Atrial Natriuretic Factor mRNA Research Articles

Specific and sustained down-regulation of genes involved in fatty acid metabolism is not a hallmark of progression to cardiac failure in mice

Preferential and specific down-regulation of genes involved in fatty acid (FA) uptake and metabolism is considered a hallmark of severe hypertrophic remodeling and progression to cardiac failure. Therefore, we investigated the time course of changes in cardiac metabolic gene expression (1) in mice subjected to regional myocardial infarction (MI) for 4 days, 1 month, or 3 months and (2) in mice overexpressing calcineurin (Cn) which initially develop concentric hypertrophy progressing after the age of 4 weeks to dilated cardiomyopathy and failure. In both models, hypertrophy was characterized by increased expression of β-myosin heavy chain protein and atrial natriuretic factor mRNA, indicative of marked structural remodeling. Fractional shortening progressively decreased from 31% to 15.1% and 3.7% 1 and 3 months after MI, respectively. One month post-MI, the expression of several metabolic genes, i.e., acyl-CoA synthetase (−50%), muscle-type carnitine palmitoyl transferase 1 (−37%) and citrate synthase (−28%), was significantly reduced in the surviving myocardium. Despite overt signs of cardiac failure 3 months post-MI, the expression of these genes had returned to normal levels. In hearts of both 4- and 6-week-old Cn mice, genes involved in both FA and glucose metabolism and mitochondrial citrate synthase were down-regulated, reflecting an overall decline in metabolic gene expression, rather than a specific and preferential down-regulation of genes involved in FA uptake and metabolism. These findings challenge the concept that specific and sustained down-regulation of genes involved in FA uptake and metabolism represents a hallmark of the development of cardiac hypertrophy and progression to failure.

Effects of salt loading and various therapies on cardiac hypertrophy and fibrosis in young spontaneously hypertensive rats

We investigated the effects of salt loading on blood pressure, cardiac hypertrophy and fibrosis as well as on the effectiveness of various antihypertensive therapies in young spontaneously hypertensive rats (SHR). Twenty-five male SHR were salt-stimulated by drinking 1% NaCl from 3 to 6 months of age. Eighteen of them were treated for the last 2 weeks of salt loading with either the angiotensin-converting enzyme inhibitor captopril, the β-adrenergic receptor blocker propranolol or the calcium-channel antagonist verapamil. Age-matched male Wistar-Kyoto (WKY) rats and SHR drinking only water served as controls. At the age of 6 months, SHR had significantly elevated blood pressure that was unchanged by salt loading. Relative heart weight was increased in SHR without (3.3) and even more so with salt intake (3.6 vs. 2.4 in WKY). Left ventricular (LV) hypertrophy was accompanied by a 17-fold increase in the expression of mRNA for atrial natriuretic factor (ANF) both in untreated and salt-loaded SHR compared to WKY ( p < 0.001). Collagen I and III mRNA increased 1.7–1.8-fold in SHR without and with additional salt intake ( p < 0.01). None of the therapies significantly reduced blood pressure or hypertrophy. Although captopril had no antihypertensive effect, it reduced ANF, collagen I and III mRNA in LV to control level. Less pronounced effects were achieved with verapamil. These findings emphasize the cardioprotective role of captopril which may not be fully expressed in the presence of elevated salt intake.

High Fat/Low Carbohydrate Feeding Prevents Cardiac Hypertrophy, LV Remodeling and ANF Gene Induction, Independent of Hypertension

