Genetic inhibition of serum glucocorticoid kinase 1 prevents obesity-related atrial fibrillation

Obesity is an important risk factor for atrial fibrillation (AF), but a better mechanistic understanding of obesity-related atrial fibrillation is required. Serum glucocorticoid kinase 1 (SGK1) is a kinase positioned within multiple obesity-related pathways, and prior work has shown a pathologic role of SGK1 signaling in ventricular arrhythmias. We validated a mouse model of obesity-related AF using wild-type mice fed a high-fat diet. RNA sequencing of atrial tissue demonstrated substantial differences in gene expression, with enrichment of multiple SGK1-related pathways, and we showed upregulated of SGK1 transcription, activation, and signaling in obese atria. Mice expressing a cardiac specific dominant-negative SGK1 were protected from obesity-related AF, through effects on atrial electrophysiology, action potential characteristics, structural remodeling, inflammation, and sodium current. Overall, this study demonstrates the promise of targeting SGK1 in a mouse model of obesity-related AF.


Intra-peritoneal Glucose Tolerance Testing
Mice were fasted for 6 hours in the morning, immediately following the dark cycle. After the fast, they were intraperitoneally injected with glucose at 2g/kg body weight. Glucose was measured using a commercially available glucometer (Contour Next EZ, Bayer) at baseline, and then at 15, 30, 60, and 120 minutes after glucose injection. Glucose levels were plotted against time, and using Graphpad Prism, area under the curve (AUC) could be measured.

Immunoblotting
Western blot analysis was performed on snap-frozen atrial tissue. Tissue samples were lysed using a Tissue Lyser (Qiagen) in protein lysis buffer supplemented with protease and phosphatase inhibitors (Boston BioProducts). Protein content was measured using a commercially available kit (DC Protein Assay, Bio-Rad). Equal amounts (15-25 g) were treated with Laemmli buffer and βmercaptoethanol and incubated at 100° C for 5 minutes. The lysate was then electrophoresed on a 4-20% SHS-polyacrylamide resolving gel and transferred to a PVDF membrane. The membrane was incubated overnight at 4° C with relevant antibodies, and then hybridization was completed with secondary antibody for 2 hours at room temperature. Antibody signal detection was achieved by employing the Clarity Western ECL Substrate (BioRad #1705061). Imaging and image quantification were done via BioRad Chemidoc Touch Imaging System and ImageLab, respectively. A detailed list of antibodies is provided in supplemental table 10.
Data were acquired on an LSRII (BD Biosciences) and analyzed with FlowJo software.

Histology and Fibrosis Quantification
Mice hearts were dissected and perfused with PBS. Hearts were then embedded in Tissue-Tek O.C.T. compound (Sakura Finetek), snap-frozen in 2-methylbutane on dry ice and sectioned into fibrosis was performed according to the manufacturer's instructions (Sigma). Quantification of fibrosis in the atria was performed using the BZX Analyzer software.

Cytokine ELISAs
Cardiac puncture was performed during heart extraction to collect plasma. Commercially available kits were utilized to determine levels of IL-6 and CRP (R&D systems).