Exchanger Current Research Articles

Identification and characterization of two ankyrin-B isoforms in mammalian heart

Excitation-contraction coupling in cardiomyocytes requires the proper targeting and retention of membrane proteins to unique domains by adaptor proteins like ankyrin-B. While ankyrin-B has been shown to interact with a variety of membrane and structural proteins located at different subcellular domains in cardiomyocytes, what regulates the specificity of ankyrin-B for particular interacting proteins remains elusive. Here, we report the identification of two novel ankyrin-B isoforms AnkB-188 and AnkB-212 in human, rat, and mouse hearts. Novel cDNAs for both isoforms were isolated by long-range PCR of reverse-transcribed mRNA isolated from human ventricular tissue. The isoforms can be discriminated based on their function and subcellular distribution in cardiomyocytes. Heterologous overexpression of AnkB-188 increases sodium-calcium exchanger (NCX) membrane expression and current, while selective knockdown of AnkB-188 in cardiomyocytes reduces NCX expression and localization in addition to causing irregular contraction rhythms. Using an isoform-specific antibody, we demonstrate that the expression of AnkB-212 is restricted to striated muscles and is localized to the M-line of cardiomyocytes by interacting with obscurin. Selective knockdown of AnkB-212 significantly attenuates the expression of endogenous ankyrin-B at the M-line but does not disrupt NCX expression at transverse tubules in cardiomyocytes. The identification and characterization of two functionally distinct ankyrin-B isoforms in heart provide compelling evidence that alternative splicing of the ANK2 gene regulates the fidelity of ankyrin-B interactions with proteins.

Regulation of calcium clock-mediated pacemaking by inositol-1,4,5-trisphosphate receptors in mouse sinoatrial nodal cells.

Inositol-1,4,5-trisphosphate receptors (IP3 Rs) modulate pacemaking in embryonic heart, but their role in adult sinoatrial node (SAN) pacemaking is uncertain. We found that stimulation of IP3 Rs accelerates spontaneous pacing rate in isolated mouse SAN cells, whereas inhibition of IP3 Rs slows pacing. In atrial-specific sodium-calcium exchanger (NCX) knockout (KO) SAN cells, where the Ca(2+) clock is uncoupled from the membrane clock, IP3 R agonists and antagonists modulate the rate of spontaneous Ca(2+) waves, suggesting that IP3 R-mediated Ca(2+) release modulates the Ca(2+) clock. IP3 R modulation also regulates Ca(2+) spark parameters, a reflection of ryanodine receptor open probability, consistent with the effect of IP3 signalling on Ca(2+) clock frequency. Modulation of Ca(2+) clock frequency by IP3 signalling in NCX KO SAN cells demonstrates that the effect is independent of NCX. These findings support development of IP3 signalling modulators for regulation of heart rate, particularly in heart failure where IP3 Rs are upregulated. Cardiac pacemaking initiated by the sinus node is attributable to the interplay of several membrane currents. These include the depolarizing 'funny current' (If ) and the sodium-calcium exchanger current (INCX ). The latter is activated by ryanodine receptor (RyR)-mediated calcium (Ca(2+) ) release from the sarcoplasmic reticulum (SR). Another SR Ca(2+) release channel, the inositol-1,4,5-triphosphate receptor (IP3 R), has been implicated in the generation of spontaneous Ca(2+) release in atrial and ventricular cardiomyocytes. Whether IP3 R-mediated Ca(2+) release also influences SAN automaticity is controversial, in part due to the confounding influence of periodic Ca(2+) flux through the sarcolemma accompanying each beat. We took advantage of atrial-specific sodium-calcium exchanger (NCX) knockout (KO) SAN cells to study the influence of IP3 signalling on cardiac pacemaking in a system where periodic intracellular Ca(2+) cycling persists despite the absence of depolarization or Ca(2+) flux across the sarcolemma. We recorded confocal line scans of spontaneous Ca(2+) release in WT and NCX KO SAN cells in the presence or absence of an IP3 R blocker (2-aminoethoxydiphenyl borate, 2-APB), or during block of IP3 production by the phospholipase C inhibitor U73122. 2-APB and U73122 decreased the frequency of spontaneous Ca(2+) transients and waves in WT and NCX KO cells, respectively. Alternatively, increased IP3 production induced by phenylephrine increased Ca(2+) transient and wave frequency. We conclude that IP3 R-mediated SR Ca(2+) flux is crucial for initiating and modulating the RyR-mediated Ca(2+) cycling that regulates SAN pacemaking. Our results in NCX KO SAN cells also demonstrate that RyRs, but not NCX, are required for IP3 to modulate Ca(2+) clock frequency.

