Mouse Ventricular Myocytes Research Articles

Palmitate attenuates myocardial contractility through augmentation of repolarizing Kv currents

There is considerable evidence to support a role for lipotoxicity in the development of diabetic cardiomyopathy, although the molecular links between enhanced saturated fatty acid uptake/metabolism and impaired cardiac function are poorly understood. In the present study, the effects of acute exposure to the saturated fatty acid, palmitate, on myocardial contractility and excitability were examined directly. Exposure of isolated (adult mouse) ventricular myocytes to palmitate, complexed to bovine serum albumin (palmitate:BSA) as in blood, rapidly reduced (by 54 ± 4%) mean (± SEM) unloaded fractional cell shortening. The amplitudes of intracellular Ca 2+ transients decreased in parallel. Current–clamp recordings revealed that exposure to palmitate:BSA markedly shortened action potential durations at 20%, 50%, and 90% repolarization. These effects were reversible and were occluded when the K + in the recording pipettes was replaced with Cs +, suggesting a direct effect on repolarizing K + currents. Indeed, voltage-clamp recordings revealed that palmitate:BSA reversibly and selectively increased peak outward voltage-gated K + (Kv) current amplitudes by 20 ± 2%, whereas inwardly rectifying K + (Kir) currents and voltage-gated Ca 2+ currents were unaffected. Further analyses revealed that the individual Kv current components I to,f, I K,slow and I ss, were all increased (by 12 ± 2%, 37 ± 4%, and 34 ± 4%, respectively) in cells exposed to palmitate:BSA. Consistent with effects on both components of I K,slow ( I K,slow1 and I K,slow 2) the magnitude of the palmitate-induced increase was attenuated in ventricular myocytes isolated from animals in which the Kv1.5 ( I K,slow 1) or the Kv2.1 ( I K,slow 2) locus was disrupted and I K,slow 1 or I K,slow2 is eliminated. Both the enhancement of I K,slow and the negative inotropic effect of palmitate:BSA were reduced in the presence of the Kv1.5 selective channel blocker, diphenyl phosphine oxide-1 (DPO-1).Taken together, these results suggest that elevations in circulating saturated free fatty acids, as occurs in diabetes, can directly augment repolarizing myocardial Kv currents and impair excitation–contraction coupling.

Calcineurin and cardiac function: is more or less better for the heart?

calcineurin (cn) is a Ca2+-calmodulin-activated serine-threonine phosphatase that is ubiquitously expressed and plays an important role in transducing Ca2+-dependent signals. Cn is a heterodimer composed of calmodulin binding catalytic subunit A (CnA) and a Ca2+ binding regulatory subunit B (CnB) ([

Regulation of excitation-contraction coupling in mouse cardiac myocytes: integrative analysis with mathematical modelling

BackgroundThe cardiomyocyte is a prime example of inherently complex biological system with inter- and cross-connected feedback loops in signalling, forming the basic properties of intracellular homeostasis. Functional properties of cells and tissues have been studied e.g. with powerful tools of genetic engineering, combined with extensive experimentation. While this approach provides accurate information about the physiology at the endpoint, complementary methods, such as mathematical modelling, can provide more detailed information about the processes that have lead to the endpoint phenotype.ResultsIn order to gain novel mechanistic information of the excitation-contraction coupling in normal myocytes and to analyze sophisticated genetically engineered heart models, we have built a mathematical model of a mouse ventricular myocyte. In addition to the fundamental components of membrane excitation, calcium signalling and contraction, our integrated model includes the calcium-calmodulin-dependent enzyme cascade and the regulation it imposes on the proteins involved in excitation-contraction coupling. With the model, we investigate the effects of three genetic modifications that interfere with calcium signalling: 1) ablation of phospholamban, 2) disruption of the regulation of L-type calcium channels by calcium-calmodulin-dependent kinase II (CaMK) and 3) overexpression of CaMK. We show that the key features of the experimental phenotypes involve physiological compensatory and autoregulatory mechanisms that bring the system to a state closer to the original wild-type phenotype in all transgenic models. A drastic phenotype was found when the genetic modification disrupts the regulatory signalling system itself, i.e. the CaMK overexpression model.ConclusionThe novel features of the presented cardiomyocyte model enable accurate description of excitation-contraction coupling. The model is thus an applicable tool for further studies of both normal and defective cellular physiology. We propose that integrative modelling as in the present work is a valuable complement to experiments in understanding the causality within complex biological systems such as cardiac myocytes.

