Mitochondrial involvement in transhemispheric diaschisis following hypoxia-ischemia: Clomethiazole-mediated amelioration.

Mitochondria play a central role in both the physiological and pathophysiological regulation of cell survival/death. Increasing evidence places mitochondrial dysfunction at the center of many neuropathological conditions. The present study investigates the extent of mitochondrial dysfunction in cortical, hippocampal and cerebellar tissues in a rat model of hypoxia-ischemia (HI). We hypothesized that; mitochondrial dysfunction in situ may be prevented by treatment with clomethiazole (CMZ), a GABA(A) receptor agonist. Assessment of mitochondrial FAD-linked respiration at both 1- and 3-day post-HI revealed a marked decrease in activity from ipsilateral cortical and hippocampal regions (P<0.001). In addition, small changes were seen in contralateral cortical and hippocampal tissues as well as in the cerebellum at 3-days (P<0.05). Assessment of the mitochondrial electron transport chain (complexes I-V), and mitochondrial markers of integrity (citrate synthase) and oxidative stress (aconitase) confirmed mitochondrial impairment in ipsilateral regions following HI. Complexes I, II-III, V and citrate synthase were also impaired in contralateral regions and cerebellum 3-days post-HI. Treatment with CMZ (414 mg/kg/day via minipumps) provided marked protection to all aspects of neuronal tissue assessed. Circulating cytokine (interleukin [IL]-1alpha, IL-1beta, tumor necrosis factor [TNF]-alpha, granulocyte macrophage colony-stimulating factor [GM-CSF], IL-4 and IL-10) levels were also assessed in these animals 3-days post-HI. Plasma IL-1alpha, IL-1beta, TNF-alpha and GM-CSF levels were significantly increased post-HI. Treatment with CMZ ameliorated the increases in IL-1alpha, IL-1beta, TNF-alpha and GM-CSF levels while increasing plasma IL-4 and IL-10 levels. This study provides evidence of the extent of mitochondrial damage following an HI-insult. In addition, we have shown that protection afforded by CMZ extends to preservation of mitochondrial function and integrity via anti-inflammatory mediated pathways.

Cell transplantation for the treatment of acute myocardial infarction using vascular endothelial growth factor-expressing skeletal myoblasts.

BACKGROUND: Vascular endothelial growth factor (VEGF) is a promising reagent for inducing myocardial angiogenesis. Skeletal myoblast transplantation has been shown to improve cardiac function in chronic heart failure models by regenerating muscle. We hypothesized that transplantation of VEGF-expressing myoblasts could effectively treat acute myocardial infarction by providing VEGF-induced cardioprotection through vasodilatation in the early phase, followed by angiogenesis effects in salvaging ischemic host myocardium combined with the functional benefits of newly formed, skeletal myoblast-derived muscle in the later phase. METHODS AND RESULTS: Primary rat skeletal myoblasts were transfected with the human VEGF(165) gene using hemagglutinating virus of Japan-liposome with >95% transfection efficiency. Four million of these myoblasts (VEGF group), control-transfected myoblasts (control group), or medium only (medium group) was injected into syngeneic rat hearts 1 hour after left coronary artery occlusion. Myocardial VEGF-expression increased for 2 weeks in the VEGF group, resulting in enhanced angiogenesis without the formation of tumors. Grafted myoblasts had differentiated into multinucleated myotubes within host myocardium. Infarct size (33.3+/-1.4%, 38.1+/-1.4%, and 43.7+/-1.6% for VEGF, control, and medium groups, respectively; P=0.0005) was significantly reduced with VEGF treatment, and cardiac function improved in the VEGF group (maximum dP/dt: 4072.0+/-93.6, 3772.5+/-101.1, and 3482.5+/-90.6 mm Hg/s in the 3 groups, respectively; P=0.0011; minimum dP/dt: -504.2+/-68.5, -2311.3+/-57.0, and -2124.0+/-57.9 mm Hg/s, respectively; P=0.0008). CONCLUSIONS: This combined strategy of cell transplantation with gene therapy could be of importance for the treatment of acute myocardial infarction.

Overexpression of interleukin-1 receptor antagonist provides cardioprotection against ischemia-reperfusion injury associated with reduction in apoptosis.

