Time and charge/pH-dependent activation of K+ channel-mediated K+ influx and K+/H+ exchange in guinea pig heart isolated mitochondria; role in bioenergetic stability.

Mitochondria play an important role not only in producing energy for the cell but also for regulating mitochondrial and cell function depending on the cell's needs and environment. Uptake of cations, anions, and substrates requires a stable, polarized transmembrane charge potential (ΔΨm). Chemiosmosis requires ion exchangers to remove Na+, K+, Ca2+, PO43-, and other charged species that enter mitochondria. Knowledge of the kinetics of mitochondrial (m) cation channels and exchangers is important in understanding their roles in regulating mitochondrial chemiosmosis and bioenergetics. The influx/efflux of K+, the most abundant mitochondrial cation, alters mitochondrial volume and shape by bringing in anions and H2O by osmosis. The effects of K+ uptake through ligand-specific mK+ channels stimulated/inhibited by agonists/antagonists on mitochondrial volume (swelling/contraction) are well known. However, a more important role for K+ influx is likely its effects on H+ cycling and bioenergetics facilitated by mitochondrial (m) K+/H+ exchange (mKHE), though the kinetics and consequences of K+ efflux by KHE are not well described. We hypothesized that a major role of K+ influx/efflux is stimulation of respiration via the influx of H+ by KHE. We proposed to modulate KHE activity by energizing guinea pig heart isolated mitochondria and by altering the mK+ cycle to capture changes in mitochondrial volume, pHm, ΔΨm, and respiration that would reflect a role for H+ influx via KHE to regulate bioenergetics. To test this, mitochondria were suspended in a 150 mM K+ buffer at pH 6.9, or in a 140 mM Cs+ buffer at pH 7.6 or 6.9 with added 10 mM K+, minimal Ca2+ and free of Na+. O2 content was measured by a Clark electrode, and pHm, ΔΨm, and volume, were measured by fluorescence spectrophotometry and light-scattering. Adding pyruvic acid (PA) alone caused increases in volume and respiration and a rapid decrease in the transmembrane pH gradient (ΔpHm = pHin-pHext) at pHext 6.9> > 7.6, so that ΔΨm was charged and maintained. BKCa agonist NS1619 and antagonist paxilline modified these effects, and KHE inhibitor quinine and K+ ionophore valinomycin depolarized ΔΨm. We postulate that K+ efflux-induced H+ influx via KHE causes an inward H+ leak that stimulates respiration, but at buffer pH 6.9 also utilizes the energy of ΔpHm, the smaller component of the overall proton motive force, ΔµH+. Thus ΔpHm establishes and maintains the ΔΨm required for utilization of substrates, entry of all cations, and for oxidative phosphorylation. Thus, K+ influx/efflux appears to play a pivotal role in regulating energetics while maintaining mitochondrial ionic balance and volume homeostasis.

Hypothermia Prevents Cardiac Dysfunction during Acute Ischemia Reperfusion by Maintaining Mitochondrial Bioenergetics and by Promoting Hexokinase II Binding to Mitochondria.

