Environmentally persistent free radicals decrease cardiac function before and after ischemia/reperfusion injury in vivo

Exposure to airborne particles is associated with increased cardiovascular morbidity and mortality. During the combustion of chlorine-containing hazardous materials and fuels, chlorinated hydrocarbons chemisorb to the surface of transition metal-oxide-containing particles, reduce the metal, and form an organic free radical. These radical-particle systems can survive in the environment for days and are called environmentally persistent free radicals (EPFRs). This study determined whether EPFRs could decrease left ventricular function before and after ischemia and reperfusion (I/R) in vivo. Male Brown–Norway rats were dosed (8?mg/kg, intratracheal) 24?h prior to testing with particles containing the EPFR of 1, 2-dichlorobenzene (DCB230). DCB230 treatment decreased systolic and diastolic function. DCB230 also produced pulmonary and cardiac inflammation. After ischemia, systolic, but not diastolic function was significantly decreased in DCB230-treated rats. Ventricular function was not affected by I/R in control rats. There was greater oxidative stress in the heart and increased 8-isoprostane (biomarker of oxidative stress) in the plasma of treated vs. control rats after I/R. These data demonstrate for the first time that DCB230 can produce inflammation and significantly decrease cardiac function at baseline and after I/R in vivo. Furthermore, these data suggest that EPFRs may be a risk factor for cardiac toxicity in healthy individuals and individuals with ischemic heart disease. Potential mechanisms involving cytokines/chemokines and/or oxidative stress are discussed.     

Donepezil provides neuroprotective effects against brain injury and Alzheimer's pathology under conditions of cardiac ischemia/reperfusion injury

Cardiac ischemia/reperfusion (I/R) injury induces brain pathology. Donepezil, a well-known acetylcholine esterase (AChE) inhibitor, has been proven to exert neuroprotective effects against several neurodegenerative diseases. However, the comprehensive mechanism regarding the therapeutic potential of donepezil on the brain under cardiac I/R injury remains obscure. Here, we hypothesized that treatment with donepezil ameliorates brain pathology following cardiac I/R injury by decreasing blood brain barrier (BBB) breakdown, oxidative stress, neuroinflammation, mitochondrial dysfunction, mitochondrial dynamics imbalance, microglial activation, amyloid-beta (Aβ) accumulation, neuronal apoptosis, and dendritic spine loss. Forty-eight adult male Wistar rats were subjected to surgery for cardiac I/R injury. Then, rats were randomly divided into four groups to receive either (1) saline (vehicle group), donepezil 3 mg/kg via intravenously administered (2) before ischemia (pretreatment group), (3) during ischemia (ischemia group), or (4) at the onset of reperfusion (reperfusion group). At the end of cardiac I/R paradigm, the brains were evaluated for BBB breakdown, brain inflammation, oxidative stress, mitochondrial function, mitochondrial dynamics, microglial morphology, Aβ production, neuronal apoptosis, and dendritic spine density. Administration of donepezil at all time points equally showed an attenuation of brain damage in response to cardiac I/R injury, as indicated by increased expression of BBB junction protein, reduced brain inflammation and oxidative stress, improved mitochondrial function and mitochondrial dynamics, and alleviated Aβ accumulation and microglial activation, resulting in protection of neuronal apoptosis and preservation of dendritic spine number. These findings suggest that donepezil potentially protects brain pathology caused by cardiac I/R injury regardless the timing of treatment.     

Nitric oxide mediates cardiac protection of tissue kallikrein by reducing inflammation and ventricular remodeling after myocardial ischemia/reperfusion

