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 sliding-with or without human cardiac troponin (Tn) and α-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.