The HERG K+ Channel increases Intracellular Calcium in myotubes by modulation of Calsequestrin.

The ERG1A K+ channel upregulates protein degradation in skeletal muscle atrophying in response to disuse1 and is found upregulated in muscle atrophying in response to cancer1 and denervation2. We have shown that overexpression of the HERG channel increases basal intracellular calcium concentration ([Ca2+]i) and calpain activity in C2C12 myotubes3; however, it is not known how HERG modulates [Ca2+]i. Indeed, we find that Ltype calcium current is not changed by HERG expression. Thus, to explore this mechanism, we increased [Ca2+]i by depolarization with 100 mM KCl and used Fura2 dyes and immunoblot to reveal that HERG does not alter myotube calcium levels by affecting L-type calcium channels. Instead, using the SERCA blocking agent thapsigargin with our Fura2 assay, we discovered that the HERG-mediated increase in calcium occurs through modulation of intracellular calcium stores4. Therefore, we hypothesized that HERG may be modulating[Ca2+]i by modulation of ryanodine receptor (RyR1) activity. To investigate this, we transduced myotubes in a 96-well plate with either a control or HERG-encoded adenovirus and after 48 hours loaded these with QBT Fura (Molecular Devices; San Jose, CA). At 1 hour post-loading, we treated the myotubes with either ryanodine (90 uM to block RyR1 receptors) or vehicle for 30 minutes. We then treated the cells with caffeine (5 mM) to activate ryanodine receptors and evaluated the [Ca2+]i over time by fluorescence (340 and 380 excitement; 508 nm emission). The 340/380 ratios were determined and normalized to baseline. The ratios were plotted over time, the area under the curve (AUC) was calculated, and these were analyzed by ANOVA with means separated by Tukey's test. Interestingly, in response to caffeine, the significant increase in [Ca2+]i was similar in control (73.3%, p<0.005) and HERGexpressing (71.7%, p<.001) myotubes. However, when the cells were treated with ryanodine to block RyR1, the [Ca2+]i increase was lower in the HERG-expressing (46.4%, p<0.02) relative to control (24.9%, p<0.8) myotubes. The data suggest that the increase in [Ca2+]i in the HERG-expressing cells is, at least in part, a result of RyR1 activation. Because calsequestrin 1 (CaSeq1) is an integral part of RyR1 modulation of [Ca2+]i and was reported mildly downregulated in HERG-expressing myotubes by Next Generation Sequencing, 5 we performed an immunoblot on control and HERGexpressing myotubes (n=8, 4 replicates each HERG and control), in which we detected a full length CaSeq ~63 kD protein along with ~50 kD and ~40 kD CaSeq proteins which appear to be CaSeq1 degradation products. Each protein was decreased in the HERGexpressing myotubes relative to control cells: ~63 kD decreased 47.2%, p<0.001; ~50 kD decreased 44.9%, p<0.01; and ~40 kD decreased 49.8%, p<0.01 (Student's t-test). CaSeq1 is known to function as a calcium buffer in skeletal muscle sarcoplasmic reticulum (SR), storing calcium when the concentration is high. Thus, we suggest that HERG expression produces a decrease in CaSeq protein, which removes this calcium binding protein and increases free calcium in intracellular calcium stores (Figure). Dysregulation of normal calcium buffering is known to contribute to pathologies in both skeletal muscle and heart. This is an exciting finding which merits further exploration. [ABSTRACT FROM AUTHOR]