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Irisin Controls Growth, Intracellular Ca2+ Signals, and Mitochondrial Thermogenesis in Cardiomyoblasts.

Xie C, Zhang Y, Tran TD, Wang H, Li S, George EV, Zhuang H, Zhang P, Kandel A, Lai Y, Tang D, Reeves WH, Cheng H, Ding Y, Yang LJ - PLoS ONE (2015)

Bottom Line: Remarkably, irisin is highly expressed in myocardium, but its physiological effects in the heart are unknown.In conclusion, irisin in a certain concentration rage increased myocardial cell metabolism, inhibited cell proliferation and promoted cell differentiation.These effects might be mediated through PI3K-AKT and Ca2+ signaling, which are known to activate expression of exercise-related genes such as follistatin and myocardin.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Immunology, and Laboratory Medicine, University of Florida College of Medicine, Gainesville, FL, 32610, United States of America.

ABSTRACT
Exercise offers short-term and long-term health benefits, including an increased metabolic rate and energy expenditure in myocardium. The newly-discovered exercise-induced myokine, irisin, stimulates conversion of white into brown adipocytes as well as increased mitochondrial biogenesis and energy expenditure. Remarkably, irisin is highly expressed in myocardium, but its physiological effects in the heart are unknown. The objective of this work is to investigate irisin's potential multifaceted effects on cardiomyoblasts and myocardium. For this purpose, H9C2 cells were treated with recombinant irisin produced in yeast cells (r-irisin) and in HEK293 cells (hr-irisin) for examining its effects on cell proliferation by MTT [3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay and on gene transcription profiles by qRT-PCR. R-irisin and hr-irisin both inhibited cell proliferation and activated genes related to cardiomyocyte metabolic function and differentiation, including myocardin, follistatin, smooth muscle actin, and nuclear respiratory factor-1. Signal transduction pathways affected by r-irisin in H9C2 cells and C57BL/6 mice were examined by detecting phosphorylation of PI3K-AKT, p38, ERK or STAT3. We also measured intracellular Ca2+ signaling and mitochondrial thermogenesis and energy expenditure in r-irisin-treated H9C2 cells. The results showed that r-irisin, in a certain concentration rage, could activate PI3K-AKT and intracellular Ca2+ signaling and increase cellular oxygen consumption in H9C2 cells. Our study also suggests the existence of irisin-specific receptor on the membrane of H9C2 cells. In conclusion, irisin in a certain concentration rage increased myocardial cell metabolism, inhibited cell proliferation and promoted cell differentiation. These effects might be mediated through PI3K-AKT and Ca2+ signaling, which are known to activate expression of exercise-related genes such as follistatin and myocardin. This work supports the value of exercise, which promotes irisin release.

No MeSH data available.


Related in: MedlinePlus

Effects of r-irisin on cellular oxygen consumption.A: Effects of r-irisin on cellular OCR was measured with the XF96 Extracellular Flux Analyzer. H9C2 cells were incubated with control (the supernatant of transformed with empty expression vector yeast cells) (blue line) or 25 nM (red line) of r-irisin for 72 h. Three inhibitors, oligomycin (1 μM), FCCP (0.75 μM) and rotenone (1 μM), were added at the indicated points. B: Effect of different r-irisin concentrations on H9C2 baseline OCR. H9C2 cells were incubated with control (the supernatant of transformed with empty expression vector yeast cells) or r-irisin 25 nM, 100 nM and 400 nM for 72 h. Results represent the means of eight independent experiments carried out in triplicate. (*, **, and *** indicating p<0.05, p<0.01, and p<0.001 statistical differences compared to control, respectively.)
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pone.0136816.g004: Effects of r-irisin on cellular oxygen consumption.A: Effects of r-irisin on cellular OCR was measured with the XF96 Extracellular Flux Analyzer. H9C2 cells were incubated with control (the supernatant of transformed with empty expression vector yeast cells) (blue line) or 25 nM (red line) of r-irisin for 72 h. Three inhibitors, oligomycin (1 μM), FCCP (0.75 μM) and rotenone (1 μM), were added at the indicated points. B: Effect of different r-irisin concentrations on H9C2 baseline OCR. H9C2 cells were incubated with control (the supernatant of transformed with empty expression vector yeast cells) or r-irisin 25 nM, 100 nM and 400 nM for 72 h. Results represent the means of eight independent experiments carried out in triplicate. (*, **, and *** indicating p<0.05, p<0.01, and p<0.001 statistical differences compared to control, respectively.)

