Ultrastructural changes in rabbit heart mitochondria during the perinatal period: Neonatal transition to aerobic metabolism

Ultrastructural changes in mitochondria of rabbit left ventricular myocardial cells were measured during the interval from 3 days before to 4 days after birth. This interval is characterized by a transition from partially anaerobic to aerobic metabolism and by a concomitant increase in the work of contraction. Measurements were made by recently developed morphometric techniques [Smith, H. E., and Page, E. (1976). J. Ultrastruct. Res.55, 31–41]. It was shown that the perinatal transition was associated with a rapid and large accumulation of mitochondria and myofibrils, reflected by increases of 29–35% and 37–41% in the mitochondrial and myofibrillar fractions of cell volume. The mitochondria thus accumulated were packed more densely with cristae. The membrane area of cristae + inner membrane per unit of mitochondrial volume rose from an antenatal value of 47 μm²/μm³ to a peak of 64 μm²/μm³ 2 days after birth, then declined significantly. The relative and absolute increases in the membrane area and volume of respiratory membrane were associated with a relative decrease in matrix volume. These measurements also permitted the calculation of the area of mitochondrial respiratory membrane per unit myofibrillar volume. This physiologically important index, which relates the amount of ATP-producing membrane to the volume of the principal ATP-consuming organelle, increased progressively throughout the perinatal period.     

Myocardial prostacyclin and thromboxane A2 synthetase activities during ischemia and reperfusion in isolated rabbit heart

The aim of the study was to determine the prostacyclin (PGI2) and thromboxane A2 (TXA2) synthetase activities of myocardial tissue and their variation during ischemia and reperfusion. Regional ischemia was induced by 10 min occlusion of the left anterior descending coronary artery in isolated Langendorff rabbit hearts. Biosynthesis of PGI2 and TXA2 were carried out by using arachidonic acid as substrate and left ventricle microsomes (LVM) from ischemic and non-ischemic areas as sources of PGI2 and TXA2 synthetase. 6-keto-PGF1 alpha and TXB2, stable metabolites of PGI2 and TXA2 respectively, were determined by radioimmunoassay. Experiments carried out under the adopted conditions showed that LVM were able to synthetise PGI2 as well as TXA2 from arachidonic acid. On the other hand, ischemia depressed both PGI2 and TXA2 synthetase activities of cardiac tissue: the depression was more pronounced on TXA2 synthetase than on PGI2 synthetase with no significant difference between ischemic and non-ischemic regions. Moreover, ischemia increased the ratio 6-keto-PGF1 alpha/TXB2 indicating therefore that it can facilitate the formation of PGI2. The post ischemic reperfusion of the heart counteracted the decrease in PGI2 synthetase induced by ischemia which returned to the normal level: reperfusion also slightly reversed the decrease in TXA2 the decrease in TXA2 synthetase. However, the diminution in TXA2 synthetase of non-ischemic myocardium was attenuated but it remained lower than the normal level. These results suggested that the whole left ventricle is affected by regional ischemia. Furthermore it appears that myocardial TXA2 synthetase is more vulnerable than PGI2 synthetase to a lack of oxygen and nutrients.     

Myocardial prostacyclin and thromboxane A2 synthetase activities during ischemia and reperfusion in isolated rabbit heart

