Functional expression of L- and T-type Ca2+ channels in murine HL-1 cells

Xia, Menghang; Salata, Joseph J.; Figueroa, David J.; Lawlor, A.-M.; Liang, Hongyu; Liu, Y.; Connolly, Thomas

doi:10.1016/j.yjmcc.2003.10.007

Cited by 43 publications

(41 citation statements)

References 27 publications

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“…This suggests that TLR4 activation of the myocardium conscripts PIP 2 and reduces its availability for I Kr activation; however, we not aware of reports demonstrating a role for PIP2 in the regulation of I Na and voltage-regulated Na ϩ channels (37). Finally, the magnitude and expression of various ionic currents varied markedly among cells reported herein and by others (29,42,43). This may reflect variability in the differentiation of the contractile phenotype among HL-1 cells depending on the age of the culture and the degree of cell confluence.…”

Section: Discussionmentioning

confidence: 53%

Lipopolysaccharide prolongs action potential duration in HL-1 mouse cardiomyocytes

Wondergem

Graves

et al. 2012

American Journal of Physiology-Cell Physiology

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Wondergem R, Graves BM, Li C, Williams DL. Lipopolysaccharide prolongs action potential duration in HL-1 mouse cardiomyocytes. Am J Physiol Cell Physiol 303: C825-C833, 2012. First published August 15, 2012; doi:10.1152/ajpcell.00173.2012.-Sepsis has deleterious effects on cardiac function including reduced contractility. We have shown previously that lipopolysaccharides (LPS) directly affect HL-1 cardiac myocytes by inhibiting Ca 2ϩ regulation and by impairing pacemaker "funny" current, I f. We now explore further cellular mechanisms whereby LPS inhibits excitability in HL-1 cells. LPS (1 g/ml) derived from Salmonella enteritidis decreased rate of firing of spontaneous action potentials in HL-1 cells, and it increased their pacemaker potential durations and decreased their rates of depolarization, all measured by whole cell current clamp. LPS also increased action potential durations and decreased their amplitude in cells paced at 1 Hz with 0.1 nA, and 20 min were necessary for maximal effect. LPS decreased the amplitude of a rapidly inactivating inward current attributed to Na ϩ and of an outward current attributed to K ϩ ; both were measured by whole cell voltage clamp. The K ϩ currents displayed a resurgent outward tail current, which is characteristic of the rapid delayed-rectifier K ϩ current, IKr. LPS accordingly reduced outward currents measured with pipette Cs ϩ substituted for K ϩ to isolate IKr. E-4031 (1 M) markedly inhibited IKr in HL-1 cells and also increased action potential duration; however, the direct effects of E-4031 occurred minutes faster than the slow effects of LPS. We conclude that LPS increases action potential duration in HL-1 mouse cardiomyocytes by inhibition of IKr and decreases their rate of firing by inhibition of INa. This protracted time course points toward an intermediary metabolic event, which either decreases available mouse ether-a-go-go (mERG) and Na ϩ channels or potentiates their inactivation.delayed rectifier K ϩ current; patch-clamp electrophysiology; Salmonella enteritidis; E-4031 SEPSIS IS A COMPLEX CLINICAL problem that can progress into multiorgan dysfunction syndrome (MODS) and death. At least one-half of the high mortality rates of severe-sepsis, sepsis, and MODS result from cardiovascular failure (27). Approximately 40% of patients in septic shock also experience myocardial dysfunction (36, 44), which comprises reduced systolic contractility, impaired diastolic relaxation, and ventricular dilatation. The initial clinical pattern, however, ironically is termed "hyperdynamic," because it often presents with high cardiac output, elevated heart rate, and vasodilatation of the dermis (27). This is attributable in part to the volume loading that is frequently the initial treatment for septic shock patients to maintain cardiac output and organ perfusion (15, 28). As is often the case in the complexity of sepsis, the prevailing symptoms can mask organ and system failure occurring at a deeper level in the clinical spectrum.The most prevalent sign of myocardial depressio...

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Section: Discussionmentioning

confidence: 53%

Lipopolysaccharide prolongs action potential duration in HL-1 mouse cardiomyocytes

Wondergem

Graves

et al. 2012

American Journal of Physiology-Cell Physiology

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“…Procedures for the current measurement in HL-1 cells were essentially the same as described previously. 19 The membrane potential was held at Ϫ50 mV to inactivate the T-type Ca 2ϩ channel current (I Ca,T ) and avoid the hyperpolarization-activated cation current (I f ) activation, 20,21 depolarized by 1-second test pulses (from Ϫ40 and ϩ40-in 10-mV increments), then repolarized back to the holding potential; 0.4 mol/L nisoldipine was included in the bath solution to block the L-type Ca 2ϩ channel current (I Ca,L ). 20 Action potentials were also measured in the current-clamp mode, elicited at a rate of 0.5 Hz by 5-ms square current pulses of 1 nA, and sampled at 20 kHz in the absence or presence of 10 mol/L E4031 (WAKO, Japan).…”