BACKGROUND: Interleukin-1 (IL-1) plays a role in mediating acute inflammation during ischemia-reperfusion (I/R) injury in the heart, which leads to both necrosis and apoptosis of cardiomyocytes. IL-1 receptor antagonist (IL-1ra) is known to inhibit the effects of IL-1alpha and IL-1beta, resulting in attenuated inflammatory injury, and to protect cells from IL-1beta-induced apoptosis in vitro. We hypothesized that IL-1ra overexpression would provide cardioprotection by reducing inflammation-mediated myocardial damage including apoptosis after I/R injury in vivo. METHODS AND RESULTS: Rat hearts were transfected with human secreted-type IL-1ra gene by intracoronary infusion of Hemagglutinating Virus of Japan liposome and were heterotopically transplanted. IL-1ra overexpression in these hearts was confirmed by enzyme immunoassay and immunohistochemistry. Myocardial tolerance of the transplanted heart was evaluated with the use of a novel system in which the heart, existing within the recipient's abdomen, was given 30 minutes of ischemia by left coronary artery occlusion and 24 hours of reperfusion. Consequently, infarct size was decreased in IL-1ra-transfected hearts compared with control-transfected ones (26.9+/-3.2% versus 46.2+/-3.0%, P=0.001), corresponding to lower myocardial myeloperoxidase activity (2.20+/-0.69 versus 6.82+/-1.19 U/g wet wt, P<0.001) and decreased neutrophil infiltration in histological study. Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling and DNA-laddering studies demonstrated that cardiomyocyte apoptosis was attenuated in IL-1ra-transfected hearts (21.4+/-3.3 versus 41.4+/-3.4%, P=0.002), correlating with reduced post I/R upregulation of Bax, Bak, and caspase-3. CONCLUSIONS: IL-1ra introduced by gene transfection protected myocardium from I/R injury by attenuating the inflammatory response, which was associated with decreased apoptosis. This suggests a potentially important role of IL-1/IL-1ra in myocardial I/R injury and the value of IL-1ra-gene therapy for myocardial preservation.

Gene therapy for myocardial protection: transfection of donor hearts with heat shock protein 70 gene protects cardiac function against ischemia-reperfusion injury.

BACKGROUND: Heat shock protein 70 (HSP70) gene transfection has been shown to enhance myocardial tolerance after normothermic ischemia-reperfusion. We investigated the effect of HSP70 gene transfection on mechanical and endothelial function in a protocol mimicking clinical heart preservation. METHODS AND RESULTS: Rat hearts were infused ex vivo with Hemagglutinating Virus of Japan-liposome complex containing HSP70 gene (HSP, n=8) or no gene (CON, n=8), and heterotopically transplanted into recipient rats. Four days after surgery, transfected hearts were perfused on a Langendorff apparatus for 45 minutes, arrested with St Thomas' No. 1 cardioplegia for 4 hours at 4 degrees C, and reperfused for 1 hour. Mechanical and endothelial function was studied before and after ischemia. Creatine kinase was measured in reperfusion effluent. Hearts underwent Western blotting and immunohistochemistry to confirm HSP70 overexpression. Postischemic recovery of mechanical function (% preischemic+/-SEM) was greater in HSP versus CON: Left ventricular developed pressure recovery was 76.7+/-3.9% versus 60. 5+/-3.1% (P:<0.05); dP/dtmax recovery was 79.4+/-4.9% versus 56. 2+/-3.2% (P:<0.05); dP/dtmin recovery was 74.8+/-4.6% versus 57. 3+/-3.6% (P:<0.05). Creatine kinase release was attenuated in HSP versus CON: 0.22+/-0.02 versus 0.32+/-0.04 IU/min/g wet wt. (P:<0. 05). Recovery of coronary flow was greater in HSP versus CON: 76. 5+/-3.8% versus 59.2+/-3.2% (P:<0.05). Recovery of coronary response to 5-hydroxytryptamine (5 x 10(-)(5) mol/L) was 55.6+/-4.7% versus 23. 9+/-3.2% (P:<0.05); recovery of coronary response to glyceryltrinitrate (15 mg/L) was not different between HSP and CON: 87.4+/-6.9% versus 84.3+/-5.8% (NS). CONCLUSIONS: In a clinically relevant donor heart preservation protocol, HSP70 gene transfection protects both mechanical and endothelial function.

Heat shock protein 70 gene transfection protects mitochondrial and ventricular function against ischemia-reperfusion injury.

