Creatine transporter (SLC6A8) knockout mice exhibit reduced muscle performance, disrupted mitochondrial Ca2+ homeostasis, and severe muscle atrophy

Creatine (Cr) is essential for cellular energy homeostasis, particularly in muscle and brain tissues. Creatine Transporter Deficiency (CTD), an X-linked disorder caused by mutations in the

SLC6A8 gene, disrupts Cr transport, leading to intellectual disability, speech delay, autism, epilepsy, and various non-neurological symptoms. In addition to neurological alterations,

Creatine Transporter knockout (CrT−/y) mice exhibit severe muscle atrophy and functional impairments. This study provides the first characterization of the skeletal muscle phenotype in

CrT−/y mice, revealing profound ultrastructural abnormalities accompanied by reduced fiber cross-sectional area and muscle performance. Notably, mitochondria are involved, as evidenced by

disrupted cristae, increased mitochondrial size, impaired Ca2+ uptake, reduced membrane potential and ATP production. Mechanistically, the expression of atrophy-specific E3 ubiquitin ligases

and suppression of the IGF1-Akt/PKB pathway, regulated by mitochondrial Ca2+ levels, further support the atrophic phenotype. These findings highlight the profound impact of Cr deficiency on

skeletal muscle, emphasizing the need for targeted therapeutic strategies to address both the neurological and peripheral manifestations of CTD. Understanding the underlying mechanisms,

particularly mitochondrial dysfunction, could lead to novel interventions for this disorder.

Creatine (Cr) plays a crucial role in regulating cellular energy homeostasis, particularly in high-demanding tissues such as the muscle and the brain [1]. Creatine Kinase (CK) converts Cr

into phospho-creatine (PCr), a metabolically inert phosphagen used for temporal and spatial buffering of ATP levels. The PCr/CK system is vital for high-intensity physical exercise in

striated muscle and heart contraction when ATP hydrolysis exceeds production by other metabolic pathways [2, 3].

Approximately one-half of the daily Cr needs in humans comes from the diet, with the remaining one-half synthesized endogenously through a two-step enzymatic pathway involving

L-arginine:glycine amidinotransferase (AGAT) and S-adenosyl-L-methionine:N-guanidinoacetate methyltransferase (GAMT) mostly in the liver, pancreas, and kidneys [3, 4]. After synthesis or

nutritional supply, Cr is released into the bloodstream to nourish all body cells. However, Cr cannot cross the lipid membrane and requires a specific Na+/Cl−-dependent transporter (Cr

transporter, CrT). In humans, CrT is predominantly expressed in the muscle, kidney, heart, and other tissues, including the brain [5,6,7].

Creatine Transporter Deficiency (CTD, OMIM #300352) is a monogenic X-linked disorder [8], associated with multiple mutations in the solute carrier family 6-member 8 (Slc6a8) gene that

encodes the protein responsible for cellular Cr transport [8, 9]. Recent studies suggest that the prevalence of this disorder might range between 1% and 3% in males with intellectual

disability [8, 10]. However, the lack of prenatal and perinatal screening makes CTD a still underdiagnosed disorder. Clinical hallmarks include intellectual disability, severe speech delay,

autistic features, epilepsy, and movement disorders [9]. Patients also experience non-neurological symptoms, including gastrointestinal dysfunction, bladder dysfunction, cardiomyopathy, and

ophthalmologic abnormalities [11, 12]. Skeletal muscle mass reduction and mild muscle weakness have been described [12, 13], primarily as late onset deficits [14,15,16]. While Cr

concentration in skeletal muscle appears to be within the normal range, this has only been analyzed for a few specific mutations [17, 18].

The absence of a functional CrT renders Cr supplementation ineffective for CTD [8]. Consequently, effective treatments for CTD remain elusive, posing ongoing challenges for patients and

their families.

To understand the causative mechanisms and the main molecular pathways altered in CTD, animal models lacking the Slc6a8 gene or engineered to express an allele with the point mutation found

in patients have been generated [19,20,21,22]. These rodent lines have significantly contributed to dissecting the pathological determinants of this disorder [8]. They revealed that the

loss-of-function of Slc6a8 does not result in overt alterations in brain structure and neuronal density but rather in a subtle reorganization of cerebral circuits and cellular metabolic

processes [19, 22,23,24]. However, the cellular and molecular players involved in the development and progression of the CTD phenotype, not only in the brain but also in skeletal muscle,

remain largely unexplored [8].

