Enhanced Ca(2+) release and Na/Ca exchange activity in hypertrophied canine ventricular myocytes: potential link between contractile adaptation and arrhythmogenesis

BACKGROUND: Ventricular arrhythmias are a major cause of sudden death in patients with heart failure and hypertrophy. The dog with chronic complete atrioventricular block (CAVB) has biventricular hypertrophy and ventricular arrhythmias and is a useful model to study underlying cellular mechanisms. We investigated whether changes in Ca(2+) homeostasis are part of the contractile adaptation to CAVB and might contribute to arrhythmogenesis. METHODS AND RESUTLS: In enzymatically isolated myocytes, cell shortening, Ca(2+) release from the sarcoplasmic reticulum (SR), and SR Ca(2+) content were enhanced at low stimulation frequencies. Ca(2+) influx through L-type Ca(2+) channels was unchanged, but Ca(2+) influx via the Na/Ca exchanger was increased and contributed to Ca(2+) loading of the SR. Inward Na/Ca exchange currents were also larger. Changes in Ca(2+) fluxes were less pronounced in the right versus left ventricle. CONCLUSIONS: Enhanced Na/Ca exchange activity may improve contractile adaptation to CAVB but at the same time facilitate arrhythmias by (1) increasing the propensity to Ca(2+) overload, (2) providing more inward current leading to (nonhomogeneous) action potential prolongation, and (3) enhancing (arrhythmogenic) currents during spontaneous Ca(2+) release.     

Ca(2+) transients and Ca(2+) waves in purkinje cells : role in action potential initiation

Purkinje cells contain sarcoplasmic reticulum (SR) directly under the surface membrane, are devoid of t-tubuli, and are packed with myofibrils surrounded by central SR. Several studies have reported that electrical excitation induces a biphasic Ca(2+) transient in Purkinje fiber bundles. We determined the nature of the biphasic Ca(2+) transient in aggregates of Purkinje cells. Aggregates (n=12) were dispersed from the subendocardial Purkinje fiber network of normal canine left ventricle, loaded with Fluo-3/AM, and studied in normal Tyrode's solution (24 degrees C). Membrane action potentials were recorded with fine-tipped microelectrodes, and spatial and temporal changes in [Ca(2+)](i) were obtained from fluorescent images with an epifluorescent microscope (x20; Nikon). Electrical stimulation elicited an action potential as well as a sudden increase in fluorescence (L(0)) compared with resting levels. This was followed by a further increase in fluorescence (L(1)) along the edges of the cells. Fluorescence then progressed toward the Purkinje cell core (velocity of propagation 180 to 313 microm/s). In 62% of the aggregates, initial fluorescent changes of L(0) were followed by focally arising Ca(2+) waves (L(2)), which propagated at 158+/-14 microm/s (n=13). Spontaneous Ca(2+) waves (L(2)*) propagated like L(2) (164+/-10 microm/s) occurred between stimuli and caused slow membrane depolarization; 28% of L(2)* elicited action potentials. Both spontaneous Ca(2+) wave propagation and resulting membrane depolarization were thapsigargin sensitive. Early afterdepolarizations were not accompanied by Ca(2+) waves. Action potentials in Purkinje aggregates induced a rapid rise of Ca(2+) through I(CaL) and release from a subsarcolemmal compartment (L(0)). Ca(2+) release during L(0) either induced further Ca(2+) release, which propagated toward the cell core (L(1)), or initiated Ca(2+) release from small regions and caused L(2) Ca(2+) waves, which propagated throughout the aggregate. Spontaneous Ca(2+) waves (L(2)*) induce action potentials.     