Chronic hypertension (HTN) causes left ventricular hypertrophy (LVH) and dilation, which leads to heart failure. Diets high in fat and low in carbohydrate are recommended to patients at risk, but their impact on LV adaptations to HTN are unknown. Male Dahl salt sensitive rats were fed for 12 wk with either 1) low fat (10% kcal fat), 2) low fat/high salt (6% NaCl), or 3) high fat/high salt (60% kcal fat, 67% saturated, 33% unsaturated; 6% NaCl) (n=10/group). Tail cuff blood pressure (BP) measurements and echocardiographic assessment of LV dimensions were performed before & after 11wks on diets. There were no differences in these parameters between groups prior to feeding, and body mass was not different among groups. BP increased with salt feeding, with no differences between low fat & high fat groups (P<0.05, 126±2, 201±3 and 193±3 mmHg respectively). Compared to the low fat/low salt group, HTN lead to an increase in LV mass (3.38±0.19 vs 2.13±0.05 g/kg) and end diastolic area (EDA; 0.80±0.03 vs 0.66±0.02 cm2), which corresponded with an 11-fold induction of atrial natriuretic factor (ANF) mRNA expression (10.7±0.2 vs 1.0±0.1%) in the low fat/high salt group. In contrast, despite similar elevations in BP, the high fat/high salt group had significantly reduced LV mass (2.74±0.11 g/kg), LV EDA (0.66±0.03), and ANF expression (3.8±0.1%) compared to the low fat/high salt group. Conclusion High fat low carbohydrate feeding prevented HTN induced LVH and LV remodeling. We speculate that the mechanism for this dramatic effect may be reduced cardiac protein synthesis secondary to a reduction in plasma insulin associated with chronic low carbohydrate/high fat diets.

Glycated Proteins Stimulate Reactive Oxygen Species Production in Cardiac Myocytes

Nonenzymatic glycation that results in the production of early-glycation Amadori-modified proteins and advanced-glycation end products may be important in the pathogenesis of diabetic complications. However, the effects of early-glycated proteins, such as glycated serum albumin (Gly-BSA), are poorly defined. In this study, we investigated the effects of Gly-BSA on reactive oxygen species (ROS) production by cardiomyocytes. Cultured neonatal rat cardiomyocytes were incubated with Gly-BSA or vehicle (bovine serum albumin [BSA]) for up to 48 hours. Gly-BSA dose-dependently increased in situ ROS production (whole-cell dichlorodihydrofluorescein fluorescence), with an optimum effect at 400 microg/mL after 24-hour incubation (152+/-10% versus BSA 100%; P<0.01). Treatment with the NADPH oxidase inhibitor apocynin, a Nox2 (gp91phox) antisense oligonucleotide (Nox2 AS), or the peptide gp91ds-tat significantly reduced Gly-BSA-induced ROS production at 24 hours (68.5+/-2.2%, 61.4+/-8.3%, and 53.2+/-5.4% reduction, respectively). NADPH-dependent activity in cell homogenates was also significantly increased by Gly-BSA at 24 hours (161+/-8% versus BSA) and was inhibited by diphenyleneiodonium, apocynin, NOX2AS, and the protein kinase C inhibitor bisindolylmaleimide I but not by a nitric oxide synthase inhibitor or mitochondrial inhibitors. Furthermore, bisindolylmaleimide I prevented Gly-BSA-stimulated Rac1 translocation, an essential step for NADPH oxidase activation. Gly-BSA-induced increases in ROS were associated with apocynin-inhibitable nuclear translocation of nuclear factor-kappaB and an increase in atrial natriuretic factor mRNA expression. Gly-BSA stimulates cardiomyocyte ROS production through a protein kinase C-dependent activation of a Nox2-containing NADPH oxidase, which results in nuclear factor-kappaB activation and upregulation of atrial natriuretic factor mRNA. These findings suggest that early-glycated Amadori products may play a role in the development of diabetic heart disease.

Pitavastatin Inhibits Cardiac Hypertrophy in a Rat Model of Progressive Renal Injury

Increased cardiovascular mortality is an unresolved problem of chronic renal failure. Cardiac hypertrophy, observed in many patients with chronic renal failure, is a major risk factor for cardiovascular death. The purpose of the present study was to examine the effects of pitavastatin on cardiac hypertrophy in a progressive renal injury rat model by subtotal nephrectomy (SNx). Because we previously reported that angiotensin II played a pivotal role in cardiac hypertrophy of SNx rats, we first investigated the effects of pitavastatin on angiotensin II-induced activation of extracellular signal-regulated kinase (ERK) and serum response element (SRE) DNA-binding activity using neonatal rat cardiomyocytes. Angiotensin II-induced ERK activation was attenuated by pretreatment with pitavastatin. Luciferase assay revealed that angiotensin II-induced increase in SRE DNA-binding activity was inhibited by pitavastatin. We next examined the effect of pitavastatin on cardiac hypertrophy of SNx rats in vivo. Treatment with pitavastatin prevented ERK activation and cardiac hypertrophy in SNx rats without changes in blood pressure. The increased expression of atrial natriuretic factor mRNA in SNx rat hearts was significantly attenuated by the treatment with pitavastatin. These results suggest that pitavastatin has a beneficial effect on cardiac hypertrophy in renal failure through preventing the activation of ERK.