The aim of the study was to determine the prostacyclin (PGI₂) and thromboxane A₂ (TXA₂) synthetase activities of myocardial tissue and their variation during ischemia and reperfusion. Regional ischemia was induced by 10 min occlusion of the left anterior descending coronary artery in isolated Langendorff rabbit hearts. Biosynthesis of PGI₂ and TXA₂ were carried out by using arachidonic acid as substrate and left ventricle microsomes (LVM) from ischemic and non-ischemic areas as sources of PGI₂ and TXA₂ synthetase. 6-keto-PGF_1α and TXB₂, stable metabolites of PGI₂ and TXA₂ respectively, were determined by radioimmunoassay. Experiments carried out under the adopted conditions showed that LVM were able to synthetise PGI₂ as well as TXA₂ from arachidonic acid. On the other hand, ischemia depressed both PGI₂ and TXA₂ synthetase activities of cardiac tissue: the depression was more pronounced on TXA₂ synthetase than on PGI₂ synthetase with no significant difference between ischemic and non-ischemic regions. Moreover, ischemia increased the ratio indicating therefore that it can facilitate the formation of PGI₂. The post ischemic reperfusion of the heart counteracted the decrease in PGI₂ synthetase induced by ischemia which returned to the normal level: reperfusion also slightly reversed the decrease in TXA₂ synthetase. However, the diminution in TXA₂ synthetase of non-ischemic myocardium was attenuated but it remained lower than the normal level. These results suggested that the whole left ventricle is affected by regional ischemia. Furthermore it appears that myocardial TXA₂ synthetase is more vulnerable than PGI₂ synthetase to a lack of oxygen and nutrients.     

Hydrogen sulfide post-conditioning preserves interfibrillar mitochondria of rat heart during ischemia reperfusion injury

Cardiac mitochondrial dysfunction is considered to be the main manifestation in the pathology of ischemia reperfusion injury, and by restoring its functional activity, hydrogen sulfide (H₂S), a novel endogenous gaseotransmitter renders cardioprotection. Given that interfibrillar (IFM) and subsarcolemmal (SSM) mitochondria are the two main types in the heart, the present study investigates the specific H₂S-mediated action on IFM and SSM during ischemic reperfusion in the Langendorff rat heart model. Rats were randomly divided into five groups, namely normal, ischemic control, reperfusion control (I/R), ischemic post-conditioning (POC), and H₂S post-conditioning (POC_H₂S). In reperfusion control, cardiac contractility decreased, and lactate dehydrogenase, creatine kinase, and infracted size increased compared to both normal and ischemic group. In hearts post-conditioned with H₂S and the classical method improved cardiac mechanical function and decreased cardiac markers in the perfusate and infarct size significantly. Both POC and POC_H₂S exerts its cardioprotective effect of preserving the IFM, as evident by significant improvement in electron transport chain enzyme activities and mitochondrial respiration. The in vitro action of H₂S on IFM and SSM from normal and I/R rat heart supports H₂S and mediates cardioprotection via IFM preservation. Our study indicates that IFM play an important role in POC_H₂S mediated cardioprotection from reperfusion injury.     

Phosphorylation of myocardial sarcolemma proteins during induction of the phosphatidylinositol cycle in isolated perfused rabbit heart]

Phosphorylation of cardiac sarcolemma proteins under stimulation of M-receptors by agonist carbacholine used to stimulate phosphatidylinositide cycle, was investigated in the isolated, rabbit heart perfused with 32Pi. Carbacholine (10(-7) stimulates the polyphosphoinositide metabolism which is expressed in the activated incorporation of 32P from [gamma-32P]ATP in polyphosphoinositide as well as in the increased content of the labelled inositol trisphosphate released through phosphatidylinositol-4,5-bisphosphate break-down by phospholipase C. The diacylglycerol produced simultaneously with inositol triphosphate as a second messenger activates the protein kinase C. This was confirmed by considerable activation of phosphorylation sarcolemma proteins-substrates of protein kinase C, with Mr 94, 87, 78, 51 and 46 kDa.     

Reasons for disorders of fatty acid oxidation in isolated heart mitochondria during ischemia

The influence of malate and cytochrome c on fatty acid oxidation under control and ischemic conditions was investigated. In the medium without malate, cytochrome did not make fatty acid oxidation decreased during ischemia return to normal. Oxidation in the media containing malate and cytochrome did not differ from control only when it was measured after preliminary oxidation of endogenous substrates. The ratio of palmitoyl-CoA and palmitoyl carnitine to the respiration rates at state 3 was unchanged at 60 min ischemia. Apparently, no changes in carnitine acyltransferase playing a role in oxidation of palmitoyl-CoA took place. Thus, the decrease of fatty acid oxidation at early periods of ischemia is largely caused by a reduction in the content of cytochrome c and intermediates of Krebs cycle in the mitochondria.     

