Characterization of the rapidly activating delayed rectifier potassium current, I (Kr), in HL-1 mouse atrial myocytes.
ABSTRACT 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.
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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.Journal of Biomedical Science 06/2012; 19(1). DOI:10.1186/1423-0127-19-59 · 2.74 Impact FactorThis article is viewable in ResearchGate's enriched formatRG Format enables you to read in context with side-by-side figures, citations, and feedback from experts in your field.
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ABSTRACT: Mdivi-1 is an inhibitor of dynamin related protein 1- (drp1)-mediated mitochondrial fission. However, the mechanisms through which this compound interacts directly with ion currents in heart cells remain unknown. In this study, its effects on ion currents and membrane potential in murine HL-1 cardiomyocytes were investigated. In whole-cell recordings, the addition of mdivi-1 decreased the amplitude of tail current (I(tail)) for the rapidly activating delayed-rectifier K⁺ current (I(Kr)) in a concentration-dependent manner with an IC₅₀ value at 11.6 μM, a value that resembles the inhibition requirement for mitochondrial division. It shifted the activation curve of I(tail) to depolarized voltages with no change in the gating charge. However, mdivi-1 did not alter the rate of recovery from current inactivation. In cell-attached configuration, mdivi-1 inside the pipette suppressed the activity of acetylcholine-activated K⁺ channels without modifying the single-channel conductance. Mdivi-1 (30 μM) slightly depressed the peak amplitude of Na⁺ current with no change in the overall current-voltage relationship. Under current-clamp recordings, addition of mdivi-1 resulted in prolongation for the duration of action potentials (APs) and to increase the firing of spontaneous APs in HL-1 cells. Similarly, in pituitary GH₃ cells, mdivi-1 was effective in directly suppressing the amplitude of ether-à-go-go-related gene-mediated K⁺ current. Therefore, the lengthening of AP duration and increased firing of APs caused by mdivi-1 can be primarily explained by its inhibition of these K⁺ channels enriched in heart cells. The observed effects of mdivi-1 on ion currents were direct and not associated with its inhibition of mitochondrial division.European journal of pharmacology 02/2012; 683(1-3):1-9. DOI:10.1016/j.ejphar.2012.02.012 · 2.59 Impact Factor
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ABSTRACT: 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(2+) 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 μg/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(+) and of an outward current attributed to K(+); both were measured by whole cell voltage clamp. The K(+) currents displayed a resurgent outward tail current, which is characteristic of the rapid delayed-rectifier K(+) current, I(Kr). LPS accordingly reduced outward currents measured with pipette Cs(+) substituted for K(+) to isolate I(Kr). E-4031 (1 μM) markedly inhibited I(Kr) 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 I(Kr) and decreases their rate of firing by inhibition of I(Na.) This protracted time course points toward an intermediary metabolic event, which either decreases available mouse ether-a-go-go (mERG) and Na(+) channels or potentiates their inactivation.AJP Cell Physiology 08/2012; 303(8):C825-33. DOI:10.1152/ajpcell.00173.2012 · 3.71 Impact Factor