Methods Myocardial Infarction Research Articles

Ventricular arrhythmia inducibility in porcine infarct model after stereotactic body radiation therapy

BackgroundVentricular arrhythmia (VA) is the primary mechanism of sudden death in patients with structural heart disease. Cardiac stereotactic body radiation therapy (SBRT) delivered to the scar in the left ventricle significantly reduces the burden of VA. ObjectiveThe goal of this study was to investigate the impact of SBRT on scar morphology and VA inducibility in a porcine infarct model. MethodsMyocardial infarction (MI) was created in 10 Yorkshire pigs involving the left anterior descending artery territory. Cardiac positron emission tomography and computed tomography were performed for targeted SBRT. Alternative pigs received SBRT at 25 Gy in a single fraction. The terminal experiment included endocardial mapping, programmed ventricular stimulation, and tissue harvesting. ResultsOf the 10 pigs infarcted, 2 died prematurely after MI and 8 (4 MI and 4 MI+SBRT) survived. Mean time from MI to SBRT was 48 ± 12 days, and mean time from SBRT to harvest was 32 ± 12 days. Scar was localized on intracardiac mapping in all pigs, and the scar was denser in the MI+SBRT compared with the MI-only group (33% ± 20% vs 14% ± 11%; P = .07). All 4 MI pigs had inducible VA during programmed stimulation, whereas only 1 of 4 pigs had inducible VA in the MI+SBRT arm (100% vs 25%; P = .07). No myocardial fibrosis was seen in the remote areas in either group. ConclusionSBRT reduced VA inducibility in pigs with scarring after MI. Endocardial mapping revealed denser scar in pigs receiving SBRT compared with those that did not, suggesting that SBRT suppresses VA inducibility through better scar homogenization.

Clusterin regulates TRPM2 to protect against myocardial injury induced by acute myocardial infarction injury

BackgroundClusterin and transient receptor potential melastatin 2 (TRPM2) play significant roles in acute myocardial infarction (AMI), but their interactions in AMI are unclear. MethodsMyocardial infarction was induced by ligation of the left anterior descending coronary artery in wild-type C57BL/6J male mice. Infarct size and myocardium pathology were evaluated after 6, 12, and 24 h of ischemia. The expression levels of clusterin and TRPM2 were measured in the myocardium. Furthermore, myocardial infarction was induced in TRPM2 knockout (TRPM2-/-) C57BL/6J male mice to evaluate the expression of clusterin. H9C2 cells with various levels of TRPM2 expression were used to analyze the effects of clusterin under hypoxic conditions. ResultsFollowing AMI, myocardial hypertrophy and TRPM2 expression increased in a time-dependent manner. In contrast, the expression of clusterin decreased in an infarct time-dependent manner. Knockout of TRPM2 protected against myocardial injury and resulted in upregulation of clusterin. In the H9C2 cells, cultured under hypoxic conditions treatment with clusterin or silencing of TRPM2 significantly increased cell viability and decreased TRPM2 expression. Treatment with clusterin protected against TRPM2 overexpression-induced damage in hypoxia-treated H9C2 cells. ConclusionThis study characterized the effects of clusterin on TRPM2 in AMI, which may guide development of new treatment strategies for AMI.

Chronic catestatin treatment reduces atrial fibrillation susceptibility via improving calcium handling in post-infarction heart failure rats

BackgroundAbnormal Ca2+ handling is a pivotal element of atrial fibrillation (AF) substrates. Catestatin (CST) modulates intracellular Ca2+ handling in cardiomyocytes (CMs). We investigated the effects of CST administration on atrial Ca2+ handling and AF susceptibility in rats with post-infarction heart failure (HF). MethodsMyocardial infarction (MI) was established by ligation of the left anterior descending coronary artery in rats. Two-week later, rats with post-infarction HF were randomly treated with saline (MI group) or CST (MI + CST group) for 4-week. Cellular Ca2+ imaging was performed by incubating atrial CMs with Fura-2 AM. An in vitro electrophysiological study was performed to assess the vulnerability to action potential duration (APD) alternans and AF. Ca2+ handling proteins expression was determined using western blotting. ResultsIn atrial CMs, compared with the sham group, the sarcoplasmic reticulum (SR) Ca2+ load, Ca2+ transient (CaT) amplitude, and threshold for Ca2+ alternans were significantly decreased, but the diastolic intracellular Ca2+ level, SR Ca2+ leakage, and spontaneous Ca2+ events were markedly increased in the MI group. However, CST attenuated these Ca2+-handling abnormalities induced by post-infarction HF. Moreover, vulnerability to atrial APD alternans and AF was significantly increased in isolated hearts from the MI group compared to the sham group, whereas all effects were prevented by CST. CST treatment also preserved SR Ca2+-ATPase protein expression but decreased the protein levels of phosphorylated-ryanodine receptor 2 and phosphorylated-Ca2+/calmodulin-dependent protein kinase II in atria from post-infarction HF rats. ConclusionChronic CST treatment reduces AF vulnerability in rats with MI-induced HF by improving Ca2+ handling.

