Min Of Reperfusion Research Articles

Arginase 1 in erythrocytes is a critical modulator of cardioprotective signalling by nitric oxide and soluble guanylyl cyclase

Abstract Background Arginase is involved in the development of ischemia-reperfusion injury by regulating nitric oxide (NO) bioactivity via competition with NO synthase (NOS) for their common substrate L-arginine. Erythrocytes are known to contain high levels of arginase that may reduce export of cardioprotective NO bioactivity. Aim To determine the role of arginase 1 in erythrocytes for cardiac protection against myocardial ischemia-reperfusion injury. Methods A tie2 cre-flox mouse model, in which arginase 1 was deleted (Arg 1-KO) in hematopoietic and endothelial cells, was developed. In vivo ischemia-reperfusion was performed in anaesthetized mice by left anterior descending coronary artery ligation (30 min ischemia and 120 min reperfusion). To determine the specific role of erythrocytes, an isolated perfused mouse heart model was applied. Erythrocytes from Arg 1-KO and wild type (WT) mice were given to isolated hearts from WT mice at the onset of global ischemia. After 40 min ischemia, the recovery of left ventricular developed pressure (LVDP) during 60 min reperfusion was recorded as an indicator of cardiac functional recovery. All animal experiments and procedures were performed according to the guidelines by the U.S National Institutes of Health (NIH publication no 85–23, revised 1996) Results Following in vivo ischemia-reperfusion, infarct size was smaller in Arg 1-KO mice than in WT mice (Fig. A). A similar degree of infarct size reduction was obtained by i.v. administration of an arginase inhibitor in WT mice. The cardioprotective effect observed in Arg 1-KO mice was abolished by the NOS inhibitor L-NMMA (Fig. A). In isolated buffer-perfused hearts, there was no difference in the post-ischemic recovery of LVDP between Arg 1-KO and WT hearts. When erythrocytes from Arg 1-KO mice were administrated to isolated WT mouse hearts, the post-ischemic recovery of LVDP was significantly improved compared to hearts given erythrocytes from WT mice. Also, the cardioprotective effect of erythrocytes from Arg 1-KO mice was abolished by pre-incubation of the erythrocytes with the NOS inhibitor L-NAME (Fig. B) or the inhibitor of soluble guanylyl cyclase ODQ (Fig. C). Conclusion Arginase 1 in erythrocyte plays an important role in regulating cardioprotection mediated via the NO-soluble guanylyl cyclase pathway. Funding Acknowledgement Type of funding sources: Foundation. Main funding source(s): Swedish Heart and Lung Foundation (20190266),The Swedish Research Council (2020-01372)

Acute Effects of Ischemic Intra-Conditioning on 30 m Sprint Performance.

The present study aimed to evaluate the effects of ischemic intra-conditioning applied during rest intervals on 30 m sprint performance. Thirty-four trained male (n = 12) and female (n = 22) track and field and rugby athletes volunteered to participate in the study (age = 19.6 ± 4 years; training experience = 5.3 ± 1.9 years). In a randomized and counterbalanced order, participants performed six sets of 30 m sprints under three different testing conditions: without ischemic intra-conditioning, and with ischemic intra-conditioning at 60% or 80% arterial occlusion pressure applied bilaterally before the first trial of the sprint and during the rest periods between all sprint trials. During experimental sessions, subjects perform 6 × 30 m sprints with a 7 min rest interval between attempts. The cuffs were applied following a 1 min rest period and lasted for 5 min before being released at the 6th minute to allow for reperfusion (1 min + 5 min ischemic intra-conditioning + 1 min reperfusion). The two-way repeated measures ANOVA did not show statistically significant condition × set interaction for time of the sprint (p = 0.06; η2 = 0.05). There was also no main effect of ischemic intra-conditioning for any condition (p = 0.190; η2 = 0.05). This study indicates that ischemic intra-conditioning did not enhance the performance of 30 m sprints performed by athletes. However, ischemic intra-conditioning did not decrease performance either.

Sympathetic nerve innervation and metabolism in ischemic myocardium in response to remote ischemic perconditioning

