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.)
Background: SIRT1 is a deacetylase that has diverse roles in intracellular Ca2+ signaling, metabolism, and cardiovascular disease. SIRT1 increases sarco-endoplasmic reticulum Ca2+ ATPase (SERCA) activity that is essential to buffer the increase in Ca2+ induced by release from the sarcoplasmic reticulum (SR). Our lab has shown that metabolic syndrome (MetS) impairs SERCA activity in coronary smooth muscle cells and causes coronary artery disease in Ossabaw miniature swine. We hypothesized that SIRT1 inhibition and MetS would impair Ca2+ buffering. 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) and mimic the naturally occurring mutation in humans and decrease 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. The cells were exposed to 5 mM caffeine to maximally release stores of Ca2+ from the SR. Ca2+ buffering capacity of each cell was analyzed after the caffeine-induced peak increase to assess Ca2+ efflux and SERCA activity. 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. The rapid phase of Ca2+ buffering due to Ca2+ efflux was not affected by SIRT1 mutation or MetS. The slower phase of Ca2+ buffering due to SERCA activity was impaired only by SIRT1 mutation (p<0.0005), not by MetS. Conclusion: SIRT1 mutation alone inhibited SERCA buffering of Ca2+ in coronary smooth muscle. (Support: NIH T35HL110854, DK120240, DK09751.)
Background Metabolic syndrome (MetS) impairs sarco‐endoplasmic reticulum Ca2+ ATPase (SERCA) activity that may decrease the ER Ca2+ store and increase store‐operated Ca2+ entry (SOCE) in a variety of cell types. Similar Ca2+ signaling via SOCE causes fibrosis, vascular disease, and may trigger immunogenic (type 1) diabetes by action on peripheral blood mononuclear cells (PBMCs). Sirtuin 1 (SIRT1) is a deacetylase that maintains healthy metabolism and generally attenuates vascular disease. SIRT1 impairment exacerbates MetS and vascular disease by hyper‐acetylation of SERCA, decreasing its activity and thereby potentially increasing SOCE. Hypothesis The SIRT1 mutation made in Ossabaw miniature swine would mimic the mutation found in humans and increase PBMC SOCE. Methods Using CRISPR/Cas9 methodology a point mutation (SIRT1L100P) was made in Ossabaw miniature swine to mimic the naturally occurring mutation in humans and decrease SIRT1 activity, thereby resulting in hyperacetylation of Ca2+transporters and impaired function. Four groups of pigs were used to analyze genotype and diet interactions: wild type lean, SIRT1 lean, wild type MetS, and SIRT1 MetS. Pigs were age 4 months at the start and fed normal chow (lean) or atherogenic diet (MetS) for 7 months. PBMCs were isolated and Ca2+ was measured with fura‐2. The ER Ca2+store was depleted by 10‐5M cyclopiazonic acid to inhibit SERCA in a Ca2+‐free extracellular solution to fully deplete the ER Ca2+store and then Ca2+ was returned to the solution to induce maximal SOCE. Cells were recovered in Ca2+‐free solution to partially re‐fill the ER Ca2+ store, then Ca2+ returned to the solution to observe submaximal SOCE. Results Both MetS diet groups (wild type MetS, SIRT1 MetS) showed impaired glucose tolerance. Fasting glucose in SIRT1 mutants was not different from wild type pigs, thus SIRT1 does not cause immunogenic diabetes at this early stage. Robust coronary atherosclerosis was shown by angiography, intravascular ultrasound, and gross imaging in MetS groups, but there was no additive or synergistic effect of SIRT1 mutation. There was no effect of MetS or SIRT1 mutation on maximal SOCE. Two‐way ANOVA showed that MetS diet increased submaximal SOCE (p<0.009), but there was no effect of SIRT1 genotype on submaximal SOCE (p=0.266). The percentage of PBMCs that responded to partial ER Ca2+ depletion was 45% in lean pigs compared to 81% in wild type and 63% in SIRT1 pigs fed MetS diet. Conclusion SIRT1L100P mutation is likely to require longer duration to manifest robust effects on PBMC SOCE that were found with MetS diet. PBMC activation and can be easily studied longitudinally in the SIRT1L100P mutant swine model due to the ease of blood sampling.
