Myocardial Mitochondria Research Articles

Preservation of Mitochondrial Structure and Function After Cardioplegic Arrest in the Neonate Using a Selective Mitochondrial K ATP Channel Opener

Mitochondrial dysfunction may contribute to early postoperative neonatal heart dysfunction. Diazoxide, a mitochondrial-selective adenosine triphosphate-sensitive potassium-channel opener, is associated with mitochondrial preservation after cardioplegic arrest. We evaluated the mitochondrial-protective effect of diazoxide in terms of mitochondrial structure and function after neonatal cardioplegic arrest. Newborn piglets (age, approximately 14 days) underwent cardiopulmonary bypass and 60 minutes of cardioplegic arrest using cold crystalloid cardioplegic solution (CCP, n = 5) or cold crystalloid cardioplegic solution with diazoxide (CCP+D, n = 5). After 6 hours of recovery, myocardium was harvested. Control myocardium from piglets that did not undergo cardiopulmonary bypass (non-CPB, n = 5) was obtained. Cardioplegic arrest was associated with translocation of Bax to the mitochondria, which was not prevented by diazoxide. Nevertheless, by electron microscopy, CCP-associated remodeling of mitochondrial structure was subjectively diminished in CCP+D hearts. In addition, CCP-associated mitochondrial permeabilization and cytochrome c release into the cytosol were prevented with CCP+D (p < 0.05). In vitro oxygen consumption of isolated mitochondria demonstrated deficient function of mitochondrial complex I in CCP, but it was preserved in the CCP+D myocardial mitochondria (p < 0.05). Complex II and IV activity was not different among groups. In parallel with impaired complex I function, the cardiac adenosine triphosphate content was diminished in CCP hearts, but well maintained in CCP+D hearts (p < 0.05). Although early apoptotic signaling events (Bax translocation) are not prevented by diazoxide, addition of the mitochondrial-selective adenosine triphosphate-sensitive potassium-channel opener to the cardioplegic solution is associated with protection of mitochondrial structural and functional integrity in a clinically relevant model of neonatal cardiac surgery. The mitochondrial-protective effects of diazoxide may contribute to improved postoperative myocardial function in the neonate.

Association of HLA class II DRB1, DPA1 and DPB1 polymorphism with genetic susceptibility to idiopathic dilated cardiomyopathy in Chinese Han nationality

Although the aetiology of idiopathic dilated cardiomyopathy (IDC) remains unclear, many immunological abnormalities involving changes in cell-mediated and humoral immunity may be associated with cardiac impairment in IDC. Autoimmune mechanisms are likely to participate in the pathogenesis of at least a subgroup of IDC and components of the major histocompatibility complex may serve as markers for the propensity to develop immune-mediated myocardial damage. Human leukocyte antigen (HLA) class II genes, which are highly polymorphic, play an important role in the activating of immune responses and thus control the predisposition for or protection from IDC. This study explores the possible contribution of HLA-DRB and DP polymorphisms to IDC susceptibility. DNA genotyping for HLA-DRB1, DPA1 and DPB1 was performed using polymerase chain reaction-sequencing based typing (PCR-SBT) method in 198 IDC patients and 136 random selected healthy Han ethnic individuals living in Northern China. IDC patients were, sub-grouped into asymptomatics (subgroup A), with arrhythmia (subgroup B) and with overt congestive heart failure (subgroup C) according to the clinical manifestations and electrocardiogram or echocardiographic characteristics. ADP/ATP autoantibody was detected in IDC group by immunoblot analysis. The results revealed that HLA-DR15, -DPB*0601 frequencies were significantly elevated in IDC group compared with normal control. The DPB1*0601 allele in homozygous form or in combination with allele DPB1*2301 or *3901, was found present more often in IDC patients. The predominance of HLA-DR4 allele was observed in subgroup B after stratification. However, the frequency of DPB1*0101 allele increased in the control than in the IDC group. The frequency of HLA-DPB1*0601 allele was significantly higher in IDC patients with positive autoantibody against ADP/ATP carrier of myocardial mitochondria in contrast to those with negative autoantibody. We conclude that HLA-DR4, -DR15, -DPB1*0601 alleles confers susceptibility to, while DPB1*0101 allele confers protection from IDC among individuals of northern Chinese Han nationality. The glutamate at position 69 in the second exon of DPB1*0601, as a key residue for special conformation of HLA-DP, may confer predisposition to IDC. HLA-DR and -DP alleles polymorphisms may serve as genetic markers for IDC and be involved in the regulation of the immune specific response to auto or exterior anti-myocardium antibodies.