Modulation of Ca(2+) signaling by microtubule disruption in rat ventricular myocytes and its dependence on the ruptured patch-clamp configuration

In the absence of hypertrophic proliferation of microtubules, microtubule disruption by colchicine does not modulate contraction of adult cardiac myocytes. However, Gomez et al (Circ Res. 2000;86:30-36) recently reported that disruption of microtubules by colchicine in ruptured patch-clamped myocytes increased I(Ca,L) density and [Ca(2+)](i) transient amplitude and depressed the response of these parameters to the beta-adrenoceptor agonist isoproterenol. These effects were ascribed to stimulation of adenylyl cyclase by increased intracellular free tubulin. In the present study, we show that in intact rat ventricular myocytes, 2 to 4 hours of exposure to 10 micromol/L colchicine had no effect on shortening or [Ca(2+)](i) transient amplitude or on the amplitude of I(Ca,L) in perforated patch-clamped cells, under basal conditions and after stimulation with 1 micromol/L isoproterenol. However, in ruptured patch-clamped myocytes, basal I(Ca,L) was 2-fold higher after treatment with colchicine compared with vehicle and, in contrast to vehicle-treated cells, I(Ca,L) did not increase in response to isoproterenol. Cell width decreased during ruptured patch-clamp experiments in colchicine-treated but not vehicle-treated myocytes. We conclude that in cells with intact sarcolemma, colchicine does not modulate Ca(2+) signaling or the response to beta stimulation. However, the combination of microtubule disruption by colchicine and the ruptured patch configuration activates I(Ca,L) and attenuates the response to beta stimulation. We propose that these effects may be due to loss of free tubulin by intracellular dialysis or to increased sensitivity to mechanical stimulation as a result of microtubule disruption. These findings have important implications for cardiomyopathies associated with decreased free tubulin or a diminished microtubular network. The full text of this article is available at http://www.circresaha.org.     

Spatiotemporal characteristics of SR Ca(2+) uptake and release in detubulated rat ventricular myocytes

In cardiac ventricular myocytes, sarcoplasmic reticulum (SR) Ca(2+) load is a key determinant of SR Ca(2+) release. This release normally occurs predominantly from SR junctions at sarcolemmal invaginations (t-tubules), ensuring synchronous SR Ca(2+) release throughout the cell. However under conditions of Ca(2+) overload, spontaneous SR Ca(2+) release and propagating Ca(2+) waves can occur, which are pro-arrhythmic. We used detubulated rat ventricular myocytes to determine the dependence of Ca(2+) wave propagation on SR Ca(2+) load, and the role of t-tubules in SR Ca(2+) uptake and spontaneous release. After SR Ca(2+) depletion, recovery of Ca(2+) transient amplitude (and SR Ca(2+) load) was slower in detubulated than control myocytes (half-maximal recovery: 9.9+/-1.4 vs. 5.5+/-0.7 beats). In detubulated myocytes the extent and velocity of Ca(2+) propagation from the cell periphery increased with each beat and depended steeply on SR Ca(2+) load. Isoproterenol (ISO) accelerated recovery, increased maximal propagation velocity and reduced the threshold SR Ca(2+) load for propagation. Ca(2+) spark frequency was uniform across control cell width and was similar at the periphery of detubulated cells. However, internal Ca(2+) spark frequency in detubulated cells was 75% lower (despite comparable local SR Ca(2+) load); this transverse spark frequency profile was similar to that in atrial myocytes. We conclude that: (1) t-tubule Ca(2+) fluxes normally control SR Ca(2+) refilling; (2) Ca(2+) wave propagation depends steeply on SR Ca(2+) content (3) SR-t-tubule junctions are important in initiating SR Ca(2+) release and (4) ISO enhances propagation of SR Ca release, but not the initiation of SR Ca release events (for given SR Ca(2+) loads).     