Abstract 17570: Rescue of Dilated Cardiomyopathy in Nav1.5 Mutant Mice Defective in Calmodulin Binding by Inhibition of Late Na+ Current

Introduction: Nav1.5, the main voltage-gated Na+ channel in the heart, has been shown to be involved in many cardiac diseases such as long QT syndrome, Brugada syndrome, and heart failure. Na+ channels are importantly regulated by Ca2+ calmodulin (CaM) mediated signaling: however, a fundamental understanding and physiological significance of CaM regulation of Na+ channel is incomplete. Here, we have created a transgenic mouse that harbors a mutated CaM binding motif (NaV1.5-IQ/AA), which is critical for Na+ channel regulation by Ca2+-CaM. Methods: Ventricle mass and function were analyzed with electrocardiogram, echocardiogram, and detailed invasive pressure-volume analysis. Additionally, single ventricular myocytes were obtained. Whole cell patch clamp was used to record membrane ionic currents, including sodium current, Ca2+ current, K+ currents and NCX current. Results: Homozygous mice are embryonic lethal and IQ/AA+/- mice exhibit a dramatic phenotype consisting of dilated cardiomyopathy (DCM) at 4-6 months of age with prolongation of QT. The Na+ current in IQ mice exhibits an enhanced slowly inactivating late component (INa,L) with concomitant up regulation of Na+/Ca2+ exchanger currents. Consistent with other models of DCM and heart failure, DCM in IQ/AA+/- mice was associated with a down regulation of transient outward K+ currents (Ito) and an increase in T-type Ca2+ currents. Chronic treatment with ranolazine designed to block INa,L prevented electrical remodeling of the hearts including an increase in INa,L and a down regulation of Ito. Consistent with the changes in INa,L and Ito, in ranolazine-fed IQ/AA+/- mice, the QT interval was decreased compared to vehicle (p<0.05). Further, the contractile dysfunction, cardiac hypertrophy, and myocardial fibrosis were attenuated in all ranolazine- fed animals, while ventricular dysfunction persisted in animals not fed drug (p<0.05). Conclusions: The data suggest that loss of CaM-mediated regulation of Na+ channel induces dilated cardiomyopathy by enhancing late Na+ current. Taken together, our data demonstrate a dynamic interplay for Ca2+ and Na+ signaling via the CaM binding motif of Na+ channels and highlight the critical importance of late Na+ currents to myopathy and arrhythmia.

Histone Deacetylase Inhibition Reduces Pulmonary Vein Arrhythmogenesis through Calcium Regulation

Pulmonary veins (PVs) play a critical role in the pathophysiology of atrial fibrillation (AF). Histone deacetylases (HDACs) are vital to calcium homeostasis and AF genesis. However, the electrophysiological effects of HDAC inhibition were unclear. This study evaluated whether HDAC inhibition can regulate PV electrical activity through calcium modulation.Whole-cell patch-clamp, confocal microscopic with fluorescence, and Western blot were used to evaluate electrophysiological characteristics and Ca2+ dynamics in isolated rabbit PV cardiomyocytes with and without MPT0E014 (a pan HDAC inhibitor), MS-275 (HDAC1 and 3 inhibitor), and MC-1568 (HDAC4 and 6 inhibitor) for 5~8h. Atrial electrical activity and induced-AF (rapid atrial pacing and acetylcholine infusion) were measured in rabbits with and without MPT0E014 (10mg/kg treated for 5hours) in vivo.MPT0E014 (1μM)-treated PV cardiomyocytes (n=12) had slower beating rates (2.1±0.2 vs. 2.8±0.1Hz, p<0.05) than control PV cardiomyocytes. However, control (n=11) and MPT0E014 (1μM)-treated (n=12) SAN cardiomyocytes had similar beating rates (3.2±0.2 vs. 2.9±0.3Hz). MS-275-treated PV cardiomyocytes (n=12, 2.3±0.2Hz), but not MC-1568-treated PV cardiomyocytes (n=14, 3.1±0.3Hz) had slower beating rates than control PV cardiomocytes. MPT0E014-treated PV cardiomyocytes (n=14) had a lower frequency (2.4±0.6 vs. 0.3±0.1 spark/mm/s, p<0.05) of Ca2+ sparks than control PV (n=17) cardiomyocytes. As compared to control, MPT0E014-treated PV cardiomyocytes had reduced Ca2+ transient amplitudes, sodium-calcium exchanger currents, and ryanodine receptor expressions. Moreover, MPT0E014-treated rabbits had less AF and shorter AF duration than control rabbits.In conclusions, HDAC inhibition reduced PV arrhythmogenesis and AF inducibility with modulation on calcium homeostasis.