HLA‐DQA1, ‐DQB1 Polymorphism and Genetic Susceptibility to Idiopathic Dilated Cardiomyopathy in Hans of Northern China

Autoimmune mechanisms are likely to participate in the pathogenesis of at least a subgroup of idiopathic dilated cardiomyopathy (IDC), and components of the major histocompatibility complex (MHC) may serve as markers for the propensity to develop immune-mediated myocardial damage. Human leukocyte antigen (HLA) class II genes, especially HLA-DQ genes, which are highly polymorphic, play an important role in the activation of immune responses and thus control the predisposition to, or protection from, IDC. This study was conducted to investigate the association of HLA-DQA1, -DQB1 allele polymorphisms with an autoantibody against the myocardial mitochondria ADP/ATP carrier, and to explore susceptibility to idiopathic dilated cardiomyopathy (IDC) among the Han ethnic group in northern China and the immunological mechanisms and hereditary susceptibility to IDC. Polymerase chain reaction sequence-specific primer (PCR-SSP) techniques were used to analyze polymorphisms of the second exon of HLA-DQA1 and -DQB1 alleles among 68 unrelated IDC patients, 4 probands of IDC pedigrees, and 100 healthy controls, all of Han nationality and having lived in northern China for a long time. Following echocardiography examination the IDC subjects were stratified according to ejection fraction (EF) values. Those with EF values higher than 50% were placed in subgroup 1, subgroup 2 included the patients with an EF value of 15-35%, and subgroup 3 consisted of those whose EF values were less than 15%. An autoantibody against the myocardial mitochondria ADP/ATP carrier was examined using immunoblot analysis. The frequencies of HLA-DQA1*0501 and HLA-DQB1*0303 were 0.3889 and 0.1806 in the IDC group, significantly higher than those of the healthy controls (0.0900 and 0.0364 respectively, both P < 0.05). The OR was 5.20 (95% CI: 3.60-8.50) and 4.85 (95% CI: 2.56-9.39) respectively. Further analysis of the three subgroups showed a higher frequency of HLA-DQA1*0501 among patients whose EF was less than 15% than those whose EF values were > or =15%. Conversely, the frequencies of HLA-DQA1*0201 and -DQB1*0502, *0504 were significantly lower in the IDC group (0.0139, 0.0139 and 0.0417 respectively) than in the control group (0.2000, 0.0727 and 0.1091 respectively) (P < 0.05). The frequency of the HLA-DQA1*0501 allele was significantly higher in IDC patients whose autoantibody is positive in contrast with those whose autoantibody is negative (18.57% vs. 5.86%, P < 0.05); the relative risk (RR) was 4.32. The other frequencies of HLA-DQA1 and -DQB1 alleles showed no significant difference in the antibody positive and negative groups of IDC patients. The alleles of HLA-DQA1*0501 and HLA-DQB1*0303 were closely associated with poor EF values in the IDC group, and may be involved in susceptibility to the disease. The DQA1*0201 and DQB1*0502, *0504 alleles may confer protection to IDC among individuals of northern Chinese Han nationality. The SER(57) residue in the second exon of DQB1*0502 and *0504 may confer resistance to IDC, and defects or substitution of this amino acid residue at position 57 of the DQbeta chain may be associated with IDC susceptibility. HLA-DQ allele polymorphisms may serve as genetic markers for IDC and be involved in the regulation of the immune specific response to auto or exterior anti-myocardium antibodies.

