Table1_The effects of temperature and donor piglet age on the transcriptomic profile and energy metabolism of myoblasts.xlsx

Rapid climate change is associated with frequent extreme heat events and the resulting thermal stress has consequences for the health, welfare, and growth of farm animals. The aim of this study was to characterize the transcriptional changes and the effects on energy metabolism in proliferating porcine myoblasts derived from piglets of different ages, representing differences in thermoregulatory abilities, and cultivated below (35°C) and above (39°C, 41°C) the standard cultivation temperature (37°C). Satellite cells originating from Musculus rhomboideus of piglets isolated on days 5 (P5, thermolabile) and 20 (P20, thermostable) of age were used. Our expression analyses highlighted differentially expressed genes in porcine myoblasts cultures under heat or cold induced stress. These gene sets showed enrichment for biological processes and pathways related to organelle fission, cell cycle, chromosome organization, and DNA replication. Culture at 35°C resulted in increased metabolic flux as well as a greater abundance of transcripts of the cold shock protein-encoding gene RBM3 and those of genes related to biological processes and signaling pathways, especially those involving the immune system (cytokine–cytokine receptor interaction, TNF and IL-17 signaling pathways). For cultivation at 39°C, differences in the expression of genes related to DNA replication and cell growth were identified. The highest glutathione index ratio was also found under 39°C. Meanwhile, cultivation at 41°C induced a heat stress response, including the upregulation of HSP70 expression and the downregulation of many biological processes and signaling pathways related to proliferative ability. Our analysis also identified differentially expressed genes between cells of donors with a not yet (P5) and already fully developed (P20) capacity for thermoregulation at different cultivation temperatures. When comparing P5 and P20, most of the changes in gene expression were detected at 37°C. At this optimal temperature, muscle cells can develop to their full capacity. Therefore, the most diverse molecular signaling pathways, including PI3K-Akt signaling, Wnt signaling, and EGFR tyrosine kinase inhibitor, were found and are more pronounced in muscle cells from 20-day-old piglets. These results contribute to a better understanding of the mechanisms underlying the adaptation of skeletal muscle cells to temperature stress in terms of their thermoregulatory ability.</p

A regulatory network of differentially-expressed miRNAs and genes in C2C12 myogenesis, focusing on mitochondrial energy-metabolism pathways.

<p>Pearson correlation coefficient analysis identified a number of miRNAs-mRNA correlation interactions. Only those interactions in which both miRNAs and target mRNAs significantly associated with differentiation and ATP level (FDR < 0.1) during C2C12 myotube induction were utilized to model an miRNA-regulatory network (IPA pathway designer tool). White nodes indicate mRNA target; edges indicate regulatory interaction between miRNA and target gene. Solid line label denotes negative regulation with negative correlation interaction; dashed line denotes indirect positive regulation with positive correlation interaction.</p

Knockdown of <i>Cox6a2</i> by RNA interference reveals regulation of ATP levels.

<p>siRNAs were designed to target <i>Cox6a2</i> and transfected into murine C2C12 muscle cells in vitro. Relative mRNA expression of Cox6a2 was measured by qPCR 48 hours after transfection. Expression was normalized to Hprt1 and Ppia internal controls. (A) Expression of <i>Cox6a2</i> was significantly reduced relative to its expression in control cells at 48 hours post-transfection of siRNA. (B) ATP levels and (C) ADP/ATP ratio were measured at D0, D4, and D8. All values are presented as mean ± SEM (n = 3).</p

Characteristics of myogenic differentiation.

<p>(A) Morphological change of C2C12 myoblasts differentiating into myotubes at the post-induction D0 (undifferentiated mononucleated cells), D4 (elongated confluent cells), and D8 (long multinucleated myotubes). (B) Myogenic differentiation was accompanied by the up-regulation of Tnnt1, Myh1, and Myh3. (C) An increase in intracellular ATP level (μmoles) during the course of myogenic differentiation. The ATP level is shown as mean ± SEM (n = 5).</p

Predicted binding sites for miRNAs in the 3′-UTRs of their correlated mRNAs (RNAhybrid).

<p>The number of binding sites at the 3´-UTR of significant correlated mRNAs is presented with the free energy hybridization of miRNA and target in parenthesis. n/a denotes no binding site available (predicted) at the 3´-UTR.</p><p>* denotes that binding sites were predicted while the negative correlation between miRNA and mRNA was not significant.</p><p>Predicted binding sites for miRNAs in the 3′-UTRs of their correlated mRNAs (RNAhybrid).</p

miR-423-3p as a candidate miRNA for functional validation.

<p>(A) Conservation of miRNA-423-3p across species; Bos taurus (bta), Pan troglodytes (ptr), Mus musculus (mmu), Homo sapiens (hsa), Rattus norvegicus (rno), and Sus scrofa (ssc). The seed region (2–8 nt) is highlighted in a gray box and underlined. (B) Predicted targets of mmu-miR-423-3p in the 3′-UTR of Cox6a2, Ndufb7, and Ndufs5 (RNAhybrid). The prediction criteria include free energy ≥ -20 kcal/mol and allowing the G:U wobble base-pair and bulging nucleotides in the seed region. The seed match is underlined.</p

Alteration of miRNA expression in C2C12 myoblasts post-myotube induction.

<p>(A) 15 down-regulated miRNAs that were negatively correlated with the ATP level (correlation coefficient, r = -0.67 to -0.83, P< 0.05). (B) 9 up-regulated miRNAs that were positively correlated with the ATP level (r = 0.67 to 0.79, P< 0.05).</p

Hierarchical clustering of the top 100 miRNA probe sets which were differentially expressed at different time point of D0, D4, and D8 of C2C12 myoblasts cell.

<p>Hierarchical clustering of the top 100 miRNA probe sets which were differentially expressed at different time point of D0, D4, and D8 of C2C12 myoblasts cell.</p

Confirmation of microRNA-microarray results by qPCR.

<p>miR-423-3p, miR-128-3p, and miR-301a-3p were selected for qPCR validation. Mean ± SEM (n = 4) of the log2 transformed microarray result (–•– on primary y-axis) and relative expression (2-ΔΔCt) derived from qPCR (—•—secondary y-axis) are overlaid. A correlation coefficient (r) and p-value are indicated.</p

List of differentially-expressed genes affecting ATP level during myogenic differentiation in C2C12 cells.

<p>Data are shown as fold regulation levels compared to control group (D0) and normalized to mean of housekeeping genes (<i>Actb</i>, <i>B2m</i>, <i>Gapdh</i>, <i>Gusb</i>, and <i>Hsp90ab1</i>), then assessed for correlation with ATP levels.</p><p>List of differentially-expressed genes affecting ATP level during myogenic differentiation in C2C12 cells.</p