Sympathetic nerve denervation after myocardial infarction (MI) predicts risk of sudden cardiac death. Therefore, therapeutic approaches limit infarct size, improving adverse remodeling and restores sympathetic innervation have a great clinical potential. Remote ischemic perconditioning (RIPerc) could markedly attenuate MI-reperfusion (MIR) injury. In this study, we aimed to assess its effects on cardiac sympathetic innervation and metabolism. Transient myocardial ischemia is induced by ligature of the left anterior descending coronary artery (LAD) in male Sprague–Dawley rats, and in vivo cardiac 2-[18F]FDG and [11C]mHED PET scans were performed at 14–15 days after ischemia. RIPerc was induced by three cycles of 5-min-long unilateral hind limb ischemia and intermittent 5 min of reperfusion during LAD occlusion period. The PET quantitative parameters were quantified in parametric polar maps. This standardized format facilitates the regional radioactive quantification in deficit regions to remote areas. The ex vivo radionuclide distribution was additionally identified using autoradiography. Myocardial neuron density (tyrosine hydroxylase positive staining) and chondroitin sulfate proteoglycans (CSPG, inhibiting neuron regeneration) expression were assessed by immunohistochemistry. There was no significant difference in the mean hypometabolism 2-[18F]FDG uptake ratio (44.6 ± 4.8% vs. 45.4 ± 4.4%) between MIR rats and MIR + RIPerc rats (P > 0.05). However, the mean [11C]mHED nervous activity of denervated myocardium was significantly elevated in MIR + RIPerc rats compared to the MIR rats (35.9 ± 7.1% vs. 28.9 ± 2.3%, P < 0.05), coupled with reduced denervated myocardium area (19.5 ± 5.3% vs. 27.8 ± 6.6%, P < 0.05), which were associated with preserved left-ventricular systolic function, a less reduction in neuron density, and a significant reduction in CSPG and CD68 expression in the myocardium. RIPerc presented a positive effect on cardiac sympathetic-nerve innervation following ischemia, but showed no significant effect on myocardial metabolism.

Acetate, a gut bacterial product, ameliorates ischemia-reperfusion induced acute lung injury in rats

Recent data suggest that short-chain fatty acids (SCFAs), the major fermentation product from gut microbial degradation of dietary fiber, have protective effects against renal ischemia–reperfusion (IR) injury, colitis, and allergic asthma. However, the effect of SCFAs on acute lung injury (ALI) caused by IR is still unclear. In this study, we examine whether SCFAs have protective effects against IR-induced ALI and explore possible protective mechanisms. IR-induced ALI was established by 40 min ischemia followed by 60 min reperfusion in isolated perfused rat lungs. Rats were randomly assigned to one of six groups: control, control + acetate (400 mg/kg), IR, and IR + acetate at one of three dosages (100, 200, 400 mg/kg). Bronchoalveolar lavage fluids (BALF) and lung tissues were obtained and analyzed at the end of the experiment. In vitro, mouse lung epithelial cells (MLE-12) subjected to hypoxia-reoxygenation (HR) were pretreated with acetate (25 mmol/L) and GPR41 or GPR43 siRNA. Acetate decreased lung weight gain, lung weight/body weight ratios, wet/dry weight ratios, pulmonary artery pressure, and protein concentration of the BALF in a dose-dependent manner for IR-induced ALI. Acetate also significantly inhibited the production of TNF-α, IL-6 and CINC-1 in the BALF. Moreover, acetate treatment restored suppressed IκB-α levels and reduced nuclear NF-κB p65 levels in lung tissues. In addition, acetate mitigated IR-induced apoptosis and tight junction disruption in injured lung tissue. In vitro analyses showed that acetate attenuated NF-κB activation and KC/CXCL-1 levels in MLE-12 cells exposed to HR. The protective effects of acetate in vitro were significantly abrogated by GPR41 or GPR43 siRNA. Acetate ameliorates IR-induced acute lung inflammation and its protective mechanism appears to be via the GPR41/43 signaling pathway. Based on our findings, acetate may provide a novel adjuvant therapeutic approach for IR-induced lung injury.

Remote ischemic conditioning for acute ischemic stroke part 2: Study protocol for a randomized controlled trial.

BackgroundRemote ischemic conditioning (RIC) refers to the application of repeated short periods of ischemia intended to protect remote areas against tissue damage during and after prolonged ischemia.AimWe aim to evaluate the efficacy of RIC, determined by the modified Rankin Scale (mRS) score at 90 days after stroke onset.Design and methodsThis study is an investigator-initiated, multicenter, prospective, randomized, open-label, parallel-group clinical trial. The sample size is 400, comprising 200 patients who will receive RIC and 200 controls. The patients will be divided into three groups according to their National Institutes of Health Stroke Scale score at enrollment: 5–9, mild; 10–14, moderate; 15–20, severe. The RIC protocol will be comprised of four cycles, each consisting of 5 min of blood pressure cuff inflation (at 200 mmHg or 50 mmHg above the systolic blood pressure) followed by 5 min of reperfusion, with the cuff placed on the thigh on the unaffected side. The control group will only undergo blood pressure measurements before and after the intervention period. This trial is registered with the UMIN Clinical Trial Registry (https://www.umin.ac.jp/: UMIN000046225).Study outcomeThe primary outcome will be a good functional outcome as determined by the mRS score at 90 days after stroke onset, with a target mRS score of 0–1 in the mild group, 0–2 in the moderate group, and 0–3 in the severe group.DiscussionThis trial may help determine whether RIC should be recommended as a routine clinical strategy for patients with ischemic stroke.

Cardioprotective effects of chloroquine pretreatment on ischemic and reperfusion injury via activation of ERK1/2 in isolated rat hearts.