A monounsaturated fatty acid (oleic acid) modulates electrical activity in atrial myocytes with calcium and sodium dysregulation

BackgroundObesity and metabolic syndrome are important risk factors for atrial fibrillation. High plasma concentrations of monounsaturated fatty acids, including oleic acid (OLA), are frequently noted in obese individuals and patients with metabolic syndrome. However, it is not clear whether monounsaturated fatty acids (MUFAs) can directly modulate the electrophysiological characteristics of atrial myocytes. MethodsWhole-cell patch clamp, indo-1 fluorescence, and Western blot analyses were used to record the action potentials (APs), ionic currents, and protein expressions of HL-1 myocytes incubated with and without (control) OLA (0.5mM) for 24h. ResultsCompared to control myocytes (n=14), OLA-treated myocytes (n=16) had shorter APD90 (65±6 vs. 85±6ms, p<0.05) and APD50 (24±6 vs. 38±4ms, p<0.05) with a higher incidence of delayed afterdepolarizations (35.7% vs. 7%, p<0.05), which were suppressed by 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS, a blocker of the calcium-activated chloride current). In addition, OLA-treated myocytes (n=19) exhibited larger calcium transients (0.54±0.06 vs. 0.38±0.05 R410/485, p<0.05), and sarcoplasmic reticular calcium contents (0.91±0.05 vs. 0.64±0.08 R410/485, p<0.05) than control myocytes (n=15). OLA-treated myocytes had larger late sodium currents, smaller sodium–calcium exchanger currents, and smaller sodium–potassium pump currents. Moreover OLA-treated myocytes had higher expressions of sarcoplasmic reticular Ca2+-ATPase and calmodulin kinase II, but lower expression of the sodium–potassium ATPase protein than control myocytes. ConclusionsMUFAs can regulate atrial electrophysiological characteristics with calcium and sodium dysregulation, which may contribute to atrial arrhythmogenesis.

Distinctive electrophysiological characteristics of right ventricular out-flow tract cardiomyocytes.

Ventricular arrhythmias commonly originate from the right ventricular out-flow tract (RVOT). However, the electrophysiological characteristics and Ca2+ homoeostasis of RVOT cardiomyocytes remain unclear. Whole-cell patch clamp and indo-1 fluorometric ratio techniques were used to investigate action potentials, Ca2+ homoeostasis and ionic currents in isolated cardiomyocytes from the rabbit RVOT and right ventricular apex (RVA). Conventional microelectrodes were used to record the electrical activity before and after (KN-93, a Ca2+/calmodulin-dependent kinase II inhibitor, or ranolazine, a late sodium current inhibitor) treatment in RVOT and RVA tissue preparations under electrical pacing and ouabain (Na+/K+ ATPase inhibitor) administration. In contrast to RVA cardiomyocytes, RVOT cardiomyocytes were characterized by longer action potential duration measured at 90% and 50% repolarization, larger Ca2+ transients, higher Ca2+ stores, higher late Na+ and transient outward K+ currents, but smaller delayed rectifier K+, L-type Ca2+ currents and Na+-Ca2+ exchanger currents. RVOT cardiomyocytes showed significantly more pacing-induced delayed afterdepolarizations (22% versus 0%, P < 0.05) and ouabain-induced ventricular arrhythmias (94% versus 61%, P < 0.05) than RVA cardiomyocytes. Consistently, it took longer time (9 ± 1 versus 4 ± 1 min., P < 0.05) to eliminate ouabain-induced ventricular arrhythmias after application of KN-93 (but not ranolazine) in the RVOT in comparison with the RVA. These results indicate that RVOT cardiomyocytes have distinct electrophysiological characteristics with longer AP duration and greater Ca2+ content, which could contribute to the high RVOT arrhythmogenic activity.

Effects of endogenous cannabinoid anandamide on cardiac Na+/Ca2+ exchanger

Endocannabinoid anandamide (N-arachidonoyl ethanolamide; AEA) has been shown to cause negative inotropic and antiarrhythmic effects in ventricular myocytes. In this study, using whole-cell patch clamp technique, we have investigated the effects of AEA on cardiac Na+/Ca2+ exchanger (NCX1)-mediated currents. AEA suppressed NCX1 with an IC50 value of 4.7μM. Both inward and outward components of exchanger currents were suppressed by AEA equally. AEA inhibition was mimicked by the metabolically stable analogue, methanandamide (metAEA, 10μM) while it was not influenced by inhibition of fatty acid amide hydrolase with 1μM URB597 incubation. The effect of AEA, was not altered in the presence of cannabinoid receptor 1 and 2 antagonists AM251 (1μM) and AM630 (1μM), respectively. In addition, inhibition by AEA remained unchanged after pertussis toxin (PTX, 2μg/ml) treatment or following the inclusion of GDP-β-S (1mM) in pipette solution. Currents mediated by NCX1 expressed in HEK-293cells were also inhibited by 10μM AEA a partially reversible manner. Confocal microscopy images indicated that the intensity of YFP-NCX1 expression on cell surface was not altered by AEA. Collectively, the results indicate that AEA directly inhibits the function of NCX1 in rat ventricular myocytes and in HEK-293cells expressing NCX1.

