Ischemic-reperfused Hearts Research Articles

Effectiveness of Some Vitamins in the Prevention of Cardiovascular Disease: A Narrative Review.

By virtue of their regulatory role in various metabolic and biosynthetic pathways for energy status and cellular integrity, both hydro-soluble and lipo-soluble vitamins are considered to be involved in maintaining cardiovascular function in health and disease. Deficiency of some vitamins such as vitamin A, B6, folic acid, C, D, and E has been shown to be associated with cardiovascular abnormalities whereas supplementation with these vitamins has been claimed to reduce cardiovascular risk for hypertension, atherosclerosis, myocardial ischemia, arrhythmias, and heart failure. However, the data from several experimental and clinical studies for the pathogenesis of cardiovascular disease due to vitamin deficiency as well as therapy due to different vitamins are conflicting. In this article, we have attempted to review the existing literature on the role of different vitamins in cardiovascular disease with respect to their deficiency and supplementation in addition to examining some issues regarding their involvement in heart disease. Although both epidemiological and observational studies have shown some merit in the use of different antioxidant vitamins for the treatment of cardiovascular disorders, the results are not conclusive. Furthermore, in view of the complexities in the mechanisms of different cardiovascular disorders, no apparent involvement of any particular vitamin was seen in any specific cardiovascular disease. On the other hand, we have reviewed the evidence that deficiency of vitamin B6 promoted KCl-induced Ca2+ entry and reduced ATP-induced Ca2+-entry in cardiomyocytes in addition to decreasing sarcolemmal (SL) ATP binding. The active metabolite of vitamin B6, pyridoxal 5′-phosphate, attenuated arrhythmias due to myocardial infarction (MI) as well as cardiac dysfunction and defects in the sarcoplasmic reticulum (SR) Ca2+-transport in the ischemic-reperfused hearts. These observations indicate that both deficiency of some vitamins as well as pretreatments with different vitamins showing antioxidant activity affect cardiac function, metabolism and cation transport, and support the view that antioxidant vitamins or their metabolites may be involved in the prevention rather than the therapy of cardiovascular disease.

Pyridoxal-5-phosphate induced cardioprotection in aging associated with up-expression of cystathionine-γ-lyase, 3-mercaptopyruvate sulfurtransferase, and ATP-sensitive potassium channels.

In the present work, we investigated the cardioprotective potential of pyridoxal-5-phosphate (PLP) in old rats as a cofactor of enzymes that synthesize hydrogen sulphide (H2 S). PLP was administered per os in a dose of 0.7mg per kg daily for 2weeks. Rats were divided into three groups (adult, old and old +PLP) of 20 animals. The cardiac mRNA levels of genes encoding H2 S-synthesizing enzymes cystathionine-γ-lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (3-MST), uncoupling proteins (UCP3), subunits of ATP-sensitive potassium (KATP ) channels were determined using real-time polymerase chain reaction analysis. We also studied the effect of PLP-administration on the content of H2 S, oxidative stress, the activities of inducible and constitutive NO-synthase (iNOS, cNOS), arginase and nitrate reductase in the heart homogenates as well as cardiac resistance to ischemia-reperfusion in Langendorff-isolated heart model. It was shown that PLP restored mRNA levels of CSE, 3-MST and UCP3genes, and H2 S content and also significantly increased the expression of SUR2 and Kir6.1 (2.2 and 3.3 times, respectively) in the heart of old rats. PLP significantly reduced the formation of superoxide, malondialdehyde, diene conjugates as well as the activity of iNOS and arginase. PLP significantly increased constitutive synthesis of NO and prevented reperfusion disturbances of the heart function after ischemia. Thus, PLP-administration in old rats was associated with up-expression of CSE, 3-MST, UCP3 and SUR2 and Kir6.1subunits of KATP channels, and also increased cNOS activity and reduced oxidative stress and prevented reperfusion dysfunction of the heart in ischemia-reperfusion.

