, which is used to distinguish myocardial tissue (red) from fibrous tissue (blue), revealed striking differences in infarct region composition across groups. (Fig. 5A). The MI group exhibited extensive fibrous tissue deposition in the infarcted area, indicating severe myocardial fibrosis. In contrast, the collagen volume fraction (CVF) in the infarcted region of the MMHP group was significantly lower than that in the MI control group, the SHP group, and the RHP group, with more myocardial tissue preserved, suggesting that MMHP mitigates fibrosis and preserves functional myocardial tissue (Fig. S7).

Fig. 5.

The MMHP group exhibited a significant reduction in infarct size and a decrease in apoptosis after MI. (A) Representative images of Masson’s trichrome staining of cardiac tissue from five groups of rats after 4 weeks of surgery (Scale bar, 1 mm). Blue and red represent fibrous tissue and myocardium, respectively. (B) Statistical analysis of infarct size in four groups of rats (n = 6 per group). (C) Statistical analysis of Ventricular wall thickness in five groups of rats (n = 6 per group). (D) Representative images of TUNEL staining in cardiac tissue slices from five groups of rats (Scale bar, 100 μm). (E) The quantitative statistics of the number of TUNEL positive cells based on the TUNEL staining (n = 4 per group). (F) Representative images of WGA immunofluorescence in cardiac tissue slices from five groups of rats (Scale bar, 20 μm). (G) The quantitative statistics of cell size based on the WGA immunofluorescence (n = 3 per group). Data are means ± standard deviation. ∗p < 0.05, ∗∗p < 0.01.

Quantitative analysis of infarct size further validated these observations (Fig. 5B). The MI group had an infarct size of 34.06 % ± 1.37 %, while the SHP group showed no significant improvement (34.74 % ± 4.80 %) and the RHP group only a modest, non-significant reduction (31.94 % ± 0.92 %). Notably, the MMHP group exhibited a marked reduction in infarct size to 19.57 % ± 2.10 %. Ventricular wall thinning is a critical consequence of remodeling, as it impairs cardiac contractility and increases the risk of ventricular rupture. Histological analysis showed that the MI and SHP groups had significantly thinned infarcted ventricular walls, while the MMHP group exhibited no obvious thinning. Quantitatively, the infarcted wall thickness increased from 0.77 ± 0.19 mm (MI group) and 0.94 ± 0.12 mm (SHP group) to 1.56 ± 0.17 mm (MMHP group), suggesting that it possess stronger contractile capacity (Fig. 5C). Interestingly, the RHP group also showed increased wall thickness (1.46 ± 0.16 mm), with no statistically significant difference from MMHP. However, the RHP group exhibited severe collagen deposition and fibrosis (Fig. S7). This phenotype probably attributed to excessive mechanical stress exerted by the rigid hydrogel, which activates fibroblasts and promotes pathological collagen synthesis [26,27]. This contrast highlights unique advantage of MMHP, as it provides sufficient mechanical support to prevent wall thinning while avoiding the pro-fibrotic effects of excessive stiffness.

Alpha-actinin (α-actinin) is a key cytoskeletal protein exclusive to viable cardiomyocytes with intact sarcomeric structures, and it was used to assess myocardial tissue viability [28]. After MI, sarcomeric structures in infarcted cardiomyocytes disintegrate, leading to loss of α-actinin expression. Cardiomyocytes in the infarct border zone may retain partial structures but exhibit weak, discontinuous staining. Immunofluorescence results showed that the MMHP group had significantly higher α-actinin expression in the infarct zones compared to the MI group (Fig. S8A and B), directly confirming that MMHP preserves sarcomeric integrity and promotes viable myocardial tissue retention.

Given that cardiomyocyte apoptosis is a major driver of post-MI tissue loss and remodeling, we quantified apoptotic cells via Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining. The MMHP group exhibited a significant reduction in TUNEL-positive cells relative to the MI control group (Fig. 5D and E). This observation is particularly significant given that the loss of functional cardiomyocytes via apoptosis exacerbates the imbalance between cardiac workload and pumping capacity, accelerating progression to heart failure, while MMHP exerts a cardioprotective effect by inhibiting cardiomyocyte apoptosis.

