Mitochondrial dysfunction in cardiac ischemia-reperfusion injury: ROS from complex I, without inhibition.

A key pathologic event in cardiac ischemia reperfusion (I-R) injury is mitochondrial energetic dysfunction, and several studies have attributed this to complex I (CxI) inhibition. In isolated perfused rat hearts, following I-R, we found that CxI-linked respiration was inhibited, but isolated CxI enzymatic activity was not. Using the mitochondrial thiol probe iodobutyl-triphenylphosphonium in conjunction with proteomic tools, thiol modifications were identified in several subunits of the matrix-facing 1alpha sub-complex of CxI. These thiol modifications were accompanied by enhanced ROS generation from CxI, but not complex III. Implications for the pathology of cardiac I-R injury are discussed.

Multiple treatment approach to limit cardiac ischemia-reperfusion injury.

BACKGROUND: This study evaluates a multiple treatment approach (ie, pharmacologic preconditioning [diazoxide], sodium-proton exchange inhibition [cariporide], and controlled reperfusion) to improve the outcome from severe cardiac ischemia-reperfusion injury that occurs during a cardiac operation. METHODS: Five groups of 10 pigs (group 1: control, group 2: diazoxide, group 3: cariporide, group 4: controlled reperfusion, and group 5: combination of diazoxide and cariporide-controlled reperfusion) underwent 75 minutes of left anterior descending occlusion, 1 hour of cardioplegic arrest, and 2 hours of reperfusion. Prior to occlusion, each group received an infusion of vehicle alone (ie, dimethylsulfoxide for the control and the controlled reperfusion groups) or vehicle with drug (ie, diazoxide or cariporide, or both for all other groups). Infarct size (primary outcome) was measured and was normalized to the region at risk. Regional function (secondary outcome) was measured using preload recruitable work area. RESULTS: Infarct size as a function of area at risk was decreased by cariporide-controlled reperfusion, and combination treatment compared with the control group (14 +/- 6%, 15 +/- 8%, and 9 +/- 4% vs 24 +/- 9%; p < 0.02), and variation in infarct size was decreased by combination treatment compared with the controlled reperfusion group alone (p < 0.02). Recovery of systolic function during reperfusion significantly improved in the left anterior descending region in the cariporide and combination groups compared with the control, controlled reperfusion, or diazoxide groups (group-time effect, p < 0.05). CONCLUSIONS: Combined use of controlled reperfusion, cariporide, and diazoxide decreases myocyte necrosis and loss of systolic function compared with an untreated control group. Combination treatment has the potential to improve the results of cardiac surgery, however further improvements are needed before clinical application.

Evidence of cardiovascular protection by moderate alcohol: role of nitric oxide.

Epidemiological evidence indicates that moderate alcohol consumption reduces the incidence of heart disease. Endothelial nitric oxide synthase (eNOS) is a key regulator of vascular homeostasis and myocardial functions through the controlled production of nitric oxide (*NO). These studies were conducted to determine if the apparent alcohol-associated cardioprotection is mediated, in part, through modulation of the eNOS protein and activity in the cardiovascular system. Rats were fed alcohol and eNOS protein and *NO production were evaluated at the end of 8 weeks. Myocardial and vascular function was assessed ex vivo in a subset of animals. Moderate alcohol improved postischemic myocardial systolic and diastolic function and attenuated the postischemic reduction in coronary vascular resistance. Moderate alcohol also enhanced maximum vascular relaxation by 26 +/- 0.2% and increased plasma *NO production concomitant with a greater than 2.5-fold increase in eNOS protein. Higher levels of alcohol impaired maximum vascular relaxation by 22 +/- 0.1%. These results suggest that moderate alcohol improves postischemic myocardial functions and increases *NO production by vascular endothelium. An increase in *NO may explain, at least in part, the cardioprotective benefits of moderate alcohol consumption.

Early reperfusion levels of Na(+) and Ca(2+) are strongly associated with postischemic functional recovery but are disassociated from K(ATP) channel-induced cardioprotection.

