Mitochondrial Bioenergetics During Ischemia and Reperfusion.

During ischemia and reperfusion (I/R) mitochondria suffer a deficiency to supply the cardiomyocyte with chemical energy, but also contribute to the cytosolic ionic alterations especially of Ca2+. Their free calcium concentration ([Ca2+]m) mainly depends on mitochondrial entrance through the uniporter (UCam) and extrusion in exchange with Na+ (mNCX) driven by the electrochemical gradient (ΔΨm). Cardiac energetic is frequently estimated by the oxygen consumption, which determines metabolism coupled to ATP production and to the maintaining of ΔΨm. Nevertheless, a better estimation of heart energy consumption is the total heat release associated to ATP hydrolysis, metabolism, and binding reactions, which is measurable either in the presence or the absence of oxygenation or perfusion. Consequently, a mechano-calorimetrical approach on isolated hearts gives a tool to evaluate muscle economy. The mitochondrial role during I/R depends on the injury degree. We investigated the role of the mitochondrial Ca2+ transporters in the energetic of hearts stunned by a model of no-flow I/R in rat hearts. This chapter explores an integrated view of previous and new results which give evidences to the mitochondrial role in cardiac stunning by ischemia o hypoxia, and the influence of thyroid alterations and cardioprotective strategies, such as cardioplegic solutions (high K-low Ca, pyruvate) and the phytoestrogen genistein in both sex. Rat ventricles were perfused in a flow-calorimeter at either 30 °C or 37 °C to continuously measure the left ventricular pressure (LVP) and total heat rate (Ht). A pharmacological treatment was done before exposing to no-flow I and R. The post-ischemic contractile (PICR as %) and energetical (Ht) recovery and muscle economy (Eco: P/Ht) were determined during stunning. The functional interaction between mitochondria (Mit) and sarcoplasmic reticulum (SR) was evaluated with selective mitochondrial inhibitors in hearts reperfused with Krebs-10 mM caffeine-36 mM Na+. The caffeine induced contracture (CIC) was due to SR Ca2+ release, while relaxation mainly depends on mitochondrial Ca2+ uptake since neither SL-NCX nor SERCA are functional under this media. The ratio of area-under-curves over ischemic values (AUC-ΔHt/AUC-ΔLVP) estimates the energetical consumption (EC) to maintain CIC. Relaxation of CIC was accelerated by inhibition of mNCX or by adding the aerobic substrate pyruvate, while both increased EC. Contrarily, relaxation was slowed by cardioplegia (high K-low Ca Krebs) and by inhibition of UCam. Thus, Mit regulate the cytosolic [Ca2+] and SR Ca2+ content. Both, hyperthyroidism (HpT) and hypothyroidism (HypoT) reduced the peak of CIC but increased EC, in spite of improving PICR. Both, CIC and PICR in HpT were also sensitive to inhibition of mNCX or UCam, suggesting that Mit contribute to regulate the SR store and Ca2+ release. The interaction between mitochondria and SR and the energetic consequences were also analyzed for the effects of genistein in hearts exposed to I/R, and for the hypoxia/reoxygenation process. Our results give evidence about the mitochondrial regulation of both PICR and energetic consumption during stunning, through the Ca2+ movement.

Cardiac Strain in a Swine Model of Regional Hibernating Myocardium: Effects of CoQ10 on Contractile Reserve Following Bypass Surgery.

There is conflicting clinical evidence whether administration of coenzyme Q10 (CoQ10) improves function following coronary artery bypass graft surgery (CABG). Using a swine model of hibernating myocardium, we tested whether daily CoQ10 would improve contractile function by MRI at 4-week post-CABG. Twelve pigs underwent a thoracotomy and had a constrictor placed on the left anterior descending (LAD). At 12 weeks, they underwent off-pump bypass and received daily dietary supplements of either CoQ10 (10 mg/kg/day) or placebo. At 4-week post-CABG, circumferential strain measurements in the hibernating LAD region from placebo and CoQ10 groups were not different and increased to a similar extent with dobutamine (-14.7 ± 0.6 versus -14.8 ± 0.1, respectively (NS)). Post-sacrifice, oxidant stress markers were obtained in the mitochondrial isolates and protein carbonyl in the placebo, and CoQ10 groups were 6.14 ± 0.36 and 5.05 ± 0.32 nmol/mg, respectively (NS). In summary, CoQ10 did not improve contractile reserve or reduce oxidant stress at 4-week post-CABG.

