Lecithinized copper, zinc-superoxide dismutase ameliorates prolonged hypoxia-induced injury of cardiomyocytes

Recent studies have suggested that prolonged hypoxia results in increased production of reactive oxygen species in cardiomyocytes, which leads to apoptosis of these cells. We previously showed that lecithinized recombinant human copper, zinc-superoxide dismutase (rhSOD) showed increased bioavailability through greater membrane affinity and a longer half-life than unmodified SOD. The purpose of this study was to investigate whether lecithinized SOD plays a protective role against hypoxic injury in cardiomyocytes. Cultured rat cardiomyocytes incubated with lecithinized SOD (100 U/ml), unmodified SOD (100 U/ml), or vehicle alone were subjected to hypoxia for up to 72 h. Lecithinized SOD, but not unmodified SOD, was successfully delivered intracellularly, which was verified by Western blot and confocal laser-scanning microscopy. Treatment of cells with lecithinized SOD significantly suppressed hypoxia-induced cell damage. Since lecithinized SOD also suppressed hypoxia-induced DNA fragmentation, the improved cell survival provided by lecithinized SOD is thought to be mediated by its antiapoptotic effect. In summary, lecithinization resulted in a facilitated rhSOD delivery into cultured cardiomyocytes, which reduced mortality of cardiomyocytes exposed to prolonged hypoxia.     

Phenylephrine protects neonatal rat cardiomyocytes from hypoxia and serum deprivation-induced apoptosis

Previous studies have shown that alpha-adrenergic activation reduces myocardial damages caused by ischemia/reperfusion. However, the molecular mechanisms of how alpha-adrenergic activation protects the myocardium are not completely understood. The objective of this study was to test the hypothesis that alpha-adrenergic activation protects the myocardium by, at least in part, inhibiting apoptosis in cardiomyocytes. The current data has shown that apoptosis in neonatal rat cardiomyocytes, induced by 24 h treatment with hypoxia (95% N2 and 5% CO2) and serum deprivation, was inhibited by co-treatment with phenylephrine. Pre-treatment with phenylephrine for 24 h also protected cardiomyocytes against subsequent 24 h treatment with hypoxia and serum deprivation. Exposure of cardiomyocytes to phenylephrine for up to 9 days under normoxic conditions did not cause apoptosis. The phenylephrine-mediated cytoprotection was blocked by an alpha-adrenergic antagonist, phentolamine. beta-adrenergic activation with isoproterenol did not protect cardiomyocytes against hypoxia and serum deprivation-induced apoptosis. Under hypoxic conditions, phenylephrine prevented the down-regulation of Bcl-2 and Bcl-X mRNA/protein and induced hypertrophic growth. Phenylephrine-mediated protection was abrogated by the phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor wortmannin and was mimicked by the caspase-9 peptidic inhibitor LEHD-fmk. These results suggest that alpha-adrenergic activation protects cardiomyocytes against hypoxia and serum deprivation-induced apoptosis through regulating the expression of mitochondrion-associated apoptosis regulatory genes, preventing activation of mitochondrial damage-induced apoptosis pathway (cytochrome C-caspase-9), and activating hypertrophic growth.     

Effect of sarcolemmal rupture on myocardial electrical impedance during oxygen deprivation

Plasma membrane disruption is a characteristic feature of cell death induced by hypoxia or ischemia. Here, we investigated whether analysis of tissue electrical impedance allows detection of ongoing cell membrane rupture and necrotic cell death in hypoxic or ischemic myocardium. Twenty-eight isolated rat hearts were submitted to 5 h of ischemia (n = 8) or hypoxia (n = 20). Myocardial electrical impedance and lactate dehydrogenase (LDH) release were monitored. The time course of hypoxia-induced cell death was modified by altering pH (pH 7.4 or 6.4, 5 h) or by adding 3 or 10 mM glycine. Ischemia and hypoxia induced an increase in electrical impedance, followed by a plateau, and later a reduction. During hypoxia, LDH release started after a prolonged lapse of time (80.00 +/- 8.37 min at pH 7.4 and 122.50 +/- 11.82 min at pH 6.4). The onset of LDH release was followed by the onset of the late reduction in electrical impedance, and both were delayed by acidic pH (P < 0.05) and by glycine (P < 0.05). The times of onset of LDH release and of late electrical changes were significantly correlated (r = 0.752, P < 0.001). In separate experiments, induction of sarcolemmal rupture with Triton X-100 (n = 6) mimicked the late effects of ischemia or hypoxia on tissue impedance. The protective effects of glycine and acidosis on membrane disruption were confirmed (propidium iodide) in energy-deprived HL-1 cardiomyocytes. These results describe for the first time a late fall in electrical impedance in myocardium submitted to prolonged oxygen deprivation and demonstrate that this fall allows detection of ongoing cell necrosis.     

