Involvement of Src in L-type Ca channel depression induced by macrophage migration inhibitory factor in atrial myocytes

Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine that controls inflammatory processes, and inflammation is known to play an important role in the pathogenesis of atrial fibrillation (AF). The present study sought to investigate whether MIF expression is responsible for the changes in L-type Ca²⁺ currents (I_Ca,L) seen in AF. Whole-cell voltage-clamp recordings and biochemical assays were used to study the regulation and expression of I_Ca,L in human atrial myocytes and in HL-1 cells. Basal I_Ca,L was reduced in AF compared to sinus rhythm (SR) controls, mRNA and protein levels of the pore-forming α_1C subunit of L-type Ca²⁺ channel (LCC α_1C) were also decreased, while MIF expression levels were increased in AF. Levels of Src and activated Src (p-Src Y⁴¹⁶) were higher in AF than in SR. Treatment of atrial myocytes from a patient with SR with human recombinant MIF (rMIF) (40 nM, 1 h) was found to depress I_Ca,L amplitudes, while mouse rMIF (20 or 40 nM, 24 h) suppressed peak I_Ca,L in HL-1 cells by  69% and  83% in a concentration-dependent manner. Mouse rMIF impaired the time-dependent recovery from inactivation of I_Ca,L and down-regulated LCC α_1C subunit levels. The depression of I_Ca,L and decrease of LCC protein levels induced by rMIF were prevented by the Src inhibitors genistein and PP1. These results implicate MIF in the electrical remodeling that accompanies AF, probably by decreasing I_Ca,L amplitudes through impairment of channel function, down-regulation of LCC α_1C subunit levels, and the activation of c-Src kinases in atrial myocytes.     

巨噬细胞移动抑制因子对HL-1细胞T型钙电流的调控

目的 观察巨噬细胞移动抑制因子（macrophage migration inhibitory factor,MIF）对心房肌细胞T型钙电流（T-type calcium channel current,ICa,T）的调控.方法 使用全细胞膜片钳和分子生物分析方法检测心房肌细胞ICa,T的表达.结果 在体外培养的心房肌细胞株（HL-1细胞）中,小鼠重组MIF（20、40 nmol/L,24 h）可明显抑制ICa,T的峰值电流,与对照组比较,差异均有统计学意义[峰值内向电流：（-17.5±2.9）pA/pF vs.（-27.9±3.4） pA/pF,P＜0.05;（-11.3±1.7）pA/pF vs.（-27.9±3.4）pA/pF,P＜0.01];并可损伤电压依赖的ICa,T激活,使T型钙通道α1G和α1H亚单位mRNA表达下调.而Src非特异性抑制剂genistein和特异性抑制剂PP1可逆转40 nmol/L MIF所致的ICa,T下调[genistein：（-11.3±1.7）pA/pF vs.（-16.1±0.8）,P＜0.05;PPI：（-11.3±1.7）pA/pFvs.（-19.0±3.2）pA/pF,P＜0.05].结论 MIF可能通过影响ICa,T参与心房颤动的病理过程,Src可能参与该信号转导途径.     

Involvement of macrophage migration inhibitory factor in pathogenesis of atrial fibrillation as a redox-sensitive cytokine

Background Macrophage migration inhibitory factor(MIF)is a pleiotropic cytokine that controls inflammatory processes,and inflammation is known to play an important role in the pathogenesis of atrial fibrillation(AF).The present study sought to investigate whether MIF played an important role in the pathogenesis of AF.Methods MIF protein and mRNA levels in specimens of human right atrial appendage(from patients with AF or sinus rhythm)or atrium myocytes(HL-1 cells)were assayed using enzyme-linked immunosorbe...     

