Mohammed Aldakkak

Ranolazine reduces Ca2+ overload and oxidative stress and improves mitochondrial integrity to protect against ischemia reperfusion injury in isolated hearts

Ranolazine is a clinically approved drug for treating cardiac ventricular dysrhythmias and angina. Its mechanism(s) of protection is not clearly understood but evidence points to blocking the late Na+ current that arises during ischemia, blocking mitochondrial complex I activity, or modulating mitochondrial metabolism. Here we tested the effect of ranolazine treatment before ischemia at the mitochondrial level in intact isolated hearts and in mitochondria isolated from hearts at different times of reperfusion. Left ventricular (LV) pressure (LVP), coronary flow (CF), and O2 metabolism were measured in guinea pig isolated hearts perfused with Krebs-Ringers solution; mitochondrial (m) superoxide (O2·-), Ca2+, NADH/FAD (redox state), and cytosolic (c) Ca2+ were assessed on-line in the LV free wall by fluorescence spectrophotometry. Ranolazine (5 μM), infused for 1 min just before 30 min of global ischemia, itself did not change O2·-, cCa2+, mCa2+ or redox state. During late ischemia and reperfusion (IR) O2·- emission and m[Ca2+] increased less in the ranolazine group vs. the control group. Ranolazine decreased c[Ca2+] only during ischemia while NADH and FAD were not different during IR in the ranolazine vs. control groups. Throughout reperfusion LVP and CF were higher, and ventricular fibrillation was less frequent. Infarct size was smaller in the ranolazine group than in the control group. Mitochondria isolated from ranolazine-treated hearts had mild resistance to permeability transition pore (mPTP) opening and less cytochrome c release than control hearts. Ranolazine may provide functional protection of the heart during IR injury by reducing cCa2+ and mCa2+ loading secondary to its effect to block the late Na+ current. Subsequently it indirectly reduces O2·- emission, preserves bioenergetics, delays mPTP opening, and restricts loss of cytochrome c, thereby reducing necrosis and apoptosis.

Mitochondrial matrix K+ flux independent of large-conductance Ca2+-activated K+ channel opening

Large-conductance Ca(2+)-activated K(+) channels (BK(Ca)) in the inner mitochondrial membrane may play a role in protecting against cardiac ischemia-reperfusion injury. NS1619 (30 microM), an activator of BK(Ca) channels, was shown to increase respiration and to stimulate reactive oxygen species generation in isolated cardiac mitochondria energized with succinate. Here, we tested effects of NS1619 to alter matrix K(+), H(+), and swelling in mitochondria isolated from guinea pig hearts. We found that 30 microM NS1619 did not change matrix K(+), H(+), and swelling, but that 50 and 100 microM NS1619 caused a concentration-dependent increase in matrix K(+) influx (PBFI fluorescence) only when quinine was present to block K(+)/H(+) exchange (KHE); this was accompanied by increased mitochondrial matrix volume (light scattering). Matrix pH (BCECF fluorescence) was decreased slightly by 50 and 100 microM NS1619 but markedly more so when quinine was present. NS1619 (100 microM) caused a significant leak in lipid bilayers, and this was enhanced in the presence of quinine. The K(+) ionophore valinomycin (0.25 nM), which like NS1619 increased matrix volume and increased K(+) influx in the presence of quinine, caused matrix alkalinization followed by acidification when quinine was absent, and only alkalinization when quinine was present. If K(+) is exchanged instantly by H(+) through activated KHE, then matrix K(+) influx should stimulate H(+) influx through KHE and cause matrix acidification. Our results indicate that KHE is not activated immediately by NS1619-induced K(+) influx, that NS1619 induces matrix K(+) and H(+) influx through a nonspecific transport mechanism, and that enhancement with quinine is not due to the blocking of KHE, but to a nonspecific effect of quinine to enhance current leak by NS1619.

