Left Atrial Myocytes Research Articles

Association between connexin 40 and potassium voltage-gated channel subfamily A member 5 expression in the atrial myocytes of patients with atrial fibrillation.

Structural and electrical remodeling within the atrium mediate the pathogenesis of atrial fibrillation (AF). Two key genes that sever a role in this remodeling are connexin 40 (Cx40) and potassium voltage-gated channel subfamily A member 5 (KCNA5), respectively. Electrical remodeling is considered to induce structural remodeling during AF. In the present study, the left atrial appendage section and atrial myocytes of patients with AF were evaluated. It was observed that Cx40 and KCNA5 mRNA (P<0.05) and protein (P<0.01) expression was significantly downregulated in AF compared with rheumatic heart disease. In addition, a positive correlation between the mRNA expression Cx40 and KCNA5 was observed in the atrial myocytes of patients with AF (P<0.05; r=0.42). The association between Cx40 and KCNA5 expression was subsequently investigated in primary cultured atrial myocytes using siRNA transfection. In atrial myocytes, downregulation of Cx40 inhibited the expression of KCNA5. Similarly, silencing of KCNA5 suppressed the expression of Cx40. These results indicate that synergistic regulation may occur between Cx40 and KCNA5 expression. Furthermore, the combined effects of electrical and structural remodeling in the atrial myocytes of patients with AF may contribute to the pathogenesis of AF.

Triggered intracellular calcium waves in dog and human left atrial myocytes from normal and failing hearts.

Abnormal intracellular Ca2+ cycling contributes to triggered activity and arrhythmias in the heart. We investigated the properties and underlying mechanisms for systolic triggered Ca2+ waves in left atria from normal and failing dog hearts. Intracellular Ca2+ cycling was studied using confocal microscopy during rapid pacing of atrial myocytes (36 °C) isolated from normal and failing canine hearts (ventricular tachypacing model). In normal atrial myocytes (NAMs), Ca2+ waves developed during rapid pacing at rates ≥ 3.3 Hz and immediately disappeared upon cessation of pacing despite high sarcoplasmic reticulum (SR) load. In heart failure atrial myocytes (HFAMs), triggered Ca2+ waves (TCWs) developed at a higher incidence at slower rates. Because of their timing, TCW development relies upon action potential (AP)-evoked Ca2+ entry. The distribution of Ca2+ wave latencies indicated two populations of waves, with early events representing TCWs and late events representing conventional spontaneous Ca2+ waves. Latency analysis also demonstrated that TCWs arise after junctional Ca2+ release has occurred and spread to non-junctional (cell core) SR. TCWs also occurred in intact dog atrium and in myocytes from humans and pigs. β-adrenergic stimulation increased Ca2+ release and abolished TCWs in NAMs but was ineffective in HFAMs making this a potentially effective adaptive mechanism in normals but potentially arrhythmogenic in HF. Block of Ca-calmodulin kinase II also abolished TCWs, suggesting a role in TCW formation. Pharmacological manoeuvres that increased Ca2+ release suppressed TCWs as did interventions that decreased Ca2+ release but these also severely reduced excitation-contraction coupling. TCWs develop during the atrial AP and thus could affect AP duration, producing repolarization gradients and creating a substrate for reentry, particularly in HF where they develop at slower rates and a higher incidence. TCWs may represent a mechanism for the initiation of atrial fibrillation particularly in HF.

221 Altered biophysical properties of the voltage-gated sodium channels in mouse atrial and ventricular cardiomyocytes

IntroductionSeveral antiarrhythmic drugs target the cardiac sodium current INA. There is an increasing interest in atrial-specific ion channel inhibition, e.g. to allow selective antiarrhythmic drug development for the treatment of...

Icariin, a Novel Blocker of Sodium and Calcium Channels, Eliminates Early and Delayed Afterdepolarizations, As Well As Triggered Activity, in Rabbit Cardiomyocytes.