Chronic Intermittent Hypobaric Hypoxia Ameliorates Ischemia/Reperfusion-Induced Calcium Overload in Heart via Na+/Ca2+ Exchanger in Developing Rats

Background/Aims: Chronic intermittent hypobaric hypoxia (CIHH) protects the heart against ischemia/reperfusion (I/R) injury. This study investigated the calcium homeostasis mechanism and the role of Na⁺/Ca²⁺ exchanger (NCX) in the cardiac protective effect of CIHH in developing rats. Methods: Neonatal male rats received CIHH treatment or no treatment (control) in a hypobaric chamber simulating 3000-meter altitude for 42 days. The left ventricular function of isolated hearts was evaluated after 30 minutes of ischemia and 60 minutes of reperfusion. Myocardial infarct size, intracellular Ca²⁺ concentration ([Ca²⁺]_i), Na⁺-Ca²⁺ exchanger currents (I_Na/Ca) in ventricular myocytes, and NCX1 protein level in the sarcolemmal membrane were determined. Results: The recovery of cardiac function after I/R was improved, with the myocardial infarct size reduced, in CIHH rats compared with control rats (p<0.05). These effects were attenuated by Bay K8644, an L-type Ca²⁺ channel agonist, or ryanodine, a sarcoplasmic reticulum Ca²⁺ channel receptor activator. Furthermore, the increases in [Ca²⁺]_i during I/R were blunted in CIHH rats, but this effect was abolished by Bay K8644 or chelerythrine, a protein kinase C (PKC) inhibitor. The I_Na/Ca was decreased and the reversal potential of I_Na/Ca was shifted toward negative potential during simulated ischemia in the control cardiomyocytes (p<0.05). The inhibition of NCX1 protein expression during I/R was smaller in the CIHH rats than in the control rats (p<0.05). Conclusion: These data suggest that CIHH protects developing rat hearts during I/R by enhancing the resistance against calcium overload and by preserving normal I_Na/Ca and NCX1 protein. PKC activation might be involved in this protective process of CIHH.

The Human Red Blood Cell K+/Ca2+ Exchanger: Effect of Internal pH

The Human Red Blood Cell (hRBC) is a highly specialized cell in oxygen transport and has an intracellular pH of 7.2, a Vm of -8 mV, and an average life span of 120 days. A rise of [Ca2+]i has been associated with the senescence process of the hRBC and it has been related with the mechanical stress at the microcirculation level. We have presented evidence for the existence of a mechano-activated K+ channel (HEMKCA) and a voltage-dependent K+/Ca2+-Exchanger (1)(2). The activation process of the K+/Ca2+-Exchanger has sigmoid voltage dependence and a permeability sequence of: K+>Rb+>>Cs+ and Ca2+>Ba2+>>Mg2+. We have proposed a new hypothesis for the senescence of hRBC (The hypothesis of K+). Here, when the hRBC passes throughout the capillary suffers mechanical stress, increasing its K+ permeability, depolarizing its membrane, and consequently activating the K+/Ca2+-Exchanger, letting the Ca2+ enters the hRBC. Using the Patch-Clamp technique, we measured K+/Ca2+-Exchanger currents and the effect of pH change (from 7.2 to 5.9) in the cytoplasmic side. At an acidic internal pH we registered a complex effect, i/e. the current increased when the exchanger was in the Ca2+ entry mode, while in the reverse mode a current decrease was observed. On the other hand (i/e. at pH 6.7), the temporal development of the inactivation was slowed and saturated, from about 430ms to 490ms (14%) at 160mV. These results suggest the existence of at least one histidine residue in the exchanger, exposed to the cell interior which affects its activity, and also, the existence of two different binding sites for intracellular K+ and Ca2+. We present new evidences for the existence of a K+/Ca2+-Exchanger in the hRBC and a complex effect of changes of internal pH.(1)(2005) Biophys.J.88(1):593.(2)(2006) Biophys.J.89(1):456.

Spatial Imperfection Encodes Functional Perfection: Success and Failure of Calcium Release to Propagate Regulate Pacemaker Cell Function

