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Myocyte Cultures (myocyte + culture)
Selected AbstractsSimultaneous Optical Mapping of Transmembrane Potential and Intracellular Calcium in Myocyte CulturesJOURNAL OF CARDIOVASCULAR ELECTROPHYSIOLOGY, Issue 5 2000VLADIMIR G. FAST Ph.D. Simultaneous Mapping of Vm and Cai2+. Introduction: Fast spatially resolved measurements of transmembrane potential (Vm) and intracellular calcium (Cai2+) are important for studying mechanisms of arrhythmias and defibrillation. The goals of this work were (1) to develop an optical technique for simultaneous multisite optical recordings of Vm and Cai2+, and (2) to determine the relationship between Vm and Cai2+ during normal impulse propagation in myocyte cultures. Methods and Results: Monolayers of neonatal rat myocytes were stained with fluorescent dye RH-237 (Vm) and Fluo-3AM (Cai2+). Both dyes were excited at the same wavelength range. The emitted fluorescent was optically separated into components corresponding to changes in Vm, and Cai2+ and measured using two 16 × 16 photodiode arrays at a spatial resolution of up to 27.5 ,m per diode and sampling rate of 2.5 kHz. The optical setup was adjusted so that there was no optical cross-talk between the two types of measurements, which was validated in experiments involving staining with either RH-237 or Fluo-3. The amplitude of Fluo-3 signals rapidly decreased during experiments due to dye leakage. Dye leakage was substantially reduced by application of 1 mM probenecid, a blocker of organic anion transport, which had no effect on action potential duration and only minor effect on conduction velocity. In double-stained preparations, during regular pacing Cai2+ transients had a rise time of 14.2 ± 2 msec, and they followed Vm upstrokes with a delay of 5.3 ± 1 msec (n = 9). Durations of Vm, and Cai2+ transients determined at 50% level of signal recovery were 54.6 ± 10 msec and 136 ± 8 msec, respectively. Application of 2 ,M nifedipine reduced the amplitude and duration of Cai2+ transients without significantly affecting conduction velocity. Conclusion: The results demonstrate feasibility of simultaneous optical recordings of Vm and Cai2+ transients with high spatial and temporal resolution. [source] In vitro characterization of HCN channel kinetics and frequency dependence in myocytes predicts biological pacemaker functionalityTHE JOURNAL OF PHYSIOLOGY, Issue 7 2009Xin Zhao The pacemaker current, mediated by hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, contributes to the initiation and regulation of cardiac rhythm. Previous experiments creating HCN-based biological pacemakers in vivo found that an engineered HCN2/HCN1 chimeric channel (HCN212) resulted in significantly faster rates than HCN2, interrupted by 1,5 s pauses. To elucidate the mechanisms underlying the differences in HCN212 and HCN2 in vivo functionality as biological pacemakers, we studied newborn rat ventricular myocytes over-expressing either HCN2 or HCN212 channels. The HCN2- and HCN212-over-expressing myocytes manifest similar voltage dependence, current density and sensitivity to saturating cAMP concentrations, but HCN212 has faster activation/deactivation kinetics. Compared with HCN2, myocytes expressing HCN212 exhibit a faster spontaneous rate and greater incidence of irregular rhythms (i.e. periods of rapid spontaneous rate followed by pauses). To explore these rhythm differences further, we imposed consecutive pacing and found that activation kinetics of the two channels are slower at faster pacing frequencies. As a result, time-dependent HCN current flowing during diastole decreases for both constructs during a train of stimuli at a rapid frequency, with the effect more pronounced for HCN2. In addition, the slower deactivation kinetics of HCN2 contributes to more pronounced instantaneous current at a slower frequency. As a result of the frequency dependence of both instantaneous and time-dependent current, HCN2 exhibits more robust negative feedback than HCN212, contributing to the maintenance of a stable pacing rhythm. These results illustrate the benefit of screening HCN constructs in spontaneously active myocyte cultures and may provide the basis for future optimization of HCN-based biological pacemakers. [source] Mechanical load induced by glass microspheres releases angiogenic factors from neonatal rat ventricular myocytes cultures and causes arrhythmiasJOURNAL OF CELLULAR AND MOLECULAR MEDICINE, Issue 5b 2008D. Y. Barac Abstract In the present study, we tested the hypothesis that similar to other mechanical loads, notably cyclic stretch (simulating pre-load), glass microspheres simulating afterload will stimulate the secretion of angiogenic factors. Hence, we employed glass microspheres (average diameter 15.7 ,m, average mass 5.2 ng) as a new method for imposing mechanical load on neonatal rat ventricular myocytes (NRVM) in culture. The collagen-coated microspheres were spread over the cultures at an estimated density of 3000 microspheres/mm2, they adhered strongly to the myocytes, and acted as small weights carried by the cells during their contraction. NRVM were exposed to either glass microspheres or to cyclic stretch, and several key angiogenic factors were measured by RT-PCR. The major findings were: (1) In contrast to other mechanical loads, such as cyclic stretch, microspheres (at 24 hrs) did not cause hypertrophy. (2) Further, in contrast to cyclic stretch, glass microspheres did not affect Cx43 expression, or the conduction velocity measured by means of the Micro-Electrode-Array system. (3) At 24 hrs, glass microspheres caused arrhythmias, probably resulting from early afterdepolarizations. (4) Glass microspheres caused the release of angiogenic factors as indicated by an increase in mRNA levels of vascular endothelial growth factor (80%), angiopoietin-2 (60%), transforming growth factor-, (40%) and basic fibroblast growth factor (15%); these effects were comparable to those of cyclic stretch. (5) As compared with control cultures, conditioned media from cultures exposed to microspheres increased endothelial cell migration by 15% (P<0.05) and endothelial cell tube formation by 120% (P<0.05), both common assays for angiogenesis. In conclusion, based on these findings we propose that loading cardiomyocytes with glass microspheres may serve as a new in vitro model for investigating the role of mechanical forces in angiogenesis and arrhythmias. [source] | ||||||||||