L-tyrosine supplementation is not therapeutic for skeletal muscle dysfunction in zebrafish and mouse models of dominant skeletal muscle α-actin nemaline myopathy

Nemaline myopathy (NM) is a skeletal muscle disorder with no curative treatment. Although L-tyrosine administration has been indicated to provide benefit to patients, previous studies have been limited due to sample size or not testing for raised L-tyrosine levels. We evaluated the efficacy of L-tyrosine treatment to improve skeletal muscle function in three animal models of NM caused by skeletal muscle α-actin (ACTA1) mutations. Firstly we determined the maximum safest L-tyrosine concentration for inclusion in the water of wildtype zebrafish. We then treated NM TgACTA1D286G-eGFP zebrafish from 24 hours post fertilization with the highest safe L-tyrosine dose (10 µM). At 6 days post fertilization, no significant improvement was detected in skeletal muscle function (swimming distance). We also determined the highest safe L-tyrosine dose for dietary L-tyrosine supplementation to wildtype mice. Next we treated the NM TgACTA1D286G mouse model continuously from preconception with 2% L-tyrosine supplemented to regular feed. We examined skeletal muscles at 6–7 weeks using indicators of skeletal muscle integrity: bodyweight, voluntary running wheel and rotarod performance, all parameters previously shown to be reduced in TgACTA1D286G mice. The L-tyrosine treatment regime did not result in any improvement of these parameters, despite significant elevation of free L-tyrosine levels in sera (57%) and quadriceps muscle (45%) of treated TgACTA1D286G mice. Additionally, we assessed the effects of 4 weeks of 2% L-tyrosine dietary supplementation on skeletal muscle function of older (6-7 month old) NM TgACTA1D286G and KIActa1H40Y mice. This dosing regime did not improve decreased bodyweight, nor the mechanical properties, energy metabolism, or atrophy of skeletal muscles in these NM models. Together these findings demonstrate that with the treatment regimes and doses evaluated, L-tyrosine does not therapeutically modulate dysfunctional skeletal muscles in NM animal models with dominant ACTA1 mutations. Therefore this study yields important information on aspects of the clinical utility of L-tyrosine for ACTA1 NM. Summary statement Despite previous encouraging reports, this study utilising zebrafish and mouse models of nemaline myopathy shows no therapeutic benefit on skeletal muscle functionality in response to L-tyrosine supplementation.


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in total bodyweight was detected in L-tyrosine treated male mice compared to untreated 2 1 4 TgACTA1 D286G mice (Fig. 4A). Likewise, reduced total bodyweight was not negated at 6-7 months 2 1 5 for ACTA1-NM mice from either mouse model treated for 1 month (TgACTA1 D286G 2 1 6 treated=32.8±4.4g; untreated=34.6±2.7g and KIActa1 H40Y treated=22.6±1.7g; untreated=23.8±2.4g; 2 1 7 Fig. 4B). Additionally, at this older age the hindlimb muscle volume was not different between 2 1 8 treated and untreated mice for both models (Fig. 4C). TgACTA1 D286G mice are impaired compared to WT mice. TgACTA1 D286G mice treated from prior to 2 2 4 conception did not exhibit any significant improvement for any voluntary running wheel activity 2 2 5 parameters relative to untreated mice of the same sex (Fig. 5). Similarly, none of the rotarod 2 2 6 measurements were significantly improved for treated versus untreated male TgACTA1 D286G mice 2 2 7 ( Fig. 6). Absolute maximal force at 6-7 months of age was unchanged for the two NM mouse models after 1 2 3 2 month of treatment ( Fig. 7A & 7B). Force production during the fatiguing protocol was comparable 2 3 3 for the treated and untreated mice for each model (Fig. 7C & 7D). Consequently, the fatigue index (data not shown) and pH variations were also similar between the treated and untreated mice for 2 3 8 each model (Fig. 8A). Prior reports of L-tyrosine supplementation to NM patients describe potential positive effects of 2 4 3 improved skeletal muscle strength, decreased pharyngeal/oral secretions, and increased 2 4 4 stamina/energy levels (Kalita, 1989;Ryan et al., 2008;Olukman et al., 2013), but lacked sufficient 2 4 5 numbers for statistical evaluation. The purpose of this study was to evaluate the therapeutic 2 4 6 usefulness of L-tyrosine supplementation on one of these previously reported potential benefits, 2 4 7 skeletal muscle function, using three dominant ACTA1-NM animal models and multiple measures. We utilised one zebrafish (TgACTA1 D286G -eGFP) and two mouse (TgACTA1 D286G and 2 4 9 KIACTA1 H40Y ) models, encompassing all known laboratory animal models of dominant ACTA1-2 5 0 NM. There are a very small number of patients (usually only one individual) with a particular 2 5 1 mutation in any of the twelve NM genes, including nebulin and actin, the two most common NM 2 5 2 genes. Therefore animal models provide a means to thoroughly investigate a possible therapeutic in 2 5 3 multiple individuals with the same genetic composition.

