Changes in the dehydrogenase activity of cardiomyocytes during acute experimental arterial occlusion of extremities

The results of quantitative histoenzymologic investigations of succinate dehydrogenase, lactate dehydrogenase and NAD-diaphorase in cardiomyocytes of dogs with acute experimental arterial occlusion in ischemic and postischemic periods are reviewed. An increased activity of dehydrogenases in the early periods (3,6 h) of ischemia and during recirculation was established, with its noticeable reduction at later terms (9,12 h). Medical correction of postischemic disorders was shown to improve cardiomyocyte metabolism.     

A histochemical analysis of the oxidative-reductive enzymes in the cardiomyocytes of mature and old rats following low-dose external gamma irradiation

The effect of gamma rays in low dose (0.5 Gy) on enzymes activity of glycolysis, tricarboxylic acid cycle, diaphorases in cardiomyocytes of adult and old albino rats at 3-, 10- and 30-day after irradiation was studied. It was established that the diaphorases activity decreased, the lactate dehydrogenase activity increased whereas that of other tricarboxylic acid cycle enzymes underwent phase changes. In result discoordination of redox enzymes in cardiomyocytes of rats hypoxic state develops. The question of the age sensitivity of heart to irradiation in low dose is discussed.     

The structuro-functional state of the ischemic myocardium under the action of a terrilitin-nicotinic acid mixture in animal experiments

Histochemical evidence of the activity and distribution of glycolysis redox enzymes, tissue respiration and terminal oxidation pattern (dehydrogenase of lactic, malic, succinic and isocitric acids, NAD-N- and NADPh-N-ase, cytochrome oxidase) as well as the levels of the major carbohydrates (glycogen, neutral aminopolysaccharides, glucose) were experimentally studied in the cardiomyocytes of myocardial necrotic, perinecrotic and intact areas in the control and in the experimental material under the administration of terrilitin-nicotinic acid mixture. It was stated that the use of aforementioned mixture contributed to the retention of enzymatic activity and optimal levels of energy formation in the cardiomyocytes of the marginal infarction zone and noticeably prevented the destructive involvement of the considered area as well as the impairment of functional activity of oscillating cardiomyocytes. Therefore, the application of the mixture improved the outcome prognosis in acute myocardial infarction.     

磷脂酰肌醇3激酶抑制剂LY294002对缺血缺氧心肌细胞的作用研究

目的:明确P13K/Akt信号通路在缺血缺氧心肌细胞损害中的作用。方法:建立心肌细胞缺血缺氧模型,施加磷脂酰肌醇3激酶抑制剂LY294002干预,观察心肌细胞活力、培养液中乳酸脱氢酶(LDH)含量及碘化丙啶(PI)染色阳性细胞比例的变化。结果:模拟缺血缺氧后细胞活力下降,LDH及PI染色阳性细胞比例显著增加。LY294002干预复合缺血缺氧后,细胞活力急剧下降,LDH含量及PI染色阳性细胞比例进一步显著增加(P<0.01)。结论:应用LY294002加重了缺血缺氧对心肌细胞的损伤效应,提示PI3K/Akt通路参与了缺血缺氧心肌细胞的内源性保护反应,减轻了缺血缺氧损害。     

一氧化氮抑制血管紧张素Ⅱ和内皮素—1诱导的心肌细胞原癌基因c—fo …

在原代培养的新生大鼠心肌细胞上,探讨一氧化氮（ＮＯ）对血管紧张素Ⅱ（ＡⅡ）和内皮素－１（ＥＴ－１）诱导的心肌细胞肥大和原癌基因ｃ－ｆｏｓ表达的影响。用Ｂｒａｄｆｏｒｄ法测定心肌细胞总蛋白含量（作为心肌细胞肥大的指标）;用基因特异性引物和ＳｕｐｅｒＳｃｒｉｐｔ一步法进行逆转录聚合酶链式反应（ＲＴ－ＰＣＲ）,检测大鼠心肌细胞原癌基因ｃ－ｆｏｓ的表达（以ＧＡＰＤＨ为内标）。结果显示,ＡⅡ和ＥＴ－１分别作     