Icariin, a flavonoid monomer from Herba Epimedii, has confirmed pharmacological and biological effects. However, its effects on arrhythmias and cardiac electrophysiology remain unclear. Here we investigate the effects of icariin on ion currents and action potentials (APs) in the rabbit myocardium. Furthermore, the effects of icariin on aconitine-induced arrhythmias were assessed in whole rabbits. Ion currents and APs were recorded in voltage-clamp and current-clamp mode in rabbit left ventricular myocytes (LVMs) and left atrial myocytes (LAMs), respectively. Icariin significantly shortened action potential durations (APDs) at 50 and 90% repolarization (APD50 and APD90) and reduced AP amplitude (APA) and the maximum upstroke velocity (Vmax) of APs in LAMs and LVMs; however, icariin had no effect on resting membrane potential (RMP) in these cells. Icariin decreased the rate-dependence of the APD and completely abolished anemonia toxin II (ATX-II)-induced early afterdepolarizations (EADs). Moreover, icariin significantly suppressed delayed afterdepolarizations (DADs) and triggered activities (TAs) elicited by isoproterenol (ISO, 1 μM) and high extracellular calcium concentrations ([Ca2+]o, 3.6 mM) in LVMs. Icariin also decreased INaT in a concentration-dependent manner in LAMs and LVMs, with IC50 values of 12.28 ± 0.29 μM (n = 8 cells/4 rabbits) and 11.83 ± 0.92 μM (n = 10 cells/6 rabbits; p > 0.05 vs. LAMs), respectively, and reversed ATX-II-induced INaL in a concentration-dependent manner in LVMs. Furthermore, icariin attenuated ICaL in a dose-dependent manner in LVMs. The corresponding IC50 value was 4.78 ± 0.89 μM (n = 8 cells/4 rabbits), indicating that the aforementioned current in LVMs was 2.8-fold more sensitive to icariin than ICaL in LAMs (13.43 ± 2.73 μM; n = 9 cells/5 rabbits). Icariin induced leftward shifts in the steady-state inactivation curves of INaT and ICaL in LAMs and LVMs but did not have a significant effect on their activation processes. Moreover, icariin had no effects on IK1 and IKr in LVMs or Ito and IKur in LAMs. These results revealed for the first time that icariin is a multichannel blocker that affects INaT, INaL and ICaL in the myocardium and that the drug had significant inhibitory effects on aconitine-induced arrhythmias in whole rabbits. Therefore, icariin has potential as a class I and IV antiarrhythmic drug.

Andrographolide inhibits arrhythmias and is cardioprotective in rabbits.

Andrographolide has a protective effect on the cardiovascular system. To study its cardic-electrophysiological effects, action potentials and voltage-gated Na+ (INa), Ca2+ (ICaL), and K+ (IK1, IKr, Ito and IKur) currents were recorded using whole-cell patch clamp and current clamp techniques. Additionally, the effects of andrographolide on aconitine-induced arrhythmias were assessed on electrocardiograms in vivo. We found that andrographolide shortened action potential duration and reduced maximum upstroke velocity in rabbit left ventricular and left atrial myocytes. Andrographolide attenuated rate-dependence of action potential duration, and reduced or abolished delayed afterdepolarizations and triggered activities induced by isoproterenol (1 μM) and high calcium ([Ca2+]o=3.6 mM) in left ventricular myocytes. Andrographolide also concentration-dependently inhibited INa and ICaL, but had no effect on Ito, IKur, IK1, or IKr in rabbit left ventricular and left atrial myocytes. Andrographolide treatment increased the time and dosage thresholds of aconitine-induced arrhythmias, and reduced arrhythmia incidence and mortality in rabbits. Our results indicate that andrographolide inhibits cellular arrhythmias (delayed afterdepolarizations and triggered activities) and aconitine-induced arrhythmias in vivo, and these effects result from INa and ICaL inhibition. Andrographolide may be useful as a class I and IV antiarrhythmic therapeutic.

Biophysical and molecular comparison of sodium current in cells isolated from canine atria and pulmonary vein.

The collar of the pulmonary vein (PV) is the focal point for the initiation of atrial arrhythmias, but the mechanisms underlying how PV cells differ from neighboring left atrial tissue are unclear. We examined the biophysical and molecular properties of INa in cells isolated from the canine pulmonary sleeve and compared the properties to left atrial tissue. PV and left atrial myocytes were isolated and patch clamp techniques were used to record INa. Action potential recordings from either tissue type were made using high-resistance electrodes. mRNA was determined using quantitative RT-PCR and proteins were determined by Western blot. Analysis of the action potential characteristics showed that PV tissue had a lower Vmax compared with left atrial tissue. Fast INa showed that current density was slightly lower in PV cells compared with LA cells (-96±18.7 pA/pF vs. -120±6.7 pA/pF, respectively, p<0.05). The recovery from inactivation of INa in PV cells was slightly slower but no marked difference in steady-state inactivation was noted. Analysis of late INa during a 225-ms pulse showed that late INa was significantly smaller in PV cells compared to LA cells at all measured time points into the pulse. These results suggest PV cells have lower density of both peak and late INa. Molecular analysis of Nav1.5 and the four beta subunits showed lower levels of Nav1.5 as well as Navβ1 subunits, confirming the biophysical findings. These data show that a lower density of INa may lead to depression of excitability and predispose the PV collar to re-entrant circuits under pathophysiological conditions.