Enhanced Ca(2+)-activated Na(+)-Ca(2+) exchange activity in canine pacing-induced heart failure

Defective excitation-contraction coupling in heart failure is generally associated with both a reduction in sarcoplasmic reticulum (SR) Ca(2+) uptake and a greater dependence on transsarcolemmal Na(+)-Ca(2+) exchange (NCX) for Ca(2+) removal. Although a relative increase in NCX is expected when SR function is impaired, few and contradictory studies have addressed whether there is an absolute increase in NCX activity. The present study examines in detail NCX density and function in left ventricular midmyocardial myocytes isolated from normal or tachycardic pacing-induced failing canine hearts. No change of NCX current density was evident in myocytes from failing hearts when intracellular Ca(2+) ([Ca(2+)](i)) was buffered to 200 nmol/L. However, when [Ca(2+)](i) was minimally buffered with 50 micromol/L indo-1, Ca(2+) extrusion via NCX during caffeine application was doubled in failing versus normal cells. In other voltage-clamp experiments in which SR uptake was blocked with thapsigargin, both reverse-mode and forward-mode NCX currents and Ca(2+) transport were increased >2-fold in failing cells. These results suggest that, in addition to a relative increase in NCX function as a consequence of defective SR Ca(2+) uptake, there is an absolute increase in NCX function that depends on [Ca(2+)](i) in the failing heart.     

Coordinated control of cell Ca(2+) loading and triggered release from the sarcoplasmic reticulum underlies the rapid inotropic response to increased L-type Ca(2+) current

The aim of this study was to investigate how sarcoplasmic reticulum (SR) Ca(2+) content and systolic Ca(2+) are controlled when Ca(2+) entry into the cell is varied. Experiments were performed on voltage-clamped rat and ferret ventricular myocytes loaded with fluo-3 to measure intracellular Ca(2+) concentration ([Ca(2+)](i)). Increasing external Ca(2+) concentration ([Ca(2+)](o)) from 1 to 2 mmol/L increased the amplitude of the systolic Ca(2+) transient with no effect on SR Ca(2+) content. This constancy of SR content is shown to result because the larger Ca(2+) transient activates a larger Ca(2+) efflux from the cell that balances the increased influx. Decreasing [Ca(2+)](o) to 0.2 mmol/L decreased systolic Ca(2+) but produced a small increase of SR Ca(2+) content. This increase of SR Ca(2+) content is due to a decreased release of Ca(2+) from the SR resulting in decreased loss of Ca(2+) from the cell. An increase of [Ca(2+)](o) has two effects: (1) increasing the fraction of SR Ca(2+) content, which is released on depolarization and (2) increasing Ca(2+) entry into the cell. The results of this study show that the combination of these effects results in rapid changes in the amplitude of the systolic Ca(2+) transient. In support of this, the changes of amplitude of the transient occur more quickly following changes of [Ca(2+)](o) than following refilling of the SR after depletion with caffeine. We conclude that the coordinated control of increased Ca(2+) entry and greater fractional release of Ca(2+) is an important factor in regulating excitation-contraction coupling.     

Effects of cytosolic ATP on Ca(2+) sparks and SR Ca(2+) content in permeabilized cardiac myocytes

Confocal imaging was used to study the influence of cytosolic ATP on the properties of spontaneous Ca(2+) sparks in permeabilized ventricular myocytes. Cells were perfused with mock intracellular solutions containing fluo 3. Reducing [ATP] to <0.5 mmol/L decreased the frequency but increased the amplitude of spontaneous Ca(2+) sparks. In the presence of 20 micromol/L ATP, the amplitude increased by 48.7+/-10.9%, and the frequency decreased by 77.07+/-3.8%, relative to control responses obtained at 5 mmol/L ATP. After exposure to a solution containing zero ATP, the frequency of Ca(2+) sparks decreased progressively and approached zero within 90 seconds. As ATP washed out of the cell, the sarcoplasmic reticulum (SR) Ca(2+) content increased, until reaching a maximum after 3 minutes. Subsequent introduction of adenylyl imidodiphosphate precipitated a burst of large-amplitude Ca(2+) sparks. This was accompanied by a rapid decrease in SR Ca(2+) content to 80% to 90% of the steady-state value obtained in the presence of 5 mmol/L ATP. Thereafter, the SR Ca(2+) content declined much more slowly over 5 to 10 minutes. The effects of ATP withdrawal on Ca(2+) sparks may reflect reduced occupancy of the adenine nucleotide site on the SR Ca(2+) channel. These effects may contribute to previously reported changes in SR function during myocardial ischemia and reperfusion, in which ATP depletion and Ca(2+) overload occur.     

