Coding sequences of sarcoplasmic reticulum calcium ATPase regulatory peptides and expression of calcium regulatory genes in recurrent exertional rhabdomyolysis

Abstract

Background Sarcolipin (SLN), myoregulin (MRLN), and dwarf open reading frame (DWORF) are transmembrane regulators of the sarcoplasmic reticulum calcium transporting ATPase (SERCA) that we hypothesized played a role in recurrent exertional rhabdomyolysis (RER). Objectives Compare coding sequences of SLN, MRLN, DWORF across species and between RER and control horses. Compare expression of muscle Ca2+ regulatory genes between RER and control horses. Animals Twenty Thoroughbreds (TB), 5 Standardbreds (STD), 6 Quarter Horses (QH) with RER and 39 breed‐matched controls. Methods Sanger sequencing of SERCA regulatory genes with comparison of amino acid (AA) sequences among control, RER horses, human, mouse, and rabbit reference genomes. In RER and control gluteal muscle, quantitative real‐time polymerase chain reaction of SERCA regulatory peptides, the calcium release channel (RYR1), and its accessory proteins calsequestrin (CASQ1), and calstabin (FKBP1A). Results The SLN gene was the highest expressed horse SERCA regulatory gene with a uniquely truncated AA sequence (29 versus 31) versus other species. Coding sequences of SLN, MRLN, and DWORF were identical in RER and control horses. A sex‐by‐phenotype effect occurred with lower CASQ1 expression in RER males versus control males (P < .001) and RER females (P = .05) and higher FKBP1A (P = .01) expression in RER males versus control males. Conclusions and Clinical Importance The SLN gene encodes a uniquely truncated peptide in the horse versus other species. Variants in the coding sequence of SLN, MLRN, or DWORF were not associated with RER. Males with RER have differential gene expression that could reflect adaptations to stabilize RYR1.

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

“…RNA-seq data further indicated that PLN gene expression was undetected in rabbit muscle (SRA SAMN00013649 2 ). Thus, RNA-seq from horse and rabbit muscles provides results consistent with previous qRT-PCR studies which report that in non-rodent mammals such as rabbit, pig, and horse, SLN is the predominantly regulatory gene expressed in fast-twitch muscle (26,40). On the other hand, MRLN is the primary regulatory peptide gene expressed in mouse muscles (21).…”

“…We previously used quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) to quantify transcription of four genes (SLN, PLN, MRLN, and DWORF) encoding SERCA regulatory peptides in horse gluteus muscle, finding that SLN is the predominant regulatory peptide gene expressed in the middle gluteal muscle of Thoroughbred horses (40). Here we mined RNA-seq data (64) to quantify transcription of the same four genes encoding SERCA regulatory peptides (SLN, PLN, MRLN, and DWORF) in the middle gluteal muscle of six Arabian horses.…”

“…In comparison, horse MRLN is a 46-residue transmembrane peptide, similar to human, rabbit, and mouse orthologs, with 5-7 potential phosphorylation sites (Ser or Thr) in the N-terminus, as deduced per amino acid sequences (40). Before the MRLN gene product was identified as a sORF coding for a translated MLRN peptide that inhibits SERCA in mouse muscle, the MRLN transcript was identified as a functional long non-noncoding RNA (lncRNA) that controls myogenic differentiation, classified as Linc-RAM and LINC00948 in mouse and human genomes, respectively (21,45,46).…”

“…The molecular etiology of RER in Thoroughbred racehorses has been suggested to involve SR Ca 2+ release and excitation-contraction coupling (57,58,61,62). We determined recently that gene transcription of CASQ1 (skeletal muscle isoform) is downregulated in the gluteal muscle of male or female horses with RER compared to healthy male or females horses (controls), while CASQ1 was also downregulated to a greater extent in the gluteal muscle of male horses with RER compared to female horses with RER (40).…”

“…The horse SLN peptide encodes 29 residues (40) versus a consensus 31-residue length for orthologous SLN peptides encoded by 100+ species from the five vertebrate classes: mammal, bird, reptile, amphibian, and fish (18,41,42). Uniquely, horse SLN is the only reported ortholog missing four regulatory sites (18,(40)(41)(42) that are common in other species: Ser4 and Thr5 for SLN phosphorylation and relief of SERCA inhibition (17,33), Cys9 for SLN acylation and relief of SERCA uncoupling (18,34), and Tyr31 for SERCA interaction, organelle targeting of SLN, and potential luminal nitration of SLN (31,43,44), with residues numbered using the consensus 31-residue length, e.g., rabbit, mouse, and human (18,(40)(41)(42). Salmon processing, were used to compile gene expression between each individual samples (N ≥ 5 horses per transcript) and for relative comparative analyses with rabbit gene expression.…”