Cannabidiol Protects the Heart From Ischemia-Reperfusion Injury Through SIRT-1/PGC-1α Activation and Nuclear Factor Kappa B Modulation: Experimental Insights.

Background: Myocardial ischemia-reperfusion (IR) injury limits the beneficial effects of early reperfusion therapy for acute myocardial infarction. Emerging evidence suggests that 7-ketocholesterol (7-KC), one of the most common dietary oxysterols, has pro-inflammatory properties and correlates with cardiovascular diseases. However, the underlying mechanisms remain scant. Here we investigated the effects of 7-KC on myocardial IR injury in mice. Methods and Results: Wild type mice were fed either a control high-fat high-cholesterol diet (HFHCD) or HFHCD containing 7-KC (7KC-HFHCD) for three weeks. In a murine model of 30-min myocardial ischemia and subsequent reperfusion, dietary 7KC-HFHCD increased plasma 7-KC level (145 ± 89.7 ng/mL vs 399 ± 175 ng/mL, P<0.0005, N=8) and infarct size after myocardial IR (52 ± 7.3% vs 64 ± 6.9%, P<0.05, N=8-10) without affecting blood pressure and heart rate. The ratio of Ly-6C high inflammatory monocytes to total monocytes increased in 7KC-HFHCD group as assessed by flow cytometric analysis (49 ± 15% vs 61 ± 9.3%, P<0.05, N=6). We then took a systems approach to explore the pro-inflammatory effects of 7-KC on macrophages and performed unbiased RNA-sequencing using murine peritoneal macrophages stimulated with 7-KC. Pathway analysis of differentially expressed transcripts revealed that 7-KC regulated the expression of transcripts related to inflammation, cholesterol biosynthesis and endoplasmic reticulum (ER) stress. We further validated in vitro that 7-KC induced ER stress, mitochondrial reactive oxygen species, and nuclear factor-kappa B activation associated with increased mRNA levels of pro-inflammatory cytokines such as MCP-1 and TNF-α in murine peritoneal macrophages. Administration of N-acetyl-L-cysteine (5mM), an antioxidant, decreased 7-KC-induced pro-inflammatory cytokines, and this decrease was not observed in Tlr4 -/- murine macrophages, or in macrophages transfected with liver X receptor siRNA. Conclusions: Dietary 7-KC exacerbated myocardial IR injury through macrophage-mediated inflammation in mice. Oxidative stress is involved in the 7-KC-induced pro-inflammatory response in macrophages. Dietary oxysterols are a promising therapeutic target for IR injury.

Diabetes and myocardial ischemia reperfusion (MIR) injury are characterized by oxidative stress, inflammation, autophagy disorders, and cardiac contractile dysfunction. Klotho and SIRT1 regulate the level of oxidative stress to participate in the regulation of many physiological functions such as cell survival, aging, apoptosis, autophagy, mitochondrial biogenesis, and inflammation. We hypothesized that the activation of Klotho/SIRT1 signaling pathway could attenuate MIR in diabetic rats. Type 1 diabetes and MIR injury model were established to examine this hypothesis in vivo . Primary rat cardiomyocytes and H9c2 cells were exposed to high glucose conditions and hypoxia/reoxygenation (H/R) insult in vitro . Hemodynamic parameters of heart function, myocardial infarct size, oxidative stress, markers of MIR injury or cell viability, and the mRNA and protein expression of Klotho and SIRT1 were measured. There was lower expression of Klotho and SIRT1 in diabetic MIR hearts than in nondiabetic rats, as well as significantly increased oxidative stress levels and decreased autophagy levels. Recombinant Klotho (rKlotho) protein and the SIRT1 agonist SRT1720 could significantly attenuate MIR injury in diabetes by activating Klotho/SIRT1 signaling pathway to reduce oxidative stress and restore autophagy levels. These findings suggest that the Klotho/SIRT1 pathway plays an important role in MIR injury in diabetic rats, and rKlotho protein and agonist SRT1720 have therapeutic potential for alleviating diabetic myocardial IR injury by activating Klotho/SIRT1 to reduce oxidative stress and restore autophagy levels.

