A specific pattern of phosphodiesterases controls the cAMP signals generated by different Gs-coupled receptors in adult rat ventricular myocytes

Compartmentation of cAMP is thought to generate the specificity of Gs-coupled receptor action in cardiac myocytes, with phosphodiesterases (PDEs) playing a major role in this process by preventing cAMP diffusion. We tested this hypothesis in adult rat ventricular myocytes by characterizing PDEs involved in the regulation of cAMP signals and L-type Ca2+ current (I(Ca,L)) on stimulation with beta1-adrenergic receptors (beta1-ARs), beta2-ARs, glucagon receptors (Glu-Rs) and prostaglandin E1 receptors (PGE1-Rs). All receptors but PGE1-R increased total cAMP, and inhibition of PDEs with 3-isobutyl-1-methylxanthine strongly potentiated these responses. When monitored in single cells by high-affinity cyclic nucleotide-gated (CNG) channels, stimulation of beta1-AR and Glu-R increased cAMP, whereas beta2-AR and PGE1-R had no detectable effect. Selective inhibition of PDE3 by cilostamide and PDE4 by Ro 20-1724 potentiated beta1-AR cAMP signals, whereas Glu-R cAMP was augmented only by PD4 inhibition. PGE1-R and beta2-AR generated substantial cAMP increases only when PDE3 and PDE4 were blocked. For all receptors except PGE1-R, the measurements of I(Ca,L) closely matched the ones obtained with CNG channels. Indeed, PDE3 and PDE4 controlled beta1-AR and beta2-AR regulation of I(Ca,L), whereas only PDE4 controlled Glu-R regulation of I(Ca,L) thus demonstrating that receptor-PDE coupling has functional implications downstream of cAMP. PGE1 had no effect on I(Ca,L) even after blockade of PDE3 or PDE4, suggesting that other mechanisms prevent cAMP produced by PGE1 to diffuse to L-type Ca2+ channels. These results identify specific functional coupling of individual PDE families to Gs-coupled receptors as a major mechanism enabling cardiac cells to generate heterogeneous cAMP signals in response to different hormones.     

Ahnak, a new player in beta-adrenergic regulation of the cardiac L-type Ca2+ channel

Ahnak, originally identified as a giant, tumour-related phosphoprotein, has emerged as an important signalling molecule in a wide range of physiological activities. In this article, current knowledge will be reviewed that places ahnak into the context of cardiac L-type Ca2+ channel function by its interaction with the beta2 subunit. Beginning with an overview on structural and functional properties of ahnak, basic features of beta subunits are highlighted. The review characterizes multiple ahnak/beta2 subunit binding sites and focuses on recent progress in understanding their functional role in Cav1.2 channel conductance (I(CaL)). Three main aspects of ahnak function in I(CaL) of cardiomyocytes emerge from available experimental data. First, ahnak acts as repressor towards I(CaL) by beta2 subunit sequestration. Second, PKA phosphorylation relieves the inhibition imposed by the C-terminal ahnak domain, ahnak-C1. Third, this action is mimicked by ahnak-derived fragments carrying a naturally occurring missense mutation Ile5236Thr. This paradigm introduces ahnak as a player in beta-adrenergic control of I(CaL) and sheds new light upon the molecular mechanism underlying this fundamental process of Cav1.2 channel physiology.     

Evidence for regulation of mitochondrial function by the L-type Ca channel in ventricular myocytes

The L-type Ca²⁺ channel is responsible for initiating contraction in the heart. Mitochondria are responsible for meeting the cellular energy demands and calcium is required for the activity of metabolic intermediates. We examined whether activation of the L-type Ca²⁺ channel alone is sufficient to alter mitochondrial function. The channel was activated directly with the dihydropyridine agonist BayK(−) or voltage-clamp of the plasma membrane and indirectly by depolarization of the membrane with high KCl. Activation of the channel increased superoxide production (assessed as changes in dihydroethidium fluorescence), NADH production and metabolic activity (assessed as formation of formazan from tetrazolium) in a calcium-dependent manner. Activation of the channel also increased mitochondrial membrane potential assessed as changes in JC-1 fluorescence. The response was reversible upon inactivation of the channel during voltage-clamp of the plasma membrane and did not appear to require calcium. We examined whether the response may be mediated through movement of cytoskeletal proteins. Depolymerization of actin or exposing cells to a peptide directed against the alpha-interacting domain of the α_1C-subunit of the channel (thereby preventing movement of the β-subunit) attenuated the increase in mitochondrial membrane potential. We conclude that activation of the L-type Ca²⁺ channel can regulate mitochondrial function and the response appears to be modulated by movement through the cytoskeleton.     

