Delayed ischaemic contracture onset by empagliflozin associates with NHE1 inhibition and is dependent on insulin in isolated mouse hearts.

AIMS: Sodium glucose cotransporter 2 (SGLT2) inhibitors have sodium-hydrogen exchanger (NHE) inhibition properties in isolated cardiomyocytes, but it is unknown whether these properties extend to the intact heart during ischaemia-reperfusion (IR) conditions. NHE inhibitors as Cariporide delay time to onset of contracture (TOC) during ischaemia and reduce IR injury. We hypothesized that, in the ex vivo heart, Empagliflozin (Empa) mimics Cariporide during IR by delaying TOC and reducing IR injury. To facilitate translation to in vivo conditions with insulin present, effects were examined in the absence and presence of insulin. METHODS AND RESULTS: Isolated C57Bl/6NCrl mouse hearts were subjected to 25 min I and 120 min R without and with 50 mU/L insulin. Without insulin, Empa and Cari delayed TOC by 100 and 129 s, respectively, yet only Cariporide reduced IR injury [infarct size (mean ± SEM in %) from 51 ± 6 to 34 ± 5]. Empa did not delay TOC in the presence of the NHE1 inhibitor Eniporide. Insulin perfusion increased tissue glycogen content at baseline (from 2 ± 2 µmol to 42 ± 1 µmol glycosyl units/g heart dry weight), amplified G6P and lactate accumulation at end-ischaemia, thereby decreased mtHKII and exacerbated IR injury. Under these conditions, Empa (1 µM) and Cariporide (10 µM) were without effect on TOC and IR injury. Empa and Cariporide both inhibited NHE activity, in isolated cardiomyocytes, independent of insulin. CONCLUSIONS: In the absence of insulin, Empa and Cariporide strongly delayed the time to onset of contracture during ischaemia. In the presence of insulin, both Empa and Cari were without effect on IR, possibly because of severe ischaemic acidification. Insulin exacerbates IR injury through increased glycogen depletion during ischaemia and consequently mtHKII dissociation. The data suggest that also in the ex vivo intact heart Empa exerts direct cardiac effects by inhibiting NHE during ischaemia, but not during reperfusion.

Cyclophilin D ablation is associated with increased end-ischemic mitochondrial hexokinase activity.

Both the absence of cyclophilin D (CypD) and the presence of mitochondrial bound hexokinase II (mtHKII) protect the heart against ischemia/reperfusion (I/R) injury. It is unknown whether CypD determines the amount of mtHKII in the heart. We examined whether CypD affects mtHK in normoxic, ischemic and preconditioned isolated mouse hearts. Wild type (WT) and CypD-/- mouse hearts were perfused with glucose only and subjected to 25 min ischemia and reperfusion. At baseline, cytosolic and mtHK was similar between hearts. CypD ablation protected against I/R injury and increased ischemic preconditioning (IPC) effects, without affecting end-ischemic mtHK. When hearts were perfused with glucose, glutamine, pyruvate and lactate, the preparation was more stable and CypD ablation-resulted in more protection that was associated with increased mtHK activity, leaving little room for additional protection by IPC. In conclusion, in glucose only-perfused hearts, deletion of CypD is not associated with end-ischemic mitochondrial-HK binding. In contrast, in the physiologically more relevant multiple-substrate perfusion model, deletion of CypD is associated with an increased mtHK activity, possibly explaining the increased protection against I/R injury.

Reducing mitochondrial bound hexokinase II mediates transition from non-injurious into injurious ischemia/reperfusion of the intact heart