Decreased circulating levels of hydrogen sulfide (H2S) are associated with higher mortality following myocardial ischemia. This study aimed at determining the long-term dose-dependent effects of sodium hydrosulfide (NaSH) administration on myocardial ischemia-reperfusion (IR) injury. Male rats were divided into control and NaSH groups that were treated for 9 weeks with daily intraperitoneal injections of normal saline or NaSH (0.28, 0.56, 1.6, 2.8, and 5.6 mg/kg), respectively. At the end of the study, hearts from all rats were isolated and hemodynamic parameters were recorded during baseline and following IR. In isolated hearts, infarct size, oxidative stress indices as well as mRNA expression of H2S-, nitric oxide (NO)-producing enzymes, and inflammatory markers were measured. In heart tissue following IR, low doses of NaSH (0.28 and 0.56 mg/kg) had no effect, whereas an intermediate dose (1.6 mg/kg), improved recovery of hemodynamic parameters, decreased infarct size, and decreased oxidative stress. It also increased expression of cystathionine γ-lyase (CSE), Raf kinase inhibitor protein (RKIP), endothelial NO synthase (eNOS), and neuronal NOS (nNOS), as well as decreased expression of inducible NOS (iNOS) and nuclear factor kappa-B (NF-κB). At the high dose of 5.6 mg/kg, NaSH administration was associated with worse recovery of hemodynamic parameters and increased infarct size as well as increased oxidative stress. This dose also decreased expression of CSE, RKIP, and eNOS and increased expression of iNOS and NF-κB. In conclusion, chronic treatment with NaSH has a U-shaped concentration effect on IR injury in heart tissue. An intermediate dose was associated with higher CSE-derived H2S, lower iNOS-derived NO, lower oxidative stress, and inflammation in heart tissue following IR.

Objective To investigate the correlative of autophagy flux and hydrogen sulfide postconditioning protecting against myocardial ischemia reperfusion (I/R) injury in type 2 diabetic rats in vivo. Methods Sixty adult male Sprague-Dawley rats were randomly divided into five groups (n=12): sham group, I/R group, chloroquine (CQ) group, sodium hydrosulfide (NaHS) group and CQ + NaHS group. Rats in the sham group only gave thoracotomy and separation of the left anterior descending coronary artery; In the I/R group rats were occluded the left anterior descending coronary artery for 30 min, followed by 4 h of reperfusion; In the CQ group rats received CQ 10 mg/kg by intraperitoneal injection at 1 h before the I/R operation; In the NaHS group rats were injected NaHS 0.05 mg/kg intravenously within 1 min after releasing the left anterior descending coronary artery undergoing 30 min occlusion, then followed by 4 h of reperfusion; and in the CQ + NaHS group rats received CQ 10 mg/kg by intraperitoneal injection at 1 h before the I/R operation additionally on the basis of NaHS group. The heart rate, mean arterial pressure and rate-pressure product (RPP) were detected and recorded at 15 min of balance period, ischemic period, and 1, 2, 4 h after reperfusion. After reperfusion for 4 h, the rats were sacrificed and the hearts were used to calculate the range of myocardial infarction and the express of microtubule-associated protein 1 light chain 3 (LC3), Cathepsin B, Beclin-1 and P62 were determined by Western blotting. Results The heart rates in the five groups had no siginficant differences at each time point (F=0.854, P=0.512), but the mean arterial pressure and RPP showed siginficant differences among the five groups at each time point (F=5.182, P=0.007; F=5.082, P=0.008). Furthermore, the mean arterial pressure[(87 ± 8) mmHg vs. (72 ± 10) mmHg, (91±10) mmHg vs. (63 ± 6) mmHg] and RPP [(35.4 ± 4.6)·103 mmHg·beat/min vs. (28.7 ± 5.8)·103 mmHg·beat/min, (36.2 ± 5.8)·103 mmHg·beat/min vs. (26.8 ± 3.8)·103 mmHg·beat/min] in the NaHS group at 2, 4 h after reperfusion were much higher than those in the I/R group (all P<0.05). The range of myocardial infarction and the express of LC3, Cathepsin B, Beclin-1 and P62 at 4 h after reperfusion all had statistical significance obviously among five groups (F=96.907, 71.164, 43.594, 57.180, 35.967, all P<0.05) , and above indicators in the NaHS group were much lower than those in the I/R group, CQ group and CQ + NaHS group (all P<0.05). Conclusion Hydrogen sulfide postconditioning play a protective role through repairinng autophagy flux in type 2 diabetic rats with I/R injury. Key words: Hydrogen sulfide; Ischemic postconditioning; Myocardial reperfusion injury; Diabetes mellitus, type 2; Autophagy; Rats

