Hypertrophied Rat Hearts Research Articles

Vitamin D Attenuates Cardiac Hypertrophy in Rats through mRNA Regulation of Interleukin-6 and Its Receptor

Abstract Context: Interleukin-6 (IL-6), a pro-inflammatory cytokine, plays an important role in the pathogenesis of myocardial hypertrophy. By integrating its membrane receptor complex (gp80), IL-6 activates the signal guidance components (gp130) and activates the hypertrophic signaling pathways. There is some evidence that 1,25 dihydroxyvitamin D exerts antihypertrophic effects, but the cellular and molecular mechanisms are not fully understood. The aim of this study was to evaluate the effect of calcitriol on the level of IL-6 and its receptor components in hypertrophied rat heart. Subjects and Methods: Male rats were divided into control, hypertrophy, Vitamin D + hypertrophy, and propylene glycol + hypertrophy groups. The groups receiving Vitamin D and propylene glycol were treated 2 weeks before induction of hypertrophy and 2 weeks after hypertrophy. Myocardial hypertrophy was induced by abdominal aortic stenosis. Mean arterial blood pressure was measured by cannulation of the left carotid artery, and expression of genes was determined by reverse transcription-polymerase chain reaction. Results: Blood pressure and heart-to-body weight ratio increased in hypertrophic groups compared to the control group (P < 0.01), but Vitamin D administration decreased these parameters (P < 0.05). Abdominal aortic stenosis increased IL-6 expression levels (P < 0.001) and Vitamin-D decreased IL-6 mRNA levels (P < 0.01). The expression of gp80 in the hypertrophic group increased compared to the control group (P < 0.05), but Vitamin D did not affect the expression of receptor subunits genes. Conclusions: The data from this study suggest a possible mechanism for the antihypertrophic effects of Vitamin D through the regulation of inflammatory responses during hypertrophy. Thus, Vitamin D can reduce IL-6 expression levels, thereby reducing hypertrophy.

Ectopic Acsl6 Overexpression Partially Improves Isoproterenol-Induced Cardiac Hypertrophy In Vivo and Cardiomyocyte Hypertrophy In Vitro.

The increase in cardiac myocyte size is a critical issue in cardiac hypertrophy development. In this study, 61 differentially expressed genes between hypertrophic rats and normal controls were enriched in the positive modulation of fatty acid uptake, fatty acid metabolism and degradation, cardiac conduction, and the oxidation of carbohydrates and other processes. Acsl6 was significantly downregulated in hypertrophic rat and mouse hearts according to online data. Based on the experimental data, Acsl6 was underexpressed in ISO-induced cardiac hypertrophy mouse model and isoproterenol (ISO)-induced cardiomyocyte hypertrophy cell model. In vivo, Acsl6 overexpression partially attenuated ISO-induced increases in the cross-sectional area and cardiac hypertrophy, elevated hypertrophic markers, and caused impairment of cardiac function. In vitro, Acsl6 overexpression partially attenuated ISO-induced cardiomyocyte hypertrophy and increased hypertrophic markers. Conclusively, Ascl6 is downregulated in ISO-induced cardiac hypertrophy mouse model and ISO-induced cardiomyocyte hypertrophy cell model. Acsl6 overexpression could partially improve cardiac hypertrophy in vivo and cardiomyocyte hypertrophy in vitro, possibly through the regulation of HIF-1α/Hippo pathway.

MCU overexpression evokes disparate dose-dependent effects on mito-ROS and spontaneous Ca2+ release in hypertrophic rat cardiomyocytes.

