Casale, Veronica (2025) Role of transsulfuration pathway in the physiopathology of skeletal muscle. [Tesi di dottorato]

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Abstract

Skeletal muscle (SKM) is not only responsible for movement but also acts as a central organ for whole-body metabolism and overall health. When muscle health is compromised, whether due to genetic disorders like Duchenne muscular dystrophy (DMD) or acquired conditions such as obesity, its structure and function progressively decline, leading to severe metabolic and functional consequences that impair the quality of life. In both cases, SKM exhibits oxidative stress, chronic inflammation, and impaired energetic efficiency. A key player of SKM homeostasis is the transsulfuration pathway (TSP), which generates bioactive sulfur species, including hydrogen sulfide (H₂S). Far from being a simple byproduct, H₂S acts as a signaling molecule with antioxidant, anti-inflammatory, and cytoprotective properties. However, the contribution of TSP to SKM physiology and pathology remains poorly understood. During my Ph.D., I investigated how alterations in TSP, and specifically in its end-product, H2S, affect muscle function in obesity and DMD, and whether restoring this pathway can improve SKM health. In the first part, I explored obesity-induced muscle dysfunction using animal models, cellular systems, advanced proteomics, and human biopsies. In obese db/db mice, excessive lipid accumulation in SKM was associated with reduced expression of 3-mercaptopyruvate sulfurtransferase (MPST), one of the three H2S-producing enzymes. This impairment leads to diminished protein persulfidation, including the key metabolic regulator SIRT1, and reduced locomotor performance. Mpst⁻/⁻ mice recapitulated these deficits, confirming the essential role of MPST-derived H₂S in preserving muscle function. Notably, exogenous H₂S supplementation improved locomotor activity in db/db mice and restored protein persulfidation, including that of SIRT-1. Consistently, myotubes exposed to an “obese environment” displayed MPST downregulation and reduced SIRT1 persulfidation, promoting inflammation. Exogenous H₂S reversed these effects, highlighting its therapeutic potential against obesity-related SKM dysfunction. The translational relevance of these findings was further supported by human muscle biopsies from obese individuals, which exhibited reduced MPST expression, underscoring the clinical significance of the MPST/H₂S axis in maintaining SKM health. In the second part, I evaluated the therapeutic potential of H₂S in DMD using mdx mice, a well-known preclinical model of DMD. Treatment with the slow-releasing H₂S donor erucin improved muscle strength and coordination, restored redox balance, reduced fibrosis, and activated SKM regenerative pathways. Moreover, it promoted a fiber-type shift toward oxidative, fatigue-resistant phenotypes. Mechanistically, these benefits were linked to the activation of the AMPK–PGC1α–SIRT3 axis and enhancement of mitochondrial oxidative phosphorylation, supporting improved bioenergetic capacity in dystrophic muscle. Taken together, these findings identify the H₂S and more generally the TSP pathway as a crucial determinant of SKM function, with relevance for both metabolic and genetic myopathies. By clarifying the molecular mechanisms through which altered H₂S signaling contributes to muscle dysfunction and by demonstrating that H₂S donors can effectively restore redox balance, metabolic efficiency, and performance, this work provides a strong conceptual and experimental basis for the development of future therapeutic strategies. Targeting this pathway may represent a promising strategy to preserve muscle health and counteract disease progression.

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