LAD Coronary Artery Ligation Research Articles

Abstract We043: MYCN is a regulator of cardiomyocyte proliferation and regeneration in the mammalian heart

The identification of new factors and mechanisms that can promote the repair and regeneration of the adult injured mammalian heart are necessary as the adult heart has limited ability to repair itself. We identified MYCN as a downstream effector of the sonic hedgehog (SHH) signaling cascade during cardiomyocyte proliferation in the neonatal heart. Although the protooncogene, MYCN, has been shown to be an essential factor during embryonic heart development, its role in regenerating the adult heart has not been established. Here, we define MYCN as a critical regulator of cardiomyocyte (CM) proliferation and regeneration using genetic mouse models and in vivo modified RNA (modRNA) delivery. In these studies, we conditionally deleted Mycn in the neonatal heart (P0). Immunohistochemical (IHC) analysis of P3 neonatal hearts demonstrated significantly (p < 0.05) decreased expression of Ki67 and pH3 in the absence of Mycn compared to control (no 4-OHT) hearts. This observation was further validated in isolated CMs and neonatal P7 hearts pulsed with EdU which demonstrated a significant decrease in EdU+ CMs in the absence of Mycn compared to control. Single-nucleus RNA-sequencing results from control and Mycn CKO P4 ventricles revealed a severe depletion of the proliferative and regenerative cardiomyocyte population in the Mycn CKO hearts. We then ligated the left anterior descending coronary artery (LAD) to produce an ischemic cardiac injury in control and Mycn CKO P2 neonatal mice. Echocardiography one-month following the LAD surgeries demonstrated that the Mycn CKO failed to recover baseline cardiac function compared to their control counterparts who achieved complete recovery. RNAseq analysis of control and Mycn CKO hearts 1 month following LAD ligations demonstrated that muscle developmental pathways were significantly downregulated in the Mycn CKO group compared to the control. We then delivered either GFP (control) or Mycn modRNA intramuscularly into the border zone following LAD coronary artery ligation surgeries in adult (2-6 month old) mice. We observed that the Mycn modRNA treated mice had significantly improved cardiac function at one month and two months following injury/delivery compared to controls. These results identify MYCN as an important regulator of CM proliferation and heart regeneration.

Abstract 17134: Mycn is a Regulator of Cardiomyocyte Proliferation and Regeneration in the Mammalian Heart

The adult mammalian heart has limited regeneration in response to injury. Therefore, we focused on defining new factors and mechanisms that could enhance repair and regeneration in the adult mouse heart. MYCN is a protooncogene that regulates embryonic heart development. We identified MYCN as a downstream effector of the sonic hedgehog (SHH) signaling cascade during cardiomyocyte proliferation. However, its role in heart regeneration is unknown and warrants further investigation. Here, we describe a novel role for MYCN as a regulator of cardiomyocyte (CM) proliferation and regeneration using genetic mouse models and in vivo modified RNA (modRNA) delivery. By crossing Mycn floxed mice with the Myh6 -MerCreMer mice ( Mycn CKO), we conditionally deleted Mycn in the neonatal heart following administration of 4-hydroxy-tamoxifen (4-OHT) starting at P0. Successful deletion of Mycn was demonstrated by qPCR analysis of P2 isolated CMs. Immunohistochemical (IHC) analyses of P3 neonatal hearts in the absence of MYCN demonstrated a significant decrease in Ki67 expression and pH3 detection. This observation was validated in isolated P7 CMs and hearts pulsed with EdU which demonstrated a significant decrease in EdU+ CMs in the absence of MYCN compared to control. We then performed LAD coronary artery ligation surgeries in control and Mycn CKO P2 neonatal mice. Echocardiography one month following LAD coronary artery ligations demonstrated that the Mycn CKO failed to recover baseline cardiac function compared to their control counterparts who achieved complete recovery measured by ejection fraction (EF) and fractional shortening (FS). RNAseq analysis of control and Mycn CKO hearts one month following LAD ligations demonstrated that muscle developmental pathways were significantly downregulated in the Mycn CKO group compared to the control. We then delivered either GFP (control) or Mycn modRNA intramuscularly into the border zone following LAD coronary artery ligation injuries in adult (2-6 month old) mice. We observed that the Mycn modRNA treated mice had significantly improved cardiac function at one month and two months following injury/delivery compared to controls. These results identify MYCN as a regulator of CM proliferation and heart regeneration.

Abstract P3138: Potentiation Mechanism Of SR-Ca2+ Uptake During Myocardial Infarction; A Molecular Explanation Of Ca2+ Arrhythmogenicity Of Purkinje Fibers In The Ischemic Heart.

