Elevated expression of activated Na+/H+ exchanger protein induces hypertrophy in isolated rat neonatal ventricular cardiomyocytes

The plasma membrane protein the Na(+)/H(+) exchanger isoform1 (NHE1) has been implicated in various cardiac pathologies including ischemia/reperfusion damage to the myocardium and cardiac hypertrophy. Levels of NHE1 protein and activity are elevated in cardiac disease; however, the mechanism by which these factors contribute to the accompanying hypertrophy in the myocardium is still not clear. To investigate the mechanism of NHE1-induced hypertrophy in the myocardium we constructed two adenoviral vectors expressing either wild type NHE1 protein or a constitutively active NHE1 protein. Infection of neonatal rat ventricular cardiomyocytes (NRVM) resulted in elevated expression of both wild type NHE1 or constitutively active NHE1. Only expression of activated NHE1 protein resulted in an increase in cell size and in an increase in protein synthesis in isolated cardiomyocyte cells. The results demonstrate that expression of activated NHE1 promotes cardiac hypertrophy in isolated cardiac cells and that simple elevation of levels of wild type NHE1 protein does not have a significant hypertrophic effect in NRVM. The results suggest that regulation of NHE1 activity is a critical direct effector of the hypertrophic effect induced in the myocardium by the NHE1 protein.     

Progression from hypertrophic to dilated cardiomyopathy in mice that express a mutant myosin transgene

A mouse model of hypertrophic cardiomyopathy (HCM) was created by expression of a cardiac alpha-myosin transgene including the R(403)Q mutation and a deletion of a segment of the actin-binding domain. HCM mice show early histopathology and hypertrophy, with progressive hypertrophy in females and ventricular dilation in older males. To test the hypothesis that dilated cardiomyopathy (DCM) is part of the pathological spectrum of HCM, we studied chamber morphology, exercise tolerance, hemodynamics, isolated heart function, adrenergic sensitivity, and embryonic gene expression in 8- to 11-mo-old male transgenic animals. Significantly impaired exercise tolerance and both systolic and diastolic dysfunction were seen in vivo. Contraction and relaxation parameters of isolated hearts were also decreased, and lusitropic responsiveness to the beta-adrenergic agonist isoproterenol was modestly reduced. Myocardial levels of the G protein-coupled beta-adrenergic receptor kinase 1 (beta-ARK1) were increased by more than twofold over controls, and total beta-ARK1 activity was also significantly elevated. Induction of fetal gene expression was also observed in transgenic hearts. We conclude that transgenic male animals have undergone cardiac decompensation resulting in a DCM phenotype. This supports the idea that HCM and DCM may be part of a pathological continuum rather than independent diseases.     

Blocking cardiac growth in hypertrophic cardiomyopathy induces cardiac dysfunction and decreased survival only in males

Mutations in myosin heavy chain (MyHC) can cause hypertrophic cardiomyopathy (HCM) that is characterized by hypertrophy, histopathology, contractile dysfunction, and sudden death. The signaling pathways involved in the pathology of HCM have not been elucidated, and an unresolved question is whether blocking hypertrophic growth in HCM may be maladaptive or beneficial. To address these questions, a mouse model of HCM was crossed with an antihypertrophic mouse model of constitutive activated glycogen synthase kinase-3beta (caGSK-3beta). Active GSK-3beta blocked cardiac hypertrophy in both male and female HCM mice. However, doubly transgenic males (HCM/GSK-3beta) demonstrated depressed contractile function, reduced sarcoplasmic (endo) reticulum Ca(2+)-ATPase (SERCA) expression, elevated atrial natriuretic factor (ANF) expression, and premature death. In contrast, female HCM/GSK-3beta double transgenic mice exhibited similar cardiac histology, function, and survival to their female HCM littermates. Remarkably, dietary modification from a soy-based diet to a casein-based diet significantly improved survival in HCM/GSK-3beta males. These findings indicate that activation of GSK-3beta is sufficient to limit cardiac growth in this HCM model and the consequence of caGSK-3beta was sexually dimorphic. Furthermore, these results show that blocking hypertrophy by active GSK-3beta in this HCM model is not therapeutic.     