Electrophysiological Development of Transplanted Embryonic Stem Cell-derived Cardiomyocytes in the Hearts of Syngeneic Mice

We undertook this study in order to investigate the electrophysiological properties of grafted mouse embryonic stem cell (ES)-derived cardiomyocytes in mouse hearts. We generated transgenic D3 ES cells that carry the alpha-myosin heavy-chain promoter-driven enhanced green fluorescent protein (EGFP) gene. ES-derived cardiomyocytes (10 days) were purified by fluorescent-activated cell sorting and then transplanted into the left ventricle of syngeneic mice. Finally, hearts were removed and the EGFP+ cardiomyocytes were dissociated from the host heart for patch clamp study. Morphological studies showed that EGFP+ cardiomyocytes were round and small before transplantation. Majority of cells were larger and longer with clear cross striations at the fourth week. Colocalization of EGFP and 4',6-diamidino-2-phenylindole-labeled nuclei of transplanted cells with cardiomyocyte markers for cardiac troponin T, as detected by immunofluorescent microscopy, indicated the survival of grafted cells. The patch clamp study revealed that the ES-derived cardiomyocytes possessed pacemaker-like action potential (AP) before transplantation. Four weeks after transplantation, grafted cells retained the characteristic of intermediate embryonic ventricular-like AP distinct from triangular AP of adult mouse ventricular myocytes, along with the loss of cellular excitability and downregulation of pacemaker current, suggesting that these grafted cells were not as mature as native ventricular cells. Transplanted ES-derived cardiomyocytes display accelerated differentiation and loss of automaticity, indicating that the long-term effectiveness of ES cell-based biological pacemakers can also be problematic.

L-Type Calcium Channel C Terminus Autoregulates Transcription

Calcium homeostasis is critical for cardiac myocyte function and must be tightly regulated. The guiding hypothesis of this study is that a carboxyl-terminal cleavage product of the cardiac L-type calcium channel (Ca(V)1.2) autoregulates expression. First, we confirmed that the Ca(V)1.2 C terminus (CCt) is cleaved in murine cardiac myocytes from mature and developing ventricle. Overexpression of full-length CCt caused a 34+/-8% decrease of Ca(V)1.2 promoter activity, and truncated CCt caused an 80+/-3% decrease of Ca(V)1.2 promoter (n=12). The full-length CCt distributes into cytosol and nucleus. A deletion mutant of CCt has a greater relative affinity for the nucleus than full-length CCt, and this is consistent with increased repression of Ca(V)1.2 promoter activity by truncated CCt. Chromatin immunoprecipitation analysis revealed that CCt interacts with the Ca(V)1.2 promoter in adult ventricular cardiac myocytes at promoter modules containing Nkx2.5/Mef2, C/EBp, and a cis regulatory module. The next hypothesis tested was that CCt contributes to transcriptional signaling associated with cellular hypertrophy. We explored whether fetal cardiac myocyte Ca(V)1.2 was regulated by serum in vitro. We tested atrial natriuretic factor promoter activity as a positive control and measured the serum response of Ca(V)1.2 promoter, protein, and L-type current (I(Ca,L)) from fetal mouse ventricular myocytes. Serum increased atrial natriuretic factor promoter activity and cell size as expected. Serum withdrawal increased Ca(V)1.2 promoter activity, mRNA, and I(Ca,L). Moreover, serum withdrawal decreased the relative nuclear localization of CCt. A combination of promoter deletion mutant analyses, and the response of promoter mutants to serum withdrawal support the conclusion that CCt, a proteolytic fragment of Ca(V)1.2, autoregulates Ca(V)1.2 expression in cardiac myocytes. These data support the novel mechanism that a mobile segment of Ca(V)1.2 links Ca handling to nuclear signaling.

Erythropoietin Protects the Heart from Ventricular Arrhythmia during Ischemia and Reperfusion via Neuronal Nitric-Oxide Synthase

