Expression In Left Ventricle Research Articles

S-12-4: CITE-SEQ IDENTIFIES TREM2-DEPENDENT GENE PROGRAM DRIVING CARDIAC REMODELING IN A HYPERTENSIVE MODEL OF HEART FAILURE WITH PRESERVED EJECTION FRACTION

Objective: The deoxycorticosterone acetate (DOCA)-salt model of hypertension has been used to study diastolic dysfunction and heart failure with preserved ejection fraction (HFpEF) in rats. Our goal was to validate DOCA-salt as a mouse model of HFpEF and to identify immune mediators of cardiac remodeling using CITE-Seq, a novel single cell technique that provides surface marker and transcriptomic data. Methods: DOCA-salt mice underwent uninephrectomy, DOCA pellet implantation, and 1% NaCl supplementation of the drinking water for three weeks. Control mice underwent a sham procedure and received normal water. Echocardiography and invasive hemodynamics were performed to examine cardiac function, and urine was collected to examine renal function. CITE-seq was performed on sorted leukocytes from four sham and DOCA-salt left ventricles. To test whether Trem2 has a causal role in HFpEF, wild type and Trem2 deficient animals were subjected to DOCA-salt. To evaluate translational potential, soluble Trem2 was measured in plasma of patients with HFpEF and healthy controls. Results: Compared to control mice, DOCA-salt mice exhibit significantly increased systolic blood pressure, albuminuria, and cardiac hypertrophy. On echocardiography, DOCA-salt mice exhibited a preserved ejection fraction. Invasive hemodynamic studies revealed an increased tau constant (5.70 ± .24, 8.22 ± .45; n = 8,5) in DOCA-salt mice, indicating diastolic dysfunction. CITE-seq revealed major transcriptional changes in cardiac macrophages between groups with upregulation of genes known to be involved in HFpEF and cardiac remodeling, such as galectin-3 (Lgals3) and osteopontin (Spp1), as well as novel genes including Trem2. Using an independent cohort of Sham and DOCA-salt treated mice, we validated a two-fold increase in Trem2 gene expression in left ventricles by qRT-PCR and a significant increase in Trem2 mRNA in Cd68+ macrophages by RNAscope. After DOCA-salt treatment, Trem2-deficient mice displayed increased heart weight to tibia length ratios (9.39 ± 0.20, 11.21 ± 0.80; n = 6–7) and albumin to creatinine ratios (5.5 ± 1.2, 14.2 ± 2.9; n = 6–7) compared to wild-type controls. No difference in albuminuria was observed at baseline (0.36 ± 0.05, 0.46 ± 0.10; n = 10). Bone marrow-derived macrophages (BMDMs) from Trem2 deficient mice expressed significantly less Lgals3 and Spp1 compared to wild type BMDMs suggesting that Trem2 signaling is upstream of galectin 3 and osteopontin in HFpEF. Plasma levels of soluble Trem2 were elevated in patients with HFpEF compared to healthy controls (51 ± 9.5; 22.4 ± 2.4 ng/mL; n = 9,12). Conclusions: The DOCA-salt mouse model induces key features of human HFpEF and identifies a Trem2-dependent gene program involved in cardiac remodeling. Loss of Trem2 resulted in increased cardiac hypertrophy and renal damage in the DOCA-salt model of HFpEF.

Abstract P3015: Cite-seq Identifies A Novel Role For Macrophage Trem2 In A Murine Model Of Hfpef

