Min Of Ischemia Research Articles

Abstract 11471: Engineered Bacterial Sodium Channel Gene Therapy Prevents Contractile Deficit and Reduces Ventricular Arrhythmias in a Non-Human Primate Model of Myocardial Infarction

Introduction: Current therapies for myocardial infarction (MI) and sudden cardiac death are limited. The ability to enhance amplitudes of sodium current and calcium transient in cardiomyocytes could improve electrical synchrony and contractions of infarcted heart tissue to both prevent arrhythmias and improve pumping function. We hypothesized that adeno-associated virus (AAV) delivery of prokaryotic sodium channel (BacNa v ) can improve electrical and contractile function of infarcted hearts. Methods: MI in non-human primates (NHPs) was induced by 60-min ischemia / reperfusion injury. Animals were randomized to sham surgery (n=6) or MI followed by intramyocardial injection of AAV-BacNa v (n=6) or AAV-GFP (n=6) into the infarct area and border zone. Left ventricular (LV) function was assessed by echocardiogram at baseline, 1- and 4-week post-MI. Spontaneous arrhythmias were tracked by a subcutaneously implanted loop recorder during the 4-week study period. Results: At 4 weeks post-MI, BacNa v and GFP expression were detected in all treated animals (Fig. 1). Compared to sham animals, GFP-treated animals showed lower LV ejection fraction (LVEF, 37.4±3.4% vs 58.2±1.2%, p<0.0001) and increased LV end-systolic volume (3.8±0.57mL vs 2.1±0.25mL, p<0.05). BacNa v -treated animals showed significant increase in LVEF compared to the GFP group (48.3±3.4% vs 37.4±3.4%, p<0.05) and did not differ from the sham group. Compared to the GFP group, fewer BacNa v -treated animals displayed spontaneous arrhythmias (16.7% vs 100%, p<0.05) and had less arrhythmia episodes per animal (0.17±0.17 vs 2.17±0.6, p<0.01), while not differing in any measured electrophysiological parameters from the sham group. Conclusion: The AAV-mediated BacNa v expression in cardiomyocytes can alleviate cardiac contractile deficit and reduce ventricular arrhythmias in the setting of MI in an NHP model. BacNa v gene delivery may represent a promising therapeutic strategy for the treatment of MI.

Abstract 11926: Selective Inhibition of Rho Kinase 2 Limits No Re-Flow in Rat Hearts Following Ischemia/Reperfusion

Introduction: Myocardial infarction causes several types of injury to the myocardium including lethal cell injury and ‘no-reflow’ (NRF) /microvascular obstruction (MVO). Nonselective Rho Kinase (ROCK1/2) inhibitors such as Fasudil, ameliorate myocardial ischemia/reperfusion (I/R) injury but cause unwanted hypotension. Selective ROCK2 inhibitors (e.g.: KD025) are safe in clinical trials without causing haemodynamic compromise, however they have not been investigated in myocardial I/R. ROCK inhibitors prevent vascular smooth muscle cell (VSMC) contraction; such VSMC contraction/coronary spasm being features of ‘no-reflow’ (NRF) and microvascular obstruction (MVO). At present, there are limited therapies to improve ischemic MVO outcomes, and prognosis is poor. Hypothesis: Using a rat model, we hypothesised that, i) ROCK2 mRNA is expressed in myocardium and coronary vasculature and ii) The selective ROCK2 inhibitor KD025, would reduce infarct size (IS%) and NRF% (MVO) following I/R. Methods: RNA scope in-situ hybridisation was performed with a fluorescent, multiplex assay for ROCK1/2 & VSMC mRNA in myocardium and coronary vasculature. Male SD rats underwent in-vivo myocardial infarction with 30min ischemia, 180min reperfusion. 15min prior to reperfusion, the ROCK inhibitors Fasudil and KD025 or vehicle (DMSO) were administered i.p. For IS%, myocardium was stained with TTC, and regions not perfused with 1.5% Thioflavin S (NRF%), were visualised under UV light. Results: RNAscope confirmed the presence of ROCK2 mRNA within myocardium and VSMC of coronary arteries. Fasudil ( 10mg/kg ) vs control significantly reduced regional IS% ( 30.3 ± 4.4 vs 52.9 ± 3.8 , p=0.02, n=15 ) and area of NRF% ( 12.4 ± 2.8 vs 28.6 ± 2.2 , p=0.001, n=15 ). However, there was significant hypotension;- Mean BP (mmHg) (72±3.9) vs control (84±2.3, p=0.007 ). KD025 ( 100mg/kg) did not reduce IS%, but significantly reduced the area of NRF% vs control ( 18.4 ± 2.8 vs 28.6 ± 2.2, p=0.02, n=14 ) without hypotensive effect. Conclusions: Our results suggest that ROCK2 may be a prospective target in the management of coronary circulation reperfusion injury and ischemic MVO.