The Protective Role of Bmal1-Regulated Autophagy Mediated by HDAC3/SIRT1 Pathway in Myocardial Ischemia/Reperfusion Injury of Diabetic Rats

Histone deacetylase 3 (HDAC3) and silent information regulator 1 (SIRT1) are histone deacetylases that regulate important metabolic pathways and play important roles in diabetes and myocardial ischemia/reperfusion (IR) injury. In this study, we explored the protective mechanism of Bmal1-regulated autophagy mediated by the HDAC3/SIRT1 pathway in myocardial IR injury of diabetic rats. Type 1 diabetes was established by administering an intraperitoneal injection of streptozotocin. After 8 weeks, the left anterior descending coronary artery was ligated for 30 min and reperfused for 120 min to establish a myocardial IR injury model in diabetic rats. H9c2 cardiomyocytes were exposed to high glucose concentration (30 mM) and hypoxia/reoxygenation (H/R) stimulation in vitro. The myocardial infarct size and levels of serum cTn-I, CK-MB, and LDH in diabetic rats subjected to myocardial IR injury were significantly higher. Upregulated HDAC3 and downregulated SIRT1 expression were observed in diabetic and IR hearts, along with a lower Bmal1 level. Autophagy was rapidly increased in the hearts of diabetic or non-diabetic rats in the IR group compared with the sham group, but significantly attenuated in the hearts of diabetic rats compared with the hearts of non-diabetic rats after IR insult. Consistent with decreased autophagy, we observed increased HDAC3 expression and decreased SIRT1 and Bmal1 levels in the myocardial tissue of diabetic rats after IR. Inhibition of HDAC3 by the inhibitor RGFP966 and activation of SIRT1 by the agonist SRT1720 could significantly attenuate myocardial IR injury in diabetic rats by restoring Bmal1-regulated autophagy. Based on these findings, the disordered HDAC3/SIRT1 circuit (upregulated HDAC3 and downregulated SIRT1 levels) plays an important role in aggravating myocardial IR injury in diabetic rats by downregulating Bmal1-mediated autophagy. Treatments targeting HDAC3/SIRT1 to activate the autophagy may represent a novel strategy to alleviate myocardial IR injury in diabetes.

Neurochemical alterations of different cerebral regions in rats with myocardial ischemia-reperfusion injury based on proton nuclear magnetic spectroscopy analysis.

Background: Recent studies have demonstrated a complex and dynamic neural crosstalk between the heart and brain. A heart-brain interaction has been described regarding cardiac ischemia, but the cerebral metabolic mechanisms involved are unknown.Methods: Male Sprague Dawley rats were randomly allocated into 2 groups: those receiving myocardial ischemia-reperfusion surgery (IR group, n =10) and surgical controls (Con group, n=10). These patterns of metabolic abnormalities in different brain regions were assessed using proton magnetic resonance spectroscopy (PMRS).Results: Results assessed by echocardiography showed resultant cardiac dysfunction following heart ischemia-reperfusion. Compared with the control group, the altered metabolites in the IR group were taurine and choline, and differences mainly occurred in the thalamus and brainstem.Conclusions: Alterations in cerebral taurine and choline are important findings offering new avenues to explore neuroprotective strategies for myocardial ischemia-reperfusion injury. These results provide preliminary evidence for understanding the cerebral metabolic process underlying myocardial ischemia-reperfusion injury in rats.

Association Between L-OPA1 Cleavage and Cardiac Dysfunction During Ischemia-Reperfusion Injury in Rats.

Background/Aims:Structural and functional alterations in mitochondria, particularly, the inner mitochondrial membrane (IMM) plays a critical role in mitochondria-mediated cell death in response to cardiac ischemia-reperfusion (IR) injury. The integrity of IMM can be affected by two potential intra-mitochondrial factors: i) mitochondrial matrix swelling, and ii) proteolytic cleavage of the long optic atrophy type 1 (L-OPA1), an IMM-localized dynamin-like GTPase engaged in the regulation of structural organization and integrity of the mitochondrial cristae. However, the relationship between these two factors in response to oxidative stress remains unclear. Here, we elucidated the effects of cardiac IR injury on L-OPA1 cleavage and OMA1 activity.Methods:Langendorff-mode perfused isolated rat hearts were subjected to 25-min of global ischemia followed by 90-min reperfusion in the presence or absence of XJB-5-131 (XJB, a mitochondria-targeting ROS scavenger) and sanglifehrin A (SfA, a permeability transition pore inhibitor).Results:XJB in combination with SfA increased post-ischemic recovery of cardiac function and reduced mitochondrial ROS production at 30- and 60-min reperfusion and affected mitochondrial swelling. L-OPA1 levels were reduced in IR hearts; however, neither XJB, SfA, and their combination prevented IR-induced reduction of L-OPA1 cleavage. Likewise, IR increased the OMA1 enzymatic activity, which remained unchanged in the presence of XJB and/or SfA.Conclusion:IR-induced cardiac and mitochondrial dysfunctions are associated with OMA1 activation and L-OPA1 cleavage. However, XJB, SfA, and their combination do not prevent these changes despite improved heart and mitochondria function, thus, suggesting that different mechanisms can be implicated in L-OPA1 processing in response to cardiac IR injury.