Ultrastructural changes in the sarcolemma in the early stages of immune damage to the heart

Early ultrastructural alterations were studied in cardiomyocyte sarcolemma on the model of acute local immune injury after the treatment of the dog's heart with anticardiac cytotoxic serum. It has been shown that immunotherapy results in the formation of numerous liposomal structures originating from the membrane material and located in subsarcolemmal space and near cardiomyocyte mitochondria. Depending on the method of fixation liposomes had the form of circular membrane vesicles or multilaminar structures. The structure and location of these liposomes near plasmalemma or mitochondrial membranes and sarcoplasmic reticulum have led to the assumption that they originate from unstable membrane phospholipids. The present data give evidence of the early and rapid phospholipid degradation in cardiomyocyte membranes during immune heart damage.     

An assessment of anaerobic metabolism during ischemia and reperfusion in isolated guinea pig heart

The effects of total ischemia and subsequent reperfusion on the formation of anaerobic metabolism products and their release into myocardial effluent were studied in isolated guinea pig hearts. During 30-min ischemia myocardial ATP and phosphocreatine decreased to 34 and 15% of the initial levels, respectively; this was accompanied by alanine formation and approximately stoichiometric glutamate loss. The increase in malate in ischemic myocardium corresponded to the anaplerotic flux aspartate----oxaloacetate----malate; the succinate production being commensurable to alpha-ketoglutarate formation in the alanine aminotransferase reaction. The release of lactate, alanine, succinate, creatine and pyruvate trace amounts into the myocardial effluent was observed during an early phase of the reperfusion using 1H-NMR. The rates of metabolite release reduced as follows: lactate much greater than alanine greater than succinate greater than creatine. By the 30th min of the reperfusion the decrease in these metabolites tissue contents was accompanied by the recovery of ATP and phosphocreatine levels up to 65 and 90% of the initial ones, respectively. The data obtained demonstrate that the formation and the release of succinate, alanine and creatine from the heart as well as of lactate may indicate profound disturbances in energy metabolism.     

The anti-thromboxane A2 synthetase activity of myocardial tissue and its variation during ischemia and reperfusion in isolated rabbit heart.

In this investigation, an anti-thromboxane A2 (TXA2) synthetase activity in the myocardial tissue, which can be modulated by ischemia and reperfusion, was observed. Regional ischemia was induced by 60 min occlusion of the left anterior descending coronary artery in isolated Langendorff rabbit hearts. Biosynthesis of TXA2 was carried out by using arachidonic acid (AA) as substrate, horse platelet microsomes (HPM) as the source of TXA2 synthetase and left ventricle microsomes (LVM) from ischemic and non-ischemic areas as effectors TXB2, the stable metabolite of TXA2, was determined by radioimmunoassay. Experiments carried out under the adopted conditions showed that LVM from control hearts were able to inhibit by up to 50% the biosynthesis of TXA2 from HPM. This anti-TXA2 synthetase activity was more pronounced when LVM from the non-ischemic area were used, rather then LVM from the ischemic one. A 60 min reperfusion decreased the anti-TXA2 activity. A superfused rabbit aorta strip was also used as a cascade bioassay to study the effect of LVM on the TX2-synthetase activity of HPM, and this confirmed our findings. These results suggest that the left ventricle possesses a self-defense mechanism against acute myocardial ischemia, independently from the circulation. The postulated mechanism may be initiated in the non-ischemic area.     