L-type Ca2+ channel density and regulation are altered in failing human ventricular myocytes and recover after support with mechanical assist devices

Ca2+ influx through the L-type calcium channel (LTCC) induces Ca2+ release from the sarcoplasmic reticulum (SR) and maintains SR Ca2+ loading. Alterations in LTCC properties, their contribution to the blunted adrenergic responsiveness in failing hearts and their recovery after support with LV assist devices (LVAD) were studied. L-type Ca2+ current (I(Ca,L)) was measured under basal conditions and in the presence of isoproterenol (ISO), dibutyryl-cAMP (db-cAMP), Bay K 8644 (BayK), Okadaic acid (OA, a phosphatase inhibitor), and phosphatase 2A (PP2A) in nonfailing (NF), failing (F), and LVAD-supported human left ventricular myocytes (HVMs). Basal I(Ca,L) density was not different in the 3 groups but I(Ca,L) was activated at more negative voltages in F- and LVAD- versus NF-HVMs (V(0.5): -7.18+/-1.4 and -7.0+/-0.9 versus 0.46+/-1.1 mV). Both ISO and db-cAMP increased I(Ca,L) in NF- and LVAD- significantly more than in F-HVMs (NF >LVAD> F: ISO: 90+/-15% versus 77+/-19% versus 24+/-12%; db-cAMP: 235%>172%>90%). ISO caused a significant leftward shift of the I(Ca,L) activation curve in NF- and LVAD- but not in F-HVMs. After ISO and db-cAMP, the I(Ca,L) activation was not significantly different between groups. BayK also increased I(Ca,L) more in NF- (81+/-30%) and LVAD- (70+/-15%) than in F- (51+/-8%) HVMs. OA increased I(Ca, L) by 85.6% in NF-HVMs but had no effect in F-HVMs, while PP2A decreased I(Ca, L) in F-HVMs by 35% but had no effect in NF-HVMs. These results suggest that the density of LTCC is reduced in F-HVMs but basal I(Ca,L) density is maintained by increasing in LTCC phosphorylation.     

Use-dependent facilitation and depression of L-type Ca2+ current in guinea-pig ventricular myocytes: modulation by Ca2+ and isoprenaline

OBJECTIVE: An increase in stimulation frequency can facilitate or depress cardiac Ca2+ current (ICa). The aim was to examine the Ca2+ dependence of these effects, to determine if facilitation is sustained, and to elucidate the mechanism by which isoprenaline modulates facilitation. METHODS: We examined the effects of increasing the stimulation frequency for 1 min, from 0.05 to 1 Hz, on ICa recorded from guinea-pig ventricular myocytes, using the whole-cell, voltage-clamp technique. RESULTS: 1 Hz stimulation caused a facilitation of ICa that peaked in 5 s and was followed by depression towards the basal level. Metabolic inhibitors or replacement of extracellular Ca2+ with Ba2+ abolished facilitation without affecting depression, implying that they are independent processes and that facilitation required ATP and Ca2+. Subtraction of the depression observed in either condition, from the response to 1 Hz stimulation recorded under control conditions, revealed that ICa facilitation was well maintained during 1 Hz stimulation. Increased intracellular Ca2+ buffering reduced both phases of the response. Furthermore, varying the extracellular Ca2+ concentration ([Ca2+]o) revealed a Ca(2+)-dependent enhancement of depression and a bell-shaped dependence of facilitation on [Ca2+]o. Facilitation increased with [Ca2+]o up to 1 mM, then declined at higher concentrations due to partial masking by the overlaping depression. Isoprenaline produced concentration-dependent inhibition of facilitation and enhancement of depression when pipettes contained 2 mM EGTA, but not BAPTA. For an equivalent increase in ICa amplitude, the effects of isoprenaline and elevated [Ca2+]o on the response to 1 Hz stimulation were quantitatively the same. CONCLUSIONS: Facilitation is sustained during increased activity, but appears transient due to overlapping depression. Both responses are promoted by increased submembrane [Ca2+]. Isoprenaline appears to modulate facilitation and depression as a consequence of increased Ca2+ influx, rather than cAMP-dependent phosphorylation. The apparent block of facilitation by isoprenaline may result from masking by the enhanced depression.     

