Myokine Musclin Is Critical for Exercise-Induced Cardiac Conditioning

Figures

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  Figure 1. Musclin is required for exercise-mediated protection from ischemia-reperfusion injury. Representative transverse sections of individual TTC-stained hearts (Left) (scale bar 2 mm) and summary statistics of infarct size (Right) from sedentary and exercised WT and Ostn-KO mice (n = 7–12). Data are means ± SD; * p < 0.05.

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  Figure 2. Musclin promotes exercise training-induced enhancements in respiratory chain enzyme expression. (A) Representative western blots of respiratory chain enzymes and GAPDH, and (B) summary statistics for respiratory complex expression normalized to GAPDH in the hearts of sedentary and exercised WT mice (n = 4). (C) Representative western blots of respiratory chain enzymes and GAPDH, and (D) summary statistics for respiratory complex expression normalized to GAPDH in the hearts of sedentary and exercised Ostn-KO mice (n = 4). (E) Representative western blots of respiratory chain enzymes and GAPDH, and (F) summary statistics for respiratory complex expression normalized to GAPDH in the hearts of exercised WT and Ostn-KO mice (n = 4). Data are means ± SD; * p < 0.05.

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  Figure 3. Musclin promotes exercise training-induced enhancements in mitochondrial biogenesis and respiratory capacity. (A) Summary statistics for mitochondrial content by tissue weight in hearts from sedentary and exercised WT and Ostn-KO mice (n = 6). (B) Summary statistics for expression of ND-1 normalized to hexokinase 2 in hearts from sedentary and exercise WT and Ostn-KO mice (n = 6). (C) Summary statistics for citrate synthase activity in hearts from sedentary and exercised WT and Ostn-KO mice (n = 6). (D) Summary statistics for carbohydrate-driven mitochondrial respiration in semi-permeabilized fibers normalized to tissue weight in hearts from sedentary and exercised WT and Ostn-KO mice (n = 5). Data are means ± SD; * p < 0.05.

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  Figure 4. Musclin regulates cardiac cGMP/Protein Kinase G-dependent signaling. (A) Representative western blots of PKG1, CREB1, PGC1α and Lamin B1 and (B) summary statistics for PKG1, CREB1, and PGC1α expression normalized to Lamin B1 in nuclear extracts from hearts of sedentary and exercised WT mice (n = 4). (C) Representative western blots of PKG1, CREB1, PGC1α and Lamin B1 and (D) summary statistics for PKG1, CREB1, and PGC1α expression normalized to Lamin B1 in nuclear extracts from hearts of sedentary and exercised Ostn-KO mice (n = 4). (E) Representative western blots of PKG1, CREB1, PGC1α and TATA binding protein (TBP) and (F) summary statistics for PKG1, CREB1, and PGC1α expression normalized to TBP in nuclear extracts from hearts of exercised WT and Ostn-KO mice (n = 4). Data are means ± SD; * p < 0.05.

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  Figure 5. Musclin upregulates cardiac transcription of genes involved in mitochondrial biogenesis. Summary statistics for mRNA expression of Creb1, Erra, Nrf1, Nrf2, Ppargc1a, and Tfam in hearts from (A) sedentary and exercised WT mice (n = 6); (B) sedentary and exercised Ostn-KO mice (n = 6); and (C) exercised WT and Ostn-KO mice (n = 6). Data are means ± SD; * p < 0.05.

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  Figure 6. Synthetic musclin infusion pharmacokinetics. (A) Chemically synthesized [13C,15N-A26-V27]-labeled musclin peptide. (B) 15N/1H-HSQC spectrum. (C) 2D slice of the HNCO spectrum. (D) 13C/1H-HSQC-CT spectrum. (E) Half-life is determined from the data fit of the peak intensities measured in the HNCO (2D) spectra. Similar half-life values were also obtained from data in the 15N/1H-HSQC or 13C/1H-HSQC-CT spectra.

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  Figure 7. Synthetic musclin infusion mimics exercise-mediated protection against IR injury. (A,B)—Representative transverse sections of TTC stained hearts (scale bar = 2 mm) and (C) summary statistics of infarct size in WT and Ostn-KO mice treated with osmotic pumps containing control or musclin peptide for 14 days or 28 (musclin ext) days (n = 7–8). Data are means ± SD; * p < 0.05.

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  Figure 8. Molecular mechanisms by which exercise-induced musclin promotes cardiac conditioning. In the heart, musclin competitively interferes with binding to NPRc, allowing increased NP/NPRa binding, which in turn increases cGMP production in the heart. cGMP in turn activates PKG1 which leads to a signaling cascade that results in increased CREB1 nuclear localization and transcription of PGC1α which promotes enhancement of mitochondrial biogenesis and respiratory capacity that support cardiac stress resistance.