Catecholamine metabolism inhibitors and receptor blockades only partially suppress cardiac hypertrophy of juvenile visceral steatosis mice with systemic carnitine deficiency

To clarify the mechanism of cardiac hypertrophy in carnitine-deficient JVS mice, we studied the possible role of catecholamine metabolism. Cardiac hypertrophy occurs 2 weeks after birth. The turnover of norepinephrine in the ventricles of JVS mice at 2 weeks was 3 times that of control, but it was not different from control at 5 days when the heart weight was not changed. To evaluate the accelerated norepinephrine turnover, we examined the effects of catecholamine metabolism inhibitors (alpha-methyltyrosine and 6-hydroxydopamine) and catecholamine receptor blockades (propranolol, prazosin and yohimbine) on the ratio of heart weight to body weight (HW/BW) and on the augmented expression of atrial natriuretic peptide (ANP) and the down-regulated carnitine deficiency-associated gene expressed in ventricle (CDV-1). The HW/BW ratio in JVS mice treated with catecholamine metabolism inhibitors and receptor blockades was significantly lower than in JVS mice without treatment, but still higher than in controls treated with each drug and in JVS mice treated with carnitine. The HW/BW ratio of JVS mice with propranolol was not significantly different from that of JVS mice treated with catecholamine metabolism inhibitors and was significantly lower than that of JVS mice treated with prazosin and yohimbine. Northern blot analysis showed that the altered expression of ANP and CDV-1 was not corrected in the ventricles of JVS mice treated with any of the drugs except carnitine. These results suggest that the catecholamine metabolism accelerated in JVS mice ventricles at 2 weeks is not the major cause of cardiac hypertrophy, but probably promotes cardiac hypertrophy mainly through the beta-adrenergic signaling pathway. The aberrant gene expression of ANP and CDV-1 found in JVS mice seems to be independent of catecholamine metabolism, and mediated primarily by the systemic carnitine deficiency.     

Pyruvate dehydrogenase kinase 4 mRNA is increased in the hypertrophied ventricles of carnitine-deficient juvenile visceral steatosis (JVS) mice

We isolated a mouse homologue cDNA of pyruvate dehydrogenase (PDH) kinase 4 (PDK4) with differential mRNA display as an up-regulated gene in the hypertrophied ventricles of juvenile visceral steatosis (JVS) mice with systemic carnitine deficiency. The PDK4 mRNA level was 5 times higher in JVS mice than in control mice under fed conditions. After 24 h starvation, this level increased to 20 times in JVS and 7 times in control, compared with the control fed level. On the other hand, carnitine administration reduced the high level of PDK4 mRNA in JVS mice to the control fed level. In control mice, the change in PDK4 mRNA was inversely correlated with the change in PDH activity. In JVS mice, however, the PDK4 mRNA level was not always correlated with the active-form PDH level.     

Attenuation of cardiac hypertrophy in carnitine-deficient juvenile visceral steatosis (JVS) mice achieved by lowering dietary lipid

We examined the development of cardiac hypertrophy in juvenile visceral steatosis (JVS) mice, a model of systemic carnitine deficiency, by varying the amount of lipid in the diet. Cardiac hypertrophy was markedly attenuated by decreasing soy bean oil (SBO) from 5% (w/w) to 1%. Triglyceride contents of the ventricles of JVS mice fed 1% SBO were significantly lower than in JVS mice fed 5% SBO. The addition of medium-chain triglycerides metabolically utilized by JVS mice did not affect the development of cardiac hypertrophy. On the other hand, the mRNA levels of atrial natriuretic peptide and skeletal alpha-actin, which are related to cardiac hypertrophy, were also attenuated by decreasing lipid in the diet. Adenylate energy charge and creatine phosphate in the heart of JVS mice at the early stage of hypertrophy were not significantly different from control mice given the same laboratory chow (4.6% of lipid). Although urinary prostaglandin F(2alpha) levels were found to be increased in JVS mice at 15 days of age when they developed cardiac hypertrophy, administration of aspirin was not efficacious. We, therefore, propose that the proportion of lipid in the diet is important in the development of cardiac hypertrophy in carnitine-deficient JVS mice, and that this is not related to prostaglandin formation.     

