Biochemical and physiological characterisation of a mouse model knocked-in for the RyR1 p.F4976L mutation identified in a severely affected child

The muscular system is one of the most important systems of our body. Muscles are not only essential for movements, but also for many other purposes, such as maintenance of posture, generation of heat and more. Moreover, the muscular system is in continuous communication with other systems, playing an important role for essential mechanisms such as breathing, blood circulation, digestion, immune response, etc. In general, we can distinguish different type of muscles: skeletal muscles, cardiac muscles or smooth muscles. While cardiac muscles and smooth muscles are under involuntary control, skeletal muscles carry out mainly voluntary movements (dynamic work), keep posture (isokinetic work) and are also involved in other processes, such as shivering for heat production, glucose storage, and metabolism.
On a molecular level, skeletal muscle contraction sees the Ryanodine receptor 1 (RyR1) as the main molecular player, responsible for the release of calcium from the sarcoplasmic reticulum into the cytosol and for enabling the contraction process. The process that converts an electrical stimulation to the generation of the contraction, is named Excitation-Contraction Coupling (ECC). The RyR1 plays a pivotal role in the ECC process, being the physical calcium release effector, starter and modulator of the contraction. Mutations in the RYR1 gene lead often to deleterious phenotypes, and are the most frequent cause of Congenital Myopathies (CMs). CMs are a group of early onset, neuromuscular disorders of variable severity, normally present at birth, and characterized by a stable or pejorative progressive phenotype with typical muscle biopsy findings. Patients affected by CMs present typical symptoms, including weakness of axial and proximal muscles, and in some cases cardiorespiratory and extraocular muscles (EOMs) involvement. Clinicians have re-classified CMs due to mutations in the RyR1, as “Ryanodine receptor type 1-related myopathies” (RYR1-RMs).
RYR1-RMs can be divided in four classes, depending on: i) histological features, ii) position of the mutation inside the protein sequence, iii) inheritance pattern of the mutation, which can be either dominant or recessive. Depending on the combinations of these characteristics, mutations can affect the RyR1 differently, changing its biophysical behaviour, stability and expression levels. Consequently, RYR1-RMs can be divided in three main classes: 1) Gain of function mutations, which are present as dominant mutations and are mostly related to Malignant hyperthermia susceptibility (MH – MIM #145600); 2) Loss of function CMs, such as Central core disease (CCD – MIM #117000) which is also caused by dominant mutations; 3) Centronuclear myopathy (CNM) and Multiminicore disease (MmD – MIM #255320), which are mainly due to recessive mutations and characterized by a pathogenic decrease of the RyR1 protein amount with consequent muscle weakness. While MH and CCD causative mutations are pathogenic at the heterozygous level, CNM or MmD patients comes either as homozygous or compound heterozygous. RYR1 dominant mutations are usually present within specific hotspots, while recessive mutations are evenly distributed throughout the whole protein sequence, with no specific pathogenic hotspots. While RYR1-RMs due to dominant mutations have been extensively studied and their pathological molecular implications are better defined, the way of action of recessive RYR1-RMs remains under investigation and more elusive. A specific isogenic mouse model is an important tool to elucidate the pathomechanisms and clinical relevance of a mutation.
Many mouse models have been created to better understand the molecular and pathological modifications brought by RYR1-RM mutations. Our lab extensively concentrates on the study of recessive RYR1 mutations, and in order to increase the knowledge around them, we created several mouse models isogenic to mutations found in severely affected patients.
In this study we characterized a novel transgenic mouse model, homozygous for the Ryr1p.F4976L (referable throughout the work as Ho). This mouse is knocked-in for an isogenic mutation found in a severely affected patient. In fact, the Ho mouse was created to understand if the molecular modifications brought by this homozygous RyR1 mutation could impact the correct muscle performance of the mouse, and ultimately to draw a correlation between the animal model and the clinical cases.
From a clinical point of view, the male proband, first son of Caucasian nonconsanguineous parents, was born preterm at around 29 weeks and was exhibiting sever hypotonia, together with respiratory distress. Moreover, the child was presenting a “floppy baby” phenotype, with general weakness. Initially he was proposed to present a myotonic dystrophy, but then he was found to carry the RYR1 gene variant c.14928C>G (p.Phe4976Leu) in exon 104 (via Next- Generation and Sanger sequencing). Parents were found to be heterozygous for the same mutation, but with a normal and healthy phenotype. At the moment, while still presenting a myopathic phenotype, the patient’s conditions have improved, being able to maintain a sitting position and walk with assistance.
Here, we describe the characterization of the Ho Ryr1p.F4976L mouse model and show that the mice have an impaired in vivo and ex vivo muscle performance, more prominently affecting fast twitch muscles, and impairing the calcium release after electrical stimulation. Interestingly, muscle fibres from Ho mice show higher levels of resting calcium, which were decreased to basal WT levels after administration of the SOCE inhibitor BTP2, leading to the hypothesis of a RyR1 leakage and SOCE involvement. Ultrastructural changes, such as decrease of calcium release units (CRU), increased number of dyads or presence of myofibrillar degeneration, portray a picture of general modifications that are probably not causative but consecutive to the presence of the mutation. Interestingly, with this model we can once more see the involvement of extraocular muscles (EOMs) in recessive RYR1-RMs. Significant changes in the biochemical composition of these type of muscles were majorly exemplified by the reduction of the specific EOM Myosin heavy chain (MyHC): MyHC13/EO. We already reported a similar situation in our double knock-in mouse model (RyR1p.Q1970fsX16+A4329D), in which almost the complete absence of this myosin isoform was shown. This is particularly important, since in the case of the Ho mouse the reduction is only of around 50%. Comparing the two animal models, since the RyR1Q1970fsX16+A4329D exhibited a more severe muscle impairment, we can hypothesize a MYHC13 dosage effect in the clinical outcome.
In general, we can conclude that the new p.F4976L mouse model recapitulates in part but on a milder level the phenotype of the affected child.
Despite many steps forward in a better understanding of RYR1 recessive diseases, studies like this must still be performed to expand our knowledge on recessive RYR1-RMs, which will help in the development of therapeutic approaches aimed to treat congenital myopathies linked to recessive RYR1 mutations.