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Molecular and Cellular Mechanisms and the Pathophysiology of Skeletal Muscle Diseases

A special issue of International Journal of Molecular Sciences (ISSN 1422-0067). This special issue belongs to the section "Molecular Pathology, Diagnostics, and Therapeutics".

Deadline for manuscript submissions: 20 January 2025 | Viewed by 1221

Special Issue Editor

Dr. Gaetano Vattemi

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Guest Editor
Department of Neurosciences, Biomedicine and Movement Sciences, Section of Clinical Neurology, University of Verona, Piazzale L.A. Scuro 10, 37134 Verona, Italy
Interests: idiopathic inflammatory myopathies; sporadic inclusion body myositis (sIBM); protein aggregates myopathies; nuclear envelopathies; amyotrophic lateral sclerosis
Special Issues, Collections and Topics in MDPI journals

Special Issue Information

Dear Colleagues,

Skeletal muscle is one of the most extended organs in the human body and is characterized by an elaborate architecture of multinucleated contractile myofibers within a complex microenvironment that includes endothelial and immune cells, motor neurons, perivascular and connective tissue, and muscle stem cells.

Several disorders affect the structure and/or function of skeletal muscle and can be classified into two broad categories: genetically determined and acquired myopathies. Many of these diseases are still awaiting effective treatments because their pathological mechanisms are not well understood. Recent advances in biological knowledge and technologies, such as stem cell protocols and high-throughput platforms, are making the study of molecular and cellular dynamics in skeletal muscle possible at unprecedented depths.

This Special Issue will update the latest findings on the biomolecular and cellular processes underlying primary muscle diseases, with the aim of improving understanding of their pathophysiological mechanisms and finding potential drug targets. Original research articles, reviews, and short communications related to this topic are all welcome.

Dr. Gaetano Vattemi
Guest Editor

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Keywords

  • acquired muscle diseases
  • inherited muscle diseases
  • pathogenesis
  • molecular and cellular mechanisms
  • in vitro and in vivo models
  • treatment strategies

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Published Papers (1 paper)

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Research

22 pages, 7966 KiB  
Article
P38α MAPK Coordinates Mitochondrial Adaptation to Caloric Surplus in Skeletal Muscle
by Liron Waingerten-Kedem, Sharon Aviram, Achinoam Blau, Tony Hayek and Eyal Bengal
Int. J. Mol. Sci. 2024, 25(14), 7789; https://doi.org/10.3390/ijms25147789 - 16 Jul 2024
Viewed by 853
Abstract
Excessive calorie intake leads to mitochondrial overload and triggers metabolic inflexibility and insulin resistance. In this study, we examined how attenuated p38α activity affects glucose and fat metabolism in the skeletal muscles of mice on a high-fat diet (HFD). Mice exhibiting diminished p38α [...] Read more.
Excessive calorie intake leads to mitochondrial overload and triggers metabolic inflexibility and insulin resistance. In this study, we examined how attenuated p38α activity affects glucose and fat metabolism in the skeletal muscles of mice on a high-fat diet (HFD). Mice exhibiting diminished p38α activity (referred to as p38αAF) gained more weight and displayed elevated serum insulin levels, as well as a compromised response in the insulin tolerance test, compared to the control mice. Additionally, their skeletal muscle tissue manifested impaired insulin signaling, leading to resistance in insulin-mediated glucose uptake. Examination of muscle metabolites in p38αAF mice revealed lower levels of glycolytic intermediates and decreased levels of acyl-carnitine metabolites, suggesting reduced glycolysis and β-oxidation compared to the controls. Additionally, muscles of p38αAF mice exhibited severe abnormalities in their mitochondria. Analysis of myotubes derived from p38αAF mice revealed reduced mitochondrial respiratory capacity relative to the myotubes of the control mice. Furthermore, these myotubes showed decreased expression of Acetyl CoA Carboxylase 2 (ACC2), leading to increased fatty acid oxidation and diminished inhibitory phosphorylation of pyruvate dehydrogenase (PDH), which resulted in elevated mitochondrial pyruvate oxidation. The expected consequence of reduced mitochondrial respiratory function and uncontrolled nutrient oxidation observed in p38αAF myotubes mitochondrial overload and metabolic inflexibility. This scenario explains the increased likelihood of insulin resistance development in the muscles of p38αAF mice compared to the control mice on a high-fat diet. In summary, within skeletal muscles, p38α assumes a crucial role in orchestrating the mitochondrial adaptation to caloric surplus by promoting mitochondrial biogenesis and regulating the selective oxidation of nutrients, thereby preventing mitochondrial overload, metabolic inflexibility, and insulin resistance. Full article
(This article belongs to the Special Issue Molecular and Cellular Mechanisms and the Pathophysiology of Skeletal Muscle Diseases)
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Figure 1