Age-related ultrastructural characteristics of various cells in acute hypoxia

Ultrastructural peculiarities, concerning reaction of cells in various populations brain-sensorimotor cortex, mammillary nuclei, vascular plexus; myocardium; liver; jejunum) in mature (6-8 months) and old (24-30 months) male white Wistar rats have been studied at an experimental acute hypobaric hypoxic hypoxia. Both general changes, that are manifested in various degree in different cell groups and organo-specific ones are noted. The manifestation degree of the reactive changes is evidently connected with certain metabolic and functional peculiarities, as well as with the character of cell restoration. In the old animals hypoxic hypoxia produces more profound destructive processes in all cell populations studied. This is accompanied with a restriction in ability to adaptation.     

Effects of a polypeptide drug on the state of energy metabolism of myocardial cells in hypoxic and ischemic conditions]

Cordialin, the agent extracted from the heart, is known to inhibit hyperoxidation of succinic acid, increasing NADH oxidation speed in suspension of cardiomyocytes in hypoxia. Cordialin presence in oxygenated cells' suspension oxidating succinate, doesn't change oxygen consumption speed. The results received may be a theoretical basis for cordialin utilization in therapy of myocardial diseases, associated with hypoxia and ischemia. Cordialin utilization may be recommended for the treatment of acute myocardial infarction and for prolongation of time-period for thrombolytic therapy, treatment of IMD, angina and other pathological states, in which oxygen transport disturbance to myocardium cells occurs.     

Xenogenic cardiomyocytes transplantation for the treatment of curing acute myocardial infarction

The autogenic cardiomyocytes transplantation presents numerous challenges in clinical application, such as the difficulty to obtain the autogenic cells, etc. Therefore, it is necessary to investigate allogenic or xenogenic cardiomyocytes transplantation. In this study, the experimental rabbits with acute infarcted myocardium were randomly divided into 3 groups: the 7-day cultured cardiomyocytes group, the 2-day cultured cardiomyocytes group and the control group. Neonate rat cardiomyocytes were labeled by DAPI and then injected into the acute infarcted myocardium of rabbits. After transplantation, results showed that, compared to the control group, the survival number of grafted cardiomyocytes in the cultured group is significantly larger (P < 0.05), with the implanted cardiomyocytes parallel to the host myocardium in an aligning direction. However, compared to the control group, the ventricular wall of the two experimental groups is thicker and the condition of myocardial fibrosis is better, especially to 7-day cultured cardiomyocytes group. These results suggested that the transplantation of xenogenic cardiomyocytes into curing acute ischemic heart of animal model is possible.     

Activity of neutral peptide hydrolases and oxidative enzymes in the myocardium and blood during hypoxic hypoxia

The citric cycle dehydrogenases and neutral peptide hydrolases (NPH) activity was determined in the myocardium and blood serum of Wistar rats under acute and prolonged hypoxic hypoxia. It was shown that the NPH activity and the activity of the most oxidative enzymes change in the same direction during adaptation to the high-altitude conditions. The role of the NPH in the oxidative metabolism reconstruction under acute and chronic hypoxia was discussed.     