We assessed the role of nitric oxide (NO) and the kinin B2 receptor in mediating tissue kallikrein's actions in intramyocardial inflammation and cardiac remodeling after ischemia/reperfusion (I/R) injury. Adenovirus carrying the human tissue kallikrein gene was delivered locally into rat hearts 4 days prior to 30-minute ischemia followed by 24-hour or 7-day reperfusion with or without administration of icatibant, a kinin B2 receptor antagonist, or N(omega)-nitro-L-arginine methyl ester (L-NAME), a nitric oxide synthase inhibitor. Kallikrein gene delivery improved cardiac contractility and diastolic function, reduced infarct size at 1 day after I/R without affecting mean arterial pressure. Kallikrein treatment reduced macrophage/monocyte and neutrophil accumulation in the infarcted myocardium in association with reduced intercellular adhesion molecule-1 levels. Kallikrein increased cardiac endothelial nitric oxide synthase phosphorylation and NO levels and decreased superoxide formation, TGF-beta1 levels and Smad2 phosphorylation. Furthermore, kallikrein reduced I/R-induced JNK, p38MAPK, IkappaB-alpha phosphorylation and nuclear NF-kappaB activation. In addition, kallikrein improved cardiac performance, reduced infarct size and prevented ventricular wall thinning at 7 days after I/R. The effects of kallikrein on cardiac function, inflammation and signaling mediators were all blocked by icatibant and L-NAME. These results indicate that tissue kallikrein through kinin B2 receptor and NO formation improves cardiac function, prevents inflammation and limits left ventricular remodeling after myocardial I/R by suppression of oxidative stress, TGF-beta1/Smad2 and JNK/p38MAPK signaling pathways and NF-kappaB activation.     

Dietary magnesium intake influences circulating pro-inflammatory neuropeptide levels and loss of myocardial tolerance to postischemic stress

Severe dietary Mg restriction (Mg(9), 9% of recommended daily allowance [RDA], plasma Mg = 0.25 mM) induces a pro-inflammatory neurogenic response in rats (substance P [SP]), and the associated increases in oxidative stress in vivo and cardiac susceptibility to ischemia/reperfusion (I/R) injury were previously shown to be attenuated by SP receptor blockade and antioxidant treatment. The present study assessed if less severe dietary Mg restriction modulates the extent of both the neurogenic/oxidative responses in vivo and I/R injury in vitro. Male Sprague-Dawley rats maintained on Mg(40) (40% RDA, plasma Mg = 0.6 mM) or Mg(100) (100% RDA, plasma Mg = 0.8 mM) diets were assessed for plasma SP levels (CHEM-ELISA) during the first 3 weeks and were compared with the Mg(9) group; red blood cell (RBC) glutathione and plasma malondialdehyde levels were compared at 3 weeks in Mg(9), Mg(20) (plasma Mg = 0.4 mM), Mg(40), and Mg(100) rats; and 40-min global ischemia/30-min reperfusion hearts from 7-week-old Mg(20), Mg(40), and Mg(100) rats were compared with respect to functional recovery (cardiac work, and diastolic, systolic, and developed pressures), tissue LDH release, and free radical production (ESR spectroscopy and alpha-phenyl-N-tert butylnitrone [PBN; 3 mM] spin trapping). The Mg(40) diet induced smaller elevations in plasma SP (50% lower) compared with Mg(9), but with a nearly identical time course. RBC glutathione and plasma malondialdehyde levels revealed a direct relationship between the severity of oxidative stress and hypomagnesemia. The dominant lipid free radical species detected in all I/R groups was the alkoxyl radical (PBN/alkoxyl: alpha(H) = 1.93 G, alpha(N) = 13.63 G); however, Mg(40) and Mg(20) hearts exhibited 2.7- and 3.9-fold higher alkoxyl levels, 40% and 65% greater LDH release, and lower functional recovery (Mg(20) < Mg(40)) compared with Mg(100). Our data suggest that varying dietary Mg intake directly influences the magnitude of the neurogenic/oxidative responses in vivo and the resultant myocardial tolerance to I/R stress.     

Protective effects of melatonin on myocardial ischemia/reperfusion induced infarct size and oxidative changes

Free radicals, calcium overloading and loss of membrane phospholipids play an important role in the development of ischemia/reperfusion (I/R) injury. Melatonin is a well-known antioxidant and free radical scavenger. Melatonin may also reduce the intracellular calcium overloading and inhibit lipid peroxidation. This study was designed to investigate the effects of melatonin on the I/R-induced cardiac infarct size in an in vivo rat model. We also investigated glutathione (GSH) levels, an antioxidant the levels of which are influenced by oxidative stress, and malondialdehyde (MDA) levels, which is an index of lipid peroxidation. To produce cardiac damage, the left main coronary artery was occluded for 30 min, followed by 120 min reperfusion, in anesthetized rats. Melatonin (10 mg/kg) or vehicle was given 10 min before ischemia via the jugular vein. Infarct size, expressed as the percentage of the risk zone, was found significantly greater in I/R group than in the melatonin-treated I/R group. MDA levels were significantly higher, but GSH levels were lower in the I/R group than in the control group. Melatonin significantly reduced the MDA values and increased the GSH levels. These results suggest that oxidative stress contributes to myocardial I/R injury and melatonin administration exerts a mitigating effect on infarct size. Furthermore, the results indicated that melatonin improves the antioxidant capacity of the heart and attenuates the degree of lipid peroxidation after I/R.     

