Tissue kallikrein infusion prevents cardiomyocyte apoptosis, inflammation and ventricular remodeling after myocardial infarction

We investigated the effect of tissue kallikrein infusion on cardiac protection at acute and sub-acute phases after myocardial infarction (MI). Immediately after MI, rats were infused with purified tissue kallikrein, with or without icatibant (a kinin B2 receptor antagonist). Intramyocardial injection of kallikrein reduced myocardial infarct size and inhibited cardiomyocyte apoptosis at 1 day after MI associated with increased nitric oxide levels, Akt and glycogen synthase kinase-3beta phosphorylation and decreased caspase-3 activation. Kallikrein infusion for 7 days improved cardiac function, normalized left ventricular wall thickness and decreased monocyte/macrophage infiltration in the infarct heart. Kallikrein treatment reduced NADH oxidase expression and activity, superoxide formation and malondialdehyde levels, and reduced MAPK and Ikappa-Balpha phosphorylation, NF-kappaB activation and MCP-1 and VCAM-1 expression. Kallikrein's effects were all blocked by icatibant. These results indicate that kallikrein through kinin B2 receptor activation prevents apoptosis, inflammation and ventricular remodeling by increased nitric oxide formation and suppression of oxidative stress-mediated signaling pathways.     

RASSF1 promotes cardiomyocyte apoptosis after acute myocardial infarction and is regulated by miR-125b

Cardiomyocyte apoptosis is a common pathological injury in association with acute myocardial infarction (AMI). In the current study, the relationship between Ras-association domain family 1 (RASSF1) and cardiomyocyte apoptosis was investigated. RASSF1 was significantly over expressed in infarcted myocardial tissues as well as in cardiomyocytes induced by hypoxia. Inhibition of RASSF1 expression alleviated cardiomyocytes apoptosis induced by hypoxia in vitro and reduced cardiomyocytes apoptosis after AMI in vivo. RASSF1 expression was directly modulated by miR-125b, which was further confirmed by luciferase reporter assay. The current study verified that the miR-125b/RASSF1 axis was involved in cardiomyocytes apoptosis. To sum up, these results suggest that RASSF1 downregulation alleviated infarction-induced cardiomyocytes apoptosis and was regulated by miR-125b.     

Endothelium and myocyte cellular insulin receptor alterations in a rat model of myocardial infarction

Ischemic heart disease is considered to be one of the leading causes of death in adults. While extensive research on mechanisms contributing to the pathogenesis of myocardial infarction (MI) has been underway, it is not known whether insulin receptor characteristics and postreceptor signaling have been fully addressed as yet. Present work attempts to investigate whether the remodeling process effectively induces alteration(s) in insulin-binding characteristics at the coronary endothelium and cardiomyocytes using a rat heart model of MI. MI was induced by ligation of the left anterior descending coronary artery of adult male Sprague-Dawley rats. Two animal groups were used in the study: (i) sham-operated CHAPS-untreated and CHAPS-treated, and (ii) MI CHAPS-untreated and MI CHAPS-treated. A physical model describing 1:1 stoichiometry of reversible insulin binding to its receptors present on the endothelium and at cardiomyocytes after CHAPS treatment was considered for data analysis. Quantitation of the collected effluents after heart perfusion, the inlet at the aortic and outlet at the coronary sinus sites, were curve fitted using a first-order Bessel function, which determines the binding constants (k(n)), the reversible constant (k(-n)), the dissociation constant (k(d) = k(-n)/k(n)), and the residency time constant (tau = 1/k(-n)). In addition, hearts were excised, separated into right and left ventricles, and individually weighed, and areas of infarcted regions were measured. Results of the MI group showed significant increases in relative heart mass, left ventricle mass, and right ventricle mass normalized to total body mass. MI induced severe ischemia and irreversible myocardial injury as assessed by planimetry and histologic studies. The data showed differences in insulin receptor affinities at the endothelial and cardiac myocytes in the sham and in the MI-operated rats. The observed reduction in the binding affinity of insulin at the myocyte postinfarction may explain the pathogenic role of insulin in ischemic heart disease and, hence, resistance. Therefore, insulin administration during and post MI might be cardioprotective.     

