Maha Abdellatif

Simvastatin Induces Regression of Cardiac Hypertrophy and Fibrosis and Improves Cardiac Function in a Transgenic Rabbit Model of Human Hypertrophic Cardiomyopathy

Background—Hypertrophic cardiomyopathy is a genetic disease characterized by cardiac hypertrophy, myocyte disarray, interstitial fibrosis, and left ventricular (LV) dysfunction. We have proposed that hypertrophy and fibrosis, the major determinants of mortality and morbidity, are potentially reversible. We tested this hypothesis in &bgr;-myosin heavy chain–Q403 transgenic rabbits. Methods and Results—We randomized 24 &bgr;-myosin heavy chain–Q403 rabbits to treatment with either a placebo or simvastatin (5 mg · kg−1 · d−1) for 12 weeks and included 12 nontransgenic controls. We performed 2D and Doppler echocardiography and tissue Doppler imaging before and after treatment. Demographic data were similar among the groups. Baseline mean LV mass and interventricular septal thickness in nontransgenic, placebo, and simvastatin groups were 3.9±0.7, 6.2±2.0, and 7.5±2.1 g (P <0.001) and 2.2±0.2, 3.1±0.5, and 3.3±0.5 mm (P =0.002), respectively. Simvastatin reduced LV mass by 37%, interventricular septal thickness by 21%, and posterior wall thickness by 13%. Doppler indices of LV filling pressure were improved. Collagen volume fraction was reduced by 44% (P <0.001). Disarray was unchanged. Levels of activated extracellular signal-regulated kinase (ERK) 1/2 were increased in the placebo group and were less than normal in the simvastatin group. Levels of activated and total p38, Jun N-terminal kinase, p70S6 kinase, Ras, Rac, and RhoA and the membrane association of Ras, RhoA, and Rac1 were unchanged. Conclusions—Simvastatin induced the regression of hypertrophy and fibrosis, improved cardiac function, and reduced ERK1/2 activity in the &bgr;-myosin heavy chain–Q403 rabbits. These findings highlight the need for clinical trials to determine the effects of simvastatin on cardiac hypertrophy, fibrosis, and dysfunction in humans with hypertrophic cardiomyopathy and heart failure.

Adenoviral delivery of E2F-1 directs cell cycle reentry and p53-independent apoptosis in postmitotic adult myocardium in vivo.

Irreversible exit from the cell cycle precludes the ability of cardiac muscle cells to increase cell number after infarction. Using adenoviral E1A, we previously demonstrated dual pocket protein- and p300-dependent pathways in neonatal rat cardiac myocytes, and have proven that E2F-1, which occupies the Rb pocket, suffices for these actions of E1A. By contrast, the susceptibility of adult ventricular cells to viral delivery of exogenous cell cycle regulators has not been tested, in vitro or in vivo. In cultured adult ventricular myocytes, adenoviral gene transfer of E2F-1 induced expression of proliferating cell nuclear antigen, cyclin-dependent protein kinase 4, cell division cycle 2 kinase, DNA synthesis, and apoptosis. In vivo, adenoviral delivery of E2F-1 by direct injection into myocardium induced DNA synthesis, shown by 5-bromodeoxyuridine incorporation, and accumulation in G2/M, by image analysis of Feulgen-stained nuclei. In p53(-)/- mice, the prevalence of G1 exit was more than twofold greater; however, E2F-1 evoked apoptosis and rapid mortality comparably in both backgrounds. Thus, the differential effects of E2F-1 on G1 exit in wild-type versus p53-deficient mice illustrate the combinatorial power of viral gene delivery to genetically defined recipients: E2F-1 can override the G1/S checkpoint in postmitotic ventricular myocytes in vitro and in vivo, but leads to apoptosis even in p53(-)/- mice.

β2 adrenergic receptor 5′ haplotypes influence promoter activity

Transcriptional control of the human β2 adrenergic receptor gene (ADRB2) predominantly resides within a 549 base pair region immediately 5′ to the start of translation. Within this region, four naturally occurring polymorphisms, −468 C→G, −367 T→C, −47 T→C, and −20 T→C, have been identified. To determine the individual site and haplotype effects of these polymorphisms, we generated 16 luciferase‐based mutant constructs which were transiently transfected into HEK293 cells, and measured ADRB2 promoter‐driven luciferase activity. Two of the 16 mutant constructs, GCCT (−468G, −367C, −47C, −20T) and CTCT, showed a highly significant 3 fold decrease in luciferase induction relative to the reference CTTT. These haplotype effects could not be accounted for by the separate and additive effects of each site. These findings indicate that promoter polymorphisms interact to significantly alter β2 adrenergic receptor expression, and should be examined further for their association with disease‐related phenotypes.

