Prohibitin protects against oxidative stress-induced cell injury in cultured neonatal cardiomyocyte

Oxidative stress is one of the main causes of myocardial injury, which is associated with cardiomyocyte death. Mitochondria play a key role in triggering the necrosis and apoptosis pathway of cardiomyocytes under oxidative stress. Although prohibitin (PHB) has been acknowledged as a mitochondrial chaperone, its functions in cardiomyocytes are poorly characterized. The present research was designed to investigate the cardioprotective role of PHB in mitochondria. Oxidative stress can increase the PHB content in mitochondria in a time-dependent manner. Overexpression of PHB in cultured cardiomyocytes by transfection of recombinant adenovirus vector containing PHB sense cDNA resulted in an increase of PHB in mitochondria. Compared with the non-transfection cardiomyocytes, PHB overexpression could protect the mitochondria from oxidative stress-induced injury. The mitochondria-mediated apoptosis pathway was consistently suppressed in PHB-overexpressed cardiomyocytes after hydrogen peroxide (H₂O₂) treatment, including a reduced change in mitochondrial membrane permeability transition and an inhibited release of cytochrome c from mitochondria to cytoplasma. As a result, the oxidative stress-induced cardiomyocyte apoptosis was suppressed. These data indicated that PHB protected the cardiomyocytes from oxidative stress-induced damage, and that increasing PHB content in mitochondria constituted a new therapeutic target for myocardium injury. XiaoHua Liu and Zhe Ren contributed equally to this work. ● Prohibitin is an evolutionarily conserved and ubiquitously expressed protein involved in mitochondrial structure, function, and inheritance whose function in cardiomyocyte is not known. In this study, we found oxidative stress could induce increased expression in cardiomyocytes and mitochondrial translocation of PHB, and PHB can protect against oxidative stress in cultured neonatal cardiomyocyte.     

Mitochondrial oxidative stress-induced apoptosis and radioprotection in proton-irradiated rat retina

There is concern about possible radiation damage to the eyes from occupational exposure and medical procedures. In this study, molecular mechanisms of proton radiation-induced oxidative damage to retinal cells were evaluated, with and without a cell-permeable superoxide dismutase (SOD) mimetic, metalloporphyrin compound (MnTE-2-PyP). Retinal mitochondria-associated genes and protein expression profiles were studied. Rats were treated with MnTE-2-PyP at 2.5 μg/injection into one eye 1 h before irradiation. Proton irradiation was delivered to the same eye at doses of 1 or 4 Gy and assays were done at 6 h. Levels of Bax, Bcl-2 and Sod2 proteins were evaluated by Western blot and caspase-3 immunohistochemistry was performed to confirm the occurrence of apoptosis. Expression of several genes playing central roles in regulating the mitochondrial apoptotic pathway were significantly increased after radiation exposure, including Bbc3, Bax, Bak1, Bid, and Bcl2. Among genes involved in radiation-induced oxidative stress, Sod2, Gpx and Ucp3 were up-regulated, whereas Ucp2 was down-regulated. In addition, irradiation caused changes in various proteins involved in apoptosis (caspase-3, Bax and Bcl2). Reduction in pro-apoptotic and increase in anti-apoptotic protein levels were documented after treatment with MnTE-2-PyP. Decreased activity of cytochrome c, which is involved in initiation of mitochondrial apoptosis, was also revealed after irradiation and MnTE-2-PyP. Data demonstrated that proton radiation induced mitochondrial apoptosis and altered mitochondrial function in retina. MnTE-2-PyP protected, or at least ameliorated, radiation-induced oxidative damage. These insights prompt further study of this compound as a potential therapeutic candidate for retinal protection against degenerative ocular damage induced by ionizing radiation.     

Integrating miRNA and mRNA expression profiles in response to heat stress-induced injury in rat small intestine

The molecular mechanisms underlying the pathophysiology of heat stress in the small intestine remain undefined. Furthermore, little information is available concerning changes in microRNA (miRNA) expression following heat stress. The present study sought to evaluate miRNA and mRNA expression profiles in the rat small intestine in response to heat stress. Male Sprague-Dawley rats were subjected to 2?h of heat stress daily for ten consecutive days. Rats were sacrificed at specific time points immediately following heat treatment, and morphological changes in the small intestine were determined. The miRNA and mRNA expression profiles from sample of small intestine were evaluated by microarray analysis. Heat stress caused pronounced morphological damage in the rat small intestine, most severe within the jejunum after 3?days of heat treatment. A mRNA microarray analysis found 270 genes to be up-regulated and 122 genes down-regulated (P?≤?0.01, ≥2.0-fold change) in the jejunum after heat treatment. A miRNA microarray analysis found 18 miRNAs to be up-regulated and 11 down-regulated in the jejunum after heat treatment (P?≤?0.05). Subsequent bioinformatic analyses of the differentially expressed mRNAs and miRNAs were carried out to integrate miRNA and mRNA expression and revealed that alterations in mRNA following heat stress were negatively correlated with miRNA expression. These findings significantly advance our understanding of the regulatory mechanisms underlying the pathophysiology of heat stress-induced injury in the small intestine, specifically with regard to miRNAs.     