Regulation of L-type calcium channel by phospholemman in cardiac myocytes

We evaluated whether phospholemman (PLM) regulates L-type Ca^2 + current (I_Ca) in mouse ventricular myocytes. Expression of α₁-subunit of L-type Ca^2 + channels between wild-type (WT) and PLM knockout (KO) hearts was similar. Compared to WT myocytes, peak I_Ca (at − 10 mV) from KO myocytes was ~ 41% larger, the inactivation time constant (τ_inact) of I_Ca was ~ 39% longer, but deactivation time constant (τ_deact) was similar. In the presence of isoproterenol (1 μM), peak I_Ca was ~ 48% larger and τ_inact was ~ 144% higher in KO myocytes. With Ba^2 + as the permeant ion, PLM enhanced voltage-dependent inactivation but had no effect on τ_deact. To dissect the molecular determinants by which PLM regulated I_Ca, we expressed PLM mutants by adenovirus-mediated gene transfer in cultured KO myocytes. After 24 h in culture, KO myocytes expressing green fluorescent protein (GFP) had significantly larger peak I_Ca and longer τ_inact than KO myocytes expressing WT PLM; thereby independently confirming the observations in freshly isolated myocytes. Compared to KO myocytes expressing GFP, KO myocytes expressing the cytoplasmic domain truncation mutant (TM43), the non-phosphorylatable S68A mutant, the phosphomimetic S68E mutant, and the signature PFXYD to alanine (ALL5) mutant all resulted in lower peak I_Ca. Expressing PLM mutants did not alter expression of α₁-subunit of L-type Ca^2 + channels in cultured KO myocytes. Our results suggested that both the extracellular PFXYD motif and the transmembrane domain of PLM but not the cytoplasmic tail were necessary for regulation of peak I_Ca amplitude. We conclude that PLM limits Ca^2 + influx in cardiac myocytes by reducing maximal I_Ca and accelerating voltage-dependent inactivation.     

Phosphodiesterase 5 restricts NOS3/Soluble guanylate cyclase signaling to L-type Ca current in cardiac myocytes

Endothelial nitric oxide synthase (NOS3) regulates the functional response to β-adrenergic (β-AR) stimulation via modulation of the L-type Ca²⁺ current (I_Ca). However, the NOS3 signaling pathway modulating I_Ca is unknown. This study investigated the contribution of soluble guanylate cyclase (sGC) and phosphodiesterase type 5 (PDE5), a cGMP-specific PDE, in the NOS3-mediated regulation of I_Ca. Myocytes were isolated from NOS3 knockout (NOS3^−/−) and wildtype (WT) mice. We measured I_Ca (whole-cell voltage-clamp), and simultaneously measured Ca²⁺ transients (Fluo-4 AM) and cell shortening (edge detection). Zaprinast (selective inhibitor of PDE5), decreased β-AR stimulated (isoproterenol, ISO)-I_Ca, and Ca²⁺ transient and cell shortening amplitudes in WT myocytes. However, YC-1 (NO-independent activator of sGC) only reduced ISO-stimulated I_Ca, but not cardiac contraction. We further investigated the NOS3/sGC/PDE5 pathway in NOS3^−/− myocytes. PDE5 is mislocalized in these myocytes and we observed dissimilar effects of PDE5 inhibition and sGC activation compared to WT. That is, zaprinast had no effect on ISO-stimulated I_Ca, or Ca²⁺ transient and cell shortening amplitudes. Conversely, YC-1 significantly decreased both ISO-stimulated I_Ca, and cardiac contraction. Further confirming that PDE5 localizes NOS3/cGMP signaling to I_Ca; YC-1, in the presence of zaprinast, now significantly decreased ISO-stimulated Ca²⁺ transient and cell shortening amplitudes in WT myocytes. The effects of YC-1 on I_Ca and cardiac contraction were blocked by KT5823 (a selective inhibitor of the cGMP-dependent protein kinase, PKG). Our data suggests a novel physiological role for PDE5 in restricting the effects of NOS3/sGC/PKG signaling pathway to modulating β-AR stimulated I_Ca, while limiting effects on cardiac contraction.     

Diastolic transient inward current in long QT syndrome type 3 is caused by Ca overload and inhibited by ranolazine

Long QT syndrome variant 3 (LQT-3) is a channelopathy in which mutations in SCN5A, the gene coding for the primary heart Na⁺ channel alpha subunit, disrupt inactivation to elevate the risk of mutation carriers for arrhythmias that are thought to be calcium (Ca²⁺)-dependent. Spontaneous arrhythmogenic diastolic activity has been reported in myocytes isolated from mice harboring the well-characterized ΔKPQ LQT-3 mutation but the link to altered Ca²⁺ cycling related to mutant Na⁺ channel activity has not previously been demonstrated. Here we have investigated the relationship between elevated sarcoplasmic reticulum (SR) Ca²⁺ load and induction of spontaneous diastolic inward current (I_TI) in myocytes expressing ΔKPQ Na⁺ channels, and tested the sensitivity of both to the antianginal compound ranolazine. We combined whole-cell patch clamp measurements, imaging of intracellular Ca²⁺, and measurement of SR Ca²⁺ content using a caffeine dump methodology. We compared the Ca²⁺ content of ΔKPQ^+/− myocytes displaying I_TI to those without spontaneous diastolic activity and found that I_TI induction correlates with higher sarcoplasmic reticulum (SR) Ca²⁺. Both spontaneous diastolic I_TI and underlying Ca²⁺ waves are inhibited by ranolazine at concentrations that preferentially target I_NaL during prolonged depolarization. Furthermore, ranolazine I_TI inhibition is accompanied by a small but significant decrease in SR Ca²⁺ content. Our results provide the first direct evidence that induction of diastolic transient inward current (I_TI) in ΔKPQ^+/− myocytes occurs under conditions of elevated SR Ca²⁺ load.     