Background Normal metabolic health and sirtuin1 (SIRT1) by its deacetylase activity maintain voltage‐gated Ca2+ channel (VGCC) function that promotes gene expression to maintain contractile coronary smooth muscle (CSM) phenotype. MetS impairs VGCC function and sarcoplasmic reticulum (SR) Ca2+ release, contributing to dedifferentiation of CSM to proliferative and osteogenic phenotypes. Hypothesis MetS and impaired SIRT1 will impair VGCC and SR Ca2+ release channels in CSM. Methods Using CRISPR/Cas9 methodology a point mutation (SIRT1L100P) was made in Ossabaw miniature swine to mimic the naturally occurring mutation in humans and decrease SIRT1 activity, thereby resulting in hyperacetylation (Ac) of Ca2+transporters and impaired function. Four groups of pigs were used to analyze genotype and diet interactions: wild type lean, SIRT1 lean, wild type MetS, SIRT1 MetS. Pigs were age 4 months at the start and fed normal chow (lean) or atherogenic diet (MetS) for 7 months. CSM cells were enzymatically dispersed and Ca2+ measured with fura‐2. Depolarization with 80 mM K+ assessed Ca2+ influx through VGCC and the peak Ca2+ response to 5 mM caffeine to open SR Ca2+ release channels assessed the caffeine‐sensitive SR Ca2+ store. Results Two‐way ANOVA showed SIRT1 mutation (p=0.02) and MetS diet (p<0.0001) significantly decreased VGCC function independently, but not additively or synergistically. Interaction of SIRT1 genotype and MetS diet was significant (p<0.0001). No significant effect of genotype or diet was observed on SR calcium store release. Conclusion SIRT1L100P mutation is likely to contribute to coronary atherosclerosis and SIRT1 activators may be effective therapies.
Background Metabolic syndrome (MetS) impairs sarco‐endoplasmic reticulum Ca2+ ATPase (SERCA) activity in coronary smooth muscle (CSM) and is implicated in coronary atherosclerosis in Ossabaw miniature swine. Sirtuin 1 (SIRT1) is a deacetylase that has diverse roles in intracellular Ca2+ signaling, metabolism, and cardiovascular disease. SIRT1 impairment exacerbates MetS and vascular disease, including calcification. After Ca2+ is increased in CSM by Ca2+ influx or release from the sarcoplasmic reticulum (SR), the Ca2+ is then buffered by either Ca2+ efflux from the cell by the plasmalemmal Ca2+ ATPase or sequestration back into the SR via SERCA. SIRT1 inhibition hyper‐acetylates SERCA and decreases activity, thereby impairing the Ca2+ buffering capacity of the cell. Hypothesis MetS and SIRT1 mutation will impair Ca2+ buffering in CSM. Methods Using CRISPR/Cas9 methodology a point mutation (SIRT1L100P) was made in Ossabaw miniature swine to mimic the naturally occurring mutation in humans and decrease SIRT1 activity, thereby resulting in hyperacetylation of Ca2+transporters and impaired function. Four groups of pigs were used to analyze genotype and diet interactions: wild type lean, SIRT1 lean, wild type MetS, and SIRT1 MetS. Pigs were age 4 months at the start and fed normal chow (lean) or atherogenic diet (MetS) for 7 months. CSM cells were enzymatically dispersed and Ca2+ was measured with fura‐2. The main Ca2+ regulation protocol was the maximal release of Ca2+ from the SR by 5 mM caffeine and then assessment of Ca2+ efflux and sequestration. Time to half recovery of Ca2+ to baseline during caffeine exposure was measured to assess Ca2+ efflux (rapid phase Ca2+buffering) and deviation from baseline was measured to assess SERCA function (slow phase Ca2+buffering). Results There was no effect of MetS or SIRT1 mutation on Ca2+ efflux. Two‐way ANOVA showed SIRT1 mutation alone inhibits SERCA buffering of Ca2+ in CSM (p=0.0005). There was no effect of MetS diet (p=0.184) and no interactions of SIRT1 genotype and MetS diet (p=0.113) on SERCA activity. Conclusion SIRT1L100P mutation is likely to contribute to coronary atherosclerosis and SIRT1 activators may be effective therapies.
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.
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