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.     

Ca2+ current-mediated regulation of action potential by pacing rate in rat ventricular myocytes

OBJECTIVE: Pacing rate regulates the duration of the cardiac action potential (AP). It also regulates the decay kinetics of the L-type Ca(2+) current (I(Ca-L)) which occurs via modulation of Ca(2+)-dependent inactivation. We investigated whether and how this latter process contributes to frequency-dependent (FD) changes in the AP waveform in rat ventricular cells. METHODS: We recorded APs using a microelectrode technique in rat papillary muscles, and using the whole-cell current patch-clamp technique in single rat ventricular cells. RESULTS: The AP duration (APD) was increased by high rates encompassing the physiological range (0.1-5.7 Hz) in both papillary muscles and single cells. This prolongation was accompanied by concomitant depolarisation (approximately 7 mV at 5.7 Hz) of the membrane potential (MP) in papillary muscles. Equivalent artificial depolarisation of the MP enhanced the FD prolongation in single cells. The FD prolongation was enhanced in presence of the K(+) current blocker 4-aminopyridine (5 mmol/l), and decreased in absence of extracellular Ca(2+). It was antagonised by Ca(2+) channel blockers (Co(2+), nifedipine, nitrendipine) and decreased by use of high EGTA (10 vs. 0.5 mmol/l EGTA) or BAPTA (20 mmol/l) in the patch-pipette. It was prevented by ryanodine or thapsigargin, two drugs that reduce or abolish SR-Ca(2+) function. CONCLUSION: I(Ca-L) contributes to the FD modulation of the AP, which occurs following a sudden change in cardiac frequency in rat ventricular cells. This highly dynamic physiological process is related to SR-Ca(2+) release and occurs through beat-to-beat adaptation of Ca(2+)-dependent inactivation of I(Ca-L).     

Differential modulation of L-type Ca2+ current by SR Ca2+ release at the T-tubules and surface membrane of rat ventricular myocytes

We have characterized modulation of ICa by Ca2+ at the t-tubules (ie, in control cells) and surface sarcolemma (ie, in detubulated cells) of cardiac ventricular myocytes, using the whole-cell patch clamp technique to record ICa. ICa inactivation was significantly slower in detubulated cells than in control cells (27.1+/-7.8 ms, n=22, versus 16.4+/-7.9 ms, n=22; P<0.05). In atrial myocytes, which lack t-tubules, ICa inactivation was not changed by the treatment used to produce detubulation. In the presence of ryanodine or BAPTA, or when Ba2+ was used as the charge carrier, the rate of inactivation was not significantly different in control and detubulated cells. Frequency-dependent facilitation occurred in control cells but not in detubulated cells, and was abolished by ryanodine. These results suggest that Ca2+ released from the SR has a greater effect on ICa in the t-tubules than at the surface sarcolemma. This does not appear to be due to differences in local Ca2+ release from the SR, because the gain of Ca2+ release was not significantly different in control and detubulated cells. These data suggest that the t-tubules are a key site for the regulation of transsarcolemmal Ca2+ flux by Ca2+ release from the SR; this could play a role in altered Ca2+ homeostasis in pathological conditions. The full text of this article is available online at http://circres.ahajournals.org.     

Ca2+ sparks in rabbit ventricular myocytes evoked by action potentials: involvement of clusters of L-type Ca2+ channels

It is not clear how many L-type Ca2+ channels (LCCs) are required to ensure that a Ca2+ spark is triggered during a normal mammalian action potential (AP). We investigated this in rabbit ventricular myocytes by examining both the properties of sparks evoked by APs and the activity of LCCs. We measured Ca2+ sparks evoked by repeated APs with pipettes containing 2 mmol/L EGTA and single LCC activity in cell-attached patches depolarized to +50 mV using pipettes containing 110 mmol/L Ba2+. With 2 mmol/L Ca2+ in the external solution, we observed sparks at the beginning of every evoked AP at numerous locations. Each spark was observed repeatedly at a fixed location and began during a limited interval after the AP peak. These sparks occurred with a probability of approximately unity. However, the chance that an LCC does not open during the interval when a spark is triggered is quite high ( approximately 0.13). Therefore, because single channels open with a probability significantly lower than 1, more than one LCC must be available to ensure that sparks are triggered with a probability of approximately unity. We conclude that it is likely that a cluster of LCCs is involved in gating a cluster of ryanodine receptors at the beginning of an AP.     

