Christian Zuppinger

Modulation of Anthracycline-Induced Myofibrillar Disarray in Rat Ventricular Myocytes by Neuregulin-1β and Anti-erbB2 Potential Mechanism for Trastuzumab-Induced Cardiotoxicity

Background—There is an increased incidence of heart failure in patients treated concurrently with anthracyclines and the chemotherapeutic anti-erbB2 agent trastuzumab (Herceptin). On the basis of our previous studies with recombinant neuregulin-1&bgr; (NRG-1&bgr;), a ligand for the erbB2 receptor tyrosine kinase, we hypothesized that activation of erbB2 by anti-erbB2 versus NRG-1 would cause differential effects on myocyte intracellular signaling as well as anthracycline-induced myofibrillar injury and might potentially account for the clinical toxicity of trastuzumab in the setting of concurrent anthracycline therapy. Methods and Results—We tested this hypothesis using adult rat ventricular myocytes (ARVMs) in culture, assessing myofibrillar structure by immunostaining for myomesin and filamentous actin. Activation of erbB2, extracellular signal–regulated kinase 1/2 (Erk1/2), and Akt was assessed by use of antibodies to phosphorylated activated receptor or kinase detected by immunoblot. ARVMs treated with doxorubicin (0.1 to 0.5 &mgr;mol/L) showed a concentration-dependent increase in myofilament disarray. NRG-1&bgr; (10 ng/mL) activated erbB2, Erk1/2, and Akt in ARVMs and significantly reduced anthracycline-induced disarray. In contrast to NRG-1&bgr;, anti-erbB2 (1 &mgr;g/mL) caused rapid phosphorylation of erbB2 but not Erk1/2 or Akt, with downregulation of erbB2 by 24 hours. Concomitant treatment of myocytes with anti-erbB2 and doxorubicin caused a significant increase in myofibrillar disarray versus doxorubicin alone. Conclusions—NRG-1&bgr;/erbB signaling regulates anthracycline-induced myofilament injury. The increased susceptibility of myofilaments to doxorubicin in the presence of antibody to erbB2 may explain the contractile dysfunction seen in patients receiving concurrent trastuzumab and anthracyclines.

Pathophysiology and diagnosis of cancer drug induced cardiomyopathy

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.

Multiply Attenuated, Self-Inactivating Lentiviral Vectors Efficiently Deliver and Express Genes for Extended Periods of Time in Adult Rat Cardiomyocytes In Vivo

Background—Among retroviral vectors, lentiviral vectors are unique in that they transduce genes into both dividing and nondividing cells. However, their ability to provide sustained myocardial transgene expression has not been evaluated. Methods and Results—Multiply attenuated, self-inactivating lentivectors based on human immunodeficiency virus-1 contained the enhanced green fluorescent protein (EGFP) gene under the transcriptional control of either the cytomegalovirus (CMV) immediate-early enhancer/promoter, the elongation factor-1&agr; (EF-1&agr;) promoter, or the phosphoglycerate-kinase (PGK) promoter. Lentivectors transduced adult rat cardiomyocytes in a dose-dependent manner (transduction rates, >90%; multiplicity of infection, ≈5). The CMV promoter achieved higher EGFP expression levels than the EF-1&agr; and PGK promoters. Insertion of the central polypurine tract pol sequence improved gene transfer efficiency by ≈2-fold. In vivo gene transfer kinetics was studied by measuring the copy number of integrated lentivirus DNA and EGFP concentrations in cardiac extracts by real-time polymerase chain reaction and ELISA, respectively. With CMV promoter-containing lentivectors, vector DNA peaked at day 3, declined by ≈4-fold at day 14, but then remained stable up to week 10. Similarly, EGFP expression peaked at day 7, decreased by ≈7-fold at day 14, but was essentially stable thereafter. In contrast, vector DNA and EGFP expression declined rapidly with EF-1&agr; promoter–containing lentivectors. Peak EGFP expression with titer-matched adenovectors was ≈35% higher than with CMV lentivectors but was lost rapidly over time. Conclusions—Lentivectors efficiently transduce and express genes for extended periods of time in cardiomyocytes in vivo. Lentivectors provide a useful tool for studying myocardial biology and a potential system for gene heart therapy.

