Publications by authors named "Francesco Timolati"

7 Publications

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Effects of doxorubicin cancer therapy on autophagy and the ubiquitin-proteasome system in long-term cultured adult rat cardiomyocytes.

Cell Tissue Res 2012 Nov 4;350(2):361-72. Epub 2012 Aug 4.

Cardiology, Swiss Cardiovascular Center Bern, Bern University Hospital and University of Bern, Bern, Switzerland.

The clinical use of anthracyclines in cancer therapy is limited by dose-dependent cardiotoxicity that involves cardiomyocyte injury and death. We have tested the hypothesis that anthracyclines affect protein degradation pathways in adult cardiomyocytes. To this aim, we assessed the effects of doxorubicin (Doxo) on apoptosis, autophagy and the proteasome/ubiquitin system in long-term cultured adult rat cardiomyocytes. Accumulation of poly-ubiquitinated proteins, increase of cathepsin-D-positive lysosomes and myofibrillar degradation were observed in Doxo-treated cardiomyocytes. Chymotrypsin-like activity of the proteasome was initially increased and then inhibited by Doxo over a time-course of 48 h. Proteasome 20S proteins were down-regulated by higher doses of Doxo. The expression of MURF-1, an ubiquitin-ligase specifically targeting myofibrillar proteins, was suppressed by Doxo at all concentrations measured. Microtubule-associated protein 1 light chain 3B (LC3)-positive punctae and both LC3-I and -II proteins were induced by Doxo in a dose-dependent manner, as confirmed by using lentiviral expression of green fluorescence protein bound to LC3 and live imaging. The lysosomotropic drug chloroquine led to autophagosome accumulation, which increased with concomitant Doxo treatment indicating enhanced autophagic flux. We conclude that Doxo causes a downregulation of the protein degradation machinery of cardiomyocytes with a resulting accumulation of poly-ubiquitinated proteins and autophagosomes. Although autophagy is initially stimulated as a compensatory response to cytotoxic stress, it is followed by apoptosis and necrosis at higher doses and longer exposure times. This mechanism might contribute to the late cardiotoxicity of anthracyclines by accelerated aging of the postmitotic adult cardiomyocytes and to the susceptibility of the aging heart to anthracycline cancer therapy.
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http://dx.doi.org/10.1007/s00441-012-1475-8DOI Listing
November 2012

The role of cell death and myofibrillar damage in contractile dysfunction of long-term cultured adult cardiomyocytes exposed to doxorubicin.

Cytotechnology 2009 Nov 5;61(1-2):25-36. Epub 2009 Nov 5.

Swiss Cardiovascular Center, Inselspital, Bern University Hospital, 3010, Bern, Switzerland.

In failing hearts cardiomyocytes undergo alterations in cytoskeleton structure, contractility and viability. It is not known presently, how stress-induced changes of myofibrils correlate with markers for cell death and contractile function in cardiomyocytes. Therefore, we have studied the progression of contractile dysfunction, myofibrillar damage and cell death in cultured adult cardiomyocytes exposed to the cancer therapy doxorubicin. We demonstrate, that long-term cultured adult cardiomyocytes, a well-established model for the study of myofibrillar structure and effects of growth factors, can also be used to assess contractility and calcium handling. Adult rat ventricular myocytes (ARVM) were isolated and cultured for a total of 14 days in serum containing medium. The organization of calcium-handling proteins and myofibrillar structure in freshly isolated and in long-term cultured adult cardiomyocytes was studied by immunofluorescence and electron microscopy. Excitation contraction-coupling was analyzed by fura 2 and video edge detection in electrically paced cardiomyocytes forming a monolayer, and cell death and viability was measured by TUNEL assay, LDH release, MTT assay, and Western blot for LC3. Adult cardiomyocytes treated with Doxo showed apoptosis and necrosis only at supraclinical concentrations. Treated cells displayed merely alterations in cytoskeleton organization and integrity concomitant with contractile dysfunction and up-regulation of autophagosome formation, but no change in total sarcomeric protein content. We propose, that myofibrillar damage contributes to contractile dysfunction prior to cell death in adult cardiomyocytes exposed to clinically relevant concentrations of anthracyclines.
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http://dx.doi.org/10.1007/s10616-009-9238-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2795142PMC
November 2009

The tight junction protein cingulin regulates gene expression and RhoA signaling.