Tumstatin (69–88) alleviates heart failure via attenuating oxidative stress in rats with myocardial infarction

BackgroundThis study aimed to elucidate the effects of tumstatin (69–88) on heart failure and the underlying mechanism. Materials and methodsMyocardial infarction (MI) was induced by ligating the left coronary artery in rats to trigger heart failure. ResultsTumstatin (69–88) can reduce cardiac insufficiency in rats with heart failure. The increased cardiac fibrosis in MI rat was attenuated by tumstatin (69–88). Increase of cardiac atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) in rats with myocardial infarction, and Ang II-treated NRCMs or H9C2 cells was inhibited by tumstatin (69–88). In the heart of MI rats, and Ang II-treated NRCMs or H9C2 cells, the superoxide anions and NADPH oxidase (Nox) activity rose and the superoxide dismutase (SOD) activity was reduced, which was inhibited by tumstatin (69–88). Diethyldithiocarbamate, an SOD inhibitor, increased the ANP and BNP in NRCMs or H9C2 cells. Tumstatin (69–88) inhibited the Ang II-induced raises of ANP and BNP in NRCMs or H9C2 cells, which was reversed by DETC. ConclusionsThese results indicate that tumstatin (69–88) alleviates cardiac dysfunction of heart failure. Tumstatin (69–88) improves the hypertrophy of cardiomyocytes via attenuation of oxidative stress. Tumstatin (69–88) may be a potential drug for heart failure in the future.

Zerumbone, a humulane sesquiterpene from Syringa pinnatifolia, attenuates cardiac fibrosis by inhibiting of the TGF-β1/Smad signaling pathway after myocardial infarction in mice

BackgroundZerumbone (ZER) is a humulane sesquiterpene isolated from Syringa pinnatifolia Hemsl., a representative Mongolian herbal medicine that is used to treat cardiovascular diseases. Cardiac fibrosis is a common pathological process in cardiovascular disease that results from the excessive accumulation of extracellular matrix (ECM), and the transforming growth factor (TGF)-β/Smad pathway is a canonical signaling pathway that directly induces expressions of ECM-related genes. Currently, the cardioprotective effect and underlying mechanisms of ZER on the inhibition of cardiac fibrosis are not well known. PurposeTo explore the cardioprotective properties and pharmacological mechanism of ZER against cardiac fibrosis via the TGF-β1/Smad signaling pathway. MethodsMyocardial infarction (MI) model was induced by ligation of the left anterior descending coronary artery in ICR mice. The mice were randomly divided into six groups: sham, model, low-dose ZER (ZER-L), medium-dose ZER (ZER-M), high-dose ZER (ZER-H) and fosinopril. Mice in each group were intragastrically administered treatments for 21 days, and cardiac function was evaluated by 2D echocardiography. The pathological structure of the heart was examined by hematoxylin and eosin (HE) and Masson staining. Content of collagen I and collagen III were assessed by immunofluorescence methods. The inhibitory effect of ZER on TGF-β1 protein expression was predicted by molecular docking technology. Reverse transcriptase polymerase chain reaction (RT–PCR) and western blotting were used to measure the levels of genes and proteins expressed in the TGF-β1/Smad signaling pathway and MMPs. TGF-β1-treated cardiac fibroblasts (CFs) of neonatal SD rats were adopted for in vitro studies. ResultsCardiac ejection fraction (EF) and fractional shortening (FS) in the model group were markedly decreased compared with those in the sham group, indicating that the MI model was successfully established. ZER and fosinopril elevated EF and FS values, suggesting cardioprotective effects. Pathological staining and immunofluorescence analysis showed that the content of collagen I and collagen III increased in the cardiac tissue of mice in model group, while ZER treatment obviously reduced collagen levels. The molecular docking simulations predicted the hydrophobic interactions between ZER and TGF-β1. In addition, the expression of TGF-β1, p-Smad2/3 and MMPs in the ZER treatment group was significantly decreased compared with the model group. In vitro studies further confirmed that α-smooth muscle actin (α-SMA) and p-Smad2/3 increased markedly in cardiac fibroblasts after incubation with TGF-β1, and treatment with ZER suppressed the expression of α-SMA and TGF-β1 downstream proteins in cardiac fibroblasts. ConclusionZER rescues cardiac function by attenuating cardiac fibrosis, and the antifibrotic effect may be mediated by blocking the TGF-β1/Smad pathway.

Uptake of [68Ga]-NODAGA-E[(cRGDyK)]2 is related to improvement in pump function in rats with chronic ischemic cardiomyopathy treated with cell therapy