Calcium-sensing receptor induces rat neonatal ventricular cardiomyocyte apoptosis

The calcium-sensing receptor (CaSR) exists in many tissues, and its expression has been identified in rat cardiac tissue. However, the physiological importance and pathophysiological involvement of CaSR in homeostatic regulation of cardiac function are unclear. To investigate the relation of CaSR and apoptosis in cardiomyocytes, we examined the role of the CaSR activator gadolinium chloride (GdCl(3)) in rat neonatal ventricular cardiomyocytes. Expression of the CaSR protein was observed by Western blot. The apoptotic ratio of rat neonatal ventricular cardiomyocytes was measured with flow cytometry and immunofluorescence techniques. A laser scan confocal microscope was used to detect the intracellular concentration of calcium ([Ca(2+)](i)) in rat neonatal ventricular cardiomyocytes using the acetoxymethyl ester of fluo-3 (fluo-3/(AM)) as a fluorescent dye. The results showed that GdCl(3) increased the phosphorylation of extracellular signal-regulated protein kinase (ERK), c-Jun NH(2)-terminal protein kinases (JNK), and p38. GdCl(3) also activated caspase 9 and increased apoptosis in myocyte by increasing [Ca(2+)](i). In conclusion, these results suggest that CaSR promotes cardiomyocyte apoptosis in rat neonatal ventricular cardiomyocytes through activation of mitogen-activated protein kinases and caspase 9 signaling pathways.     

Endothelin-1 receptor subtypes expression and binding in a perfused rat model of myocardial infarction

Endothelin-1 (ET-1) pathophysiologic actions are mediated via binding with two receptor subtypes, ET(A) and ET(B). Release of ET-1 from endocardial endothelial cells and cardiac myocytes can modulate heart tissue necrosis and alterations. This study investigates the remodeling processes in Sprague-Dawley rats of myocardial infarction (MI) induced by ligating the left anterior descending coronary artery. Histological studies were done on cell type distribution using cell specific markers and Western blot analysis to localize ET-1 receptor subtypes and assess their expression post-MI. In addition, the binding kinetics of ET-1 with its receptors in heart perfusion, inlet via the aortic lumen and effluent outlet via the right atrium, between two animal model-subgroups were done: (1) sham-operated, and sham-operated-CHAPS (3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate)-treated; and (2) MI-operated, and MI-operated-CHAPS-treated. Effluent ET-1 concentration was plotted vs. time using a physical model for 1:1 ligand-receptor binding at coronary endothelium and myocytes. First order impulse function was used to calculate the affinity constants. In MI hearts, fluorescence activity increased for ET(A) vs. ET(B) across areas of the muscle compared to normal hearts. Western blotting showed upregulation of ET(A) and ET(B) receptors in MI compared with normal hearts. Results of ET-1 binding affinity post-MI indicated drastic reduction in spite the upregulation of ET(B) on coronary endothelium. Furthermore, substantial affinity increase was observed between ET-1 binding with ET(A) at the myocyte site. These findings stipulate that during 1 month post-MI some biochemical and hormonal effects could alter ET-1 receptor subtype(s) regulation and pharmacodynamics thus predisposing to cardiac hypertrophy and mitogenesis.     

