Growing evidence indicates that microRNAs (miRNAs or miRs) are involved in basic cell functions and oncogenesis. Here we report that miR-133 has a critical role in determining cardiomyocyte hypertrophy. We observed decreased expression of both miR-133 and miR-1, which belong to the same transcriptional unit, in mouse and human models of cardiac hypertrophy. In vitro overexpression of miR-133 or miR-1 inhibited cardiac hypertrophy. In contrast, suppression of miR-133 by 'decoy' sequences induced hypertrophy, which was more pronounced than that after stimulation with conventional inducers of hypertrophy. In vivo inhibition of miR-133 by a single infusion of an antagomir caused marked and sustained cardiac hypertrophy. We identified specific targets of miR-133: RhoA, a GDP-GTP exchange protein regulating cardiac hypertrophy; Cdc42, a signal transduction kinase implicated in hypertrophy; and Nelf-A/WHSC2, a nuclear factor involved in cardiogenesis. Our data show that miR-133, and possibly miR-1, are key regulators of cardiac hypertrophy, suggesting their therapeutic application in heart disease.

Cardiomyocyte hypertrophy differs according to the stress exerted on the myocardium. While pressure overload-induced cardiomyocyte hypertrophy is associated with depressed contractile function, physiological hypertrophy after exercise training associates with preserved or increased inotropy. We determined the activation state of myocardial Akt signaling with downstream substrates and fetal gene reactivation in exercise-induced physiological and pressure overload-induced pathological hypertrophies. C57BL/6J mice were either treadmill trained for 6 weeks, 5 days/week, at 85-90% of maximal oxygen uptake (VO(2max)), or underwent transverse aortic constriction (TAC) for 1 or 8 weeks. Total and phosphorylated protein levels were determined with SDS-PAGE, and fetal genes by real-time RT-PCR. In the physiologically hypertrophied heart after exercise training, total Akt protein level was unchanged, but Akt was chronically hyperphosphorylated at serine 473. This was accompanied by activation of the mammalian target of rapamycin (mTOR), measured as phosphorylation of its two substrates: the ribosomal protein S6 kinase-1 (S6K1) and the eukaryotic translation initiation factor-4E binding protein-1 (4E-BP1). Exercise training did not reactivate the fetal gene program (beta-myosin heavy chain, atrial natriuretic factor, skeletal muscle actin). In contrast, pressure overload after TAC reactivated fetal genes already after 1 week, and partially inactivated the Akt/mTOR pathway and downstream substrates after 8 weeks. In conclusion, changes in opposite directions of the myocardial Akt/mTOR signal pathway appears to distinguish between physiological and pathological hypertrophies; exercise training associating with activation and pressure overload associating with inactivation of the Akt/mTOR pathway.

The apicidin derivative, API-D, is capable of reducing hypertrophy and, consequently, the transition to heart failure in mice subjected to TAC. Treatment with this substance, therefore, holds promise as an important therapeutic option for heart failure.

Protein-based therapies are useful in a variety of diseases; however, their potential for immunogenicity is a disadvantage. Neutralizing antibodies (NAbs) that develop to interferon beta (IFNbeta) products (IFNbeta-1b, IFNbeta-1a-Avonex((R)), or IFNbeta-1a-Rebif((R))), which are first-line therapies for the treatment of multiple sclerosis, are reported to reduce the clinical efficacy of these agents. In individual clinical studies of each commercially available IFNbeta product, 28% to 47% of patients develop NAbs to IFNbeta-1b, 12% to 28 % to IFNbeta-1a-Rebif, and 2% to 6% to IFNbeta-1a-Avonex. Problems exist in comparing the incidence of NAbs among IFNbeta products across studies because of differences in study methodology, including assay methods, treatment duration, and the definition of NAb positive. Results from studies that have directly compared these products are consistent with results from the respective clinical trials of IFNbetas. Both the clinical trials and the independent studies have shown that NAbs develop more frequently with IFNbeta-1b treatment than with IFNbeta-1a treatment and that, among IFNbeta-1a products, NAbs develop more frequently with IFNbeta-1a-Rebif treatment than with IFNbeta-1a-Avonex treatment. Factors that may affect the immunogenicity of IFNbetas, including the dosing regimens and the biochemical properties of the products, are discussed.

Abstract-MicroRNAs play important roles in many cellular and biological functions via the regulation of mRNA target translation. In the cardiovascular field, microRNAs are now acknowledged as fundamental in regulating the expression of genes that governs physiological and pathological myocardial adaptation to stress. Here, we review recent progress in the understanding of microRNA functions and their involvement in heart disease. (Circ Cardiovasc Genet. 2009;2: 402-408.)