Protection Against Cardiac Injury by Small Ca 2 + -Sensitive K + Channels Identified in Guinea Pig Cardiac Inner Mitochondrial Membrane

We tested if small conductance, Ca(2+)-sensitive K(+) channels (SK(Ca)) precondition hearts against ischemia reperfusion (IR) injury by improving mitochondrial (m) bioenergetics, if O(2)-derived free radicals are required to initiate protection via SK(Ca) channels, and, importantly, if SK(Ca) channels are present in cardiac cell inner mitochondrial membrane (IMM). NADH and FAD, superoxide (O(2)(-)), and m[Ca(2+)] were measured in guinea pig isolated hearts by fluorescence spectrophotometry. SK(Ca) and IK(Ca) channel opener DCEBIO (DCEB) was given for 10 min and ended 20 min before IR. Either TBAP, a dismutator of O(2)()(-), NS8593, an antagonist of SK(Ca) isoforms, or other K(Ca) and K(ATP) channel antagonists, were given before DCEB and before ischemia. DCEB treatment resulted in a 2-fold increase in LV pressure on reperfusion and a 2.5 fold decrease in infarct size vs. non-treated hearts associated with reduced O(2)(-) and m[Ca(2+)], and more normalized NADH and FAD during IR. Only NS8593 and TBAP antagonized protection by DCEB. Localization of SK(Ca) channels to mitochondria and IMM was evidenced by a) identification of purified mSK(Ca) protein by Western blotting, immuno-histochemical staining, confocal microscopy, and immuno-gold electron microscopy, b) 2-D gel electrophoresis and mass spectroscopy of IMM protein, c) [Ca(2+)]-dependence of mSK(Ca) channels in planar lipid bilayers, and d) matrix K(+) influx induced by DCEB and blocked by SK(Ca) antagonist UCL1684. This study shows that 1) SK(Ca) channels are located and functional in IMM, 2) mSK(Ca) channel opening by DCEB leads to protection that is O(2)(-) dependent, and 3) protection by DCEB is evident beginning during ischemia.

Enhanced Na+/H+ Exchange During Ischemia and Reperfusion Impairs Mitochondrial Bioenergetics and Myocardial Function

Inhibition of Na+/H+ exchange (NHE) during ischemia reduces cardiac injury due to reduced reverse mode Na+/Ca2+ exchange. We hypothesized that activating NHE-1 at buffer pH 8 during ischemia increases mitochondrial oxidation, Ca2+ overload, and reactive O2 species (ROS) levels and worsens functional recovery in isolated hearts and that NHE inhibition reverses these effects. Guinea pig hearts were perfused with buffer at pH 7.4 (control) or pH 8 +/- NHE inhibitor eniporide for 10 minutes before and for 10 minutes after 35- minute ischemia and then for 110 minutes with pH 7.4 buffer alone. Mitochondrial NADH and FAD, [Ca2+], and superoxide were measured by spectrophotofluorometry. NADH and FAD were more oxidized, and cardiac function was worse throughout reperfusion after pH 8 versus pH 7.4, Ca2+ overload was greater at 10-minute reperfusion, and superoxide generation was higher at 30-minute reperfusion. The pH 7.4 and eniporide groups exhibited similar mitochondrial function, and cardiac performance was most improved after pH 7.4+eniporide. Cardiac function on reperfusion after pH 8+eniporide was better than after pH 8. Percent infarction was largest after pH 8 and smallest after pH 7.4+eniporide. Activation of NHE with pH 8 buffer and the subsequent decline in redox state with greater ROS and Ca2+ loading underlie the poor functional recovery after ischemia and reperfusion.

