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Bridging the muscle genome to phenome across multiple biological scales

ABSTRACT: Muscle is highly hierarchically organized, with functions shaped by genetically controlled expression of protein ensembles with different isoform profiles at the sarcomere scale. However, it remains unclear how isoform profiles shape whole-muscle performance. We compared two mouse hindlimb muscles, the slow, relatively parallel-fibered soleus and the faster, more pennate-fibered tibialis anterior (TA), across scales: from gene regulation, isoform expression and translation speed, to force-length-velocity-power for intact muscles. Expression of myosin heavy-chain (MHC) isoforms directly corresponded with contraction velocity. The fast-twitch TA with fast MHC isoforms had faster unloaded velocities (actin sliding velocity, Vactin; peak fiber velocity, Vmax) than the slow-twitch soleus. For the soleus, Vactin was biased towards Vactin for purely slow MHC I, despite this muscle's even fast and slow MHC isoform composition. Our multi-scale results clearly identified a consistent and significant dampening in fiber shortening velocities for both muscles, underscoring an indirect correlation between Vactin and fiber Vmax that may be influenced by differences in fiber architecture, along with internal loading due to both passive and active effects. These influences correlate with the increased peak force and power in the slightly more pennate TA, leading to a broader length range of near-optimal force production. Conversely, a greater force-velocity curvature in the near-parallel fibered soleus highlights the fine-tuning by molecular-scale influences including myosin heavy and light chain expression along with whole-muscle characteristics. Our results demonstrate that the individual gene, protein and whole-fiber characteristics do not directly reflect overall muscle performance but that intricate fine-tuning across scales shapes specialized muscle function.

ORGANISM(S): Mus musculus

PROVIDER: GSE203400 | GEO | 2022/05/23

REPOSITORIES: GEO

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Project description:Stimulation of the mouse hindlimb via the sciatic nerve was used to induce contractions for 4 hours to investigate acute muscle gene activation in a model of muscle phenotype conversion. Initial force production (1.6 + 0.1 g/g body weight) declined 45% within 10 min and was maintained for the remainder of the experiment. Force returned to initial levels upon completion of the study. An immediate-early growth response was present in the EDL (FOS, JUN, ATF3, MAFK) with a similar but attenuated pattern in the soleus. Transcript profiles showed decreased fast fiber specific mRNA (myosin heavy chains 2A, 2B; troponins T3, I; -tropomyosin, m-creatine kinase) and increased slow transcripts (myosin heavy chain slow/1, troponin C, tropomyosin 3) in the EDL. Histological analysis of the EDL revealed glycogen depletion without inflammatory cell infiltration or myofiber damage in stimulated vs. control muscles. Several fiber type specific transcription factors (EYA1, TEAD1, NFATc1 and c4, PPARG, PPARGC1 and β, BHLHB2) increased in the EDL along with transcription factors characteristic of embryogenesis (KLF4, SOX17, TCF15, PKNOX1, ELAV). No established in vivo satellite cell markers or the genes activated during our parallel studies of satellite cell proliferation in vitro (CYCLINS A2, B2, C, E1, MyoD) increased in the stimulated muscles. These data indicated that onset of fast to slow phenotype conversion occurred in the EDL within 4 hours of stimulation without satellite cell recruitment or muscle injury but was driven by phenotype specific transcription factors from resident fiber myonuclei including activation of nascent developmental transcriptional programs. Adult male Swiss Webster mice (30-35 g) were anesthetized, a bipolar electrode was implanted adjacent to the sciatic nerve and the hindlimb immobilized. The voltage-force relation was determined to establish supramaximal stimulation conditions and the length-tension relation was determined to set the resting length for maximum twitch tension. Contractions were induced by sciatic nerve stimulation (0.5 msec duration, 2-5 volts). The muscles were allowed to rest 15 minutes for full metabolic recovery at physiologic temperatures. Supramaximal stimulation was applied at a rate of 10 Hz for 4 hours. At the end of each experiment the soleus muscles were carefully dissected and flash frozen in liquid nitrogen for analysis of mRNA expression via microarray analysis. The contralateral, unstimulated Soleus provided a genetically matched, paired control for each specimen.

Project description:Background: skeletal muscle is a complex, versatile tissue composed of a variety of functionally diverse fiber types. Although the biochemical, structural and functional properties of myofibers have been the subject of intense investigation for the last decades, understanding molecular processes regulating fiber type diversity is still complicated by the heterogeneity of cell types present in the whole muscle organ. Methodology/Principal Findings: we have produced a first catalogue of genes expressed in mouse slow-oxidative (type 1) and fast-glycolytic (type 2B) fibers through transcriptome analysis at the single fiber level (microgenomics). Individual fibers were obtained from murine soleus and EDL muscles and initially classified by myosin heavy chain isoform content. Gene expression profiling on high density DNA oligonucleotide microarrays showed that both qualitative and quantitative improvements were achieved, compared to results with standard muscle homogenate. First, myofiber profiles were virtually free from non-muscle transcriptional activity. Second, thousands of muscle-specific genes were identified, leading to a better definition of gene signatures in the two fiber types as well as the detection of metabolic and signaling pathways that are differentially activated in specific fiber types. Several regulatory proteins showed preferential expression in slow myofibers. Discriminant analysis revealed novel genes that could be useful for fiber type functional classification. Conclusions/Significance: as gene expression analyses at the single fiber level significantly increased the resolution power, this innovative approach would allow a better understanding of the adaptive transcriptomic transitions occurring in myofibers under physiological and pathological conditions. EDL and soleus muscles were incubated with type I collagenase to dissociate intact myofibres that were separated under stereo microscope from the bulk of hyper contracted fibres. Isolated myofibres were divided in two parts: one was immersed in Laemmli buffer for fibre typing; the other was placed in RNA extraction buffer for RNA amplification. We analyzed the transcription profiles of 10 biological replicas of type 2B single muscle fibres from EDL and 10 biological replicas of type 1 single muscle fibres from soleus. Microarray competitive hybridizations were carried out against an artificial control with a balanced composition of type 1 and type 2B fibres (20 hybridizations). Each oligonucleotide is spotted in two replicates on the glass slide, so for every data two intra-slide replicas are present. The control was created as follows: three couples of soleus and EDL muscles were removed from 3 different mice and treated with collagenase. Total RNA was extracted separately from EDL and soleus muscles. By mixing about 1/3 RNA from EDL and 2/3 RNA from soleus muscles the control had approximately the same contributions of type 1 and type 2B fibres. Purified RNA samples from single fibres and from control were amplified twice using the Amino Allyl MessageAmpM-bM-^DM-" II aRNA Amplification Kit (Ambion).

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Bridging the muscle genome to phenome across multiple biological scales

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