Erythropoietin (EPO) is a potent cardioprotective agent in models of myocardial ischemia and reperfusion (I/R). It has been suggested recently that EPO may also reduce ventricular arrhythmia after I/R. The present study investigated the role of neuronal nitric oxide synthase (nNOS) on the antiarrhythmic effects of EPO. EPO treatment increased nNOS expression in isolated neonatal mouse ventricular myocytes. Cotreatment with the phosphatidylinositol 3 (PI3)-kinase inhibitor, LY294002 [2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride], or treatment of cardiomyocytes infected with a dominant negative adenovirus targeted to Akt1 (ADV-dnAkt1) blocked the effects of EPO on nNOS expression, suggesting that EPO regulates nNOS expression via PI3-kinase and Akt. To examine the in vivo antiarrhythmic effects of EPO, wild-type (WT) and nNOS(-/-) mice were anesthetized and, after a baseline measurement, subjected to myocardial I/R to provoke ventricular arrhythmias. Pretreatment with EPO 24 h before ischemia increased nNOS expression and significantly reduced the number of premature ventricular contractions (PVCs) and the incidence of ventricular tachycardia (VT) in WT mice. In contrast, treatment with EPO had no effect on PVCs or the incidence of VT in nNOS(-/-) mice. Furthermore, EPO treatment after ischemia significantly reduced the threshold dose of cesium chloride (CsCl) to induce VT. We conclude that EPO via nNOS protects the heart from spontaneous and CsCl-induced ventricular arrhythmia during myocardial I/R.

Cardiac Ca2+ Release Channel/RyR2 -Molecular Mechanism Of Green Tea Extract epigallocatechin-3-gallate

Consumption of green tea has been found to closely correlate with a lower risk of heart disease and reduced cardiovascular mortality. Extracted from green leaves, a group of catechin polyphenols appears to be responsible for these beneficial cardiovascular effects. Studies have shown that epigallocatechin-3-gallate (EGCG), one of the most abundant and potent catechins, can modulate myocardial contractility through some unknown mechanisms. Recent evidence suggests a role for Na+/H+ and Na+/Ca2+ exchangers in the ionotropic effects of EGCG in rat heart. Here we report that cardiac Ca2+ release channel - RyR2 serves as a possible molecular target of EGCG that may be responsible for amplified Ca2+ transients, enhanced ventricular contractility and increased cell shortening in cardiac myocytes. In single isolated mouse ventricular myocytes EGCG (5μM) increased peak systolic Ca2+ transient amplitude, increased decay of the Ca2+ transient, increased fractional shortening and increased contraction and relaxation velocities. To assess the direct interaction of EGCG with RyR2, both purified and membrane-bound RyR2 single channels were reconstituted in planar lipid bilayers. In the presence of 0.5μM EGCG, the open probability (Po) increased from Po=0.012 in the control state to Po=0.387. Using [3H]ryanodine as a probe to assess channel conformational states, our data further indicate that (1) EGCG dose-dependently enhanced junctional SR membrane-bound RyR2 channel activity and (2) EGCG increased Ca2+-induced Ca2+ activation of RyR2 (EC50 = 7.0 and 1.7 μM for control and EGCG, respectively). In summary, our data are consistent with previous reports for positive inotropic and lusitropic effects by EGCG on heart cells. In addition, our data clearly supports a role of EGCG in modulating cardiac E-C coupling through interacting with RyR2 channel complex. Supported by K01 AR51519 to GR and P01 AR52354.

Effect of Stem Cell Transplantation on the Calcium Signaling in Adult Ventricular Myocytes

Bone marrow derived stem cells (MSCs) are often discussed as a potential source for cardiac replacement tissue. Transplantation of undifferentiated cells into cardiac infarct regions has been shown to decrease infarct size and preserve cardiac function but the impact the cells have through paracrine effects or intercellular coupling remains to be determined. To determine how MSCs influence the excitability of cardiac myocytes we established a co-culture between freshly isolated mouse ventricular myocytes and dissociated MSCs. After 3 hrs of co-culture the cells were loaded with the Ca2+ indicator Fluo-4/AM and the Ca2+ handling properties of ventricular myocytes were analyzed at a stimulation frequency of 0.5 Hz. In comparison to control myocytes (ctrl) cardiomyocytes that co-localized with MSCs (co-MSC) exhibited a significantly increased Ca2+-transient amplitude (F/Fo: ctrl: 2.3 ± 0.5, n =8; co-MSC: 3.5 ± 1.2, n=4). In addition, the transient duration at 50% (APD50: ctrl: 457 ± 61 ms to co-MSC: 360 ± 33 ms); and 90% inactivation (APD90: ctrl: 1.31 ± 0.15 s; co-MSC: 1.08 ± 0.16 s) was significantly shortened. We have previously demonstrated that stem cell derived cardiomyocytes and adult myocytes can establish intercellular coupling within 1 hour of co-culture. However, in heterocellular pairs of ventricular myocytes and MSCs no change MSC [Ca2+]i could be determined upon stimulation of the myocyte. The data indicate that MSCs modulate substantially the Ca2+ signaling properties of adult ventricular myocytes and therefore could have as substantial anti-arrhythmic effect upon transplantation. It remains to be determined if intercellular coupling is necessary to establish this effect.