Buffer glucose adjustment affects myocardial function after ischemia-reperfusion in long-term diabetic rat isolated hearts.

Due to its comorbidities type 2 diabetes mellitus (T2DM) and hypertension, the Zucker Spontaneous Hypertensive Fatty (ZSF1) rat is a clinically relevant animal model when assessing ischemia-reperfusion (IR) injury. Most IR studies in hearts isolated from diabetic animals have been conducted at normal glucose concentrations, providing a different environment compared to in-vivo. We hypothesized IR injury to be attenuated in isolated hearts of diabetic ZSF1 rats when adjusting the Krebs-buffer (KB) to their in-vivo, i.e., elevated blood glucose (BG) levels. Diabetic and non-diabetic ZSF1 rats were anesthetized, hearts isolated and Langendorff-prepared. While standard KB was used for the non-diabetic and diabetic unadjusted groups, KB with glucose levels increased to each rat's prior BG level was used for the adjusted diabetic group. All hearts underwent 30 min ischemia and 120 min reperfusion. Diastolic contracture during ischemia and early reperfusion was delayed and temporarily attenuated in the adjusted compared to the unadjusted diabetic and the non-diabetic groups. The decrease in coronary flow on reperfusion was attenuated in diabetic animals. Left ventricular developed pressure and contractility were not different among the three groups. Infarct size was significantly lower in non-diabetic animals; buffer adjustment made no difference in diabetic animals. In our study, T2DM did not worsen myocardial function in ZSF1 rat isolated hearts. Since our results reveal that hearts with an adjusted glucose level exhibit an at least temporary improvement of function following IR, further studies should consider adapting glucose levels to create more realistic conditions in isolated, perfused hearts.

Cardioprotective effect of cinnamaldehyde pretreatment on ischemia/ reperfusion injury via inhibiting NLRP3 inflammasome activation and gasdermin D mediated cardiomyocyte pyroptosis

Cinnamaldehyde (CD) is one of the most important active compounds derived from Cinnamomum cassia and showed multiple biological activities. Accumulating evidence has shown that the nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome significantly contributes to sterile inflammatory response and gasdermin D (GSDMD)-mediated pyroptosis in myocardial ischemia/reperfusion injury (MI/RI). Whether CD has any influence on NLRP3 inflammasome activation and pyroptosis during myocardial I/R injury remains unknown. In the present study, we investigated the cardioprotective effect of CD via establishing the MI/RI rats’ model by ligating the left anterior descending coronary artery for 30 min ischemia followed by 120 min reperfusion. Sprague-Dawley rats were intragastrically administered with CD (45 and 90 mg/kg/d) or vehicle for 7 successive days before ligation of the coronary artery to evoke MI/RI. The results found that CD significantly improved cardiac diastolic function, decreased cardiac infarct size and myocardial injury enzymes, inhibited cardiomyocyte apoptosis, attenuated cardiac structure abnormality, and mitigated oxidative stress and inflammatory response. We also found that MI/RI activated the NLRP3 inflammasome as evidenced by the upregulation levels of NLRP3, pro-caspase-1, caspase-1, and ASC proteins and mRNA. Importantly, MI/RI could trigger cardiomyocyte pyroptosis by increased DNA fragmentation, membrane pore formation, and mitochondrial swelling as well as increased levels of pyroptosis-related proteins and mRNA, including GSDMD, IL-18, and IL-1β. As expected, all these deleterious alterations were reversed by CD pretreatment. Our findings demonstrated that CD showed an outstanding cardioprotective effect via inhibiting NLRP3 inflammasome activation and GSDMD-mediated cardiomyocyte pyroptosis, which has a promising application value and development prospect against myocardial I/R injury in the future.