Experimental and clinical evidence attributes lethal VTs and VFs associated with ischemic myocardial infarctions ( MI ) to abnormal electric signaling in the Purkinje network. In cardiac Purkinje cells ( Pcells ), spontaneous Ca 2+ releases from the sarcoplasmic reticulum ( SR-CaR ) generate depolarizing Ca 2+ waves which, in turn, can mediate DADs and non-driven APs. Increase of SR-CaR was reported in Pcells of dog heart with 48Hrs MI. An augmentation of SR-Ca 2+ uptake ( SR-CaU ) accelerates SR-Ca 2+ recycling and is a possible cause of larger (pro-arrhythmic) SR-CaR in Pcells of ischemic heart. We tested this hypothesis in canine and porcine models of acute MI.Pcells and heart tissues were prepared 48Hrs after LAD coronary artery ligation. Intracellular Ca 2+ activity was captured by confocal microscopy. SR-CaU was studied by analyzing Purkinje-typical Ca 2+ events, namely Ca 2+ -sparks, peripheral Ca 2+ -wavelets and cell-wide Ca 2+ -waves ( CWW s). Relevance of our analysis was tested with a Purkinje-specific model of Ca 2+ mobilization. Protein/gene expressions of SR-Ca 2+ pump isoforms were studied by immunofluorescence, WBs, and qPCR in dog and pig infarcted tissues.We found that (1) individual Ca 2+ release sites in MI Pcells fired sparks at 61% larger frequency, (2) the duration of wavelet Ca 2+ decay was reduced by 25% due to 37% acceleration of cytosolic Ca 2+ removal (time constant: 156±21ms (Normal; n=10) vs 98±9ms (MI; n=16; P<0.01)), (3) wavelet amplitude was increased by 50%, and (4) larger waves (CWWs) showed similar alterations. Numerical replication of MI wavelets succeeded with 52% increase of Ca 2+ uptake rate in the model of normal wavelets. Our results are consistent with an augmentation of SR-CaU taking place in Pcells shortly after the onset of MI. Molecular analyses done in the same MI model in pig showed that post-MI increase of SR-CaU results from the substitution of cardiac isoform SERCA2a by the ubiquitous and more potent SERCA2b in the infarcted cardiac tissues including Purkinje fibers. Our study reveals, for the first time, the molecular foundation of Purkinje arrhythmogenicity in ischemic heart and highlights novel targets for diagnosis, prevention, and treatment of risk of sudden cardiac death associated with ischemic MIs.

Investigation of Cardioprotective Effect of 2, 4-thiazolidinedione Derivative Using HFD-STZ Fed Rats

Abstract: Background: Myocardial infarction is a 2nd most important origin of universal death and after coronary revascularization, cardiovascular events occurs further a lot in subjects through DM: 3.5- as well as 2-fold fatality ranks correspondingly behind percutaneous coronary intervention or coronary bypass graft surgery. Objectives: The aim of an attendance research was to discover usefulness of 2, 4- thiazolidinedione derivatives lying on diabetic MI and HFD-STZ diabetic rats. Materials and Methods: In in-vivo investigation, Type-2 diabetes was elicited by higher fatty meal plus little dosage for streptozotocin and MI was provoked by LAD Coronary artery ligation model. The rats were divided into a variety of groups, including treatment groups (Composite An epalrestat, Composite B pioglitazone). And after 15 days of treatment a variety of factors were evaluated, i.e. serum glucose, total cholesterol, serum triglycerides, lipid profile antioxidant enzymes, ECG analysis, and Histopathological examination of heart were done. Results: In in-vivo investigation, levels of serum glucose, triglycerides, LDL level, LDH level, CK-MB, Troponin I, malondialdehyde (MDA), total cholesterol, infarct area, were considerably decreased in composite A and B treated animals. Whereas the HDL level, antioxidant enzymes, had drastically risen in composite, treated animals balanced to disease control rats. In ECG analysis ST elevation was restored in drug treated animals. Composite treated heart indicated mild congestion and inflammation in histopathological examination. Homogenate of drug treated animals indicated less apoptotic signals and nearer to normal animals. Conclusion: The treatment with Composite A and Composite B notably ameliorated the alterations in MI in a dose dependant manner. Keywords: 2, 4-Thiazolidinedione, Myocardial infarction, Diabetic MI, Type-2 diabetes, LAD, Macrovascular complication.