A Heteroplasmic Mitochondrial DNA 3310 Mutation in the ND1 Gene in a Patient with Type 2 Diabetes, Hypertrophic Cardiomyopathy, and Mental Retardation

A mentally retarded 57-year-old Japanese man with maternally-inherited type 2 diabetes was found to have hypertrophic cardiomyopathy (HCM) that was associated with pathological changes in the myocardial mitochondria. The mitochondrial DNA (mtDNA) of this patient was examined and a C3310 T mutation was found in the ND1 gene, which resulted in the substitution of serine for proline. The normal 3310 mtDNA band could not be detected by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) in mtDNA from his myocardium, pancreas, cerebral tissue, skeletal muscle, and lymphocytes. However two clones sequenced from his pancreatic tissue did not show this C3310 T mutation while forty-eight did. Mitochondria isolated from the lymphocytes of his two sisters also had this mutation. mtDNA point mutations in the ND1 gene region reported thus far have been mostly homoplasmic. However, the C3310 T point mutation that was found in this patient was heteroplasmic, which is a high level of mutation and may represent the pathogenic gene that was responsible for causing mitochondrial disease.

Localization and function of the brain excitatory amino acid transporter type 1 in cardiac mitochondria

Glutamate is the only amino acid extracted by healthy myocardium in net amounts, with uptake further increased during hypoxic or ischemic conditions. Glutamate supplementation provides cardioprotection from hypoxic and reperfusion injury through several metabolic pathways that depend upon adequate transport of glutamate into the mitochondria. Glutamate transport across the inner mitochondrial membrane is a key component of the malate/aspartate shuttle. Glutamate transport in the brain has been well characterized since the discovery of the excitatory amino acid transporter (EAAT) family. We hypothesize that a protein similar to EAAT1 found in brain may function as a glutamate transporter in cardiac mitochondria. Rat heart total RNA was screened by reverse transcriptase-polymerase chain reaction with an array of primer pairs derived from the rat brain EAAT1 cDNA sequence, yielding a 3786-bp cDNA comprising a 1638-bp open reading frame identical to rat brain EAAT1 with flanking 5′- and 3′-untranslated regions. Northern blot analysis confirmed a 4-kb mRNA product in rat heart and brain, with greater abundance in brain. A protein of the predicted approximate 60-kD size was recognized in myocardial lysates by an anti-EAAT1 polyclonal antibody produced against an amino-terminal peptide from human EAAT1. The protein enriched in rat heart mitochondria by immunoblot, co-localized with the mitochondrial protein cytochrome c by immunohistochemistry, and further localized to the inner mitochondrial membrane upon digitonin fractionation of the mitochondria. In myocytes overexpressing EAAT1, activity of the malate/aspartate shuttle increased by 33% compared to non-transfected cells ( P = 0.004). These data indicate that EAAT1 is expressed in myocardial mitochondria, and functions in the malate/aspartate shuttle, suggesting a role for EAAT1 in myocardial glutamate metabolism.

Protective effect of urinary trypsin inhibitor on myocardial mitochondria during hemorrhagic shock and reperfusion.

To examine the mitochondrial function in the myocardium after hemorrhagic shock and reperfusion and to evaluate the protective effect of urinary trypsin inhibitor (UTI) on mitochondria. Animal experiment. University research laboratory. Wistar rats receiving 50,000 units/kg/hr of UTI (n = 27; UTI group) and control rats (n = 26; control group). Rats were subjected to low-perfusion ischemia with the left ventricular systolic pressure maintained at 50 mm Hg for 60 mins by bleeding, followed by a 60-min reperfusion by transfusion of shed blood. UTI was infused continuously from 10 mins before bleeding. Cardiac function was measured before bleeding, after bleeding, and after transfusion; at each determination point, the myocardial contents of adenosine triphosphate (ATP), creatine phosphate (P-Cr), pyruvate (Pyr), and lactate (Lac) were measured enzymatically. The cytosolic phosphorylation potential (PP) as well as the redox potential of the oxidized form of nicotinamide adenine dinucleotide/reduced form of nicotinamide adenine dinucleotide couple in mitochondria (Eh(NAD+/NADH)) and change of Gibbs free energy in ATP hydrolysis (deltaG(ATP hydrolysis) energy) were calculated. Cardiac function decreased during hemorrhagic shock but improved significantly in the UTI group after transfusion compared with the control group. Lac and the Lac/Pyr ratio were significantly lower in the UTI group than in the control group after transfusion. ATP and P-Cr were significantly higher in the UTI group than in the control group after transfusion. PP (x10(3) M-1), Eh(NAD+/NADH) (x - 1 mV), and deltaG(ATP hydrolysis) (x - 1 kcal/mol) were 1.9 +/- 0.4, 266 +/- 4, and 9.7 +/- 0.2, respectively, in the control group and 4.0 +/- 0.9, 274 +/- 5 and 13.0 +/- 0.2, respectively, in the UTI group after transfusion (p <.001, p <.001, and p <.001, respectively). In reperfusion after hemorrhagic shock, oxidative phosphorylation in myocardial mitochondria is impaired and energy production remains reduced, even after reperfusion. UTI contributed to the recovery of cardiac function after reperfusion, probably by reducing the severity of mitochondrial dysfunction during a state of shock and by maintaining energy production.