Ca(2+) signaling in cardiac myocytes overexpressing the alpha(1) subunit of L-type Ca(2+) channel

Voltage-gated L-type Ca(2+) channels (LCCs) provide Ca(2+) ingress into cardiac myocytes and play a key role in intracellular Ca(2+) homeostasis and excitation-contraction coupling. We investigated the effects of a constitutive increase of LCC density on Ca(2+) signaling in ventricular myocytes from 4-month-old transgenic (Tg) mice overexpressing the alpha(1) subunit of LCC in the heart. At this age, cells were somewhat hypertrophic as reflected by a 20% increase in cell capacitance relative to those from nontransgenic (Ntg) littermates. Whole cell I(Ca) density in Tg myocytes was elevated by 48% at 0 mV compared with the Ntg group. Single-channel analysis detected an increase in LCC density with similar conductance and gating properties. Although the overexpressed LCCs triggered an augmented SR Ca(2+) release, the "gain" function of EC coupling was uncompromised, and SR Ca(2+) content, diastolic cytosolic Ca(2+), and unitary properties of Ca(2+) sparks were unchanged. Importantly, the enhanced I(Ca) entry and SR Ca(2+) release were associated with an upregulation of the Na(+)-Ca(2+) exchange activity (indexed by the half decay time of caffeine-elicited Ca(2+) transient) by 27% and SR Ca(2+) recycling by approximately 35%. Western analysis detected a 53% increase in the Na(+)-Ca(2+) exchanger expression but no change in the abundance of ryanodine receptor (RyR), SERCA2, and phospholamban. Analysis of I(Ca) kinetics suggested that SR Ca(2+) release-dependent inactivation of LCCs remains intact in Tg cells. Thus, in spite of the modest cardiac hypertrophy, the overexpressed LCCs form functional coupling with RyRs, preserving both orthograde and retrograde Ca(2+) signaling between LCCs and RyRs. These results also suggest that a modest but sustained increase in Ca(2+) influx triggers a coordinated remodeling of Ca(2+) handling to maintain Ca(2+) homeostasis.     

Reduction of Ca(2+) channel activity by hypoxia in human and porcine coronary myocytes.

OBJECTIVE: Oxygen (O(2)) tension is a major regulator of blood flow in the coronary circulation. Hypoxia can produce vasodilation through activation of ATP regulated K(+) (K(ATP)) channels in the myocyte membrane, which leads to hyperpolarization and closure of voltage-gated Ca(2+) channels. However, there are other O(2)-sensitive mechanisms intrinsic to the vascular smooth muscle since hypoxia can relax vessels precontracted with high extracellular K(+), a condition that prevents hyperpolarization following opening of K(+) channels. The objective of the present study was to determine whether inhibition of Ca(2+) influx through voltage-dependent channels participates in the response of coronary myocytes to hypoxia. METHODS: Experiments were performed on porcine anterior descendent coronary arterial rings and on enzymatically dispersed human and porcine myocytes of the same artery. Cytosolic [Ca(2+)] was measured by microfluorimetry and whole-cell currents were recorded with the patch clamp technique. RESULTS: Hypoxia (O(2) tension approximately 20 mmHg) dilated endothelium-denuded porcine coronary arterial rings precontracted with high K(+) in the presence of glibenclamide (5 microM), a blocker of K(ATP) channels. In dispersed human and porcine myocytes, low O(2) tension decreased basal cytosolic [Ca(2+)] and transmembrane Ca(2+) influx independently of K(+) channel activation. In patch clamped cells, hypoxia reversibly inhibited L-type Ca(2+) channels. RT-PCR indicated that rHT is the predominant mRNA variant of the alpha(1C) Ca(2+) channel subunit in human coronary myocytes. CONCLUSION: Our study demonstrates, for the first time in a human preparation, that voltage-gated Ca(2+)channels in coronary myocytes are under control of O(2) tension.     