Ann N Y Acad Sci 2009 May;1165:88-98

Department of Molecular Biology, University of Geneva, Geneva, Switzerland.

Tight junctions (TJ) regulate the passage of solutes across epithelial sheets, contribute to the establishment and maintenance of epithelial apico-basal polarity and are involved in the regulation of gene expression and cell proliferation. Cingulin, a Mr 140 kDa protein localized in the cytoplasmic region of TJ, is not directly required for TJ formation and epithelial polarity but regulates RhoA signaling, through its interaction with the RhoA activator GEF-H1, and gene expression. Here we describe in more detail the effect of cingulin mutation in embryoid bodies (EB) on gene expression, by identifying the genes that show the highest degree of up- or downregulation, and the putative canonical pathways that might be affected by cingulin. Furthermore, we show that full-length canine GEF-H1, produced in baculovirus-infected insect cells, interacts with regions both in the cingulin globular head, and in the coiled-coil rod domain. These results extend our previous studies and provide new perspectives for the mechanistic analysis of cingulin function.
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http://dx.doi.org/10.1111/j.1749-6632.2009.04053.xDOI Listing
May 2009

Inhibition of ErbB2 by receptor tyrosine kinase inhibitors causes myofibrillar structural damage without cell death in adult rat cardiomyocytes.

Exp Cell Res 2009 Apr 12;315(7):1302-12. Epub 2009 Feb 12.

Swiss Cardiovascular Center, Bern University Hospital, Bern, Switzerland.

Inhibition of ErbB2 (HER2) with monoclonal antibodies, an effective therapy in some forms of breast cancer, is associated with cardiotoxicity, the pathophysiology of which is poorly understood. Recent data suggest, that dual inhibition of ErbB1 (EGFR) and ErbB2 signaling is more efficient in cancer therapy, however, cardiac safety of this therapeutic approach is unknown. We therefore tested an ErbB1-(CGP059326) and an ErbB1/ErbB2-(PKI166) tyrosine kinase inhibitor in an in-vitro system of adult rat ventricular cardiomyocytes and assessed their effects on 1. cell viability, 2. myofibrillar structure, 3. contractile function, and 4. MAPK- and Akt-signaling alone or in combination with Doxorubicin. Neither CGP nor PKI induced cardiomyocyte necrosis or apoptosis. PKI but not CGP caused myofibrillar structural damage that was additive to that induced by Doxorubicin at clinically relevant doses. These changes were associated with an inhibition of excitation-contraction coupling. PKI but not CGP decreased p-Erk1/2, suggesting a role for this MAP-kinase signaling pathway in the maintenance of myofibrils. These data indicate that the ErbB2 signaling pathway is critical for the maintenance of myofibrillar structure and function. Clinical studies using ErbB2-targeted inhibitors for the treatment of cancer should be designed to include careful monitoring for cardiac dysfunction.
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http://dx.doi.org/10.1016/j.yexcr.2009.02.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4991362PMC
April 2009

Pathophysiology and diagnosis of cancer drug induced cardiomyopathy.

Cardiovasc Toxicol 2007 ;7(2):61-6

Swiss Cardiovascular Center Bern, Cardiology, Inselspital, University Hospital, 3010 Bern, Switzerland.

The clinical manifestations of anti-cancer drug associated cardiac side effects are diverse and can range from acutely induced cardiac arrhythmias to Q-T interval prolongation, changes in coronary vasomotion with consecutive myocardial ischemia, myocarditis, pericarditis, severe contractile dysfunction, and potentially fatal heart failure. The pathophysiology of these adverse effects is similarly heterogeneous and the identification of potential mechanisms is frequently difficult since the majority of cancer patients is not only treated with a multitude of cancer drugs but might also be exposed to potentially cardiotoxic radiation therapy. Some of the targets inhibited by new anti-cancer drugs also appear to be important for the maintenance of cellular homeostasis of normal tissue, in particular during exposure to cytotoxic chemotherapy. If acute chemotherapy-induced myocardial damage is only moderate, the process of myocardial remodeling can lead to progressive myocardial dysfunction over years and eventually induce myocardial dysfunction and heart failure. The tools for diagnosing anti-cancer drug associated cardiotoxicity and monitoring patients during chemotherapy include invasive and noninvasive techniques as well as laboratory investigations and are mostly only validated for anthracycline-induced cardiotoxicity and more recently for trastuzumab-associated cardiac dysfunction.
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http://dx.doi.org/10.1007/s12012-007-0016-2DOI Listing
October 2007

Inhibition of ErbB2/neuregulin signaling augments paclitaxel-induced cardiotoxicity in adult ventricular myocytes.