Ischemia/reperfusion (I/R) of the heart becomes injurious when duration of the ischemic insult exceeds a certain threshold (approximately ≥20 min). Mitochondrial bound hexokinase II (mtHKII) protects against I/R injury, with the amount ofmtHKII correlating with injury. Here, we examine whether mtHKII can induce the transition from non-injurious to injurious I/R, by detaching HKII from mitochondria during a non-injurious I/R interva l . Additionally, we examine possible underlying mechanisms (increased reactive oxygen species (ROS), increased oxygen consumption (MVO2) and decreased cardiac energetics) associated with this transition. Langendorff perfused rat hearts were treated for 20 min with saline, TAT-only or 200 nM TAT-HKII, a peptide that translocates HKII from mitochondria. Then, hearts were exposed to non-injurious 15-min ischemia, followed by 30-min reperfusion. I/R injury was determined by necrosis (LDH release) and cardiac mechanical recovery. ROS were measured by DHE fluorescence. Changes in cardiac respiratory activity (cardiac MVO2 and efficiency and mitochondrial oxygen tension (mitoPO2) using protoporphyrin IX) and cardiac energetics (ATP, PCr, ΔGATP) were determined following peptide treatment.When exposed to 15-min ischemia, control hearts had no necrosis and 85% recovery of function. Conversely, TAT-HKII treatment resulted in significant LDH release and reduced cardiac recovery (25%), indicating injurious I/R. This was associated with increased ROS during ischemia and reperfusion. TAT-HKII treatment reducedMVO2 and improved energetics (increased PCr) before ischemia, without affecting MVO2/RPP ratio or mitoPO2. In conclusion, a reduction in mtHKII turns non-injurious I/R into injurious I/R. Loss of mtHKII was associated with increased ROS during ischemia and reperfusion, but not with increased MVO2 or decreased cardiac energetics before damage occurs

Reducing mitochondrial bound hexokinase II mediates transition from non-injurious into injurious ischemia/reperfusion of the intact heart.

Ischemia/reperfusion (I/R) of the heart becomes injurious when duration of the ischemic insult exceeds a certain threshold (approximately ≥20 min). Mitochondrial bound hexokinase II (mtHKII) protects against I/R injury, with the amount of mtHKII correlating with injury. Here, we examine whether mtHKII can induce the transition from non-injurious to injurious I/R, by detaching HKII from mitochondria during a non-injurious I/R interval. Additionally, we examine possible underlying mechanisms (increased reactive oxygen species (ROS), increased oxygen consumption (MVO2) and decreased cardiac energetics) associated with this transition. Langendorff perfused rat hearts were treated for 20 min with saline, TAT-only or 200 nM TAT-HKII, a peptide that translocates HKII from mitochondria. Then, hearts were exposed to non-injurious 15-min ischemia, followed by 30-min reperfusion. I/R injury was determined by necrosis (LDH release) and cardiac mechanical recovery. ROS were measured by DHE fluorescence. Changes in cardiac respiratory activity (cardiac MVO2 and efficiency and mitochondrial oxygen tension (mitoPO2) using protoporphyrin IX) and cardiac energetics (ATP, PCr, ∆GATP) were determined following peptide treatment. When exposed to 15-min ischemia, control hearts had no necrosis and 85% recovery of function. Conversely, TAT-HKII treatment resulted in significant LDH release and reduced cardiac recovery (25%), indicating injurious I/R. This was associated with increased ROS during ischemia and reperfusion. TAT-HKII treatment reduced MVO2 and improved energetics (increased PCr) before ischemia, without affecting MVO2/RPP ratio or mitoPO2. In conclusion, a reduction in mtHKII turns non-injurious I/R into injurious I/R. Loss of mtHKII was associated with increased ROS during ischemia and reperfusion, but not with increased MVO2 or decreased cardiac energetics before damage occurs.

Increased in vivo mitochondrial oxygenation with right ventricular failure induced by pulmonary arterial hypertension: mitochondrial inhibition as driver of cardiac failure?