Following myocardial injury, widespread cardiomyocyte loss triggers a series of compensatory responses aimed at maintaining basal cardiac function. Among these, left ventricular hypertrophy and left ventricular dilation are the most prominent and extensively studied adaptive mechanisms [29]. While these changes can temporarily offset insufficient myocardial contractility, prolonged hypertrophy increases cardiac wall stress and metabolic burden, ultimately leading to further deterioration of cardiac function and progression to decompensated heart failure [30,31]. To evaluate the effect of MMHP on cardiomyocyte hypertrophy, we used wheat germ agglutinin (WGA), a lectin that specifically labels myocardial cell membranes, to assess the cross-sectional area of viable cardiomyocytes in the infarct border zone. WGA staining results revealed a substantial increase in cardiomyocyte size in the MI group, which directly reflects the development of compensatory hypertrophy (Fig. 5F and G). In sharp contrast, the MMHP group exhibited a significantly smaller cardiomyocyte cross-sectional area compared to the MI group. The result confirms that MMHP attenuates compensatory cardiomyocyte hypertrophy, thereby reducing prolonged cardiac stress and preserving myocardial function.

2.5. MMHP suppresses cardiomyocyte apoptosis through the mitochondrial apoptotic pathway

Cardiomyocyte apoptosis after MI is mediated by two primary signaling cascades, the intrinsic (mitochondrial) apoptotic pathway and the extrinsic (death receptor) apoptotic pathway. These pathways often act synergistically to amplify apoptotic cell loss, ultimately exacerbating myocardial dysfunction and pathological remodeling. To dissect the specific molecular mechanism by which MMHP inhibits cardiomyocyte apoptosis, we first investigated the mRNA expression of key apoptotic regulators via qPCR (Fig. S9). Among these regulators, caspases constitute a family of proteases with a cysteine active center. Caspase-3 functions as a key executioner that drives the final steps of apoptosis through the cleavage of downstream cellular substrates [32,33]. Additionally, Bcl-2 is a key anti-apoptotic protein localized primarily to the mitochondrial outer membrane, where it inhibits apoptosis by maintaining mitochondrial membrane integrity and preventing the release of pro-apoptotic factors [34,35]. qPCR results showed distinct expression patterns in the MMHP group compared to the MI group. Caspase-3 mRNA expression was significantly lower in the MMHP group, indicating reduced activation of the apoptotic execution cascade, while Bcl-2 mRNA expression was significantly higher in the MMHP group, reflecting enhanced anti-apoptotic capacity. (Fig. 6A). These transcriptional changes provided initial molecular evidence that MMHP modulates the expression of key apoptosis regulators, with a bias toward suppressing apoptotic signaling.

Fig. 6.

The MMHP suppressed the apoptosis after MI. (A) qPCR analysis of Caspase-3 and Bcl-2 mRNA expression in five groups of rats (n = 3 per group). (B) Immunoblots of Cleaved caspase-3 and Bcl-2 in ventricular myocardium from sham-operated rats or infarcted zone of cardiac tissue from other four groups of rats. (C) Immunoblots of Bcl-2 in mitochondria from five groups of rats. (D) Representative images of Cleaved caspase-3 (red), and DAPI (blue) immunofluorescence in ventricular myocardium from sham-operated rats or infarcted zone of cardiac tissue from other four groups of rats (Scale bar, 50 μm). (E) The quantification of mean fluorescence intensity for Cleaved caspase-3 (n = 4 per group). (F) Representative images of Bcl-2 (green), and DAPI (blue) immunofluorescence in ventricular myocardium from sham-operated rats or infarcted zone of cardiac tissue from other four groups of rats (Scale bar, 50 μm). (G) The quantification of mean fluorescence intensity for Bcl-2 (n = 4 per group). Data are means ± standard deviation. ∗p < 0.05, ∗∗p < 0.01. C-caspase-3: Cleaved caspase-3, MFI: Mean Fluorescence Intensity.

To validate the qPCR findings at the protein level, we performed Western blot analysis on myocardial tissue from the infarct zone. Results confirmed that the MMHP group exhibited a significant downregulation in the expression of cleaved caspase-3 (C-caspase-3, the activated form of caspase-3) and a significant upregulation in Bcl-2 protein levels, relative to the MI group (Fig. 6B). This finding was consistent with the expression trend observed in the qPCR assay. We isolated mitochondria from the infarcted myocardial tissue of five groups of rats and subsequently extracted mitochondrial proteins, with Cytochrome c oxidase IV (COX IV) selected as the loading control for normalization. Notably, Bcl-2 upregulation was more pronounced in the isolated mitochondrial of the MMHP group (Fig. 6C). This observation holds biological significance because the selective enrichment of Bcl-2 in mitochondria strongly suggests that MMHP specifically targets the mitochondrial apoptotic pathway, rather than exerting a non-specific global effect on apoptosis. To further validate these results, we performed immunofluorescence staining for C-caspase-3 and Bcl-2, followed by quantitative analysis (Fig. 6D–G). Consistent with Western blot results, immunofluorescence images showed a marked reduction in C-caspase-3-positive signals in the MMHP group, reflecting fewer actively apoptotic cardiomyocytes, along with a significant increase in Bcl-2-positive signals in the MMHP group. Quantitative analysis of fluorescence intensity further corroborated these observations, thereby providing histological confirmation of MMHP’s regulatory effect on the expression of apoptotic proteins. All the preceding data indicate that four weeks after MI, the application of MMHP has demonstrated multiple positive effects, including marked enhancement of cardiomyocyte retention, reduction in cardiomyocyte loss, and effective alleviation of cardiomyocyte hypertrophy. Taken together, the evidence indicate that cardiac patches with moderate mechanical properties, acting without support from any additional biological agents or cells, have a substantial reversal effect on pathological cardiac remodeling after MI.