Dietary green tea extract increases phase 2 enzyme activities in protecting against myocardial ischemia-reperfusion

Introduction: Menopause is associated with reduced nitric oxide (NO) bioavailability and lower tolerance against myocardial ischemia-reperfusion (IR) injury. This study investigated whether long-term nitrate administration provides resistance against myocardial IR injury in ovariectomized (OVX) rats.Method: After ovariectomy, female rats were assigned to the OVX and the OVX + nitrate groups (n = 14/group); the latter group consumed nitrate (100 mg/L) for 9 months. At month 9, each group was divided into two subgroups (n = 7/subgroup), of which one subgroup was exposed to myocardial IR (IR+ hearts) and the other was not exposed (IR− hearts). The hearts of rats were isolated, and NO metabolite (NOx), oxidative stress indices, and mRNA expressions of endothelial (eNOS), inducible (iNOS), and neuronal (nNOS) NO synthases, as well as markers of apoptosis, were measured in the IR− and IR+ hearts. In the IR+ hearts, cardiac function indices (CFI) and the infarct size were also measured.Results: Nitrate increased catalase activity (97%) and eNOS expression (2.94-fold) in the IR− hearts. In the IR+ hearts, nitrate reduced left ventricular (LV) end-diastolic pressure (11.6%) and infarct size (26.2%) and increased recovery of LV developed pressure (44.0%) and peak rate of positive (28.9%) and negative (15.4%) changes in LV pressure. In addition, in the IR+ hearts, nitrate increased eNOS and B-cell lymphoma-2 (Bcl-2) as well as decreased iNOS, Bcl-2 associated X protein (Bax), caspase-3, caspase-8, caspase-9, and tumor necrosis factor-α (TNF-α) expression. Nitrate increased total antioxidant capacity (TAC) and catalase (CAT) activity and decreased malondialdehyde (MDA) levels at month nine in serum and IR+ hearts.Conclusion: The favorable effects of nitrate against IR injury were associated with higher eNOS and Bcl-2 expression, CAT activity, TAC, and lower iNOS, Bax, caspase-3, caspase-8, caspase-9 and TNF-α expression, and MDA in the heart tissue. Nitrate preconditioning alleviated IR-induced myocardial injury in OVX rats; this effect was associated with eNOS upregulation before IR and the blunting of OVX-induced eNOS downregulation, iNOS upregulation, apoptosis, and oxidative stress in heart tissue after IR.

Myocardial ischemia-reperfusion (MI/R) injury is a leading cause of morbidity and mortality around the world, characterized by injury to cardiomyocytes that leads to various forms of cell death, including necrosis, apoptosis, autophagy, and ferroptosis. Preventing cell death is crucial for preserving cardiac function after ischemia-reperfusion injury. Ferroptosis, a novel type of cell death, has recently been identified as a key driver of cardiomyocyte death following MI/R. However, the complex regulatory mechanisms involved in ferroptosis remain unclear. Here, we found that SENP2 expression decreased following myocardial ischemia reperfusion injury. Deletion of SENP2 increased cardiomyocyte ferroptosis and hindered cardiac function recovery after MI/R injury, whereas overexpression of SENP2 significantly reduced cardiomyocyte ferroptosis and mitigated MI/R injury. Mechanistically, SENP2 removed the SUMOylation of NCOA4 modified by SUMO1 at K81, K343, and K600 sites. The level of NCOA4 SUMOylation regulated ferritinophagy-dependent ferroptosis through affecting NCOA4 protein stability. SENP2-mediated NCOA4 deSUMOylation alleviated the interaction between NCOA4 and OTUB1, which directly deubiquitinated NCOA4 and maintained its protein stability. Furthermore, administration of SENP2 in the animal MI/R model reduced ferroptosis events, protected the injured myocardium and promoted cardiac function recovery. Collectively, our results demonstrate that SENP2 catalyzes deSUMOylation of NCOA4, alleviates ferritinophagy-mediated ferroptosis in an OTUB1-dependent manner, thereby facilitating cardiac function recovery following MI/R. These findings suggest a potential therapeutic strategy for MI/R treatment. Abbreviations: I/R: ischemia-reperfusion; MI/R: myocardial ischemia-reperfusion; NCOA4: nuclear receptor coactivator 4; OTUB1: OTU domain, ubiquitin aldehyde binding 1; SENP2: SUMO/sentrin specific peptidase 2