BACKGROUND: The leading cause of mortality due to pulmonary arterial hypertension (PAH) is failure of the cardiac right ventricle. It has long been hypothesized that during the development of chronic cardiac failure the heart becomes energy deprived, possibly due to shortage of oxygen at the level of cardiomyocyte mitochondria. However, direct evaluation of oxygen tension levels within the in vivo right ventricle during PAH is currently lacking. Here we directly evaluated this hypothesis by using a recently reported technique of oxygen-dependent quenching of delayed fluorescence of mitochondrial protoprophyrin IX, to determine the distribution of mitochondrial oxygen tension (mitoPO2) within the right ventricle (RV) subjected to progressive PAH. METHODS: PAH was induced through a single injection of monocrotaline (MCT). Control (saline-injected), compensated RV hypertrophy (30 mg/kg MCT; MCT30), and RV failure (60 mg/kg MCT; MCT60) rats were compared 4 wk after treatment. The distribution of mitoPO2 within the RV was determined in mechanically-ventilated, anaesthetized animals, applying different inspired oxygen (FiO2) levels and two increment dosages of dobutamine. RESULTS: MCT60 resulted in RV failure (increased mortality, weight loss, increased lung weight), MCT30 resulted in compensated RV hypertrophy. At 30% or 40% FiO2, necessary to obtain physiological arterial PO2 in the diseased animals, RV failure rats had significantly less mitochondria (15% of total mitochondria) in the 0-20 mmHg mitoPO2 range than hypertrophied RV rats (48%) or control rats (54%). Only when oxygen supply was reduced to 21% FiO2, resulting in low arterial PO2 for the MCT60 animals, or when oxygen demand increased with high dose dobutamine, the number of failing RV mitochondria with low oxygen became similar to control RV. In addition, metabolic enzyme analysis revealed similar mitochondrial mass, increased glycolytic hexokinase activity following MCT, with increased lactate dehydrogenase activity only in compensated hypertrophied RV. CONCLUSIONS: Our novel observation of increased mitochondrial oxygenation suggests down-regulation of in vivo mitochondrial oxygen consumption, in the absence of hypoxia, with transition towards right ventricular failure induced by pulmonary arterial hypertension.

Targeting hexokinase II to mitochondria to modulate energy metabolism and reduce ischaemia-reperfusion injury in heart.

Mitochondrially bound hexokinase II (mtHKII) has long been known to confer cancer cells with their resilience against cell death. More recently, mtHKII has emerged as a powerful protector against cardiac cell death. mtHKII protects against ischaemia-reperfusion (IR) injury in skeletal muscle and heart, attenuates cardiac hypertrophy and remodelling, and is one of the major end-effectors through which ischaemic preconditioning protects against myocardial IR injury. Mechanisms of mtHKII cardioprotection against reperfusion injury entail the maintenance of regulated outer mitochondrial membrane (OMM) permeability during ischaemia and reperfusion resulting in stabilization of mitochondrial membrane potential, the prevention of OMM breakage and cytochrome C release, and reduced reactive oxygen species production. Increasing mtHK may also have important metabolic consequences, such as improvement of glucose-induced insulin release, prevention of acidosis through enhanced coupling of glycolysis and glucose oxidation, and inhibition of fatty acid oxidation. Deficiencies in expression and distorted cellular signalling of HKII may contribute to the altered sensitivity of diabetes to cardiac ischaemic diseases. The interaction of HKII with the mitochondrion constitutes a powerful endogenous molecular mechanism to protect against cell death in almost all cell types examined (neurons, tumours, kidney, lung, skeletal muscle, heart). The challenge now is to harness mtHKII in the treatment of infarction, stroke, elective surgery and transplantation. Remote ischaemic preconditioning, metformin administration and miR-155/miR-144 manipulations are potential means of doing just that.

Hexokinase cellular trafficking in ischemia-reperfusion and ischemic preconditioning is altered in type I diabetic heart.

Diabetes mellitus (DM) has been reported to alter the cardiac response to ischemia-reperfusion (IR). In addition, cardioprotection induced by ischemic preconditioning (IPC) is often impaired in diabetes. We have previously shown that the subcellular localisation of the glycolytic enzyme hexokinase (HK) is causally related to IR injury and IPC protective potential. Especially the binding of HK to mitochondria and prevention of HK solubilisation (HK detachment from mitochondria) during ischemia confers cardioprotection. It is unknown whether diabetes affects HK localisation during IR and IPC as compared to non-diabetes. In this study we hypothesize that DM alters cellular trafficking of hexokinase in response to IR and IPC, possibly explaining the altered response to IR and IPC in diabetic heart. Control (CON) and type I diabetic (DM) rat hearts (65 mg/kg streptozotocin, 4 weeks) were isolated and perfused in Langendorff-mode and subjected to 35 min I and 30 min R with or without IPC (3 times 5 min I). Cytosolic and mitochondrial fractions were obtained at (1) baseline, i.e. after IPC but before I, (2) 35 min I, (3) 5 min R and (4) 30 min R. DM improved rate-pressure product recovery (RPP; 71 ± 10 % baseline (DM) versus 9 ± 1 % baseline (CON) and decreased contracture (end-diastolic pressure: 24 ± 8 mmHg (DM) vs 77 ± 4 mmHg (CON)) after IR as compared to control, and was associated with prevention of HK solubilisation at 35 min I. IPC improved cardiac function in CON but not in DM hearts. IPC in CON prevented HK solubilisation at 35 min I and at 5 min R, with a trend for increased mitochondrial HK. In contrast, the non-effective IPC in DM was associated with solubilisation of HK and decreased mitochondrial HK at early reperfusion and a reciprocal behaviour at late reperfusion. We conclude that type I DM significantly altered cellular HK translocation patterns in the heart in response to IR and IPC, possibly explaining altered response to IR and IPC in diabetes.