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)

Perioperative outcomes following robot-assisted partial nephrectomy for complex renal masses: A Vattikuti Collective Quality Initiative database study.

Outcomes of robot-assisted partial nephrectomy (RAPN) depend on tumor complexity, surgeon experience and patient profile among other variables. We aimed to study the perioperative outcomes of RAPN for patients with complex renal masses using the Vattikuti Collective Quality Initiative (VCQI) database that allowed evaluation of multinational data. From the VCQI, we extracted data for all the patients who underwent RAPN with preoperative aspects and dimensions used for an anatomical (PADUA) score of ≥10. Multivariate logistic regression was conducted to ascertain predictors of trifecta (absence of complications, negative surgical margins, and warm ischemia times [WIT] <25 min or zero ischemia) outcomes. Of 3,801 patients, 514 with PADUA scores ≥10 were included. The median operative time, WIT, and blood loss were 173 (range 45-546) min, 21 (range 0-55) min, and 150 (range 50-3500) ml, respectively. Intraoperative complications and blood transfusions were reported in 2.1% and 6%, respectively. In 8.8% of the patients, postoperative complications were noted, and surgical margins were positive in 10.3% of the patients. Trifecta could be achieved in 60.7% of patients. Clinical tumor size, duration of surgery, WIT, and complication rates were significantly higher in the group with a high (12 or 13) PADUA score while the trifecta was significantly lower in this group (48.4%). On multivariate analysis, surgical approach (retroperitoneal vs. transperitoneal) and high PADUA score (12/13) were identified as predictors of the trifecta outcomes. RAPN may be a reasonable surgical option for patients with complex renal masses with acceptable perioperative outcomes.

Magnoflorine Attenuates Cerebral Ischemia-Induced Neuronal Injury via Autophagy/Sirt1/AMPK Signaling Pathway.

Ischemic stroke is a common cause of permanent disability worldwide. Magnoflorine has been discovered to have good antioxidation, immune regulation, and cardiovascular system protection functions. However, whether magnoflorine treatment protects against cerebral ischemic stroke and the mechanism of such protection remains unknown. Here, we investigated the effect of magnoflorine on the development of ischemic stroke disorder in rats. A middle cerebral artery occlusion (MCAO) model followed by 24 h reperfusion after 90 min ischemia was used. The rats were treated with magnoflorine (10 mg/kg or 20 mg/kg) for 15 consecutive days. The neurological deficit scores, cerebral infarct volume, and brain water content were measured. The neuronal density was determined using Nissl and NeuN staining. The oxidative stress levels were determined using commercial kits. Immunofluorescence staining of LC3 and western blot assay for LC3 and p62 were used to assess autophagy. Magnoflorine treatment significantly reduced the cerebral infarct volume and brain water content and improved the neurological deficit scores in the rat MCAO model. In addition, magnoflorine ameliorated neuronal injury and neuron density in the cortex of rats. Magnoflorine also prevented oxidative damage following ischemia, reflected by the decrement of nitric oxide and malondialdehyde and the increase of glutathione (GSH) and GSH peroxidase. Moreover, the fluorescence intensity of LC3 and the ratio of LC3-II to LC3-I were remarkably downregulated in ischemic rat administration of magnoflorine. Finally, the expression levels of p62, sirtuin 1 (Sirt1), and phosphorylated-adenosine monophosphate-activated protein kinase (AMPK) were upregulated with magnoflorine. Magnoflorine attenuated the cerebral ischemia-induced neuronal damage, which was possibly associated with antioxidative stress, suppression of autophagy, and activation of the Sirt1/AMPK pathway in the rats.