BACKGROUND: Upregulation of heat shock protein 70 (HSP70) is beneficial in cardioprotection against ischemia-reperfusion injury, but the mechanism of action is unclear. We studied the role of HSP70 overexpression through gene therapy on mitochondrial function and ventricular recovery in a protocol that mimics clinical donor heart preservation. METHODS AND RESULTS: Hemagglutinating virus of Japan (HVJ)-liposome technique was used to transfect isolated rat hearts via intracoronary infusion of either the HSP70 gene (HSP group, n=16) or no gene (CON group, n=16), which was heterotopically transplanted into recipient rats. Four days after surgery, hearts were either perfused on a Langendorff apparatus for 30 minutes at 37 degrees C (preischemia studies [n=8/group]) or perfused for 30 minutes at 37 degrees C, cardioplegically arrested for 4 hours at 4 degrees C, and reperfused for 30 minutes at 37 degrees C (postischemia studies [n=8/group]). Western blotting and immunohistochemistry confirmed HSP70 upregulation in the HSP group. Postischemic mitochondrial respiratory control indices (RCIs) were significantly better preserved in HSP than in CON hearts: NAD(+)-linked RCI values were 9.54+/-1.1 versus 10.62+/-0.46 before ischemia (NS) but 7.98+/-0.69 versus 1.28+/-0.15 after ischemia (P<0.05), and FAD-linked RCI values were 6.87+/-0.88 versus 6.73+/-0.93 before ischemia (NS) but 4.26+/-0.41 versus 1.34+/-0.13 after ischemia (P<0.05). Postischemic recovery of mechanical function was greater in HSP than in CON hearts: left ventricular developed pressure recovery was 72.4+/-6.4% versus 59.7+/-5.3% (P<0.05), maximum dP/dt recovery was 77.9+/-6.6% versus 52.3+/-5.2% (P<0.05), and minimum dP/dt recovery was 72.4+/-7.2% versus 54.8+/-6.9% (P<0.05). Creatine kinase release in coronary effluent after reperfusion was 0.20+/-0.04 versus 0.34+/-0.06 IU. min(-1). g wet wt(-1) (P<0.05) in HSP versus in CON hearts. CONCLUSIONS: HSP70 upregulation protects mitochondrial function after ischemia-reperfusion injury; this was associated with improved preservation of ventricular function. Protection of mitochondrial function may be important in the development of future cardioprotective strategies.

Atenolol offers better protection than clonidine against cardiac injury in kainic acid-induced status epilepticus.

BACKGROUND AND PURPOSE: Status epilepticus is increasingly associated with cardiac injury in both clinical and animal studies. The current study examined ECG activity for up to 48 h following kainic acid (KA) seizure induction and compared the potential of atenolol and clonidine to attenuate this cardiac pathology. EXPERIMENTAL APPROACH: Sprague-Dawley rats (male, 300-350 g) were implanted with ECG and electrocorticogram electrodes to allow simultaneous telemetric recordings of cardiac and cortical responses during and after KA-induced seizures. Animals were randomized into saline controls, and saline vehicle-, clonidine- or atenolol-pretreated KA groups. KEY RESULTS: KA administration in the saline-pretreated group produced an immediate bradycardic response (maximal decrease of 28 ± 6%), coinciding with low-level seizure activity. As high-level seizure behaviours and EEG spiking increased, tachycardia also developed, with a maximum heart rate increase of 38 ± 7% coinciding with QTc prolongation and T wave elevation. Both clonidine and atenolol pretreatment attenuated seizure activity and reduced KA-induced changes in heart rate, QTc interval and T wave amplitude observed during both bradycardic and tachycardic phases in saline-pretreated KA animals. Clonidine, however, failed to reduce the power of EEG frequencies. Atenolol and to a lesser extent clonidine attenuated the cardiac hypercontraction band necrosis, inflammatory infiltration, and oedema at 48 h after KA, relative to the saline-KA group. CONCLUSIONS AND IMPLICATIONS: Severe seizure activity in this model was clearly associated with altered ECG activity and cardiac pathology. We suggest that modulation of sympathetic activity by atenolol provides a promising cardioprotective approach in status epilepticus.

Time-dependent impairment of mitochondrial function after storage and transplantation of rabbit kidneys.