Pathophysiological consequences of TAT-HKII peptide administration are independent of impaired vascular function and ensuing ischemia.

RATIONALE: We have shown that partial dissociation of hexokinase II (HKII) from mitochondria in the intact heart using low-dose transactivating transcriptional factor (TAT)-HKII (200 nmol/L) prevents the cardioprotective effects of ischemic preconditioning, whereas high-dose TAT-HKII (10 µmol/L) administration results in rapid myocardial dysfunction, mitochondrial depolarization, and disintegration. In this issue of Circulation Research, Pasdois et al argue that the deleterious effects of TAT-HKII administration on cardiac function are likely because of vasoconstriction and ensuing ischemia. OBJECTIVE: To investigate whether altered vascular function and ensuing ischemia recapitulate the deleterious effects of TAT-HKII in intact myocardium. METHODS AND RESULTS: Using a variety of complementary techniques, including mitochondrial membrane potential (ΔΨm) imaging, high-resolution optical action potential mapping, analysis of lactate production, nicotinamide adenine dinucleotide epifluorescence, lactate dehydrogenase release, and electron microscopy, we provide direct evidence that refutes the notion that acute myocardial dysfunction by high-dose TAT-HKII peptide administration is a consequence of impaired vascular function. Moreover, we demonstrate that low-dose TAT-HKII treatment, which abrogates the protective effects of ischemic preconditioning, is not associated with ischemia or ischemic injury. CONCLUSIONS: Our findings challenge the notion that the effects of TAT-HKII are attributable to impaired vascular function and ensuing ischemia, thereby lending further credence to the role of mitochondria-bound HKII as a critical regulator of cardiac function, ischemia-reperfusion injury, and cardioprotection by ischemic preconditioning.

Deletion of the innate immune NLRP3 receptor abolishes cardiac ischemic preconditioning and is associated with decreased Il-6/STAT3 signaling.

OBJECTIVE: Recent studies indicate that the innate immune system is not only triggered by exogenous pathogens and pollutants, but also by endogenous danger signals released during ischemia and necrosis. As triggers for the innate immune NLRP3 inflammasome protein complex appear to overlap with those for cardiac ischemia-reperfusion (I/R) and ischemic preconditioning (IPC), we explored the possibility that the NLRP3 inflammasome is involved in IPC and acute I/R injury of the heart. PRINCIPAL FINDINGS: Baseline cardiac performance and acute I/R injury were investigated in isolated, Langendorff-perfused hearts from wild-type (WT), ASC(-/-) and NLRP3(-/-) mice. Deletion of NLRP3 inflammasome components ASC(-/-) or NLRP3(-/-) did not affect baseline performance. The deletions exacerbated I/R-induced mechanical dysfunction, but were without effect on I/R-induced cell death. When subjected to IPC, WT and ASC(-/-) hearts were protected against I/R injury (improved function and less cell death). However, IPC did not protect NLRP3(-/-) hearts against I/R injury. NLRP3(-/-) hearts had significantly decreased cardiac IL-6 levels with a trend towards lower IL-1ß levels at end reperfusion, suggesting abrogation of IPC through diminished IL-6 and/or IL-1ß signaling. Subsequent experiments showed that neutralising IL-6 using an antibody against IL-6 abrogated IPC in WT hearts. However, inhibition of the IL-1r receptor with the IL-1 receptor inhibitor Anakinra (100 mg/L) did not abrogate IPC in WT hearts. Analysis of survival kinases after IPC demonstrated decreased STAT3 expression in NLRP3(-/-) hearts when compared to WT hearts. CONCLUSIONS: The data suggest that the innate immune NLRP3 protein, in an NLRP3-inflammasome-independent fashion, is an integral component of IPC in the isolated heart, possibly through an IL-6/STAT3 dependent mechanism.

