NO directly activates mitoK(ATP) channels and potentiates the ability of diazoxide to open these channels. These results provide novel mechanistic links between NO-induced cardioprotection and mitoK(ATP) channels.

Recent studies implicate increased cGMP synthesis as a postreceptor contributor to reduced cardiac sympathetic responsiveness. Here we provide the first evidence that modulation of this interaction by cGMP-specific phosphodiesterase PDE5A is also diminished in failing hearts, providing a novel mechanism for blunted beta-adrenergic signaling in this disorder. In normal conscious dogs chronically instrumented for left ventricular pressure-dimension analysis, PDE5A inhibition by EMD82639 had modest basal effects but markedly blunted dobutamine-enhanced systolic and diastolic function. In failing hearts (tachypacing model), however, EMD82639 had negligible effects on either basal or dobutamine-stimulated function. Whole myocardium from failing hearts had 50% lower PDE5A protein expression and 30% less total and EMD92639-inhibitable cGMP-PDE activity. Although corresponding myocyte protein and enzyme activity was similar among groups, the proportion of EMD82639-inhibitable activity was significantly lower in failure cells. Immunohistochemistry confirmed PDE5A expression in both the vasculature and myocytes of normal and failing hearts, but there was loss of z-band localization in failing myocytes that suggested altered intracellular localization. Thus, PDE5A regulation of cGMP in the heart can potently modulate beta-adrenergic stimulation, and alterations in enzyme localization and reduced synthesis may blunt this pathway in cardiac failure, contributing to dampening of the beta-adrenergic response.

Abstract-Mitochondria can either enhance or suppress cell death. Cytochrome c release from mitochondria and depolarization of the mitochondrial membrane potential (⌬⌿) are crucial events in triggering apoptosis. In contrast, activation of mitochondrial ATP-sensitive potassium (mitoK ATP ) channels prevents lethal ischemic injury in vivo, implicating these channels as key players in the process of ischemic preconditioning. We probed the relationship between mitoK ATP channels and apoptosis in cultured neonatal rat cardiac ventricular myocytes. Incubation with 200 mol/L hydrogen peroxide induced TUNEL positivity, cytochrome c translocation, caspase-3 activation, poly(ADPribose) polymerase cleavage, and dissipation of ⌬⌿. Key Words: apoptosis Ⅲ ischemia Ⅲ oxidative stress A cute coronary syndromes remain the leading causes of death in developed countries. The classical notion that interruption of blood flow kills cells solely by necrosis (catastrophic cell rupture) has been challenged by evidence that apoptosis contributes to ischemic injury in the heart. 1,2 Apoptosis is a genetically encoded, highly orchestrated mode of cell death in which mitochondria play a key role. 3,4 Release of the electron transport protein cytochrome c into the cytosol activates caspases, culminating in DNA fragmentation and cytolysis. 5 In contrast, another mitochondrial pathway promotes cell survival rather than cell death in "ischemic preconditioning." 6 This endogenous process, welldocumented to be operative in vivo in diverse species and tissues, refers to the paradoxical protection against lethal ischemia by brief episodes of prior "conditioning" ischemia. Similar cardioprotection can be recruited by drugs such as diazoxide that open mitochondrial ATP-sensitive potassium (mitoK ATP ) channels; conversely, mitoK ATP channel blockers (5-hydroxydecanoate [5-HD] or glibenclamide) prevent both preconditioning and pharmacological cardioprotection. [7][8][9] Thus, mitochondria are key determinants both of cell death and of cell survival.These observations prompted us to investigate whether the cytoprotective effect of mitoK ATP channel activation is related to inhibition of apoptosis. Oxidative stress induces cardiac myocyte apoptosis in vitro 10,11 and contributes to tissue injury in ischemic syndromes 12,13 and congestive heart failure. 14 To test our hypothesis, we have investigated apoptosis induced by oxidative stress in cardiac myocytes. Our findings reveal that activation of mitoK ATP channels suppresses programmed cell death. Materials and Methods Primary Culture of Neonatal Rat Cardiac Ventricular MyocytesCardiac ventricular myocytes were prepared from 1-to 2-day-old Sprague-Dawley rats and cultured as described. 15 In brief, the hearts were removed, and the ventricles were minced in calcium-and bicarbonate-free Hanks' buffer with HEPES. These tissue fragments were digested by stepwise trypsin dissociation. The dissociated cells were preplated for 1 hour to enrich the culture with myocytes. The nonadherent myocytes were then plate...

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