L-tyrosine supplementation does not ameliorate skeletal muscle dysfunction in zebrafish and mouse models of dominant skeletal muscle α-actin nemaline myopathy

Figures

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  Figure 1. Toxicity analyses for L-tyrosine treatment of WT zebrafish. (A) Percentage survival, and (B) resting heart rate in beats per minute (bpm) of zebrafish treated with increasing L-tyrosine concentrations (from 0.1 μM to 10 mM) or H2O (used as a control). Error bars represent ± s.e.m. for four independent experiments with 30 zebrafish per experiment for survival assays and 10 zebrafish per experiment for heart rate assays, *p < 0.05, **p < 0.01 compared to H20 treatment. (C) Normalised distance travelled by 6 dpf zebrafish treated with increasing L-tyrosine concentrations (from 0.1 μM to 1 mM) or H2O. Error bars represent ± s.e.m. for four independent experiments with 19–24 zebrafish per dose per experiment. **p < 0.01.

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  Figure 2. L-tyrosine treatment of TgACTA1D286G-eGFP zebrafish. Normalised distance travelled by 6 dpf TgACTA1D286G-eGFP and WT sibling control zebrafish treated with 10 μM L-tyrosine or H2O. Error bars represent ± s.e.m for five independent experiments with n = 238 for TgACTA1D286G-eGFP, n = 168 WT siblings, 16–39 zebrafish per treatment, per genotype, per experiment. *p < 0.05, **p < 0.01.

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  Figure 3. Levels of free L-tyrosine in sera and quadriceps femoris of TgACTA1D286G mice. The concentration of free L-tyrosine was quantified via LC/MS in (A) sera samples (untreated, n = 4; L-tyrosine, n = 8), and (B) quadriceps femoris muscles (untreated, n = 6; L-tyrosine, n = 11) of 6-week old TgACTA1D286G mice receiving a 2% L-tyrosine supplemented diet from preconception compared to those fed control diets. Each data point represents an individual mouse ± s.d. *p < 0.05, **p < 0.01.

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  Figure 4. Total body mass and hindlimb muscles volume for NM mice treated with a 2% L-tyrosine supplemented diet. Total body mass was determined in 6-week old TgACTA1D286G mice (A) (untreated = 10 males, 12 females; L-tyrosine = 14 males, 17 females) receiving a 2% L-tyrosine supplemented diet or an untreated diet, as well as C57BL/6 J mice (untreated = 22 males, 14 females). (B) Total body mass of 6–7 month old mice. TgACTA1D286G (untreated = 9 males, L-tyrosine = 8 males); KIActa1H40Y (untreated = 13 females, L-tyrosine = 13 females). (C) Hindlimb muscles volume for mice: male 6–7 month old TgACTA1D286G (untreated = 8; L-tyrosine = 7), female KIActa1H40Y (untreated = 13; L-tyrosine = 13). Each data point represents an individual mouse ± s.d. *p < 0.05, ***p < 0.001.

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  Figure 5. Voluntary running wheel activity in 6-week old TgACTA1D286G mice receiving a 2% L-tyrosine supplemented diet from preconception compared to TgACTA1D286G and WT C57BL/6 J mice fed an untreated diet. Parameters of voluntary running wheel performance measured included (A) maximum speed (m/min), (B) distance travelled (km/d), (C) average speed (m/min), and (D) duration spent running (min/d). Untreated TgACTA1D286G mice (10 males, 12 females), L-tyrosine treated TgACTA1D286G mice (14 males, 17 females), untreated WT C57BL/6 J mice (15 males, 10 females). Each data point represents an individual mouse average calculated over days 4, 5, and 6 of voluntary wheel access ± s.d. *p < 0.05, **p < 0.01, ***p < 0.001.

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  Figure 6. Accelerated rotarod performance in TgACTA1D286G and C57BL/6 J male mice. Performance on an accelerated rotarod apparatus was determined in 6-week old TgACTA1D286G male mice receiving a 2% L-tyrosine supplemented diet (n = 14) compared to TgACTA1D286G (n = 9) and C57BL/6 J (n = 10) mice fed untreated diets. (A) Average speed at fall (rpm), and (B) latency to fall (s). Each data point represents the average of 3 attempts by an individual mouse ± s.d. ****p < 0.0001.

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  Figure 7. In vivo gastrocnemius skeletal muscle performance of TgACTA1D286G and KIActa1H40Y mice. Absolute maximal force production (A,B) and force production during the stimulation protocol (C,D) from 6–7 month old TgACTA1D286G (B,D) and KIActa1H40Y (A,C) mice fed either an untreated diet or a diet supplemented with 2% L-tyrosine for one month. TgACTA1D286G (untreated = 8 males, L-tyrosine = 7 males,); KIActa1H40Y (untreated = 13 females, L-tyrosine = 13 females). For (A,B) each data point represents an individual mouse ± s.d. Data points for parts (C,D) are represented by the mean force for 5 contractions of all mice in each diet group ± s.d. ***p < 0.001 (significantly different from first five contractions), which demonstrates the effect of time on muscle force performance during exercise.

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  Figure 8. Changes in gastrocnemius PCr and pH during the exercise stimulation in TgACTA1D286G and KIActa1H40Y mice. [PCr] (A,B) and pH (C,D) for hindlimb skeletal muscles from 6–7 month old TgACTA1D286G (B,D) and KIActa1H40Y (A,C) mice fed either an untreated diet or supplemented with a 2% L-tyrosine diet, at rest versus at the end of the exercise stimulation protocol. The [PCr] and pH values significantly differed between rest and exercised states for each strain, however there was no significant difference detected due to L-tyrosine treatment. TgACTA1D286G (untreated = 8 males, L-tyrosine = 7 males); KIActa1H40Y (untreated = 13 females, L-tyrosine = 13 females). Each data point represents an individual mouse value expressed as mean ± s.d. ***p < 0.001.