Duchenne muscular dystrophy (DMD) is a devastating disease caused by mutations in the dystrophin gene that result in the complete absence of dystrophin protein. We have shown previously that recombinant mouse Galectin-1 treatment improves physiological and histological outcome measures in the mdx mouse model of DMD. Because recombinant human Galectin-1 (rHsGal1) will be used to treat DMD patients, we performed a dose-ranging study and intraperitoneal or intravenous delivery to determine the efficacy of rHsGal1 to improve preclinical outcome measures in mdx mice. Our studies showed that the optimal dose of rHsGal1 delivered intraperitoneally was 20 mg/kg and that this treatment improved muscle strength, sarcolemma stability, and capillary density in skeletal muscle. We next examined the efficacy of intravenous delivery and found that a dose of 2.5 mg/kg rHsGal1 was well tolerated and improved outcome measures in the mdx mouse model. Our studies identified that intravenous doses of rHsGal1 exceeding 2.5 mg/kg resulted in toxicity, indicating that dosing using this delivery mechanism will need to be carefully monitored. Our results support the idea that rHsGal1 treatment can improve outcome measures in the mdx mouse model and support further development as a potential therapeutic agent for DMD.

Peptidyl-tRNA hydrolase 2 (PTRH2) regulates integrin-mediated pro-survival and apoptotic signaling. PTRH2 is critical in muscle development and regulates myogenic differentiation. In humans a biallelic mutation in the PTRH2 gene causes infantile-onset multisystem disease with progressive muscle weakness. We report here that the Ptrh2 knockout mouse model recapitulates the progressive congenital muscle pathology observed in patients. Ptrh2 null mice demonstrate multiple degenerating and regenerating muscle fibers, increased central nuclei, elevated creatine kinase activity and endomysial fibrosis. This progressive muscle pathology resembles the muscular dystrophy phenotype in humans and mice lacking the α7 integrin. We demonstrate that in normal muscle Ptrh2 associates in a complex with the α7β1 integrin at the sarcolemma and Ptrh2 expression is decreased in α7 integrin null muscle. Furthermore, Ptrh2 expression is altered in skeletal muscle of classical congenital muscular dystrophy mouse models. Ptrh2 levels were up-regulated in dystrophin deficient mdx muscle, which correlates with the elevated levels of the α7β1 integrin observed in mdx muscle and Duchenne muscular dystrophy patients. Similar to the α7 integrin, Ptrh2 expression was decreased in laminin-α2 dyW null gastrocnemius muscle. Our data establishes a PTRH2 mutation as a novel driver of congenital muscle degeneration and identifies a potential novel target to treat muscle myopathies.

Muscle differentiation requires a complex signaling cascade that leads to the production of multinucleated myofibers. Genes regulating the intrinsic mitochondrial apoptotic pathway also function in controlling cell differentiation. How such signaling pathways are regulated during differentiation is not fully understood. Bit-1 (also known as PTRH2) mutations in humans cause infantile-onset multisystem disease with muscle weakness. We demonstrate here that Bit-1 controls skeletal myogenesis through a caspase-mediated signaling pathway. Bit-1-null mice exhibit a myopathy with hypotrophic myofibers. Bit-1-null myoblasts prematurely express muscle-specific proteins. Similarly, knockdown of Bit-1 expression in C2C12 myoblasts promotes early differentiation, whereas overexpression delays differentiation. In wildtype mice, Bit-1 levels increase during differentiation. Bit-1-null myoblasts exhibited increased levels of caspase 9 and caspase 3 without increased apoptosis. Bit-1 re-expression partially rescued differentiation. In Bit-1-null muscle, Bcl-2 levels are reduced, suggesting that Bcl-2-mediated inhibition of caspase 9 and caspase 3 is decreased. Bcl-2 re-expression rescued Bit-1-mediated early differentiation in Bit-1-null myoblasts and C2C12 cells with knockdown of Bit-1 expression. These results support an unanticipated yet essential role for Bit-1 in controlling myogenesis through regulation of Bcl-2.

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The receptor for advanced glycation end products (RAGE) is a key contributor to the immune and inflammatory response in a myriad of diseases. RAGE is a transmembrane pattern recognition receptor with special interest in pulmonary anomalies due to its naturally abundant expression in the lungs. Our previous studies demonstrated a role for RAGE in inflammation following acute exposure to secondhand smoke (SHS). However, chronic inflammatory mechanisms associated with RAGE have yet to be fully elucidated. In this study, we address the impact of long-term SHS exposure on RAGE signaling. RAGE knockout (RKO) and wild type (WT) mice were exposed to SHS five times weekly via a nose-only delivery system (Scireq Scientific, Montreal, Canada) for six months. SHS animals were compared to mice exposed to room air only. Immunoblot and colorimetric high throughput FACE assays (Active Motif) were used to assess phospho-AKT and NF-κB, respectively. A mouse cytokine antibody array (Abcam) was used to screen secreted cytokines in bronchoalveolar lavage fluid (BALF). Phospho-AKT was decreased and NF-κB was elevated in both groups of SHS exposed mice, with RKO+SHS mice demonstrating tempered outcomes for both intermediates compared to WT+SHS exposed mice. BALF contained increased levels of pro-inflammatory cytokines including IFNγ, IL-13, MIP-1γ and Eotaxin1 in exposed groups and diminished secretion was observed in exposed RKO mice. These results validate a role for RAGE in the mediation of chronic pulmonary inflammatory responses and suggest AKT signaling as a viable pathway of RAGE dependent inflammatory responses. Additional characterization of RAGE-mediated pulmonary responses to prolonged exposure will provide valuable insight into cellular mechanisms of lung diseases such as chronic obstructive pulmonary disease. This work was supported by funding from the National Institutes of Health (NIH 1R15-HL152257, PRR and JAA). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.