Increased Myofilament Ca2+ Sensitivity Decreases Sarcomere Length and Increases Spark-Spark Interactions

People with familial hypertrophic cardiomyopathy (FHC) harboring mutations of cardiac troponin T (cTnT) are often at a high risk of sudden cardiac death. Transgenic mice harboring some of these cTnT mutations show increased myofilament sensitivity to Ca2+ and also shortened diastolic sarcomere length (SL). Our computational studies predicted that decreasing the distances between Ca2+ release units (CRUs) of the sarcoplasmic reticulum (SR) by decreasing SL can destabilize the Ca2+ control system and increase the probability of spontaneous Ca2+ waves. Destabilization results from enhanced crosstalk between neighboring CRUs. In this study we mimic the greater myofilament Ca2+ sensitivity conferred by cTnT mutations using the myofilament Ca2+ sensitizer EMD 57033 (EMD). At concentrations up to 3 μM, EMD had no effect on either the peak Ca2+ transient or the diastolic Ca2+ levels and did not alter the SR Ca2+ load. To test the prediction that SL shortening increases the coupling between CRUs, we loaded myocytes with Di8-ANEPPS and Fluo-4 and simultaneously measured SL and Ca2+ sparks in 2 spatial dimensions using the Zeiss 5 Live high-speed 2-D scanning confocal microscope. EMD (1.5 μM) decreased SL significantly compared to the control cells in normal Tyrode (1.58 μm vs.1.69 μm, p<0.05). The spark coupling strength measures the influence of one CRU on another and is derived from an analysis of the spatio-temporal distribution of Ca2+ sparks. EMD treatment significantly increased the spark coupling strength 2.5 fold. The enhanced spark-spark coupling as diastolic SL decreases may contribute to increased frequency of spontaneous Ca2+ waves during diastole that can lead to triggered arrhythmias and sudden cardiac death in FHC.