Background: Hypothermia (H), cardioplegia (CP), and both combined (HCP) are known to be protective against myocardial ischemia reperfusion (IR) injury. Mitochondria have molecular signaling mechanisms that are associated with both cell survival and cell death. In this study, we investigated the dynamic changes in proapoptotic and prosurvival signaling pathways mediating H, CP, or HCP-induced protection of mitochondrial function after acute myocardial IR injury. Methods: Rats were divided into five groups. Each group consists of 3 subgroups based on a specific reperfusion time (5, 20, or 60 min) after a 25-min global ischemia. The time control (TC) groups were not subjected to IR but were perfused with 37 °C Krebs-Ringer's (KR) buffer, containing 4.5 mM K+, in a specific perfusion protocol that corresponded with the duration of each IR protocol. The IR group (control) was perfused for 20 min with KR, followed by 25-min global ischemia, and then KR reperfusion for 5, 20, or 60 min. The treatment groups were exposed to 17 °C H, 37 °C CP (16 mM K+), or HCP (17 °C + CP) for 5 min before ischemia and for 2 min on reperfusion before switching to 37 °C KR perfusion for the remainder of each of the reperfusion times. Cardiac function and mitochondrial redox state (NADH/FAD) were monitored online in the ex vivo hearts before, during, and after ischemia. Mitochondria were isolated at the end of each specified reperfusion time, and changes in O2 consumption, membrane potential (ΔΨ m), and Ca2+ retention capacity (CRC) were assessed using complex I and complex II substrates. In another set of hearts, mitochondrial and cytosolic fractions were isolated after a specified reperfusion time to conduct western blot assays to determine hexokinase II (HKII) and Bax binding/translocation to mitochondria, cytosolic pAkt levels, and cytochrome c (Cyto-c) release into the cytosol. Results: H and HCP were more protective of mitochondrial integrity and, concomitantly, cardiac function than CP alone; H and HCP improved post-ischemic cardiac function by (1) maintaining mitochondrial bioenergetics, (2) maintaining HKII binding to mitochondria with an increase in pAkt levels, (3) increasing CRC, and (4) decreasing Cyto-c release during reperfusion. Bax translocation/binding to mitochondria was unaffected by any treatment, regardless of cardiac functional recovery. Conclusions: Hypothermia preserved mitochondrial function and cardiac function, in part, by maintaining mitochondrial bioenergetics, by retaining HKII binding to mitochondria via upstream pAkt, and by reducing Cyto-c release independently of Bax binding to mitochondria.

Modulation of peroxynitrite produced via mitochondrial nitric oxide synthesis during Ca2+ and succinate-induced oxidative stress in cardiac isolated mitochondria.

We hypothesized that NO• is generated in isolated cardiac mitochondria as the source for ONOO- production during oxidative stress. We monitored generation of ONOO- from guinea pig isolated cardiac mitochondria subjected to excess Ca2+ uptake before adding succinate and determined if ONOO- production was dependent on a nitric oxide synthase (NOS) located in cardiac mitochondria (mtNOS). Mitochondria were suspended in experimental buffer at pH 7.15, and treated with CaCl2 and then the complex II substrate Na-succinate, followed by menadione, a quinone redox cycler, to generate O2•-. L-tyrosine was added to the mitochondrial suspension where it is oxidized by ONOO- to form dityrosine (diTyr) in proportion to the ONOO- present. We found that exposing mitochondria to excess CaCl2 before succinate resulted in an increase in diTyr and amplex red fluorescence (H2O2) signals, indicating that mitochondrial oxidant stress, induced by elevated mtCa2+ and succinate, increased mitochondrial ONOO- production via NO• and O2•-. Changes in mitochondrial ONOO- production dependent on NOS were evidenced by using NOS inhibitors L-NAME/L-NNA, TEMPOL, a superoxide dismutase (SOD) mimetic, and PTIO, a potent global NO• scavenger. L-NAME and L-NNA decreased succinate and menadione-mediated ONOO- production, PTIO decreased production of ONOO-, and TEMPOL decreased ONOO- levels by converting more O2•- to H2O2. Electron microscopy showed immuno-gold labeled iNOS and nNOS in mitochondria isolated from cardiomyocytes and heart tissue. Western blots demonstrated iNOS and nNOS bands in total heart tissue, bands for both iNOS and nNOS in ß-tubulin-free non-purified (crude) mitochondrial preparations, and a prominent iNOS band, but no nNOS band, in purified (Golgi and ER-free) mitochondria. Prior treatment of guinea pigs with lipopolysacharride (LPS) enhanced expression of iNOS in liver mitochondria but not in heart mitochondria. Our results indicate that release of ONOO- into the buffer is dependent both on O2•- released from mitochondria and NO• derived from a mtCa2+-inducible nNOS isoform, possibly attached to mitochondria, and a mtNOS isoform like iNOS that is non-inducible.