Abstract Background An increasing number of patients living with chronic ischemic cardiomyopathy (ICM) are potential candidates for cell therapy, including adipose tissue-derived mesenchymal stromal cells (ASC). However, proper assessment of clinical effects in relation to cellular effector mechanisms, such as angiogenesis, is currently lacking. Purpose To investigate the prognostic value of assessing angiogenesis non-invasively using novel PET imaging in terms of functional outcome after cell therapy in a homologous animal model of ICM. Methods Myocardial infarction was induced by permanent ligation of the left anterior descending coronary artery. Four weeks after infarction, the rats were scanned with [18F]-FDG and echocardiography, and based on left ventricular ejection fraction (LVEF) and infarct size randomized to allogeneic ASC treatment (n=14) or saline (n=9). Animals were treated using echo-guided trans-thoracic intramyocardial injections. Follow-up echocardiography was performed four weeks after treatment. Angiogenesis was assessed non-invasively using the [68Ga]-NODAGA-E[(cRGDyK)]2 (RGD) PET-tracer before treatment and two weeks after the treatment (Figure). The output was RGD-uptake by maximum standardized uptake value (SUVmax). Results RGD-uptake in the infarct area significantly decreased in the saline group (p=0.04), while this decline was not significant in the ASC group (p=0.58). There was no effect on LVEF of the cell therapy in this study (p=0.70). When the rats were grouped by RGD-uptake post treatment, the high RGD-uptake group (n=8) improved LVEF compared to the rats with medium or low RGD-uptake (n=15, p=0.04) irrespective of the treatment. This could indicate that non-invasive detection of a high degree of myocardial angiogenesis measured using RGD is predictive of improvement in cardiac pump function in ICM. Conclusions Following injection of ASC or saline, a high RGD-uptake precluded improvement in pump function. This is the first evidence of how uptake of the novel RGD tracer relates to changes in pump function in ICM and the results could affect the design of future clinical trials using regenerative therapy for ICM patients. Funding Acknowledgement Type of funding sources: Private grant(s) and/or Sponsorship. Main funding source(s): Aase and Ejnar Danielsens FondDoctor Sofus Carl Emil Friis and wife Olga Doris Friis' Scholarship Study design, RGD-uptake, and results

Nanoparticles loaded with hepatic growth factor and insulin-like growth factor-1 improve left ventricular repair in a porcine model of myocardial Ischemia reperfusion injury

Abstract Introduction Nanomedicine offers great potential for treatment of cardiovascular disease. We tested whether intramyocardial (IM) injection of pro-angiogenic hepatocyte growth factor (HGF) and anti-apoptotic, pro-myogenic insulin-like growth factor 1 (IGF-1) encapsulated in Alginate-Sulfate nanoparticles (AlgS-NP) improves left ventricular (LV) functional recovery in a porcine ischemia-reperfusion (I/R) model. Methods Myocardial infarction (MI) was induced by 75min balloon occlusion of the mid-LAD followed by reperfusion. After 1w, pigs (n=12) with marked LV dysfunction (EF<45%) were randomized to fusion imaging-guided IM injections of 8 mg Cy5-labelled AlgS-NP loaded with 200μg HGF and IGF-1 (GF) or with phosphate-buffered saline (CON) using the MYOSTAR injection catheter. AlgS-NP retention in the heart was determined by measuring Cy5 levels in peripheral blood. At 8w, treatment effect was evaluated using cardiac magnetic resonance imaging and coronary flow reserve (CFR) measurements, and further assessed using sirius red staining to measure myocardial fibrosis. Results At 1w after MI, LV ejection frqction (LVEF) was 37±5% (range 27–45%) and infarct size (IS)/LV mass 24±6% (range 19–38%). Myocardial retention of AlgS-NP was comparable between 2 groups (maximal systemic leakage after IM injection: 9% CON vs 20% GF, P=0.25). After 8 w, IS/LV mass decreased by one third in GF-treated pigs compared with 14% in CON (P=0.03, Fig. A) and was associated with a trend towards improvement in CFR (P=0.05, Fig. B). LVEF significantly increased in GF-treated pigs (6±2% vs. −1±1%, P=0.02, Fig. C), which was attributable to a greater reduction in end-systolic volume. The improvement in LVEF was also consistent with significant reduction of fibrosis (P=0.01, Fig. D) in the peri-infarct zone (PI). Conclusions Intramyocardial injection of AlgS-nanoparticle-encapsulated HGF and IGF-1 to the ischemic myocardium significantly improves LV repair, and offers the prospect of innovative treatment for patients with refractory ischemic heart disease. Funding Acknowledgement Type of funding source: Public grant(s) – EU funding. Main funding source(s): EuroNanoMed, Horizon 2020

How do Extracellular Vesicles Protect the Ischemic Myocardium?

Background & Aim Introduction Cell therapy for chronic heart failure has demonstrated that Cardiac Progenitor Cells (CPCs) delivered to the heart exert their beneficial effects through a paracrine mechanism harnessing endogenous repair pathways largely mediated by extracellular vesicles (EVs). We used a bi-transgenic fate-mapping MerCreMer/ZEG mouse model to assess whether the cardioprotection could be partly explained by de novo cardiogenesis. Methods, Results & Conclusion Methods Myocardial infarction (MI) was induced by permanent left anterior descending coronary artery ligation in MerCreMer/ZEG mice. Surviving animals (n=30) with a left ventricular ejection fraction (LVEF) ≤ 45% were treated 3 weeks after MI with either EV (from 1.4 × 106 human iPS-derived CPC; 10 × 109 particles; n=15) or PBS (n=15) delivered by echo-guided intra-myocardial injections targeted to the peri-infarcted area. To track a putative proliferation of endogenous cardiomyocytes, osmotic pumps delivering EdU were implanted for 10-13 days after EV or PBS treatnent. Cardiac function was assessed 4-6 weeks after injections by echocardiography (n=15 p/group) and MRI (n=6-7 p/group), blinded to treatment group. Hearts were harvested and subjected to histological and transcriptomic analyses by qRT-PCR and genome-wide microarrays (Affymetrix). Results In controls, echocardiographically assessed EF slightly decreased over time whereas in the EV-treated group, it increased so that at the end-study time point, the between-group difference in changes from baseline averaged 20.7 % ± 10.5 % (p=0.048). This improvement was confirmed by MRI (11%, p=0.05). EV-treated hearts did not show a different number of new cardiomyocytes (TnT+/EdU+/GFP+) compared to controls (32 analyzed sections/mouse). However, EV-treatment was associated with a reduction in infarct size (-11.9% ± 5.75%), and reduced expression of 4 pro-fibrotic genes (Col1a2, Col3a1, Lox, Col1a2) as assessed by qRT-PCR (n=5-6 p/group). Further, the EV-treatment group showed an over-expression of the anti-fibrotic miRNA 133a-1 (2.13 fold) as compared to controls (n=3 p/group; p=0.0015). Conclusions EV secreted by iPSC-CPC improve cardiac function in chronic heart failure by mechanisms which seem to be largely related to the modulation of fibrosis without participation of a de novo cardiogenesis. Funding Labex Revive, Fondation pour la Recherche Medicale, Fonds Marion Elizabeth BRANCHER

