The Role of Myofilament Modifications in Regulation of Cardiac Function under Acute and Chronic Stress

The central theme of this thesis is to examine the role of myofilament modifications in the regulation of cardiac function under acute and chronic stress. Specifically, our data address the hypothesis that acute ceramide accumulation associated with altered lipid uptake can directly regulate cardiomyocyte contractile function through post-translational modifications of the sarcomeric proteins and that persistent modifications associated with hypertrophic cardiomyopathy (HCM) can trigger maladaptive remodeling leading to cardiac disease. Metabolic perturbations associated with obesity and type 2 diabetes are emerging as a major contributor to heart disease, termed cardiac lipotoxicity, particularly in industrialized countries. Increases in circulating lipids promote greater uptake of fatty acids in excess of mitochondrial fatty acid oxidation. As a result, fatty acids are shuttled to non-oxidative pathways resulting in the accumulation of triglycerides and ceramide. These toxic lipid intermediates have been associated with reduced contractile function and disease pathogenesis, yet a direct link is lacking. The first part of this thesis tests the hypothesis that increased ceramide can directly alter ventricular cardiomyocyte contractility through transient modifications of the myofilament proteins. Our studies demonstrate that ceramide treatment to isolated rat ventricular cardiomyocytes leads to a depression in both the peak and the rate of cell shortening without altering intracellular Ca2+ transients. These functional effects were associated with PKCε-dependent phosphorylation of the myofilament proteins troponin I, troponin T and myosin binding protein-C. Our data provide the first evidence that ceramide can directly depress contractility and further suggest that ceramide be considered as a significant contributor to the contractile dysfunction ascribed to cardiac lipotoxicity. The second part of this thesis sought to test the hypothesis that chronic increases in myofilament Ca2+ sensitivity due to the HCM-linked missense mutation in tropomyosin at position 70 (Tm70) is causal of arrhythmias and increased risk for sudden cardiac death apart from the development of hypertrophy and fibrosis. HCM is a genetically-linked form of cardiomyopathy caused primarily by mutations in sarcomeric proteins. It remains today the leading cause of sudden cardiac death in young individuals, yet the clinical phenotype is heterogeneous making understanding the pathogenesis of disease development difficult. Our findings from Tm70 transgenic mice not only recapitulates the human disease phenotype but further demonstrate that persistence of a single charge change within the tropomyosin coiled-coil structure is sufficient to alter the inherent myofilament response to Ca2+ leading to age-dependent alterations in diastolic dysfunction, intracellular Ca2+ homeostasis and increased susceptibility to arrhythmia. Moreover data show a gender-dependent difference in disease onset and severity. Collectively, this thesis provides new insight into the reciprocity between the sarcomere and the intracellular environment. It demonstrates how acutely, metabolic alterations can directly regulate contractile function through post-translational modifications of the sarcomeric proteins and how long-term charge modifications within the sarcomere can provoke maladaptive remodeling leading to cardiac disease and increased risk for sudden cardiac death.