Telomerase Deficiency Predisposes to Heart Failure and Ischemia-Reperfusion Injury.

Introduction: Elevated levels of mitochondrial reactive oxygen species (ROS) contribute to the development of numerous cardiovascular diseases. TERT, the catalytic subunit of telomerase, has been shown to translocate to mitochondria to suppress ROS while promoting ATP production. Acute overexpression of TERT increases survival and decreases infarct size in a mouse model of myocardial infarct, while decreased telomerase activity predisposes to mitochondrial defects and heart failure. In the present study, we examined the role of TERT on cardiac structure and function under basal conditions and conditions of acute or prolonged stress in a novel rat model of TERT deficiency. Methods: Cardiac structure and function were evaluated via transthoracic echocardiogram. Langendorff preparations were used to test the effects of acute global ischemia reperfusion injury on cardiac function and infarction. Coronary flow and left ventricular pressure were measured during and after ischemia/reperfusion (I/R). Mitochondrial DNA integrity was measured by PCR and mitochondrial respiration was assessed in isolated mitochondria using an Oxygraph. Angiotensin II infusion was used as an established model of systemic stress. Results: No structural changes (echocardiogram) or coronary flow/left ventricle pressure (isolated hearts) were observed in TERT-/- rats at baseline; however, after I/R, coronary flow was significantly reduced in TERT-/- compared to wild type (WT) rats, while diastolic Left Ventricle Pressure was significantly elevated (n = 6 in each group; p < 0.05) in the TERT-/-. Interestingly, infarct size was less in TERT-/- rats compared to WT rats, while mitochondrial respiratory control index decreased and mitochondrial DNA lesions increased in TERT-/- compared to WT. Angiotensin II treatment did not alter cardiac structure or function; however, it augmented the infarct size significantly more in TERT-/- compared to the WT. Conclusion: Absence of TERT activity increases susceptibility to stress like cardiac injury. These results suggest a critical role of telomerase in chronic heart disease.

Peroxynitrite nitrates adenine nucleotide translocase and voltage-dependent anion channel 1 and alters their interactions and association with hexokinase II in mitochondria.

Cardiac ischemia and reperfusion (IR) injury induces excessive emission of deleterious reactive O2 and N2 species (ROS/RNS), including the non-radical oxidant peroxynitrite (ONOO-) that can cause mitochondria dysfunction and cell death. In this study, we explored whether IR injury in isolated hearts induces tyrosine nitration of adenine nucleotide translocase (ANT) and alters its interaction with the voltage-dependent anion channel 1 (VDAC1). We found that IR injury induced tyrosine nitration of ANT and that exposure of isolated cardiac mitochondria to ONOO- induced ANT tyrosine, Y81, nitration. The exposure of isolated cardiac mitochondria to ONOO- also led ANT to form high molecular weight proteins and dissociation of ANT from VDAC1. We found that IR injury in isolated hearts, hypoxic injury in H9c2 cells, and ONOO- treatment of H9c2 cells and isolated mitochondria, each decreased mitochondrial bound-hexokinase II (HK II), which suggests that ONOO- caused HK II to dissociate from mitochondria. Moreover, we found that mitochondria exposed to ONOO- induced VDAC1 oligomerization which may decrease its binding with HK II. We have reported that ONOO- produced during cardiac IR injury induced tyrosine nitration of VDAC1, which resulted in conformational changes of the protein and increased channel conductance associated with compromised cardiac function on reperfusion. Thus, our results imply that ONOO- produced during IR injury and hypoxic stress impeded HK II association with VDAC1. ONOO- exposure nitrated mitochondrial proteins and also led to cytochrome c (cyt c) release from mitochondria. In addition, in isolated mitochondria exposed to ONOO- or obtained after IR, there was significant compromise in mitochondrial respiration and delayed repolarization of membrane potential during oxidative (ADP) phosphorylation. Taken together, ONOO- produced during cardiac IR injury can nitrate tyrosine residues of two key mitochondrial membrane proteins involved in bioenergetics and energy transfer to contribute to mitochondrial and cellular dysfunction.