Figure 1
<p>p38α<sup>AF</sup> mice presented worse metabolic parameters than control mice. (<b>A</b>) Six-week-old mice were fed with ND or an HFD for 10 weeks, and GC muscles were isolated (<span class="html-italic">n</span> = 5) from the control and p38α<sup>AF</sup> mice. Protein lysates from three of the mice per treatment were randomly analyzed by Western blotting with the designated antibodies. α Tubulin was used as the loading control. The quantification of relative p38α phosphorylation is presented in the histogram. (<b>B</b>) The mice underwent the diets described in (A), and the weight of each mouse was measured weekly (<span class="html-italic">n</span> = 5). The graphs represent the average percent change in the body weight of the two mouse groups (control and p38α<sup>AF</sup>), which were fed with ND or HFD. The weight was set to 100 on the first day of the diet. (<b>C</b>) The hematological parameters of control mice and p38α<sup>AF</sup> on an HFD. The glucose and cholesterol levels were measured in the serum of control and p38α<sup>AF</sup> mice after 10 weeks on an HFD (AML-central lab services). Insulin was measured (<span class="html-italic">n</span> = 3) using an ELISA kit (Millipore RAB0817). The significance probabilities between treatments were designated as numbers. (<b>D</b>) Insulin tolerance test (ITT): the graph displays the relative average glucose levels at 0, 30, 45, 60, 90, and 120 min following insulin injection (0.5 U/kg BW) in the blood of control and p38α<sup>AF</sup> mice after a 10-week HFD (<span class="html-italic">n</span> = 4 mice per group). The mice were deprived of chaw for 6 h before insulin was IP-injected. The glucose level before insulin injection was set to 100 percent, and all values were relative to 100. Data are presented as the mean ± SE. One-way ANOVA was followed by Tukey post-tests (<b>A</b>), two-way ANOVA was followed by Bonferroni post-tests (<b>B</b>,<b>D</b>) and a Student t-test (<b>C</b>). The <span class="html-italic">p</span> values for group difference are designated as follows: * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, and *** <span class="html-italic">p</span> &lt; 0.001.</p> Full article ">Figure 2
<p>Block of the insulin-mediated 2 deoxy-glucose (2DG) uptake by the Tibialis muscle of p38α<sup>AF</sup> mice. (<b>A</b>) Experimental layout: saline or insulin (1 unit/kg) was IP-injected following a 3 h fasting of the mice previously fed with an HFD for 10 weeks. Ten min later, 5% 2DG was IP-injected (10 μL to 1 g weight). The mice were sacrificed one hour later, and the Tibialis muscles were frozen and used in the mass spectrometry (MS) analysis of metabolites, or to extract proteins for Western blotting analysis. (<b>B</b>) Peak area were analyzed by the MS values of 2- Deoxy –D Glucose (<span class="html-italic">n</span> = 4) that were normalized to mg tissue. (<b>C</b>) Protein extracts from the Tb muscles (<span class="html-italic">n</span> = 3) were analyzed by Western blotting with antibodies directed to phosphorylated Akt (Serine 473) and Pan Akt. Quantification of the relative phosphorylation (pAkt/Akt) is presented in the histogram. Data are presented as the mean ± SE. The Wilcoxon test and significance probabilities between treatments are designated as numbers in (<b>B</b>). One-way ANOVA was followed by Tukey post-tests. The <span class="html-italic">p</span> values for group difference are designated as follows: * <span class="html-italic">p</span> &lt; 0.05 (<b>C</b>).</p> Full article ">Figure 3
