Reduced Heart Weight Research Articles

Effect of feed restriction on organs and intestinal mucosa of growing rabbits

This experiment was carried out to evaluate the effect of feed restriction on internal organs with respect to their weight and size, and the development of the intestinal mucosa of growing rabbits. Sixty 33-day-old New Zealand White rabbits were used in a randomized block with four treatments and five blocks. The treatments consisted of ad libitum feeding; feed restriction from 33 to 40 days of age; feed restriction from 54 to 61 days of age; and feed restriction from 33 to 40 and from 54 to 61 days of age. There was no effect of feed restriction on weight, length and width of internal organs, except for heart weight, which decreased when feeding was restricted from 54-61 days and from 33-40 and 54-61 days of age. There was no effect of feed restriction at the different ages on duodenal morphometry. In jejunum, the villi of rabbits fed ad libitum or restricted from 33 to 40 days were higher, and rabbits restricted from 33-40 days had wider villi and higher absorption surface. In the ileum, only rabbits fed ad libitum had higher villi. Feeding restriction reduced heart weight, but not its size, and negatively affected jejunum morphometry when performed in later stages.

Choline Protects Against Cardiac Hypertrophy Induced by Increased After-load

Background: Although inadequate intake of essential nutrient choline has been known to significantly increase cardiovascular risk, whether additional supplement of choline offering a protection against cardiac hypertrophy remain unstudied.Methods: The effects of choline supplements on pathological cardiac hypertrophic growth induced by transverse aorta constriction (TAC) for three weeks and cardiomyocyte hypertrophy in cultured cells induced by isoproterenol (ISO) 10 μM for 48 h stimulation were investigated. Western blot analysis and real-time PCR were used to determine the expression of ANP, BNP, β-MHC, miR-133a and Calcineurin.Results: Administration of 14 mg/kg choline to mice undergone TAC effectively attenuated the cardiac hypertrophic responses, as indicated by the reduced heart weight, left ventricular weight, ventricular thickness, and reduced expression of biomarker genes of cardiac hypertrophy. This anti-hypertrophic efficacy was reproduced in a cellular model of cardiomyocyte hypertrophy induced by isoproterenol in cultured neonatal cardiomyocytes. Our results further showed that choline rescued the aberrant downregulation of the muscle-specific microRNA miR-133a expression, a recently identified anti-hypertrophic factor, and restored the elevated calcineurin protein level, the key signaling molecule for the development of cardiac hypertrophy. These effects of choline were abolished by the M3 mAChR-specific antagonist 4-DAMP.Conclusion: Our study unraveled for the first time the cardioprotection of choline against cardiac hypertrophy, with correction of expression of miR-133a and calcineurin as a possible mechanism. Our findings suggest that choline supplement may be considered for adjunct anti-hypertrophy therapy.

Abstract 70: mTOR Is Essential for Normal Heart Development and Compensatory Cardiac Growth in Fetal Mice

Remarkable growth plasticity enables the prenatal mammalian heart to counteract various unfavorable intrauterine conditions and build a functional and normally sized organ at birth. We have recently shown that the murine embryonic and fetal heart has a substantial regenerative capacity in response to tissue mosaicism for mitochondrial dysfunction caused by heart specific inactivation of the X-linked holocytochrome c synthase (Hccs) gene. In heterozygous Hccs knockout (Hccs+/-) embryos, hyperproliferation of healthy cardiomyocytes compensates for the functional loss of 50% cardiac cells, ensuring formation of a functional heart at birth. However, we hypothesized that embryonic heart regeneration alters peri- and postnatal cardiac growth mechanisms. Indeed, neonatal Hccs+/- hearts are hypoplastic containing a reduced number of cardiomyocytes, whereas in adult Hccs+/- hearts compensatory cellular hypertrophy normalizes morphology and size. We aimed at identifying postnatal adaptive growth mechanisms utilized by the hypoplastic Hccs+/- heart to restore organ size and allow normal heart function throughout lifetime. Microarray RNA expression analyses revealed numerous genes involved in amino acid metabolism, protein homeostasis and translational control being differentially expressed in neonatal Hccs+/- hearts. Subsequently, western blot analyses evidenced significantly increased mammalian target of rapamycin (mTOR) activity, a major regulator of translation and cell growth, in Hccs+/- hearts compared to controls. To clarify its role for compensatory growth of the Hccs+/- myocardium, we inhibited mTOR in fetal and neonatal mice by rapamycin treatment of pregnant dams. Rapamycin treated Hccs+/- neonates (n=17) show significantly reduced heart weight to body weight ratios compared to controls (n=18) or vehicle treated animals (n=12 and n=13). Furthermore, Hccs+/- hearts after prenatal mTOR inhibition are morphologically characterized by developmental delay. In conclusion, our data revealed mTOR being essential for normal as well as compensatory growth of the fetal heart. Thus, metabolic adaptations converging on mTOR might be required to prevent postnatal heart disease after impaired intrauterine development.

