ERG1A K+ channel increases intracellular calcium concentration through modulation of calsequestrin1 in C2C12 myotubes

Abstract

The ERG1A K⁺ channel modulates the protein degradation that contributes to skeletal muscle atrophy by increasing intracellular calcium concentration ([Ca²⁺]i) and enhancing calpain activity, but the mechanism by which the channel regulates the [Ca²⁺]i is not known. Here, we have investigated the effect of human ERG1A (HERG) on [Ca²⁺]i in C₂C₁₂ myotubes, using Fura-2 calcium assays, immunoblot, RT-qPCR, and electrophysiology. The data show that the rise in [Ca²⁺]_i induced by KCl-stimulated depolarization is of greater amplitude in C₂C₁₂ myotubes over-expressing HERG relative to controls, but this difference does not result from an increase in L-type channel (Ca_v1.1) Ca²⁺ influx because there is no statistical difference in the nifedipine-sensitive response upon depolarization between the expression groups. Indeed, HERG overexpression in C₂C₁₂ myotubes has no effect on the amplitude of L-type channel current nor does it affect the mRNA levels nor protein abundance of the Cav1.1 channel. This finding suggests that HERG modulates excitation coupled calcium entry (ECCE). Indeed, the HERG-enhanced increase in [Ca²⁺]i induced by depolarization is blocked by 2-aminoethoxydiphenyl borate, an inhibitor of ECCE. Further, HERG also modulates the activity of ryanodine receptors (RYR1, a component of ECCE) as well as store operated calcium entry (SOCE). Therefore, we investigated the effect of HERG on calsequestrin1, a calcium buffering/binding protein known to modulate RYR1 and SOCE activities. Indeed, we find that calsequestrin1 mRNA levels are decreased 0.83-fold (p < 0.05) and the total protein abundance is lowered 77% (p < 0.05) in myotubes over-expressing HERG relative to controls. In conclusion, the data show that ERG1A overexpression modulates [Ca²⁺]i in skeletal muscle cells by lowering the abundance of the calcium buffering/binding protein calsequestrin1 which interacts with RyR1 and SOCE pathways. Indeed, we report that overexpression of HERG in myotubes increases [Ca²⁺]_i by modulation of RyR1 as well as ECCE and SOCE activities. It is likely that HERG enhancement of RyR1 activity, through decreased Casq1 abundance, is increasing [Ca²⁺]i. This study provides a potential mechanism to explain how upregulation of ERG1A contributes to increased [Ca²⁺]i and, thus, atrophy in skeletal muscle.

Keywords: Ether-a-gogo related K+ channel; Intracellular calcium; RyR1; Skeletal muscle; Store operated calcium entry; calsequestrin1.

Fig. 1

HERG does not enhance intracellular calcium concentration ([Ca²⁺]i) through modulation of the L-type channel in skeletal muscle. (A) The increase in intracellular Ca²⁺ ([Ca²⁺]i) initiated by depolarization (with 100 mM KCl treatment) is significantly greater in myotubes expressing HERG relative to controls. (B) Change of [Ca²⁺]i in control and HERG treated cells in response to KCl treatment over time (cells from Figure A). (C) Nifedipine (10 µM) has a significant inhibitory effect on the increase in [Ca²⁺]i that occurs in control cells in response to depolarization over 90 s after addition of KCl. (D) Change of [Ca²⁺]i in control and HERG treated cells in response to KCl and nifedipine treatments over time (cells from Figure C). (E) Nifedipine also inhibits a portion of the HERG-induced increase in [Ca²⁺]i for up to 40 s. (F) Nifedipine-sensitive fluorescence ratios do not differ significantly between the control and HERG-expressing myotubes, demonstrating that the HERG-modulated increase in [Ca²⁺]i does not result from activation of L-type Ca²⁺ channels. [Ca²⁺]i was evaluated by the ratiometric fluorescent Fura-2 dye. The 340/380 nm ratios were determined, normalized to baseline, and analyzed by a 2 × 2 ANOVA design for repeated measures. There was no statistically significant interaction between HERG and treatment. The bars (A,C,E) and symbols (B,D,F) represent means. The error bars represent standard error of the mean. n = 16 (8 control and 8 HERG-expressing myotubes). *p < 0.05, **p < 0.01.

Fig. 2

HERG Expression does not change peak Cav channel current density in C₂C₁₂ myotubes. (A,B) Example ensembles of current traces elicited by stepping from − 80 mV to 50 mV in 10 mV increments for 150 ms, from a holding potential of − 60 mV in a control transduced myotube (A) or a HERG transduced myotube (B). Currents in (A) and (B) were recorded in a bath solution containing 140 mM Na⁺ and 10 mM Ba²⁺. (C) Compiled IV curves for peak Ba²⁺ (slow) currents measured in control or HERG expressing myotubes. The current density differed only at the two most positive voltages (40 and 50 mV), *p < 0.05.

