Elasticity of Developing Cardiac Tissue and Influence on Early Cardiomyocyte Beating

Abstract

Recent studies have demonstrated the effects of cardiomyocyte mechanosensitivity to ECM elasticity on beating physiology of 10 day chick embryo cardiomyocytes[1] and also of neonatal rat cardiomyocytes[2], developmental stages for which the heart mechanics are established and the Young's modulus is E∼10-20 kPa. Here we study the evolution of embryonic cardiac tissue mechanics in parallel with the effects of microenvironment mechanics on individual cardiomyocyte function throughout early development. To measure the changing mechanical properties of cardiac tissue, we used micropipette aspiration to measure local and average bulk elastic moduli of embryonic avian heart tissue. We observe stiffening of the looping heart tube (days 2-4) from 1 kPa average elasticity to 2 kPa. We measure a greater local elastic modulus in the inner curvature of the looping heart tube than the outer curvature. Treating heart tubes with blebbistatin led to a uniform 30% decrease in elasticity, indicating that the local elasticity is not solely due to actomyosin contractility. We performed a proteomic analysis of various anatomical positions along the 2-4 day heart tubes by mass spectrometry, to measure the relative spaciotemporal changes in protein expression. To explore the effects of the immediate microenvironment mechanics on early cardiomyocyte function, heart cells were isolated from 2-4 day chicken embryos, cultured on collagen-coated polyacrylamide gels of varying stiffness, and observed for effects of substrate elasticity on spontaneous beating and morphology. After 24 hours in culture, cardiomyocyte beating magnitude was larger on softer 1 kPa gels than on stiffer 5-34 kPa gels, with no measurable difference in behavior with developmental stage. [1] A. Engler et al. Journal of Cell Science 121: 3794-3802 (2008). [2] J. Jacot, et al. Biophysical Journal 95(7): 3479-3487 (2008).