Abstract 412: DJ-1 Deficiency Impairs Post-Ischemic Cardiac Fatty Acid Oxidation

Background: DJ-1 is a cytoprotective protein implicated in many cellular processes. We have recently shown that DJ-1 plays a protective role in the setting of acute myocardial ischemia-reperfusion injury and heart failure. However, specific mechanisms of action remain unknown. Here, we sought to determine if DJ-1 maintains fatty acid oxidation following myocardial I/R injury via the regulation of PPARalpha transcriptional activity. Methods and Results: WT and DJ-1 KO hearts were subjected to 45 minutes of ischemia via coronary artery ligation followed by up to 72 hours of reperfusion. Initial studies revealed that PPARalpha activity (PPARalpha Transcription Factor Assay Kit; abcam) was lower in DJ-1 KO hearts under both basal (sham) and I/R conditions. Next, we performed an analysis of PPAR genes using a qPCR array (Mouse PPAR Targets RT2 Profiler PCR Array from Qiagen) that profiled the expression of 84 key genes involved in PPAR activation and response. Marked differences in genes involved in FA metabolism were evident in the hearts of DJ-1 KO mice. Further studies using qPCR validated a significant decrease in the expression of acsl1 (protein ACSL1), acsl3, acsl5, cpt1b (protein CPT1), slc25a20 (protein CACT), and cpt2. The protein expression of ACSL1, CPT1, and CACT were also decreased in the hearts of DJ-1 KO mice following I/R injury. These changes were accompanied by higher cardiac lipid content and depressed mitochondrial fatty-acid β-oxidation and ATP synthesis. Conclusion: These data demonstrate that DJ-1 plays an essential role in regulating post-I/R cardiac FAO. Future studies will garner mechanistic insight for the impaired function of PPARalpha with the loss of DJ-1.

The role of ferroptosis in chronic diseases

Recently, ferroptosis, an iron-dependent novel type of cell death, has been characterized as an excessive accumulation of lipid peroxides and reactive oxygen species. Emerging studies demonstrate that ferroptosis not only plays an important role in the pathogenesis and progression of chronic diseases, but also functions differently in the different disease context. Notably, it is shown that activation of ferroptosis could potently inhibit tumor growth and increase sensitivity to chemotherapy and immunotherapy in various cancer settings. As a result, the development of more efficacious ferroptosis agonists remains the mainstay of ferroptosis-targeting strategy for cancer therapeutics. By contrast, in non-cancerous chronic diseases, including cardiovascular & cerebrovascular diseases and neurodegenerative diseases, ferroptosis functions as a risk factor to promote these diseases progression through triggering or accelerating tissue injury. As a matter of fact, blocking ferroptosis has been demonstrated to effectively prevent ischemia-reperfusion heart disease in preclinical animal models. Therefore, it is a promising field to develope potent ferroptosis inhibitors for preventing and treating cardiovascular & cerebrovascular diseases and neurodegenerative diseases. In this article, we summarize the most recent progress on ferroptosis in chronic diseases, and draw attention to the possible clinical impact of this recently emerged ferroptosis modalities.

NLRP3 Inflammasome and Its Central Role in the Cardiovascular Diseases.

Materials The NLRP3 inflammasome controls the activation of the proteolytic enzyme caspase-1. Caspase-1 in turn regulates the maturation of the proinflammasome cytokines IL-1β and IL-18, which leads to an inflammatory response. We made a mini-review on the association of regulatory mechanisms of NLRP3 inflammasome with the development of cardiovascular diseases systematically based on the recent research studies. Discussion. The inflammasome plays an indispensable role in the development of atherosclerosis, coronary heart diseases (CHD), and heart ischemia-reperfusion (I/R) injury, and NLRP3 inflammasome may become a new target for the prevention and treatment of cardiovascular diseases. Effective regulation of NLRP3 may help prevent or even treat cardiovascular diseases. Conclusion This mini-review focuses on the association of regulatory mechanisms of NLRP3 inflammasome with the development of cardiovascular diseases, which may supply some important clues for future therapies and novel drug targets for cardiovascular diseases.