BACKGROUND: The mitochondrial respiratory chain is implicated as a major target of kidney damage after ischemia-reperfusion. This study measures changes in integrated mitochondrial function and in the activity of enzymes of the respiratory chain after cold storage and transplantation-reperfusion in vivo. METHODS: Mitochondrial oxygen consumption and activities of respiratory chain enzymes and citrate synthase were measured in cortical mitochondria isolated from rabbit kidneys after 1-48 hr of cold ischemia with or without transplantation-reperfusion. RESULTS: State 4 mitochondrial oxygen consumption was significantly increased after 48 hr of ischemia or 24-48 hr of ischemia with transplantation. Prolonged (24 or 48 hr) ischemic storage with and without transplantation caused a significant decrease in state 3 oxygen consumption, as did transplantation after 1, 24, and 48 hr of cold storage. Complex I and complex II-III activity decreased after 24 or 48 hr of ischemia, with transplantation having little additional effect. Complex IV activity was significantly decreased after 48 hr of ischemia, this decrease being exacerbated by transplantation-reperfusion. Complex V activity decreased significantly after 1 hr of ischemia and continued to decrease after 24-48 hr of ischemia. Transplantation after 1-24 hr (but not 48 hr) of ischemia resulted in partial recovery of complex V activity. Citrate synthase activity was decreased significantly only after 48 hr of ischemia and reperfusion, consistent with the loss of mitochondrial membrane integrity seen in electron micrographs of the transplanted 48-hr group. CONCLUSIONS: These data suggest that individual rabbit kidney mitochondrial complexes have different susceptibilities to cold ischemic and reperfusion damage.

Carbon monoxide is a major contributor to the regulation of vascular tone in aortas expressing high levels of haeme oxygenase-1.

The contribution of haeme oxygenase-derived carbon monoxide (CO) to the regulation of vascular tone in thoracic aorta was investigated following induction of the inducible isoform of haeme oxygenase (HO-1). Isometric smooth muscle contractions were recorded in isolated rat aortic ring preparations. Rings were incubated in the presence of the nitric oxide (NO) donor S-nitroso-N-acetyl penicillamine (SNAP, 500 microM) for 1 h, then repetitively washed and maintained for a further 4 h prior to producing a concentration-response curve to phenylephrine (PE, 1-3000 nM). Treatment with SNAP resulted in increased mRNA and protein expression of aortic HO-1 and was associated with a significant suppression of the contractile response to PE (P<0.05 vs control). Immunohistochemical staining procedures revealed marked HO-1 expression in the endothelial layer and, to a lesser extent, in smooth muscle cells. Induction of HO-1 in SNAP-treated rings was associated with a higher 14CO release compared to control, as measured by scintillation counting after incubation of aortas with [2-14C]-L-glycine, the precursor of haeme. Guanosine 3',5'-monophosphate (cyclic GMP) content was also greatly enhanced in aortas expressing high levels of HO-1. Incubation of aortic rings with the NO synthase inhibitor, NG-monomethyl-L-arginine (100 microM), significantly (P<0.05) increased the contractile response to PE in controls but failed to restore PE-mediated contractility in SNAP-treated rings. In contrast, the selective inhibitor of haeme oxygenase, tin protoporphyrin IX (SnPP-IX, 10 microM), restored the pressor response to PE in SNAP-treated rings whilst markedly reducing CO and cyclic GMP production. We conclude that up-regulation of the HO-1/CO pathway significantly contributes to the suppression of aortic contractility to PE. This effect appears to be mediated by the elevation of cyclic GMP levels and can be reversed by inhibition of the haeme oxygenase pathway.

Pyruvate/dichloroacetate supply during reperfusion accelerates recovery of cardiac energetics and improves mechanical function following cardioplegic arrest.