Kinetic properties of extracted lactate dehydrogenase and creatine kinase from mouse embryonic stem cell- and neonatal-derived cardiomyocytes

Embryonic stem cells (ESCs), representing a population of undifferentiated pluripotent cells with both self-renewal and multilineage differentiation characteristics, are capable of spontaneous differentiation into cardiomyocytes. The present study sought to define the kinetic characterization of lactate dehydrogenase (LDH) and creatine kinase (CK) of ESC- and neonatal-derived cardiomyocytes. Spontaneously differentiated cardiomyocytes from embryoid bodies (EBs) derived from mouse ESC line (Royan B1) and neonatal cardiomyocytes were dispersed in a buffer solution. Enzymes were extracted by sonication and centrifugation for kinetic evaluation of LDH and CK with spectrophotometric methods. While a comparison between the kinetic properties of the LDH and CK of both groups revealed not only different Michaelis constants and optimum temperatures for LDH but also different Michaelis constants and optimum pH for CK, the pH profile of LDH and optimum temperature of CK were similar. In defining some kinetic properties of cardiac metabolic enzymes of ESC-derived cardiomyocytes, our results are expected to further facilitate the use of ESCs as an experimental model.     

Cell-permeable superoxide dismutase and glutathione peroxidase mimetics afford superior protection against doxorubicin-induced cardiotoxicity: the role of reactive oxygen and nitrogen intermediates.

The use of the potent antitumor antibiotic doxorubicin (DOX) is hampered because of its severe cardiac toxicity that leads to the development of cardiomyopathy and heart failure. In this study, we have developed a cell culture model for DOX-induced myocardial injury using primary adult rat cardiomyocytes that were cultured in serum-free medium and exposed to 1 to 40 microM DOX. DOX caused a dose-dependent release of sarcosolic enzyme lactate dehydrogenase (LDH) from cultured myocytes. The release of LDH was prevented by the cell-permeable superoxide dismutase (SOD) mimetic (MnTBAP), but was unaffected by either cell-impermeable SOD enzyme, or manganese (II) sulfate. Ebselen, a glutathione peroxidase (GPx) mimetic, enhanced the protection of cardiomyocytes afforded by MnTBAP. DOX caused the increased formation of oxidants in cardiomyocytes, and MnTBAP lowered the amount of intracellular oxidants induced by DOX. In addition, DOX selectively inactivated aconitase in cardiomyocytes, and MnTBAP partially reversed this inactivation. Ebselen further amplified the protective effect of MnTBAP on aconitase activity. These results suggest that the SOD mimetic MnTBAP prevents DOX-induced damage to cardiomyocytes and that the GPx mimetic ebselen synergistically enhanced the cardioprotection afforded by MnTBAP. Relevance of these findings to minimizing cardiotoxicity in cancer treatment is discussed.     

细胞粘附分子在中性粒细胞介导的缺氧／复氧心肌细胞损伤中的作用

目的观察缺氧/复氧对心肌细胞与中性粒细胞粘附效应的影响及细胞间粘附分子-1(intercellularadhensionmolecule-1,ICAM-1)和淋巴细胞功能相关抗原-1(lymphocytefunctionassociatedantigen-1,LFA-1)在中性粒细胞介导的心肌细胞损伤的作用。方法计数不同实验条件下与心肌细胞粘附的中性粒细胞;以及抗ICAM-1单抗和抗LFA-1单抗阻断后中性粒细胞粘附数的改变,检测心肌细胞乳酸脱氢酶释放量。结果中性粒细胞与缺氧/复氧心肌细胞粘附数较缺氧组和正常对照组显著增加(P＜0.01);心肌细胞释放LDH明显增高(P＜0．01),单纯缺氧组与正常对照组相比无显著差异(P＞0．05)。加入抗ICAM-1单抗和抗LFA-1单抗后,缺氧/复氧组与心肌细胞粘附的中性粒细胞数较正常对照组显著降低(P<0．01),心肌细胞释放LDH也明显下降(P＜0．01)。缺氧组与正常对照组相比则无显著差异(P＞0.05)。结论缺氧/复氧使心肌细胞与中性粒细胞粘附效应增加,心肌细胞损伤加重,ICAM-1和LFA-1参与这一过程。抗ICAM-1和抗LFA-1单抗可减轻中性粒细胞对缺氧/复氧心肌细胞的损伤。     

