LAD Reperfusion Research Articles

Effects of pirfenidone on scar size and ventricular remodeling after myocardial infarction: a preclinical study

Abstract Background An intense fibrotic response after myocardial infarction (MI) may lead to scar expansion and left ventricular (LV) remodeling, increasing the risk for ventricular arrhythmias and heart failure. Pirfenidone is a drug currently approved for the treatment of idiopathic pulmonary fibrosis, and reduces LV fibrosis in patients with heart failure and preserved ejection fraction. We aimed to characterize the different profiles or protein expression in the infarct zone in rats receiving or not receiving pirfenidone on top of standard therapy for MI. Methods Male Wistar rats were randomized to: sham procedure (group 1; n=13), reperfused MI induced by surgical ligation of the left anterior descending artery (LAD) for 45 minutes (group 2; n=17), reperfused MI plus standard therapy (aspirin, angiotensin-converting enzyme inhibitor, beta-blocker, and also mineralocorticoid receptor antagonist [MRA]) (group 3; n=17), reperfused MI plus pirfenidone alone (group 4; n=17), reperfused MI plus standard therapy and pirfenidone (group 5; n=17). Rats surviving LAD ligation and reperfusion underwent cardiac magnetic resonance (CMR) after 72 hours and 30 days from MI, and were sacrificed the day after the second CMR exam. The primary endpoint was the change in late gadolinium enhancement (LGE) mass as a percentage of LV mass. Results Forty-six rats survived until the second CMR. Percent changes in LV end-diastolic and end-systolic volume (p=0.370 and 0.423, respectively), ejection fraction (p=0.236) and mass (p=0.815) did not differ significantly between rats on pirfenidone plus standard therapy compared with those receiving standard therapy alone. The decrease in LGE mass was significantly greater in rats on pirfenidone plus standard therapy vs. those on standard therapy alone: -22.0% (interquartile range, -37.1 to -5.7, range -51.1 to +37.5) vs. +1.9% (-7.7 to +12.5, range -19.8 to +56.5), p=0.029. Conclusions Pirfenidone had additive effects to standard therapy for MI (including an MRA) in reducing LV fibrosis in a rat model of reperfused MI, while it did not further improve LV function, volumes or mass.

Mitochondrial succinate transport as a novel therapeutic target for cardiac ischemia/reperfusion injury

Abstract Background The accumulation of the mitochondrial metabolite succinate during ischemia and its rapid oxidation by succinate dehydrogenase (SDH) upon reperfusion drives ischemia/reperfusion injury (IRI) in myocardial infarction. Succinate oxidation leads to the production of reactive oxygen species (ROS) by reverse electron transport (RET) at mitochondrial complex I, orchestrating a cascade of events leading to cardiomyocyte death. Modulating succinate metabolism, such as SDH inhibition, is a promising therapeutic strategy. During ischemia, the mitochondria produced succinate is transported into the cytosol, enabling the accumulation of a large succinate pool. On reperfusion, the succinate is transported back into mitochondria and oxidised by SDH, thus we hypothesised that targeting succinate mitochondrial transport could be a novel therapeutic target to prevent cardiac IRI. Purpose To assess the role of succinate mitochondrial transport as a novel therapeutic target to prevent cardiac IRI. Methods Isolated heart mitochondria were used to assess mitochondrial respiration by Oroboros, RET-ROS production by Amplex Red and butylmalonate delivery. Cultured H9C2 myoblasts and isolated primary adult cardiomyocytes from C57BL/6J mice were used to assess butylmalonate uptake and delivery from esters, as well as their impact on succinate accumulation and oxidation in vitro. LAD ligation and reperfusion in C57BL/6J mice was used as an in vivo MI model. Metabolite levels were measured by LC-MS/MS. Results Butylmalonate, an inhibitor of the mitochondrial dicarboxylate carrier (DIC), blocks succinate-dependent respiration and RET-ROS production in isolated mitochondria. However, butylmalonate entry into cells was inefficient and does not impact infarct size after IRI in vivo. We developed novel butylmalonate esters to improve butylmalonate delivery to cardiomyocytes in vivo. We found that diacetoxymethyl butylmalonate (DAB) effectively delivered butylmalonate and could prevent succinate accumulation and oxidation in vitro. DAB could also prevent succinate-mediated respiration and RET-ROS at lower concentrations than butylmalonate alone. Finally, DAB reduced acute infarct size in vivo when administered at reperfusion. Conclusions Blocking succinate transport back into mitochondria at reperfusion prevents the damage in IRI by preventing the oxidation of succinate by SDH and downstream RET-ROS production. Therefore, targeting the mitochondrial transport of succinate may be a promising novel strategy in preventing cardiac IRI.

Spinal neuromodulation mitigates myocardial ischemia-induced sympathoexcitation by suppressing the intermediolateral nucleus hyperactivity and spinal neural synchrony.