Local Diastolic Ca2+releases (LCRs) contribute to sinoatrial-node-cell pacemaker function by activation of Na+/Ca2+exchanger current that accelerates diastolic depolarization. LCRs are generated by clusters of ryanodine receptors, Ca2+release units (CRUs), residing in the sarcoplasmic reticulum. CRU distribution in pacemaker cells exhibits substantial heterogeneity vs. a crystal-like distribution of cardiac muscle cells. While this heterogeneity can be noticed in images of ryanodine receptor immunofluorescence, its functional importance has never been addressed. We approached the problem by performing numerical simulations in a new sinoatrial-node-cell model that has membrane ion currents and a sub-membrane sarcoplasmic reticulum with a Ca2+pump and two-dimensional array of stochastic, diffusively coupled CRUs. With an equally-spaced, crystal-like distribution of CRUs, synchronization of Ca2+releases during diastolic depolarization undergoes an explosive, phase-like transition from individual Ca2+sparks to simultaneous occurrence of merging numerous LCRs over the entire cell, i.e. an all-or-none behavior. Since almost all CRUs fire during the phase transition (leaving no reserve), the model is not amenable to autonomic modulation of pacemaker rate. Allowing each CRU position to vary around its original crystalline position fundamentally changes the model behavior: the transition from individual sparks to propagating LCRs becomes smooth with the number and sizes of LCRs observed experimentally. Furthermore, some distant CRUs do not fire during diastolic depolarization under resting conditions, but become engaged (as a reserve) during beta-adrenergic stimulation. Thus, the model with the CRU positional variability is easily tuned to respond to autonomic modulation. We conclude: the irregularity in CRU distribution in sinoatrial-node-cells is not a noise or imperfection, but rather a functional modality of the pacemaker cells that allows them to gradually engage CRUs to satisfy the chronotropic demand of the heart at a given condition.

Mechanisms of Beat-to-Beat Regulation of Cardiac Pacemaker Cell Function by Ca2+ Cycling Dynamics

Whether intracellular Ca2+ cycling dynamics regulate cardiac pacemaker cell function on a beat-to-beat basis remains unknown. Here we show that under physiological conditions, application of low concentrations of caffeine (2–4 mM) to isolated single rabbit sinoatrial node cells acutely reduces their spontaneous action potential cycle length (CL) and increases Ca2+ transient amplitude for several cycles. Numerical simulations, using a modified Maltsev-Lakatta coupled-clock model, faithfully reproduced these effects, and also the effects of CL prolongation and dysrhythmic spontaneous beating (produced by cytosolic Ca2+ buffering) and an acute CL reduction (produced by flash-induced Ca2+ release from a caged Ca2+ buffer), which we had reported previously. Three contemporary numerical models (including the original Maltsev-Lakatta model) failed to reproduce the experimental results. In our proposed new model, Ca2+ releases acutely change the CL via activation of the Na+/Ca2+ exchanger current. Time-dependent CL reductions after flash-induced Ca2+ releases (the memory effect) are linked to changes in Ca2+ available for pumping into sarcoplasmic reticulum which, in turn, changes the sarcoplasmic reticulum Ca2+ load, diastolic Ca2+ releases, and Na+/Ca2+ exchanger current. These results support the idea that Ca2+ regulates CL in cardiac pacemaker cells on a beat-to-beat basis, and suggest a more realistic numerical mechanism of this regulation.

Distinctive sodium and calcium regulation associated with sex differences in atrial electrophysiology of rabbits

BackgroundSex and sodium/calcium regulation play critical roles in cardiac electrophysiology and atrial arrhythmogenesis. We investigated whether sodium and calcium contributed to sex differences in atrial electrophysiology. MethodsWhole-cell patch clamp techniques and the indo-1 fluorometric ratio technique were used to investigate the ionic current and intracellular calcium in single isolated male and female rabbit myocytes from the left atrium posterior wall (LAPW) and right atrium (RA). ResultsFemale LAPW (n=95) and RA (n=49) myocytes had larger cell widths (15.1±0.4 vs. 13.8±0.4μm, p<0.05; 14.9±0.6 vs. 13.5±0.4μm, p<0.05) than male LAPW (n=142) and RA (n=57) myocytes. Male LAPW myocytes (n=26) had a higher incidence (57 vs. 16%, p<0.05) of delayed afterdepolarizations (DADs) than female LAPW myocytes (n=24) but there were similar incidences (20 vs. 20%, p>0.05) of DADs in male and female RA myocytes. The late sodium current, calcium transients, and sarcoplasmic reticulum calcium contents were larger in male than female LAPW myocytes but were similar in male and female RA myocytes. However, the ICa-L and nickel-sensitive sodium/calcium exchanger currents were similar between two groups. Different from those in female myocytes, ouabain (10μM) only induced repeated atrial beats (0 to 45%, p<0.05) in male myocytes (n=11). Moreover, ranolazine (3μM) perfusion (4.5±0.6 vs. 1min, p<0.05) was required to decrease the amplitude of DADs in male but not female LAPW myocytes. ConclusionsIncreased late sodium currents and calcium contents may contribute to higher arrhythmogenesis in male LAPW myocytes.