Section: Methodsmentioning

confidence: 99%

“…The possible contamination of other voltage-dependent currents was minimized by adding 0.4mol/L nisoldipine to bath solution to block I Ca,L 20 and by setting a holding potential at Ϫ50mV to inactivate I Ca,T and to prevent activation of I f . 20,21 Figure 7A shows whole-cell membrane currents recorded in HL-1. Depolarizing pulses activated time-dependent outward currents which increased with depolarization up to 0 mV (Control, None).…”

Section: Regulation Of Endogenous Mouse Erg and Cardiac Action Potentmentioning

confidence: 99%

Reciprocal Control of hERG Stability by Hsp70 and Hsc70 With Implication for Restoration of LQT2 Mutant Stability

Ninomiya

Kurata

et al. 2011

Circulation Research

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Rationale:The human ether-a-go-go-related gene (hERG) encodes the ␣ subunit of the potassium current I Kr . It is highly expressed in cardiomyocytes and its mutations cause long QT syndrome type 2. Heat shock protein (Hsp)70 is known to promote maturation of hERG. Hsp70 and heat shock cognate (Hsc70) 70 has been suggested to play a similar function. However, Hsc70 has recently been reported to counteract Hsp70.Objective: We investigated whether Hsc70 counteracts Hsp70 in the control of wild-type and mutant hERG stability. Methods and Results:Coexpression of Hsp70 with hERG in HEK293 cells suppressed hERG ubiquitination and increased the levels of both immature and mature forms of hERG. Immunocytochemistry revealed increased levels of hERG in the endoplasmic reticulum and on the cell surface. Electrophysiological studies showed increased I Kr . All these effects of Hsp70 were abolished by Hsc70 coexpression. Heat shock treatment of HL-1 mouse cardiomyocytes induced endogenous Hsp70, switched mouse ERG associated with Hsc70 to Hsp70, increased I Kr , and shortened action potential duration. Channels with disease-causing missense mutations in intracellular domains had a higher binding capacity to Hsc70 than wild-type channels and channels with mutations in the pore region. Knockdown of Hsc70 by small interfering RNA or heat shock prevented degradation of mutant hERG proteins with mutations in intracellular domains. T he human ether-a-go-go-related gene (hERG) encodes the ␣ subunit of a rapidly activating delayed-rectifier K ϩ current (I Kr ), 1-3 which controls the action potential duration in cardiomyocytes. Mutations in the gene cause long-QT syndrome type 2 (LQT2), a disorder that leads to life-threatening arrhythmia. To date, more than 200 naturally occurring mutations of hERG have been identified. Functional analysis of mutant proteins showed that most of them had an impairment of protein maturation and/or trafficking. 4 -6 They are recognized by the quality control machinery of the endoplasmic reticulum (ER), ubiquitinated, and eventually degraded by the proteasomal degradation system. 6 -8 The maturation of hERG can be evaluated by comparing the levels of the 2 forms of this protein; a core-glycosylated, immature form of 135-kDa localized in the ER, and a fully glycosylated mature form of 155 kDa localized either in the Golgi apparatus or on cell surface. 7,9 Molecular chaperones participate in every step of hERG biogenesis, including synthesis, folding, assembly, and translocation. 8,10,11 The heat shock protein (Hsp)70 family, including stress-induced Hsp70 and constitutively expressed heat shock cognate protein (Hsc)70, interact with the core-glycosylated form of hERG. 8,12 Hsp70 increases the levels of both immature and mature forms of hERG, 8 whereas the role of Hsc70 remains unknown. In other channel proteins, such as the murine epithelial sodium channel, Hsc70 has been shown to counteract the action of Hsp70 and, thus, decreases the level of the channel protein. 13 The primary purpose of this stud...

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“…The finding that C-BID and BID suppressed functional Ca V 1.2 channels raised the important question of whether this dominant-negative effect was Ca V 1.2-specific, or whether it extended to other types of Ca 2ϩ channels. In particular, L-type Ca 2ϩ currents contributed by Ca V 1.3 channels (␣ 1D ) have been reported in HL-1 cells (Xia et al, 2004). The Ca V 1.3 channels can be distinguished from high-threshold Ca V 1.2 channels by their activation at mid-voltages and their lower sensitivity to nifedipine at negative membrane potentials (Koschak et al, 2001;Xu and Lipscombe, 2001;Xia et al, 2004;Zhang et al, 2005;Striessnig et al, 2006).…”

Section: ؉mentioning

confidence: 97%

“…The Ca V 1.3 channels (␣ 1D ) mediate L-type current at mid-voltages, and they are approximately 10-fold less sensitive to 1,4-dihydro-2,6-dimethyl-4-(2-nitrophenyl)-3,5-pyridinedicarboxylic acid dimethyl ester (nifedipine)-induced block than Ca V 1.2 channels at negative membrane potentials (Koschak et al, 2001). HL-1 cells also express Ca V 3 channels that activate at much lower voltages than L-type channels, and they mediate transient ("T-type") current associated with normal pacemaker activity in cardiac cells (Xia et al, 2004;Zhang et al, 2005).…”

mentioning

confidence: 99%

Design of Mutant β₂Subunits as Decoy Molecules to Reduce the Expression of Functional Ca²⁺Channels in Cardiac Cells