Role of the Transient Outward Current in Regulating Mechanical Properties of Canine Ventricular Myocytes

I_to and Myocyte Contractility. Introduction: The transient outward current (I_to) is a major repolarizing current in the heart. Reduction of I_to density is consistently observed in human heart failure (HF) and animal HF models. It has been proposed that I_to, via its influence on phase‐1 repolarization of the action potential, facilitates L‐type Ca²⁺ current (I_Ca–L) activation and sarcoplasmic reticulum Ca²⁺ release, and that its down‐regulation may contribute to the impaired contractility in failing heart. Methods and Results: We used the dynamic clamp to quantitatively examine the influence of I_to on the mechanical properties of canine left ventricular myocytes at 34°C. In endocardial myocytes, where the native I_to is small, simulation of an epicardial‐level artificial I_to accentuated the phase‐1 repolarization and significantly suppressed cell shortening. The peak amplitude of Ca²⁺ transient was also reduced in the presence of simulated I_to, although the rate of rise of the Ca²⁺ transient was increased. Conversely, subtraction, or “blockade” of the native I_to enhanced contractility in epicardial cells. These results agree with the inverse correlation between I_to levels and myocyte contractility and Ca²⁺ transient amplitude in epicardial and endocardial myocytes. Action potential clamp studies showed that the phase‐1 repolarization/I_to versus I_Ca–L relationship had an inverted‐J shape; small I_to enhanced peak I_Ca–L while moderate‐to‐large I_to decreased peak I_Ca–L and markedly reduced early Ca²⁺ influx. Conclusion: Our results show that epicardial‐level of I_to acts as a negative, rather than positive regulator of myocyte mechanical properties in canine ventricular myocytes. (J Cardiovasc Electrophysiol, Vol. 21, pp. 697‐703, June 2010)     

Sarcoplasmic Reticulum Function in Murine Ventricular Myocytes Overexpressing SR CaATPase

To examine the effects of the overexpression of sarcoplasmic reticulum (SR) CaATPase on function of the SR and Ca²⁺homeostasis, we measured [Ca²⁺]_itransients (fluo-3), and L-type Ca²⁺currents (I_Ca,L), Na/Ca exchanger currents (I_Na/Ca), and SR Ca²⁺content with voltage clamp in ventricular myocytes isolated from wild type (WT) mice and transgenic (SRTG) mice. The amplitude of [Ca²⁺]_itransients was insignificantly increased in SRTG myocytes, while the diastolic [Ca²⁺]_itended to be lower. The initial and terminal declines of [Ca²⁺]_itransients were significantly accelerated in SRTG myocytes, implying a functional upregulation of the SR CaATPase. We examined the functional contribution of only the SR CaATPase to the initial and the terminal phase of the decline of [Ca²⁺]_i, by abruptly inhibiting Na/Ca exchange with a rapid switcher device. The rate of [Ca²⁺] decline mediated by the SR CaATPase was increased by 40% in SRTG compared with WT myocytes. The function of the L-type Ca²⁺channel was unchanged in SRTG myocytes, while INa/Ca density was slightly (10%) decreased. Measured SR Ca²⁺content was significantly increased by 29% in SRTG myocytes. Thus, overexpression of SR CaATPase markedly accelerates the decline of [Ca²⁺]_itransients, and induces an increase in SR Ca²⁺content, with some downregulation of the Na/Ca exchanger.     

