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Purkinje fibers play an essential role in transmitting electrical impulses through the heart, but they may also serve as triggers for arrhythmias linked to defective intracellular calcium (Ca(2+)) regulation. Although prior studies have extensively characterized spontaneous Ca(2+) release in nondriven Purkinje cells, little attention has been paid to rate-dependent changes in Ca(2+) transients. Therefore we explored the behaviors of Ca(2+) transients at pacing rates ranging from 0.125 to 3 Hz in single canine Purkinje cells loaded with fluo3 and imaged with a confocal microscope. The experiments uncovered the following novel aspects of Ca(2+) regulation in Purkinje cells: 1) the cells exhibit a negative Ca(2+)-frequency relationship (at 2.5 Hz, Ca(2+) transient amplitude was 66 ± 6% smaller than that at 0.125 Hz); 2) sarcoplasmic reticulum (SR) Ca(2+) release occurs as a propagating wave at very low rates but is localized near the cell membrane at higher rates; 3) SR Ca(2+) load declines modestly (10 ± 5%) with an increase in pacing rate from 0.125 Hz to 2.5 Hz; 4) Ca(2+) transients show considerable beat-to-beat variability, with greater variability occurring at higher pacing rates. Analysis of beat-to-beat variability suggests that it can be accounted for by stochastic triggering of local Ca(2+) release events. Consistent with this hypothesis, an increase in triggering probability caused a decrease in the relative variability. These results offer new insight into how Ca(2+) release is normally regulated in Purkinje cells and provide clues regarding how disruptions in this regulation may lead to deleterious consequences such as arrhythmias., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Cardiac Purkinje fibers, due to their unique anatomical location, cell structure and electrophysiologic characteristics, play an important role in cardiac conduction and arrhythmogenesis. Purkinje cell action potentials are longer than their ventricular counterpart, and display two levels of resting potential. Purkinje cells provide for rapid propagation of the cardiac impulse to ventricular cells and have pacemaker and triggered activity, which differs from ventricular cells. Additionally, a unique intracellular Ca2+ release coordination has been revealed recently for the normal Purkinje cell. However, since the isolation of single Purkinje cells is difficult, particularly in small animals, research using Purkinje cells has been restricted. This review concentrates on comparison of Purkinje and ventricular cells in the morphology of the action potential, ionic channel function and molecular determinants by summarizing our present day knowledge of Purkinje cells.

Ca(2+)/calmodulin-dependent protein kinase II is a multifunctional serine/threonine kinase with diverse cardiac roles including regulation of excitation contraction, transcription, and apoptosis. Dynamic regulation of CaMKII activity occurs in cardiac disease and is linked to specific disease phenotypes through its effects on ion channels, transporters, transcription and cell death pathways. Recent mathematical models of the cardiomyocyte have incorporated limited elements of CaMKII signaling to advance our understanding of how CaMKII regulates cardiac contractility and excitability. Given the importance of CaMKII in cardiac disease, it is imperative that computer models evolve to capture the dynamic range of CaMKII activity. In this study, using mathematical modeling combined with biochemical and imaging techniques, we test the hypothesis that CaMKII signaling in the canine infarct border zone (BZ) contributes to impaired calcium homeostasis and electrical remodeling. We report that the level of CaMKII autophosphorylation is significantly increased in the BZ region. Computer simulations using an updated mathematical model of CaMKII signaling reproduce abnormal Ca(2+) transients and action potentials characteristic of the BZ. Our simulations show that CaMKII hyperactivity contributes to abnormal Ca(2+) homeostasis and reduced action potential upstroke velocity due to effects on I(Na) gating kinetics. In conclusion, we present a new mathematical tool for studying effects of CaMKII signaling on cardiac excitability and contractility over a dynamic range of kinase activities. Our experimental and theoretical findings establish abnormal CaMKII signaling as an important component of remodeling in the canine BZ.