<p>Reduced glycolytic metabolites and increased lactate-to-pyruvate ratio in the muscles of HFD-fed p38α<sup>AF</sup> mice. Extracted metabolites from the Tibialis muscles of 10-week HFD-fed mice that were IP-injected without or with insulin (<span class="html-italic">n</span> = 4). (<b>A</b>) The normalized peak areas (to mg tissue) that were analyzed by the MS of several glycolytic metabolites. (<b>B</b>) The normalized peak areas (to mg tissue) that were analyzed by the MS of pyruvate, lactate, and the ratio of lactate to pyruvate. (<b>C</b>) Analysis of the expression and the phosphorylation on serine 293 of the E1 subunit of pyruvate dehydrogenase (PDH) in the Tb muscles of control and p38α<sup>AF</sup> mice (<span class="html-italic">n</span> = 5) by Western blotting using antibodies to phospho-PDH (Ser293) and PDH. The quantification of relative phosphorylation (pPDH/PDH) is presented in the histogram. Data are presented as the mean ± SE. The Wilcoxon test and significance probabilities between treatments are designated as numbers in (<b>B</b>).</p> Full article ">Figure 4
<p>Reduced β oxidation in the muscles of p38α<sup>AF</sup> mice relative to the muscles of control mice following a high-fat diet. Metabolites were extracted from the Tibialis muscles of 10-week HFD-fed mice that were IP-injected without or with insulin (<span class="html-italic">n</span> = 4). (<b>A</b>) The peak areas (normalized to mg tissue) of glycerol analyzed by MS are presented. (<b>B</b>) Analysis of the mRNA levels of FABP3 in the muscles of control and p38α<sup>AF</sup> mice by qPCR (<span class="html-italic">n</span> = 5). The β-actin housekeeping gene was used to normalize the mRNA levels. (<b>C</b>) Analysis of the mRNA levels of ACC2 in the muscles of control and p38α<sup>AF</sup> mice by qPCR (<span class="html-italic">n</span>= 4). The β-actin housekeeping gene was used to normalize mRNA levels. (<b>D</b>) Analysis of the expression and the phosphorylation on serine 212 of Acetyl CoA Carboxylase 2 (ACC2) in the muscles of control and p38α<sup>AF</sup> mice (<span class="html-italic">n</span> = 5) by Western blotting using antibodies to phospho-ACC2 (Ser212), ACC2, and αTubulin (which served as a loading control). The histograms present the relative expression of ACC2 (ACC2/Tubulin) and relative ACC2 phosphorylation on serine 212 (pACC2/ACC2). (<b>E</b>) The peak areas (normalized to mg tissue) of acyl-carnitines are presented. Values represent the means ± SEM. The Wilcoxon test and significance probabilities between treatments are designated as numbers (<b>A</b>,<b>E</b>). One-way ANOVA followed by Tukey post-tests (<b>B</b>,<b>C</b>). The <span class="html-italic">p</span> values for group difference are designated as follows: * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, and *** <span class="html-italic">p</span> &lt; 0.001.</p> Full article ">Figure 5
<p>Severe mitochondrial defects in the muscles of p38α<sup>AF</sup> mice. (<b>A</b>) Transmission electron microscopy (TEM) analysis of the representative muscles from control and p38α<sup>AF</sup> mice fed with NDs and HFDs. The Tibialis muscles were isolated, and longitudinal sections were processed for TEM analysis (see <a href="#sec4dot10-ijms-25-07789" class="html-sec">Section 4.10</a>). Representative images are shown. Scale bar: 1 μm. Asterisks are adjacent to the mitochondria (<b>B</b>) Analysis of the mRNA levels of PGC1α in the muscles of control and p38α<sup>AF</sup> mice fed with NDs and HFDs by qPCR (<span class="html-italic">n</span> = 5). The β-actin housekeeping gene was used to normalize the mRNA levels. Data represent the means ± SEM. One-way ANOVA was followed by Tukey post-tests (B). The <span class="html-italic">p</span> values for group differences are designated as follows: * <span class="html-italic">p</span> &lt; 0.05.</p> Full article ">Figure 6