Stretch Current‐Induced Abnormal Impulses in CaMKIIδ Knockout Mouse Ventricular Myocytes

CaMKII and Arrhythmias. Background: CaMKII activation is proarrhythmic in heart failure where myocardium is stretched. However, the arrhythmogenic role of CaMKII in stretched ventricle has not been well understood. Objective: We tested abnormal impulse inducibility by stretch current in myocytes isolated from CaMKIIδ knockout (KO) mouse left ventricle (LV) where CaMKII activity is reduced by ≈ 62%. Methods and Results: Action potentials were recorded by whole‐cell patch clamp, and abnormal impulses were induced in LV myocytes by a simulation of stretch‐activated channel (SAC) current. SAC activation failed to induce abnormal impulses in wild type (WT) myocytes but steadily produced early after‐depolarizations and automaticity in KO myocytes in which an increase in L‐type calcium channel (LTCC) current (I_Ca) and a reduction of sarcoplasmic reticulum Ca²⁺ leak and action potential duration (APD) were observed. The abnormal impulses were not suppressed by CaMKII inhibitor AIP whereas a low concentration of nifedipine eliminated abnormal impulses without shortening APD, implicating I_Ca in promoting stretch‐induced abnormal impulses. In addition, APD prolongation by LTCC opener S(‐)Bay K 8644 or isoproterenol facilitated abnormal impulse induction in WT ventricular myocytes even in the presence of CaMKII inhibitor AIP, whereas APD prolongation by K⁺ channel blocker 4‐aminopyridine promoted abnormal impulses in KO myocytes but not in WT myocytes. Conclusion: I_Ca activation plays a central role in stretch‐induced abnormal impulses and APD prolongation is arrhythmogenic only when I_Ca is highly activated. At increased I_Ca activation, CaMKII inhibition cannot suppress abnormal impulse induction. (J Cardiovasc Electrophysiol, Vol. 24, pp. 457‐463, April 2013)     

Specificities of atrial electrophysiology: Clues to a better understanding of cardiac function and the mechanisms of arrhythmias

The electrical properties of the atria and ventricles differ in several aspects reflecting the distinct role of the atria in cardiac physiology. The study of atrial electrophysiology had greatly contributed to the understanding of the mechanisms of atrial fibrillation (AF). Only the atrial L-type calcium current is regulated by serotonine or, under basal condition, by phosphodiesterases. These distinct regulations can contribute to I_Ca down-regulation observed during AF, which is an important determinant of action potential refractory period shortening. The voltage-gated potassium current, I_Kur, has a prominent role in the repolarization of the atrial but not ventricular AP. In many species, this current is based on the functional expression of K_V1.5 channels, which might represent a specific therapeutic target for AF. Mechanisms regulating the trafficking of K_V1.5 channels to the plasma membrane are being actively investigated. The resting potential of atrial myocytes is maintained by various inward rectifier currents which differ with ventricle currents by a reduced density of I_K1, the presence of a constitutively active I_KACh and distinct regulation of I_KATP. Stretch-sensitive or mechanosensitive ion channels are particularly active in atrial myocytes and are involved in the secretion of the natriuretic peptide. Integration of knowledge on electrical properties of atrial myocytes in comprehensive schemas is now necessary for a better understanding of the physiology of atria and the mechanisms of AF.     

Signal transduction by cGMP in heart

Summary Early studies in whole heart indicated that cGMP antagonized the positive inotropic effects of catecholamines and cAMP. However, the regulation of cGMP levels by a variety of agents was not always consistent with their effects on contractility. It is now clear that at least two major cell types in whole heart, cardiac myocytes and vascular smooth muscle cells, differ markedly in their mechanisms of cGMP regulation and response to cGMP. Furthermore, experiments on isolated cardiac myocytes indicate that the mechanism of cGMP action even in this single cell type can be multifaceted. Cyclic GMP inhibits the L-type calcium channel current (I_Ca), which is the major source of Ca⁺⁺ entry into heart cells, and which plays a predominant role in the initiation and regulation of cardiac electrical and contractile activities. Patch-clamp measurements of I_Ca indicate that in isolated frog myocytes cGMP inhibits I_Ca by stimulation of cAMP phosphodiesterase (cGS-PDE), whereas in purified rat ventricular myocytes, cGMP predominantly inhibits I_Ca via a mechanism involving cGMP-dependent protein kinase (cGMP-PK). Under certain conditions, cGMP can also inhibit a cGMP-inhibited cAMP phosphodiesterase (cGI-PDE) and thereby produce a stimulatory effect on I_Ca. Biochemical characterization of the endogenous PDEs and cGMP-PK in purified cardiac myocytes provided further evidence in support of these mechanisms of cGMP action on I_Ca.     