Cardiac dysfunction in heart failure (HF) and diabetic cardiomyopathy (DCM) is associated with aberrant intracellular Ca2+ handling and impaired mitochondrial function accompanied with reduced mitochondrial calcium concentration (mito-[Ca2+]). Pharmacological or genetic facilitation of mito-Ca2+ uptake was shown to restore Ca2+ transient amplitude in DCM and HF, improving contractility. However, recent reports suggest that pharmacological enhancement of mito-Ca2+ uptake can exacerbate ryanodine receptor-mediated spontaneous sarcoplasmic reticulum (SR) Ca2+ release in ventricular myocytes (VMs) from diseased animals, increasing propensity to stress-induced ventricular tachyarrhythmia. To test whether chronic recovery of mito-[Ca2+] restores systolic Ca2+ release without adverse effects in diastole, we overexpressed mitochondrial Ca2+ uniporter (MCU) in VMs from male rat hearts with hypertrophy induced by thoracic aortic banding (TAB). Measurement of mito-[Ca2+] using genetic probe mtRCamp1h revealed that mito-[Ca2+] in TAB VMs paced at 2 Hz under β-adrenergic stimulation is lower compared with shams. Adenoviral 2.5-fold MCU overexpression in TAB VMs fully restored mito-[Ca2+]. However, it failed to improve cytosolic Ca2+ handling and reduce proarrhythmic spontaneous Ca2+ waves. Furthermore, mitochondrial-targeted genetic probes MLS-HyPer7 and OMM-HyPer revealed a significant increase in emission of reactive oxygen species (ROS) in TAB VMs with 2.5-fold MCU overexpression. Conversely, 1.5-fold MCU overexpression in TABs, that led to partial restoration of mito-[Ca2+], reduced mitochondria-derived reactive oxygen species (mito-ROS) and spontaneous Ca2+ waves. Our findings emphasize the key role of elevated mito-ROS in disease-related proarrhythmic Ca2+ mishandling. These data establish nonlinear mito-[Ca2+]/mito-ROS relationship, whereby partial restoration of mito-[Ca2+] in diseased VMs is protective, whereas further enhancement of MCU-mediated Ca2+ uptake exacerbates damaging mito-ROS emission.NEW & NOTEWORTHY Defective intracellular Ca2+ homeostasis and aberrant mitochondrial function are common features in cardiac disease. Here, we directly compared potential benefits of mito-ROS scavenging and restoration of mito-Ca2+ uptake by overexpressing MCU in ventricular myocytes from hypertrophic rat hearts. Experiments using novel mito-ROS and Ca2+ biosensors demonstrated that mito-ROS scavenging rescued both cytosolic and mito-Ca2+ homeostasis, whereas moderate and high MCU overexpression demonstrated disparate effects on mito-ROS emission, with only a moderate increase in MCU being beneficial.

Non-Selective Cation Currents Mediated by Cx43 Hemichannel-P2X4 Receptor Signaling Pathway in Rat Atrial Myocytes under Shear Stress

Cardiac myocytes are exposed to shear stress under physiological conditions and hemodynamic disturbances. Shear stress has been thought to trigger two distinct global Ca2+ waves in atrial myocytes through autocrine activation of P2X4 receptor (P2X4R) and P2Y1-receptor (P2Y1R) via connexin 43 (Cx43)-mediated ATP release. In this study, we examined whether such shear stress-triggered Cx43-ATP-P2 receptor signaling generates depolarizing currents in atrial myocytes. Whole-cell patch clamp and dye-flux assays were used with genetic and pharmacological interventions in rat atrial myocytes and HL-1 cells. We found that shear stress activated a non-selective inward cation (Cs+) current (IS,Cs) at resting potential which was eliminated by Cx43 blockade using siRNA, antibodies, carbenoxolone or La3+. IS,Cs was enhanced by removal of external Ca2+, whereas internal Ca2+ buffering suppressed IS,Cs. Hemichannel-mediated dye (calcein red-orange) flux was induced by shear stress. This response was augmented by removing external Ca2+ but was not affected by pannexin blocker (probenecid). The shear-activated current was significantly reduced when Cs+ was replaced with NMDG+, and this was enhanced by quinine and suppressed by La3+. P2X4R antagonists (5-BDBD and PSB-12054), P2XR-blocker (iso-PPADS) and anti-P2X4R antibodies each blocked IS,Cs by approximately 50%. About 25% of IS,Cs was suppressed by P2Y1R inhibitor (MRS2179) and/or transient receptor potential melastatin 4 blocker (9-phenanthrol). P2X4R, but not P2X5R, was co-localized with Cx43 at the cell ends. P2X4R-dependent IS,Cs was augmented in atrial myocytes from hypertrophied rat hearts by short-term aortic constriction. Our data suggest that shear stress triggers Cx43 hemichannels to gate P2X4Rs in their vicinity. This shear stress signaling may play an important role in membrane depolarization at negative potentials and in atrial remodeling under increased afterload.

