Age affects myosin relaxation states in skeletal muscle fibers of female but not male mice

Figures

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  Fig 1. Schematic summary of myosin biochemical and structural states in muscle. Yellow: actin, blue: myosin heavy chain, green: essential light chain (ELC), red: regulatory light chain (RLC), orange: MyBP-C, Ca2+: calcium ion, T: temperature, Phos: phosphorylation of RLC and/or MyBP-C. Actin filaments are activated by Ca2+ (which binds to troponin, not shown) allowing myosin heads to be activated by actin binding followed by rapid turnover of ATP and molecular force generation. When Ca2+ is removed and ADP is replaced by ATP, myosin heads return to DRX, which can then cycle to and from SRX. When myosin heads are not ordered on the thick filament backbone (as in SRX), they are disordered and can rotate about in the interfilament space, as depicted by gray double-headed arrows in DRX.

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  Fig 2. Fluorescence imaging of a muscle fiber in chase experiments. (A) Fluorescence image of a single mouse skeletal muscle fiber loaded with mantATP prior to start of ATP chase. One large rectangular region of interest (ROI) encompassing a major portion of the fiber (red) was used to monitor the time course of mantATP fluorescence intensity decay during the ATP chase. Rectangular ROI’s outside the fiber (green) were used for background subtraction, a method adapted from studies of mouse skeletal muscle fibers [25]. (B) Average fluorescence intensity of fiber (red) and background (green) ROI throughout the experiment. MantATP binds to the muscle thick filament (A bands) upon addition to the culture dish. The fiber was incubated in 250 μM mantATP for 5 min. Chase was started after removal of mantATP and 4mM of non-fluorescent nucleotides were exchanged in. Chase ends when fiber fluorescence intensity returns to baseline.

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  Fig 3. Fitting nucleotide exchange data with exponential decay functions. (A) Representative normalized fluorescence trace (black) and best fit to a 3-exponenetial decay (Eq 1) from a young female. Inset: χ2 errors, showing that a 3-exponential fit is optimal. (B) Residual plots (fit minus data) from fits to n-exponential functions. (C) Representative trace of mantATP fluorescence decay during the time-course of nucleotide exchange with MgATP. Data from young female. All decay processes start at t = 0. The color shading corresponds to the total time it takes for each kinetic process to go 63% of completion. That time is also reflected as the lifetime (τ) of each respective decay component.

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  Fig 4. Validation of modeling nucleotide exchange with three-exponential decay function. (A) Fiber nucleotide exchange with MgATP (black and blue) or MgADP (pink). (B) Fiber nucleotide exchange in the presence (blue) and absence of Mg2+ using ATP (black) or ADP (red).

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  Fig 5. Effects of age on relaxation states of skeletal muscle myosin in fibers from female mice. The fraction of myosin (A) in DRX (X2 in Eq 2) and (B) in SRX (X3 in Eq 3). Lifetime of nucleotide turnover (C) in DRX (τ2) and (D) in SRX (τ3). Values are reported as average ± SEM. Individual dots are muscle fibers depicted as the average values from each chase for that fiber.

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  Fig 6. Effects of age on relaxation states of skeletal muscle myosin in fibers from male mice. The fraction of (A) DRX myosin (X2 in Eq 2) and (B) SRX myosin (X3 in Eq 3). Lifetime (τ) of nucleotide turnover of (C) DRX myosin (τ2) and (D) SRX myosin (τ3). Values are reported as average ± SEM. Individual dots are the average values from each tested muscle fiber.