Several therapeutic agents have been found to prevent myocardial ischemic and reperfusion (I/R) injury after cardiac surgery; however, no drug is routinely used to afford cardioprotective benefits in clinical settings. Herein, we aimed to determine whether chloroquine (CQ) pretreatment attenuates I/R injury after global ischemia in isolated rat hearts and elucidate mechanisms underlying the effects of CQ. Isolated rat hearts were subjected to 30-min global ischemia, followed by 60-min reperfusion with Krebs-Henseleit buffer (KHB). Immediately before ischemia, 10mL of pretreatment solutions (KHB, n = 4 or KHB + CQ [100μM], n = 4) were injected through the aortic root. Cardiac function was examined based on the rate pressure product (RPP). Myocardial apoptosis was evaluated using TUNEL staining. To assess the reperfusion ischemia salvage kinase pathway, protein expression levels of AKT and extracellular signal-regulated kinase (ERK1/2) were determined using western blotting. To investigate the role of ERK1/2, an ERK1/2 selective inhibitor was used in eight additional rats. The recovery rate of the RPP was higher in the KHB + CQ group than in the KHB group 60min after I/R (KHB, 44 ± 3% vs. KHB + CQ, 69 ± 7%; P = 0.019, d = 2.2). CQ pretreatment reduced apoptosis and enhanced the phosphorylation of ERK1/2; however, AKT phosphorylation was unaltered. In addition, the ERK1/2 inhibitor abolished CQ-mediated cardioprotective effects. CQ pretreatment showed protective effects on cardiac function after I/R by activating ERK1/2.

Short-Term Administration of Lemon Balm Extract Ameliorates Myocardial Ischemia/Reperfusion Injury: Focus on Oxidative Stress.

We aimed to investigate the cardioprotective effects of ethanolic Melissa officinalis L. extract (ME) in the rat model of myocardial ischemia/reperfusion (I/R) injury. Thirty-two Wistar rats were randomly divided into a CTRL non-treated control group with myocardial I/R injury and three experimental groups of rats treated with 50, 100, or 200 mg/kg of ME for 7 days per os. Afterward, hearts were isolated, and cardiodynamic function was assessed via the Langendorff model of global 20 min ischemia and 30 min reperfusion. Oxidative stress parameters were determined spectrophotometrically from the samples of coronary venous effluent (O2−, H2O2, TBARS, and NO2−,) and heart tissue homogenate (TBARS, NO2−, SOD, and CAT). H/E and Picrosirius red staining were used to examine cardiac architecture and cardiac collagen content. ME improved cardiodynamic parameters and achieved to preserve cardiac architecture after I/R injury and to decrease fibrosis, especially in the ME200 group compared to CTRL. ME200 and ME100 markedly decreased prooxidants TBARS, O2−, and H2O2 while increasing NO2−. Hereby, we confirmed the ME`s ability to save the heart from I/R induced damage, even after short-term preconditioning in terms of preserving cardiodynamic alterations, cardiac architecture, fibrosis, and suppressing oxidative stress, especially in dose of 200 mg/kg.

Proteomics Revealed That Mitochondrial Function Contributed to the Protective Effect of Herba Siegesbeckiae Against Cardiac Ischemia/Reperfusion Injury.

BackgroundMyocardial ischemia/reperfusion (I/R) injury is the main obstacle to percutaneous coronary intervention, lacking effective therapeutic measures in a clinical setting. Herba Siegesbeckiae (HS) is a traditional herb with multiple pharmacological activities and evidence of cardiovascular protection. However, few data are available regarding the role of HS in cardiac I/R. This study aimed to explore the effect and underlying mechanism of HS aqueous extract on cardiac I/R injury.Materials and MethodsHerba Siegesbeckiae aqueous extract was prepared and analyzed by UHPLC-MS/MS. After intragastric administration of HS once daily for 7 days, male Sprague-Dawley rats were subjected to 30 min occlusion of the left anterior descending coronary artery followed by 120 min reperfusion to elicit I/R. Various parameters like myocardial infarction and apoptosis, 12-lead ECG and hemodynamics, cardiac morphology and myocardial enzymes, quantitative proteomics, mitochondrial ultrastructure and electron transport chain (ETC) function, oxidative stress and antioxidation, and NLRP3 inflammasome and inflammation were evaluated.ResultsThe chemical constituents of HS aqueous extract were mainly divided into flavonoids, diterpenoids, and organic acids. In vivo, HS aqueous extract notably alleviated myocardial I/R injury, as evidenced by a reduction in infarct size, apoptotic cells, and cardiac lesion enzymes; decline of ST-segment elevation; improvement of cardiac function; and preservation of morphology. Quantitative proteomics demonstrated that HS reversed the alteration in the expression of Adgb, Cbr1, Decr1, Eif5, Uchl5, Lmo7, Bdh1, Ckmt2, COX7A, and RT1-CE1 after I/R. In addition, HS preserved myocardial ultrastructure and restored the function of mitochondrial ETC complexes following exposure to I/R; HS significantly suppressed I/R-elicited increase of ROS, RNS, MDA, and 8-OHdG, restrained the acetylation of MnSOD, and recovered the activity of MnSOD; and HS reversed I/R-induced elevation of NLRP3 inflammasome and inhibited the release of inflammatory factors and pyroptosis.ConclusionHerba Siegesbeckiae aqueous extract ameliorated cardiac I/R injury, which is associated with mitigating oxidative stress, suppressing NLRP3 inflammasome, and restoring mitochondrial function by regulating the expression of Adgb, Cbr1, Decr1, Eif5, Uchl5, Lmo7, Bdh1, Ckmt2, COX7A, and RT1-CE1.