Rab11b GTPase Is Expressed In Ventricular Myocardium And Regulates Trafficking Of L-type Ca2+ Channels

Alterations in the density of L-type Ca2+ channels (LTCCs) have been implicated in several cardiac diseases; however, the mechanisms directing the trafficking of LTCCs have not been well defined. Nearly all steps of intracellular transport are regulated by Rab GTPases, a family of over 60 proteins with varying tissue distributions. The purpose of the present study was to determine if Rab11b, a protein localized to the endocytic recycling compartment, is expressed in the heart, and if it regulates membrane trafficking of LTCCs. RT-PCR using primers specific for Rab11b yielded a 280 base pair product from cDNA prepared from purified mouse ventricular myocytes and Western blot analysis using Rab11b-specific antibody detected a single band at 28 kDa in left ventricular lysate. To test whether Rab11b regulates trafficking of LTCCs, we expressed WT Rab11b or a dominant negative GDP-locked S25N mutant fused to GFP in HEK293 cells along with the pore forming Cav1.2 and auxiliary β2CN4 subunits. L-type currents were recorded in 10 mM Ba2+ (IBa,L) using the whole cell ruptured patch clamp technique. Expression of GFP-Rab11bS25N increased mean peak inward IBa,L from 42±3 pA/pF (n=14) in GFP-expressing control cells to 68±6 pA/pF (n=12), a 64.2% increase (p<0.005). Mean peak IBa,L of GFP-Rab11bWT cells (43±6 pA/pF, n=4) was not significantly different than the GFP control group. To determine whether Cav1.2 and Rab11b exist in a protein complex, HEK293 cells were transfected with HA-tagged Cav1.2, β2CN4, and GFP-Rab11bWT or S25N and lysates were used for immunoprecipitation with HA antibody. Interestingly, GFP-Rab11bS25N but not GFP-Rab11bWT associated with HA-Cav1.2, suggesting Rab11b preferentially interacts with Cav1.2 in its GDP-bound form. These data demonstrate that a Rab11b-dependent pathway is important for proper maintenance of LTCC density at the surface membrane.

Direct toxic effects of aqueous extract of cigarette smoke on cardiac myocytes at clinically relevant concentrations

Aims Our goal was to determine if clinically relevant concentrations of aqueous extract of cigarette smoke (CSE) have direct deleterious effects on ventricular myocytes during simulated ischemia, and to investigate the mechanisms involved. Methods CSE was prepared with a smoking chamber. Ischemia was simulated by metabolic inhibition (MI) with cyanide (CN) and 0 glucose. Adult rabbit and mouse ventricular myocyte [Ca 2+] i was measured by flow cytometry using fluo-3. Mitochondrial [Ca 2+] was measured with confocal microscopy, and Rhod-2 fluorescence. The mitochondrial permeability transition (MPT) was detected by TMRM fluorescence and myocyte contracture. Myocyte oxidative stress was quantified by dichlorofluorescein (DCF) fluorescence with confocal microscopy. Results CSE 0.1% increased myocyte contracture caused by MI. The nicotine concentration (HPLC) in 0.1% CSE was 15 ng/ml, similar to that in humans after smoking cigarettes. CSE 0.1% increased mitochondrial Ca 2+ uptake, and increased the susceptibility of mitochondria to the MPT. CSE 0.1% increased DCF fluorescence in isolated myocytes, and increased [Ca 2+] i in paced myocytes exposed to 2.0 mM CN, 0 glucose (P-MI). These effects were inhibited by the superoxide scavenger Tiron. The effect of CSE on [Ca 2+] i during P-MI was also prevented by ranolazine. Conclusions CSE in clinically relevant concentrations increases myocyte [Ca 2+] i during simulated ischemia, and increases myocyte susceptibility to the MPT. These effects appear to be mediated at least in part by oxidative radicals in CSE, and likely contribute to the effects of cigarette smoke to increase myocardial infarct size, and to decrease angina threshold.

