The Effect of ACTN3 Gene Doping on Skeletal Muscle Performance

Abstract

Loss of expression of ACTN3, due to homozygosity of the common null polymorphism (p.Arg577X), is underrepresented in elite sprint/power athletes and has been associated with reduced muscle mass and strength in humans and mice. To investigate ACTN3 gene dosage in performance and whether expression could enhance muscle force, we performed meta-analysis and expression studies. Our general meta-analysis using a Bayesian random effects model in elite sprint/power athlete cohorts demonstrated a consistent homozygous-group effect across studies (per allele OR = 1.4, 95% CI 1.3-1.6) but substantial heterogeneity in heterozygotes. In mouse muscle, rAAV-mediated gene transfer overexpressed and rescued α-actinin-3 expression. Contrary to expectation, in vivo "doping" of ACTN3 at low to moderate doses demonstrated an absence of any change in function. At high doses, ACTN3 is toxic and detrimental to force generation, to demonstrate gene doping with supposedly performance-enhancing isoforms of sarcomeric proteins can be detrimental for muscle function. Restoration of α-actinin-3 did not enhance muscle mass but highlighted the primary role of α-actinin-3 in modulating muscle metabolism with altered fatiguability. This is the first study to express a Z-disk protein in healthy skeletal muscle and measure the in vivo effect. The sensitive balance of the sarcomeric proteins and muscle function has relevant implications in areas of gene doping in performance and therapy for neuromuscular disease.

Keywords: ACTN3; Z-disk; Z-line; actinin-3; alpha actinin 3; fast fibers; gene doping; muscle; rAAV; skeletal muscle.

Figure 1

Meta-analysis of the ACTN3 Arg577X Genotype Effect on Elite Power Performance (A) Forrest plots with estimates of the posterior median and 90% credible interval of the odds ratios (OR) for each study are shown to demonstrate both the homozygote and dominance effects from the general meta-analysis model. Studies are grouped based on the ancestry of their participants, according to the key provided, and overall estimates are shown in blue. (B) The same OR estimates are plotted jointly, with homozygote and dominance effects on the x and y axes, respectively. Diagonal gray lines correspond to dominant and recessive models. The point and interval estimates are identical to those in (A) and are numbered by study according to the key provided.

Figure 2

High Doses of rAAV-ACTN3 Cause Muscle Damage (A) Schematics of the rAAV6 vector constructs. Expression of FLAG-tagged human ACTN3, or empty vector (MCS control), is driven by a CMV promoter, flanked by a SV-40 poly(A) tail, and capped by short inverted terminal repeats (ITRs). (B) rAAV-ACTN3 vector expression, as shown by FLAG expression, increase with dose (5E8, 1E9, 1E10, and 1E11 vg) in WT (n = 3) muscles, indicative of efficient product expression. However, total ACTN3 protein did not increase with dose. (C) Six weeks after high-dose injection (1E11 vg rAAV-ACTN3) into the WT TA muscles, cross-section shows positive flag staining accompanied by substantial centralized nuclei and cellular infiltration (H&E) and fibrosis (wheat germ agglutinin [WGA] staining).

Figure 3

High Doses of rAAV-ACTN3 Intramuscular Injection (1E11 vg) Did Not Enhance Muscle Force in WT-ACTN3 Muscles (A) α-Atinin-3 levels varied across injections in WT-ACTN3 TA (n = 8) compared to WT-MCS (n = 8), with no mean changes in α-actinin-3 or 2. (B) No change in WT-ACTN3 TA force (1,227 ± 225 mN, n = 8) compared to WT-MCS (1,412 ± 152 mN, n = 10) 5 weeks after injection. (C) Fatigue response and force recovery at 1 and 10 min (arrows) was also not different. (D) α-Actinin-3 was increased in WT-ACTN3 EDL muscles compared to WT-MCS at 1E11 vg while α-actinin-2 is decreased. (E) EDL force was lower in WT-ACTN3 (177 ± 16 mN, n = 13) compared to WT-MCS (207 ± 17 mN, n = 23) (p = 0.001). (F) WT-ACTN3 EDL muscles showed improved recovery at 1 min and 10 min after fatigue compared to WT-MCS. Boxplots represent the median, whiskers; min-max. Mean ± SEM. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001 Mann Whitney U test.

