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Published on: August 1, 2016
Cardiac Troponin C E135A Variant Impairs Myofilament Response to PKA Phosphorylation and Is Associated With Autosomal
Maicon Landim-Vieira1, Robin M Perelli2,3, Michelle Rodriguez-Garcia1
1Department of Biomedical Sciences, College of Medicine (M.L.-V., M.R.-G., R.C.C., J.H.L., S.P.C., J.R.P.), Duke University School of Medicine, Durham, NC.
Insights
A novel TNNC1 variant (cTnC-E135A) causes dilated cardiomyopathy by disrupting cardiac muscle function and response to PKA phosphorylation. This genetic defect impairs crossbridge cycling, leading to reduced ejection fraction and increased ventricular stiffness.
Area of Science:
- Cardiovascular Genetics
- Molecular Cardiology
- Biophysics
Background:
- Dilated cardiomyopathy (DCM) involves left ventricular enlargement and systolic dysfunction, often linked to genetic variants affecting cardiac contractility.
- TNNC1 (cardiac troponin C) variants are associated with cardiomyopathies, but their functional impact, particularly concerning PKA phosphorylation, remains unclear.
- Understanding TNNC1 variant mechanisms is crucial for elucidating DCM pathogenesis, especially how post-translational modifications like PKA phosphorylation influence sarcomere function.
Purpose of the Study:
- To investigate the functional consequences of a novel TNNC1 variant (c.404A>C, cTnC-E135A) associated with dilated cardiomyopathy.
- To determine how the cTnC-E135A variant affects myofilament function, specifically in response to protein kinase A (PKA)-mediated phosphorylation.
- To elucidate the impact of this variant on cardiac crossbridge kinetics and calcium handling.
Main Methods:
- Identified a multigenerational family with autosomal dominant DCM carrying the TNNC1-c.404A>C variant.
- Utilized reconstituted human cardiac muscle preparations with recombinant cTnC-E135A to measure isometric force and crossbridge kinetics.
- Employed in silico mathematical modeling to analyze crossbridge cycling kinetics before and after PKA incubation.
Main Results:
- The TNNC1-c.404A>C variant was identified in a family with autosomal dominant DCM exhibiting both systolic and diastolic dysfunction.
- Functional assays demonstrated that the cTnC-E135A variant abolishes the myofilament response to PKA-mediated phosphorylation.
- In silico modeling revealed that the variant decreases Ca2+ off-rate and myosin detachment, potentially increasing ventricular stiffness and reducing ejection fraction.
Conclusions:
- The cTnC-E135A variant is associated with DCM, disrupting cardiac function through altered Ca2+ and crossbridge kinetics.
- The variant abolishes the myofilament's response to PKA phosphorylation, highlighting a novel mechanism in DCM pathogenesis.
- Combined clinical, genetic, and functional data provide mechanistic insights into TNNC1-associated cardiomyopathies.
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+ kOFF-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.
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