Glucocorticoid stimulation increases cardiac contractility by SGK1-dependent SOCE-activation in rat cardiac myocytes

Abstract

Aims: Glucocorticoid (GC) stimulation has been shown to increase cardiac contractility by elevated intracellular [Ca] but the sources for Ca entry are unclear. This study aims to determine the role of store-operated Ca entry (SOCE) for GC-mediated inotropy.

Methods and results: Dexamethasone (Dex) pretreatment significantly increased cardiac contractile force ex vivo in Langendorff-perfused Sprague-Dawley rat hearts (2 mg/kg BW i.p. Dex 24 h prior to experiment). Moreover, Ca transient amplitude as well as fractional shortening were significantly enhanced in Fura-2-loaded isolated rat ventricular myocytes exposed to Dex (1 mg/mL Dex, 24 h). Interestingly, these Dex-dependent effects could be abolished in the presence of SOCE-inhibitors SKF-96356 (SKF, 2 μM) and BTP2 (5 μM). Ca transient kinetics (time to peak, decay time) were not affected by SOCE stimulation. Direct SOCE measurements revealed a negligible magnitude in untreated myocytes but a dramatic increase in SOCE upon Dex-pretreatment. Importantly, the Dex-dependent stimulation of SOCE could be blocked by inhibition of serum and glucocorticoid-regulated kinase 1 (SGK1) using EMD638683 (EMD, 50 μM). Dex preincubation also resulted in increased mRNA expression of proteins involved in SOCE (stromal interaction molecule 2, STIM2, and transient receptor potential cation channels 3/6, TRPC 3/6), which were also prevented in the presence of EMD.

Conclusion: Short-term GC-stimulation with Dex improves cardiac contractility by a SOCE-dependent mechanism, which appears to involve increased SGK1-dependent expression of the SOCE-related proteins. Since Ca transient kinetics were unaffected, SOCE appears to influence Ca cycling more by an integrated response across multiple cardiac cycles but not on a beat-to-beat basis.

Fig 1. Dex significantly increased inotropy in Langendorff-perfused rat hearts.

(A) Exemplary original tracings of left ventricular pressure (LVP) as a function of time of spontaneously beating Langendorff-perfused rat hearts. Mean data for systolic developed pressure (devel. LVP, B) and maximal pressure development rate (dPdt_max), (C). 24 h Dex pre-treatment (i.p., 2 mg/kg BW) significantly increased devel. LVP and dPdt_max consistent with a strong positive inotropic effect. (D) Mean data for total time to peak systolic LVP (time to peak). In contrast to systolic function, diastolic function was not affected by Dex pretreatment as indicated by mean data for left ventricular end-diastolic pressure (LVEDP, E) and relaxation time (F). *—p<0.05 vs. vehicle (unpaired t-test, n = 5 hearts in each group).

Fig 2. Dex increased Ca transient amplitude by a SOCE-dependent mechanism.

(A) exemplary original tracings of electrical field-stimulated Ca transients (1 Hz, 20 V; marked by ticks) and caffeine-transients (10 mM) of FURA-2 loaded isolated ventricular myocytes cultured for 24 h with either vehicle, Dex or Dex in the presence of SOCE inhibitor BTP2. Mean data for fractional shortening (B), Ca transient amplitude (D), Caffeine-transient amplitude (D), time to peak Ca transient (peak time, E), diastolic Ca (F), and time to 90% relaxation of the Ca transient (G) are also shown. 24 h Dex-pretreatment (1 mg/mL) significantly increased fractional shortening, Ca transient amplitude, and caffeine-induced Ca transient. This could be prevented by addition of SOCE inhibitors SKF (2 μM) or BTP2 (5 μM). Peak time, diastolic Ca, and time to 90% baseline were unaltered in all groups. n = 5–10 animals for each group. *P<0.05 vs vehicle, #P<0.05 vs Dex (one-way ANOVA).

Fig 3. Dex stimulates SOCE via an SGK1-dependent mechanism.

(A) Schematic representation of the SOCE-protocol. Isolated rat ventricular myocytes loaded with FURA2 were electrical-field stimulated (1 Hz, 20V) in the presence of 1 mM extracellular Ca concentration. After addition of 10 mM caffeine, the superfusion was changed to Ca/Na-free Tyrode's solution to empty the intracellular Ca stores. The SERCA-inhibitor thapsigargin (100 nM) and LTCC-inhibitor verapamil (10 μM) were added to prevent Ca reuptake into the SR and Ca entry via LTCC and caffeine (10 mM) was repeatedly administered to completely empty the SR. SOCE was then measured after wash in of Ca containing Tyrode. In parallel experiments, SKF (5 min., 2 μM) was added to inhibit SOCE. (B) Original traces of SOCE measured at the end of the protocol in myocytes pre-treated with either vehicle or Dex ((1 mg/mL, 24 h). (C) Mean data for SOCE amplitude. Dex exposure significantly increased SOCE amplitude. Interestingly, exposure to selective the SGK1 inhibitor EMD (24 h, 50 μM) completely abolished the Dex-dependent stimulation of SOCE amplitude. Data are from n = 4–11 animals. *P<0.05 vs vehicle, #P<0.05 vs Dex (one-way ANOVA).

Fig 4. Dex incubation increases m-RNA expression of SOCE-proteins in cardiac myocytes.

Mean data for mRNA levels measured via real-time PCR deploying the standard-curve method are shown as relative expression versus β-actin in arbitrary units (a.u.). Dex significantly increased STIM1 (A), STIM2 (B), Orai3 (C), and TRPC3 (D) and TRPC6 (E) expression. Importantly, Dex-dependent stimulation of STIM2, TRPC3 and TRPC6 expression were prevented by EMD pretreatment (B, D, and E, respectively). Data are from n = 5–18 animals. *P<0.05 vs vehicle, #P<0.05 vs Dex (one-way ANOVA).

Fig 5. Schematic presentation of the positive inotropic effect of dexamethasone.

Dex stimulates mRNA expression of SOC channels TRPC channel 3 and 6 and STIM2 via an SGK1-dependent mechanism. This results in increased SOCE mediated through Orai and TRPC channels leading to an elevation of SR Ca load and improved systolic RyR Ca release, which increases Ca transient amplitude and contractile force.