Deoxycorticosterone acetate (DOCA)-salt is a common model of hypertension and has been used to study heart failure with preserved ejection fraction (HFpEF) in rats. Our goal was to validate DOCA-salt as a mouse model of HFpEF and to identify immune mediators of cardiac remodeling using unbiased methods. DOCA-salt mice underwent uninephrectomy, implantation of a DOCA pellet, and supplementation of the drinking water with 1%NaCl for three weeks. Control mice underwent a sham procedure and received normal water. Compared to control mice, DOCA-salt mice exhibited increased systolic blood pressure, cardiac hypertrophy, and decreased time to exhaustion upon treadmill exercise testing. In addition, DOCA-salt mice exhibited albuminuria, indicative of renal glomerular injury. On echocardiography, DOCA-salt mice exhibited a preserved ejection fraction. Invasive hemodynamic studies revealed an increased tau constant (5.70±.24, 8.22±.45; n=8,5) in DOCA-salt mice, consistent with diastolic dysfunction. CITE-seq, a novel technique to obtain transcriptomic and surface marker expression on single cells, was performed on a total of 4,359 and 7,600 sorted leukocytes from four sham and four DOCA-salt left ventricles, respectively. Differential gene expression analysis revealed major transcriptional changes in cardiac macrophages between groups and identified Trem2 as being upregulated in cardiac macrophages. We validated a two-fold increase in Trem2 gene expression in left ventricles of DOCA-salt mice and observed a two-fold increase in cardiac Trem2+Mac2+ cells in DOCA-salt by immunofluorescence. To test whether Trem2 has a causal role in HFpEF, wild type and Trem2 deficient animals were subjected to DOCA-salt. After DOCA-salt, Trem2-deficient mice displayed significantly increased heart weight to tibia length ratios (9.39±0.20, 11.21±0.80; n=6-7) and albumin to creatinine ratios (5.5±1.2, 14.2±2.9; n=6-7) compared to wild-type controls. No difference in albuminuria was observed at baseline (0.36±0.05, 0.46±0.10; n=10). In conclusion, the DOCA-salt mouse model induces key features of HFpEF and identifies Trem2 as a potential novel modulator of cardiac remodeling and renal injury.

Identification of potential dilated cardiomyopathy-related targets by meta-analysis and co-expression analysis of human RNA-sequencing datasets

AimsDilated cardiomyopathy (DCM) remains among the most refractory heart diseases because of its complicated pathogenesis, and the key molecules that cause it remain unclear. Main methodsTo elucidate the molecules and upstream pathways critical for DCM pathogenesis, we performed meta-analysis and co-expression analysis of RNA-sequencing (RNA-seq) datasets from publicly available databases. We analyzed three RNA-seq datasets containing comparisons of RNA expression in left ventricles between healthy controls and DCM patients. We extracted differentially expressed genes (DEGs) and clarified upstream regulators of cardiovascular disease-related DEGs by Ingenuity Pathway Analysis (IPA). Weighted Gene Co-expression Network Analysis (WGCNA) and Protein–Protein Interaction (PPI) analysis were also used to identify the hub gene candidates strongly associated with DCM. Key findingsIn total, 406 samples (184 healthy, 222 DCM) were used in this study. Overall, 391 DEGs [absolute fold change (FC) ≥ 1.5; P < 0.01], including 221 upregulated and 170 downregulated ones in DCM, were extracted. Seven common hub genes (LUM, COL1A2, CXCL10, FMOD, COL3A1, ADAMTS4, MRC1) were finally screened. IPA showed several upstream transcriptional regulators, including activating (NFKBIA, TP73, CALR, NFKB1, KLF4) and inhibiting (CEBPA, PPARGC1A) ones. We further validated increased expression of several common hub genes in the transverse aortic constriction-induced heart failure model. SignificanceIn conclusion, meta-analysis and WGCNA using RNA-seq databases of DCM patients identified seven hub genes and seven upstream transcriptional regulators.

The effects of eight weeks high intensity interval training on the levels of endothelial nitric oxide synthase (eNOS) gene expression in left ventricle of type 2 diabetic rats

Background: According to previous research’s, the function of endothelial nitric oxide synthase (eNOS) impairs in diabetes. Therefore, the present study conducted aimed to investigate the effects of eight weeks high intensity interval training (HIIT) on the levels of eNOS gene expression in left ventricle tissue of type 2 diabetic rats. Methods: in experimental research, the 14 male Wistar rats with eight weeks old and the average weight of 160±10 g feeding of high fat and sweet diets (30% fat and 25% fructose) for seven months. The rats after confirming the type 2 diabetes, randomly assigned into two groups of six rats consist of control and HIIT groups. The rats in the HIIT group trained for eight weeks and five sessions per week. The 24 hours after last training sessions, blood samples collected and the left ventricle removed and the levels of eNOS gene expression measured by Real Time PCR assay in heart tissue. Results: There was a significant increase in eNOS gene expression in HIIT group compared to the control group (p=0.011). Moreover, the levels of glucose, insulin and insulin resistance (HOMA-IR) was significantly lower in the HIIT group compared to the control group (p&lt;0.05). Conclusion: It seems that HIIT play an important role in improving heart tissue endothelial function and improve the insulin sensitivity in diabetic rats by high fat and sweet diets.