We previously demonstrated that pinacidil does not affect Na(+)(i) accumulation, cellular energy depletion, or acidosis during myocardial ischemia, but dramatically improves the cationic/energetic status during reperfusion. We investigated the role of this latter effect in K(ATP) channel-induced cardioprotection. Employing (23)Na and (31)P nuclear magnetic resonance spectroscopy with perfused rat hearts, reperfusion Na(+)(i) was altered with brief infusions of ouabain and/or RbCl to transiently decrease or increase Na(+)/K(+) ATPase activity. The increases and decreases in functional recovery (%LVDP-R) with pinacidil or ouabain, respectively, were largely unaltered by each other's presence. Early reperfusion Na(+)(i) and cellular energy were greatly altered by ouabain and indicated linear relationships with %LVDP-R. Pinacidil shifted these relationships to higher %LVDP-R. Increasing early reperfusion Na(+)(i) decreased %LVDP-R but did not diminish pinacidil's capacity to improve %LVDP-R. Approximately 75% and 45% of the pinacidil-induced improvements in %LVDP-R, could be disassociated from early reperfusion Na(+)(i) and cellular energy, respectively. Both pinacidil and RbCl infusion blunted ouabain's elevation of reperfusion Na(+)(i), but RbCl did not improve %LVDP-R. Atomic absorption tissue Ca(2+) measurements indicated that pinacidil reduced late reperfusion Ca(2+) uptake, but did not reduce early reperfusion Ca(2+), and its beneficial effects were resistant to ouabain-induced early reperfusion Ca(2+) increases. In conclusion, K(ATP) channel-induced cardioprotection does not require moderation of Na(+)(i) accumulation, cellular energy depletion, or acidosis during ischemia. K(ATP) channel-induced cardioprotection is largely independent of the accelerated reperfusion Na(+)(i) recovery it induces and does not require early reperfusion reductions of tissue Ca(2+). A larger role for early reperfusion cellular energy cannot be excluded.

Intra-myocyte ion homeostasis during ischemia-reperfusion injury: effects of pharmacologic preconditioning and controlled reperfusion.

BACKGROUND: This study determines whether controlled reperfusion or diazoxide improves intramyocyte Na(+) homeostasis using a porcine model of severe ischemia-reperfusion injury. METHODS: Three groups (n = 10 pigs per group) had 75 minutes of left anterior descending artery occlusion during bypass. Group 1 had no treatment (control group), group 2 had controlled reperfusion (500 mL warm cardioplegia) (controlled reperfusion group), and group 3 had diazoxide (50 micromol/L before left anterior descending artery occlusion) (diazoxide group). Biopsies were taken from the left anterior descending artery region before ischemia and at 3, 5, and 10 minutes postreperfusion. Intra-myocyte Na(+) and water contents were determined using atomic absorption spectroscopy, and Na(+) concentrations were calculated. RESULTS: Intra-myocyte Na(+) increased for the diazoxide group pigs at 3-minutes postreperfusion (21.9 +/- 2.9 vs 34.0 +/- 3.4 micromol/mL; p = 0.02), but decreased to 19.9 +/- 3.2 micromol/mL at 10 minutes postreperfusion (p = 1.0 vs baseline). At 10 minutes postreperfusion, intra-myocyte Na(+) in the controlled reperfusion group was lower than baseline (22.3 +/- 2.7 vs 17.2 +/- 3.1 micromol/mL; p < 0.001). Intra-myocyte Na(+) at 10 minutes postreperfusion for the diazoxide and controlled reperfusion groups was lower than for the control group (p < 0.05). CONCLUSIONS: Diazoxide and controlled reperfusion improved intra-myocyte Na(+) homeostasis after severe ischemia-reperfusion injury.

Modulation of mitochondrial adenosine triphosphate-sensitive potassium channels and sodium-hydrogen exchange provide additive protection from severe ischemia-reperfusion injury.