Abstract 294: Atrogin-1 Is Required for Atrophic Remodeling of the Heart

The heart adapts to changes in load by remodeling both metabolically and structurally. During this process, cardiomyocytes break down unnecessary or damaged proteins and use the resulting amino acids for the synthesis of new proteins and/or energy provision. Protein degradation via the ubiquitin proteasome system is controlled by ubiquitin ligases, which determine the specific proteins to be degraded. Atrogin-1 is a muscle specific ubiquitin ligase required for skeletal muscle atrophy, and over-expressing Atrogin-1 inhibits the development of cardiac hypertrophy. We tested the hypothesis that Atrogin-1 is required for atrophic remodeling of the unloaded heart. Hearts from wild type (WT) and Atrogin-1 -/- mice were subjected to mechanical unloading by heterotopic transplantation. In WT hearts, seven days of unloading significantly reduced heart weight and myocyte cross-sectional area, while hearts lacking Atrogin-1 significantly hypertrophied. Conventional markers of atrophic remodeling, such as the reactivation of the fetal gene program were detected in both WT and Atrogin-1 -/-transplanted hearts. Proteasome activity and markers of autophagy were increased after unloading, although not significantly different between WT and Atrogin-1 -/- hearts. Pathways regulating protein synthesis were enhanced in the absence of Atrogin-1; there was an increase in activated Akt and its downstream pathway including mTOR, 4E-BP1, and p70 S6 kinase. Additionally, calcinuerin, a known target of Atrogin-1 involved in hypertrophy and protein synthesis, was upregulated in unloaded Atrogin-1 deficient hearts. Consequently, μunloaded” cardiomyocytes lacking Atrogin-1 in vitro exhibit increased basal rates of protein synthesis. While inhibition of calcineurin decreased rates of protein synthesis in unloaded cardiomyocytes in the absence of Atrogin-1, protein synthesis rates were still higher than in WT unloaded cardiomyocytes. These results suggest that more than one pathway regulating protein synthesis is controlled by Atrogin-1 in the heart. Furthermore, the data provide evidence that Atrogin-1 not only enhances protein degradation, but also keeps protein synthesis in check. Thus Atrogin-1 has a duel role in regulating cardiac mass.

Short-term therapy with simvastatin reduces inflammatory mediators and heart inflammation during the acute phase of experimental Chagas disease

Trypanosoma cruzi infection induces progressive cardiac inflammation that leads to fibrosis and modifications in the heart architecture and functionality. Statins, such as 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase inhibitors, have been studied due to their pleiotropic roles in modulating the inflammatory response. Our goal was to evaluate the effects of simvastatin on the cardiac inflammatory process using a cardiotropic strain of T. cruzi in a murine model of Chagas cardiomyopathy. C57BL/6 mice were infected with 500 trypomastigotes of the Colombian strain of T. cruzi and treated with an oral dose of simvastatin (20 mg/Kg/day) for one month and inflammatory and morphometric parameters were subsequently evaluated in the serum and in the heart, respectively. Simvastatin reduced the total cholesterol and inflammatory mediators (interferon-gamma, tumour necrosis factor-alpha, CCL2 and CCL5) in the serum and in the heart tissue at 30 days post-infection. Additionally, a proportional reduction in heart weight and inflammatory infiltration was observed. Simvastatin also reduced epimastigote proliferation in a dose-dependent manner in vitro and was able to reduce blood trypomastigotes and heart amastigote nests during the acute phase of Chagas disease in vivo. Based on these data, we conclude that simvastatin exerts a modulatory effect on the inflammatory mediators that are elicited by the Colombian strain of T. cruzi and ameliorates the heart damage that is observed in a murine model of Chagas disease.