Ginsenoside-Mc1 reduces ischemia/reperfusion-induced cardiac arrhythmias through activating JAK2/STAT3 pathway and attenuating oxidative/endoplasmic reticulum stress in hyperlipidemic rats

Abstract Objectives Patients suffering from myocardial ischemia-reperfusion (IR) injuries usually have varying degrees of negatively-affecting comorbidities like hyperlipidemia. We evaluated the preconditioning effect of ginsenoside-Mc1 on reperfusion injury-induced myocardial arrhythmias, along with cardiac oxidative stress, endoplasmic reticulum stress protein expression, and histological damage in hyperlipidemic rats, and further, explore the role of JAK2/STAT3 activity. Methods Thirty-five Sprague–Dawley rats fed a high-fat diet for eight weeks. Ginsenoside-Mc1 (10 mg/dL, IP) was administered to hyperlipidemic rats daily for one month before IR injury. IR injury was induced by 35 min LAD coronary artery ligation and subsequent 60-min reperfusion. A selective JAK2 inhibitor (AG490) was injected before IR injury. Electrocardiography was recorded and myocardial arrhythmias (ventricular premature complexes, tachycardia, and fibrillation) were evaluated in the reperfusion phase according to Lambeth convention. Hematoxylin-Eosin staining, spectrophotometry, and Western blotting techniques were employed to measure the endpoints. Results IR injury in hyperlipidemic rats significantly increased the reperfusion-induced myocardial arrhythmias counts, timing, incidence, and severity. The expression of proteins of endoplasmic reticulum stress (p-PERK, p-eIF2α, CHOP), and oxidative stress marker malondialdehyde were significantly upregulated following IR induction, whereas antioxidant superoxide-dismutase and JAK2/STAT3 proteins expression significantly reduced, as compared with untreated-hyperlipidemic rats. Administration of ginsenoside-Mc1 to hyperlipidemic rats significantly corrected the arrhythmogenic feature of IR injury, reduced phosphorylation and expression of PERK, eIF2α, CHOP, and improved oxidative stress and histological changes. Interestingly, inhibition of the JAK2/STAT3 pathway via AG490 significantly abolished ginsenoside-Mc1-induced cardioprotection. Conclusions Taken together, ginsenoside-Mc1 exerts substantial anti-arrhythmogenic influences against myocardial IR injury in hyperlipidemic rats through activation of JAK2/STAT3 pathway and subsequent reduction of oxidative/endoplasmic reticulum stress.

Mdivi-1 alleviates cardiac fibrosis post myocardial infarction at infarcted border zone, possibly via inhibition of Drp1-Activated mitochondrial fission and oxidative stress

Mitochondrial division inhibitor 1(Mdivi-1) has been shown to play a beneficial role in a variety of diseases, mainly by inhibiting Drp1-mediated mitochondrial fission. The effects of Mdivi-1 on cardiac fibrosis at infarcted border zone area and its possible mechanism remain unclear. This study aimed to investigate the effects of Mdivi-1 on reactive cardiac fibrosis and cardiac function post myocardial infarction and its potential mechanisms. Mice were randomly divided into six groups (n = 9 for each group): Sham; Mdivi-1; MI 7d; MI 14d; MI 28d; MI 28d + Mdivi-1. The MI model was induced by ligation of LAD coronary artery. Mdivi-1 (1 mg/kg) was administered to mice every other day at a time from the second day until the sacrifice of the mice (total 14 injection of Mdivi-1). In vitro experiments, the effect of Mdivi-1 on TGF-β1-induced fibrosis-related pathophysiological changes of fibroblasts was examined in NIH3T3 cells. We found that Mdivi-1 significantly attenuated fibroblast activation, collagen production and fibrosis at infarcted border zone after MI, improved impaired heart function. Mechanistically, we observed that Mdivi-1 reduced the protein expression of P-Drp1-S616 and abnormal mitochondrial fission of cardiac fibroblasts in the infarcted border zone area. In addition, we found that the effects of Mdivi-1 partially relied on increasing the expression of Hmox1 and inhibiting oxidative stress. In conclusion, Mdivi-1 could attenuate cardiac fibrosis at infarcted border zone and improve impaired heart function partially through attenuation of Drp1-mediated mitochondrial fission. Moreover, inhibition of oxidative stress, which is possible due to the up-regulation of Hmox1, may be another potential mechanism of action of Mdivi-1.

Applying Vasopressin-Pre-Conditioned Human Adipose Mesenchymal Stem Cells Improves Heart Condition after Transplantation into Infarcted Myocardium.