Pressure overload selectively up-regulates Ca2+/calmodulin-dependent protein kinase II in vivo

Signals transduced by the multifunctional calcium/calmodulin-dependent protein kinases (CaMKs), have been suggested to regulate the development of hypertrophy. We address the role of the three multifunctional CaMKs, CaMK I, II, and IV, in this process using transverse aortic constriction (TAC) to induce cardiac hypertrophy in mice. We find a 33% increase in total CaMK activity 7 d after TAC. However, there are no changes in the levels of CaMKI, which is expressed in the ventricles, or CaMKIV, which is not detectable in the ventricles. Moreover, mice null for the CaMKIV gene develop ventricular hypertrophy and induce the expression of selected hypertrophy marker mRNAs, indicating that CaMKIV is not required at any time during the development of hypertrophy. On the other hand, TAC does increase both mRNA and protein levels of specific isoforms of CaMKII derived from both gamma and delta genes. Included among these isoforms are those that localize to both cytoplasm and nucleus. Collectively, the increased levels of CaMKII isoforms result in a constitutive increase in the Ca(2+)/calmodulin-independent activity of CaMKII in the ventricles. We conclude that CaMKII is the multifunctional CaMK most likely to mediate Ca(2+)- dependent protein phosphorylation events in response to TAC-induced cardiac hypertrophy.     

Failure of the feeding response to fasting in carnitine-deficient juvenile visceral steatosis (JVS) mice: Involvement of defective acyl-ghrelin secretion and enhanced corticotropin-releasing factor signaling in the hypothalamus

Carnitine-deficient juvenile visceral steatosis (JVS) mice, suffering from fatty acid metabolism abnormalities, have reduced locomotor activity after fasting. We examined whether JVS mice exhibit specific defect in the feeding response to fasting, a key process of anti-famine homeostatic mechanism. Carnitine-deficient JVS mice showed grossly defective feeding response to 24 h-fasting, with almost no food intake in the first 4 h, in marked contrast to control animals. JVS mice also showed defective acyl-ghrelin response to fasting, less suppressed leptin, and seemingly normal corticotropin-releasing factor (CRF) expression in the hypothalamus despite markedly increased plasma corticosterone. The anorectic response was ameliorated by intraperitoneal administration of carnitine or acyl-ghrelin, with decreased CRF expression. Intracerebroventricular treatment of CRF type 2 receptor antagonist, anti-sauvagine-30, recovered the defective feeding response of 24 h-fasted JVS mice. The defective feeding response to fasting in carnitine-deficient JVS mice is due to the defective acyl-ghrelin and enhanced CRF signaling in the hypothalamus through fatty acid metabolism abnormalities. In this animal model, carnitine normalizes the feeding response through an inhibition of CRF.     

Cardiomegaly in the juvenile visceral steatosis (JVS) mouse is reduced with acute elevation of heart short-chain acyl-carnitine level after L-carnitine injection

The long-term administration of L-carnitine was very effective in preventing cardiomegaly in juvenile visceral steatosis (JVS) mice, which was confirmed by heart weight as well as the lipid contents in heart tissue. After i.p. injection of L-carnitine, the concentration of free carnitine in heart remained constant, although serum free carnitine level increased up to 80-fold. On the other hand, a significant increase in short-chain acyl-carnitine level in heart was observed. These results suggest that increased levels of short-chain acyl-carnitine, not free carnitine, might be a key compound in the protective effect of L-carnitine administration in JVS mice.     

Identification of a novel aldose reductase-like gene upregulated in the failing heart of cardiomyopathic hamster

Cardiomyopathy (CM) is degenerative disease of myocardium which leads to severe cardiac failure. Although many causative genes for CM have been identified, molecular pathogenesis of CM is not fully understood. In this study, we searched for a novel pathway recruited in the development of CM by using BIO14.6 hamster as an animal model for human CM. We screened upregulated genes in the left ventricle by differential display technique and searched for a gene which had never been linked to CM. We identified a novel gene overexpressed in BIO14.6 hamster ventricles, which was considered to be a new member of aldo-keto reductase (AKR) superfamily. The cloned cDNA encoded a 316 amino acid polypeptide with calculated molecular mass of 35,804, which showed high amino acid sequence similarities to aldose reductase and its relative: 69.6% to AKR1B1 (human aldose reductase), 68.4% to AKR1B3 (mouse aldose reductase), and 85.8% to AKR1B7 (mouse vas deferens protein). The upregulation of this aldose reductase-like gene in BIO14.6 hamster ventricles (6.3 ± 0.8-fold) seemed to be influenced by the overexpression of activator protein-1 present there. With the fact that AKR1B1, AKR1B3, and AKR1B7 have synthetic activities of prostaglandin F2α, the aldose reductase-like protein could cause cardiac hypertrophy through production of prostaglandin F2α whose precursor and receptor were abundant in BIO14.6 hamster ventricles. Aldose reductase and its related proteins would give a new clue to dissect the pathogenesis of CM including oxidative stress and cardiac hypertrophy, and to develop a new drug for the treatment of CM.     

