BackgroundThe human genetic disorder Microvillus Inclusion Disease (MVID) results from inactivating mutations in Myosin Vb (MYO5B). MVID is characterized by severe, unremitting diarrhea that typically presents shortly after birth. The necessity of implementing total parenteral nutrition (TPN) and the long‐term administration of TPN in neonates with MVID presents challenges to elucidating the complex pathogenesis of MVID in humans. Thus, a large animal model of MVID that closely recapitulates human physiology is needed. To better understand the pathophysiology of MVID we created a large animal model that closely mimics human MVID.MethodsPigs were cloned using somatic cell nuclear transfer of primary pig fibroblasts edited with TALEN to express a P663L mutation of the MYO5B gene. This swine P663L mutation, referred to as MYO5B(P663L), corresponds to the Navajo P660L mutation seen in MVID patients. Neonatal pigs were euthanized within 1 day of birth and tissue was collected for analysis and generation of intestinal enteroids.ResultsThe MYO5B(P663L) pigs developed diarrhea shortly after birth, similar to patients with MVID. Immunostaining for brush border components F‐actin, ezrin, CD10 and DPPIV showed the presence of subapical intracellular inclusions in enterocytes of MYO5B P663L pigs. Transmission electron microscopy confirmed the presence of inclusions lined with microvilli in the MYO5B(P663L) pigs. Immunostaining for NHE3 and SGLT1, drivers of intestinal sodium and water absorption, showed decreased apical expression and diffuse sub‐apical expression in the MYO5B(P663L) pigs compared to control pigs. In contrast to decreased expression of sodium transporters, CFTR was maintained on the apical membrane of MYO5B(P663L) pigs. Enteroids generated from the proximal small intestine of neonatal control and MYO5B(P663L) pigs grew at similar rates, but showed differences in the brush border. Enteroids derived from MYO5B(P663L) pigs had sub‐apical expression of intestinal alkaline phosphatase and DPPIV and decreased apical expression of NHE3 compared to control enteroids. Administration of forskolin resulted in increased swelling in MYO5B(P663L) enteroids (170.8% ± 15.9) compared to control enteroids (90.4% ± 4.6), indicative of functional Cl− secretion.ConclusionsCollectively, these data suggest that decreased apical expression of NHE3 and SGLT1, which are required for water absorption, are likely responsible for the MVID diarrhea disorder. Moreover, maintenance of functional CFTR on the apical membrane of enterocytes in the MYO5B(P663L) pig may further exacerbate the development of diarrhea by actively secreting Cl−, thus driving further water loss by facilitating fluid secretion. This novel large animal model of MVID could provide an important vehicle for testing novel therapeutic approaches for patients with MVID.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Background:
Changes in Ca2+ regulation have been implicated in various pathologies such as coronary artery disease and metabolic syndrome (MetS), thereby potentiating these diseases. Our lab has shown that MetS decreases voltage-gated Ca2+ channel (VGCC) activity and sarcoplasmic reticulum (SR) Ca2+ release in coronary smooth muscle cells and increases coronary artery disease in Ossabaw miniature swine. Furthermore, decreased SIRT1 enzyme function can impair Ca2+ signaling and increase coronary disease and MetS. We hypothesized that impaired SIRT1 and MetS would decrease VGCC function and SR calcium store.
Methods:
CRISPR/Cas9 methods delivered a leucine to proline point mutation in SIRT1 (SIRT1L100P) into the Ossabaw swine genome to compare to wild type (WT), mimicking the naturally occurring mutation in humans which decreases SIRT1 activity. Four treatment groups of juvenile swine were based on genotype and diet: WT Lean, SIRT1 Lean, WT MetS, and SIRT1 MetS. Lean swine were fed normal chow and MetS were fed a hypercaloric, atherogenic diet for 7 months. The left anterior descending coronary artery was harvested and enzymatically digested to obtain cells. Fluorescence microscopy measured the Ca2+ indicator fura-2 in single cells. Depolarization of cells with perfusion of 80 mM K+ was used to elicit Ca2+ influx through VGCC. Caffeine (5 mM) exposure activated the Ca2+ release channel (ryanodine receptor) on the SR.
Results:
MetS was confirmed by increased body weight, impaired glucose tolerance, hyperinsulinemia, and hypercholesterolemia. Coronary atherosclerosis was shown by angiography, intravascular ultrasound, and gross imaging. A two-way analysis of variance revealed statistically significant overall effects of genotype (p=0.02), diet (p<0.0001), and an interaction (p<0.0001) between these variables to decrease VGCC function. In contrast, no effect was observed on SR Ca2+ release.
Conclusion and Potential Impact:
SIRT1 inhibition and MetS decreased VGCC function independently, but not additively or synergistically. (Support: NIH T35HL110854, DK120240, DK09751.)
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