The effects of swimming training on N-cadherin, β-catenin and Emerin in cardiac left ventricle in male Wistar rat

Background: Physical activity and exercise training plays a protective role against cardiovascular disease via reducing risk factors. Swimming as one of the best aerobic activity and exercise modality recommended for preventing and treatment of cardiovascular disease. On the other hand, exercise training could effects cardiac muscle protein structure. Therefore, the propose of this study was to investigate the effects of eight-week swimming training on N-cadherin, beta catenin and Emerin gene expression in left ventricle of cardiac muscle in male Wistar rat. Methods: In this study, twenty four male Wistar rat (8-week old) with an average (weight 237±33 in gr) randomly divided to three groups: Control (n=8), 8- week control (n=8) and swimming training (n=8). Swimming group participated in a swimming training for 8- week (5 seasons per week of 30 minutes floating on the water with moderate intensity) and the control group continued their usual lives. Twenty four hours after the last training session, the heart tissue of rats were extracted and β-catenin, N-cadherin and Emerin gene expression evaluated by Real Time-PCR. Data were statistically analyzed by one-way ANOVA and post-hoc Tukey methods (P<0.05). Results: The results of the study showed the expression of genes N-cadherin and beta catenin significantly higher in the swimming group than in the control group. However, Emerin gene expression in swimming group was significantly lower than in the control group. Conclusion: According to these findings, it seems that eight-weeks of swimming training an effective way of improving the structure and function of the heart muscle cells by increasing beta catenin and N-cadherin.

Cartilage intermediate layer protein-1 alleviates pressure overload-induced cardiac fibrosis via interfering TGF-β1 signaling

Cardiac fibrosis is characterized by excessive deposition of extracellular matrix (ECM) proteins in the myocardium and results in decreased ventricular compliance and diastolic dysfunction. Cartilage intermediate layer protein-1 (CILP-1), a novel identified cardiac matricellular protein, is upregulated in most conditions associated with cardiac remodeling, however, whether CILP-1 is involved in pressure overload-induced fibrotic response is unknown. Here, we investigated whether CILP-1 was critically involved in the fibrotic remodeling induced by pressure overload. Western blot analysis and immunofluorescence staining showed that CILP-1 was predominantly detected in cardiac myocytes and to a less extent in the interstitium. In isolated adult mouse ventricular myocytes and nonmyocytes, CILP-1 was found to be mainly synthesized by myocytes. CILP-1 expression in left ventricles was upregulated in C57BL/6 mice undergoing transverse aortic constriction (TAC). Myocardial CILP-1 knockdown aggravated whereas CILP-1 overexpression attenuated TAC-induced ventricular remodeling and dysfunction, as measured by echocardiography test, morphological examination, and gene expressions of fibrotic molecules. Incubation of cardiac fibroblasts with the conditioned medium containing full-length, N-terminal, or C-terminal CILP-1 inhibited transforming growth factor (TGF)-β1-induced Smad3 phosphorylation and the subsequent profibrotic events. We first demonstrated that C-terminal CILP-1 increased Akt phosphorylation, promoted the interaction between Akt and Smad3, and suppressed Smad3 phosphorylation. Blockade of PI3K-Akt pathway attenuated the inhibitory effect of C-CILP-1 on TGF-β1-induced Smad3 activation. We conclude that CILP-1 is a novel ECM protein possessing anti-fibrotic ability in pressure overload-induced fibrotic remodeling. This anti-fibrotic effect of CILP-1 attributes to interfering TGF-β1 signaling through its N- and C- terminal fragments.

Abstract 115: Myocardial Hypertrophy Upregulates the Calcium-activated Chloride Channel TMEM16A in the Heart

Background: Transmembrane member 16A (TMEM16A), or Anoctamin-1, is a protein encoded by the Ano1 gene. TMEM16A forms a voltage-sensitive calcium-activated chloride (Cl - ) channel in the cardiac atrial and ventricular myocytes. In this study we tested our hypothesis that activation of TMEM16A may be a novel adaptive mechanism for the pressure-overload induced structural and electrical remodeling of the heart . Methods: Wild type C57BL/6 (8-10 week old, male) mice were subjected to minimally-invasive transaortic banding (MTAB) surgery to create a pressure-over load myocardial hypertrophy model. Echocardiography at 2-D and M-mode were performed weekly after MTAB or sham-operation to monitor changes in left ventricular structure (wall thickness and chamber diameters) and cardiac function such as ejection fraction (EF) and fraction shortening (%FS). TMEM16A protein expression in left ventricles of control mice subjected to sham-operation or MTAB was estimated by capillary electrophoresis immunoblotting system (or Simple Western) using Wes platform. Whole-cell voltage- or current- clamp recordings were used to examine the functional expression of TMEM16A in left ventricular myocytes. Results: MTAB caused a time-dependent increase the left ventricular wall mass (LVWM) and wall thickness in the mice while no significant changes in the EF and SF, TMEM16A expression was increased significantly, which picked at week 4 and maintained at an expression level higher than normal control at week 10 after MTAB. Electrophysiological recordings followed the similar pattern of changes as the molecular expression of TMEM16A. Conclusions: Myocardial hypertrophy caused a significant upregulation of TMEM16A Cl - channels in cardiac myocytes and it may provide a novel adaptive mechanism for the structural and electrical remodeling induced by pressure overload.

