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Spatial proteomics of skeletal muscle using thin cryosections reveals metabolic adaption at the muscle-tendon transition zone

ABSTRACT: Morphological studies of skeletal muscle tissue have provided detailed insights into the architecture of muscle fibers, the surrounding cells, and the extracellular matrix. However, a spatial proteomics analysis of the skeletal muscle, including the muscle-tendon transition zone, is lacking. Here, we prepared thin cryotome muscle sections along the longitudinal axis of the mouse soleus muscle and measured each muscle slice using short LC-MS gradients. We generated more than 3000 high-resolution longitudinal protein profiles of central to distal skeletal muscle regions and created a molecular network of different skeletal muscle regions that reveals the complex architecture of the muscle-tendon transition zone. Among the proteins that show an increasing profile from muscle to tendon, we find proteins related to neuronal activity, fatty acid biosynthesis, and the renin-angiotensin system (RAS). Blocking the RAS in cultured mouse tenocytes using losartan reduces the synthesis of extracellular matrix proteins, including collagen and fibronectin. Overall, our analysis of thin cryotome sections provides a spatial proteome of skeletal muscle and reveals that the RAS acts as an additional regulator of the matrix within muscle-tendon junctions.

INSTRUMENT(S): Orbitrap Eclipse, timsTOF Pro 2

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Skeletal Muscle, Cell Culture

SUBMITTER: Luisa Marie Schmidt

LAB HEAD: Marcus Krüger

PROVIDER: PXD046596 | Pride | 2024-05-22

REPOSITORIES: Pride

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			Action	DRS
	20210930_E1_LC6colID316_LS13_M6_Sol_M_LS909.raw	Raw
	20210930_E1_LC6colID316_LS30_M8_Sol_T_LS935.raw	Raw
	20210930_E1_LC6colID316_LS31_M8_Sol_MTJ_LS936.raw	Raw
	20210930_E1_LC6colID316_LS32_M7_Sol_MTJ_LS922.raw	Raw
	20210930_E1_LC6colID316_LS36_M8_Sol_M_LS937.raw	Raw

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Project description:Background: Niemann-Pick disease type A (NPDA), a disease caused by mutations in acid sphingomyelinase (ASM), involves severe neurodegeneration and early death. Intracellular lipid accumulation and plasma membrane alterations are implicated in the pathology. ASM is also linked to the mechanism of plasma membrane repair, so we investigated the impact of ASM deficiency in skeletal muscle, a tissue that undergoes frequent cycles of injury and repair in vivo. Methods: Utilizing the NPDA/B mouse model ASM−/− and wild type (WT) littermates, we performed excitation- contraction coupling/Ca2+ mobilization and sarcolemma injury/repair assays with isolated flexor digitorum brevis fibers, proteomic analyses with quadriceps femoris, flexor digitorum brevis, and tibialis posterior muscle and in vivo tests of the contractile force (maximal isometric torque) of the quadriceps femoris muscle before and after eccentric contraction-induced muscle injury. Results: ASM−/− flexor digitorum brevis fibers showed impaired excitation-contraction coupling compared to WT, a defect expressed as reduced tetanic [Ca2+]i in response to electrical stimulation and early failure in sustaining [Ca2+]i during repeated tetanic contractions. When injured mechanically by needle passage, ASM−/− flexor digitorum brevis fibers showed susceptibility to injury similar to WT, but a reduced ability to reseal the sarcolemma. Proteomic analyses revealed changes in a small group of skeletal muscle proteins as a consequence of ASM deficiency, with downregulation of calsequestrin occurring in the three different muscles analyzed. In vivo, the loss in maximal isometric torque of WT quadriceps femoris was similar immediately after and 2 min after injury. The loss in ASM−/− mice immediately after injury was similar to WT, but was markedly larger at 2 min after injury. Conclusions: Skeletal muscle fibers from ASM−/− mice have an impairment in intracellular Ca2+ handling that results in reduced Ca2+ mobilization and a more rapid decline in peak Ca2+ transients during repeated contraction-relaxation cycles. Isolated fibers show reduced ability to repair damage to the sarcolemma, and this is associated with an exaggerated deficit in force during recovery from an in vivo eccentric contraction- induced muscle injury. Our findings uncover the possibility that skeletal muscle functional defects may play a role in the pathology of NPDA/B disease.

Project description:Context: Exercise training is a plausible model for identification of molecular mechanisms that cause metabolic-related changes in human skeletal muscle. Objective: The goal was to explore the molecular basis of the adaptation of skeletal muscle to exercise training. Design and Intervention: Obese male subjects were subjected to an individualized supervised training program targeted in order to optimize lipid oxidation during 8 weeks. Main Outcome Measures: Primary outcome measures were gene expression profiling of skeletal muscle. Body composition, oral glucose tolerance test, Resting metabolic rate, respiratory quotient, maximal oxygen uptake and metabolic biochemistry were also assessed. Overall Design The obese (BMI 30-36) male volunteers (age 32-42) were asked to refrain from vigorous physical activity 48h before presenting to the clinical investigation centre, and ate a weight-maintaining diet consisting of 35% fat, 16% protein, and 49% carbohydrates two days before the experiment. Muscle biopsies of Vastus Lateralis weighing 60–100 mg were obtained using the Bergstrom technique, cleaned and snap-frozen in liquid nitrogen. Resting metabolic rate, respiratory quotient and maximal oxygen uptake were assessed. The subjects were investigated at baseline and after 8 weeks of supervised aerobic exercise training program consisting of daily sessions of 45-60 min of endurance exercise, 5 days a week, at least 48-72h after the last acute exercise bout. Skeletal muscle biopsies were obtained at the beginning and at the end of the protocol. Transcriptome analysis compared 8 subjects before vs. after training using arrays using a common reference design (Cy5 dye was incorporated into all muscle RNA samples, while a reference RNA pool made of the mix of commercial human liver, adipose tissue and skeletal muscle RNA was labelled with Cy3 dye (Applied Biosystems/Ambion, Foster City, USA)( and whole genome 4x44k oligonucleotide arrays (Agilent Technologies).

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Spatial proteomics of skeletal muscle using thin cryosections reveals metabolic adaption at the muscle-tendon transition zone

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