Myocardial ischemia disrupts the cardio-spinal neural network that controls the cardiac sympathetic preganglionic neurons, leading to sympathoexcitation and ventricular tachyarrhythmias (VTs). Spinal cord stimulation (SCS) is capable of suppressing the sympathoexcitation caused by myocardial ischemia. However, how SCS modulates the spinal neural network is not fully known. In this pre-clinical study, we investigated the impact of SCS on the spinal neural network in mitigating myocardial ischemia-induced sympathoexcitation and arrhythmogenicity. Ten Yorkshire pigs with left circumflex coronary artery (LCX) occlusion-induced chronic myocardial infarction (MI) were anesthetized and underwent laminectomy and a sternotomy at 4-5 weeks post-MI. The activation recovery interval (ARI) and dispersion of repolarization (DOR) were analyzed to evaluate the extent of sympathoexcitation and arrhythmogenicity during the left anterior descending coronary artery (LAD) ischemia. Extracellular in vivo and in situ spinal dorsal horn (DH) and intermediolateral column (IML) neural recordings were performed using a multichannel microelectrode array inserted at the T2-T3 segment of the spinal cord. SCS was performed for 30 min at 1 kHz, 0.03 ms, 90% motor threshold. LAD ischemia was induced pre- and 1 min post-SCS to investigate how SCS modulates spinal neural network processing of myocardial ischemia. DH and IML neural interactions, including neuronal synchrony as well as cardiac sympathoexcitation and arrhythmogenicity markers were evaluated during myocardial ischemia pre- vs. post-SCS. ARI shortening in the ischemic region and global DOR augmentation due to LAD ischemia was mitigated by SCS. Neural firing response of ischemia-sensitive neurons during LAD ischemia and reperfusion was blunted by SCS. Further, SCS showed a similar effect in suppressing the firing response of IML and DH neurons during LAD ischemia. SCS exhibited a similar suppressive impact on the mechanical, nociceptive and multimodal ischemia sensitive neurons. The LAD ischemia and reperfusion-induced augmentation in neuronal synchrony between DH-DH and DH-IML pairs of neurons were mitigated by the SCS. These results suggest that SCS is decreasing the sympathoexcitation and arrhythmogenicity by suppressing the interactions between the spinal DH and IML neurons and activity of IML preganglionic sympathetic neurons.

P251 EFFECTS OF PIRFENIDONE ON SCAR SIZE AND VENTRICULAR REMODELING AFTER MYOCARDIAL INFARCTION: A PRECLINICAL STUDY

Abstract Background An intense fibrotic response after myocardial infarction (MI) may lead to scar expansion and left ventricular (LV) remodeling, increasing the risk for ventricular arrhythmias and heart failure. Pirfenidone is a drug currently approved for the treatment of idiopathic pulmonary fibrosis, and reduces LV fibrosis in patients with heart failure and preserved ejection fraction. We aimed to characterize the different profiles or protein expression in the infarct zone in rats receiving or not receiving pirfenidone on top of standard therapy for MI. Methods Male Wistar rats were randomized to: sham procedure (group 1; n=13), reperfused MI induced by surgical ligation of the left anterior descending artery (LAD) for 45 minutes (group 2; n=17), reperfused MI plus standard therapy (aspirin, angiotensin–converting enzyme inhibitor, beta–blocker, and also mineralocorticoid receptor antagonist [MRA]) (group 3; n=17), reperfused MI plus pirfenidone alone (group 4; n=17), reperfused MI plus standard therapy and pirfenidone (group 5; n=17). Rats surviving LAD ligation and reperfusion underwent cardiac magnetic resonance (CMR) after 72 hours and 30 days from MI, and were sacrificed the day after the second CMR exam. The primary endpoint was the change in late gadolinium enhancement (LGE) mass as a percentage of LV mass. Results Forty–six rats survived until the second CMR. Percent changes in LV end–diastolic and end–systolic volume (p=0.370 and 0.423, respectively), ejection fraction (p=0.236) and mass (p=0.815) did not differ significantly between rats on pirfenidone plus standard therapy compared with those receiving standard therapy alone. The decrease in LGE mass was significantly greater in rats on pirfenidone plus standard therapy vs. those on standard therapy alone: –22.0% (interquartile range, –37.1 to –5.7, range –51.1 to +37.5) vs. +1.9% (–7.7 to +12.5, range –19.8 to +56.5), p=0.029. Conclusions Pirfenidone had additive effects to standard therapy for MI (including an MRA) in reducing LV fibrosis in a rat model of reperfused MI, while it did not further improve LV function, volumes or mass.

