Microenvironment stiffness requires extracellular matrix proteins to modulate heart regeneration

Abstract

Introduction Injecting decellularized cardiac extracellular matrix (dECM) into the myocardium stimulates heart regeneration in rodent and porcine myocardial infarction (MI) models. Early-age cardiac dECM showed higher therapeutic efficacy than adult dECM for MI in animal models. In this study, we demonstrate that decreasing microenvironment stiffness further improves the therapeutic efficacy of fetal dECM on post-ischemia heart regeneration through YAP-regulated cardiomyocyte proliferation and CAPG-regulated fibroblast differentiation. Decreasing heart stiffness has been reported to preserve the heart regenerative capacity in newborn mice. However, how microenvironment stiffness affects cardiomyocyte proliferation and heart regeneration has not been fully understood. Using in vivo and in vitro cardiac injury models of various tissue stiffness, we investigated how tissue stiffness modulates heart post-injury response and exogenous fetal dECM induced heart recovery at different time points. We also examined the total mRNA expression and identified signaling pathways regulating the synergistic effect of tissue stiffness and dECM treatment on heart regeneration. Methods P5 neonatal mouse heart stiffness was lowered by injecting BAPN, and increased by genipin. Solubilized porcine fetal dECM was injected immediately after ligating coronary artery. Heart function and histology were conducted at week 3 post-MI. Cardiomyocyte cell cycle activity, fibrosis, fibroblast differentiation, and vascularization were examined. Post-MI mice at day3 and day21 were investigated for relevant signaling pathways. A mice ventricle explant model was used to investigate the role of mechanosensitive proteins and cytoskeleton organization in cardiomyocyte proliferation regulation. Results Tissue stiffness was measured by atomic force microscopy (AFM). Heart stiffness was lowered from 50kPa to 9kPa by BAPN and increased to 142kPa by genipin. We observed that fetal dECM treatment preserved cardiac output, reduced fibrosis, and promoted cardiomyocyte cell cycle activity on week 3 post-MI. Decreasing tissue stiffness further improved the therapeutic efficacy of fetal dECM on heart post-MI response (Fig.1). Decreasing tissue stiffness lowered the ratio of fibrotic area in no-dECM treated MI control hearts, but did not affect the other aspects. Using a ventricle explant model of various stiffness, molecular evidence demonstrated that agrin-YAP signaling pathway is involved in the mechano-regulation of fetal dECM-induced heart regeneration. Agrin expression was elevated and cardiomyocyte YAP activation was not affected by changing tissue stiffness in control (no-dECM) mechano-modulated explants. However, YAP activation in fetal-dECM treated explants was increased by softening tissue. Disrupting cytoskeleton organization reduced YAP activity and cardiomyocyte cell cycle activity induced by fetal dECM. We also demonstrated that dECM inhibits fibroblast to myofibroblast differentiation. RNA sequencing and western blot revealed that macrophage-capping protein (CAPG) expression was lowered by dECM treatments. Interfering CAPG expression in fibroblasts inhibits fibroblast differentiation.