Effects of µ-Opioid Agonist on ATP-sensitive Potassium Channel Activity in Isolated Ventricular Cardiomyocytes

It is well known that opioid agonists are released from the myocardium during hypoxia or ischemia, and that ATP-sensitive potassium channels (KATP channels) exist in the cardiac cell membrane and function as a cardioprotector preventing the myocardium from ischemic damage. In the present study, therefore, to determine whether opioid agonists are involved in the regulation of ATP-sensitive potassium channel activities, effects of the μ-opioid agonist DAMGO were examined on KATP channel activities by using excised inside-out and cell-attached patch clamp techniques in enzymatically (collagenase and protease) isolated mouse ventricular cardiac myocytes. In the excised inside-out patches, DAMGO (1∼300 μmol/L) inhibited KATP channel activities in a dose-dependent manner. KATP channel activity, which had been attenuated by the addition of ATP (100 μmol/L) to the internal solution, was not reactivated by DAMGO. The fashion of the single-channel inhibition by DAMGO was that both channel opening frequency and mean open-time were decreased, but the amplitudes of single channel currents and channel conductances were not altered. The half-maximal inhibition concentration (IC50) for DAMGO was 18 μmol/L. In the cellattached patch configuration, however, DAMGO (1∼300 μmol/L) increased dinitrophenol (50 μmol/L)induced KATP channel activities. It was inferred that the μ-opioid agonist is involved in the regulation of ATP-sensitive potassium channel activity in cardiac myocardium, agonizing (through internal target) or antagonizing (through external target) the inhibitory action of ATP in a competitive manner, thereby attenuating or enhancing the channel openings.

Modulation of the Local SR Ca2+ Release by Intracellular Mg2+ in Cardiac Myocytes

In cardiac muscle, Ca2+-induced Ca2+ release (CICR) from the sarcoplasmic reticulum (SR) defines the amplitude and time course of the Ca2+ transient. The global elevation of the intracellular Ca2+ concentration arises from the spatial and temporal summation of elementary Ca2+ release events, Ca2+ sparks. Ca2+ sparks represent the concerted opening of a group of ryanodine receptors (RYRs), which are under the control of several modulatory proteins and diffusible cytoplasmic factors (e.g., Ca2+, Mg2+, and ATP). Here, we examined by which mechanism the free intracellular Mg2+ ([Mg2+]free) affects various Ca2+ spark parameters in permeabilized mouse ventricular myocytes, such as spark frequency, duration, rise time, and full width, at half magnitude and half maximal duration. Varying the levels of free ATP and Mg2+ in specifically designed solutions allowed us to separate the inhibition of RYRs by Mg2+ from the possible activation by ATP and Mg2+-ATP via the adenine binding site of the channel. Changes in [Mg2+]free generally led to biphasic alterations of the Ca2+ spark frequency. For example, lowering [Mg2+]free resulted in an abrupt increase of spark frequency, which slowly recovered toward the initial level, presumably as a result of SR Ca2+ depletion. Fitting the Ca2+ spark inhibition by [Mg2+]free with a Hill equation revealed a Ki of 0.1 mM. In conclusion, our results support the notion that local Ca2+ release and Ca2+ sparks are modulated by Mg2+ in the intracellular environment. This seems to occur predominantly by hindering Ca2+-dependent activation of the RYRs through competitive Mg2+ occupancy of the high-affinity activation site of the channels. These findings help to characterize CICR in cardiac muscle under normal and pathological conditions, where the levels of Mg2+ and ATP can change.

Abstract 1531: SCN5A Promoter and Enhancer Variants associated with Atrial Fibrillation

We have recently reported that 5.9% of patients with atrial fibrillation (AF) have rare variants in the coding region of the predominant cardiac sodium channel gene SCN5A . In this study, we tested the hypothesis that AF susceptibility is also influenced by variants in the SCN5A core promoter and short non-coding nucleotide sequences highly conserved across species (CNS) that we previously identified. We resequenced the SCN5A core promoter region (~3 kb) and CNS regions in intron 1 (&lt;1 kb for each) in 376 patients with AF (111 patients with lone AF and 234 patients with AF and cardiac disease) and 47 ethnically-defined controls. The promoter activity of variants showing different frequencies in patients with AF compared to controls was studied using promoter-reporter assays in CHO cells and in neonatal mouse ventricular myocytes. Identification of variants Four common variants were identified in the core promoter in controls, and one of the variants (c.−225– 834 T&gt;C) was significantly more common in patients with AF than in controls (minor allele frequency: lone AF, 17%; AF and heart disease, 17%; control, 7%; P&lt;0.05 vs. control for each). In addition, 10 novel rare promoter variants were identified in patients with AF and were absent in controls. In CNS28, a 406 bp region in intron 1 that includes three TEF-1 binding sites, one variant (c.−53±15307 C&gt;T) which is predicted to disrupt one of the TEF-1 binding sites was identified in 7 out of 376 patients with AF (1 with lone AF, 6 with AF and heart disease), but not in controls. Functional study There was no difference in promoter activity between wild-type and c.−225– 834 T&gt;C SCN5A core promoters expressed in CHO cells. Wild-type CNS28 coexpressed with the SCN5A core promoter increased reporter activity by 77 ± 14% (P=0.03) in CHO cells and 261 ± 8% (P=0.001) in cardiomyocytes, compared to SCN5A promoter alone. However, c.−53+15307 C&gt;T CNS28 showed smaller effects on promoter activity in both CHO cells (52 ± 2 % of wild-type, P&lt;0.05) and cardiomyocytes (62 ± 8%, P&lt;0.05) compared to wild-type CNS28. Variants in the SCN5A promoter and an enhancer region in intron 1 were associated with AF. Variability in SCN5A transcription may affect AF susceptibility.