Increment of late sodium currents in the left atrial myocytes and its potential contribution to increased susceptibility of atrial fibrillation in castrated male mice

The incidence of atrial fibrillation (AF) is correlated with decreased levels of testosterone in elderly men. Late sodium current may exert a role in AF pathogenesis. The purpose of this study was to explore the effect of testosterone deficiency on AF susceptibility and the therapeutic effect of late sodium current inhibitors in mice. Male ICR mice (5 weeks old) were castrated to establish a testosterone deficiency model. One month after castration, dihydrotestosterone 5 mg/kg was administered subcutaneously for 2 months. Serum total testosterone level was assessed by enzyme-linked immunosorbent assay. High-frequency electrical stimulation was used to induce atrial arrhythmias. Whole-cell patch-clamp technique was used to for single-cell electrophysiologic study. Serum dihydrotestosterone levels of castration mice declined significantly but recovered with administration of exogenous dihydrotestosterone. In comparison with sham mice, the number of AF episodes significantly increased by 13.5-fold, AF rate increased by 3.75-fold, and AF duration prolonged in castrated mice. Dihydrotestosterone administration alleviated the occurrence of AF. Action potential duration at both 50% and 90% repolarization were markedly increased in castrated mice compared to sham controls. The late sodium current was enhanced in castrated male mice. These alterations were alleviated by treatment with dihydrotestosterone. Systemic application of the INa-L inhibitors ranolazine, eleclazine, and GS967 inhibited the occurrence of AF in castrated mice. Testosterone deficiency contributed to the increased late sodium current, prolonged action potential repolarization, and increased susceptibility to AF. Blocking of late sodium current is beneficial against the occurrence of AF in castrated mice.

Atrial Fibrillation in Hypertensive Heart Disease is Associated with Distinct Patterns of Electrical Remodeling in the Left and Right Atria

Atrial fibrillation (AF) is a major complication in hypertensive heart disease and has been linked to alterations in angiotensin II (AngII) signaling; however, the mechanisms underlying the increase in susceptibility to AF in the presence of AngII are poorly understood. The goal of this study was to characterize changes in atrial electrophysiology in mice treated chronically with AngII. AngII treated mice developed elevated blood pressure and cardiac hypertrophy in comparison to saline treated controls. In intracardiac programmed stimulation studies, 50% of AngII treated mice (n=10) were induced into AF vs. 0% of saline treated mice (P<0.05; n=9). P wave duration, a measure of atrial conduction, was prolonged in AngII treated mice (P<0.001; n=12 mice). Electrical remodeling following AngII treatment was investigated by patch-clamping atrial myocytes. In right atrial myocytes, AngII prolonged action potential (AP) duration compared to saline (P<0.05; n=9-11 myocytes) without changes in AP upstroke velocity (Vmax; P=0.66) or overshoot (OS; P=0.53). Strikingly, in left atrial myocytes AngII treatment resulted in greater AP prolongation (P<0.001; n=12-14 myocytes) and reductions (P<0.05) in AP Vmax and AP overshoot vs. saline controls. Voltage clamp studies demonstrate that in right atrial myocytes (n=21-26) the transient outward potassium current (Ito) was reduced (P<0.05) following AngII treatment. Similarly, 4-aminopyradine (4-AP; 100 µM) reduced right atrial K+ currents (P<0.05) indicating a reduction in the ultra-rapid delayed rectifier K+ current (IKur) after AngII treatment. In left atrial myocytes, (n=11-13) Ito and 4-AP sensitivity were both reduced (P<0.05) following AngII treatment. The reduction in Ito was larger (P<0.05) in left vs. right atrial myocytes. Our study demonstrates that chronic AngII treatment creates a substrate for AF in association with distinct electrophysiological alterations in right and left atrial myocytes.