267 EFFECTS OF PIRFENIDONE ON SCAR SIZE AND VENTRICULAR REMODELING AFTER MYOCARDIAL INFARCTION: A PRECLINICAL STUDY

Abstract Background An intense fibrotic response after myocardial infarction (MI) may lead to scar expansion and left ventricular (LV) remodeling, increasing the risk for ventricular arrhythmias and heart failure. Pirfenidone is a drug currently approved for the treatment of idiopathic pulmonary fibrosis, and reduces LV fibrosis in patients with heart failure and preserved ejection fraction. We aimed to characterize the different profiles or protein expression in the infarct zone in rats receiving or not receiving pirfenidone on top of standard therapy for MI. Methods Male Wistar rats were randomized to: sham procedure (group 1; n=13), reperfused MI induced by surgical ligation of the left anterior descending artery (LAD) for 45 minutes (group 2; n=17), reperfused MI plus standard therapy (aspirin, angiotensin-converting enzyme inhibitor, beta-blocker, and also mineralocorticoid receptor antagonist [MRA]) (group 3; n=17), reperfused MI plus pirfenidone alone (group 4; n=17), reperfused MI plus standard therapy and pirfenidone (group 5; n=17). Rats surviving LAD ligation and reperfusion underwent cardiac magnetic resonance (CMR) after 72 hours and 30 days from MI, and were sacrificed the day after the second CMR exam. The primary endpoint was the change in late gadolinium enhancement (LGE) mass as a percentage of LV mass. Results Forty-six rats survived until the second CMR. Percent changes in LV end-diastolic and end-systolic volume (p=0.370 and 0.423, respectively), ejection fraction (p=0.236) and mass (p=0.815) did not differ significantly between rats on pirfenidone plus standard therapy compared with those receiving standard therapy alone. The decrease in LGE mass was significantly greater in rats on pirfenidone plus standard therapy vs. those on standard therapy alone: -22.0% (interquartile range, -37.1 to -5.7, range -51.1 to +37.5) vs. +1.9% (-7.7 to +12.5, range -19.8 to +56.5), p=0.029. Conclusions Pirfenidone had additive effects to standard therapy for MI (including an MRA) in reducing LV fibrosis in a rat model of reperfused MI, while it did not further improve LV function, volumes or mass.

Preclinical trial of a MAP4K4 inhibitor to reduce infarct size in the pig: does cardioprotection in human stem cell-derived myocytes predict success in large mammals?

Reducing infarct size (IS) by interfering with mechanisms for cardiomyocyte death remains an elusive goal. DMX-5804, a selective inhibitor of the stress-activated kinase MAP4K4, suppresses cell death in mouse myocardial infarction (MI), human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs), and 3D human engineered heart tissue, whose fidelity to human biology is hoped to strengthen the route to clinical success. Here, DMX-10001, a soluble, rapidly cleaved pro-drug of DMX-5804, was developed for i.v. testing in large-mammal MI. Following pharmacodynamic studies, a randomized, blinded efficacy study was performed in swine subjected to LAD balloon occlusion (60 min) and reperfusion (24 h). Thirty-six animals were enrolled; 12 were excluded by pre-defined criteria, death before infusion, or technical issues. DMX-10001 was begun 20 min before reperfusion (30 min, 60 mg/kg/h; 23.5 h, 17 mg/kg/h). At all times tested, beginning 30 min after the start of infusion, DMX-5804 concentrations exceeded > fivefold the levels that rescued hPSC-CMs and reduced IS in mice after oral dosing with DMX-5804 itself. No significant reduction occurred in IS or no-reflow corrected for the area at ischemic risk, even though DMX-10001 reduced IS, expressed in grams or % of LV mass, by 27%. In summary, a rapidly cleaved pro-drug of DMX-5804 failed to reduce IS in large-mammal MI, despite exceeding the concentrations for proven success in both mice and hPSC-CMs.

Quantification of cardiac kinetic energy and its changes during transmural myocardial ischemia assessed by multi-dimensional seismocardiography

Abstract Background The third law of Newton implies that if one body exerts a force on a second body, the latter exerts a force equal in magnitude and opposite in direction on the first body. This reaction to heart and blood motion elicits low frequency vibrations transmitted to the chest surface. Multi-dimensional seismocardiography (SCG) assesses these linear and rotational accelerations by means of micro-accelerometers and micro-gyroscopes. From the linear and rotational acceleration signals, kinetic energy (KE) and its temporal integration (iK) can be computed as scalar parameters, both in a linear (iKLin) and in a rotational (iKRot) dimension. Evidence demonstrate that acute myocardial ischemia (AMI) profoundly affects the SCG waveform amplitude and that unidimensional SCG is accurate for the determination of acute coronary syndrome (ACS). Whether and how AMI affects multidimensional SCG signals is unknwon. Purpose AMI upon left anterior descendent coronary artery (LAD) occlusion markedly depresses iKLin and iKRot. Methods The protocol was in accordance with the principles of laboratory animal care. After 20 minutes of steady-state, anesthetized Landrance pigs underwent a percutaneous mid-LAD occlusion by means of intra-coronary balloon inflation for 90 min., followed by deflation and reperfusion (RE). SCG was recorded at baseline (BSL), upon LAD occlusion (AMIt0), after 60 min of occlusion (AMIt60), at the onset of LAD reperfusion (REt0) and after 60 min of recovery (REt60). iKLin and iKRot were computed from the amplitudes of SCG waveforms. iKLin and iKRot were calculated during the systolic and diastolic phases of the cardiac cycle (iKSys and iKDia, respectively). Firedman's ANOVA was used to test the trend of the above variables throughout the protocol, followed by Wilcoxon paired tests. Data are presented as median [IR]. Results Of the 21 investigated pigs, 3 died before protocol termination as a result of AMI complications and 4 had ruled out during the signal processing because of technical reasons. Considering the rotational variables, when compared to baseline, iKRot_Sys decreased by 17% both at AMIt0 (p=0.05) and AMIt60 (p=0.02); by 38% and 49% at REt0 and t60 (respectively, p<0.001). Similarly, iKRot_Dia decreased by 64% and 59% at AMIt0 and AMIt60 (respectively, p<0.02), and by 74% at REt0 and REt60 (both p=0.002). Considering the linear variables, iKLin_Sys decreased by 19% and 34% at AMIt0 and AMIt60 (respectively, p<0.04), by 39% and 53% at REt0 and REt60 (respectively, p<0.02). iKLin_Dia decreased by 42% at AMIt60 (p=0.03) and by 67% at REt60 (p=0.005) compared to baseline but did not change at the other time points (figure 1). Conclusions AMI depresses profoundly cardiac kinetic energy as recorded by multidimensional SCG. Thus, this nonintrusive and operator independent tool may prove useful in the detection of AMI and provide considerable opportunities for remote non-intrusive monitoring of cardiac functions. Funding Acknowledgement Type of funding source: Private grant(s) and/or Sponsorship. Main funding source(s): Fonds Erasme, FNRS_Fonds National Recherche Scientifique, Fonds pour la Chirurgie Cardiaque, PRODEXA