Curcumin ameliorated myocardial infarction by inhibition of cardiotoxicity in the rat model

Cardiovascular diseases are the main cause of death globally. Many attempts have been done to ameliorate the pathological changes after the occurrence of myocardial infarction. Curcumin is touted as a polyphenol phytocompound with appropriate cardioprotective properties. In this study, the therapeutic effect of curcumin was investigated on acute myocardial infarction in the model of rats. Rats were classified into four groups; control, isoproterenol hydrochloride (ISO) (100 mg/kbw), curcumin (50 mg/kbw), and curcumin plus ISO treatment groups. After 9-day administration of curcumin, levels of lactate dehydrogenase (LDH), creatine kinase (CK), and cardiac troponin I (cTnI) were determined. Superoxide dismutase (SOD) and malondialdehyde (MDA) contents were measured to investigate the oxidative status in infarct rats received curcumin. By using H & E staining, tissue inflammation was performed. Masson’s trichrome staining was conducted to show cardiac remodeling and collagen deposition. The number of apoptotic cells was determined by using the terminal deoxynucleotidyl transferase dUTP nick end labeling assay. Data showed the serum decrease of LDH, CK, and cTnI in infarct rats after curcumin intake compared to the rats given (ISO) ( P < 0.05). Curcumin was found to reduce oxidative status by reducing SOD and MDA contents ( P < 0.05). Gross and microscopic examinations revealed that the decrease of infarct area, inflammation response and collagen deposition in rats given ISO plus curcumin ( P < 0.05). We noted the superior effect of curcumin to reduce the number of apoptotic cardiomyocytes after 9 days. Data point the cardioprotective effect of curcumin to diminish the complication of infarction by the reduction of cell necrosis and apoptosis in a rat model of experimental infarction.     

Phosphoproteome mapping of cardiomyocyte mitochondria in a rat model of heart failure

Mitochondria are complex organelles essential to cardiomyocyte survival. Protein phosphorylation is emerging as a key regulator of mitochondrial function. In the study reported here, we analyzed subsarcolemmal (SSM) mitochondria harvested from rats who have received 4 weeks of aldosterone/salt treatment to simulate the neurohormonal profile of human congestive heart failure. Our objective was to obtain an initial qualitative inventory of the phosphoproteins in this biologic system. SSM mitochondria were harvested, and the phosphoproteome was analyzed with a gel-free bioanalytical platform. Mitochondrial proteins were digested with trypsin, and the digests were enriched for phosphopeptides with immobilized metal ion affinity chromatography. The phosphopeptides were analyzed by ion trap liquid chromatography–tandem mass spectrometry, and the phosphoproteins identified via database searches. Based on MS/MS and MS³ data, we characterized a set of 42 phosphopeptides that encompassed 39 phosphorylation sites. These peptides mapped to 26 proteins, for example, long-chain specific acyl-CoA dehydrogenase, Complex III subunit 6, and mitochondrial import receptor TOM70. Collectively, the characterized phosphoproteins belong to diverse functional modules, including bioenergetic pathways, protein import machinery, and calcium handling. The phosphoprotein panel discovered in this study provides a foundation for future differential phosphoproteome profiling toward an integrated understanding of the role of mitochondrial phosphorylation in heart failure.     

S-allylcysteine mediates cardioprotection in an acute myocardial infarction rat model via a hydrogen sulfide-mediated pathway

