It is well known that exercise training (TRN) and ischemic preconditioning (IPC) reduce cardiomyocyte (CM) death following ischemia and reperfusion (IR), but it is unknown if protection can be elicited by moderate exercise TRN or if the effects of TRN and IPC summate to generate greater protection than either protocol alone. To address these questions, we used wheel running, a moderate-intensity paradigm, to TRN male and female rats, and two IPC protocols. Hearts were studied ex vivo with regional ischemia (RI) produced by occluding the left anterior descending coronary artery for 30 min followed by 3 h reperfusion. IPC consisted of a 5-min period of global ischemia and either 5 (PC5) or 10 (PC10) min reperfusion before the initiation of index RI. We found that wheel running and PC10, but not PC5, reduced CM death compared with sedentary, with the TRN effect greater in males. TRN, but not IPC, showed functional protection by reducing the loss of left ventricular developed pressure. Surprisingly, the TRN and PC10 protection from cell death was not additive, suggesting that these modalities converge on the same pathway. We also showed that TRN significantly improved mitochondrial respiration and ΔΨm repolarization during oxidative phosphorylation after IR when compared with SED rats; the improved bioenergetics was associated with the increased prosurvival kinase, hexokinase II (HKII), translocation to mitochondria, which was mediated by increased total Akt and the phosphorylated Akt. We conclude that TRN-induced cardioprotection is mediated, in part, by preservation of mitochondrial bioenergetics likely mediated via the Akt-pAkt-HKII signaling pathway.NEW & NOTEWORTHY Our study produced the novel findings that wheel running in rats, a form of moderate-intensity continuous training (MICT), can provide protection against IR injury as evidenced by a reduction in cardiomyocyte death following 3 h reperfusion and that the protection generated by MICT and IPC is not additive. Importantly, we demonstrate for the first time that MICT-induced cardioprotection is associated with preservations of mitochondrial bioenergetics likely by the Akt-pAkt-HKII signaling pathway.

(1) Background: Mild traumatic brain injury (mTBI), the most prevalent form of traumatic brain injury, often results from repetitive impacts to the head and is associated with long-term neurological impairment. The pathophysiology of mTBI is multifactorial and involves alterations in mitochondrial bioenergetics, a key determinant of neuronal function and survival. Although mitochondrial dysfunction is recognized as a hallmark of mTBI, its long-term effects on bioenergetics and the roles of regulatory cytosolic and mitochondrial proteins remain poorly understood. We hypothesized that repeated mTBI (rmTBI) induces sustained deficits in mitochondrial bioenergetics that are associated with long-term changes in key bioenergetic and other regulatory proteins. (2) Methods: Using the repeated CHIMERA injury model in adult male rats, randomly assigned to sham or rmTBI groups, we assessed mitochondrial respiration in isolated mitochondria and whole cerebral cortex homogenates using a Clark O2 electrode and an Oroboros O2k respirometer at time points ranging from 1 day to 2 months post-injury. Western blotting was performed for expression of regulatory proteins HKI, DRP1, MFN2, VDAC1, and ANT2. (3) Results: At 2 months post-rmTBI, respiration was faster and uncoupled, while ATP synthesis was significantly slowed compared with sham rats. This was accompanied by decreased expression of mitochondrial MFN2 and ANT2, by increased mitochondrial expression of DRP1, and by decreased translocation of HKI to mitochondria. There was no significant difference in VDAC1 expression. Earlier time points showed no significant differences in bioenergetics or protein expression, but neuro-inflammatory markers (GFAP and Iba1) were significantly elevated at these earlier time points of post-injury. (4) Conclusions: These findings indicate that rmTBI leads to a delayed long-term impairment of mitochondrial bioenergetics associated with alterations in proteins critical for bioenergetic regulation and mitochondrial control. This suggests a pathophysiologic mechanism for the persistent cognitive and behavioral deficits observed following rmTBI.