Epinephrine enhances Ca2+ current-regulated Ca2+ release and Ca2+ reuptake in rat ventricular myocytes.

The voltage dependence of the intracellular Ca2+ transients was measured in single rat ventricular myocytes with the fluorescent Ca2+ indicator dye fura-2. The whole-cell voltage clamp technique was used to measure the membrane current, and 0.9 mM fura-2 was loaded into the cell by including it in the dialyzing solution of the patch electrode. A mechanical light chopper operating at 1200 Hz was used to obtain simultaneous measurements of the intracellular Ca2+ activity with fluorescence excitation on either side of the isosbestic point (330 nm and 410 nm). The symmetry of the two optical Ca2+ signals was used as a criterion to guard against artifacts resulting, for instance, from motion. The voltage dependence of peak Ca2+ current and the Ca2+ transient measured 25 ms after depolarizing clamps from a holding potential of -40 mV were bell-shaped and virtually identical. The Ca2+ entry estimated from the integral of the Ca2+ current (0 mV, 25 ms) corresponds to a 5-10 microM increase in the total intracellular Ca2+ concentration, whereas the optical signal indicated a 100 microM increase in total intracellular Ca2+. Repolarization of clamp pulses from highly positive potentials were accompanied by a second Ca2+ transient, the magnitude of which, when summed with that measured during depolarization, was nearly constant. Ryanodine (10 microM) had little or no effect on the peak Ca2+ current but reduced the magnitude of the early Ca2+ transients by 70-90%. Epinephrine (1 microM) increased the Ca2+ current and the Ca2+ transients, accelerated the rate of decline of the Ca2+ transients at potentials between -30 and +70 mV, and reduced the intracellular [Ca2+] below baseline at potentials positive to +80 or negative to -40 mV, where clamp pulses did not elicit any Ca2+ release. Elevation of intracellular cAMP mimicked the relaxant effect of epinephrine at depolarizing potentials, whereas elevation of extracellular [Ca2+] did not. These results suggest that most of the activator Ca2+ in rat ventricular cells is released from the sarcoplasmic reticulum as a graded response to sarcolemmal Ca2+ influx. Consistent with a graded Ca2+-induced Ca2+ release we find that epinephrine increases the internal Ca2+ release by increasing the Ca2+ current. Epinephrine may also increase the Ca2+ content of the sarcoplasmic reticulum that may, in turn, increase the Ca2+-induced Ca2+ release. The relaxant effect of epinephrine appears to be caused by enhanced rate of Ca2+ resequestration and is mediated by adenylate cyclase system.     

Src family tyrosine kinases inhibit single L-type: Ca2+ channel activity in human atrial myocytes

OBJECTIVE: Tyrosine kinases (TKs) are important regulators of the L-type Ca(2+) channel (LTCC) current in various cell types. However, there are no data addressing the role of TKs in the control of single LTCC activity in human atrial cardiac myocytes, where changes in LTCC gating properties have been described in a number of disease states. METHODS AND RESULTS: Single LTCC activity was recorded in isolated human atrial myocytes. The broad-spectrum TK inhibitor genistein and the Src family-selective TK inhibitor PP1 significantly enhanced single LTCC ensemble average current, availability, and open probability; the latter was due to significant increases of mean open time and mode 2 gating. Conversely, the tyrosine phosphatase inhibitor bisperoxo-phenanthroline-vanadate inhibited single LTCC activity, indicating that LTCC gating properties in human atrial myocytes are controlled by TKs and tyrosine phosphatases in a reciprocal fashion. The effects of genistein on single LTCC activity were not affected by stimulation (8Br-cAMP) or inhibition (Rp-8-CPT-cAMPS) of protein kinase A (PKA) or by inhibition of serine/threonine phosphatases types I and IIa (okadaic acid), indicating that TKs inhibit LTCC gating in human atrial myocytes independent of PKA and phosphatases types I and IIa. However, inhibition of protein kinase C (PKC) by staurosporine or bisindolylmaleimide reversed the stimulatory effects of genistein on single LTCC gating properties, indicating that PKC is required for the inhibitory effect of TKs on single LTCC activity. CONCLUSION: Src family TKs inhibit single LTCC activity in human atrial myocytes via PKC-dependent, but PKA and phosphatase types I and IIa-independent, molecular pathways.     

