Mitochondrial production of reactive oxygen species contributes to the β-adrenergic stimulation of mouse cardiomycytes

Non-technical summary When under stress, the heart beat becomes stronger, in part due to enhanced fluxes of Ca2+ at the level of the cardiac cell. It is known that this effect is mediated by activation of beta-receptors on the cardiac cell surface. This leads to modifications of intracellular proteins that in turn increase the flux of Ca2+ within the cell. In this study we show that activation of beta-receptors increases the production of reactive oxygen species (ROS) in the heart cell. These ROS generate enhanced Ca2+ fluxes and more vigorous contraction. This finding shows a new cellular signalling route for regulating the power of the heart beat and might contribute to our understanding of diseases with defective cardiac contraction, such as heart failure.The sympathetic adrenergic system plays a central role in stress signalling and stress is often associated with increased production of reactive oxygen species (ROS). Furthermore, the sympathetic adrenergic system is intimately involved in the regulation of cardiomyocyte Ca2+ handling and contractility. In this study we hypothesize that endogenously produced ROS contribute to the inotropic mechanism of beta-adrenergic stimulation in mouse cardiomyocytes. Cytoplasmic Ca2+ transients, cell shortening and ROS production were measured in freshly isolated cardiomyocytes using confocal microscopy and fluorescent indicators. As a marker of oxidative stress, malondialdehyde (MDA) modification of proteins was detected with Western blotting. Isoproterenol (ISO), a beta-adrenergic agonist, increased mitochondrial ROS production in cardiomyocytes in a concentration- and cAMP-protein kinase A-dependent but Ca2+-independent manner. Hearts perfused with ISO showed a twofold increase in MDA protein adducts relative to control. ISO increased Ca2+ transient amplitude, contraction and L-type Ca2+ current densities (measured with whole-cell patch-clamp) in cardiomyocytes and these increases were diminished by application of the general antioxidant N-acetylcysteine (NAC) or the mitochondria-targeted antioxidant SS31. In conclusion, increased mitochondrial ROS production plays an integral role in the acute inotropic response of cardiomyocytes to beta-adrenergic stimulation. On the other hand, chronically sustained adrenergic stress is associated with the development of heart failure and cardiac arrhythmias and prolonged increases in ROS may contribute to these defects.