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. 2012 Oct 30;126(18):2208-19.
doi: 10.1161/CIRCULATIONAHA.112.115592. Epub 2012 Sep 26.

Pathological role of serum- and glucocorticoid-regulated kinase 1 in adverse ventricular remodeling

Saumya Das  1 , Takeshi Aiba, Michael Rosenberg, Katherine Hessler, Chunyang Xiao, Pablo A Quintero, Filomena G Ottaviano, Ashley C Knight, Evan L Graham, Pontus Boström, Michael R Morissette, Federica del Monte, Michael J Begley, Lewis C Cantley, Patrick T Ellinor, Gordon F Tomaselli, Anthony Rosenzweig
Affiliations

Pathological role of serum- and glucocorticoid-regulated kinase 1 in adverse ventricular remodeling

Saumya Das et al. Circulation. .

Abstract

Background: Heart failure is a growing cause of morbidity and mortality. Cardiac phosphatidylinositol 3-kinase signaling promotes cardiomyocyte survival and function, but it is paradoxically activated in heart failure, suggesting that chronic activation of this pathway may become maladaptive. Here, we investigated the downstream phosphatidylinositol 3-kinase effector, serum- and glucocorticoid-regulated kinase-1 (SGK1), in heart failure and its complications.

Methods and results: We found that cardiac SGK1 is activated in human and murine heart failure. We investigated the role of SGK1 in the heart by using cardiac-specific expression of constitutively active or dominant-negative SGK1. Cardiac-specific activation of SGK1 in mice increased mortality, cardiac dysfunction, and ventricular arrhythmias. The proarrhythmic effects of SGK1 were linked to biochemical and functional changes in the cardiac sodium channel and could be reversed by treatment with ranolazine, a blocker of the late sodium current. Conversely, cardiac-specific inhibition of SGK1 protected mice after hemodynamic stress from fibrosis, heart failure, and sodium channel alterations.

Conclusions: SGK1 appears both necessary and sufficient for key features of adverse ventricular remodeling and may provide a novel therapeutic target in cardiac disease.