Myocardial ischemia selectively depletes cardiolipin in rabbit heart subsarcolemmal mitochondria

Mitochondria contribute to myocyte injury during ischemia. After 30 and 45 min of ischemia in the isolated perfused rabbit heart, subsarcolemmal mitochondria (SSM), located beneath the plasma membrane, sustain a decrease in oxidative phosphorylation through cytochrome oxidase. In contrast, oxidation through cytochrome oxidase in interfibrillar mitochondria (IFM), located between the myofibrils, remains unaffected. Cytochrome oxidase activity in the intact membrane requires an inner mitochondrial membrane lipid environment enriched in cardiolipin. During ischemia, the content of cardiolipin decreased only in SSM, whereas the content of other phospholipids was preserved. Ischemia did not alter the composition of the cardiolipin that remained in SSM. Cardiolipin content was preserved in IFM during ischemia. Thus cardiolipin is a relatively early target of ischemic mitochondrial damage, leading to loss of oxidative phosphorylation through cytochrome oxidase in SSM.     

Effect of endogenous nitric oxide on energy metabolism of rat heart mitochondria during ischemia and reperfusion

The effects of ischemia and postischemic reperfusion on the functions of the heart and its mitochondria were studied with special attention to the effect of nitric oxide (NO) by treatment of rat hearts with the nitric oxide synthase (NOS) inhibitor N^G-nitro-L-arginine methyl ester (L-NAME) or its noninhibitory isomer N^G-nitro-D-arginine methyl ester (D-NAME). NO generated during reperfusion caused increase in coronary flow (CF), but had no effect on the left ventricular pressure (LVP) or heart rate (HR). The ATP level of the heart decreased during ischemia and was not completely restored by introduction of oxygen during reperfusion due to damage of complexes I and II of the respiratory chain of mitochondria by NO. Inhibition of the respiratory chain resulted in generation of hydrogen peroxide, and NO and NO-derived species generated after production of NO caused further damage of various proteins in mitochondria, such as complexes I and II of the respiratory chain and pyruvate dehydrogenase (PDH). These results suggested that NO generated on reperfusion was the primary cause of mitochondrial dysfunction by damage of complexes I and II of the respiratory chain, with consequent increase of CF in the heart.     

Effect of ischemia and reperfusion on protein oxidation in isolated rabbit hearts

Reactive oxygen species and other oxidants are involved in the mechanism of postischemic contractile dysfunction, known as myocardial stunning. The present study investigated the oxidative modification of cardiac proteins in isolated Langendorff-perfused rabbit hearts subjected to 15 min normothermic ischemia followed by 10 min reperfusion. Reperfusion under these conditions resulted in only 61.8+/-2.7 % recovery of developed pressure relative to preischemic values and this mechanical dysfunction was accompanied by oxidative damage to cardiac proteins. The total sulfhydryl group content was significantly reduced in both ventricle homogenates and mitochondria isolated from stunned hearts. Fluorescence measurements revealed enhanced formation of bityrosines and conjugates of lipid peroxidation-end products with proteins in cardiac homogenates, whereas these parameters were unchanged in the mitochondrial fraction. Reperfusion did not alter protein surface hydrophobicity, as detected by a fluorescent probe 1-anilino-8-naphthalenesulfonate. Our results indicate that oxidation of proteins in mitochondria and possibly in other intracellular structures occurs during cardiac reperfusion and might contribute to ischemia-reperfusion injury.     

Lipid peroxidation and alterations to oxidative metabolism in mitochondria isolated from rat heart subjected to ischemia and reperfusion.

Ischemia-reperfusion injury to cardiac myocytes involves membrane damage mediated by oxygen free radicals. Lipid peroxidation is considered a major mechanism of oxygen free radical toxicity in reperfused heart. Mitochondrial respiration is an important source of these reactive oxygen species and hence a potential contributor to reperfusion injury. We have examined the effects of ischemia (30 min) and ischemia followed by reperfusion (15 min) of rat hearts, on the kinetic parameters of cytochrome c oxidase, on the respiratory activities and on the phospholipid composition in isolated mitochondria. Mitochondrial content of malonyldialdheyde (MDA), an index of lipid peroxidation, was also measured. Reperfusion was accompanied by a significant increase in MDA production. Mitochondrial preparations from control, ischemic and reperfused rat heart had equivalent Km values for cytochrome c, although the maximal activity of the oxidase was 25 and 51% less in ischemic and reperfused mitochondria than that of controls. These changes in the cytochrome c oxidase activity were associated to parallel changes in state 3 mitochondrial respiration. The cytochrome aa3 content was practically the same in these three types of mitochondria. Alterations were found in the mitochondrial content of the major phospholipid classes, the most pronounced change occurring in the cardiolipin, the level that decreased by 28 and by 50% as function of ischemia and reperfusion, respectively. The lower cytochrome c oxidase activity in mitochondria from reperfused rat hearts could be almost completely restored to the level of control hearts by exogenously added cardiolipin, but not by other phospholipids nor by peroxidized cardiolipin. It is proposed that the reperfusion-induced decline in the mitochondrial cytochrome c oxidase activity can be ascribed, at least in part, to a loss of cardiolipin content, due to peroxidative attack of its unsaturated fatty acids by oxygen free radicals. These findings may provide an explanation for some of the factors that lead to myocardial reperfusion injury.     