Protection against in vivo focal myocardial ischemia/reperfusion injury-induced arrhythmias and apoptosis by Hesperidin

Among the heart diseases, ischemia and reperfusion (I/R) induced arrhythmias contribute to episodes of sudden death. Cardiac arrhythmias during ischemia reperfusion are believed to be related to oxidative stress. Therefore, the aim of this study was to examine whether treatment with Hesperidin alleviates arrhythmias and infarct size in experimentally-induced myocardial I/R injury using an in vivo rat model. In this study haemodynamics parameters, markers of inflammation, biomarkers of oxidative stress and tissue nitrite level and infarct size of the heart were estimated in various groups. I/R showed a significant decrease in tissue nitrite and antioxidant level and significant increase in arrhythmias, inflammation and myocardial cell apoptosis. Treatment with Hesperidin showed a significant increase in tissue nitrite, antioxidant level and reduction in inflammation, arrhythmias and apoptosis. In conclusion, the protecting effect of Hesperidin in I/R induced arrhythmias is due to reduction in inflammation and oxidative stress.     

Celecoxib prevents pressure overload‐induced cardiac hypertrophy and dysfunction by inhibiting inflammation,apoptosis and oxidative stress

To explore the effects of celecoxib on pressure overload‐induced cardiac hypertrophy (CH), cardiac dysfunction and explore the possible protective mechanisms. We surgically created abdominal aortic constrictions (AAC) in rats to induce CH. Rats with CH symptoms at 4 weeks after surgery were treated with celecoxib [2 mg/100 g body‐weight(BW)] daily for either 2 or 4 weeks. Survival rate, blood pressure and cardiac function were evaluated after celecoxib treatment. Animals were killed, and cardiac tissue was examined for morphological changes, cardiomyocyte apoptosis, fibrosis, inflammation and oxidative stress. Four weeks after AAC, rats had significantly higher systolic, diastolic and mean blood pressure, greater heart weight and enlarged cardiomyocytes, which were associated with cardiac dysfunction. Thus, the CH model was successfully established. Two weeks later, animals had impaired cardiac function and histopathological abnormalities including enlarged cardiomyocytes and cardiac fibrosis, which were exacerbated 2 weeks later. However, these pathological changes were remarkably prevented by the treatment of celecoxib, independent of preventing hypertension. Mechanistic studies revealed that celecoxib‐induced cardiac protection against CH and cardiac dysfunction was due to inhibition of apoptosis via the murine double mimute 2/P53 pathway, inhibition of inflammation via the AKT/mTOR/NF‐κB pathway and inhibition of oxidative stress via increases in nuclear factor E2‐related factor‐2‐mediated gene expression of multiple antioxidants. Celecoxib suppresses pressure overload‐induced CH by reducing apoptosis, inflammation and oxidative stress.     

A new model of congestive heart failure in rats

Current rodent models of ischemia/infarct or pressure-volume overload are not fully representative of human heart failure. We developed a new model of congestive heart failure (CHF) with both ischemic and stress injuries combined with fibrosis in the remote myocardium. Sprague-Dawley male rats were used. Ascending aortic banding (Ab) was performed to induce hypertrophy. Two months post-Ab, ischemia-reperfusion (I/R) injury was induced by ligating the left anterior descending (LAD) artery for 30 min. Permanent LAD ligation served as positive controls. A debanding (DeAb) procedure was performed after Ab or Ab + I/R to restore left ventricular (LV) loading properties. Cardiac function was assessed by echocardiography and in vivo hemodynamic analysis. Myocardial infarction (MI) size and myocardial fibrosis were assessed. LV hypertrophy was observed 4 mo post-Ab; however, systolic function was preserved. LV hypertrophy regressed within 1 mo after DeAb. I/R for 2 mo induced a small to moderate MI with mild impairment of LV function. Permanent LAD ligation for 2 mo induced large MI and significant cardiac dysfunction. Ab for 2 mo followed by I/R for 2 mo (Ab + I/R) resulted in moderate MI with significantly reduced ejection fraction (EF). DeAb post Ab + I/R to reduce afterload could not restore cardiac function. Perivascular fibrosis in remote myocardium after Ab + I/R + DeAb was associated with decreased cardiac function. We conclude that Ab plus I/R injury with aortic DeAb represents a novel model of CHF with increased fibrosis in remote myocardium. This model will allow the investigation of vascular and fibrotic mechanisms in CHF characterized by low EF, dilated LV, moderate infarction, near-normal aortic diameter, and reperfused coronary arteries.     