Association Between L‐OPA1 Cleavage/OMA1 Activity and Cardiac Function During Ischemia‐Reperfusion Injury in the Rat

Cardiac ischemia‐reperfusion (IR) injury is a complex process associated with high ROS production, intracellular Ca2+ overload and ATP depletion that together lead to cell death. Structural and functional changes in mitochondria, particularly, the inner mitochondrial membrane (IMM) permeability play a critical role in IR‐induced cell death. Two potential intra‐mitochondrial factors can diminish the structural and functional integrity of the IMM: i) mitochondrial (matrix) swelling due to increased permeability transition (PT) that triggers the opening of PT pores (PTP), and ii) proteolytic cleavage of optic atrophy type 1 (OPA1), a dynamin‐like GTPase localized in the IMM that, in addition to the role in mitochondrial fusion, maintains the structural organization/integrity of the cristae. OPA1 is present in a long IMM‐bound (L‐OPA1) and a short‐soluble (S‐OPA1) forms. The latter results from L‐OPA1 proteolytic cleavage by one of its peptidases, OMA1, which is activated during the conditions that occur in response to oxidative stress such as IR. However, the relationship between cardiac/mitochondria function and L‐OPA1/cleavage/OMA1 activity in response to oxidative stress remains unclear. Here, we elucidated the effects of cardiac IR injury on L‐OPA1 cleavage and OMA1 activity in rats. Isolated and Langendorff‐mode perfused Sprague‐Dawley rat hearts were subjected to 25‐min of global ischemia followed by 90‐min reperfusion in the following groups: i) control (no IR), ii) IR, iii) IR+XJB (XJB‐5‐131, a mitochondria‐targeting electron and ROS scavenger), iv) IR+SfA (sanglifehrin A, a PTP inhibitor), and v) IR+XJB+SfA. Hearts were perfused with Krebs‐Henseleit solution supplemented with XJB, SfA or XJB+SfA. Isolated mitochondria were analyzed for respiratory control index (RCI), calcium‐induced swelling (a marker of PTP opening), ROS production, L‐OPA/S‐OPA1 protein levels, and OMA1 activity/protein levels. Our results show that IR hearts recovered 45% of the control (no IR) group. However, XJB and XJB+SfA increased the post‐ischemic recovery to 70% and 74%, respectively, compared to IR alone. XJB+SfA group was able to reduce mitochondrial ROS production with calcium addition in comparison to IR. Both basal and calcium‐induced swelling was affected in XJB and SfA treated hearts. L‐OPA1 levels reduced by 30% in IR hearts compared to control hearts. Neither XJB, SfA nor their combination prevented IR‐induced reduction of L‐OPA cleavage. Likewise, IR increased the OMA1 enzymatic activity that was remained unchanged in the presence of XJB and/or SfA. In conclusion, our data show that although IR stimulates OMA1 activity/L‐OPA1 cleavage, beneficial effects on cardiac function and mitochondrial swelling are not associated with prevention of OMA1 activation/L‐OPA1 proteolytic cleavage. This study suggests that in addition to mitochondrial swelling other mechanisms can be involved in OMA1 activation.Support or Funding InformationThis study was supported by the NIH NIGMS (Grants SC1GM128210 and R25GM061838).

Early reductive stress and late onset overexpression of antioxidant enzymes in experimental myocardial infarction