Background/Aims: Pterostilbene (PT) exerts antidiabetic effects by decreasing blood glucose and modulating lipid metabolism and has been shown to attenuate myocardial ischemia-reperfusion (IR) injury in non-diabetic subjects. However, whether PT can protect against myocardial IR injury in diabetes is unknown. AMPK stimulation is indispensable in offering cardioprotection against myocardial IR injury in diabetes by limiting cardiac apoptosis. Thus, we hypothesized that PT may confer protection against myocardial IR injury in diabetes via AMPK activation. Methods: Sprague-Dawley rats at eight weeks of diabetes induction (induced by an intravenous dose of 65 mg/kg streptozotocin) were administered with vehicle or PT (20 and 40 mg/kg/day, p.o.) for four weeks (starting from week 9 to 12). At the end of week 12, myocardial IR injury was induced by subjecting the diabetic rats to 30 minutes of coronary artery ligation and followed by 2 hours of reperfusion. In in vitro studies, rat primary cardiomyocytes were incubated with low glucose (LG, 5.5 mM) or high glucose (HG, 30 mM) and exposed to 45 minutes hypoxia and 2 hours reoxygenation in the presence or absence of PT (0.5 µM) or the AMPK inhibitor compound C (CC, 5 µM). Results: PT significantly reduced post-ischemic cardiac infarct size, oxidative stress, plasma lactate dehydrogenase (LDH), creatine kinase-MB levels and apoptosis in diabetic rats. In cardiomyocytes, PT decreased hypoxia/ reoxygenation-induced oxidative stress, attenuated LDH and cleaved caspase3/caspase3 ratio and increased Bcl-2/Bax ratio and AMPK phosphorylation. However, CC administration blunted the cardioprotective effects of PT both in vivo and in vitro. Conclusion: Suppressing cardiac oxidative stress and apoptosis via AMPK stimulation may represent a primary mechanism whereby pterostilbene attenuates diabetic myocardial IR injury.

Mitochondria-mediated apoptosis plays a critical role in myocardial ischemia reperfusion (IR) injury and causes a negative impact on cardiac efficiency and function. The combined angiotensin receptor-neprilysin inhibitor (ARNI) is a promising cardioprotective pharmacological agent that could rescue the heart from IR injury. This study investigated the cardioprotective effect of thiorphan (TH) in combination with three different doses of irbesartan (IRB) on myocardial IR injury and detected the most effective dose combination. Male Wistar rats were used and divided into five groups (10 rats/group): (I) Sham, (II) ischemia-reperfusion I/R, (III) TH/IRB + IR (0.1/5mg/kg), (IV) TH/IRB + IR (0.1/10mg/kg), and (V) TH/IRB + IR (0.1/15 mg/kg) groups. Thiorphan and irbesartan were injected intraperitoneally 15 min before IR induction. Mean arterial blood pressure, left ventricular end diastolic pressure (LVEDP), left ventricular maximum rate of pressure (LVdp/dtmax ), and cardiac levels of creatine kinase-MB, malondialdehyde, superoxide dismutase, and endothelin-1 were measured. Cardiac mitochondria complexes activities, histopathological examination of myocardial tissues, immunohistochemistry studies for myocardial apoptosis (Bax and Bcl-2), and electron microscopy examination of left ventricle were performed. TH/IRB combination preserved cardiac functions and mitochondria complex activities and mitigated cardiac damage, oxidative stress, and apoptosis following IR. Also, there was an evident improvement in histopathological changes and electron microscopy examination of left ventricle compared with I/R group. TH/IRB in a dose of 0.1/10mg/kg showed significant improvement compared with the other treated groups. Thiorphan/irbesartan improved cardiac functions following IR injury. This could be explained by the reported improvement of mitochondria complex activities and reduction of oxidative stress, endothelin-1, and apoptosis.