Sulfur Dioxide Derivative Prevents the Prolongation of Action Potential During the Isoproterenol-Induced Hypertrophy of Rat Cardiomyocytes

Exogenous SO2 is toxic especially to the pulmonary and cardiovascular system, similar to nitric-oxide, carbon-monoxide, and hydrogen-sulfide. Endogenous SO2 is produced in many cell types. The SO2 content of the rat heart has been observed to substantially decrease during isoproterenol-induced hypertrophy. This study sought to determine whether an SO2 derivative could inhibit the prolongation of action potentials during the isoproterenol-induced hypertrophy of rat cardiomyocytes and explore the ionic currents. Alongside electrocardiogram recordings, the voltage and current-clamped measurements were conducted in the enzymatically isolated left ventricular cardiomyocytes of Wistar rats. The consistency of the results was evaluated by the novel mathematical electrophysiology model. Our results show that SO2 significantly blocked the prolongation of QT-interval and action potential duration. Furthermore, SO2 did not substantially affect the Na+ currents and did not improve the decreased steady-state and transient outward K+ currents, but it reverted the reduced L-type Ca2+ currents (I CaL) to the physiological levels. Altered inactivation of I CaL was remarkably recovered by SO2. Interestingly, SO2 significantly increased the Ca2+ transients in hypertrophic rat hearts. Our mathematical model also confirmed the mechanism of the SO2 effect. Our findings suggest that the shortening mechanism of SO2 is related to the Ca2+ dependent inactivation kinetics of the Ca2+ current.

Response of non-failing hypertrophic rat hearts to prostaglandin F2α.

BackgroundProstaglandin F2α (PGF2α) has a positively inotropic effect on right ventricular (RV) trabeculae from healthy adult rat hearts, and may therefore be therapeutically useful as a non-catecholaminergic inotrope. These provide additional contractile support for the heart without the added energetic demand of increased heart rate, and are also suitable for patients with reduced β adrenergic receptor (β-AR) responsiveness, or impaired mitochondrial energy supply. However, the response of hypertrophied rat hearts to PGF2α has not previously been examined. Our aim was therefore to determine the effect of PGF2α on isolated perfused rat hearts with RV hypertrophy following induction of pulmonary artery hypertension.MethodsMale Wistar rats (300 g) were injected with either 60 mg kg−1 of monocrotaline (MCT, n = 10) or sterile saline as control (CON, n = 11). Four weeks post injection; hearts were isolated and Langendorff-perfused in sinus rhythm. Measurement of left ventricular (LV) pressure and the electrocardiogram were made and the response to 0.3 μM PGF2α was determined.ResultsPGF2α increased LV developed pressure in CON and in 60% MCT hearts, with no change in heart rate. However, 40% of MCT hearts developed arrhythmias during the peak inotropic response. For comparison, the response to 0.03 μM isoproterenol (ISO) was also investigated. Peak LV pressure developed sooner in response to ISO compared to PGF2α in both rat groups, although the inotropic response to ISO was reduced in MCT hearts. Analysis of fixed ventricular tissue confirmed that only RV myocytes were hypertrophied in MCT hearts. Our study showed that PGF2α was positively inotropic for healthy hearts, but found it generated arrhythmias in 40% of MCT hearts at the dose investigated. However, a more physiological dose of PGF2α may be a useful alternative without the added energetic cost of catecholaminergic inotropes.

Deep ocean minerals inhibit IL-6 and IGFIIR hypertrophic signaling pathways to attenuate diabetes-induced hypertrophy in rat hearts.

We previously reported that deep sea water (DSW) prolongs the life span of streptozotocin (STZ)-induced diabetic rats by the compensatory augmentation of the insulin like growth factor (IGF)-I survival signaling and inhibition of apoptosis. Here, we investigated the effects of DSW on cardiac hypertrophy in diabetic rats. Cardiac hypertrophy was induced in rats by using STZ (65 mg/kg) administered via IP injection. DSW was prepared by mixing DSW mineral extracts and desalinated water. Different dosages of DSW-1X (equivalent to 37 mg Mg2+·kg-1·day-1), 2X (equivalent to 74 mg Mg2+·kg-1·day-1) and 3X (equivalent to 111 mg Mg2+·kg-1·day-1) were administered to the rats through gavage for 4 wk. Cardiac hypertrophy was evaluated by the heart weight-to-body weight ratio and the cardiac tissue cross-sectional area after hematoxylin and eosin staining. The protein levels of the cardiac hypertrophy signaling molecules were determined by Western blot. Our results showed that the suppressive effects of the DSW treatment on STZ-induced cardiac hypertrophy were comparable to those of MgSO4 administration and that the hypertrophic marker brain natriuretic peptide (BNP) was decreased by DSW. In addition, DSW attenuated both the eccentric hypertrophy signaling pathway, IL-6-MEK-STAT3, and the concentric signaling pathway, IGF-II-PKCα-CaMKII, in DM rat hearts. The cardiac hypertrophy-associated activation of extracellular signal-regulated kinase (ERK) and the upregulation of the transcription factor GATA binding protein 4 (GATA4) were also negated by treatment with DSW. The results from this study suggest that DSW could be a potential therapeutic agent for the prevention and treatment of diabetic cardiac hypertrophy.NEW & NOTEWORTHY Deep sea water, containing high levels of minerals, improve cardiac hypertrophy in diabetic rats through attenuating the eccentric signaling pathway, IL-6-MEK5-STAT3, and concentric signaling pathway, IGF2-PKCα-CaMKII. The results from this study suggest that deep sea water could be a potential therapeutic agent for the prevention and treatment of diabetic cardiac hypertrophy.

