Endocrine Abstracts

The nicotinamide adenine dinucleotide (NAD+) dependent Poly-(ADP-ribosyl)polymerase 1 (PARP1) generates the post-translational modification ADP-Ribosylation (ADPR). Molecular studies have identified potential for NAD+ consuming enzymes to influence metabolic function. Given that PARP1 determines cellular NAD+ concentrations and ADPR shifts target protein activity, we sought to identify the molecular actions of PARP1 within skeletal muscle. Analysis of C2C12 lysates demonstrate that PARP1 (often assumed as basally inactive) and ADPR proteins, are detectable and dynamic during differentiation days 0-5 (P < 0.001; n = 6). RNAseq of siPARP1 (n = 4) versus scrambled control (n = 5) showed that knockdown of PARP1 significantly upregulated 115 and downregulated 150 genes. Gene ontological analysis of these gene-sets showed over-representation in siPARP1 cells of those pathways regulating cell differentiation, inducing actin binding, cytoskeletal structure, and NAD+ binding genes. To identify if dynamic peaks in PARP1 and ADPR protein levels have functional consequences over fully formed myotubes, C2C12 cells were treated with a single dose of the PARP1 inhibitor BYK204165 (10uM) at initiation of differentiation. Cells were left to align and fuse, with the media being replaced on differentiation day 2; final lysate collection was on day 5. Unbiased LC-MS analyses of these lysates detected 180 significantly differential proteins in PARP inhibitor treated cells (n = 6) when compared to vehicle controls (n = 7). Pathway over-representation analyses showed that PARP inhibition impacted biological processes governing muscle development (False Detection Rate 8.99-08), muscle contraction (FDR 9.47-08, myofiber assembly (FDR 8.06-05) and metabolism (FDR 2.93-06). Cell respirometry data support influence of PARP1 over myotube function. Our experiments show PARP1 mediated ADPR is critical to ‘early phase’ events surrounding myotube formation. Reducing PARP1 expression during these critical times significantly altered the orchestration of genes crucial to achieving skeletal muscle architecture and overall phenotype. These results have importance for studies seeking to leverage PARP inhibition or NAD+ availability towards human-health.