Transcriptome analysis reveals potential mechanisms underlying differential heart development in fast- and slow-growing broilers under heat stress

BackgroundModern fast-growing broilers are susceptible to heart failure under heat stress because their relatively small hearts cannot meet increased need of blood pumping. To improve the cardiac tolerance to heat stress in modern broilers through breeding, we need to find the important genes and pathways that contribute to imbalanced cardiac development and frequent occurrence of heat-related heart dysfunction. Two broiler lines – Ross 708 and Illinois – were included in this study as a fast-growing model and a slow-growing model respectively. Each broiler line was separated to two groups at 21 days posthatch. One group was subjected to heat stress treatment in the range of 35–37 °C for 8 h per day, and the other was kept in thermoneutral condition. Body and heart weights were measured at 42 days posthatch, and gene expression in left ventricles were compared between treatments and broiler lines through RNA-seq analysis.ResultsBody weight and normalized heart weight were significantly reduced by heat stress only in Ross broilers. RNA-seq results of 44 genes were validated using Biomark assay. A total of 325 differentially expressed (DE) genes were detected between heat stress and thermoneutral in Ross 708 birds, but only 3 in Illinois broilers. Ingenuity pathway analysis (IPA) predicted dramatic changes in multiple cellular activities especially downregulation of cell cycle. Comparison between two lines showed that cell cycle activity is higher in Ross than Illinois in thermoneutral condition but is decreased under heat stress. Among the significant pathways (P < 0.01) listed for different comparisons, “Mitotic Roles of Polo-like Kinases” is always ranked first.ConclusionsThe increased susceptibility of modern broilers to cardiac dysfunction under heat stress compared to slow-growing broilers could be due to diminished heart capacity related to reduction in relative heart size. The smaller relative heart size in Ross heat stress group than in Ross thermoneutral group is suggested by the transcriptome analysis to be caused by decreased cell cycle activity and increased apoptosis. The DE genes in RNA-seq analysis and significant pathways in IPA provides potential targets for breeding of heat-tolerant broilers with optimized heart function.

Increase of the rate of \"mediator complex subunit 13\" gene expression in left ventricle due to endurance activity

Increase of the rate of "mediator complex subunit 13" gene expression in left ventricle due to endurance activity

Contribution of mammalian target of rapamycin in the pathophysiology of cirrhotic cardiomyopathy.

To explore the role of mammalian target of rapamycin (mTOR) in the pathogenesis of cirrhotic cardiomyopathy and the potential of rapamycin to improve this pathologic condition. Male albino Wistar rats weighing 100-120 g were treated with tetrachloride carbon (CCl4) for 8 wk to induce cirrhosis. Subsequently, animals were administered rapamycin (2 mg/kg per day). The QTc intervals were calculated in a 5-min electrocardiogram. Then, the left ventricular papillary muscles were isolated to examine inotropic responsiveness to β-adrenergic stimulation using a standard organ bath equipped by Powerlab system. Phosphorylated-mTOR localization in left ventricles was immunohistochemically assessed, and ventricular tumor necrosis factor (TNF)-α was measured. Western blot was used to measure levels of ventricular phosphorylated-mTOR protein. Cirrhosis was confirmed by hematoxylin and eosin staining of liver tissues, visual observation of lethargy, weight loss, jaundice, brown urine, ascites, liver stiffness, and a significant increase of spleen weight (P < 0.001). A significant prolongation in QTc intervals occurred in cirrhotic rats exposed to CCl4 (P < 0.001), while this prolongation was decreased with rapamycin treatment (P < 0.01). CCl4-induced cirrhosis caused a significant decrease of contractile responsiveness to isoproterenol stimulation and a significant increase in cardiac TNF-α. These findings were correlated with data from western blot and immunohistochemical studies on phosphorylated-mTOR expression in left ventricles. Phosphorylated-mTOR was significantly enhanced in cirrhotic rats, especially in the endothelium, compared to controls. Rapamycin treatment significantly increased contractile force and myocardial localization of phosphorylated-mTOR and decreased cardiac TNF-α concentration compared to cirrhotic rats with no treatment. In this study, we demonstrated a potential role for cardiac mTOR in the pathophysiology of cirrhotic cardiomyopathy. Rapamycin normalized the inotropic effect and altered phosphorylated-mTOR expression and myocardial localization in cirrhotic rats.

