Mammalian enabled (Mena) is a critical regulator of cardiac function.

Mammalian enabled (Mena) of the Drosophila enabled/vasodilator-stimulated phosphoprotein gene family is a cytoskeletal protein implicated in actin regulation and cell motility. Cardiac Mena expression is enriched in intercalated discs (ICD), the critical intercellular communication nexus between adjacent muscle cells. We previously identified Mena gene expression to be a key predictor of human and murine heart failure (HF). To determine the in vivo function of Mena in the heart, we assessed Mena protein expression in multiple HF models and characterized the effects of genetic Mena deletion on cardiac structure and function. Immunoblot analysis revealed significant upregulation of Mena protein expression in left ventricle tissue from patients with end-stage HF, calsequestrin-overexpressing mice, and isoproterenol-infused mice. Characterization of the baseline cardiac function of adult Mena knockout mice (Mena(-/-)) via echocardiography demonstrated persistent cardiac dysfunction, including a significant reduction in percent fractional shortening compared with wild-type littermates. Electrocardiogram PR and QRS intervals were significantly prolonged in Mena(-/-) mice, manifested by slowed conduction on optical mapping studies. Ultrastructural analysis of Mena(-/-) hearts revealed disrupted organization and widening of ICD structures, mislocalization of the gap junction protein connexin 43 (Cx43) to the lateral borders of cardiomyoycytes, and increased Cx43 expression. Furthermore, the expression of vinculin (an adherens junction protein) was significantly reduced in Mena(-/-) mice. We report for the first time that genetic ablation of Mena results in cardiac dysfunction, highlighted by diminished contractile performance, disrupted ICD structure, and slowed electrical conduction.

Role of angiotensin II AT1 receptor activation in cardiovascular diseases.

Numerous clinical studies and experimental investigations using cell culture and animal models suggest that angiotensin II (AngII) via AT(1) receptor activation might induce cardiovascular hypertrophy, fibrosis and atherosclerosis resulting in vascular events such as myocardial infarction, heart failure or stroke and in end-organ damages. However, a question still remains: which part of these damages is due to a direct effect of AngII on its target tissues and which is due to AngII-induced hypertension? In an attempt to answer this question, a new model of transgenic mice, expressing a constitutively activated AT(1A) receptor instead of the wild type receptor has been obtained by homologous recombination. These mice present with a moderate increase of blood pressure (20 mm Hg), hypertrophy of the small kidney arteries but not cardiac hypertrophy. The major phenotypic trait of these mice is the early and progressive development of a cardiovascular fibrosis. In light of these results and those from the literature, there is more and more evidence that in human hypertension, activation of the renin angiotensin system plays a minor role in the development of cardiovascular hypertrophy, but clearly participates to the development of cardiovascular fibrosis.

Identification of Nogo as a novel indicator of heart failure.

Numerous genetically engineered animal models of heart failure (HF) exhibit multiple characteristics of human HF, including aberrant beta-adrenergic signaling. Several of these HF models can be rescued by cardiac-targeted expression of the Gbetagamma inhibitory carboxy-terminus of the beta-adrenergic receptor kinase (betaARKct). We recently reported microarray analysis of gene expression in multiple animal models of HF and their betaARKct rescue, where we identified gene expression patterns distinct and predictive of HF and rescue. We have further investigated the muscle LIM protein knockout model of HF (MLP-/-), which closely parallels human dilated cardiomyopathy disease progression and aberrant beta-adrenergic signaling, and their betaARKct rescue. A group of known and novel genes was identified and validated by quantitative real-time PCR whose expression levels predicted phenotype in both the larger HF group and in the MLP-/- subset. One of these novel genes is herein identified as Nogo, a protein widely studied in the nervous system, where it plays a role in regeneration. Nogo expression is altered in HF and normalized with rescue, in an isoform-specific manner, using left ventricular tissue harvested from both animal and human subjects. To investigate cell type-specific expression of Nogo in the heart, immunofluorescence and confocal microscopy were utilized. Nogo expression appears to be most clearly associated with cardiac fibroblasts. To our knowledge, this is the first report to demonstrate the relationship between Nogo expression and HF, including cell-type specificity, in both mouse and human HF and phenotypic rescue.