葛根素对离体大鼠缺血/复灌心脏的保护作用及其作用机制

目的：探讨葛根素（puerarin,Pue）预处理抗心肌缺血／复灌（ischemia／reperfusion,I／R）损伤是否与线粒体渗透性转换孔和／或线粒体ATP敏感性钾通道有关。方法：采用离体大鼠心脏Leangendorff灌流方法,全心停灌30min,复灌120min复制I／R模型。测定心室力学指标和复灌各时间点冠脉流出液中乳酸脱氢酶（LDH）含量。实验结束测定心肌组织formazan量的变化。结果：与单纯I／R组相比,Pue（0．24mmol／L,5min）预处理明显提高心肌细胞的formazan含量,降低复灌期间冠脉流出液中LDH含量,明显促进左室发展压、左心室内压最大上升和下降速率、心率与发展压乘积和左室舒张末压力的恢复,缓解冠脉流量的减少。线粒体渗透性转换孔开放剂苍术苷（20μmol／L。复灌前给药20min）和线粒体ATP敏感性钾通道抑制剂5-羟基癸酸（100μmol／L,缺血前给药20min）能明显减弱Pue的保护作用。结论：在大鼠离体心脏灌流模型上,Pue预处理具有抗心脏缺血／复灌损伤的作用,这种保护作用可能与其抑制线粒体渗透性转换孔的开放和促进线粒体ATP敏感性钾通道的开放有关。     

Injection of isolated mitochondria during early reperfusion for cardioprotection

Previously, we demonstrated that ischemia induces mitochondrial damage and dysfunction that persist throughout reperfusion and impact negatively on postischemic functional recovery and cellular viability. We hypothesized that viable respiration-competent mitochondria, isolated from tissue unaffected by ischemia and then injected into the ischemic zone just before reperfusion, would enhance postischemic functional recovery and limit infarct size. New Zealand White rabbits (n = 52) were subjected to 30 min of equilibrium and 30 min of regional ischemia (RI) induced by snaring the left anterior descending coronary artery. At 29 min of RI, the RI zone was injected with vehicle (sham control and RI vehicle) or vehicle containing mitochondria (7.7 x 10(6) +/- 1.5 x 10(6)/ml) isolated from donor rabbit left ventricular tissue (RI-Mito). The snare was released at 30 min of RI, and the hearts were reperfused for 120 min. Our results show that left ventricular peak developed pressure and systolic shortening in RI-Mito hearts were significantly enhanced (P < 0.05 vs. RI-vehicle) to 75% and 83% of equilibrium value, respectively, at 120 min of reperfusion compared with 57% and 62%, respectively, in RI-vehicle hearts. Creatine kinase-MB, cardiac troponin I, and infarct size relative to area at risk were significantly decreased in RI-Mito compared with RI-vehicle hearts (P < 0.05). Confocal microscopy showed that injected mitochondria were present and viable after 120 min of reperfusion and were distributed from the epicardium to the subendocardium. These results demonstrate that viable respiration-competent mitochondria, isolated from tissue unaffected by ischemia and then injected into the ischemic zone just before reperfusion, significantly enhance postischemic functional recovery and cellular viability.