Ischemic preconditioning upregulates Mitofusin2 and preserves muscle strength in tourniquet-induced ischemia/reperfusion.

BackgroundTourniquet-induced ischemia and reperfusion (I/R) has been related to postoperative muscle atrophy through mechanisms involving protein synthesis/breakdown, cellular metabolism, mitochondrial dysfunction, and apoptosis. Ischemic preconditioning (IPC) could protect skeletal muscle against I/R injury. This study aims to determine the underlying mechanisms of IPC and its effect on muscle strength after total knee arthroplasty (TKA).MethodsTwenty-four TKA patients were randomized to receive either sham IPC or IPC (3 cycles of 5-min ischemia followed by 5-min reperfusion). Vastus medialis muscle biopsies were collected at 30 ​min after tourniquet (TQ) inflation and the onset of reperfusion. Western blot analysis was performed in muscle protein for 4-HNE, SOD2, TNF-ɑ, IL-6, p-Drp1ser616, Drp1, Mfn1, Mfn2, Opa1, PGC-1ɑ, ETC complex I-V, cytochrome c, cleaved caspase-3, and caspase-3. Clinical outcomes including isokinetic muscle strength and quality of life were evaluated pre- and postoperatively.ResultsIPC significantly increased Mfn2 (2.0 ​± ​0.2 vs 1.2 ​± ​0.1, p ​= ​0.001) and Opa1 (2.9 ​± ​0.3 vs 1.9 ​± ​0.2, p ​= ​0.005) proteins expression at the onset of reperfusion, compared to the ischemic phase. There were no differences in 4-HNE, SOD2, TNF-ɑ, IL-6, p-Drp1ser616/Drp1, Mfn1, PGC-1ɑ, ETC complex I–V, cytochrome c, and cleaved caspase-3/caspase-3 expression between the ischemic and reperfusion periods, or between the groups. Clinically, postoperative peak torque for knee extension significantly reduced in the sham IPC group (−16.6 [-29.5, −3.6] N.m, p ​= ​0.020), while that in the IPC group was preserved (−4.7 [-25.3, 16.0] N.m, p ​= ​0.617).ConclusionIn TKA with TQ application, IPC preserved postoperative quadriceps strength and prevented TQ-induced I/R injury partly by enhancing mitochondrial fusion proteins in the skeletal muscle.The translational potential of this articleMitochondrial fusion is a potential underlying mechanism of IPC in preventing skeletal muscle I/R injury. IPC applied before TQ-induced I/R preserved postoperative quadriceps muscle strength after TKA.

Ischemia preconditioning alleviates ischemia/reperfusion injury-induced coronary no-reflow and contraction of microvascular pericytes in rats.

Ischemia preconditioning (IPC) ameliorates coronary no-reflow induced by ischemia/reperfusion (I/R) injury, and pericytes play an important role in microvascular function. However, it is unclear whether IPC exerts a protective effect on coronary microcirculation and regulates the pericytes. The purpose of this study was to assess whether IPC improves coronary microvascular perfusion and reduces pericyte constriction after myocardial I/R injury. Rats were randomly divided into three groups: the sham group, the I/R group, and the IPC+I/R group. The left anterior descending artery (LAD) of rats in the I/R group were ligated for 45min, and the rats in the IPC+I/R group received 4 episodes of 6min occlusion followed by 6min reperfusion before the LAD was ligated. After 24h of reperfusion, the area of no-reflow, and area at risk were evaluated with thioflavin-S and Evens blue staining, and infarct size with triphenyl tetrazolium chloride staining, respectively. Besides, fluorescent microspheres were perfused to enable visualization of the non-obstructed coronary vessels. Cardiac pericytes and microvascular were observed by immunofluorescence, and the diameter of microvascular at the site of the pericyte somata was analyzed. The infarct size, and area of no-reflow in the IPC+I/R group were significantly reduced compared with the I/R group (infarct size, 33.5%±11.9% vs. 49.2%±9.4%, p=0.021；no-reflow, 12.7%±5.2% vs. 26.6%±5.0%, p<0.001). IPC improved microvascular perfusion and reduced the percentage of the blocked coronary capillary. Moreover, we found that cardiac pericytes were widely distributed around the microvascular in various regions of the heart, and expressed the contractile protein α-smooth muscle actin. The microvascular lumen diameter at pericyte somata was reduced after I/R (4.3±1.0μm vs. 6.5±1.2μm, p<0.001), which was relieved in IPC+I/R group compared with the I/R group (5.2±1.0μm vs. 4.3±1.0μm, p<0.001). Besides, IPC could reduce the proportion of apoptotic pericytes compared to the I/R group (22.1%±8.4% vs. 38.5%±7.5%, p<0.001). IPC reduced no-reflow and inhibited the contraction of microvascular pericytes induced by cardiac I/R injury, suggesting that IPC might play a protective role by regulating the pericyte function.