Alterations in mitochondrial dynamics with age‐related Sirtuin1/Sirtuin3 deficiency impair cardiomyocyte contractility

Sirtuin1 (SIRT1) and Sirtuin3 (SIRT3) protects cardiac function against ischemia/reperfusion (I/R) injury. Mitochondria are critical in response to myocardial I/R injury as disturbance of mitochondrial dynamics contributes to cardiac dysfunction. It is hypothesized that SIRT1 and SIRT3 are critical components to maintaining mitochondria homeostasis especially mitochondrial dynamics to exert cardioprotective actions under I/R stress. The results demonstrated that deficiency of SIRT1 and SIRT3 in aged (24–26 months) mice hearts led to the exacerbated cardiac dysfunction in terms of cardiac systolic dysfunction, cardiomyocytes contractile defection, and abnormal cardiomyocyte calcium flux during I/R stress. Moreover, the deletion of SIRT1 or SIRT3 in young (4–6 months) mice hearts impair cardiomyocyte contractility and shows aging‐like cardiac dysfunction upon I/R stress, indicating the crucial role of SIRT1 and SIRT3 in protecting myocardial contractility from I/R injury. The biochemical and seahorse analysis showed that the deficiency of SIRT1/SIRT3 leads to the inactivation of AMPK and alterations in mitochondrial oxidative phosphorylation (OXPHOS) that causes impaired mitochondrial respiration in response to I/R stress. Furthermore, the remodeling of the mitochondria network goes together with hypoxic stress, and mitochondria undergo the processes of fusion with the increasing elongated branches during hypoxia. The transmission electron microscope data showed that cardiac SIRT1/SIRT3 deficiency in aging alters mitochondrial morphology characterized by the impairment of mitochondria fusion under I/R stress. Thus, the age‐related deficiency of SIRT1/SIRT3 in the heart affects mitochondrial dynamics and respiration function that resulting in the impaired contractile function of cardiomyocytes in response to I/R.     

Mineralocorticoid receptor blockade improves diastolic function independent of blood pressure reduction in a transgenic model of RAAS overexpression

There is emerging evidence that aldosterone can promote diastolic dysfunction and cardiac fibrosis independent of blood pressure effects, perhaps through increased oxidative stress and inflammation. Accordingly, this investigation was designed to ascertain if mineralocorticoid receptor blockade improves diastolic dysfunction independently of changes in blood pressure through actions on myocardial oxidative stress and fibrosis. We used young transgenic (mRen2)27 [TG(mRen2)27] rats with increases in both tissue ANG II and circulating aldosterone, which manifests age-related increases in hypertension and cardiac dysfunction. Male TG(mRen2)27 and age-matched Sprague-Dawley rats were treated with either a low dose (～1 mg·kg(-1)·day(-1)) or a vasodilatory, conventional dose (～30 mg·kg(-1)·day(-1)) of spironolactone or placebo for 3 wk. TG(mRen2)27 rats displayed increases in systolic blood pressure and plasma aldosterone levels as well as impairments in left ventricular diastolic relaxation without changes in systolic function on cine MRI. TG(mRen2)27 hearts also displayed hypertrophy (left ventricular weight, cardiomyoctye hypertrophy, and septal wall thickness) as well as fibrosis (interstitial and perivascular). There were increases in oxidative stress in TG(mRen2)27 hearts, as evidenced by increases in NADPH oxidase activity and subunits as well as ROS formation. Low-dose spironolactone had no effect on systolic blood pressure but improved diastolic dysfunction comparable to a conventional dose. Both doses of spironolactone caused comparable reductions in ROS/3-nitrotryosine immunostaining and perivascular and interstitial fibrosis. These data support the notion mineralocorticoid receptor blockade improves diastolic dysfunction through improvements in oxidative stress and fibrosis independent of changes in systolic blood pressure.     