Dietary nitrate inhibits stress-induced gastric mucosal injury in the rat

Dietary nitrate is reduced to nitrite by some oral bacteria and the resulting nitrite is converted to nitric oxide (NO) in acidic gastric juice. The aim of this study is to elucidate the pathophysiological role of dietary nitrate in the stomach. Intragastric administration of nitrate rapidly increased nitrate and NO in plasma and the gastric headspace, respectively. Water-immersion-restraint stress (WIRS) increased myeloperoxidase (MPO) activity in gastric mucosa and induced hemorrhagic erosions by a nitrate-inhibitable mechanism. In animals that had received either cardiac ligation or oral treatment with povidone-iodine, a potent bactericidal agent, administration of nitrate failed to increase gastric levels of NO and to inhibit WIRS-induced mucosal injury. WIRS decreased gastric mucosal blood flow by a mechanism which was inhibited by administration of nitrate. These data suggested that the enterosalivary cycle of nitrate and related metabolites consisted of gastrointestinal absorption and salivary secretion of nitrate, its conversion to nitrite by oral bacteria and then to NO in the stomach might play important roles in the protection of gastric mucosa from hazardous stress.     

Mitochondrial transplantation ameliorates ischemia/reperfusion-induced kidney injury in rat

No real therapeutic modality is currently available for Acute kidney injury (AKI) and if any, they are mainly supportive in nature. Therefore, developing a new therapeutic strategy is crucial. Mitochondrial dysfunction proved to be a key contributor to renal tubular cell death during AKI. Thus, replacement or augmentation of damaged mitochondria could be a proper target in AKI treatment. Here, in an animal model of AKI, we auto-transplanted normal mitochondria isolated from healthy muscle cells to injured kidney cells through injection to renal artery. The mitochondria transplantation prevented renal tubular cell death, restored renal function, ameliorated kidney damage, improved regenerative potential of renal tubules, and decreased ischemia/reperfusion-induced apoptosis. Although further studies including clinical trials are required in this regard, our findings suggest a novel therapeutic strategy for treatment of AKI. Improved quality of life of patients suffering from renal failure and decreased morbidity and mortality rates would be the potential advantages of this therapeutic strategy.     

Mitochondrial permeability transition in pH-dependent reperfusion injury to rat hepatocytes

To simulateischemia and reperfusion, cultured rat hepatocytes were incubated inanoxic buffer at pH 6.2 for 4 h and reoxygenated at pH 7.4. Duringanoxia, intracellular pH (pH_i)decreased to 6.3, mitochondria depolarized, and ATP decreased to <1%of basal values, but the mitochondrial permeability transition (MPT)did not occur as assessed by confocal microscopy from theredistribution of cytosolic calcein into mitochondria. Moreover, cellviability remained >90%. After reperfusion at pH 7.4, pH_i returned to pH 7.2, the MPToccurred, and most hepatocytes lost viability. In contrast, afterreperfusion at pH 6.2 or withNa⁺-free buffer at pH 7.4, pH_i did not rise and cellviability remained >80%. After acidotic reperfusion, the MPT did notoccur. When hepatocytes were reperfused with cyclosporin A (0.5-1µM) at pH 7.4, the MPT was prevented and cell viability remained>80%, although pH_i increased to7.2. Reperfusion with glycine (5 mM) also prevented cell killing butdid not block recovery of pH_i orthe MPT. Retention of cell viability was associated with recovery of30-40% of ATP. In conclusion, preventing the rise ofpH_i after reperfusion blocked theMPT, improved ATP recovery, and prevented cell death. Cyclosporin Aalso prevented cell killing by blocking the MPT without blocking recovery of pH_i. Glycine preventedcell killing but did not inhibit recovery ofpH_i or the MPT.