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We first investigated the safety of increased L-tyrosine levels for WT zebrafish and performance. These findings suggest that the potential toxicity of high L-tyrosine dosing should be 2 5 7 considered for humans supplementing with this amino acid, for whatever therapeutic reason. For the 2 5 8 zebrafish aspect of our study, we utilised the highest concentration of L-tyrosine that did not 2 5 9 produce these negative outcomes (10 μM). Nevertheless, the L-tyrosine treated wildtype and 2 6 0 TgACTA1 D286G -eGFP zebrafish did not exhibit any improvement in the swimming distance 2 6 1 travelled.

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We also supplemented regular mouse feed with 3 levels of L-tyrosine to determine the 2 6 3 highest safe dose. Supplementation with both 4% and 8% L-tyrosine was associated with 2 6 4 deleterious side effects in WT mouse mothers as well as their pups, when the feed was 2 6 5 supplemented from pre-conception. Our pilot toxicity study in mice was not exhaustive or extensive, yet resulted in high mortality rates for two conditions. These adverse findings with the 2 6 7 4% and 8% doses, especially in combination with the findings from the zebrafish toxicity analysis, tyrosine. Tyrosine related toxicity, deleterious effects and weight loss has been previously reported in 2 7 1 the literature, e.g. Boctor and Harper, 1968. A potential explanation for the deleterious effects 2 7 2 observed in mice receiving higher doses of L-tyrosine may be due to L-tyrosine being a precursor 2 7 3 for brain catecholamines. Previous mouse studies report direct correlations between aggressive 2 7 4 activity and brain catecholamines in mice (Thurmond and Brown, 1984) with the effects proposed 2 7 5 due to the prevention of NE depletion (Deijen et al., 1999). Aggressive behaviour, defined by the 2 7 6 number of territorial-induced attacks, was reported in previously unstressed rodents receiving a diet 2 7 7 supplemented with 4% L-tyrosine when they were later put under stress (Brady et al., 1980). The facilitation of aggressive behaviour and suggested that aggressive behaviour may be related to 2 8 0 lower brain NE and serotonin levels relative to DA (Brady et al., 1980). Aggression by the mother 2 8 1 may have been the cause of death for some of the mouse pups on the 4% and 8% supplemented 2 8 2 doses in our study. As no overtly deleterious side effects were seen with 2% L-tyrosine dietary 2 8 4 supplementation, the 2% L-tyrosine supplementation dose was pursued for the efficacy studies with 2 8 5 the ACTA1-NM mouse models. The 2% L-tyrosine dose significantly increased the free L-tyrosine 2 8 6 levels in sera (>55%) and quadriceps muscle (45%) of treated mice. Other studies determined serum 2 8 7 tyrosine levels in rats receiving either a 2% or 5% casein diet for 14 days (of 40±3 nmol/ml and 2 8 8 86±8 nmol/ml respectively (Fernstrom and Fernstrom, 1995). The level of sera L-tyrosine detected 2 8 9 in untreated TgACTA1 D286G mice (52.7±7.8 nmol/ml) in this study is in accordance with these 2 9 0 previous reports. A paucity of data exists for free L-tyrosine levels in rodent skeletal muscles, 2 9 1 although baseline levels of L-tyrosine in other tissues (retina, 0.25 nmol/mg; hypothalamus, 0.55 2 9 2 nmol/mg) have been established for rats (Fernstrom and Fernstrom, 1995). The mean value for L-2 9 3 tyrosine in quadriceps muscle of untreated mice we determined (0.089±0.021 nmol.mg -1 ) was less 2 9 4 than these levels.

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We performed a two-pronged investigation with the 2% L-tyrosine supplemented feed and 2 9 6 the NM mouse models, to evaluate pre-birth versus later-onset treatment. We reasoned that if the 2 9 7 pre-birth experimental arm established breeding mice on the diet fortified with the highest safe L- milk (Rassin et al., 1978). Therefore this L-tyrosine regime would presumably provide the best their NM disease if L-tyrosine were therapeutic for this parameter. A well-known example whereby 3 0 5 1 0 taking supplements prenatally/throughout gestation has significant therapeutic effects is folic acid in 3 0 6 the prevention of neural tube defects such as spina bifida (Group, 1991). TgACTA1 D286G mice 3 0 7 treated with the pre-birth 2% L-tyrosine supplementation regime until 7 weeks of age demonstrated 3 0 8 no improvement in body weight (in fact, L-tyrosine treated 7 week old male TgACTA1 D286G mice 3 0 9 weigh significantly less than untreated males), voluntary exercise and rotarod capacity deficits  Our second experimental arm with the murine NM models assessed a dosing regime that 3 1 2 started in older mice at 5 to 6 months of age and continued for one month. This is the same L-tyrosine for the KIActa1 H40Y or the TgACTA1 D286G models. A potential factor that may account did not report L-tyrosine levels in sera or skeletal muscles from treated mice we are not able to 3 2 1 provide a direct comparison as to the efficiency of the two dosing routes. Thus to relate the dose of 3 2 2 L-tyrosine that mice were exposed to in this study to the dose in the previous study, we calculated 3 2 3 that mice consuming the 2% L-tyrosine supplemented feed would have received a dose ranging 3 2 4 from ~60-90 mg/d (based on the daily intake of 3-4.5 g/d for adult ACTA1-NM mice that we 3 2 5 determined, which fits with the published murine average daily food consumption range of 3 2 6 adolescent mice being from 3.1-6.3 g/d, Bachmanov et al., 2002). Moreover, if we normalise this 3 2 7 dose intake to body weight using the average weight for young mice (12g) treated since pre-3 2 8 conception in addition to the older mice (35g) that were treated at 6 months, this equates to 0.5 -3 2 9 0.75% and 0.17 -0.25% of total bodyweight for young and older mice respectively. Nguyen et al. and mice receiving 25 mg/d).