Dynasore Protects Mitochondria and Improves Cardiac Lusitropy in Langendorff Perfused Mouse Heart

Background

Heart failure due to diastolic dysfunction exacts a major economic, morbidity and mortality burden in the United States. Therapeutic agents to improve diastolic dysfunction are limited. It was recently found that Dynamin related protein 1 (Drp1) mediates mitochondrial fission during ischemia/reperfusion (I/R) injury, whereas inhibition of Drp1 decreases myocardial infarct size. We hypothesized that Dynasore, a small noncompetitive dynamin GTPase inhibitor, could have beneficial effects on cardiac physiology during I/R injury.

Methods and Results

In Langendorff perfused mouse hearts subjected to I/R (30 minutes of global ischemia followed by 1 hour of reperfusion), pretreatment with 1 µM Dynasore prevented I/R induced elevation of left ventricular end diastolic pressure (LVEDP), indicating a significant and specific lusitropic effect. Dynasore also decreased cardiac troponin I efflux during reperfusion and reduced infarct size. In cultured adult mouse cardiomyocytes subjected to oxidative stress, Dynasore increased cardiomyocyte survival and viability identified by trypan blue exclusion assay and reduced cellular Adenosine triphosphate(ATP) depletion. Moreover, in cultured cells, Dynasore pretreatment protected mitochondrial fragmentation induced by oxidative stress.

Conclusion

Dynasore protects cardiac lusitropy and limits cell damage through a mechanism that maintains mitochondrial morphology and intracellular ATP in stressed cells. Mitochondrial protection through an agent such as Dynasore can have clinical benefit by positively influencing the energetics of diastolic dysfunction.     

Role of oxidative stress in alterations of mitochondrial function in ischemic-reperfused hearts

To study the mechanisms of mitochondrial dysfunction due to ischemia-reperfusion (I/R) injury, rat hearts were subjected to 20 or 30 min of global ischemia followed by 30 min of reperfusion. After recording both left ventricular developed pressure (LVDP) and end-diastolic pressure (LVEDP) to monitor the status of cardiac performance, mitochondria from these hearts were isolated to determine respiratory and oxidative phosphorylation activities. Although hearts subjected to 20 min of ischemia failed to generate LVDP and showed a marked increase in LVEDP, no changes in mitochondrial respiration and phosphorylation were observed. Reperfusion of 20-min ischemic hearts depressed mitochondrial function significantly but recovered LVDP completely and lowered the elevated LVEDP. On the other hand, depressed LVDP and elevated LVEDP in 30-min ischemic hearts were associated with depressions in both mitochondrial respiration and oxidative phosphorylation. Reperfusion of 30-min ischemic hearts elevated LVEDP, attenuated LVDP, and decreased mitochondrial state 3 and uncoupled respiration, respiratory control index, ADP-to-O ratio, as well as oxidative phosphorylation rate. Alterations of cardiac performance and mitochondrial function in I/R hearts were attenuated or prevented by pretreatment with oxyradical scavenging mixture (superoxide dismutase and catalase) or antioxidants [N-acetyl-L-cysteine or N-(2-mercaptopropionyl)-glycine]. Furthermore, alterations in cardiac performance and mitochondrial function due to I/R were simulated by an oxyradical-generating system (xanthine plus xanthine oxidase) and an oxidant (H(2)O(2)) either upon perfusing the heart or upon incubation with mitochondria. These results support the view that oxidative stress plays an important role in inducing changes in cardiac performance and mitochondrial function due to I/R.     

Heart protective effects and mechanism of quercetin preconditioning on anti-myocardial ischemia reperfusion (IR) injuries in rats

