The Contribution of Chronic Protein Kinase C-Mediated Troponin I Phosphorylation to Cardiac Dysfunction

Abstract

Heart failure (HF), a leading cause of death in the United States, is characterized as dysfunction in the filling or pumping of blood from the heart. The myofilaments, a key component of the heart’s muscle machinery, are directly responsible for force production and contraction of cardiac muscle cells, that are essential for the heart’s pumping function. Myofilaments contain repeating units of sarcomeres, composed of highly organized arrangements of thin and thick filaments. In the absence of Ca2+, the thin filament molecular switch protein cardiac troponin I (cTnI), inhibits the interaction between thin and thick filaments. When Ca2+ levels increase in response to an action potential, cTnI toggles from actin towards troponin C (cTnC), the Ca2+-binding subunit of the heterotrimeric troponin (cTn) complex, allowing thick filament myosin to bind and slide the thin filament. The oscillatory function of cTnI can be modulated via phosphorylation by a number of kinases. This dissertation focuses on the impact of chronic protein kinase C (PKC) phosphorylation of cTnI at the S43/45 residues, to test whether it produces contractile dysfunction leading to HF. Multiple biochemical and in vitro studies indicate that S43/45 phosphorylation in cTnI increases during HF, and this phosphorylation diminishes contractile function. However, there is debate about whether S43/45 phosphorylation plays a similar role in vivo, primarily because of the lack of mouse models studying phosphorylation at only cTnI S43/45. One major goal of the presented work is to determine whether in vivo S43/45 phosphorylation causes cardiac dysfunction. The hypothesis tested is that acute S43/45 phosphorylation acts as a master brake on contractile function, while chronic S43/45 phosphorylation produces progressive cardiac dysfunction, and the absence of phosphorylation at this site does not change resting contractile function. As part of investigating the in vivo S43/45 phosphorylation role, we also sought to determine whether the amount of phosphorylation (i.e., phosphorylation “dose”) in this cluster had an impact on the in vivo response. To address this question, transgenic mice were generated to replace endogenous cTnI with varied levels of phospho-mimetic aspartic acid (D) at only S43/45 (cTnIS43/45D). In these mice, cTnIS43/45D causes dose-dependent, progressive cardiac dysfunction and remodeling, leading to HF and death. Additional work tests whether sarcomere-to-mitochondrial communication is an early contributor to the progressive phenotype. Specifically, studies test the idea that early accumulation of mitochondrial reactive oxygen species (ROS) disrupts mitochondrial function and lays the foundation for progressive HF. Then, studies test whether early mitigation of mitochondrial ROS improves contractile function. Parallel analyses using non-phosphorylatable substitutions at S43/45 bolsters the idea that chronic S43/45 phosphorylation is detrimental in vivo. Previously, traditional nonpolar alanine (S43/45A) substitutions at S43/45 reduced cardiomyocyte contractile function and myofilament Ca2+ sensitivity. These alterations indicated the A substitution is not functionally conservative at S43/45. Earlier studies also showed that novel non-phosphorylatable asparagine (N) used in place of A (S43/45N) does not alter in vitro contractile function. Thus, in this dissertation, in vivo cardiac function was analyzed by echocardiography in mice expressing cTnIS43/45N, with no change in cardiac morphology and function. Together, the studies presented here demonstrate chronic cTnI S43/45 phosphorylation causes HF via mitochondrial dysfunction, while the lack of phosphorylation does not impact cardiac function. These findings are relevant to a broad population of patients at-risk for HF, as many pathologies are associated with increased PKC activity and presumably, downstream cTnI phosphorylation.