Myocardial ischaemia-reperfusion (IR) injury poses a severe threat to cardiac health, particularly in the ageing population, where susceptibility to such damage is significantly heightened owing to age-related declines in mitochondrial function, thus highlighting mitochondria as crucial targets for innovative therapies. The aim of this study was to investigate the combined modality therapy involving mitochondrial transplantation and the mitochondrial boosters mitoquinone and melatonin to address myocardial IR injury in aged rats. A total of 54 male Wistar rats, aged 22-24 months, were randomly divided into groups that either received IR injury or not, and were subjected to various treatments, both individually and in combination. Myocardial IR injury was induced by temporarily blocking and reopening the left anterior descending coronary artery. Mitoquinone was given intraperitoneally for 14 days prior to ischaemia, while melatonin and isolated mitochondria were administered intraperitoneally and intramyocardially, respectively, at the onset of reperfusion. Finally, we evaluated changes in haemodynamic indices, creatine kinase-MB levels, mitochondrial function endpoints and the expression of mitochondrial biogenesis genes, including sirtuin 1 (SIRT-1), peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) and nuclear respiratory factor 2 (NRF-2). The triple therapy enhanced myocardial function, decreased creatine kinase-MB levels and improved mitochondrial function along with the expression of mitochondrial biogenesis genes in aged IR rats. This combined approach elicited significant cardioprotection in comparison to single or dual therapies. The triple therapy provided substantial cardioprotection in aged rat hearts by improving mitochondrial function and biogenesis through enhanced SIRT-1/PGC-1α/NRF-2 profiles, suggesting a promising strategy for mitigating IR injury in elderly patients.

Junctophilin-2 is a structural membrane protein that tethers T-tubules to the sarcoplasmic reticulum to allow for coordinated calcium-induced calcium release in cardiomyocytes. Defective excitation-contraction coupling in myocardial ischemia-reperfusion (IR) injury is associated with junctophilin-2 proteolysis. However, it remains unclear whether preventing junctophilin-2 proteolysis improves the recovery of cardiac contractile dysfunction in IR injury. Matrix metalloproteinase-2 (MMP-2) is a zinc and calcium-dependent protease that is activated by oxidative stress in myocardial IR injury and cleaves both intracellular and extracellular substrates. To determine whether junctophilin-2 is targeted by MMP-2, isolated rat hearts were perfused in working mode aerobically or subjected to IR injury with the selective MMP inhibitor ARP-100. IR injury impaired the recovery of cardiac contractile function which was associated with increased degradation of junctophilin-2 and damaged cardiac dyads. In IR hearts, ARP-100 improved the recovery of cardiac contractile function, attenuated junctophilin-2 proteolysis, and prevented ultrastructural damage to the dyad. MMP-2 was co-localized with junctophilin-2 in aerobic and IR hearts by immunoprecipitation and immunohistochemistry. In situ zymography showed that MMP activity was localized to the Z-disc and sarcomere in aerobic hearts and accumulated at sites where the striated JPH-2 staining was disrupted in IR hearts. In vitro proteolysis assays determined that junctophilin-2 is susceptible to proteolysis by MMP-2 and in silico analysis predicted multiple MMP-2 cleavage sites between the membrane occupation and recognition nexus repeats and within the divergent region of junctophilin-2. Degradation of junctophilin-2 by MMP-2 is an early consequence of myocardial IR injury which may initiate a cascade of sequelae leading to impaired contractile function.

Inflammatory response plays an important role in myocardial ischaemia-reperfusion (IR) injury. Up-regulation of vascular cell adhesion molecule-1 (VCAM) contributes to this. We examined the feasibility of using intravenously administered VCAM-MPIO (microparticle iron oxide) to characterize VCAM expression patterns in myocardial IR injury. Myocardial ischemia was simulated by 30 min of transient ligation of the left coronary vessel in rats. Purified, monoclonal, rat-specific, mouse VCAM antibody coupled to MPIO was administered through the tail vein at 3 h post reperfusion and the rats were sacrificed 1 h later. High resolution 3D ex vivo MRI images were acquired at 9.4 Tesla. Extensive foci of signal voids were observed on T2*-weighted gradient-echo sequences, which corresponded to focal deposits of MPIOs observed in histological sections. The spatial density of the signal voids (expressed as a percentage of pixels below a threshold value) was increased in the peri-infarct zone compared with non-infarct zone (32.5 ± 4% vs. 13.9 ± 5%; n = 6; p < 0.05) and was substantially greater than the signal loss due to non-specific binding seen in rats administered IgG control MPIO (2.0 ± 1%; n = 6; p < 0.05). The VCAM-specific MPIO signal was also seen in myocardium and pericardium in segments remote from the IR injury, but not in rats undergoing a sham operation. In conclusion, molecular imaging in a model of myocardial IR injury is possible using high field MRI and VCAM-MPIOs and may provide novel insights beyond those achieved by standard histological and molecular analysis.