Differences in K+ current components in mesenteric artery myocytes from WKY and SHR

Previous studies have documented increased K⁺ permeability of arterial smooth muscle in hypertension and suggested a role in altered arterial contractile function. To characterize the mechanisms responsible for these alterations, we determined the contribution of K⁺ current (I_K) components to whole cell I_K in freshly dispersed myocytes and tetraethylammonium (TEA)-induced contractile responses in mesenteric arteries of Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR). Tetraethylammonium produced a larger tonic contractile response in SHR with a lower threshold compared to WKY (ie, 0.1 v 1 mmol/L), which was due in part to the larger Ca²⁺ current in SHR. Whole cell I_K recorded by perforated patch methods was similar at a holding potential (HP) of −60 mV (I_K60), but were larger in SHR when recorded from a HP of −20 mV (I_K20). The selective blocker iberiotoxin (IbTX) was used to separate the contribution of voltage- (K_V) and calcium-dependent (K_Ca) components of I_K60. The I_K60 and I_K20 component inhibited by 100 nmol/L IbTX (ie, K_Ca) was larger in SHR than in WKY myocytes, whereas the IbTX-insensitive I_K60 component (ie, K_V) was larger in WKY. In the presence of IbTX, 1 and 10 mmol/L TEA inhibited a larger fraction of I_K60 in SHR myocytes compared with WKY. The activation properties of the TEA-sensitive and TEA-insensitive K_V components determined by fitting a Boltzmann activation function to the current-voltage data, exhibited both group and treatment differences in the half maximal activation voltage (V_0.5). The V_0.5 of the TEA-sensitive K_V component was more positive than that of the TEA-insensitive component in both groups, and values for the V_0.5 of both TEA-sensitive and TEA-insensitive components were more negative in SHR than WKY. These results show that SHR myocytes have larger K_Ca and smaller K_V current components compared with WKY. Furthermore, SHR myocytes have a larger TEA-sensitive K_V component. These differences may contribute to the differences in TEA contractions, resting membrane potential, Ca²⁺ influx, and K_Ca current reported in hypertensive arteries.     

Gender-related differences in ventricular myocyte repolarization in the guinea pig

Abstract. It is well established that gender-differences exist in cardiac electrophysiology and these are thought to contribute to the increased risk of women, compared to men, for the potentially lethal ventricular arrhythmia, torsades de pointes. Data from animal models with abbreviated estrus cycles suggest that androgens may play a protective role in males. However, the role of female sex hormones in gender-differences in cardiac electrophysiology is less clear. This report describes gender differences in ventricular electrophysiology, investigated using the guinea pig heart. Ionic currents and action potentials were compared between ventricular myocytes isolated from male guinea pig hearts and those from females on the day of estrus (day 0) and 4 days post-estrus (day 4). The density of inward rectifier K⁺ current (I_K1) at –120 mV was significantly greater in male myocytes than in female myocytes either at day 0 or day 4. The peak L-type Ca²⁺ current (I_Ca) at +10 mV was also significantly larger in male myocytes than in day 0 and day 4 female myocytes. Moreover, I_Ca differed significantly between day 0 and day 4 female myocytes, strongly suggesting that I_Ca density varies around the estrus cycle. Delayed rectifier (I_K) tail currents were significantly different between male and female day 4 myocytes. Action potential duration (at 90% repolarization; APD₉₀) was significantly shorter in male myocytes than in female myocytes at day 0, but not at day 4, broadly consistent with the combined differences in I_K and I_Ca between the three groups. Taken together, our data are consistent with the contribution of multiple factors, rather than a single hormone, to gender differences in ventricular repolarization. Since female guinea pigs possess a conventional estrus cycle, our data suggest that this species may be well suited to elucidating the modulatory influence of ovarian steroids on ventricular repolarization and arrhythmic risk. Our findings suggest that further work examining the basis to gender differences in ventricular repolarization in the guinea pig is warranted.     