In vitro measurement of enzymatic markers as a tool to detect mouse cardiomyocytes injury

Primary cultures of cardiomyocytes represent a useful model for analyzing cardiac cell biology as well as pathogenesis of several cardiovascular disorders. Our aim was to standardize protocols for determining the damage of cardiac cells cultured in vitro by measuring the creatine kinase and its cardiac isotype and lactate dehydrogenase activities in the supernatants of mice cardiomyocytes submitted to different protocols of cell lysis. Our data showed that due to its higher specificity, the cardiac isotype creatine kinase was the most sensitive as compared to the others studied enzymatic markers, and can be used to monitor and evaluate cardiac damage in in vitro assays.     

Protection of cardiomyocytes from ischemic/hypoxic cell death via Drbp1 and pMe2GlyDH in cardio-specific ARC transgenic mice

The ischemic death of cardiomyocytes is associated in heart disease and heart failure. However, the molecular mechanism underlying ischemic cell death is not well defined. To examine the function of apoptosis repressor with a caspase recruitment domain (ARC) in the ischemic/hypoxic damage of cardiomyocytes, we generated cardio-specific ARC transgenic mice using a mouse alpha-myosin heavy chain promoter. Compared with the control, the hearts of ARC transgenic mice showed a 3-fold overexpression of ARC. Langendoff preparation showed that the hearts isolated from ARC transgenic mice exhibited improved recovery of contractile performance during reperfusion. The cardiomyocytes cultured from neonatal ARC transgenic mice were significantly resistant to hypoxic cell death. Furthermore, the ARC C-terminal calcium-binding domain was as potent to protect cardiomyocytes from hypoxic cell death as ARC. Genome-wide RNA expression profiling uncovered a list of genes whose expression was changed (>2-fold) in ARC transgenic mice. Among them, expressional regulation of developmentally regulated RNA-binding protein 1 (Drbp1) or the dimethylglycine dehydrogenase precursor (pMe(2)GlyDH) affected hypoxic death of cardiomyocytes. These results suggest that ARC may protect cardiomyocytes from hypoxic cell death by regulating its downstream, Drbp1 and pMe(2)GlyDH, shedding new insights into the protection of heart from hypoxic damages.     

碱性成纤维细胞生长因子对乳鼠心肌细胞缺氧复氧损伤的影响

本实验在培养的乳鼠心肌细胞缺氧复氧损伤模型上观察碱性纤维细胞生长因子对Ａ／Ｒ损伤及蛋白激酶活性的影响,以探讨ｂＥＧＦ作为药物预处理心肌保护的可行性及其机理。结果表明,ｂＥＧＦ预处理呈浓度依赖地提高Ａ／Ｒ后心肌细胞存活率,减少细胞内ＡＴＰ消耗及胞浆乳酸脱氢酶漏出;ＰＫＣ抑制剂Ｈ７完全消除ｇＦＧＦ的上述保护作用;实验结果表明,ｂＦＧＦ可以直接激活心肌细胞ＰＫＣ,其激活时相的变化与缺氧预处理者相近,提示     

Autophagy mediates the secretion of macrophage migration inhibitory factor from cardiomyocytes upon serum-starvation