422.6: Effects of Argon Inhalation on Reducing Pulmonary Warm Ischemia-Reperfusion Injury in CLAWN Miniature Swine

Background: Ischemia-reperfusion injury (IRI) remains a major cause of early death in lung transplantation. In addition, warm ischemia of the donor organ is inevitable when transplanting from DCD donor, and it is important to develop new strategies for reducing warm IRI. The cytoprotective effects of chemically inert noble gases such as argon (Ar) have been demonstrated. Ar is particularly safe and cost-effective because it is non-anesthetic and the third most abundant gas in the atmosphere, suggesting higher safety levels and lower cost advantages. The inhibitory effect of IRI has been reported in small animal models of various organs, but there are conflicting reports on the effect in large animal models. In this study, we evaluated the cytoprotective effect and safety of Ar inhalation using large animal model of pulmonary warm IRI model. Methods: Ten CLAWN miniature swine were evenly divided into two groups (Ar-treated and control group). In both groups, warm ischemia was induced for 90 minutes by clamping the left bronchus, pulmonary artery and veins. Animals were inhaled with either 70% Ar (Ar-treated) or 70% nitrogen (control) in 30% oxygen for 360 minutes throughout the procedure. Lung function and structure were serially assessed via the ratio of partial pressure of oxygen to fractional inspired oxygen (pO2/FiO2) measured by both arterial blood and pulmonary vein (PV), chest X-ray (CXR) and lung biopsy, as well as the presence of side effects. Results: Ar inhalation dramatically decreased lung injury associated with ischemia and reperfusion without apparent adverse effects. In the control group, 90 minutes of warm ischemia resulted in a significant decrease in pO2/FiO2 by arterial blood, from 568 +/-12 mmHg before ischemia to 272 +/-39 mmHg 2 hours after reperfusion (p<0.05). In sharp contrast, animals in the Ar-treated group had no significant change in pO2/FiO2 by arterial blood despite 90-min ischemia (563 +/-18 mmHg before ischemia to 431 +/-49 mmHg 2 hours after reperfusion). Moreover, pO2/FiO2 by PV showed well-maintained lung function in the Ar-treated group (331 +/-40 vs. 186 +/-17 mmHg at 2 hours, p<0.05; 519 +/-19 vs. 292 +/-33 mmHg at 2 days after reperfusion, p<0.05). In addition, histological scores of lung biopsies (calculated based on four items: cellular infiltration, intralobular edema, fibrin exudation, and hemorrhage) were significantly better in the Ar-inhalation group for both 2-hour and 2-day biopsies after reperfusion. The evaluation of serum and biopsy samples suggested that anti-oxidant and anti-apoptosis were the main mechanisms of Ar inhalation. Conclusion: In this study, we demonstrated for the first time the inhibitory effect of perioperative Ar inhalation on pulmonary warm IRI in large animals. In order to apply this new therapy to clinical practice, further detailed analysis of the mechanism and evaluation using ischemic models including cold storage are required in the future.

422.7: Necroptosis Plays a Role in Acute Kidney Injury (AKI) and the Progression to Renal Fibrosis Following Ischemia Reperfusion Injury (IRI)