L-type Ca channel facilitation mediated by H2O2-induced activation of CaMKII in rat ventricular myocytes

The Ca²⁺-dependent facilitation (CDF) of L-type Ca²⁺ channels, a major mechanism for force-frequency relationship of cardiac contraction, is mediated by Ca²⁺/CaM-dependent kinase II (CaMKII). Recently, CaMKII was shown to be activated by methionine oxidation. We investigated whether oxidation-dependent CaMKII activation is involved in the regulation of L-type Ca²⁺ currents (I_Ca,L) by H₂O₂ and whether Ca²⁺ is required in this process. Using patch clamp, I_Ca,L was measured in rat ventricular myocytes. H₂O₂ induced an increase in I_Ca,L amplitude and slowed inactivation of I_Ca,L. This oxidation-dependent facilitation (ODF) of I_Ca,L was abolished by a CaMKII blocker KN-93, but not by its inactive analog KN-92, indicating that CaMKII is involved in ODF. ODF was not affected by replacement of external Ca²⁺ with Ba²⁺ or presence of EGTA in the internal solutions. However, ODF was abolished by adding BAPTA to the internal solution or by depleting sarcoplasmic reticulum (SR) Ca²⁺ stores using caffeine and thapsigargin. Alkaline phosphatase, β-iminoadenosine 5′-triphosphate (AMP-PNP), an autophosphorylation inhibitor autocamtide-2-related inhibitory peptide (AIP), or a catalytic domain blocker (CaM-KIINtide) did not affect ODF. In conclusion, oxidation-dependent facilitation of L-type Ca²⁺ channels is mediated by oxidation-dependent CaMKII activation, in which local Ca²⁺ increases induced by SR Ca²⁺ release is required.     

Influence of interleukin-2 on Ca2+ handling in rat ventricular myocytes

In the present study, we examined the effect of interleukin-2 (IL-2) on cardiomyocyte Ca(2+) handling. The effects of steady-state and transient changes in stimulation frequency on the intracellular Ca(2+) transient were investigated in isolated ventricular myocytes by spectrofluorometry. In the steady state (0.2 Hz) IL-2 (200 U/ml) decreased the amplitude of Ca(2+) transients induced by electrical stimulation and caffeine. At 1.25 mM extracellular Ca(2+) concentration ([Ca(2+)](o)), when the stimulation frequency increased from 0.2 to 1.0 Hz, diastolic Ca(2+) level and peak intracellular Ca(2+) concentration ([Ca(2+)](i)), as well as the amplitude of the transient, increased. The positive frequency relationships of the peak and amplitude of [Ca(2+)](i) transients were blunted in the IL-2-treated myocytes. The effect of IL-2 on the electrically induced [Ca(2+)](i) transient was not normalized by increasing [Ca(2+)](o) to 2.5 mM. IL-2 inhibited the frequency relationship of caffeine-induced Ca(2+) release. Blockade of sarcoplasmic reticulum (SR) Ca(2+)-ATPase with thapsigargin resulted in a significant reduction of the amplitude-frequency relationship of the transient similar to that induced by IL-2. The restitutions were not different between control and IL-2 groups at 1.25 mM [Ca(2+)](o), which was slowed in IL-2-treated myocytes when [Ca(2+)](o) was increased to 2.5 mM. There was no difference in the recirculation fraction (RF) between control and IL-2-treated myocytes at both 1.25 and 2.5 mM [Ca(2+)](o). The effects of IL-2 on frequency relationship, restitution, and RF may be due to depressed SR functions and an increased Na(+)-Ca(2+) exchange activity, but not to any change in L-type Ca(2+) channels.     