Transplantation of H-AdMSCs may improve heart function after MI. AVP is a neurohypophyseal hormone that reduces cardiovascular damage. This study investigated the role of AVP preconditioning in the survival of MSCs and their effect on myocardial repair in the MI rats. H-AMSCs were isolated and incubated for 3 days. The expression of oxytocin and vasopressin receptors was evaluated by Real-time-PCR. Forty male Wistar rats were divided into 4 groups: control, sham, ASC and AVP-ASC. Ischemia was established by ligation of LAD coronary artery. Electrocardiography, fibrosis, angiogenesis, and apoptosis in myocardium were determined after 7 days. Results showed that preconditioned MSCs significantly increased cardiac function when compared with group that received non-preconditioned MSCs. This was associated with significantly reduced fibrosis, increased vascular density, and decreased resident myocyte apoptosis. Results indicate that AVP preconditioned MSCs can be consider a novel approach to management of MI.

SH2B1 protects cardiomyocytes from ischemia/reperfusion injury via the activation of the PI3K/AKT pathway

BackgroundApoptosis, reactive oxidative stress (ROS) and inflammation act as the pivotal pathogenesis of myocardial ischemia/reperfusion (I/R) injury (MIRI). Our prior study and other investigation have demonstrated the participations of src homology 2 (SH2) B adaptor protein 1 (SH2B1) in ischemic injury and cardiac hypertrophy; whereas, the involvements of SH2B1 in MIRI and underlying mechanisms are completely unknown. MethodIn present study, MIRI model in vivo was induced by 30 min of ligation of LAD coronary artery and 24 h of reperfusion, and primary cultured cardiomyocytes were challenged with 2 h of hypoxia followed by 4 h of reoxygenation (H/R) to mimic MIRI in vitro. Adenovirus encoding for SH2B1 or GFP were pre-transfected into myocardium prior to MIRI both in vivo and in vitro. The myocardial damage, cardiac function, apoptosis, ROS and inflammation were evaluated systematically. Immunofluorescence staining and western blotting were alternatively performed to detect protein expression. ResultsThe results exhibited that H/R or I/R significantly reduced SH2B1 in cardiomyocytes, followed by impaired cell survival and function, which were strongly reversed after the adenovirus-mediated SH2B1 up-regulation. Meanwhile, I/R- and H/R-elevated inflammation, apoptosis and ROS were also alleviated by SH2B1 up-regulation. A mechanistic study suggested that the protective contributions of SH2B1 on H/R-suffered cardiomyocytes were based on the activation of the PI3K/AKT pathway. The abolishment of the PI3K/AKT via a pharmacological inhibitor (LY294002) repressed anti-H/R capabilities of SH2B1. ConclusionTherefore, SH2B1 prevents cardiomyocytes from inflammation, apoptosis and ROS in MIRI partially through the PI3K/AKT-dependent avenues. It may provide a novel therapeutic target for the treatment of MIRI.

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 .

140 Heterozygous deletion of pmca1 might serve a protective role in the heart following myocardial infarction

IntroductionCoronary artery disease (CAD) and its main consequence-myocardial infarction (MI) are the UK’s biggest killers. Most of these deaths occur either acutely post-MI or chronically as a result of pathological...

Radiofrequency Renal Denervation Improves Vascular Reactivity in a Rodent Model of Heart Failure