The cardioprotective effects of carvedilol on ischemia and reperfusion injury by AMPK signaling pathway

Carvedilol, a third generation beta blocker, is in clinical use for heart failure patients. However, besides adrenergic receptor blockade, the pharmacological effects of carvedilol on cardiomyocytes remain unknown. AMP-activated protein kinase (AMPK) is an emerging target recognized for heart failure treatment. The mechanical properties and intracellular Ca2+ properties were measured in isolated cardiomyocyte contractile functions in response to ischemic stress. Treatment of cardiomyocytes with carvedilol augmented phosphorylation of AMPK and downstream acetyl CoA carboxylase (ACC), and ameliorated hypoxia-induced impairment in maximal velocity of shortening (+dL/dt) and relengthening (-dL/dt), and the impaired peak height and peak shortening (PS) amplitude caused by hypoxia. Carvedilol treatment improved calcium homeostasis with rescuing the peak Ca2+ signal, the maximum rate of Ca2+ change during contraction (+dF/dt) and the maximum rate of Ca2+ change during relaxation (-dF/dt) under hypoxia conditions. In mouse hearts perfused ex vivo with carvedilol, the function of post-ischemia left ventricle was improved and an augmentation in myocardial glucose uptake and glucose oxidation, and inhibition of fatty acid oxidation during ischemia and reperfusion. The protective effect of carvedilol was further supported in an in vivo regional ischemia model by ligation of left anterior descending coronary artery (LAD), mice treated with carvedilol followed by LAD occlusion and reperfusion showed significant size reduction in infarcted myocardium and improved cardiac functions. Therefore, Carvedilol as a clinical drug can modulate cardiac AMPK signaling pathway to reduce ischemic insults by ischemia and reperfusion.

Effect of Telmisartan in amelioration inflammatory Responses Induced by Myocardial Ischemia/Reperfusion Injury IN male Mice

Background: Myocardial ischemia–reperfusion represents a clinically relevant problem associated with thrombolysis, angioplasty and coronary bypass surgery. Angiotensin II may contribute to reperfusion injury by increasing oxidative stress and inflammatory factors . Ang II exerts most of its effects via AT1Rs. Apoptosis of cardiomyocytes may further be influenced by Ang II. Objective: This study was undertaken to investigate the potential role of Telmisartan in amelioration of myocardial I/R injury induced by ligation of coronary artery in mouse model. Materials & method: Adult male Swiss-albino mice were randomized into 4 equal groups. Group (1) sham group: Mice underwent the same anesthetic and surgical procedure as the active control group except ligation of LAD coronary artery.Group( 2) active control group: Mice were subjected to regional ischemia for 30 min by ligation of LAD coronary artery and reperfusion for 2 hours .Group( 3) control vehicle group (1): Mice in this group injected with DMSO (vehicle for Telmisartan ) via IP route & underwent Myocardial ischemia for 30 minutes by ligation of (LAD) coronary artery & reperfusion for 2 hr . Group (4)Telmisartan treated group : Mice pretreated with Telmisartan 0.5mg/kg i.p 30 minutesbefore ligation of LAD coronary artery. Results: Compared with the sham group, Levels of TNF-α & IL-1β, IL-6,caspase 3 and plasma level of cardiac troponin I increased in control group (p<0.001).Levels of Bcl2 decreased in control group(p<0.001). Histologically ,All mice in control group showed a significant (p<0.001) cardiac injury. Telmisartan significantly counteract the increase in myocardium level of TNF-α, IL-1B,IL-6,caspase 3 ,plasma cTnI (P < 0.001). Furthermore, the Telmisartan significantly increased in myocardium level of Bcl2. Histological analysis revealed that both Telmisartan markedly reduced (P < 0.001) the severity of cardiac injury in the mice underwent LAD ligation procedure . Conclusion:The results of the present study reveal that Telmisartan may ameliorate myocardial I/R injury in Male Mice via interfering with inflammatory reactions & apoptosis which induced by I/R injury .