Macrophage migration inhibitory factor(MIF) is an inflammatory cytokine. It is elevated early in the blood of acute myocardial infarction patients. However, it is unclear whether and how MIF is released. This study investigated the cellular source and mechanism of MIF release from hearts. An ischemia-mimic treatment induced the secretion of MIF from neonatal rat cardiomyocytes but not from fibroblasts. The treatment did not cause significant leakage of lactate dehydrogenase, suggesting that ischemia induced the MIF secretion without causing severe cell damage. Plasma samples from patients with acute chest pain at the emergency department were collected for the detection of MIF. MIF levels in patients with acute coronary syndrome(ACS)increased early, when cardiac injury markers were not yet elevated, suggesting that ischemia can induce MIF secretion before the occurrence of severe myocardial damage. Serum-starvation caused MIF secretion from rat cardiomyocytes and Langendorffperfused rat hearts. The secretion was suppressed by the inhibition of autophagy by inhibitors or by silencing of Atg5. In conclusion, serum-starvation induces the secretion of MIF from cardiomyocytes via autophagy dependent pathway. Clarifying the mechanism of MIF secretion will be helpful for its application in the early diagnosis and treatment of ACS.     

缺氧复氧时肥大心肌细胞凋亡及其与能量代谢途径转换的关系

心肌细胞凋亡是心肌肥大向心力衰竭转化的重要机制,因此,抑制肥大心肌细胞凋亡可能是防治心力衰竭的有 效药物靶点之一。本研究以0．1μmol／L血管紧张素Ⅱ和1 μmol／L去甲肾上腺素刺激培养心肌细胞,复制心肌细胞肥大模 型,用三气孵箱培养。缺氧条件是95％N2和5％CO2,控制氧分压低于5 mmHg以下,8 h后常氧培养,液闪计数法测 定丙酮酸脱氢酶(pyruvate dehydrogenase,PDH)和肉碱脂酰转移酶-1(carnitine palmitoyltransferase 1,CPT-1)活性,糖氧化、 糖酵解、脂肪酸氧化率,以及细胞凋亡百分率,分析肥大心肌细胞能量代谢变化与细胞凋亡间的关系。结果如下:(1)与 常氧培养比较,缺氧8 h后,活化型丙酮酸脱氢酶(PDHa)和CPT-1活性均有显著下降,但复氧早期肥大心肌细胞PDHa活 性有轻度进一步降低(P>0．05),而CPT-1活性却较快恢复。(2)缺氧时,正常和肥大心肌细胞葡萄糖氧化代谢率均有降低[分 别下降(16±0．9)％、(48±1．1)％];复氧时,正常心肌细胞糖氧化代谢较快恢复,而肥大心肌细胞在复氧早期,糖氧化 率进一步降低,此后才逐渐恢复。(3)在缺氧时,肥大心肌细胞糖酵解率仅轻度下降,但在复氧后糖酵解率迅速升高,呈 爆发样达峰值后又逐渐恢复到缺氧前水平。(4)肥大心肌细胞在缺氧后脂肪酸代谢明显降低,但复氧后脂肪酸代谢呈爆发式 上升,并大大高于缺血前的代谢水平。(5)缺氧时肥大心肌细胞凋亡率即显著增加,在复氧早期细胞凋亡率继续大幅度上 升,此后逐渐减少。(6)预先用二氯乙酸处理肥大心肌细胞,可显著逆转缺氧复氧导致的细胞糖氧化受抑、糖酵解和脂肪 酸代谢活化,同时,抑制细胞凋亡的发生。上述结果提示,缺氧复氧后的肥大心肌细胞能量代谢途径转换是导致细胞凋 亡的重要原因。     