Interestingly, unlike CTD patients, skeletal muscles of CrT knockout (CrT−/y) animals exhibit low Cr levels and a severely compromised phenotype, characterized by impaired motor function,

muscle atrophy, increased glucose metabolism due to AMPK activation [21, 25], and susceptibility to fatigue [26]. Motor alterations are absent in mice harboring a brain-specific Slc6a8

deletion, suggesting a peripheral contribution to the onset of CTD pathology [27, 28]. Nevertheless, a complete description of the skeletal muscle phenotype and the underlying mechanisms

remain elusive. The study of this aspect is particularly important given that the PCr-CK system requires high levels of total Cr (20-40 mM) in skeletal muscle [29] that can be achieved only

with functional cellular Cr uptake [29].

Interestingly, CTD clinical phenotype closely resembles that observed in a wide range of mitochondrial disorders (e.g., developmental, or cognitive disabilities, seizures, fatigue and

cardiomyopathy). CTD patients initially suspected of having a mitochondrial disorder were later found to have a creatine transporter defect. Notably, there is a strong link between Cr and

mitochondrial function, and changes in PCr/Cr ratio finely regulate mitochondrial respiration [1, 30,31,32]. It is thus not surprising that the metabolic phenotype of Cr deficiency results

in disturbed metabolic activity, as well as structural abnormalities of mitochondrial organelles [24, 28, 33, 34]. Nevertheless, mitochondrial abnormalities and their causative role in the

pathogenesis of this disease have scarcely been assessed at functional and molecular levels. This is a crucial aspect, as preclinical evidence suggests that mitochondria might be promising

targets for therapeutic intervention in various brain and motor dysfunctions.

In this study, we describe the profound impact of Cr deficiency on the skeletal muscle of CrT−/y animals. Specifically, we found that muscles exhibit an atrophic phenotype accompanied by

fibers ultrastructural alterations, diminished performance, and increased expression of key E3 ubiquitin ligases associated with the progression of atrophy. Additionally, mitochondria

display significant morphological abnormalities, reduced membrane potential, and impaired mitochondrial Ca2+ uptake, accompanied by changes in the expression of proteins involved in

mitochondrial Ca2+ homeostasis.

The mouse model carrying the ubiquitous deletion of 5–7 exons in the Slc6a8 gene (CrT knockout, CrT−/y) [20] exhibits low Cr levels in the brain, skeletal and cardiac muscles, and kidneys

[20, 23]. Given that no comprehensive characterization of skeletal muscle structure has been performed in this model thus far and considering the crucial role of Cr in muscle metabolism, we

began by assessing the presence of qualitative and quantitative ultrastructure alterations. Using Transmission Electron Microscopy (TEM), we analyzed the Extensor Digitorum Longus (EDL)

muscle in WT and CrT−/y mice at Post Natal Day (PND) 40. In WT mice, the average cross-sectional area (CSA) of the fibers is 970 ± 16 μm2 (mean ± SEM) (Fig. 1A, gray columns and Fig. 1B),

while in CrT−/y mice the CSA of the fibers is significantly reduced to 280 ± 3 μm2 (Fig. 1Ablue columns and Fig. 1B). The number of fibers per 1000 μm2 is 0.9 ± 0.1 in WT mice, resulting in

a fractional area of the muscle occupied by the fibers of [(970 μm2/fiber)/(1000 μm2/0.9 fibers)]= 0.87 ± 0.10 (Fig. 1B). In CrT−/y mice, with 2.2 ± 0.5 fibers per 1000 μm2 (Fig. 1B), the

fractional area is the muscle occupied by the fibers is 0.62 ± 0.16 (Fig. 1B), approximately 30% lower than in WT mice. CrT−/y muscles exhibit an increased fraction of intermyofibrillar

space relative to fiber volume (0.292 ± 0.009) compared to WT muscles (0.155 ± 0.007), resulting in a decreased fractional fiber volume (0.708 ± 0.016 in CrT−/y mice and 0.845 ± 0.014 in WT

mice, Fig. 1C, D). The CSA occupied by the myofibrils within the muscle can be determined multiplying the CSA of the EDL muscle, estimated from the wet weight of the muscle (see Methods), by

a factor α = 0.87 × 0.845 = 0.74 ± 0.08 (or 74%) for the WT mice and 0.62 × 0.708 = 0.44 ± 0.10 (or 44%) for the CrT−/y mice.

A Distribution of the cross-sectional area of the fibers in the EDL muscle of WT and CrT−/y mice. Samples size: 3 EDLs for each group. B Cross-sectional area occupied by the fibers within

the muscle in WT and CrT−/y mice. C Representative electron micrographs of cross sections of EDL fibers from WT (left panel) and CrT−/y (right panel) muscles. The intermyofibrillar space and

the myofibrillar volume over the fiber volume are reported (D). Scale bar in (C): 1 μm. In (B) and (D), data are presented as mean ± SEM. n = 15. For data analysis, parametric Student

t-test (two tailed, unpaired) was used. *p