Angiotensin-(1–7) binds to specific receptors on cardiac fibroblasts to initiate antifibrotic and antitrophic effects

ANG-(1-7) improves the function of the remodeling heart. Although this peptide is generated directly within the myocardium, the effects of ANG-(1-7) on cardiac fibroblasts that play a critical role in cardiac remodeling are largely unknown. We tested the hypothesis that specific binding of ANG-(1-7) to cardiac fibroblasts regulates cellular functions that are involved in cardiac remodeling. 125I-labeled ANG-(1-7) binding assays identified specific binding sites of ANG-(1-7) on adult rat cardiac fibroblasts (ARCFs) with an affinity of 11.3 nM and a density of 131 fmol/mg protein. At nanomolar concentrations, ANG-(1-7) interacted with specific sites that were distinct from ANG II type 1 and type 2 receptors without increasing cytosolic Ca2+ concentration. At these concentrations, ANG-(1-7) had inhibitory effects on collagen synthesis as assessed by [3H]proline incorporation and decreased mRNA expression of growth factors in ARCFs. These effects of ANG-(1-7) contrasted with effects of ANG II. Pretreatment of ARCFs with ANG-(1-7) inhibited ANG II-induced increases in collagen synthesis and in mRNA expression of growth factors, including endothelin-1 and leukemia inhibitory factor. ANG-(1-7) pretreatment also inhibited the stimulatory effects of conditioned medium from ANG II-treated ARCFs on [3H]leucine incorporation and atrial natriuretic factor mRNA expression, markers of hypertrophy, in cardiomyocytes. Thus ANG-(1-7) interacted with specific receptors on ARCFs to exert potential antifibrotic and antitrophic effects that could reverse ANG II effects. These results suggest that ANG-(1-7) may play an important role in the heart in regulating cardiac remodeling.

The Antioxidant Edaravone Attenuates Pressure Overload–Induced Left Ventricular Hypertrophy

The free radical scavenger 3-methyl-1-phenyl-2-pyrazolin-5-one (edaravone) is used to treat patients with ischemic brain damage. We and others reported previously that in vitro and in vivo reactive oxygen species (ROS) act as second messengers to develop cardiac hypertrophy. In this study, we used an in vivo murine model of pressure overload-induced cardiac hypertrophy to examine the effects of edaravone on left ventricular hypertrophy. The animals were subjected to the transverse thoracic aorta constriction, and edaravone (10 mg/kg) was infused intraperitoneally twice daily. Seven days after the operation, we observed a significant increase in ROS production in hearts, which was eliminated by the treatment with edaravone. Pressure-overloaded hearts showed a significant increase in left ventricular weight/body weight ratio and the expression level of atrial natriuretic factor mRNA, which were attenuated by edaravone. It also reduced perivascular and intermuscular fibrosis and inhibited pressure overload-induced activation of apoptosis signal-regulating kinase 1 (ASK1) and its downstream kinases of c-Jun N-terminal protein kinase and p38 mitogen-activated protein kinase. Edaravone attenuated the hypertrophic response even when the treatment was started after the onset of cardiac hypertrophic response. These findings indicate that edaravone significantly attenuates pressure overload-induced cardiac hypertrophy mediated through its antioxidative function and subsequent inhibition of ASK1 signaling pathway.

Induction of Cardiac Hypertrophy by a Controlled Reproducible Sutureless Aortocaval Shunt in the Mouse

