Abstract
The sarcomeric troponin-tropomyosin complex is a critical mediator of excitation-contraction coupling, sarcomeric stability and force generation. We previously reported that induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) from patients with a dilated cardiomyopathy (DCM) mutation, troponin T (TnT)-R173W, display sarcomere protein misalignment and impaired contractility. Yet it is not known how TnT mutation causes dysfunction of sarcomere microdomains and how these events contribute to misalignment of sarcomeric proteins in presence of DCM TnT-R173W. Using a human iPSC-CM model combined with CRISPR/Cas9-engineered isogenic controls, we uncovered that TnT-R173W destabilizes molecular interactions of troponin with tropomyosin, and limits binding of PKA to local sarcomere microdomains. This attenuates troponin phosphorylation and dysregulates local sarcomeric microdomains in DCM iPSC-CMs. Disrupted microdomain signaling impairs MYH7-mediated, AMPK-dependent sarcomere-cytoskeleton filament interactions and plasma membrane attachment. Small molecule-based activation of AMPK can restore TnT microdomain interactions, and partially recovers sarcomere protein misalignment as well as impaired contractility in DCM TnT-R173W iPSC-CMs. Our findings suggest a novel therapeutic direction targeting sarcomere- cytoskeleton interactions to induce sarcomere re-organization and contractile recovery in DCM.
Highlights
The sarcomeric troponin-tropomyosin complex is a critical mediator of excitation-contraction coupling, sarcomeric stability and force generation
We identified AMPK to assist in cytoskeleton filament interactions with both sarcomere- and plasma membrane junctions via myosin heavy chain 7 (MYH7) in dilated cardiomyopathy (DCM) iPSC-CMs
We showed that AMPK activation can in part overcome destabilized microdomain interactions, as well as sarcomere protein misalignment and impaired contractility in presence of the troponin T (TnT)-R173W mutation
Summary
The sarcomeric troponin-tropomyosin complex is a critical mediator of excitation-contraction coupling, sarcomeric stability and force generation. Using a human iPSC-CM model combined with CRISPR/Cas9-engineered isogenic controls, we uncovered that TnT-R173W destabilizes molecular interactions of troponin with tropomyosin, and limits binding of PKA to local sarcomere microdomains This attenuates troponin phosphorylation and dysregulates local sarcomeric microdomains in DCM iPSC-CMs. Disrupted microdomain signaling impairs MYH7mediated, AMPK-dependent sarcomere-cytoskeleton filament interactions and plasma membrane attachment. We identified AMPK to assist in cytoskeleton filament interactions with both sarcomere- and plasma membrane junctions via myosin heavy chain 7 (MYH7) in DCM iPSC-CMs. We showed that AMPK activation can in part overcome destabilized microdomain interactions, as well as sarcomere protein misalignment and impaired contractility in presence of the TnT-R173W mutation. We contribute to novel understanding of local signal regulation in sarcomeric microdomains as well as sarcomere interactions with other cytoskeleton filament proteins and plasma membrane compartments These findings may be exploited in the future for therapeutic manipulation of molecular disease mechanisms
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