Gender-specific patterns of left ventricular and myocyte remodeling following myocardial infarction in mice deficient in the angiotensin II type 1a receptor

Left ventricular (LV) remodeling after myocardial infarction (MI) results from hypertrophy of myocytes and activation of fibroblasts induced, in part, by ligand stimulation of the ANG II type 1 receptor (AT1R). The purpose of the present study was to explore the specific role for activation of the AT 1a R subtype in post-MI remodeling and whether gender differences exist in the patterns of remodeling in wild-type and AT 1a R knockout (KO) mice. AT 1a R-KO mice and wild-type littermates underwent coronary ligation to induce MI or sham procedures; echocardiography and hemodynamic evaluation were performed 6 wk later, and LV tissue was harvested for infarct size determination, morphometric measurements, and gene expression analysis. Survival and infarct size were similar among all male and female wild-type and AT 1a R-KO mice. Hemodynamic indexes were also similar except for lower systolic blood pressure in the AT 1a R-KO mice compared with wild-type mice. Male and female wild-type and male AT 1a R-KO mice developed similar increases in LV chamber size, LV mass corrected for body weight (LV/BW), and myocyte cross-sectional area (CSA). However, female AT 1a R-KO mice demonstrated no increase in LV/BW and myocyte CSA post-MI compared with shams. Both male and female wild-type mice demonstrated higher atrial natriuretic peptide (ANP) levels after MI, with female wild types being significantly greater than males. However, male and female AT 1a R-KO mice developed no increase in ANP gene expression with MI despite an increase in LV mass and myocyte size in males. These data support that gender-specific patterns of LV and myocyte hypertrophy exist after MI in mice with a disrupted AT 1a R gene, and suggest that myocyte hypertrophy post-MI in females relies, in part, on activation of the AT 1a R. Further work is necessary to explore the potential mechanisms underlying these gender-based differences.     

Sex differences in physiological cardiac hypertrophy are associated with exercise-mediated changes in energy substrate availability

Exercise-induced cardiac hypertrophy has been recently identified to be regulated in a sex-specific manner. In parallel, women exhibit enhanced exercise-mediated lipolysis compared with men, which might be linked to cardiac responses. The aim of the present study was to assess if previously reported sex-dependent differences in the cardiac hypertrophic response during exercise are associated with differences in cardiac energy substrate availability/utilization. Female and male C57BL/6J mice were challenged with active treadmill running for 1.5 h/day (0.25 m/s) over 4 wk. Mice underwent cardiac and metabolic phenotyping including echocardiography, small-animal PET, peri-exercise indirect calorimetry, and analysis of adipose tissue (AT) lipolysis and cardiac gene expression. Female mice exhibited increased cardiac hypertrophic responses to exercise compared with male mice, measured by echocardiography [percent increase in left ventricular mass (LVM): female: 22.2 ± 0.8%, male: 9.0 ± 0.2%; P < 0.05]. This was associated with increased plasma free fatty acid (FFA) levels and augmented AT lipolysis in female mice after training, whereas FFA levels from male mice decreased. The respiratory quotient during exercise was significantly lower in female mice indicative for preferential utilization of fatty acids. In parallel, myocardial glucose uptake was reduced in female mice after exercise, analyzed by PET {injection dose (ID)/LVM [%ID/g]: 36.8 ± 3.5 female sedentary vs. 28.3 ± 4.3 female training; P < 0.05}, whereas cardiac glucose uptake was unaltered after exercise in male counterparts. Cardiac genes involved in fatty acid uptake/oxidation in females were increased compared with male mice. Collectively, our data demonstrate that sex differences in exercise-induced cardiac hypertrophy are associated with changes in cardiac substrate availability and utilization.     