Much of the understanding about the pathophysiological responses to chronic cardiac overload has been gained by the use of rat and dog models of aortocaval fistula (ACF). The use of a similar model in genetically manipulated mice may further elucidate the molecular mechanisms in these responses. The only reports about ACF in mice to date have applied a needle puncture to create the ACF, which may result in an uncontrolled and irreproducible size of the shunt, and require several weeks to induce the characteristic cardiac changes. In order to obtain a more consistent approach to characterize this mode of cardiac hyperfunction, we present a surgical murine model of ACF that results in rapid progression of the typical systemic and cardiac changes. A sutureless side-to-side infrarenal surgical anastomosis of 0.6–0.8 mm in diameter was created between the abdominal aorta and inferior vena cava in ICR (Institute of Cancer Research) mice. Six to 7 days later, significant cardiac hypertrophy developed. The heart/body weight ratio increased from 0.45 ± 0.02% in control mice to 0.77 ± 0.03% in mice with ACF (p <. 003). The dry heart weight ratio increased from 0.099 ± 0.0033% to 0.13 ± 0.008% (p <. 006). The ACF dramatically induced the atrial and ventricular expression of atrial natriuretic factor mRNA, and increased the total cardiac content of endothelin-1 (162.5 ± 50.6 vs. 83.9 ± 9.0 pg). Mean arterial pressure in anesthetized mice with ACF decreased from 69.8 ± 4.9 to 54.8 ± 5.5 mm Hg (p <. 025). Urinary sodium excretion returned to preoperative levels several days following surgery. These results demonstrate that cardiac hypertrophy could be rapidly and reproducibly achieved in mice by the placement of a surgical ACF. This model, when applied in genetically manipulated mice, may be a valuable tool for functional genomic studies about the pathogenesis of cardiac hypertrophy and heart failure.

Increased cardiac workload by closure of the ductus arteriosus leads to hypertrophy and apoptosis rather than to hyperplasia in the late fetal period.

It is generally thought that adult mammalian cardiomyocytes compensate for an increased workload by hypertrophy, whereas fetal myocardium grows by cellular proliferation. We analyzed the response of late-fetal rat hearts upon an increased workload imposed by premature constriction of the ductus arteriosus with indomethacin. Initially the fetal heart responds by proliferative growth, as both wet weight and labeling index (bromodeoxyuridine incorporation) of the ventricles increased, whereas neither a change in the fibroblast fraction, ploidy and nucleation in the ventricles is observed. However, this hyperplastic growth is abrogated by a subsequent burst in apoptosis and followed by a hypertrophic response as based on a decrease in DNA and increase in both RNA and protein concentration. This hypertrophic growth was accompanied by a 1.4-fold increase in the volume of the cardiomyocytes. Changes in the molecular phenotype characteristic of pressure-overload hypertrophic growth accompany the process. Thus, the levels of expression of beta-myosin heavy chain and atrial natriuretic factor mRNA increased, of sarcoplasmic/endoplasmic reticulum ATPase (SERCA2) mRNA decreased, and of alpha-myosin heavy chain, phospholamban, and calsequestrin mRNA did not change. In situ hybridization showed that the pattern of mRNA expression changed first in the right ventricular wall and subsequently in the left ventricular free wall as well. It is concluded that pressure-overload imposed on the late-fetal heart induces limited proliferative growth complemented by extensive hypertrophic growth.

Selective upregulation of cardiac brain natriuretic peptide at the transcriptional and translational levels by pro-inflammatory cytokines and by conditioned medium derived from mixed lymphocyte reactions via p38 MAP kinase

An increase in circulating brain natriuretic peptide (BNP) but not atrial natriuretic factor (ANF) is observed coincident with cardiac allograft rejection that is reversed upon treatment with anti-lymphocyte therapy suggesting that pro-inflammatory cytokines may uniquely modulate BNP gene expression and secretion. This study tested pro-inflammatory cytokines or conditioned medium (CM) derived from mixed– lymphocyte reaction (MLR) cultures in their ability to modulate ANF or BNP mRNA expression, secretion, as well as BNP promoter activity in cultured neonatal rat cardiocytes. IL-1β and TNF-α elicited a significant dose- and time-dependent increase in BNP mRNA, and secretion, whereas, ANF mRNA levels and secretion did not change. IL-1β and TNF-α rapidly increased phosphorylated p38 MAP kinase abundance and activity. Inhibition of p38 MAP kinase with SB203580 abolished IL-1β- and TNF-α-stimulated increase in BNP mRNA, promoter activity and secretion. MLR-CM in 20%, 50% and 100% proportions increased BNP but not ANF secretion. The MLR-induced increases in BNP secretion were completely abolished by SB203580 pre-treatment. These investigations show that exposure of cultured rat cardiocytes to specific pro-inflammatory cytokines as well as MLR-CM results in the only known instance of upregulation of cardiac BNP at the transcriptional and translational levels without a corresponding increase in ANF gene expression. Furthermore, these effects are dependent on signaling by p38 MAP kinase. In all, the findings reveal a unique dis-coordinated expression of BNP and ANF to inflammatory cytokines and offers an opportunity to better understand the differential regulation of these two cardiac-derived endocrine hormones that share receptors as well as biological properties.