Genome-wide RNAi screen of Ca(2+) influx identifies genes that regulate Ca(2+) release-activated Ca(2+) channel activity

Recent studies by our group and others demonstrated a required and conserved role of Stim in store-operated Ca(2+) influx and Ca(2+) release-activated Ca(2+) (CRAC) channel activity. By using an unbiased genome-wide RNA interference screen in Drosophila S2 cells, we now identify 75 hits that strongly inhibited Ca(2+) influx upon store emptying by thapsigargin. Among these hits are 11 predicted transmembrane proteins, including Stim, and one, olf186-F, that upon RNA interference-mediated knockdown exhibited a profound reduction of thapsigargin-evoked Ca(2+) entry and CRAC current, and upon overexpression a 3-fold augmentation of CRAC current. CRAC currents were further increased to 8-fold higher than control and developed more rapidly when olf186-F was cotransfected with Stim. olf186-F is a member of a highly conserved family of four-transmembrane spanning proteins with homologs from Caenorhabditis elegans to human. The endoplasmic reticulum (ER) Ca(2+) pump sarco-/ER calcium ATPase (SERCA) and the single transmembrane-soluble N-ethylmaleimide-sensitive (NSF) attachment receptor (SNARE) protein Syntaxin5 also were required for CRAC channel activity, consistent with a signaling pathway in which Stim senses Ca(2+) depletion within the ER, translocates to the plasma membrane, and interacts with olf186-F to trigger CRAC channel activity.     

Effects of Na(+)/Ca(2+)-exchanger overexpression on excitation-contraction coupling in adult rabbit ventricular myocytes

The Na(+)/Ca(2+)-exchanger (NCX) is the main mechanism by which Ca(2+) is transported out of the ventricular myocyte. NCX levels are raised in failing human heart, and the consequences of this for excitation-contraction coupling are still debated. We have increased NCX levels in adult rabbit myocytes by adenovirally-mediated gene transfer and examined the effects on excitation-contraction coupling after 24 and 48 h. Infected myocytes were identified through expression of green fluorescent protein (GFP), transfected under a separate promoter on the same viral construct. Control experiments were done with both non-infected myocytes and those infected with adenovirus expressing GFP only. Contraction amplitude was markedly reduced in NCX-overexpressing myocytes at either time point, and neither increasing frequency nor raising extracellular Ca(2+) could reverse this depression. Resting membrane potential and action potential duration were largely unaffected by NCX overexpression, as was peak Ca(2+) entry via the L-type Ca(2+) channel. Systolic and diastolic Ca(2+) levels were significantly reduced, with peak systolic Ca(2+) in NCX-overexpressing myocytes lower than diastolic levels in control cells at 2 m m extracellular Ca(2+). Both cell relengthening and the decay of the Ca(2+) transient were significantly slowed. Sarcoplasmic reticulum (SR) Ca(2+) stores were completely depleted in a majority of myocytes, and remained so despite increasingly vigorous loading protocols. Depressed contractility following NCX overexpression is therefore related to decreased SR Ca(2+) stores and low diastolic Ca(2+) levels rather than reduced Ca(2+) entry.     

Relative abundance of alpha2 Na(+) pump isoform influences Na(+)-Ca(2+) exchanger currents and Ca(2+) transients in mouse ventricular myocytes