Electrophysiological Effects of Triggered Calcium Waves on the Action Potential in Left Atrial Myocytes from Normal and Failing Dog Heart

We previously reported that triggered Ca waves (TCWs) occur in dog atrial myocytes during rapid pacing. The goal of this study was to investigate the effect of TCWs on APs during rapid pacing in left atrial myocytes from both normal and failing hearts (rapid RV pacing model). Intracellular Ca and AP recordings were made in atrial myocytes isolated from normal and failing (HF) dog hearts loaded with Ca-sensitive fluorescent dyes and stimulated at increasingly rapid rates (1-5Hz). Both normal and HF atrial myocytes developed TCWs which increased action potential duration (APD) and reduced maximum diastolic potential (MDP) throughout the duration of each TCW. Depending on the timing of the TCW, both MDP and APD could be affected during a single or multiple cycles. TCWs developed at slower rates in HF than in normals so changes in AP and MDP also occurred at slower rates making TCWs of potentially greater importance to arrhythmogenesis in HF. A subset of TCWs failed to propagate along the cell length but were short enough to occur during only 1-2 APs. These events (triggered Ca flares, TCFs) produce the same overall electrophysiological effects as TCWs that propagate along the cell length. In summary, TCFs and TCWs cause APD prolongation and cell depolarization in normal and diseased atrial myocytes probably by inducing inward Na+-Ca2+ exchange current in response to these Ca release events. This depolarization and slowing of repolarization is likely to establish repolarization gradients and cause reentry thus promoting atrial arrhythmogenesis such as atrial fibrillation.

Atrial Spceific Pitx2 Insufficiencyincreases the Frequency of Calcium Sparks, Waves, and After-Depolarizations in Mouse Atrial Myocytes

The transcription factor Pitx2 has been proposed as a molecular link between single nucleotide polymorphisms on chromosome 4q25 and increased risk of atrial fibrillation in carriers of the risk variant. Since atrial fibrillation has been associated with calcium handling disturbances in isolated atrial myocytes, we here tested the hypothesis that Pitx2 insufficiency alters the calcium homeostasis in atrial myocytes.To test this hypothesis, we used right atrial myocytes from a transgenic mouse model with inducible atrial specific Pitx2 deletion. Spontaneous calcium release was detected with confocal calcium imaging and resulting ion currents or membrane depolarizations were measured with patch-clamp technique in myocytes from wild-type (Pitx2+/+) and heterozygous Pitx2+/- mice.Calcium imaging revealed that the frequency of calcium sparks (2.1±0.7 vs 0.2±0.1 events/cell/s, p<0.05) and waves (3.2±1.2 vs. 0 events/min, p<0.05) were significantly higher in Pitx2+/- mice. This was also true for the frequency of transient inward currants activated by calcium waves (2.8±0.5 vs 1.2±0.5 events/min, p<0.05). The higher frequency was of spontaneous calcium release was likely due to a higher caffeine releasable sarcoplasmic reticulum calcium load in the Pitx2+/- mice (23.8±5.8 vs 14.4±1.9 amol/pF, p<0.05). Moreover, only myocytes from Pitx2+/- had spontaneous action potentials at a resting potential of −80 mV (0.5±0.4/min). At −60 mV the frequency of spontaneous action potentials was 5.3±1.8/min for Pitx2+/- and 0.4±1.2/min for Pitx2+/+ mice. Importantly, spontaneous after-potentials were also recorded in myocytes subjected to field stimulation, but only in myocytes from Pitx2+/- mice. These results were not unique to right atrial myocytes as the calcium spark frequency was also 3.9 fold higher in left atrial myocytes from Pitx2+/- mice (p<0.001).Together, these results demonstrate that Pitx2 insufficiency promote both spontaneous calcium release and spontaneous action potentials. Both of these features are hallmarks of myocytes from patients with atrial fibrillation, suggesting that Pitx2-mediated modulation of intracellular calcium handling plays an important role in electrophysiological processes associated with atrial fibrillation.

Atrial-Ventricular Differences in Voltage-Gated Na+ Currents of Rabbit Cardiomyocytes

