Troponin destabilization impairs sarcomere-cytoskeleton interactions in iPSC-derived cardiomyocytes from dilated cardiomyopathy patients

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 cellderived 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 MYH7mediated, 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.Sarcomeres are the basic contractile unit of cardiac cells, whose particularly specialized function depends on a highly organized structure. The troponin-tropomyosin (Tn-Tm) complex at sarcomeric thin filaments is a critical component for excitation-contraction coupling. Stability and anchoring of the Tn-Tm complex on sarcomeres is provided by binding of the troponin T (TnT) subunit to Tm and the troponin I subunit (TnI) on actin myofilaments. Tropomyosin (TPM) together with TnI regulates actin/myosin binding and ATPase function in presence of micromolar, cytocolic Ca 2+ , which is bound by the troponin C subunit (TnC). This highly sensitive mechanisms is fine-tuned by post-translational modifications, such as PKA-mediated phosphorylation of TnI. Mutations in the Tn-Tm complex lead to severe disease, such as dilated cardiomyopathy (DCM). DCM is featured by left ventricular dilatation, contractile dysfunction, and arrhythmias 1 and represents a frequent cause of heart failure. More than 25% of DCM cases are caused by inherited mutations, particularly in sarcomeric proteins 2 . Recently, human iPSC-derived cardiomyocytes (iPSC-CMs) have been utilized for human genetic disease modeling 3-6 and drug testing 7 . Here, we analyze a sarcomeric mutation in cardiac troponin T (TnT), TnT-R173W. This mutation is located within one of the two tropomyosin binding regions of TnT, the T1 domain 8 and was the first DCM mutation reported in a human patient-specific iPSC-derived cardiomyocyte model 9 . This report www.nature.com/scientificreports www.nature.com/scientificreports/ demonstrated DCM patient-specific iPSC-CMs to display mole...

Genetic Cardiomyopathies: The Lesson Learned from hiPSCs

“…TNNT2-R173W was proved to destabilize molecular interactions of troponin with tropomyosin, and to limit binding of PKA (protein kinase cAMP-dependent) to local sarcomere microdomains. Small molecule-based activation of AMPK can restore Troponin T microdomain interactions, and partially recover sarcomere protein misalignment as well as impaired contractility in DCM TNNT2-R173W hiPSC-CMs [60].…”

Patient-specific induced pluripotent stem cells as “disease-in-a-dish” models for inherited cardiomyopathies and channelopathies – 15 years of research

“…In-depth analysis uncovered the epigenetic activation of phosphodiesterase genes PDE2A and PDE3A as the underlying mechanism responsible for the defective βadrenergic signaling and contractile dysfunction[ 40 ]. The latest data indicates that TNNT2 -R173W hampers molecular interactions between troponin and tropomyosin and restricts the binding of PKA to local sarcomere microdomains, resulting in diminished troponin phospho-rylation and misalignment of sarcomeric proteins[ 41 ]. An R173W variant also altered the interaction between sarcomere microdomain and cytoskeleton filaments via MYH7 and AMPK, with consequent disturbance of sarcomere protein alignment and impaired contractility.…”

“…An R173W variant also altered the interaction between sarcomere microdomain and cytoskeleton filaments via MYH7 and AMPK, with consequent disturbance of sarcomere protein alignment and impaired contractility. In terms of phenotype rescue, AMPK activation by small molecules, such as A-769662, improved sarcomere-cytoskeleton attachment and partially recovered sarcomere protein misalignment and subsequent impaired contractility in mutated iPSC-CMs[ 41 ].…”

Modeling Cardiac Disease Mechanisms Using Induced Pluripotent Stem Cell-Derived Cardiomyocytes: Progress, Promises and Challenges

“…Additionally, diminished phosphorylation of troponin I (TnI) was detected as an effect of lower levels of PKA binding at sarcomeric microdomains in R173W mutated cardiomyocytes. R173W also accounts for an impaired interaction between sarcomere microdomain and cytoskeleton filaments via MYH7 and AMPK, leading to the disruption of sarcomere protein alignment and impaired contractility [ 197 ].…”