MPS001: Histological and ultrastructural findings in RYR1 -mutant fetal skeletal muscle. (A,B) Hematoxilin & Eosin stain shows increased fiber size variability in the RYR1-mutant muscle (A, foetus F1) compared to an age-matched control (B). Some RYR1-mutant fibres have intensely eosinophilic cytoplasm (A, arrows). The nuclei are localized centrally in all fibres, compatible with the gestational age. The perinuclear clear halo present in many fibres is an artefact due to formalin fixation. (C,D) Labelling against desmin reveals a similar pattern of immunoreactivity and no evident core-like structures in both RYR1-mutant (C, foetus F1) and control tissue (D). (E-H) Labelling against the myosin heavy chain fast (E,F) and slow (G,H) shows that the numbers of myosin fast-positive type II fibres is comparable between the patient (E, foetus F2) and the control (F), whereas myosin slow-positive type I fibres are markedly reduced in RYR1-mutant (G, foetus F2) compared to control muscle (H). (I) Labelling for active caspase 3 is negative, excluding apoptosis, also in atrophic RYR1-mutant muscle fibres (foetus F2). (J) Alizarin red S staining shows no detectable accumulation of calcium inside the RYR1-mutant muscle fibres (foetus F1). (K,L) Ultrastructural analysis reveals profound myofibrillar disarray with disappearance of the Z-bands in the RYR1-mutant muscle fibres (K, foetus F2). By contrast, Z-bands are easily detected in control tissue (L). Magnifications: (A-J) 400x; K,L 30000x. (lower panels): histological findings in RYR1-mutant (Family MPS003) foetal skeletal muscle, GA 23 weeks. (M,N) Hematoxilin & Eosin stain of formalin fixed and paraffin embedded psoas muscle shows loss of fibres with increased fibre size variability and mild fibrosis in the RyR1-mutant muscle (M,N) compared to an age-matched control (O).