Percutaneous cardiac support during myocardial infarction drastically reduces mortality: perspectives from a swine model.

Background/AimsAcute myocardial infarction (AMI) with cardiogenic shock (CS) remains the leading cause of in-hospital death in acute coronary syndromes. In the AMI-CS pig model we tested the efficacy of temporary percutaneous cardiorespiratory assist device (PCRA) in rescuing the failing heart and reducing early mortality.MethodsIn open-chest pigs we induced AMI by proximal left anterior descending coronary artery (LAD) ligation. Eight animals without PCRA (C group) were compared with 12 animals otherwise treated with PCRA (T group), starting approximately at 60 minutes post-occlusion and lasting 120–180 minutes. In 3 animals of the T group, regional myocardial oxygen content was also imaged by two-dimensional near infrared spectroscopy (2D-NIRS) with and without PCRA, before and after LAD reperfusion.ResultsAll animals without PCRA died despite unrelenting resuscitation maneuvers (120 minutes average survival time). Conversely, animals treated with PCRA showed a reduction in life-threatening arrhythmia and maintenance of aortic pressure, allowing interruption of PCRA in all cases early in the experiments, with sound hemodynamics at the end of the observation period. During LAD occlusion, NIRS showed severe de-oxygenation of the LAD territory that improved with PCRA. After PCRA suspension and LAD reperfusion, the residual de-oxygenated area proved to be smaller than the initial risk area.ConclusionsIn AMI, PCRA initiated during advanced CS drastically reduced early mortality from 100% to 0% in a 4–5 hour observation period. PCRA promoted oxygenation of the ischemic area during LAD occlusion. Results support the use of PCRA as first line of treatment in AMI-CS, improving myocardial rescue and short-term survival.

Role of Epinephrine and Extracorporeal Membrane Oxygenation in the Management of Ischemic Refractory Ventricular Fibrillation: A Randomized Trial in Pigs

xtracorporeal membrane oxygenation (ECMO) is used in cardiopulmonary resuscitation (CPR) of refractory cardiac arrest. We used a 2×2 study design to compare ECMO versus CPR and epinephrine versus placebo in a porcine model of ischemic refractory ventricular fibrillation (VF). Pigs underwent 5 minutes of untreated VF, 10 minutes of CPR, and were randomized to receive epinephrine versus placebo for another 35 minutes. Animals were further randomized to LAD reperfusion at minute 45 with ongoing CPR versus veno-arterial ECMO cannulation at minute 45 of CPR and subsequent LAD reperfusion. Four-hour survival was improved with ECMO while epinephrine showed no effect.

Adipocyte derived factors affect cardioprotective programs initiated by short term high fat feeding

RationaleHigh fat mediated cardioprotection is a form of protection that utilizes short‐term high fat diet to decrease infarct size post ischemia/reperfusion injury. This protection is dependent on the increase of autophagy related proteins and the activation of NF‐κB mediated gene transcription. This study addresses the hypothesis that signaling from adipose tissue can remotely influence the cardioprotective markers associated with high fat mediated (HFD) cardioprotection.MethodsHormonal activation of cardiac receptors was assessed through treatment with murine recombinant adiponectin and AdipoRon peptide. Isolation of adipocyte derived exosomes was also preformed from 3T3‐L1 (ATCC® CL‐173) cells during pre differentiation, mitotic expansion, and post differentiation states. Viability of cells pretreated with each of the previously described agents was then assessed with Hoescht 33342 and propridium iodide staining before and after the administration of an oxygen glucose assay (OGD) protocol. Autophagy measurements were performed with LC‐3A/B and Beclin‐1 western blot, and the Premo Autohpagy Sensor® transduction for each treatment condition. In vivo studies were preformed in wild type C57/B6J and AdipoQ‐KO murine models, with in vivo LAD occlusion (30m) and reperfusion (24h).ConclusionExosomes isolated from 3 phases during the differentiation of 3T3‐L1 (ATCC® CL‐173) cells into mature adipocytes did not increase the viability of cells after OGD treatment. Post OGD protocol, AdipoRon (25uM) treatment improved viability and cell survival relative to vehicle treated H9c2(2‐1) (ATCC® CRL‐1446) controls. Total serum levels of adiponectin were increased by an average of 15% in 24h HFD wild type animals relative to control fed wild type animals, and AdipoQ‐KO animals lacked cardioprotection at previously reported cardioprotective high fat fed time points. Future studies will focus on the assessment of in vivo hormonal signaling during high fat mediated cardioprotection.

