Gianluigi Condorelli

ErbB2 is essential in the prevention of dilated cardiomyopathy

Amplification of the gene encoding the ErbB2 (Her2/neu) receptor tyrosine kinase is critical for the progression of several forms of breast cancer. In a large-scale clinical trial, treatment with Herceptin (trastuzumab), a humanized blocking antibody against ErbB2, led to marked improvement in survival. However, cardiomyopathy was uncovered as a mitigating side effect, thereby suggesting an important role for ErbB2 signaling as a modifier of human heart failure. To investigate the physiological role of ErbB2 signaling in the adult heart, we generated mice with a ventricular-restricted deletion of Erbb2. These ErbB2-deficient conditional mutant mice were viable and displayed no overt phenotype. However, physiological analysis revealed the onset of multiple independent parameters of dilated cardiomyopathy, including chamber dilation, wall thinning and decreased contractility. Additionally, cardiomyocytes isolated from these conditional mutants were more susceptible to anthracycline toxicity. ErbB2 signaling in cardiomyocytes is therefore essential for the prevention of dilated cardiomyopathy.

Akt induces enhanced myocardial contractility and cell size in vivo in transgenic mice

The serine-threonine kinase Akt seems to be central in mediating stimuli from different classes of receptors. In fact, both IGF-1 and IL6-like cytokines induce hypertrophic and antiapoptotic signals in cardiomyocytes through PI3K-dependent Akt activation. More recently, it was shown that Akt is involved also in the hypertrophic and antiapoptotic effects of β-adrenergic stimulation. Thus, to determine the effects of Akt on cardiac function in vivo, we generated a model of cardiac-specific Akt overexpression in mice. Transgenic mice were generated by using the E40K, constitutively active mutant of Akt linked to the rat α-myosin heavy chain promoter. The effects of cardiac-selective Akt overexpression were studied by echocardiography, cardiac catheterization, histological and biochemical techniques. We found that Akt overexpression produced cardiac hypertrophy at the molecular and histological levels, with a significant increase in cardiomyocyte cell size and concentric LV hypertrophy. Akt-transgenic mice also showed a remarkable increase in cardiac contractility compared with wild-type controls as demonstrated by the analysis of left ventricular (dP/dtmax) in an invasive hemodynamic study, although with graded dobutamine infusion, the maximum response was not different from that in controls. Diastolic function, evaluated by left ventricular dP/dtmin, was not affected at rest but was impaired during graded dobutamine infusion. Isoproterenol-induced cAMP levels, β-adrenergic receptor (β-AR) density, and β-AR affinity were not altered compared with control mice. Moreover, studies on signaling pathway activation from myocardial extracts demonstrated that glycogen synthase kinase3-β is phosphorylated, whereas p42/44 mitogen-activated protein kinases is not, indicating that Akt induces hypertrophy in vivo by activating the glycogen synthase kinase3-β/GATA 4 pathway. In summary, our results not only demonstrate that Akt regulates cardiomyocyte cell size in vivo, but, importantly, show that Akt modulates cardiac contractility in vivo without directly affecting β-AR signaling capacity.

The knockout of miR-143 and -145 alters smooth muscle cell maintenance and vascular homeostasis in mice: Correlates with human disease

Mechanisms controlling vascular smooth muscle cell (VSMC) plasticity and renewal still remain to be elucidated completely. A class of small RNAs called microRNAs (miRs) regulate gene expression at the post-transcriptional level. Here, we show a critical role of the miR-143/145 cluster in SMC differentiation and vascular pathogenesis, also through the generation of a mouse model of miR-143 and -145 knockout (KO). We determined that the expression of miR-143 and -145 is decreased in acute and chronic vascular stress (transverse aortic constriction and in aortas of the ApoE KO mouse). In human aortic aneurysms, the expression of miR-143 and -145 was significantly decreased compared with control aortas. In addition, overexpression of miR-143 and -145 decreased neointimal formation in a rat model of acute vascular injury. An in-depth analysis of the miR-143/145 KO mouse model showed that this miR cluster is expressed mostly in the SMC compartment, both during development and postnatally, in vessels and SMC-containing organs. Loss of miR-143 and miR-145 expression induces structural modifications of the aorta, because of an incomplete differentiation of VSMCs. In conclusion, our results show that the miR-143/145 gene cluster has a critical role during SMC differentiation and strongly suggest its involvement in the reversion of the VSMC differentiation phenotype that occurs during vascular disease.