14(R,S)-[18F]Fluoro-6-thia-heptadecanoic acid as a tracer of free fatty acid uptake and oxidation in myocardium and skeletal muscle.

14( R, S)-[(18)F]Fluoro-6-thia-heptadecanoic acid ([(18)F]FTHA) is a long-chain fatty acid substrate for fatty acid metabolism. [(18)F]FTHA has been used to study fatty acid metabolism in human heart and skeletal muscle. It has been suggested that the rate of radioactivity accumulation in the myocardium reflects the beta-oxidation rate of free fatty acids (FFAs). However, the net accumulation of FFAs in tissue always represents the sum of FFA oxidation and incorporation into triglycerides. The fraction of [(18)F]FTHA entering directly into mitochondria for oxidation has not been previously measured. Eight anaesthetized pigs were studied with [(18)F]FTHA and positron emission tomography (PET). Immediately after each PET experiment, tissue samples from myocardium and skeletal muscle were taken for the isolation of mitochondria and measurements of radioactivity accumulation, and for intracellular [(18)F]FTHA metabolite analysis. Fractional [(18)F]FTHA uptake rates were calculated both by graphical analysis of PET data and by measuring (18)F in the tissue samples. Fractional [(18)F]FTHA uptake rates based on the analysis of tissue samples were 0.56+/-0.17 ml g(-1) min(-1) and 0.037+/-0.007 ml g(-1) min(-1) for myocardium and skeletal muscle (mean +/- SD), respectively. The myocardial results obtained from the PET data (0.50+/-0.11 ml g(-1) min(-1)) were similar to the values obtained from the tissue samples ( r=0.94, P=0.002). We also found that 89%+/-23% (mean+/-SD, n=7) of the (18)F entered mitochondria in myocardium, as compared with only 36%+/-15% (mean+/-SD, n=7) in skeletal muscle. Intracellular [(18)F]FTHA metabolite analysis showed that a major part of [(18)F]FTHA is metabolized in the mitochondria in the heart. Our data suggest that ~89% of [(18)F]FTHA taken up by the heart enters mitochondria. This supports the hypothesis that [(18)F]FTHA traces FFA beta-oxidation in the heart. In contrast to this, only ~36% of [(18)F]FTHA accumulated in skeletal muscle appears to directly enter mitochondria; the majority is taken up by the other cell fractions, suggesting that in skeletal muscle [(18)F]FTHA traces FFA uptake but not specifically FFA beta-oxidation.

Possible association of HLA-DRB1 gene with the autoantibody against myocardial mitochondria ADP/ATP carrier in dilated cardiomyopathy.

To probe the genetic background and immunopathogenesis of dilated cardiomyopathy (DCM) 77 patients with DCM, HLA-DRB1 gene polymorphism were analyzed by using the polymerase chain reaction/sequence specific primer (PCR/SSP) technique and autoantibody against myocardial mitochondria ADP/ATP carrier were examined by using the Immunoblot analysis. The frequency of HLA-DRB1*0901 allele was significantly higher in DCM patients in which autoantibody against ADP/ATP carrier of myocardial mitochondria is positive in contrast with those in which the autoantibody is negative (25.46% vs 3.45%, P < 0.05), the relative risk (RR) being 9.56. The other frequencies of HLA-DRB1 alleles have no significant difference in the antibody positive group and negative group. It is possible that a subset of DCM patients may exist in which autoimmunity is associated with genetic factors.

