Ischemic cardiac injury can be substantially alleviated by exposing the heart to pharmacological agents such as volatile anesthetics before occurrence of ischemia-reperfusion. A hallmark of this preconditioning phenomenon is its memory, when cardioprotective effects persist even after removal of preconditioning stimulus. Since numerous studies pinpoint mitochondria as crucial players in protective pathways of preconditioning, the aim of this study was to investigate the effects of preconditioning agent isoflurane on the mitochondrial bioenergetic phenotype. Endogenous flavoprotein fluorescence, an indicator of mitochondrial redox state, was elevated to 195 +/- 16% of baseline upon isoflurane application in intact cardiomyocytes, indicating more oxidized state of mitochondria. Isoflurane treatment also elicited partial dissipation of mitochondrial transmembrane potential, which remained depolarized even after anesthetic withdrawal (tetramethylrhodamine fluorescence intensity declined to 83 +/- 3 and 81 +/- 7% of baseline during isoflurane exposure and washout, respectively). Mild uncoupling, with preserved ATP synthesis, was also detected in mitochondria that were isolated from animals that had been previously preconditioned by isoflurane in vivo, revealing its memory nature. These mitochondria, after exposure to hypoxia and reoxygenation, exhibited better preserved respiration and ATP synthesis compared with mitochondria from nonpreconditioned animals. Partial mitochondrial depolarization was paralleled by a diminished Ca(2+) uptake into isoflurane-treated mitochondria, as indicated by the reduced increment in rhod-2 fluorescence when mitochondria were challenged with increased Ca(2+) (180 +/- 24 vs. 258 +/- 14% for the control). In conclusion, isoflurane preconditioning elicits partial mitochondrial uncoupling and reduces mitochondrial Ca(2+) uptake. These effects are likely to reduce the extent of the mitochondrial damage after the hypoxic stress.

We examined the cardioprotective profile of the new A 3 adenosine receptor (AR) agonist 903 [N 6 -(2,5-dichlorobenzyl)-3Ј-aminoadenosine-5Ј-N-methylcarboxamide] in an in vivo mouse model of infarction and an isolated heart model of global ischemia/reperfusion injury. In radioligand binding and cAMP accumulation assays using human embryonic kidney 293 cells expressing recombinant mouse ARs, CP-532,903 was found to bind with high affinity to mouse A 3 ARs (K i ϭ 9.0 Ϯ 2.5 nM) and with high selectivity versus mouse A 1 AR (100-fold) and A 2A ARs (1000-fold). In in vivo ischemia/reperfusion experiments, pretreating mice with 30 or 100 g/kg CP-532,903 reduced infarct size from 59.2 Ϯ 2.1% of the risk region in vehicle-treated mice to 42.5 Ϯ 2.3 and 39.0 Ϯ 2.9%, respectively. Likewise, treating isolated mouse hearts with CP-532,903 (10, 30, or 100 nM) concentration dependently improved recovery of contractile function after 20 min of global ischemia and 45 min of reperfusion, including developed pressure and maximal rate of contraction/relaxation. In both models of ischemia/reperfusion injury, CP-532,903 provided no benefit in studies using mice with genetic disruption of the A 3 AR gene, A 3 knockout (KO) mice. In isolated heart studies, protection provided by CP-532,903 and ischemic preconditioning induced by three brief ischemia/ reperfusion cycles were lost in Kir6.2 KO mice lacking expression of the pore-forming subunit of the sarcolemmal ATPsensitive potassium (K ATP ) channel. Whole-cell patch-clamp recordings provided evidence that the A 3 AR is functionally coupled to the sarcolemmal K ATP channel in murine cardiomyocytes. We conclude that CP-532,903 is a highly selective agonist of the mouse A 3 AR that protects against ischemia/reperfusion injury by activating sarcolemmal K ATP channels.A 3 adenosine receptor (AR) agonists have been shown to effectively limit infarct size and reduce contractile dysfunction in several different animal models of ischemia/reperfusion injury (Auchampach et al., 1997b(Auchampach et al., , 2003Tracey et al., 1997Tracey et al., , 1998Tracey et al., , 2003Jordan et al., 1999;Thourani et al., 1999;Ge et al., 2004Ge et al., , 2006. A 3 AR agonists are attractive as cardioprotective agents because they do not alter systemic hemodynamic parameters in nonrodent species and are effective if administered before the ischemic event or only during reperfusion (Auchampach et al

Background Cardioprotection by volatile anesthetic-induced preconditioning (APC) involves activation of protein kinase C (PKC). The current study investigated the importance of APC-activated PKC in delaying mitochondrial permeability transition pore (mPTP) opening. Methods Rat ventricular myocytes were exposed to isoflurane in the presence or absence of nonselective PKC inhibitor chelerythrine or isoform-specific inhibitors of PKC-δ (rottlerin) and PKC-ε (myristoylated PKC-ε V1-2 peptide), and the mPTP opening time was measured using confocal microscopy. Ca2+-induced mPTP opening was measured in mitochondria isolated from rats exposed to isoflurane in the presence and absence of chelerythrine, or in mitochondria directly treated with isoflurane after isolation. Translocation of PKC-ε was assessed in APC and control cardiomyocytes by Western blotting. Results In cardiomyocytes, APC prolonged time necessary to induce mPTP opening (261±26 s APC vs. 216±27 s control; P<0.05), while chelerythrine abolished this delay to 213±22 s. The effect of isoflurane was also abolished when PKC-ε inhibitor was applied (210±22 s), but not in the presence of PKC-δ inhibitor (269±31 s). Western blotting revealed translocation of PKC-ε toward mitochondria in APC cells. The Ca2+ concentration required for mPTP opening was significantly higher in mitochondria from APC rats (45±8 μM mg-1 control vs. 64±8 μM mg-1 APC), and APC effect was reversed with chelerythrine. In contrast, isoflurane did not protect directly treated mitochondria. Conclusion APC induces delay of mPTP opening through PKC-ε-mediated inhibition of mPTP opening, but not through PKC-δ. These results point to the connection between cytosolic and mitochondrial components of cardioprotection by isoflurane.

Photobiomodulation with near infrared light (NIR) provides cellular protection in various disease models. Previously, infrared light emitted by a low-energy laser has been shown to significantly improve recovery from ischemic injury of the canine heart. The goal of this investigation was to test the hypothesis that NIR (670 nm) from light emitting diodes produces cellular protection against hypoxia and reoxygenation-induced cardiomyocyte injury. Additionally, nitric oxide (NO) was investigated as a potential cellular mediator of NIR. Our results demonstrate that exposure to NIR at the time of reoxygenation protects neonatal rat cardiomyocytes and HL-1 cells from injury, as assessed by lactate dehydrogenase release and MTT assay. Similarly, indices of apoptosis, including caspase 3 activity, annexin binding and the release of cytochrome c from mitochondria into the cytosol, were decreased after NIR treatment. NIR increased NO in cardiomyocytes, and the protective effect of NIR was completely reversed by the NO scavengers carboxy-PTIO and oxyhemoglobin, but only partially blocked by the NO synthase (NOS) inhibitor L-NMMA. Mitochondrial metabolism, measured by ATP synthase activity, was increased by NIR, and NO-induced inhibition of oxygen consumption with substrates for complex I or complex IV was reversed by exposure to NIR. Taken together these data provide evidence for protection against hypoxia and reoxygenation injury in cardiomyocytes by NIR in a manner that is dependent upon NO derived from NOS and non-NOS sources.