Early increases in coronary vascular reserve in exercised rats are independent of cardiac hypertrophy

To evaluate the relationship between the physiological cardiac hypertrophy associated with physical training and the increases in vascular capacitance associated with this stimuli, male and female rats trained by a swimming program were studied. Both sexes were used so that the coronary vascular response to exercise could be studied in the presence (females) and absence (males) of cardiac hypertrophy. Coronary vascular reserve was assessed in isolated retrograde buffer-perfused hearts under conditions of minimal coronary resistance (15 microM adenosine or anoxia). Both groups demonstrated an increase in coronary vascular reserve after 8 wk of exercise swim training, male animals increasing flow (per g of myocardium) by 15% and females by 18%. When the time course of this response was compared in female animals with the time course of the development of myocardial hypertrophy, it was evident that the vascular changes occurred early, greater than 80% of the response was seen within the first 10 days of exercise, compared with an approximately 35% increase in cardiac mass. These data suggest that the vascular response to exercise swim training is independent of the hypertrophic response and further that the increase in coronary vascularity is an early event in the cardiac adaptation to a physiological load.     

DGAT1 Expression Increases Heart Triglyceride Content but Ameliorates Lipotoxicity

Intracellular lipid accumulation in the heart is associated with cardiomyopathy, yet the precise role of triglyceride (TG) remains unclear. With exercise, wild type hearts develop physiologic hypertrophy. This was associated with greater TG stores and a marked induction of the TG-synthesizing enzyme diacylglycerol (DAG) acyltransferase 1 (DGAT1). Transgenic overexpression of DGAT1 in the heart using the cardiomyocyte- specific α-myosin heavy chain (MHC) promoter led to approximately a doubling of DGAT activity and TG content and reductions of ∼35% in cardiac ceramide, 26% in DAG, and 20% in free fatty acid levels. Cardiac function assessed by echocardiography and cardiac catheterization was unaffected. These mice were then crossed with animals expressing long-chain acyl-CoA synthetase via the MHC promoter (MHC-ACS), which develop lipotoxic cardiomyopathy. MHC-DGAT1XMHC-ACS double transgenic male mice had improved heart function; fractional shortening increased by 74%, and diastolic function improved compared with MHC-ACS mice. The improvement of heart function correlated with a reduction in cardiac DAG and ceramide and reduced cardiomyocyte apoptosis but increased fatty acid oxidation. In addition, the survival of the mice was improved. Our study indicates that TG is not likely to be a toxic lipid species directly, but rather it is a feature of physiologic hypertrophy and may serve a cytoprotective role in lipid overload states. Moreover, induction of DGAT1 could be beneficial in the setting of excess heart accumulation of toxic lipids.     

Obesity and Elevated Plasma Leptin Concentration in oMT1A‐o Growth Hormone Transgenic Mice

OBERBAUER, A. M., JONATHAN A. RUNSTADLER, JAMES D. MURRAY, AND PETER J. HAVEL. Obesity and elevated plasma leptin concentration in oMT1A‐o growth hormone transgenic mice. Objective: This study was undertaken to evaluate plasma leptin concentration in the regulatable ovine metallothionein‐ovine growth hormone (oMT1a‐oGH) transgenic (TG) mouse model of obesity. Research Methods and Procedures: Transgene stimulus (zinc) was provided at 21 days of age to male and female wild‐type (WT) and TG mice. Plasma leptin concentrations were measured by radioimmunoassay at 42, 63, 84, and 105 days of age and from inactivated TG mice at 84 and 105 days. Results: WT and TG mice did not differ significantly in plasma leptin concentration at any of the ages examined (42, 63, 84, and 105 days), although females showed consistently higher plasma leptin concentrations than males regardless of genotype throughout the duration of the study. Male and female TG mice in which the transgene was inactivated at 63 days had a 1.5‐fold to 3.5‐fold increase in plasma leptin concentration over WT mice and continuously activated TG mice at 84 and 105 days of age. The elevated plasma leptin concentration seen in the inactivated TG mice at 84 and 105 days of age reflects the >300% increase in white adipose tissue seen in this model and correlated with all adipose depot weights and overall body lipid at these later ages. When plasma leptin was expressed per gram of total body fat, the leptin adjusted for body lipid was significantly higher in WT mice than either continuously activated TG or activated and then inactivated TG groups. Discussion: The inactivated TG mice in this study had higher plasma leptin levels with increasing total body adiposity, but the relative proportion of circulating leptin, on a total body lipid basis, was reduced when compared with the WT mice. This reduction was also observed in activated TG mice at the older ages. Although the absolute levels of circulating leptin were elevated in the inactivated TG animals, the amount of leptin produced per gram of fat was lowered. With the inactivation of the transgene, the leptin remained depressed after the removal of the elevated growth hormone. This represents a potential explanation for the ensuing hypertrophy of the fat depots and the abnormal phenotypic response of inactivated TG mice to elevated plasma leptin concentrations resulting in the development of obesity.     

