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 (plt;0.0001) significantly decreased VGCC function independently, but not additively or synergistically. Interaction of SIRT1 genotype and MetS diet was significant (plt;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.