Mitochondria regulate intracellular calcium ion (Ca2+) signaling by a fine-tuned process of mitochondrial matrix (m) Ca2+ influx, mCa2+ buffering (sequestration) and mCa2+ release (Ca2+ efflux). This process is critically important in the neurosynaptic terminal, where there is a simultaneous high demand for ATP utilization, cytosolic (c) Ca2+ regulation, and maintenance of ionic gradients across the cell membrane. Brain synaptic and non-synaptic mitochondria display marked differences in Ca2+ retention capacity. We hypothesized that mitochondrial Ca2+ handling in these two mitochondrial populations is determined by the net effects of Ca2+ uptake, buffering or efflux with increasing CaCl2 boluses. We found first that synaptic mitochondria have a more coupled respiration than non-synaptic mitochondria; this may correlate with the higher local energy demand in synapses to support neurotransmission. When both mitochondrial fractions were exposed to increasing mCa2+ loads we observed decreased mCa2+ sequestration in synaptic mitochondria as assessed by a significant increase in the steady-state free extra matrix Ca2+ (ss[Ca2+]e) compared to non-synaptic mitochondria. Since, non-synaptic mitochondria displayed a significantly reduced ss[Ca2+]e, this suggested a larger mCa2+ buffering capacity to maintain [Ca2+]m with increasing mCa2+ loads. There were no differences in the magnitude of the transient depolarizations and repolarizations of the membrane potential (ΔΨm) and both fractions exhibited similar gradual depolarization of the baseline ΔΨm during additional CaCl2 boluses. Adding the mitochondrial Na+/Ca2+ exchanger (mNCE) inhibitor CGP37157 to the mitochondrial suspensions unmasked the mCa2+ sequestration and concomitantly lowered ss[Ca2+]e in synaptic vs. non-synaptic mitochondria. Adding complex V inhibitor oligomycin plus ADP (OMN + ADP) bolstered the matrix Ca2+ buffering capacity in synaptic mitochondria, as did Cyclosporin A (CsA), in non-synaptic. Our results display distinct differences in regulation of the free [Ca2+]m to prevent collapse of ΔΨm during mCa2+ overload in the two populations of mitochondria. Synaptic mitochondria appear to rely mainly on mCa2+ efflux via mNCE, while non-synaptic mitochondria rely mainly on Pi-dependent mCa2+ sequestration. The functional implications of differential mCa2+ handling at neuronal synapses may be adaptations to cope with the higher metabolic activity and larger mCa2+ transients at synaptosomes, reflecting a distinct role they play in brain function.

Cardiovascular disease is a leading cause of morbidity and mortality, and exercise-training (TRN) is known to reduce risk factors and protect the heart from ischemia and reperfusion injury. Though the cardioprotective effects of exercise are well-documented, underlying mechanisms are not well understood. This review highlights recent findings and focuses on cardiac factors with emphasis on K+ channel control of the action potential duration (APD), β-adrenergic and adenosine regulation of cardiomyocyte function, and mitochondrial Ca2+ regulation. TRN-induced prolongation and shortening of the APD at low and high activation rates, respectively, is discussed in the context of a reduced response of the sarcolemma delayed rectifier potassium channel (IK) and increased content and activation of the sarcolemma KATP channel. A proposed mechanism underlying the latter is presented, including the phosphatidylinositol-3kinase/protein kinase B pathway. TRN induced increases in cardiomyocyte contractility and the response to adrenergic agonists are discussed. The TRN-induced protection from reperfusion injury is highlighted by the increased content and activation of the sarcolemma KATP channel and the increased phosphorylated glycogen synthase kinase-3β, which aid in preventing mitochondrial Ca2+ overload and mitochondria-triggered apoptosis. Finally, a brief section is presented on the increased incidences of atrial fibrillation associated with age and in life-long exercisers.