RBFox1-mediated RNA splicing regulates cardiac hypertrophy and heart failure

RNA splicing is a major contributor to total transcriptome complexity; however, the functional role and regulation of splicing in heart failure remain poorly understood. Here, we used a total transcriptome profiling and bioinformatic analysis approach and identified a muscle-specific isoform of an RNA splicing regulator, RBFox1 (also known as A2BP1), as a prominent regulator of alternative RNA splicing during heart failure. Evaluation of developing murine and zebrafish hearts revealed that RBFox1 is induced during postnatal cardiac maturation. However, we found that RBFox1 is markedly diminished in failing human and mouse hearts. In a mouse model, RBFox1 deficiency in the heart promoted pressure overload-induced heart failure. We determined that RBFox1 is a potent regulator of RNA splicing and is required for a conserved splicing process of transcription factor MEF2 family members that yields different MEF2 isoforms with differential effects on cardiac hypertrophic gene expression. Finally, induction of RBFox1 expression in murine pressure overload models substantially attenuated cardiac hypertrophy and pathological manifestations. Together, this study identifies regulation of RNA splicing by RBFox1 as an important player in transcriptome reprogramming during heart failure that influence pathogenesis of the disease.

E1A Can Provoke G1 Exit That Is Refractory to p21 and Independent of Activating Cdk2

E1A can evoke G1 exit in cardiac myocytes and other cell types by displacing E2F transcription factors from tumor suppressor pocket proteins and by a less well-characterized p300-dependent pathway. Bypassing pocket proteins (through overexpression of E2F-1) reproduces the effect of inactivating pocket proteins (through E1A binding); however, pocket proteins associate with a number of molecular targets apart from E2F. Hence, pocket protein binding by E1A might engage mechanisms for cell cycle reentry beyond those induced by E2F-1. To test this hypothesis, we used adenoviral gene transfer to express various E2F-1 and E1A proteins in neonatal rat cardiac myocytes that are already refractory to mitogenic serum, in the absence or presence of several complementary cell cycle inhibitors-p16, p21, or dominant-negative cyclin-dependent kinase-2 (Cdk2). Rb binding by E2F-1 was neither necessary nor sufficient for G1 exit, whereas DNA binding was required; thus, exogenous E2F-1 did not merely function by competing for the Rb pocket. E2F-1-induced G1 exit was blocked by the universal Cdk inhibitor p21 but not by p16, a specific inhibitor of Cdk4/6; p21 was permissive for E2F-1 induction of cyclins E and A, but prevented their stimulation of Cdk2 kinase activity. In addition, E2F-1-induced G1 exit was blocked by dominant-negative Cdk2. Forced expression of cyclin E induced endogenous Cdk2 activity but not G1 exit. Thus, E2F-1-induced Cdk2 function was necessary, although not sufficient, to trigger DNA synthesis in cardiac muscle cells. In contrast, pocket protein-binding forms of E1A induced G1 exit that was resistant to inhibition by p21, whereas G1 exit via the E1A p300 pathway was sensitive to inhibition by p21. Both E1A pathways-via pocket proteins and via p300-upregulated cyclins E and A and Cdk2 activity, consistent with a role for Cdk2 in G1 exit induced by E1A. However, p21 blocked Cdk2 kinase activity induced by both E1A pathways equally. Thus, E1A can cause G1 exit without an increase in Cdk2 activity, if the pocket protein-binding domain is intact. E1A also overrides p21 in U2OS cells, provided the pocket protein-binding domain is intact; thus, this novel function of E1A is not exclusive to cardiac muscle cells. In summary, E1A binding to pocket proteins has effects beyond those produced by E2F-1 alone and can drive S-phase entry that is resistant to p21 and independent of an increase in Cdk2 function. This suggests the potential involvement of other endogenous Rb-binding proteins or of alternative E1A targets.