Modulation of Mitochondrial Bioenergetics in the Isolated Guinea Pig Beating Heart by Potassium and Lidocaine Cardioplegia: Implications for Cardioprotection

Mitochondria are damaged by cardiac ischemia/reperfusion (I/R) injury but can contribute to cardioprotection. We tested if hyperkalemic cardioplegia (CP) and lidocaine (LID) differently modulate mitochondrial (m) bioenergetics and protect hearts against I/R injury. Guinea pig hearts (n = 71) were perfused with Krebs Ringers solution before perfusion for 1 minute just before ischemia with either CP (16 mM K+) or LID (1 mM) or Krebs Ringers (control, 4 mM K+). The 1-minute perfusion period assured treatment during ischemia but not on reperfusion. Cardiac function, NADH, FAD, m[Ca2+], and superoxide (reactive oxygen species) were assessed at baseline, during the 1-minute perfusion, and continuously during I/R. During the brief perfusion before ischemia, CP and LID decreased reactive oxygen species and increased NADH without changing m[Ca2+]. Additionally, CP decreased FAD. During ischemia, NADH was higher and reactive oxygen species was lower after CP and LID, whereas m[Ca2+] was lower only after LID. On reperfusion, NADH and FAD were more normalized, and m[Ca2+] and reactive oxygen species remained lower after CP and LID. Better functional recovery and smaller infarct size after CP and LID were accompanied by better mitochondrial function. These results suggest that mitochondria may be implicated, directly or indirectly, in protection by CP and LID against I/R injury.

Comparison of cumulative planimetry versus manual dissection to assess experimental infarct size in isolated hearts.

INTRODUCTION Infarct size (IS) is an important variable to estimate cardiac ischemia/reperfusion injury in animal models. Triphenyltetrazolium chloride (TTC) stains viable cells red while leaving infarcted cells unstained. To quantify IS, infarcted and non-infarcted tissue is often manually dissected and weighed (IS-DW). An alternative is to measure infarcted areas by cumulative planimetry (IS-CP). METHODS We prospectively compared these two methods in 141 Langendorff-prepared guinea pig hearts (1.44+/-0.02 g) that were part of different studies on mechanisms of cardioprotection. Hearts were perfused with Krebs-Ringers and subjected to 30 min global ischemia after various cardioprotective treatments. Two hours after reperfusion hearts were cut into 6-7 transverse sections (3mm) and stained for 5 min in 1% TTC and 0.1M KH2PO4 buffer (pH 7.4, 38 degrees C). Each slice was first scanned and its infarcted area measured with Image 1.62 software (NIH). Infarctions in individual slices of each heart were averaged (IS-CP) on the basis of their weight. After scanning, IS-DW was determined by careful manual dissection of infarcted from non-infarcted tissue and measuring their respective total weight. RESULTS We found limited tissue permeation of TTC in relation to the slice thickness leaving tissue in the center unstained, as well as significant cross-contamination of stained vs. unstained tissue after manual dissection. IS-CP and IS-DW ranged from 6.0 to 73.1% and 19.4 to 70.5%, respectively, and correlated as follows: IS-DW=(27.6+/-1.4)+(0.518+/-0.038) * IS-CP; r=0.75 (Pearson), p<0.001. In addition, IS-CP correlated better with return of function after reperfusion like developed left ventricular pressure, contractility and relaxation, and myocardial oxygen consumption. DISCUSSION Despite a good correlation between both methods, limited tissue permeation by TTC diffusion and limited precision in the ability to manually dissect stained from unstained tissue leads to an overestimation of infarct size by dissection and weighing compared to cumulative planimetry.

Mitochondrial handling of excess Ca2+ is substrate-dependent with implications for reactive oxygen species generation

