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. 2021 Oct 4;22(19):10735.
doi: 10.3390/ijms221910735.

Deletion of Neuronal CuZnSOD Accelerates Age-Associated Muscle Mitochondria and Calcium Handling Dysfunction That Is Independent of Denervation and Precedes Sarcopenia

Yu Su  1   2 , Dennis R Claflin  3   4 , Meixiang Huang  5 , Carol S Davis  2 , Peter C D Macpherson  2 , Arlan Richardson  6   7 , Holly Van Remmen  7   8   9 , Susan V Brooks  2   4
Affiliations

Deletion of Neuronal CuZnSOD Accelerates Age-Associated Muscle Mitochondria and Calcium Handling Dysfunction That Is Independent of Denervation and Precedes Sarcopenia

Yu Su et al. Int J Mol Sci. .

Abstract

Skeletal muscle suffers atrophy and weakness with aging. Denervation, oxidative stress, and mitochondrial dysfunction are all proposed as contributors to age-associated muscle loss, but connections between these factors have not been established. We examined contractility, mitochondrial function, and intracellular calcium transients (ICTs) in muscles of mice throughout the life span to define their sequential relationships. We performed these same measures and analyzed neuromuscular junction (NMJ) morphology in mice with postnatal deletion of neuronal Sod1 (i-mn-Sod1-/- mice), previously shown to display accelerated age-associated muscle loss and exacerbation of denervation in old age, to test relationships between neuronal redox homeostasis, NMJ degeneration and mitochondrial function. In control mice, the amount and rate of the decrease in mitochondrial NADH during contraction was greater in middle than young age although force was not reduced, suggesting decreased efficiency of NADH utilization prior to the onset of weakness. Declines in both the peak of the ICT and force were observed in old age. Muscles of i-mn-Sod1-/- mice showed degeneration of mitochondrial and calcium handling functions in middle-age and a decline in force generation to a level not different from the old control mice, with maintenance of NMJ morphology. Together, the findings support the conclusion that muscle mitochondrial function decreases during aging and in response to altered neuronal redox status prior to NMJ deterioration or loss of mass and force suggesting mitochondrial defects contribute to sarcopenia independent of denervation.

Keywords: NADH; calcium; denervation; oxidative stress; sarcopenia.

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Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Figures