Roles of isometric contraction training in promoting neuroprotection and angiogenesis after stroke in adult rats.

100 rats were randomly divided into a sham-operated group and middle cerebral artery occlusion (MCAO) modeling groups. The sham group after surgery was observed for 14 days. After MCAO, some rats received isometric contraction training (ICT) which was as follows: an atraumatic tourniquet was placed around left or right hind limb to achieve hind limb ischemia for 5 min, followed by 5 min of reperfusion, 4 cycles for one time, once a day, and five days per week. The MCAO modeling groups included the following four groups: i) a group only received MCAO, and was observed for seven days (MCAO-7d), ii) a group only received MCAO, and was observed for 14 days (MCAO-14d), iii) a group, after MCAO, received ICT for seven days (ICT-7d), and iv) a group, after MCAO, received ICT for 14 days (ICT-14d). Brain infarct area, behavioral outcomes, the number of neurons, apoptosis, cerebral edema and cerebral water content were assessed, respectively. The mRNA expression of vascular endothelial growth factor (VEGF) was assayed with RT-PCR, and protein expression of VEGF was quantified with western blot. compared with MCAO controls, cerebral infarction, neurological deficits and neuronal apoptosis were reduced significantly in the ICT groups, while the number of neurons was increased. Moreover, the mRNA expression of VEGF and protein expression of VEGF were enhanced after 1 and 2 weeks of ICT. ICT may promote angiogenesis and neuroprotection after ischemic stroke and this new remodeling method provide a novel strategy for rehabilitation of stroke patients.

Ischemic limb preconditioning-induced anti-arrhythmic effect in reperfusion-induced myocardial injury: is it mediated by the RISK or SAFE pathway?

The mechanism for limb ischemic precondition (RLIPC)-induced suppression of reperfusion arrhythmia remains unknown. The purpose of this study was to examine the roles of the pro-survival reperfusion injury salvage kinase (RISK) and survivor activating factor enhancement (SAFE) pathways in this RLIPC-mediated antiarrhythmic activity. Male Sprague Dawley rats were assigned to sham-operated, control, or RLIPC groups. All rats except for the sham rats had 5min of left main coronary artery occlusion with another 20min of reperfusion. RLIPC was initiated by four cycles of limb ischemia (5min) and reperfusion (5min) on the bilateral femoral arteries. Hearts in every group were taken for protein phosphorylation analysis. RLIPC ameliorated reperfusion-induced arrhythmogenesis and reduced the incidence of sudden cardiac death during the entire 20-min reperfusion period (66.7% of control rats had SCD vs. only 16.7% of RLIPC-treated rats). RLIPC enhances ventricular ERK1/2 phosphorylation after reperfusion. RLIPC-induced antiarrhythmic action and ERK1/2 phosphorylation are abolished in the presence of the ERK1/2 inhibitor U0126. Limb ischemic preconditioning protects the heart against myocardial reperfusion injury-induced lethal arrhythmia. These beneficial effects may involve the activation of ERK1/2 in the RISK signaling pathway.

Subacute Reperfusion in Ischemic Hearts: Study of Autophagy and its Possible Interconnection with Receptor-Interacting Protein Kinase 3.

The role of cardiac autophagy during ischemia and reperfusion (I/R) remains controversial. Furthermore, whether this cell death during I/R is also interconnected with other cell damaging event, such as necroptosis, is insufficiently known. Thus, the aim of this study was to investigate possible links between autophagy and necroptosis in the hearts under conditions of acute I/R injury. Langendorff-perfused male Wistar rat hearts were subjected to 30-min global ischemia followed by 10-min reperfusion in the presence of either vehicle or a drug inhibiting the pro-necroptotic receptor-interacting protein kinase 3 (RIP3). Hemodynamic parameters and lactate dehydrogenase (LDH) release were measured to assess heart function and non-specific cell death due to the disruption of plasma membrane. Immunoblot analysis of left ventricles revealed that early reperfusion suppressed the activation of autophagy as evidenced by the decreased protein expression of Beclin-1, pSer555-ULK1, pSer555-ULK1/ULK1 ratio, and LC3-II/LC3-I ratio. On the other hand, the molecular signalling responsible for autophagy inhibition did not appear to be affected in these I/R settings. RIP3 inhibition during reperfusion significantly mitigated the loss of the plasma membrane integrity but did not improve cardiac function. This pharmacological intervention targeting necroptosis-mediating protein decreased LC3-II expression in I/R hearts, suggesting some effect on autophagosome processing, but it did not significantly alter other signalling pathways involved in autophagy activation or inhibition. In summary, we showed for the first time that an early reperfusion phase does not promote autophagy and that there may be an interplay between pro-necroptotic protein RIP3 and autophagy with respect to the regulation of autophagosome processing.

Discrepancy between the Actions of Glucagon-like Peptide-1 Receptor Ligands in the Protection of the Heart against Ischemia Reperfusion Injury.