Atrial fibrillation (AF) is the most common clinical arrhythmia. Voltage-gated Na+ channel blockers (NCB) are amongst the most effective antiarrhythmic drugs for AF but carry significant risk of ventricular tachyarrhythmia. It has been suggested that differences in Na+ channel characteristics between atrial and ventricular cells can be exploited to develop atrial-selective antiarrhythmic drugs. Ranolazine, an antianginal drug, is an NCB that possesses antiarrhythmic properties and has been suggested to show atrial selectivity the basis for which remains unclear. In this study, atrial-ventricular differences in the voltage-gated Na+ current (INa) and its block by ranolazine were investigated. INa was recorded from rabbit left atrial and right ventricular myocytes at room temperature using the whole-cell patch clamp technique. Recording solutions contained low equimolar Na+ (10 mM) and nifedipine (20 μM) was used to block Ca2+ currents. INa-voltage relations and steady-state voltage-dependent inactivation curves were obtained from a holding potential (Vhold) of −120 mV. INa conductance density was greater in atrial than in ventricular cells (2.0±0.1 vs 1.1±0.1 nS/pF; P<0.0001). The voltage for half-maximal (V1/2, act) activation for atrial and ventricular myocytes respectively was −46.9±0.7 mV and −39.4±1.2 mV (P<0.0001) and the V1/2, inact inactivation was −102.1±0.4 mV and −90.4±0.2 mV (P<0.0001) respectively. The use-dependence of INa block by ranolazine (30 μM) was investigated using trains of 40 depolarising pulses at various diastolic intervals (DI; 110, 60 & 40 ms) from different Vhold (−120, −110 & −100 mV) in atrial and ventricular myocytes. While both cell types exhibited block that was both use- and voltage-dependent, ranolazine was effective at more negative voltages in atrial than in ventricular cells. In summary, INa of rabbit cardiomyocytes show atrial-ventricular differences in voltage-dependent activation and inactivation and in ranolazine block consistent with an atrial-selective antiarrhythmic action.

Sodium Current Analysis in Myocytes Obtained from Human Induced Pluripotent Stem Cells and Native Adult Heart

Background: The proposed Comprehensive in Vitro Pro-Arrhythmia Assay (CiPA) initiative aims to examine proarrhythmic drug effects on multiple cardiac ion channels in adult cardiac tissue as well as in human-induced pluripotent stem cell-derived (hiPSC) cardiomyocytes. In this study, we examined the biophysical properties of INa in hiPSC myocytes and compared them to adult canine atrial and ventricular myocytes. Methods: Left atrial and ventricular myocytes were isolated from dog hearts, whereas hiPSC myocytes were obtained from commercially available sources. Action potential recordings from all tissue types were made using high resistance electrodes. Patch clamp techniques were used to record INa from single cells. Results: Analysis of the action potential characteristics showed a higher upstroke velocity in atrial and ventricular tissue (185.4±18.4 and 183.3±14.2 V/s, respectively) compared to hiPSC (33.1±3.3 V/s) beating clusters. Voltage clamp recordings of fast INa showed that current density was lower in hiPSC myocytes compared with atrial and ventricular myocytes (−25.8±4.28 pA/pF vs. −119.6±6.7 pA/pF and −70.0±9.28 pA/pF, respectively, p<0.05). Steady state-inactivation was also significantly different between the 3 cardiac tissue types with atrial cells showing the most negative V½ and ventricular cells showing the least negative. Application of the INa blocker lidocaine (100 µM) resulted in a differential effect on Vmax and INa blockade with atrial tissue and hiPSC showing the greatest reduction in both parameters whereas ventricular tissue showing the smallest reduction. Conclusions: These data show that the density of INa in hiPSC myocytes was much lower compared to native myocytes. In addition, the response of hiPSC cells to a prototypical sodium channel blocker indicates caution should be exercised when comparing the results obtained in these cells to native ventricular cells.

Excavatolide B Modulates the Electrophysiological Characteristics and Calcium Homeostasis of Atrial Myocytes.