Introduction: In kidney transplantation, ischemia reperfusion injury (IRI), a type of acute kidney injury (AKI), is associated with poorer outcomes including delayed graft function, rejection and chronic kidney disease (CKD) with fibrosis. Therapies to reduce AKI and CKD following transplantation have been investigated but none has been translated into clinical practice. Here we examined necroptosis, a pathway of regulated necrosis that has been implicated in kidney IRI. Necroptosis is triggered by recruitment of the intracellular kinases RIPK1 and RIPK3 and activation of the pseudokinase MLKL. Active MLKL causes cell death by plasma membrane rupture, driving “necroinflammation”. RIPK1 and RIPK3 may also have a cell death-independent role in the development of kidney fibrosis. The aims of this study were to (1) investigate the role of necroptosis in a mouse IRI model of AKI, using knockout mice and small molecule inhibitors, and (2) determine whether inhibition of RIPK1 or RIPK3 after AKI interferes with the progression to CKD. Methods: AKI model: 10-12 week old male MLKL KO mice and wild type (WT) littermates underwent right nephrectomy followed by 18, 20, 22 or 24 min left kidney ischemia. Samples were collected at 24 h to assess renal injury, inflammation and necroptosis. In a separate experiment using the 18 min model, WT mice were treated either before or after IR with a RIPK1 inhibitor Nec-1s, a RIPK3 inhibitor GSK872, or vehicle.CKD-after-AKI model: This model was adapted from the AKI 18 min model by (i) omitting right nephrectomy, (ii) delaying the endpoint to 28 days, and (iii) extending the analyses to include renal fibrosis (Masson’s trichrome staining) and relevant gene expression. WT mice were treated with Nec-1s, GSK872 or vehicle daily on days 3-9. Results: In the AKI model, expression of necroptotic (RIPK1, RIPK3 and MLKL), kidney injury (KIM-1, NGAL) and inflammatory (IL-6, IL-1β, IL-33, TNFα) genes was significantly upregulated following moderate (18 min) and severe (20-24 min) ischemia. MLKL KO mice were protected from moderate but not severe IRI, with lower creatinine (µmol/L) (47.13±6.28 vs 80.78±10.45, p=0.007) and less tubular injury (%) (16.79±2.80 vs 26.95±3.68, p=0.048) compared to WT littermates. WT mice receiving Nec-1s before or after IR showed reduced creatinine and tubular injury compared to vehicle controls, whereas GSK872 was not protective.In the CKD-after-AKI model, vehicle-treated WT mice showed significant renal fibrosis at 28 days compared to sham (fibrosis score 28.68±1.25 vs 2.33±0.11, p=0.0025), with increased expression of RIPK1 (p=0.0079), RIPK3 (p=0.0025), and the pro-fibrotic genes TGFβ (p=0.0025), HIF1α (p=0.0025) and Col-I (p=0.0025). Inhibiting RIPK1 or RIPK3 on days 3-9 resulted in reduced fibrosis.Conclusion: Our data support a role for necroptosis in AKI and fibrosis following IR, and identify inhibition of the necroptosis pathway as a potential therapeutic strategy. NHMRC scholarship GNT1168307.

Intrarenal Arterial Transplantation of Dexmedetomidine Preconditioning Adipose Stem-Cell-Derived Microvesicles Confers Further Therapeutic Potential to Attenuate Renal Ischemia/Reperfusion Injury through miR-122-5p/Erythropoietin/Apoptosis Axis

Intravenous adipose mesenchymal stem cells (ADSCs) attenuate renal ischemia/reperfusion (IR) injury but with major drawbacks, including the lack of a specific homing effect after systemic infusion, cell trapping in the lung, and early cell death in the damaged microenvironment. We examined whether intrarenal arterial transplantation of dexmedetomidine (DEX) preconditioning ADSC-derived microvesicles (DEX-MVs) could promote further therapeutic potential to reduce renal IR injury. We evaluated the effect of DEX-MVs on NRK-52E cells migration, hypoxia/reoxygenation (H/R)-induced cell death, and reactive oxygen species (ROS) amount and renal IR model in rats. IR was established by bilateral 45 min ischemia followed by 4 h reperfusion. Intrarenal MVs or DEX-MVs were administered prior to ischemia. Renal oxidative stress, hemodynamics and function, western blot, immunohistochemistry, and tubular injury scores were determined. The miR-122-5p expression in kidneys was analyzed using microarrays and quantitative RT-PCR and its action target was predicted by TargetScan. DEX-MVs were more efficient than MVs to increase migration capability and to further decrease H/R-induced cell death and ROS level in NRK-52E cells. Consistently, DEX-MVs were better than MV in increasing CD44 expression, improving IR-depressed renal hemodynamics and renal erythropoietin expression, inhibiting IR-enhanced renal ROS level, tubular injury score, miR-122-5p expression, pNF-κB expression, Bax/caspase 3/poly(ADP-ribose) polymerase (PARP)-mediated apoptosis, blood urea nitrogen, and creatinine levels. The use of NRK-52E cells confirmed that miR-122-5p mimic via inhibiting erythropoietin expression exacerbated Bax-mediated apoptosis, whereas miR-122-5p inhibitor via upregulating erythropoietin and Bcl-2 expression reduced apoptosis. In summary, intrarenal arterial DEX-MV conferred further therapeutic potential to reduce renal IR injury through the miR-122-5p/erythropoietin/apoptosis axis.