Vascular Endothelial Growth Factor Antagonist Modulates Leukocyte Trafficking and Protects Mouse Livers against Ischemia/Reperfusion Injury

Objective To observe the protective effect of Salvia miltiorrhiza on myocardial ischemia reperfu-sion injury in rats.Methods 150 healthy male rats were randomly divided into 5 groups：sham operation group,model group,Radix Salviae Miltiorrhizae in high dose group（100mg/kg）,middle dose group（30mg/kg）,low dose group （10mg/kg）.Salvia miltiorrhiza group were administered orally preoperatively,once a day.On 3 consecutive days. Sham operation group and model group were treated with equal volume of saline.The coronary artery ligation rat for 30min,then sonza coronary artery for 180 min to make the model of myocardial ischemia reperfusion injury.Make determination of myocardial infarct size;make determination of serum creatine kinase （ CK ） , lactate dehydrogenase （ LDH） .Results In the model group compared with the sham operation group,myocardial infarction and serum CK and LDH activity significantly increased（t =14.382,21.460,all P〈0.05）,made up of the myocardial ischemia reperfusion injury in rats model.The different doses of Danshen group can significantly reduce the myocardial ischemia reperfusion injury in rats about the range of myocardial infarction and decrease serum CK , there were significant＆amp;nbsp;differences compared with the model group （t=7.426,6.891,11.274,all P〈0.05）.Different doses of Danshen group can significantly reduce serum LDH, there were significant differences compared with the model group （ t =22.436,10.843,16.252,all P〈0.05）.Conclusion Salvia miltiorrhiza has oxidative stress on myocardial ischemia reperfusion in rats,which has protective effect of Salvia miltiorrhiza on myocardial ischemia reperfusion injury in rats. Key words: Salvia miltiorrhiza; Myocardial ischemia reperfusion injury; Protective effect; Pharmacological experiment

Purpose: To investigate the protective effect of liraglutide on myocardial ischemia reperfusion (I/R) injury and its molecular mechanism. Methods: Ischemia reperfusion model male Sprague-Dawley (SD) rats were randomly divided into negative control group, I/R group (saline), liraglutide group (liraglutide) and PD group (liraglutide + PD98059). The weight of myocardium in ischemic and infarction areas of the heart, myocardial injury biomarker, oxidative stress, as well as expressions of mRNA molecules of apoptosis were determined. Results: The myocardial mass of ischemic and infarcted areas of the heart (relative to left ventricular mass) of I/R group were significantly higher ( p ˂ 0.05) than those of negative control group, but significantly lower in liraglutide group than in I/R group ( p > 0.05). However, the parameters were significantly higher in PD group than in liraglutide group ( p ˂ 0.05). CK, CK-MB and LDH activities, as well as levels of cTnI and cTnT in I/R group were significantly higher ( p ˂ 0.05) than those of negative control group. However, the parameters were significantly lower ( p ˂ 0.05) in liraglutide group than in I/R group, but higher in PD group ( p ˂ 0.05) than in liraglutide group. Serum SOD, GSH-Px, CAT activities and tBcl-2 mRNA expression were significantly lower in I/R group than those of negative control group ( p ˂ 0.001), while those PD group were significantly lower than those of liraglutide group ( p ˂ 0.001). Conclusion: Liraglutide alleviates myocardial ischemia-reperfusion injury and inhibits oxidative stress and apoptosis via ERK1/2 signaling pathway in rats, but further studies are required to ascertain the clinical efficacy and safety of the compound. Keywords: Ischemia-reperfusion injury, Liraglutide, ERK1/2 signal pathway, Oxidative stress, Apoptosis