2.6. MMHP triggers YAP activation in cardiomyocytes and exerts anti-apoptotic effects following hydrogen peroxide treatment

To further dissect the molecular mechanism by which MMHP mediates post-MI myocardial repair, we focused our research on mechanosensory-related proteins in the infarcted myocardial tissue and thus systematically analyzed their mRNA expression levels (Fig. S10). Interestingly, compared with the MI, SHP, and RHP groups, the mRNA expression of Yes-associated protein (YAP) was notably upregulated in the MMHP group (Fig. 7A). YAP is a key mechanosensitive effector of the Hippo signaling pathway that links extracellular mechanical cues to intracellular transcriptional regulation of cell survival and tissue remodeling [36]. To validate this transcriptional change at the protein level, we performed Western blot analysis of infarcted myocardial tissue. As consistent with qPCR findings, the MMHP group showed a significant increase in total YAP protein expression relative to the other groups (Fig. 7B). The upregulation of YAP provided initial evidence that MMHP may modulate the Hippo-YAP pathway. The functional activity of YAP is not determined solely by its expression, but more critically by its phosphorylation status and subcellular localization [37,38]. Under physiological conditions, active Hippo signaling initiates a series of kinase cascades that phosphorylates YAP. Subsequently, phosphorylated YAP (pYAP) is sequestered in the cytoplasm, rendering it inactive. In contrast, when Hippo signaling is inhibited, YAP subsequently translocates to the nucleus where it can interact with transcription factors to drive the expression of survival and anti-remodeling genes [39,40]. To evaluate YAP activation status, we quantified pYAP levels via Western blot. Results showed that both the MMHP and RHP groups exhibited significantly lower pYAP protein expression compared to the MI group. Additionally, the ratio of pYAP to total YAP (pYAP/YAP) was also reduced in these two groups (Fig. 7C). These data confirm that both MMHP and RHP can inhibit YAP phosphorylation to a certain extent, thereby promoting YAP activation.

Fig. 7.

MMHP triggered intracellular YAP activation. (A) qPCR assay of YAP mRNA expression in five groups of rats (n = 3 per group). (B) Immunoblots of YAP and pYAP in ventricular myocardium from five groups of rats. (C) The quantification of pYAP/YAP ratio (n = 3 per group). (D) Schematic for cyclic stretching on cardiomyocytes in vitro. (E) Immunoblots of various proteins in the cytoplasm and nucleus of cardiomyocytes following cyclic stretching, with or without MMHP and H₂O₂ treatment. (F) Quantification of Immunoblots results (n = 3 per group). Data are means ± standard deviation. ∗p < 0.05, ∗∗p < 0.01. C-YAP: Cytoplasmic YAP, C-caspase3: Cleaved caspase-3, N-YAP: Nuclear YAP.

