Cardiac Thin Filament Activation Modulation by Sarcomere Length

Myofilament Length Dependent Activation (LDA) forms the cellular basis of the Frank-Starling law observed on the heart. It has been previously shown in isolated myocardium that the responsiveness of cardiac myofilaments to activating calcium ions is increased with stretch. However, the molecular mechanism underlying LDA is still unknown. The aim of our study was to investigate whether LDA is modulated by increased calcium binding to the thin filaments, and whether or not thick filaments modulate this mechanism. Accordingly, experiments were performed under strict sarcomere length control on skinned guinea pig multicellular preparations isolated from the left ventricle. Myofilament calcium sensitivity was measured at both short (1.9 μm) and long (2.3 μm) sarcomere length. Simultaneously, calcium binding to cTnC was monitored using fluorescently labeled cTnC (cTnCT53C∗) with an environmentally sensitive probe (2-(4′-iodoadcetamidoanilo)-napthalene-6-sulfonic acid (IAANS)). The involvement of thick filaments in the regulation of calcium binding to cTnC was evaluated either in the presence of Blebbistatin to inhibit cross bridges formation or in rigor solution to inhibit cross-bridges detachment. Calcium dependent modulation of IAANS fluorescence showed calcium dependent decrease in IAANS fluorescence, indicating that the label was correctly incorporated into a functional TnC. The labeled TnC was then incorporated into recombinant Tn complex (Tn∗) and endogenous Tn was exchanged for Tn∗. Preliminary data show that Tn exchange did not alter the myofilament properties as demonstrated by preserved Tension-pCa curves at both short and long SL. Our data also show alteration of IAANS emitted fluorescence upon calcium activation in exchanged skinned muscles. Moreover, fluorescence was also altered by stretch in relaxed muscles. These data suggest that LDA may be due to altered thin filament activation in response to stretch secondary to a direct influence of sarcomere length on troponin structure.