Hypoxia inducible factor-1 improves the actions of positive inotropic agents in stunned cardiac myocytes.

1. In the present study, we tested hypothesis that upregulation of hypoxia-inducible factor-1 (HIF-1) would improve the actions of positive inotropic agents in cardiac myocytes after simulated ischaemia-reperfusion (I/R). 2. Hypoxia-inducible factor-1α was upregulated with deferoxamine (150 mg/kg per day for 2 days). Rabbit cardiac myocytes were subjected to simulated ischaemia (15 min, 95% N(2)-5% CO2) and reperfusion (re-oxygenation) and compared with control myocytes. Cell contraction and calcium transients were measured at baseline and after forskolin (10(-7) and 10(-6) mol/L) or ouabain (10(-5) and 10(-4) mol/L). 3. Under control conditions, high-dose forskolin and ouabain increased percentage shortening by 20 and 18%, respectively. Deferoxamine-treated control myocytes responded similarly. In stunned myocytes, forskolin and ouabain did not significantly increase shortening (increases of 8% and 9%, respectively). Deferoxamine restored the effects of forskolin (+26%) and ouabain (+28%) in stunning. The results for maximum shortening and relaxation rates were similar. The increased calcium transients caused by forskolin and ouabain were also depressed in stunned myocytes, but were maintained by HIF-1 upregulation. 4. These results suggest that simulated I/R impaired the functional and calcium transient effects of positive inotropic agents. Upregulation of HIF-1 protects cardiac myocyte function after I/R by maintaining calcium release.

Ca2+ sensitizer superior to catecholamine during myocardial stunning?

BACKGROUND: After open-chest cardiac surgery, ventricular function remains depressed (myocardial stunning). Catecholamines (epinephrine) improve ventricular function by increasing the intracellular Ca(2+) concentration. In parallel, the oxygen consumption is increased, so that the hitherto intact myocardium can be jeopardized. In the very insufficient ventricle, epinephrine can even become ineffective. Since Ca(2+) sensitizers provide another therapeutic avenue, the effects of epinephrine and levosimendan on postischemic hemodynamics were investigated. METHODS: After hemodynamic steady state, isolated, blood (erythrocyte-enriched Krebs-Henseleit solution)-perfused rabbit hearts were subjected to 25 min normothermic, no-flow ischemia and 20 min reperfusion. Heart rate (HR), cardiac output (CO), left ventricular pressure (LVP), coronary blood flow (CBF), and arterio-venous oxygen difference (AVDO(2)) were recorded during reperfusion and after administration of either epinephrine (n=16; 0.03 micromol), or levosimendan (n=11; 0.75 micromol) or epinephrine plus levosimendan (n=5). RESULTS: Epinephrine increased HR (19%, p=0.01) and improved hemodynamics in terms of CO (62%, p=0.0006), stroke volume SV (46%, p=0.02), stroke work W (158%, p=0.01), LVP(max) (58%, p=0.0001), maximal pressure increase dP/dt(max)(140%, p=0.0004), minimal pressure increase dP/dt(min) (104%, p=0.0002), LVP(ed) (-26%, p=0.02), and increased coronary resistance CR (31%, p=0.05). Epinephrine impaired hemodynamics in terms of AVDO(2) (+63%, p=0.003), myocardial oxygen consumption MVO(2) (+67%, p=0.0003) and MVO(2)/beat (+36%, p=0.01). External efficiency eta was increased by 92% (p=0.02). Levosimendan in postischemic hearts increased HR (32%, p=0.009) and improved hemodynamics in terms of CO (85%, p=0.01), SV (44%, p=0.03), W (115%, p=0.04), LVP(max) (95%, p=0.04), dP/dt(max) (133%, p=0.009), dP/dt(min) (121%, p=0.007), LVP(ed) (-63%, p=0.0006), and CR (-17%; n.s., p=0.1). It altered hemodynamics in terms of AVDO(2) (+7.0%; n.s., p=0.3) and MVO(2) (+32%, p=0.007) and MVO(2)/beat (+2.3%; n.s., p=0.4). External efficiency was increased by 307% (p=0.04). In five additional extremely dysfunctional rabbit hearts, epinephrine was ineffective. Additional levosimendan increased hemodynamics in terms of HR (56%; n.s., p=0.1), CO (159%, p=0.04), SV (89%, p=0.03), W (588%, p=0.02), LVP(max) (168%, p=0.03), dP/dt(max) (102%, p=0.005), dP/dt(min) (78%, p=0.006), LVP(ed) (-98%, p=0.0006), and CR (-50%, p=0.02). It altered hemodynamics in terms of AVDO(2) (-11%; n.s., p=0.05), MVO(2) (+131%, p=0.04) and MVO(2)/beat (+171%, p=0.03). External efficiency was increased by 212% (p=0.04). CONCLUSION: In contrast to epinephrine, levosimendan improves ventricular function without increasing oxygen demand, thereby considerably improving external efficiency. Even during epinephrine resistance in extremely dysfunctional hearts, levosimendan successfully improves ventricular function.