Notably, a striking discrepancy in therapeutic efficacy emerged despite this shared YAP activation. Only the MMHP group exerted significant myocardial repair effects, whereas the RHP group failed to elicit comparable therapeutic benefits. Concerning this phenomenon, we hypothesize that although RHP induces YAP activation, the aberrant and excessive mechanical cues it delivers might trigger cardiac fibroblast activation via alternative signaling routes [41,42]. The fibroblast activation drives pathological processes in the infarcted region, including abnormal fibrosis and excessive collagen deposition, which not only impair the integration of viable myocardial tissue but also override the pro-survival benefits of YAP activation. In contrast, MMHP precisely mimics native myocardial mechanics, enabling selective activation of signaling for pro-survival effects mediated by YAP without triggering excessive fibroblast activation. This in turn preserves the therapeutic potential of YAP activation for MI repair. To further validate that these effects are directly driven by mechanical cues of MMHP, we designed a series of in vitro experiments to simulate the in vivo myocardial mechanical microenvironment and to assess the impact of mechanical characteristics of MMHP on YAP activation and apoptotic signaling in cardiomyocytes. Given that in vivo cardiomyocytes are continuously exposed to cyclic mechanical stress generated by ventricular contractions, we applied cyclic stretching to H9c2 cardiomyocytes cultured on hydrogels to mimic physiological mechanical stimuli. The parameters for cyclic stretching were set to 10 % elongation and a frequency of 1.25 Hz [43]. The waveform was set to the heart wave that mimics the actual pressure wave generated by a heartbeat (Fig. 7D, Fig. S11). Concurrently, we established a cardiomyocyte apoptosis model via hydrogen peroxide (H₂O₂) treatment [44] (Fig. S12). After 3 days of cyclic stretching, we isolated cytoplasmic and nuclear protein fractions from cardiomyocytes and performed Western blot analysis to quantify key protein expression. Histone H3, a specific and stably expressed nuclear structural protein, was used as the internal reference for nuclear protein normalization. Results showed that cardiomyocytes seeded on MMHP exhibited significantly higher nuclear YAP (N-YAP) expression compared to control cardiomyocytes not seeded on hydrogels. This observation directly confirms that specific mechanical cues of MMHP promote YAP nuclear translocation and successfully triggered its activation. Following the induction of cardiomyocyte apoptosis by H₂O₂ treatment, the cardiomyocytes inoculated on MMHP exhibited an upregulation of N-YAP expression accompanied by marked downregulation of cytoplasmic pYAP. In addition, significantly reduced expression of C-caspase-3 and increased expression of Bcl-2 were observed, which suggests a decrease in myocardial cell apoptosis (Fig. 7E and F).

These results collectively demonstrate that mechanical cues of MMHP combined with physiological cyclic stretching significantly activate YAP in cardiomyocytes, with this activation associated with attenuated apoptosis following H₂O₂ treatment. This in vitro validation represents a key innovation as it establishes a direct causal link between mechanical properties of MMHP and YAP-mediated anti-apoptotic effects, eliminates in vivo confounding factors including systemic inflammation and vascular remodeling, and confirms that mechanical cues alone are sufficient to drive the observed therapeutic signaling.

2.7. MMHP confers cardiomyocyte protection through mechanical cue-mediated YAP activation

Building on the in vitro observation that mechanical cues of MMHP modulate YAP subcellular localization and attenuate H₂O₂-induced apoptosis, we designed targeted intervention experiments to validate the causal relationship between YAP activation and the effect of MMHP in suppressing apoptosis. We first used ML-7, a specific inhibitor of YAP nuclear translocation [19,45], at a working concentration of 20 μM to block YAP activation in cardiomyocytes seeded on MMHP. Cardiomyocytes seeded on MMHP were subjected to different treatments, including the presence or absence (P/A) of apoptosis induction by H₂O₂ and P/A of ML-7. After cyclic stretching, cytoplasmic and nuclear proteins were isolated for Western blot analysis (Fig. 8A and B). Results showed that ML-7 treatment significantly blocked YAP nuclear translocation in cardiomyocytes seeded on MMHP, as evidenced by reduced N-YAP expression. Critically, following apoptosis induction, the ML-7 group exhibited increased C-caspase-3 expression and decreased Bcl-2 expression, indicating enhanced apoptosis and reduced anti-apoptotic capacity. The inhibition of YAP activation completely abrogated the protective effects of MMHP. This experiment directly confirms that YAP nuclear translocation is necessary for the anti-apoptotic effect mediated by mechanical cues of MMHP.

Fig. 8.

MMHP drive cardiomyocyte protection through mechanical cue-mediated YAP activation. (A) Immunoblots of various proteins in the cytoplasm and nucleus of cardiomyocytes seeded on MMHP following cyclic stretching, with or without ML-7 and H₂O₂ treatment. (B) Quantification of Immunoblots results (n = 3 per group). (C) Immunoblots of various proteins in the cytoplasm and nucleus of cardiomyocytes following cyclic stretching, with or without TDI and H₂O₂ treatment. (D) Quantification of Immunoblots results (n = 3 per group). Data are means ± standard deviation. ∗p < 0.05, ∗∗p < 0.01. C-YAP: Cytoplasmic YAP, C-caspase3: Cleaved caspase-3, N-YAP: Nuclear YAP.