BackgroundWe have previously reported that radiofrequency (RF) renal denervation (RDN) exerts cardioprotective effects in the setting of myocardial ischemia‐reperfusion (MI‐R) injury. The protective actions of RF‐RDN in MI‐R injury are related in part to attenuated oxidative stress and increased nitric oxide (NO) bioavailability. More recently we have demonstrated that RF‐RDN protects the failing heart independent of reductions in blood pressure. The mechanisms by which RF‐RDN affords these benefits have not been fully described, including the effects on vascular function.MethodsMale Wistar‐Kyoto (WKY) rats and spontaneously hypertensive rats (SHR) at 20 weeks of age were subjected to 45 minutes of transient LAD coronary artery ligation followed by reperfusion for 12 weeks. Four weeks following reperfusion animals were subjected to either bilateral RF‐RDN or a sham procedure (SHAM) with n=12 in all groups. At 12 weeks after reperfusion, thoracic aorta was harvested and vascular reactivity was evaluated. Vasoactivity was assessed for the endothelium‐dependent vasodilators acetylcholine (ACh, 0.1nM–10mM) and the calcium ionophore A23187 (A23187, 10nM‐1mM). In addition, vascular reactivity to the endothelium‐independent vasodilator sodium nitroprusside (SNP, 0.1nM–10 mM) was investigated. The effects of RF‐RDN therapy on cardiac structure and function were also determined via biweekly 2‐D serial echocardiography.ResultsRF‐RDN significantly improved cardiovascular function as reflected in improved left ventricle (LV) ejection fraction at 12 weeks following reperfusion in both SHRs (46.8 ± 2.4% vs. 30.4 ± 2.5%, p<0.01) and WKYs (50.4 ± 3.0% vs 35.4 ± 2.7%; p<0.05). RF‐RDN treatment significantly improved endothelium mediated vasorelaxation in both hypertensive and normotensive animals as reflected in ACh EC50 (SHR: 98.8 ± 19.2 nM vs. 349.6 ± 25.0 nM, p<0.0001; WKY: 10.03 ± 1.32 nM vs. 44.97 ± 12.03 nM, p<0.01). Similar improvements were seen in smooth muscle mediated vasorelaxation due to SNP (SHR: 29.4 ± 4.2 nM vs. 145.3 ± 14.2 nM, p<0.0001; WKY: 11.68 ± 1.28 nM vs 39.68 ± 7.47 nM, p<0.01). RF‐RDN also improved vasorelaxation to the direct endothelium‐dependent vasodilator, A23187, in hypertensive animals (SHR: 10.8 ± 1.3 nM vs. 23.2 ± 2.7 nM, p<0.001) but not normotensive animals. We did not observe any significant reductions in blood pressure in either the SHR or WKY rats following RF‐RDN at anytime during the experimental protocol.ConclusionRF‐RDN significantly improved LV function in the setting of heart failure independent of blood pressure reductions. Therapy improved vascular function suggesting that the cardiovascular benefits afforded by RF‐RDN may be partially due to improved vascular function. The improved vascular reactivity and cardiac function observed following RF‐RDN may be a result of reduced oxidative stress and increased NO bioavailability. Experiments are currently underway to evaluate this possibility. Our results reveal a novel mechanism related to the beneficial effects of RF‐RDN in heart failure.

‐LCZ696, the First‐in‐Class Angiotensin Receptor Neprilysin Inhibitor, Improves Vascular Reactivity in Hypertensive Rats in the Setting of Heart Failure ‐

BackgroundLCZ696 combines an angiotensin receptor blocker (ARB) with a neprilysin inhibitor (NEPi). This inhibits RAAS over activity while simultaneously augmenting the natriuretic peptide system. LCZ696 has been shown to improve cardiovascular function in the setting of heart failure, however, its effects on vascular function have not been defined.MethodsMale spontaneously hypertensive rats (SHRs) at 18–20 weeks of age were subjected to 45 minutes of transient LAD coronary artery ligation followed by reperfusion for 8 weeks to induce heart failure with reduced ejection fraction. At 4 weeks post‐reperfusion animals received daily oral therapy of either vehicle (VEH, n=14) or 68 mg/kg LCZ696 (LCZ, n=14). Myocardial infarct size as measured by plasma troponin levels at 4 hours post‐reperfusion were similar between the study groups. After 4 weeks of therapy, thoracic aorta was collected and evaluated for vascular reactivity in a tissue chamber apparatus connected to force transducers. Vasoreactivity was assessed for the endothelium‐dependent vasodilator acetylcholine (ACh, 0.1nM–10mM) and the direct smooth muscle vasodilator sodium nitroprusside (SNP, 0.1nM–10 mM). In addition, we also studied the vasoconstriction response to the endothelial nitric oxide synthase (eNOS) inhibitor N(omega)‐nitro‐L‐arginine methyl ester (L‐NAME, 30mM). Effects on heart function were also determined via 2‐D echocardiography and measurements of left ventricle (LV) pressures.ResultsLCZ improved LV function with a significant reduction in LV end‐diastolic pressure (3.31 ± 0.39 mmHg vs 7.86 ± 1.10mmHg; p<0.01). LCZ also attenuated the decrease in ejection fraction at 8 weeks post reperfusion (−6.1 ± 2.5% vs −14.3 ± 3.6%; p=0.06). LCZ treatment significantly improved vascular reactivity in the dose response relaxation curve for both ACh and SNP. There was a significant improvement in maximal relaxation in response to ACh (76.1 ± 3.6% vs 52.9 ± 3.6%; p<0.0001) as well as ACh EC50 (11.0 ± 4.0 nM vs 97.3 ± 9.8 nM, p<0.0001) suggesting augmented endothelium mediated vasorelaxation. Similar changes were seen in SNP maximal relaxation (90.9 ± 3.5% vs 69.1 ± 5.9%; p<0.01) and SNP EC50 (26.8 ± 4.4nM vs 125.7 ± 13.4nM, p<0.0001) suggesting improvements in vascular smooth muscle function. Lastly, there was an increased contraction in response to administration of L‐NAME following maximal relaxation by ACh suggesting the potential role of nitric oxide in the improvements seen in vascular reactivity (60 ± 6mg vs 40 ± 6mg, p=0.06).ConclusionLCZ696 significantly improved LV function during heart failure in the setting of hypertension with concomitant improvements in vascular reactivity. These improvements in vascular reactivity are likely a result of increased activity of eNOS and increased bioavailability of nitric oxide (NO). Studies are currently underway to evaluate eNOS function and NO levels following LCZ administration in heart failure.