Differential Regulation of MicroRNAs in End-Stage Failing Hearts Is Associated with Left Ventricular Assist Device Unloading

Mechanical unloading by left ventricular assist devices (LVADs) in advanced heart failure (HF), in addition to improving symptoms and end-organ perfusion, is supposed to stimulate cellular and molecular responses which can reverse maladaptive cardiac remodeling. As microRNAs (miRNAs) are key regulators in remodeling processes, a comparative miRNA profiling in transplanted hearts of HF patients with/without LVAD assistance could aid to comprehend underlying molecular mechanisms. Next generation sequencing (NGS) was used to analyze miRNA differential expression in left ventricles of HF patients who underwent heart transplantation directly (n = 9) or following a period of LVAD support (n = 8). After data validation by quantitative real-time PCR, association with functional clinical parameters was investigated. Bioinformatics' tools were then used for prediction of putative targets of modulated miRNAs and relative pathway enrichment. The analysis revealed 13 upregulated and 10 downregulated miRNAs in failing hearts subjected to LVAD assistance. In particular, the expression level of some of them (miR-338-3p, miR-142-5p and -3p, miR-216a-5p, miR-223-3p, miR-27a-5p, and miR-378g) showed correlation with off-pump cardiac index values. Predicted targets of these miRNAs were involved in focal adhesion/integrin pathway and in actin cytoskeleton regulation. The identified miRNAs might contribute to molecular regulation of reverse remodeling and heart recovery mechanisms.

Reduction of prohibitin expression contributes to left ventricular hypertrophy via enhancement of mitochondrial reactive oxygen species formation in spontaneous hypertensive rats

Left ventricular hypertrophy (LVH) in hypertension is characterized by thickening of myocardium and decrease in heart chamber volume in response to mechanical or pathological stress, but the underlying molecular mechanisms remain to be defined. In this work, we investigate whether mitochondrial prohibitin (PHB) was involved in the progression of LVH in spontaneous hypertensive rats (SHR). First, it was found that mitochondrial dysfunction occurred in left ventricles of SHR. Through analysis using quantitative reverse transcription polymerase chain reaction and Western blotting, it was found that PHB mRNA and mitochondrial PHB levels in left ventricles of SHR were significantly lower than that in Wistar–Kyoto rats. Furthermore, PHB mRNA levels were negatively correlated to left ventricles weight-to-body weight ratio in SHR. Knockdown of PHB led to increased formation of mitochondrial reactive oxygen species (ROS) and reduced activities of complex I, mitochondrial adenosine triphosphate generation and mitochondrial membrane potential in cultured cardiomyocytes. Knockdown of PHB contributed to the cardiomyocyte hypertrophy, which could be attenuated by treatment with the Tempol. Angiotensin II (AngII) was increased in plasma and left ventricles of SHR. Incubation with AngII reduced mitochondrial PHB expression in cardiomyocytes, which was reversed when pretreated with losartan. In conclusion, reduction of PHB expression in left ventricles in SHR contributed to LVH, at least in part, through promoting mitochondrial ROS formation.

Deficient prolylcarboxypeptidase gene and protein expression in left ventricles of spontaneously hypertensive rats (SHR)

