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.)
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