Abstract 1875: L-type Calcium Channel C-terminus Suppresses L-type Calcium Channel Transcription in Cardiomyocytes

Calcium homeostasis is critical for cardiac myocyte function and must be tightly regulated. In this study we test the hypothesis that a mobile carboxyl-terminal cleavage product (CCt) of the cardiac L-type calcium channel (Ca V 1.2) auto-regulates expression. Our ongoing in vivo studies showed that persistent Ca V 1.2 block induced an up-regulation of Ca V 1.2 mRNA, protein and I Ca,L . In the present study we test the hypothesis that CCt contributes to transcriptional signaling associated with cellular hypertrophy. We created eGFP-CCt fusion constructs to explore CCt regulation of the Ca V 1.2 promoter, and to report sub-cellular localization of CCt in cardiac myocytes. Developing cardiac myocytes were cultured in 10% FBS. Over-expression of native, full-length CCt causes a 34 ± 8% decrease of Ca V 1.2 promoter activity, and truncated CCt caused 80 ± 3% decrease (n = 12). Wild-type CCt distributes into cytosol and nucleus, with a greater presence in the nucleus than cytosol. A deletion mutant of CCt was also tested. Truncated CCt has a greater relative affinity for the nucleus than wild-type CCt. This is consistent with increased suppression of Ca V 1.2 promoter activity by truncated CCt. Chromatin immunoprecipitation (ChIP) assay showed that CCt interacts with the Ca V 1.2 promoter in adult ventricular cardiac myocytes at promoter modules containing NKX2.5/MEF2, C/EBp, and a cis regulatory module identified in silico . We next explored whether fetal cardiac myocyte Ca V 1.2 was regulated by serum in vitro . We tested ANF promoter activity as a positive control, and measured serum-response, versus serum-withdrawal on Ca V 1.2 promoter, mRNA, protein, and L-type current (I Ca,L ) from fetal mouse ventricular myocytes. Serum increased ANF promoter activity and cell size. Serum withdrawal increased Ca V 1.2 promoter activity, Ca V 1.2 protein, and I Ca,L . Moreover, serum withdrawal decreased the relative nuclear localization of CCt. A combination of promoter deletion mutant analysis, and the response of promoter mutants to serum withdrawal support the conclusion that CCt, a proteolytic fragment of Ca V 1.2 auto-regulates Ca V 1.2 expression in cardiac myocytes. These data support the novel mechanism that a mobile segment of Ca V 1.2 links Ca handling to nuclear signaling.

Abstract 1503: Knockdown of Mog1 Expression Reduces Sodium Currents by Reducing the Trafficking of Cardiac Sodium Channel Na V 1.5 to the Cell Membrane

The cardiac sodium channel (Na v 1.5) plays a significant role in cardiac physiology and leads to cardiac arrhythmias and sudden death when mutated. Modulation of Na v 1.5 activity can also arise from changes to accessory subunits or proteins. Our laboratory has recently reported that MOG1, a small protein that is highly conserved from yeast to humans, is a co-factor of Na v 1.5. Increased MOG1 expression has been shown to increase Na v 1.5 current density. In adult mouse ventricular myocytes, these two proteins were found to be co-localized at the intercalated discs. Here, we further characterize the regulatory role of MOG1 using the RNA interference technique. Sodium current was recorded in voltage-clamp mode from a holding potential of −100 mV and activated to −20 mV. In 3-day old mouse neonatal ventricular cells transfected with siRNA against mouse MOG1 decreased sodium current densities (pA/pF) compared to control or scramble siRNA treated cells (−10.2±3.3, n=11 vs. −165±16, n=20 or −117.9±11.7, n=11). A similar reduction in sodium current was observed in mammalian expression system consisting of HEK293 cells stably expressing human Na v 1.5, by transfecting siRNAs against either human or mouse MOG1 (−41.7±8.3, n=7 or, −82.6±9.6, n=7 vs. −130.6±11.5, n=7; −111.5±8.5, n=7, respectively). Immunocytochemistry revealed that the expression of MOG1 and Na v 1.5 were decreased in both HEK and neonatal cells when compared to scramble siRNAs or control groups. These results show that MOG1 is an essential co-factor for Na v 1.5 by way of a channel trafficking. Such interactions between MOG1 and Na v 1.5 suggest that early localization of MOG1 on the membrane of neonatal cardiomyocytes may be necessary for proper localization and the distribution of Na v 1.5 during cardiac development. This research has received full or partial funding support from the American Heart Association, AHA National Center.

