Although Ca2+ is the principal regulator of contraction in striated muscle, in vitro evidence suggests that some actin-myosin interaction is still possible even in its absence. Whether this Ca2+-independent activation (CIA) occurs under physiological conditions remains unclear, as does its potential impact on the function of intact cardiac muscle. The purpose of this study was to investigate CIA using computational analysis. We added a structurally motivated representation of this phenomenon to an existing myofilament model, which allowed predictions of CIA-dependent muscle behavior. We found that a certain amount of CIA was essential for the model to reproduce reported effects of nonfunctional troponin C on myofilament force generation. Consequently, those data enabled estimation of ΔGCIA, the energy barrier for activating a thin filament regulatory unit in the absence of Ca2+. Using this estimate of ΔGCIA as a point of reference (∼7 kJ mol(-1)), we examined its impact on various aspects of muscle function through additional simulations. CIA decreased the Hill coefficient of steady-state force while increasing myofilament Ca2+ sensitivity. At the same time, CIA had minimal effect on the rate of force redevelopment after slack/restretch. Simulations of twitch tension show that the presence of CIA increases peak tension while profoundly delaying relaxation. We tested the model's ability to represent perturbations to the Ca2+ regulatory mechanism by analyzing twitch records measured in transgenic mice expressing a cardiac troponin I mutation (R145G). The effects of the mutation on twitch dynamics were fully reproduced by a single parameter change, namely lowering ΔGCIA by 2.3 kJ mol(-1) relative to its wild-type value. Our analyses suggest that CIA is present in cardiac muscle under normal conditions and that its modulation by gene mutations or other factors can alter both systolic and diastolic function.

Mutations in MYBPC3, the gene encoding the muscle regulatory protein cardiac myosin binding protein-C (cMyBP-C), are among the most common causes of hypertrophic cardiomyopathy (HCM) in both people and cats. However, despite the high prevalence of mutations in MYBPC3, relatively little is understood regarding how mutations lead to disease. One possibility is that some point mutations alter cMyBP-C protein structure leading to enhanced degradation and elimination of the mutant protein. If levels of cMyBP-C protein expression are reduced, then haploinsufficiency (lack of sufficient protein) can trigger disease. Here we tested this idea by analyzing the impact of the A31P mutation, linked to HCM in Maine Coon cats, on 1) the in vitro protein structure of the C0 domain of cMyBP-C, and 2) the total protein expression of cMyBP-C in myocardium of aged cats heterozygous for the A31P mutation. In vitro results demonstrated that the A31P mutation disrupts folding of the C0 domain as shown by three independent methods: altered epitope recognition on Western blots; changes in sensitivity to proteolytic degradation; and reduced b-sheet content assessed by circular dichroism. Western blots of endogenous cMyBP-C obtained from myocardial samples also suggested that C0 structure is altered in vivo because an antibody that preferentially recognizes C0 reacted less with A31P cMyBP-C compared to wild-type cMyBP-C. However, despite these significant structural differences, the A31P cMyBP-C was incorporated into sarcomeres and total cMyBP-C protein (wild-type plus mutant) was similar in wild type and heterozygous A31P cats. These results suggest that despite protein folding abnormalities, the A31P mutation does not lead to haploinsufficiency in the population of older heterozygous cats studied here.