Joon-Chul Kim

Overexpression of junctate induces cardiac hypertrophy and arrhythmia via altered calcium handling

Junctate-1 is a newly identified integral endoplasmic/sarcoplasmic reticulum Ca2+ binding protein. However, its functional role in the heart is unknown. In the present study, the consequences of constitutively overexpressed junctate in cardiomyocytes were investigated using transgenic (TG) mice overexpressing junctate-1. TG mice (8 weeks old) showed cardiac remodeling such as marked bi-atrial enlargement with intra-atrial thrombus and biventricular hypertrophy. The TG mice also showed bradycardia with atrial fibrillation, reduced amplitude and elongated decay time of Ca2+ transients, increased L-type Ca2+ current and prolonged action potential durations. Time-course study (2-8 weeks) showed an initially reduced SR function due to down-regulation of SERCA2 and calsequestrin followed by sarcolemmal protein expression and cardiac hypertrophy at later age. These sequential changes could well be correlated with the physiological changes. Adrenergic agonist treatment and subsequent biochemical study showed that junctate-1 TG mice (8 weeks old) were under local PKA signaling that could cause increased L-type Ca2+ current and reduced SR function. Junctate-1 in the heart is closely linked to the homeostasis of E-C coupling proteins and a sustained increase of junctate-1 expression leads to a severe cardiac remodeling and arrhythmias.

Fluid pressure modulates L-type Ca2+ channel via enhancement of Ca2+-induced Ca2+ release in rat ventricular myocytes.

This study examines whether fluid pressure (FP) modulates the L-type Ca(2+) channel in cardiomyocytes and investigates the underlying cellular mechanism(s) involved. A flow of pressurized (approximately 16 dyn/cm(2)) fluid, identical to that bathing the myocytes, was applied onto single rat ventricular myocytes using a microperfusion method. The Ca(2+) current (I(Ca)) and cytosolic Ca(2+) signals were measured using a whole cell patch-clamp and confocal imaging, respectively. It was found that the FP reversibly suppressed I(Ca) (by 25%) without altering the current-voltage relationships, and it accelerated the inactivation of I(Ca). The level of I(Ca) suppression by FP depended on the level and duration of pressure. The Ba(2+) current through the Ca(2+) channel was only slightly decreased by the FP (5%), suggesting an indirect inhibition of the Ca(2+) channel during FP stimulation. The cytosolic Ca(2+) transients and the basal Ca(2+) in field-stimulated ventricular myocytes were significantly increased by the FP. The effects of the FP on the I(Ca) and on the Ca(2+) transient were resistant to the stretch-activated channel inhibitors, GsMTx-4 and streptomycin. Dialysis of myocytes with high concentrations of BAPTA, the Ca(2+) buffer, eliminated the FP-induced acceleration of I(Ca) inactivation and reduced the inhibitory effect of the FP on I(Ca) by approximately 80%. Ryanodine and thapsigargin, abolishing sarcoplasmic reticulum Ca(2+) release, eliminated the accelerating effect of FP on the I(Ca) inactivation, and they reduced the inhibitory effect of FP on the I(Ca). These results suggest that the fluid pressure indirectly suppresses the Ca(2+) channel by enhancing the Ca(2+)-induced intracellular Ca(2+) release in rat ventricular myocytes.

IP3-Induced Cytosolic and Nuclear Ca2+ Signals in HL-1 Atrial Myocytes: Possible Role of IP3 Receptor Subtypes

HL-1 cells are the adult cardiac cell lines available that continuously divide while maintaining an atrial phenotype. Here we examined the expression and localization of inositol 1,4,5-trisphosphate receptor (IP3R) subtypes, and investigated how pattern of IP3-induced subcellular local Ca2+ signaling is encoded by multiple IP3R subtypes in HL-1 cells. The type 1 IP3R (IP3R1) was expressed in the perinucleus with a diffuse pattern and the type 2 IP3R (IP3R2) was expressed in the cytosol with a punctate distribution. Extracellular ATP (1 mM) elicited transient intracellular Ca2+ releases accompanied by a Ca2+ oscillation, which was eliminated by the blocker of IP3Rs, 2-APB, and attenuated by ryanodine. Direct introduction of IP3 into the permeabilized cells induced Ca2+ transients with Ca2+ oscillations at ⩾ 20 μM of IP3, which was removed by the inhibition of IP3Rs using 2-APB and heparin. IP3-induced local Ca2+ transients contained two distinct time courses: a rapid oscillation and a monophasic Ca2+ transient. The magnitude of Ca2+ oscillation was significantly larger in the cytosol than in the nucleus, while the monophasic Ca2+ transient was more pronounced in the nucleus. These results provide evidence for the molecular and functional expression of IP3R1 and IP3R2 in HL-1 cells, and suggest that such distinct local Ca2+ signaling may be correlated with the punctate distribution of IP3R2s in the cytosol and the diffuse localization of IP3R1 in the peri-nucleus.

