Loss of Unconventional Myosin VI Affects cAMP/PKA Signaling in Hindlimb Skeletal Muscle in an Age-Dependent Manner

Figures

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  FIGURE 1. Lack of myosin VI affects the muscle/body mass ratio. (A), Schematic diagram of the organization of the MVI heavy chain; detailed explanation in the text. (B), Schematic representation of the possible role of MVI in cAMP signaling. cAMP produced by adenylate cyclase (AC) binds to cAMP-dependent protein kinase PKA regulatory subunits (R), allowing dissociation of PKA catalytic subunits (c), which phosphorylate target proteins, including CREB. PDE hydrolyzes cAMP to 5′-AMP. A-kinase anchoring protein 9 (AKAP9) organizes PKA signaling components into macromolecular complexes at specific subcellular sites. MVI binds to AKAP9 and is proposed to be involved in transporting the complex to the appropriate destination. (C), Mouse experimental design. (D) and (E), Analysis of the muscle/body weight ratio of hindlimb muscles of 3-months- and 12-month-old mice, respectively. GM, gastrocnemius medialis muscle; SOL, soleus muscle; TA, tibialis anterior muscle; EDL, extensor digitorum longus muscle. Data are expressed as mean ± SD, t-test, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001; vs. control. (B) and (C) were created using Servier Medical Art - (smart.servier.com).

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  FIGURE 2. Analysis of hindlimb muscles of newborn (P0) WT and KO mice. (A), Hematoxylin/eosin staining of hindlimb muscles. (B), Size distribution of muscle fibers. (C), Cross-section area of hindlimb muscles. (D), Number of fibers per selected area. Data are expressed as mean ± SD, t-test, **p ≤ 0.01; *p ≤ 0.05 vs. control.

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  FIGURE 3. Analysis of soleus (SOL) muscles of 3-month-old WT and KO mice. (A), Size distribution of muscle fibers. (B), Cross-section area. (C), The average number of myofibers per selected area. (D), The number of nuclei per myofiber. Data are expressed as mean ± SD, t-test, ***p ≤ 0.001, ****p < 0.0001; vs. control.

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  FIGURE 4. Analysis of soleus (SOL) muscles of 12-month-old WT and KO mice. (A), Size distribution of muscle fibers. (B), Cross-section area of hindlimb muscles. (C), The average number of myofibers per selected area. (D), Number of nuclei per myofiber. Data are expressed as mean ± SD, t-test, **p ≤ 0.01, ****p < 0.0001; vs. control.

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  FIGURE 5. Analysis of gastrocnemius medialis (GM) muscles of 3-month-old WT and KO mice. (A), Size distribution of muscle fibers. (B). Cross-section area of hindlimb muscles. (C), The average number of myofibers per selected area. (D), Number of nuclei per myofiber. Data are expressed as mean ± SD, t-test, *p ≤ 0.05; **p ≤ 0.01, ***p ≤ 0.001; vs. control.

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  FIGURE 6. Analysis of gastrocnemius medialis (GM) muscles of 12-month-old WT and KO mice. (A), Size distribution of muscle fibers. (B), Cross-section area of hindlimb muscles. (C), The average number of myofibers per selected area. (D), Number of nuclei per myofiber. Data are expressed as mean ± SD.

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  FIGURE 7. Assessment of cAMP level in P0 hindlimb as well as in 3- and 12-months gastrocnemius medialis (GM) muscle. The levels were evaluated in muscle homogenates as described in Materials and methods. Data are expressed as mean ± SD, t-test, *p ≤ 0.05; vs. control.

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  FIGURE 8. Analysis of cAMP/PKA signaling pathway in hindlimb muscles isolated from P0 mice. (A), RT-qPCR analysis of mRNA levels of key genes involved in cAMP/PKA signaling (see Figure 1B). The data are expressed as mean ± SD, t-test, n = 8, *p ≤ 0.05 vs. control. (B), Representative Western blot of key proteins involved in cAMP/PKA pathway. GAPDH or β-tubulin served as the internal loading control. (C), Localization of AKAP9 (red staining) in muscles of WT (left panels) and KO (right panels) mice. Bars, 10 µm.

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  FIGURE 9. Changes in the levels of key proteins involved in cAMP/PKA pathway in soleus (SOL; (A) and (B)) and gastrocnemius medialis (GM; (C) and (D)) muscles isolated from 3- (A) and (C) and 12-month-old (B) and (D)) WT and KO mice. GAPDH or β-tubulin served as the internal loading control.

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  FIGURE 10. Interaction of myosin VI with AKAP9. (A), depiction of partner interaction sites within MVI globular tail domain and its mutants (RRL and WWY). (B), values of Pearsons’s coefficient characterizing the interaction of AKAP9 with GFP-tagged MVI and its mutants. (C), Co-localization of AKAP9 (in red, probed with anti-AKAP9 antibody) with GFP-tagged MVI and its mutants (in green) in murine C2C12 myoblasts. Insets, ∼2x magnification of the marked area. Bars, 10 µm. Further details in Materials and methods.

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  FIGURE 11. Examination of the presence of active forms of PKA (pPKA) and CREB (pCREB) in subcellular compartments. (A), Detection of pPKA and pCREB proteins in the cytoplasmic (left panel) and nucleoplasmic (right panel) fractions of hindlimb muscles homogenates of P0 WT and KO mice. β-actin and lamin B1 served as the internal loading control and fraction purity. (B), Densitometric analysis of the levels pPKA and pCREB in the examined fractions. Data are expressed as mean ± SD, t-test, *p ≤ 0.05 vs. control.