Extracellular matrix signaling in morphogenesis and repair.

The extracellular matrix (ECM) is critically important for many cellular processes including growth, differentiation, survival, and morphogenesis. Cells remodel and reshape the ECM by degrading and reassembling it, playing an active role in sculpting their surrounding environment and directing their own phenotypes. Both mechanical and biochemical molecules influence ECM dynamics in multiple ways; by releasing small bioactive signaling molecules, releasing growth factors stored within the ECM, eliciting structural changes to matrix proteins which expose cryptic sites and by degrading matrix proteins directly. The dynamic reciprocal communication between cells and the ECM plays a fundamental roll in tissue development, homeostasis, and wound healing.

Developing cardiac and skeletal muscle share fast-skeletal myosin heavy chain and cardiac troponin-I expression.

Skeletal muscle derived stem cells (MDSCs) transplanted into injured myocardium can differentiate into fast skeletal muscle specific myosin heavy chain (sk-fMHC) and cardiac specific troponin-I (cTn-I) positive cells sustaining recipient myocardial function. We have recently found that MDSCs differentiate into a cardiomyocyte phenotype within a three-dimensional gel bioreactor. It is generally accepted that terminally differentiated myocardium or skeletal muscle only express cTn-I or sk-fMHC, respectively. Studies have shown the presence of non-cardiac muscle proteins in the developing myocardium or cardiac proteins in pathological skeletal muscle. In the current study, we tested the hypothesis that normal developing myocardium and skeletal muscle transiently share both sk-fMHC and cTn-I proteins. Immunohistochemistry, western blot, and RT-PCR analyses were carried out in embryonic day 13 (ED13) and 20 (ED20), neonatal day 0 (ND0) and 4 (ND4), postnatal day 10 (PND10), and 8 week-old adult female Lewis rat ventricular myocardium and gastrocnemius muscle. Confocal laser microscopy revealed that sk-fMHC was expressed as a typical striated muscle pattern within ED13 ventricular myocardium, and the striated sk-fMHC expression was lost by ND4 and became negative in adult myocardium. cTn-I was not expressed as a typical striated muscle pattern throughout the myocardium until PND10. Western blot and RT-PCR analyses revealed that gene and protein expression patterns of cardiac and skeletal muscle transcription factors and sk-fMHC within ventricular myocardium and skeletal muscle were similar at ED20, and the expression patterns became cardiac or skeletal muscle specific during postnatal development. These findings provide new insight into cardiac muscle development and highlight previously unknown common developmental features of cardiac and skeletal muscle.

Engineered fetal cardiac graft preserves its cardiomyocyte proliferation within postinfarcted myocardium and sustains cardiac function.

The goal of cellular cardiomyoplasty is to replace damaged myocardium by healthy myocardium achieved by host myocardial regeneration and/or transplantation of donor cardiomyocytes (CMs). In the case of CM transplantation, studies suggest that immature CMs may be the optimal cell type to survive and functionally integrate into damaged myocardium. In the present study, we tested the hypothesis that active proliferation of immature CMs contributes graft survival and functional recovery of recipient myocardium. We constructed engineered cardiac tissue from gestational day 14 rat fetal cardiac cells (EFCT) or day 3 neonatal cardiac cells (ENCT). Culture day 7 EFCTs or ENCTs were implanted onto the postinfarct adult left ventricle (LV). CM proliferation rate of EFCT was significantly higher than that of ENCT at 3 days and 8 weeks after the graft implantation, whereas CM apoptosis rate remained the same in both groups. Echocardiogram showed that ENCT implantation sustained LV contraction, whereas EFCT implantation significantly increased the LV contraction at 8 weeks versus sham group (p < 0.05, analysis of variance). These results suggest that active CM proliferation may play a critical role in immature donor CM survival and the functional recovery of damaged recipient myocardium.

Directed stem cell differentiation: the role of physical forces.

A number of factors contribute to the control of stem cell fate. In particular, the evidence for how physical forces control the stem cell differentiation program is now accruing. In this review, the authors discuss the types of physical forces: mechanical forces, cell shape, extracellular matrix geometry/properties, and cell-cell contacts and morphogenic factors, which evidence suggests play a role in influencing stem cell fate.

A three-dimensional gel bioreactor for assessment of cardiomyocyte induction in skeletal muscle-derived stem cells.

Skeletal muscle-derived stem cells (MDSCs) are able to differentiate into cardiomyocytes (CMs). However, it remains to be investigated whether differentiated CMs contract similar to native CMs. Here, we developed a three-dimensional collagen gel bioreactor (3DGB) that induces a working CM phenotype from MDSCs, and the contractile properties are directly measured as an engineered cardiac tissue. Neonate rat MDSCs were isolated from hind-leg muscles via the preplate technique. Isolated MDSCs were approximately 60% positive to Sca-1 and negative to CD34, CD45, or c-kit antigens. We sorted Sca-1(-) MDSCs and constructed MDSC-3DGBs by mixing MDSCs with acid soluble rat tail collagen type-I and matrix factors. MDSC-3DGB exhibited spontaneous cyclic contraction by culture day 7. MDSC-3DGB expressed cardiac-specific genes and proteins. Histological assessment revealed that cardiac-specific troponin-T and -I expressed in a typical striation pattern and connexin-43 was expressed similar to the native fetal ventricular papillary muscle. beta-Adrenergic stimulation increased MDSC-3DGB spontaneous beat frequency. MDSC-3DGB generated contractile force and intracellular calcium ion transients similar to engineered cardiac tissue from native cardiac cells. Results suggest that MDSC-3DGB induces a working CM phenotype in MDSCs and is a useful 3D culture system to directly assess the contractile properties of differentiated CMs in vitro.

Engineered early embryonic cardiac tissue increases cardiomyocyte proliferation by cyclic mechanical stretch via p38-MAP kinase phosphorylation.

Cardiomyocyte (CM) transplantation is one therapeutic option for cardiac repair. Studies suggest that fetal CMs display the best cell type for cardiac repair, which can finitely proliferate, integrate with injured host myocardium, and restore cardiac function. We have recently developed an engineered early embryonic cardiac tissue (EEECT) using embryonic cardiac cells and have shown that EEECT contractile properties and cellular proliferative response to cyclic mechanical stretch stimulation mimic developing fetal myocardium. However, it remains unknown whether cyclic mechanical stretch-mediated high cellular proliferation activity within EEECT reflects CM or non-CM population. Studies have shown that p38-mitogen-activated protein kinase (p38MAPK) plays an important role in both cyclic mechanical stretch stimulation and cellular proliferation. Therefore, in the present study, we tested the hypothesis that cyclic mechanical stretch (0.5 Hz, 5% strain for 48 h) specifically increases EEECT CM proliferation mediated by p38MAPK activity. Cyclic mechanical stretch increased CM, but not non-CM, proliferation and increased p38MAPK phosphorylation. Treatment of EEECT with the p38MAPK inhibitor, SB202190, reduced CM proliferation. The negative CM proliferation effects of SB202190 were not reversed by concurrent stretch stimulation. Results suggest that immature CM proliferation within EEECT can be positively regulated by mechanical stretch and negatively regulated by p38MAPK inhibition.