Assessing the impact of boldine on the gastrocnemius using multiomics profiling at 7 and 28 days post-complete spinal cord injury in young male mice [RNAseq]

Spinal cord injury (SCI) results in rapid muscle loss in the acute timeframe post-injury. The principal mechanisms of general muscle atrophy have been well-described but there is limited information available specific to SCI. Exogenous drug/small molecule interventions to slow muscle atrophy in severe-to-complete SCI have been relatively ineffective due to the unique combination in loss of central nervous system function due to the spinal lesion, systemic response to the trauma itself, and resultant paralysis. The wide-ranging physiologic response to SCI requires the search for novel therapeutic targets. Connexin hemichannels (CxHCs) allow non-selective passage of small molecules into and out of the cell. Boldine, a CxHC-inhibiting aporphine found in the boldo tree (Peumus boldus), has shown promising results in slowing muscle atrophy and preserving muscle function during endotoxemia-induced sepsis and dysferlinopathy. We administered 50 mg/kg/d of boldine to spinal cord transected mice beginning at day 3 post-injury. Tissue was collected at 7 and 28 d post-injury. Boldine treatment did not alter the loss of body or muscle mass. We then performed molecular analysis at three different levels on the gastrocnemius muscle. Metabolomic analysis revealed that boldine attenuated the SCI-induced changes in proline, phenylalanine, leucine, and isoleucine, as well as glucose, 7 d post-SCI in the gastrocnemius, but not at 28 d. RNA-sequencing further found that SCI resulted in the differential expression of 7,705 and 2,991 genes at 7 and 28 d compared to sham animals, with enrichment for pathways associated with ribosome biogenesis, translation and oxidative phosphorylation. Boldine altered the expression of 146 genes at 7 d and 110 genes at 28 d post-SCI. Additionally, DNA methylation analyses of reduced representation bisulfite sequencing highlighted distinct methylation patterns at both 7 and 28 d following SCI both with and without boldine, though most methylation changes did not associate with genes that were differentially expressed. Taken together, boldine is not an efficacious therapy to preserve body and muscle mass after complete SCI, though boldine was able to preserve or attenuate SCI-induced changes across the metabolome, transcriptome and methylome. Additional research is needed to determine if treatment could be modified or used in combination for long-term recovery or if boldine would be a more efficacious therapy in mild or moderate forms of injury. Comparative gene expression analysis of RNA-seq data from gastrocnemius tissue using Sham, SCI, and SCI-boldine mice.