The possible cardioprotective effect of valsartan in the amelioration of myocardial I/R injury induced by ligation of coronary artery in a rat model

Ischemia/reperfusion injury describes the experimentally and clinically prevalent finding that tissue ischemia with inadequate oxygen followed by successful reperfusion initiates a wide and complex array of inflammatory responses that may aggravate local injury as well as induce impairment of remote organ function by mechanisms that involves oxidative stress, inflammation, and apoptosis. In this study, we investigated the cardio protective effects of Valsartan in regional ischemia/ reperfusion injury. adult male Albino rats were randomized into 4 equal groups. Group (1) sham group: rats underwent the same anesthetic and surgical procedure as the control group except ligation of LAD coronary artery , Group( 2) control group: rats subjected to regional ischemia for 25 min by ligation of LAD coronary artery and reperfusion for 2 hours ,Group( 3) control vehicle group: rats received (normal saline) vehicle of valsartan via I.P injection and subjected to regional ischemia for 25 min and reperfusion for 2 hours , Group( 4) valsartan treated group : rats pretreated with valsartan 10mg/kg I.P 30 minutes before ligation of LAD coronary artery then subjected to the surgical procedure with ligation of LAD for 30 minutes followed by 120 minutes reperfusion. At the end of reperfusion, Heart was divided into two parts, the apex for histopathology and the remaining part used for determination of tissue TNF-α,and IL-6 It has been found that Valsartan treated group showed significant reduction (P˂0.05) in TNFα with respect to the control groups.Histopathology study revealed that the treatment with Valsartan significantly (P˂0.05) improved cardiac injury as compared with control groups, we concluded that Valsartan reduces inflammatory reaction associated with ischemia/reperfusion injury induced by LAD ligation in addition to its reduction for cardiac injury induced by ischemia reperfusion.

MicroRNA-155 aggravates ischemia-reperfusion injury by modulation of inflammatory cell recruitment and the respiratory oxidative burst.

The inflammatory sequelae of ischemia-reperfusion injury (IRI) are a major causal factor of tissue injury in various clinical settings. MicroRNAs (miRs) are short, non-coding RNAs, which regulate protein expression. Here, we investigated the role of miR-155 in IR-related tissue injury. Quantifying microRNA-expression levels in a human muscle tissue after IRI, we found miR-155 expression to be significantly increased and to correlate with the increased expression of TNF-α, IL-1β, CD105, and Caspase3 as well as with leukocyte infiltration. The direct miR-155 target gene SOCS-1 was downregulated. In a mouse model of myocardial infarction, temporary LAD ligation and reperfusion injury resulted in a smaller area of necrosis in miR-155-/- animals compared to wildtype animals. To investigate the underlying mechanisms, we evaluated the effect of miR-155 on inflammatory cell recruitment by intravital microscopy and on the generation of reactive oxygen species (ROS) of macrophages. Our intravital imaging results demonstrated a decreased recruitment of inflammatory cells in miR-155-/- animals during IRI. The generation of ROS in leukocytic cells of miR-155-/- animals was also reduced. RNA silencing of the direct miR-155 target gene SOCS-1 abrogated this effect. In conclusion, miR-155 aggravates the inflammatory response, leukocyte infiltration and tissue damage in IRI via modulation of SOCS-1-dependent generation of ROS. MiR-155 is thus a potential target for the treatment or prevention of IRI.

Abstract 15983: Increased Mitochondrial Reactive Oxygen Species Lead to Deleterious JNK Signaling and Ischemia-Reperfusion Injury in AMPK-Inactivated Hearts

AMP-activated kinase (AMPK) is a key stress responsive kinase that regulates cellular adaptation to metabolic stress. Inactivation of AMPK in “kinase-dead” (KD) mice increases myocardial damage following ischemia-reperfusion (IR). We have also shown decreased mitochondrial respiration and increased mitochondrial reactive oxygen species (ROS) production and susceptibility to mitochondrial transition pore opening in KD hearts following IR. The aim of this study was to establish the importance of mitochondrial ROS production and downstream deleterious signaling to mediate tissue damage in absence of active AMPK in the heart. Detoxification of mitochondrial ROS requires conversion of hydrogen peroxide to water, therefore, we studied the effect of transgenic expression of mitochondrial catalase (MCAT) in wild type (WT) and KD mice. MCAT prevents mitochondrial hydrogen peroxide production independent of mitochondrial energy production. Myocardial necrosis was assessed in vitro after global ischemia-reperfusion and in vivo after LAD ligation and reperfusion. Mitogen activated protein kinase kinase 4 (MKK4) and downstream c-Jun terminal kinase (JNK) expression and phosphorylation was assessed in vivo. Increased necrosis in KD hearts following mild global ischemia (15 minutes) - reperfusion (10 minutes) in vitro, was prevented by expression of MCAT (WT vs. KD 17.8±4.1 vs. 50±4.1%, p<0.05 and MCAT-WT vs. MCAT-KD 16.9±4.8 vs. 29.3±4.4%, n.s., and factorial p<0.05). Total JNK protein was not increased in WT and KD hearts with or without expression of MCAT. After coronary occlusion in vivo, KD mice showed increased cardiac activation of MKK4/JNK pathway (p<0.05) as well as greater myocardial necrosis (p<0.05). MCAT expression prevented the excessive cardiac JNK activation observed during IR in KD mice in vivo. Inhibition of JNK with SP600125 (10μM) during in vivo IR also resulted in a significant decrease in necrosis in KD hearts (WT vs. KD 9.1±0.7 vs. 28.2±2.9%, p<0.05 and WT vs. KD with SP600125 8.5±0.5 vs. 10.2±1.1%, n.s. and factorial p<0.05, percentage of equivalent area at risk). Thus, AMPK activation during IR prevents excess mitochondrial reactive oxygen production and consequent JNK signaling, thus protecting against myocardial injury.