S-allylcysteine (SAC) is an organosulfur-containing compound derived from garlic. Studies have shown that garlic is beneficial in the treatment of cardiovascular diseases. This study aims to elucidate if SAC is responsible for this cardioprotection using acute myocardial infarction (AMI) rat models. In addition, we hypothesized that SAC may mediate cardioprotection via a hydrogen sulfide (H(2)S)-related pathway. Rats were pretreated with saline, SAC (50 mg x kg(-1) x day(-1)), SAC + propagylglycine (PAG; 50 mg + 10 mg x kg(-1) x day(-1)) or PAG (10 mg x kg(-1) x day(-1)) for 7 days before AMI induction and killed 48 h after. Our results showed that SAC significantly lowered mortality (12.5% vs. 33.3%, P < 0.05) and reduced infarct size. SAC + PAG- and PAG-treated rats had larger infarct sizes than controls (60.9 +/- 0.01 and 62.0 +/- 0.03%, respectively, vs. 50.0 +/- 0.03%; P < 0.05). Pretreatment with SAC did not affect BP, but BP was significantly elevated in SAC + PAG and PAG-treated groups (P < 0.05). In addition, plasma H(2)S levels and left ventricular cystathionine-gamma-lyase (CSE) activities were analyzed to investigate the involvement of H(2)S. CSE is the enzyme responsible for H(2)S production in the heart. SAC increased left ventricular CSE activity in AMI rats (2.75 +/- 0.34 vs. 1.23 +/- 0.16 micromol x g protein(-1) x h(-1); P < 0.01). SAC + PAG-treated rats had significantly lower CSE activity compared with the SAC-treated group (1.22 +/- 0.27 vs. 2.75 +/- 0.34 micromol x g protein(-1) x h(-1); P < 0.05). Similarly, SAC-treated rats had higher plasma H(2)S concentration compared with controls and the SAC + PAG-treated group. Protein expression studies revealed that SAC upregulated CSE expression (1.1-fold of control; P < 0.05), whereas SAC + PAG and PAG downregulated its expression (0.88-fold of control in both groups; P < 0.005). In conclusion, our study provides novel evidence that SAC is protective in myocardial infarction via an H(2)S-related pathway.     

Chrysin attenuates interstitial fibrosis and improves cardiac function in a rat model of acute myocardial infarction

Interstitial fibrosis after acute myocardial infarction (MI) leads to cardiac structural remodeling and dysfunction. The peroxisome proliferator-activated receptor-gamma (PPAR-γ) agonist chrysin has been shown to protect injured myocardium through suppression of oxidative stress and inflammation. This study was designed to investigate the effect and mechanism of chrysin on myocardial fibrosis. A rat MI model was created by ligating the left coronary artery. The rats with MI were treated with chrysin (40 mg/kg/day) or 0.5% carboxymethylcellulose sodium by intragastric administration for 4 weeks after operation. The effect of chrysin on cardiac fibroblasts (CFs) were also assessed in vitro. Echocardiography showed that cardiac function was significantly improved after chrysin treatment. Chrysin reduced the levels of MDA and SOD and GSH-Px in myocardial tissue. Chrysin attenuated the interstitial and perivascular fibrosis and the expression of collagenlin the peri-infarcted zone and remarkably decreased the levels of matrix metalloproteinase-2 (MMP-2) and MMP-9. Chrysin up-regulated PPAR-γ and inhibited the nuclear factor-kappa B (NF-κB) pathway by suppressing inhibitor kappa B kinase β phosphorylation. Immunohistochemistry analysis and PCR detected downregulated expression of AP-1 after chrysin treatment. Chrysin also markedly reduced the increased α-SMA, typeland type III collagen expression of CFs mediated by AngII in vitro. In conclusion, chrysin has an antifibrosis cardioprotective effect on the infarct peripheral zone after MI. The underlined mechanism may be the up-regulation of PPAR-γ and inhibition of the NF-κB and AP1 pathway.     

Post-infarct sleep disruption and its relation to cardiac remodeling in a rat model of myocardial infarction

