Sucla2 Knock-Out in Skeletal Muscle Yields Mouse Model of Mitochondrial Myopathy With Muscle Type-Specific Phenotypes

Abstract

Background: Pathogenic variants in subunits of succinyl-CoA synthetase (SCS) are associated with mitochondrial encephalomyopathy in humans. SCS catalyses the conversion of succinyl-CoA to succinate coupled with substrate-level phosphorylation of either ADP or GDP in the TCA cycle. This report presents a muscle-specific conditional knock-out (KO) mouse model of Sucla2, the ADP-specific beta subunit of SCS, generating a novel in vivo model of mitochondrial myopathy.

Methods: The mouse model was generated using the Cre-Lox system, with the human skeletal actin (HSA) promoter driving Cre-recombination of a CRISPR-Cas9-generated Sucla2 floxed allele within skeletal muscle. Inactivation of Sucla2 was validated using RT-qPCR and western blot, and both enzyme activity and serum metabolites were quantified by mass spectrometry. To characterize the model in vivo, whole-body phenotyping was conducted, with mice undergoing a panel of strength and locomotor behavioural assays. Additionally, ex vivo contractility experiments were performed on the soleus (SOL) and extensor digitorum longus (EDL) muscles. SOL and EDL cryosections were also subject to imaging analyses to assess muscle fibre-specific phenotypes.

Results: Molecular validation confirmed 68% reduction of Sucla2 transcript within the mutant skeletal muscle (p < 0.001) and 95% functionally reduced SUCLA2 protein (p < 0.0001). By 3 weeks of age, Sucla2 KO mice were 44% the size of controls by body weight (p < 0.0001). Mutant mice also exhibited 34%-40% reduced grip strength (p < 0.01) and reduced spontaneous exercise, spending about 88% less cumulative time on a running wheel (p < 0.0001). Contractile function was also perturbed in a muscle-specific manner; although no genotype-specific deficiencies were seen in EDL function, SUCLA2-deficient SOL muscles generated 40% less specific tetanic force (p < 0.0001), alongside slower contraction and relaxation rates (p < 0.001). Similarly, a SOL-specific threefold increase in mitochondria (p < 0.0001) was observed, with qualitatively increased staining for both COX and SDH, and the proportion of Type 1 myosin heavy chain expressing fibres within the SOL was nearly doubled (95% increase, p < 0.0001) in the Sucla2 KO mice compared with that in controls.

Conclusions: SUCLA2 loss within murine skeletal muscle yields a model of SCS-deficient mitochondrial myopathy with reduced body weight, muscle weakness and exercise intolerance. Physiological and morphological analyses of hindlimb muscles showed remarkable differences in ex vivo function and cellular consequences between the EDL and SOL muscles, with SOL muscles significantly more impacted by Sucla2 inactivation. This novel model will provide an invaluable tool for investigations of muscle-specific and fibre type-specific pathogenic mechanisms to better understand SCS-deficient myopathy.

Keywords: contractility; extensor digitorum longus; fibre‐type switching; mitochondrial myopathy; soleus; succinyl‐CoA synthetase.

FIGURE 1

Functional variants in Sucla2 lead to unique patterns of SCS expression and activity in skeletal muscle tissues. (a) Relative gene expression of SCS components measured via RT‐qPCR in hindlimb muscles of Sucla2 wild‐type (WT) mice (blue) and conditional knock‐out (KO) mice (red) at postnatal day zero (p0), n = 3–4. (b–c) ADP‐ and GDP‐specific enzymatic activity of SCS in whole‐cell lysates of the (b) soleus (SOL), n = 3, and (c) extensor digitorum longus (EDL), n = 4, of adult mice. Total activity is defined as the sum of nucleotide‐specific activities (Table S3). (d) Composite image of western blot analyses of SCS components in the SOL (d) and EDL (Table S4). (e–f) Densitometry quantitation calculated using ImageJ software in both the SOL (e) and EDL (f), n = 9. Densitometry results are plotted on a logarithmic axis. All data presented in bar graphs are represented as means ± SD. For all data shown, the WT (controls) genotype Sucla2 ^+/+ , HSA‐Cre positive, and KO (mutants) mice are either Sucla2 ^−/− , HSA‐Cre positive. Sucla2 alleles and equal levels of KO between the Sucla2 mutant genotypes are outlined in Figure S2. Significant differences are depicted by asterisks, where * = p < 0.05, ** = p < 0.01, *** = p < 0.001 and **** = p < 0.0001 by multiple unpaired t‐tests, with p‐values corrected for multiple testing via false discovery rate (FDR).

