Transplantation studies in mice and rats have shown that human embryonic stem cell-derived cardiomyocytes (hESC-CMs) can improve the function of infarcted hearts1–3, but two critical issues related to their electrophysiological behavior in vivo remain unresolved. First, the risk of arrhythmias following hESC-CM transplantation in injured hearts has not been determined. Second, the electromechanical integration of hESC-CMs in injured hearts has not been demonstrated, so it is unclear if these cells improve contractile function directly through addition of new force-generating units. Here we use a guinea pig model to show hESC-CM grafts in injured hearts protect against arrhythmias and can contract synchronously with host muscle. Injured hearts with hESC-CM grafts show improved mechanical function and a significantly reduced incidence of both spontaneous and induced ventricular tachycardia (VT). To assess the activity of hESC-CM grafts in vivo, we transplanted hESC-CMs expressing the genetically-encoded calcium sensor, GCaMP34, 5. By correlating the GCaMP3 fluorescent signal with the host ECG, we found that grafts in uninjured hearts have consistent 1:1 host-graft coupling. Grafts in injured hearts are more heterogeneous and typically include both coupled and uncoupled regions. Thus, human myocardial grafts meet physiological criteria for true heart regeneration, providing support for the continued development of hESC-based cardiac therapies for both mechanical and electrical repair.

Anthracyclines remain among the most widely prescribed and effective anticancer agents. Unfortunately, life-threatening cardiotoxicity continues to compromise their usefulness. Despite more than four decades of investigation, the pathogenic mechanisms responsible for anthracycline cardiotoxicity have not been completely elucidated. In addition, new drugs and combination therapies often exacerbate the toxicity. The First International Workshop on Anthracycline Cardiotoxicity, held in fall 2006, in Como, Italy, focused on the state-of-the-art knowledge and discussed the research needed to address the cardiotoxicity of these drugs. Here, we incorporate these discussions into the framework of a broader review of preclinical and clinical issues.

Voltage-gated Na + channels (VGSC) have been implicated in the metastatic potential of human breast, prostate, and lung cancer cells. Specifically, the SCN5A gene encoding the VGSC isotype Na v 1.5 has been defined as a key driver of human cancer cell invasion. In this study, we examined the expression and function of VGSCs in a panel of colon cancer cell lines by electrophysiologic recordings. Na + channel activity and invasive potential were inhibited pharmacologically by tetrodotoxin or genetically by small interfering RNAs (siRNA) specifically targeting SCN5A. Clinical relevance was established by immunohistochemistry of patient biopsies, with strong Na v 1.5 protein staining found in colon cancer specimens but little to no staining in matched-paired normal colon tissues. We explored the mechanism of VGSC-mediated invasive potential on the basis of reported links between VGSC activity and gene expression in excitable cells. Probabilistic modeling of loss-of-function screens and microarray data established an unequivocal role of VGSC SCN5A as a high level regulator of a colon cancer invasion network, involving genes that encompass Wnt signaling, cell migration, ectoderm development, response to biotic stimulus, steroid metabolic process, and cell cycle control. siRNA-mediated knockdown of predicted downstream network components caused a loss of invasive behavior, demonstrating network connectivity and its function in driving colon cancer invasion. Cancer Res; 70(17); 6957-67. ©2010 AACR.

Localization and staining features of the oxidant-sensitive fluorescent probe 2'7'-dichlorofluorescin (DCFH) were evaluated in isolated cardiac muscle cells. Cardiomyocytes rapidly accumulated the probe and retained steady levels of DCFH and its highly fluorescent oxidized product dichlorofluorescein (DCF) in probe-free medium for 1.5 h. DCF was associated with mitochondria and was released by the proton ionophore carbonyl cyanide m-chlorophenylhydrazone but not by saponin, which permeabilizes the plasma membrane. A mitochondrial distribution of DCF was also suggested by experiments with the mitochondrial marker MitoTracker Red, in which quenching was observed between DCF and MitoTracker Red in live cells. Isolated cardiac mitochondria rapidly accumulated DCF, and high micromolar concentrations of the probe inhibited ADP-stimulated respiration rate. The study provides an information base essential for the interpretation and design of experiments with DCF as a marker of oxidative stress in cardiac muscle and reveals preferential localization of the probe in mitochondria.