Severe bacterial infections caused by sepsis always result in profound physiological changes, including fever, hypotension, arrhythmia, necrosis of tissue, systemic multi-organ dysfunction, and finally death. The lipopolysaccharide (LPS) provokes an inflammatory response under sepsis, which may increase propensity to arrhythmogenesis. Excavatolide B (EXCB) possesses potent anti-inflammatory effects. However, it is not clear whether EXCB could modulate the electrophysiological characteristics and calcium homeostasis of atrial myocytes. This study investigated the effects of EXCB on the atrial myocytes exposed to lipopolysaccharide. A whole-cell patch clamp and indo-1 fluorimetric ratio technique was employed to record the action potential (AP), ionic currents, and intracellular calcium ([Ca2+]i) in single, isolated rabbit left atrial (LA) cardiomyocytes, with and without LPS (1 μg/mL) and LPS + EXCB administration (10 μM) for 6 ± 1 h, in order to investigate the role of EXCB on atrial electrophysiology. In the presence of LPS, EXCB-treated LA myocytes (n = 13) had a longer AP duration at 20% (29 ± 2 vs. 20 ± 2 ms, p < 0.05), 50% (52 ± 4 vs. 40 ± 3 ms, p < 0.05), and 90% (85 ± 5 vs. 68 ± 3 ms, p < 0.05), compared to the LPS-treated cells (n = 12). LPS-treated LA myocytes showed a higher late sodium current, Na+/Ca2+ exchanger current, transient outward current, and delayed rectifier potassium current, but a lower l-type Ca2+ current, than the control LA myocytes. Treatment with EXCB reversed the LPS-induced alterations of the ionic currents. LPS-treated, EXCB-treated, and control LA myocytes exhibited similar Na+ currents. In addition, the LPS-treated LA myocytes exhibited a lower [Ca2+]i content and higher sarcoplasmic reticulum calcium content, than the controls. EXCB reversed the LPS-induced calcium alterations. In conclusion, EXCB modulates LPS-induced LA electrophysiological characteristics and calcium homeostasis, which may contribute to attenuating LPS-induced arrhythmogenesis.

Reduced density and altered regulation of rat atrial L-type Ca2+ current in heart failure.

Constitutive regulation by PKA has recently been shown to contribute to L-type Ca2+ current (ICaL) at the ventricular t-tubule in heart failure. Conversely, reduction in constitutive regulation by PKA has been proposed to underlie the downregulation of atrial ICaL in heart failure. The hypothesis that downregulation of atrial ICaL in heart failure involves reduced channel phosphorylation was examined. Anesthetized adult male Wistar rats underwent surgical coronary artery ligation (CAL, N=10) or equivalent sham-operation (Sham, N=12). Left atrial myocytes were isolated ~18 wk postsurgery and whole cell currents recorded (holding potential=-80 mV). ICaL activated by depolarizing pulses to voltages from -40 to +50 mV were normalized to cell capacitance and current density-voltage relations plotted. CAL cell capacitances were ~1.67-fold greater than Sham (P ≤ 0.0001). Maximal ICaL conductance (Gmax ) was downregulated more than 2-fold in CAL vs. Sham myocytes (P < 0.0001). Norepinephrine (1 μmol/l) increased Gmax >50% more effectively in CAL than in Sham so that differences in ICaL density were abolished. Differences between CAL and Sham Gmax were not abolished by calyculin A (100 nmol/l), suggesting that increased protein dephosphorylation did not account for ICaL downregulation. Treatment with either H-89 (10 μmol/l) or AIP (5 μmol/l) had no effect on basal currents in Sham or CAL myocytes, indicating that, in contrast to ventricular myocytes, neither PKA nor CaMKII regulated basal ICaL Expression of the L-type α1C-subunit, protein phosphatases 1 and 2A, and inhibitor-1 proteins was unchanged. In conclusion, reduction in PKA-dependent regulation did not contribute to downregulation of atrial ICaL in heart failure.NEW & NOTEWORTHY Whole cell recording of L-type Ca2+ currents in atrial myocytes from rat hearts subjected to coronary artery ligation compared with those from sham-operated controls reveals marked reduction in current density in heart failure without change in channel subunit expression and associated with altered phosphorylation independent of protein kinase A.

Hyperaldosteronism induces left atrial systolic and diastolic dysfunction.

Patients with hypertension and hyperaldosteronism show an increased risk of stroke compared with patients with essential hypertension. Aim of the study was to assess the effects of aldosterone on left atrial function in rats as a potential contributor to thromboembolism. Osmotic mini-pumps delivering 1.5 μg aldosterone/h were implanted in rats subcutaneously (Aldo, n = 39; controls, n = 38). After 8 wk, left ventricular pressure-volume analysis of isolated working hearts was performed, and left atrial systolic and diastolic function was also assessed by atrial pressure-diameter loops. Moreover, left atrial myocytes were isolated to investigate their global and local Ca2+ handling and contractility. At similar heart rates, pressure-volume analysis of isolated hearts and in vivo hemodynamic measurements revealed neither systolic nor diastolic left ventricular dysfunction in Aldo. In particular, atrial filling pressures and atrial size were not increased in Aldo. Aldo rats showed a significant reduction of atrial late diastolic A wave, atrial active work index, and increased V waves. Consistently, in Aldo rats, sarcomere shortening and the amplitude of electrically evoked global Ca2+ transients were substantially reduced. Sarcoplasmic reticulum-Ca2+ content and fractional Ca2+ release were decreased, substantiated by a reduced sarcoplasmic reticulum calcium ATPase activity, resulting from a reduced CAMKII-evoked phosphorylation of phospholamban. Hyperaldosteronism induced atrial systolic and diastolic dysfunction, while atrial size and left ventricular hemodynamics, including filling pressures, were unaffected in rats. The described model suggests a direct causal link between hyperaldosteronism and decreased atrial contractility and diastolic compliance.