In this study, we investigated the effects and mechanism of quercetin preconditioning on anti-myocardial ischemia reperfusion (IR) injuries in vivo. Meanwhile, their potential anti-oxidative stress and anti-inflammation effect were assessed. SD rats were orally given quercetin 250 mg/kg. Myocardium apoptosis was determined with TUNEL staining. The biomarkers related to myocardial ischemia injury were determined. Simultaneously, hemodynamic parameters were monitored as left ventricular systolic pressure (LVSP), LV end-diastolic pressure (LVEDP) and maximal rate of increase and decrease of left ventricular pressure (dP/dtmax). The oxidative stress indicators and inflammatory factors were also evaluated. Western blot method was used for analysis of PI3K, Akt, p-Akt, Bax and Bcl-2 protein expressions. The results showed that quercetin significantly reduced apoptosis rate, improved cardiac function, decreased levels of creatine kinase (CK), aspartate aminotransferase (AST) and lactate dehydrogenase (LDH). Quercetin also restrained the oxidative stress related to myocardial ischemia injury as evidenced by decreased malondialdehyde (MDA), and elevated GSH, superoxide dismutase (SOD), catalase (CAT), glutathione-peroxidase (GSH-Px), glutathione reductase (GR) activity. Meanwhile, the inflammatory cascade was inhibited as evidenced by decreased cytokines such as tumor necrosis factor-α (TNF-α), C-reactive protein (CRP) and interleukin-1β (IL-1β). Our results still showed that quercetin pretreatment significantly inhibited the apoptosis by decreasing the number of apoptotic cells, decreasing the level of cleaved Bax, and increasing the level of Bcl-2 in rats subjected to I/R injury. Simultaneously, quercetin pretreatment markedly increased the phosphorylation of Akt. Blockade of PI3K activity by LY294002, dramatically abolished its anti-apoptotic effect and lowered Akt phosphorylation level. It can be concluded that quercetin pretreatment was protected against myocardium IR injury by decreasing oxidative stress, repressing inflammatory cascade, inhibiting apoptosis in vivo and PI3K/Akt pathway involved in the anti-apoptotic effect.     

Diabetic animal fed with high‐fat diet prevents the protective effect of myocardial ischemic preconditioning effect in isolated rat heart perfusion model

Diabetic heart (diabetes mellitus [DM]) has been shown to attenuate the beneficial effect of ischemic preconditioning (IPC) in rat heart. But the effect of IPC on diabetic rat heart that develops myopathy remains unclear. This study was designed to test the impact of IPC on diabetic cardiomyopathy (DCM) rat heart. Male Wistar rats were grouped as (a) normal, (b) DM (streptozotocin: 65 mg/kg; fed with normal diet), and (c) DCM (streptozotocin: 65 mg/kg; fed with high‐fat diet). Isolated rat hearts from each group were randomly subjected to (a) normal perfusion, (b) ischemia‐reperfusion (I/R), and (c) IPC procedure. At the end of the perfusion experiments, hearts were analyzed for injury, contractile function, mitochondrial activity, and oxidative stress. The results obtained from hemodynamics, cardiac injury markers, and caspase‐3 activity showed that DCM rat displayed prominent I/R‐associated cardiac abnormalities than DM rat heart. But the deteriorated physiological performance and cardiac injury were not recovered in both DM and DCM heart by IPC procedure. Unlike normal rat heart, IPC did not reverse mitochondrial dysfunction (determined by electron transport chain enzymes activity, ATP level, and membrane integrity, expression levels of genes like PGC‐1ɑ, GSK3β, complex I, II, and V) in DCM and DM rat heart. The present study demonstrated that IPC failed to protect I/R‐challenged DCM rat heart, and the underlying pathology was associated with deteriorated mitochondrial function.     

Berberine reduces ischemia/reperfusion-induced myocardial apoptosis via activating AMPK and PI3K–Akt signaling in diabetic rats

Diabetes increases the risk of cardiovascular diseases. Berberine (BBR), an isoquinoline alkaloid used in Chinese medicine, exerts anti-diabetic effect by lowering blood glucose and regulating lipid metabolism. It has been reported that BBR decreases mortality in patients with chronic congestive heart failure. However, the molecular mechanisms of these beneficial effects are incompletely understood. In the present study, we sought to determine whether BBR exerts cardioprotective effect against ischemia/reperfusion (I/R) injury in diabetic rats and the underlying mechanisms. Male Sprague-Dawley rats were injected with low dose streptozotocin and fed with a high-fat diet for 12 weeks to induce diabetes. The diabetic rats were intragastrically administered with saline or BBR (100, 200 and 400 mg/kg/d) starting from week 9 to 12. At the end of week 12, all rats were subjected to 30 min of myocardial ischemia and 3 h of reperfusion. BBR significantly improved the recovery of cardiac systolic/diastolic function and reduced myocardial apoptosis in diabetic rats subjected to myocardial I/R. Furthermore, in cultured neonatal rat cardiomyocytes, BBR (50 μmol/L) reduced hypoxia/reoxygenation-induced myocardial apoptosis, increased Bcl-2/Bax ratio and decreased caspase-3 expression, together with enhanced activation of PI3K–Akt and increased adenosine monophosphate-activated protein kinase (AMPK) and eNOS phosphorylation. Pretreatment with either PI3K/Akt inhibitor wortmannin or AMPK inhibitor Compound C blunted the anti-apoptotic effect of BBR. Our findings demonstrate that BBR exerts anti-apoptotic effect and improves cardiac functional recovery following myocardial I/R via activating AMPK and PI3K–Akt–eNOS signaling in diabetic rats.     