Reduced hexokinase II impairs muscle function 2 wk after ischemia-reperfusion through increased cell necrosis and fibrosis.

We previously demonstrated that hexokinase (HK) II plays a key role in the pathophysiology of ischemia-reperfusion (I/R) injury of the heart (Smeele et al. Circ Res 108: 1165-1169, 2011; Wu et al. Circ Res 108: 60-69, 2011). However, it is unknown whether HKII also plays a key role in I/R injury and healing thereafter in skeletal muscle, and if so, through which mechanisms. We used male wild-type (WT) and heterozygous HKII knockout mice (HKII(+/-)) and performed in vivo unilateral skeletal muscle I/R, executed by 90 min hindlimb occlusion using orthodontic rubber bands followed by 1 h, 1 day, or 14 days reperfusion. The contralateral (CON) limb was used as internal control. No difference was observed in muscle glycogen turnover between genotypes at 1 h reperfusion. At 1 day reperfusion, the model resulted in 36% initial cell necrosis in WT gastrocnemius medialis (GM) muscle that was doubled (76% cell necrosis) in the HKII(+/-) mice. I/R-induced apoptosis (29%) was similar between genotypes. HKII reduction eliminated I/R-induced mitochondrial Bax translocation and oxidative stress at 1 day reperfusion. At 14 days recovery, the tetanic force deficit of the reperfused GM (relative to control GM) was 35% for WT, which was doubled (70%) in HKII(+/-) mice, mirroring the initial damage observed for these muscles. I/R increased muscle fatigue resistance equally in GM of both genotypes. The number of regenerating fibers in WT muscle (17%) was also approximately doubled in HKII(+/-) I/R muscle (44%), thus again mirroring the increased cell death in HKII(+/-) mice at day 1 and suggesting that HKII does not significantly affect muscle regeneration capacity. Reduced HKII was also associated with doubling of I/R-induced fibrosis. In conclusion, reduced muscle HKII protein content results in impaired muscle functionality during recovery from I/R. The impaired recovery seems to be mainly a result of a greater susceptibility of HKII(+/-) mice to the initial I/R-induced necrosis (not apoptosis), and not a HKII-related deficiency in muscle regeneration.

Disruption of hexokinase II-mitochondrial binding blocks ischemic preconditioning and causes rapid cardiac necrosis.

RATIONALE: Isoforms I and II of the glycolytic enzyme hexokinase (HKI and HKII) are known to associate with mitochondria. It is unknown whether mitochondria-bound hexokinase is mandatory for ischemic preconditioning and normal functioning of the intact, beating heart. OBJECTIVE: We hypothesized that reducing mitochondrial hexokinase would abrogate ischemic preconditioning and disrupt myocardial function. METHODS AND RESULTS: Ex vivo perfused HKII(+/-) hearts exhibited increased cell death after ischemia and reperfusion injury compared with wild-type hearts; however, ischemic preconditioning was unaffected. To investigate acute reductions in mitochondrial HKII levels, wild-type hearts were treated with a TAT control peptide or a TAT-HK peptide that contained the binding motif of HKII to mitochondria, thereby disrupting the mitochondrial HKII association. Mitochondrial hexokinase was determined by HKI and HKII immunogold labeling and electron microscopy analysis. Low-dose (200 nmol/L) TAT-HK treatment significantly decreased mitochondrial HKII levels without affecting baseline cardiac function but dramatically increased ischemia-reperfusion injury and prevented the protective effects of ischemic preconditioning. Treatment for 15 minutes with high-dose (10 µmol/L) TAT-HK resulted in acute mitochondrial depolarization, mitochondrial swelling, profound contractile impairment, and severe cardiac disintegration. The detrimental effects of TAT-HK treatment were mimicked by mitochondrial membrane depolarization after mild mitochondrial uncoupling that did not cause direct mitochondrial permeability transition opening. CONCLUSIONS: Acute low-dose dissociation of HKII from mitochondria in heart prevented ischemic preconditioning, whereas high-dose HKII dissociation caused cessation of cardiac contraction and tissue disruption, likely through an acute mitochondrial membrane depolarization mechanism. The results suggest that the association of HKII with mitochondria is essential for the protective effects of ischemic preconditioning and normal cardiac function through maintenance of mitochondrial potential.