Cardiomyocytes induce endothelial cells to trans-differentiate into cardiac muscle: Implications for myocardium regeneration

The concept of tissue-restricted differentiation of postnatal stem cells has been challenged by recent evidence showing pluripotency for hematopoietic, mesenchymal, and neural stem cells. Furthermore, rare but well documented examples exist of already differentiated cells in developing mammals that change fate and trans-differentiate into another cell type. Here, we report that endothelial cells, either freshly isolated from embryonic vessels or established as homogenous cells in culture, differentiate into beating cardiomyocytes and express cardiac markers when cocultured with neonatal rat cardiomyocytes or when injected into postischemic adult mouse heart. Human umbilical vein endothelial cells also differentiate into cardiomyocytes under similar experimental conditions and transiently coexpress von Willebrand factor and sarcomeric myosin. In contrast, neural stem cells, which efficiently differentiate into skeletal muscle, differentiate into cardiomyocytes at a low rate. Fibroblast growth factor 2 and bone morphogenetic protein 4, which activate cardiac differentiation in embryonic cells, do not activate cardiogenesis in endothelial cells or stimulate trans-differentiation in coculture, suggesting that different signaling molecules are responsible for cardiac induction during embryogenesis and in successive periods of development. The fact that endothelial cells can generate cardiomyocytes sheds additional light on the plasticity of endothelial cells during development and opens perspectives for cell autologous replacement therapies.

Adult c-kitpos Cardiac Stem Cells Are Necessary and Sufficient for Functional Cardiac Regeneration and Repair

The epidemic of heart failure has stimulated interest in understanding cardiac regeneration. Evidence has been reported supporting regeneration via transplantation of multiple cell types, as well as replication of postmitotic cardiomyocytes. In addition, the adult myocardium harbors endogenous c-kit(pos) cardiac stem cells (eCSCs), whose relevance for regeneration is controversial. Here, using different rodent models of diffuse myocardial damage causing acute heart failure, we show that eCSCs restore cardiac function by regenerating lost cardiomyocytes. Ablation of the eCSC abolishes regeneration and functional recovery. The regenerative process is completely restored by replacing the ablated eCSCs with the progeny of one eCSC. eCSCs recovered from the host and recloned retain their regenerative potential in vivo and in vitro. After regeneration, selective suicide of these exogenous CSCs and their progeny abolishes regeneration, severely impairing ventricular performance. These data show that c-kit(pos) eCSCs are necessary and sufficient for the regeneration and repair of myocardial damage.

Human p300 Protein Is a Coactivator for the Transcription Factor MyoD

Human p300 protein is a cellular target of adenoviral E1A oncoprotein and a potential transcriptional coactivator. Both p300 and Rb family protein-binding regions of E1A are required for the repression of muscle gene expression, which is regulated by MyoD family transactivators. This implies that p300 is involved in MyoD-dependent transactivation. We show that the repression of MyoD-mediated E box (MyoD consensus) reporter activity by E1A is correlated with its interaction with p300, indicating that p300 participates in MyoD-dependent transactivation. In addition, p300 is able to interact both in vivo and in vitro with MyoD through a portion at the carboxyl-terminal cysteine/histidine-rich domain and associates with the components of the basal transcriptional complex through its two separate transactivation domains at the amino and carboxyl termini. Consistent with its role as a coactivator, p300 potentiates MyoD-activated transcription.

Reciprocal Regulation of MicroRNA-1 and Insulin-Like Growth Factor-1 Signal Transduction Cascade in Cardiac and Skeletal Muscle in Physiological and Pathological Conditions

Background— MicroRNAs (miRNAs/miRs) are small conserved RNA molecules of 22 nucleotides that negatively modulate gene expression primarily through base paring to the 3′ untranslated region of target messenger RNAs. The muscle-specific miR-1 has been implicated in cardiac hypertrophy, heart development, cardiac stem cell differentiation, and arrhythmias through targeting of regulatory proteins. In this study, we investigated the molecular mechanisms through which miR-1 intervenes in regulation of muscle cell growth and differentiation. Methods and Results— On the basis of bioinformatics tools, biochemical assays, and in vivo models, we demonstrate that (1) insulin-like growth factor-1 (IGF-1) and IGF-1 receptor are targets of miR-1; (2) miR-1 and IGF-1 protein levels are correlated inversely in models of cardiac hypertrophy and failure as well as in the C2C12 skeletal muscle cell model of differentiation; (3) the activation state of the IGF-1 signal transduction cascade reciprocally regulates miR-1 expression through the Foxo3a transcription factor; and (4) miR-1 expression correlates inversely with cardiac mass and thickness in myocardial biopsies of acromegalic patients, in which IGF-1 is overproduced after aberrant synthesis of growth hormone. Conclusions— Our results reveal a critical role of miR-1 in mediating the effects of the IGF-1 pathway and demonstrate a feedback loop between miR-1 expression and the IGF-1 signal transduction cascade.