     

Dehydrosilybin attenuates the production of ROS in rat cardiomyocyte mitochondria with an uncoupler-like mechanism

Reactive oxygen species (ROS) originating from mitochondria are perceived as a factor contributing to cell aging and means have been sought to attenuate ROS formation with the aim of extending the cell lifespan. Silybin and dehydrosilybin, two polyphenolic compounds, display a plethora of biological effects generally ascribed to their known antioxidant capacity. When investigating the cytoprotective effects of these two compounds in the primary cell cultures of neonatal rat cardiomyocytes, we noted the ability of dehydrosilybin to de-energize the cells by monitoring JC-1 fluorescence. Experiments evaluating oxygen consumption and membrane potential revealed that dehydrosilybin uncouples the respiration of isolated rat heart mitochondria albeit with a much lower potency than synthetic uncouplers. Furthermore, dehydrosilybin revealed a very high potency in suppressing ROS formation in isolated rat heart mitochondria with IC₅₀ = 0.15 μM. It is far more effective than its effect in a purely chemical system generating superoxide or in cells capable of oxidative burst, where the IC₅₀ for dehydrosilybin exceeds 50 μM. Dehydrosilybin also attenuated ROS formation caused by rotenone in the primary cultures of neonatal rat cardiomyocytes. We infer that the apparent uncoupler-like activity of dehydrosilybin is the basis of its ROS modulation effect in neonatal rat cardiomyocytes and leads us to propose a hypothesis on natural ischemia preconditioning by dietary polyphenols.     

Calpain-2 protects against heat stress-induced cardiomyocyte apoptosis and heart dysfunction by blocking p38 mitogen-activated protein kinase activation

Cardiovascular dysfunction is a common complication among heatstroke patients, but its underlying mechanism is unclear. This study was designed to investigate the role of calpain-2 and its downstream signal pathway in heat stress-induced cardiomyocyte apoptosis and heart dysfunction. In cultured primary mouse neonatal cardiomyocytes (MNCs), heat stress (43°C for 2 hr) induced a heat-shock response, as indicated by upregulated heat-shock protein 27 (HSP27) expression and cellular apoptosis, as indicated by increased caspase-3 activity, DNA fragmentation and decreased cell viability. Meanwhile, heat stress decreased calpain activity, which was accompanied by downregulated calpain-2 expression and increased phosphorylation of p38, extraceIIuIar signaI-reguIated protein kinase (ERK1/2) and c-Jun N-terminaI kinase (JNK). Calpain-2 overexpression abrogated heat stress-induced apoptosis and phosphorylation of p38 and JNK, but not of ERK1/2. Blocking only p38 prevented heat stress-induced apoptosis in MNCs. In cardiac-specific calpain-2 overexpressing transgenic mice, p38 phosphorylation and cardiomyocyte apoptosis were decreased in the heart tissue of heatstroke mice, as revealed by western blot and terminal deoxynucleotidyl transferase dUTP nick end labelling assays, respectively. M-mode echocardiography also demonstrated that calpain-2 overexpression significantly improved heatstroke-induced decreases in ventricular end-diastolic volume and cardiac output. In conclusion, our study suggests that heat stress reduces calpain-2 expression, which then activates p38, leading to cardiomyocyte apoptosis and heart dysfunction.     

Dysregulation of hepatic iron with aging: implications for heat stress-induced oxidative liver injury

Environmental heat stress is associated with an age-related increase in hepatic oxidative damage and an exaggerated state of oxidative stress. The purpose of this investigation was to evaluate the regulation of hepatic iron after heat stress. A secondary aim was to determine a potential role for iron in heat stress-induced liver injury. Hyperthermia-induced alterations in hepatic iron were evaluated in young (6 mo) and old (24 mo) Fischer 344 rats by exposing them to a two-heat stress protocol. Livers were harvested at several time points after the second heating and assayed for labile and nonheme iron. In the control condition, there was no difference in labile iron between age groups. Both labile iron and storage iron were not altered by hyperthermia in young rats, but both were increased immediately after heating in old rats. To evaluate a role for iron in liver injury, hepatic iron content was manipulated in young and old rats, and then both groups were exposed to heat stress. Iron administration to young rats significantly increased hepatic iron content and ferritin but did not affect markers of lipid peroxidation under control conditions or after heat stress. In old rats, iron chelation with deferoxamine prevented the increase in nonheme iron, labile iron, ferritin, and lipid peroxidation after heat stress. These results suggest that iron may play a role in hepatic injury after hyperthermia. Thus, dysregulation of iron may contribute to the gradual decline in cellular and physiological function that occurs with aging.     

Mitochondrial injury in pancreatitis

Pancreatitis is an increasingly common disease that carries a significant mortality and which lacks specific therapy. Pathological calcium signalling is an important contributor to the initiating cell injury, caused by or acting through mitochondrial inhibition. A principal effect of disordered cell signalling and impaired mitochondrial function is cell death, either by apoptosis that is primarily protective, or by necrosis that is deleterious, both locally and systemically. Mitochondrial calcium overload is particularly important in necrotic injury, which may include damage mediated by the mitochondrial permeability transition pore. The role of reactive oxygen species remains controversial. Present understanding of the part played by disordered pancreatic acinar calcium signalling and mitochondrial inhibition offers several new potential therapeutic targets.     