Introduction: Histone deacetylase (HDAC) 6 functions to remove acetyl groups from lysine residues on histone and non-histone proteins. We showed that the augmented activity of HDAC6 in diabetic mice undergoing myocardial ischemia/reperfusion injury (MIRI) was associated with mitochondrial damage. However, it remains unclear how the inhibition of HDAC6 activity affects post-MIRI cardiac remodeling and function in type 2 diabetes. Hypothesis: HDAC6 inhibition suppresses adverse cardiac remodeling and improves mitochondrial dynamics and cardiac function after MIRI in type 2 diabetic mice. Methods: Type 2 diabetic db/db, db/+, and C57BL/6 mice underwent coronary artery occlusion for 20 min followed by reperfusion. Tubastatin A, a selective inhibitor of HDAC6, was injected intraperitoneally 60 min before coronary artery occlusion and once daily after surgery. Mouse hearts were evaluated with echocardiography 28 days after surgery. Myocardium was imaged using electron microscopy, and the expression of mitochondrial dynamin-related protein 1 (DRP1) and fission 1 was measured by Western blotting analysis. H9c2 cardiomyocytes were subjected to hypoxia for 3 hours followed by normoxia for 24 hours in the presence of 5.5- or 25.0-mM D-dextrose and tubastatin A or vehicle. Results: There were no significant differences in the activity of HDAC6, left ventricular diameters and fractional shortening, mitochondrial density volume and surface area, and the ratios of DRP1/GAPDH and fission 1/GAPDH between the db/+ and C57BL/6 groups. Compared to both db/+ and C57BL/6 groups, HDAC6 activity was lower, left ventricular diameters at both end diastole and end systole were longer, fractional shortening and mitochondrial surface area were smaller, and the expression of DRP1 and fission 1 was increased in the db/db group 28 days after MIRI. Interestingly, 10 mg/kg Tubastatin A significantly mitigated these effects of MIRI in db/db mice. Hypoxia/reoxygenation in the presence of 25.0-mM D-dextrose augmented HDAC6 activity and increased the expression of DRP1 and FIS1, which were blocked by Tubastatin A. Conclusions: Tubastatin A prevents post-MIRI cardiac remodeling and improves cardiac function by limiting mitochondrial fission in type 2 diabetic mice.

The histone deacetylase 6 (HDAC6) inhibitor, tubastatin A, reduces myocardial ischemia/reperfusion injury (MIRI) in type 1 diabetic rats. It remains unclear whether HDAC6 regulates MIRI in type 2 diabetic animals. Diabetes augments activity of HDAC6 and generation of tumor necrosis factor α (TNFα) and impairs mitochondrial complex I (mCI). Here we examined how HDAC6 regulates TNFα production, mCI activity, mitochondria, and cardiac function in type 1 and type 2 diabetic mice undergoing MIRI.

Aims The histone deacetylase 6 (HDAC6) inhibitor, tubastatin A (TubA), reduces myocardial ischaemia/reperfusion injury (MIRI) in type 1 diabetic rats. It remains unclear whether HDAC6 regulates MIRI in type 2 diabetic animals. Diabetes augments the activity of HDAC6 and the generation of tumour necrosis factor alpha (TNF-alpha) and impairs mitochondrial complex I (mCI). Here, we examined how HDAC6 regulates TNF-alpha production, mCI activity, mitochondria, and cardiac function in type 1 and type 2 diabetic mice undergoing MIRI.Methods and results HDAC6 knockout, streptozotocin-induced type 1 diabetic, and obese type 2 diabetic db/db mice underwent MIRI in vivo or ex vivo in a Langendorff-perfused system. We found that MIRI and diabetes additively augmented myocardial HDAC6 activity and generation of TNF-alpha, along with cardiac mitochondrial fission, low bioactivity of mCI, and low production of adenosine triphosphate. Importantly, genetic disruption of HDAC6 or TubA decreased TNF-alpha levels, mitochondrial fission, and myocardial mitochondrial nicotinamide adenine dinucleotide levels in ischaemic/reperfused diabetic mice, concomitant with augmented mCI activity, decreased infarct size, and improved cardiac function. Moreover, HDAC6 knockout or TubA treatment decreased left ventricular dilation and improved cardiac systolic function 28 days after MIRI. H9c2 cardiomyocytes with and without HDAC6 knockdown were subjected to hypoxia/reoxygenation injury in the presence of high glucose. Hypoxia/reoxygenation augmented HDAC6 activity and TNF-alpha levels and decreased mCI activity. These negative effects were blocked by HDAC6 knockdown.Conclusion HDAC6 is an essential negative regulator of MIRI in diabetes. Genetic deletion or pharmacologic inhibition of HDAC6 protects the heart from MIRI by limiting TNF-alpha-induced mitochondrial injury in experimental diabetes. Graphical Abstract