Sleep disruption after myocardial infarction (MI) by affecting ubiquitin–proteasome system (UPS) is thought to contribute to myocardial remodeling and progressive worsening of cardiac function. The aim of current study was to test the hypothesis about the increased risk of developing heart failure due to experience of sleep restriction (SR) after MI. Male Wistar rats (n = 40) were randomly assigned to four experimental groups: (1) Sham, (2) MI, (3) MI and SR (MI + SR) (4) Sham and SR (Sham + SR). MI was induced by permanent ligation of left anterior descending coronary artery. Twenty-four hours after surgery, animals were subjected to chronic SR paradigm. Blood sampling was performed at days 1, 8 and 21 after MI for determination of serum levels of creatine kinase-MB (CK-MB), corticosterone, malondialdehyde (MDA) and nitric oxide (NO). Finally, at 21?days after MI, echocardiographic parameters and expression of MuRF1, MaFBx, A20, eNOS, iNOS and NF-kB in the heart were evaluated. We used H&;E staining to detect myocardial hypertrophy. We found out that post infarct SR increased corticosterone levels. Our results highlighted deteriorating effects of post-MI SR on NO production, oxidative stress, and echocardiographic indexes (p < 0.05). Moreover, its detrimental effects on myocardial damage were confirmed by overexpression of MuRF1, MaFBx, iNOS and NF-kB (p < 0.001) in left ventricle and downregulation of A20 and eNOS (p < 0.05). Furthermore, histological examination revealed that experience of SR after MI increased myocardial diameter as compared to Sham subjects (p < 0.05). Our data suggest that SR after MI leads to an enlargement of the heart within 21?days, marked by an increase in oxidative stress and NO production as well as an imbalance in UPS that ultimately results in cardiac dysfunction and heart failure.     

Mesenchymal stem cells modified with angiopoietin-1 improve remodeling in a rat model of acute myocardial infarction

We used human angiopoietin-1 (hAng1)-modified mesenchymal stem cells (MSCs) to treat acute myocardial infarction (AMI) in rats. The hAng1 gene was transfected into cultured rat MSCs using an adenoviral vector. Five million hAng-transfected MSCs (MSC(Ang1)) or green fluorescent protein transfected MSCs (MSC(GFP)) or PBS only (PBS group) were injected intramyocardially into the inbred Lewis rat hearts immediately after myocardial infarction. MSC(Ang1) survived in the infarcted myocardium, and expressed hAng1 at both mRNA and protein levels. The vascular density was higher in the MSC(Ang1) and MSC(GFP) groups than in the PBS group. The measurements of infarcted ventricular wall thickness, infarction area, and left ventricular diameter indicated that heart remodeling was inhibited and heart function was improved in both the MSC(Ang1) and MSC(GFP) groups. However, in contrast to the MSC(GFP) group, the MSC(Ang1) group showed enhanced angiogenesis and arteriogenesis (by 11-35%), infarction area was reduced by 30% and the left ventricular wall was 46% thicker (P<0.05). The results indicated that hAng1-modified MSCs improved heart function, followed by angiogenic effects in salvaging ischemic myocardium and reduced cardiac remodeling.     

Attenuation of left ventricular dysfunction by an ACE inhibitor after myocardial infarction in a kininogen-deficient rat model

Bradykinin (BK) coronary outflow and left ventricular (LV) performance of kininogen-deficient Brown Norway Katholiek (BNK) rats and Brown Norway Hannover (BNH) controls were investigated. We analyzed whether the angiotensin-converting enzyme (ACE) inhibitor ramipril is able to attenuate LV dysfunction after induction of myocardial infarction (MI) in this animal model. Ex vivo, the basal BK content in the coronary outflow of buffer-perfused, isolated hearts was measured by specific radioimmunoassay. In vivo, left ventricular pressure (LVP), the maximal rate of LVP increase, LV end-diastolic pressure, the maximal rate of LVP decrease and heart rate were determined using a tip catheter 3 weeks after induction of MI. Compared to BNK rats, basal BK outflow was increased 30-fold in controls (p<0.01). In vivo, we found no significant differences between sham-ligated BNK and BNH rats in basal LV function. After MI, the impairment of LV function was significantly worse in BNK rats when compared to BNH rats. ACE inhibition significantly attenuated this LV dysfunction in both groups, when compared to untreated animals. Reduced basal BK level resulting from kininogen deficiency has no effect on basal LV function, but remains to be a risk factor for the ischemic heart. However, ACE inhibition is sufficient to improve LV function despite kininogen deficiency.     