Tirzepatide is a dual glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) receptor agonist and a promising therapy for type 2 diabetes mellitus (T2DM). GLP-1 is an incretin hormone with therapeutic potential beyond type 2 diabetes mellitus. However, GLP-1 is rapidly degraded by dipeptdyl peptidase-IV (DPP-IV) to GLP-1 (9-36). Exendin-4 (Ex-4) is a DPP-IV-resistant GLP-1 receptor agonist which, when truncated to Ex-4 (9-39), acts as a GLP-1 receptor antagonist. In the present study, hearts isolated from Wistar rats (n = 8 per group) were perfused with a modified Langendorff preparation. Left ventricular (LV) contractility and cardiovascular hemodynamics were evaluated by a data acquisition program and infarct size was evaluated by 2,3,5-Triphenyl-2H-tetrazolium chloride (TTC) staining and cardiac enzyme levels. Hearts were subjected to 30 min regional ischemia, produced by ligation of the left anterior descending (LAD) coronary artery followed by 30 min reperfusion. Hearts were treated during reperfusion with either the non-lipidated precursor of tirzepatide (NLT), GLP-1, GLP-1 (9-36), or Ex-4 in the presence or absence of Ex-4 (9-39). Infusion of GLP-1 (9-36) or Ex-4 protected the heart against I/R injury (p > 0.01) by normalizing cardiac hemodynamic and enzyme levels. Neither GLP-1, NLT, nor Ex-4 (9-39) showed any protection. Interestingly, Ex-4 (9-39) blocked Ex-4-mediated protection but not that of GLP-1 (9-36). These data suggest that Ex-4-mediated protection is GLP-1-receptor-dependent but GLP-1 (9-36)-mediated protection is not.

Effects of intracardiac delivery of aldehyde dehydrogenase 2 gene in myocardial salvage.

Intrinsic activity of aldehyde dehydrogenase (ALDH)2, a cardiac mitochondrial enzyme, is vital in detoxifying 4-hydroxy-2-nonenal (4HNE) like cellular reactive carbonyl species (RCS) and thereby conferring cardiac protection against pathological stress. It was also known that a single point mutation (E487K) in ALDH2 (prevalent in East Asians) known as ALDH2*2 reduces its activity intrinsically and was associated with increased cardiovascular diseases. We and others have shown that ALDH2 activity is reduced in several pathologies in WT animals as well. Thus, exogenous augmentation of ALDH2 activity is a good strategy to protect the myocardium from pathologies. In this study, we will test the efficacy of intracardiac injections of the ALDH2 gene in mice. We injected both wild type (WT) and ALDH2*2 knock-in mutant mice with ALDH2 constructs, AAv9-cTNT-hALDH2-HA tag-P2A-eGFP or their control constructs, AAv9-cTNT-eGFP. We found that intracardiac ALDH2 gene transfer increased myocardial levels of ALDH2 compared to GFP alone after 1 and 3 weeks. When we subjected the hearts of these mice to 30 min global ischemia and 90 min reperfusion (I-R) using the Langendorff perfusion system, we found reduced infarct size in the hearts of mice with ALDH2 gene vs GFP alone. A single time injection has shown increased myocardial ALDH2 activity for at least 3 weeks and reduced myocardial 4HNE adducts and infarct size along with increased contractile function of the hearts while subjected to I-R. Thus, ALDH2 overexpression protected the myocardium from I-R injury by reducing 4HNE protein adducts implicating increased 4HNE detoxification by ALDH2. In conclusion, intracardiac ALDH2 gene transfer is an effective strategy to protect the myocardium from pathological insults.

Effects of ischemic conditioning on head and neck free flap oxygenation: a randomized controlled trial

Flap failure after microvascular reconstructive surgery is a rare but devastating complication caused by reperfusion injury and tissue hypoperfusion. Remote ischemic conditioning (RIC) provides protection against ischemia/reperfusion injury and reduces tissue infarction. We hypothesized that RIC would enhance flap oxygenation and exert organ-protective effects during head and neck free flap reconstructive surgery. Adult patients undergoing free flap transfer surgery for head and neck cancer were randomized to receive either RIC or sham-RIC during surgery. RIC consisted of four cycles of 5-min ischemia and 5-min reperfusion applied to the upper or lower extremity. The primary endpoint, tissue oxygen saturation of the flap, was measured by near-infrared spectroscopy on the first postoperative day. Organ-protective effects of RIC were evaluated with infarct size of rat hearts perfused with plasma dialysate from patients received RIC or sham-RIC. Between April 2018 and July 2019, 50 patients were randomized (each n = 25) and 46 were analyzed in the RIC (n = 23) or sham-RIC (n = 23) groups. Tissue oxygen saturation of the flap was similar between the groups (85 ± 12% vs 83 ± 9% in the RIC vs sham-RIC groups; P = 0.471). Myocardial infarct size after treatment of plasma dialysate was significantly reduced in the RIC group (44 ± 7% to 26 ± 6%; P = 0.018) compared to the sham-RIC group (42 ± 6% to 37 ± 7%; P = 0.388). RIC did not improve tissue oxygenation of the transferred free flap in head and neck cancer reconstructive surgery. However, there was evidence of organ-protective effects of RIC in experimental models.Trial registration: Registry number of ClinicalTrials.gov: NCT03474952.