一氧化氮抑制血管紧张素Ⅱ和内皮素-1诱导的心肌细胞原癌基因c-fos表达

在原代培养的新生大鼠心肌细胞上, 探讨一氧化氮 (NO)对血管紧张素Ⅱ (AⅡ)和内皮素-1 (ET-1)诱导的心肌细胞肥大和原癌基因c-fos表达的影响.用Bradford 法测定心肌细胞总蛋白含量 (作为心肌细胞肥大的指标); 用基因特异性引物和 SuperScript一步法进行逆转录聚合酶链式反应 (RT-PCR), 检测大鼠心肌细胞原癌基因c-fos的表达 (以GAPDH为内标).结果显示, AⅡ和ET-1分别作用5 d和3 d后, 心肌细胞总蛋白含量显著增加; 硝普钠 (NO供体)可抑制AⅡ或ET-1诱导的心肌细胞总蛋白增加.AⅡ,ET-1和PMA (蛋白激酶C激动剂)均可诱导心肌细胞原癌基因c-fos的表达; L-精氨酸可抑制AⅡ,ET-1和PMA诱导心肌细胞原癌基因c-fos的表达, L-NAME (NOS抑制剂)可抑制L-精氨酸的这一作用; 硝普钠对可抑制AⅡ,ET-1和PMA诱导心肌细胞原癌基因c-fos的表达.结果表明, NO可抑制AⅡ或ET-1诱导的心肌细胞肥大和原癌基因c-fos表达, 其作用机制可能与蛋白激酶C这一环节有关.     

The biochemical and morphological changes in the myocardium of patients with an acute surgical vascular pathology (based on data from early autopsies)]

Biochemical and morphological disorders of glucose 6-phosphate dehydrogenase (G-6-PDH) were studied in the myocardium of 9 patients who had died from different vascular surgical diseases. The inhibition of G-6-PDH activity most prominent in lung artery thromboembolism was shown biochemically. Histological and histoenzymological findings demonstrate low G-6-PDH activity in different myocardial regions and solitary defects of cardiomyocytes. The data obtained evidence significant sensibility of the myocardium in surgical patients to different influences.     

Adrenergic innervation of the myocardium in rats with toxic exposures

Chronic intraperitoneal injection of cadmium and copper salts produces cardiotoxic effects of various degree. The degree of the muscular tissue lesion in the ventricles and atria is inverse to the number of luminescent nervous terminals. Changes in the adrenergic fibers are accompanied with certain metabolic shifts in the muscular tissue of the heart; this is evident from decreasing succinate dehydrogenase activity in cardiomyocytes and accumulation of lipids. Certain disorders are also revealed in cardiomyocytes, in vessels and in interstitial connective tissue demonstrated as: plethora, phenomena of stasis in the capillary bed, moderate perivascular edema, myocardial dystrophy. The foci of lesions are found more often in the left ventricle in myocardial tissue and under epicardium, sometimes near plethoric vessels and less often in the right ventricle and in the atria. The dependence between location of the myocardial lesions and vascular disorders is not always noted. This is observed more often under effect of cadmium sulfate and, evidently, is dependent not only on hypoxia, connected with congestive plethora, but with neurohumoral influences, too.     

In vivo regulation of mitochondrial respiration in cardiomyocytes: specific restrictions for intracellular diffusion of ADP

Relative diffusivities of ADP and creatine in cardiomyocytes were studied. The isolated rat cardiomyocytes were lysed with saponin (40 micrograms/ml) to perforate or completely disrupt sarcolemma that was evidenced by leakage of 80-100% lactate dehydrogenase. In these cardiomyocytes mitochondria were used as 'enzymatic probes' to determine the average local concentration of substrates exerting acceptor control of respiration--ADP or creatine (the latter activates respiration via mitochondrial creatine kinase reaction)--when their concentrations in the surrounding medium were changed. The kinetic parameters for ADP and creatine in control of respiration of saponin-treated cardiomyocytes were compared with those determined in isolated mitochondria and skinned cardiac fibers. The apparent Km for creatine (at 0.2 mM ATP) was very close and in a range of 6.0-6.9 mM in all systems studied, showing the absence of diffusion difficulties for this substrate. On the contrary, the apparent Km for ADP increased from 18 +/- 1 microM for isolated mitochondria to 250 +/- 59 microM for cardiomyocytes with the lysed sarcolemma and to 264 +/- 57 microM for skinned fibers. This elevation of Km was not eliminated by inhibition of myokinase with diadenosine pentaphosphate. When 25 mM creatine was present, the apparent Km for ADP decreased to 36 +/- 6 microM. These data are taken to indicate specific restrictions of diffusion of ADP most probably due to its interaction with intermediate binding sites in cardiomyocytes. The important role of phosphocreatine-creatine kinase system of energy transport is to overcome the restrictions in regulation of energy fluxes due to decreased diffusivity of ADP.     