Effect of Curcuma longa and Ocimum sanctum on myocardial apoptosis in experimentally induced myocardial ischemic-reperfusion injury.

BACKGROUND: In the present investigation, the effect of Curcuma longa (Cl) and Ocimum sanctum (Os) on myocardial apoptosis and cardiac function was studied in an ischemia and reperfusion (I-R) model of myocardial injury. METHODS: Wistar albino rats were divided into four groups and orally fed saline once daily (sham, control IR) or Cl (100 mg/kg; Cl-IR) or Os (75 mg/kg; Os-IR) respectively for 1 month. On the 31st day, in the rats of the control IR, Cl-IR and Os-IR groups LAD occlusion was undertaken for 45 min, and reperfusion was allowed for 1 h. The hemodynamic parameters{mean arterial pressure (MAP), heart rate (HR), left ventricular end-diastolic pressure (LVEDP), left ventricular peak positive (+) LVdP/dt (rate of pressure development) and negative (-) LVdP/dt (rate of pressure decline)} were monitored at pre-set points throughout the experimental duration and subsequently, the animals were sacrificed for immunohistopathological (Bax, Bcl-2 protein expression & TUNEL positivity) and histopathological studies. RESULTS: Chronic treatment with Cl significantly reduced TUNEL positivity (p < 0.05), Bax protein (p < 0.001) and upregulated Bcl-2 (p < 0.001) expression in comparison to control IR group. In addition, Cl demonstrated mitigating effects on several myocardial injury induced hemodynamic {(+)LVdP/dt, (-) LVdP/dt & LVEDP} and histopathological perturbations. Chronic Os treatment resulted in modest modulation of the hemodynamic alterations (MAP, LVEDP) but failed to demonstrate any significant antiapoptotic effects and prevent the histopathological alterations as compared to control IR group. CONCLUSION: In the present study, significant cardioprotection and functional recovery demonstrated by Cl may be attributed to its anti-apoptotic property. In contrast to Os, Cl may attenuate cell death due to apoptosis and prevent the impairment of cardiac performance.

Cardioprotective effects of Ilex paraguariensis extract: evidence for a nitric oxide-dependent mechanism.

AIM: To examine the effects of an Ilex paraguariensis (Ip) extract on postischemic alterations derived from 20 min of global ischemia and 30 min of reperfusion. METHODS: Isolated rat hearts were treated 10 min before ischemia and the first 10 min of reperfusion with Ip 30 microg/ml. In other hearts, chelerythrine (1 microM), a protein kinase C blocker, or l(G)-nitro l-arginine methyl ester (l-NAME), a nitric oxide synthase inhibitor, were administered prior to Ip infusion. Left ventricular developed pressure (LVDP), +dP/dt(max), -dP/dt(max), and left ventricular end diastolic pressure (LVEDP) were used to assess myocardial function. Thiobarbituric acid reactive substances (TBARS) were measured. RESULTS: Ip treatment produced an improvement of postichemic recovery (LVDP=96+/-8%; +dP/dt(max)=95+/-10%; -dP/dt(max)=90+/-12% vs. 57+/-6%, 53+/-6% and 57+/-8%, respectively, in untreated hearts) and an attenuation of the increase of LVEDP and TBARS content. Chelerythrine did not modify and l-NAME abolished the protection induced by Ip. CONCLUSIONS: These data are the first demonstration that Ip extract attenuates the myocardial dysfunction provoked by ischemia and reperfusion and that this cardioprotection involves a diminution of oxidative damage through a nitric oxide-dependent mechanism.

Effect of levosimendan on balance between ATP production and consumption in isolated perfused guinea-pig heart before ischemia or after reperfusion.