The effect of Simvastatin in attenuation of Myocardial Ischemia/ Reperfusion injury

Background: Myocardial ischemia–reperfusion represents a clinically relevant problem associated with thrombolysis, angioplasty and coronary bypass surgery. Injury of myocardium due to ischemia–reperfusion includes cardiac contractile dysfunction, arrhythmias as well as irreversible myocytes damage.Objective: This study was undertaken to investigate the potential role of simvastatin in amelioration of myocardial I/R injury induced by ligation of coronary artery in a rat model. Materials & methods: adult male Swiss Albino rats were randomized into 4 equal groups. Group (I) sham group: rats underwent the same anesthetic and surgical procedure as the control group except ligation of LAD coronary artery , Group(II) control group: rats subjected to regional ischemia for 25 min by ligation of LAD coronary artery and reperfusion for 2 hours ,Group(III ) control vehicle group: rats received vehicle of simvastatin (normal saline) via I.P injection and subjected to regional ischemia for 25 min by ligation of LAD coronary artery and reperfusion for 2 hours , Group(IV) simvastatin treated group :rats pretreated with simvastatin 1mg/kg i.p 1 hr before ligation of LAD coronary artery. At the end of experiment (2 hr of reperfusion), the heart was harvested and the part of the heart just below the site of ligation was divided into two parts ,the upper part was homogenized for the measurement of interleukin-1β (IL-1β) and the lower part of tissues fixed in 10% formalin and embedded in paraffin the sections were stained with hematoxylin and eosin (H&E) then used for histopathological study .Results: Compared with the sham group, levels of myocardial IL-1β, were increased (p<0.001) in the control group. Simvastatin significantly counteract the increase in myocardium level of IL-1B (P < 0.001). Histological analysis revealed that simvastatin markedly reduced (P < 0.001) the severity of cardiac injury in the rats underwent LAD ligation procedure .Conclusions:The results of the present study reveal that simvastatin may ameliorate myocardial I/R injury in rats via interfering with inflammatory responses which induced by I/R injury.

Expressions of eNOS3 and Ve-Cadherin in Microvascular Endothelium at 7-Day of Reperfused Acute Myocardial Infarction

To investigate the expressions of eNOS3 and Ve-cadherin at the first week of reperfused acute myocardial infarction (AMI). 16 of mini-swines (20 to 30 Kg) were randomly assigned to the sham-operated group and the AMI group. Pathologic myocardial tissue was collected at 7-day of LAD reperfusion and assessed by immunofluorescence and laser co-focus microscope scan, in-situ hybridization, real-time quantitative polymerase chain reaction and western blot. At 7-day of reperfusion, the eNOS3 mRNA and protein expressions in the infarcted and marginal areas were lower than those in the normal area and sham-operated area (all P<0.05), similarly the infarcted and marginal areas had lower Ve-cadherin mRNA expression than the normal area and the sham-operated area (all P<0.05), and Ve-cadherin protein expression was lower in the infarcted area compared with the marginal area, the normal area and the sham-operated area (all P<0.05). Low expressions of eNOS3 and Ve-cadherin in the salvaged sub-healthy microvascular endothelium of infarcted and marginal areas suggest that endothelial system is impaired at 7-day of reperfused acute myocardial infarction.

Angiogenesis and Vasculogenesis at 7-Day of Reperfused Acute Myocardial Infarction

Abstract Objectives This study is to investigate the angiogenesis and vasculogenesis at the first week of reperfused acute myocardial infarction (AMI).Methods 16 of mini-swines (20 to 30 Kg) were randomly assigned to the sham-operated group and the AMI group. The acute myocardial infarction and reperfusion model was created and the pig tail catheter was performed to monitor hemodynamics before left anterior descending coronary artery (LAD) occlusion, 90 min of LAD occlusion and 120 min of LAD reperfusion. Pathologic myocardial tissue was collected at 7-day of LAD reperfusion and further assessed by immunochemistry, dual immunochemistry, in-situ hybridization, real-time quantitative polymerase chain reaction and western blot. Results The infarcted area had higher FLK1 mRNA expression than sham-operated area and the normal area (all P&lt;0.05), and the infarcted and marginal areas showed higher CD146 protein expression than the sham-operated area (all P&lt;0.05), but the microvessel density (CD31 positive expression of microvessels/HP) was not significantly different between the infarcted area and the sham-operated area (8.92±3.05 vs 6.43±1.54) at 7-day of reperfused acute myocardial infarction (P&gt;0.05). Conclusions FLK1 and CD146 expression significantly increase in the infarcted and marginal areas, and the microvessel density is not significantly different between the infarcted area and the sham-operated area, suggesting that angiogenesis and vasculogenesis in the infarcted area appear to high frequency of increase in 7-day of reperfused myocardial infarction.