Functional Role and Mechanism of microRNA-28b in Atrial Myocyte in a Persistent Atrial Fibrillation Rat Model.

BackgroundPersistent atrial fibrillation has been indicated to be related with microRNA-28b. However, the exact role of microRNA-28b in persistent atrial fibrillation needs to be further elucidated. Therefore, this study aimed to establish a rat model of persistent atrial fibrillation to investigate the level of microRNA-28b in atrial myocytes and to explore the molecular mechanism involved.Material/MethodsA persistent atrial fibrillation model was established in rats by using chronic rapid atrial pacing induction. The size of the heart was measured by ultrasonic method. The expression of microRNA-28b in left atrial myocytes was quantified by RT-PCR. Cardiomyocytes were isolated and cultured to detect cell proliferation and apoptosis by MTT and flow cytometry, respectively. The specific inhibitor of ERK signaling pathway, PD98059, was used to further illustrate the role of ERK signaling pathway in the modulation of cardiomyocytes in persistent atrial fibrillation.ResultsMicroRNA-28b was up-regulated in the experimental rat model with persistent atrial fibrillation. The proliferation of cardiomyocytes was significantly inhibited with potentiated apoptosis. Blockage of the ERK pathway suppressed the microRNA-28b expression and inhibited cell apoptosis.ConclusionsmicroRNA-28b-induced growth inhibition and cell apoptosis of atrial myocytes was observed in the rat model with persistent atrial fibrillation, via activation of the ERK signaling pathway.

Thin filament length in the cardiac sarcomere varies with sarcomere length but is independent of titin and nebulin

Thin filament length (TFL) is an important determinant of the force-sarcomere length (SL) relation of cardiac muscle. However, the various mechanisms that control TFL are not well understood. Here we tested the previously proposed hypothesis that the actin-binding protein nebulin contributes to TFL regulation in the heart by using a cardiac-specific nebulin cKO mouse model (αMHC Cre Neb cKO). Atrial myocytes were studied because nebulin expression has been reported to be most prominent in this cell type. TFL was measured in right and left atrial myocytes using deconvolution optical microscopy and staining for filamentous actin with phalloidin and for the thin filament pointed-end with an antibody to the capping protein Tropomodulin-1 (Tmod1). Results showed that TFLs in Neb cKO and littermate control mice were not different. Thus, deletion of nebulin in the heart does not alter TFL. However, TFL was found to be ~0.05μm longer in the right than in the left atrium and Tmod1 expression was increased in the right atrium. We also tested the hypothesis that the length of titin's spring region is a factor controlling TFL by studying the Rbm20ΔRRM mouse which expresses titins that are ~500kDa (heterozygous mice) and ~1000kDa (homozygous mice) longer than in control mice. Results revealed that TFL was not different in Rbm20ΔRRM mice. An unexpected finding in all genotypes studied was that TFL increased as sarcomeres were stretched (~0.1μm per 0.35μm of SL increase). This apparent increase in TFL reached a maximum at a SL of ~3.0μm where TFL was ~1.05μm. The SL dependence of TFL was independent of chemical fixation or the presence of cardiac myosin-binding protein C (cMyBP-C). In summary, we found that in cardiac myocytes TFL varies with SL in a manner that is independent of the size of titin or the presence of nebulin.