Based on the above results, we further hypothesize that promoting YAP nuclear translocation may alleviate H₂O₂-induced apoptosis, even in the absence of mechanical cues of MMHP. To verify this hypothesis, we used TDI-011536 (TDI), a potent and specific Lats kinase inhibitor [46,47], at a working concentration of 3 μM to activate YAP in cardiomyocytes. Cardiomyocytes not seeded on MMHP were subjected to different treatments, including P/A of apoptosis induction and P/A of TDI. Western blot results showed that TDI treatment significantly reduced cytoplasmic pYAP levels and increased N-YAP expression, mimicking the same YAP activation pattern induced by mechanical cues of MMHP (Fig. 8C and D). Concomitantly, TDI treatment led to a significant decrease in C-caspase-3 expression and an increase in Bcl-2 expression. This partial reversal of apoptosis demonstrates that YAP activation via Lats kinase inhibition can directly replicate anti-apoptotic effect of MMHP, independent of mechanical cues from the hydrogel. This finding is particularly impactful, as it confirms that YAP activation is not only necessary for protective effects of MMHP but also sufficient to drive the anti-apoptotic response. It further validates that mechanical cues of MMHP act as a “natural activator” of the YAP, achieving the same protective outcome as targeted pharmacological inhibition of the Hippo pathway via small-molecule inhibitors and without the potential side effects.

3. Conclusion

Myocardial infarction (MI) triggers massive cardiomyocyte loss, which drives pathological ventricular remodeling and subsequent progression to heart failure. Conventional therapeutic interventions including percutaneous coronary intervention and pharmacotherapy primarily restore myocardial perfusion but rarely achieve effective reversal of ventricular remodeling after MI [48]. Tissue engineering strategies have emerged as promising alternatives, with SilMA hydrogel patches gaining attention due to their superior biocompatibility and tunable mechanical properties, which are key attributes for accommodating the cyclic deformation of myocardial tissue during contraction.

To dissect the role of mechanical signals in MI repair, this study fabricated three SilMA hydrogel cardiac patches with distinct elastic moduli. A critical finding emerged that only the mechano-matching hydrogel patch (MMHP), with a modulus comparable to native myocardial tissue, exerted significant therapeutic effects. These effects included reducing local abnormal mechanical stress in the infarcted region, markedly improving cardiac function in MI rats, decreasing infarct size and myocardial fibrosis, and suppressing cardiomyocyte apoptosis. The result underscores a pivotal principle for cardiac biomaterial design that the mechanical properties of biomaterials must match those of the host tissues to optimize repair efficacy. Specifically, such mechanical intervention avoids the activation of fibrotic signals caused by rigid hydrogels and compensates for the defect of soft hydrogels that fail to effectively improve ventricular remodeling due to insufficient mechanical support. Collectively, these data confirm that excessively high or low elastic moduli both lead to therapeutic failure, highlighting the necessity of precise mechanical tuning.

Mechanistically, this study identified Yes-associated protein (YAP) as the central hub linking mechanical cues of MMHP to anti-apoptotic protection. MMHP triggered YAP dephosphorylation and nuclear translocation, which in turn upregulated the expression of the anti-apoptotic protein Bcl-2 and downregulated cleaved caspase-3, ultimately inhibiting the mitochondrial apoptotic pathway. In vitro validation further confirmed this mechanism. Under physiological cyclic stretching, MMHP induced YAP nuclear translocation in cardiomyocytes and reduced H₂O₂-induced apoptosis. Crucially, treatment with the YAP nuclear translocation inhibitor ML-7 completely abrogated this protective effect, directly verifying YAP as the obligate mediator between mechanical signals and apoptosis regulation (Fig. 9). Notably, while RHP also activated YAP in vivo, it failed to improve myocardial repair likely due to its excessive stiffness triggering alternative signaling pathways that drive fibroblast proliferation and pathological collagen deposition, overriding its pro-survival benefits.

Fig. 9.

Mechanistic schematic of cardiomyocyte protection via activation of theYAP-Bcl-2 pathway triggered by mechanical cues from mechano-matching hydrogel patch (Created with Biorender.com).

A major innovation of this study lies in addressing key limitations of current cardiac patch strategies, in that existing patches predominantly rely on cell or biofactor delivery for MI repair, and this approach is plagued by challenges including low post-implantation cell viability, metabolic inactivation of exogenous biofactors, immune rejection, and ethical concerns. By integrating in vivo efficacy assessments, in vitro mechanistic experiments, and molecular intervention studies, we established a complete evidence chain encompassing phenomenon, mechanism, and validation. This chain demonstrates that acellular SilMA hydrogels can mediate myocardial repair exclusively through mechanical signals, representing a paradigm shift that enables the development of “ready-to-use” cardiac repair materials. This strategy eliminates the need for complex preparation of bioactive components, reducing production costs and mitigating clinical application risks. Despite its significance, this study has limitations that warrant consideration. Efficacy was only verified within 4 weeks in a rat MI model, with no long-term tracking of cardiac function or potential late-stage complications. The molecular details of YAP-mediated Bcl-2 regulation remain incompletely elucidated. Given that large animal models including pigs and sheep exhibit cardiac physiology and anatomy more analogous to those of humans, validation in such models is essential to confirm the translational potential of this mechanical regulation strategy.

