Reduced efficiency of sarcolipin-dependent respiration in myocytes from humans with severe obesity

Abstract

Objective: Sarcolipin (SLN) regulates muscle energy expenditure through its action on sarco/endoplasmic reticulum Ca(2+) -ATPase (SERCA) pump. It is unknown whether SLN-dependent respiration has relevance to human obesity, but whole-transcriptome gene expression profiling revealed that SLN was more highly expressed in myocytes from individuals with severe obesity (OB) than in lean controls (LN). The purpose of this study was to examine SLN-dependent cellular respiratory rates in LN and OB human muscles.

Methods: Primary myocytes were isolated from muscle biopsy from seven LN and OB Caucasian females. Cellular respiration was assessed with and without lentivirus-mediated SLN knockdown in LN and OB myocytes.

Results: SLN mRNA and protein abundance was greater in OB compared to LN cells. Despite elevated SLN levels in wild-type OB cells, respiratory rates among SLN-deficient cells were higher in OB compared to LN. Obesity-induced reduction in efficiency of SLN-dependent respiration was associated with altered sarcoplasmic reticulum phospholipidome.

Conclusions: SLN-dependent respiration is reduced in muscles from humans with severe obesity compared to lean controls. Identification of the molecular mechanism that affects SLN efficiency might lead to interventions that promote an increase in skeletal muscle energy expenditure.

Figure 1. HSkMCs retain metabolic properties ex vivo

A: Representative images of LN and OB HSkMCs. B: Insulin-stimulated pAkt (n=6/group). *: Effect of insulin, p < 0.05. †: Effect of obesity, p < 0.05. C: Rates of fatty acid oxidation (n=5/group). *: Effect of obesity, p < 0.05. D: Volcano plot of differential expression profile of genes with RPKM > 1 (n=3/group). Dotted lines indicate cutoff lines for fold-difference (2) and p (0.05). All data were collected from day 4 of differentiation. Filled bars: LN, empty bars: OB. Data are shown mean ±SEM.

Figure 2. Higher SLN expression in OB HSkMCs does not yield elevated respiratory rates

A: SLN expression by RNAseq. RPKM: reads per kilobase of target region per millions mapped. B: Gene expression of SLN and other SR calcium uptake enzymes in LN and OB HSkMCs by quantitative PCR (n=6/group). C: Protein expression of SLN, SERCA1, SERCA2 and COXIV in LN and OB HSkMCs by western blotting (n=6/group). D: Representative western blots from LN and OB HSkMCs. E: Gene expression of SLN and other SR calcium uptake enzymes in LN and OB muscle biopsies by quantitative PCR (n=6/group). F: Representative western blots of SLN, SERCA1 and COXIV protein from LN and OB muscle biopsies. G: Cellular respiratory rates of LN and OB HSkMCs (n=5/group). Measurements for all 10 samples were performed on a single Seahorse plate. H: Mean values for four phases of respirations in LN and OB HSkMCs (n=5/group). OCR: oxygen consumption rate. Oligo: oligomycin. FCCP: carbonyl cyanide-p-trifluoromethoxyphenylhydrazone. All data were collected from day 4 of differentiation. Filled bars/dots: LN, empty bars/dots: OB. *: p < 0.05. Data are shown mean ±SEM.

Figure 3. SLN deficiency promotes reduction in cellular respiratory rates

A: Representative images of sc and shSLN C2C12 cells. SLN knockdown did not affect differentiation efficiency or morphology. B: Quantitative PCR of SLN in sc and shSLN C2C12 myotubes. C: Cellular respiration rates of sc and shSLN C2C12 myotubes. D: Mean values for four phases of respirations. All data were collected from day 4 of differentiation. Filled bars/dots: sc, empty bars/dots: shSLN. sc: scrambled, shSLN: short-hairpin SLN (SLN-knockdown). n=6/group. *: p < 0.05. Data are shown mean ±SEM.

Figure 4. Reduced SLN efficiency in OB HSkMCs

A: Quantitative PCR of SLN in LN sc, LN shSLN, OB sc and OB shSLN HSkMCs (n=6/group). B: Representative western blot of SLN protein in LN sc, LN shSLN, OB sc and OB shSLN HSkMCs. C: Cellular respiration rates. Representative data from a single Seahorse plate (n=4 or 5 technical replicates/group). D: Mean values for four phases of respirations (n=6 biological replicates/group). Data from multiple Seahorse plates were each normalized to the first value of LN sc, due to variability in OCR values inherent with HSkMCs and plates. Filled bars/dots: LN sc, dark gray bars/dots: LN shSLN, empty bars/dots: OB sc, light gray bars/dots: OB shSLN. E: OCR_SLN: SLN-dependent OCR contribution, calculated with an equation OCR_SLN = [OCR_sc – OCR_shSLN] / [OCR_sc]. All data were collected from day 4 of differentiation. Filled bars: LN, empty bars: OB. sc: scrambled, shSLN: short-hairpin SLN (SLN-knockdown). *: p < 0.05. Data are shown mean ±SEM.

Figure 5. Differential SR phospholipidome in LN and OB HSkMCs

A: Total PC, PE and PS in isolated SR of LN and OB HSkMCs. PC: phosphatidylcholine, PE: phosphatidylethanolamine, PS: phosphatidylserine. B: Individual PC species in isolated SR of LN and OB cells. C: Individual PE species in isolated SR of LN and OB cells. D: Individual PS species in isolated SR of LN and OB cells. E: SR’s PC/PE ratio in LN and OB cells. F: SR lipid saturation index (nmol saturated acyl-chains/nmol unsaturated acyl-chains) in LN and OB cells. N=4/group. All data were collected from day 4 of differentiation. Filled bars: LN, empty bars: OB. *: p < 0.05. Data are shown mean ±SEM.