201 Stage-dependent activation of cell cycle and apoptosis mechanisms in the right ventricle by pressure overload

induction and development of cardiac hypertrophy (CH). In this study we investigated, whether calcineurin might be involved in the development of the left/right ventricle differences in NE-induced CH. Female Sprague-Dawley rats were treated with continuous NaCl-infusion (controls, C) or NE-infusion (0.1 mg/kg.h i.v.) for 12 and 96 hours. The effect of NE-treatment was documented by changes in the LV and RV function and in the change in the LV weight to body weight (LVW/BW) and RV weight to body weight (RVW/BW) ratios. Western blots were done with calcineurin-A (Cn-A) antibody (Stressgene Biotechnologies). We used the rat brain tissue extract as positive control for calcineurin antibody. After 12h of NE-treatment the RV systolic pressure (C 28.7±4.1, NE 65.9±2.7 mm Hg), heart rate (C 381±10.5, NE 490±12.7 beats/min), RV dP/dt max (C 1927±462.2, NE 5380±508 mm Hg/s) as well as LV dP/dt max (C 9276±1192, NE 18405±1538 mm Hg/s) were significantly increased (all p < 0.01). The LVW/BW ratio, but not the RVW/BW ratio was significantly increased in NE-treated rats (p < 0.05). Also the mRNA for atrial natriuretic factor (ANP), a marker of hypertrophy, was significantly increased in the LV only as shown previously by Barth et al. (J Mol Cell Cardiol 32, 2000, 273-284). At this time point neither in the LV, nor in the RV was the Cn-A protein significantly changed after NEtreatment. After 96h of NE-treatment heart function remained elevated to a similar extent as after 12h. The LVW/BW, but not the RVW/BW ratio was significantly increased (from 2.19±0.07 to 2.5±0.05, p < 0.01). There was a significant increase in the Cn-A protein in the LV only (factor of change for LV 1.49±0.26, p < 0.05). This increase was also significantly different from the change in the RV (factor of change 1.01 ± 0.15; p < 0.05). Thus, calcineurin seems to be differently expressed in the left and right ventricle in the NE-induced hypertrophy and may play a role in the signalling for ventricle-specific cardiac hypertrophy.

Expression of cVg1 mRNA during chicken embryonic development.

Using degenerated PCR-primers to identify known and novel BMPs that are expressed in the developing chicken heart, we identified not only BMP2, -4, and -7 mRNA, but also the TGFbeta superfamily member cVg1. The expression pattern of cVg1 mRNA was determined during chicken development from HH4 to HH44. In early developmental stages, cVg1 mRNA is expressed in the primitive streak, paraxial mesoderm, developing somites, and developing neural tube. Subsequently, cVg1 mRNA is expressed in the developing central and peripheral nervous system, retina, auditory vesicle, notochord, lung alveoli, and olfactory mucosa. In the heart, cVg1 is initially expressed through the linear heart tube, but becomes restricted to the forming chamber myocardium, in an expression domain similar to that of atrial natriuretic factor (ANF) mRNA.

Minimally invasive aortic banding in mice: effects of altered cardiomyocyte insulin signaling during pressure overload.

We developed a minimally invasive method for producing left ventricular (LV) pressure overload in mice. With the use of this technique, we quickly and reproducibly banded the transverse aorta with low surgical morbidity and mortality. Minimally invasive transverse aortic banding (MTAB) acutely and chronically increased LV systolic pressure, increased heart weight-to-body weight ratio, and induced myocardial fibrosis. We used this technique to determine whether reduced insulin signaling in the heart altered the cardiac response to pressure overload. Mice with cardiac myocyte-restricted knockout of the insulin receptor (CIRKO) have smaller hearts than wild-type (WT) controls. Four weeks after MTAB, WT and CIRKO mice had comparably increased LV systolic pressure, increased cardiac mass, and induction of mRNA for beta-myosin heavy chain and atrial natriuretic factor. However, CIRKO hearts were more dilated, had depressed LV systolic function by echocardiography, and had greater interstitial fibrosis than WT mice. Expression of connective tissue growth factor was increased in banded CIRKO hearts compared with WT hearts. Thus lack of insulin signaling in the heart accelerates the transition to a more decompensated state during cardiac pressure overload. The use of the MTAB approach should facilitate the study of the pathophysiology and treatment of pressure-overload hypertrophy.