Prolylcarboxypeptidase (PRCP), an endothelial cell membrane serine peptidase that inactivates angiotensin II and activates pre-kallikrein, is thought to have anti-hypertensive and anti-proliferative roles in cardiovascular homeostasis. We hypothesized that PRCP function may be altered in heart tissue under conditions that predispose to left ventricle hypertrophy (LVH) in rats. We therefore used real-time PCR and western-blotting to examine the mRNA and protein expression of PRCP in the hearts of spontaneously hypertensive rats (SHR) at pre-hypertensive (5-week-old) and hypertensive (16-week-old) stages compared with age-matched hypertensive (2 kidney-1 clip; 2K-1C) rats and normotensive Wistar rats. PRCP mRNA expression was significantly reduced in hearts of 5- and 16-week-old SHR compared with age-matched Wistar controls, 2K-1C hypertensive rats and sham-operated normotensive rats. There were no significant differences in the PRCP mRNA and protein expression levels in hearts from hypertensive renovascular and sham-operated normotensive rats. Prolonged treatment of SHR with the AT1 receptor antagonist losartan (40mg/kg, gavage for 8 weeks) reduced the left ventricular weight/body weight ratio (LVW/BW), as well as the mRNA expression of collagen type 1, collagen type 3 and MMP9 in left ventricular tissue, without affecting PRCP gene and protein expression. Our results suggest that diminished PRCP gene and protein expression might be constitutionally involved in the SHR phenotype. In addition, since neither the development of arterial hypertension in the 2K-1C model nor its successful treatment in SHR altered PRCP gene and protein expression in heart tissue, it appears unlikely that PRCP function is regulated by the renin–angiotensin system or by afterload conditions.

Bioinformatics analysis of time series gene expression in left ventricle (LV) with acute myocardial infarction (AMI)

This study is to investigate the key genes and their possible function in acute myocardial infarction (AMI). The data of GSE4648 downloaded from the Gene Expression Omnibus (GEO) database include 6 time points (15 min, 60 min, 4h, 12h, 24h and 48 h) of 12 left ventricle (LV) samples, 12 surviving LV free wall (FW) samples, 12 inter-ventricular septum (IVS) samples after AMI operation and corresponding sham-operated samples. The data of each sample were analyzed with Affy and Bioconductor packages, and differentially expressed genes (DEGs) were screened out using BETR package with false discovery rate (FDR)<0.01. Then, functional enrichment analysis for DEGs was conducted with Database for Annotation, Visualization and Integrated Discovery (DAVID). Totally 194 DEGs were identified in LV, and only the gene tubulin beta 2a (Tubb2a) and natriuretic peptide B (Nppb) were respectively up-regulated in surviving FW tissue and IVS tissue. The biological process response to wounding and inflammatory response were significantly enriched, as well as leukocyte transendothelial migration pathway. Besides, the expression pattern analysis showed the DEGs mostly up-regulated at 4h after AMI, and these genes were mainly associated with immunity. Additionally, in transcriptional regulatory network, early growth response 1 (Egr1), activating transcription factor 3 (Atf3), Atf4, Myc and Fos were considered as the key transcription factors related to immune response. The key transcription factors and potential target genes might provide new information for the development of AMI, and leukocyte transendothelial migration pathway might play a vital role in AMI.

TRPM4 channel re-expression after myocardial infarction is essential for survival in mice

Introduction: TRPM4 is a Ca2+-activated non selective cation channel involved in conduction disorders. TRPM4 is expressed in sinoatrial and atrial cells but only poorly in ventricles. However, in vitro studies detected this current in cardiac hypertrophy concomitantly with fetal gene re-expression. The aim of our study was to investigate whether TRPM4 plays a functional role in ventricular cardiomyocytes in hypertrophy following Myocardial Infarction (MI). Methods: MI was induced by left coronary artery ligation in Trpm4-gene knock-out (Trpm4-/-) and Trpm4+/+ mice (23 and 12, respectively). Post-MI heart failure was verified using Doppler-echocardiography. Electrocardiograms (ECGs) were recorded by telemetry during 12h. Left Ventricular (LV) cardiomyocytes were isolated 4 weeks after MI. Action Potential (AP) and TRPM4-current were recorded using the patch-clamp technique. The propensity of cells to trigger spontaneous electrical activities was assessed. Store-Operated Ca2+-Entry (SOCE), negatively regulated by TRPM4, was measured using fluorescence microscopy. TRPM4 transcript and protein expression in left ventricle were studied using qPCR and western blot analysis. Results: Kaplan-Meier analysis evidenced a higher mortality rate for Trpm4-/–MI mice when compared with Trpm4+/+-MI animals (56% vs. 91%, P=0.03). Echocardiograms showed increased LV mass (3.85±0.4 vs. 2.49±0.2 mg/g, P=0.009) while fractional shortening was reduced (15.2±1.5 vs. 22.7±2.2, P=0.013) in Trpm4-/–MI vs. Trpm4+/+-MI mice. The number of premature ventricular complexes was increased in Trpm4-/–MI vs. Trpm4+/+-MI mice (57±5 vs. 35±3, P=0.028). Surprisingly, there was no difference in the lengthening of AP in LV myocytes following MI between Trpm4-/- and Trpm4+/+ (APD90: 94.4±23 vs. 88±15 ms, P=0.58, respectively). However, Trpm4-/–MI cells exhibited more EADs and DADs than Trpm4+/+-MI (EADs: 11/25 vs. 6/30, P=0.02; DADs: 8/25 vs. 2/30, P=0.008). Moreover, an increase of Trpm4 transcript was found in Trpm4+/+-MI ventricle, associated with TRPM4 protein presence (western blot) and function (TRPM4 current: 1.28±0.1 vs. 0.01±0.06 pA/pF at +40 mV, P=0.001). This expression correlated to a decrease of SOCE in cardiomyocytes from MI group vs. sham group (P=0.001). Conclusion: Lack of TRPM4 channel re-expression in the LV worsened the deleterious effects of post-MI cardiac remodeling on survival, hypertrophy, heart function, and arrhythmic events. Negative regulation of SOCE, involved in cardiac remodeling, and occurrence of spontaneous cellular arrhythmias suggests that TRPM4 channel plays a critical role in post-MI adaptations.

