Characterization of the Rapidly Activating Delayed Rectifier Potassium Current, I Kr, in HL-1 Mouse Atrial Myocytes
Department of Physiology, Shiga University of Medical Science, Otsu, Japan. Journal of Membrane Biology
(Impact Factor: 2.46).
06/2010; 235(2):73-87. DOI: 10.1007/s00232-010-9257-2
HL-1 is the adult murine cardiac cell line that can be passaged repeatedly in vitro without losing differentiated phenotype. The present study was designed to characterize the rapidly activating delayed rectifier potassium current, I (Kr), endogenously expressed in HL-1 cells using the whole-cell patch-clamp technique. In the presence of nisoldipine, depolarizing voltage steps applied from a holding potential of -50 mV evoked the time-dependent outward current, followed by slowly decaying outward tail current upon return to the holding potential. The amplitude of the current increased with depolarizations up to 0 mV but then progressively decreased with further depolarizations. The time-dependent outward current as well as the tail current were highly sensitive to block by E-4031 and dofetilide (IC(50) of 21.1 and 15.1 nM, respectively) and almost totally abolished by micromolar concentrations of each drug, suggesting that most of the outward current in HL-1 cells was attributable to I (Kr). The magnitude of I (Kr) available from HL-1 cells (18.1 +/- 1.5 pA pF(-1)) was sufficient for reliable measurements of various gating parameters. RT-PCR and Western blot analysis revealed the expression of alternatively spliced forms of mouse ether-a-go-go-related genes (mERG1), the full-length mERG1a and the N-terminally truncated mERG1b isoforms. Knockdown of mERG1 transcripts with small interfering RNA (siRNA) dramatically reduced I (Kr) amplitude, confirming the molecular link of mERG1 and I (Kr) in HL-1 cells. These findings demonstrate that HL-1 cells possess I (Kr) with properties comparable to those in native cardiac I (Kr) and provide an experimental model suitable for studies of I (Kr) channels.
Available from: Chuanfu Li
- "As noted, we have observed marked variation in the magnitude of outward K+ currents in HL-1 cells under these conditions. Indeed, we have been able to elicit robust Ca2+ transients in otherwise quiescent cells by perfusing the cells with an inhibitor of the delayed-rectifier K+ channels (E-4031, 10 μM; Graves and Wondergem, unpublished observations); which are prevalent in HL-1 cells [8,33]. Thus, variation among HL-1 cells in the strength of repolarizing K+ current during action potentials or in cells at rest may account for the different rates and amplitudes of Ca2+ transients as well as [Ca2+i. "
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ABSTRACT: The phosphoinositide 3-kinases (PI3K/Akt) dependent signaling pathway plays an important role in cardiac function, specifically cardiac contractility. We have reported that sepsis decreases myocardial Akt activation, which correlates with cardiac dysfunction in sepsis. We also reported that preventing sepsis induced changes in myocardial Akt activation ameliorates cardiovascular dysfunction. In this study we investigated the role of PI3K/Akt on cardiomyocyte function by examining the role of PI3K/Akt-dependent signaling on [Ca2+]i, Ca2+ transients and membrane Ca2+ current, I
Ca, in cultured murine HL-1 cardiomyocytes. LY294002 (1–20 μM), a specific PI3K inhibitor, dramatically decreased HL-1 [Ca2+]i, Ca2+ transients and I
Ca. We also examined the effect of PI3K isoform specific inhibitors, i.e. α (PI3-kinase α inhibitor 2; 2–8 nM); β (TGX-221; 100 nM) and γ (AS-252424; 100 nM), to determine the contribution of specific isoforms to HL-1 [Ca2+]i regulation. Pharmacologic inhibition of each of the individual PI3K isoforms significantly decreased [Ca2+]i, and inhibited Ca2+ transients. Triciribine (1–20 μM), which inhibits AKT downstream of the PI3K pathway, also inhibited [Ca2+]i, and Ca2+ transients and I
Ca. We conclude that the PI3K/Akt pathway is required for normal maintenance of [Ca2+]i in HL-1 cardiomyocytes. Thus, myocardial PI3K/Akt-PKB signaling sustains [Ca2+]i required for excitation-contraction coupling in cardiomyoctyes.
Available from: Brian D Green
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ABSTRACT: HL-1 is a line of immortalized cells of cardiomyocyte origin that are a useful complement to native cardiomyocytes in studies of cardiac gene regulation. Several types of ion channel have been identified in these cells, but not the physiologically important inward rectifier K(+) channels. Our aim was to identify and characterize inward rectifier K(+) channels in HL-1 cells. External Ba(2+) (100 µM) inhibited 44 ± 0.05% (mean ± s.e.m., n = 11) of inward current in whole-cell patch-clamp recordings. The reversal potential of the Ba(2+)-sensitive current shifted with external [K(+)] as expected for K(+)-selective channels. The slope conductance of the inward Ba(2+)-sensitive current increased with external [K(+)]. The apparent Kd for Ba(2+) was voltage dependent, ranging from 15 µM at -150 mV to 148 µM at -75 mV in 120 mM external K(+). This current was insensitive to 10 µM glybenclamide. A component of whole-cell current was sensitive to 150 µM 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), although it did not correspond to the Ba(2+)-sensitive component. The effect of external 1 mM Cs(+) was similar to that of Ba(2+). Polymerase chain reaction using HL-1 cDNA as template and primers specific for the cardiac inward rectifier K(ir)2.1 produced a fragment of the expected size that was confirmed to be K(ir)2.1 by DNA sequencing. In conclusion, HL-1 cells express a current that is characteristic of cardiac inward rectifier K(+) channels, and express K(ir)2.1 mRNA. This cell line may have use as a system for studying inward rectifier gene regulation in a cardiomyocyte phenotype.
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ABSTRACT: The human ether-a-go-go-related gene (hERG) encodes the α 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.
We investigated whether Hsc70 counteracts Hsp70 in the control of wild-type and mutant hERG stability.
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.
These results indicate reciprocal control of hERG stability by Hsp70 and Hsc70. Hsc70 is a potential target in the treatment of LQT2 resulting from missense hERG mutations.
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