Atrial local Ca2+ signaling and inositol 1,4,5-trisphosphate receptors.

In atrial myocytes lacking t-tubules, action potential triggers junctional Ca(2+) releases in the cell periphery, which propagates into the cell interior. The present article describes growing evidence on atrial local Ca(2+) signaling and on the functions of inositol 1,4,5-trisphosphate receptors (IP(3)Rs) in atrial myocytes, and show our new findings on the role of IP(3)R subtype in the regulation of spontaneous focal Ca(2+) releases in the compartmentalized areas of atrial myocytes. The Ca(2+) sparks, representing focal Ca(2+) releases from the sarcoplasmic reticulum (SR) through the ryanodine receptor (RyR) clusters, occur most frequently at the peripheral junctions in isolated resting atrial cells. The Ca(2+) sparks that were darker and longer lasting than peripheral and non-junctional (central) sparks, were found at peri-nuclear sites in rat atrial myocytes. Peri-nuclear sparks occurred more frequently than central sparks. Atrial cells express larger amounts of IP(3)Rs compared with ventricular cells and possess significant levels of type 1 IP(3)R (IP(3)R1) and type 2 IP(3)R (IP(3)R2). Over the last decade the roles of atrial IP(3)R on the enhancement of Ca(2+)-induced Ca(2+) release and arrhythmic Ca(2+) releases under hormonal stimulations have been well documented. Using protein knock-down method and confocal Ca(2+) imaging in conjunction with immunocytochemistry in the adult atrial cell line HL-1, we could demonstrate a role of IP(3)R1 in the maintenance of peri-nuclear and non-junctional Ca(2+) sparks via stimulating a posttranslational organization of RyR clusters.

Shear stress induces a longitudinal Ca2+ wave via autocrine activation of P2Y1 purinergic signalling in rat atrial myocytes

Cardiac myocytes are subjected to fluid shear stress during the cardiac cycle and haemodynamic disturbance. A longitudinally propagating, regenerative Ca2+ wave is initiated in atrial myocytes under shear stress. Here we determine the cellular mechanism for this shear‐induced Ca2+ wave using two‐dimensional confocal Ca2+ imaging combined with pressurized fluid flow. Our data suggest that shear stress triggers the Ca2+ wave through ryanodine receptors via P2Y1 purinoceptor–phospholipase C‐type 2 inositol 1,4,5‐trisphosphate receptor signal transduction in atrial myocytes, and that this mechanotransduction is activated by gap junction hemichannel‐mediated ATP release. Shear‐specific mechanotransduction and the subsequent regenerative Ca2+ wave may be one way for atrial myocytes to assess mechanical stimuli directly and alter their Ca2+ signalling accordingly.

Shear stress activates monovalent cation channel TRPM4 in rat atrial myocytes via type 2 inositol 1,4,5-trisphosphate receptors and Ca(2+) release.

During each contraction and haemodynamic disturbance, cardiac myocytes are subjected to fluid shear stress as a result of blood flow and the relative movement of sheets of myocytes. The present study aimed to characterize the shear stress‐sensitive membrane current in atrial myocytes using the whole‐cell patch clamp technique, combined with pressurized fluid flow, as well as pharmacological and genetic interventions of specific proteins. The data obtained suggest that shear stress indirectly activates the monovalent cation current carried by transient receptor potential melastatin subfamily 4 channels via type 2 inositol 1,4,5‐trisphosphate receptor‐mediated Ca2+ release in subsarcolemmal domains of atrial myocytes. Ca2+‐mediated interactions between these two proteins under shear stress may be an important mechanism by which atrial cells measure mechanical stress and translate it to alter their excitability.

Shear stress activates monovalent cation channel transient receptor potential melastatin subfamily 4 in rat atrial myocytes via type 2 inositol 1,4,5-trisphosphate receptors and Ca2+ release

During each contraction and haemodynamic disturbance, cardiac myocytes are subjected to fluid shear stress as a result of blood flow and the relative movement of sheets of myocytes. The present study aimed to characterize the shear stress‐sensitive membrane current in atrial myocytes using the whole‐cell patch clamp technique, combined with pressurized fluid flow, as well as pharmacological and genetic interventions of specific proteins. The data obtained suggest that shear stress indirectly activates the monovalent cation current carried by transient receptor potential melastatin subfamily 4 channels via type 2 inositol 1,4,5‐trisphosphate receptor‐mediated Ca2+ release in subsarcolemmal domains of atrial myocytes. Ca2+‐mediated interactions between these two proteins under shear stress may be an important mechanism by which atrial cells measure mechanical stress and translate it to alter their excitability.