Delta‐6 desaturase inhibition reverses cardiolipin remodeling and hypertrophy, but not mitochondrial dysfunction, in the aged mouse heart

Alterations in the fatty acid composition of cardiolipin (CL), a mitochondrial phospholipid, may contribute to mitochondrial dysfunction associated with myocardial aging, but its mechanism and role in this process remains unclear. We hypothesized that CL remodeling in the aged heart, characterized by a loss of linoleic acid (LA) and increase in highly unsaturated fatty acids (HUFAs), results from increased activity of delta 6‐desaturase (D6D), which catalyzes the production of HUFAs from essential fatty acids such as LA. Aged (24 mo) C57Bl/6 mice were administered a selective D6D inhibitor (SC‐26196, 100 mpk/d; SC) or no drug for 4 weeks, followed by isolation of cardiac subsarcolemmal (SSM) and interfibrillar (IFM) mitochondria for assessment of respiratory function and CL composition. Aged hearts exhibited a classic pattern of CL remodeling in IFM, but not SSM, which paralleled a 25% loss in state 3 respiration in IFM only. SC reversed CL remodeling in IFM, but had no effect on mitochondrial respiration. Interestingly, SC also reversed left ventricular dilatation, reduced heart weight and attenuated contractile dysfunction in aged mice. These studies indicate that D6D plays a pivotal role in CL remodeling in the aged heart, but argue against the role of this process in mitochondrial dysfunction. The mechanism by which D6D influences cardiac remodeling and function warrants further investigation. Funding: AHA

Abstract P180: Atrogin-1, a Muscle-Specific Ubiquitin Ligase, Drives Atrophic Remodeling of the Heart

The heart adapts to changes in load by remodeling both metabolically and structurally. During this process, cardiomyocytes break down unnecessary or damaged proteins and use the resulting amino acids for the synthesis of new proteins and/or energy provision. Protein degradation via the ubiquitin proteasome system is controlled by ubiquitin ligases, which determine the specific proteins to be degraded. Atrogin-1, a muscle specific ubiquitin ligase, is required for skeletal muscle atrophy, and over-expressing Atrogin-1 inhibits the development of cardiac hypertrophy. We now tested the hypothesis that Atrogin-1 is required for atrophic remodeling of the unloaded heart. Hearts from wild type (WT) and Atrogin-1 -/- mice (8-10 weeks old, n =8-12) were subjected to mechanical unloading by heterotopic transplantation. In WT hearts, seven days of unloading significantly reduced heart weight and myocyte cross-sectional area, while hearts lacking Atrogin-1 significantly hypertrophied (at least a 1.5-fold increase in heart weight, 2-fold increase in myocyte area). Conventional markers of atrophic remodeling, such as the reactivation of the fetal gene program (ANF, MHC isoform switch), were detected in both WT and Atrogin-1 -/- transplanted hearts. Proteasome activity and markers of autophagy were increased after unloading, although not significantly different between WT and Atrogin-1 -/- hearts. Pathways regulating protein synthesis were enhanced in the absence of Atrogin-1; there was an increase in activated Akt and its downstream pathway including mTOR, 4E-BP1, and p70 S6 kinase. Additionally, two known targets of Atrogin-1 involved in hypertrophy and protein synthesis, calcineurin and eukaryotic initiation factor 3f, were upregulated in unloaded Atrogin-1 deficient hearts. Consequently, “unloaded” cardiomyocytes lacking Atrogin-1 in vitro exhibit increased basal rates of protein synthesis. The results suggest that Atrogin-1 not only enhances protein degradation, but also keeps protein synthesis in check. Thus Atrogin-1 has a duel role in regulating cardiac mass.

Epigallocatechin gallate, the major component of polyphenols in green tea, inhibits telomere attrition mediated cardiomyocyte apoptosis in cardiac hypertrophy