P1646Do Extracellular Vesicles repair chronic ischemic heart disease by replenishing the cardiomyocyte pool?

Abstract Introduction Extracellular Vesicles (EV) seem to mediate the benefits of cell therapy for ischemic heart failure. Although their mechanism of action remains poorly understood, one hypothesis is that they might trigger the generation of new cardiomyocytes. The doubly transgenic fate-mapping MerCreMer/ZEG mice model was thus used to distinguish whether these putative new cardiomyocytes originated from the division of preexisting ones (GFP+, Troponin T [TnT+], EdU+) or differentiated from endogenous progenitors, in which case they would stain positive for TnT+/EdU+ but negative for GFP. Methods Myocardial infarction was induced in 35 MerCreMer/ZEG mice by permanent occlusion of the left anterior descending coronary artery. Three weeks later, the surviving mice (n=18) with a left ventricular ejection fraction (LVEF) ≤45% received transcutaneous echo-guided injections in the peri-infarct myocardium of either EV (from 1.4 million human iPS-derived cardiovascular progenitor cells; 10 billion particles, n=9) or PBS (n=9); osmotic pumps were implanted to deliver EdU for 7 days in order to track the proliferation of new and native cardiomyocytes. Four-6 weeks after treatment all mice were evaluated by echocardiography (n=9 per group) and MRI (7 in each group), and then sacrificed for histological assessment, blindly. Results Based on echocardiography (MRI data pending), EV improved LVEF by 16% relative to baseline while a decrease of 4% was observed in the PBS group (p=0.46). The number of new cardiomyocytes (TnT+/EdU+/GFP+) did not significantly differ between the EV-treated hearts and the controls, and averaged 0.54% of the total heart cell content in infarct, peri-infarct and remote areas. However, EV treatment better preserved preexisting GFP+/WGA+/TnT+ cardiomyocytes in the peri-infarct area as their number was greater by 5.15% compared to PBS (32 sections analyzed for each mouse). Compared to the PBS control group, EV delivery was also associated with a 2.5% decrease in fibrosis, a reduction of infarct size by 14.9%, and an increase in angiogenesis in the peri-infarct area (with a between-group absolute difference of 71 capillaries, on the basis of isolectin staining). Conclusions EV secreted by iPS-derived cardiovascular progenitors improve the function of chronically infarcted hearts. Preservation of the existing cardiomyocyte pool and limitation of adverse remodeling and scarred tissue, likely favored by increased neoangiogenesis, are the main mechanisms mediated by the EV, while fate mapping allowed to exclude the generation of new cardiomyocytes.

5234KAND567, the first selective small molecule CX3CR1 antagonist in clinical development, mediates anti-inflammatory cardioprotective effects in rodent models of atherosclerosis and myocardial infarction

Abstract Background Fractalkine is a chemokine that mediates recruitment and extravasation of CX3CR1-expressing subsets of leukocytes and monocytes and has been implicated in the inflammation-driven pathology of cardiovascular disease. More specifically, fractalkine signaling has been proposed to contribute to increased infarct size and enhanced atherosclerotic plaque vulnerability in patients and experimental models. Blocking fractalkine/CX3CR1 signaling is suggested as a promising anti-inflammatory strategy for the treatment of both acute and chronic cardiovascular disease. KAND567 is a small molecule, selective, non-competitive, allosteric antagonist of the fractalkine receptor CX3CR1, that is under preparation for a clinical phase IIa study in AMI patients. Purpose To explore the therapeutic effects of the short and long term administration of KAND567 in experimental rodent models of acute myocardial infarction and atherosclerosis, respectively. Methods Myocardial infarction was induced in Wistar rats (N=6–8 per group) by ligation of the left anterior descending (LAD) coronary artery for 30 minutes followed by 2 h of reperfusion. The drug or vehicle infusion started either 5 min before or 30 min after start of reperfusion and continued during the remainder of the experiment. Hearts were collected and subjected to triphenyl tetrazolium chlorine (TTC) staining and the infarction area/area at risk of the left ventricle was determined by planimetry and compared against vehicle group. Atherosclerosis-prone LDL-receptor deficient mice on a high-cholesterol diet, (N=15–25 per group) were treated with KAND567 for 15–23 weeks. Atherosclerotic plaque development in the thoracic arch was determined by ultrasound imaging and histology. Immunohistochemistry was used to follow changes in the cellular composition in the atherosclerotic lesions. Results In the acute myocardial infarction study, the infusion of KAND567 before the start of reperfusion significantly reduced infarcted/risk area (by up to 50%) as compared to the vehicle group. However, the infusion had no effect on the infarct size when administration was initiated 30 min after start of reperfusion. In the atherogenesis study, oral treatment with KAND567 significantly reduced vascular macrophage infiltration by 50% and reduced intima media thickness. Furthermore, reduced plaque volume and a more stable plaque phenotype was noted following treatment with KAND567. KAND567 experimental results Conclusion Specific inhibition of fractalkine-driven inflammation by KAND567 provides cardioprotective, anti-atherosclerotic and plaque stabilizing effects via mechanisms related to immune cell infiltration, in rodent models. Further studies should be initiated to test if KAND567 is a potential candidate drug, targeting the excessive inflammatory injury associated with ischemia/reperfusion in myocardial infarction and providing plaque stabilization by reducing inflammatory risk for recurrent coronary events.

