Myopathy-linked mutations in TPM2 and their impact on troponin-mediated regulation of actomyosin contractility.

Abstract

In striated muscle, the regulatory complex of tropomyosin (Tpm) and troponin (Tn) governs the Ca²⁺-dependent interactions between myosin heads and actin, controlling muscle contraction. The N-terminal and central regions of Tpm are crucial for Tn binding, yet their roles in regulating contraction in concert with Tn remain poorly understood. To explore this, we selected four pathogenic missense mutations in the TPM2 gene encoding the skeletal Tpm2.2 isoform (β-tropomyosin): D20H and E181K, associated with hypercontractility, and E41K and N202K, linked to hypocontractility. Using in vitro functional assays, we characterized these Tpm2.2 variants to unravel details of the molecular mechanisms underlying disease phenotypes. At low, non-activating Ca²⁺, all Tpm2.2 variants inhibited the Tn-regulated steady-state actomyosin ATPase reaction without affecting Tn binding to actin. At activating Ca²⁺, hypocontractile mutants suppressed actomyosin ATPase activity and motor function more than wild-type, suggesting interference with normal Tn regulation. Conversely, hypercontractile mutants enhanced myosin-driven actin translocation. Arrhenius plots revealed a limited ability of E181K mutant to activate Tn-regulated actomyosin ATPase at temperatures below 30 °C, which was fully recovered, and even enhanced, at physiological temperatures. In the absence of actin, the N-terminal mutations enhanced high-affinity Tpm2.2-Tn interactions, whereas central mutations had no effect. Additionally, although hypercontractile mutations increased Ca²⁺-sensitivity of the cross-bridge cycle, hypocontractile mutants decreased it. These findings demonstrate that mutations in the N-terminal and central segments of Tpm2.2 disrupt Tn-dependent regulation of actomyosin contractility through distinct mechanisms, providing new insights into the pathophysiology of Tpm-related myopathies.