Reactive oxygen species (ROS) and/or Ca 2؉ overload can trigger depolarization of mitochondrial inner membrane potential (⌬⌿ m ) and cell injury. Little is known about how loss of ⌬⌿ m in a small number of mitochondria might influence the overall function of the cell. Here we employ the narrow focal excitation volume of the two-photon microscope to examine the effect of local mitochondrial depolarization in guinea pig ventricular myocytes. Remarkably, a single local laser flash triggered synchronized and self-sustained oscillations in ⌬⌿ m , NADH, and ROS after a delay of ϳ40s, in more than 70% of the mitochondrial population. Oscillations were initiated only after a specific threshold level of mitochondrially produced ROS was exceeded, and did not involve the classical permeability transition pore or intracellular Ca 2؉ overload. The synchronized transitions were abolished by several respiratory inhibitors or a superoxide dismutase mimetic. Anion channel inhibitors potentiated matrix ROS accumulation in the flashed region, but blocked propagation to the rest of the myocyte, suggesting that an inner membrane, superoxide-permeable, anion channel opens in response to free radicals. The transitions in mitochondrial energetics were tightly coupled to activation of sarcolemmal K ATP currents, causing oscillations in action potential duration, and thus might contribute to catastrophic arrhythmias during ischemia-reperfusion injury.Mitochondria play a multifunctional role as key arbiters of cell life and death. In addition to oxidative phosphorylation, mitochondria are involved in thermogenesis, free radical production, and intracellular Ca 2ϩ homeostasis. Impairment of mitochondrial function during and after ischemia causes rapid energy depletion, contractile failure, and loss of cellular integrity, which may lead to necrotic or apoptotic cell death (1-3). ROS 1 have been implicated in ischemic dysfunction; however, they play a dual role as determinants of cell survival, on the one hand contributing to Ca 2ϩ overload and the induction of a mitochondrial permeability transition, but on the other acting as second messengers that protect cells against injury (4 -6). Mitochondria are a major site of physiological ROS production in the cardiomyocyte, with ϳ1-5% of the electrons flowing through the electron transport chain leaking into the production of ROS (7, 8). The negative effects of ROS on metabolism are evident in several studies showing rapid and spatiotemporally heterogeneous discharge of ⌬⌿ m in response to oxidative stress (1, 2) and/or Ca 2ϩ overload (9), including protocols employing laser-induced photooxidation (10, 11) and mitochondrial ROS-induced ROS release (12). In light of our previous work showing that substrate deprivation can initiate synchronized oscillations of mitochondrial redox and membrane potential (13), and that a diffusible cytoplasmic messenger may be involved (14), the present study tests whether similar global self-organizing behavior can be triggered by a highly localized perturbation of ...

Subcellular localization of nitric oxide (NO) synthases with effector molecules is an important regulatory mechanism for NO signalling. In the heart, NO inhibits L-type Ca2+ channels but stimulates sarcoplasmic reticulum (SR) Ca2+ release, leading to variable effects on myocardial contractility. Here we show that spatial confinement of specific NO synthase isoforms regulates this process. Endothelial NO synthase (NOS3) localizes to caveolae, where compartmentalization with beta-adrenergic receptors and L-type Ca2+ channels allows NO to inhibit beta-adrenergic-induced inotropy. Neuronal NO synthase (NOS1), however, is targeted to cardiac SR. NO stimulation of SR Ca2+ release via the ryanodine receptor (RyR) in vitro, suggests that NOS1 has an opposite, facilitative effect on contractility. We demonstrate that NOS1-deficient mice have suppressed inotropic response, whereas NOS3-deficient mice have enhanced contractility, owing to corresponding changes in SR Ca2+ release. Both NOS1-/- and NOS3-/- mice develop age-related hypertrophy, although only NOS3-/- mice are hypertensive. NOS1/3-/- double knockout mice have suppressed beta-adrenergic responses and an additive phenotype of marked ventricular remodelling. Thus, NOS1 and NOS3 mediate independent, and in some cases opposite, effects on cardiac structure and function.

Summary The origins and developmental mechanisms of coronary arteries are incompletely understood. We showed here by fate mapping, clonal analysis and immunohistochemistry that endocardial cells generate the endothelium of coronary arteries. Dye tracking, live imaging, and tissue transplantation also revealed that ventricular endocardial cells are not terminally differentiated; instead, they are angiogenic and form coronary endothelial networks. Myocardial Vegf-a or endocardial Vegfr-2 deletion inhibited coronary angiogenesis and arterial formation by ventricular endocardial cells. In contrast, lineage and knockout studies showed that endocardial cells make a small contribution to the coronary veins, the formation of which is independent of myocardial-to-endocardial Vegf signaling. Thus, contrary to the current view of a common source for the coronary vessels, our findings indicate that the coronary arteries and veins have distinct origins and are formed by different mechanisms. This information may help develop better cell therapies for coronary artery disease.

Ion channels on the mitochondrial inner membrane influence cell function in specific ways that can be detrimental or beneficial to cell survival. At least one type of potassium (K+) channel, the mitochondrial adenosine triphosphate-sensitive K+ channel (mitoKATP), is an important effector of protection against necrotic and apoptotic cell injury after ischemia. Here another channel with properties similar to the surface membrane calcium-activated K+ channel was found on the mitochondrial inner membrane (mitoKCa) of guinea pig ventricular cells. MitoKCa significantly contributed to mitochondrial K+ uptake of the myocyte, and an opener of mitoKCa protected hearts against infarction.