Biology subjects

Dalton, N. D.

Publications and source records attributed to Dalton, N. D..

3 recordsLinked to original sources

bioRxiv2022

Combined loss of obscurin and obscurin-like 1 in murine hearts results in impaired diastolic dysfunction, altered metabolism and deregulated mitophagy.

Muscle proteins of the obscurin protein family play important roles in sarcomere organization, sarcoplasmic reticulum (SR) and T-tubule architecture and function. However, their precise molecular functions and redundancies between protein family members as well as their involvement in cardiac diseases remain to be fully understood. To investigate the functional roles of obscurin and its close homologue obscurin-like 1 (Obsl1) in the heart, we generated and analyzed knockout mice for obscurin, Obsl1, as well as obscurin/Obsl1 double-knockouts (dKO). We show that dKO mice are viable but show postnatal deficits in cardiac muscle SR and mitochondrial architecture and function at the microscopic, biochemical and cellular level. Altered SR structure resulted in perturbed calcium cycling, while mitochondrial ultrastructure deficits were linked to decreased levels of Chchd3, a Micos complex protein. Hearts of dKO mice also show increased expression of Atg4d, a novel Obsl1 interacting protein, resulting in abnormal mitophagy and increased unfolded protein response. At the physiological level, loss of obscurin and Obsl1 resulted in a profound delay of cardiac relaxation, associated with metabolic signs of heart failure. Taken together, our data suggest that obscurin and Obsl1 play crucial roles in cardiac SR structure, calcium cycling, mitochondrial function, turnover and metabolism.

cell biology↗

bioRxiv2022

CRISPR-Mediated Generation and Characterization of a Gaa Homozygous c.1935C>A (p.D645E) Pompe Disease Knock-in Mouse Model Recapitulates Human Infantile Onset-Pompe Disease

Pompe disease (PD) is an autosomal recessive disorder caused by deficient lysosomal acid -glucosidase (GAA), leading to reduced degradation and subsequent accumulation of intra-lysosomal glycogen in tissues, especially skeletal and oftentimes cardiac muscle. The c.1935C>A (p.Asp645Glu) variant is the most frequent GAA pathogenic mutation in people of Taiwanese and Southern Chinese ethnicity, causing infantile-onset PD (IOPD), which presents neonatally with severe hypertrophic cardiomyopathy, profound muscle hypotonia, and respiratory failure leading to premature death if untreated. To further investigate the pathogenic mechanism and facilitate development of therapies pertaining to this variant, we applied CRISPR-Cas9 homology-directed repair (HDR) using a novel dual sgRNA approach flanking the target site to generate a GaaEm1935C>A knock-in mouse model as well as a myoblast cell line carrying the Gaa c.1935C>A mutation. Herein we describe the molecular, biochemical, physiological, histological, and behavioral characterization of 3-month-old homozygous GaaEm1935C>A mice. Homozygous GaaEm1935C>A knock-in mice exhibited normal Gaa mRNA expression levels relative to wild-type mice, but GAA enzymatic activity was almost completely abolished, leading to a substantial increase in tissue glycogen storage, and significant concomitant impairment of autophagy. Echocardiography of 3-month-old knock-in mice revealed significant cardiac hypertrophy. The mice also demonstrated skeletal muscle weakness but, paradoxically, not early mortality. Longitudinal studies of this model, including assessment of its immune response to exogenously supplied GAA enzyme, are currently underway. In summary, the GaaEm1935C>A knock-in mouse model recapitulates the molecular, biochemical, histopathologic, and phenotypic aspects of human IOPD caused by the GAA c.1935C>A pathogenic variant. It is an ideal model to assess innovative therapies to treat IOPD, including personalized therapeutic strategies that correct pathogenic variants, restore GAA activity and produce functional phenotypes.

genetics↗

bioRxiv2019

CRISPR-Cas9 generated Pompe knock-in murine model exhibits early-onset cardiomyopathy and impaired skeletal muscle function

Infantile-onset Pompe Disease (IOPD), caused by mutations in lysosomal acid alpha-glucosidase (Gaa), manifests rapidly progressive fatal cardiac and skeletal myopathy incompletely attenuated by synthetic GAA intravenous infusions. The currently available murine model does not fully simulate human IOPD, displaying skeletal myopathy with late-onset hypertrophic cardiomyopathy. Bearing a Cre-LoxP induced exonic disruption of the murine Gaa gene, this model is also not amenable to genome-editing based therapeutic approaches. We report the early onset of severe hypertrophic cardiomyopathy in a novel murine IOPD model generated utilizing CRISPR-Cas9 homology-directed recombination to harbor the orthologous Gaa mutation c.1826dupA (p.Y609*), which causes human IOPD. We demonstrate the dual sgRNA approach with a single-stranded oligonucleotide donor is highly specific for the Gaac.1826 locus without genomic off-target effects or rearrangements. Cardiac and skeletal muscle were deficient in Gaa mRNA and enzymatic activity and accumulated high levels of glycogen. The mice demonstrated skeletal muscle weakness but did not experience early mortality. Altogether, these results demonstrate that the CRISPR-Cas9 generated Gaac.1826dupA murine model recapitulates hypertrophic cardiomyopathy and skeletal muscle weakness of human IOPD, indicating its utility for evaluation of novel therapeutics.

molecular biology↗