The mitochondrial electron transport chain is the major source of reactive oxygen species (ROS) during cardiac ischemia. Several mechanisms modulate ROS production; one is mitochondrial Ca(2+) uptake. Here we sought to elucidate the effects of extramitochondrial Ca(2+) (e[Ca(2+)]) on ROS production (measured as H(2)O(2) release) from complexes I and III. Mitochondria isolated from guinea pig hearts were preincubated with increasing concentrations of CaCl(2) and then energized with the complex I substrate Na(+) pyruvate or the complex II substrate Na(+) succinate. Mitochondrial H(2)O(2) release rates were assessed after giving either rotenone or antimycin A to inhibit complex I or III, respectively. After pyruvate, mitochondria maintained a fully polarized membrane potential (ΔΨ; assessed using rhodamine 123) and were able to generate NADH (assessed using autofluorescence) even with excess e[Ca(2+)] (assessed using CaGreen-5N), whereas they remained partially depolarized and did not generate NADH after succinate. This partial ΔΨ depolarization with succinate was accompanied by a large release in H(2)O(2) (assessed using Amplex red/horseradish peroxidase) with later addition of antimycin A. In the presence of excess e[Ca(2+)], adding cyclosporin A to inhibit mitochondrial permeability transition pore opening restored ΔΨ and significantly decreased antimycin A-induced H(2)O(2) release. Succinate accumulates during ischemia to become the major substrate utilized by cardiac mitochondria. The inability of mitochondria to maintain a fully polarized ΔΨ under excess e[Ca(2+)] when succinate, but not pyruvate, is the substrate may indicate a permeabilization of the mitochondrial membrane, which enhances H(2)O(2) emission from complex III during ischemia.

Low-flow Perfusion of Guinea Pig Isolated Hearts With 26°C Air-saturated Lifor Solution for 20 Hours Preserves Function and Metabolism

BACKGROUND Donor human hearts cannot be preserved for >5 hours between explantation and recipient implantation. A better approach is needed to preserve transplantable hearts for longer periods, ideally at ambient conditions for transport. We tested whether Lifor solution could satisfactorily preserve guinea pig isolated hearts perfused at low flow with no added oxygen at room temperature for 20 hours. METHODS Hearts were isolated from 18 guinea pigs and perfused initially with oxygenated Krebs-Ringer (KR) solution at 37 degrees C. Hearts were then perfused with recirculated Lifor or cardioplegia (CP) solution (K(+) 15 mmol/liter) equilibrated with room air at 20% of control flow at 26 degrees C for 20 hours. Hearts were then perfused at 100% flow with KR for 2 hours at 37 degrees C. RESULTS Lifor and CP arrested all hearts. During the 20-hour low-flow perfusion with Lifor coronary pressure increased by 6 +/- 2 mm Hg and percent oxygen extraction by 29 +/- 2%, whereas oxygen consumption (MVo(2)) decreased by 74 +/- 4%. Similar changes were noted for CP, except that MVo(2) was decreased by 86 +/- 7%. After 20-hour low-flow perfusion with Lifor and 2 hours of warm reperfusion with KR solution, diastolic left ventricular pressure (LVP), maximal dLVP/dt and percent oxygen extraction returned completely to baseline values, whereas heart rate returned to 80 +/- 3%, developed LVP to 76 +/- 3%, minimal dLVP/dt (relaxation) to 65 +/- 4%, coronary flow to 80 +/- 4%, oxygen consumption to 82 +/- 4% and cardiac efficiency to 85 +/- 4% of baseline values. Flow responses to adenosine and nitroprusside after Lifor treatment were 65 +/- 3% and 64 +/- 3% of their baseline values. After cardioplegia, treatment there was no cardiac activity, with a diastolic pressure of 35 +/- 14 mm Hg and a return of coronary flow to only 45 +/- 3% of baseline value. CONCLUSIONS Compared with a cardioplegia solution at ambient air and temperature conditions, Lifor solution is a much better medium for long-term cardiac preservation in this model.