Vitamin C inhibits hypoxia-induced damage and apoptotic signaling pathways in cardiomyocytes and ischemic hearts

Reactive oxygen species play a central role in myocardial ischemic injury and are a target for therapeutic intervention. Vitamin C is an essential antioxidant yet difficult to deliver in pharmacologic concentration to the myocardium. We found that adult rat cardiomyocytes accumulate vitamin C by transporting dehydroascorbic acid (DHA), the oxidized form of vitamin C, but do not transport ascorbic acid. Loading cells with vitamin C by DHA treatment resulted in resistance to hypoxia- and hypoxia/reoxygenation-induced cell death associated with the quenching of reactive oxygen species. When rats were injected with DHA before coronary occlusion, the ascorbic acid content in the heart was six to eight times higher than in untreated controls and myocardial infarction was reduced by 62%. DHA also provided significant protection when administered intravenously 2 h after coronary occlusion. In cardiomyocytes subjected to hypoxia/reoxygenation, DHA treatment resulted in decreased apoptosis associated with inhibition of Bax expression, caspase-3 activation, and cytochrome c translocation into the cytoplasm. DHA treatment also inhibited Jak2, STAT1, and STAT5 phosphorylation, and increased STAT3 phosphorylation, in hypoxic cardiomyocytes and ischemic myocardial tissue. Our findings suggest that DHA may be useful as a cardioprotectant in ischemic heart disease.     

Death of myocardiocytes and destruction of their structures during embryonic histogenesis

By means of light and electron microscopy, death of cardiomyocytes and their structures has been studied in the myocardium of chick (the 4th-20th days of incubation) and in rat (the 14th-20th days of embryogenesis) embryos. The same processes have been studied after a single and repeated injections of insulin and 5-fluorauracyl to the chick embryos (into the air chamber of the egg). In the myocardium of the chick and rat embryos atrophy of some muscle cells takes place as a demonstration of histogenetic (physiological) death characterized by changes in nucleo- and cytoplasm according to the "dark" type. Under the effect of 5-fluorauracyl the same type of cardiomyocyte death is observed; differences with the control are but quantitative ones. When the chick embryo is affected with cytotoxic concentrations of insulin, together with histogenetic death of the cardiomyocytes (the "dark" type) the death according to the "light" type also takes place. Under the effect of both insulin and 5-fluorauracyl the destructive processes in the cardiomyocytes are manifested greater than in the control. This is a morphological manifestation of certain disturbances in processes of physiological restoration at the cellular level.     

Characteristics of ultrastructure of the myocardium of rats with different resistance to oxygen deficiency after acute and periodic effects of hypoxia]

In experiments on rats with different resistance to oxygen deficiency (high-resistant--HR, and low-resistant LR animals) the myocardium ultrastructure of nonadapted and adapted rats was studied. It was shown that there were more glycogen granules and lipid drops initially in cardiomyocytes of nonadapted HR animals in comparison with LR ones. After a long-term adaptation to hypoxia the hypertrophia and hyperplasia of mitochondria, the nucleus and endoplasmatic reticulum hypertrophy were observed. Moreover, the increase of glycogen and lipids content was more pronounced in the myocardium of LR rats. Besides, the activation of protein-synthesizing processes was observed not only as a result of long-term adaptation, but also after single acute hypoxic effect. The results of submicroscopic cardiomyocyte studies of HR and LR rats are in good correlation with the peculiarities of energetic metabolism.     

Estrogen prevents cardiomyocyte apoptosis through inhibition of reactive oxygen species and differential regulation of p38 kinase isoforms

From human and animal studies, estrogen is known to protect the myocardium from an ischemic insult. However, there is limited knowledge regarding mechanisms by which estrogen directly protects cardiomyocytes. In this report, we employed an in vitro model, in which cultured rat cardiomyocytes underwent prolonged hypoxia followed by reoxygenation (H/R), to study the cardioprotective mechanism of estrogen. 17-beta-estradiol (E2) acting via estrogen receptors inhibited H/R-induced apoptosis of cardiomyocytes. Mitochondrial reactive oxygen species (ROS) generated from H/R activated p38alpha MAPK, and inhibition of p38alpha with SB203580 significantly prevented H/R-induced cell death. E2 suppressed ROS formation and p38alpha activation by H/R and concomitantly augmented the activity of p38beta. Unlike p38alpha, p38beta was little affected by H/R. Dominant negative p38beta protein expression decreased E2-mediated cardiomyocyte survival and ROS suppression during H/R stress. The prosurvival signaling molecule, phosphoinositol-3 kinase (PI3K), has previously been linked to cell survival following ischemia-reperfusion injury. Here, E2-activated PI3K was found to inhibit ROS generated from H/R injury, leading to inhibition of downstream p38alpha. We further linked these signaling pathways in that p38beta was activated by E2 stimulation of PI3K. Thus, E2 differentially modulated two major isoforms of p38, leading to cardiomyocyte survival. This was achieved by signaling through PI3K, integrating cell survival mediators.     