Figure 4

Postnatal Replacement of α-Actinin-3 in Actn3 KO Muscles Does Not Enhance Force, Muscle Mass, or Fast Fiber Size (A) Intramuscular delivery of ACTN3 resulted in robust expression of α-actinin-3 in all fiber types. KO-ACTN3 TA muscle is immuno-labeled for myosin heavy chain 2B (red) and 2A (green) to denote fiber types. A longitudinally cut KO-ACTN3 muscle section stained for α-actinin-3 antibody (green), nuclei (blue) demonstrates localization to the Z-disk. (B) KO-ACTN3 muscles show α-actinin-3 protein expression and reciprocal downregulation of α-actinin-2. Myosin and actin are loading controls. (C) EDL force was increased in KO-ACTN3 (192 ± 5 mN) versus KO-MCS (177 ± 5 mN) but was lower than WT-MCS (210 ± 17 mN). (D) EDL mass was not different between groups. (E) KO-ACTN3 TA force is unchanged (1,044 ± 72 mN) compared to KO-MCS (1,099 ± 78 mN) and 18% lower than WT-MCS. (F) TA mass. (G–I) Mean 2B fiber size of KO-MCS and KO-ACTN3 TA muscles was not different, but 2X fiber size was reduced in KO-ACTN3 compared to KO-MCS, likely due to increased numbers of smaller 2X fibers. (J) Total fiber number. (K) KO-ACTN3 TA muscles have increased 2B and decreased 2X fiber proportions relative to KO-MCS. N = 7–13 for all groups. Mean ± 95%CI. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗∗p < 0.0001 Mann Whitney U.

Figure 5

Postnatal α-Actinin-3 Replacement in Actn3 KO Muscles Did Not Alter Calcineurin Activity or Expression of Other Z-disk Proteins (A) Calcineurin activity (as shown by RCAN1-4 expression) was unaltered in KO-ACTN3 muscles, despite reciprocal downregulation of α-actinin-2. Similarly, calcium handling proteins SERCA1 and sarcalumenin were unchanged in KO-ACTN3 muscles relative to KO-MCS. (B) Expression of the Z-disk proteins. Myotilin, ZASP, calsarcin-2, and the remodeling protein, Desmin, was not altered in KO-ACTN3 muscles. Mean ± 95%CI, N = 7–13 for all groups. Boxplots represent the median, whiskers; min-max. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, Mann Whitney U.

Figure 6

Postnatal Replacement of α-Actinin-3 in Actn3 KO Muscles Increases Muscle Fatigability and Reduces Force Recovery after Fatigue (A and B) Force is reduced with repeated muscle stimulation to fatigue, and force recovery is assessed at 1, 2, 3, 5, and 10 min post-stimulation in EDL (A) and TA (B) muscles (mean ± SEM). Arrows indicate time points of cohort comparisons in the corresponding column graphs. (C) KO-ACTN3 EDL demonstrated reduced force recovery at 10 min compared to the KO-MCS. (D) KO-ACTN3 TA fatigued more rapidly than KO-MCS and were slower to recover after 1 min, similar to the WT-MCS. Mean ± 95% CI ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, ^∗∗∗∗p < 0.0001, Mann Whitney U, N = 6–13 for all groups.

Figure 7

α-Actinin-3 Replacement in KO TA Muscles Reduces Mitochondrial Oxidative Metabolism (A) Succinate dehydrogenase activity staining in WT-MCS, KO-MCS, and KO-ACTN3 TA muscle. (B) Intensity of positive blue staining is reduced in KO-ACTN3 relative to KO-MCS (indicative of lower SDH activity) but remains different compared to the WT-MCS, N = 6 all groups. (C) KO-ACTN3 TA muscle demonstrated decreases in mitochondrial complexes I, II, III, IV, and V to WT levels in response to α-actinin-3 expression. N = 5 for all groups. Boxplots represent the median, whiskers; min-max. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001.