Myotrophin/V-1, a Protein Up-regulated in the Failing Human Heart and in Postnatal Cerebellum, Converts NFκB p50-p65 Heterodimers to p50-p50 and p65-p65 Homodimers

Myotrophin/V-1 is a cytosolic protein found at elevated levels in failing human hearts and in postnatal cerebellum. We have previously shown that it disrupts nuclear factor of κB (NFκB)-DNA complexes in vitro. In this study, we demonstrated that in HeLa cells native myotrophin/V-1 is predominantly present in the cytoplasm and translocates to the nucleus during sustained NFκB activation. Three-dimensional alignment studies indicate that myotrophin/V-1 resembles a truncated IκBα without the signal response domain (SRD) and PEST domains. Co-immunoprecipitation studies reveal that myotrophin/V-1 interacts with NFκB proteinsin vitro; however, it remains physically associated only with p65 and c-Rel proteins in vivo during NFκB activation. In vitro studies indicate that myotrophin/V-1 can promote the formation of p50-p50 homodimers from monomeric p50 proteins and can convert the preformed p50-p65 heterodimers into p50-p50 and p65-p65 homodimers. Furthermore, adenovirus-mediated overexpression of myotrophin/V-1 resulted in elevated levels of both p50-p50 and p65-p65 homodimers exceeding the levels of p50-p65 heterodimers compared with Adβgal-infected cells, where the levels of p50-p65 heterodimers exceeded the levels of p50-p50 and p65-p65 homodimers. Thus, overexpression of myotrophin/V-1 during NFκB activation resulted in a qualitative shift by quantitatively reducing the level of transactivating heterodimers while elevating the levels of repressive p50-p50 homodimers. Correspondingly, overexpression of myotrophin/V-1 resulted in significantly reduced κB-luciferase reporter activity. Because myotrophin/V-1 is found at elevated levels during NFκB activation in postnatal cerebellum and in failing human hearts, this study cumulatively suggests that myotrophin/V-1 is a regulatory protein for modulating the levels of activated NFκB dimers during this period.

Leading the Way Using Microarray A More Comprehensive Approach for Discovery of Gene Expression Patterns

Cells react to various stimuli by modulating their biological functions through selective changes in the activities of their constituent proteins. These changes are rendered by secondary modifications, translocation, or interaction with other molecules or cofactors or by altering a protein’s concentration. The latter event occurs via regulated modifications in the rate of gene transcription, RNA translation, or degradation of RNA or protein. In the premicroarray era, the number of genes that were subject to differential expression was seriously underestimated; this is demonstrated in the study by Stanton et al1 in this issue of Circulation Research . Using microarray technology, Stanton et al identified more than 700 genes whose expression was altered during myocardial infarction. Expression profiling is not merely a descriptive method; disclosure of the temporal and spatial changes in gene expression provides insight into cellular functions and underlying mechanisms in disease pathogenesis.nnDNA microarrays, or gene chips, are usually comprised of micron-range–sized spots of genomic DNA, cDNA, or oligonucleotides arrayed on a glass slide. They are used for a wide scope of applications, including sequencing, detection of mutations or polymorphisms, identification of drug targets, and gene expression profiling (reviewed by Wilgenbus and Lichter2 ). The latter application has gained wide use for monitoring differences in gene expression patterns in normal versus pathological conditions. Different clustering methods have been devised for the management of the large number of data …

Rethinking Ras: p21 Ras Proteins and Cardiac Signal Transduction

Hypertrophy of cardiac muscle occurs both as the normal mode of ventricular growth after birth and, as a pathophysiological response, in adaptation to mechanical overload [1–3]. Classically, cardiac myocytes lose their proliferative capacity shortly after birth, and subsequent enlargement is due to an increase in cell size, mediated in turn by an increase in total cellular protein. During “adaptive” hypertrophy, the resulting increase in protein synthesis and protein content is not merely a generalized effect, but is instead a highly regulated event marked by preferential modulation of specific subsets of genes [1–3]. For example, in rodent models, which have been the most thoroughly investigated for evidence of this plasticity, a switch occurs in the expression of myosin heavy chain (MHC) isoforms: selective reactivation of the “fetal” βMHC gene, whereas αMHC ordinarily predominates [4,5]. Expression of α-skeletal actin — a second contractile protein gene that is preferentially transcribed in the embryonic ventricle — is likewise specifically enhanced in hypertrophied hearts [5,6]. Altered expression of these contractile protein genes has been linked to altered mechanical performance of ventricular myocardium, as increased fractional content of PMHC is held to improve the economy of contraction, at the expense of the maximum velocity of unloaded shortening [7].