Computational tool to study perturbations in muscle regulation and its application to heart disease

Abstract

25Striated muscle contraction occurs when myosin thick filaments bind to thin filaments in 26 the sarcomere and generate pulling forces. This process is regulated by calcium, and it 27 can be perturbed by pathological conditions (e.g., myopathies), physiological adaptations 28 (e.g., b-adrenergic stimulation), and pharmacological interventions. Therefore, it is 29 important to have a methodology to robustly determine the mechanism of these 30 perturbations and statistically evaluate their effects. Here, we present an approach to 31 measure the equilibrium constants that govern muscle activation, estimate uncertainty in 32 these parameters, and statistically test the effects of perturbations. We provide a 33 MATLAB-based computational tool for these analyses, along with easy-to-follow tutorials 34 that make this approach accessible. The hypothesis testing and error estimation 35 approaches described here are broadly applicable, and the provided tools work with other 36 types of data, including cellular measurements. To demonstrate the utility of the 37 approach, we apply it to determine the biophysical mechanism of a mutation that causes 38 familial hypertrophic cardiomyopathy. This approach is generally useful for studying the 39 mechanisms of muscle diseases and therapeutic interventions that target muscle 40 contraction. 41 42

Disrupted mechanobiology links the molecular and cellular phenotypes in familial dilated cardiomyopathy

“…Five technical replicates were used to define each curve. As described in SI Appendix, Materials and Methods, we modified the fitting and analysis procedure used by McKillop and Geeves to better define the values of the fitted variables (50). We performed an additional titration at an intermediate calcium concentration (pCa 6.25) and used an annealing algorithm to globally fit the binding curves of the fractional change in pyrene fluorescence ðaÞ as a function of myosin concentration for all 3 calcium concentrations ( Fig.…”

“…In vitro motility assays were conducted as previously described (36). The equilibrium constants that govern thin filament activation were determined using stopped-flow and steady-state fluorescence techniques (47,50). Full details are provided in SI Appendix, Materials and Methods.…”

Mechanical dysfunction induced by a hypertrophic cardiomyopathy mutation is the primary driver of cellular adaptation

“…To test whether the shift in calcium sensitivity can be explained by a change in the distribution of positions assumed by tropomyosin along the thin filament ( Fig. 6A), we measured the equilibrium constants that define the fraction of thin filament regulatory units in each state (21,33). The equilibrium constant between the blocked and closed states, KB, was determined by rapidly mixing fluorescently labeled regulated thin filaments together with myosin and then measuring the rate of myosin binding (seen as quenching of the fluorescence signal) in the presence and absence of calcium (see Materials and…”

Hypertrophic cardiomyopathy ß-cardiac myosin mutation (P710R) leads to hypercontractility by disrupting super-relaxed state

“…Finally, computational models can provide key insights into the interactions between related dynamic parameters and the resultant implications for the total production of force. Computational models incorpating different degrees of detail and different components of sarcomere structure have been used for decades to answer questions relating to fundamental muscle mechanics [25][26][27][28][29][30][31][32] and alterations to thin filament regulation in the context of cardiac disease and HCM [33,34]. A new model of myosin activity which incorporates an OFF state representative of the SRX state has recently been validated against experimental measurements of cardiac muscle [35].…”