BACKGROUND:Diabetes augments activity of histone deacetylase 6 (HDAC6) and generation of tumor necrosis factor α (TNFα) and impairs the physiological function of mitochondrial complex I (mCI) which oxidizes reduced nicotinamide adenine dinucleotide (NADH) to nicotinamide adenine dinucleotide to sustain the tricarboxylic acid cycle and β-oxidation. Here we examined how HDAC6 regulates TNFα production, mCI activity, mitochondrial morphology and NADH levels, and cardiac function in ischemic/reperfused diabetic hearts.METHODS:HDAC6 knockout, streptozotocin-induced type 1 diabetic, and obese type 2 diabetic db/db mice underwent myocardial ischemia/reperfusion injury in vivo or ex vivo in a Langendorff-perfused system. H9c2 cardiomyocytes with and without HDAC6 knockdown were subjected to hypoxia/reoxygenation injury in the presence of high glucose. We compared the activities of HDAC6 and mCI, TNFα and mitochondrial NADH levels, mitochondrial morphology, myocardial infarct size, and cardiac function between groups.RESULTS:Myocardial ischemia/reperfusion injury and diabetes synergistically augmented myocardial HDCA6 activity, myocardial TNFα levels, and mitochondrial fission and inhibited mCI activity. Interestingly, neutralization of TNFα with an anti-TNFα monoclonal antibody augmented myocardial mCI activity. Importantly, genetic disruption or inhibition of HDAC6 with tubastatin A decreased TNFα levels, mitochondrial fission, and myocardial mitochondrial NADH levels in ischemic/reperfused diabetic mice, concomitant with augmented mCI activity, decreased infarct size, and ameliorated cardiac dysfunction. In H9c2 cardiomyocytes cultured in high glucose, hypoxia/reoxygenation augmented HDAC6 activity and TNFα levels and decreased mCI activity. These negative effects were blocked by HDAC6 knockdown.CONCLUSIONS:Augmenting HDAC6 activity inhibits mCI activity by increasing TNFα levels in ischemic/reperfused diabetic hearts. The HDAC6 inhibitor, tubastatin A, has high therapeutic potential for acute myocardial infarction in diabetes.

The voltage-dependent anion channel 1 (VDAC1) is the primary pathway for metabolite and ion flux across the outer mitochondrial membrane. Oligomerization of VDAC1 that results in the formation of a mega-pore is a critical step in the release of cytochrome c and mitochondrial DNA that triggers apoptosis and inflammasome activation, respectively. However, the underlying mechanism that leads to mega-pore formation is unresolved. Previously, we identified phosphorylation of VDAC1 at S35 to be associated with cardioprotection. Here we report a novel finding that S35 phosphorylation regulates VDAC1 oligomerization and, subsequently, mega-pore formation. In 90 min recordings of channel activity of recombinant VDAC1 in planar lipid bilayers, wild-type (WT) VDAC1 exhibited cumulative current increases due to multiple channel insertions. In 50% of the recordings, a dramatic increase in cumulative channel conductance was observed (>1.8 µS). This was accompanied by a progressive shift at baseline, suggestive of high local ionic accumulation at the bilayer surface. These distinct biophysical characteristics were attributed to mega-pore formation. The probability of similar cumulative large conductances with associated baseline shifts was reduced in the phosphomimetic S35E VDAC1 (20% vs 50% in WT), while it was increased in a non-phosphorylatable mutant S35A (90% vs 50% in WT). In VDAC1 knock-out H9c2 cells expressing WT or mutant (S35A/S35E) VDAC1, 400 µM H2O2 induced the formation of VDAC1 oligomers. However, compared to WT, the formation of trimers and higher order oligomers was significantly decreased in cells expressing the S35E mutant, while it was significantly increased in cells expressing the S35A mutant. These effects are consistent with the corresponding biophysical characteristics observed in the bilayer experiments. Our results suggest that phosphorylation of S35 decreases, while dephosphorylation increases, the susceptibility of VDAC1 to oligomerization and mega-pore formation.