239 THE ROLE OF CA2+/CALMODULIN-DEPENDENT PROTEIN KINASE II IN NECROPTOTIC AND APOPTOTIC CELL DEATH IN MYOCARDIUM SUBJECTED TO ISCHEMIA-REPERFUSION

BackgroundNecroptosis, alternatively termed programmed necrosis, is a relatively recently discovered form of cell death. Cellular pathways responsible for necroptosis execution are currently being slowly elucidated and based on current findings...

Myocardial AT1 gene expression is increased in a model of left ventricular hypertrophy induced by high salt intake.

ObjectiveTo evaluate gene expression of the renin‐angiotensin system components in a model of left ventricular hypertrophy induced by high salt intake demonstrated in a previous study.MethodsWistar rats were fed normal (NS1.3% NaCl) or high (HS 8%) salt diet since weaning. From the 7th week of age, two HS subgroups received hydralazine or losartan (HZ 15 and LOS 20mg/kg/day). Angiotensinogen, renin, ACE, ACE2, AT1 and AT2 gene expression in left ventricle were measured.Results (n=8/group) NS HS HS+HZ HS+LOS Angiotensinogen/GAPDH (IDV) 0.21±0.017a 0.19±0.017a 0.21±0.013a 0.19±0.024a Renin/GAPDH (IDV) 0.71±0.02a 0.68±0.02a 0.67±0.02a 0.67±0.02a ACE/GAPDH (IDV) 0.87±0.03a 0.83±0.05a 0.97±0.06a 097±0.05 a ACE2/GAPDH (IDV) 0.41±0.02a 0.43±0.02a 0.37±0.03a 0.34±0.02a AT1/GAPDH (IDV) 0.23±0.00a 0.46±0.06b 0.45±0.06b 0.22±0.00a AT2/GAPDH (IDV) 0.52±0.04a 0.51±0.03a 0.45±0.03a 0.44±0.02a Data are reported as mean±SEM. Means in row with superscripts without a common letter differ, P&lt;0.05 ConclusionHigh salt intake increases AT1 gene expression, however it is prevented by losartan treatment. Supported by FAPESP and CNPq.

Physiological regulation of pro-inflammatory cytokines expression in rat cardiovascular tissues by sympathetic nervous system and angiotensin II

Pro-inflammatory cytokines regulation by sympathetic nervous system (SNS) and angiotensin II (ANG II) was widely described in cardiovascular system, but the role of such neuro-humoral interaction needs further investigation in this context. We tested SNS-ANG II interaction on IL-6 and TNF-α mRNA expression in left ventricle and aorta from normotensive rats by sympathectomy with guanethidine and blockade of the ANG II AT1 receptors (AT1R) antagonist with losartan. mRNA synthesis of IL-6 and TNF-α were performed by Q-RT-PCR. In the left ventricle, IL-6 mRNA increased by 63% (p < 0.01) after sympathectomy, still unchanged after losartan treatment and decreased by 38% (p < 0.05) after combined treatment. TNF-α mRNA decreased by 44% (p < 0.01), only after combined treatment. In the aorta, IL-6 mRNA increased equally by 65% (p < 0.05) after sympathectomy or losartan treatment. TNF-α mRNA decreased by 28, 41, and 42% (p < 0.05) after sympathectomy, losartan and combined treatments, respectively. Our data suggest that ANG II stimulates directly (via AT1R) and indirectly (via SNS) IL-6 mRNA synthesis in left ventricle and aorta and TNF-α mRNA in left ventricle. ANG II seems unable to influence directly TNF-α mRNA synthesis in the aorta but can stimulate this cytokine via SNS. The results are relevant to prevent or reduce proinflammatory cytokines overexpression seen in cardiovascular diseases.