Development and Characterization of a Scaffold-Free 3D Spheroid Model of Induced Pluripotent Stem Cell-Derived Human Cardiomyocytes.

Cardiomyocytes (CMs) are terminally differentiated cells in the adult heart, and ischemia and cardiotoxic compounds can lead to cell death and irreversible decline of cardiac function. As testing platforms, isolated organs and primary cells from rodents have been the standard in research and toxicology, but there is a need for better models that more faithfully recapitulate native human biology. Hence, a new in vitro model comprising the advantages of 3D cell culture and the availability of induced pluripotent stem cells (iPSCs) of human origin was developed and characterized. Human CMs derived from iPSCs were studied in standard 2D culture and as cardiac microtissues (MTs) formed in hanging drops. Two-dimensional cultures were examined using immunofluorescence microscopy and western blotting, while the cardiac MTs were subjected to immunofluorescence, contractility, and pharmacological investigations. iPSC-derived CMs in 2D culture showed well-formed myofibrils, cell-cell contacts positive for connexin-43, and other typical cardiac proteins. The cells reacted to prohypertrophic growth factors with a substantial increase in myofibrils and sarcomeric proteins. In hanging drop cultures, iPSC-derived CMs formed spheroidal MTs within 4 days, showing a homogeneous tissue structure with well-developed myofibrils extending throughout the whole spheroid without a necrotic core. MTs showed spontaneous contractions for more than 4 weeks that were recorded by optical motion tracking, sensitive to temperature and responsive to electrical pacing. Contractile pharmacology was tested with several agents known to modulate cardiac rate and viability. Calcium transients underlay the contractile activity and were also responsive to electrical stimulation, caffeine-induced Ca(2+) release, and extracellular calcium levels. A three-dimensional culture using iPSC-derived human CMs provides an organoid human-based cellular platform that is free of necrosis and recapitulates vital cardiac functionality, thereby providing a new and promising relevant model for the evaluation and development of new therapies and detection of cardiotoxicity.

Cancer therapy-associated cardiotoxicity and signaling in the myocardium

The cardiotoxic potential of cytotoxic cancer chemotherapy is well known. Prime examples are the anthracyclines, which are highly efficacious agents for hemopoietic malignancies and solid tumors, but their clinical use is limited primarily by cardiotoxicity. Besides the conventional chemotherapeutics, new cancer drugs were developed in the last decade with the goal to specifically inhibit selected molecular targets such as growth factor receptors or intracellular tyrosine kinases in cancer cells. However, the outcome of combining conventional and newer cancer therapies could have unexpected side effects not anticipated so far and the long-term outcome is not known. Sometimes, however, unexpected side effects also shed light on previously unknown physiological functions. For example, the anti-HER2 cancer therapeutic trastuzumab (Herceptin), which can induce cardiac dysfunction, has demonstrated the importance of the ErbB/neuregulin signaling system in the adult heart. Subsequently, the role of endothelial-myocardial communication in maintaining phenotype and survival of adult cardiomyocytes has increasingly been recognized.

Characterization of cytoskeleton features and maturation status of cultured human iPSC-derived cardiomyocytes.

Recent innovations in stem cell technologies and the availability of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have opened new possibilities for studies and drug testing on human cardiomyocytes in vitro. Still, there are concerns about the precise nature of such ‘reprogrammed’ cells. We have performed an investigation using immunocytochemistry and confocal microscopy on several cellular features using commercially available hiPSC-CMs. For some selected developmentally regulated or cardiac chamber-specific proteins, we have compared the results from hiPSC-derived cardiomyocytes with freshly isolated, ventricular cardiomyocytes from adult rats. The results show that all typical cardiac proteins are expressed in these hiPSC-CMs. Furthermore, intercalated disclike structures, calcium cycling proteins, and myofibrils are present. However, some of these proteins are only known from early developmental stages of the ventricular myocardium or the diseased adult heart. A heterogeneous expression pattern in the cell population was noted for some muscle proteins, such as for myosin light chains, or incomplete organization in sarcomeres, such as for telethonin. These observations indicate that hiPSC-CMs can be considered genuine human cardiomyocytes of an early developmental state. The here described marker proteins of maturation may become instrumental in future studies attempting the improvement of cardiomyocyte in vitro models.