Roles of I(f) and intracellular Ca2+ release in spontaneous activity of ventricular cardiomyocytes during murine embryonic development

In recent years, the contribution of I(f), an important pacemaker current, and intracellular Ca(2+) release (ICR) from sarcoplasmic reticulum to pacemaking and arrhythmia has been intensively studied. However, their functional roles in embryonic heart remain uncertain. Using patch clamp, Ca(2+) imaging, and RT-PCR, we found that I(f) regulated the firing rate in early and late stage embryonic ventricular cells, as ivabradine (30 µM), a specific blocker of I(f), slowed down action potential (AP) frequency. This inhibitory effect was even stronger in late stage cells, though I(f) was down-regulated. In contrast to I(f), ICR was found to be indispensable for the occurrence of APs in ventricular cells of different stages, because abolishment of ICR with ryanodine and 2-aminoethoxydiphenyl borate (2-APB), specific blockers of ryanodine receptors (RyRs) and inositol trisphosphate receptors (IP3Rs), completely abolished APs. In addition, we noticed that RyR- and IP3R-mediated ICR coexisted in early-stage ventricular cells and RyRs functionally dominated. While at late stage RyRs, but not IP3Rs, mediated ICR. In both early and late stage ventricular cells, Na-Ca exchanger current (I(Na/Ca)) mediated ICR-triggered depolarization of membrane potential and resulted in the initiation of APs. We also observed that different from I(f), which presented as the substantial component of the earlier diastolic depolarization current, application of ryanodine, and/or 2-APB slowed the late phase of diastolic depolarization. Thus, we conclude that in murine embryonic ventricular cells I(f) regulates firing rate, while RyRs and IP3Rs (early stage) or RyRs (late stage)-mediated ICR determines the occurrence of APs.

Anandamide Reduces Intracellular Ca2+ Concentration through Suppression of Na+/Ca2+ Exchanger Current in Rat Cardiac Myocytes

PurposeAnandamide, one of the endocannabinoids, has been reported to exhibit cardioprotective properties, particularly in its ability to limit the damage produced by ischemia reperfusion injury. However, the mechanisms underlying the effect are not well known. This study is to investigate whether anandamide alter Na+/Ca2+ exchanger and the intracellular free Ca2+ concentration ([Ca2+]i).MethodsNa+/Ca2+ exchanger current (INCX) was recorded and analysed by using whole-cell patch-clamp technique and [Ca2+]i was measured by loading myocytes with the fluorescent Ca2+ indicator Fura-2/AM.ResultsWe found that INCX was enhanced significantly after perfusion with simulated ischemic external solution; [Ca2+]i was also significantly increased by simulated ischemic solution. The reversal potential of INCX was shifted to negative potentials in simulated ischemic external solution. Anandamide (1–100 nM) failed to affect INCX and [Ca2+]i in normal solution. However, anandamide (1–100 nM) suppressed the increase in INCX in simulated ischemic external solution concentration-dependently and normalized INCX reversal potential. Furthermore, anandamide (100 nM) significantly attenuated the increase in [Ca2+]i in simulated ischemic solution. Blocking CB1 receptors with the specific antagonist AM251 (500 nM) failed to affect the effects of anandamide on INCX and [Ca2+]i in simulated ischemic solution. CB2 receptor antagonist AM630 (100 nM) eliminated the effects of anandamide on INCX and [Ca2+]i in simulated ischemic solution, and CB2 receptor agonist JWH133 (100 nM) simulated the effects of anandamide that suppressed the increase in INCX and [Ca2+]i in simulated ischemic solution. In addition, pretreatment with the Gi/o-specific inhibitor pertussis toxin (PTX, 500 ng/ml) eliminated the effects of anandamide and JWH133 on INCX in simulated ischemic solution.ConclusionsCollectively, these findings suggest that anandamide suppresses calcium overload through inhibition of INCX during perfusion with simulated ischemic solution; the effects may be mediated by CB2 receptor via PTX-sensitive Gi/o proteins. This mechanism is importantly involved in the anti-ischemia injury caused by endocannabinoids.

Fractional Ca2+ Currents through TRP and TRPL Channels in Drosophila Photoreceptors

Light responses in Drosophila photoreceptors are mediated by two Ca2+ permeable cation channels, transient receptor potential (TRP) and TRP-like (TRPL). Although Ca2+ influx via these channels is critical for amplification, inactivation, and light adaptation, the fractional contribution of Ca2+ to the currents (Pf) has not been measured. We describe a slow (τ ∼ 350 ms) tail current in voltage-clamped light responses and show that it is mediated by electrogenic Na+/Ca2+ exchange. Assuming a 3Na:1Ca stoichiometry, we derive empirical estimates of Pf by comparing the charge integrals of the exchanger and light-induced currents. For TRPL channels, Pf was ∼17% as predicted by Goldman-Hodgkin-Katz (GHK) theory. Pf for TRP (29%) and wild-type flies (26%) was higher, but lower than the GHK prediction (45% and 42%). As predicted by GHK theory, Pf for both channels increased with extracellular [Ca2+], and was largely independent of voltage between –100 and –30 mV. A model incorporating intra- and extracellular geometry, ion permeation, diffusion, extrusion, and buffering suggested that the deviation from GHK predictions was largely accounted for by extracellular ionic depletion during the light-induced currents, and the time course of the Na+/Ca2+ exchange current could be used to obtain estimates of cellular Ca2+ buffering capacities.