BackgroundTelomere signaling plays a role in regulating cardiomyocyte apoptosis during cardiac dysfunction. In this study, we investigated the effects of epigallocatechin gallate (EGCG), the major component of polyphenols in green tea, on telomere dependent apoptotic signal in pressure overload cardiac hypertrophy. Methods and resultsCardiac hypertrophy in rats was established by abdominal aortic constriction (AC). EGCG 50, 100mg/kg, quercetin (Que) 100mg/kg, captopril (Cap) 50mg/kg, losartan (Los) 30mg/kg and carvedilol (Carv) 30mg/kg was intragastrically administered for 6weeks. Three, five and 7weeks after aortic constriction, the heart weight indices increased progressively. Malondialdehyde (MDA) contents progressively increased, while superoxide dismutase (SOD) activities decreased. Progressive cardiomyocyte apoptosis and telomere attrition were also found. Although no significant alteration of telomerase reverse transcriptase (TERT) mRNA was found till 7weeks after aortic constriction, progressive upregulation of p53, c-myc and downregulation of bcl-2, telomere repeat-binding factor 2(TRF2) were seen. EGCG, quercetin, captopril, losartan and carvedilol markedly reduced heart weight indices and apoptotic cardiomyocyte in hypertrophic myocardium, but they had different effects on apoptotic related proteins bcl-2, p53 and c-myc. EGCG, quercetin and carvedilol, have potent antioxidant effects as evidenced by reduction of MDA contents and resumption of SOD activities. EGCG, quercetin and carvedilol could prevent telomere attrition and telomere repeat-binding factor 2 (TRF2) loss remarkably, whereas captopril and losartan had no effect on oxidative stress and telomere signal. ConclusionsPressure overload induced cardiac hypertrophy initiates oxidative stress, induces telomere repeat-binding factor 2 loss and accelerates telomere shortening in hypertrophic myocardium. EGCG, quercetin and carvedilol with potent antioxidant effect, may inhibit cardiac myocyte apoptosis by preventing telomere shortening and telomere repeat-binding factor 2 (TRF2) loss.

Long-term cardiac pro-B-type natriuretic peptide gene delivery prevents the development of hypertensive heart disease in spontaneously hypertensive rats.

Diastolic dysfunction associated with high blood pressure (BP) leads to cardiac remodeling and fibrosis and progression to congestive heart failure. B-type natriuretic peptide (BNP) has BP-lowering, antifibrotic, and antihypertrophic properties, which makes BNP an attractive agent for attenuating the adverse cardiac remodeling associated with hypertension. In the current study, we tested the effects of sustained cardiac proBNP gene delivery on BP, cardiac function, and remodeling in spontaneously hypertensive rats (SHR). We used the myocardium-tropic adeno-associated virus serotype 9 (AAV9) vector to achieve continuously enhanced cardiac rat proBNP expression. In SHR, a single systemic administration of AAV9 vector allowed long-term cardiac BNP overexpression, resulting in reductions in systolic and diastolic BP for 9 months after injection. Left ventricular (LV) thickness, LV end-systolic dimensions, and LV mass were reduced, whereas ejection fraction was significantly increased, in BNP-treated compared with untreated SHR. Circumferential systolic strain and strain rate of the early phase of diastole were improved in BNP-treated compared with untreated SHR. Noncardiac overexpression of BNP via AAV2 vector was not associated with changes in BP and plasma BNP in SHR. Furthermore, normal Wistar rats injected with AAV9 proBNP vector showed significantly reduced heart weights 4 weeks after injection without BP reduction. AAV9 vector facilitates sustained cardiac proBNP overexpression and improves LV function in hypertensive heart disease. Long-term proBNP delivery improved both systolic and diastolic function. The effects on cardiac structure and function occurred independently of BP-lowering effects in normal Wistar rats.

Regular Grape Intake Reduces Cardiac and Renal Dysfunction and Metabolic Syndrome in Obesity‐Prone Rats

Metabolic Syndrome is characterized by increased systemic inflammation, abdominal fat, high triglycerides, low HDL, hypertension, and insulin resistance. The presence of some or all of these risk factors predisposes one to Type II diabetes and cardiovascular disease. Epidemiologic studies suggest that higher intake of fruits/vegetables could modify risk factors for metabolic syndrome. Phytochemicals may be vital to the observed health effects. Grapes are a rich, whole‐food source of phytochemicals. In the obesity‐prone Zucker Fatty rat, we tested the effect of a higher fat diet(45% kcal) with added whole table grape powder(3% w:w). Rats were fed for 90 days. Compared to a macronutrient and calorie‐matched control group, the grape diet had no significant effect on body weight or DEXA‐determined body composition. Grape intake reduced blood pressure and proteinuria while improving cardiac output. The grape‐fed group had reduced heart weight, kidney weight, and liver weight. The grape‐fed animals had reduced fasting serum triglycerides and hyperinsulinemia but not total cholesterol(p=0.12). Finally, grape intake reduced plasma inflammatory markers C‐reactive protein, TNF‐α and IL‐6, and reduced plasma oxidative stress marker 8‐isoprostane. The results suggest that regular intake of grapes reduced several risk factors for cardiovascular disease and metabolic syndrome.