Expression and characterization of the Na+/H+ exchanger in the mammalian myocardium

We examined two expression systems for studying the Na⁺/H⁺ exchanger in the mammalian myocardium. Mammalian NHE1 with a hemagglutinin (HA) tag and was cloned behind the alpha myosin heavy chain promoter. Transgenic mice were made with wild type NHE1 protein or with a hyperactive NHE1 protein mutated at the calmodulin-binding domain. Three lines of transgenic mice were made of each cDNA with expression levels of each type varying from high to low. Higher levels and activity of the Na⁺/H⁺ exchanger were associated with decreased long-term survival of mice, and with dilated or hypertrophic cardiomyopathy. The exogenous NHE1 protein was present in freshly made cardiomyocytes from transgenic mice, however, expression from the alpha myosin heavy chain promoter declined rapidly and little exogenous NHE1 was apparent on the fourth day after cardiomyocyte isolation. To express NHE1 protein in isolated cardiomyocytes, we transferred a mutated form of the protein into an adenoviral expression system. Infection of neonatal rat cardiomyocytes resulted in robust expression of the exogenous NHE1 protein. The mutant form of the NHE1 protein could be distinguished from the endogenous Na⁺/H⁺ exchanger by its resistance to inhibition by amiloride analogs. Our results suggest that for in vivo studies on intact hearts and animals, expression in transgenic mice is an appropriate system, however for long-term studies on cardiomyocytes, this model is inappropriate due to waning expression from the alpha myosin heavy chain promoter. Therefore, infection by adenovirus is a superior system for long-term studies on cardiomyocytes in culture.     

c-Jun N-terminal kinases (JNK) antagonize cardiac growth through cross-talk with calcineurin-NFAT signaling

The c-Jun N-terminal kinase (JNK) branch of the mitogen-activated protein kinase (MAPK) signaling pathway regulates cellular differentiation, stress responsiveness and apoptosis in multicellular eukaryotic organisms. Here we investigated the functional importance of JNK signaling in regulating differentiated cellular growth in the post-mitotic myocardium. JNK1/2 gene-targeted mice and transgenic mice expressing dominant negative JNK1/2 were determined to have enhanced myocardial growth following stress stimulation or with normal aging. A mechanism underlying this effect was suggested by the observation that JNK directly regulated nuclear factor of activated T-cell (NFAT) activation in culture and in transgenic mice containing an NFAT-dependent luciferase reporter. Moreover, calcineurin Abeta gene targeting abrogated the pro-growth effects associated with JNK inhibition in the heart, while expression of an MKK7-JNK1 fusion protein in the heart partially reduced calcineurin-mediated cardiac hypertrophy. Collectively, these results indicate that JNK signaling antagonizes the differentiated growth response of the myocardium through direct cross-talk with the calcineurin-NFAT pathway. These results also suggest that myocardial JNK activation is primarily dedicated to modulating calcineurin-NFAT signaling in the regulation of differentiated heart growth.     

Gender differences in β‐adrenoceptor system in cardiac hypertrophy due to arteriovenous fistula