Effects of short-term and prolonged aerogenic hypoxia on gamma-glutamyl transpeptidase activity in the brain,liver, and biological fluids of young rats

Posthypoxic fluctuations in the levels of two excitatory amino acids, glutamate and aspartate, may be related to changes in mechanisms(s) which are responsible for their reuptake. As gamma-glutamyl transpeptidase (GGT) plays a role in mediating the uptake of glutamate and aspartate into various compartments of the brain, we studied changes in the activity of this enzyme in main regions of the brain in young and adult rats. We found a posthypoxic increase in bound GGT activity in some brain regions of 18-day-old animals after acute exposure, but no changes were observed after prolonged altitude hypoxia, with the exception of a decrease in cortical GGT activity. In contrast, acute hypoxia decreased GGT activity in the cortical capillaries to 59%, but prolonged hypoxic exposure was ineffective. However, the activity of soluble GGT in the cerebrospinal fluid of both groups of rats was several-times elevated in comparison with controls. At the same time, bound GGT activity was increased in the liver after acute or prolonged altitude hypoxia. The soluble GGT activity in plasma was only increased after prolonged exposure. Ninety days after prolonged hypoxic exposure the bound GGT activity was reduced in all brain regions to about 60–70% of controls (significantly higher in females than in males) as long-term developmental sequel from early postnatal hypoxia.     

Overexpression of the NHE1 isoform of the Na+/H+ exchanger causes elevated apoptosis in isolated cardiomyocytes after hypoxia/reoxygenation challenge

The mammalian Na⁺/H⁺ exchanger isoform 1 (NHE1) is a ubiquitously expressed membrane protein that regulates intracellular pH in the myocardium and other tissues. NHE1 is an important mediator of myocardial damage that occurs after ischemia–reperfusion injury. It has also been implicated in apoptotic damage in many tissues and its expression and activity are elevated in disease states in the myocardium. In this study, we examined the effect of additional exogenous NHE1 expression on isolated cardiomyocytes susceptibility to ischemia/reperfusion damage. Exogenous NHE1 elevated Na⁺/H⁺ exchanger expression and activity when introduced into isolated cardiomyocytes through an adenoviral system. Isolated cardiomyocytes were subjected to simulated ischemia and reperfusion after infection with either control or NHE1-containing adenovirus. Cells were placed into an anaerobic chamber and effects of NHE1 expression after hypoxia/reoxygenation were examined. Hypoxia/reoxygenation increased caspase-3-like activity in controls, and the effect was greatly magnified in cells expressing NHE1 protein. It also elevated the percentage of apoptotic cardiomyocytes, which was also aggravated by expression of NHE1 protein. Hypoxia/reoxygenation also increased phospho-ERK levels. Elevated NHE1 expression was coincidental with increased expression of the ER stress protein, protein disulfide isomerase (PDI) and calreticulin (CRT). Our results demonstrate that increased NHE1 protein expression makes cells more susceptible to damage induced by hypoxia/reoxygenation in isolated cardiomyocytes. They suggest that elevated NHE1 in cardiovascular disease could predispose the human myocardium to enhanced apoptotic damage.     

外源性精胺对缺氧诱导乳鼠心肌细胞凋亡的影响及其机制

目的：观察外源性精胺对缺氧所致的乳鼠心肌细胞凋亡的影响,并探讨其机制。方法：复制原代培养乳鼠心肌细胞缺氧损伤模型（使用pH=6.8的Hank's平衡盐溶液作为细胞培养基,排出氧气,然后在缺氧箱中培养24 h）,细胞随机分为正常对照（Control）组、缺氧（Hypoxia）组和精胺干预（Hypoxia+Sp）组。Western blot检测心肌细胞多胺代谢关键酶（ODC、SSAT）蛋白质表达;CCK-8,Hoechst 33342染色观察细胞凋亡情况;光吸收法检测细胞（或培养液）内T-SOD和Caspase-3/-9活性,MDA、GSH含量;DCFH-DA染色观察细胞内活性氧（ROS）生成。结果：与正常组相比,Hypoxia组SSAT蛋白质表达、细胞凋亡率、MDA含量以及细胞内ROS生成增加,而ODC蛋白质表达、SOD活性、GSH含量降低;与Hypoxia组比较,Sp处理可减轻上述指标的变化。结论：外源性精胺可减轻缺氧引起的乳鼠心肌细胞损伤和凋亡,其机制与恢复多胺稳态和清除活性氧有关。     

