Minimal expression of dysferlin prevents development of dysferlinopathy in dysferlin exon 40a knockout mice.

Dysferlin is a Ca2+-activated lipid binding protein implicated in muscle membrane repair. Recessive variants in DYSF result in dysferlinopathy, a progressive muscular dystrophy. We showed previously that calpain cleavage within a motif encoded by alternatively spliced exon 40a releases a 72 kDa C-terminal minidysferlin recruited to injured sarcolemma. Herein we use CRISPR/Cas9 gene editing to knock out murine Dysf exon 40a, to specifically assess its role in membrane repair and development of dysferlinopathy. We created three Dysf exon 40a knockout (40aKO) mouse lines that each express different levels of dysferlin protein ranging from ~ 90%, ~ 50% and ~ 10-20% levels of wild-type. Histopathological analysis of skeletal muscles from all 12-month-old 40aKO lines showed virtual absence of dystrophic features and normal membrane repair capacity for all three 40aKO lines, as compared with dysferlin-null BLAJ mice. Further, lipidomic and proteomic analyses on 18wk old quadriceps show all three 40aKO lines are spared the profound lipidomic/proteomic imbalance that characterises dysferlin-deficient BLAJ muscles. Collective results indicate that membrane repair does not depend upon calpain cleavage within exon 40a and that ~ 10-20% of WT dysferlin protein expression is sufficient to maintain the muscle lipidome, proteome and membrane repair capacity to crucially prevent development of dysferlinopathy.

A genetic basis is identified in 74% cases of paediatric hyperCKaemia without weakness presenting to a tertiary paediatric neuromuscular centre.

Paediatric hyperCKaemia without weakness presents a clinical conundrum. Invasive investigations with low diagnostic yields, including muscle biopsy, may be considered unjustifiable. Improved access to genome-wide genetic testing has shifted first-line investigations towards genetic studies in neuromuscular disease. This research aims to provide an evidence-based diagnostic approach to paediatric hyperCKaemia without weakness, a current gap in the literature. We identified 47 individuals (10-months to 16-years-old; 34 males, 13 females) from 43 families presenting with hyperCKaemia on two or more occasions, without weakness, from The Children's Hospital at Westmead Neuromuscular Clinic Database. Clinical features, investigations and outcomes were analysed via retrospective chart review. Genetic testing has been performed in 34/43. Genetic variants explaining hyperCKaemia were identified in 25/34 (74%) using multiplex ligation-dependent probe amplification, massive parallel sequencing, single gene testing and exome sequencing. Pathogenic/likely pathogenic variants were identified in 19 neuromuscular disease genes and six metabolic myopathy genes. Individuals with metabolic diagnoses had higher peak creatine kinase levels that sometimes normalized. Conversely, creatine kinase levels remained persistently elevated those with neuromuscular diagnoses. In summary, a genetic cause is found in most paediatric patients with hyperCKaemia without weakness informing clinical management and counselling. Thus, we propose a diagnostic algorithm for this cohort.

Regeneration and repair in the healing lung.

The lung achieves an efficient gas exchange between a complex non-sterile atmosphere and the body via a delicate and extensive epithelial surface, with high efficiency because of elastic deformation allowing for an increase and decrease in volume during the process of breathing and because of an extensive vasculature which aids rapid gas diffusion. The lungs' large surface area exposes the organ to a continual risk of damage from pathogens, toxins or irritants; however, lung damage can be rapidly healed via regenerative processes that restore its structure and function. In response to sustained and extensive damage, the lung is healed via a non-regenerative process resulting in scar tissue which locally stiffens its structure, which over time leads to a serious loss of lung function and to increasing morbidities. This review discusses what is known about the factors which influence whether a lung is healed by regeneration or repair and what potential new therapeutic approaches may positively influence lung healing.

Loss of calpains-1 and -2 prevents repair of plasma membrane scrape injuries, but not small pores, and induces a severe muscular dystrophy.

The ubiquitous calpains, calpain-1 and -2, play important roles in Ca2+-dependent membrane repair. Mechanically active tissues like skeletal muscle are particularly reliant on mechanisms to repair and remodel membrane injury, such as those caused by eccentric damage. We demonstrate that calpain-1 and -2 are master effectors of Ca2+-dependent repair of mechanical plasma membrane scrape injuries, although they are dispensable for repair/removal of small wounds caused by pore-forming agents. Using CRISPR gene-edited human embryonic kidney 293 (HEK293) cell lines, we established that loss of both calpains-1 and -2 (CAPNS1-/-) virtually ablates Ca2+-dependent repair of mechanical scrape injuries but does not affect injury or recovery from perforation by streptolysin-O or saponin. In contrast, cells with targeted knockout of either calpain-1 (CAPN1-/-) or -2 (CAPN2-/-) show near-normal repair of mechanical injuries, inferring that both calpain-1 and calpain-2 are equally capable of conducting the cascade of proteolytic cleavage events to reseal a membrane injury, including that of the known membrane repair agent dysferlin. A severe muscular dystrophy in a murine model with skeletal muscle knockout of Capns1 highlights vital roles for calpain-1 and/or -2 for health and viability of skeletal muscles not compensated for by calpain-3 (CAPN3). We propose that the dystrophic phenotype relates to loss of maintenance of plasma membrane/cytoskeletal networks by calpains-1 and -2 in response to directed and dysfunctional Ca2+-signaling, pathways hyperstimulated in the context of membrane injury. With CAPN1 variants associated with spastic paraplegia, a severe dystrophy observed with muscle-specific loss of calpain-1 and -2 activity identifies CAPN2 and CAPNS1 as plausible candidate neuromuscular disease genes.