Changes of myocardial mitochondrial Ca2+ transport and mechanism in the early stage after severe burns

Objective: To investigate the change of myocardial mitochondrial Ca2+ transport and its mechanism in the early stage after burns. Methods: Forty-eight Wistar rats were randomized into a normal control group (n=8) and a burns group (n=40). The rats of the burns group were given a 30%TBSA full-thickness. Myocardial mitochondria were isolated from normal and scalded rats which were sacrificed at the 1st, 3rd, 6th, 12th and 24th hour post-burn. Mitochondrial Ca2+ transport velocity, membrane potential (MP), ATP content and cytosolic Ca2+ concentration [Ca2+]c were determined. The effects of exogenous ATP on mitochondrial Ca2+ transport velocity were also investigated. Results: Mitochondrial Ca2+ uptake velocity of the 1st hour post-burn was higher than that of the control, and Ca2+ release velocity did not change significantly, but mitochondrial Ca2+ transport velocity, MP and ATP content were all decreased at the 3rd, 6th, 12th and 24th hour post-burn. Mitochondrial Ca2+ uptake velocity was positively correlated with MP after burn, and Ca2+ release velocity with mitochondrial ATP content. [Ca2+]c was increased at the 3rd, 6th, 12th and 24th hour post-burn. Exogenous ATP increased myocardial mitochondrial Ca2+ uptake velocity of rats at the 3rd and 6th hour post-burn and Ca2+ release velocity at the 3rd, 6th and 12th hour post-burn. Conclusions: Increase of [Ca2+]c led to reinforcement of mitochondrial Ca2+ uptake at the beginning of the post-burns period. ATP depletion and MP collapse cause myocardial mitochondrial Ca2+ transport disorder in the following stages.

Calcium induced the damage of myocardial mitochondrial respiratory function in the early stage after severe burns

Objective: To explore the role of Ca 2+ in the damage to myocardial mitochondrial respiratory function in the early stage after severe burns. Methods: An experimental model of 30%TBSA full-thickness skin scalding was reproduced in rats. Myocardial mitochondria were isolated from control and burned rats in the 1st, 3rd, 6th, 12th and 24th hour post-burn. The mitochondrial respiratory function, contents of mitochondrial calcium ([Ca 2+] m), activities of mtPLA 2, mtNOS, F 0F 1-ATPase and cytochrome c oxidase were determined. Results: (1) At the 1st hour post-burn, [Ca 2+] m was increased significantly and the myocardial mitochondrial respiratory function was significantly reinforced. At the same time, mitochondrial respiratory control rate (RCR) was elevated and positively correlated with [Ca 2+] m ( r=0.8415, P<0.01). At the 3rd, 6th, 12th and 24th hour post-burn, [Ca 2+] m increased further to a higher level, however, the mitochondrial respiratory function was decreased from the peak value at 6 h, and RCR was negatively correlated with [Ca 2+] m. (2) The activities of mtNOS and mtPLA 2 were higher significantly at the 3rd, 6th, 12th and 24th hour post-burn than that of the control. After severe burns, mtNOS and mtPLA 2 activities were both positively correlated with [Ca 2+] m ( r=0.8945, P<0.05; r=0.9271, P<0.01, respectively). (3) The F 0F 1-ATPase synthetic activity increased at the 1st hour post-burn, but it decreased to 51.4, 44.9, 77.6 and 87.4% of that of the control at the 3rd, 6th, 12th and 24th hour post-burn respectively. The F 0F 1-ATPase hydrolytic activity decreased at the 1st hour post-burn and increased at the 3rd, however, it decreased again at the 6th, 12th and 24th hour post-burn. The activity of cytochrome c oxidase at the 3rd, 6th, 12th and 24th hour was low compared to the control. Conclusions: The changes of [Ca 2+] m were involved in damage to or regulation of mitochondrial respiratory function after severe burns. Appropriate increase of [Ca 2+] m reinforced the mitochondrial respiration at 1st hour after of burn injury, but Ca 2+ severe overload impairing F 0F 1-ATPase and cytochrome c oxidase directly, or, indirectly by activation of mtPLA 2 and mtNOS, might play an important role in damage to myocardial mitochondrial respiratory function at later stages after severe burns.