Optical metabolic imaging of irradiated rat heart exposed to ischemia-reperfusion injury.

Whole thoracic irradiation (WTI) is known to cause deterioration in cardiac function. Whether irradiation predisposes the heart to further ischemia and reperfusion (IR) injury is not well known. The aim of this study is to examine the susceptibility of rat hearts to IR injury following a single fraction of 15 Gy WTI and to investigate the role of mitochondrial metabolism in the differential susceptibility to IR injury. After day 35 of irradiation, ex vivo hearts from irradiated and nonirradiated rats (controls) were exposed to 25-min global ischemia followed by 60-min IR, or hearts were perfused without IR for the same protocol duration [time controls (TC)]. Online fluorometry of metabolic indices [redox state: reduced nicotinamide adenine dinucleotide (NADH), oxidized flavin adenine dinucleotide (FAD), and NADH/FAD redox ratio] and functional variables [systolic left ventricular pressure (LVP), diastolic LVP (diaLVP), coronary flow (CF), and heart rate were recorded in the beating heart; developed LVP (dLVP) and rate pressure product (RPP)] were derived. At the end of each experimental protocol, hearts were immediately snap frozen in liquid N2 for later three-dimensional imaging of the mitochondrial redox state using optical cryoimaging. Irradiation caused a delay in recovery of dLVP and RPP after IR when compared to nonirradiated hearts but recovered to the same level at the end of reperfusion. CF in the irradiated hearts recovered better than the control hearts after IR injury. Both fluorometry and 3-D cryoimaging showed that in WTI and control hearts, the redox ratio increased during ischemia (reduced) and decreased on reperfusion (oxidized) when compared to their respective TCs; however, there was no significant difference in the redox state between WTI and controls. In conclusion, our results show that although irradiation of rat hearts compromised baseline cardiovascular function, it did not alter cardiac mitochondrial redox state and induce greater susceptibility of these hearts to IR injury.

Endogenous and Agonist-induced Opening of Mitochondrial Big Versus Small Ca2+-sensitive K+ Channels on Cardiac Cell and Mitochondrial Protection.

Both big (BKCa) and small (SKCa) conductance Ca-sensitive K channels are present in mammalian cardiac cell mitochondria (m). We used pharmacological agonists and antagonists of BKCa and SKCa channels to examine the importance of endogenous opening of these channels and the relative contribution of either or both of these channels to protect against contractile dysfunction and reduce infarct size after ischemia reperfusion (IR) injury through a mitochondrial protective mechanism. After global cardiac IR injury of ex vivo perfused Guinea pig hearts, we found the following: both agonists NS1619 (for BKCa) and DCEB (for SKCa) improved contractility; BKCa antagonist paxilline (PAX) alone or with SKCa antagonist NS8593 worsened contractility and enhanced infarct size; both antagonists PAX and NS8593 obliterated protection by their respective agonists; BKCa and SKCa antagonists did not block protection afforded by SKCa and BKCa agonists, respectively; and all protective effects by the agonists were blocked by scavenging superoxide anions (O2) with Mn(III) tetrakis (4-benzoic acid) porphyrin (TBAP). Contractile function was inversely associated with global infarct size. In in vivo rats, infusion of NS8593, PAX, or both antagonists enhanced regional infarct size while infusion of either NS1619 or DCEB reduced infarct size. In cardiac mitochondria isolated from ex vivo hearts after IR, combined SKCa and BKCa agonists improved respiratory control index and Ca retention capacity compared with IR alone, whereas the combined antagonists did not alter respiratory control index but worsened Ca retention capacity. Although the differential protective bioenergetics effects of endogenous or exogenous BKCa and SKCa channel opening remain unclear, each channel likely responds to different sensing Ca concentrations and voltage gradients over time during oxidative stress-induced injury to individually or together protect cardiac mitochondria and myocytes.