Cardioprotection by selective SGLT-2 inhibitors in a non-diabetic mouse model of myocardial ischemia/reperfusion injury: a class or a drug effect?

Major clinical trials with sodium glucose co-transporter-2 inhibitors (SGLT-2i) exhibit protective effects against heart failure events, whereas inconsistencies regarding the cardiovascular death outcomes are observed. Therefore, we aimed to compare the selective SGLT-2i empagliflozin (EMPA), dapagliflozin (DAPA) and ertugliflozin (ERTU) in terms of infarct size (IS) reduction and to reveal the cardioprotective mechanism in healthy non-diabetic mice. C57BL/6 mice randomly received vehicle, EMPA (10mg/kg/day) and DAPA or ERTU orally at the stoichiometrically equivalent dose (SED) for 7days. 24h-glucose urinary excretion was determined to verify SGLT-2 inhibition. IS of the region at risk was measured after 30min ischemia (I), and 120min reperfusion (R). In a second series, the ischemic myocardium was collected (10th min of R) for shotgun proteomics and evaluation of the cardioprotective signaling. In a third series, we evaluated the oxidative phosphorylation capacity (OXPHOS) and the mitochondrial fatty acid oxidation capacity by measuring the respiratory rates. Finally, Stattic, the STAT-3 inhibitor and wortmannin were administered in both EMPA and DAPA groups to establish causal relationships in the mechanism of protection. EMPA, DAPA and ERTU at the SED led to similar SGLT-2 inhibition as inferred by the significant increase in glucose excretion. EMPA and DAPA but not ERTU reduced IS. EMPA preserved mitochondrial functionality in complex I&II linked oxidative phosphorylation. EMPA and DAPA treatment led to NF-kB, RISK, STAT-3 activation and the downstream apoptosis reduction coinciding with IS reduction. Stattic and wortmannin attenuated the cardioprotection afforded by EMPA and DAPA. Among several upstream mediators, fibroblast growth factor-2 (FGF-2) and caveolin-3 were increased by EMPA and DAPA treatment. ERTU reduced IS only when given at the double dose of the SED (20mg/kg/day). Short-term EMPA and DAPA, but not ERTU administration at the SED reduce IS in healthy non-diabetic mice. Cardioprotection is not correlated to SGLT-2 inhibition, is STAT-3 and PI3K dependent and associated with increased FGF-2 and Cav-3 expression.

Improved microvascular reactivity after aged garlic extract intake is not mediated by hydrogen sulfide in older adults at risk for cardiovascular disease: a randomized clinical trial.

This study aimed to investigate the effects of AGE on microvascular reactivity, systolic blood pressure (SBP), and diastolic blood pressure (DBP) in older individuals at high risk for cardiovascular disease (CVD). Urinary thiosulfate was also investigated as an indirect marker of endogenous hydrogen sulfide (H2S) synthesis. The study was conducted in a randomized, double-blind, crossover, and placebo-controlled way. Twenty-eight participants (14 male), 67 ± 6years old with CVD risk factors, ingested 2.4g of AGE or placebo (PLA). Near-infrared spectroscopy evaluated tissue oxygen saturation (StO2) during a vascular occlusion test (30s baseline, 5min occlusion, and 2min reperfusion). The upslope of StO2 signal after cuff release was calculated to measure microvascular reactivity. Urinary thiosulfate levels were measured using a high-performance liquid chromatography system. The upslope of StO2 was significantly faster after AGE (1.01 ± 0.37%s-1) intake compared to PLA (0.83 ± 0.35%s-1; P < 0.001; d = 0.50). Relative changes in Δ% SBP from pre- to post-AGE intake (-5.17 ± 5.77%) was significantly different compared to Δ% PLA (0.32 ± 5.99%; P = 0.001; d = 0.93). No significant changes in urinary thiosulfate concentrations were observed between interventions. Moreover, no significant gender effect in any parameter assessed was found. This study demonstrated that a single dose of AGE improved microvascular reactivity in older adults at risk of CVD despite such an effect was not linked with urinary thiosulfate levels. This trial was registered at clinicaltrials.gov as NCT04008693 (May 19, 2020).

Insulin reduces pyroptosis-induced inflammation by PDHA1 dephosphorylation-mediated NLRP3 activation during myocardial ischemia-reperfusion injury.

