Regulation of skeletal muscle metabolism and contraction performance via teneurin-latrophilin action

Skeletal muscle regulation is responsible for voluntary muscular movement in vertebrates. The genes of two essential proteins, teneurins and latrophilins (LPHN), evolving in ancestors of multicellular animals, form a ligand-receptor pair, and are now shown to be required for skeletal muscle function. Teneurins possess a bioactive peptide, termed the teneurin C-terminal associated peptide (TCAP) that interacts with the LPHNs to regulate skeletal muscle contractility strength and fatigue by an insulin-independent glucose importation mechanism. CRISPR-based knockouts and siRNA-associated knockdowns of LPHN-1 and-3 shows that TCAP stimulates an LPHN-mediated cytosolic Ca2+ signal transduction cascade to increase energy metabolism and enhance skeletal muscle function via increases in type-1 oxidative fiber formation and reduce the fatigue response. Thus, the teneurin/TCAP-LPHN system is presented as a novel mechanism likely to regulate the energy requirements and performance of skeletal muscle.


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Skeletal muscle is critical for all voluntary behaviours and is derived from the earliest contractile 47 proteins present in the ancestral single-celled heterotrophs. Enhanced contractile strength and 48 efficient energy metabolism among these primitive skeletal muscle cells were critical for both 49 locomotion and feeding [1,2]. Because of these integrated requirements for the evolutionary 50 success of early metazoans, we have postulated that essential intercellular signaling systems 51 originating phylogenetically early, conferred a selective advantage upon these basal heterotrophs  [19][20][21]. As type-II proteins, their carboxyl terminus is displaced extracellularly. The most 63 distal region contains a β-barrel structure unique to metazoans, but is similar to that found in 64 prokaryotic Tc-toxins [9,14,[22][23][24][25]. Associated with this structure lies an extended amino-acid TCAP-1 where each residue, with the exception of the initial pyroglutamyl residue (pE), was 126 randomized in its placement within the peptide (Fig. 1B). This sc-TCAP-1 has been used in 127 previous studies to establish an additional level of controls to ensure that TCAP-1 is not affecting 128 non-specific (e.g. oligopeptide transporters; non-target receptors) actions. The vehicle included sc-129 TCAP solubilized in 0.9% saline or cell culture medium, unless otherwise stated. The short-term application of TCAP-1 utilized 16 male adult SD rats (250g) that were acclimated  TA muscle was excised then flash-frozen in liquid nitrogen cooled-isopentane. The tissue was 204 cryo-sectioned at 10 μm at -20˚C and transferred to coverslides and fixed using ice-cold methanol.

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Following blocking for 1h with 10% normal goat serum (NGS: Cell Signaling, Inc.), the primary 206 antibody (Table 2), diluted in 1% NGS, was added and incubated overnight (ON) at 4˚C.  cDNA prepared above (see Table 3).  with mRNA extracted from differentiated mouse C2C12 cells using the method described above 308 using the primer sequences indicated in Table 1.   Radioactive glucose uptake 354 The 3 H-2-deoxyglucose uptake protocol was followed as previously described with minor  subsequently treated with secondary antibody conjugated with chemiluminescent tags (Table 2). were added to a microELISA plate coated with purified mouse DAG or IP3 antibodies.

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The absorbance change was measured at 450 nm by spectrophotometry (SpectraMax Plus, NH,  using the green GFP filter set using the same experimental configuration as previously described.