P6349Inflammation mitigation improves post-infarction functional recovery of the heart

Abstract Aim The in vitro evaluation of the cardiac functional effects of TNF-α antagonist administration in rats after isoproterenol induced myocardial infarction. Material and methods Myocardial infarction was reproduced using a proven model based on isoproterenol i/p administration in rats in 2 consecutive days in a similar dose, 150 mg/kg. In another group the animals after isoproterenol induced myocardial infarction (series IMI) have received daily TNF-α antagonist, a specific monoclonal antibody (ma-TNF-α) i/p in dose of 50 mg/kg during 8 days (series IMI+ma-TNF-α). In both series the animals were sacrificed after 10 days from the 1st injection and their isolated hearts ware perfused with Krebs solution according to Langendorff and Neely-Rovetto models. Results The most remarkable traits of left ventricle dysfunction in IMI in comparison to control were following: (1) diminution of cardiac output (CO), systolic pressure (SP) and +dP/dT max by respectively 28,7 and 34,7 and 23,3%; (2) negative inotropic effect to action of endothelin-1 manifested by decrease of SP and aortic jet during stimulation up to 13,9%; (3) increased cardiac arrhythmogenic activity in response to calcium overload; (4) increasing by 45,2% of ischemia induced contracture as well as decreasing by 37,5% of SP during reperfusion. The ma-TNF-α administration in post-infarction period led to noticeable benefits such as: significant enhancement of SP and CO respectively by 17,3 and 18,6% as well as positive inotropic effect developing to ET-1 action as well as significant increase of time regarding the appearance of ventricular extrasystole and ventricular tachyarrhythmia by respectively 12,9 and 11,7% as well as perceptible improvement of ischemia-reperfusion syndrome. Conclusion A sustained inflammation inhibition by ma-TNF-α administration in post-infarction period improves tangibly the cardiac functioning that proves the role of inflammatory response in myocardial infarction induced functional and structural myocardial remodeling and underlines the inflammation as a therapeutic target.

Effects of exercise training and stem cell therapy on the left ventricle of infarcted rats

Introduction and ObjectivesStem cell therapy and aerobic exercise are non-pharmacological therapies following myocardial infarction. The aim of this study was to test whether aerobic exercise training enhances the benefits of mesenchymal stem cell (MSC) therapy on remodeling of the extracellular matrix and fetal gene expression in the left ventricle of infarcted rats. MethodsMyocardial infarction was surgically induced in six-week old male Wistar rats. Animals were divided into four groups: sedentary control (SC) and sedentary and stem cell treated (SCMSC); exercised (EX) and exercised and stem cell treated (EXMSC). Bone marrow-derived MSCs were immediately transplanted via the tail vein (concentration: 1×106 cells). Exercise training (five days/week, 60 min/day; 60% of maximal running speed) started 24 hours after myocardial infarction and lasted for 12 weeks. ResultsExercise capacity was higher in exercised than in sedentary groups. Animals in the SCMSC, EX and EXMSC groups exhibited better cardiac function than those in SC. Collagen content was lower in the SCMSC, EX and EXMSC groups than in SC and skeletal α-actin expression was lower in EX and EXMSC than in SC. The α/β-MHC ratio was higher in EX and EXMSC than in SC. The combination of therapies further reduced collagen content in the remote region of the infarct (∼24%) and skeletal α-actin expression (∼30%). ConclusionAerobic exercise training appears to enhance the beneficial effects of stem cell therapy on remodeling of the extracellular matrix and fetal gene expression in the left ventricle of rats with moderate infarction.

Combination of Ligusticum Chuanxiong and Radix Paeonia Promotes Angiogenesis in Ischemic Myocardium through Notch Signalling and Mobilization of Stem Cells.