The threshold response of neonatal rat cardiomyocytes to gradual enhancement of cryptosporidial invasion

One common cause of excessive cardiac loading in children is infectious gastroenteritis that produces malabsorption and tachyarrythmia. Our recent studies have shown that neonatal cryptosporidial gastroenteritis causes the long-term pathology of cardiomyocytes. In the present work, we studied how cryptosporidiosis of different degrees of severity is reflected on the heart anatomy and polyploidization and remodeling of cardiomyocytes of neonatal rats, as well as on the expression of their gene encoding factor sensitive to hypoxia (HIF-1α). By methods of Real-Time PCR, cytometry, immunocytochemistry, image analysis, and visual study of interatrial septum, we have established that gradual enhancement of infestation by cryptosporidiosis is accompanied by threshold changes in heart. With weak infection, the interatrial septum preserves integrity, while changes in cardiomyocytes are absent. With moderate and strong infection, the changes are expressed approximately equally: in the interatrial septum, foramen ovale appears and the heart becomes atrophied and elongated, while cardiomyocytes lose protein and become thinned and hyperpolyploid. In addition, an excessive level of mRNA of gene HIF-1α appears in the myocardium. It is important to note that the threshold response to the gradual increase of the action is a criterion of triggering of the developmental programming of cardiovascular diseases, as such response is based on irreversible disturbance of the anatomy of the organ and failure of cell differentiation. In our case, the disturbance of the heart is the patent foramen ovale in the interatrial septum, while the disturbance of differentiation is hyperpolyploidization of cardiomyocytes. Our data may be of interest to physicians, as they show for the first time a connection between gastroenteritis, disturbance of the integrity of the interatrial septum, and pathological alterations in cardiomyocytes.     

Properties and functions of KATP during mouse perinatal development

BackgroundPrevailing data suggest that ATP-sensitive potassium channels (K_ATP) contribute to a surprising resistance to hypoxia in mammalian embryos, thus we aimed to characterize the developmental changes of K_ATP channels in murine fetal ventricular cardiomyocytes.MethodsPatch clamp was applied to investigate the functions of K_ATP. RT-PCR, Western blot were used to further characterize the molecular properties of K_ATP channels.ResultsSimilar K_ATP current density was detected in ventricular cardiomyocytes of late development stage (LDS) and early development stage (EDS). Molecular–biological study revealed the upregulation of Kir6.1/SUR2A in membrane and Kir6.2 remained constant during development. Kir6.1, Kir6.2, and SUR1 were detectable in the mitochondria without marked difference between EDS and LDS. Acute hypoxia–ischemia led to cessation of APs in 62.5% of tested EDS cells and no APs cessation was observed in LDS cells. SarcK_ATP blocker glibenclamide rescued 47% of EDS cells but converted 42.8% of LDS cells to APs cessations under hypoxia-ischemic condition. MitoK_ATP blocker 5-HD did not significantly influence the response to acute hypoxia–ischemia at either EDS or LDS. In summary, sarcK_ATP played distinct functional roles under acute hypoxia-ischemic condition in EDS and LDS fetal ventricular cardiomyocytes, with developmental changes in sarcK_ATP subunits. MitoK_ATP were not significantly involved in the response of fetal cardiomyocytes to acute hypoxia–ischemia and no developmental changes of K_ATP subunits were found in mitochondria.     