The purpose of this study was to determine whether IP(3)Rs contribute to the generation of wide long lasting perinuclear Ca(2+) release events in canine Purkinje cells. Spontaneous Ca(2+) release events (elevations of basal [Ca(2+)] equivalent to F/F(0) 3.4SD over F(0)) were imaged using Fluo-4AM and 2D confocal microscope. Only cells free of Ca(2+) waves were analyzed. Subsarcolemmal region (SSL) was defined as 5 microm from cell edges. Core was the remaining cell. The majority of events (94%, 0.0035+/-0.0007 events (ev)/microm(2)/s, N=34 cells) were detected within a single frame (typical events, TE). However, a subpopulation (6.0%, 0.00022+/-0.00005 ev/microm(2)/s, N=41 cells: wide long lasting events, WLE) lasted for several frames, showed a greater spatial extent (51.0+/-3.9 vs. TE 9.0+/-0.3 microm(2), P<0.01) and higher amplitude (F/F(0) 1.38+/-0.02 vs. TE 1.20+/-0.003, P<0.01). WLE event rate was increased by phenylephrine (10 microM, P<0.01), inhibited by 2APB and U73122 (P<0.05), and abolished by tetracaine (1 mM) and ryanodine (100 microM). While SSL WLEs were scattered randomly, Core WLEs (n=69 events) were predominantly distributed longitudinally 18.2+/-1.6 microm from the center of nuclei. Immunocytochemistry showed that IP(3)R1s were located not only at SSL region but also near both ends of nucleus overlapping with RyRs. In Purkinje cells, wide long lasting Ca(2+) release events occur in SSL and in specific perinuclear regions. They are likely due to RyRs and IP(3)R1s evoked Ca(2+) release and may play a role in Ca(2+) dependent nuclear processes.

Cardiac calcium channels play a pivotal role in the proper functioning of cardiac cells. In response to various pathologic stimuli, they become remodeled, changing how they function, as they adapt to their new environment. Specific features of remodeled channels depend upon the particular disease state. This review will summarize what is known about remodeled cardiac calcium channels in three disease states: hypertrophy, heart failure and atrial fibrillation. In addition, it will review the recent advances made in our understanding of the function of the various molecular building blocks that contribute to the proper functioning of the cardiac calcium channel.

Studies from our laboratory have defined alterations in Ca(i)handling in the non-dialyzed subepicardial cells that have survived in the 5 day infarcted heart (IZs). To determine whether changes in the action potential profile contributed to the observed Ca(i)changes we have used a combined voltage clamp/epifluorescent technique to determine and compare changes in fura 2 ratios in IZs compared to those of epicardial cells from the non-infarcted canine hearts (NZs). We found that Ca(i)changes in voltage clamped IZs persisted. In NZs, Ca(i)transients showed the expected voltage dependence while IZs did not. To determine whether altered NaCa exchanger activity contributed to the observed changes in Ca(i)in IZs, we measured NaCa exchanger Ca(2+)fluxes (reverse and forward mode) and ionic currents in both cell types and under different Na(i)loads (10 and 20 m m). We found that there were no significant differences in resting, peak or magnitude of fura 2 ratio changes or in outward current densities between NZs and IZs even under the different Na(i)loads. Thus, we suggest that chronic up- or downregulation of the NaCa exchanger protein does not underlie observed Ca(i)changes in IZs. Additionally, Ca(2+)released with paced voltage steps represented 79% of that released by caffeine in NZs while, in IZs, caffeine releasable Ca(2+)was equivalent to that released with step depolarization. Thus, abnormalities in Ca(i)handling in IZs appear not to arise secondarily to changes in action potential configuration nor do they appear to be due to disease-induced alteations in NaCa exchanger function., (Copyright 2000 Academic Press.)

Previous studies have shown that the muscle fibers that survive on the epicardial surface of a 5- or 14-day-old infarct in canine hearts have reduced or absent plateau phase during repolarization. Since the absence of a plateau phase could be related to a decrease in the slow inward current, we determined whether norepinephrine (NE) (3.13 x 10(-5) M) or caffeine (2.5 to 5.0 mM) restored the reduced plateau phase of action potentials of fibers from the 5- to 14-day infarct (4 mM K+). In addition, we compared the slow inward current-mediated slow response action potentials induced by NE or caffeine in K+ depolarized fibers from the 5- and 14-day infarcts to those induced in control fibers. Our results show that NE and caffeine had little or no significant effects on repolarization or the plateau area of action potentials of fibers from infarcted preparations. In addition, NE and caffeine both induced slow response action potentials in normal fibers, but in some fibers from infarcts, these agents failed to elicit slow responses. In other fibers from infarcts, NE and caffeine induced slow response action potentials but they had reduced mean areas when compared to the control potentials. In conclusion, the alteration and absence of slow responses in fibers from infarcted preparations suggest that there may be a chronic abnormality in the slow Ca current channel in fibers from the 5- and 14-day infarct.