不同旋体氨氯地平对大鼠心室肌细胞L型钙离子流和细胞内钙离子浓度的影响

目的探讨不同旋体氨氯地平(Aml)对大鼠心室肌细胞L型钙离子流(ICa-L)及细胞内钙离子浓度的影响。方法采用酶消化法分离大鼠心室肌细胞,以全细胞膜片钳技术分别记录加入0.1,0.5,1,5和10μmol/L左旋、右旋和混旋Aml后大鼠心室肌细胞ICa-L及通道动力学参数的变化;采用荧光探针Fura-2/AM测定细胞内钙离子浓度,观察加入1,5,10,50和100μmol/L左旋、右旋和混旋Aml后细胞内钙离子浓度的变化。结果加入不同浓度左旋和混旋Aml后,随着浓度增加,ICa-L及峰值电流呈浓度依赖性阻滞、I-V曲线上移、稳态激活曲线和稳态失活曲线左移、失活后恢复时间延长(P<0.05);细胞内钙离子浓度逐渐降低,左旋和混旋Aml对细胞内钙离子浓度影响的半效抑制浓度分别为8.13和16.19μmol/L(P<0.05),但不同浓度右旋Aml对ICa-L、通道动力学参数和细胞内钙离子浓度均无影响(P>0.05)。结论左旋和混旋Aml对ICa-L有阻滞作用,而右旋Aml对ICa-L无阻滞作用。     

Spatiotemporal characteristics of junctional and nonjunctional focal Ca2+ release in rat atrial myocytes

Atrial myocytes have two functionally separate Ca2+ release sites: those in peripheral sarcoplasmic reticulum (SR) adjacent to the Ca2+ channels of surface membrane and those in central SR not associated with Ca2+ channels. Recently, we have reported on the gating of these two different Ca2+ release sites by Ca2+ current. In the present study, we report on the spatiotemporal properties of focal Ca2+ releases (sparks) occurring spontaneously in central and peripheral sites of voltage-clamped rat atrial myocytes, using rapid 2-dimensional (2-D) confocal Ca2+ imaging. Peripheral and central sparks were similar in size and release time (approximately 300 000 Ca2+ ions for congruent with 12 ms), but significantly larger and longer than ventricular sparks. Both sites were resistant to Cd2+ and inhibited by ryanodine. Peripheral sparks were brighter and flattened against surface membrane, had approximately 5-fold higher frequency, approximately 2 times faster diffusion coefficient, and dissipated abruptly. Central sparks, in contrast, occurred less frequently, were elongated along the cellular longitudinal axis, and dissipated slowly. Compound sparks (composed of 2 to 5 unitary focal releases) aligned longitudinally and occurred more frequently at the center. The diversity of peripheral and central sparks with respect to shape, frequency, and speed of spatial development and decay is consistent with regional ultrastructural heterogeneity of SR. The retarded dissipation of central atrial sparks, and high prevalence of compound sparks in cell center may be critical in facilitating the propagation of Ca2+ waves in atrial myocytes lacking t-tubular system and provide the atrial myocytes with functional Ca2+ signaling diversity. The full text of this article is available at http://www.circresaha.org.     

Interaction between autoantibodies against the beta1-adrenoceptor and isoprenaline in enhancing L-type Ca2+ current in rat ventricular myocytes

Autoantibodies against the beta1-adrenoceptor (beta1-AAB) in the serum of patients with dilated cardiomyopathy (DCM) are associated with stimulatory effects at cardiac beta1-adrenoceptors. They enhance cardiomyocyte shortening and increase the amplitude of L-type Ca2+ current, ICa. However, in contrast to the unselective beta-adrenoceptor agonist (-)-isoprenaline, beta1-AAB produce positive responses in a fraction of myocytes (responder cells) only and fail to do so in the remaining ones (non-responder cells). To understand this peculiar behaviour, the electrophysiological characteristics of ICa in response to beta1-AAB and (-)-isoprenaline were investigated in responder and non-responder cells. The immunoglobulin G (IgG) fractions containing beta1-AAB (beta1-IgG) were obtained from patients with DCM undergoing immunoabsorption therapy. Only antibody preparations that tested positive in the neonatal rat cardiomyocyte bio-assay by enhancing beating rate were used for further experimentation. Calcium currents were measured with the standard patch clamp technique in adult rat ventricular myocytes. Less than half of all cells exposed to beta1-IgG or purified beta1-AAB were responder cells in which ICa amplitude increased. ICa increase by beta1-IgG or (-)-isoprenaline was reversed by addition of carbachol. Exposure to subtype-selective beta-adrenoceptor blockers indicated that the effects of IgG were mediated via beta1-adrenoceptors. In responder cells, there were no differences between beta1-IgG- and (-)-isoprenaline-induced changes in current-voltage relationship of ICa, in the time constants of fast inactivation, and in steady-state activation and steady-state inactivation curves. (-)-Isoprenaline (1 microM) effectively increased ICa after wash-out of antibody in all cells including non-responder cells. However, when non-responder cells were challenged with (-)-isoprenaline in the presence of beta1-IgG, any further increase in ICa was completely suppressed. Conversely, in responder cells, the cumulative concentration-response curves for (-)-isoprenaline on top of the autoantibodies reached the same maximum ICa amplitude as in control cells. From these interactions we conclude that beta1-AAB not only may enhance ICa via stimulation of beta1-adrenoceptors but also may inhibit beta1-adrenoceptor-mediated increase upon stimulation with catecholamines suggesting a receptor interaction distinct from that with (-)-isoprenaline.     

