Stretch Synchronizes Sarcomere Shortening In Isolated Cardiomyocytes

According to the Frank-Starling mechanism, stretching cardiac muscle augments contractile force. However, it is unclear how this phenomenon occurs regionally within cardiomyocytes, at the level of individual sarcomeres and their local Ca2+ signals. To investigate this issue, we simultaneously monitored cytosolic Ca2+ and sarcomere striations in isolated rat cardiomyocytes by recording Fluo-4 fluorescence (515–560 nm) and brightfield signals respectively, using a high-speed sCMOS camera. Cells were affixed to glass rods (CellTester, World Precision Instruments) to allow control of cell length and measurement of force production during electrical stimulation at 1 Hz. At resting sarcomere length (SL), differential sarcomere deformation was observed despite uniform Ca2+ transients corresponding to individual sarcomeres. Specifically, while most sarcomeres contracted during the stimulus, a minority of sarcomeres (~13%) were observed to be stretched or were stationary. Sarcomeres which exhibited stretch during the Ca2+ transient tended to be shorter than those that contracted, and were significantly shorter than their neighbours. Step-wise stretch of the cell generated an expected increase in force development, but also revealed that more sarcomeres exhibited contraction, reaching nearly 100% of sarcomeres at 20% stretch. During cell lengthening, resting SL increased in all sarcomeres, suggesting that the shortest sarcomeres surpassed a critical length which enabled sufficient force generation and shortening. SL variability in unstretched cells was not caused by regional differences in Ca2+, as it remained apparent during permeabilization in Ca2+-free conditions. Rather, electron microscopy analysis traced differences in SL to the I-band, where dimensions are regulated by titin. We postulate, therefore, that slackened titin in unstretched cells allows greater variability in I-band length, cross-bridge formation, and active force generation between sarcomeres. When the cell is lengthened, sarcomere shortening is synchronized, which may contribute to augmented force production defined by the Frank-Starling mechanism.