Background and Purpose: In cardiac myocytes, cyclic AMP (cAMP) produced by both β 1-and β 2-adrenoceptors increases L-type Ca 2+ channel activity and myocyte contraction. However, only cAMP produced by β 1-adrenoceptors enhances myocyte relaxation through phospholamban-dependent regulation of the sarco/endoplasmic reticulum Ca 2+ ATPase 2 (SERCA2). Here we have tested the hypothesis that stimulation of β 2-adrenoceptors produces a cAMP signal that is unable to reach SERCA2 and determine what role, if any, phosphodiesterase (PDE) activity plays in this compartmentation. Experimental Approach: The cAMP responses produced by β 1-and β 2-adrenoceptor stimulation were studied in adult rat ventricular myocytes using two different fluorescence resonance energy transfer (FRET)-based biosensors, the Epac2-camps, which is expressed uniformly throughout the cytoplasm of the entire cell and the Epac2-αKAP, which is targeted to the SERCA2 signalling complex.

During the cardiac thin filament activation process, Tropomyosin (Tm) oscillates over the surface of actin filament in the azimuthal direction to regulate the access of Myosin heads to actin binding sites. These dynamical modes of oscillation and the resultant cooperative activation effects depend critically on the stiffness characteristics and the mobility of Tm molecules. In this study, we developed a stochastic coarse-grained computational model to describe the Tm motions over the surface of actin filament using Langevin-Brownian dynamics. The model represents the structural arrangement of Tm molecules as a flexible chain with variable torsional stiffness using a crystal elastic network approach. The model accounts for the spatial interactions among nearest-neighbor regulatory units (RUs), which are thought to appear from the structural coupling of adjacent Tropomyosins. This elastic coupling between RUs is accomplished by assigning a multiwell potential energy for each RU. The model is then used to study the effects of Tm torsional stiffness variations on the cooperative activation of the thin filament. The results suggest that small perturbations in Tm torsional stiffness can lead to a significant effect on force-Ca 2þ sensitivity, the rate of tension redevelopment, relaxation rate, and other contraction characteristics. The present stochastic computational model draws for the first time a more detailed molecular connection between Tm torsional stiffness, Tm modes of oscillations over actin surface, cooperativity among RUs, and dynamic muscle twitches. Thus, this coarse-graining approach may be useful in explaining many cardiomyopathies induced by structural remodeling and stiffening in Tm molecules as a result of point mutations in the human gene TPM1. Familial cardiomyopathies are the leading cause of sudden cardiac death among young people, and pediatric-onset disease is particularly devastating. Dilated cardiomyopathy (DCM) is a familial cardiomyopathy characterized by dilation of the left ventricular chamber of the heart. DCM can be caused by mutations in proteins involved in cardiac muscle contraction, including troponin T; however, it is not well understood how these mutations affect cardiac contraction and contribute to the disease phenotype. To address this critical gap in our knowledge, we examined the molecular-level impact of a troponin T mutation implicated in pediatric-onset DCM, R134G. In vitro motility assays, in which troponin/tropomyosin-regulated thin filaments are propelled across a myosin-coated surface, were carried out over a range of physiologically relevant calcium concentrations. These measurements revealed decreased calcium sensitivity in the troponin complex containing the R134G mutant. To elucidate the molecular mechanism underlying the altered calcium sensitivity conferred by the R134G mutation, we used stopped-flow and steady-state fluorescence measurements to determine the equilibrium constants that define binding of myosin to regulated thin filaments. By inputting these equil...

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