Melatonin protects ADSCs from ROS and enhances their therapeutic potency in a rat model of myocardial infarction

Myocardial infarction (MI) is a major cause of death and disability worldwide. In the last decade, mesenchymal stem cells (MSCs) based cell therapy has emerged as a promising therapeutic strategy. Although great advance have been made using MSCs to treat MI, the low viability of transplanted MSCs severely limits the efficiency of MSCs therapy. Here, we show evidence that ex vivo pre‐treatment with melatonin, an endogenous hormone with newly found anti‐oxidative activity, could improve survival and function of adipose tissue derived MSCs (ADSCs) in vitro as well as in vivo. ADSCs with 5 μM melatonin pre‐treatment for 24 hrs showed increased expression of the antioxidant enzyme catalase and Cu/Zn superoxide dismutase (SOD‐1), as well as pro‐angiogenic and mitogenic factors like insulin‐like growth factor 1, basic fibroblast growth factor, hepatocyte growth factor (HGF), epidermal growth factor. Furthermore, melatonin pre‐treatment protected MSCs from reactive oxygen species (ROS) induced apoptosis both directly by promoting anti‐apoptosis kinases like p‐Akt as well as blocking caspase cascade, and indirectly by restoring the ROS impaired cell adhesion. Using a rat model of MI, we found that melatonin pre‐treatment enhanced the viability of engrafted ADSCs, and promoted their therapeutic potency. Hopefully, our results may shed light on the design of more effective therapeutic strategies treating MI by MSCs in clinic.     

Layer-specific strain analysis: investigation of regional deformations in a rat model of acute versus chronic myocardial infarction

Myocardial infarction (MI) injury extends from the endocardium toward the epicardium. This phenomenon should be taken into consideration in the detection of MI. To study the extent of damage at different stages of MI, we hypothesized that measurement of layer-specific strain will allow better delineation of the MI extent than total wall thickness strain at acute stages but not at chronic stages, when fibrosis and remodeling have already occurred. After baseline echocardiography scans had been obtained, 24 rats underwent occlusion of the left anterior descending coronary artery for 30 min followed by reperfusion. Thirteen rats were rescanned at 24 h post-MI and eleven rats at 2 wk post-MI. Next, rats were euthanized, and histological analysis for MI size was performed. Echocardiographic scans were postprocessed by a layer-specific speckle tracking program to measure the peak circumferential strain (S(C)(peak)) at the endocardium, midlayer, and epicardium as well as total wall thickness S(C)(peak). Linear regression for MI size versus S(C)(peak) showed that the slope was steeper for the endocardium compared with the other layers (P < 0.001), meaning that the endocardium was more sensitive to MI size than the other layers. Moreover, receiver operating characteristics analysis yielded better sensitivity and specificity in the detection of MI using endocardial S(C)(peak) instead of total wall thickness S(C)(peak) at 24 h post-MI (P < 0.05) but not 2 wk later. In conclusion, at acute stages of MI, before collagen deposition, scar tissue formation, and remodeling have occurred, damage may be nontransmural, and thus the use of endocardial S(C)(peak) is advantageous over total wall thickness S(C)(peak).     

Lipopolysaccharide preconditioning enhances the efficacy of mesenchymal stem cells transplantation in a rat model of acute myocardial infarction

Background  

Mesenchymal stem cells (MSCs)-based regenerative therapy is currently regarded as an alternative approach to salvage the acute myocardial infarcted hearts. However, the efficiency of MSCs transplantation is limited by lower survival rate of engrafted MSCs. In previous study, we found that 1.0 μg/ml Lipopolysaccharide (LPS) could protect MSCs against apoptosis induced by oxidative stress and meanwhile enhance the proliferation of MSCs. Therefore, in the present study, we firstly preconditioned MSCs with 1.0 μg/ml LPS, then transplanted MSCs into ischemic myocardium, and observed the survival and cardiac protective capacity of MSCs in a rat model of acute myocardial infarction. Furthermore, we tried to explore the underlying mechanisms and the role of Toll-like receptor-4 (TLR4) in the signal pathway of LPS-induced cardiac protection.