Organization of the human myostatin gene and expression in healthy men and HIV-infected men with muscle wasting

Abstract

Myostatin, a member of the transforming growth factor-beta superfamily, is a genetic determinant of skeletal muscle growth. Mice and cattle with inactivating mutations of myostatin have marked muscle hypertrophy. However, it is not known whether myostatin regulates skeletal muscle growth in adult men and whether increased myostatin expression contributes to wasting in chronic illness. We examined the hypothesis that myostatin expression correlates inversely with fat-free mass in humans and that increased expression of the myostatin gene is associated with weight loss in men with AIDS wasting syndrome. We therefore cloned the human myostatin gene and cDNA and examined the gene's expression in the skeletal muscle and serum of healthy and HIV-infected men. The myostatin gene comprises three exons and two introns, maps to chromosomal region 2q33.2, has three putative transcription initiation sites, and is transcribed as a 3.1-kb mRNA species that encodes a 375-aa precursor protein. Myostatin is expressed uniquely in the human skeletal muscle as a 26-kDa mature glycoprotein (myostatin-immunoreactive protein) and secreted into the plasma. Myostatin immunoreactivity is detectable in human skeletal muscle in both type 1 and 2 fibers. The serum and intramuscular concentrations of myostatin-immunoreactive protein are increased in HIV-infected men with weight loss compared with healthy men and correlate inversely with fat-free mass index. These data support the hypothesis that myostatin is an attenuator of skeletal muscle growth in adult men and contributes to muscle wasting in HIV-infected men.

Figure 1

Human myostatin gene and its products. (A) Position and size of introns 1 and 2. (B) Myostatin cDNA indicating the positions of the intron/exon junctions (vertical arrows) and forward (F) and reverse (R) human primers; P3 is the only mouse primer. (C) Cloning strategy indicating Pflm1 and PstI sites. (D) Human skeletal muscle mRNA with its transcriptional initiation sites. (E) Myostatin protein precursor with positions of synthetic peptides used to raise the antibodies. (F) Sequence of exon/intron junctions limited to the first 60 bases in the intron and the 10 contiguous bases in the exon.

Figure 2

Chromosomal mapping of the human myostatin gene. (A) DNA fragments amplified from the human somatic cell hybrid panel by using primers F10/R14. Chromosomes are indicated at the top of each lane. G, human genomic DNA; C; human myostatin cDNA; N, hamster control. (B) Schematic representation of genetic markers and the YACs corresponding to chromosome region where myostatin was mapped by the radiation-reduced somatic cell hybrid panel. Marker D2S2027 also is named WI3652. The framework markers at 144 cM are shown; map distances are given in centiRays (cR) or in the lower YAC map as MB (conversion factor, 3.7 cR/MB).

Figure 3

Expression of myostatin mRNA in the human skeletal muscle. (A, Upper) Northern blot with poly(A)⁺ RNA hybridized with a P3/R13 myostatin cDNA probe. (A, Lower) The same blot hybridized with β-actin cDNA probe. (B) 5′ rapid amplification of cDNA ends/PCR-amplified products obtained with primers R19B/AP1. The use of vector primer AP1 results in a cDNA 49 nt longer than the original reverse-transcribed RNA. (C) Schematic representation of the 5′ untranslated region of the human skeletal muscle myostatin mRNA.

Figure 4

Western blot of myostatin-related protein in the skeletal muscle and human serum. Tissue extracts were prepared from mouse (Gel 1) and human skeletal muscle (Gel 2) and fractionated into supernatant (S) and membrane extracts (E). H, unfractionated homogenate. (Gel 1) The Western blot membranes were cut into three sections and reacted with either nonimmune IgG (NI) or antibody B directly (D), or in the presence of an excess of the corresponding peptide (P). (Gel 2) The S fraction also was prepared from human prostate, bladder, or penile corpora cavernosa. (Gel 3) The S fraction from the human skeletal muscle was incubated with either Con A-Sepharose (Con-A) or Sepharose (Seph). After centrifugation, both the supernatant (Spnt) and the eluates with increasing concentrations of α-methyl-d-mannoside (Elution) were submitted to Western blot assays with antibody B. (Gel 4, Left) The S fraction of the human skeletal muscle and increasing amounts of human serum (lane 1, 0.2 μl; lane 2, 0.5 μl; lane 3, 1 μl, lane 4, 2 μl) were subjected to Western blot assays with antibody B. (Gel 4, Right) The blot from Gel 4, Left was stripped and reacted with antibody A.

Figure 5

Immunohistochemical staining. (a) Sections of skeletal muscle were incubated with antibody B (×50). (b) Higher magnification (×100) of a. (c) Serial sections were stained with myosin antibody to identify type I and II fibers. (d) Negative control with antibody B preadsorbed with 7 μg/liter of peptide B.

Figure 6

Serum and intramuscular concentrations of myostatin-related protein in healthy and HIV-infected men. (A) Correlation of serum myostatin immunoreactivity and fat-free mass index (fat free mass/height²) in healthy and HIV-infected men. r = 0.33; P < 0.003. (B) Serum myostatin-related protein levels in healthy and HIV-infected men. The HIV-infected men without wasting (HIV+ NWL) either had not lost weight or had lost less than 10% body weight in the preceding 6 months. The HIV-infected men with wasting (HIV+ WL) had lost more than 10% of their premorbid weight in the preceding 6 months. (C) Western blot of the 26-kDa myostatin-related protein in skeletal muscle biopsy from healthy (C, lanes 1–3) and HIV-infected men (HIV, lanes 4–7).