The Role of Cardiac Myosin Light Chain 2V Phosphorylation in the Healthy and Failing Myocardium

While a number of factors underlie cardiac dysfunction in HF, recent attention has fallen on phosphorylation changes in myofilament proteins, notably the regulatory proteins. Cardiac myosin regulatory light chain (cMLC2) is a 166 amino acid protein located on the hinge region of myosin heavy chain and optimally positioned to modulate contraction. Recent studies have shown that cMLC2v contributes to regulating cardiac pump function and allows for beat-to-beat tuning of the myosin motor arm. Humans, as well as large rodents such as rabbits, have high sequence homology and a single phosphorylation site at ser15. We hypothesize that human MLC2v phosphorylation is a critical regulator of myocyte contraction that is dysregulated in HF and that increasing MLC2v phosphorylation with myosin light chain kinase (MLCK) can partially reverse the dysfunction seen with HF. Human donor or HF permeabilized cardiac tissue were incubated with either alkaline phosphatase to reduce phosphorylation or PKA to induce maximal phosphorylation of the other myofilament proteins. Maximal cMLC2v phosphorylation was then achieved by incubation with MLCK. Force-calcium relationship, rate of tension redevelopment, tension cost and length dependent activation was determined. To assess the role of cMLC2v phosphorylation on dynamic regulation of length dependent activation, intact rabbit cardiomyocytes were isolated from rabbit hearts and transfected with MLCK to provide maximal cMLC2v phosphorylation. Cells were then attached using a force transducer and length controller and a simulated length-force relationship, which approximate the pressure-volume relationship in vivo, was evaluated at different diastolic sarcomere lengths. We show that MLC2v phosphorylation is a critical regulator of myocyte contraction that is dysregulated in the failing heart and treatment with MLCK can partially reverse the dysfunction seen with HF.