Stomatin-like protein-2 relieve myocardial ischemia/reperfusion injury by adenosine 5′-monophosphate-activated protein kinase signal pathway

Previous studies have shown that stomatin-like protein-2 (SLP-2) could regulate mitochondrial biogenesis and function. The study was designed to explore the contribution of SLP-2 to the myocardial ischemia and reperfusion (I/R) injury. Anesthetized rats were treated with SLP-2 and subjected to ischemia for 30 minutes before 3 hours of reperfusion. An oxygen-glucose deprivation/reoxygenation model of I/R was established in H9C2 cells. In vivo, SLP-2 significantly improved cardiac function recovery of myocardial I/R injury rats by increasing fractional shortening and ejection fraction. SLP-2 pretreatment alleviated infarct area and myocardial apoptosis, which was paralleled by decreasing the level of cleaved caspase-3 and the ratio of Bax/Bcl-2, increasing the content of superoxide dismutase and reducing oxidative stress damage in serum. In addition, SLP-2 increased the level of ATP and stabilized mitochondrial potential (Ψm). The present in vitro study revealed that overexpression with SLP-2 reduced H9C2 cells apoptosis, accompanied by an increased level of ATP, the ratio of mitochondrial DNA/nuclear DNA, activities of complex II and V, and decreased the production of mitochondrial reactive oxygen species. Simultaneously, SLP-2 activated the adenosine 5′-monophosphate-activated protein kinase (AMPK) signaling pathway in myocardial I/R injury rats and H9C2 cells. This study revealed that SLP-2 mediates the cardioprotective effect against I/R injury by regulating AMPK signaling pathway.     

Downregulation of p300/CBP-associated factor inhibits cardiomyocyte apoptosis via suppression of NF-κB pathway in ischaemia/reperfusion injury rats

Cardiomyocyte apoptosis is the main reason of cardiac injury after myocardial ischaemia-reperfusion (I/R) injury (MIRI), but the role of p300/CBP-associated factor (PCAF) on myocardial apoptosis in MIRI is unknown. The aim of this study was to investigate the main mechanism of PCAF modulating cardiomyocyte apoptosis in MIRI. The MIRI model was constructed by ligation of the rat left anterior descending coronary vessel for 30 min and reperfusion for 24 h in vivo. H9c2 cells were harvested after induced by hypoxia for 6 h and then reoxygenation for 24 h (H/R) in vitro. The RNA interference PCAF expression adenovirus was transfected into rat myocardium and H9c2 cells. The area of myocardial infarction, cardiac function, myocardial injury marker levels, apoptosis, inflammation and oxidative stress were detected respectively. Both I/R and H/R remarkably upregulated the expression of PCAF, and downregulation of PCAF significantly attenuated myocardial apoptosis, inflammation and oxidative stress caused by I/R and H/R. In addition, downregulation of PCAF inhibited the activation of NF-κB signalling pathway in cardiomyocytes undergoing H/R. Pretreatment of lipopolysaccharide, a NF-κB pathway activator, could blunt these protective effects of PCAF downregulation on myocardial apoptosis in MIRI. These results highlight that downregulation of PCAF could reduce cardiomyocyte apoptosis by inhibiting the NF-κB pathway, thereby providing protection for MIRI. Therefore, PCAF might be a promising target for protecting against cardiac dysfunction induced by MIRI.     

Calcineurin B subunit acts as a potential agent for preventing cardiac ischemia/reperfusion injury

Calcineurin B subunit (CnB) is the regulatory subunit of calcineurin (Cn), a Ca²⁺/calmodulin-dependent serine/threonine protein phosphatase. It has been reported that mice deleting the CnB gene lose nearly all Cn activity and show poor tolerance to cardiac stress; CnB gene expression is downregulated in the hearts of rats that have suffered ischemia/reperfusion (I/R) injury. Therefore, we wonder whether injection of exogenous CnB protein can prevent the rats from suffering I/R injury. In cardiomyocytes, fluorogenic labeling shows that exogenous CnB quickly enters the cell. Pretreatment of cardiomyocytes with CnB reduces apoptosis in response to hypoxia/reoxygenation injury (an in vitro model mimicking ischemia/reperfusion injury), and CsA reverses this effect by inhibiting Cn activity. Furthermore, CnB upregulates Bcl-2 and Bcl-XL expression in the process of hypoxia/reoxygenation injury, which may contribute to protecting cardiomyocytes against apoptosis. In vivo experiments shows that pretreatment with CnB improves cardiac contractile function and reduces the frequency of arrhythmias induced by global I/R injury. These findings reveal a novel function for CnB protein in cardiac stress response and suggest a possible application of CnB in coronary disease therapy.     