Regional Gradation of L-Type Calcium Currents in the Feline Heart with a Healed Myocardial Infarct

I_Ca in Healed Myocardial Infarction. Introduction: Abnormal action potentials in myocytes adjacent to > 2-month-uld feline LV myocardial infarcts (MI) may reflect alterations in Ca²⁺ currents (I_Ca). Methods and Results: We compared I_Ca, at 36°C, in subendocardial myocytes isolated from areas adjacent to MI and to I_Ca in cells from remote areas (> 4 mm away; REM) and control cells from similar regions in normal hearts. Control (CON) myocytes had membrane capacitance of 234 ± 10 pF (n = 81 cells) compared to 305 ± 14 pF in REM (71 cells; P < 0.05 from CON) and 237 ± 11 pF (n = 55 cells) in MI (not different from CON). From V_h=?40 mV, peak I_Ca elicited by test potentials (?35 to +70 mV) were significantly larger in CON (?1746 ± 123 pA) and REM (?1795 ± 142 pA) compared to Ml (?1352 ± 129 pA) (P < 0.05). Peak 1(11 density was significantly reduced in REM (?6.0 ± 0.4 pA/pF) or MI (?5.7 ± 0.4 pA/pF, P < 0.05) compared to CON (?7.5 ± 0.4 pA/pF). Double exponential I_Ca decay was similar among groups. Half-inactivation potential (V_0.5) was significantly shifted (hyperpolarizing direction) for MI (?29.1 ± 2.6 mV) and REM (?24.6 ± 1.2 mV) myocytes compared to ?20.3 ± 1.0 mV in CON. MI slope factor (k; 9.0 ± 0.5) was significantly different from CON (6.8 ± 0.3) and RKM (7.3 ± 0.4). No differences in time course of recovery from inactivation were noted. Five millimolar Ba²⁺₀ produced significant increases in I_Ca in CON and REM but an attenuated response in MI. Bay k8644 (1 μM) produced similar I_Ca increase in all groups. I_Ca increase due to isoproterenol (1 I_CaM) in MI and REM was half that in CON, but there were no differences in increased I_Ca responses among groups following phenylephrine (10 μM). Conclusion: Reduced I_Ca density in REM reflects cell hypertrophy, whereas altered I_Ca of MI may reflect altered channel structure and/or function.     

Chronic obstructive sleep apnea causes atrial remodeling in canines: mechanisms and implications

Obstructive sleep apnea (OSA) is closely related to atrial fibrillation (AF). However, the roles and mechanisms of chronic OSA in atrial remodeling are still unclear. Canine model of chronic OSA was simulated by stopping the ventilator and closing the airway for 4 h per day and lasting for 12 weeks. AF inducibility and duration was increased while atrial effective refractory period (AERP) was shortened after chronic apnea. Meanwhile, upregulation of proteins encoding inward rectifier K⁺ current (I _K1), delayed rectifier K⁺ current (I _Kr and I _Ks), acetylcholine activated K⁺ current (I _KACh), transient outward K⁺ current (I _to) and ultra-rapid delayed rectifier potassium current (I _Kur) as well as downregulation of protein encoding L-type Ca²⁺ current (I _Ca,L) were found after chronic OSA. Besides abnormal electrical activity, chronic OSA induced apoptosis and interstitial fibrosis of atrial myocytes, which was partly mediated by caspase 9, phosphorylation of extracellular-regulated kinase 1/2, and α-smooth muscle actin. In addition, atrial sympathetic and parasympathetic hyperinnervation were found manifesting by enhanced growth-associated protein 43, tyrosine hydroxylase and elevated choline acetyltransferase. Moreover, protein expression of β₁, β₂, and M₂ receptor were markedly increased by chronic OSA. In summary, we firstly demonstrated in canine model that chronic OSA could shorten AERP and lead to altered expression of important channel proteins, moreover, induce atrial structure remodeling by increased atrial apoptosis, fibrosis, and autonomic remodeling, eventually promoting the development of a substrate of AF. Our findings suggested that reversing atrial remodeling might be a potential therapeutic strategy for OSA-induced AF.     