Effects of SM-20550, a selective Na+-H+ exchange inhibitor, on the ion transport of myocardial mitochondria.

The effect of a novel Na+-H+ exchange inhibitor, SM-20550 [N-(aminoiminomethyl)-1,4-dimethyl-1H-indole-2-carboxamide methanesulfonate] (SM) on the ion transport of myocardial mitochondria was studied using ion fluorometry and superfusion techniques. Isolated mitochondria from the guinea-pig heart were pre-loaded with fluoroprobes of either BCECF AM for H+, SBFI AM for Na+ or fura-2 AM for Ca2+. Initially, the treated mitochondria were superfused with a normal medium (MOPS-buffer, pH 7.4, 24 degrees C), subsequently fluorometric experiments on the Na+, H+, Ca2+ mobilization across the mitochondrial membrane were performed. The intramitochondrial pH (pHm) was increased by the superfusion of Na+ at physiological cytosolic concentrations of 10 mM, indicating the existence of a Na+-H+ exchange in mitochondrial membranes. The Na+ induced elevation of pH was dose-dependently inhibited by SM 1 microM (delta pHm; 45% as drug-free 100%), and 10 microM (delta pHm; 70%), as observed in our experiments with the myocardial sarcolemmal membrane. The selective Na+-H+ exchange inhibitor SM reduced such pHm elevations more markedly than that of EIPA [5-(N-ethyl-N-isopropyl) amiloride]. The Na+-H+ exchange inhibitors, SM and EIPA suppressed the intramitochondrial Ca2+ elevation ([Ca2+]m) brought on by external Ca2+ concentration changes: The pretreatment with SM 1 microM, 10 microM and EIPA 10 microM reduced the [Ca2+]m influx by 28.3, 56.5 and 63%, respectively. Additionally, the [Ca2+]m elevation induced by acidification of the perfusate was reduced by the prior infusion of SM and EIPA. Pretreatment of mitochondria with SM or EIPA which had beneficial effects on the left ventricular developed pressure (LVDP) in the ischemia-reperfusion injury of Langendorff hearts, reduced the intramitochondrial Na+ and pHm levels, indicating interplay of the inhibitory mechanism of Ca2+-uptake into mitochondria coupled with Na+-H+ exchange. These findings suggested that protective effects of Na+-H+ exchange inhibitors on reperfused myocardium are due in part to the Ca2+-paradox at the mitochondria level.

Nicorandil metabolism in rat myocardial mitochondria.

The in vitro study using rats was carried out to clarify the hypothesis that nicorandil is denitrated and then may produce nitric oxide (NO) in myocardial mitochondria. In the presence of a NADPH-generating system, [14C]nicorandil, which was incubated in mitochondrial and microsomal fractions of the lung, heart, or liver, was converted to its main denitrated metabolite, SG-86 and other metabolites. Apparent Km and Vmax for nicorandil in mitochondrial and microsomal fractions of the heart were considerably similar to those of the lung, but completely different from those of the liver. It seems that glutathione-S-transferases (GSTs) are not primarily involved in the conversion of nicorandil to SG-86, because a known GST inhibitor, indomethacin, did not affect the nicorandil degradation in the mitochondrial fraction. Nitrite, the stable metabolite of NO, was measured by the Griess reaction. In the presence of an NADPH-generating system, nicorandil significantly increased nitrite production in myocardial mitochondria, but SG-86 did not. These data strongly indicate that nicorandil is metabolized to SG-86 in myocardial mitochondria, then releasing NO, and that GSTs are not primarily responsible for the conversion of nicorandil to SG-86.