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.

Methamphetamine use during the first or second half of pregnancy worsens cardiac ischemic injury in adult female offspring

There is growing evidence that methamphetamine use during pregnancy may produce detrimental cardiovascular effects in the adult offspring. Prior work demonstrated that chronic methamphetamine exposure throughout the gestational period causes adult female offspring to become hypersensitive to myocardial ischemic injury. The goal of the present study was to determine whether this methamphetamine-induced effect occurs early or late in the gestational period. Pregnant female rats were divided into 4 experimental groups. Groups 1 and 2 received subcutaneous injections of saline (group 1) or methamphetamine (5 mg/kg) (group 2) throughout the gestational period. Group 3 received methamphetamine injections on days 1-11 and saline on days 12-22, and group 4 received saline on days 1-11 and methamphetamine on days 12-22. Hearts were isolated from adult (8 weeks) female offspring and subjected to 30 min ischemia and 2 hours reperfusion on a Langendorff isolated heart apparatus. Contractile function was measured via an intraventricular balloon, and infarct size was measured by triphenyltetrazolium chloride staining. Infarcts were significantly larger in methamphetamine exposed offspring regardless of whether they had been exposed to methamphetamine during the first half or the second half of the gestational period. Prenatal exposure to methamphetamine had no effect on preischemic contractile function or postischemic recovery of contractile function. These data indicate that methamphetamine use during either the first half or second half of pregnancy increases susceptibility to myocardial infarction in adult female offspring. These data provide further evidence that prenatal exposure to methamphetamine may increase the risk of developing cardiovascular diseases during adulthood.

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.

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.

Protective effect of sacubitril/valsartan (Entresto) on kidney function and filtration barrier injury in a porcine model of partial nephrectomy.

Kidney surgery often includes organ ischaemia with a risk of acute kidney injury. The present study tested if treatment with the combined angiotensin II-angiotensin II receptor type 1 and neprilysin blocker Entresto (LCZ696, sacubitril/valsartan) protects filtration barrier and kidney function after ischaemia and partial nephrectomy (PN) in pigs. Single kidney glomerular filtration rate (GFR) by technetium-99m diethylene-triamine-pentaacetate clearance was validated (n=6). Next, four groups of pigs were followed for 15 days (n=24) after PN (one-third right kidney, 60min ischaemia) + Entresto (49/51mg/day; n=8), PN+vehicle (n=8), sham+Entresto (49/51mg/day; n=4) and sham+vehicle (n=4). GFR, diuresis and urinary albumin were measured at baseline and from each kidney after 15 days. The sum of single-kidney GFR (right 25± 6mL/min, left 31± 7mL/min) accounted for the total GFR (56± 14mL/min). Entresto had no effect on baseline blood pressure, p-creatinine, mid-regional pro-atrial natriuretic peptide (MR-proANP), heart rate and diuresis. After 15 days, Entresto increased GFR in the uninjured kidney (+23± 6mL/min, P<.05)and reduced albuminuria from both kidneys. In the sham group, plasma MR-proANP was not altered by Entresto; it increased to similar levels 2 h after surgery with and without Entresto. Fractional sodium excretion increased with Entresto. Kidney histology and kidney injury molecule-1 in cortex tissue were not different. In conclusion, Entresto protects the filtration barrier and increases the functional adaptive response of the uninjured kidney.