Limitation of Angiography to Identify the Culprit Plaque in Acute Myocardial Infarction With Coronary Total Occlusion: Utility of Coronary Plaque Temperature Measurement to Identify the Culprit Plaque

The purpose of this study was to test the hypothesis that the maximal temperature (Tmax) site, as measured by thermal wire, coincides with the culprit plaque by intravascular ultrasound (IVUS) in patients with acute myocardial infarction (AMI). Subsequent thrombosis developing to the proximal region from the site of plaque rupture or erosion can potentially complicate the ability of coronary angiography to identify the accurate culprit plaque in patients with coronary total occlusion. In 45 consecutive patients with a first anterior AMI, the Tmax site by thermal wire and the culprit plaque by IVUS were evaluated in the left anterior descending coronary artery (LAD). Twenty-five patients had LAD total occlusion, and the remaining 20 had LAD reperfusion. In both groups of patients, the Tmax site was significantly more distal to the angiographically most stenotic site or occlusive site (reperfusion: mean distance [MD] = 1.1 mm distal, 95% confidence interval [CI] 0.3 to 1.9 mm, p = 0.01; total occlusion: MD = 8.8 mm distal, 95% CI 8.0 to 9.6 mm, p < 0.0001). The culprit plaques by IVUS approximately coincided with those by angiography or thermal wire in patients with reperfusion. However, the angiographic occlusive site was significantly more proximal to the culprit plaque by IVUS (MD = 9.2 mm, 95% CI 7.9 to 10.6 mm, p < 0.0001), but the Tmax site coincided with the culprit plaque by IVUS (MD = 0.3 mm distal, 95% CI 0.3 mm proximal to 1.0 mm distal, p = 0.293) in patients with total occlusion. Temperature measurement of coronary plaque enables accurate localization of the culprit plaque in AMI with coronary total occlusion.

Correlation of limb and myocardial ischemia postconditioning with acute myocardial reperfusion injury

We tested the hypothesis that remote postconditioning, which is induced by a single 5-min episode of femoral artery occlusion and reperfusion applied just before the onset of coronary artery reperfusion, protects the myocardium from reperfusion injury. In anesthetized open-chest rabbits, the left anterior descending artery (LAD) was occluded for 30 min and reperfused for 3 hrs. All rabbits were randomly divided into four groups (n = 10 in each group): (1) CONTROL: LAD occlusion and reperfusion only, with no other intervention; (2) Myocardial ischemic preconditioning (Pre-con): Three cycles of myocardial ischemia (5 min) and reperfusion (5 min) preceded the index ischemia/reperfusion protocol; (3) Myocardial ischemic postconditionng (Post-con): After 30 min of LAD occlusion, reperfusion was initiated for 30 s followed by 30 s reocclusion. Three cycles of myocardial ischemia (30 s) and reperfusion (30 s) followed the index ischemia/reperfusion protocol. (4) Remote postconditioning (Re Post-con): After 24 min of LAD occlusion, the femoral artery was occluded for 5 min and released for 1 min before 3 hrs of LAD reperfusion. Myocardial infarct size and tissue myeloperoxidase (MPO) activity were determined at the end of the experiment. Plasma creatine kinase (CK) activity and malondialdehyde (MDA) activity were measured at baseline, the end of ischemia, and after 3 hrs of reperfusion respectively. Myocardial infarct size was significantly reduced in Pre-con (15.5% +/- 1.7%, P < 0.01), Post-con (16.15% +/- 2.05%, P < 0.01) and Re Post-con (17.11% +/- 1.70%, P < 0.01) groups as compared to CONTROL (31.46% +/- 1.28%). Results were confirmed by plasma CK activity (in Re Post-con 18.0 IU/g +/- 1.6 IU/g vs. CONTROL 45.6 IU/g +/- 5.5 IU/g). Plasma MDA was significantly less at 3 hrs of reperfusion in Pre-con (2.12 micromol/ml +/- 0.30 micromol/ml, P < 0.01), Post-con (2.17 micromol/ml +/- 0.24 micromol/ml, P < 0.01) and Re Post-con (2.16 micromol/ml +/- 0.33 micromol/ml, P < 0.01) than that in CONTROL (3.49 micromol/ml +/- 0.32 micromol/ml). Neutrophil accumulation (MPO activity) in the area at risk was less in Pre-con (1.43 U/100 g +/- 0.32 U/100 g, P < 0.01), Post-con (2.26 U/100 g +/- 0.28 U/100 g, P < 0.01) and Re Post-con (2.45 U/100 g +/- 0.28 U/100 g, P < 0.01) than that in CONTROL (5.44 U/100 g +/- 0.46 U/100 g). Remote limb postconditioing applied just before the onset of coronary artery reperfusion provides potent myocardial infarct size reduction, which is similar to the cardioprotective effect of myocardial postconditioning exerted during the first minutes of coronary reperfusion. The potential mechanism of this inter-organ remote postconditioning phenomenon might be associated with decreasing the injury caused by oxygen free radicals and strengthening the action of antioxidation.