Télémaque

Sonkusare²,

Grain³

et al. 2008

J Pharmacol Exp Ther

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Calcium influx through long-lasting ("L-type") Ca 2ϩ channels (Ca V ) drives excitation-contraction in the normal heart. Dysregulation of this process contributes to Ca 2ϩ overload, and interventions that reduce expression of the pore-forming ␣ 1 subunit may alleviate cytosolic Ca 2ϩ excess. As a molecular approach to disrupt the assembly of Ca V 1.2 (␣ 1C ) channels at the cell membrane, we targeted the Ca 2ϩ channel ␤ 2 subunit, an intracellular chaperone that interacts with ␣ 1C via its ␤ interaction domain (BID) to promote Ca V 1.2 channel expression. Recombinant adenovirus expressing either the full ␤ 2 subunit (Full-␤ 2 ) or truncated ␤ 2 subunit constructs lacking either the C terminus, N terminus, or both (N-BID, C-BID, and BID, respectively) fused to green fluorescent protein were developed as potential decoys and overexpressed in HL-1 cells. Fluorescence microscopy revealed that the localization of Full-␤ 2 at the surface membrane was associated with increased Ca 2ϩ current mainly attributed to Ca V 1.2 channels. In contrast, truncated N-BID and C-BID constructs showed punctate intracellular expression, and BID showed a diffuse cytosolic distribution. Total expression of the ␣ 1C protein of Ca V 1.2 channels was similar between groups, but HL-1 cells overexpressing C-BID and BID exhibited reduced Ca 2ϩ current. C-BID and BID also attenuated Ca 2ϩ current associated with another L-type Ca 2ϩ channel, Ca V 1.3, but they did not reduce transient Ca 2ϩ currents attributed to Ca V 3 channels. These results suggest that ␤ 2 subunit mutants lacking the N terminus may preferentially disrupt the proper localization of L-type Ca 2ϩ channels in the cell membrane. Cardiac-specific delivery of these decoy molecules in vivo may represent a gene-based treatment for pathologies involving Ca 2ϩ overload.The predominant voltage-gated Ca 2ϩ channels (Ca V ) in cardiac cells are the dihydropyridine-sensitive Ca V 1.2 (␣ 1C ) channels that mediate long-lasting ("L-type") Ca 2ϩ current as a critical step in excitation-contraction coupling (Bers, 2002). The Ca V 1.2 channels are multiprotein complexes composed of a large, pore-forming ␣ 1C subunit and accessory ␤ and ␣ 2 -␦ subunits. The expression level of functional Ca V 1.2 channels is regulated by the ␤ subunits (␤ 1 , ␤ 2 , ␤ 3 , and ␤ 4 ), which are cytoplasmic proteins encoded by four different genes, including multiple splice variants (Foell et al., 2004). The ␤ 2 subunit is predominantly expressed in rat ventricular myocytes, although other gene families have been reported. It is noteworthy that ␤ 2 acts as a molecular chaperone that escorts ␣ 1C to the sarcolemma to localize functional Ca V 1.2 channels at the surface membrane De Waard et al., 1996;Opatowsky et al., 2003). Indeed, we recently reported that overexpression of ␤ 2 in isolated feline ventricular myocytes significantly increases Ca 2ϩ influx and cardiac contractility (Chen et al., 2005). Although mutations This work was supported in part by the

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Functional expression of L- and T-type Ca2+ channels in murine HL-1 cells

Cited by 43 publications

References 27 publications

Lipopolysaccharide prolongs action potential duration in HL-1 mouse cardiomyocytes

Lipopolysaccharide prolongs action potential duration in HL-1 mouse cardiomyocytes

Reciprocal Control of hERG Stability by Hsp70 and Hsc70 With Implication for Restoration of LQT2 Mutant Stability

Design of Mutant β₂Subunits as Decoy Molecules to Reduce the Expression of Functional Ca²⁺Channels in Cardiac Cells

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Functional expression of L- and T-type Ca2+ channels in murine HL-1 cells

Cited by 43 publications

References 27 publications

Lipopolysaccharide prolongs action potential duration in HL-1 mouse cardiomyocytes

Lipopolysaccharide prolongs action potential duration in HL-1 mouse cardiomyocytes

Reciprocal Control of hERG Stability by Hsp70 and Hsc70 With Implication for Restoration of LQT2 Mutant Stability

Design of Mutant β2Subunits as Decoy Molecules to Reduce the Expression of Functional Ca2+Channels in Cardiac Cells

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Design of Mutant β₂Subunits as Decoy Molecules to Reduce the Expression of Functional Ca²⁺Channels in Cardiac Cells