Levosimendan is a novel drug developed for treatment of decompensated heart failure. Levosimendan is a calcium sensitizer that increases contractile force of the myocardium by enhancing the sensitivity of myofilaments to calcium without increasing intracellular calcium concentration. The present study was carried out to investigate whether levosimendan induces any changes in the phosphorylation potential (ie, the balance between ATP production and consumption) in the normal heart and in the post-ischemic heart while exerting its positive inotropic effect. We show that 0.1 microM levosimendan increased the left ventricle developed pressure in the pre-ischemic and in the post ischemic hearts by 16 and 18% respectively, and the +dP/dt by 16 and 19%, respectively. At that concentration levosimendan did not cause any effect on the phosphorylation potential (1 x 10(5) M(-1) and 0.2 x 10(5) M(-1) in the pre-ischemic and post-ischemic heart, respectively) as assessed by P-NMR, although an increased beating rate (13%) and oxygen consumption (10%) was observed when adding the drug post-ischemically. Our findings are consistent with the results of a recent clinical trial (RUSSLAN), which showed that levosimendan does not induce ischemia and reduces the risk of worsening heart failure and death, in patients with left ventricular failure complicating acute myocardial infarction.

Postresuscitation stunning: postfibrillatory myocardial dysfunction caused by reduced myofilament Ca2+ responsiveness after ventricular fibrillation-induced myocyte Ca2+ overload.

INTRODUCTION: Resuscitation from ventricular fibrillation (VF), particularly from prolonged VF, frequently is complicated by postfibrillatory myocardial dysfunction (postresuscitation stunning). We tested whether this dysfunction can be caused by reduced myofilament Ca2+ responsiveness after VF-induced myocyte Ca2+ overload. We also tested whether electrical defibrillation shocks contribute to this dysfunction. METHODS AND RESULTS: Myofilament Ca2+ responsiveness was estimated as ratio of left ventricular developed pressure over myocyte Ca2+ transient amplitudes (assessed as indo-1 fluorescence) in isolated perfused rat hearts before, during, and after VF (1.5 or 10 min) comparing three modes of defibrillation (biphasic electrical shocks, lidocaine, or spontaneous). We found that, independent of these defibrillation modes, myofilament Ca2+ responsiveness was significantly reduced, particularly after prolonged VF, although hearts were not ischemic or acidotic during and after VF (unchanged coronary flow, myocardial oxygen consumption, and pH of the coronary effluent). This reduction was associated with VF-induced myocyte Ca2+ overload and increasing or decreasing Ca2+ overload during VF (using 1 microM diltiazem or 6 mM extracellular calcium) led to parallel changes of myofilament Ca2+ responsiveness. However, myofilament Ca2+ responsiveness was not associated with the defibrillation shock energy (range 0.1-15.0 J/g wet heart weight). CONCLUSION: Postfibrillatory myocardial dysfunction can be caused by reduced myofilament Ca2+ responsiveness after VF-induced myocyte Ca2+ overload. Electrical defibrillation shocks (up to 15 J/g wet heart weight), however, do not significantly contribute to this dysfunction. Our findings suggest that early additional therapy targeting intracellular Ca2+ overload may normalize myocyte Ca2+ and partially prevent postresuscitation stunning.

Enhancement of glucose uptake in stunned myocardium: role of glucose transporter.

This study quantifies the myocardial glucose uptake and clarifies the pathway of augmented glucose uptake in myocardium reperfused after a brief period of ischemia (stunned myocardium). The glucose uptake rate was determined from the time course of the sugar phosphate (SP) resonance in rat myocardium (d[SP]/dt) with 31P nuclear magnetic resonance after the substitution of glucose with its analog 2-deoxyglucose. The d[SP]/dt in stunned myocardium [1.03 +/- 0.05 (SE) micromol x g wet wt(-1) x min(-1); n = 8] increased significantly compared with nonischemic control myocardium (0.18 +/- 0.03 micromol x g wet wt(-1) x min(-1); n = 8; P < 0.0001), reaching the maximal stimulatory uptake rate during exposure to insulin (1.05 +/- 0.04 micromol x g wet wt(-1) x min(-1); n = 8). Twenty minutes after reperfusion, the d[SP]/dt was still augmented (0.41 +/- 0.05 micromol x g wet wt(-1) x min(-1); n = 5; P < 0.05 vs. control myocardium). To elucidate further the mechanism of augmented glucose uptake, N6-(L-2-phenylisopropyl)-adenosine (PIA; 100 micromol/l), a potent blocker of the glucose transporter, was administered to stunned hearts and, as a control, to insulin-stimulated hearts. PIA significantly and comparably inhibited the increase in d[SP]/dt in stunned myocardium (0.36 +/- 0.07 micromol x g wet wt(-1) x min(-1); n = 4; P < 0.0001 vs. without PIA) and in insulin-stimulated myocardium (0.38 +/- 0.02 micromol x g wet wt(-1) x min(-1); n = 4; P < 0.0001 vs. without PIA). These results indicate that the augmented glucose uptake in stunned myocardium is maintained by the glucose transporter, the amount of which is almost equal to that which can be maximally recruited by insulin.