Mentions: Mitochondria supply the energy for a variety of cardiac functions, and cellular consumption of oxygen mainly reflects mitochondrial metabolism. To assess the effect of irisin on cardiomyoblast energetics, we measured OCR of H9C2 cells treated with different concentrations of r-irisin (or negative control) for 72 h using the Seahorse XF96 Extracellular Flux Analyzer. As shown in Fig 4A, 25nM r-irisin significantly increased baseline OCR values. However, higher concentrations of r-irisin (100 and 400 nM) showed no significant increase compared with the control (Fig 4B). To dissect the metabolic pathway leading to the OCR increase, oligomycin (1μg/ml) was added as an ATP synthase inhibitor. Inhibition of ATP synthesis by oligomycin significantly reduces electron flow through the electron transport chain [30]. The decrease in oxygen consumption rate upon injection of the oligomycin therefore represents the portion of basal respiration used to drive ATP production. Under this condition, OCR of H9C2 cells treated with r-irisin and the negative control was reduced to about 30% of baseline, indicating that about two-thirds (68%) was due to oxygen consumption linked to ATP production (Fig 4A). Strikingly, OCR improvement by r-irisin was reduced by oligomycin, although its value remained higher than in control cells. This result suggested that increasing ATP synthesis was also one function of r-irisin signaling. Next, we examined whether r-irisin affected mitochondrial maximum respiration. FCCP uncouples mitochondrial oxidative phosphorylation by dissipating membrane potential that drives ATP synthesis, and is thus commonly used to examine maximum respiration. Treatment with FCCP (0.75 μM) dramatically improved OCR values of H9C2 cells treated with r-irisin or negative control (Fig 4A). However, OCR differences between the two groups were not detected. Furthermore, incubation with 1 μM of the Complex I inhibitor, rotenone, induced a more glycolytic phenotype; OCR was reduced to about 20% of the baseline, representing non-mitochondrial respiration (Fig 4A). This result suggests that ~85% of the mitochondrial respiration was coupled to ATP synthesis and the rest was associated with proton leak. The higher OCR of H9C2 cells treated with r-irisin vs. control may indicate a potential up-regulation of the glycolysis pathway. Collectively, these data demonstrated that irisin increased mitochondrial metabolism in cardiomyoblasts.


Irisin Controls Growth, Intracellular Ca2+ Signals, and Mitochondrial Thermogenesis in Cardiomyoblasts.

Xie C, Zhang Y, Tran TD, Wang H, Li S, George EV, Zhuang H, Zhang P, Kandel A, Lai Y, Tang D, Reeves WH, Cheng H, Ding Y, Yang LJ - PLoS ONE (2015)

Effects of r-irisin on cellular oxygen consumption.A: Effects of r-irisin on cellular OCR was measured with the XF96 Extracellular Flux Analyzer. H9C2 cells were incubated with control (the supernatant of transformed with empty expression vector yeast cells) (blue line) or 25 nM (red line) of r-irisin for 72 h. Three inhibitors, oligomycin (1 μM), FCCP (0.75 μM) and rotenone (1 μM), were added at the indicated points. B: Effect of different r-irisin concentrations on H9C2 baseline OCR. H9C2 cells were incubated with control (the supernatant of transformed with empty expression vector yeast cells) or r-irisin 25 nM, 100 nM and 400 nM for 72 h. Results represent the means of eight independent experiments carried out in triplicate. (*, **, and *** indicating p<0.05, p<0.01, and p<0.001 statistical differences compared to control, respectively.)
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4549318&req=5

