The miRNA Transcriptome Directly Reflects the Physiological and Biochemical Differences between Red, White, and Intermediate Muscle Fiber Types

Abstract

MicroRNAs (miRNAs) are small non-coding RNAs that can regulate their target genes at the post-transcriptional level. Skeletal muscle comprises different fiber types that can be broadly classified as red, intermediate, and white. Recently, a set of miRNAs was found expressed in a fiber type-specific manner in red and white fiber types. However, an in-depth analysis of the miRNA transcriptome differences between all three fiber types has not been undertaken. Herein, we collected 15 porcine skeletal muscles from different anatomical locations, which were then clearly divided into red, white, and intermediate fiber type based on the ratios of myosin heavy chain isoforms. We further illustrated that three muscles, which typically represented each muscle fiber type (i.e., red: peroneal longus (PL), intermediate: psoas major muscle (PMM), white: longissimus dorsi muscle (LDM)), have distinct metabolic patterns of mitochondrial and glycolytic enzyme levels. Furthermore, we constructed small RNA libraries for PL, PMM, and LDM using a deep sequencing approach. Results showed that the differentially expressed miRNAs were mainly enriched in PL and played a vital role in myogenesis and energy metabolism. Overall, this comprehensive analysis will contribute to a better understanding of the miRNA regulatory mechanism that achieves the phenotypic diversity of skeletal muscles.

Figure 1

The characterization of various skeletal muscles. (a) Hierarchical clustering analysis for the ratios of myosin heavy chain isoforms in 15 muscle tissues based on Pearson correlation. M, masseter; T, trapezius; S, soleus; LD, latissimus dorsi; GMED, gluteus medius; PL, peroneal longus; BF, biceps femoris; PMM, psoas major muscle; SC, semispinalis capitis; SM, semimembranosus; TB, triceps brachii; GC, gastrocnemius; OEA, obliquus externus abdominis; LDM, longissimus dorsi muscle; ST, semitendinosus; (b) mtDNA copies per cell in 15 muscle tissues (p < 0.01); (c) The relative expression of lactose dehydrogenase A (LDHA) in 15 muscle tissues (p < 0.01); (d) The colors of LDM, PMM, and PL. Data are means ± SD. Statistical significance was calculated by one-way repeated-measures ANOVA (n = 3 per individual). ** p < 0.001.

Figure 2

Analysis of miRNAs universally abundant across the three muscles. (a) The top 10 miRNA reads as a percentage of total unique miRNAs; (b) Distribution of the top 10 miRNAs in each muscle; (c) Fold change of miRNAs abundantly expressed in all three muscles. The relative expression levels, measured by qRT-PCR, of: (d) miRNAs shown in (c); (e) HK2; (f) PDH1; (g) myogenic regulatory factors; and (h) angiogenesis-related genes. Data are means ± SD. Statistical significance was calculated by one-way repeated-measures analysis of variance (n = 3 per individual). * p < 0.05, ** p < 0.001.

Figure 3

Distribution of differentially expressed miRNAs among three tissues.

Figure 4

Schematic of the regulatory mechanism for myogenesis by PL-enriched miRNAs via targeting (a) myogenic regulatory factors and (b) insulin-like growth factor (IGF) pathways. The blue and black circles in (a) indicated satellite cells and cell nucleus in the myofiber (light brown tube), respectively.

Figure 5

Energy metabolism-related miRNAs enriched in PL. miRNAs involved in (a) angiogenesis and (b) reducing hypoxic damage; (c) Abundance of energy metabolism-related miRNAs.