The Effects of Diet Induced Obesity (DIO) on Skeletal Muscle Transcription in MuRF1 KO Mice

Background: As obesity and Type II Diabetes rise globally, it is important to understand the similarities and differences in the response of metabolic tissues between males and females. We wanted to evaluate the impact of prolonged diet induced obesity (DIO) on the skeletal muscle transcriptome of our MuRF1 KO (KO) mice. Methods: RNA was isolated from the gastrocnemius muscle of male and female WT and KO mice that were fed either standard chow (Envigo 2918) or a 45% HFD (Research Diets D12451) for 22 weeks (n = 4). RNA was enriched for mRNA prior to library preparation. RNA sequencing was performed using 150 bp paired-end reads (~ 31.6 M reads per sample). Differentially expressed genes (DEGs) were identified using DESeq2 with an FDR set to 5%. Results: At baseline (chow diet), both male and female KO mice had DEGs compared to their WT counterparts (male, 1174; female, 105). Most DEGs were found to be unique by sex (male, 1151; female, 82), though 23 genes were found to be changed in common. After obesity was induced by 22 weeks of 45% HFD feeding, KO animals showed a greater transcriptional response than their WT counterparts. Males had 1821 DEGs (v. 179 in WT) while females had 4425 DEGs (v. 2090 in WT). In males, 78 genes were changed in common between WT and KO in response to DIO, with 76 of those genes changing in the same direction (Slc282a and Gm15427 did not). In females, 1445 genes were changed in common between WT and KO, with all but 2 genes (Pla2g7 and Zfp385b) changing in the same direction. In both male and female KO animals, oxidative phosphorylation and ribosomal pathways were most significant, though the direction of change in the DEGs was opposite. Conclusion: In skeletal muscle, sex highly influences the genes and pathways changed in response to DIO. Even among common pathways identified, the response between males and females differed. Loss of MuRF1 results in common and unique transcript changes in and between males and females under normal conditions and in DIO.

Divergent transcriptomic profiles in skeletal muscle of diabetics with and without heart failure

Funding Acknowledgements Type of funding sources: None. Background Patients with type 2 diabetes mellitus (DM) that have coexistent heart failure (HF) have exacerbated symptoms and prognosis, however beside cardiac dysfunction the mechanisms governing these features are incompletely understood. Evidence indicates abnormalities in the periphery could contribute to this worse clinical phenotype, including a role for skeletal muscle whereby disturbances in the transcriptome could disrupt muscle homeostasis/repair to offer a novel therapeutic approach. Purpose Is the skeletal muscle transcriptome distinguishable between DM patients with and without HF? Methods DM patients without (n = 11) or with HF with reduced left ventricular ejection fraction (LVEF) (n = 16) were included. Muscle biopsies were collected from the pectoralis major during pacemaker implantation. Following RNA extraction and cDNA synthesis, non-bias RNA sequencing (RNAseq) was performed (Cambridge Genomic Services, UK) followed by targeted RT-PCR gene expression of relevant targets. DESeq2 identified differentially expressed genes (DEGs) with a false discovery rate (p < 0.05). Gene enrichment analysis was performed with clusterProfiler v3.16.0 to interrogate the gene ontology database, while pathway analysis was conducted using ReactomePA v1.32.0 to interrogate the Reactome database, using an adjusted p value. Values of p < 0.05 were accepted as significant. Results Groups were not different (p > 0.05) for age (74 ± 11 vs. 66 ± 10 years), BMI (31 ± 7 vs 29 ± 6), sex (n = 2 females per group), or HbA1c (56 ± 10 vs. 57 ± 8 mmol/mol), although LVEF was lower in the group with HF (27 ± 8 vs. 54 ± 2%; p < 0.05). Of the 19,544 genes analysed, RNAseq identified 53 DEGs between DM patients with and without HF, with several relevant targets related to myofiber homeostasis such as autophagy (RUBCN), protein synthesis (DGKζ), and inflammation/apoptosis (TLE1). Follow-up RT-PCR analysis confirmed a trend towards upregulation of the autophagy-related machinery p62 (p = 0.043) and BNIP3 (p = 0.085) in the HF group, but not ubiquitin-proteasome (MuRF1, MAFbx; p > 0.05). Gene-enrichment analysis of DEGs identified 7 overrepresented terms (P < 0.05), including lipid metabolism/signalling alongside epigenetic modifications related to histone deacetylases (HDAC6/10). Furthermore, pathway analysis identified 4 terms (p < 0.05) related to NOTCH signalling and phosphatidyl inositol-bisphosphate (PIP2) hydrolysis thus indicating alterations to muscle repair and lipid signalling respectively. Conclusion(s): This study confirms that DM patients with and without HF demonstrate distinct skeletal muscle transcriptome profiles. Key differences related to skeletal muscle myogenesis, autophagy, epigenetic regulation, and lipid signalling were identified that could form part of important therapeutic targets. Whether these underlying muscle transcriptome differences contribute to poorer clinical outcomes in DM patients with HF remains to be determined.