Cardiovascular disease is the leading cause of death in developed countries. It is now clear that many cardiomyopathies leading to the progression of heart failure result from genetic mutations or alterations in the cytoskeletal network/basic structural components of the cardiac muscle. For the last five decades, the simplistic obligatory step of calcium binding to actin bound regulatory proteins (eg., troponin-tropomyosin complex) has contributed to the prevailing view that the control of calcium cycling and actin-bound regulatory proteins (thin filament proteins) dominates regulation of muscle contraction as well as underlie the dysregulation of contractile dynamics in heart failure. Several studies have shown that dysregulation of myosin regulatory proteins (thick filament proteins), such as myosin light chain-2 (MLC2), via dephosphorylation is associated with human cardiomyopathies and heart failure; however, mechanistic insights into its underlying role in cardiac muscle and heart disease remain poorly understood. Using integrative gene-targeted mouse and multi-scale computational models, we identify the mechanisms underlying an indispensable role for ventricular MLC2 (MLC2v) phosphorylation in regulating cardiac myosin cycling kinetics, which directly control actin-myosin interactions, while also surprisingly, feeding back to cooperatively influence calcium-dependent activation of the thin filament. Loss of these mechanisms uncovers previously unrecognized early cardiac muscle defects in the rates of twitch relaxation and ventricular torsion, which strikingly precede the left ventricular dysfunction of heart disease and failure in a novel non-phosphorylatable MLC2v mouse model. We show that in contrast to conventional views, there is a direct and early role for MLC2 phosphorylation in regulating actin-myosin interactions in striated muscle contraction and that dephosphorylation of MLC2 and loss of these mechanisms play a critical role in heart failure progression.