Proteome and transcriptome profiling of equine myofibrillar myopathy identifies diminished peroxiredoxin 6 and altered cysteine metabolic pathways

Abstract

Equine myofibrillar myopathy (MFM) causes exertional muscle pain and is characterized by myofibrillar disarray and ectopic desmin aggregates of unknown origin. To investigate the pathophysiology of MFM, we compared resting and 3 h postexercise transcriptomes of gluteal muscle and the resting skeletal muscle proteome of MFM and control Arabian horses with RNA sequencing and isobaric tags for relative and absolute quantitation analyses. Three hours after exercise, 191 genes were identified as differentially expressed (DE) in MFM vs. control muscle with >1 log2 fold change (FC) in genes involved in sulfur compound/cysteine metabolism such as cystathionine-beta-synthase (CBS, ↓4.51), a cysteine and neutral amino acid membrane transporter (SLC7A10, ↓1.80 MFM), and a cationic transporter (SLC24A1, ↓1.11 MFM). In MFM vs. control at rest, 284 genes were DE with >1 log2 FC in pathways for structure morphogenesis, fiber organization, tissue development, and cell differentiation including > 1 log2 FC in cardiac alpha actin (ACTC1 ↑2.5 MFM), cytoskeletal desmoplakin (DSP ↑2.4 MFM), and basement membrane usherin (USH2A ↓2.9 MFM). Proteome analysis revealed significantly lower antioxidant peroxiredoxin 6 content (PRDX6, ↓4.14 log2 FC MFM), higher fatty acid transport enzyme carnitine palmitoyl transferase (CPT1B, ↑3.49 MFM), and lower sarcomere protein tropomyosin (TPM2, ↓3.24 MFM) in MFM vs. control muscle at rest. We propose that in MFM horses, altered cysteine metabolism and a deficiency of cysteine-containing antioxidants combined with a high capacity to oxidize fatty acids and generate ROS during aerobic exercise causes chronic oxidation and aggregation of key proteins such as desmin.

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

“…Here RNA-seq analysis of middle gluteal muscle from the same six Arabian horses determined that the ATP2A1 gene encoding SERCA1 is expressed at 7.3 ± 2.8 fold greater level (mean ± SD) than the ATP2A2 gene encoding SERCA2 (Figure 1A,C). These novel RNA-seq results (Figure 1) are consistent with histological analysis of myocyte type (64), and it is well-documented that the SERCA1 protein is expressed at higher density than the SERCA2 protein in fast-twitch muscles from a wide range of nonequine mammalian species (26). The middle gluteal muscle showed insignificant expression of the horse ATP2A3 gene, which is transcribed at a level comprising ~ 0.04% of total transcripts from the ATP2A gene family (TPM/TPM) ( Figure 1A).…”

“…RNA transcriptome shotgun sequencing (RNA-seq) of the middle gluteal muscle of Arabian horses (63) was used to assess expression of the ATP2A gene family, per quantification in transcripts per million reads (TPM) reported as mean ± standard deviation (SD) (Figure 1), including comparison to muscle myocyte composition. Histological analysis determined a mean ± SD of 4.9 ± 0.9 fold greater abundance of fast-twitch myocytes (type 2A = 52 ± 8% plus type 2X = 31 ± 8%) versus slow-twitch myocytes (type 1 = 17 ± 2%) in the middle gluteal muscle of six healthy Arabian horses (N = 6) (64). Here RNA-seq analysis of middle gluteal muscle from the same six Arabian horses determined that the ATP2A1 gene encoding SERCA1 is expressed at 7.3 ± 2.8 fold greater level (mean ± SD) than the ATP2A2 gene encoding SERCA2 (Figure 1A,C).…”

“…The middle gluteal muscle showed insignificant expression of the horse ATP2A3 gene, which is transcribed at a level comprising ~ 0.04% of total transcripts from the ATP2A gene family (TPM/TPM) ( Figure 1A). RNA-seq data generated from gluteal muscle of the set of Arabian horses (64) were deposited in the National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA) database (65) with accession number SRP082284 2 .…”

“…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. RNA-seq determined that SLN is the predominant regulatory peptide gene transcribed in the gluteal muscle of Arabian horses, with >55-fold greater expression than PLN and DWORF genes, and 780 ± 420 fold greater expression than MRLN (Figure 1B,C).…”

“…The horse SLN gene product encodes a 29-residue transmembrane peptide that is missing four canonical regulatory residues (S4, T5, C9, and Y31; numbered per the consensus amino acid sequence), as determined for Thoroughbred, Standardbred, Quarter Horse, and Przewalski's Horse, plus zebra and donkey (40,41,64). Here RNA-seq was used to deduce the amino acid sequence of SLN of the Arabian horse (N = 6) ( Figure 2), which encodes the identical 29-residue sequence found in other members of the genus Equus (40).…”