Sarcolipin Exhibits Abundant RNA Transcription and Minimal Protein Expression in Horse Gluteal Muscle

Abstract

Ca2+ regulation in equine muscle is important for horse performance, yet little is known about this species-specific regulation. We reported recently that horse encode unique gene and protein sequences for the sarcoplasmic reticulum (SR) Ca2+-transporting ATPase (SERCA) and the regulatory subunit sarcolipin (SLN). Here we quantified gene transcription and protein expression of SERCA and its inhibitory peptides in horse gluteus, as compared to commonly-studied rabbit skeletal muscle. RNA sequencing and protein immunoblotting determined that horse gluteus expresses the ATP2A1 gene (SERCA1) as the predominant SR Ca2+-ATPase isoform and the SLN gene as the most-abundant SERCA inhibitory peptide, as also found in rabbit skeletal muscle. Equine muscle expresses an insignificant level of phospholamban (PLN), another key SERCA inhibitory peptide expressed commonly in a variety of mammalian striated muscles. Surprisingly in horse, the RNA transcript ratio of SLN-to-ATP2A1 is an order of magnitude higher than in rabbit, while the corresponding protein expression ratio is an order of magnitude lower than in rabbit. Thus, SLN is not efficiently translated or maintained as a stable protein in horse muscle, suggesting a non-coding role for supra-abundant SLN mRNA. We propose that the lack of SLN and PLN inhibition of SERCA activity in equine muscle is an evolutionary adaptation that potentiates Ca2+ cycling and muscle contractility in a prey species domestically selected for speed.

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

“…All animal research was reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Minnesota, with IACUC protocol # 1511-33199A for horses and IACUC protocol # 1611-34327A for rabbits. Palliative care and euthanasia protocols [9][10][11] were consistent with guidelines from the American Veterinary Medical Association.…”

“…SR vesicles from horse gluteus (predominantly fast-twitch myofibers) and rabbit skeletal muscle (fast-twitch muscles pooled from back and legs) were isolated using mechanical homogenization and differential centrifugation. Horse SR vesicles are defined as the pellet from the centrifugation of a gluteal muscle homogenate at 10,000× g for 20 min at 4 • C, following an initial clarification spin of 3800× g for 20 min at 4 • C. Rabbit SR vesicles are defined as the pellet from the centrifugation of a muscle homogenate (pooled back muscle and hind leg muscle) at 23,000× g for 60 min, following initial clarification spins of 4000× g for 20 min at 4 • C and 11,800× g for 20 min at 4 • C [10,11,36]. The pooled muscle tissue from the rabbit was white colored (indicating a fast-twitch muscle).…”

“…These proteins include the sarcoplasmic reticulum (SR) Ca 2+transporting ATPase protein (SERCA = 110 kDa), the regulatory peptide subunits sarcolipin (SLN = 3.3 kDa), and phospholamban (PLN = 5.2 kDa), plus the luminal Ca 2+ -storage protein calsequestrin (CASQ ~55 kDa). RNA-seq determined that (i) the SLN transcript is the predominant regulatory peptide expressed in horse muscle, as compared to the PLN transcript, and (ii) the SLN transcript is expressed at a many-fold greater level than the ATP2A1 transcript, which produces the SERCA1 protein expressed in fast-twitch skeletal muscle (hereafter, SERCA1 will be referred to as 'SERCA') [9,10]. RNA-seq also determined that the gene expression of the CASQ1 transcript, which produces the fast-and slow-twitch skeletal muscle protein isoform (hereafter referred to as CASQ) is downregulated in the gluteal muscle of male or female horses with RER, as compared to healthy male or female horses (controls).…”

“…This new horse SR prep allowed for an improved characterization of the protein profile of horse SR using quantitative immunoblotting and Stains-all in-gel staining [11]. The RNA-seq data demonstrated the supra-abundant expression of the SLN transcript in horse muscle, yet quantitative immunoblotting detected only a trace level of SLN peptide in horse SR vesicles, as compared to SERCA protein (0.06 SLN/SERCA mol/mol) [9][10][11]. SLN is the primary regulatory peptide of SERCA expressed in skeletal muscles from larger mammals, e.g., rabbits, dogs, pigs, and humans [12][13][14][15].…”

“…Therefore, the use of SLN knockout transgenic mice as experimental model muscles to study the lack of SLN is limited. Thus, an investigation of Ca 2+ uptake activity using a SR skeletal muscle system that lacks known SERCA inhibitors, i.e., the minimal or insignificant expression of SLN, PLB, and MRLN, has been limited to in vitro assays of Ca 2+ -activated ATPase activity by SR vesicles purified from horse gluteus [10,11].…”