Nitric oxide inhibits Kv4.3 and human cardiac transient outward potassium current (Ito1)

Chronic atrial fibrillation (CAF) is characterized by a shortening of the plateau phase of the action potentials (AP) and a decrease in the bioavailability of nitric oxide (NO). In this study, we analysed the effects of NO on Kv4.3 (I(Kv4.3)) and on human transient outward K(+) (I(to1)) currents as well as the signalling pathways responsible for them. We also analysed the expression of NO synthase 3 (NOS3) in patients with CAF. I(Kv4.3) and I(to1) currents were recorded in Chinese hamster ovary cells and in human atrial and mouse ventricular dissociated myocytes using the whole-cell patch clamp. The expression of NOS3 was analysed by western blotting. AP were recorded using conventional microelectrode techniques in mouse atrial preparations. NO and NO donors inhibited I(Kv4.3) and human I(to1) in a concentration- and voltage-dependent manner (IC(50) for NO: 375.0 +/- 48 nM) as a consequence of the activation of adenylate cyclase and the subsequent activation of the cAMP-dependent protein kinase and the serine-threonine phosphatase 2A. The density of the I(to1) recorded in ventricular myocytes from wild-type (WT) and NOS3-deficient mice (NOS3(-/-)) was not significantly different. Furthermore, the duration of atrial AP repolarization in WT and NOS3(-/-) mice was not different. The increase in NO levels to 200 nM prolonged the plateau phase of the mouse atrial AP and lengthened the AP duration measured at 20 and 50% of repolarization of the human atrial CAF-remodelled AP as determined using a mathematical model. However, the expression of NOS3 was not modified in left atrial appendages from CAF patients. Our results suggested that the increase in the atrial NO bioavailability could partially restore the duration of the plateau phase of CAF-remodelled AP by inhibiting the I(to1) as a result of the activation of non-canonical enzymatic pathways.

Syntaxin-1A inhibition of P-1075, cromakalim, and diazoxide actions on mouse cardiac ATP-sensitive potassium channel

Syntaxin (Syn)-1A binds sulfonylurea receptor (SUR) nucleotide binding folds of cardiac myocyte (SUR2A) and islet beta-cells (SUR1) to inhibit ATP-sensitive potassium (K(ATP)) channels. We further reported that Syn-1A reduced the potency and efficacy of beta-cell-specific K(ATP) channel openers (KCOs). Here, we examined whether Syn-1A would influence non-specific (diazoxide) and SUR2-specific KCOs [N-cyano-N'-(1,1-dimethylpropyl)-N''-3-pyridylguanidine (P-1075) and cromakalim] on cardiac myocyte K(ATP) channels activation. Confocal microscopy and Western blotting verified the presence of both Syn-1A and -1B expressions on rodent cardiac ventricular myocytes. Inside-out patch-clamp electrophysiology was utilized to examine the effects of these syntaxins on K(ATP) macroscopic currents activated by various KCOs from a stable cell line expressing the potassium inward rectifier 6.2 (Kir6.2)/SUR2A and from C57BL/6 male mouse ventricular myocytes. Syn-1A inhibited the current amplitude activated by P-1075, cromakalim and diazoxide via its H3 but not Habc domain. Syn-1B exhibited similar inhibitory effects on P-1075 activation of K(ATP) currents. In examining for direct effects of Syn-1A on the KCO binding to cardiac SUR2 receptors, we found that Syn-1A did not directly affect [(3)H]-P-1075 binding to rat cardiac membrane SUR2A at maximum binding capacity, but was able to mildly reduce the affinity of cold P-1075 and cromakalim to displace [(3)H]-P-1075 binding. In conclusion, Syn-1A (and Syn-1B) could inhibit K(ATP) currents activated by SUR2A-acting KCOs. Potential fluctuations in the levels of these syntaxins in the myocardium may affect the therapeutic effectiveness of cardiac KCOs.