Objective To study the cardioprotective mechanism by which the combination of Chuanxiong (CX) and Chishao (CS) promotes angiogenesis. Methods Myocardial infarction (MI) mouse models were induced by ligation of the left anterior descending coronary artery. The effects on cardiac function were evaluated in the perindopril tert-butylamine group (PB group) (3 mg/kg/d), CX group (55 mg/kg/d), CS group (55 mg/kg/d), and CX and CS combination (CX-CS) group (27.5 mg/kg/d CX plus 27.5 mg/kg/d CS). RO4929097, an inhibitor of Notch γ secretase, was used (10 mg/kg/d) to explore the role of Notch signalling in the CX-CS-induced promotion of angiogenesis in the myocardial infarcted border zone (IBZ). The left ventricular ejection fraction (LVEF) and percentage of MI area were evaluated with animal ultrasound and Masson staining. The average optical densities (AODs) of CD31 and vWF in the myocardial IBZ were detected by immunofluorescence. Angiogenesis-related proteins including hypoxia-inducible factor 1-alpha (HIF-1α), fibroblast growth factor receptor 1 (FGFR-1), Notch1 and Notch intracellular domain (NICD), and stem cell mobilization-related proteins including stromal cell-derived factor 1 (SDF-1), C-X-C chemokine receptor type 4 (CXCR-4), and cardiotrophin1 were detected by western blot analysis. Results Compared with the model group, the CX-CS and PB groups both showed markedly improved LVEF and decreased percentage of MI area after 21 days of treatment. Although the CX group and CS group showed increased LVEF and decreased MI areas compared with the model group, the difference was not significant. The AOD of CD31 in the IBZ in both the model and the CX-CS-I group was markedly reduced compared with that in the sham group. CX-CS significantly increased the CD31 AOD in the IBZ and decreased the AODs of CD31 and vWF in the infarct zone compared with those in the model group. The expression of HIF-1α in both the model group and the CX-CS group was higher than that in the sham group. Compared with the model group, the expression of FGFR-1, SDF-1, cardiotrophin1, Notch1, and NICD was increased in the CX-CS group. Notch1 and NICD expression in the CX-CS-I group was reduced compared with that in the CX-CS group. Conclusions The combination of CX and CS protected cardiomyocytes in the IBZ better than CX or CS alone. The mechanism by which CX-CS protects ischemic myocardium may be related to the proangiogenesis effect of CX-CS exerted through Notch signalling and the mobilization of stem cells to the IBZ.

Involvement of lncR-30245 in Myocardial Infarction–Induced Cardiac Fibrosis Through Peroxisome Proliferator-Activated Receptor-γ–Mediated Connective Tissue Growth Factor Signalling Pathway

BackgroundLong noncoding RNAs (lncRNAs) are emerging as important mediators of cardiac pathophysiology. The aim of the present study is to investigate the effects of lncR-30245, an lncRNA, on cardiac fibrogenesis and the underlying mechanism. MethodsMyocardial infarction (MI) and transforming growth factor (TGF)-β1 were used to induce fibrotic phenotypes. Cardiac fibrosis was detected by Masson’s trichrome staining. Cardiac function was evaluated by echocardiography. Western blot, quantitative reverse transcription-polymerase chain reaction, and pharmacological approaches were used to investigate the role of lncR-30245 in cardiac fibrogenesis. ResultsExpression of lncR-30245 was significantly increased in MI hearts and TGF-β1–treated cardiac fibroblasts (CFs). LncR-30245 was mainly located in the cytoplasm. Overexpression of lncR-30245 promoted collagen production and CF proliferation. Knockdown of lncR-30245 significantly inhibited TGF-β1–induced collagen production and CF proliferation. LncR-30245 overexpression inhibited the antifibrotic role of peroxisome proliferator-activated receptor (PPAR)-γ and increased connective tissue growth factor (CTGF) expression, whereas lncR-30245 knockdown exerted the opposite effects. Rosiglitazone, a PPAR-γ agonist, significantly inhibited lncR-30245–induced CTGF upregulation and collagen production in CFs. In contrast, T0070907, a PPAR-γ antagonist, attenuated the inhibitory effects of lncR-30245 small interfering RNA (siRNA) on TGF-β1–induced CTGF expression and collagen production. LncR-30245 knockdown significantly enhanced ejection fraction and fractional shortening and attenuated cardiac fibrosis in MI mice. ConclusionOur study indicates that the lncR-30245/PPAR-γ/CTGF pathway mediates MI-induced cardiac fibrosis and might be a therapeutic target for various cardiac diseases associated with fibrosis.

Grape seed proanthocyanidin extract improves myocardial function in rats with myocardial infarction by inhibiting PARP-1 activity