A4989 Induction of endoplasmic reticulum stress and variation in expression levels of Zip14 in hypertrophic rat heart

A4989 Induction of endoplasmic reticulum stress and variation in expression levels of Zip14 in hypertrophic rat heart

Effect of Calcium on Slow Force Responses in Isolated Right Ventricle Preparations of Healthy and Hypertrophied Myocardium in Male and Female Rats.

Effect of different Ca2+ concentrations in the bathing solution [Ca2+]o on the parameters of single isometric contraction and slow force response to stretching was studied in isolated preparations of healthy and hypertrophied myocardium of male and female Wistar rats. In all groups of experimental animals, the increase in calcium concentration was followed by a decrease in the myocardium slow response intensity. We revealed a complementary relationship between the current and medium-term systems of myocardial contractility regulation by the length of the myocardium aimed at the maintenance of the constant level during adaptation to the load. Slow responses of the hypertrophied rat heart myocardium were suppressed in comparison with those in the healthy myocardium and their intensity did not depend on animal sex.

Enhanced Redox State and Efficiency of Glucose Oxidation With miR Based Suppression of Maladaptive NADPH-Dependent Malic Enzyme 1 Expression in Hypertrophied Hearts.

Metabolic remodeling in hypertrophic hearts includes inefficient glucose oxidation via increased anaplerosis fueled by pyruvate carboxylation. Pyruvate carboxylation to malate through elevated ME1 (malic enzyme 1) consumes NADPH necessary for reduction of glutathione and maintenance of intracellular redox state. To elucidate upregulated ME1 as a potential maladaptive mechanism for inefficient glucose oxidation and compromised redox state in hypertrophied hearts. ME1 expression was selectively inhibited, in vivo, via non-native miR-ME1 (miRNA specific to ME1) in pressure-overloaded rat hearts. Rats subjected to transverse aortic constriction (TAC) or Sham surgery received either miR-ME1 or PBS. Effects of ME1 suppression on anaplerosis and reduced glutathione (GSH) content were studied in isolated hearts supplied 13C-enriched substrate: palmitate, glucose, and lactate. Human myocardium collected from failing and nonfailing hearts during surgery enabled RT-qPCR confirmation of elevated ME1 gene expression in clinical heart failure versus nonfailing human hearts (P<0.04). TAC induced elevated ME1 content, but ME1 was lowered in hearts infused with miR-ME1 versus PBS. Although Sham miR-ME1 hearts showed no further reduction of inherently low anaplerosis in normal heart, miR-ME1 reduced anaplerosis in TAC to baseline: TAC miR-ME1=0.034±0.004; TAC PBS=0.081±0.005 (P<0.01). Countering elevated anaplerosis in TAC shifted pyruvate toward oxidation in the tricarboxylic acid cycle. Importantly, via the link to NADPH consumption by pyruvate carboxylation, ME1 suppression in TAC restored GSH content, reduced lactate production, and ultimately improved contractility. A maladaptive increase in anaplerosis via ME1 in TAC is associated with reduced GSH content. Suppressing increased ME1 expression in hypertrophied rat hearts, which is also elevated in failing human hearts, reduced pyruvate carboxylation thereby normalizing anaplerosis, restoring GSH content, and reducing lactate accumulation. Reducing ME1 induced favorable metabolic shifts for carbohydrate oxidation, improving intracellular redox state and enhanced cardiac performance in pathological hypertrophy.

