During neonatal development, there is an increase in myocardial stiffness that coincides with an increase in the contractility of the heart. In vitro assays have shown that substrate stiffness plays a role in regulating the twitch forces produced by immature cardiomyocytes. However, its effect on twitch power is unclear due to difficulties in measuring the twitch velocity of cardiomyocytes. Here, we introduce what we consider a novel approach to quantify twitch power by combining the temporal resolution of optical line scanning with the subcellular force resolution of micropost arrays. Using this approach, twitch power was found to be greater for cells cultured on stiffer posts, despite having lower twitch velocities. The increased power was attributed in part to improved myofibril structure (increased sarcomere length and Z-band width) and intracellular calcium levels. Immunofluorescent staining of α-actin revealed that cardiomyocytes had greater sarcomere length and Z-band width when cultured on stiffer arrays. Moreover, the concentration of intracellular calcium at rest and its rise with each twitch contraction was greater for cells on the stiffer posts. Altogether, these findings indicate that cardiomyocytes respond to substrate stiffness with biomechanical and biochemical changes that lead to an increase in cardiac contractility.
As cardiomyocytes mature, their sarcomeres and Z-band widths increase in length in order for their myofibrils to produce stronger twitch forces during a contraction. In this study, we tested the hypothesis that tensional homeostasis is affected by altering myofibril forces. To assess this hypothesis, neonatal rat cardiomyocytes were cultured on arrays of microposts to measure cellular contractility. An optical line scanning technique was used to measure the deflections in the microposts with high temporal resolution, enabling the analysis of twitch force, twitch velocity, and twitch power. Myofibril force production was elevated by vector-mediated overexpression of ribonucleotide reductase (RR) to increase cellular dATP content or by adding the inotropic agent EMD 57033 (EMD). We found that RR and EMD treatment did not affect cardiomyocyte twitch force, but it did lead to reduced twitch velocity and twitch power. Immunofluorescent analysis of α-actinin revealed that RR-over-expressing cardiomyocytes and EMD-treated cardiomyocytes had lower spread area, sarcomere length, and Z-band width as compared to control cells. These results indicate a correlation between myofibril structure and cardiac power. This correlation was confirmed by exposing the cells to the myosin II inhibitor blebbistatin, and then subsequently washing it out. After wash-out, cardiomyocytes exhibited a reduction in twitch force, velocity, and power due to shorter sarcomere length and Z-band widths. Our results suggest that cardiac myofibril structure is regulated by tensional homeostasis. If myofibril-generated forces in cardiomyocytes are elevated, a state of tensional homeostasis is maintained by producing sufficient twitch forces with a lower degree myofibril structure.
Heart failure was induced in adult male ferrets by ascending aortic coarctation. All procedures accorded with The United Kingdom Animals (Scientific Procedures) Act, 1986. Protein extracts from sham and failing hearts were separated by 2% SDS PAGE. The mean ratio of the major titin isoforms N2BA:N2B increased from 0.3 in control to 0.5 in failing hearts (n=6, p<0.01). Titin molecules were isolated from left ventricle, aligned and stretched by a receding liquid meniscus (which applied a tensile force of ~60pN) 3 and visualised by atomic force microscopy. Combed titin molecules exhibited a straightened and beaded appearance. The mean molecular diameter of titin decreased in failing hearts compared to control (0.2650.001nm vs 0.3350.001nm, p<0.001, n=104-130 molecules, 3 animals per group). This difference was more pronounced in the shorter molecules (<3.5 mm). The mean distance between beads was increased in failing hearts (49.351.5nm vs 126.854.5nm, p<0.001, n=370-429, 3 animals per group). The decreased titin molecular diameter combined with an increased inter-bead distance suggests that titin from failing hearts is less resistant to tensile forces when compared to control, and may help to explain the decreased titin-based passive tension observed in diseased hearts.
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