Micromechanical thermal assays of Ca 2+-regulated thin-filament function and modulation by hypertrophic cardiomyopathy mutants of human cardiac troponin

Abstract

Microfabricated thermoelectric controllers can be employed to investigate mechanisms underlying myosin-driven sliding of Ca 2+-regulated actin and disease-associated mutations in myofilament proteins. Specifically, we examined actin filament slidingwith or without human cardiac troponin (Tn) and -tropomyosin (Tm)propelled by rabbit skeletal heavy meromyosin, when temperature was varied continuously over a wide range (∼20-63°C ). At the upper end of this temperature range, reversible dysregulation of thin filaments occurred at pCa 9 and 5; actomyosin function was unaffected. Tn-Tm enhanced sliding speed at pCa 5 and increased a transition temperature (T t) between a high activation energy (E a) but low temperature regime and a low E a but high temperature regime. This was modulated by factors that alter cross-bridge number and kinetics. Three familial hypertrophic cardiomyopathy (FHC) mutations, cTnI R145G, cTnI K206Q, and cTnT R278C, cause dysregulation at temperatures ∼5-8°C lower; the latter two increased speed at pCa 5 at all temperatures. Copyright 2012 Nicolas M. Brunet et al.