Upregulation of α-enolase protects cardiomyocytes from phenylephrine-induced hypertrophy.

Cardiac hypertrophy often refers to the abnormal growth of heart muscle through a variety of factors. The mechanisms of cardiomyocyte hypertrophy have been extensively investigated using neonatal rat cardiomyocytes treated with phenylephrine. α-Enolase is a glycolytic enzyme with "multifunctional jobs" beyond its catalytic activity. Its possible contribution to cardiac dysfunction remains to be determined. The present study aimed to investigate the change of α-enolase during cardiac hypertrophy and explore its role in this pathological process. We revealed that mRNA and protein levels of α-enolase were significantly upregulated in hypertrophic rat heart induced by abdominal aortic constriction and in phenylephrine-treated neonatal rat cardiomyocytes. Furthermore, knockdown of α-enolase by RNA interference in cardiomyocytes mimicked the hypertrophic responses and aggravated phenylephrine-induced hypertrophy without reducing the total glycolytic activity of enolase. In addition, knockdown of α-enolase led to an increase of GATA4 expression in the normal and phenylephrine-treated cardiomyocytes. Our results suggest that the elevation of α-enolase during cardiac hypertrophy is compensatory. It exerts a catalytic independent role in protecting cardiomyocytes against pathological hypertrophy.

Induction of endoplasmic reticulum stress and changes in expression levels of Zn2+-transporters in hypertrophic rat heart.

Clinical and experimental studies have shown an association between intracellular free Zn2+ ([Zn2+]i)-dyshomeostasis and cardiac dysfunction besides [Ca2+]i-dyshomeostasis. Since [Zn2+]i-homeostasis is regulated through Zn2+-transporters depending on their subcellular distributions, one can hypothesize that any imbalance in Zn2+-homeostasis via alteration in Zn2+-transporters may be associated with the induction of ER stress and apoptosis in hypertrophic heart. We used a transverse aortic constriction (TAC) model to induce hypertrophy in young male rat heart. We confirmed the development of hypertrophy with a high ratio of heart to body weight and cardiomyocyte capacitance. The expression levels of ER stress markers GRP78, CHOP/Gadd153, and calnexin are significantly high in TAC-group in comparison to those of controls (SHAM-group). Additionally, we detected high expression levels of apoptotic status marker proteins such as the serine kinase GSK-3β, Bax-to-Bcl-2 ratio, and PUMA in TAC-group in comparison to SHAM-group. The ratios of phospho-Akt to Akt and phospho-NFκB to the NFκB are significantly higher in TAC-group than in SHAM-group. Furthermore, we observed markedly increased phospho-PKCα and PKCα levels in TAC-group. We, also for the first time, determined significantly increased ZIP7, ZIP14, and ZnT8 expressions along with decreased ZIP8 and ZnT7 levels in the heart tissue from TAC-group in comparison to SHAM-group. Furthermore, a roughly calculated total expression level of ZIPs responsible for Zn2+-influx into the cytosol (increased about twofold) can be also responsible for the markedly increased [Zn2+]i detected in hypertrophic cardiomyocytes. Taking into consideration the role of increased [Zn2+]i via decreased ER-[Zn2+] in the induction of ER stress in cardiomyocytes, our present data suggest that differential changes in the expression levels of Zn2+-transporters can underlie mechanical dysfunction, in part due to the induction of ER stress and apoptosis in hypertrophic heart via increased [Zn2+]i- besides [Ca2+]i-dyshomeostasis.

Abstract 431: Upregulation of Small Conductance Ca 2+ Activated K + Channels Promotes Formation of Mitochondrial Supercomplexes and Increases Respiration Reserve