The second member of transient receptor potential-melastatin channel family protects hearts from ischemia-reperfusion injury

The second member of the transient receptor potential-melastatin channel family (TRPM2) is expressed in the heart and vasculature. TRPM2 channels were expressed in the sarcolemma and transverse tubules of adult left ventricular (LV) myocytes. Cardiac TRPM2 channels were functional since activation with H2O2 resulted in Ca(2+) influx that was dependent on extracellular Ca(2+), was significantly higher in wild-type (WT) myocytes compared with TRPM2 knockout (KO) myocytes, and inhibited by clotrimazole in WT myocytes. At rest, there were no differences in LV mass, heart rate, fractional shortening, and +dP/dt between WT and KO hearts. At 2-3 days after ischemia-reperfusion (I/R), despite similar areas at risk and infarct sizes, KO hearts had lower fractional shortening and +dP/dt compared with WT hearts. Compared with WT I/R myocytes, expression of the Na(+)/Ca(2+) exchanger (NCX1) and NCX1 current were increased, expression of the α1-subunit of Na(+)-K(+)-ATPase and Na(+) pump current were decreased, and action potential duration was prolonged in KO I/R myocytes. Post-I/R, intracellular Ca(2+) concentration transients and contraction amplitudes were equally depressed in WT and KO myocytes. After 2 h of hypoxia followed by 30 min of reoxygenation, levels of ROS were significantly higher in KO compared with WT LV myocytes. Compared with WT I/R hearts, oxygen radical scavenging enzymes (SODs) and their upstream regulators (forkhead box transcription factors and hypoxia-inducible factor) were lower, whereas NADPH oxidase was higher, in KO I/R hearts. We conclude that TRPM2 channels protected hearts from I/R injury by decreasing generation and enhancing scavenging of ROS, thereby reducing I/R-induced oxidative stress.

Effects of phosphodiesterase type 5A inhibition on intracellular calcium handling and its implications for cardioprotection and antiarrhythmogenesis

Phosphodiesterase type 5A inhibition with sildenafil improves cardiac function in heart failure. In addition, sildenafil in animal models of myocardial infarction has direct cardioprotective and antiarrhythmic effects. Sildenafil reduces L-type calcium current (Ica-L) and attenuates adrenergically driven inotropism, but effects on calcium handling are largely undetermined.Isolated adult rat ventricular myocytes were voltage clamped and calcium fluorescence measured with the indicator fura-2. Cells were paced at 0·5 Hz with depolarisations from −60 mV to +10 mV. Sarcoplasmic reticulum (SR) content was determined by application of caffeine (10–20 mmol/L) and integration of inward sodium-calcium exchanger current. Rate constants for calcium extrusion from the cell (Kcaff) and calcium uptake into the SR (KSERCA) were determined by fitting first order exponentials to decay phases of the respective calcium transients. Following the initial control protocol, a therapeutically relevant dose of sidenafil (1 μM) was applied. Differences were determined with student's paired t tests.Sildenafil reduced SR content by 26·5% (n=9, p<0·01). To a lesser extent, sildenafil also reduced calcium transient amplitude (by −13·6%, n=9, p<0·05); this was not accompanied by a reduction in KSERCA (–2.3% with sildenafil, p=0·97, n=5). Peak and integrated Ica-L were also reduced with sildenafil (–9·1% and −6·0%, respectively, n=9, p<0·05). The effect on Ica-L was also seen in adult dog ventricular myocytes (reducing peak and integrated Ica-L by 15·9% and 26·4%, respectively, p<0·05 and p<0·01, n=6, 23°C). These effects cannot be attributed to run-down effects.Sildenafil substantially reduced SR content with no reduction in KSERCA, and thus may be mediated through ryanodine receptor modulation. Such reduction in SR load may reduce proarrhythmic SR calcium release, indicating a novel mechanism through which sildenafil exerts an antiarrhythmic effect. Acute reductions in calcium transient amplitude and Ica-L with sildenafil indicate acute negative inotropic effects and may contribute to our understanding of its cardioprotective effects in the setting of hyperadrenergic drive in heart failure. FundingBritish Heart Foundation.