In the mouse, genetic reduction in the Na(+), K(+)-ATPase alpha1 or alpha2 isoforms results in different functional phenotypes: heterozygous alpha2 isolated hearts are hypercontractile, whereas heterozygous alpha1 hearts are hypocontractile. We examined Na(+)/Ca(2+) exchange (NCX) currents in voltage clamped myocytes (pipette [Na(+)]=15 mM) induced by abrupt removal of extracellular Na(+). In wild-type (WT) myocytes, peak exchanger currents were 0.59+/-0.04 pA/pF (mean+/-S.E.M., n=10). In alpha1(+/-) myocytes (alpha2 isoform increased by 54%), NCX current was reduced to 0.33+/-0.05 (n=9, P<0.001) indicating a lower subsarcolemmal [Na(+)]. In alpha2(+/-) myocytes (alpha2 isoform reduced by 54%), the NCX current was increased to 0.89+/-0.11 (n=8, P=0.03). The peak sarcolemmal Na(+) pump currents activated by abrupt increase in [K(+)](o) to 4 mM in voltage clamped myocytes in which the Na(+) pump had been completely inhibited for 5 min by exposure to 0 [K(+)](o) were similar in alpha1(+/-) (0.86+/-0.12, n=10) and alpha2(+/-) myocytes (0.94+/-0.08 pA/pF, n=16), and were slightly but insignificantly reduced relative to WT (1.03+/-0.05, n=24). The fluo-3 [Ca(2+)](i) transient (F/F(o)) in WT myocytes paced at 0.5 Hz was 2.18+/-0.09, n=34, was increased in alpha2(+/-) myocytes (F/F(o)=2.56+/-0.14, n=24, P=0.02), and was decreased in alpha1(+/-) myocytes (F/F(o)=1.93+/-0.08, n=28, P<0.05). Thus the alpha2 isoform rather than the alpha1 appears to influence Na(+)/Ca(2+) exchanger currents [Ca(2+)](i) transients, and contractility. This finding is consistent with the proposal that alpha2 isoform of the Na pump preferentially alters [Na(+)] in a subsarcolemmal micro-domain adjacent to Na(+)/Ca(2+) exchanger molecules and SR Ca(2+) release sites.     

Beat-to-beat Ca(2+)-dependent regulation of sinoatrial nodal pacemaker cell rate and rhythm

Whether intracellular Ca²⁺ regulates sinoatrial node cell (SANC) action potential (AP) firing rate on a beat-to-beat basis is controversial. To directly test the hypothesis of beat-to-beat intracellular Ca²⁺ regulation of the rate and rhythm of SANC we loaded single isolated SANC with a caged Ca²⁺ buffer, NP-EGTA, and simultaneously recorded membrane potential and intracellular Ca²⁺. Prior to introduction of the caged Ca²⁺ buffer, spontaneous local Ca²⁺ releases (LCRs) during diastolic depolarization were tightly coupled to rhythmic APs (r² = 0.9). The buffer markedly prolonged the decay time (T₅₀) and moderately reduced the amplitude of the AP-induced Ca²⁺ transient and partially depleted the SR load, suppressed spontaneous diastolic LCRs and uncoupled them from AP generation, and caused AP firing to become markedly slower and dysrhythmic. When Ca²⁺ was acutely released from the caged compound by flash photolysis, intracellular Ca²⁺ dynamics were acutely restored and rhythmic APs resumed immediately at a normal rate. After a few rhythmic cycles, however, these effects of the flash waned as interference with Ca²⁺ dynamics by the caged buffer was reestablished. Our results directly support the hypothesis that intracellular Ca²⁺ regulates normal SANC automaticity on a beat-to-beat basis.     

Compartmentation of cAMP in adult canine ventricular myocytes. Relation to single-cell free Ca2+ transients.

Isolated adult canine ventricular myocytes were used to study the role of compartmentation of cAMP in the diverse functional responses to various drugs that elevate cAMP. Myocytes presented with the beta-agonist isoproterenol accumulated cAMP with a half maximally effective concentration (EC50) of 3.55 x 10(-8) M. Approximately 45% of the total cAMP was recovered in the particulate fraction of digitonin-lysed myocytes under these conditions. With phosphodiesterase inhibition (10 microM isobutylmethylxanthine), isoproterenol-stimulated cAMP production was up to 3.4-fold greater, but the proportion of total cAMP residing in the particulate fraction declined to less than 20%. Similar results were obtained with forskolin, a direct activator of adenylate cyclase. Treatment with isoproterenol shortened the duration at 50% maximum peak height (T 1/2) and increased the peak fluorescence ratio of electrically triggered single-cell free Ca2+ transients in fura-2-loaded canine myocytes. Isoproterenol dose-response curves gave EC50 values of 1.7 x 10(-9) and 4.4 x 10(-9) M for effects on T 1/2 and peak height, respectively. Alterations in peak height and T 1/2 of Ca2+ transients also showed a dose dependency on isobutylmethylxanthine and forskolin. Comparison of myocyte cAMP content with the corresponding changes in free Ca2+ transients demonstrated a close correlation between particulate cAMP and the extent of shortening or increase in peak height of the fura-2 Ca2+ transients (r = 0.92 for each). However, when these two parameters were plotted as a function of total cAMP, the resulting curves were nonlinear and divergent for each agent tested. The results support the hypothesis that differences in responses to agents that augment cAMP can be explained in part by compartmentation of cAMP. Furthermore, Ca2+ mobilization seems to be most affected by cAMP located in the particulate compartment of canine cardiac myocytes.     