Exp Cell Res 2007 May 22;313(8):1588-601. Epub 2007 Feb 22.

Swiss Cardiovascular Center Bern, University Hospital, CH-3010 Bern, Switzerland.

Paclitaxel (Taxol) has been successfully combined with the monoclonal antibody trastuzumab (Herceptin) in the treatment of ErbB2 overexpressing cancers. However, this combination therapy showed an unexpected synergistic increase in cardiac dysfunction. We have studied the mechanisms of paclitaxel/anti-ErbB2 cardiotoxicity in adult rat ventricular myocytes (ARVM). Myofibrillar organization was assessed by immunofluorescence microscopy and cell viability was tested by the TUNEL-, LDH- and MTT-assay. Oxidative stress was measured by DCF-fluorescence and myocyte contractile function by video edge-detection and fura-2 fluorescence. Treatment of ARVM with paclitaxel or antibodies to ErbB2 caused a significant increase in myofilament degradation, similarly as observed with an inhibitor of MAPK-signaling, but not apoptosis, necrosis or changes in mitochondrial activity. Paclitaxel-treatment and anti-ErbB2 reduced Erk1/2 phosphorylation. Paclitaxel increased diastolic calcium, shortened relaxation time and reduced fractional shortening in combination with anti-ErbB2. A minor increase in oxidative stress by paclitaxel or anti-ErbB2 was found. We conclude, that concomitant inhibition of ErbB2 receptors and paclitaxel treatment has an additive worsening effect on adult cardiomyocytes, mainly discernible in changes of myofibrillar structure and function, but in the absence of cell death. A potential mechanism is the modulation of the MAPK/Erk1/2 signaling by both drugs.
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http://dx.doi.org/10.1016/j.yexcr.2007.02.007DOI Listing
May 2007

Neuregulin-1 beta attenuates doxorubicin-induced alterations of excitation-contraction coupling and reduces oxidative stress in adult rat cardiomyocytes.

J Mol Cell Cardiol 2006 Nov 26;41(5):845-54. Epub 2006 Sep 26.

Swiss Cardiovascular Center, University Hospital, CH-3010 Bern, Switzerland.

Treatment of metastatic breast cancer with doxorubicin (Doxo) in combination with trastuzumab, an antibody targeting the ErbB2 receptor, results in an increased incidence of heart failure. Doxo therapy induces reactive oxygen species (ROS) and alterations of calcium homeostasis. Therefore, we hypothesized that neuregulin-1 beta (NRG), a ligand of the cardiac ErbB receptors, reduces Doxo-induced alterations of EC coupling by triggering antioxidant mechanisms. Adult rat ventricular cardiomyocytes (ARVM) were isolated and treated for 18-48 h. SERCA protein was analyzed by Western blot, EC coupling parameters by fura-2 and video edge detection, gene expression by RT-PCR, and ROS by DCF-fluorescence microscopy. At clinically relevant doses Doxo reduced cardiomyocytes contractility, SERCA protein and SR calcium content. NRG, similarly as the antioxidant N-acetylcystein (NAC), did not affect EC coupling alone, but protected against Doxo-induced damage. NRG and Doxo showed an opposite modulation of glutathione reductase gene expression. NRG, similarly as NAC, reduced peroxide- or Doxo-induced oxidative stress. Specific inhibitors showed, that the antioxidant action of NRG depended on signaling via the ErbB2 receptor and on the Akt- and not on the MAPK-pathway. Therefore, NRG attenuates Doxo-induced alterations of EC coupling and reduces oxidative stress in ARVM. Inhibition of the ErbB2/NRG signaling pathway by trastuzumab in patients concomitantly treated with Doxo might prevent beneficial effects of NRG in the myocardium.
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http://dx.doi.org/10.1016/j.yjmcc.2006.08.002DOI Listing
November 2006