The protective effect of lipoxygenase inhibitor FLM 5011 on mitochondria ultrastructure of ischemic and reperfused cardiomyocytes

Lipoxygenase inhibitor FLM 5011 was used in experimental ischemia and reperfusion with dogs to investigate its ultrastructure-preserving effects on the mitochondria of myocardium. Ischemic and non-ischemic areas of the heart were ultrastructural-morphometric analysed, which revealed that FLM 5011 was able to diminish ischemic damage especially of mitochondria. The protective effects on mitochondria consisted mainly in reduction of defective intramitochondrial areas and in excellent protection of the structural integrity of cristae and matrix. The injury of mitochondria by ischemia/reperfusion in unprotected condition was partly more pronounced in the non-ischemic than in the ischemic area of the hearts probably caused by compensatory overload of the residual myocardium.

Myocardial Distribution and Biotransformation In Vitro and In Vivo of Nicorandil in Rats, with Special Reference to Mitochondria

This study reports subcellular localization of nicorandil in the myocardium and metabolism in mitochondria after oral dosing of 3 mg/kg nicorandil to rats. In the in vitro experiments, nicorandil, which was incubated with tissue homogenates (liver, kidney, heart, and small intestine), was metabolized to its denitrated compound, SG-86, and unknown substances. In the absence of a NADPH-generating system in the heart, the metabolic activity existed only in the mitochondrial fraction, but not in cytosolic and microsomal fractions. In the presence of the system, the activity in the mitochondrial fraction became much higher. To examine subcellular distribution of nicorandil in the myocardium, [14C]nicorandil was orally given to rats. Fifteen minutes after oral dosing of 3 mg/kg [14C]nicorandil, of which myocardial concentration reached a peak, nicorandil and SG-86 were found in mitochondrial fractions as well as in cytosolic and microsomal ones of the heart. Electron-microscopic autoradiograms, 15 min after oral dosing of 3 mg/kg [3H]nicorandil to rats, also showed the existence of the silver grains (showing radioactivity) in mitochondria of the heart. We conclude that nicorandil given orally is distributed in mitochondria of the heart, being partly transformed into SG-86, and that the myocardial mitochondria may be a potential site of action of nicorandil, an opener of KATP channels, which have been demonstrated to be present in this subcellular particle.

P–213 - Protective role of nitric oxide synthase in myocardial mitochondria against ischemia-reperfusion injury in guinea pigs.

P–213 - Protective role of nitric oxide synthase in myocardial mitochondria against ischemia-reperfusion injury in guinea pigs.

A delivery circuit for normothermic reperfusion model.

In order to facilitate the introduction and assessment of Injectio Radix Ginseng Compositae in normothermic reperfusion model, a simplified delivery circuit is described herein. The circuit added mainly a venous reservoir collecting Injectio Radix Ginseng Compositae with cardiopulmonary bypass. Injectio Radix Ginseng Compositae was pumped into the ascending aorta of the animal after mixing with cardioplegia in the venous reservoir. The circuit was acceptable in terms of price by most inland medical laboratories. Perfusion as well as biochemical results monitored were satisfactory. Significant difference was noted between the average damage rates of the reperfused myocardial mitochondria in groups by ultrastructural analysis (70.12 +/- 2.56% vs. 25.33 +/- 17.62%, p = 0.023). No complications associated with perfusion technique occurred in either group. The circuit could offer a constant arterial perfusion, and was proved to be simple, safe and effective in cohort.

Protection of myocardial mitochondria against oxidative damage by selenium-containing abzyme m4G3.

Selenium-containing abzyme (m4G3) was prepared and its protection of myocardial mitochondria against oxidative damage was studied using the swelling of mitochondria, quantity of lipid peroxidation products, and change in cytochrome-c oxidase activity as a measure of mitochondrial damage. The results showed that m4G3 could inhibit mitochondrial damage caused by the hypoxanthine-xanthine oxidase system in vitro. Electronic spin resonance (ESR) studies demonstrated that m4G3 could decrease the amount of free radicals generated in the damage system.

Cytochemistry and ultrastructure of canine myocardium undergoing global ischemia and reperfusion injury.