Pretreatment with low-dose gadolinium chloride attenuates myocardial ischemia/reperfusion injury in rats.

We have shown that low-dose gadolinium chloride (GdCl3) abolishes arachidonic acid (AA)-induced increase of cytoplasmic Ca(2+), which is known to play a crucial role in myocardial ischemia/reperfusion (I/R) injury. The present study sought to determine whether low-dose GdCl3 pretreatment protected rat myocardium against I/R injury in vitro and in vivo. Cultured neonatal rat ventricular myocytes (NRVMs) were treated with GdCl3 or nifedipine, followed by exposure to anoxia/reoxygenation (A/R). Cell apoptosis was detected; the levels of related signaling molecules were assessed. SD rats were intravenously injected with GdCl3 or nifedipine. Thirty min after the administration the rats were subjected to LAD coronary artery ligation followed by reperfusion. Infarction size, the release of serum myocardial injury markers and AA were measured; cell apoptosis and related molecules were assessed. In A/R-treated NRVMs, pretreatment with GdCl3 (2.5, 5, 10 μmol/L) dose-dependently inhibited caspase-3 activation, death receptor-related molecules DR5/Fas/FADD/caspase-8 expression, cytochrome c release, AA release and sustained cytoplasmic Ca(2+) increases induced by exogenous AA. In I/R-treated rats, pre-administration of GdCl3 (10 mg/kg) significantly reduced the infarct size, and the serum levels of CK-MB, cardiac troponin-I, LDH and AA. Pre-administration of GdCl3 also significantly decreased the number of apoptotic cells, caspase-3 activity, death receptor-related molecules (DR5/Fas/FADD) expression and cytochrome c release in heart tissues. The positive control drug nifedipine produced comparable cardioprotective effects in vitro and in vivo. Pretreatment with low-dose GdCl3 significantly attenuates I/R-induced myocardial apoptosis in rats by suppressing activation of both death receptor and mitochondria-mediated pathways.

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.

The Long-Term Consumption of Ginseng Extract Reduces the Susceptibility of Intermediate-Aged Hearts to Acute Ischemia Reperfusion Injury.

BackgroundA large number of experimental studies using young adult subjects have shown that ginseng (Panax ginseng C.A. Meyer) protects against ischemia heart disease. However, ginseng has not been explored for its anti-I/R effect and mechanism of action in the aged myocardium. The present study was designed to evaluate the effects of the long-term consumption of ginseng extract on myocardial I/R in an in vivo rat model and explore the potential underlying mechanism.Methods and ResultsYoung (6-mo-old) and intermediate-aged (18-mo-old) rats were gavaged with either standardized ginseng extract (RSE) at 80 mg/kg or vehicle for 90 days. The rats were sacrificed after LAD coronary artery ligation was performed to induce 30 min of ischemia, followed by 90 min of reperfusion. The myocardial infarct size was measured. Left ventricular function was evaluated using pressure-volume loops. The levels of survival, apoptotic and longevity protein expression were assessed through Western blot analysis. Myocardial pathology was detected through H&E or Masson’s trichrome staining. We observed higher infarct expansion with impairment in the LV functional parameters, such as LVSP and LVEDP, in aged rats compared with young rats. Enhanced Akt phosphorylation and eNOS expression in RSE-treated aged hearts were accompanied with reduced infarct size, improved cardiac performance, and inducted survival signals. In contrast, p-Erk and caspase 7 were significantly downregulated in aged rats, suggesting that cardiomyocyte apoptosis was suppressed after RSE treatment. RSE also inhibited caspase-3/7 activation and decreased Bax/Bcl-2 ratio. Consistent with the results of apoptosis, Sirt1 and Sirt3 were significantly increased in the RSE-treated aged heart compared with vehicle-treated I/R, suggesting that the anti-aging effect was correlated with the anti-apoptotic activity of RSE.ConclusionThese findings suggest that the long-term consumption of ginseng extract reduced the susceptibility of intermediate-aged hearts to acute ischemia reperfusion injury in rats. These effects might be mediated through the activation of Akt/eNOS, suppression of Erk/caspase 7 and upregulation of Sirt1 and Sirt3 in intermediate-aged rats.

