scholarly journals SIRT1 Inhibition Impairs Ca2+ Buffering in Coronary Smooth Muscle Cells of Ossabaw Miniature Swine

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
John Reed ◽  
Aish Thamba ◽  
John Strobel ◽  
James Byrd ◽  
Mouhamad Alloosh ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Coronary Artery ◽  
Smooth Muscle Cells ◽  
Single Cells ◽  
Muscle Cells ◽  
Atherogenic Diet ◽  
Miniature Swine ◽  
Rapid Phase ◽  
Coronary Smooth Muscle ◽  
Normal Chow

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.) 

scholarly journals Metabolic Syndrome and SIRT1 Mutation Impair Ca2+ Channels in Coronary Smooth Muscle Cells of Ossabaw Miniature Swine

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Aish Thamba ◽  
John Reed ◽  
John S. Strobel ◽  
James Byrd ◽  
Mouhamad Alloosh ◽  
...  
Keyword(s):  
Coronary Artery Disease ◽  
Metabolic Syndrome ◽  
Smooth Muscle ◽  
Coronary Artery ◽  
Smooth Muscle Cells ◽  
Single Cells ◽  
Muscle Cells ◽  
Miniature Swine ◽  
Coronary Smooth Muscle ◽  
Artery Disease

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.)

Endothelin mediates Ca influx and release in porcine coronary smooth muscle cells

1991 ◽  
Vol 260 (4) ◽  
pp. C771-C777 ◽  
Author(s):  
C. Wagner-Mann ◽  
M. Sturek
Keyword(s):  
Smooth Muscle ◽  
Coronary Artery ◽  
Smooth Muscle Cells ◽  
External Solution ◽  
Muscle Cells ◽  
Free Solution ◽  
Coronary Smooth Muscle ◽  
Rapid Release ◽  
Baseline Values ◽  
Induced Changes

Endothelin (ET)-induced changes in intracellular free Ca (Cai) in freshly dispersed coronary artery smooth muscle cells were determined using fura-2 microfluorometry to differentiate the action of ET on Ca influx vs. release from internal stores. Comparison was made with caffeine (CAF)-induced Ca release from the sarcoplasmic reticulum (SR) to determine whether ET acts on the same Ca store. In physiological external solution, ET (5 x 10(-8) M) induced a rapid (within 90 s), transient (less than 2.5-min duration) 70% increase in Cai above baseline (n = 20). Pretreatment with diltiazem (10(-4) M; n = 10) did not change the amplitude or shape of the ET-induced Cai transient. In Ca-free solution, ET elicited a Cai response similar in duration but smaller (P less than 0.05) in peak magnitude (31% increase; n = 7). CAF (5 x 10(-3) M) also elicited a rapid (less than 60 s), transient 82% increase in Cai (n = 7). In the continued presence of CAF, ET caused no change in Cai. In contrast, ET elicited a transient 69% increase in Cai (n = 8), and in the continued presence of ET, CAF caused a 24% increase in Cai. Ryanodine (5 x 10(-5) M) suppressed the subsequent ET-induced Cai transient. These data on porcine cells suggest ET induces a rapid release of Ca from a CAF- and ryanodine-sensitive store and causes rapid influx of Ca, which is different from bovine smooth muscle cells. The return of Cai to baseline values in the continued presence of ET suggests the ET-sensitive store is depleted and increased Ca efflux matches Ca influx.

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