Many arrhythmias are believed to be triggered by ectopic sources arising from the border of the ischemic tissue. However, the development of ectopic activity from individual sources to a larger mass of cardiac tissue remains poorly understood. To address this critical issue, we used monolayers of neonatal rat cardiomyocytes to create conditions that promoted progression of ectopic activity from single cells to the network that consisted of hundreds of cells. To explain complex spatiotemporal patterns observed in these experiments we introduced a new theoretical framework. The framework's main feature is a parameter space diagram, which uses cell automaticity and coupling as two coordinates. The diagram allows one to depict network behavior, quantitatively address the heterogeneity factor, and evaluate transitions between different regimes. The well-organized wave trains were observed at moderate and high cell coupling values and network heterogeneity was found to be qualitatively unimportant for these regimes. In contrast, at lower values of coupling, spontaneous ectopic activity led to the appearance of fragmented ectopic waves. For these regimes, network heterogeneity played an essential role. The ectopic waves occasionally gave rise to spiral activity in two different regions within the parameter space via two distinct mechanisms. Together, our results suggest that localized ectopic waves represent an essential step in the progression of ectopic activity. These studies add to the understanding of initiation and progression of arrhythmias and can be applied to other phenomena that deal with assemblies of coupled oscillators.

The paper presents high-resolution fluorescence images obtained using laser-scanning confocal microscopy. Isolated cells from adult rat hearts were preloaded with 2',7'-dichlorofluorescin (an oxidant-sensitive fluorescent probe) and exposed to doxo- rubicin, an important anticancer drug with prominent cardioxicity. Fluorescence images were collected from live cells simultaneously on two channels: 1) 515-530 nm emission range was used to monitor an increase in dichlorofluorescein, the oxidized product of dichlorofluorescein, and 2) emission > 610 nm was used to visualize the intracellular distribution of doxorubicin. The images reveal intracellular oxidation close to the mitochondria after only 20 min of exposure of isolated cardiomyocytes to 40-160 microM doxorubicin. The data confirm an oxidative mechanism of doxorubicin cardiotoxicity and demonstrate the capability of a new technique to monitor intracellular oxidation in living cardiomyocytes.

Di(2-ethylhexyl) phthalate (DEHP) is a widely used plasticizer found in a variety of polyvinyl chloride (PVC) medical products. The results of studies in experimental animals suggest that DEHP leached from flexible PVC tubing may cause health problems in some patient populations. While the cancerogenic and reproductive effects of DEHP are well recognized, little is known about the potential adverse impact of phthalates on the heart. This study examined the effects of clinically relevant concentrations of DEHP on neonatal rat cardiomyocytes. It was found that application of DEHP to a confluent, synchronously beating cardiac cell network, leads to a marked, concentrationdependent decrease in conduction velocity and asynchronous cell beating. The mechanism behind these changes was a loss of gap junctional connexin-43, documented using western blot analysis, dye-transfer assay and immunofluorescence. In addition to its effect on electrical coupling, DEHP treatment also affected the mechanical movement of myocyte layers. The latter was linked to the decreased stiffness of the underlying fibroblasts, as the amount of triton-insoluble vimentin was significantly decreased in DEHP-treated samples. The data indicate that DEHP, in clinically relevant concentrations, can impair the electrical and mechanical behavior of a cardiac cell network. Applicability of these findings to human patients remains to be established.

Blebbistatin is a recently discovered myosin II inhibitor. It is rapidly becoming a compound of choice to reduce motion artifacts during cardiac optical mapping, as well as to study cell motility and cell invasion. Although blebbistatin has a number of advantages over other electromechanical uncouplers, many of its properties have yet to be addressed. Here we describe several methodological issues associated with the use of blebbistatin, including its spectral properties, reversibility, and its effect on tissue metabolic state. We show that if precautions are not taken, perfusion with blebbistatin may result in blebbistatin precipitate that accumulates in the vasculature. Although such precipitate is fluorescent, it is not detectable within wavelength bands that are typically used for transmembrane voltage fluorescence imaging (i.e., emission wavelengths >600 nm). Therefore, blockage of the microcirculation by blebbistatin may cause data misinterpretation in studies that use voltage-sensitive dyes. Blebbistatin may also impact imaging of green fluorophores due to the spectral shift it causes in endogenous tissue fluorescence. 3D excitation–emission matrices of blebbistatin in precipitate form and in various solutions (DMSO, water, and 1 % aqueous albumin) revealed significant changes in the fluorescence of this molecule in different environments. Finally, we examined the reversibility of blebbistatin’s uncoupling effect on cardiac contraction. Our findings provide important new information about the properties of this myosin II inhibitor, which will aid in the proper design and interpretation of studies that use this compound.