Abstract 57: Platelet Extracellular-signal-regulated Kinase 5 is a Redox Switch which Regulates Myocardial Infarct Expansion via Matrix Metalloproteinases

Background: Dysregulated platelet activation in an ischemic microvascular environment may play a role in myocardial infarction (MI). Platelet receptor signaling is well-characterized, but mechanisms of receptor-independent activation, such as by reactive oxygen species (ROS) generated in ischemic conditions, are less well understood. We discovered that ERK5, a nuclear protein which is ROS-activated in others cells, is abundantly present in platelets. We investigated whether ERK5 could regulate platelet activation and thrombosis in healthy and diseased states. Methods: Human and mouse platelets were stimulated with agonists including ADP, U46619, TRAP, convulxin, or ROS (H 2 O 2 or 5% O 2 ). ERK5 activity was assessed by immunoblotting. Platelet activation was assessed via fluorescent-activated cell sorting (FACS) for P-selectin or activated GPIIb/IIIa. Intravascular thrombus (pulmonary embolus) or mesenteric thrombus (oxidative injury) formation was assessed by ex vivo fluorescent imaging and in vivo intravital microscopy, respectively. MI was performed in wild-type (WT) and in platelet specific ERK5 deficient (ERK5 -/- ) mice by LAD coronary artery ligation. Left ventricular (LV) function was determined by echocardiography. Matrix metalloproteinase (MMP) activity was determined by in-gel zymography. Results: Human and platelet ERK5 was activated by ROS and via the thrombin and thromboxane receptors, but not via the purinergic or collagen receptors. Murine in vivo thrombosis was regulated by platelet ERK5 only if the injury involved oxidative stress. MI in mice promoted sustained platelet activation over one week in an ERK5-dependent manner. Following MI, platelet ERK5 -/- mice had less reactive platelets, less platelet MMP activity and thromboxane production, attenuated MMP activity in the LV, less remodeling with smaller infarcts, and enhanced myocardial systolic performance. Conclusions: ERK5 is an ischemic sensor in platelets which regulates ongoing platelet activation after MI as well as remodeling via myocardial microvasculature. These observations may explain ischemic microvascular aberrations like the no-reflow phenomenon following percutaneous coronary intervention, suggesting a novel pharmacologic target.

Abstract 19220: Potential of Human Cardiac Progenitor Cells from Young versus Old Donor Hearts in Cardiac Protection against Ischemic Left Ventricular Dysfunction and Late Remodeling

Introduction: Recent discovery of c-Kit + resident cardiac progenitor cells (CPC) has demonstrated cardiac regenerative capacity, which is lost upon increasing age in mice. Accordingly, we hypothesized that c-KIT + -selected CPC from young human hearts (yCPC) have a greater myocardial repair potential after myocardial infarction (MI) than CPC from old patients (oCPC). Methods: After Ethics Committee approval, cardiac biopsies from young (1-15y old) and old (58-80y old) patients, undergoing corrective or valve surgery were obtained. After collagenase-based digestion, cells were culture-expanded for 2 passages prior to c-KIT + selection using magnetic beads. Purity was confirmed using qRT-PCR for c-KIT, GATA4, CD90, CD105, DDR2, CD31 and MYH11 expression levels. To trace transplanted cells, CPC were lentivirally tagged with GFP. After permanent LAD coronary artery ligation in immunodeficient mice, 250,000 CPCs were injected in the infarct border zone (4x 62,500 cells/2.5 μl; yCPC, n =8; oCPC, n =6; or PBS, n =6). Cardiac ultrasound (VisualSonics, 3D 30 MHz probe) was performed at baseline, 1 and 35 days after MI. At 35 days post-MI, infarct remodeling and cell survival was evaluated using Sirius red and CPC engraftment by anti-GFP staining. Results: In contrast to low MYH11 and CD31 expression levels, c-KIT and GATA4 were markedly and equally expressed in yCPC and oCPC, whereas transcript levels of DDR2, CD105 and CD90 were 89, 109 and 56% higher in oCPC compared to yCPC. Systolic function was better preserved in yCPC than in oCPC and PBS-treated mice 24h after MI (LVEF from baseline - D1: 53±1 - 38±3 vs. 52±3 - 30±5 vs. 50±3 - 27±4%; ESVi from baseline - D1: 3.7±0.2 - 5.1±0.4 vs. 3.8±0.5 - 6.2±0.7 vs. 3.9±0.5 - 6.5±0.6ml/mm 2 ). Moreover, after 35 days, the dilatory response was reduced in yCPC compared to oCPC and PBS-treated mice (EDVi from D1 - D35: 8.3±0.5 - 13.5±1.3 vs. 8.8±0.5 - 16.7±1.8 vs. 8.5±0.5 - 16.6±1.3ml/mm 2 ). Improved remodeling was consistent with a markedly greater rim of surviving myocardium while anti-GFP staining showed only rarely retained CPC. Conclusions: In summary, these findings suggest enhanced protection by c-KIT + CPC isolated from young donor hearts against acute systolic left ventricular dysfunction and late remodeling after MI.