BACKGROUND: Preconditioning and inhibition of sodium-proton exchange attenuate myocardial ischemia-reperfusion injury by means of independent mechanisms that might act additively when used together. The hypothesis of this study is that treatment with a sodium-proton exchange inhibitor and a mitochondrial adenosine triphosphate-sensitive potassium channel opener produces superior functional recovery and a greater decrease in left ventricular infarct size compared with treatment with either drug alone in a model of severe global ischemia. METHODS: Isolated crystalloid-perfused rat hearts (n = 8 hearts per group) were administered vehicle (control, 0.04% dimethyl sulfoxide), diazoxide (100 micromol/L in 0.04% dimethyl sulfoxide), cariporide (10 micromol /L in 0.04% dimethyl sulfoxide), or diazoxide and cariporide before 40 minutes of ischemia at 35.5 degrees C to 36.5 degrees C and 30 minutes of reperfusion. RESULTS: The combination group had superior postischemic systolic function compared with that seen in the cariporide, diazoxide, and control groups (recovery of developed pressure: 91% +/- 7% vs 26% +/- 5%, 35% +/- 6%, and 16% +/- 3%, respectively; P <.05). Postischemic diastolic function in the combination group was superior compared with that seen in the other groups (change(pre-post) diastolic pressure of 67 +/- 4 mm Hg with control, 49 +/- 11 mm Hg with diazoxide, 59 +/- 10 mm Hg with cariporide, and 3 +/- 3 mm Hg with diazoxide and cariporide combination; P <.05). The left ventricular infarct area was less in the combination group compared with that in the cariporide, diazoxide, and control groups (6% +/- 2% vs 35% +/- 7%, 25% +/- 3%, and 37% +/- 9%, respectively; P <.05). CONCLUSIONS: Combining a selective mitochondrial adenosine triphosphate-sensitive potassium channel opener with a selective reversible inhibitor of sarcolemmal sodium-proton exchange improves recovery of contractile function from severe global ischemia in the isolated buffer-perfused rat heart. The putative mechanism for this benefit is superior protection of mitochondrial function.

Mitochondrial function in response to cardiac ischemia-reperfusion after oral treatment with quercetin.

Polyphenolic compounds present in red wines, such as the flavonol quercetin, are thought capable of cardioprotection through mechanisms not yet clearly defined. It has been established that mitochondria play a critical role in myocardial recovery from ischemia-reperfusion (I-R) damage, and in vitro experiments indicate that quercetin can exert a variety of direct effects on mitochondrial function. The effects of quercetin at concentrations typically found in 1-2 glasses of red wine on cardiac I-R and mitochondrial function in vivo are not known. Quercetin was administered to rats (0.033 mg/kg per day by gavage for 4 d). Isolated Langendorff perfused hearts were subjected to I-R, and cardiac functional parameters determined both before and after I-R. Mitochondria were isolated from post-I-R hearts and their function assessed. Compared to an untreated control group, quercetin treatment significantly decreased the impairment of cardiac function following I-R. This protective effect was associated with improved mitochondrial function after I-R. These results indicate that oral low dose quercetin is cardioprotective, possibly via a mechanism involving protection of mitochondrial function during I-R.

Ischemic preconditioning and Na+/H+ exchange inhibition improve reperfusion ion homeostasis.

BACKGROUND: Intramyocyte sodium (Na+) increases during ischemia and reperfusion, which causes myocardial calcium (Ca2+) uptake and leads to myocyte injury or death. This study determines if ischemic preconditioning and myocyte sodium-hydrogen ion (Na+-H+) exchange (NHE) inhibition decreases Na+ gain that otherwise occurs with cardioplegic arrest and reperfusion. METHODS: Pigs had 1 hour of cardioplegic arrest followed by reperfusion. Group 1 had no intervention (controls). Group 2 received dimethyl amiloride (DMA, an NHE inhibitor), and group 3 had ischemic preconditioning before cardioplegic arrest. Precardioplegia to postreperfusion change in intramyocyte ion content was measured with atomic absorption spectrometry. The time to initial electrical activity and number of defibrillations needed to establish an organized rhythm postreperfusion were used as electrophysiologic variables to measure ischemia-reperfusion injury. RESULTS: Intramyocyte Na+ content for group 1 increased from 45.9+/-6.7 to 61.9+/-22.5 micromol/g (p = 0.02). Group 2 had an insignificant decrease in intramyocyte Na+ of 27.7+/-19.58 micromol/g (p = 0.06), and group 3 had an insignificant decrease of 10.8+/-46.33 micromol/g (p = 0.48). Interstitial water increased significantly in all groups, but there were no significant increases in intramyocyte water content. Electrophysiologic recovery was similar for all three groups. CONCLUSIONS: The NHE inhibition and ischemic preconditioning each eliminated the increase in intramyocyte Na+ content that otherwise occurred with cardioplegic arrest and reperfusion in this porcine model. Because their mechanisms are distinct, it is possible that an additive beneficial effect against ischemia-reperfusion injury can be achieved by using NHE inhibition together with a preconditioning stimulus as prereperfusion therapy.