Reduction in hexokinase II levels results in decreased cardiac function and altered remodeling after ischemia/reperfusion injury.

RATIONALE: Cardiomyocytes switch substrate utilization from fatty acid to glucose under ischemic conditions; however, it is unknown how perturbations in glycolytic enzymes affect cardiac response to ischemia/reperfusion (I/R). Hexokinase (HK)II is a HK isoform that is expressed in the heart and can bind to the mitochondrial outer membrane. OBJECTIVE: We sought to define how HKII and its binding to mitochondria play a role in cardiac response and remodeling after I/R. METHODS AND RESULTS: We first showed that HKII levels and its binding to mitochondria are reduced 2 days after I/R. We then subjected the hearts of wild-type and heterozygote HKII knockout (HKII(+/)⁻) mice to I/R by coronary ligation. At baseline, HKII(+/)⁻ mice have normal cardiac function; however, they display lower systolic function after I/R compared to wild-type animals. The mechanism appears to be through an increase in cardiomyocyte death and fibrosis and a reduction in angiogenesis; the latter is through a decrease in hypoxia-inducible factor-dependent pathway signaling in cardiomyocytes. HKII mitochondrial binding is also critical for cardiomyocyte survival, because its displacement in tissue culture with a synthetic peptide increases cell death. Our results also suggest that HKII may be important for the remodeling of the viable cardiac tissue because its modulation in vitro alters cellular energy levels, O2 consumption, and contractility. CONCLUSIONS: These results suggest that reduction in HKII levels causes altered remodeling of the heart in I/R by increasing cell death and fibrosis and reducing angiogenesis and that mitochondrial binding is needed for protection of cardiomyocytes.

Partial hexokinase II knockout results in acute ischemia-reperfusion damage in skeletal muscle of male, but not female, mice.

Cellular studies have demonstrated a protective role of mitochondrial hexokinase against oxidative insults. It is unknown whether HK protective effects translate to the in vivo condition. In the present study, we hypothesize that HK affects acute ischemia-reperfusion injury in skeletal muscle of the intact animal. Male and female heterozygote knockout HKII (HK(+/-)), heterozygote overexpressed HKII (HK(tg)), and their wild-type (WT) C57Bl/6 littermates mice were examined. In anesthetized animals, the left gastrocnemius medialis (GM) muscle was connected to a force transducer and continuously stimulated (1-Hz twitches) during 60 min ischemia and 90 min reperfusion. Cell survival (%LDH) was defined by the amount of cytosolic lactate dehydrogenase (LDH) activity still present in the reperfused GM relative to the contralateral (non-ischemic) GM. Mitochondrial HK activity was 72.6 +/- 7.5, 15.7 +/- 1.7, and 8.8 +/- 0.9 mU/mg protein in male mice, and 72.7 +/- 3.7, 11.2 +/- 1.4, and 5.9 +/- 1.1 mU/mg in female mice for HK(tg), WT, and HK(+/-), respectively. Tetanic force recovery amounted to 33 +/- 7% for male and 17 +/- 4% for female mice and was similar for HK(tg), WT, and HK(+/-). However, cell survival was decreased (p = 0.014) in male HK(+/-) (82 +/- 4%LDH) as compared with WT (98 +/- 5%LDH) and HK(tg) (97 +/- 4%LDH). No effects of HKII on cell survival was observed in female mice (92 +/- 2% LDH). In conclusion, in this mild model of acute in vivo ischemia-reperfusion injury, a partial knockout of HKII was associated with increased cell death in male mice. The data suggest for the first time that HKII mediates skeletal muscle ischemia-reperfusion injury in the intact male animal.