Optical Cryoimaging Reveals a Heterogeneous Distribution of Mitochondrial Redox State in ex vivo Guinea Pig Hearts and Its Alteration During Ischemia and Reperfusion.

Oxidation of substrates to generate ATP in mitochondria is mediated by redox reactions of NADH and FADH2. Cardiac ischemia and reperfusion (IR) injury compromises mitochondrial oxidative phosphorylation. We hypothesize that IR alters the metabolic heterogeneity of mitochondrial redox state of the heart that is only evident in the 3-D optical cryoimaging of the perfused heart before, during, and after IR. The study involved four groups of hearts: time control (TC: heart perfusion without IR), global ischemia (Isch), global ischemia followed by reperfusion (IR) and TC with PCP (a mitochondrial uncoupler) perfusion. Mitochondrial NADH and FAD autofluorescence signals were recorded spectrofluorometrically online in guinea pig ex vivo-perfused hearts in the Langendorff mode. At the end of each specified protocol, hearts were rapidly removed and snap frozen in liquid N2 for later 3-D optical cryoimaging of the mitochondrial NADH, FAD, and NADH/FAD redox ratio (RR). The TC hearts revealed a heterogeneous spatial distribution of NADH, FAD, and RR. Ischemia and IR altered the spatial distribution and caused an overall increase and decrease in the RR by 55% and 64%, respectively. Uncoupling with PCP resulted in the lowest level of the RR (73% oxidation) compared with TC. The 3-D optical cryoimaging of the heart provides novel insights into the heterogeneous distribution of mitochondrial NADH, FAD, RR, and metabolism from the base to the apex during ischemia and IR. This 3-D information of the mitochondrial redox state in the normal and ischemic heart was not apparent in the dynamic spectrofluorometric data.

Stretch-induced increase in cardiac contractility is independent of myocyte Ca2+ while block of stretch channels by streptomycin improves contractility after ischemic stunning.

Stretching the cardiac left ventricle (LV) enhances contractility but its effect on myoplasmic [Ca(2+)] is controversial. We measured LV pressure (LVP) and [Ca(2+)] as a function of intra-LV stretch in guinea pig intact hearts before and after 15 min global stunning ± perfusion with streptomycin (STM), a stretch-activated channel blocker. LV wall [Ca(2+)] was measured by indo-1 fluorescence and LVP by a saline-filled latex balloon inflated in 50 µL steps to stretch the LV. We implemented a mathematical model to interpret cross-bridge dynamics and myofilament Ca(2+) responsiveness from the instantaneous relationship between [Ca(2+)] and LVP ± stretching. We found that: (1) stretch enhanced LVP but not [Ca(2+)] before and after stunning in either control (CON) and STM groups, (2) after stunning [Ca(2+)] increased in both groups although higher in STM versus CON (56% vs. 39%), (3) STM-enhanced LVP after stunning compared to CON (98% vs. 76% of prestunning values), and (4) stretch-induced effects on LVP were independent of [Ca(2+)] before or after stunning in both groups. Mathematical modeling suggested: (1) cooperativity in cross-bridge kinetics and myofilament Ca(2+) handling is reduced after stunning in the unstretched heart, (2) stunning results in depressed myofilament Ca(2+) sensitivity in the presence of attached cross-bridges regardless of stretch, and (3) the initial mechanism responsible for increased contractility during stretch may be enhanced formation of cross-bridges. Thus stretch-induced enhancement of contractility is not due to increased [Ca(2+)], whereas enhanced contractility after stunning in STM versus CON hearts results from improved Ca(2+) handling and/or enhanced actinomyosin cross-bridge cycling.

Reversible blockade of complex I or inhibition of PKCß reduces activation and mitochondria translocation of p66Shc to preserve cardiac function after ischemia.