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R123 was excited with a wavelength of 480 nm for 100 ms every 5s and fluorescence emission 433 was measured at 516 nm. The primary structure of rat and mouse TCAP-1 possesses a high degree of homology among the 478 other three paralogues (Fig 1A). Because the primary structure of rat and mouse TCAP-1 is In rat TA muscle mRNA extracts, all 4 teneurin mRNAs were identified based on the PCR primers 485 indicated in Table 1. Teneurins-3 and-4 showed the strongest response, although both teneurins-1 486 and -2 were present, albiet weakly expressed. In contrast, TCAP-1 and -2 showed a strong signal 487 relative to that indicated by teneurins-1 and -2 whereas TCAP-4 showed a signal consistent with 488 teneurin-4. Although these studies were not quantitative, they do establish that both teneurins and (p<0.05) (Fig. 5 A,B), slower contraction velocity (p<0.05) (Fig 5C), and potentially higher faster 555 relaxation rates (Fig. 5D) compared to vehicle-treated animals. Following baseline measurements, 556 a 6-m fatigue protocol was induced in the muscle where contractile kinetics were recorded at 0, 1, 557 and 5m after the fatigue protocol. TCAP-1 enhanced recovery from the twitch stimulation (Fig.   558 5E-G). Although TCAP-1 did not influence peak twitch force (Fig. 5E), it significantly (p<0.05) 559 maintained twitch max dx/dt (Fig. 5F) and 1/2RT (Fig. 5G) over the course of the fatigue protocol 25 560 which was diminished in vehicle-treated animals. All data was normalized to muscle mass. The 561 treatment did not affect muscle mass (Fig. 5H), tetanic force (Fig. 5I) or the fatigue force curve 562 (Fig. 5J). Thus, TCAP-1 enhanced the efficiency of the existing muscle morphology, rather than The initial PCR screen of C2C12 cells indicated that, although only teneurin-3 was highly 580 expressed (Fig.6A), all 4 TCAP transcripts could be discerned (Fig. 6B). In both undifferentiated 581 C2C12 myoblasts, and 6-d myotubules, the transcripts for LPHN-1 and -3 were present (Fig. 6C). to highlight actin fibers (Fig. 6E). This resulted in a major increase in actin polymerization in the 588 TCAP-1-treated cells at both 30m (p<0.01) and 2d (p<0.001).  Increased glucose importation increases ATP and NADH turnover in cells due to glycolytic and 652 tricylic acid (TCA) cycle activity. Therefore, this was examined with respect to TCAP-1 treatment.

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Similarly, the LPHN-3 siRNA-associated oligonucleotides significantly (p<0.01) decreased its 695 mRNA expression about 65% relative to the WT cells. There were no significant changes in 696 mRNA expression of the LPHN-1 transcript in either the LPHN-1 KD or the NT cells (Fig. 10B).

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KOs did not show an increase after TCAP-1 treatment, whereas the NT control cells showed a 736 significant (p<0.01 increase after 120m (Fig. 11I). FCCP treatment, indicating cell viability 737 induced significant increases (p<0.001; p<0.0001) across all cell types (Fig. 11 F,G,H,I). about a 50% increase (p<0.05) in PGC-1α expression (Fig. 11K). This indicated that TCAP-1 has 745 the potential to directly influence MCH fibre expression at the transcriptional level.   in the sarcolemma rather than being spread throughout all regions of DG labelling (see Fig. 3B).

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Our data indicates that teneurin-3 is the dominant teneurin in skeletal muscle tissue. However, the  showed that the transgenic expression of teneurin-1 TCAP co-precipitated with the transgenic localized in the sarcolemma (Fig.3). These data corroborate with the PCR expression data 932 indicating that these mRNA transcripts show the highest expression, but are not meant to suggest 933 that this is indicative of cognitive ligand and receptor pairs, per se.

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There is evidence that LPHN paralogues interact with each other. In our LPHN-1 and -3 935 attenuation studies, the reduction of one receptor inhibited the TCAP-1 mediated Ca 2+ actions of 936 the other receptor. It is possible that there is a minor Ca 2+ response mediated by the non-target 937 LPHN, but too low to detect with our assay conditions, although this seems unlikely. Homo-and 938 heterophilic oligomerization is a characteristic of the GPCRs, but this has not been well-studied in studies showing its presence in as a circulating hormone in serum has been equivocal (Lovejoy,958 unpublished observations). As this is a critical aspect of TCAP action, this is a goal for upcoming 959 studies.

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In summary, our data in this study indicate that TCAP-1 regulates energy metabolism in skeletal 961 muscle via an insulin-independent mechanism, and by doing so, modulates contractile kinetics, via 962 Ca 2+ dynamics and ATP production. Together, these data describe a previously unknown 963 mechanism to regulate skeletal muscle dynamics. These data provide the foundation for a proposed and NADH production, as well as increased SDH-ATP levels. Ca 2+ is subsequently pumped out 972 of the mitochondria likely via Na + /Ca 2+ exchangers (NCX), thus restoring homeostatic levels of 973 Ca 2+ . Moreover, we showed that the TCAP-1 increased cellular energy availability by increased 974 glucose importation into cells likely due to increased GLUT4 expression. The TCAP-1 mediated 975 mechanism is likely due to its interactions with LPHNs -1 and -3.