OBJECTIVE:We sought to investigate the biological effects of pre-reperfusion treatments of the liver after warm and cold ischemic injuries in a porcine donation after circulatory death model.SUMMARY OF BACKGROUND DATA:Donation after circulatory death represents a severe form of liver ischemia and reperfusion injury that has a profound impact on graft function after liver transplantation.METHODS:Twenty donor pig livers underwent 60 minutes of in situ warm ischemia after circulatory arrest and 120 minutes of cold static preservation prior to simulated transplantation using an ex vivo perfusion machine. Four reperfusion treatments were compared: Control-Normothermic (N), Control- Subnormothermic (S), regulated hepatic reperfusion (RHR)-N, and RHR-S (n = 5 each). The biochemical, metabolic, and transcriptomic profiles, as well as mitochondrial function were analyzed.RESULTS:Compared to the other groups, RHR-S treated group showed significantly lower post-reperfusion aspartate aminotransferase levels in the reperfusion effluent and histologic findings of hepatocyte viability and lesser degree of congestion and necrosis. RHR-S resulted in a significantly higher mitochondrial respiratory control index and calcium retention capacity. Transcriptomic profile analysis showed that treatment with RHR-S activated cell survival and viability, cellular homeostasis as well as other biological functions involved in tissue repair such as cytoskeleton or cytoplasm organization, cell migration, transcription, and microtubule dynamics. Furthermore, RHR-S inhibited organismal death, morbidity and mortality, necrosis, and apoptosis.CONCLUSION:Subnormothermic RHR mitigates IRI and preserves hepatic mitochondrial function after warm and cold hepatic ischemia. This organ resuscitative therapy may also trigger the activation of protective genes against IRI. Sub- normothermic RHR has potential applicability to clinical liver transplantation.