AIM: Excess mitochondrial reactive oxygen species (mROS) play a vital role in cardiac ischemia reperfusion (IR) injury. P66Shc, a splice variant of the ShcA adaptor protein family, enhances mROS production by oxidizing reduced cytochrome c to yield H2O2. Ablation of p66Shc protects against IR injury, but it is unknown if and when p66Shc is activated during cardiac ischemia and/or reperfusion and if attenuating complex I electron transfer or deactivating PKCß alters p66Shc activation during IR is associated with cardioprotection. METHODS: Isolated guinea pig hearts were perfused and subjected to increasing periods of ischemia and reperfusion with or without amobarbital, a complex I blocker, or hispidin, a PKCß inhibitor. Phosphorylation of p66Shc at serine 36 and levels of p66Shc in mitochondria and cytosol were measured. Cardiac functional variables and redox states were monitored online before, during and after ischemia. Infarct size was assessed in some hearts after 120 min reperfusion. RESULTS: Phosphorylation of p66Shc and its translocation into mitochondria increased during reperfusion after 20 and 30 min ischemia, but not during ischemia only, or during 5 or 10 min ischemia followed by 20 min reperfusion. Correspondingly, cytosolic p66Shc levels decreased during these ischemia and reperfusion periods. Amobarbital or hispidin reduced phosphorylation of p66Shc and its mitochondrial translocation induced by 30 min ischemia and 20 min reperfusion. Decreased phosphorylation of p66Shc by amobarbital or hispidin led to better functional recovery and less infarction during reperfusion. CONCLUSION: Our results show that IR activates p66Shc and that reversible blockade of electron transfer from complex I, or inhibition of PKCß activation, decreases p66Shc activation and translocation and reduces IR damage. These observations support a novel potential therapeutic intervention against cardiac IR injury.

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.

Adding ROS quenchers to cold K+ cardioplegia reduces superoxide emission during 2-hour global cold cardiac ischemia.

We reported that the combination of reactive oxygen species (ROS) quenchers Mn(III) tetrakis (4-benzoic acid) porphyrin (MnTBAP), catalase, and glutathione (MCG) given before 2 hours cold ischemia better protected cardiac mitochondria against cold ischemia and warm reperfusion (IR)-induced damage than MnTBAP alone. Here, we hypothesize that high K(+) cardioplegia (CP) plus MCG would provide added protection of mitochondrial bioenergetics and cardiac function against IR injury. Using fluorescence spectrophotometry, we monitored redox balance, ie reduced nicotinamide adenine dinucleotide and flavin adenine dinucleotide (NADH/FAD), superoxide (O(2) (•-)), and mitochondrial Ca(2+) (m[Ca(2+)]) in the left ventricular free wall. Guinea pig isolated hearts were perfused with either Krebs Ringer's (KR) solution, CP, or CP + MCG, before and during 27°C perfusion followed immediately by 2 hours of global ischemia at 27°C. Drugs were washed out with KR at the onset of 2 hours 37°C reperfusion. After 120 minutes warm reperfusion, myocardial infarction was lowest in the CP + MCG group and highest in the KR group. Developed left ventricular pressure recovery was similar in CP and CP + MCG and was better than in the KR group. O(2) (•-), m[Ca(2+)], and NADH/FAD were significantly different between the treatment and KR groups. O(2) (•-) was lower in CP + MCG than in the CP group. This study suggests that CP and ROS quenchers act in parallel to improve mitochondrial function and to provide protection against IR injury at 27°C.

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.

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-Ringer's 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.

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 Ringer's solution before perfusion for 1 minute just before ischemia with either CP (16 mM K) or LID (1 mM) or Krebs Ringer's (control, 4 mM K). The 1-minute perfusion period assured treatment during ischemia but not on reperfusion. Cardiac function, NADH, FAD, m[Ca], 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[Ca]. Additionally, CP decreased FAD. During ischemia, NADH was higher and reactive oxygen species was lower after CP and LID, whereas m[Ca] was lower only after LID. On reperfusion, NADH and FAD were more normalized, and m[Ca] 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.

