Developmentally regulated alternative splicing is perturbed in type 1 diabetic skeletal muscle

Abstract

Introduction: Type 1 diabetic patients can develop skeletal muscle weakness and atrophy by molecular mechanisms that are not well understood. Alternative splicing (AS) is critical for gene expression in the skeletal muscle, and its dysregulation is implicated in muscle weakness and atrophy. Therefore, we investigated whether AS patterns are affected in type 1 diabetic skeletal muscle contributing to skeletal muscle defects.

Methods: AS patterns were determined by reverse transcription-polymerase chain reaction and levels of RNA binding proteins were assessed by Western blot in type 1 diabetic mouse skeletal muscle and during normal mouse skeletal muscle development.

Results: Five genes with critical functions in the skeletal muscle are misspliced in type 1 diabetic skeletal muscle, resembling their AS patterns at embryonic stages. AS of these genes undergoes dramatic transitions during skeletal muscle development, correlating with changes in specific RNA binding proteins.

Conclusion: Embryonic spliced variants are inappropriately expressed in type 1 diabetic skeletal muscle. Muscle Nerve 56: 744-749, 2017.

Keywords: RNA binding proteins; alternative splicing; muscle development; skeletal muscle; type 1 diabetes.

FIGURE 1

Alternative splicing is dysregulated in skeletal muscle of non-obese diabetic mice. Alternative splicing analysis of (A) and (B), Ppp3ca exon 13, (C) Atp2b1 exon 21, (D) Fxr1 exon 15 + 16, (E) Mtmr3 exon 16, and (F) Gpx8 exon 2 in ICR/HaJ (control) (white bars) or type 1 non-obese diabetic (T1D:NOD) (black bars) mouse gastrocnemius muscle, as determined by qRT-PCR (n ≥ 4; *P < 0.05, **P < 0.01, ***P < 0.005).

FIGURE 2

Alternative splicing events affected in type 1 diabetic skeletal muscle undergo changes during mouse skeletal muscle development. Representative gel images of (A) Ppp3ca and (B) Atp2b1 AS in normal FVB mouse skeletal muscle during development. Quantification of alternative exon inclusions of (A) Ppp3ca, (B) Atp2b1, (C) Fxr1, (D) Mtmr3, and (E) Gpx8 in embryonic day 18 (E18) limb, newborn limb, and adult mouse quadriceps (n = 3; *P < 0.05, **P < 0.01, ***P < 0.005).

FIGURE 3

Expression of splicing regulators is dynamically controlled during skeletal muscle development. Western blot (WB) analysis of CELF1, RBFOX1, MBNL1, and Histone H3 in wild-type E18 embryonic mouse limb, newborn mouse limb, and adult mouse quadriceps. Ponceau S staining and Histone H3 WB were used to monitor protein loading.

FIGURE 4

Protein levels of splicing regulators are modulated in non-obese diabetic skeletal muscle. Western blot analysis of (A) RBFOX1 and MBNL1 in ICR (control) or non-obese diabetic (T1D:NOD) gastrocnemius muscle of mice. (B) CELF1, MYOD1, and MYF5 in control (n = 3) and NOD (n = 7) mice gastrocnemius muscle. Ponceau S staining was used to monitor protein loading. Fold-change in each protein in NOD mice skeletal muscle was quantified after normalizing to the loading control and compared with control ICR mice (*P < 0.05, **P < 0.01, ***P < 0.005, NS = not significant). Fold changes in protein levels were as follows: RBFOX1 protein 2.65 ± 0.15 (P < 0.01); MBNL1 protein 1.1 ± 0.1 (not significant); CELF1 protein 2.78 ± 0.36 (P < 0.05); MYOD1 protein −2.17 ± 0.06 (P < 0.005); and MYF5 protein −1.79 ± 0.09 (P < 0.05).