The role of the troponin T interactions with actin in regulation of cardiac thin filament revealed by the troponin T pathogenic variant Ile79Asn

Abstract

Cardiac muscle contraction/relaxation cycle depends on the rising and falling Ca²⁺ levels in sarcomeres that control the extent of interactions between myosin-based thick and actin-based thin filaments. Cardiac thin filament (cTF) consists of actin, tropomyosin (Tm) that regulates myosin binding to actin, and troponin complex that governs Tm position upon Ca²⁺-binding. Troponin has three subunits – Ca²⁺-binding troponin C (TnC), Tm stabilizing troponin T (TnT), and inhibitory troponin I (TnI). TnT N-terminus (TnT1) interactions with actin stabilize the inhibited state of cTF. TnC, TnI, and Tm work in concert to control actomyosin interactions. Cryo-electron microscopy (cryo-EM) provided factual structures of healthy cTF, but structures of cTF carrying missense mutations linked to human cardiomyopathy are unknown. Variant Ile79Asn in human cardiac TnT (TnT-I79N) increases myofilament Ca²⁺ sensitivity and slows cross-bridge kinetics, leading to severe hypertrophic/restrictive cardiomyopathy. Here, we used TnT-I79N mutation as a tool to examine the role of TnT1 in the complex mechanism of cTF regulation. Comparison of the cryo-EM structures of murine wild type and TnT-I79N native cTFs at systolic Ca²⁺ levels (pCa = 5.8) demonstrates that TnT-I79N causes 1) dissociation of the TnT1 loop from its actin interface that results in Tm release to a more activated position, 2) reduced interaction of TnI C-terminus with actin-Tm, and 3) increased frequency of Ca²⁺-bound regulatory units. Our data indicate that the TnT1 loop is a crucial element of the allosteric regulatory network that couples Tn subunits and Tm to maintain adequate cTF response to physiological Ca²⁺ levels during a heartbeat.