MicroRNA-133a Protects Against Myocardial Fibrosis and Modulates Electrical Repolarization Without Affecting Hypertrophy in Pressure-Overloaded Adult Hearts

Rationale: MicroRNA (miR)-133a regulates cardiac and skeletal muscle differentiation and plays an important role in cardiac development. Because miR-133a levels decrease during reactive cardiac hypertrophy, some have considered that restoring miR-133a levels could suppress hypertrophic remodeling. Objective: To prevent the “normal” downregulation of miR-133a induced by an acute hypertrophic stimulus in the adult heart. Methods and Results: miR-133a is downregulated in transverse aortic constriction (TAC) and isoproterenol-induced hypertrophy, but not in 2 genetic hypertrophy models. Using MYH6 promoter-directed expression of a miR-133a genomic precursor, increased cardiomyocyte miR-133a had no effect on postnatal cardiac development assessed by measures of structure, function, and mRNA profile. However, increased miR-133a levels increased QT intervals in surface electrocardiographic recordings and action potential durations in isolated ventricular myocytes, with a decrease in the fast component of the transient outward K+ current, Ito,f, at baseline. Transgenic expression of miR-133a prevented TAC-associated miR-133a downregulation and improved myocardial fibrosis and diastolic function without affecting the extent of hypertrophy. Ito,f downregulation normally observed post-TAC was prevented in miR-133a transgenic mice, although action potential duration and QT intervals did not reflect this benefit. miR-133a transgenic hearts had no significant alterations of basal or post-TAC mRNA expression profiles, although decreased mRNA and protein levels were observed for the Ito,f auxiliary KChIP2 subunit, which is not a predicted target. Conclusions: These results reveal striking differences between in vitro and in vivo phenotypes of miR expression, and further suggest that mRNA signatures do not reliably predict either direct miR targets or major miR effects.

Inhibition of cellular ras prevents smooth muscle cell proliferation after vascular injury in vivo

Proliferation of smooth muscle cells of the arterial wall in response to local injury is an important aetiologic factor of vascular proliferative disorders such as atherosclerosis and restenosis after angioplasty. Ras proteins are key transducers of mitogenic signals from membrane to nucleus in many cell types. We investigated the role of ras proteins in the vascular response to arterial injury by inactivating cellular ras of rats in which the common carotid artery was subjected to balloon injury. DNA vectors expressing ras transdominant negative mutants, which interfere with ras function, reduced neointimal formation after injury. Our results indicate a key role for ras in smooth muscle cell proliferation and show that the local delivery of transdominant negative mutants of ras in vivo might prevent some of the acute vascular injury caused by balloon injury.

Cardiovascular side effects of cancer therapies: a position statement from the Heart Failure Association of the European Society of Cardiology

The reductions in mortality and morbidity being achieved among cancer patients with current therapies represent a major achievement. However, given their mechanisms of action, many anti‐cancer agents may have significant potential for cardiovascular side effects, including the induction of heart failure. The magnitude of this problem remains unclear and is not readily apparent from current clinical trials of emerging targeted agents, which generally under‐represent older patients and those with significant co‐morbidities. The risk of adverse events may also increase when novel agents, which frequently modulate survival pathways, are used in combination with each other or with other conventional cytotoxic chemotherapeutics. The extent to which survival and growth pathways in the tumour cell (which we seek to inhibit) coincide with those in cardiovascular cells (which we seek to preserve) is an open question but one that will become ever more important with the development of new cancer therapies that target intracellular signalling pathways. It remains unclear whether potential cardiovascular problems can be predicted from analyses of such basic signalling mechanisms and what pre‐clinical evaluation should be undertaken. The screening of patients, optimization of therapeutic schemes, monitoring of cardiovascular function during treatment, and the management of cardiovascular side effects are likely to become increasingly important in cancer patients. This paper summarizes the deliberations of a cross‐disciplinary workshop organized by the Heart Failure Association of the European Society of Cardiology (held in Brussels in May 2009), which brought together clinicians working in cardiology and oncology and those involved in basic, translational, and pharmaceutical science.