Neuropeptide Substance-P Modulates Electrical Characteristics of Rabbit Atrial Myocytes

Background: The cardiac nervous system plays an important role in the initiation and maintenance of atrial fibrillation (AF). Besides the classical neurotransmitters, the nerves innervating the atria also release a wide range of neuropeptides that may play a role in the pathophysiology of AF. Since the direct effect of these neuropeptides on atrial myocytes is largely unknown, we investigated the electrophysiological response of atrial myocytes to several neuropeptides, importantly substance-P (Sub-P).Methods and Results: Single left atrial myocytes from rabbit hearts were obtained by enzymatic dissociation and the effects of Sub-P, neuropeptide-Y (NPY), somatostatin-14 (SOM-14) and vasoactive intestinal peptide (VIP), were studied using patch-clamp and indo-1 fluorescence methodologies. NPY, SOM-14 and VIP (1 μM) did not affect atrial action potential (AP) characteristics. Sub-P however, reduced both resting membrane potential (RMP) and action potential amplitude, and increased action potential duration at 90% of repolarisation (APD90) by 40%. The effects on APD90 were dose-dependent and occurred from a concentration of 10 nM. Voltage-clamp analysis revealed that Sub-P significantly diminished L-type calcium current, inward rectifier K+ current (IK1) and an unknown steady-state outward current. Transient outward current, Ca2+- activated chloride current and Na+-Ca2+-exchanger current were unaffected, as was the intracellular calcium handling. The reduction in RMP and increase in APD90 are likely due to the decrease in IK1 and steady-state outward current, respectively. Our experiments indicate that the latter is probably carried by background-like potassium channels.Conclusion: Atrial myocyte electrophysiology is significantly modulated by the neuropeptide Sub-P, mainly characterized by a substantial AP prolongation due to inhibition of a background potassium current. Since lengthening of atrial repolarization is potentially anti-arrhythmic, we hypothesize that release of Sub-P may be beneficial to the prevention and/or termination of AF.

TRPM2 Channels Protect Hearts from Ischemia-Reperfusion Injury

TRPM2 channels are expressed in the heart and vasculature. To elucidate the function of TRPM2, we generated a global TRPM2-KO mouse. To evaluate whether TRPM2 channels were functional in the heart, we activated TRPM2 channels with H2O2 and demonstrated that Ca2+ influx was dependent on extracellular Ca2+ and significantly higher in WT compared to TRPM2-KO myocytes. We then examined the effects of TRPM2 channels on cardiac function, both at rest and after ischemia-reperfusion (I/R) injury. At rest, there were no differences in LV mass, heart rate, fractional shortening (FS) and +dP/dt between WT and TRPM2-KO hearts. At 2-3 days post-I/R injury, despite similar areas-at-risk and infarct sizes, TRPM2-KO hearts had lower FS and +dP/dt when compared to WT hearts. Compared to WT I/R myocytes, expression of Na+/Ca2+ exchanger (NCX1) and NCX1 current were increased, and action potential duration was prolonged in TRPM2-KO I/R myocytes. After 2 h of hypoxia followed by 30 min of reoxygenation to simulate I/R in vitro, levels of reactive oxygen species (ROS) were significantly higher in TRPM2-KO when compared to WT myocytes. Oxygen radical scavenging enzymes (SOD1 and SOD2) and their upstream regulators (FoxO1, FoxO3a and HIF-1α) were lower while NADPH oxidase (Nox2) was higher in TRPM2-KO I/R hearts. We conclude that TRPM2 channels protected hearts from I/R injury by enhancing expression of oxygen radical scavenging enzymes, thereby reducing oxidative stress associated with ischemia-reperfusion.

Effect of Internal ATP-Mg2+ Concentration on the Human Red Blood Cell K+/Ca2+ Exchanger

Little is known about the process of senescence of human erythrocytes, however it is accepted that the [Ca2 +]i increases with age. We have previously presented evidence of the existence of a novel transporter capable to account with the Ca2+ intake during the ageing process, the K+/ Ca2+ Exchanger (1)(2). The Activation process of the K+/ Ca2+ Exchanger has a sigmoidal voltage dependence and a permeability sequence: K+>Rb+>>Cs+ and Ca2+>Ba2+>> Mg2+. The ATP-Mg2+ complex is known to regulate the activity of different transporters. In order to determine the effect of the internal ATP-Mg2+ concentration on the K+/ Ca2+ Exchanger's permeability, activation and deactivation, we used Patch-clamp, changing the ATP concentration in the internal solution from 0 to 4mM. We found that exchanger currents increase in the presence of ATP-Mg2+. Interestingly, in the Ca2+ output mode the effect is dose-dependent, and no saturation is observed even at 4mM ATP-Mg2+, increasing from 48% to 200% (500µM to 4mM). On the other hand, in the Ca2+ entry mode, the permeability increases by 36% and this effect is already saturated at 500µM. Also, in this mode there is an increment of 20% in the time course of the deactivation, saturated at 500µM, whereas no such effect on the Ca2+ output mode was observed. These results suggest the existence of either two different binding sites for ATP, or one binding site that changes its affinity with the mode of exchange: Site A with low affinity in the order of mM, affecting permeability in the Ca2+ output mode, and site B, with high affinity in the order of µM, affecting permeability and the deactivation in the Ca2+ entry mode.(1)(2005) Biophys.J.88(1):593(2)(2006) Biophys.J.89(1):456