0514 : Isolation of cardiac myocytes from human heart

The investigation of single cardiac myocytes from healthy and diseased hearts of various species is a valuable tool to explore cardiac physio/ pathophysiology. The application of cell isolation to human donor tissue has been proofed to be difficult due to the limited amount of human tissue (mainly human right atrial appendages during cardiac surgery). Another limitation is the low viability of cardiomyocytes after isolation. In this study, we present a method to obtain single cardiac myocytes from different regions of human heart. Human hearts rejected for transplantation were obtained from Bordeaux hospital. This protocol was approved by the Agence de la Biomédecine. Left atrial (LA) and ventricular (LV) myocytes were obtained by enzymatic dissociation. The ventricles and right atrium were removed and used for other studies (e.g. high resolution optical mapping). LA was cannulated by the circumflex artery and mounted into a Langendorff perfusion system after suture of the leaky atrial branches. LA was perfused with a Ca2+-free solution (~10 min), then collagenase and protease solution (0.08 mM Ca2+) and recirculated for ~25 min. Enzymes were washed out with a 0.2 mM Ca2+ solution. LA was separated into 4 regions: Endocardium, Epicardium, roof and pulmonary vein; LV myocytes were also obtained. Cells were re-suspended into a 1.8 mM Ca2+ solution by steps. Ca2+ transients were recorded (Fura-2, field stimulation) using an IonOptix system and cell membrane was stained with di-8 ANEPPS and visualized under confocal microscopy. Ca2+ tolerant myocytes were obtained from the 4 LA regions and LV. Human cardiac myocytes respond to electrical stimulation and Ca2+ transient can be recorded. Analysis of functional and structural data will be presented. Isolation of single cardiac myocytes from human samples is a tedious task, but we present data showing reliable method to obtain functional and structural insights. The author hereby declares no conflict of interest

Down Regulation of L-Type Calcium Current in Rat Atrial Myocytes during Heart Failure

Heart failure causes down-regulation of the atrial L-type Ca2+ current (ICaL) that has consequences on contractile function and susceptibility to atrial fibrillation. The reduction in atrial ICaL density has been suggested to involve changes in both the expression and phosphorylation of channel protein. The objective of this project was to examine the mechanism of the decrease in atrial ICaL in a surgical model of heart failure. Heart failure was induced in adult male Wistar rats by surgical ligation of the left anterior descending coronary artery (CAL) under general anesthesia. Control animals were subject to equivalent sham operation (Sham). Left atrial myocytes were isolated ∼18 weeks after surgery and superfused with a Tyrode's solution (37 °C). Whole-cell L-type Ca currents (ICaL) were recorded during depolarisation to 0 mV from a holding potential of −80 mV and normalised to whole-cell capacitance, which was also used as an index of cell size. CAL cells were hypertrophied (94.7±5.2 pF, n=63) compared to Sham (56.6±2.1 pF, n=47; P≤0.0001). ICaL density at 0 mV was reduced in CAL compared to Sham (3.3±0.4 pA/pF; n=31 vs 7.3±0.7 pA/pF; n=33; P≤0.001), but the response of ICaL to 1 μmol/L norepinephrine was greater in CAL (x6.8) than in Sham (x3.2) so that there was no difference in ICaL density between the two groups in the presence of norepinephrine. These data indicate a change in channel protein phosphorylation without a change in channel density. The regulation of L-type Ca2+ channel phosphorylation may be a potential therapeutic target for atrial arrhythmias in heart failure.This work was supported by the British Heart Foundation

Type 2 Diabetes Induces Prolonged P-wave Duration without Left Atrial Enlargement.

Prolonged P-wave duration has been observed in diabetes. However, the underlying mechanisms remain unclear. The aim of this study was to elucidate the possible mechanisms. A rat model of type 2 diabetes mellitus (T2DM) was used. P-wave durations were obtained using surface electrocardiography and sizes of the left atrium were determined using echocardiography. Cardiac inward rectifier K+ currents (Ik1), Na+ currents (INa), and action potentials were recorded from isolated left atrial myocytes using patch clamp techniques. Left atrial tissue specimens were analyzed for total connexin-40 (Cx40) and connexin-43 (Cx43) expression levels on western-blots. Specimens were also analyzed for Cx40 and Cx43 distribution and interstitial fibrosis by immunofluorescent and Masson trichrome staining, respectively. The mean P-wave duration was longer in T2DM rats than in controls; however, the mean left atrial sizes of each group of rats were similar. The densities of Ik1 and INa were unchanged in T2DM rats compared to controls. The action potential duration was longer in T2DM rats, but there was no significant difference in resting membrane potential or action potential amplitude compared to controls. The expression level of Cx40 protein was significantly lower, but Cx43 was unaltered in T2DM rats. However, immunofluorescent labeling of Cx43 showed a significantly enhanced lateralization. Staining showed interstitial fibrosis was greater in T2DM atrial tissue. Prolonged P-wave duration is not dependent on the left atrial size in rats with T2DM. Dysregulation of Cx40 and Cx43 protein expression, as well as fibrosis, might partly account for the prolongation of P-wave duration in T2DM.