Previous studies proved that pyrin domain-containing protein 3 (NLRP3)-induced pyroptosis plays an important role in Myocardial ischemia-reperfusion injury (MIRI). Insulin can inhibit the activation of NLRP3 inflammasome, although the exact mechanism remains unclear. The aim of this study was to determine whether insulin reduces NLRP3-induced pyroptosis by regulating pyruvate dehydrogenase E1alpha subunit (PDHA1) dephosphorylation during MIRI. Rat hearts were subject to 30 min global ischemia followed by 60 min reperfusion, with or without 0.5 IU/L insulin. Myocardial ischemia-reperfusion injury was evaluated by measuring myocardial enzymes release, Cardiac hemodynamics, pathological changes, infarct size, and apoptosis rate. Cardiac aerobic glycolysis was evaluated by measuring ATP, lactic acid content, and pyruvate dehydrogenase complex (PDHc) activity in myocardial tissue. Recombinant adenoviral vectors for PDHA1 knockdown were constructed. Pyroptosis-related proteins were measured by Western blotting analysis, immunohistochemistry staining, and ELISA assay, respectively. It was found that insulin significantly reduced the area of myocardial infarction, apoptosis rate, and improved cardiac hemodynamics, pathological changes, energy metabolism. Insulin inhibits pyroptosis-induced inflammation during MIRI. Subsequently, Adeno-associated virus was used to knock down cardiac PDHA1 expression. Knockdown PDHA1 not only promoted the expression of NLRP3 but also blocked the inhibitory effect of insulin on NLRP3-mediated pyroptosis in MIRI. Results suggest that insulin protects against MIRI by regulating PDHA1 dephosphorylation, its mechanism is not only to improve myocardial energy metabolism but also to reduce the NLRP3-induced pyroptosis.

Spinal cord stimulation mitigates the myocardial ischemia induced sympathoexcitation by suppressing the spinal neural synchrony and intermediolateral nucleus hyperactivity

BackgroundMyocardial ischemia disrupts the cardiospinal neural network which leads to cardiac sudden death. Spinal cord stimulation (SCS) suppresses the sympathoexcitation caused by myocardial ischemia. However, how SCS modulates the spinal neural network is not known.HypothesisWe hypothesize that SCS decreases myocardial ischemia‐induced sympathoexcitation by suppressing the spinal neural synchrony and intermediolateral nucleus (IML) activity.Materials and MethodsEight Yorkshire pigs (50‐55 kg) with chronic left circumflex coronary artery myocardial infarction (MI) were anesthetized and underwent laminectomy and a sternotomy, 5 weeks post MI. Extracellular in‐vivo spinal neural recordings were done by using a 64 channel microelectrode array inserted at the T2 segment of the spinal cord. SCS was performed at 1 kHz, 0.03 ms, 90% motor threshold for 30 mins. Acute (3 min) left anterior descending coronary artery occlusion (LAD CAO) was performed pre‐ and 1 min post‐SCS to evaluate the impact of SCS on spinal neural network processing during myocardial ischemia. Dorsal horn (DH) and IML neural interactions, including normalized neuronal synchrony, activation recovery interval (ARI), and dispersion of repolarization (DOR) (markers of cardiac arrhythmogenesis) were evaluated during myocardial ischemia pre‐ vs. post‐ SCS.ResultsFiring rate of 1283 recorded spinal neurons increased during 3 mins LAD CAO and 1 min reperfusion pre‐SCS (baseline(BL): 0.516 ± 0.038 Hz; LAD CAO: 0.559 ± 0.038 Hz ,P&lt;0.001 vs. BL; reperfusion: 0.524 ± 0.037 Hz ,P&lt;0.001 vs. BL). SCS at 1kHz, mitigated the spinal neural network response to the LAD CAO (BL: 0.509 ± 0.039 Hz; LAD CAO: 0.486 ± 0.036 Hz; reperfusion: 0.489 ± 0.038 Hz, P&lt;0.001 vs. BL). This suppression of neural firing by SCS was observed in both IML (pre‐SCS: BL: 0.208 ± 0.033 Hz; LAD CAO: 0.308 ± 0.042Hz ,P&lt;0.001 vs. BL; reperfusion: 0.374 ± 0.048 Hz ,P&lt;0.001 vs. BL; post‐SCS: BL: 0.069 ± 0.010 Hz; LAD CAO: 0.080 ± 0.011Hz; reperfusion: 0.109 ± 0.018 Hz) and DH neurons (pre‐SCS: BL: 0.033 ± 0.003 Hz; LAD CAO: 0.039 ± 0.003 Hz ,P&lt;0.001 vs. BL; reperfusion: 0.041 ± 0.003 Hz ,P&lt;0.001 vs. BL; post‐SCS: BL: 0.036 ± 0.003 Hz; LAD CAO: 0.035 ± 0.003 Hz; reperfusion: 0.029 ± 0.002 Hz, P&lt;0.001 vs. BL). ARI shortening due to LAD CAO was mitigated by SCS (pre‐SCS: ‐43.200 ± 5.206 ms; post‐SCS: ‐29.810 ± 6.495 ms, P&lt; 0.05 vs. pre‐SCS). SCS decreased the LAD CAO induced augmented DOR (pre‐SCS: 2461.00 ± 350.40 ms2; post‐SCS: 1996.00 ± 302.00 ms2, P&lt;0.05 vs. pre‐SCS)ConclusionMyocardial ischemia induces sympathoexcitation by increasing the IML activity which is caused by activation of DH neurons and increased spinal neural synchrony. SCS mitigates the adverse effect of myocardial ischemia by suppressing the spinal neural synchrony and IML hyperactivity.