Alterations in muscle mass and contractile phenotype in response to unloading models: role of transcriptional/pretranslational mechanisms

Abstract

Skeletal muscle is the largest organ system in mammalian organisms providing postural control and movement patterns of varying intensity. Through evolution, skeletal muscle fibers have evolved into three phenotype clusters defined as a motor unit which consists of all muscle fibers innervated by a single motoneuron linking varying numbers of fibers of similar phenotype. This fundamental organization of the motor unit reflects the fact that there is a remarkable interdependence of gene regulation between the motoneurons and the muscle mainly via activity-dependent mechanisms. These fiber types can be classified via the primary type of myosin heavy chain (MHC) gene expressed in the motor unit. Four MHC gene encoded proteins have been identified in striated muscle: slow type I MHC and three fast MHC types, IIa, IIx, and IIb. These MHCs dictate the intrinsic contraction speed of the myofiber with the type I generating the slowest and IIb the fastest contractile speed. Over the last ~35 years, a large body of knowledge suggests that altered loading state cause both fiber atrophy/wasting and a slow to fast shift in the contractile phenotype in the target muscle(s). Hence, this review will examine findings from three different animal models of unloading: (1) space flight (SF), i.e., microgravity; (2) hindlimb suspension (HS), a procedure that chronically eliminates weight bearing of the lower limbs; and (3) spinal cord isolation (SI), a surgical procedure that eliminates neural activation of the motoneurons and associated muscles while maintaining neurotrophic motoneuron-muscle connectivity. The collective findings demonstrate: (1) all three models show a similar pattern of fiber atrophy with differences mainly in the magnitude and kinetics of alteration; (2) transcriptional/pretranslational processes play a major role in both the atrophy process and phenotype shifts; and (3) signaling pathways impacting these alterations appear to be similar in each of the models investigated.

Keywords: hindlimb suspension (unloading); myosin isoforms; non-coding RNAs; spaceflight; spinal cord isolation.

Figure 1

Functional specialization of skeletal muscle. The brain initiates the motor command for a motor neuron to fire and stimulate a group of muscle fibers to contract. A motor unit consists of a single motor neuron together with all the muscle fibers it innervates. Different types of motor units express different MHC phenotypes, having a specialized function. Note that each myofiber can express either a single MHC isoform, or a hybrid mix of two or more isoforms. Slow motor unit expresses type I and are engaged in antigravity postures. The fast glycolytic motor units express IIb and IIx, and are recruited during burst power like during weight lifting. (Weight lifting image is Wikimedia Commons depicting Andrei Rybakou of Belarus Weightlifting at the 2008 Summer Olympics in the 85 kg category. This image is licensed under the Creative Commons Attribution 2.0 Generic license).

Figure 2

Flow of genetic information and key steps in the regulation of gene expression. The level of protein expressed in the cell results from the net balance between protein synthesis and protein degradation. Protein synthesis can be regulated via several processes including those operating at the transcriptional, post-transcriptional, pre-translational, translational, and post-translational levels. The product of each step is subjected to degradation control.

Figure 3

Protein degradation via the ubiquitin/proteasome system. Multiple Ub molecules are covalently conjugated to amino groups of the protein. Ub is first activated by E1 (Ub activating enzyme), then transferred to E2 (Ub carrier protein or Ub-conjugating enzyme) to be ligated to an amino group of the target protein with the help of E3 (Ub-protein ligase). This is followed by a sequential conjugation of additional Ub molecules each linked to an NH2 group of a lysine of the previously added Ub, thereby generating a polyubiquitinated protein that becomes recognized by the 26S proteasome machinery and targeted for degradation.

Figure 4

Both soleus and medial gastrocnemius (MG) atrophy in response to unloading. Change in muscle mass relative to control of both MG and Soleus in response to 16 days of hindlimb suspension in rats.

Figure 5

Signaling Pathways Leading to Altered Protein Balance Affecting Muscle Fiber Size. A simplified schematic of signaling pathways affecting protein balance in muscle fibers. Signals are initiated by either growth factors, nerve, or muscle contractile activity and are transmitted into the cells to affect protein synthesis via mTOR/Akt signals, protein transcription via MAPK ERKs, and protein degradation through Foxo/Atrogin/Murf1 action. For further information, the readers are referred to excellent reviews by Sandri (2008); Elkina et al. (2011); Bonaldo and Sandri (2013) and by Frost and Lang (2012).

Figure 6

Muscle atrophy in antigravity muscle is rapid and preceded by transcriptional repression of myosin and actin. Change in soleus mass, actin and type I MHC pre-mRNA in response to 7 days hindlimb suspension (HS) unloading. ^* is for p < 0.05 vs. 0 Day time point for each variable.

Figure 7

The organization of the sarcomeric myosin heavy chain (MHC) gene family. At least 8 MHC genes are expressed in striated muscle and are found in two clusters: (1) the cardiac MHC gene cluster on rat chromosome 15, which consists of the type I also called β and the α cardiac MHC genes. Type I is the slow MHC expressed in slow skeletal muscle fibers; (2) the skeletal MHC gene cluster on rat chromosome 10, the embryonic (Emb), fast IIa, IIx, IIb, neonatal (Neo) and extraocular (Eo) genes are located in tandem in the order depicted. This MHC gene organization, order, head to tail orientation, and spacing has been conserved through millions of years of evolution and could be of great significance to the way these genes are regulated in response to various stimuli. Human and mouse cardiac MHCs are found on chromosome 14; whereas human skeletal MHCs are found on chromosome 17, and the mouse skeletal MHCs are found on chromosome 11.

Figure 8

Relationship between Sense and Antisense Transcripts in Soleus Muscle. IIa mRNA is inversely proportional to IIa NAT (A); whereas IIx pre-mRNA is correlated positively to IIa NAT (B). Lines are generated by regression analyses (GraphPad Prism). r, Pearson coefficient determined with correlation analyses for each set. Open triangle, control; closed square, SI. Also shown is a schematic of the IIa and IIx genes, and transcriptional activity in the intergenic region depicting a bidirectional promoter (BPR) in the IIx 5′ proximal region (Pandorf et al., 2006).

Figure 9

Chromatin state and gene transcription. Model for chromatin factors interacting with transcription factors to regulate transcription of a gene. Histone modifications and DNA methylation are important factors in regulating the chromatin from active to repressed and vice versa. Histone H3 acetylation and histone H3 methylation and lysine 4, are both associated with an active chromatin state. In contrast, histone H3 methylation at lysine 9 or lysine 27 as well as DNA methylation are associated with repressive chromatin state. Chromatin is in a dynamic equilibrium between the two states.