Despite aggressive treatment, heart failure (HF) remains a major clinical problem with high mortality. HF is characterized by depressed cardiac pump function. Recent studies have shown that depressed contractile protein function underlies, in part, depressed pump function in HF; however, the molecular mechanisms are largely unknown. Studies proposed in project 3 emphasize ?Control of Sarcomere Dynamics?, with a strong focus on cardiac myosin light chain 2 (cMLC2) phosphorylation. Strong synergistic interactions of project 3 continue with project 1 (biased ligand signaling targeting sarcomeres) and project 2 (sarcomere growth and remodeling). In addition project 3 relies critically on the support from Core B (Human Tissue;
aims 1 and 2) and Core C (Proteomics and Analytical Biochemistry;
aim 3). The significance of cMLC2 phosphorylation was substantially enhanced by the recent identification of a cardiac specific isoform of myosin light chain kinase (cMLCK), which is upregulated in HF. However, both the extent and regional variation of cMLC2 phosphorylation in health and disease, as well as the functional impact of this post-translational modification on cardiac contractile biology has not been studied in detail. Accordingly, the overall goal of project 3 is to define the role of cMLC2 on human myofilament function in health and disease. We hypothesize that human cMLC2 is a critical regulator of myocyte contraction that is dysregulated in HF, and that increasing MLC2 phosphorylation will reverse myofilament dysfunction and improve cardiac pump function. To test this hypothesis, we have developed three independent specific aims.
In aim 1 we will determine the role of cMLC2 in skinned myocardium from healthy and failing human hearts. Biophysical experiments will focus on skinned human isolated myocardium where we will alter contractile protein phosphorylation status by treatment with kinases or phosphatase, or recombinant protein exchange.
In aim 2 we will determine the role of cMLC2 in healthy and diseased intact cells under simulated length-force relationships that approximate in-situ pressure- volume loops. Finally, in aim 3 we will determine the extent of cMLC2 biochemical alterations by using state of the art proteomic analysis. Specific attention will be on MLC2 phosphorylation on S15 as well as the extent of N14 deamidation, and the regional variation of this parameter. We expect to identify decreased cMLC2 phosphorylation as a significant mechanism underlying sarcomeric dysfunction in heart failure. Our results may provide a path to a novel therapeutic target in the treatment of heart failure, where restoration of cardiac myosin light chain 2 phosphorylation levels is expected to improve cardiac pump performance.
Project 3: Narrative Depressed cardiac pump function in heart failure (HF) is caused, in part, by depressed contractile protein function; the underlying mechanisms are incompletely understood. Recent evidence indicates a significant role for myosin light chain 2 (cMLC2) phosphorylation in HF. Studies proposed here will employ state of the art biophysical techniques to define human cMLC2 function in health and disease, as well as state of the art proteomic approaches to determine cMLC2 phosphorylation in human HF. Our studies may provide a path to novel therapeutic strategies to combat depressed cardiac pump function in human HF.
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