This study was undertaken to determine alterations in the β‐adrenoceptor (β‐AR) signaling system in male and female rats at 4 weeks after the induction of arteriovenous (AV) fistula or shunt. AV shunt produced a greater degree of cardiac hypertrophy and larger increase in cardiac output in male than in female animals. Increases in plasma levels of norepinephrine and epinephrine (EPI) due to AV shunt were also higher in male than females. While no difference in the β₁‐AR affinity was seen in males and females, AV shunt induced increase in β₁‐AR density in female rats was higher than that in males. Furthermore, no changes in basal adenylyl cyclase (AC) V/VI mRNA levels were seen; however, the increase in EPI‐stimulated AC activities was greater in AV shunt females than in males. AV shunt decreased myocardial β₁‐AR mRNA level in male rats and increased β₂‐AR mRNA level in female hearts; an increase in G_i‐protein mRNA was detected only in male hearts. Although GRK2 gene expression was increased in both sexes, an increase in GRK3 mRNA was seen only in AV shunt female rats. β‐arrestin1 mRNA was elevated in females whereas β‐arrestin 2 gene expression was increased in both male and female AV shunt rats. While these data demonstrate gender associated differences in various components of the β‐AR system in cardiac hypertrophy due to AV shunt, only higher levels of plasma catecholamines may account for the greater increase in cardiac output and higher degree of cardiac hypertrophy in males. J. Cell. Physiol. 226: 181–186, 2010. © 2010 Wiley‐Liss, Inc.     

Sex-related changes in cardiac function following myocardial infarction in mice

Recent awareness of cardiovascular diseases as a number one killer of the middle-aged women has prompted interest in sex differences leading to heart failure (HF). Therefore, we evaluated cardiac function in female and male mice following myocardial infarction (MI) using the Millar pressure-volume (P-V) conductance system in vivo, at time points corresponding to early (2 wk), late compensatory hypertrophy (4 wk), and decompensation (10 wk) to HF. A significant deterioration of the load dependent and independent hemodynamic measurements occurred in both female and male mice during the early phase of hypertrophy. Later, compensatory hypertrophy was marked by a normalization of volumes to control levels in females compared with males. The most notable differences between sexes occurred in the measurements of cardiac contractility during the decompensation to HF. In females, there was a significant improvement in contractility compared with males, which was apparent in the load-independent measurements of preload recruitable stroke work (10 wk post-MI, female=48.7+/-8.0 vs. male=25.2+/-1.8 mmHg, P<0.05) and maximum dP/dt vs. maximum end-diastolic volume (10 wk post-MI, female=359+/-58 vs. male=149+/-28 mmHg.s(-1).microl(-1), P<0.05). Despite these differences, there were no differences in the heart weight to body weight ratio and infarct size between the sexes. These data demonstrate that compensatory hypertrophy is associated with an improvement in contractility and a delayed decompensation to HF in females. However, compensatory hypertrophy in males appears to be undermined by a steady decline in contractility associated with decompensation to HF.     

Extracellular high‐mobility group box 1 mediates pressure overload‐induced cardiac hypertrophy and heart failure

Inflammation plays a key role in pressure overload‐induced cardiac hypertrophy and heart failure, but the mechanisms have not been fully elucidated. High‐mobility group box 1 (HMGB1), which is increased in myocardium under pressure overload, may be involved in pressure overload‐induced cardiac injury. The objectives of this study are to determine the role of HMGB1 in cardiac hypertrophy and cardiac dysfunction under pressure overload. Pressure overload was imposed on the heart of male wild‐type mice by transverse aortic constriction (TAC), while recombinant HMGB1, HMGB1 box A (a competitive antagonist of HMGB1) or PBS was injected into the LV wall. Moreover, cardiac myocytes were cultured and given sustained mechanical stress. Transthoracic echocardiography was performed after the operation and sections for histological analyses were generated from paraffin‐embedded hearts. Relevant proteins and genes were detected. Cardiac HMGB1 expression was increased after TAC, which was accompanied by its translocation from nucleus to both cytoplasm and intercellular space. Exogenous HMGB1 aggravated TAC‐induced cardiac hypertrophy and cardiac dysfunction, as demonstrated by echocardiographic analyses, histological analyses and foetal cardiac genes detection. Nevertheless, the aforementioned pathological change induced by TAC could partially be reversed by HMGB1 inhibition. Consistent with the in vivo observations, mechanical stress evoked the release and synthesis of HMGB1 in cultured cardiac myocytes. This study indicates that the activated and up‐regulated HMGB1 in myocardium, which might partially be derived from cardiac myocytes under pressure overload, may be of crucial importance in pressure overload‐induced cardiac hypertrophy and cardiac dysfunction.     