Discoordinate re-expression of cardiac fetal genes in N(omega)-nitro-L-arginine methyl ester (L-NAME) hypertension.

Hypertension produced by chronic inhibition of nitric oxide (NO) synthase by N(omega)-nitro-L-arginine methyl ester (L-NAME) was used to determine the effect of severe pressure overload with or without left ventricular (LV) hypertrophy on the transcriptional activation of the cardiac fetal genes encoding for the natriuretic peptides (NP) atrial natriuretic factor (ANF) and brain natriuretic peptide (BNP), and for beta-myosin heavy chain (MHC) in both atrial and ventricular muscle. A previously reported association of LV hypertrophy with the activation of cardiac renin and angiotensin-converting enzyme (ACE) in this hypertension model was also investigated. Male Sprague-Dawley rats received L-NAME (75 mg/kg/day) or were left untreated for 4 (n=12) or 8 (n=12) weeks. L-NAME-treated rats became severely hypertensive in both treatment groups but only five out of 12 8-week treatment animals showed a significantly increased LV weight to body weight (BW) ratio (LVW/BW). LV ANF mRNA, but not LV BNP mRNA, correlated significantly with LVW/BW only in animals showing LV hypertrophy. No changes were observed in atrial gene expression or plasma concentration of ANF or BNP. A significant correlation was found between LVW/BW and LV renin mRNA and LV ACE activity in rats with LV hypertrophy. LV beta-MHC mRNA levels were significantly increased in the LV of rats with or without LV hypertrophy at both 4 and 8 weeks of treatment. It is concluded that pressure overload per se does not promote NP or cardiac renin-angiotensin system gene expression while increased beta-MHC expression is a marker of LV pressure overload even in the absence of LV hypertrophy. It is apparent that L-NAME causes a disruption in the coordinated transcriptional activation of cardiac fetal genes expected of hypertrophic stimuli acting on the LV.

Endothelin-A receptor blockade prevents left ventricular hypertrophy and dysfunction in salt-sensitive experimental hypertension.

Salt-sensitive hypertension represents a major cause of left ventricular (LV) dysfunction. We therefore explored the potential effects of the selective endothelin-A (ETA) receptor antagonist darusentan on the development of hypertension, LV hypertrophy (LVH), and dysfunction in a genetic rat model of salt-sensitive hypertension. Animals from the salt-sensitive Sabra rat strain (SBH/y) and the salt-resistant strain (SBN/y) were treated with either normal diet (SBH/y and SBN/y) or with deoxycorticosterone-acetate (DOCA) and salt (SBN/y-DOCA and SBH/y-DOCA). Additional groups were treated with 50 mg x kg(-1) x d(-1) of darusentan (SBH/y-DOCA-DA and SBN/y-DOCA-DA). Systolic blood pressure and LV weight increased in response to DOCA only in the SBH/y strain (+75 mm Hg and +30%; P<0.05). LV end-diastolic pressure increased and -dP/dtmax decreased in SBH/y-DOCA compared with SBH/y (P<0.05). This was paralleled by a 5-fold upregulation of LV mRNA expression of atrial natriuretic factor (ANF) and a significant reduction of sarcoplasmic reticulum (SR) Ca2+-reuptake and the SR Ca2+-ATPase to phospholamban protein ratio (-30%). Whereas treatment with darusentan in SBH/y-DOCA-DA reduced the SBP increase by 50%, LVH elevation of ANF mRNA and LV dysfunction were completely prevented (P<0.05); this was associated with a normalization of SR Ca2+-reuptake and SR Ca2+-ATPase to phospholamban ratio by darusentan (P<0.05). A moderate elevation of interstitial fibrosis in SBH/y-DOCA (P<0.05) remained unaffected by darusentan treatment. In the Sabra model of salt-sensitive hypertension, ETA-receptor blockade demonstrated striking effects on the prevention of LVH and LV dysfunction beyond its considerable antihypertensive effect.