Low-flow perfusion of guinea pig isolated hearts with 26 degrees 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.

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.

Ten-hour preservation of guinea pig isolated hearts perfused at low flow with air-saturated Lifor solution at 26{degrees}C: comparison to ViaSpan solution.

There is no suitable solution to preserve hearts for longer than 5 h between donor explant and recipient implant. Lifor is a fully artificial preservation medium containing both a nonprotein oxygen and nutrient carrier (nanoparticles) and cellular nutrients, including amino acids and sugars. We proposed that recirculated Lifor solution would satisfactorily preserve guinea pig isolated hearts perfused at low flow with no added O(2) at room temperature for 10 h. Hearts were isolated from 21 guinea pigs and perfused with Krebs-Ringer (KR) solution (97% O(2) and 3% CO(2)) at 37 degrees C. Heart rate, inflow and outflow O(2) tension, coronary flow, left ventricular pressure (LVP), and maximal and minimal rate of change in LVP (dLVP/dt) were measured. After baseline measurements, hearts were perfused with recirculated Lifor or ViaSpan equilibrated with room air at 15% of control flow at 26 degrees C for 10 h. Hearts were then perfused at 100% flow with KR for 2 h at 37 degrees C. A time control (untreated) group was perfused only with KR solution for 15 h. Lifor arrested and protected hearts against diastolic contracture and maintained a low O(2) extraction. Compared with time controls, Lifor led to a higher developed LVP and coronary flow; %O(2) extraction and cardiac efficiency were similar between these two groups. Hearts similarly treated with ViaSpan exhibited diastolic contracture and lower %O(2) extraction during treatment and, upon reperfusion with KR, exhibited continued diastolic contracture, no return of heart rate or contractility, low coronary flow, low %O(2) extraction, and marked infarction. For long-term cardiac protection, a suitable preservation solution recirculated at low flow and room temperature without supplemental O(2) would reduce the support apparatus required for transport. Lifor was far superior to ViaSpan in meeting these requirements.

ROS scavenging before 27 degrees C ischemia protects hearts and reduces mitochondrial ROS, Ca2+ overload, and changes in redox state.

We have shown that cold perfusion of hearts generates reactive oxygen and nitrogen species (ROS/RNS). In this study, we determined 1) whether ROS scavenging only during cold perfusion before global ischemia improves mitochondrial and myocardial function, and 2) which ROS leads to compromised cardiac function during ischemia and reperfusion (I/R) injury. Using fluorescence spectrophotometry, we monitored redox balance (NADH and FAD), O(2)(*-) levels and mitochondrial Ca(2+) (m[Ca(2+)]) at the left ventricular wall in 120 guinea pig isolated hearts divided into control (Con), MnTBAP (a superoxide dismutase 2 mimetic), MnTBAP (M) + catalase (C) + glutathione (G) (MCG), C+G (CG), and N(G)-nitro-L-arginine methyl ester (L-NAME; a nitric oxide synthase inhibitor) groups. After an initial period of warm perfusion, hearts were treated with drugs before and after at 27 degrees C. Drugs were washed out before 2 h at 27 degrees C ischemia and 2 h at 37 degrees C reperfusion. We found that on reperfusion the MnTBAP group had the worst functional recovery and largest infarction with the highest m[Ca(2+)], most oxidized redox state and increased ROS levels. The MCG group had the best recovery, the smallest infarction, the lowest ROS level, the lowest m[Ca(2+)], and the most reduced redox state. CG and L-NAME groups gave results intermediate to those of the MnTBAP and MCG groups. Our results indicate that the scavenging of cold-induced O(2)(*-) species to less toxic downstream products additionally protects during and after cold I/R by preserving mitochondrial function. Because MnTBAP treatment showed the worst functional return along with poor preservation of mitochondrial bioenergetics, accumulation of H(2)O(2) and/or hydroxyl radicals during cold perfusion may be involved in compromised function during subsequent cold I/R injury.