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SIRT1 Mutation Impairs Ca 2+ Buffering in Coronary Smooth Muscle Cells of Ossabaw Miniature Swine
Author(s) -
Reed John,
Thamba Aish,
Strobel John,
Byrd James,
Alloosh Mouhamad,
Coutts Alex,
Sturek Michael
Publication year - 2022
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2022.36.s1.r6317
Subject(s) - serca , endoplasmic reticulum , medicine , endocrinology , caffeine , chemistry , intracellular , atpase , homeostasis , microbiology and biotechnology , biology , biochemistry , enzyme
Background Metabolic syndrome (MetS) impairs sarco‐endoplasmic reticulum Ca 2+ 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 Ca 2+ signaling, metabolism, and cardiovascular disease. SIRT1 impairment exacerbates MetS and vascular disease, including calcification. After Ca 2+ is increased in CSM by Ca 2+ influx or release from the sarcoplasmic reticulum (SR), the Ca 2+ is then buffered by either Ca 2+ efflux from the cell by the plasmalemmal Ca 2+ ATPase or sequestration back into the SR via SERCA. SIRT1 inhibition hyper‐acetylates SERCA and decreases activity, thereby impairing the Ca 2+ buffering capacity of the cell. Hypothesis MetS and SIRT1 mutation will impair Ca 2+ buffering in CSM. Methods Using CRISPR/Cas9 methodology a point mutation (SIRT1 L100P ) was made in Ossabaw miniature swine to mimic the naturally occurring mutation in humans and decrease SIRT1 activity, thereby resulting in hyperacetylation of Ca 2+ 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 Ca 2+ was measured with fura‐2. The main Ca 2+ regulation protocol was the maximal release of Ca 2+ from the SR by 5 mM caffeine and then assessment of Ca 2+ efflux and sequestration. Time to half recovery of Ca 2+ to baseline during caffeine exposure was measured to assess Ca 2+ efflux (rapid phase Ca 2+ buffering) and deviation from baseline was measured to assess SERCA function (slow phase Ca 2+ buffering). Results There was no effect of MetS or SIRT1 mutation on Ca 2+ efflux. Two‐way ANOVA showed SIRT1 mutation alone inhibits SERCA buffering of Ca 2+ 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 SIRT1 L100P mutation is likely to contribute to coronary atherosclerosis and SIRT1 activators may be effective therapies.