伊布利特对兔急性心肌梗死后梗死周边区心外膜心室肌细胞L型钙电流的影响

目的观察伊布利特对急性心肌梗死(AMI)后一周心室肌细胞L型钙通道电流(ICa-L)的影响。方法兔开胸,左前降支结扎造成AMI,1周后酶解分离梗死周边区心外膜心室肌细胞,用全细胞膜片钳技术记录10-6mol/L伊布利特细胞外液(伊布利特组)对梗死周边区心外膜心室肌细胞ICa-L活性的影响,并与正常对照组(对照组)及AMI但未灌流伊布利特组(AMI组)比较。结果①AMI 1周时兔梗死周边区心室肌细胞ICa-L受到抑制,电流密度-电压曲线(I-V)上移,ICa-L电流密度峰值降低[-3.52±0.91 pA/pF(n=10)vs-5.68±1.53 pA/pF(n=10),P<0.05];伊布利特组电流密度峰值为-4.84±1.22 pA/pF(n=8),较AMI组显著增大(P<0.05),与对照组比较,虽有减小,但无差异(P>0.05)。②AMI组、伊布利特组ICa-L失活曲线明显左移,以AMI组左移更加明显,对照组半数失活电压(V0.5)为-32±4 mV(n=10),AMI组V0.5增加为-46±7 mV(n=10,P<0.05),伊布利特组V0.5为-36±6mV(n=8),与对照组比较无差异(P>0.05)。结论AMI后1周梗死周边带心室肌细胞L型钙通道受阻滞,伊布利特对缺血引起的ICa-L的异常有明显改善作用。     

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).     

Differential effects of docosahexaenoic acid on contractions and L-type Ca2+ current in adult cardiac myocytes

Beneficial effects of n-3 polyunsaturated fatty acids in Ca2+ overload have been attributed to blockade of L-type Ca2+ current (I(Ca-L)). However, cardiac contractions may be maintained despite block of I(Ca-L). OBJECTIVE: This study investigates the cellular basis by which docosahexaenoic acid (DHA), a representative n-3 polyunsaturated fatty acid, inhibits I(Ca-L) while preserving contraction. METHODS: Experiments were conducted in adult guinea pig ventricular myocytes with Na+ currents blocked. Contractions initiated by the voltage-sensitive release mechanism (VSRM) and calcium-induced calcium release (CICR) triggered by I(Ca-L), were activated separately with voltage clamp techniques. RESULTS: DHA (10 microM) inhibited I(Ca-L) and CICR contractions but not VSRM contractions. CICR contractions exhibited a bell-shaped voltage-dependence. However, in the presence of DHA, only contractions with a sigmoidal voltage-dependence characteristic of the VSRM remained. These contractions exhibited inactivation properties characteristic of the VSRM. DHA abolished I(Ca-L) elicited by test steps from -40 mV. Block was voltage-dependent, as residual I(Ca-L) was elicited by steps from -70 mV. Cd2+ inhibited residual current, but not contractions initiated by the same activation steps. CONCLUSION: Preservation of VSRM contractions during block of I(Ca-L), may explain the ability of n-3 polyunsaturated fatty acids to inhibit Ca2+ influx while preserving cardiac contractile function.