Thyroid hormone control of contraction and the Ca(2+)-ATPase/phospholamban complex in adult rat ventricular myocytes

Thyroid hormones may have important long-term effects on cellular Ca2+ handling in the heart. We investigated isolated adult rat cardiomyocytes in a primary culture exposed (T3-cells) or not exposed to (control cells) 10(-8) M triiodothyronine (T3) for 48 h. Northern blot analysis revealed reciprocal alterations in the expression of SERCA2 and phospholamban. The ratio of the SERCA2/phospholamban signal was approximately 10 times higher in the T3-cells as compared with the control cells (P < 0.05). Phospholamban protein content was significantly reduced by 33% but SR-Ca(2+)-ATPase protein content was not significantly altered in T3-cells. These results were associated with functional alterations measured by an inverted microscope equipped to monitor fluorescence at two excitation wavelengths as well as cell shortening by a video edge detection unit. The peak calcium transients as measured by fura-2 acetoxymethyl ester (AM) were increased significantly during stimulation at 0.25 and 0.5 Hz in T3-cells compared with control cells (P < 0.05). The monoexponential decline of the fura-2 transient was significantly faster at all frequencies in the T3-cells as compared with control cells (P < 0.05). Interestingly, we observed blunted responses to both isoproterenol stimulation and post rest potentiation in the T3-cells. The intracellular level of sodium as represented by SBFI-AM was significantly lower in the T3-cells compared with the control cells (P < 0.05). The increased SR-Ca(2+)-ATPase/phospholamban ratio and decrease in phospholamban protein content in T3-treated cells was reflected in a parallel increase of contraction and calcium transients and more rapid Ca2+ reuptake, but the post-rest potentiation and response to isoproterenol were reduced.     

Voltage dependence of intracellular [Ca2+]i transients in guinea pig ventricular myocytes

[Ca2+]i transients, elicited by voltage-clamp depolarization of single guinea pig cardiac ventricular cells, were observed through use of the fluorescent Ca2+ indicator, fura-2. Individual cells, loaded with fura-2 either by internal perfusion or by exposure to fura-2/AM, were studied with the use of an inverted microscope that was equipped with ultraviolet epifluorescence illumination, an intensified silicon intensifier target camera, and a photomultiplier tube. Variation of membrane voltage and exposure of cells to verapamil (a Ca2+ channel blocker) and ryanodine (which was assumed to abolish selectively the release of Ca2+ from the sarcoplasmic reticulum) were used to investigate the cellular processes that determine the [Ca2+]i transient. The principal results of the study are: When appropriate methods are used, the properties of cytosolic fura-2 inside guinea pig cells are similar to those of fura-2 in solution, irrespective of the method of loading. The amplitude (at 100 msec) of verapamil-sensitive fluorescence transients elicited by pulse depolarization (range -30 to 80 mV) has a bell-shaped dependence on membrane voltage (maximum at 10 mV). Rapid, ryanodine-sensitive and verapamil-sensitive "tail transients" are elicited on repolarization from membrane potentials greater than 30 mV; their amplitude increases as the amplitude of the preceding pulse increases. The amplitude of slow fluorescence transients that are insensitive to verapamil and ryanodine increases continuously with membrane potential throughout the range -20 to 80 mV. The voltage dependence and pharmacology of the rapid transients elicited by pulse depolarization or by repolarization are consistent with their having arisen from Ca2+ released from the sarcoplasmic reticulum, via Ca2+-induced Ca2+ release.(ABSTRACT TRUNCATED AT 250 WORDS)     