6A3-5/Osa2 is an early activated gene implicated in the control of vascular smooth muscle cell functions.

Vascular smooth muscle cells (VSMC) growth plays a key role in the pathophysiology of vascular diseases. However, the molecular mechanisms controlling gene transcription in VSMC remain poorly understood. We previously identified, by differential display, a new gene (6A3-5) overexpressed in proliferating rat VSMC. In this study, we have cloned the full-length cDNA by screening a rat foetal brain cDNA library and investigated its functions. The 6A3-5 protein shows 4 putative conserved functional motifs: a DNA binding domain called ARID (AT-rich interaction domain), two recently described motifs (Osa Homology Domain), and a nuclear localization signal. The deduced protein sequence was observed to be 85% identical to the recently described human Osa2 gene. Immunolabelling, using an anti-6A3-5/Osa2 monoclonal antibody, showed a nuclear localization of the 6A3-5/Osa2 protein. In addition, PDGF upregulated 6A3-5/Osa2 expression at both the transcript and protein levels in a dose and time-dependent fashion. The pattern of upregulation by PDGF was reminiscent of the early responsive gene c-fos. The PDGF-induced upregulation of 6A3-5/Osa2 and proliferation of VSMC were significantly inhibited in a dose and sequence-dependent fashion by an antisense, but not by sense, scrambled or mismatched oligonucleotides directed against 6A3-5/Osa2. In VSMC of aortas derived from hypertensive (LH) rats, 6A3-5/Osa2 is overexpressed as compared to that in normotensive (LL) rats. The 6A3-5/Osa2-gene expression is downregulated by an ACE inhibitor and upregulated by exogenous AngiotensinII in LH rats. In summary, these results indicate that 6A3-5/Osa2 is an early activated gene that belongs to a new family of proteins involved in the control of VSMC growth.

Hypersensitivity of the adrenal cortex to trophic and secretory effects of angiotensin II in Lyon genetically-hypertensive rats.

In Lyon hypertensive (LH) rats, a model of low-renin genetic hypertension, we investigated adrenal sensitivity to angiotensin II in terms of angiotensin II receptor (AT1 and AT2 receptors) regulation, morphological changes, and aldosterone and corticosterone secretion. Twelve-week-old LH rats, compared with normotensive LN and LL rats, were either untreated or treated for 4 weeks with AT1 receptor antagonist irbesartan (50 mg/kg/d), angiotensin-converting enzyme inhibitor perindopril (3 mg/kg/d), or perindopril (3 mg/kg/d) plus angiotensin II infusion (200 ng/kg/min). At 16 weeks, untreated LH rats had high systolic blood pressure (P<0.05), low aldosterone (P<0.05), and increased corticosterone (P<0.05) plasma levels. AT1-receptor binding density in the zona glomerulosa was similar in the three strains. In LH rats, angiotensin II infusion increased the relative adrenal weight from 10.5+/-0.3 to 16.7+/-0.7 mg/100g (P<0.05), whereas this change was very modest in normotensive rats. Zona glomerulosa enlarged and plasma aldosterone increased after angiotensin II infusion in the 3 strains, but more markedly in LH versus normotensive rats (2.4- versus 1.3- and 1.6-fold, respectively; 20- versus 10-fold in normotensive rats, P<0.05). Surprisingly, after angiotensin II infusion, despite the absence of angiotensin II receptors in the three strains, the zona fasciculata-reticularis enlarged 1.5-fold and plasma corticosterone increased 1.7-fold only in LH rats (P<0.05), suggesting an indirect control of this compartment by angiotensin II. The hypertrophy and hypersecretory activity of both zona glomerulosa and zona fasciculata-reticularis in LH rats in response to angiotensin II point to the adrenal cortex as a pivotal tissue in the pathophysiology of hypertension in LH rats.