Tropomyosin Phosphorylation and Oxidative Stress in Hypertrophic Cardiomyopathy

The work described within this dissertation investigates the general hypothesis that post-translational modifications of α-Tm elicit a progression of a pathophysiological phenotype through a mechanism involving altered redox signals. Hypertrophic cardiomyopathy (HCM) is a genetic disease of the sarcomere that ultimately results in left ventricular hypertrophy and diastolic dysfunction. This disease affects 1 in 500 and is the leading cause of sudden cardiac death in young, especially athletes. The exact mechanism by which these mutations elicit hypertrophy is unknown. In order to gain a better understanding of this disorder and how to devise effective treatments, a mouse model has been generated that mimics human HCM where cardiac specific α-tropomyosin (α-Tm) glutamic acid (E) at position 180 was exchanged with uncharged glycine (G) (α-cMyBP-C). This well characterized model displays severely enlarged left atria, fibrosis, myocyte disarray, hypertrophy, and diastolic dysfunction associated with an increased Ca2+-sensitivity of myofilaments. Human HCM hearts have been shown to exhibit increases in oxidation that inappropriately activate kinases and phosphatases. When we examined the HCM myofibrils, we detected an increase in oxidative markers that target myofilament proteins. Moreover, in the adult stages of this model, the 30-60% reduction in the phosphorylation status of α-Tm was not evident compared to non-transgenic (NTG) littermates. Collectively these data led us to hypothesize that decreasing phosphorylation of α-Tm at S283 would facilitate the rescue of the phenotype, including preventing the oxidative modifications associated with cMyBP-C. We further hypothesized that antioxidant treatment using N-acetylcysteine (NAC) would reverse the HCM phenotype. Experiments reported in chapter one uses a double mutant transgenic (DMTG) mouse model incorporating α-cMyBP-C with dephosphorylated α-Tm (S283A) to understand the role phosphorylation plays in disease progression. Dephosphorylation prevented the hypertrophic remodeling, decreased the Ca2+ sensitivity, improved relaxation, and reduced oxidative glutathionylation of myosin binding protein C (cMyBP-C) and carbonylation of myofibrillar proteins. These data suggest that altered charge status and post-translational modification of α-Tm causes a strain on the heart that elicits oxidative modifications. Chapter two examines the direct functional effect and specific sites of glutathionylation of myofilament proteins. Employing Western blotting and mass spectrometry, we induced glutathionylation in vitro by treatment of isolated myofibrils and detergent extracted fiber bundles (skinned fibers) with oxidized glutathione (GSSG). Results indicate that there are at least three cysteines on cMyBP-C that are susceptible to oxidative modification. Force generating skinned fiber bundles showed an increase in Ca2+-sensitivity when treated with oxidized glutathione, which was reversed with the reducing agent, dithiothreitol. These results indicated that antioxidants would be beneficial to reduce Ca2+-sensitivity in HCM. The object of experiments reported in chapter three aimed to determine whether antioxidant treatment could reverse HCM. In our approach, we treated NTG and cMyBP-C mice with N-Acetylcysteine (NAC) for one month. NAC is a precursor to the major intracellular antioxidant glutathione. Glutathione is found in every mammalian cell at a concentration between 1-10 mM. Oxidative stress has been demonstrated to decrease the cellular antioxidant capacity and alter the redox potential. We found that oral administration of NAC reverses redox mediated hypertrophic signaling, remodeling, and improves the relaxation kinetics by a mechanism that involves modifying key sarcomeric and Ca2+ handling proteins that mediate contraction and relaxation. Our findings provide a new appreciation of the role of tropomyosin phosphorylation in control of hypertrophic and oxidative signaling. Results of this thesis work show the complexity of the relation between myofilament modifications, oxidative stress, and the hypertrophic phenotype in HCM. HCM mutations inducing oxidative myofilament modifications could be resolved by treatment with antioxidants that relieve the constraint placed on the molecular motors that drive the cardiac cycle.