乙醛脱氢酶2可减少氧化损伤诱导的心肌细胞凋亡

乙醛脱氢酶2 (aldehyde dehydrogenase 2, ALDH2)是线粒体特异性酶,已被证明参与氧化应激诱导的细胞凋亡,而在心肌细胞中的作用知之甚少。本研究旨在通过用特异性ALDH2抑制剂大豆苷抑制ALDH2活性来研究ALDH2在抗霉素A诱导的心肌细胞凋亡中的作用。应用抗霉素A和大豆苷诱导小鼠心肌细胞,然后测定ALDH2酶活性、细胞内活性氧(reactive oxy gen species, ROS)含量和细胞凋亡,应用RT-PCR和蛋白质印迹法(Western blotting)检测ALDH2 m RNA和蛋白表达。结果表明,抗霉素A (40μg/mL)可诱导新生心肌细胞凋亡,而大豆苷(50μmol/L)能有效地抑制ALDH2活性而对细胞凋亡没有影响,并且可显著增强抗霉素A诱导的心肌细胞凋亡(53.72%～71.33%, p<0.05)。与单独用抗霉素A处理的细胞相比,抗霉素A和大豆苷共处理的心肌细胞中活化的丝裂原活化蛋白激酶(mitogen-activated protein kinase, MAPK)信号传导途径(p38-MAPK)的磷酸化也显著增加。本研究初步表明,改变线粒体ALDH2活性可能是减少氧化损伤诱导的心肌细胞凋亡的潜在选择。     

Activation of peroxisomal acyl-CoA oxidase and lipid peroxidation in the rat myocardium as affected by prolonged administration of ethanol

The influence of chronic alcoholic intoxication on the activity of peroxisomal acyl-CoA oxidase and antioxidative defensive enzymes (catalase, glutathione reductase, glutathione-S-transferase, superoxide dismutase, glucose-6-phosphate dehydrogenase) was studied in the rat myocardium. The parameters of lipid peroxidation in cardiomyocytes (the level of spontaneous chemiluminescence, accumulation of thiobarbituric acid-reactive material), as well as reduced glutathione content were also examined. The data obtained suggest that ethanol-induced activation of lipid peroxidation in the myocardium may be due to the elevation of hydrogen peroxide-generating activity of peroxisomes.     

MiR-181a-5p is involved in the cardiomyocytes apoptosis induced by hypoxia–reoxygenation through regulating SIRT1

ABSTRACT

MiR-181a-5p’s mechanism in hypoxia–reoxygenation (H/R)-induced cardiomyocytes apoptosis has not been clarified. This study verified that SIRT1 was the target of miR-181a-5p. MiR-181a-5p expression was up-regulated or down-regulated in H/R-induced cardiomyocytes, and SIRT1 was transfected into cells alone or in combination with miR-181a-5p. Cell viability, apoptosis, levels of released lactate dehydrogenase (LDH), malondialdehyde (MDA), and superoxide dismutase (SOD), as well as the Bcl-2, Bax, and Caspase 3 levels in treated cells were tested. On the one hand, down-regulated miR-181a-5p promoted cell viability, reduced released LDH and MDA, and increased SOD level in H/R-induced cardiomyocytes. On the other hand, miR-181a-5p inhibited apoptosis and elevated Bcl-2 expression while decreasing the expressions of Bax and Caspase 3 in treated cells, but the effects of miR-181a-5p could be rescued by SIRT1. In conclusion, miR-181a-5p involved in H/R-induced cardiomyocytes apoptosis through regulating SIRT1, which might become a novel direction for related diseases.