Reactive Oxygen Species-Induced Activation of p90 Ribosomal S6 Kinase Prolongs Cardiac Repolarization Through Inhibiting Outward K + Channel Activity

p90 ribosomal S6 kinase (p90RSK) is activated in cardiomyopathies caused by conditions such as ischemia/reperfusion injury and diabetes mellitus in which prolongation of cardiac repolarization and frequent arrhythmias are common. Molecular mechanisms underlying the electric remodeling in cardiac diseases are largely unknown. In the present study, we determined the role of p90RSK activation in the modulation of voltage-gated K+ channel activity determining cardiac repolarization. Mice with increased cardiac p90RSK activity due to transgenic expression of p90RSK (p90RSK-Tg) had prolongation of QT intervals and of ventricular myocyte action potential durations. Fast transient outward K+ current (I(to,f)), slow delayed outward K+ current (I(K,slow)), and steady-state K+ current (I(SS)) were significantly decreased in p90RSK-Tg mouse ventricular myocytes. mRNA levels of Kv4.3, Kv4.2, Kv1.5, Kv2.1, and KChIP2 from ventricles between p90RSK-Tg and nontransgenic littermate control mice were similar, as assessed by quantitative reverse transcriptase-polymerase chain reaction, indicating that p90RSK regulates voltage-gated K+ channels through posttranslational modification. Kv4.3- and Kv1.5- rather than Kv4.2- and Kv2.1-encoded channels in HEK 293 cells were inhibited by p90RSK. In vitro phosphorylation analysis showed that Kv4.3 was phosphorylated by p90RSK at 2 conserved sites, Ser516 and Ser550. p90RSK expression significantly inhibited Kv4.3- and Kv4.3 and KChIP2-encoded channel activities in HEK 293 cells, whereas p90RSK's effects were blocked by amino acid mutation(s) at phosphorylation site(s) in Kv4.3. Hydrogen peroxide, a mediator of induced cardiac p90RSK activation in ischemia/reperfusion injury and diabetes mellitus, had effects similar to those of p90RSK on Kv4.3- or Kv4.3- and KChIP2-encoded channels. Fluoromethylketone, a specific p90RSK inhibitor, abolished hydrogen peroxide effects. These findings indicate that p90RSK activation is critical for reactive oxygen species-mediated inhibition of voltage-gated K+ channel activity and leads to prolongation of cardiac repolarization.

S1P1 Receptor Localization Confers Selectivity for Gi-mediated cAMP and Contractile Responses

Adult mouse ventricular myocytes express S1P(1), S1P(2), and S1P(3) receptors. S1P activates Akt and ERK in adult mouse ventricular myocytes through a pertussis toxin-sensitive (G(i/o)-mediated) pathway. Akt and ERK activation by S1P are reduced approximately 30% in S1P(3) and 60% in S1P(2) receptor knock-out myocytes. With combined S1P(2,3) receptor deletion, activation of Akt is abolished and ERK activation is reduced by nearly 90%. Thus the S1P(1) receptor, while present in S1P(2,3) receptor knock-out myocytes, is unable to mediate Akt or ERK activation. In contrast, S1P induces pertussis toxin-sensitive inhibition of isoproterenol-stimulated cAMP accumulation in both WT and S1P(2,3) receptor knock-out myocytes demonstrating that the S1P(1) receptor can functionally couple to G(i). An S1P(1) receptor selective agonist, SEW2871, also decreased cAMP accumulation but failed to activate ERK or Akt. To determine whether localization of the S1P(1) receptor mediates this signaling specificity, methyl-beta-cyclodextrin (MbetaCD) treatment was used to disrupt caveolae. The S1P(1) receptor was concentrated in caveolar fractions, and associated with caveolin-3 and this localization was disrupted by MbetaCD. S1P-mediated activation of ERK or Akt was not diminished but inhibition of cAMP accumulation by S1P and SEW2871 was abolished by MbetaCD treatment. S1P inhibits the positive inotropic response to isoproterenol and this response is also mediated through the S1P(1) receptor and lost following caveolar disruption. Thus localization of S1P(1) receptors to caveolae is required for the ability of this receptor to inhibit adenylyl cyclase and contractility but compromises receptor coupling to Akt and ERK.

PDE4B is the predominant PDE4 isoform regulating the L-type Ca 2+ current in mouse ventricular myocytes

PDE4B is the predominant PDE4 isoform regulating the L-type Ca 2+ current in mouse ventricular myocytes