Cardiac Troponin C E135A Variant Impairs Myofilament Response to PKA Phosphorylation and Is Associated With Autosomal Dominant Dilated Cardiomyopathy With Diastolic Dysfunction.

Abstract

Background: Dilated cardiomyopathy (DCM) is a heart muscle disease in which the left ventricle is enlarged, resulting in systolic dysfunction. Pathogenic variants in genes encoding proteins involved in cardiac contractility, cytoskeleton structure, and Ca²⁺ handling have been associated with DCM. TNNC1 (cTnC [cardiac troponin C]) variants are implicated in DCM, hypertrophic, and restrictive cardiomyopathies. Unlike other sarcomere genes, most reports of TNNC1 variants lack segregation or pedigree data, partly because the majority of the variants described, to date, have been reported as de novo. Therefore, a critical need is warranted to further understand the mechanisms by which TNNC1 variants could impact myofilament function, especially in response to PKA (protein kinase A)-mediated phosphorylation as this posttranslational modification modulates sarcomere function in response to β-adrenergic stimulation.

Methods: Probands with the novel TNNC1-c.404A>C variant (cTnC-E135A) and family members were identified and consented. cTnC-depleted donor human cardiac muscle preparations were reconstituted with recombinant exogenous human cTnC-E135A. Steady-state isometric force and crossbridge kinetics were measured before and after PKA incubation. We used in silico modeling to further investigate crossbridge cycling kinetics.

Results: We identified a multigenerational family carrying the TNNC1-c.404A>C variant with autosomal dominant DCM with both systolic and diastolic dysfunctions. Using reconstituted human cardiac muscle preparations, we showed that the cTnC-E135A abolishes the myofilament response to PKA-mediated phosphorylation. Furthermore, in silico mathematical modeling showed that this variant affects crossbridge kinetics by decreasing both Ca²⁺ k_OFF-rate constant and myosin detachment rate, which could result in increased ventricular stiffness and reduced ejection fraction.

Conclusions: Our clinical and genetics data, combined with the in silico modeling and functional assays, suggest that cTnC-E135A is associated with DCM and disrupts kinetics of Ca²⁺ and crossbridge cycling by abolishing the myofilament response to PKA phosphorylation.