当归多糖通过上调VEGF抑制细胞凋亡

本研究旨在探讨当归多糖减轻糖尿病缺血再灌注(I/R)诱导大鼠心肌细胞凋亡的作用机制。通过构建糖尿病I/R大鼠模型,再将大鼠随机分为4组(n=10):假手术组(Sham)、糖尿病I/R组(I/R)、I/R+10 mg/kg当归多糖组(I/R+ANG)、I/R+10 mg/kg当归多糖+15μg/kg阿柏西普组(I/R+ANG+AF);通过TTC染色法分析不同实验组大鼠心肌梗死面积差异;使用ELISA试剂盒分析当归多糖干预对I/R大鼠心肌酶水平和氧化应激反应的影响;借助TUNEL/DAPI双重染色分析各组大鼠心肌细胞凋亡情况;通过Western blotting检测当归多糖对血管内皮生长因子A (VEGFA)蛋白表达的影响。TTC染色检测结果表明,与糖尿病I/R组相比,当归多糖可显著减少糖尿病I/R大鼠心肌梗塞面积(p<0.05)。ELISA检测结果显示,与I/R组相比,当归多糖显著降低了糖尿病I/R组大鼠心肌酶--LDH和CK血清水平(p<0.05),并降低TNF-α(p<0.05)、IL-6 (p<0.05)水平,以及上调SOD活性(p<0.05)。TUNEL/DAPI双重染色镜检观察到当归多糖组的TUNEL阳性心肌细胞百分比显著低于I/R组(p<0.05);蛋白免疫印迹分析表明,与假手术组相比,在糖尿病I/R大鼠中检测到p-eNOS蛋白表达下调;而与I/R相比,当归多糖显著减轻了I/R对p-eNOS蛋白表达的抑制作用;与I/R组和阿柏西普组相比,当归多糖处理组的Caspase-3活化水平较低(p<0.05)。而VEGF抑制剂--阿柏西普处理均明显减轻上述当归多糖在糖尿病I/R大鼠中的所有有益作用。当归多糖通过减轻糖尿病I/R大鼠的氧化应激以及炎症反应,以及上调VEGFA表达和抑制Caspase-3活化来减弱糖尿病缺血/再灌注诱导的心肌细胞凋亡,从而在大鼠体内发挥心脏保护作用。     

Activation of proteolytic enzymes and depression of the sarcolemmal Na+/K+-ATPase in ischemia-reperfused heart may be mediated through oxidative stress

We tested whether the activation of proteolytic enzymes, calpain, and matrix metalloproteinases (MMPs) during ischemia-reperfusion (I/R) is mediated through oxidative stress. For this purpose, isolated rat hearts were subjected to a 30?min global ischemia followed by a 30?min reperfusion. Cardiac function was monitored and the activities of Na(+)/K(+)-ATPase, Mg(2+)-ATPase, calpain, and MMP were measured. Depression of cardiac function and Na(+)/K(+)-ATPase activity in I/R hearts was associated with increased calpain and MMP activities. These alterations owing to I/R were similar to those observed in hearts perfused with hypoxic medium, H(2)O(2) and xanthine plus xanthine oxidase. The I/R-induced changes were attenuated by ischemic preconditioning as well as by perfusing the hearts with N-acetylcysteine or mercaptopropionylglycine. Inhibition of MMP activity in hearts treated with doxycycline depressed the I/R-induced changes in cardiac function and Na(+)/K(+)-ATPase activity without affecting the calpain activation. On the other hand, inhibition of calpain activity upon treatment with leupeptin or MDL 28170 significantly reduced the MMP activity in addition to attenuating the I/R-induced alterations in cardiac function and Na(+)/K(+)-ATPase activity. These results suggest that the I/R-induced depression in Na(+)/K(+)-ATPase and cardiac function may be a consequence of the increased activities of both calpain and MMP because of oxidative stress in the heart.