Fig. 3

Cav1.1 L-type Ca²⁺ channel gene expression and protein abundance are not affected by HERG expression in C₂C₁₂ myotubes. (A,B) rtPCR assays reveal that mRNA levels of Cav1.1 (adult [A] and embryonic [Cav1.1e; B]) are not affected by HERG expression for up to 60 h post transduction. Expression of genes encoding either Cav1.2 or 1.3 was not detected in the myotubes. (C,D) Immunoblot (C) and optical density measures of protein bands (D) show that Cav1.1 protein abundance is not significantly affected at 48 h post HERG expression. (A,B,D) Bars represent mean fold changes in HERG-expressing cells over control cells, error bars represent standard deviations, and filled circles represent a single data point/replicate. A one-way ANOVA was used to analyze the data in panels (A and B). Data displayed in panel (D) were analyzed by a Student’s t-test. (A,B) The data in panels (A) and (B) were derived from the same sample set. There were 9 replicates for each the control and the HERG-expressing cells, which were assayed at the three different time points (0, 46,and 60 h post-transduction). The data were calculated as HERG/control (see Methods) to produce ninefold change data points, three per time point. Thus, n = 9 data points total (i.e., ninefold changes—HERG-expressing over control cells). Two different sample sets were analyzed and yielded the same basic results; only one set is represented here. (C,D) The data shown in panels (C) and (D) were derived from a set of 6 cellular lysates (n = 6) composed of 3 control and 3 HERG-expressing myotube lysate samples. This experiment was performed with two different sample sets of which one is shown here. (See Additional file 2 for the second immunoblot and all full length Cav1.1 immunoblots.)

Fig. 4

Excitation coupled calcium entry (ECCE) is a source of the greater increase in intracellular calcium concentration ([Ca²⁺]i) that is elicited in response to depolarization by KCl in HERG-expressing cells. (A) Initially, in control cells, 2-APB appears to have a mild, but statistically insignificant inhibitory effect on the initial mean increase in [Ca²⁺]i that occurs in response to depolarization. (B) The 2-APB has a statistically significant inhibitory effect on the HERG-modulated increase in [Ca²⁺]i that occurs in response to depolarization. (C) The initial 2-APB-sensitive currents differ significantly between the depolarized control and HERG-expressing myotubes, suggesting that a source of the initial HERG-modulated increase in [Ca²⁺]i is extracellular. The [Ca²⁺]i was evaluated by the ratiometric fluorescent Fura-2 dye and the 340/380 ratios were determined and normalized to baseline. The data of panels (A) and (B) were analyzed by a 2 × 2 ANOVA design for repeated measures; the interaction between HERG and treatment was not statistically significant. For panel (C), the mean difference and standard error of the mean difference were used to estimate p values using a comparison of means calculator (see Methods). The bars (A,B) and symbols (C) represent means of time frame units and error bars represent standard deviations. n = 24 (12 control and 12 HERG-expressing myotubes).

Fig. 5

Sarcoplasmic reticulum Ca²⁺ stores is a source of the greater increase in intracellular Ca²⁺ concentration ([Ca²⁺]i) that occurs in response to depolarization by KCl (100 mM) in HERG-expressing cells. (A) In control cells, thapsigargin (Tg, 1 µM) has no statistically significant effect on [Ca²⁺]i in response to depolarization. (B) Change of [Ca²⁺]i in control cells in response to KCl treatment with and without Tg over time (cells from Figure A). (C) Thapsigargin has a significant inhibitory effect on the HERG-modulated increase in [Ca²⁺]i that occurs in response to depolarization. (D) Change of [Ca²⁺]i in HERG-expressing cells in response to KCl treatment with and without Tg over time (cells from Figure C). (E) Initial Tg-sensitive fluorescent ratios differ significantly between the depolarized control and HERG-expressing myotubes, suggesting that HERG may also affect release of Ca²⁺ from intracellular stores. The [Ca²⁺]i was evaluated by the ratiometric fluorescent Fura-2 dye and the 340/380 ratios were determined and normalized to baseline. The fluorescence ratios at noted timepoints (in panels A and C) were analyzed by a 2 × 2 ANOVA design for repeated measures. There was no significant interaction between HERG and treatment. For panel (E), the mean difference and standard error of the mean difference were used to estimate p values using a comparison of means calculator (see Methods). The bars (A,C) and symbols (B,D,E) represent mean fluorescence ratios (i.e., [Ca²⁺]i levels) and error bars represent standard deviation. n = 20 (10 control and 10 HERG-expressing myotubes). *p < 0.05.

