Background-Renin released by ischemia/reperfusion from cardiac mast cells activates a local renin-angiotensin system (RAS). This exacerbates norepinephrine release and reperfusion arrhythmias (ventricular tachycardia and fibrillation), making RAS a new therapeutic target in myocardial ischemia. Methods and Results-We investigated whether ischemic preconditioning (IPC) prevents cardiac RAS activation in guinea pig hearts ex vivo. When ischemia/reperfusion (20 minutes of ischemia/30 minutes of reperfusion) was preceded by IPC (two 5-minute ischemia/reperfusion cycles), renin and norepinephrine release and ventricular tachycardia and fibrillation duration were markedly decreased, a cardioprotective anti-RAS effect. Activation and blockade of adenosine A 2b /A 3 receptors and activation and inhibition of protein kinase C⑀ (PKC⑀) mimicked and prevented, respectively, the anti-RAS effects of IPC. Moreover, activation of A 2b /A 3 receptors or activation of PKC⑀ prevented degranulation and renin release elicited by peroxide in cultured mast cells (HMC-1). Activation and inhibition of mitochondrial aldehyde dehydrogenase type-2 (ALDH2) also mimicked and prevented, respectively, the cardioprotective anti-RAS effects of IPC. Furthermore, ALDH2 activation inhibited degranulation and renin release by reactive aldehydes in HMC-1. Notably, PKC⑀ and ALDH2 were both activated by A 2b /A 3 receptor stimulation in HMC-1, and PKC⑀ inhibition prevented ALDH2 activation. Conclusions-The

Renin released by ischemia/reperfusion (I/R) from cardiac mast cells (MCs) activates a local renin-angiotensin system (RAS) causing arrhythmic dysfunction. Ischemic preconditioning (IPC) inhibits MC renin release and consequent activation of this local RAS. We postulated that MC histamine H 4 -receptors (H 4 Rs), being Ga i/o -coupled, might activate a protein kinase C isotype-« (PKC«)-aldehyde dehydrogenase type-2 (ALDH2) cascade, ultimately eliminating MC-degranulating and renin-releasing effects of aldehydes formed in I/R and associated arrhythmias. We tested this hypothesis in ex vivo hearts, human mastocytoma cells, and bone marrow-derived MCs from wild-type and H 4 R knockout mice. We found that activation of MC H 4 Rs mimics the cardioprotective anti-RAS effects of IPC and that protection depends on the sequential activation of PKC« and ALDH2 in MCs, reducing aldehyde-induced MC degranulation and renin release and alleviating reperfusion arrhythmias. These cardioprotective effects are mimicked by selective H 4 R agonists and disappear when H 4 Rs are pharmacologically blocked or genetically deleted. Our results uncover a novel cardioprotective pathway in I/R, whereby activation of H 4 Rs on the MC membrane, possibly by MC-derived histamine, leads sequentially to PKC« and ALDH2 activation, reduction of toxic aldehyde-induced MC renin release, prevention of RAS activation, reduction of norepinephrine release, and ultimately to alleviation of reperfusion arrhythmias. This newly discovered protective pathway suggests that MC H 4 Rs may represent a new pharmacologic and therapeutic target for the direct alleviation of RAS-induced cardiac dysfunctions, including ischemic heart disease and congestive heart failure.

During myocardial ischemia/reperfusion, lipid peroxidation leads to the formation of toxic aldehydes that contribute to ischemic dysfunction. Mitochondrial aldehyde dehydrogenase type 2 (ALDH2) alleviates ischemic heart damage and reperfusion arrhythmias via aldehyde detoxification. Because excessive norepinephrine release in the heart is a pivotal arrhythmogenic mechanism, we hypothesized that neuronal ALDH2 activation might diminish ischemic norepinephrine release. Incubation of cardiac sympathetic nerve endings with acetaldehyde, at concentrations achieved in myocardial ischemia, caused a concentration-dependent increase in norepinephrine release. A major increase in norepinephrine release also occurred when sympathetic nerve endings were incubated in hypoxic conditions. ALDH2 activation substantially reduced acetaldehyde-and hypoxia-induced norepinephrine release, an action prevented by inhibition of ALDH2 or protein kinase C (PKC). Selective activation of G i/o -coupled adenosine A 1 , A 3 , or histamine H 3 receptors markedly inhibited both acetaldehyde-and hypoxia-induced norepinephrine release. These effects were also abolished by PKC and/or ALDH2 inhibition. Moreover, A 1 -, A 3 -, or H 3 -receptor activation increased ALDH2 activity in a sympathetic neuron model (differentiated PC12 cells stably transfected with H 3 receptors). This action was prevented by the inhibition of PKC and ALDH2. Our findings suggest the existence in sympathetic neurons of a protective pathway initiated by A 1 -, A 3 -, and H 3 -receptor activation by adenosine and histamine released in close proximity of these terminals. This pathway comprises the sequential activation of PKC and ALDH2, culminating in aldehyde detoxification and inhibition of hypoxic norepinephrine release. Thus, pharmacological activation of PKC and ALDH2 in cardiac sympathetic nerves may have significant protective effects by alleviating norepinephrine-induced life-threatening arrhythmias that characterize myocardial ischemia/reperfusion.

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