Objective To observe the improvement effect of grape seed proanthocyanidin extract (GSPE) on myocardial function in rats with myocardial infarction and to explore its mechanism. Methods Myocardial infarction model rats were randomly divided into five groups: model group, GSPE low, middle and high group, poly ADP-ribose polymerase-1 (PARP-1) inhibitor BSI-201 group, with 15 rats in each group, and another 15 rats were as the sham operation control group. Rats in GSPE low, medium and high dose groups were administrated with 100 mg/ml GSPE, 200 mg/ml GSPE and 400 mg/ml GSPE respectively; BSI-201 group was given 120 μmol/L BSI-201 of gavage, and the control group and model group were treated with equal volume of normal saline of gavage, 1 times a day for 4 weeks. Doppler echocardiography was used to evaluate cardiac function in rats; myocardial infarction volume was detected by three-phenyl tetrazolium chloride (TTC) staining; hematoxylin and eosin (HE) staining was used to detect histopathological changes of myocardial tissue; terminal dexynucleotidyl transferase (TdT) mediated dUTP nick end labeling (TUNEL) assay was used to detect the apoptosis of myocardial cells; Western blot was used to analyze the expressions of Bax, cleaved-caspase3, Bcl-2 and PAR in myocardial tissue. Results Compared with the control group, the heart rate, left ventricular end diastolic pressure, myocardial infarction area and cell apoptosis rate were significantly increased in the model group, and the mean arterial pressure and left ventricular systolic pressure were decreased significantly (P<0.05); Myocardial cells were broken and necrotic, irregular fibers arranged in the myocardium, and a large number of inflammatory cells were infiltrated; the protein expressions of Bax, cleaved-caspase3 and PAR in myocardial tissues were increased significantly (P<0.05), and the protein expression of Bcl-2 was significantly decreased (P<0.05). Compared with the model group, the heart rate, the left ventricular end diastolic pressure, myocardial infarction area and cell apoptosis rate in the GSPE low, middle and high dose groups and the BSI-201 group were significantly decreased, and the mean arterial pressure and the left ventricular systolic pressure were increased significantly (P<0.05); the degree of histopathological damage was alleviated; the expressions protein of Bax, cleaved-caspase3 and PAR in myocardial tissues were decreased significantly (P<0.05), and the protein expression of Bcl-2 was increased significantly (P<0.05). Conclusions Grape seed proanthocyanidin extract may inhibit cardiomyocyte apoptosis and improve myocardial function of rats with myocardial infarction by inhibiting PARP-1 activation. Key words: Proanthocyanidins/PD; Poly(ADP-ribose) polymerases/DE; Myocardial infarction/DT; Apoptosis; Rats

Long non-coding RNA GAS5 reduces cardiomyocyte apoptosis induced by MI through sema3a

ObjectiveTo investigate the role of GAS5 on cardiomyocyte apoptosis. MethodsMyocardial infarction (MI) model was established by the left-anterior descending coronary artery ligation. Norepinephrine (NE) was used to induce cardiomyocyte apoptosis. GAS5 levels and mRNA expressions of Semaphorin 3a (sema3a) were measured by qRT-PCR. Protein level of sema3a was detected by Western blotting. Cardiomyocyte apoptosis was detected by flow cytometry assay. RNA pull-down and RIP assay were used to verify the combination between GAS5 and sema3a. Infarct size was measured by TTC staining. ResultsGAS5 expression was increased in infarct boundary zone of MI group, while sema3a protein expression was decreased. Moreover, GAS5 expression in cardiomyocyte induced by NE was higher than control group, while sema3a protein expression was lower than control group. In addition, GAS5 could negatively regulate sema3a protein expression. pcDNA-GAS5 reversed cardiomyocyte apoptosis induced by NE, while pcDNA-sema3a countered the inhibitory effect. In animal experiment, overexpression of GAS5 decreased sema3a protein expression and reduced infarct size. ConclusionGAS5 could ameliorate cardiomyocyte apoptosis induced by MI via down-regulating sema3a.

WITHDRAWN: Suppression of lncR-30245 alleviates myocardial infarction induced cardiac fibrosis via the PPAR-γ/CTGF pathway

Abstract Background Long noncoding RNAs (lncRNAs) are emerging as important mediators of cardiac pathophysiology. The aim of the present study is to investigate the effects of an lncRNA lncR-30245 on cardiac fibrogenesis and the underlying mechanism. Methods Myocardial infarction (MI) and transforming growth factor beta1 (TGF-β1) were used to induce fibrotic phenotypes. Cardiac fibrosis was detected by Masson's trichrome staining. Cardiac function was evaluated by echocardiography. Western blot, quantitative RT-PCR, and pharmacological approaches were employed to investigate the role of lncR-30245 in cardiac fibrogenesis. Results Expression of lncR-30245 was significantly increased in MI hearts and TGF-β1-treated cardiac fibroblasts (CFs). LncR-30245 was mainly located in the cytoplasm. Overexpression of lncR-30245 promoted collagen production and CFs proliferation. Knockdown of lncR-30245 significantly inhibited TGF-β1-induced collagen production and CFs proliferation. LncR-30245 overexpression inhibited the anti-fibrotic role of peroxisome proliferator-activated receptor gamma (PPAR-γ) and increased connective tissue growth factor (CTGF) expression, while lncR-30245 knockdown exerted the opposite effects. Rosiglitazone, a PPAR-γ agonist, significantly inhibited lncR-30245-induced CTGF upregulation and collagen production in CFs. In contrast, T0070907, a PPAR-γ antagonist, attenuated the inhibitory effects of lncR-30245 siRNA on TGF-β1-induced CTGF expression and collagen production. Importantly, lncR-30245 knockdown significantly enhanced ejection fraction and fractional shortening and attenuated cardiac fibrosis in MI mice. Conclusion Our study indicates that the lncR-30245/PPAR-γ/CTGF pathway mediates MI-induced cardiac fibrosis and might be a therapeutic target for various cardiac diseases associated with fibrosis.