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In conclusion, we determined safe concentrations of L-tyrosine for dosing WT zebrafish (water) and mice (dietary supplementation), noting higher concentrations had deleterious effects. The dose evaluated in the dominant ACTA1-NM mouse models significantly increased the free L- from pre-conception (TgACTA1 D286G ), or for one month from 5 to 6 months of age (TgACTA1 D286G and KIActa1 H40Y ). The amassed data from our multi-pronged evaluation study demonstrate that All animal experiments were performed in agreement with the guidelines of the respective countries    independent treatments were performed for each experiment with 30 fish per treatment. Zebrafish swimming assays and resting heart rate determination 3 7 0 The resting heart rates were measured at 2 dpf by counting the number of heart beats in 10 sec.

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Heart rate measurements were performed in triplicate with 10 fish per experiment. Assay of 3 7 2 swimming ability, as well as the subsequent data analyses performed on 6 dpf wild type zebrafish a 10-minute period in the dark was measured in mm using zebraboxes (Viewpoint Life Sciences).

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The values for each genotype and treatment were then normalised to the average of the wildtype 3 7 7 untreated siblings in the same replicate. For swimming assays on wild type zebrafish, four analyses were completed the treatments groups were revealed. mouse strains were used for the initial L-tyrosine safety dosing study (FVB/NJArc), and as a  France; basal L-tyrosine level of 0.45%) contained all nutritional dietary parameters either meeting 3 9 5 or exceeding the maintenance guidelines for rats and mice outlined by the National Research conducted a pilot study with normal mice to compare the Australian standard feed (containing 0.7% 3 9 8 L-tyrosine) to the same feed supplemented with an additional 2%, 4% or 8% L-tyrosine. Breeding pairs were established on their respective ad libitum diets so that all offspring mice were conceived 4 0 0 and maintained on these until they were sacrificed at ~7 weeks of age. Once the 2% L-tyrosine supplemented feed was established as the highest safe concentration regime commenced when mice were 5 to 6 months of age and continued for 4 weeks upon which weighing feed each week for 3 or more weeks prior to addition of the L-tyrosine supplemented feed Blood was collected from L-tyrosine treated and non-treated mice via cardiac puncture at 4 1 5 euthanasia at ~7 weeks of age. Immediately afterwards, quadriceps femoris muscles were excised, applied to an Agilent 1290 UPLC coupled to a 6560 QQQ for the measurement of free L-tyrosine. acquisition method of the kit was followed. The internal standard was homophenylalanine. Data Male 6-week old TgACTA1 D286G mice treated from pre-conception were weighed prior to 4 2 9 individually being housed with access to Low-Profile Wireless Running Wheels (Med Associates 4 3 0 Inc, USA) for 6 consecutive days. For both TgACTA1 D286G and KIActa1 H40Y mice, body weight was 4 3 1 measured after 1 month of exposure to the 2% L-tyrosine supplemented diet or to the normal diet. The daily distance travelled, daily time spent running (summary of 1 min intervals in which at least 4 3 5 one wheel revolution was recorded), average speed and maximum speed values were calculated. The mean values for all wheel activity traits on days 4, 5 and 6 were used for data analyses. At ~6 4 3 7 weeks of age, male TgACTA1 D286G mice treated from pre-conception were acclimatised to the rpm for 2 minutes. The following day, mice were tested with placement on the rotarod at 4 rpm, times on the same day, being allowed at least 10 minutes to rest in between assessments. The for each test. Data were expressed as the averaged value across the three tests. Investigations of similarly treated 6 -7 months of age KIActa1 H40Y female mice were performed at Bruker, Ettlingen, Germany) was used for image acquisition. Metabolic changes were investigated using 31 P-MRS at rest and during the fatiguing protocol.

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Spectra from the gastrocnemius region were continuously acquired at rest and throughout the System, Inc., Boulder, CO, USA). The first 180 FID were acquired at rest and summed together. The next 317 FID were acquired during the stimulation period and summed together. Relative          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.