Effects of pre- and postnatal protein deprivation and postnatal refeeding on atrial natriuretic peptide (ANP) granules of auricular cardiocytes

Background & aims: Atrial natriuretic peptide (ANP-granules) of auricular cardiocytes was examined by morphometry in undernourished and refed rats. Methods: The study was conducted on 21 and 42-day-old rats from mothers exposed to 73% protein restriction during the gestation and lactation with and without refeeding and on age-matched control animals. The body and the heart were weighted and the ANP-granules are located by transmission electron microscopy. Results: Protein deprivation in the 42 postnatal days reduced the body weights and the heart weights of the pups. Refeeding did not restore body weight or heart weight to control. In the undernourished group, the number of ANP-granules/field (27.3 ± 6.22) was significantly (p < 0.05) greater at 42 days than in the control (10.5 ± 1.89). In the refed group, the number of ANP-granules was also significantly greater, than that in the group on undernutrition. The diameter of ANP-granules in the undernourished animals was similar to the refed rats at 42 days and significantly greater than in the control (p < 0.05). Conclusion: Refeeding between 21 and 42 days increased the number of ANP-granules and maintained their diameter even when there is a reduction in heart weight caused by protein deprivation. © 2010 European Society for Clinical Nutrition and Metabolism.

Signal pathways involved in reverse remodeling of the hypertrophic rat heart after pressure unloading

Background The purpose of this study was to gain a better understanding of molecular changes associated with the beneficial reverse remodeling through heterotopic transplantation model of rat hypertrophic hearts. Methods Stable cardiac hypertrophy was induced by abdominal aortic constriction (AAC) in Lewis rats (6 weeks). Left ventricular (LV) pressure unloading was induced by heterotopic transplantation of hypertrophic hearts (AAC–HT) (2 weeks). We measured heart weight (HW), LV weight (LVW) and the LV-to-final body weight ratio (LVW/BW). Cross-sectional areas of cardiomyocyte and collagen content were assessed by hematoxylin/eosin staining and picrosirius red staining, respectively. We further analyzed the signaling pathways of mitogen-activated protein kinases (MAPKs), Akt/GSK3β, NF-κB, metalloproteinase-2,9 (MMP-2, 9) and tissue inhibitors of metalloproteinase-1 (TIMP-1) by Western blot. Results The HW, LVW and LVW/BW in AAC hearts were higher than normal hearts, but the transplanted hearts showed a significant reduction in HW, LVW and LVW/BW compared to AAC hearts. Unloading induced a decrease in cardiomyocyte size and an increase in collagen content in AAC–HT hearts. A significant decrease in phosphorylation of p44/p42 MAP kinases (ERK), Akt, GSK3β and NF-κB was detected in AAC–HT hearts, but the phosphorylation of p38 MAP kinase and Jun-N-terminal kinase (JNK) was not changed. MMP-2, MMP-9 and TIMP-1 activity also increased accompanied by unloading. Conclusions Pressure unloading of the hypertrophic heart caused a reverse remodeling through regulating the ERK, Akt/GSK3β, and NFκB signal pathways, revealing these as potential target pathways for reversal of LV hypertrophy.

Cardiac myocyte‐specific caveolin‐3 overexpression modulates ANP production and attenuates cardiac hypertrophy in vivo

IntroductionThe organization of signaling molecules in cardiac hypertrophy are incompletely understood. Caveolae, invaginations of the sarcolemmal membrane localize signaling molecules involved in cardiac hypertrophy. Atrial naturetic peptide (ANP) regulates endothelin‐1 release and cardiac hypertrophy. Furthermore, ANP is secreted via and stored within caveolae. We hypothesized that cardiac myocyte‐specific overexpression of Cav‐3 (Cav‐3 OE) modulates ANP production attenuating cardiac hypertrophy in vivo.MethodsCav‐3 OE and littermate controls (C) were subjected to transverse aortic constriction (TAC) for 4 weeks. Echocardiography, histology and molecular analysis were performed.ResultsCav‐3OE mice had reduced heart weights post TAC compared to C. Furthermore, Cav‐3 OE and C mice had similar fractional shortening (FS) pre TAC but had greater FS compared to C post TAC. mRNA levels of ANP were 7‐fold greater in Cav‐3 OE at baseline compared to C, resulting in increased protein expression of ANP in the cardiac myocyte and blood. Disruption of caveolae with colchicine in adult cardiac myocytes isolated from Cav‐3 OE resulted in a decrease in ANP.ConclusionWe show that Cav‐3 OE alters ANP production in cardiac myocytes blunting cardiac hypertrophy while preserving cardiac function post TAC. This suggests a therapeutic role for overexpression of Cav‐3 in pathological cardiac hypertrophy.