Chronic hydrogen-rich saline treatment reduces oxidative stress and attenuates left ventricular hypertrophy in spontaneous hypertensive rats

In hypertensive animals and patients, oxidative stress represents the primary risk factor for progression of left ventricular hypertrophy. Recently, it has been demonstrated that hydrogen, as a novel antioxidant, can selectively reduce hydroxyl radicals and peroxynitrite anion to exert therapeutic antioxidant activity. In the current study, we explored the effect of chronic treatment with hydrogen-rich saline (HRS) on left ventricular hypertrophy in spontaneously hypertensive rats (SHR). The 8-week-old male SHR and age-matched Wistar-Kyoto rats (WKY) were randomized into HRS-treated (6 ml/kg/day for 3 months, i.p.) and vehicle-treated groups. HRS treatment had no significant effect on blood pressure, but it effectively attenuated left ventricular hypertrophy in SHR. HRS treatment abated oxidative stress, restored the activity of antioxidant enzymes including GPx, GST, catalase, and SOD, suppressed NADPH oxidase activity and downregulated Nox2 and Nox4 expression in left ventricles of SHR. HRS treatment suppressed pro-inflammatory cytokines including IL-1β, IL-6, TNF-α, and MCP-1, and inhibited NF-κB activation through preventing IκBα degradation in left ventricles of SHR. HRS treatment preserved mitochondrial function through restoring electron transport chain enzyme activity, repressing ROS formation, and enhancing ATP production in left ventricles of SHR. Moreover, HRS treatment suppressed ACE expression and locally reduced angiotensin II generation in left ventricles of SHR. In conclusion, HRS treatment attenuates left ventricular hypertrophy through abating oxidative stress, suppressing inflammatory process, preserving mitochondrial function, in which suppression of HRS on angiotensin II in left ventricles locally might be involved.

252 Cardiac β3-adrenoceptors as a new therapeutic target in dilated cardiomyopathy

Introduction β-adrenergic system is altered in heart failure (HF) due to non-ischemic dilated cardiomyopathy (DCM). There are few data concerning the relative contribution of β1- and β3-adrenoceptor subtypes (β1- and β3-AR) during DCM development. We evaluated the expression and the role of each β-AR subtype in this pathology. Methods DCM rat model is performed by doxorubicin injections (cumulative dose: 15 mg.kg-1) and validated by in vivo measurements with echocardiography-doppler. The variations of β1- and β3-AR transcript expression in left ventricle (LV) are evaluated by real-time RT-PCR. The ex vivo cardiac responses induced by selective β3-AR or non-selective β-AR stimulations are evaluated on isolated perfused heart. Results DCM rats present LV dilation, systolic and diastolic dysfunction (see table). Compared to controls, β1-AR transcripts and β3-AR transcripts are increased in DCM LV (+36%, n=8, p Table: Basal parameters and maximum values obtained by non-selective β1-AR stimulation (isoproterenol) or selective β3-AR stimulation (SR58611A). Results are expressed by mean ± SEM.*: p Control rats (n=9-31) DCM rats (n=10-31) Basal in vivo LV end-diastolic diameter (mm) 8.82±0.33 7.61±0.17 * LV ejection fraction (%) 83.0±1.9 71.2±2.8* LV Isovolumic relaxation time (ms) 21.41±1.13 32.39±1.10* Basal ex vivo DP/dt max (mmHg.s-1) 2035±365 2669±504* DP/dt min (mmHg.s-1) –1258±226 –1847±349* Isoproterenol ex vivo (1 μM) DP/dt max (mmHg.s-1) 5263±1754 4373±1383* DP/dt min (mmHg.s-1) –3815±1271 –3227±1020* SR58611A ex vivo (1 μM) DP/dt max (mmHg.s-1) 1722±497 1930±611£ DP/dt min (mmHg.s-1) –1027±297 –1138±360£ Conclusion DCM induces a β3-AR gene over-expression, associated to an increase of β3AR-induced negative inotropic and lusitropic effects. Those results could partly explain the alteration of isoproterenol response in our model, suggesting that β3-AR could be a new therapeutic target in DCM.