FIGURE 2

Metabolic markers of myopathy are observed in the serum of mice deficient for SUCLA2 in the skeletal muscle. (a) Serum levels of methylmalonic acid, n = 6–7. (b) Serum levels of significantly altered acylcarnitines (Dataset S1), n = 6–7. (c) Serum concentrations of lactate, n = 7. (d) Serum concentrations of FGF‐21, n = 10. (e) Serum concentrations of creatinine, n = 6–7. All data are presented as means ± SD and normalized to the average control. For all data shown, the control genotype is Sucla2 ^+/+ , HSA‐Cre positive, and mutant mice are Sucla2 ^−/− , HSA‐Cre positive. Significant differences are depicted by asterisks, where * = p < 0.05, ** = p < 0.01, *** = p < 0.001 and **** = p < 0.0001 by standard unpaired t‐tests. Figure (b) depicts only acylcarnitines species that were significantly different between genotypes following multiple unpaired t‐tests with FDR correction. All measured metabolites are provided in Dataset S1.

FIGURE 3

Skeletal muscle‐specific Sucla2 deficiency yields an in vivo model of myopathy with significant growth deficiency. (a) Representative image depicting WT and KO female mice at p21. (b) Body weights of Sucla2 WT and KO mice from p21 to p77 (3 to 11 weeks of age), n = 21. p‐value indicates statistically significant effects of both genotype and time via mixed‐effects model. (c) Gait analysis of mice at p21, n = 4 consecutive steps of four mice per genotype, and stride length (data not shown, not significant) and (c) stride width of both forelimbs and hindlimbs were measured. (d–e) Two‐paw grip strength measured on both forelimb (d) and hindlimb paws, n = 7–8. All measured behavioural phenotypes are provided in Dataset S2 and Figure S4. All data are presented as means ± SD, and significant differences are depicted by asterisks, where * = p < 0.05, ** = p < 0.01 and *** = p < 0.001 by standard unpaired t‐tests, with a p‐value correction via FDR calculated for the gait analysis. For all data shown, the control genotype is Sucla2 ^+/+ , HSA‐Cre positive, and Mutant mice are Sucla2 ^−/− , HSA‐Cre positive.

FIGURE 4

Sucla2 skeletal muscle‐specific knock‐out mice exhibit reduced physical activity. Mice were observed in an open field with access to both a light and dark area. (a) Total movement time and (b) distance travelled were measured in both the light and dark environments, n = 7–8. Mice were housed in a telemetry cage with unrestricted access to a running wheel, food and water for approximately 10 days, and running wheel activity (c–e) and feeding behaviour (f) were monitored, n = 7–8. For figures (e)–(f), the white and grey columns indicate alternating 12‐h periods of light and dark, respectively. All data presented in bar graphs are means ± SD, and significant differences are depicted by asterisks, where * = p < 0.05, ** = p < 0.01 and **** = p < 0.0001 by multiple unpaired t‐tests with p‐value correction for multiple testing via FDR where necessary. Statistical comparison of longitudinal data was conducted by t‐test comparisons following area under the curve (AUC) analysis, and consumption behaviour was conducted via linear regression analysis, with the p‐value representing significantly differing slopes. For all data shown, the WT genotype is Sucla2 ^+/+ , HSA‐Cre positive, and KO mice are Sucla2 ^−/− , HSA‐Cre positive. All behavioural data measured are shown in Figure S4 with raw data provided in Dataset S2.