MMI-0100 inhibits cardiac fibrosis in myocardial infarction by direct actions on cardiomyocytes and fibroblasts via MK2 inhibition

The cell-permeant peptide inhibitor of MAPKAP kinase 2 (MK2), MMI-0100, inhibits MK2 and downstream fibrosis and inflammation. Recent studies have demonstrated that MMI-0100 reduces intimal hyperplasia in a mouse vein graft model, pulmonary fibrosis in a murine bleomycin-induced model and development of adhesions in conjunction with abdominal surgery. MK2 is critical to the pathogenesis of ischemic heart injury as MK2−/− mice are resistant to ischemic remodeling. Therefore, we tested the hypothesis that inhibiting MK2 with MMI-0100 would protect the heart after acute myocardial infarction (AMI) in vivo. AMI was induced by placing a permanent LAD coronary ligation. When MMI-0100 peptide was given 30min after permanent LAD coronary artery ligation, the resulting fibrosis was reduced/prevented ~50% at a 2week time point, with a corresponding improvement in cardiac function and decrease in left ventricular dilation. In cultured cardiomyocytes and fibroblasts, MMI-0100 inhibited MK2 to reduce cardiomyocyte caspase 3/7 activity, while enhancing primary cardiac fibroblast caspase 3/7 activity, which may explain MMI-0100's salvage of cardiac function and anti-fibrotic effects in vivo. These findings suggest that therapeutic inhibition of MK2 after acute MI, using rationally-designed cell-permeant peptides, inhibits cardiac fibrosis and maintains cardiac function by mechanisms that involve inhibiting cardiomyocyte apoptosis, while enhancing primary cardiac fibroblast cell death.

Qishenyiqi protects ligation-induced left ventricular remodeling by attenuating inflammation and fibrosis via STAT3 and NF-κB signaling pathway.

AimQi-shen-yi-qi (QSYQ), a formula used for the routine treatment of heart failure (HF) in China, has been demonstrated to improve cardiac function through down-regulating the activation of the Renin-Angiotensin-Aldosterone System (RAAS). However, the mechanisms governing its therapeutic effects are largely unknown. The present study aims to demonstrate that QSYQ treatment can prevent left ventricular remodeling in heart failure by attenuating oxidative stress and inhabiting inflammation.MethodsSprague-Dawley (SD) rats were randomly divided into 6 groups: sham group, model group (LAD coronary artery ligation), QSYQ group with high dosage, middle dosage and low dosage (LAD ligation and treated with QSYQ), and captopril group (LAD ligation and treated with captopril as the positive drug). Indicators of fibrosis (Masson, MMPs, and collagens) and inflammation factors were detected 28 days after surgery.ResultsResults of hemodynamic alterations (dp/dt value) in the model group as well as other ventricular remodeling (VR) markers, such as MMP-2, MMP-9, collagen I and III elevated compared with sham group. VR was accompanied by activation of RAAS (angiotensin II and NADPHoxidase). Levels of pro-inflammatory cytokines (TNF-α, IL-6) in myocardial tissue were also up-regulated. Treatment of QSYQ improved cardiac remodeling through counter-acting the aforementioned events. The improvement of QSYQ was accompanied with a restoration of angiotensin II-NADPHoxidase-ROS-MMPs pathways. In addition, “therapeutic” QSYQ administration can reduce both TNF-α-NF-B and IL-6-STAT3 pathways, respectively, which further proves the beneficial effects of QSYQ.ConclusionsOur study demonstrated that QSYQ protected LAD ligation-induced left VR via attenuating AngII -NADPH oxidase pathway and inhabiting inflammation. These findings provide evidence as to the cardiac protective efficacy of QSYQ to HF and explain the beneficial effects of QSYQ in the clinical application for HF.

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.