Figure 1
Figure 1
Lumbrical muscle contractile properties. (A) Representative traces of force generation during twitch (blue) and tetanic (red) contractions, with parameters used to describe muscle contractile properties annotated on the graph. Data shown for (B) peak specific force, (C) time to peak twitch tension (TPT), and (E) half relaxation time (HRT) for lumbrical muscles from young (n = 18, white, cricle), middle (n = 10, blue, triangle) and old (n = 5, orange, square) aged wild type mice. Specific force was calculated by normalizing the maximum isometric tetanic force by maximum muscle cross-sectional area. Additionally, shown are correlations between (D) age and TPT and (F) age and HRT analyzed by Pearson correlation (TPT: R2 = 0.865, p < 0.0001; HRT: R2 = 0.672, p < 0.0001). Data in (B,C,E) are presented as individual data points with box plots and error bars indicating the minimum, first quartile, median, third quartile, and maximum number of the dataset. Data were analyzed by one-way ANOVA, ** p < 0.01, *** p < 0.001, **** p < 0.0001.
Figure 2
Figure 2
Muscle calcium handling properties. (A) Representative traces of intracellular calcium transient reported by mag-fura-2 during a single twitch contraction, with parameters used to describe calcium handling properties annotated on the graph. Data are shown for (B) the peak of the intracellular calcium transient (ICT peak), (C) time during which the ICT remains at or above its half maximum width (full width at half-maximum, ICT FWHM), (D) time for ICT transient increased to its peak, and (E) time for ICT transient to decrease from 90% to 10% of its maximum for lumbrical muscles of young (n = 10, white, circle), middle (n = 10, blue, triangle) and old (n = 5, orange, square) aged wild type mice. Additionally, shown is (F) the correlation between age and ICT FWHM analyzed by Pearson correlation (R2 = 0.689, p < 0.0001). Data in (B–E) are presented as individual data points with box plots and error bars indicating the minimum, first quartile, median, third quartile, and maximum number of the dataset. Data were analyzed by one-way ANOVA, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.
Figure 3
Figure 3
NADH fluorescence response to a 5s tetanic contraction. (A) A representative trace of NADH fluorescence dynamics illustrating parameters used to quantify the NADH fluorescence response scaled to maximum and minimum NADH levels. Times to each peak (TP1, TP2, TP3) were measured relative to t = 0; peak amplitudes (ΔP1, ΔP2, ΔP3) were measured relative to pre-contraction resting level. (B) Representative NADH fluorescence records for lumbrical muscles of young (black), middle (blue), and old (orange) aged mice. Data are shown for (C) basal NADH level prior to contraction, (D) time for NADH fluorescence increase to the 1st peak with contraction, (E) time between NADH fluorescence peaks P1 and P2 (TP2 minus TP1), (F) amplitude of changes in NADH fluorescence between P1 and P2, (G) rate of change in NADH fluorescence between P1 to P2, and (H) NADH level recorded 100 s after the end of the contraction relative to rest level for lumbrical muscles of young (n = 10, white), middle (n = 10, blue) and old (n = 5, orange) aged wild type mice. Data in (C–H) are presented as individual data points with box plots and error bars indicating the minimum, first quartile, median, third quartile, and maximum number of the dataset. Data were analyzed by one-way ANOVA, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.
Figure 4
Figure 4
Comparison of lumbrical muscle contractile properties between control and i-mn-Sod1-/- mice. Data are shown for (A) peak specific force for muscles of middle-aged and old control (blue) and i-mn-Sod1-/- (red) mice. Additionally, shown are (B) representative twitch contractions for muscles of control (blue) and i-mn-Sod1-/- (red) mice at middle-age as well as data for (C) time to peak twitch tension (TPT), (D) half relaxation time (HRT) for muscles of middle- and old-aged control (blue; n = 10 for middle age, n = 5 for old age) and i-mn-Sod1-/- (red; n = 12 for middle age, n = 7 for old age) mice. Data in A, C, and D are presented as individual data points (circle) with box plots and error bars indicating the minimum, first quartile, median, third quartile, and maximum number of the dataset. Data were analyzed by two-way ANOVA, ** p < 0.01, *** p < 0.001, **** p < 0.0001.
Figure 5
Figure 5
Comparison of calcium handling and mitochondrial function between control and i-mn-Sod1-/- mice. Representative traces are shown for (A) intracellular calcium transients (ICT) and (D) NADH fluorescence response to a tetanic contraction for lumbrical muscles of control (blue) and i-mn-Sod1-/- (red) mice at middle-age. Data are shown for (B) the peak of the intracellular calcium transient (ICT peak), (C) time during which the ICT remains at or above its half maximum width (full width at half-maximum, ICT FWHM), (E) time for NADH fluorescence increase to the 1st peak with contraction, and (F) basal NADH level prior to contraction for control (blue; n=10 for middle age, n=5 for old age, blue) and i-mn-Sod1-/- (red; n = 12 for middle age, n = 7 for old age) mice. Data in (B,C,E,F) are presented as individual data points with box plots and error bars indicating the minimum, first quartile, median, third quartile, and maximum number of the dataset. Data were analyzed by two-way ANOVA, * p < 0.05, ** p < 0.01.
Figure 6
Figure 6
NMJ morphology in control and i-mn-Sod1-/- mice. (A) Representative immunofluorescent images of NMJs in lumbrical muscles of 20-month-old control and i-mn-Sod1-/- mice. Muscles were stained for nerve with anti-βIII Tubulin antibody (green) and acetylcholine receptors, AChR with BTX (red) to visualize pre- and post-synaptic structure, respectively. Scale bar (100 µm) is labeled in image. Data are shown for the number of (B) fully innervated, (C) partially denervated, and (D) fully denervated endplates for control (n = 3, blue, circle) and i-mn-Sod1-/- (n = 4, red, triangle) mice. Data in (B–D) are presented as mean+SEM including individual data points and analyzed by Student’s t-test. There were no differences between any groups.
Figure 7
Figure 7
Fiber type composition in lumbrical muscle between control and i-mn-Sod1-/- mice. (A) Representative immunofluorescence images of lumbrical muscle cross sections for control and i-mn-Sod1-/- mice at middle- and old-age. Fiber types are represented in different pseudo-color as type 1 (blue), type 2a (green), type 2x (black), type 2b (red). Scale bar (100 µm) is labeled in image. Data are shown for (B) total fiber number and (C) total lumbrical muscle cross-sectional area for muscles of controls (blue; n = 7 for middle age, n = 4 for old age) and i-mn-Sod1-/- mice (red; n = 10 for middle age, n = 7 for old age) and are presented as individual data points with box plots and error bars indicating the minimum, first quartile, median, third quartile, and maximum number of the dataset. These data were analyzed by two-way ANOVA, ** p < 0.01. Additionally, shown are (D) the number of each type of fiber and (E) the fraction of the total muscle cross-sectional area occupied by each type of fiber in lumbrical muscles from control and i-mn-Sod1-/- mice at middle and old-age. Data are presented as means+SEM and analyzed by two-way ANOVA, Turkey’s multiple comparisons test. * p < 0.05 compared to control littermates and # p < 0.05 compared to same genotype mice at middle-age. Sample sizes for control mice are n = 7 for middle age and n = 4 for old age and n = 10 for middle age and n = 7 for old age for i-mn-Sod1-/- mice.
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