Ischemic preconditioning affects hexokinase activity and HKII in different subcellular compartments throughout cardiac ischemia-reperfusion.

The glycolytic enzyme hexokinase (HK) is suggested to play a role in ischemic preconditioning (IPC). In the present study we determined how ischemic preconditioning affects HK activity and HKI and HKII protein content at five different time points and three different subcellular fractions throughout cardiac ischemia-reperfusion. Isolated Langendorff-perfused rat hearts (10 groups of 7 hearts each) were subjected to 35 min ischemia and 30 min reperfusion (control groups); the IPC groups were pretreated with 3 times 5-min ischemia. IPC was without effect on microsomal HK activity, and only decreased cytosolic HK activity at 35 min ischemia, which was mimicked by decreased cytosolic HKII, but not HKI, protein content. In contrast, mitochondrial HK activity at baseline and during reperfusion was elevated by IPC, without changes during ischemia. No effect of IPC on mitochondrial HK I protein content was observed. However, mitochondrial HK II protein content during reperfusion was augmented by IPC, albeit not following the IPC stimulus. It is concluded that IPC results in decreased cytosolic HK activity during ischemia that could be explained by decreased HKII protein content. IPC increased mitochondrial HK activity before ischemia and during reperfusion that was only mimicked by increased HK II protein content during reperfusion. IPC was without effect on the phosphorylation status of HK before ischemia. We conclude that IPC is associated with 1) a biphasic response of increased mitochondrial HK activity before and after ischemia, 2) decreased cytosolic HK activity during ischemia, and 3) cellular redistribution of HKII but not HKI.

Mitochondrial oxygen tension within the heart.

By using a newly developed optical technique which enables non-invasive measurement of mitochondrial oxygenation (mitoPO(2)) in the intact heart, we addressed three long-standing oxygenation questions in cardiac physiology: 1) what is mitoPO(2) within the in vivo heart?, 2) is mitoPO(2) heterogeneously distributed?, and 3) how does mitoPO(2) of the isolated Langendorff-perfused heart compare with that in the in vivo working heart? Following calibration and validation studies of the optical technique in isolated cardiomyocytes, mitochondria and intact hearts, we show that in the in vivo condition mean mitoPO(2) was 35+/-5 mm Hg. The mitoPO(2) was highly heterogeneous, with the largest fraction (26%) of mitochondria having a mitoPO(2) between 10 and 20 mm Hg, and 10% between 0 and 10 mm Hg. Hypoxic ventilation (10% oxygen) increased the fraction of mitochondria in the 0-10 mm Hg range to 45%, whereas hyperoxic ventilation (100% oxygen) had no major effect on mitoPO(2). For Langendorff-perfused rat hearts, mean mitoPO(2) was 29+/-5 mm Hg with the largest fraction of mitochondria (30%) having a mitoPO(2) between 0 and 10 mm Hg. Only in the maximally vasodilated condition, did the isolated heart compare with the in vivo heart (11% of mitochondria between 0 and 10 mm Hg). These data indicate 1) that the mean oxygen tension at the level of the mitochondria within the heart in vivo is higher than generally considered, 2) that mitoPO(2) is considerably heterogeneous, and 3) that mitoPO(2) of the classic buffer-perfused Langendorff heart is shifted to lower values as compared to the in vivo heart.

Ischemic preconditioning, insulin, and morphine all cause hexokinase redistribution.