Long-chain triglycerides improve recovery from myocardial stunning in conscious dogs.

OBJECTIVES: Free fatty acid (FFA) oxidation is depressed in the postischaemic stunned myocardium and recovers in parallel with the normalization of contractile performance. Assuming a causal role for this metabolic disturbance in the pathogenesis of stunning, we questioned whether exogenous administration of high dose triglycerides during reperfusion of postischaemic myocardium, could improve its functional recovery. METHODS: Thirteen dogs were chronically instrumented to measure global and regional haemodynamics and to produce a 10 min episode of regional myocardial ischaemia. In 7 dogs, Intralipid 20% was administered i.v. during the reperfusion phase. Contractile recovery of stunned myocardium was compared with control saline treatments. The series were repeated in another 6 animals, but oxfenicine (CPT I inhibitor) preceeded Intralipid during reperfusion. RESULTS: Contractile recovery of stunned myocardium was faster and more extensive when Intralipid was administered during reperfusion than with saline treatment (wall thickening fraction 86 +/- 6% of preischaemic controls versus 52 +/- 11% at 90 min post-reperfusion; P < 0.05). Oxfenicine pretreatment completely abolished this beneficial effect. CONCLUSIONS: Exogenous administration of triglycerides during reperfusion of postischaemic myocardium improves functional recovery from stunning. This beneficial effect most likely operates through enhanced FFA availability and/or oxidation since it could be abolished by selective inhibition of the carnitine palmitoyl I enzyme.

Effects of cold and warm blood cardioplegia assessed by myocardial pH and release of metabolic markers.

The optimal temperature of blood cardioplegia remains controversial. Interstitial myocardial pH was monitored online with a probe that was inserted in the anterior wall of the left ventricle. Venous pH, lactate production, and creatine kinase and troponin T release were measured in coronary sinus blood obtained in 14 dogs after ischemic arrest periods of 5, 10, 20, and 40 minutes with warm (n = 7; mean myocardial temperature, 35 degrees +/- 2 degrees C) and cold (n = 7; mean myocardial temperature, 12 degrees +/- 1 degree C) blood cardioplegic protection. Blood cardioplegic solution was delivered at a rate of 100 mL/min during the 10 minutes between each ischemic arrest. The interstitial myocardial pH decreased significantly (p < 0.05) from 7.1 +/- 0.3 to 6.53 +/- 0.3 after ischemia in animals perfused with warm blood cardioplegia and from 7.04 +/- 0.3 to 6.64 +/- 0.1 in those receiving cold blood cardioplegic protection; however, the difference between the groups was not significant (p > 0.05). Lactate production and creatine kinase and troponin T release increased significantly after ischemia, but there was no difference in the changes between the warm and cold blood cardioplegia groups. In conclusion, ischemia caused significant changes in all variables measured, and these changes were directly proportional to the duration of ischemia. However, there was no significant difference (p > 0.05) in the myocardial metabolic changes between the warm and cold blood cardioplegia groups in terms of the duration of ischemic arrest studied.(ABSTRACT TRUNCATED AT 250 WORDS)

Cardiac preconditioning. Induction of endogenous tolerance to ischemia-reperfusion injury.

Cardiac preconditioning is a phenomenon by which a brief exposure to ischemia renders the heart more tolerant of a subsequent sustained ischemic insult. Understanding the mechanism involved may allow pharmacologic access to this protective state before cardiopulmonary bypass surgery and transplantation. We discuss herein the preconditioning phenomenon, the potential mechanisms involved, and the therapeutic implications of cardiac preconditioning.