Notably, this study also sets multiple directions for follow-up research in cardiac mechanobiology and regenerative medicine. For one, it provides a starting point to explore whether combining mechano-matching hydrogels with non-invasive mechanical stimulation amplifies YAP-Bcl-2 pathway activation, potentially boosting mechanical therapy’s anti-apoptotic effect without hydrogel modification. For another, the confirmed YAP-Bcl-2 mechanical response axis enables screening synergistic regulators, and future studies may combine mechanical cues with targeted molecules to reinforce anti-apoptotic effects. Additionally, the acellular mechanical therapy paradigm can extend to other myocardial diseases or repair of organs with mechanical microenvironment disorders (such as liver fibrosis, lung emphysema), guiding cross-field mechanical regulation research in tissue repair.

In summary, this study establishes that the mechanical properties can serve as a single variable to modulate myocardial repair process after MI. The mechano-matching hydrogel patch achieves cardiac protection by activating YAP-mediated anti-apoptotic signaling, without relying on cells or exogenous growth factors. This finding provides a novel design principle for acellular myocardial repair materials without growth factors and holds great promise for advancing the clinical translation of implantable cardiac patches for MI therapy.

4. Methods

4.1. Synthesis of SilMA hydrogel

40 g of cut cocoon was boiled in 4L of 0.05 M sodium carbonate (Na₂CO₃) at 100 °C for 1 h and then rinsed four or five times with distilled water followed by dehydration in a constant temperature oven at 60 °C. 20 g of dried silk was dissolved in 100 mL of 9.3 M lithium bromide (LiBr) at 55 °C for 1 h and filtered. Subsequently, glycidyl methacrylate (GMA) was dropped into SF solution and stirred at 350 rpm for 3 h at 60 °C to allow the reaction between SF and GMA. Then, the reaction mixture was dialyzed against distilled water for 7 days, followed by reverse osmosis concentration using a 20 % (w/v) polyethylene glycol (PEG) solution to obtain the target concentration of 20 % (w/v) SilMA. The SilMA solution within 0.1 % alpha ketoglutaric acid was injected into well plates (120 μL/well for 48-well plates, and 250 μL/well for 24-well plates) and was irradiated with a 405 nm light source for 1min to gel. SHP was made of 10 % (w/v) SilMA, MMHP was made of 20 % (w/v) SilMA and RHP was made of 10 % (w/v) SilMA followed by soaking in 75 % ethanol dehydration overnight at room temperature.

4.2. The characterization of SilMA hydrogel

SF and SilMA were examined through 1H nuclear magnetic resonance (¹H NMR) at a frequency of 400 MHz using a Bruker DPX FT-NMR spectrometer (Bruker, Germany) and 700 μL of deuterium oxide (D2O, Sigma-Aldrich) as the solvent per 5 mg of sample. The formula for the degree of methacrylation (DoM) is as follows:

$$\text{DoM}(\%)=1–\left(\frac{\text{Lysine}\text{integrated}\text{signal}\text{of}\text{functionalized}\text{sample}}{\text{Lysine}\text{integrated}\text{signal}\text{of}\text{RSF}}\right)\times 100\%$$

Fourier transform infrared spectra (FTIR) of the samples were collected with a FTIR spectrometer (Thermo Electron Instruments Co., Ltd., USA) in the frequency range of 4000-400 cm⁻¹ with a total of 32 scans and resolution of 4 cm⁻¹. The mechanical properties of different hydrogels were studied by compression test with LS1 materials testing system (AMETEK, America), compressive ramp up to 80 % strain and the elastic modulus of the hydrogels was calculated from the slope of the stress versus strain curves. The electrical properties of three different SilMA hydrogels were conducted on an electrochemical workstation (CorrTest Instruments, China). The morphologies of hydrogel were observed by scanning electron microscope (SEM, H-7650, Hitachi, Japan). The equilibrium swelling ratio (ESR) of the SilMA hydrogels was investigated by the gravimetric method. The different SilMA hydrogel scaffolds were freeze-dried and weighed, and the initial mass of dried hydrogel (m0) was recorded. The hydrogels were incubated in PBS solution to reach the swelling equilibrium state at 37 °C, and weighed at 30 min, 1 h, 2 h, 4 h, 24 h and 48 h. The hydrogel weight was recorded as (mt). The following equation was used to calculate the ESR: ESR =(mt-m0)/m0 × 100 %. Three types of hydrogel patches were implanted into the infarcted myocardial region of rats. At 1, 2, 3, and 4 weeks post-implantation, the residual hydrogel was carefully separated from the myocardial tissue and weighed after lyophilization (W_t). The initial dry weight of each hydrogel patch before implantation was recorded as W₀. The degradation ratio of the hydrogel patches was calculated as follows:

Degradation ratio (%) = (W_t - W₀)/W₀ × 100 %

4.3. Cell culture, cytocompatibility test and cyclic stretching

H9c2 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) with 10 % fetal bovine serum (Gibco, Carlsbad, CA, United States) at 37 °C in 5 % CO₂. For cytocompatibility test, the hydrogels were incubated overnight in cardiomyocyte growth medium. Then the H9c2 cells were seeded onto each hydrogel at a total number of 2 × 10⁵ cells and incubated for 3 days, the growth medium was changed every day. The live/dead cell staining were conducted for cytocompatibility tests according to the manufacturers’ instructions. The assay solution was prepared by adding 2 μL of 2 mM EthD-1 and 0.5 μL of 5 mM calcein AM to 1 mL DPBS. The cell culture medium was removed, and the assay solution was added. After 10 min of incubation, samples were washed twice with DPBS for 5 min. Live (green)/dead (red) staining images were acquired using a fluorescence microscope. The 20 % SilMA solution within 0.1 % alpha ketoglutaric acid was injected into BioFlex Culture Plate and was irradiated with a 405 nm light source to gel. After seeding cells onto the MMHP and culturing them for 24 h to achieve adequate spreading, the stretching experiment was then initiated. The parameters for cyclic stretching were set to 10 % elongation and a frequency of 1.25 Hz. The waveform was set to the heart wave that mimics the actual pressure wave generated by a heartbeat. The cell stretching experiment was conducted twice a day, 6 h per session, for a total of 3 days. At 6 h before the termination of stretching, 600 μM hydrogen peroxide was administered to trigger cell apoptosis. Afterward, the cells were collected, with the subsequent extraction and isolation of nuclear and cytoplasmic proteins.

4.4. Establishment of myocardial infarction model

The acquisition and use of these laboratory animals were conducted in strict compliance with the guidelines of the ethics committee and obtained approval from the Animal Ethics Committee of Southern Medical University (SMUL202311025). The experiments were performed in line with the Regulations for Laboratory Animal Management promulgated by the Ministry of Science and Technology of China. Adult male Sprague-Dawley rats (220 g) underwent left anterior descending artery (LAD) ligation surgery or sham surgery and were randomly divided into five groups. The groups were: (1) sham-operated rat; (2) rats with myocardial infarction, (3) rats patched with soft hydrogel after MI, (4) rats patched with moderate-stiffness hydrogel after MI, (5) and rats patched with stiff hydrogel after MI. All rats were injected with pentobarbital sodium and mechanically ventilated. Then, the LAD branch was permanently ligation in all rats except the sham group, and the myocardial tissue at the ligation site showed ischemic phenomenon. Immediately, three SilMA hydrogels were transplanted to the infarct area. All patches are circular with a diameter of 8 mm and thickness of 1.5 mm and carefully fixed to the infarct area by suture. After surgery, rats were housed in plastic cages at a controlled temperature of 20°C-22 °C, on a 12-h light/12-h dark cycle with lights on from 06:00–18:00 throughout the study.

4.5. Echocardiography examination

The left heart function was assessed by echocardiography (Vevo 2100, Visual Sonics) in experimental rats. At 1 week and 4 weeks after surgery, transthoracic echocardiography was performed, and left ventricular long-axis view, and B-mode echocardiography were recorded, respectively. Left ventricular function parameters were measured, including LVIDs, LVIDd, EF, and FS.

4.6. Histomorphometric analysis

Four weeks after myocardial infarction, myocardial tissues of a subset of rats were processed and embedded in paraffin and 3 μm sagittal serial sections were prepared for histological analysis. For morphological analysis, Masson’s trichrome staining was performed on the sections. Images were obtained with an Axio Scope A1 microscope (Carl Zeiss Microscopy GmbH, Jena, Germany). The infarct size was assessed morphologically and calculated as the area of myocardial necrosis as a percentage of the whole myocardium by Masson’s trichrome staining.

4.7. Immunostaining

The rats were euthanized, myocardial tissues were collected and embedded in OCT. Then immunofluorescence (IF) staining was performed on frozen sections of myocardial tissue which were cryosectioned at a thickness of 6 μm using a Leica CM3050 S cryostat (Leica Biosystems). Sections were fixed with 4 % paraformaldehyde for 30 min and then washed with PBS three times, each time for 5 min. After incubating in 5 % bovine serum albumin for 1 h, sections were incubated overnight at 4 °C with Bcl-2 antibody (1:300, 60178-1-IG, Proteintech, Wuhan, China), Cleaved caspase-3 antibody (1:200, 25128-1-AP, Proteintech, Wuhan, China) and α-actinin antibody (1:500, ab9465, Abcam, Cambridge, UK), then with the relevant secondary antibody for 1 h at 37 °C. Nuclei were counterstained with diamidino phenylindole (Thermo Fisher Scientific). Images were obtained with a confocal laser scanning microscope (Olympus FV1000, Tokyo, Japan), equipped with a Hamamatsu camera.

