Horse gluteus is a null-sarcolipin muscle with enhanced sarcoplasmic reticulum calcium transport

Abstract

We have analyzed gene transcription, protein expression, and enzymatic activity of the Ca 2+ -transporting ATPase (SERCA) in horse gluteal muscle. Horses are bred for peak athletic performance but exhibit a high incidence of exertional rhabdomyolysis, with myosolic Ca 2+ suggested as a correlative linkage. To assess Ca 2+ regulation in horse gluteus, we developed an improved protocol for isolating horse sarcoplasmic reticulum (SR) vesicles. RNA-seq and immunoblotting determined that the ATP2A1 gene (protein product SERCA1) is the predominant Ca 2+ -ATPase expressed in horse gluteus, as in rabbit muscle. Gene expression was assessed for four regulatory peptides of SERCA, finding that sarcolipin (SLN) is the predominant regulatory peptide transcript expressed in horse gluteus, as in rabbit muscle. Surprisingly, the RNA transcription ratio of SLN-to-ATP2A1 in horse gluteus is an order of magnitude higher than in rabbit muscle, but conversely, the protein expression ratio of SLN-to-SERCA1 in horse gluteus is an order of magnitude lower than in rabbit. Thus, the SLN gene is not translated to a stable protein in horse gluteus, yet the suprahigh level of SLN RNA suggests a non-coding role. Gel-stain analysis revealed that horse SR expresses calsequestrin (CASQ) protein abundantly, with a CASQ-to-SERCA ratio ~3-fold greater than rabbit SR. The Ca 2+ transport rate of horse SR vesicles is ~2-fold greater than rabbit SR, suggesting horse myocytes have enhanced luminal Ca 2+ stores that increase intracellular Ca 2+ release and muscular performance. The absence of SLN inhibition of SERCA and the abundant expression of CASQ may potentiate horse muscle contractility and susceptibility to exertional rhabdomyolysis. The horse has been selectively bred for thousands of years to achieve remarkable athletic ability, in part conferred by a naturally-high proportion (75-95%) of fast-twitch myofibers in locomotor muscles that provide powerful contractions and high speed (1). Calcium (Ca 2+ ) 1 is the signaling molecule that controls muscle contraction by activating the actomyosin sliding-filament mechanism (2). In turn, myosolic Ca 2+ is controlled predominantly by the sarcoplasmic reticulum (SR), a membrane organelle that connects with transverse tubules as membrane junctions and also surrounds actomyosin myofibrils as longitudinal sheaths (3). Thus, SR is a dual structure/function membrane system comprising junctional SR with the ryanodine receptor Ca 2+ release channel (RYR) acting to initiate contraction, and longitudinal SR with the Ca 2+ transporting ATPase (SERCA) acting to induce relaxation (4,5). For muscle contraction, the bolus of released Ca 2+ originates almost solely from SR through the RYR channel, and this released Ca 2+ is subsequently re-sequestered in the SR lumen by SERCA (6). The strength of muscle contraction is controlled by the amount of Ca 2+ released from SR, which is in turn modulated by the Ca 2+ load in the SR lumen (7). Calsequestrin (CASQ), which binds up to 80 Ca 2+ ions (mol/mol), is the high-capacity ...

Uncoupling of sarcoendoplasmic reticulum calcium ATPase pump activity by sarcolipin as the basis for muscle non-shivering thermogenesis

“…In a recent study by Autry et al [123], it was shown that the gluteus muscle of horses is devoid of SLN protein in spite of the high amount of mRNA expression. This is interesting in the light that horses lack UCP1 and are very cold tolerant (they lived side by side with woolly mammoths and woolly rhinos during the Ice Ages), therefore bringing the role of SLN-based NST into question.…”

Sarcoplasmic Reticulum from Horse Gluteal Muscle Is Poised for Enhanced Calcium Transport

“…The differences in the V max of the horse versus rabbit SERCA orthologs could potentially be attributed to the lack of SLN peptide in horse muscle [10,11]. Another possibility for increased transport is sequence variation in the horse versus rabbit SERCA proteins that affect the equilibrium distribution among biochemical intermediates, i.e., the activation energy for conformational transitions in steps along the enzymatic cycle [65]. For example, the single-residue variations R164H and R559C-each of which individually decrease the activity of bovine SERCA-are sufficient to induce congenital pseudomyotonia syndrome in cattle [66][67][68].…”