Prevention of hypertrophy by overexpression of Kv4.2 in cultured neonatal cardiomyocytes.

Prolonged action potentials (APs) and decreased transient outward K+ currents (I(to)) are consistent findings in hypertrophic myocardium. However, the connection of these changes with cardiac hypertrophy is unknown. The present study investigated the effects of changes in I(to) and the associated alterations in AP on myocyte hypertrophy induced by phenylephrine. Chronic incubation of cultured neonatal ventricular rat myocytes (NVRMs) with phenylephrine (PE) reduced I(to) density and prolonged AP duration, leading to a 2-fold increase in the net Ca2+ influx per beat and a 1.4-fold increase in Ca2+-transient amplitude. PE treatment of chronically paced (2-Hz) NVRM also induced increases in cell size, protein/DNA ratio, atrial natriuretic factor mRNA expression, as well as beta/alpha myosin mRNA ratio. These hypertrophic changes were associated with a 2.4-fold increase in activation of nuclear factor of activated T-cells (NFAT), indicating increased activity of the Ca2+-dependent phosphatase calcineurin. Overexpression of Kv4.2 channels using adenovirus prevented the AP duration prolongation as well as the increases in Ca2+ influx and Ca2+-transient amplitude induced by PE. Kv4.2 overexpression also prohibited the PE-induced increases in cell size, protein/DNA ratio, atrial natriuretic factor expression, beta/alpha myosin mRNA ratio, and NFAT activation. Our results demonstrate that PE-mediated hypertrophy in NRVMs seems to require I(to) reductions and AP prolongation associated with increased Ca2+ influx and Ca2+ transients as well as calcineurin activation. The clinical implications of these studies and the possible involvement of other signaling pathways are discussed.

Left ventricular unloading alters receptor tyrosine kinase expression in the failing human heart

Background: Experimental and clinical data suggest that the loss of membrane receptor tyrosine kinase (RTK) activity in cardiac myocytes results in increased frequency of apoptotic cell death and progression of heart failure. The goal of our study was to examine the expression characteristics of RTKs in ventricular myocardium obtained from patients before and after mechanical unloading. Methods: We extracted RNA from paired formalin-fixed, paraffin-embedded left ventricular tissue blocks obtained at the time of left ventricular assist device (LVAD) implantation and explantation from a cohort of 36 patients (median age 51 years). The duration of LVAD support ranged from 1 to 314 days (median 95 days), 17 patients had ischemic and 19 non-ischemic cardiomyopathy at the time of LVAD implantation. Using real-time reverse transcription–polymerase chain reaction (RT-PCR) we quantitated transcripts for atrial natriuretic factor (ANF) and tumor necrosis factor-alpha (TNF-α), markers of heart failure, and the RTKs Her2/neu, Her4 and gp130, regulators of cardiac cell survival. Results: In patients undergoing mechanical unloading, ANF and TNF-α mRNA levels were independently suppressed. Her2/neu, along with Her4 was upregulated, mostly in cases of ischemic cardiomyopathy, whereas gp130 levels decreased. Post-LVAD transcript levels of Her2 correlated tightly with gp130 in patients with non-pathologic entry values of gp130. Duration of treatment and age were also determining factors in the change of expression of these genes. Conclusion: Real-time quantitative (Q)-RT-PCR can be used to quantitate gene expression in archival myocardial tissue blocks. Mechanical unloading leads to a re-adjustment of RTK transcript levels, but not their reverting to control values in heart failure patients.

