Ion Channel Activity in a 3‐hydroxyacyl‐coA dehydratase 1 (HACD1) Deficient Muscle Cell Line

Abstract

Centronuclear myopathy in Labrador retrievers is the most common inherited neuromuscular disorder in dogs: this is caused by a mutation in a fatty acid processing gene, HACD1. The mutation is carried by 15–20% of the breed worldwide (Pele et al., 2005). These dogs present with generalised progressive weakness. A congenital myopathy due to a HACD1 mutation has also been documented in a human family, where infants have hypotonia and delayed motor milestones (Muhammed et al., 2013). The cellular mechanisms by which HACD1 mutation lead to muscle weakness are unknown, but since fatty acids interact with membrane ion channels we hypothesised that dysregulation of membrane function could be involved. To investigate this we have used a muscle cell line (C2C12) with shRNA knock down (KD) of HACD1 in combination with ion channel gene qPCR and patch‐clamp electrophysiology.To quantify changes in C2C12 myogenic differentiation following HACD1 KD, we used a fusion index calculation on day 8 myotubes stained for the differentiation marker MF20 and counted nuclei that were present in MF20 positive myotubes against those that were in undifferentiated, MF20 negative cells. No significant difference in myotube fusion was observed in HACD1‐KD cells compared to control (n=2).Before beginning patch‐clamp studies on C2C12 cells we used qPCR to identify potential ion channel targets in control C2C12 myoblasts. We found expression of (<30ct) of 21 ion channels, including those of particular interest to calcium homeostasis such as the voltage‐gated Ca2+ channels; Cacna1c, cacna1g, the non‐selective cation (NSC) channels; TRPV4, TRPV2, TRPC1 and a Ca2+‐activated K+ channel; Kcnn3.To functionally identify channel gating we used cell‐attached patch‐clamp electrophysiology of control and HACD1‐KD C2C12 myoblasts. K‐means clustering revealed five statistically significant clusters of ion channel activity in these cells (p<0.05). Assuming an RMP of −15mV (Tanaka et al., 2017) clusters had centeroids of;(i) conductance 21 ± 0.4pS, Vrev 30 ± 6mV (ii) 28 ± 6pS, −38 ± 4mV (iii) 29 ± 0.7pS, −78 ± 4mV (iv) 40 ± 6pS, 5 ± 3mV (v) 94 ± 13, 16 ± 44mV.In conclusion, the phenotype of the smallest ion channel conductance (28pS) would be consistent with the Ca2+‐activated K+ channel KCNN3, and the 94pS consistent with any of the three TRP channels identified by qPCR. These will be further characterised with pharmacological inhibitors and in the future we will examine if the populations change as the control and HACD1‐KD myoblasts differentiate into myotubes.Support or Funding InformationAuthors would like to thank J Blondelle & L Tiret from the Alfort Veterinary School for the cells used in these experiments.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.