Cardiac overexpression of myotrophin triggers myocardial hypertrophy and heart failure in transgenic mice

Cardiac hypertrophy and heart failure remain leading causes of death in the United States. Many studies have suggested that, under stress, myocardium releases factors triggering protein synthesis and stimulating myocyte growth. We identified and cloned myotrophin, a 12-kDa protein from hypertrophied human and rat hearts. Myotrophin (whose gene is localized on human chromosome 7q33) stimulates myocyte growth and participates in cellular interaction that initiates cardiac hypertrophy in vitro. In this report, we present data on the pathophysiological significance of myotrophin in vivo, showing the effects of overexpression of cardio-specific myotrophin in transgenic mice in which cardiac hypertrophy occurred by 4 weeks of age and progressed to heart failure by 9-12 months. This hypertrophy was associated with increased expression of proto-oncogenes, hypertrophy marker genes, growth factors, and cytokines, with symptoms that mimicked those of human cardiomyopathy, functionally and morphologically. This model provided a unique opportunity to analyze gene clusters that are differentially up-regulated during initiation of hypertrophy versus transition of hypertrophy to heart failure. Importantly, changes in gene expression observed during initiation of hypertrophy were significantly different from those seen during its transition to heart failure. Our data show that overexpression of myotrophin results in initiation of cardiac hypertrophy that progresses to heart failure, similar to changes in human heart failure. Knowledge of the changes that take place as a result of overexpression of myotrophin at both the cellular and molecular levels will suggest novel strategies for treatment to prevent hypertrophy and its progression to heart failure.     

Increased levels of myosin mRNAs in spontaneously hypertensive rat heart

RNAs isolated from normal and spontaneously hypertensive (SHR) rat heart tissues were examined by direct hybridization assay using cloned DNA probes containing chicken cardiac myosin light chain and heavy chain specific cDNA sequences. In 7 weeks old SHR heart, the level of mRNA hybridizable to these probes is the same as in normal rat heart. However, at 18 weeks of age, when hypertrophy in SHR is well established as a consequence of age-related increase in blood pressure and cardiac mass, there is an increase in SHR mRNA levels consistent with the increase in the corresponding proteins. Thus, the increase in mRNAs for major myofibrillar proteins and the onset of hypertrophic state in SHR appear to occur simultaneously.     

Myotrophin-kappaB DNA interaction in the initiation process of cardiac hypertrophy

To investigate how cardiac hypertrophy and heart failure develop, we isolated and characterized a candidate initiator, the soluble 12-kDa protein myotrophin, from rat and human hearts. Myotrophin stimulates protein synthesis and myocardial cell growth associated with increased levels of hypertrophy marker genes. Recombinant myotrophin from the cloned gene showed structural/functional motifs, including ankyrin repeats and putative phosphorylation sites for protein kinase C (PKC) and casein kinase II. One repeat, homologous with I kappaB, interacts with rel/NF-kappaB in vitro. We analyzed the interaction of recombinant myotrophin and nuclear extracts prepared from neonatal and adult cardiomyocytes; gel mobility shift assay showed that myotrophin bound to kappaB DNA. To define PKC's role in myotrophin-induced myocyte growth, we incubated neonatal rat myocytes (normal and stretch) with specific inhibitors and found that myotrophin inhibits [3H]leucine incorporation into myocytes and different hypertrophic gene expression in neonatal myocytes. Using confocal microscopy, we observed that a basal level of myotrophin was present in both cytoplasm and nucleus under normal conditions, but under cyclic stretch, myotrophin levels became elevated in the nucleus. Myotrophin gene levels were upregulated when myocytes underwent cyclic stretch or were treated with tumor necrosis factor-alpha (TNF-alpha) or interleukin-1beta and also when excised beating hearts were exposed to high pressure. Our data showed that the myotrophin-kappaB interaction was increased with age in spontaneously hypertensive rats (SHRs) only. Our data provide evidence that myotrophin-kappaB DNA interaction may be an important step in initiating cardiac hypertrophy.     

运动性和高血压性心肌肥大重塑时心肌初级和次级应答基因表达的差异

我们前期研究表明运动性和高血压性心肌肥大细胞表型变化在结构、功能和代谢方面均表现不同,但两者基因表达的不同特征尚不清楚。本实验采用Ｎｏｒｔｈｅｒｎ分子杂交方法对游泳运动１２周大鼠和自发性高血压大鼠（ＳＨＲ）肥大心脏心肌初级和次级应答基因表达进行比较研究。结果表明,游泳大鼠心系数比对照大鼠提高２６％（Ｐ〈０．０１）,心肌ｃ－ｆｏｓ和心房钠尿肽（ＡＮＦ）基因表达在最后一次运动后即刻明显增强,在运动后２     

Cloning and expression of the atrial natriuretic factor gene

Atrial natriuretic factor (ANF) is a 28-amino acid peptide hormone with potent natriuretic, diuretic and vasodilator properties. Isolation and DNA sequence analysis of rat and human cDNA clones revealed that ANF is synthesized from a 126-amino acid precursor which is highly conserved in both species. Southern blot analysis indicated that the ANF gene is present in a single copy per haploid genome. Both human and rat ANF genes were isolated and showed a similar structural organization which consisted of three exons and two introns. The ANF gene was localized to the short arm of human chromosome 1 and mouse chromosome 4. While atria are the major site of expression of the ANF gene in adult heart, other tissues like ventricles, lung, anterior pituitary, hypothalamus and adrenal synthesize ANF albeit to a much lower extent. In ventricles, ANF mRNA levels are 150 times lower than in atria. However, in cardiac hypertrophy or in congestive heart failure, ventricular ANF mRNA and peptide levels are dramatically (100-fold) increased both in animal models and in humans. This suggests that ventricles are a major site of ANF gene expression in certain pathophysiological conditions and that ANF is not an exclusively atrial peptide as was originally thought.     

Inhibition of nuclear factor κB regresses cardiac hypertrophy by modulating the expression of extracellular matrix and adhesion molecules

Myocardial remodeling denotes a chronic pathological condition of dysfunctional myocardium that occurs in cardiac hypertrophy (CH) and heart failure (HF). Reactive oxygen species (ROS) are major initiators of excessive collagen and fibronectin deposition in cardiac fibrosis. Increased production of ROS and nuclear factor κB (NF-κB) activation provide a strong link between oxidative stress and extracellular matrix (ECM) remodeling in cardiac hypertrophy. The protective inhibitory actions of pyrrolidine dithiocarbamate (PDTC), a pharmacological inhibitor of NF-κB and a potent antioxidant, make this a good agent to evaluate the role of inhibition of NF-κB and prevention of excessive ECM deposition in maladaptive cardiac remodeling during HF. In this report, we used a transgenic mouse model (Myo-Tg) that has cardiac-specific overexpression of myotrophin. This overexpression of myotrophin in the Myo-Tg model directs ECM deposition and increased NF-κB activity, which result in CH and ultimately HF. Using the Myo-Tg model, our data showed upregulation of profibrotic genes (including collagen types I and III, connective tissue growth factor, and fibronectin) in Myo-Tg mice, compared to wild-type mice, during the progression of CH. Pharmacological inhibition of NF-κB by PDTC in the Myo-Tg mice resulted in a significant reduction in cardiac mass, NF-κB activity, and profibrotic gene expression and improved cardiac function. To the best of our knowledge, this is the first report of ECM regulation by inhibition of NF-κB activation by PDTC. The study highlights the importance of the NF-κB signaling pathway and therapeutic benefits of PDTC treatment in cardiac remodeling.