Signaling Pathway for Endothelin-1- and Phenylephrine-Induced cAMP Response Element Binding Protein Activation in Rat Ventricular Myocytes: Role of Inositol 1,4,5-Trisphosphate Receptors and CaMKII

Background/Aims: Endothelin-1 (ET-1) and the α1-adrenoceptor agonist phenylephrine (PE) activate cAMP response element binding protein (CREB), a transcription factor implicated in cardiac hypertrophy. The signaling pathway involved in CREB activation by these hypertrophic stimuli is poorly understood. We examined signaling pathways for ET-1- or PE-induced cardiac CREB activation. Methods: Western blotting was performed with pharmacological and genetic interventions in rat ventricular myocytes. Results: ET-1 and PE increased CREB phosphorylation, which was inhibited by blockade of phospholipase C, the extracellular-signal-regulated kinase 1/2 (ERK1/2) pathway, protein kinase C (PKC) or Ca2+-calmodulin-dependent protein kinase II (CaMKII). Intracellular Ca2+ buffering decreased ET-1- and PE-induced CREB phosphorylation by ≥80%. Sarcoplasmic reticulum Ca2+ pump inhibitor, inositol 1,4,5-trisphosphate receptor (IP3R) blockers, or type 2 IP3R (IP3R2) knock-out abolished ET-1- or PE-induced CREB phosphorylation. ET-1 and PE increased phosphorylation of CaMKII and ERK1/2, which was eliminated by IP3R blockade/knock-out or PKC inhibition. Activation of CaMKII, but not ERK1/2, by these agonists was sensitive to Ca2+ buffering or to Gö6976, the inhibitor of Ca2+-dependent PKC and protein kinase D (PKD). Conclusion: CREB phosphorylation by ET-1 and PE may be mainly mediated by IP3R2/Ca2+-PKC-PKD-CaMKII signaling with a minor contribution by ERK1/2, linked to IP3R2 and Ca2+-independent PKC, in ventricular myocytes.

Alterations of contractions and L-type Ca2+ currents by murrayafoline-A in rat ventricular myocytes

We examined the effects of murrayafoline-A (1-methoxy-3-methylcarbazole, Mu-A), which is isolated from the dried roots of Glycosmis stenocarpa, on cell shortenings and L-type Ca2+ currents (ICa,L) in rat ventricular myocytes. Cell shortenings and ICa,L were measured using the video edge detection method and patch-clamp techniques, respectively. Mu-A transiently increased cell shortenings in a concentration-dependent manner with an EC50 of ~20 μM. The maximal effect of Mu-A, approximately 175% of the control, was observed at ≥100 μM. The positive inotropic effect of Mu-A (25 μM) reached a maximum after ~2-min exposures, and then decayed after a ~1-min steady-state. During the Mu-A-induced positive inotropy, the rate of contraction was accelerated, whereas the rate of relaxation was not significantly altered. To understand the possible mechanism for the Mu-A-induced positive inotropy, the ICa,L was assessed. Mu-A transiently enhanced the ICa,L. Concentration-dependence of the increase in ICa,L by Mu-A was similar to that of positive inotropic effect of Mu-A. The maximal effect of Mu-A (25 μM) on ICa,L was observed at 2-3 min after the application of Mu-A. A partial inhibition of ICa,L using verapamil (1 μM) induced a right shift of concentration-response curve of the positive inotropic effect of Mu-A and significantly attenuated the effect. These results suggest that Mu-A may transiently enhance contractility, at least in part, by increasing the Ca2+ influx through the L-type Ca2+ channels in rat ventricular myocytes.

Shear stress enhances Ca2 + sparks through Nox2-dependent mitochondrial reactive oxygen species generation in rat ventricular myocytes☆

Shear stress enhances diastolic and systolic Ca2+ concentration in ventricular myocytes. Here, using confocal Ca2+ imaging in rat ventricular myocytes, we assessed the effects of shear stress (~16dyn/cm2) on the frequency of spontaneous Ca2+ sparks and explored the mechanism underlying shear-mediated Ca2+ spark regulation. The frequency of Ca2+ sparks was immediately increased by shear stress (by ~80%), and increased further (by ~150%) during prolonged exposure (20s). The 2-D size and duration of individual sparks were increased by shear stimulation. Inhibition of nitric oxide synthase (NOS) only partially attenuated the prolonged shear-mediated enhancement in spark frequency. Pretreatment with antioxidants significantly attenuated the short- and long-term effects of shear on spark frequency. Microtubule or nicotinamide adenine dinucleotide phosphate oxidase 2 (Nox2) inhibition abolished the immediate shear-induced increase in spark frequency and suppressed the effects of prolonged exposure to shear stress by ~70%. Scavenging of mitochondrial reactive oxygen species (ROS) and mitochondrial uncoupling also abolished the effect of short-term shear on spark occurrence, and markedly reduced (by ~80%) the effects of prolonged shear. Mitochondrial ROS levels increased under shear; this was eliminated by blocking Nox2. Sarcoplasmic reticulum Ca2+ content was increased only by prolonged shear. Our data suggest that shear stress enhances ventricular spark frequency mainly via ROS generated from mitochondria through Nox2, and that NOS and higher sarcoplasmic reticulum Ca2+ concentrations may also contribute to the enhancement of Ca2+ sparks under shear stress. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.