Gender and aging in a transgenic mouse model of hypertrophic cardiomyopathy

Mutations in the cardiac myosin heavy chain (MHC) can cause familial hypertrophic cardiomyopathy (FHC). A transgenic mouse model has been developed in which a missense (R403Q) allele and an actin-binding deletion in the alpha-MHC are expressed in the heart. We used an isovolumic left heart preparation to study the contractile characteristics of hearts from transgenic (TG) mice and their wild-type (WT) littermates. Both male and female TG mice developed left ventricular (LV) hypertrophy at 4 mo of age. LV hypertrophy was accompanied by LV diastolic dysfunction, but LV systolic function was normal and supranormal in the young TG females and males, respectively. At 10 mo of age, the females continued to present with LV concentric hypertrophy, whereas the males began to display LV dilation. In female TG mice at 10 mo of age, impaired LV diastolic function persisted without evidence of systolic dysfunction. In contrast, in 10-mo-old male TG mice, LV diastolic function worsened and systolic performance was impaired. Diminished coronary flow was observed in both 10-mo-old TG groups. These types of changes may contribute to the functional decompensation typically seen in hypertrophic cardiomyopathy. Collectively, these results further underscore the potential utility of this transgenic mouse model in elucidating pathogenesis of FHC.     

Myocardial Na+/H+ exchanger-1 (NHE1) content is decreased by exercise training

The effect of exercise training on myocardial Na⁺/H⁺ exchanger-1 (NHE1) protein expression was examined. Adult female Sprague–Dawley rats were randomly divided into sedentary (S; n?=?8) and exercised (E; n?=?9) groups. Twenty-four hours after the last exercise bout, hearts were weighed and connected to an isolated perfused working heart apparatus for evaluation of cardiac functional performance. Heart weight and heart weight/body weight from E rats was significantly increased by 7.1 and 7.2 % (P?<?0.05), respectively, compared with S hearts. The E hearts displayed 15 % greater cardiac output and 35 % external cardiac work compared with the S group at both low and high workloads (P?<?0.05 for both parameters). Left ventricular tissue from the same hearts was homogenized and NHE1 and Na⁺/Ca²⁺ exchanger (NCX) content determined by Western blotting. E hearts had a 38 % (P?<?0.001) reduction in NHE1 content related to S hearts, and there was no difference in NCX content between groups. Cytochrome c oxidase activity in plantaris increased by 100 % (P?<?0.05) and was assessed as a marker of mitochondria content and to verify training status. Our data indicate that exercise training at an intensity that results in cardiac hypertrophy and improved performance is accompanied by decreased NHE1 content in heart.     

Differential expression of the tetracycline-controlled transactivator-driven human CYP1B1 gene in double-transgenic mice is due to androgens: application for detecting androgens and antiandrogens

Differential expression of the tetracycline-controlled transactivator (tTA)-driven human cytochrome p450 (CYP) 1B1 gene was found in the livers of male mice, at high levels in neonates, but at low levels in adults. The goals of this study were to determine whether the differential expression of the tTA-driven human CYP1B1 (hCYP1B1) gene in neonates and adults was testosterone dependent and whether flutamide, a representative potent antiandrogen, led to the induction of hCYP1B1. This was tested by treating castrated transgenic mice with testosterone propionate and musk extracts. It was concluded that: (i). the levels of expression of both tTA and hCYP1B1 gradually declined, with clear changes being apparent between 2 and 4 weeks of age, (ii). castration of adult males resulted in the increased expressions of both tTA and hCYP1B1 to levels similar to those found in adult females, (iii). treatment of castrated male and adult female mice with testosterone propionate and musk extracts led to the restoration of the levels of expression of hCYP1B1 in the adult males, and (iv). treatment of adult males with flutamide caused an increase in the levels of expression of hCYP1B1 in the adult females, as indicated by the antiandrogenic activity. Thus, the differential expression of the tTA-driven hCYP1B1 gene in the transgenic mice was caused by androgen, and it is possible that castrated male and adult female mice expressing the tTA-controlled hCYP1B1 could be used as the basis for a strategy for the detection of androgens and antiandrogens.