EGCG Inhibits Proliferation of Cardiac Fibroblasts in Rats with Cardiac Hypertrophy

This study was carried out in order to investigate the effects of epigallocatechin gallate (EGCG) on myocardial fibrosis and cell proliferation in cardiac hypertrophy. Cardiac hypertrophy was established in rats by abdominal aortic constriction, and EGCG at doses of 25, 50 and 100 mg/kg was administered intragastrically for 6 weeks. The results showed that in the rats with cardiac hypertrophy, EGCG at 25 - 100 mg/kg dose-dependently reduced heart weight indices, decreased atrial natriuretic polypeptide and endothelin levels in plasma, but increased nitrite (the oxidative product of NO) levels in the serum and in the myocardium. EGCG also reduced the hydroxyproline concentration and decreased the proliferating cell nuclear antigen expression in the hypertrophic myocardium. EGCG remarkably inhibited pressure overload-induced c-myc increase in Western blot analysis. In cultured newborn rat cardiac fibroblasts, treatment with EGCG at 12.5 - 200 mg/L for 6 - 48 h decreased cell proliferation induced by serum. EGCG at 12.5 - 100 mg/L dose-dependently inhibited cell proliferation and DNA synthesis of fibroblasts induced by angiotensin II (Ang II) at 1 mumol/L. EGCG also significantly increased nitrite levels in culture medium, and up-regulated inducible nitric oxide synthase protein expression if compared with the Ang II group. The inhibitory effect of EGCG on cell proliferation induced by Ang II was partly blocked by pretreatment with N(omega)-nitro- L-arginine methyl ester hydrochloride. These results suggest that EGCG inhibits the proliferation of cardiac fibroblasts both in vivo and in vitro, thereby preventing myocardial fibrosis in cardiac hypertrophy. EGCG might exert its cardiac protective action through induction of NO production.

Effects of Novel Vasopressin Receptor Antagonists on Renal Function and Cardiac Hypertrophy in Rats with Experimental Congestive Heart Failure

Arginine vasopressin (AVP) plays an important role in renal hemodynamic alterations, water retention, and cardiac remodeling in congestive heart failure (CHF). The present study evaluated the acute and chronic effects of vasopressin V(1a) receptor subtype (V(1a)) and vasopressin V(2) receptor subtype (V(2)) antagonists on renal function and cardiac hypertrophy in rats with CHF. The effects of acute administration of SR 49059 [(2S)1-[(2R,3S)-5-chloro-3-(2-chlorophenyl)-1-(3,4-dimethoxybenzene-sulfonyl)-3-hydroxy-2,3-dihydro-1H-indole-2-carbonyl]-pyrrolidine-2-carboxamide)] (0.1 mg/kg) and SR 121463B (1-[4-(N-tert-butylcarbamoyl)-2-methoxybenzenesulfonyl]-5-ethoxy-3-spiro-[4-(2-morpholinoethoxy)cyclohexane]indol-2-one, fumarate; equatorial isomer) (0.3 mg/kg), V(1a) and V(2) antagonists, respectively, on renal function, and of chronic treatment (3.0 mg/kg/day for 7 or 28 days, via osmotic minipumps or p.o.), on water excretion and cardiac hypertrophy were studied in rats with aortocaval fistula and control rats. CHF induction increased plasma AVP (12.8 +/- 2.5 versus 32.2 +/- 8.3 pg/ml, p < 0.05). Intravenous bolus injection of SR 121463B to controls produced dramatic diuretic response (from 5.5 +/- 0.8 to 86.3 +/- 21.9 microl/min; p < 0.01). In contrast, administration of SR 49059 did not affect urine flow. Likewise, administration of SR 121463B, but not SR 49059, to rats with CHF significantly increased urinary flow rate from 20.8 +/- 6.4 to 91.6 +/- 26.5 microl/min (p < 0.01). The diuretic effects of SR 121463B were associated with a significant decline in urinary osmolality and insignificant change of Na+ excretion. In line with its acute effects, chronic administration of SR 121463B to CHF rats increased daily urinary volume 2 to 5-fold throughout the treatment period. Both SR 121463B and SR 49059 significantly reduced heart weight in CHF rats when administered for 4 weeks, but not 1 week. These results suggest that V(2) and V(1a) antagonists improve water balance and cardiac hypertrophy in CHF and might be beneficial for the treatment of water retention and cardiac remodeling in CHF.