Introduction: Small conductance calcium (Ca 2+ )-activated K + (SK) channels are present in the inner mitochondria membrane of ventricular cardiomyocytes. We recently reported that pharmacological enhancement of mito-SKs protects from Ca 2+ dependent arrhythmia by reducing production of reactive oxygen species (ROS) by mitochondria thereby normalizing oxidation state and activity of Ca 2+ release channels ryanodine receptors in hypertrophic rat hearts. Recent advances demonstrate that respiratory components of the mitochondrial electron transport chain can organize into supercomplexes (SCs) resulting in more efficient electron transfer and reduced generation of ROS. Hypothesis: Functional upregulation of mito-SK channels protects from Ca 2+ -dependent arrhythmia via promotion of mitochondrial SC formation and improved respiration. Methods: H9C2 myoblasts were infected with adenoviral vectors to overexpress SK2 and Cox7rp. After 48 hours in culture, cell respiration was measured using Seahorse XFe Analyzer; ROS was measured using general ROS indicator CFDA; and mitochondria were isolated to assess SC formation using Blue-Native electrophoresis (BN-PAGE). The effects of SK enhancer NS309 (2 μM, 10 min.) on SC formation were studied in Langendorff perfused rat hearts with hypertrophy induced by thoracic aortic banding (TAB). Results: rSK2 overexpression in H9C2s reduced intracellular ROS ~ 30%, enhanced maximum respiration rate vs. controls (0.74±0.04 and 0.58±0.03 pM/min/μg respectively, p&lt;0.05); and caused a robust shift (&gt;2 fold) of complex IV towards large mitochondrial SCs (~1.2 mDa). As a control, similar results were obtained with overexpression of Cox7rp protein that promotes SC formation. In addition, BN-PAGE experiments in isolated rat hearts demonstrated that robust decrease in high MW Complexes I and IV in hypertrophy was fully restored to control levels upon treatment with SK enhancer NS309. Conclusion: Upregulation of SK channels reduces mito-ROS by an increase in respiration reserve which is linked to enhanced mitochondrial SC formation. Our data suggest that targeting mito-SK channels has therapeutic potential to treat cardiac arrhythmia in conditions accompanied by oxidative stress.

SK channel enhancers attenuate Ca2+-dependent arrhythmia in hypertrophic hearts by regulating mito-ROS-dependent oxidation and activity of RyR.

Plasmamembrane small conductance Ca2+-activated K+ (SK) channels were implicated in ventricular arrhythmias in infarcted and failing hearts. Recently, SK channels were detected in the inner mitochondria membrane (IMM) (mSK), and their activation protected from acute ischaemia-reperfusion injury by reducing intracellular levels of reactive oxygen species (ROS). We hypothesized that mSK play an important role in regulating mitochondrial function in chronic cardiac diseases. We investigated the role of mSK channels in Ca2+-dependent ventricular arrhythmia using rat model of cardiac hypertrophy induced by banding of the ascending aorta thoracic aortic banding (TAB). Dual Ca2+ and membrane potential optical mapping of whole hearts derived from TAB rats revealed that membrane-permeable SK enhancer NS309 (2 μM) improved aberrant Ca2+ homeostasis and abolished VT/VF induced by β-adrenergic stimulation. Using whole cell patch-clamp and confocal Ca2+ imaging of cardiomyocytes derived from TAB hearts (TCMs) we found that membrane-permeable SK enhancers NS309 and CyPPA (10 μM) attenuated frequency of spontaneous Ca2+ waves and delayed afterdepolarizations. Furthermore, mSK inhibition enhanced (UCL-1684, 1 μM); while activation reduced mitochondrial ROS production in TCMs measured with MitoSOX. Protein oxidation assays demonstrated that increased oxidation of ryanodine receptors (RyRs) in TCMs was reversed by SK enhancers. Experiments in permeabilized TCMs showed that SK enhancers restored SR Ca2+ content, suggestive of substantial improvement in RyR function. These data suggest that enhancement of mSK channels in hypertrophic rat hearts protects from Ca2+-dependent arrhythmia and suggest that the protection is mediated via decreased mitochondrial ROS and subsequent decreased oxidation of reactive cysteines in RyR, which ultimately leads to stabilization of RyR-mediated Ca2+ release.

Proteomic analysis of the protective effects of aqueous bark extract of Terminalia arjuna (Roxb.) on isoproterenol-induced cardiac hypertrophy in rats