Cardiomyocytes adapt to physical stress by increasing their size while maintaining cell function. The serine/threonine kinase Akt plays a critical role in this process of adaptation. We previously reported that transgenic overexpression of an active form of Akt (Akt-E40K) in mice results in increased cardiac contractility and cell size, as well as improved sarcoplasmic reticulum (SR) Ca 2؉ handling. Because it is not fully elucidated, we decided to study the molecular mechanism by which Akt-E40K overexpression improves SR Ca 2؉ handling. To this end, SR Ca 2؉ uptake and the phosphorylation status of phospholamban (PLN) were evaluated in heart extracts from wild-type and Akt-E40K mice and mice harboring inducible and cardiac specific knock-out of phosphatidylinositol-dependent kinase-1, the upstream activator of Akt. Moreover, the effect of Akt was assessed in vitro by overexpressing a mutant Akt targeted preferentially to the SR, and by biochemical assays to evaluate potential interaction with PLN. We found that when activated, Akt interacts with and phosphorylates PLN at Thr Furthermore, overexpression of SR-targeted Akt in cardiomyocytes improved Ca2؉ handling without affecting cell size. Thus, we describe here a new mechanism whereby the preferential translocation of Akt to the SR is responsible for enhancement of contractility without stimulation of hypertrophy.Akt, also referred to as protein kinase B, is a serine/threonine kinase found as part of the insulin, insulin-like growth factor-1 (IGF-1) 4 /phosphatidylinositol 3-kinase (PI3K)/phosphatidylinositol-dependent kinase-1 (PDK1) pathway (1). Upon activation, Akt phosphorylates a broad range of substrates involved in metabolism, transcription, translation, cell growth, differentiation, proliferation, and survival (2, 3). In the heart the IGF-1/ Akt axis is implicated in the control of physiological cardiac hypertrophy, contractile function, and Ca 2ϩ handling (4 -11). The cardiac effects of Akt have been described in many transgenic (Tg) mouse models, and cardiac specific overexpression of different mutated forms of Akt results in varying phenotypes (5,(12)(13)(14)(15)(16). A common effect observed in most of these Tg mouse lines is increased cardiomyocyte (CM) size and either maintained or improved cardiac function (5,(12)(13)(14), with modest or no activation of either mitogen-activated protein kinases (MAPKs) or fetal genes, two hallmarks of maladaptation to stress (5, 12, 13). Our group has previously described a Tg mouse model (Akt-E40K Tg) expressing an Akt with a mutation (E40K) located in its pleckstrin homology domain. This mutation renders Akt constitutively active in a manner similar to stimulation with growth factor (5). The occurrence of increased CM size with preservation of cardiac contractility constitutes an intriguing aspect of this mouse model. Single cell studies revealed not only increased inotropism but also enhanced lusitropism and an increase in systolic calcium (Ca 2ϩ ) transients in adult CMs isolated from this Tg mouse (5, 15)....

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AIMTo treated with electrochemotherapy (ECT) a prospective case series of patients with liver cirrhosis and Vp3-Vp4- portal vein tumor thrombus (PVTT) from hepatocellular carcinoma (HCC), in order to evaluate the feasibility, safety and efficacy of this new non thermal ablative technique in those patients.METHODSSix patients (5 males and 1 female), aged 61-85 years (mean age, 70 years), four in Child-Pugh A and two in Child-Pugh B class, entered our study series. All patients were studied with three-phase computed tomography (CT), contrast enhanced ultrasound (CEUS) and ultrasound-guided percutaneous biopsy of the thrombus before ECT. All patients underwent ECT treatment (Cliniporator Vitae®, IGEA SpA, Carpi, Modena, Italy) of Vp3-Vp4 PVTT in a single session. At the end of the procedure a post-treatment biopsy of the thrombus was performed. Scheduled follow-up in all patients entailed: CEUS within 24 h after treatment; triphasic contrast-enhanced CT and CEUS at 3 mo after treatment and every six months thereafter.RESULTSPost-treatment CEUS showed complete absence of enhancement of the treated thrombus in all cases. Post-treatment biopsy showed apoptosis and necrosis of tumor cells in all cases. The follow-up ranged from 9 to 20 mo (median, 14 mo). In 2 patients, the follow-up CT and CEUS demonstrated complete patency of the treated portal vein. Other 3 patients showed a persistent avascular non-tumoral shrinked thrombus at CEUS and CT during follow-up. No local recurrence was observed at follow-up CT and CEUS in 5/6 patients. One patient was lost to follow-up because of death from gastrointestinal hemorrage 5 wk after ECT.CONCLUSIONIn patients with cirrhosis, ECT seems effective and safe for curative treatment of Vp3-Vp4 PVTT from HCC.