Toll-Like Receptor-4 Modulates Survival by Induction of Left Ventricular Remodeling after Myocardial Infarction in Mice

Left ventricular (LV) remodeling is known to contribute to morbidity and mortality after myocardial infarction (MI). Because LV remodeling is strongly associated with an inflammatory response, we investigated whether or not TLR-4 influences LV remodeling and survival in a mice model of MI. Six days after MI induction, TLR4 knockout (KO)-MI mice showed improved LV function 32 and reduced LV remodeling as indexed by reduced levels of atrial natriuretic factor and total collagen as well as by a reduced heart weight to body weight ratio when compared with WT-MI mice. This was associated with a reduction of protein levels of the intracellular TLR4 adapter protein MyD88 and enhanced protein expression of the anti-hypertrophic JNK in KO-MI mice when compared with wild-type (WT)-MI mice. In contrast, protein activation of the pro-hypertrophic kinases protein kinase Cdelta and p42/44 were not regulated in KO-MI mice when compared with WT-MI mice. Improved LV function, reduced cardiac remodeling, and suppressed intracellular TLR4 signaling in KO-MI mice were associated with significantly improved survival compared with WT-MI mice (62 vs 23%; p < 0.0001). TLR4 deficiency led to improved survival after MI mediated by attenuated left ventricular remodeling.

Thymocytes, Pre‐B Cells, and Organ Changes in a Mouse Model of Chronic Ethanol Ingestion—Absence of Subset‐Specific Glucocorticoid‐Induced Immune Cell Loss

The well-known immune deficiency of the chronic alcoholic dictates the need for a long-term rodent ethanol administration model to evaluate the baseline immunologic effects of chronic ethanol abuse, and investigate the genetic determinants of those effects. Much published work with rodents has shown clearly that acute ethanol administration and short-term ethanol-containing liquid diets both cause elevated corticosterone and can cause significant thymocyte, pre-B cell and peripheral lymphocyte losses. Such losses may mask more subtle alterations in immune homeostasis, and in any case are generally short-lived compared with the span of chronic ethanol abuse. Thus, it is important to have a model in which long-term immune alterations can be studied free of corticosteroid-induced cell losses. We have utilized chronic 20% (w/v) ethanol in water administration to several mouse strains for prolonged periods of time and evaluated serum corticosterone, immunologic stress parameters, and other organ changes by standard methods. We now confirm earlier reports that chronic ethanol in water administration to mice does not produce net elevations of corticosterone, although diurnal variation is altered. Importantly, there is neither selective loss of immune cell populations known to be corticosteroid sensitive, CD4+CD8+ thymocytes and pre-B cells, nor are changes observed in the histologic appearance of the thymus. Nonetheless, there are significant chronic ethanol effects in other tissues, including reduced heart weight, mild hepatic steatosis, alterations of gut flora, increased serum peptidoglycan, and as published elsewhere, immune system abnormalities. This model of ethanol administration is convenient, sustainable for up to 1 year, demonstrably feasible in several mouse strains, permits good weight gains in most strains, and results in significant changes in a number of organs. The administration method also will permit modeling of long-term steady abuse punctuated by major binges, and is suitable for supplementation studies using water soluble additives. Overall, the method is useful for a wide range of studies requiring a chronic low-stress method of ethanol administration.