High glucose protects single beating adult cardiomyocytes against hypoxia

In the heart, the opening of sarcolemmal ATP-sensitive K(+) (K(ATP)) channels seems to be crucial for the cardiac protection against hypoxia/ischaemia. In the present study, we have exposed cardiomyocytes under hypoxia to high extracellular glucose (30 mM). Under these conditions, intracellular concentration of 1,3-bisphosphoglycerate has increased confirming stimulation of glycolysis. Perforated patch-clamp electrophysiology revealed that hypoxia induces whole-cell K(+) current in cardiomyocytes more efficiently in the presence than in the absence of high glucose. Glucose significantly promoted survival of cardiomyocytes exposed to hypoxia. HMR 1098, an antagonist of sarcolemmal K(ATP) channels, inhibited glucose-induced activation of whole-cell K(+) current during hypoxia as well as glucose-mediated cytoprotection. An inhibitor of glyceraldehyde 3-phosphate dehydrogenase, iodoacetate, inhibited glycolysis in hypoxia and blocked the activation of sarcolemmal K(ATP) channels. Based on the obtained results, we conclude that the activation of sarcolemmal K(ATP) channels is involved in glucose-mediated cardioprotection.     

The action of short-range low-intensity infrared laser irradiation on the functional and metabolic parameters of the isolated rat myocardium in hypoxia

It was found that infrared laser radiation (IRL) reduces the sparing action of acute hypoxia on ventricular transport function of low-resistant animals and accelerates the recovery of the function during the post-hypoxia period. The effect was caused by the IRL affecting directly the speed of perfusion through the myocardium and thus the latter's breathing rate. The protective effect of the IRL was practically absent in highly resistant animals, which may be indicative of the existence of basic differences in the regulatory systems which is responsible for local vasodilation and supply of oxygen to cells, and which participates in the formation of resistance of cardiomyocytes to oxygen shortage.     

Cardiomyocytes of chronically ischemic pig hearts express the MDR-1 gene-encoded P-glycoprotein.

The multidrug-resistant (MDR)-1 gene-encoded P-glycoprotein (Pgp-170) is not normally present in the cardiomyocyte. Given that in other tissues Pgp-170 is not found under normoxic conditions but is expressed during hypoxia, we searched for Pgp-170 in chronically ischemic porcine cardiomyocytes. Pgp-170 was detected and localized via immunohistochemistry in ischemic and nonischemic cardiomyocytes of eight adult pigs 8 weeks after placement of an Ameroid constrictor at the origin of the left circumflex artery (Cx). Regional myocardial ischemia in the Cx bed was documented with nuclear perfusion scans. Pgp-170 mass was quantified using Western blot analysis. In all pigs, Pgp-170 was consistently present in the sarcolemma and T invaginations of the cardiomyocytes of the ischemic zone. Pgp-170 expression decreased toward the border of the ischemic zone and was negative in nonischemic regions as well as in the myocardium of sham-operated animals. Western blot analysis yielded significantly higher Pgp-170 mass in ischemic than in nonischemic areas. We conclude that Pgp-170 is consistently expressed in the cardiomyocytes of chronically ischemic porcine myocardium. Its role in the ischemic heart as well as in conditions such as myocardial hibernation, stunning, and preconditioning may have potentially relevant clinical implications and merits further investigation.     

间歇性低压低氧对过氧化氢心肌细胞损伤的保护作用

目的:观察慢性间歇性低压低氧对过氧化氢所致心肌细胞损伤的保护作用及其机制。方法:雄性豚鼠20只,随机分为两组(n=10):对照组(non-IHH)、低氧组(IHH)。低氧组豚鼠于低压氧舱接受28 d(海拔5 000 m、每天6 h)的低压低氧处理。胶原酶方法急性分离心肌细胞。细胞动缘探测系统测定过氧化氢对各组细胞收缩力的变化。生化方法测定各组丙二醛(MDA)、乳酸脱氢酶(LDH)及超氧化物歧化酶(SOD)和过氧化氢酶(CAT)的变化。结果:①过氧化氢可使心肌细胞出现收缩、舒张紊乱,但IHH处理使其出现的潜伏期明显延长。②给予过氧化氢(300μmol/L,10 min)使来自于non-IHH或IHH的心肌细胞LDH、MDA含量均明显增加,但IHH心肌细胞LDH、MDA含量明显低于non-IHH心肌细胞的LDH、MDA含量。③经IHH处理组的心肌细胞SOD,CAT活性均明显高于non-IHH组。给予过氧化氢使来自于non-IHH或IHH的心肌细胞SOD,CAT活性均明显降低,但IHH心肌细胞SOD,CAT活性明显高于non-IHH心肌细胞的SOD,CAT活性。结论:IHH具有对抗过氧化氢心肌细胞损伤的作用,可能与其增强抗氧化酶活性有关。