Adult zebrafish heart as a model for human heart? An electrophysiological study

The zebrafish has recently emerged as an excellent model for studies of heart development and regeneration. The physiology of the zebrafish heart has been suggested to resemble that of the human heart in many aspects, whereas, in contrast to mammals, the zebrafish has a remarkable ability to regenerate after heart injury. Thus, zebrafish have been proposed as a cost-effective model for genetic and pharmacological screens of factors affecting heart function and repair. However, realizing the full potential of the zebrafish heart as a model will require a better understanding of the electrophysiology of the adult zebrafish myocardium. Here, we characterize action potentials (APs) from intact adult atria and ventricles and find that the overall shape of zebrafish APs is similar to that of humans. We show that zebrafish, like most mammals, display functional acetylcholine-activated K⁺ channels in the atrium, but not in the ventricle. Furthermore, the zebrafish AP upstroke is dominated by Na⁺ channels, L-type Ca²⁺ channels contribute to the plateau phase and I_Kr channels are involved in repolarization. However, despite these similarities between zebrafish and mammalian electrophysiology, we also identified important differences. In particular, zebrafish display a robust T-type Ca²⁺ current in both atrial and ventricular cardiomyocytes. Interestingly, in most mammals T-type Ca²⁺ channels are only expressed in the developing heart or under pathophysiological conditions, indicating that adult zebrafish cardiomyocytes display a more immature phenotype.     

Calcium influx through Cav1.2 is a proximal signal for pathological cardiomyocyte hypertrophy

Pathological cardiac hypertrophy (PCH) is associated with the development of arrhythmia and congestive heart failure. While calcium (Ca²⁺) is implicated in hypertrophic signaling pathways, the specific role of Ca²⁺ influx through the L-type Ca²⁺ channel (I_Ca-L) has been controversial and is the topic of this study. To determine if and how sustained increases in I_Ca-L induce PCH, transgenic mouse models with low (LE) and high (HE) expression levels of the β2a subunit of Ca²⁺ channels (β2a) and in cultured adult feline (AF) and neonatal rat (NR) ventricular myocytes (VMs) infected with an adenovirus containing a β2a-GFP were used. In vivo, β2a LE and HE mice had increased heart weight to body weight ratio, posterior wall and interventricular septal thickness, tissue fibrosis, myocyte volume, and cross-sectional area and the expression of PCH markers in a time- and dose-dependent manner. PCH was associated with a hypercontractile phenotype including enhanced I_Ca-L, fractional shortening, peak Ca²⁺ transient, at the myocyte level, greater ejection fraction, and fractional shortening at the organ level. In addition, LE mice had an exaggerated hypertrophic response to transverse aortic constriction. In vitro overexpression of β2a in cultured AFVMs increased I_Ca-L, cell volume, protein synthesis, NFAT, and HDAC translocations and in NRVMs increased surface area. These effects were abolished by the blockade of I_Ca-L, intracellular Ca²⁺, calcineurin, CaMKII, and SERCA. In conclusion, increasing I_Ca-L is sufficient to induce PCH through the calcineurin/NFAT and CaMKII/HDAC pathways. Both cytosolic and SR/ER-nuclear envelop Ca²⁺ pools were shown to be involved.     

Altered distribution of ICa impairs Ca release at the t-tubules of ventricular myocytes from failing hearts

In mammalian cardiac ventricular myocytes, Ca influx and release occur predominantly at t-tubules, ensuring synchronous Ca release throughout the cell. Heart failure is associated with disrupted t-tubule structure, but its effect on t-tubule function is less clear. We therefore investigated Ca influx and release at the t-tubules of ventricular myocytes isolated from rat hearts ~ 18 weeks after coronary artery ligation (CAL) or corresponding Sham operation. L-type Ca current (I_Ca) was recorded using the whole-cell voltage-clamp technique in intact and detubulated myocytes; Ca release at t-tubules was monitored using confocal microscopy with voltage- and Ca-sensitive fluorophores. CAL was associated with cardiac and cellular hypertrophy, decreased ejection fraction, disruption of t-tubule structure and a smaller, slower Ca transient, but no change in ryanodine receptor distribution, L-type Ca channel expression, or I_Ca density. In Sham myocytes, I_Ca was located predominantly at the t-tubules, while in CAL myocytes, it was uniformly distributed between the t-tubule and surface membranes. Inhibition of protein kinase A with H-89 caused a greater decrease of t-tubular I_Ca in CAL than in Sham myocytes; in the presence of H-89, t-tubular I_Ca density was smaller in CAL than in Sham myocytes. The smaller t-tubular I_Ca in CAL myocytes was accompanied by increased latency and heterogeneity of SR Ca release at t-tubules, which could be mimicked by decreasing I_Ca using nifedipine. These data show that CAL decreases t-tubular I_Ca via a PKA-independent mechanism, thereby impairing Ca release at t-tubules and contributing to the altered excitation–contraction coupling observed in heart failure.