4.8. Wheat germ agglutinin (WGA) staining

The cross-sectional areas of the cardiomyocytes in the left ventricle were observed by fluorescein-conjugated WGA (25530, AAT Bioquest, United States) staining. Briefly, after dewaxing the paraffin sections of rat hearts, directly added the WGA working solution and incubated at room temperature for 30 min. After washing three times with PBS, imaging was performed under a fluorescence microscope. The single myocyte cross-sectional area was measured by ImageJ software.

4.9. TUNEL staining

Paraffin-embedded heart sections (4 μm) were permeabilized with 0.5 % Triton X-100 for 30 min at room temperature, followed by staining with TUNEL reagent (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling) to detect apoptotic cell and with DAPI to stain all cell nuclei. Fluorescent staining was observed using a FluoView FV1000 microscope (Olympus). The number of TUNEL-positive cells was counted in at least three randomly selected fields within the infarcted area of each slide and analyzed using ImageJ software.

4.10. Protein extraction and mitochondria isolation

Total protein was extracted from myocardial tissue and H9c2 cells following lysis in RIPA buffer. Nuclear and cytoplasmic protein extraction kit was purchased from Beyotime Biotechnology (P0027, Shanghai, China). Tissue mitochondria isolation kit was purchased from Beyotime Biotechnology (C3606, Shanghai, China). BCA assay was used to determine the concentration of protein. Lysates were boiled in 2 × SDS sample buffer. Proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes (Bio-Rad, CA, United States) for immunoblotting with relevant antibodies. The primary antibodies were anti-YAP (1:2000, 13584-1-AP, Proteintech, Wuhan, China), anti-Cleaved caspase-3 (1:1000, 25128-1-AP, Proteintech, Wuhan, China), anti-Bcl-2 (1:2000, 60178-1-IG, Proteintech, Wuhan, China), anti-COXIV (1:5000, 11242-1-AP, Proteintech, Wuhan, China), anti-pYAP (1:2000, 13008, Cell Signaling Technology, Boston, United States), anti-Histone H3 (1:5000, 17168-1-AP, Proteintech, Wuhan, China) and anti-GAPDH (1:5000, 60004-1-IG, Proteintech, Wuhan, China).

4.11. RT-qPCR

Total RNA from myocardial tissue was extracted with Trizol reagent (Life Technologies, CA, United States) according to the manufacturer’s protocol. Trizol reagent was added to the tissue samples (1 mL/50–100 mg of tissue). cDNA was synthesized from 1 μg of total RNA. The RNA was reverse transcribed with HIScript QRT MIX for qPCR (Vazyme Biotech, Nanjing, China). The resulting cDNAs were amplified using SYBR-Green Master PCR Mix (Takara Bio Inc., Shiga, Japan). QPCR was performed in an ABI StepOnePlusTM Real-Time PCR System (Thermo Fisher Scientific, MA, USA). Briefly, cDNA template was mixed with forward and reverse primers and 2 × SYBR Green PCR Master Mix. The mixture was run under the following conditions: 95 °C for 10 min, then 40 cycles at 95 °C for 10 s, 60 °C for 20 s, and 72 °C for 35 s. The data were normalized against endogenous GAPDH controls of each sample. The primers are listed in the Supplementary Table.

4.12. Statistical analysis

Statistical analysis was performed with SPSS 23.0 (IBM Corp, Armonk, NY, United States). Data were reported as means ± standard deviation of at least three independent experiments. Data were first tested for normality using the Shapiro-Wilk test and for homogeneity of variance using Levene’s test. For comparisons between two groups, two-tailed t-tests were used if data met the assumptions of normality and equal variance. P-values <0.05 were considered statistically significant. Photomicrographs and western blot images are representative of at least three independent experiments.

CRediT authorship contribution statement

Yanyan Zhao: Writing – review & editing, Writing – original draft, Investigation, Data curation. Jie Shen: Software, Formal analysis. Rurong Lin: Software, Data curation. Jianxing Huang: Software, Data curation. Xiaoming Zou: Resources, Methodology. Honghao Hou: Funding acquisition. Xiaozhong Qiu: Funding acquisition.

Additional resources

This study did not generate any additional resources.

Declaration of competing interest

The authors declare that they have no competing interests.

Appendix A. Supplementary data

The following is the supplementary data to this article:

Data availability

Data will be made available on request.