Ethnopharmacological relevanceAqueous bark extract of Terminalia arjuna (TA) has been in use as an ethnomedicine for cardiovascular ailments in the Indian subcontinent for centuries. Studies using hemodynamic, ROS scavenging and anti-inflammatory parameters in animal models have shown its anti-atherogenic, hypotensive, inotropic, anti-inflammatory effects. However, details analysis on its effects on established molecular and cell biological markers are a prerequisite for its wider acceptance to the medical community. Aims of the studyTo test the efficacy of TA extract in ameliorating cardiac hypertrophy induced by ISO in rats. MethodsCardiac hypertrophy was induced by ISO (5mg/kg/day s.c. for 14 days) in rats and a standardized aqueous extract of TA stem bark was orally administered by gavage. Total RNA and protein were isolated from control, ISO, ISO plus TA and TA treated rat hearts and analyzed for the transcripts for the markers of hypertrophy, signaling kinases, transcription factors and total protein profile. ResultsTA extract reversed the induction of fetal genes like β-myosin heavy chain, skeletal α-actin and brain natriuretic peptide in hypertrophic rat hearts. While ISO slightly increased the level of phospho-ERK, TA repressed it to about one third of the base line level. Survival kinase Akt, ER stress marker Grp78 and epigenetic regulator HDAC5 were augmented by ISO and TA restored them by various extents. ISO administration moderately increased the transcription factor NFκB binding activity, while coadministration of TA further increased it. AP-1 binding activity was largely unchanged by ISO treatment but it was upregulated when administered along with TA. MEF2D binding activity was increased by ISO and TA restored it to the baseline level. Global proteomic analysis revealed that TA treatment restored a subset of proteins up- and down-regulated in the hypertrophied hearts. Amongst those restored by TA were purinergic receptor X, myosin light chain 3, tropomyosin, and kininogen; suggesting a nodal role of TA in modulating cardiac function. ConclusionsThis study for the first time reveals that TA partially or completely restores the marker mRNAs, signaling kinases, transcription factors and total protein profile in rat heart, thereby demonstrating its efficacy in preventing ISO-induced cardiac hypertrophy.

The H3K9 dimethyltransferases EHMT1/2 protect against pathological cardiac hypertrophy.

Cardiac hypertrophic growth in response to pathological cues is associated with reexpression of fetal genes and decreased cardiac function and is often a precursor to heart failure. In contrast, physiologically induced hypertrophy is adaptive, resulting in improved cardiac function. The processes that selectively induce these hypertrophic states are poorly understood. Here, we have profiled 2 repressive epigenetic marks, H3K9me2 and H3K27me3, which are involved in stable cellular differentiation, specifically in cardiomyocytes from physiologically and pathologically hypertrophied rat hearts, and correlated these marks with their associated transcriptomes. This analysis revealed the pervasive loss of euchromatic H3K9me2 as a conserved feature of pathological hypertrophy that was associated with reexpression of fetal genes. In hypertrophy, H3K9me2 was reduced following a miR-217-mediated decrease in expression of the H3K9 dimethyltransferases EHMT1 and EHMT2 (EHMT1/2). miR-217-mediated, genetic, or pharmacological inactivation of EHMT1/2 was sufficient to promote pathological hypertrophy and fetal gene reexpression, while suppression of this pathway protected against pathological hypertrophy both in vitro and in mice. Thus, we have established a conserved mechanism involving a departure of the cardiomyocyte epigenome from its adult cellular identity to a reprogrammed state that is accompanied by reexpression of fetal genes and pathological hypertrophy. These results suggest that targeting miR-217 and EHMT1/2 to prevent H3K9 methylation loss is a viable therapeutic approach for the treatment of heart disease.

Mitochondrial SK Channels Attenuate Ca-Dependent Arrhythmia in Hypertrophic Hearts by Regulating Mito-ROS-Dependent Oxidation and Activity of RyR

Rationale: Small conductance Ca2+-activated K+ (SK) channels are implicated in ventricular arrhythmias in the context of heart failure and myocardial infarct. In addition to their plasmalemmal localization, SK channels were recently discovered in mitochondria inner membrane (mSK). Activation of these channels is protective against ischemia-reperfusion injury by reducing intracellular levels of reactive oxygen species (ROS). We hypothesized that mSK may also play an important role in regulating mitochondrial function in cardiac disease as well.Objective: We investigated the role of mSK channels in Ca2+-dependent ventricular arrhythmia using a rat model of hypertrophy induced by banding of the ascending aorta (TAB).Methods and Results: Dual Ca2+ and membrane potential optical mapping of whole hearts from TAB rats revealed that membrane-permeable SK activator improved aberrant Ca2+ homeostasis and abolished VT/VF induced by b-adrenergic stimulation. Using whole cell patch-clamp and confocal Ca2+ imaging we found that membrane-permeable SK enhancers attenuated frequency of spontaneous Ca2+ waves and delayed afterdepolarizations in hypertrophic cardiomyocytes. Furthermore, mSK inhibition enhanced; and activation reduced mitochondrial ROS production in TAB myocytes measured with MitoSOX. Monobromobimane assay demonstrated that increased oxidation of ryanodine receptors (RyRs) in TAB cells was reversed by SK enhancers. Experiments in permeabilized TAB myocytes showed that SK enhancers restored SR Ca2+ content, suggestive of substantial improvement in RyR function.Conclusions: These data suggest that enhancement of mSK channels in hypertrophic rat hearts protects from Ca2+-dependent arrhythmia. This protection is mediated via decreased mitochondrial ROS and subsequent decreased oxidation of reactive cysteines in RyR, which ultimately leads to stabilization of RyR-mediated Ca2+ release.