Reductive stress is defined as a pathophysiological situation in which the cell becomes more reduced than in the normal, resting state. It represents a disturbance in the redox state that is harmful to biological systems. Our aim was to study the occurrence of reductive stress in the early phases of experimental myocardial infarction and to determine the mechanisms leading to such stress using a swine model. During the ischemic period, we found a decrease in the oxidized to reduced glutathione ratio (GSSG/GSH) (0.7–0.3), in the lactate to pyruvate ratio (42.7–132.4), in protein glutathionylation (111.8–96.1), and in p38 phosphorylation (0.9–0.4). This was accompanied by a significant increase in the expression of Thioredoxin (TXN) (0.6–1.9) and peroxiredoxin (PRDX6) (0.6–1.6) in different left ventricle areas. After reperfusion, there was a massive increase in oxidative damage markers including lipid peroxidation (0.2–0.4), protein carbonylation (144.9–462.8), and glutathionylation (111.8–176.8). Concomitantly, we found an activation of nuclear factor erythroid 2-related factor 2 (Nrf2) (1.2–6.1) and of a set of antioxidant enzymes including TXN, PRDX6, glutathione peroxidase (GPX1), glutathione reductase (GSR), and glucose 6 phosphate dehydrogenase (G6PD). We describe an early reductive, followed by a late onset oxidative stress (1 week and 1 month after reperfusion) in a swine myocardial infarction model. The occurrence of an early reductive phase may explain the lack of effectiveness of antioxidant therapies when administered in the early phases after reperfusion of ischemic hearts.

Abstract 266: The Effect of Different Kinds of Chest Compression Waveforms on the Heart Ischemia-Reperfusion Injury After Cardiac Arrest

Purpose: This study was aimed to compare the effect of different kinds of chest compression waveforms on the heart ischemia-reperfusion injury after cardiac arrest (CA) in a rat model. Materials and methods: After the CA model was successfully established with continuous electrical stimulation of ventricular fibrillation, rats were divided into four different chest compression groups randomly, including manual group, triangular wave group, sine wave group and trapezoidal wave group. The success rates of return of spontaneous circulation (ROSC) of those four groups were calculated, and all ROSC rats were executed 30 minutes after ROSC. Further comparisons were made among those four groups, including pathological changes, superoxide dismutase (SOD) activities, malondialdehyde (MDA) and superoxide (O 2 - ) content. Results: The success ROSC rates for those four groups (manual group, triangular wave group, sine wave group and trapezoidal wave group) were 30%, 40%, 40% and 90% respectively. There were inflammatory cell infiltration and edema in the heart tissue of those four groups. The activities of SOD in trapezoidal wave group were 85.91±1.91 U/mg, much higher than those of the other three groups (P <0.05). On the other hand, the MDA and O 2 - levels of trapezoidal wave group were much lower than those of the other three groups (p<0.05). Conclusion: Our study demonstrated that trapezoidal wave chest compression may be beneficial in improving the success rate of ROSC and alleviate heart injury. Its possible mechanism might lie in its anti-oxidative stress capability during the chest compression.

Complex Effects of Putative DRP-1 Inhibitors on Stress Responses in Mouse Heart and Rat Cardiomyoblasts.

Dynamin-related protein-1 (DRP-1)-dependent mitochondrial fission may influence cardiac tolerance to ischemic or oxidative stress, presenting a potential "cardioprotective" target. Effects of dynamin inhibitors [mitochondrial division inhibitor 1 (MDIVI-1) and dynasore] on injury, mitochondrial function, and signaling proteins were assessed in distinct models: ischemia-reperfusion (I-R) in mouse hearts and oxidative stress in rat H9c2 cardiomyoblasts. Hearts exhibited substantial cell death [approx. 40 IU lactate dehydrogenase (LDH) efflux] and dysfunction (approx. 40 mmHg diastolic pressure, approx. 40% contractile recovery) following 25 minutes' ischemia. Pretreatment with 1 μM MDIVI-1 reduced dysfunction (30 mmHg diastolic pressure, approx. 55% recovery) and delayed without reducing overall cell death, whereas 5 μM MDIVI-1 reduced overall death at the same time paradoxically exaggerating dysfunction. Postischemic expression of mitochondrial DRP-1 and phospho-activation of ERK1/2 were reduced by MDIVI-1. Conversely, 1 μM dynasore worsened cell death and reduced nonmitochondrial DRP-1. Postischemic respiratory fluxes were unaltered by MDIVI-1, although a 50% fall in complex-I flux control ratio was reversed. In H9c2 myoblasts stressed with 400 μM H2O2, treatment with 50 μM MDIVI-1 preserved metabolic (MTT assay) and mitochondrial (basal respiration) function without influencing survival. This was associated with differential signaling responses, including reduced early versus increased late phospho-activation of ERK1/2, increased phospho-activation of protein kinase B (AKT), and differential changes in determinants of autophagy [reduced microtubule-associated protein 1 light chain 3b (LC3B-II/I) vs. increased Parkinson juvenile disease protein 2 (Parkin)] and apoptosis [reduced poly-(ADP-ribose) polymerase (PARP) cleavage vs. increased BCL2-associated X (BAX)/B-cell lymphoma 2 (BCL2)]. These data show MDIVI-1 (not dynasore) confers some benefit during I-R/oxidative stress. However, despite mitochondrial and metabolic preservation, MDIVI-1 exerts mixed effects on cell death versus dysfunction, potentially reflecting differential changes in survival kinase, autophagy, and apoptosis pathways. SIGNIFICANCE STATEMENT: Inhibition of mitochondrial fission is a novel approach to still elusive cardioprotection. Assessing effects of fission inhibitors on responses to ischemic or oxidative stress in hearts and cardiomyoblasts reveals mitochondrial division inhibitor 1 (MDIVI-1) and dynasore induce complex effects and limited cardioprotection. This includes differential impacts on death and dysfunction, survival kinases, and determinants of autophagy and apoptosis. Although highlighting the interconnectedness of fission and these key processes, results suggest MDIVI-1 and dynasore may be of limited value in the quest for effective cardioprotection.