Effects of Streptozotocin‐Induced Diabetes on Contraction and Calcium Transport in Rat Ventricular Cardiomyocytes

Cardiovascular diseases are the major cause of morbidity and mortality in diabetic patients. Contractile function of the heart is frequently compromised in the clinical setting and in experimental models of diabetes mellitus (DM). This article investigated the effect of streptozotocin (STZ)-induced type 1 DM on contraction, L-type calcium (Ca2+) current (I(Ca(2+)L)), and on cytosolic calcium concentrations [Ca2+]i in ventricular myocytes of the rat heart. After 4-10 weeks of STZ treatment, blood glucose levels in diabetic animals were significantly (P < 0.05) higher compared to age-matched controls. Diabetic rats have significantly (P < 0.05) reduced body, reduced heart weight, and reduced viability of ventricular myocytes compared to controls. The amplitude of I(Ca(2+)L) and amplitude of contraction were significantly reduced (P < 0.05) at test potentials in the range -10 mV to +20 mV and -30 mV to +40 mV, respectively, in myocytes from diabetic animals compared to age-matched controls. Moreover, there was a significant (P < 0.05) delay in electrically stimulated and caffeine-evoked time to half relaxation of the Ca2+ transient in myocytes from diabetic animals compared to controls. A similar effect was obtained in myocytes treated with a combination of caffeine and nickel chloride (NiCl2). It is concluded that the diabetes-induced voltage-dependent decrease in contraction is associated with reduced Ca2+ channel activities and prolonged diastolic cytosolic Ca2+ compared to age-matched control. Taken together, the results suggest that Ca2+ homeostasis is deranged during DM and this may be expressed at the level of the Na+/Ca2+ exchanger.

Gata4 is required for maintenance of postnatal cardiac function and protection from pressure overload-induced heart failure.

An important event in the pathogenesis of heart failure is the development of pathological cardiac hypertrophy. In cultured cardiomyocytes, the transcription factor Gata4 is required for agonist-induced hypertrophy. We hypothesized that, in the intact organism, Gata4 is an important regulator of postnatal heart function and of the hypertrophic response of the heart to pathological stress. To test this hypothesis, we studied mice heterozygous for deletion of the second exon of Gata4 (G4D). At baseline, G4D mice had mild systolic and diastolic dysfunction associated with reduced heart weight and decreased cardiomyocyte number. After transverse aortic constriction (TAC), G4D mice developed overt heart failure and eccentric cardiac hypertrophy, associated with significantly increased fibrosis and cardiomyocyte apoptosis. Inhibition of apoptosis by overexpression of the insulin-like growth factor 1 receptor prevented TAC-induced heart failure in G4D mice. Unlike WT-TAC controls, G4D-TAC cardiomyocytes hypertrophied by increasing in length more than width. Gene expression profiling revealed up-regulation of genes associated with apoptosis and fibrosis, including members of the TGF-beta pathway. Our data demonstrate that Gata4 is essential for cardiac function in the postnatal heart. After pressure overload, Gata4 regulates the pattern of cardiomyocyte hypertrophy and protects the heart from load-induced failure.

Second-Hand Smoke–Induced Cardiac Fibrosis Is Related to the Fas Death Receptor Apoptotic Pathway without Mitochondria-Dependent Pathway Involvement in Rats

Exposure to environmental tobacco smoke has been epidemiologically linked to heart disease among nonsmokers. However, the molecular mechanism behind the pathogenesis of cardiac disease is unknown. In this study, we found that Wistar rats, exposed to tobacco cigarette smoke at doses of 5, 10, or 15 cigarettes for 30 min twice a day for 1 month, had a dose-dependently reduced heart weight to body weight ratio and enhanced interstitial fibrosis as identified by histopathologic analysis. The mRNA and activity of matrix metalloprotease-2 (MMP-2), representing the progress of cardiac remodeling, were also elevated in the heart. In addition, we used reverse-transcriptase polymerase chain reaction and Western blotting to demonstrate significantly increased levels of the apoptotic effecter caspase-3 in treated animal hearts. Dose-dependently elevated mRNA and protein levels of Fas, and promoted apoptotic initiator caspase-8 (active form), a molecule of a death-receptor–dependent pathway, coupled with unaltered or decreased levels of cytosolic cytochrome c and the apoptotic initiator caspase-9 (active form), molecules of mitochondria-dependent pathways, may be indicative of cardiac apoptosis, which is Fas death-receptor apoptotic-signaling dependent, but not mitochondria pathway dependent in rats exposed to second-hand smoke (SHS). With regard to the regulation of survival pathway, using dot blotting, we found cardiac insulin-like growth factor-1 (IGF-1) and IGF-1 receptor mRNA levels to be significantly increased, indicating that compensative effects of IGF-1 survival signaling could occur. In conclusion, we found that the effects of SHS on cardiomyocyte are mediated by the Fas death-receptor–dependent apoptotic pathway and might be related to the epidemiologic incidence of cardiac disease of SHS-exposed non-smokers.