In order to highlight severity of myocardial injury during the course of global ischemia and reperfusion, cytochemistry of glycogen and succinate dehydrogenase (SDH) as well as hematoxylin and eosin staining (H & E) and electron microscopy were observed in canine myocardium. Seven mongrel dogs were selected for reperfusion injury after global ischemia in this study. Myocardial biopsies were taken from the anterior wall of the left ventricle (a) after cardiopulmonary bypass (the first biopsy); (b) at the end of the aortic crossclamp (the second biopsy); and (c) 30 minutes after crossclamp removal (the third biopsy). All biopsies were cytochemically assessed, and the latter two, for electron microscopic studies. The averages of myocardial necrotic rate and surface to volume ratio of myocardial mitochondria were calculated under electron microscopy and in electron microscopic slices, respectively. Myofibrillae were of normal morphology in the first biopsy; in wave-shape and partly vacuolated, with large and deformed nuclei in the second one; and in wave-shape and severely vacuolated in the third one, in H & E. Glycogen granules were variously stained in moderate, weak and intensive positive reactions in the three biopsies respectively in glycogen staining. SDH was stained in intensive, weak, and moderate positive reactions in three, respectively. By electron microscopy, Z bands twisted severely, and local dissolution of cristae and matrix occurred in a minority of the mitochondria in the second biopsy; and majority of the Z bands in necrotic region had disappeared, the myofibrillae were obscure and patchily dissolved. Clustered and deformed mitochondria could be found in the third biopsy. Significant difference could be noted between the averages of the second and third biopsies (14.88 +/- 3.09% vs. 60.25 +/- 8.55%, p < 0.001). The surface to volume ratio of the ischemic mitochondria was much bigger than that of the reperfused (3.95 +/- 1.09 micron-1 vs. 2.77 +/- 0.93 micron-1, p = 0.041). Myocardial injury was more severe in reperfusion than in ischemia myocardium. There were correlations between histobiochemical and ultrastructural alterations in damaged canine myocardium.

In VivoHeat Shock Protects Rat Myocardial Mitochondria

Heat shock (HS)/stress proteins (HSP) provide protection from a variety of stresses other than HS, including oxidative stress and mitochondria have been implicated as the target of HS-related protection in stressed cultured cells. Here we investigated whether mitochondria also are targets for the HS-mediated protection in vivo. Sprague Dawley rats were exposed, or not, to HS (41°C, 15 min). After a 21h recovery period, hearts were excised and perfused with or without H2O2(0.15 mM). Myocardial mitochondria were then isolated, and their oxygen consumption was analyzed. HS prevented H2O2-induced alterations in state 3 respiration while increasing the expression of Hsp70 and heme oxygenase (HO). Thus,in vivoHS protects rat myocardial mitochondrial respiration against the deleterious effects of oxidative injury, a protection relating to Hsp70 and/or HO and targeting state 3 respiration.

Interaction of electron leak and proton leak in respiratory chain of mitochondria--proton leak induced by superoxide from an electron leak pathway of univalent reduction of oxygen.

By incubating the isolated rat myocardial mitochondria with xanthine-xanthine oxidase, an exogenous superoxide (O2.-) generating system, and by ischemia-reperfusion procedure of isolated rat heart as an endogenous O2.- generating system, it was found that both sources O2.- showed the same injurious effects on mitochondrial function resulting in (i) increasing proton leak rate, lowering proton pumping activity and H+/2e ratio of respiratory chain, and (ii) decreasing transmembrane potential of energized mitochondrial inner membrane by succinate oxidation. The injurious effects of O2.- on these mitochondrial bioenergitical parameters mentioned above exhibited a dosage- or reaction time-dependent mode. O2.- has no effects on the electron transfer activity and transmembrane potential of nonenergized mitochondria. Being a superoxide scavenger, 3,4-dihydroxylphenyl lactate showed obvious protection effects against damage of both exogenous superoxide sources from xanthine-xanthine oxidase system and endogenous O2.- source from ischemia-reperfused heart on proton pumping activity, H+/2e ratio and transmembrane potential of energized mitochondria. The experimental results presented here clearly showed that O2.- radicals induce an increase of proton leak in mitochondria directly related to the molecular mechanism of early phase damage of ischemia-reperfused heart, and also provide evidence to support our recent proposed hypothesis that electron leakage to oxygen to form O2.- in respiratory chain is one of the mechanical cause of proton leakage in mitochondria.