Identification and validation of novel contraction-regulated myokines released from primary human skeletal muscle cells.

Proteins secreted by skeletal muscle, so called myokines, have been shown to affect muscle physiology and additionally exert systemic effects on other tissues and organs. Although recent profiling studies have identified numerous myokines, the amount of overlap from these studies indicates that the secretome of skeletal muscle is still incompletely characterized. One limitation of the models used is the lack of contraction, a central characteristic of muscle cells. Here we aimed to characterize the secretome of primary human myotubes by cytokine antibody arrays and to identify myokines regulated by contraction, which was induced by electrical pulse stimulation (EPS). In this study, we validated the regulation and release of two selected myokines, namely pigment epithelium derived factor (PEDF) and dipeptidyl peptidase 4 (DPP4), which were recently described as adipokines. This study reveals that both factors, DPP4 and PEDF, are secreted by primary human myotubes. PEDF is a contraction-regulated myokine, although PEDF serum levels from healthy young men decrease after 60 min cycling at VO2max of 70%. Most interestingly, we identified 52 novel myokines which have not been described before to be secreted by skeletal muscle cells. For 48 myokines we show that their release is regulated by contractile activity. This profiling study of the human skeletal muscle secretome expands the number of myokines, identifies novel contraction-regulated myokines and underlines the overlap between proteins which are adipokines as well as myokines

DPP4 protein level and release from myotubes and serum level after acute exercise.

<p>A. Primary human myotubes were differentiated and DPP4 protein level during differentiation was analyzed by SDS-PAGE and Western blot. Data were normalized to the protein level of tubulin and are expressed relative to day 2 of differentiation. n = 4, *p<0.05 vs. day 2 of differentiation. B. Release of DPP4 during differentiation of myotubes was analyzed by ELISA. Data were normalized to day 2 of differentiation and are expressed relative to day 2 of differentiation. n = 9−10, *p<0.001 vs. day 2 of differentiation. C. Relative gene expression of DPP4 after 24 h EPS (1 Hz, 2 ms, 11.5 V) compared to non-stimulated cells was measured by real-time PCR as described, n = 4. D. DPP4 released by human myotubes was measured after 4, 8, 12 and 24 hours in non-stimulated cells compared to cells stimulated by EPS using ELISA, n≥11. White symbols, control; black symbols, EPS. E. Serum samples were taken before and after 60 min cycling (70% VO₂max) at indicated time points. Serum DPP4 was analyzed by ELISA, n = 8. All data are mean values ± SEM.</p

DPP4 and PEDF protein level during differentiation and release by human primary myotubes.

<p>A. Primary human myoblasts were differentiated for 0 to 6 days. Protein level of DPP4 and PEDF were analyzed in total cell lysates by SDS-PAGE and Western blot. MHC served as a positive control for differentiation. B+C. To analyze the secretion mechanism, myotubes were incubated with 1 µg/µl BFA for 24 h on day 5 of differentiation. The release of DDP4 was analyzed by ELISA (B, n = 10) and the release of PEDF was analyzed by Western blot (C, n = 6, *p<0.001).</p

PEDF expression, protein level, and release in myotubes and serum concentration after acute exercise.

<p>A. Relative gene expression of PEDF during differentiation of myotubes was measured by real-time PCR as described and is expressed relative to day 2 of differentiation, n = 3−4, *p<0.05 vs. day 0 of differentiation. B. PEDF protein level of primary human myotubes was analyzed during differentiation by SDS-PAGE and Western blot. Data were normalized to the protein level of tubulin and are expressed relative to day 2 of differentiation. n = 5−6, *p<0.05 vs. day 0 of differentiation. C. Secretion of PEDF during differentiation of myotubes was analyzed by ELISA. Data are normalized to day 2 of differentiation, n = 5. D. Relative gene expression of PEDF after 24 h EPS (1 Hz, 2 ms, 11.5V) compared to non-stimulated cells was measured by real-time PCR as described, n = 4. E. PEDF secretion of human myotubes was measured after 4, 8, 12 and 24 hours in non-stimulated cells compared to cells stimulated by ELISA, n = 3−4, *p<0.05 vs. control. White symbols, control; black symbols, EPS. F. Serum samples were taken before and after 60 min cycling (70% VO2max) at indicated time points and PEDF level was analyzed by ELISA, n = 8, *p<0.05. All data are mean values ± SEM.</p

Identification of novel myokines by cytokine antibody arrays.

<p>CM of myotubes was collected after 24 h and analysed as described using three different cytokine antibody arrays.</p

Impact of hyperinsulinemia and hyperglycemia on valvular interstitial cells – A link between aortic heart valve degeneration and type 2 diabetes

Type 2 diabetes is a known risk factor for cardiovascular diseases and is associated with an increased risk to develop aortic heart valve degeneration. Nevertheless, molecular mechanisms leading to the pathogenesis of valve degeneration in the context of diabetes are still not clear. Hence, we hypothesized that classical key factors of type 2 diabetes, hyperinsulinemia and hyperglycemia, may affect signaling, metabolism and degenerative processes of valvular interstitial cells (VIC), the main cell type of heart valves. Therefore, VIC were derived from sheep and were treated with hyperinsulinemia, hyperglycemia and the combination of both. The presence of insulin receptors was shown and insulin led to increased proliferation of the cells, whereas hyperglycemia alone showed no effect. Disturbed insulin response was shown by impaired insulin signaling, i.e. by decreased phosphorylation of Akt/GSK-3α/β pathway. Analysis of glucose transporter expression revealed absence of glucose transporter 4 with glucose transporter 1 being the predominantly expressed transporter. Glucose uptake was not impaired by disturbed insulin response, but was increased by hyperinsulinemia and was decreased by hyperglycemia. Analyses of glycolysis and mitochondrial respiration revealed that VIC react with increased activity to hyperinsulinemia or hyperglycemia, but not to the combination of both. VIC do not show morphological changes and do not acquire an osteogenic phenotype by hyperinsulinemia or hyperglycemia. However, the treatment leads to increased collagen type 1 and decreased α-smooth muscle actin expression. This work implicates a possible role of diabetes in early phases of the degeneration of aortic heart valves

Evidence against a Beneficial Effect of Irisin in Humans

Brown adipose tissue has gained interest as a potential target to treat obesity and metabolic diseases. Irisin is a newly identified hormone secreted from skeletal muscle enhancing browning of white fat cells, which improves systemic metabolism by increasing energy expenditure in mice. The discovery of irisin raised expectations of its therapeutic potential to treat metabolic diseases. However, the effect of irisin in humans is unclear. Analyses of genomic DNA, mRNA and expressed sequence tags revealed that FNDC5, the gene encoding the precursor of irisin, is present in rodents and most primates, but shows in humans a mutation in the conserved start codon ATG to ATA. HEK293 cells transfected with a human FNDC5 construct with ATA as start codon resulted in only 1% full-length protein compared to human FNDC5 with ATG. Additionally, in vitro contraction of primary human myotubes by electrical pulse stimulation induced a significant increase in PGC1a mRNA expression. However, FNDC5 mRNA level was not altered. FNDC5 mRNA expression in muscle biopsies from two different human exercise studies was not changed by endurance or strength training. Preadipocytes isolated from human subcutaneous adipose tissue exhibited differentiation to brite human adipocytes when incubated with bone morphogenetic protein (BMP) 7, but neither recombinant FNDC5 nor irisin were effective. In conclusion, our findings suggest that it is rather unlikely that the beneficial effect of irisin observed in mice can be translated to humans. © 2013 Raschke et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited