Microenvironment Stiffness Amplifies Post-ischemia Heart Regeneration in Response to Exogenous Extracellular Matrix Proteins in Neonatal Mice.

The cardiogenesis of the fetal heart is absent in juveniles and adults. Cross-transplantation of decellularized extracellular matrix (dECM) can stimulate regeneration in myocardial infarct (MI) models. We have previously shown that dECM and tissue stiffness have cooperative regulation of heart regeneration in transiently regenerative day 1 neonatal mice. To investigate underlying mechanisms of mechano-signaling and dECM, we pharmacologically altered heart stiffness and administered dECM hydrogels in non-regenerative mice after MI. The dECM combined with softening exhibits preserved cardiac function, LV geometry, increased cardiomyocyte mitosis and lowered fibrosis while stiffening further aggravated ischemic damage. Transcriptome analysis identified a protein in cardiomyocytes, CLCA2, confirmed to be upregulated after MI and downregulated by dECM in a mechanosensitive manner. Synthetic knock-down of CLCA2 expression induced mitosis in primary rat cardiomyocytes in the dish. Together, our results indicate that therapeutic efficacy of extracellular molecules for heart regeneration can be modulated by heart microenvironment stiffness in vivo.

Wang X ，Pierre V ，Senapati S ，Park PS ，Senyo SE ... -  《Frontiers in Cardiovascular Medicine》

Exogenous extracellular matrix proteins decrease cardiac fibroblast activation in stiffening microenvironment through CAPG.

Controlling fibrosis is an essential part of regenerating the post-ischemic heart. In the post-ischemic heart, fibroblasts differentiate to myofibroblasts that produce collagen-rich matrix to physically stabilize the infarct area. Infarct models in adult mice result in permanent scarring unlike newborn animals which fully regenerate. Decellularized extracellular matrix (dECM) hydrogels derived from early-aged hearts have been shown to be a transplantable therapy that preserves heart function and stimulates cardiomyocyte proliferation and vascularization. In this study, we investigate the anti-fibrotic effects of injectable dECM hydrogels in a cardiac explant model in the context of age-associated tissue compliance. Treatments with adult and fetal dECM hydrogels were tested for molecular effects on cardiac fibroblast activation and fibrosis. Altered sensitivity of fibroblasts to the mechanosignaling of the remodeling microenvironment was evaluated by manipulating the native extracellular matrix in explants and also with elastomeric substrates in the presence of dECM hydrogels. The injectable fetal dECM hydrogel treatment decreases fibroblast activation and contractility and lowers the stiffness-mediated increases in fibroblast activation observed in stiffened explants. The anti-fibrotic effect of dECM hydrogel is most observable at highest stiffness. Experiments with primary cells on elastomeric substrates with dECM treatment support this phenomenon. Transcriptome analysis indicated that dECM hydrogels affect cytoskeleton related signaling including Macrophage capping protein (CAPG) and Leupaxin (LPXN). CAPG was down-regulated by the fetal dECM hydrogel. LPXN expression was decreased by stiffening the explants; however, this effect was reversed by dECM hydrogel treatment. Pharmacological disruption of cytoskeleton polymerization lowered fibroblast activation and CAPG levels. Knocking down CAPG expression with siRNA inhibited fibroblast activation and collagen deposition. Collectively, fibroblast activation is dependent on cooperative action of extracellular molecular signals and mechanosignaling by cytoskeletal integrity.

Wang X ，Pierre V ，Liu C ，Senapati S ，Park PS ，Senyo SE ... -  《-》

Microenvironment stiffness requires decellularized cardiac extracellular matrix to promote heart regeneration in the neonatal mouse heart.

The transient period of regeneration potential in the postnatal heart suggests molecular changes with maturation influence the cardiac response to damage. We have previously demonstrated that injury and exercise can stimulate cardiomyocyte proliferation in the adult heart suggesting a sensitivity to exogenous signals. Here, we consider whether exogenous fetal ECM and mechanically unloading interstitial matrix can drive regeneration after myocardial infarction (MI) surgery in low-regenerative hearts of day5 mice. Compared to controls, exogenous fetal ECM increases cardiac function and lowers fibrosis at 3 weeks post-injury and this effect can be augmented by softening heart tissue. In vitro experiments support a mechano-sensitivity to exogenous ECM signaling. We tested potential mechanisms and observed that fetal ECM increases nuclear YAP localization which could be enhanced by pharmacological stabilization of the cytoskeleton. Blocking YAP expression lowered fetal ECM effects though not completely. Lastly we observed mechanically unloading heart interstitial matrix increased agrin expression, an extracellular node in the YAP signaling pathway. Collectively, these data support a combined effect of exogenous factors and mechanical activity in altering agrin expression, cytoskeletal remodeling, and YAP signaling in driving cardiomyocyte cell cycle activity and regeneration in postnatal non-regenerative mice. STATEMENT OF SIGNIFICANCE: With the purpose of developing regenerative strategies, we investigate the influence of the local niche on the cardiac injury response. We conclude tissue stiffness, as anticipated in aging or disease, impairs regenerative therapeutics. Most novel, mechanical unloading facilitates enhanced cardiac regeneration only after cells are pushed into a permissive state by fetal biomolecules. Specifically, mechanical unloading appears to increase extracellular agrin expression that amplifies fetal-stimulation of nuclear YAP signaling which correlates with observed increases of cell cycle activity in cardiomyocytes. The results further suggest the cytoskeleton is critical to this interaction between mechanical unloading and independently actived YAP signaling. Using animal models, tissue explants, and cells, this work indicates that local mechanical stimuli can augment proliferating-permissive cardiomyocytes in the natural cardiac niche.

Wang X ，Senapati S ，Akinbote A ，Gnanasambandam B ，Park PS ，Senyo SE ... -  《-》

Injectable Extracellular Matrix Microparticles Promote Heart Regeneration in Mice with Post-ischemic Heart Injury.

Ischemic heart injury causes permanent cardiomyocyte loss and fibrosis impairing cardiac function. Tissue derived biomaterials have shown promise as an injectable treatment for the post-ischemic heart. Specifically, decellularized extracellular matrix (dECM) is a protein rich suspension that forms a therapeutic hydrogel once injected and improves the heart post-injury response in rodents and pig models. Current dECM-derived biomaterials are delivered to the heart as a liquid dECM hydrogel precursor or colloidal suspension, which gels over several minutes. To increase the functionality of the dECM therapy, an injectable solid dECM microparticle formulation derived from heart tissue to control sizing and extend stability in aqueous conditions is developed. When delivered into the infarcted mouse heart, these dECM microparticles protect cardiac function promote vessel density and reduce left ventricular remodeling by sustained delivery of biomolecules. Longer retention, higher stiffness, and slower protein release of dECM microparticles are noted compared to liquid dECM hydrogel precursor. In addition, the dECM microparticle can be developed as a platform for macromolecule delivery. Together, the results suggest that dECM microparticles can be developed as a modular therapy for heart injury.

Wang X ，Ansari A ，Pierre V ，Young K ，Kothapalli CR ，von Recum HA ，Senyo SE ... -  《-》

Injectable Decellularized Extracellular Matrix Hydrogel Containing Stromal Cell-Derived Factor 1 Promotes Transplanted Cardiomyocyte Engraftment and Functional Regeneration after Myocardial Infarction.

Wu K ，Wang Y ，Yang H ，Chen Y ，Lu K ，Wu Y ，Liu C ，Zhang H ，Meng H ，Yu Q ，Zhang Y ，Shen Z ... -  《-》