Preventive effect of omega-3 fatty acids in a rat model of stress-induced liver injury

Omega-3 fatty acids are gaining attention as a therapeutic agent of many diseases. Their protective effect in a variety of diseases has been demonstrated. To the best of our knowledge, this is the first study on omega-3 fatty acids related to acute cold-restraint stress (CRS) induced hepatic dysfunction in rats. Forty adult male Sprague–Dawley albino rats were used and classified into: control, omega-3 group, each rat was pretreated with omega-3 fatty acids; CRS group, rats were subjected to acute CRS for 6 hr; and CRS group pretreated with omega-3 fatty acids. Serum was obtained to determine corticosterone (CORT), alanine aminotransferase (ALT), aspartate aminotransferase (AST), and tumor necrosis factor-α (TNF-α) levels. Hepatic malondialdehyde (MDA) and total antioxidant capacity (TAC) levels were measured. Also, liver tissues were taken for histological examination and immunohistochemical assessment of the apoptotic marker, caspase-3. Results showed that pretreatment of stressed rats with omega-3 fatty acids led to significant decrease in hepatic MDA and increase in TAC levels. They reduced serum levels of CORT, ALT, AST, and TNF-α. Also, they improved liver damage and suppressed hepatic caspase-3 expression. In conclusion, pretreatment of stressed rats with omega-3 fatty acids has ameliorated stress-induced liver damage due to their antioxidant, anti-inflammatory, and antiapoptotic effects. So, they can be used to minimize stress complications on the liver.     

Sphingolipids signal heat stress-induced ubiquitin-dependent proteolysis

Sphingolipids are essential eukaryotic membrane lipids that are structurally and metabolically conserved through evolution. Sphingolipids have also been proposed to regulate eukaryotic stress responses as novel second messengers. Here we show that, in Saccharomyces cerevisiae, phytosphingosine, a putative sphingolipid second messenger, mediates heat stress signaling and activates ubiquitin-dependent proteolysis via the endocytosis vacuolar degradation and 26 S proteasome pathways. Inactivation of serine palmitoyltransferase, a key enzyme in generating endogenous phytosphingosine, prevents proteolysis during heat stress. Addition of phytosphingosine bypasses the requirement for serine palmitoyltransferase and restores proteolysis. Phytosphingosine-induced proteolysis requires multiubiquitin chain formation through the stress-responsive lysine 63 residue of ubiquitin. We propose that heat stress increases phytosphingosine and activates ubiquitin-dependent proteolysis.     

Ammonia aggravates stress-induced gastric mucosal oxidative injury through the cancellation of cytoprotective heat shock protein 70

The relationship between Helicobacter pylori colonization and the formation of stress-induced gastric mucosal injury remains unknown. Since ammonia (NH(3)) is known as one of the injurious factors in H. pylori-colonized gastric mucosa, the present study is designed to investigate the level of stress-induced gastric mucosal oxidative injury with or without intragastric NH(3) overloading. To apply emotional stress, the communication box paradigm was used in the mouse model. Mice (C57BL/6, male) were pretreated with distilled water (responder-H(2)O) or 0.01% NH(3) (responder-NH(3)) through a gastric tube once a day for a week. Emotional stress was then applied to the responder mice for 3 h per day for 3 d by watching and hearing the behavior of the sender mice subjected to electric shocks to the feet (2 mA, 10 s, 50 s interval). After the communication box protocol, the tissue MPO activity, the contents of TBA-reactive substances (TBARS), and the level of gastric mucosal HSP70 were examined. Responder-NH(3) mice developed more severe gastric lesions than the responder-H(2)O subjects. MPO activity and TBARS contents were enhanced significantly in the responder-NH(3) group compared with the responder-H(2)O subjects. Although the contents of HSP70 in the gastric mucosa increased in the responder-H(2)O group compared with the control-H(2)O animals, they were significantly attenuated in the responder-NH(3) mice. Excess intragastric NH(3) was able to enhance the formation of emotional stress-induced gastric mucosal lesions. This injury may be associated with the enhanced production of oxygen free radicals from accumulated neutrophils under the NH(3)-mediated cancellation of gastric mucosal cytoprotective HSP70.     

