Substrate Stiffness Affects the Functional Maturation of Neonatal Rat Ventricular Myocytes

Abstract

Cardiac cells mature in the first postnatal week, concurrent with altered extracellular mechanical properties. To investigate the effects of extracellular stiffness on cardiomyocyte maturation, we plated neonatal rat ventricular myocytes for 7 days on collagen-coated polyacrylamide gels with varying elastic moduli. Cells on 10 kPa substrates developed aligned sarcomeres, whereas cells on stiffer substrates had unaligned sarcomeres and stress fibers, which are not observed in vivo. We found that cells generated greater mechanical force on gels with stiffness similar to the native myocardium, 10 kPa, than on stiffer or softer substrates. Cardiomyocytes on 10 kPa gels also had the largest calcium transients, sarcoplasmic calcium stores, and sarcoplasmic/endoplasmic reticular calcium ATPase2a expression, but no difference in contractile protein. We hypothesized that inhibition of stress fiber formation might allow myocyte maturation on stiffer substrates. Treatment of maturing cardiomyocytes with hydroxyfasudil, an inhibitor of RhoA kinase and stress fiber-formation, resulted in enhanced force generation on the stiffest gels. We conclude that extracellular stiffness near that of native myocardium significantly enhances neonatal rat ventricular myocytes maturation. Deviations from ideal stiffness result in lower expression of sarcoplasmic/endoplasmic reticular calcium ATPase, less stored calcium, smaller calcium transients, and lower force. On very stiff substrates, this adaptation seems to involve RhoA kinase.

Hippo Pathway Effectors Control Cardiac Progenitor Cell Fate by Acting as Dynamic Sensors of Substrate Mechanics and Nanostructure

“…28,29 The responsiveness of adult and undifferentiated cells to substrate stiffness and topology has been the subject of a number of investigations so far, showing that cell-specific responses can be triggered by the mechanics of the substrate. 12,20,30,41,42 Moreover, a specific response of tissue resident cardiac progenitor cells to substrate mechanical properties has been highlighted. 25,43 Recently, cell sensitivity to substrate composition has been associated with the ability of the Hippo pathway downstream effectors YAP and TAZ to act as transcriptional coactivators directly binding to lineage-specific effectors, thus acting as onÀoff relays in mechano-transduction.…”

Substrate stiffness-modulated registry phase correlations in cardiomyocytes map structural order to coherent beating

“…The position of the peak in registry corresponds in our theory to the substrate elasticity at which this particular type of cardiomyocytes (of embryonic chicks) become fully contractile, E*. The value of E* ¼ 4 kPa is comparable to the corresponding saturation rigidity of 10 kPa for cardiomyocytes in neonatal rats 5 . Figure 2 shows that besides differing in their peak positions, the shape and width of the registry and strain curves plotted versus substrate elasticity are significantly different, the width at half maximum being about 50 and 8 kPa, respectively.…”

“…In particular, both the morphology and beating of embryonic heart muscle cells or cardiomyocytes have been observed to depend sensitively on the rigidity of the substrate on which they are cultured [4][5][6][7][8] .…”

Tissue Contraction Force Microscopy for Optimization of Engineered Cardiac Tissue

“…[12][13][14] However, these assays do not capture the complex three-dimensional environment that cells experience in vivo or in an engineered tissue construct. Not only do extracellular matrix (ECM) properties regulate cellular function, 15 cell-cell interactions are important determinants of tissue properties as well.…”