<p>Biochemical and metabolic analysis of the myotubes derived from control and p38α<sup>AF</sup> mice. (<b>A</b>) p38 MAPK phosphorylation: Myotubes were grown for 24 h in the absence or presence of 0.4 mM of palmitate. Insulin (10 μg/mL) was added 30 min before the proteins were extracted and analyzed by Western blotting using the designated antibodies. (<b>B</b>) Insulin signaling pathway: The same protein samples as in (A) were analyzed by Western blotting using the designated antibodies. (<b>C</b>) Metabolism of the (U-<sup>13</sup>C<sub>6</sub>) glucose in myotubes: (U-<sup>13</sup>C<sub>6</sub>) glucose was introduced to the myotube media with or without 0.4 mM of palmitate for 24 h. The relative levels of glucose 6-phosphate (+6), fructose 6-phosphate (+6), and ribose phosphate (+5) isotopologues are presented. The peak area was normalized to protein concentration. (<b>D</b>) Medium acidification (ECAR) of myotubes in a “Seahorse” analysis: Myotubes were grown in glucose, or glucose and palmitate, for 24 h before analysis. (<b>E</b>) Metabolism of the (U-<sup>13</sup>C<sub>6</sub>) glucose in myotubes: The relative levels of the isotopologues of citrate are presented. The peak areas were normalized to protein concentration. (<b>F</b>) Mitochondrial enzymes: The same protein samples as in (A) were analyzed by Western blotting. The histograms present the relative expression and phosphorylation of PDH (Ser293), and the expression of citrate synthase. Data represent the means ± SEM. The Wilcoxon test and significance probabilities between treatments are designated as follows: * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, and *** <span class="html-italic">p</span> &lt; 0.001 (<b>C</b>,<b>E</b>). One-way ANOVA was followed by Tukey post-tests (<b>D</b>). The <span class="html-italic">p</span> values for group differences are designated as follows: * <span class="html-italic">p</span> &lt; 0.05 and *** <span class="html-italic">p</span> &lt; 0.001.</p> Full article ">Figure 7
<p>Metabolism of palmitate in the myotubes derived from control and p38α<sup>AF</sup> mice. Myotubes were grown in a low-glucose DMEM supplemented with 0.4 mM of palmitate-<sup>13</sup>C<sub>16</sub> for 6 and 24 h. (<b>A</b>) The peak area (normalized to protein concentration) of palmitate (+16), the isotopologues of the TCA cycle, and the derived amino acids that originated from palmitate-<sup>13</sup>C<sub>16.</sub> FC: fold change in the palmitate derived (<sup>13</sup>C ≥ 2) metabolite abundance relative to a WT of 6 h or WT of 24 h. Dashed arrows indicate of missing stages in the TCA-cycle. (<b>B</b>) Myotubes were grown for 24 h in the absence or presence of 0.4 mM of palmitate. Insulin (10 μg/mL) was added 30 min before proteins were extracted and analyzed by Western blotting with the designated antibodies. The histograms present the relative expression of ACC2, the phosphorylation of ACC2 (Ser212), and the phosphorylation of AMPKα (Thr172). (<b>C</b>) The oxygen consumption rate (OCR) at the maximal respiration of myotubes that were grown on glucose, or glucose and palmitate, for 24 h. (<b>D</b>) Comparison of the mitochondrial membrane electrochemical potential in myotubes that were grown on glucose, or glucose and palmitate, for 24 h. JC-1 dye was used to monitor the mitochondrial membrane potential. FCCP disrupts the mitochondrial membrane potential. Data represent the means ± SEM. One-way ANOVA was followed by Tukey post-tests (<b>A</b>,<b>C</b>). The <span class="html-italic">p</span> values for group difference are designated as follows: * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, and *** <span class="html-italic">p</span> &lt; 0.001.</p> Full article ">Figure 8
<p>A model for the role of p38α in insulin sensitivity. In the left panel, a high-fat diet activates p38α in wild-type mice, leading to an increased expression and activity of PGC1α and ACC2 in the skeletal muscles. PGC1α acts as a co-activator, increasing mitochondrial biogenesis and activity, while ACC2 regulates fatty acid transport into mitochondria. These activities of p38α help coordinate glucose and fat oxidation, preserving metabolic flexibility and preventing mitochondrial damage. Under these conditions, both energy balance and insulin sensitivity are preserved.</p> Full article ">
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