Maturation of Cultured Neonatal Rat Ventricular Myocytes. Introduction: Cultured neonatal rat ventricular myocytes (NRVM) reestablish gap junctions as they form synchronously and spontaneously beating monolayers, thus providing a useful model for studying activation and repolarization.
Methods and Results: We used the multielectrode array data acquisition system with 60 unipolar electrodes to investigate the functional organization of cultured NRVM, by determining propagation and repolarization patterns. Activation maps were constructed from the local activation times at each electrode. During days 3 to 8 in culture, QRS amplitude and dV/dtmax increased with age. Concomitantly, with the culture maturation, QT interval (representing action potential duration) decreased, and T wave amplitude and slopes of the T wave ascending and descending limbs progressively increased. The changes in conduction velocity were different than those of the electrogram properties, slightly increasing during the first 3 to 5 days and gradually declining toward day 8 in culture.
Conclusion: Establishment of uniform activation patterns in spontaneously firing or driven myocytes in monolayer cultures is accompanied by organization of activation and repolarization whose evolution appears in concert with that of a mature connexin43 staining pattern. The experimental techniques developed in this study provide useful tools to investigate the complex relations among gap junctions, conduction velocity, and propagation patterns, as well as a means to learn how gap junctional remodeling under pathophysiologic conditions predisposes the myocardium to arrhythmias.
"Hence, the TVP1022-induced augmentation in phosphorylated Cx43 probably contributed to the increased intercellular coupling , resulting in increased activation and propagation parameters. This conclusion is supported by the following findings: 1) an increase in phosphorylated Cx43 protein expression with no parallel increase in NP Cx43 is positively correlated with synchronized contraction, reflecting better activation and propagation patterns in NRVM (Oyamada et al., 1994); 2) we previously reported that, in NRVM, increased Cx43 density is associated with higher conduction velocity, QRS amplitude, and dV/dt max (Meiry et al., 2001); 3) slower conduction velocity was demonstrated in myocytes from the Cx43 homozygote neonatal (Beauchamp et al., 2004) and heterozygote neonatal and adult (Guerrero et al., 1997) knockout mice compared with wild-type mice; 4) the conduction velocity in Cx43 mutant gene-modified NRVM was more than 3-fold slower relative to controls, indicating the central role of Cx43 in electrical impulse conduction (Kizana et al., 2007). However, in contrast with these findings, it should be noted that Thomas et al. (2003) showed that a 50% reduction in Cx43 levels did not result in a reduced conduction velocity. "
[Show abstract][Hide abstract] ABSTRACT: Our recent studies demonstrated that propargylamine derivatives such as rasagiline (Azilect, Food and Drug Administration-approved anti-Parkinson drug) and its S-isomer TVP1022 protect cardiac and neuronal cell cultures against apoptotic-inducing stimuli. Studies on structure-activity relationship revealed that their neuroprotective effect is associated with the propargylamine moiety, which protects mitochondrial viability and prevents apoptosis by activating Bcl-2 and protein kinase C-epsilon and by down-regulating the proapoptotic protein Bax. Based on the established cytoprotective and neuroprotective efficacies of propargylamine derivatives, as well as on our recent study showing that TVP1022 attenuates serum starvation-induced and doxorubicin-induced apoptosis in neonatal rat ventricular myocytes (NRVMs), we tested the hypothesis that TVP1022 will also provide protection against doxorubicin-induced NRVM functional derangements. The present study demonstrates that pretreatment of NRVMs with TVP1022 (1 microM, 24 h) prevented doxorubicin (0.5 microM, 24 h)-induced elevation of diastolic [Ca(2+)](i), the slowing of [Ca(2+)](i) relaxation kinetics, and the decrease in the rates of myocyte contraction and relaxation. Furthermore, pretreatment with TVP1022 attenuated the doxorubicin-induced reduction in the protein expression of sarco/endoplasmic reticulum calcium (Ca(2+)) ATPase, Na(+)/Ca(2+) exchanger 1, and total connexin 43. Finally, TVP1022 diminished the inhibitory effect of doxorubicin on gap junctional intercellular coupling (measured by means of Lucifer yellow transfer) and on conduction velocity, the amplitude of the activation phase, and the maximal rate of activation (dv/dt(max)) measured by the Micro-Electrode-Array system. In summary, our results indicate that TVP1022 acts as a novel cardioprotective agent against anthracycline cardiotoxicity, and therefore potentially can be coadmhence, theinistered with doxorubicin in the treatment of malignancies in humans.
Journal of Pharmacology and Experimental Therapeutics 11/2009; 332(2):413-20. DOI:10.1124/jpet.109.161158 · 3.97 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We have previously shown that left atrial-pulmonary vein tissue (LA-PV) can generate reentrant arrhythmias (atrial fibrillation, AF) in wild-type (mXinα+/+) but not in mXinα-null (mXinα-/-) mice. With the present experiments, we investigated the arrhythmogenic activity and the underlying mechanisms in mXinα+/+ vs. mXinα-/- LA-PV. Electrical activity and conduction velocity (CV) were recorded in LA-PV by means of a MED64 system. CV was significantly faster in mXinα+/+ than in mXinα-/- LA-PV and it was increased by 1 μM isoproterenol (ISO). AF could be induced by fast pacing in the mXinα+/+ but not in mXinα-/- LA-PV where automatic rhythms could occur. ISO increased the incidence of AF in Xinα+/+ whereas it increased that of automatic rhythms in mXinα-/- LA-PV. In LA-PV with the right atrium attached (RA-LA-PV), automatic rhythms occurred in all preparations. In mXinα+/+ RA-LA-PV simultaneously treated with ISO, strophanthidin and atropine, the incidence of the automatic rhythm was about the same, but AF increased significantly. In contrast, in mXinα-/- RA-LA-PV under the same condition, the automatic rhythm was markedly enhanced, but still no AF occurred. Conventional microelectrode techniques showed a longer APD90 and a less negative maximum diastolic potential (MDP) in mXinα-/- than mXinα+/+ LA-PV tissues. Whole-cell current clamp experiments also showed a less negative MDP in mXinα-/- vs. mXinα+/+ LA-PV cardiomyocytes. The fact that AF could be induced by fast pacing under several conditions in mXinα+/+ but not in mXinα-/- LA-PV preparations appears to be due to a slower CV, a prolonged APD90, a less negative MDP and possibly larger areas of conduction block in mXinα-/- myocardial cells. In contrast, the non-impairment of automatic and triggered rhythms in mXinα-/- preparations may be due to the fact that the mechanisms underlying these rhythms do not involve cell-to-cell conduction.
Life Sciences 08/2008; 83(7-8):272-83. DOI:10.1016/j.lfs.2008.06.020 · 2.70 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Left ventricular hypertrophy induces remodeling for various ion channels. We modified a model of electrical activity of rat ventricular cell by incorporating available experimental data on the kinetics and conductances of various currents. We simulated hypertrophy by incorporating experimental data of changes in channel kinetics, cell size and Ca<sup>2+</sup> handling. The simulated action potentials (AP) and calcium transients consistent with experimental data. We developed virtual ventricular strands to investigate the rate dependent propagation in homogenous tissue under normal and hypertrophic conditions. A continuous increase in APD and corresponding decrease in conduction velocity (CV) with subsequent beats was observed, resulting in irregular conduction block at low values of stimulus intervals (SI), for which the simulated action potential duration (APD) restitution of the cell models has negative slope.
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