Radiation therapy (RT) is a commonly used treatment for thoracic cancers. However, the risk of coronary events increase by 4-16% per Gy of heart dose. Our prior studies show that localized heart RT leads to radiation-induced heart disease (RIHD) and increased mortality in Dahl Salt Sensitive (SS) rats starting at 3mo post-RT. We have also shown that whole thoracic RT increases susceptibility to ischemia-reperfusion (IR) injury in Wistar rats. Taken together, we hypothesized that 10weeks after targeted cardiac RT (prior to RIHD) SS rat hearts would have worsened cardiac function as measured by echocardiography and be more susceptible to IR injury. Echocardiograms were performed 10weeks after 24Gy dose of CT-guided localized cardiac RT and in age-matched controls (C). Next, hearts were isolated and perfused ex vivo using the Langendorf method - without ischemia reperfusion (time controls; TC; n=3 in C and RT) or exposed to 25min of global ischemia followed by 60min reperfusion (IR; n=6 in C and RT). During TC and IR, cardiac function, mitochondrial redox state, and vascular reactivity were assessed; the hearts were then sectioned and stained to assess infarct size. Echocardiography of the RT group when compared to C showed an increase in interventricular septal thickness (1.32±0.1 vs 0.78±0.07 cm, p<0.0001; mean±SD) and left ventricular posterior wall thickness (1.03±0.08 vs 0.75±0.06 cm, p<0.005). Accordingly, a decrease in end diastolic (1.79±0.46 vs 2.38±0.35 ml/kg, p<0.05) and end systolic (0.10±0.04 vs 0.23±0.08 ml/kg, p<0.05) volume was also seen, with no significant differences in ejection fraction (94.5±2.1 vs 90.3±3.9 %) or stroke volume (1.69±0.44 vs 2.15±0.27 ml; p>0.05). Interestingly, in the ex vivo perfused hearts, TC RT had a 2 times higher rate-pressure product - a measure of cardiac work - compared to C (p<0.05), and following IR, the RT group recovered to 2.5x higher levels to C (p<0.0001). Following IR, the RT group compared to C also showed a greater recovery of coronary flow rate (1.6x, p<0.05) and rates of contractility (2.25x) and relaxation (lusitropy; 2.05x) (p<0.0001). No difference in mitochondrial redox state, infarct size, and endothelium-dependent and independent vascular reactivity were seen between the groups. In conclusion, our results show for the first time that 10weeks post-targeted cardiac RT, ex vivo hearts show better recovery and cardiac function compared to C after IR through mechanisms other than differences in redox state and vascular reactivity.

Phosphorylation of the voltage-dependent anion channel 1 (VDAC1), an outer mitochondrial membrane protein that is the primary pathway for metabolite and ion flux, has been reported but its physiological impact is not well characterized. Here we demonstrate that site-specific phosphorylation of VDAC1 has differential effects on cell fate during oxidative stress. Using a rat cardiac model of ischemia/reperfusion (IR) injury and quantitative mass spectrometry approach, we identified two phosphorylated residues on VDAC1, S35 and T165, associated with cardioplegia and hypothermia-mediated cardioprotection.

Though angiogenesis has been investigated in depth, vascular regression and rarefaction remain poorly understood. Regression of renal vasculature accompanies many pathological states such as diabetes, hypertension, atherosclerosis, and radiotherapy. Radiation decreases microvessel density in multiple organs, though the mechanism is not known. By using a whole animal (rat) model with a single dose of partial body irradiation to the kidney, changes in the volume of renal vasculature were recorded at two time points, 60 and 90 days after exposure. Next, a novel vascular and metabolic imaging (VMI) technique was used to computationally assess 3D vessel diameter, volume, branch depth, and density over multiple levels of branching down to 70 µm. Four groups of rats were studied, of which two groups received a single dose of 12.5 Gy X-rays. The kidneys were harvested after 60 or 90 days from one irradiated and one non-irradiated group at each time point. Measurements of the 3D vasculature showed that by day-90 post-radiation, when renal function is known to deteriorate, total vessel volume, vessel density, maximum branch depth, and the number of terminal points in the kidneys decreased by 55%, 57%, 28%, and 53%, respectively. Decreases in the same parameters were not statistically significant at 60 days post-irradiation. Smaller vessels with internal diameters of 70-450 µm as well as large vessels of diameter 451-850 µm, both decreased by 90 days post-radiation. Vascular regression in the lungs of the same strain of irradiated rats has been reported to occur before 60 days supporting the hypothesis that this process is regulated in an organ-specific manner and occurs by a concurrent decrease in luminal diameters of small as well as large blood vessels.

Mitochondria pump protons to create a transmembrane pH gradient (ΔpHm) that contributes to polarizing ΔΨm; proton reentry tends to depolarize ΔΨm. Electrophoretic influx of other cations, i.e., Na+, K+, and Ca2+, can also depolarize ΔΨm and must be removed by exchanging one ion for another (chemiosmosis). The importance of regulated Km+ influx in bioenergetics is unclear but Km+ influx can lead to rapid mitochondrial swelling so the added Km+ must be extruded, primarily in exchange for Hm+ entry (KHEm).