T-Type and tetrodotoxin-sensitive Ca(2+) currents coexist in guinea pig ventricular myocytes and are both blocked by mibefradil

Under Na(+)-free conditions, low-voltage-activated Ca(2+) currents in cardiomyocytes from various species have been described either as Ni(2+)-sensitive T-type Ca(2+) current (I(Ca(T))) or as tetrodotoxin (TTX)-sensitive Ca(2+) current (I(Ca(TTX))). So far, coexistence of the 2 currents within the same type of myocyte has never been reported. We describe experimental conditions under which I(Ca(T)) and I(Ca(TTX)) can be separated and studied in the same cell. Rat and guinea pig ventricular myocytes were investigated with the whole-cell voltage-clamp technique in Na(+)-free solutions. Whereas rat myocytes lack I(Ca(T)) and exhibit I(Ca(TTX)) only, guinea pig myocytes possess both of these low-voltage-activated Ca(2+) currents, which are separated pharmacologically by superfusion with TTX or Ni(2+). I(Ca(T)) and I(Ca(TTX)) were of similar amplitude but significantly differed in their electrophysiological properties: I(Ca(TTX)) activated at more negative potentials than did I(Ca(T)), the potential for half-maximum steady-state inactivation was more negative, and current deactivation and recovery from inactivation were faster. I(Ca(TTX)) but not I(Ca(T)) increased after membrane rupture ("run-up"). Isolation of I(Ca(TTX)) by application of the bivalent cation Ni(2+) is critical because of possible shifts in voltage dependence. Therefore, we investigated whether the T-type Ca(2+) channel blocker mibefradil (10 micromol/L) is a suitable tool for the study of I(Ca(TTX)). However, mibefradil not only blocked I(Ca(T)) by 85+/-2% but also decreased I(Ca(TTX)) by 48+/-8%. We conclude that under Na(+)-free conditions I(Ca(T)) and I(Ca(TTX)) coexist in guinea pig ventricular myocytes and that both currents are sensitive to mibefradil. Future investigations of I(Ca(T)) will have to consider the TTX-sensitive current component to avoid possible interference.     

Ca2+ sparks and waves in canine purkinje cells: a triple layered system of Ca2+ activation

We have investigated the subcellular spontaneous Ca2+ events in canine Purkinje cells using laser scanning confocal microscopy. Three types of Ca2+ transient were found: (1) nonpropagating Ca2+ transients that originate directly under the sarcolemma and lead to (2) small Ca2+ wavelets in a region limited to 6-microm depth under the sarcolemma causing (3) large Ca2+ waves that travel throughout the cell (CWWs). Immunocytochemical studies revealed 3 layers of Ca2+ channels: (1) channels associated with type 1 IP3 receptors (IP3R1) and type 3 ryanodine receptors (RyR3) are prominent directly under the sarcolemma; (2) type 2 ryanodine receptors (RyR2s) are present throughout the cell but virtually absent in a layer between 2 and 4 microm below the sarcolemma (Sub-SL); (3) type 3 ryanodine receptors (RyR3) is the dominant Ca2+ release channel in the Sub-SL. Simulations of both nonpropagating and propagating transients show that the generators of Ca2+ wavelets differ from those of the CWWs with the threshold of the former being less than that of the latter. Thus, Purkinje cells contain a functional and structural Ca2+ system responsible for the mechanism that translates Ca2+ release occurring directly under the sarcolemma into rapid Ca2+ release in the Sub-SL, which then initiates large-amplitude long lasting Ca2+ releases underlying CWWs. The sequence of spontaneous diastolic Ca2+ transients that starts directly under the sarcolemma and leads to Ca2+ wavelets and CWWs is important because CWWs have been shown to cause nondriven electrical activity.