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

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.     

Involvement of K+ channel-dependant pathways in lipoxin A4-induced protective effects on hypoxia/reoxygenation injury of cardiomyocytes

Studies have shown that lipoxin A₄ (LXA₄) and activation of LXA₄ receptor provided protection against myocardial ischemia/reperfusion injury in animal models. However, the mechanisms by which LXA₄ induced protective role on myocardial ischemia/reperfusion injury remains unclear. In the present studies, we investigated the protective effects of LXA₄ on H9c2 cardiomyocytes exposed to hypoxia/reoxygenation (H/R) injury and involvement of heme oxygenase-1 (HO-1)- and K⁺ channel-dependant pathways in the LXA₄ action. H9c2 cardiomyocytes were pretreated with or without LXA₄ or HO-1 specific interfering RNA (siRNA) or various blockers and openers of K⁺ channels before exposing to H/R injury. The levels of lactate dehydrogenase (LDH) and creatine kinase (CK) in cellular supernatants and necrosis factor-α (TNF-α) in cellular lysates were measured by using ELISA. Expressions of HO-1 mRNA and protein were analyzed by using RT-PCR and Western blot respectively. Pretreatment of the cells undergoing H/R injury with LXA₄ significantly reduced the LDH and CK levels induced by H/R injury, and increased the expressions and activity of HO-1. However, the protective effects of LXA₄ were completely blocked by transfection of the cells with HO-1 siRNA, and were partially but significantly blocked by pretreatment of the cells with various blockers of K⁺ channels. The LXA₄-induced expressions of HO-1 in the cells were also inhibited by HO-1 siRNA and various blockers of K⁺ channels. The inhibitory effects of LXA₄ on enhanced TNF-α levels induced by H/R injury were abolished by transfection of the cells with HO-1 siRNA. In conclusion, the protective role of LXA₄ on cardiomyocytes against H/R injury is related to upregulation of HO-1 via reduced production of TNF-α and activation of ATP-sensitive K⁺ channels and calcium-sensitive K⁺ channel.     

A cell-based sensor system for toxicity testing using multiwavelength fluorescence spectroscopy

A novel cell-based fluorometric sensor system for toxicity monitoring is described, which uses functional spontaneously contracting cardiomyocytes (HL-1 cell line) as the biological recognition element. Based on these highly specialized cells, it has the potential of providing a sensitive and relevant analytical in vitro toxicity testing method. The system was configured by propagating the surface-attaching HL-1 cardiomyocytes in the wells of a 96-well microtiter plate and connecting the plate via an optical fiber to a fluorescence spectrometer capable of excitation-emission matrix scanning. The fluorescence data were analyzed using a conventional spectral analysis software program. The performance of the system for detection of general cytotoxicity to the cells was evaluated using three well-known drugs: verapamil, quinidine, and acetaminophen. The dose-response curves were assessed and the EC₅₀ values were determined (0.10 ± 0.007, 0.23 ± 0.025, and 12.32 ± 2.40 mM, respectively). Comparison with in vitro and in vivo reference data for the drugs showed good correlations, suggesting that this cell-based sensor system could be a useful tool in pharmacological in vitro drug testing.     

Isolation and Cryopreservation of Neonatal Rat Cardiomyocytes

Cell culture has become increasingly important in cardiac research, but due to the limited proliferation of cardiomyocytes, culturing cardiomyocytes is difficult and time consuming. The most commonly used cells are neonatal rat cardiomyocytes (NRCMs), which require isolation every time cells are needed. The birth of the rats can be unpredictable. Cryopreservation is proposed to allow for cells to be stored until needed, yet freezing/thawing methods for primary cardiomyocytes are challenging due to the sensitivity of the cells. Using the proper cryoprotectant, dimethyl sulfoxide (DMSO), cryopreservation was achieved. By slowly extracting the DMSO while thawing the cells, cultures were obtained with viable NRCMs. NRCM phenotype was verified using immunocytochemistry staining for α-sarcomeric actinin. In addition, cells also showed spontaneous contraction after several days in culture. Cell viability after thawing was acceptable at 40-60%. In spite of this, the methods outlined allow one to easily cryopreserve and thaw NRCMs. This gives researchers a greater amount of flexibility in planning experiments as well as reducing the use of animals.     

Detection and quantitation of iron in ferritin,transferrin and labile iron pool (LIP) in cardiomyocytes using 55Fe and storage phosphorimaging

Dysregulated iron metabolism has a detrimental effect on cardiac function. The importance of iron homeostasis in cardiac health and disease warrants detailed studies of cardiomyocyte iron uptake, utilization and recycling at the molecular level. In this study, we have performed metabolic labeling of primary cultures of neonatal rat cardiomyocytes with radioactive iron coupled with separation of labeled iron-containing molecules by native electrophoresis followed by detection and quantification of incorporated radioiron by storage phosphorimaging. For the radiolabeling we used a safe and convenient beta emitter ⁵⁵Fe which enabled sensitive and simultaneous detection and quantitation of iron in cardiomyocyte ferritin, transferrin and the labile iron pool (LIP). The LIP is believed to represent potentially dangerous redox–active iron bound to uncharacterized molecules. Using size-exclusion chromatography spin micro columns, we demonstrate that iron in the LIP is bound to high molecular weight molecule(s) (≥5000?Da) in the neonatal cardiomyocytes.