Junctophilin-2 is a target of matrix metalloproteinase-2 in myocardial ischemia-reperfusion injury.

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.

Myocardial MMP-2 contributes to SERCA2a proteolysis during cardiac ischaemia-reperfusion injury.

Matrix metalloproteinase-2 (MMP-2) is a zinc-dependent protease which contributes to cardiac contractile dysfunction when activated during myocardial ischaemia-reperfusion (IR) injury. MMP-2 is localized to several subcellular sites inside cardiac myocytes; however, its role in the sarcoplasmic reticulum (SR) is unknown. The Ca2+ ATPase SERCA2a, which pumps cytosolic Ca2+ into the SR to facilitate muscle relaxation, is degraded in cardiac IR injury; however, the protease responsible for this is unclear. We hypothesized that MMP-2 contributes to cardiac contractile dysfunction by proteolyzing SERCA2a, thereby impairing its activity in IR injury. Isolated rat hearts were subjected to IR injury in the presence or absence of the selective MMP inhibitor ARP-100, or perfused aerobically as a control. Inhibition of MMP activity with ARP-100 significantly improved the recovery of cardiac mechanical function and prevented the increase of a 70 kDa SERCA2a degradation fragment following IR injury, although 110 kDa SERCA2a and phospholamban levels appeared unchanged. Electrophoresis of IR heart samples followed by LC-MS/MS confirmed the presence of a SERCA2a fragment of ∼70 kDa. MMP-2 activity co-purified with SR-enriched microsomes prepared from the isolated rat hearts. Endogenous SERCA2a in SR-enriched microsomes was proteolyzed to ∼70 kDa products when incubated in vitro with exogenous MMP-2. MMP-2 also cleaved purified porcine SERCA2a in vitro. SERCA activity in SR-enriched microsomes was decreased by IR injury; however, this was not prevented with ARP-100. This study shows that MMP-2 activity is found in SR-enriched microsomes from heart muscle and that SERCA2a is proteolyzed by MMP-2. The cardioprotective actions of MMP inhibition in myocardial IR injury may include the prevention of SERCA2a degradation.

CaMKII mediates myocardial ischemia/reperfusion injury‑induced contracture in isolated rat heart.