Association of hexokinase (HK) with mitochondria preserves mitochondrial integrity and is an important mechanism by which cancer cells are protected against hypoxic conditions. Maintenance of mitochondrial integrity also figures prominently as a major characteristic of many cardioprotective manipulations. In this study, we provide evidence that cardioprotective interventions may promote HK redistribution from the cytosol to the mitochondria in the heart. Isolated Langendorff-perfused rat hearts (n = 6/group) were subjected to normoxic perfusion (control, Con), three 5-min ischemia-reperfusion periods (ischemic preconditioning, IPC), 1 U/l insulin (Ins), or 1 microM morphine (Mor). Hearts were immediately homogenized and centrifuged to obtain whole cell, cytosolic, and mitochondrial fractions. HK, lactate dehydrogenase (LDH), and citrate synthase (CS) enzyme activities were determined. No change in LDH or CS present in the cytosol fraction relative to whole cell activity was observed with any of the cardioprotective interventions. By contrast, HK present in the cytosol fraction relative to whole cell activity decreased significantly (P < 0.05) with all cardioprotective interventions, from 0.58 +/- 0.03 (Con) to 0.46 +/- 0.04 (IPC), 0.41 +/- 0.01 (Ins), and 0.45 +/- 0.02 (Mor). In addition, HK relative to CS activity in the mitochondrial fraction increased significantly with cardioprotection, from 0.15 +/- 0.001 (Con) to 0.21 +/- 0.002 (IPC), 0.18 +/- 0.003 (Ins), and 0.21 +/- 0.005 (Mor). Our novel data suggest that well-known cardioprotective interventions share a common end-effector mechanism of cytosolic HK translocation. Association of HK with mitochondria may promote inhibition of the mitochondrial permeability transition pore and thereby reduce cell death and apoptosis.

Ratiometric intracellular calcium imaging in the isolated beating rat heart using indo-1 fluorescence.

Abnormalities in intracellular calcium (Ca(i)(2+)) handling have been implicated as the underlying mechanism in a large number of pathologies in the heart. Study into the relation between Ca(i)(2+) behavior and performance of the whole heart function could provide detailed information into the cellular basis of heart function. In this study we describe an optical ratio imaging setup and an analysis method for the beat-to-beat Ca(i)(2+) videofluorescence images of an indo-1 loaded, isolated Tyrode-perfused beating rat heart. The signal-to-noise ratio and the spatiotemporal resolution (with an optimum of 1 ms and 0.6 mm, respectively) made it possible to register different temporal Ca(i)(2+) transients together with left ventricle pressure changes. The Ca(i)(2+) transients showed that Ca(i)(2+) activation propagates horizontally from left to right during sinus rhythm or from the stimulus site during direct left ventricle stimulation. The indo-1 ratiometric video technique developed allows the imaging of ratio changes of Ca(i)(2+) with a high temporal (1 ms) and spatial (0.6 mm) resolution in the isolated Tyrode-perfused beating rat heart.

Commonly used numbers of microspheres affect cardiac vascular resistance.

The main goal of the study was to examine how the microsphere technique affects the hemodynamics and mitochondrial energy status of the Langendorff-perfused rat heart. The hearts were perfused at a constant flow with Tyrode solution. NADH videofluorometry of the surface of the left ventricle was used to record the mitochondrial energy status as indication of regional ischemia. The effects of seven successive (separated by 10 min) injections of 0.1 ml of saline or (0.05% Tween 20; polysorbate 20, Sigma-Aldrich, St. Louis, MO, U.S.A.) or (0.05% Tween 20 + microspheres) were studied. The number of microspheres per injection were: #1 (2,500), #2 (5,000), #3 (10,000), #4 (20,000), #5 (40,000), #6 (40000), and #7 (80000). The anti-aggregation agent Tween always caused a biphasic response in perfusion pressure. Compared with the Tween effect, the injection of microspheres caused an initial change (mm Hg) in perfusion pressure of #1 (-10), #2 (NS), #3 (NS), #4 (+7.5), #5 (+12.3), #6 (+14.4), #7 (+18.3), and a delayed change (10 min after injection) of #1 (-22.2), #2 (-6.0), #3 (-4.1), #4 (-4.5), #5 (NS), #6 (NS), and #7 (+5.9). The microspheres caused a significant delayed increase in NADH only for injection #6 and #7. Similar results were found for different durations of the input function or when hearts were perfused at constant perfusion pressure. In hearts without flow reserve (10 microM adenosine), Tween injections were without effect, whereas three successive injections of 60,000 microspheres each only caused increases in perfusion pressure and NADH. The data demonstrate that in hearts with flow reserve present, even very low numbers of microspheres (2,000/g heart) cause large decreases in perfusion pressure without obvious signs of ischemia. When flow reserve was exhausted by either microsphere loading or adenosine addition, microspheres only caused increases in perfusion pressure and resulted in detectable ischemia (NADH). It is concluded that microspheres affect the vascular resistance of the heart and that these effects are flow reserve dependent.