Polyethylene glycol capped gold nanoparticles ameliorate renal ischemia–reperfusion injury in diabetic mice through AMPK-Nrf2 signaling pathway

The aim of this study is to investigate the protective effect of polyethylene glycol capped gold nanoparticles (PEG-AuNPs) on renal ischemia–reperfusion injury (I/R)–induced acute kidney injury (AKI) in diabetic mice via the activation of adenosine 5′ monophosphate–activated protein kinase—nuclear factor erythroid-2-related factor-2 (AMPK-Nrf2) pathway. Diabetes was induced in male mice (12/group) by streptozotocin (50 mg/kg) for 5 consecutive days. After 4 weeks, the mice have intravenously received doses of PEG-AuNPs (40, 150, and 400 µg/kg body weight) for 3 consecutive days, and then animals were subjected to 30 min ischemia and 48 h reperfusion. Following the treatment with three different doses of PEG-AuNPs, the levels of blood urea nitrogen (BUN) and creatinine were reduced. Obvious reduction in renal tubular atrophy, glomerular damage, mitochondrial damage, and necrotic area were ultra-structurally detected, and renal interstitial inflammation and apoptosis were diminished. Moreover, PEG-AuNPs increased the recovering of damaged renal cells, suppressed significantly levels of malondialdehyde (MDA), downregulated significantly the level of inflammatory cytokines (TNF-α and IL-1β), and upregulated the AMPK-Nrf2 pathway. PEG-AuNPs exhibited a promising alternative therapeutic target for diabetic renal I/R-induced AKI through upregulation of AMPK/PI3K/AKT path which additionally stimulated Nrf2-regulated antioxidant enzymes in a dose-dependent manner.Graphical abstract

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.

The temporal impact of erythropoietin administration on mitochondrial function and dynamics in cardiac ischemia/reperfusion injury

Erythropoietin (EPO) has been shown to provide protection against ischemia/reperfusion (I/R) injury in various experimental models. However, clinical trials revealed unsatisfactory results when EPO was given to patients with myocardial infarction following reperfusion. The timing of EPO administration and its relation to mitochondrial function may largely involve in this controversy. We hypothesized that EPO given at different time points exert varying cardioprotective effects in terms of myocardial infarct size, left ventricular (LV) function, arrhythmia, apoptosis, mitochondrial function, and mitochondrial dynamics in rats with cardiac I/R injury. Male Wistar rats were subjected to either sham (n = 6) or cardiac I/R operation (n = 48). Rats undergoing cardiac I/R operation (30-min ischemia, followed by 120-min reperfusion) were allotted into 4 subgroups (n = 12/group): vehicle, EPO pretreatment, EPO given during ischemia, and EPO given at reperfusion. EPO was administered intravenously at 5000 unit/kg. Arrhythmia and LV function were monitored throughout the protocol. Next, the hearts were collected to determine infarct size, mitochondrial function, mitochondrial dynamics, gap junction protein, and apoptosis. Cardiac I/R promoted arrhythmias, LV dysfunction, infarct size expansion, apoptosis, mitochondrial dysfunction and increased mitochondrial fission. EPO given either before or during ischemia, but not at reperfusion, attenuated arrhythmia scores, LV dysfunction, infarct size, and apoptosis. Only EPO given either before or during ischemia alleviated mitochondrial swelling, mitochondrial depolarization, and reduced the levels of p-Drp1ser616/Drp1. Data from in vitro study also confirmed that EPO directly attenuated mitochondrial dysfunction in H9c2 cells subjected to hypoxia/reoxygenation. In conclusion, the EPO administration, either before or during ischemia, exerted cardioprotection against I/R injury by attenuating mitochondrial dynamic imbalance, mitochondrial dysfunction, and apoptosis, leading to reduced infarct size and improved LV function.

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.