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Figures

Figure 1
Figure 1
SGK1 activity is increased in murine and human heart disease. (A) SGK1 kinase assays were performed on SGK1 immunoprecipitated from heart lysates. Data shown are mean±SEM normalized to WT sham-treated values in each group from 3 independent experiments (*p<0.05, Welch’s t-test with Bonferroni adjustment). (B) Immunoblots of ventricular lysates from humans with healthy hearts who died of other causes (CTRL), patients with dilated cardiomyopathy (DCM) at the time of transplant, or patients with hypertension and increased LV mass (HHD), using an antibody against total- or phospho-SGK1. (C) Cumulative quantitated data (mean±SD for n=6-10 samples/group) normalized to GAPDH are represented as fold-change over control (*p<0.05, **p<0.001; one-way ANOVA; Fischer-Hayter post hoc test).
Figure 2
Figure 2
SGK1-DN mice are protected from heart failure after aortic constriction. (A-E): Gravimetric, echocardiographic, and histological data from SGK1-DN and WT littermates 7 weeks after TAC or SHAM operation. (A): HW/BW ratios showed significantly more LV mass in WT mice compared with SGK1-DN mice after TAC; (B-D): Wall thickness, FS, and LVDD as measured by echocardiography 7 weeks after TAC showed SGK1-DN mice are protected from LV dilatation and cardiac dysfunction compared with WT mice; (E): Fibrosis is reduced in SGK1-DN compared to WT mice after TAC (inset shows representative Masson-Trichrome stain). *p<0.05 (two-way ANOVA with post hoc Bonferroni) n=10 in TAC groups, n=5 in sham groups.
Figure 3
Figure 3
Propensity to ventricular arrhythmias, action potential prolongation, and afterdepolarizations are increased in SGK1-CA mice. (A) Representative tracings for WT and SGK1-CA mice from the distal (RV) pole during rapid pacing protocol (with pacing cycle lengths [CL] down to 60 msec in WT mice and 90 msec in the SGK1-CA mice). Asterisks denote pacing, double line indicates duration of pacing and scale bar denotes 200 msec. (B) Cumulative results for VT/VF inducibility from age-matched WT (0/5), SGK1-CA (3/6) and SGK1-DN (0/4) mice are shown (*p<0.05 by Z-test of proportions), in the absence of fibrosis or structural abnormalities (Supplemental Fig. S3). (C) Superimposed action potentials (APs) at CL 0.5-4 Hz in WT and SGK1-CA ventricular cardiomyocytes. Inset: APD at 90% repolarization (APD90) was longer in SGK1-CA compared with WT ventricular CMs, *p<0.05 by repeated measures ANOVA. (D) Representative EADs or DADs in SGK1-CA myocytes paced at 0.5 Hz. For quantification of EADs/DADs in comparison to WT see Fig. 5D.
Figure 4
Figure 4
Sodium current density, activation and inactivation are altered in SGK1-CA cardiomyocytes. (A) Representative INa currents in WT and SGK1-CA cardiomyocytes (10mM [Na+]o). (B) Current-Voltage (I-V) relationship for WT (n=7) and SGK1-CA (n=10) cardiomyocytes. (C) Cumulative quantitation reveals peak INa density is increased in SGK1-CA n=10) compared with WT (n=7) cardiomyocytes (*p<0.05, T-test). (D) Superimposed normalized conductance-voltage (GNa-V) and steady state inactivation curves from WT (open circles; n=6 and open triangles; n=7 respectively) and SGK1-CA (closed circles; n=7 and closed triangles; n=9 respectively) cardiomyocytes. There is a significant hyperpolarizing shift noted both in the voltage dependence of activation and steady-state inactivation of INa in SGK1-CA compared with WT.
Figure 5
Figure 5
SGK1 activation increases INaL while ranolazine normalizes APD and suppresses afterdepolarizations in SGK1-CA cardiomyocytes. (A) Representative normalized INaL (% of peak) superimposed for WT and SGK1-CA cardiomyocytes. Inset shows that normalized INaL was larger in SGK1-CA (n=6) than in WT cardiomyocytes (n=5). (B) Superimposed APs in cardiomyocytes from WT and SGK1-CA mice before (baseline: solid line) and after ranolazine (dotted line) at 0.5Hz pacing. Ranolazine (1μmol/L) normalized APD and suppressed EAD in SGK but did not affect APs in WT myocytes. (C) Ranolazine (1μmol/L) normalized APD90 in SGK1-CA cardiomyocytes (*p<0.05, repeated measures ANOVA), but did not affect APD90 in WT cardiomyocytes. (D) EADs and DADs were more frequent in SGK1-CA compared with WT cardiomyocytes. Ranolazine (1 μmol/L) reduced after-depolarizations in SGK1-CA cardiomyocytes (*p<0.05 by χ2 test) to levels seen in WT cardiomyocytes.
Figure 6
Figure 6
Increased susceptibility to ventricular arrhythmias and cardiac dysfunction in SGK1-CA mice can be reversed by ranolazine. (A) Mortality is increased in SGK1-CA mice after ischemia-reperfusion (I/R: 30 minute LAD ligation/24 hour reperfusion) Left panel: mortality in SGK1-CA (8/10), WT (7/29) and SGK1-DN (2/16) mice, *p<0.005 by Z-test of proportions. Right panel: Telemetry demonstrated that SGK1-CA mice develop lethal ventricular arrhythmia (VA) (4/5) after I/R, that were not seen in WT mice (0/4) *p=0.02 by Z-test. (B) Representative tracings of VA in SGK1-CA mice during reperfusion. Lower tracing shows ventricular bigeminy (asterisks) prior to onset of ventricular tachycardia with AV dissociation (arrows denote p-waves). (C) Schema for in vivo ranolazine experiments. (D) Ranolazine decreases QT and QTc intervals in SGK1-CA mice in comparison to placebo (*p<0.05 by unequal variance Ttest or Mann-Whitney test). (E) Fractional shortening was better in SGK1-CA mice after 7 days of ranolazine treatment than in mice treated with placebo (*p<0.05 by two-way repeated measures ANOVA). In paired T-tests, mice treated with ranolazine showed a non-significant trend towards improved fractional shortening (p=0.055), while there was no change in placebotreated mice (p=0.59). (F) Incidence of lethal VA after I/R was decreased in ranolazine-treated SGK1-CA mice in comparison to placebo (*p<0.05 by Z-test of proportions).
Figure 7
Figure 7
SGK1 activation is necessary and sufficient for alterations in cardiac sodium channel seen in failing hearts. (A) Cardiomyocytes were infected with either Ad.SGK1-CA or Ad.GFP, and biotin labeled to tag surface (e.g. Nav1.5, Glut4) but not cytoplasmic proteins (e.g. GSK3β). Biotin-labeled proteins were captured using an avidin column and subjected to immunoblotting. SGK1-CA expression increased surface expression of Nav1.5, *p<0.05, n=3 independent experiments. (B) Total heart lysates from WT (sham-operated), TAC-HF, or unoperated SGK1-CA mice were immunoprecipitated with a pan-Na+-channel antibody (SP-19). Immunoblotting with antibodies to Nav1.5 or Nedd4-2 showed a decrease in Nedd4-2 binding in TAC-HF and SGK1-CA hearts. (C) Cardiac lysates from SGK1-DN transgenic or WT mice subjected to TAC or sham-operation were immunoprecipitated with SP-19 and immunoblotted with the antibody specific Nav1.5 or Nedd4-2 (bottom panel). The decrease in Nedd4-2 binding to Nav1.5 seen in failing WT hearts is completely prevented in SGK1-DN hearts after TAC. (D) SGK1 directly associates with Nav1.5: Sodium channel was immunoprecipitated from heart lysates using SP-19 or control IgG antibody-coupled columns and subjected to immunoblotting as labeled. Figures are representative of 3 independent experiments.
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