Contracture or diastolic dysfunction is a primary cause of injury following ischemia/reperfusion (IR). The present study examined whether Ca2+/calmodulin‑dependent kinaseII (CaMKII) is involved in contracture. Isolated rat hearts were subjected to either global IR or Ca2+ paradox(CaP), which is characterized by contracture. Left ventricular end‑diastolic pressure, electron microscopy and troponinI TnI) in coronary effluent were examined to indicate the extent of contracture. Compared with control hearts, both the IR and CaPgroups exhibited an increase in necrosis and apoptosis, and a marked depression in contractile function. Western blot analysis showed that IR stimulated the phosphorylation (Thr287) and oxidation (Met281/282) of CaMKII, and the phosphorylation of phospholamban (PLN), a substrate of CaMKII. By contrast, only the phosphorylation of CaMKII was increased in the CaPgroup. Treatment with either 3µM KN‑62, an inhibitor of CaMKII, or 5µM KB‑R7943, an inhibitor of the Na+/Ca2+ exchanger, mitigated the damage and the post‑translational modification of both CaMKII and PLN. Similar to the effect of the negative inotropic agent 2,3‑butanedione‑monoxime, the increased cell survival after treatment with KN‑62 was associated with improved diastolic function. Examination using electron microscopy and a biochemical test showed the development of contraction bands, disruption of the sarcolemmal membrane and an increase in the release of TnI in both IR and CaP hearts; these results indicated the occurrence of contracture. Furthermore, these changes were inhibited by either KN‑62 or KB‑R7943. Taken together, these data provided evidence that CaMKII mediates reperfusion‑elicited contracture, and that the activation of CaMKII via phosphorylation is involved in this process.

Mapping Changes of Whole Brain Blood Flow in Rats with Myocardial Ischemia/Reperfusion Injury Assessed by Positron Emission Tomography.

18F-labeled fluorodeoxyglucose positron emission tomography (18F-FDG PET) is the most sensitive tool for studying brain metabolism in vivo. We investigated the image patterns of 18F-FDG PET during reperfusion injury and correlated changes of whole brain blood flow utilizing a rat myocardial ischemia/reperfusion injury (MIRI) model. The results assessed by echocardiography indicated resultant cardiac dysfunction after ischemia-reperfusion in the rat heart. It was found that the average standardized uptake value (SUVaverage) of the whole brain was significantly decreased in model rats, and the glucose uptake of different brain regions including accumbens core/shell (Acb), left caudate putamen (LCPu), hippocampus (HIP), left hypothalamus (LHYP), olfactory (OLF), superior colliculus (SC), right midbrain (RMID), ventral tegmental area (VTA), inferior colliculus (IC) and left thalamus whole (LTHA) was significantly decreased in MIRI rats whereas no significant difference was found in the SUVaverage of amygdala (AMY), right CPu, RHYP, right HYP, left MID, right THA, pons and medulla oblongata (MO). These 18F-FDG PET data provide a reliable identification method for brain metabolic changes in rats with MIRI.

LncRNA H19 Alleviated Myocardial I/RI via Suppressing miR-877-3p/Bcl-2-Mediated Mitochondrial Apoptosis

Ischemic cardiac disease is the leading cause of morbidity and mortality in the world. Despite the great efforts and progress in cardiac research, the current treatment of cardiac ischemia reperfusion injury (I/RI) is still far from being satisfactory. This study was performed to investigate the role of long non-coding RNA (lncRNA) H19 in regulating myocardial I/RI. We found that H19 expression was downregulated in the I/R hearts of mice and cardiomyocytes treated with H2O2. Overexpression of H19 alleviated myocardial I/RI of mice and cardiomyocyte injury induced by H2O2. We found that H19 functioned as a competing endogenous RNA of miR-877-3p, which decreased the expression of miR-877-3p through the base-pairing mechanism. In parallel, miR-877-3p was upregulated in H2O2-treated cardiomyocytes and mouse ischemia reperfusion (I/R) hearts. miR-877-3p exacerbated myocardial I/RI and cardiomyocyte apoptosis. We further established Bcl-2 as a downstream target of miR-877-3p. miR-877-3p inhibited the mRNA and protein expression of Bcl-2. Furthermore, H19 decreased the Bcl-2/Bax ratio at mRNA and protein levels, cytochrome c release, and activation of caspase-9 and caspase-3 in myocardial I/RI mice, which were canceled by miR-877-3p. In summary, the H19/miR-877-3p/Bcl-2 pathway is involved in regulation of mitochondrial apoptosis during myocardial I/RI, which provided new insight into molecular mechanisms underlying regulation of myocardial I/RI.