gp130 plays a critical role in pressure overload-induced cardiac hypertrophy

gp130, a common receptor for the interleukin 6 family, plays pivotal roles in growth and survival of cardiac myocytes. In the present study, we examined the role of gp130 in pressure overload-induced cardiac hypertrophy using transgenic (TG) mice, which express a dominant negative mutant of gp130 in the heart under the control of alpha myosin heavy chain promoter. TG mice were apparently healthy and fertile. There were no differences in body weight and heart weight between TG mice and littermate wild type (WT) mice. Pressure overload-induced increases in the heart weight/body weight ratio, ventricular wall thickness, and cross-sectional areas of cardiac myocytes were significantly smaller in TG mice than in WT mice. Northern blot analysis revealed that pressure overload-induced up-regulation of brain natriuretic factor gene and down-regulation of sarcoplasmic reticulum Ca(2+) ATPase 2 gene were attenuated in TG mice. Pressure overload activated ERKs and STAT3 in the heart of WT mice, whereas pressure overload-induced activation of STAT3, but not of ERKs, was suppressed in TG mice. These results suggest that gp130 plays a critical role in pressure overload-induced cardiac hypertrophy possibly through the STAT3 pathway.     

LncRNA TUG1 alleviates cardiac hypertrophy by targeting miR-34a/DKK1/Wnt-β-catenin signalling

The current study was designed to explore the role and underlying mechanism of lncRNA taurine up-regulated gene 1 (TUG1) in cardiac hypertrophy. Mice were treated by transverse aortic constriction (TAC) surgery to induce cardiac hypertrophy, and cardiomyocytes were treated by phenylephrine (PE) to induce hypertrophic phenotype. Haematoxylin-eosin (HE), wheat germ agglutinin (WGA) and immunofluorescence (IF) were used to examine morphological alterations. Real-time PCR, Western blots and IF staining were used to detect the expression of RNAs and proteins. Luciferase assay and RNA pull-down assay were used to verify the interaction. It is revealed that TUG1 was up-regulated in the hearts of mice treated by TAC surgery and in PE-induced cardiomyocytes. Functionally, overexpression of TUG1 alleviated cardiac hypertrophy both in vivo and in vitro. Mechanically, TUG1 sponged and sequestered miR-34a to increase the Dickkopf 1 (DKK1) level, which eventually inhibited the activation of Wnt/β-catenin signalling. In conclusion, the current study reported the protective role and regulatory mechanism of TUG1 in cardiac hypertrophy and suggested that TUG1 may serve as a novel molecular target for treating cardiac hypertrophy.     

Targeted Sprouty1 overexpression in cardiac myocytes does not alter myocardial remodeling or function

The mitogen activated protein kinase (MAPK) signaling pathway regulates multiple events leading to heart failure including ventricular remodeling, contractility, hypertrophy, apoptosis, and fibrosis. The regulation of conserved intrinsic inhibitors of this pathway is poorly understood. We recently identified an up-regulation of Sprouty1 (Spry1) in a targeted approach for novel inhibitors of the MAPK signaling pathway in failing human hearts following reverse remodeling. The goal of this study was to test the hypothesis that up-regulated expression of Spry1 in cardiac myocytes would be sufficient to inhibit ERK1/2 activation and tissue remodeling. We established a murine model with up-regulated Spry1 expression in cardiac myocytes using the alpha-myosin heavy chain promoter (α-MHC). Heart weight and cardiac myocyte morphology were unchanged in adult male α-MHC–Spry1 mice compared to control mice. Ventricular function of α-MHC–Spry1 mice was unaltered at 8 weeks or 1 year of age. These findings were consistent with the lack of an effect of Spry1 on ERK1/2 activity. In summary, targeted up-regulation of Spry1 in cardiac myocytes is not sufficient to alter cell or tissue remodeling consistent with the lack of an effect on ERK1/2 activity.     