El suero de seno coronario tras un infarto de miocardio induce angiogénesis y reparación de la obstrucción microvascular. Implicación del factor inducible por hipoxia-1A

Introduction and objectivesMicrovascular obstruction (MVO) exerts deleterious effects following acute myocardial infarction (AMI). We investigated coronary angiogenesis induced by coronary serum and the role of hypoxia-inducible factor-1A (HIF-1A) in MVO repair. MethodsMyocardial infarction was induced in swine by transitory 90-minute coronary occlusion. The pigs were divided into a control group and 4 AMI groups: no reperfusion, 1minute, 1 week and 1 month after reperfusion. Microvascular obstruction and microvessel density were quantified. The proangiogenic effect of coronary serum drawn from coronary sinus on endothelial cells was evaluated using an in vitro tubulogenesis assay. Circulating and myocardial HIF-1A levels and the effect of in vitro blockade of HIF-1A was assessed. ResultsCompared with control myocardium, microvessel density decreased at 90-minute ischemia, and MVO first occurred at 1minute after reperfusion. Both peaked at 1 week and almost completely resolved at 1 month. Coronary serum exerted a neoangiogenic effect on coronary endothelial cells in vitro, peaking at ischemia and 1minute postreperfusion (32 ± 4 and 41 ± 9 tubes vs control: 3 ± 3 tubes; P < .01). Hypoxia-inducible factor-1A increased in serum during ischemia (5-minute ischemia: 273 ± 52 pg/mL vs control: 148 ± 48 pg/mL; P < .01) being present on microvessels of all AMI groups (no reperfusion: 67% ± 5% vs control: 15% ± 17%; P < .01). In vitro blockade of HIF-1A reduced the angiogenic response induced by serum. ConclusionsCoronary serum represents a potent neoangiogenic stimulus even before reperfusion; HIF-1A might be crucial. Coronary neoangiogenesis induced by coronary serum can contribute to understanding the pathophysiology of AMI.Full English text available from:www.revespcardiol.org/en

Coronary Serum Obtained After Myocardial Infarction Induces Angiogenesis and Microvascular Obstruction Repair. Role of Hypoxia-inducible Factor-1A

Introduction and objectivesMicrovascular obstruction (MVO) exerts deleterious effects following acute myocardial infarction (AMI). We investigated coronary angiogenesis induced by coronary serum and the role of hypoxia-inducible factor-1A (HIF-1A) in MVO repair. MethodsMyocardial infarction was induced in swine by transitory 90-minute coronary occlusion. The pigs were divided into a control group and 4 AMI groups: no reperfusion, 1minute, 1 week and 1 month after reperfusion. Microvascular obstruction and microvessel density were quantified. The proangiogenic effect of coronary serum drawn from coronary sinus on endothelial cells was evaluated using an in vitro tubulogenesis assay. Circulating and myocardial HIF-1A levels and the effect of in vitro blockade of HIF-1A was assessed. ResultsCompared with control myocardium, microvessel density decreased at 90-minute ischemia, and MVO first occurred at 1minute after reperfusion. Both peaked at 1 week and almost completely resolved at 1 month. Coronary serum exerted a neoangiogenic effect on coronary endothelial cells in vitro, peaking at ischemia and 1minute postreperfusion (32 ± 4 and 41 ± 9 tubes vs control: 3 ± 3 tubes; P < .01). Hypoxia-inducible factor-1A increased in serum during ischemia (5-minute ischemia: 273 ± 52 pg/mL vs control: 148 ± 48 pg/mL; P < .01) being present on microvessels of all AMI groups (no reperfusion: 67% ± 5% vs control: 15% ± 17%; P < .01). In vitro blockade of HIF-1A reduced the angiogenic response induced by serum. ConclusionsCoronary serum represents a potent neoangiogenic stimulus even before reperfusion; HIF-1A might be crucial. Coronary neoangiogenesis induced by coronary serum can contribute to understanding the pathophysiology of AMI.

Naoxintong inhibits myocardial infarction injury by VEGF/eNOS signaling-mediated neovascularization

Ethnopharmacological relevanceNaoxintong capsules (NXT), a traditional Chinese Medical preparation, are widely used for treatment of cardiovascular diseases, while the mechanism is still unclear. Materials and methodsMyocardial infarction (MI) was induced by ligation of the left coronary artery in mice. Echocardiographic measurements were performed to do physiological assessments of left ventricle (LV) function. Histological and immunohistochemical staining was used to determine infarct size, capillary density, tissue endothelial nitric oxide synthase (eNOS) expression. Bone Marrow Transplantation (BMT) model and flow cytometric (FCM) analyses were applied to assay endothelial progenitor cells (EPCs) mobilization. Quantitative Real-Time Reverse Transcription Polymerase Chain Reaction (qRT-PCR), Western blotting and enzyme-linked immunosorbent assay (ELISA) were performed to detect the expressions of vascular endothelial growth factor (VEGF), kinase domain region (KDR), phosphorylated-Akt (p-Akt), phosphorylated-eNOS (p-eNOS). ResultsNXT administration reduced myocardium fibrosis and increased myocardium capillary density in response to MI. NXT increased circulating Sca1+/ Fetal liver kinase 1 (Flk1)+ mononuclear cells (MNCs) and soluble Kit ligand (sKitL) of bone marrow (BM) in response to MI. In mice transplanted with green fluorescent protein (GFP) BM cells, NXT increased the numbers of GFP-positive cells at the border zone of the ischemic region in MI-induced mice. NXT increased the numbers of eNOS-expressing BM-derived cells in tissues, which was involved in increased the expressions of VEGF, KDR, p-eNOS, p-Akt in the myocardium. ConclusionNXT-mediated recovery in MI-induced mice was involved in mobilization and incorporation of bone marrow–derived EPCs/circulating angiogenic cells (CACs) leading to enhancement of neovascularization via VEGF/eNOS signaling.