GENETIC VARIABILITY IN FORCED AND VOLUNTARY ENDURANCE EXERCISE PERFORMANCE IN SEVEN INBRED MOUSE STRAINS

PURPOSE: The goal of this study was to characterize the genetic contribution to both forced and voluntary exercise performance and to determine whether performance in these two paradigms is controlled by similar genetic influences. METHODS: Seven inbred mouse strains (Balbc/ByJ, C3H/HeJ, C57BL/6J, DBA/1J, DBA/2J, FVB/NJ, and SW) were assessed for treadmill exercise performance and voluntary wheel performance. Expression of two molecular markers of cardiac hypertrophy (beta myosin heavy chain (β-MyHC) and atrial natriuretic factor (ANF)) was determined for all strains. Cardiac function was assessed via echocardiography and isolated work-performing heart preparations. RESULTS: There were marked strain differences in treadmill exercise performance, with SW and FVB/NJ mice showing elevated performance and C57BL/6J animals showing decreased performance compared to all other strains. Analysis of voluntary free-wheel performance also showed significant strain differences, with SW and C57BL/6J animals showing increased voluntary wheel activity compared to all other strains and DBA/1J animals showing decreased performance compared to all other strains. There was no correlation between treadmill performance and voluntary free-wheel performance. For treadmill and voluntary wheel performance, two estimates of heritability in the broad-sense, the intraclass correlation (rI) and the coefficient of genetic determination (g2) ranged from rI = 0.38 and g2 = 0.24 (average speed on the voluntary wheel) to rI = 0.90 and g2 = 0.82 (treadmill endurance test). DBA/1J and SW mice exhibited significantly greater cardiac contractility than all other analyzed strains while Balbc/ByJ mice exhibited significantly reduced cardiac contractility compared to all other strains. ANF and β-MyHC mRNA levels were significantly elevated in the DBA/2J myocardium compared to all other analyzed strains. CONCLUSIONS: The inbred mouse strains analyzed demonstrate markedly distinct endurance exercise capacities for both treadmill-based and voluntary wheel-based exercise paradigms. For the strains analyzed, treadmill performance did not predict voluntary wheel performance, and there was no correlation across strains between the relative performances for these two distinct exercise paradigms. There were significant strain differences for cardiac gene expression and cardiac function, but there was no significant correlation between any of these measures and either forced or voluntary exercise performance.

Targeted myocardial transgenic expression of HIV Tat causes cardiomyopathy and mitochondrial damage.

Cardiac effects of human immunodeficiency virus (HIV) transactivator (Tat) are unclear, but Tat decreases liver glutathione (an important mitochondrial antioxidant) when ubiquitously expressed in transgenic mice (TG). With an alpha-myosin heavy chain promoter, Tat was selectively targeted to murine cardiac myocytes. One high-expression hemizygous ((+/-)Tat(high); 12 copies) and two low-expression ((+/-)Tat(lowA,B); 2-5 copies) TG lines were created. Cardiomyopathy was documented with increased left ventricle (LV) mass, ventricular expression of atrial natriuretic factor (ANF) mRNA, mitochondrial ultrastructural defects, and myocardial depletion of glutathione. In (+/-)Tat(high) TGs, normalized LV mass (determined echocardiographically) increased 46% (90 days), 134% (240 days), and 96% (365 days) compared with wild-type littermates (WT). LV fractional shortening was decreased to 28% (90 days), 27% (240 days), and 19% (365 days). (+/-)Tat(low) LV mass was unchanged (<or=365 days). ANF in (+/-)Tat(high) ventricles (180 days) was twofold WT values. Glutathione was selectively decreased in (+/-)Tat(high) hearts (120 days). (+/-)Tat(high) hearts contained damaged mitochondria (>or=210 days); however, profound mitochondrial destruction occurred in homozygous (+/+)Tat(high) hearts (10 days) and the pups died (14 days). Tat caused cardiac dysfunction in this TG and may impact on cardiomyopathy in acquired immunodeficiency syndrome.

Gene expression of adrenomedullin in failing myocardium: comparison to atrial natriuretic peptide

The expression of adrenomedullin (AM) and atrial natriuretic factor (ANF) were investigated in the myocardium of a rat model of chronic ischemic heart failure (CHF) compared with sham-operated controls. In addition, human myocardium of patients with end-stage heart failure due to idiopathic dilated cardiomyopathy compared with myocardium of normal subjects (NF) was studied. In CHF, similar AM levels but increased ANF expression were observed in left ventricular myocardium, as assessed by semiquantitative PCR. Functional experiments with freshly excised papillary muscles showed no influence of AM on myocardial contractility. In NF human myocardium, the expression of AM mRNA was threefold higher in atrial compared with ventricular tissue. In analogy, ANF mRNA was increased by approximately 15-fold in atrial tissue. In dilated cardiomyopathy, the expression of AM was significantly increased in right and left ventricles compared with NF. In parallel, ventricular ANF expression was enhanced.