Reduced mitochondrial Ca2+ loading and improved functional recovery after ischemia-reperfusion injury in old vs. young guinea pig hearts

Oxidative damage and impaired cytosolic Ca(2+) concentration ([Ca(2+)](cyto)) handling are associated with mitochondrial [Ca(2+)] ([Ca(2+)](mito)) overload and depressed functional recovery after cardiac ischemia-reperfusion (I/R) injury. We hypothesized that hearts from old guinea pigs would demonstrate impaired [Ca(2+)](mito) handling, poor functional recovery, and a more oxidized state after I/R injury compared with hearts from young guinea pigs. Hearts from young (∼4 wk) and old (>52 wk) guinea pigs were isolated and perfused with Krebs-Ringer solution (2.1 mM Ca(2+) concentration at 37°C). Left ventricular pressure (LVP, mmHg) was measured with a balloon, and NADH, [Ca(2+)](mito) (nM), and [Ca(2+)](cyto) (nM) were measured by fluorescence with a fiber optic probe placed against the left ventricular free wall. After baseline (BL) measurements, hearts were subjected to 30 min global ischemia and 120 min reperfusion (REP). In old vs. young hearts we found: 1) percent infarct size was lower (27 ± 9 vs. 57 ± 2); 2) developed LVP (systolic-diastolic) was higher at 10 min (57 ± 11 vs. 29 ± 2) and 60 min (55 ± 10 vs. 32 ± 2) REP; 3) diastolic LVP was lower at 10 and 60 min REP (6 ± 3 vs. 29 ± 4 and 3 ± 3 vs. 21 ± 4 mmHg); 4) mean [Ca(2+)](cyto) was higher during ischemia (837 ± 39 vs. 541 ± 39), but [Ca(2+)](mito) was lower (545 ± 62 vs. 975 ± 38); 5) [Ca(2+)](mito) was lower at 10 and 60 min REP (129 ± 2 vs. 293 ± 23 and 122 ± 2 vs. 234 ± 15); 6) reduced inotropic responses to dopamine and digoxin; and 7) NADH was elevated during ischemia in both groups and lower than BL during REP. Contrary to our stated hypotheses, old hearts showed reduced [Ca(2+)](mito), decreased infarction, and improved basal mechanical function after I/R injury compared with young hearts; no differences were noted in redox state due to age. In this model, aging-associated protection may be linked to limited [Ca(2+)](mito) loading after I/R injury despite higher [Ca(2+)](cyto) load during ischemia in old vs. young hearts.

Differential effects of buffer pH on Ca2+-induced ROS emission with inhibited mitochondrial complexes I and III

Excessive mitochondrial reactive oxygen species (ROS) emission is a critical component in the etiology of ischemic injury. Complex I and complex III of the electron transport chain are considered the primary sources of ROS emission during cardiac ischemia and reperfusion (IR) injury. Several factors modulate ischemic ROS emission, such as an increase in extra-matrix Ca2+, a decrease in extra-matrix pH, and a change in substrate utilization. Here we examined the combined effects of these factors on ROS emission from respiratory complexes I and III under conditions of simulated IR injury. Guinea pig heart mitochondria were suspended in experimental buffer at a given pH and incubated with or without CaCl2. Mitochondria were then treated with either pyruvate, a complex I substrate, followed by rotenone, a complex I inhibitor, or succinate, a complex II substrate, followed by antimycin A, a complex III inhibitor. H2O2 release rate and matrix volume were compared with and without adding CaCl2 and at pH 7.15, 6.9, or 6.5 with pyruvate + rotenone or succinate + antimycin A to simulate conditions that may occur during in vivo cardiac IR injury. We found a large increase in H2O2 release with high [CaCl2] and pyruvate + rotenone at pH 6.9, but not at pHs 7.15 or 6.5. Large increases in H2O2 release rate also occurred at each pH with high [CaCl2] and succinate + antimycin A, with the highest levels observed at pH 7.15. The increases in H2O2 release were associated with significant mitochondrial swelling, and both H2O2 release and swelling were abolished by cyclosporine A, a desensitizer of the mitochondrial permeability transition pore (mPTP). These results indicate that ROS production by complex I and by complex III is differently affected by buffer pH and Ca2+ loading with mPTP opening. The study suggests that changes in the levels of cytosolic Ca2+ and pH during IR alter the relative amounts of ROS produced at mitochondrial respiratory complex I and complex III.