pone.0136816.g004: Effects of r-irisin on cellular oxygen consumption.A: Effects of r-irisin on cellular OCR was measured with the XF96 Extracellular Flux Analyzer. H9C2 cells were incubated with control (the supernatant of transformed with empty expression vector yeast cells) (blue line) or 25 nM (red line) of r-irisin for 72 h. Three inhibitors, oligomycin (1 μM), FCCP (0.75 μM) and rotenone (1 μM), were added at the indicated points. B: Effect of different r-irisin concentrations on H9C2 baseline OCR. H9C2 cells were incubated with control (the supernatant of transformed with empty expression vector yeast cells) or r-irisin 25 nM, 100 nM and 400 nM for 72 h. Results represent the means of eight independent experiments carried out in triplicate. (*, **, and *** indicating p<0.05, p<0.01, and p<0.001 statistical differences compared to control, respectively.)
Mentions: Mitochondria supply the energy for a variety of cardiac functions, and cellular consumption of oxygen mainly reflects mitochondrial metabolism. To assess the effect of irisin on cardiomyoblast energetics, we measured OCR of H9C2 cells treated with different concentrations of r-irisin (or negative control) for 72 h using the Seahorse XF96 Extracellular Flux Analyzer. As shown in Fig 4A, 25nM r-irisin significantly increased baseline OCR values. However, higher concentrations of r-irisin (100 and 400 nM) showed no significant increase compared with the control (Fig 4B). To dissect the metabolic pathway leading to the OCR increase, oligomycin (1μg/ml) was added as an ATP synthase inhibitor. Inhibition of ATP synthesis by oligomycin significantly reduces electron flow through the electron transport chain [30]. The decrease in oxygen consumption rate upon injection of the oligomycin therefore represents the portion of basal respiration used to drive ATP production. Under this condition, OCR of H9C2 cells treated with r-irisin and the negative control was reduced to about 30% of baseline, indicating that about two-thirds (68%) was due to oxygen consumption linked to ATP production (Fig 4A). Strikingly, OCR improvement by r-irisin was reduced by oligomycin, although its value remained higher than in control cells. This result suggested that increasing ATP synthesis was also one function of r-irisin signaling. Next, we examined whether r-irisin affected mitochondrial maximum respiration. FCCP uncouples mitochondrial oxidative phosphorylation by dissipating membrane potential that drives ATP synthesis, and is thus commonly used to examine maximum respiration. Treatment with FCCP (0.75 μM) dramatically improved OCR values of H9C2 cells treated with r-irisin or negative control (Fig 4A). However, OCR differences between the two groups were not detected. Furthermore, incubation with 1 μM of the Complex I inhibitor, rotenone, induced a more glycolytic phenotype; OCR was reduced to about 20% of the baseline, representing non-mitochondrial respiration (Fig 4A). This result suggests that ~85% of the mitochondrial respiration was coupled to ATP synthesis and the rest was associated with proton leak. The higher OCR of H9C2 cells treated with r-irisin vs. control may indicate a potential up-regulation of the glycolysis pathway. Collectively, these data demonstrated that irisin increased mitochondrial metabolism in cardiomyoblasts.

Bottom Line: Remarkably, irisin is highly expressed in myocardium, but its physiological effects in the heart are unknown.In conclusion, irisin in a certain concentration rage increased myocardial cell metabolism, inhibited cell proliferation and promoted cell differentiation.These effects might be mediated through PI3K-AKT and Ca2+ signaling, which are known to activate expression of exercise-related genes such as follistatin and myocardin.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Immunology, and Laboratory Medicine, University of Florida College of Medicine, Gainesville, FL, 32610, United States of America.

ABSTRACT
Exercise offers short-term and long-term health benefits, including an increased metabolic rate and energy expenditure in myocardium. The newly-discovered exercise-induced myokine, irisin, stimulates conversion of white into brown adipocytes as well as increased mitochondrial biogenesis and energy expenditure. Remarkably, irisin is highly expressed in myocardium, but its physiological effects in the heart are unknown. The objective of this work is to investigate irisin's potential multifaceted effects on cardiomyoblasts and myocardium. For this purpose, H9C2 cells were treated with recombinant irisin produced in yeast cells (r-irisin) and in HEK293 cells (hr-irisin) for examining its effects on cell proliferation by MTT [3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay and on gene transcription profiles by qRT-PCR. R-irisin and hr-irisin both inhibited cell proliferation and activated genes related to cardiomyocyte metabolic function and differentiation, including myocardin, follistatin, smooth muscle actin, and nuclear respiratory factor-1. Signal transduction pathways affected by r-irisin in H9C2 cells and C57BL/6 mice were examined by detecting phosphorylation of PI3K-AKT, p38, ERK or STAT3. We also measured intracellular Ca2+ signaling and mitochondrial thermogenesis and energy expenditure in r-irisin-treated H9C2 cells. The results showed that r-irisin, in a certain concentration rage, could activate PI3K-AKT and intracellular Ca2+ signaling and increase cellular oxygen consumption in H9C2 cells. Our study also suggests the existence of irisin-specific receptor on the membrane of H9C2 cells. In conclusion, irisin in a certain concentration rage increased myocardial cell metabolism, inhibited cell proliferation and promoted cell differentiation. These effects might be mediated through PI3K-AKT and Ca2+ signaling, which are known to activate expression of exercise-related genes such as follistatin and myocardin. This work supports the value of exercise, which promotes irisin release.

No MeSH data available.


Related in: MedlinePlus