Myocardial Preconditioning Potential of Hedgehog Activator Purmorphamine (Smoothened Receptor Agonist) against Ischemia-Reperfusion in Deoxycortisone Acetate Salt-Induced Hypertensive Rat Hearts

Aim: To investigate the myocardial preconditioning potential of hedgehog activator smoothened (SMO) receptor agonist purmorphamine (PUR) against ischemia-reperfusion (I/R) injury in deoxycortisone acetate (DOCA) salt-induced hypertensive rat heart. Methods: Hypertension in rats was produced by surgical removal of the kidney and DOCA salt administration for 6 weeks. Isolated rat heart was subjected to 30 min ischemia followed by 120 min of I/R. The heart was subjected to pharmacological preconditioning with the hedgehog (Hh) activator PUR (1 μM), PUR (2 μM), and atractyloside (4 μM), an mPTP opener, in the last episode of reperfusion before I/R. Elevated blood pressure was evaluated through tail-cuff method. Myocardial infarction was assessed in terms of the increase in lactate dehydrogenase, creatinine kinase-muscle/brain, and infarct size through triphenyltetrazolium chloride staining. Further, there were a decrease in the release of nitrite and coronary flow rate. Immunohistochemistry was performed for the assessment of tumor necrosis factor-α level in cardiac tissue. Results: Pharmacological preconditioning with PUR significantly attenuated I/R-induced myocardial infarction. However, atractyloside limits the ameliorative preconditioning potential of PUR and confirmed the role of Hh pathway in ischemic preconditioning. Conclusion: It may be concluded that the Hh activator PUR (SMO receptor agonist) prevents I/R injury in DOCA salt-induced hypertensive rat heart.

KPC1 alleviates hypoxia/reoxygenation-induced apoptosis in rat cardiomyocyte cells though BAX degradation.

Bax triggers cell apoptosis by permeabilizing the outer mitochondrial membrane, leading to membrane potential loss and cytochrome c release. However, it is unclear if proteasomal degradation of Bax is involved in the apoptotic process, especially in heart ischemia‐reperfusion (I/R)‐induced injury. In the present study, KPC1 expression was heightened in left ventricular cardiomyocytes of patients with coronary heart disease (CHD), in I/R‐myocardium in vivo and in hypoxia and reoxygenation (H/R)‐induced cardiomyocytes in vitro. Overexpression of KPC1 reduced infarction size and cell apoptosis in I/R rat hearts. Similarly, the forced expression of KPC1 restored mitochondrial membrane potential (MMP) and cytochrome c release driven by H/R in H9c2 cells, whereas reducing cell apoptosis, and knockdown of KPC1 by short‐hairpin RNA (shRNA) deteriorated cell apoptosis induced by H/R. Mechanistically, forced expression of KPC1 promoted Bax protein degradation, which was abolished by proteasome inhibitor MG132, suggesting that KPC1 promoted proteasomal degradation of Bax. Furthermore, KPC1 prevented basal and apoptotic stress‐induced Bax translocation to mitochondria. Bax can be a novel target for the antiapoptotic effects of KPC1 on I/R‐induced cardiomyocyte apoptosis and render mechanistic penetration into at least a subset of the mitochondrial effects of KPC1.

Attenuation of Cardiac Ischaemia-reperfusion Injury by Treatment with Hydrogen-rich Water.

Background: Hydrogen has been shown to exert a bioactive effect on the myocardium. This study examined the signalling pathways for hydrogen attenuating ischaemia-reperfusion injury.Methods: In total, 20 male Wistar rats were evaluated for the effects of hydrogen-rich water on ischaemia-reperfusion in hearts. Left ventricular tissue was taken for screening and analysis of active protein factors by protein chip technology. The enrichment of the KEGG pathway was obtained by using the Gene Ontology (GO) enrichment principle. The expression of JAK2, STAT1, STAT3, p-STAT1, p-JAK2, p-STAT3 in rat myocardium was detected by Western blot analysis and immunohistochemistry. The apoptosis rates of the control and hydrogen-rich water groups were detected by TUNEL staining.Results: The expression levels of 25 proteins, including five transduction pathways, were downregulated in the hydrogen-rich water group. The expression levels of p-JAK2/JAK2, p-STAT3/STAT3 were upregulated in the hydrogen-rich water group compared with the control group, and p-STAT1/STAT1 was downregulated in the hydrogen-rich water group compared with the control group. Furthermore, the apoptosis rate was significantly decreased in the hydrogen-rich water group, as well.Conclusion: Hydrogen-rich water may inhibit the apoptosis of cardiomyocytes after ischaemia-reperfusion by upregulating the expression of the JAK2-STAT3 signalling pathway, which reduces ischaemia-reperfusion injury.