Mitochondria pump protons resulting in an alkaline matrix (ΔpHm) important for maintaining ΔΨm. Electrophoretic influx of other cations, Na+, K+, and Ca2+, can depolarize ΔΨm and must be removed by exchanging one ion for another (chemiosmosis). The importance of regulated Km+ influx in bioenergetics is unclear but Km+ influx leads to swelling and efflux of excess Km+, primarily in exchange for Hm+ entry (KHEm). Knowledge of the kinetics of K+ uptake by Km+ channels and its efflux by KHEm exchange is important in understanding how K+ modulates mitochondrial volume and function. To examine this we isolated guinea pig cardiac mitochondria and monitored changes in volume (light scattering), pHm (BCECF), and ΔΨm (TMRM) over 15 min in K+ depleted (Cs+-substituted), Na+ and Ca2+-free buffers at pH 6.9 and 7.6; added were: substrate (control), ±NS1619, a Ca2+-sensitive big conductance K+ channel (BKCa) agonist ±paxilline (PAX), a BKCa antagonist, quinine (QUIN), a KHE inhibitor, valinomycin (VAL), a K+ ionophore, and ADP. We tested if endogenous/NS1619-induced Km+ influx is coupled to KHEm and buffer pH and time-dependent. We found an abrupt rise in pHm (proton pumping) followed by slight swelling, but with enhanced swelling after NS1619 coupled to a slow fall in pHm (KHEm) and a small decrease in ΔΨm. PAX decreased swelling by NS1619; QUIN and VAL increased swelling, and ADP caused volume contraction; all effects were greater at pH 6.9 vs 7.6. In our model, Km+ influx and KHEm activity can be better defined using K+-depleted buffer and pharmacologic tools. Since NS1619 enhances respiration, Km+ influx via Km+ channels, coupled with KHEm to influx H+, may play a role in regulating bioenergetics as well as maintaining volume. These insights may guide understanding of the cardioprotective effects of Km+ influx/efflux.

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 (ΔΨm), 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.

Mitochondrial ionic homeostasis is critical for maintaining mitochondrial morphology, membrane potential, bioenergetics, and calcium handling in the regulation of cell survival and death. Ion fluxes in and out of mitochondria are facilitated by ion channels and transporters in the outer and the inner mitochondrial membranes (OMM and IMM, respectively). We characterized the ionic current at the contact sites between the OMM and IMM using the patch clamp technique. Mitoplasts, mitochondria devoid of the OMM, were prepared by incubating isolated mitochondria in hypotonic solution.

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 (delta psi(m)). Chemiosmosis requires ion ex-changers 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, pH(m), delta psi(m), and respiration that would reflect a role for H+ influx via KHE to regulate bio-energetics. 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+. O-2 content was measured by a Clark electrode, and pH(m), delta psi(m), 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 (delta pH(m) = pH(in)-pH(ext)) at pH(ext )6.9 > > 7.6, so that delta psi(m) was charged and maintained. BKCa agonist NS1619 and antagonist paxilline modified these effects, and KHE inhibitor quinine and K+ iono-phore valinomycin depolarized delta psi(m). 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 delta pH(m), the smaller component of the overall proton motive force, delta mu H+. Thus delta pH(m) establishes and maintains the delta psi(m) 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.

Mitochondrial calcium (Ca2+) plays a key role in regulating normal cardiac function. A physiological increase in mitochondrial matrix calcium [Ca2+]m drives mitochondrial ATP production to meet the high-energy demands during excitation-contraction coupling. However, a pathological increase in [Ca2+]m leads to increased oxidative stress, impaired bioenergetics, and the opening of mitochondrial permeability transition pore (mPTP), a hallmark of the failing heart. Therefore, a better understanding of the [Ca2+]m handling and its role in heart function and dysfunction is of great importance. Here, we describe a detailed protocol for measuring mitochondrial Ca2+ handling in the isolated functionally intact mitochondria from cardiac tissue of the guinea pig.