OBJECTIVES: Cardioplegic arrest during cardiac surgery induces severe abnormalities of the pyruvate metabolism, which may affect functional recovery of the heart. We aimed to evaluate the effect of pyruvate and dichloroacetate administration during reperfusion on recovery of mechanical function and energy metabolism in the heart subjected to prolonged cardioplegic arrest. METHODS: Four groups of rat hearts perfused in working mode were subjected to cardioplegic arrest (St. Thomas' No. 1), 4 h of ischaemia at 8 degrees C and reperfusion with either Krebs buffer alone (C) or with 2.8 mM pyruvate (P), with 1 mM dichloroacetate (D), or with a combination of both (PD). Mechanical function was recorded before cardioplegic arrest and at the end of experiments. In groups C and PD, additional experiments were performed using (31)P nuclear magnetic resonance spectroscopy in non-working Langendorff mode to evaluate cardiac high-energy phosphate concentration changes throughout the experiment. RESULTS: Improved recovery of cardiac output (% of the preischaemic value+/-SEM, n=9-12) was observed in all three treated groups (65.7+/-4.3, 59.5+/-5.2 and 59.5+/-5.3% in PD, P and D, respectively) as compared with C (42.2+/-4.6%; P<0.05). Recovery of coronary flow was improved from 66.4+/-3.8 in C to 94.9+/-8.6% in PD (P<0.05). The phosphocreatine recovery rate in the first minutes of reperfusion was increased from 9.9+/-1.5 in C to 31.5+/-4.3 micromol/min per g dry wt in PD (P<0.001). No differences were observed in ATP or phosphocreatine concentrations at the end of experiment. CONCLUSIONS: The administration of pyruvate and dichloroacetate improves the recovery of mechanical function following hypothermic ischaemia. Accelerated restoration of the energy equilibrium in the initial phase of reperfusion may underlie the metabolic mechanism of this effect.

Impairment of hepatic mitochondrial respiratory function following storage and orthotopic transplantation of rat livers.

Prolonged storage of organs for transplant results in tissue damage which may be compounded on reperfusion of the graft tissue. The effect of storage times was examined on hepatic mitochondrial oxygen consumption and activities of complexes I, II-III, IV, and V in mitochondria isolated from rat liver isografts stored for 25 min and 24 h pre- and posttransplantation. While Complex I activity was significantly (P < 0.05) inhibited under all the conditions studied, Complex II-III activity was only significantly (P < 0.05) reduced following transplantation of 24-h stored tissue. Complex IV activity remained unchanged under all the conditions studied. Although Complex V activity was significantly damaged within the first 25 min of ischemia, activity values were partially recovered to control levels following 3 h of reperfusion after transplantation. Prolonged (24 h) storage induced decreases in Complex V activity which were irrecoverable. Mitochondria subjected to 25 min ischemia alone also showed a significant (P < 0.01) decrease in NAD(+)-linked respiratory control indices due to a stimulated state 4 rate. The 24-h storage and transplantation brought about a significantly (P < 0.001) greater inhibition of respiratory control and state 3 respiration. FAD-linked respiration parameters were significantly (P < 0.05) affected in livers subjected to prolonged (24 h) storage or transplantation. These data suggest that a loss of membrane integrity coupled with an inhibition of Complexes I and V and an involvement of Complex II-III in 24-h stored hepatic transplants accounts for mitochondrial respiratory dysfunction in hepatic transplantation injury. No indication of Complex IV damage was found in this study. This study shows that damage to specific mitochondrial complexes occurs as a consequence of hypothermic ischemic injury.

Heat stress contributes to the enhancement of cardiac mitochondrial complex activity.

Hyperthermic stress is known to protect against myocardial dysfunction after ischemia-reperfusion injury. It is unclear however, what energetic mechanisms are affected by the molecular adaptation to heat stress. We hypothesized that mild hyperthermic stress can increase mitochondrial respiratory enzyme activity, affording protection to mitochondrial energetics during prolonged cardiac preservation for transplantation. Rat hearts were excised after heat-stress or sham treatment and subjected to cold cardioplegic arrest and ischemia followed by reperfusion in an ex vivo perfusion system. Cardiac function, mitochondrial respiratory, and complex activities were assessed before and after ischemia. Heat shock protein (Hsp 32, 60, and 72) expression was increased in heat-stressed hearts. This was associated with increased mitochondrial complex activities in heat-stress versus sham-treated groups for complex I-V. During reperfusion, higher complex activities and respiratory control ratios were observed in heat-stressed versus sham-treated groups. Recovery of ventricular function was improved in heat-stressed hearts. Furthermore, mitochondria in reperfused heat-stressed myocardium exhibited intact membranes with packed, parallel, lamellar cristae, whereas in sham-treated myocardium, mitochondria were severely disrupted. This study provides the first evidence of heat-stress-mediated enhancement of mitochondrial energetic capacity. This is associated with increased tolerance to ischemia-reperfusion injury. Protection by heat stress against myocardial dysfunction may be partially due to enhancement of mitochondrial energetics.