FIGURE 5

SUCLA2 loss in skeletal muscle creates different phenotypic effects on slow‐ and fast‐twitch muscles. (a) Twitch and tetanic specific force and (b) the rate of muscle contraction and relaxation of the SOL and EDL were measured ex vivo. The data are presented as means ± SD, and statistically significant differences are depicted by asterisks, where * = p < 0.05, ** = p < 0.02, *** = p < 0.001 and **** = p < 0.0001 by multiple unpaired t‐tests, with p‐values corrected via FDR. (c–d) The relationships between frequency of stimulus and resultant force recorded in the (c) EDL and (d) SOL of WT (blue) and KO (red) mice. (e–f) The force measured in response to a repeated pulse frequency over 5 min to assess muscle fatigue in the EDL (e) and SOL (f). For figures (c)–(f), p‐values indicate a statistically significant effect of genotype via a mixed‐effects model. For all data shown, the WT genotype is Sucla2 ^+/+ , HSA‐Cre positive, and KO mice are Sucla2 ^−/− , HSA‐Cre positive. All ex vivo muscle function data are provided in Dataset S3, n = 9–10.

FIGURE 6

Evidence for mitochondrial proliferation is observed in SUCLA2‐deficient SOL. (a) The copy number of the mitochondrial chromosome (mtDNA) measured via qPCR in the EDL and SOL, n = 6–7. (b) Densitometry quantification of western blots against citrate synthase conducted using ImageJ in the EDL and SOL, n = 3. (c) Citrate synthase enzymatic activity in the EDL and SOL, n = 4. (d) Representative transmission electron microscopy (TEM) images taken of the EDL and SOL. For both muscles, the representative images were taken at a magnification of 6800×, scale bar = 2 μm. (e) ImageJ software was used to quantify the total lipid (top) and mitochondrial (bottom) area of Sucla2 WT and KO mice of representative TEM images (n = 4 images per mouse, 4 mice per genotype). All TEM quantification data are provided in Table S6. Data in bar graphs are means ± SD, and significant differences are depicted by asterisks, where * = p < 0.05, ** = p < 0.01, *** = p < 0.001 and **** = p < 0.0001 by multiple unpaired t‐tests with p‐values corrected via FDR. For all data shown, the WT genotype is Sucla2 ^+/+ , HSA‐Cre positive, and KO mice are Sucla2 ^−/− , HSA‐Cre positive.

FIGURE 7

Histochemical staining of mitochondrial enzymes shows different levels of mitochondrial proliferation in skeletal muscles. Histochemical staining of mitochondrial cytochrome c oxidase (COX) and succinate dehydrogenase (SDH), and lipid staining via oil red O (ORO) in WT and KO mice in the EDL and SOL. All images were taken at 40× magnification, scale bar = 100 μm. The WT genotype is Sucla2 ^+/+ , HSA‐Cre positive, and KO mice are Sucla2 ^−/− , HSA‐Cre positive.

FIGURE 8

Loss of SUCLA2 in the skeletal muscle leads to muscle‐specific fibre‐type switching and Type 1 fibre predominance in the SOL. (a) Immunofluorescent staining for myosin heavy chains (MyHC) conducted to classify fibre‐type distribution in the EDL and SOL. (b–c) Percent fibre‐type contributions to the total surface area of the muscle sections calculated for both the (b) EDL (n = 6 animals per genotype, totalling 3407 fibres in WT and 4.894 fibres in KO). and (c) SOL (n = 6 animals per genotype, totalling 3603 fibres in WT and 5595 fibres in KO). The colour legend for both the staining and bar graphs is included at the bottom of the figure. Statistical differences between the distributions of fibre‐type percentages were calculated via two‐way ANOVA, with Šídák multiple comparisons test revealing statistically distinct proportions of Type 1 (n = 1538 fibres in WT and 4153 fibres in KO) and Type 2A (n = 1567 fibres in WT and 1117 fibres in KO) fibres within the SOL (*p < 0.0001), and no genotype‐specific changes of fibre proportions within the EDL. Individual analyses of all fibre types are provided in Figure S6. (d) The relative frequency of various cross‐sectional fibre areas (μm²) in the SOL of the Sucla2 controls (blue) and mutants (red), with p‐value representing statistical differences between the distributions as calculated by Chi‐squared analysis. The WT genotype is Sucla2 ^+/+ , HSA‐Cre positive, and KO mice are Sucla2 ^−/− , HSA‐Cre positive.