Cardioprotective and Anti-arrhythmic Effects of Magnesium Pretreatment Against Ischaemia/Reperfusion Injury in Isoprenaline-Induced Hypertrophic Rat Heart.

The effects of magnesium (Mg2+) on ischaemic complications of pathological cardiac hypertrophy are unclear. In this study, we investigated effects of Mg2+ pretreatment on ischaemia/reperfusion (I/R) injury in isoprenaline (ISO)-induced hypertrophic hearts. Wistar rats were treated for 7days with different combinations of ISO (1.25mg/kg) subcutaneously, MgSO4 (270mg/kg) intraperitoneally, or vehicle (saline). On the eighth day, hearts were either subjected to regional I/R during Langendorff perfusion or histologically stained with haematoxylin and eosin and Masson's trichrome. Haemodynamic and electrocardiographic parameters were recorded using the PowerLab data-acquisition system. Infarcts were identified by triphenyltetrazolium chloride staining. Plasma Mg2+ was measured using photometric assays. Mg2+ pretreatment significantly decreased I/R-induced infarct size (p=0.001) and the overall arrhythmia score (p<0.001) of I/R-induced ventricular ectopics, ventricular tachycardia, and ventricular fibrillation in hypertrophic hearts, but not non-hypertrophied hearts. Mg2+ also improved post-I/R left ventricular developed pressure in hypertrophic hearts. However, Mg2+ did not reverse the ISO-induced myocyte thickening and interstitial fibrosis or increases in heart weight. Plasma Mg2+ was not different among treatment groups. These results suggest that Mg2+ pretreatment may protect against I/R-induced injury and malignant arrhythmias in hypertrophic hearts, possibly via mechanisms unrelated to long-lasting changes in plasma Mg2+ or prevention of structural changes such as fibrosis.

Biochemical and physiological studies of rat heart hypertrophy experimentaly induced with Deoxicorticosterone acetate(DOCA)

Biochemical and physiological studies of rat heart hypertrophy experimentaly induced with Deoxicorticosterone acetate(DOCA)

Abstract 15571: Suppressing Increased Expression of NADPH-Dependent Malic Enzyme 1 in Hypertrophied Hearts Eliminates Maladaptive Anaplerosis and Improves Redox Conditions

The remodeled metabolic phenotype of hypertrophic hearts includes inefficient glucose oxidation, due in part to pyruvate carboxylation into anaplerosis. Increased carboxylation of pyruvate to malate via elevated malic enzme-1 (ME1) also consumes NADPH, which is necessary for maintaining glutathione reduction. Therefore, we studied the consequences of selective inhibition of ME1 expression via non-native miRNA targeted to the ME1 gene (miRME1) in hypertrophied rat hearts after transverse aortic constriction (TAC). TAC elevated ME1 content, but ME1 was lowered in hearts receiving miRME1 vs PBS infusion (*P&lt;0.01). The effects of ME1 suppression on anaplerosis and GSH content were studied in isolated hearts supplied 13C palmitate, glucose, and lactate. While Sham miRME1 hearts showed no further reduction of inherently low baseline ratios of anaplerosis to citrate synthase flux from normal hearts, miRME1 reduced anaplerosis in TAC to baseline: TAC miRME1=0.034±0.004; TAC PBS=0.081±0.005 (*P&lt;0.01, **P&lt;0.001). Importantly, ME1 suppression restored GSH in TAC (†P&lt;0.05). The findings demonstrate the maladaptive increase in anaplerosis via ME1 in TAC is associated with reduced GSH content. Suppressing increased ME1 expression in hypertrophied hearts, and thus consumption of NADPH for anaplerotic malate, produced favorable metabolic shifts, improving intracellular redox state.