Fig. 6

HERG modulates ryanodine receptor-mediated Ca²⁺ release. (A) HERG-over-expressing myotubes exhibit a significantly greater increase in intracellular concentration ([Ca²⁺]i) than control cells when treated with caffeine (10 mM); this response is significantly inhibited by ryanodine (90 uM) in both HERG-expressing and control myotubes, but the inhibition is more statistically significant in the HERG-over-expressing cells. n = 36 wells (18 wells of HERG-expressing myotubes and 18 wells of controls). (B) Representative line graph of a single assay of 24 wells. n = 24; the 24 wells consisted of 3 wells per each of six groups. The [Ca²⁺]i was evaluated by ratiometric fluorescent Fura-2 dye and the 340/380 ratios were determined and normalized to baseline. Areas under the curve (AUCs) were analyzed by one-way ANOVA and means were separated by Tukey’s test. The bars (A) and symbols (B) represent means and error bars represent the standard deviation. Con = control cells transduced with the control adenovirus rather than the one encoding HERG. Buf = cells treated with the vehicle buffer rather than ryanodine. Ryano = ryanodine. Caff = caffeine.

Fig. 7

HERG modulates SOCE. (A) SOCE Activity in control and HERG over-expressing myotubes. Graphic shows changes in 340/380 nm ratios over time as measured by fluorescence (Fura-2 dye) in response to thapsigargin (Tg, 1 µM, 20 min) and then CaCl₂ (2.5 mM, 10 min) treatments with and without 2-APB (100 µM). Ratios represent intracellular calcium concentration ([Ca²⁺]i). All time points were normalized (by subtraction) to the average 1 min initial baseline (except panel E). (B) SOCE activity in Control and HERG over-expressing myotubes in the absence of 2-APB. HERG over-expressing cells exhibit a significantly greater increase in [Ca²⁺]i than control cells when treated with high calcium after depletion of SR calcium stores. (C) SOCE activity in control and HERG overexpressing myotubes treated with 2-APB. 2-APB blocks the rise in [Ca²⁺]i that would occur upon addition of extracellular Ca²⁺. (D) [Ca²⁺]i response to Tg. Graphic showing the areas under the curve (AUCs) resulting from Tg treatment (panel A data). For each treatment group, the 340/380 nm ratios were determined at each timepoint after Tg addition and before calcium treatment. The timepoint ratios were normalized and the AUCs were determined. (E) HERG enhances SOCE activity. For each treatment group, the 340/380 nm ratios were determined at each timepoint after calcium addition and normalized (by subtraction) to the average “baseline” measured for two minutes prior to calcium addition to correct for differences in [Ca²⁺]i occurring during incubation with Tg. (F) SOCE activity normalized to initial one min baseline. Here, for each treatment group, the 340/380 nm ratios were determined at each timepoint after calcium addition and normalized to the initial baseline. Statistics. The AUCs for time points after treatments were calculated per treatment group and analyzed by one-way ANOVA using GraphPad Prism. When different, means were separated by Tukey’s test. The symbols (A–C) and bars (D–F) represent means and error bars represent the standard deviation. n = 12 wells per viral treatment (6 wells of HERG-expressing myotubes and 6 wells of controls; per viral treatment, 3 wells received 2-APB and 3 received vehicle). For panels (D) and (F), the change in AUCs resulting from 2-APB within cell expression group (control or HERG) were calculated by subtracting all 2-APB treated replicates from all non-treated replicates within a cell expression group and then compared between expression group (control versus HERG) using a Student’s t-test.

Fig. 8

Calsequestrin 1 (Casq1) mRNA and protein levels are reduced in C₂C₁₂ myotubes at 48 h after transduction with 400 MOI HERG encoded virus relative to myotubes transduced with control virus. (A) Fold changes in HERG and Casq1 mRNA levels in response to transduction with HERG-encoded adenovirus. HERG mRNA increased 2.6-fold (p < 0.02) in HERG-transduced myotubes and Casq1 mRNA decreased 0.83-fold (p < 0.05) in the HERG-expressing cells. (B) Control and HERG-expressing myotube lysates immunoblotted with antibody specific for Casq1 protein. (C) Control and HERG-expressing myotube lysates immunoblotted with antibody specific for the “house-keeping” protein GAPDH. (D) PVDF membrane stained with Coomassie blue to confirm equal sample loading in lanes. (E) Normalized optical densities (OD) of individual Casq1 proteins and of Casq1 proteins combined (“Total”) from immunoblot. Mean ODs of control and HERG-expressing cells (within each protein) were analyzed by Student’s T-test. Bars represent average OD and error bars denote the standard deviation. n = 8, 4 control groups and 4 HERG-expressing groups. Skm = mouse skeletal muscle. All full blots are available in Additional file 3.

Fig. 9

HERG increases intracellular Ca²⁺ concentration by decreasing abundance of the Ca²⁺ binding/buffering protein Calsequestrin1. Two important questions are: (A) How does ERG1A decrease Calsequestrin1?; and (B) What induces increased levels of the ERG1A channel itself?