HSP70 inhibits stress-induced cardiomyocyte apoptosis by competitively binding to FAF1

Stress-induced cardiomyocyte apoptosis plays an important role in the pathogenesis of a variety of cardiovascular diseases. Our early studies showed that HSP70 effectively inhibited apoptosis, but the underlying mechanism remained unclear. Fas-associated factor 1 (FAF1) is a member of the Fas death-inducing signaling complex (Fas-DISC) that acts upstream of caspase-8. We investigated the interactions among FAF1, HSP70, and FAS in stressed cardiomyocytes to elucidate the protective mechanism of HSP70. FAS and caspase-3/8 activity was higher in cardiomyocytes undergoing stress-induced apoptosis in restraint-stressed rats compared with cardiomyocytes in non-stressed rats, which indicated that the Fas signaling pathway was activated after restraint stress. Geranylgeranylacetone (GGA) induced an increase in HSP70 expression, which reduced stress-induced apoptosis. Additionally, overexpression of HSP70 via transfection with the pEGFP-rHSP70 plasmid attenuated norepinephrine (NE)-induced apoptosis. FAF1 expression increased during stress-induced apoptosis, and overexpression of FAF1 exacerbated NE-induced apoptosis. We also found that HSP70 interacted with FAF1. Overexpression of HSP70 inhibited the binding of FAF1 to FAS in H9C2 cells, which indicated that HSP70 suppressed NE-induced apoptosis by competitively binding to FAF1. An N-terminal deletion mutant of HSP70 (HSP70-△N) was unable to interact with FAF1. After HSP70-△N was transfected into H9C2 cells, the cells were unable to attenuate the NE-induced increases in caspase-8 and apoptosis. These results indicate that the 1–120 sequence of HSP70 binds to FAF1, which alters the interactions between FAS and FAF1 and inhibits the activation of the Fas signaling pathway and apoptosis.

Electronic supplementary material

The online version of this article (doi:10.1007/s12192-015-0589-9) contains supplementary material, which is available to authorized users.     

Research of cardiomyocyte precursors in adult rat heart

Recent reports supported the existence of stem cells in adult hearts. However, phenotype and localization of these cells have not been completely described and it is unknown if cardiac regenerative potential differs from one subject to another. The aims of our work were to identify different populations of cardiac stem cells by the analysis of specific markers and to evaluate the expression variability of these markers in 12 adult rat hearts. The expression of CD9, taube nuss and nanog suggests the presence of stem cells from the earliest stages of embryogenesis in adult myocardium. Their different expression could be associated to the degree of stem cell differentiation. CD34 and c-Kit antibodies were used to detect stem cells committed to one or more specific tissue lineages and we found a strong immunoreactivity for CD34 exclusively in the endothelial cells and a low positivity for c-Kit in the interstitium and next to the vessels. Moreover, as c-Kit expression highly differed within all examined hearts, we suggest that cardiomyogenic potential is different among the various subjects. Undifferentiated cells with myogenic-committed phenotype expressing GATA-4 and nestin were found, respectively, in the interstitial and myocardial cells and in few interstitial cells. Therefore, the physiologic turn over of cardiomyocytes may occur in adult hearts as it has been shown in many others organs. The study of myogenic potential could be important to identify markers specific of stem cells in in vivo adult myocardium that may be used to purify these cells and evaluate their regenerative ability.     

LPS induces cardiomyocyte injury through calcium-sensing receptor

The development of atherosclerosis (AS) is a multifactorial process in which elevated plasma cholesterol levels play a central role. As a new class of players involved in AS, the regulation and function of microRNAs (miR) in response to AS remain poorly understood. This study analyzed the effects of miR-1 (antagomir and mimic) on endothelial permeability and myosin light chain kinase (MLCK) expression and activity in the artery wall of apoE knock-out mice after feeding them a high-cholesterol diet. Further, we tested to determine whether that effects are involved in ERK phosphorylation. Here, we show that a high-cholesterol diet induces a significant decrease of miR-1 expression. Histopathologic examination demonstrated that miR-1 antagomir enhances endothelial permeability induced by high cholesterol and miR-1 mimic attenuated endothelial barrier dysfunction. Consistent with endothelial permeability, Western blotting, qPCR, and γ-³²P-ATP phosphate incorporation showed that MLCK expression and activity were further increased in miR-1 antagomir-treated mice and decreased in miR-1 mimic-treated mice compared with those of mice receiving control miR. Further mechanistic studies showed that high-cholesterol-induced extracellular signal regulated kinase (ERK) activation was enhanced by miR-1 antagomir and attenuated by miR-1 mimic. Collectively, those results indicate that miR-1 contributes to endothelial barrier function via mechanisms involving not only MLCK expression and activity but also ERK phosphorylation.