Substrate uptake and metabolism are preserved in hypertrophic caveolin-3 knockout hearts

Caveolin-3 (Cav3), the primary protein component of caveolae in muscle cells, regulates numerous signaling pathways including insulin receptor signaling and facilitates free fatty acid (FA) uptake by interacting with several FA transport proteins. We previously reported that Cav3 knockout mice (Cav3KO) develop cardiac hypertrophy with diminished contractile function; however, the effects of Cav3 gene ablation on cardiac substrate utilization are unknown. The present study revealed that the uptake and oxidation of FAs and glucose were normal in hypertrophic Cav3KO hearts. Real-time PCR analysis revealed normal expression of lipid metabolism genes including FA translocase (CD36) and carnitine palmitoyl transferase-1 in Cav3KO hearts. Interestingly, myocardial cAMP content was significantly increased by 42%; however, this had no effect on PKA activity in Cav3KO hearts. Microarray expression analysis revealed a marked increase in the expression of genes involved in receptor trafficking to the plasma membrane, including Rab4a and the expression of WD repeat/FYVE domain containing proteins. We observed a fourfold increase in the expression of cellular retinol binding protein-III and a 3.5-fold increase in 17beta-hydroxysteroid dehydrogenase type 11, a member of the short-chain dehydrogenase/reductase family involved in the biosynthesis and inactivation of steroid hormones. In summary, a loss of Cav3 in the heart leads to cardiac hypertrophy with normal substrate utilization. Moreover, a loss of Cav3 mRNA altered the expression of several genes not previously linked to cardiac growth and function. Thus we have identified a number of new target genes associated with the pathogenesis of cardiac hypertrophy.     

大鼠不同心肌肥厚模型左心室基因表达谱变化的比较

为了解心肌肥厚时基因表达谱的变化规律,本实验复制了三种大鼠心肌肥厚模型：肾上腹主动脉缩窄(suprarenal abdominal aortic stenosis,SRS)、动静脉瘘(arterial-vein fistula,AVF)和去甲。肾上腺素持续静脉输注(jugular vein infusion of norepinephrine,NEi),并应用组织化学方法和超声心动术检测大鼠心脏结构和功能指标,应用cDNA基因芯片技术检测心脏基因表达水平的变化。SRS和NEi引起大鼠向心性心肌肥厚,AVF引起大鼠离心性心肌肥厚,其中NEi大鼠心肌纤维化明显。对不同心肌肥厚模型间大鼠左心室基因表达谱的变化进行两两比较。结果显示,有部分基因在不同模型中表达水平均发生变化,其中多数基因在两种模型中表达水平改变的方向相同,也有少部分基因在两种模型中表达水平改变方向相反。综合比较三种心肌肥厚模型的基因表达谱,各种模型都有特异的基因表达变化,但是有19个基因在三种心肌肥厚模型中表达水平均发生改变。研究结果有可能成为心肌肥厚的标志性基因或治疗靶点,为心肌肥厚发生机制的深入研究提供了新的线索。     

Comparative analysis of mRNA isoform expression in cardiac hypertrophy and development reveals multiple post-transcriptional regulatory modules

Cardiac hypertrophy is enlargement of the heart in response to physiological or pathological stimuli, chiefly involving growth of myocytes in size rather than in number. Previous studies have shown that the expression pattern of a group of genes in hypertrophied heart induced by pressure overload resembles that at the embryonic stage of heart development, a phenomenon known as activation of the "fetal gene program". Here, using a genome-wide approach we systematically defined genes and pathways regulated in short- and long-term cardiac hypertrophy conditions using mice with transverse aortic constriction (TAC), and compared them with those regulated at different stages of embryonic and postnatal development. In addition, exon-level analysis revealed widespread mRNA isoform changes during cardiac hypertrophy resulting from alternative usage of terminal or internal exons, some of which are also developmentally regulated and may be attributable to decreased expression of Fox-1 protein in cardiac hypertrophy. Genes with functions in certain pathways, such as cell adhesion and cell morphology, are more likely to be regulated by alternative splicing. Moreover, we found 3'UTRs of mRNAs were generally shortened through alternative cleavage and polyadenylation in hypertrophy, and microRNA target genes were generally de-repressed, suggesting coordinated mechanisms to increase mRNA stability and protein production during hypertrophy. Taken together, our results comprehensively delineated gene and mRNA isoform regulation events in cardiac hypertrophy and revealed their relations to those in development, and suggested that modulation of mRNA isoform expression plays an importance role in heart remodeling under pressure overload.