SK channel enhancers attenuate Ca2+-dependent arrhythmia in hypertrophic hearts by regulating mito-ROS-dependent oxidation and activity of RyR

Abstract

Aims: Plasmamembrane small conductance Ca2+-activated K+ (SK) channels were implicated in ventricular arrhythmias in infarcted and failing hearts. Recently, SK channels were detected in the inner mitochondria membrane (IMM) (mSK), and their activation protected from acute ischaemia-reperfusion injury by reducing intracellular levels of reactive oxygen species (ROS). We hypothesized that mSK play an important role in regulating mitochondrial function in chronic cardiac diseases. We investigated the role of mSK channels in Ca2+-dependent ventricular arrhythmia using rat model of cardiac hypertrophy induced by banding of the ascending aorta thoracic aortic banding (TAB).

Methods and results: Dual Ca2+ and membrane potential optical mapping of whole hearts derived from TAB rats revealed that membrane-permeable SK enhancer NS309 (2 μM) improved aberrant Ca2+ homeostasis and abolished VT/VF induced by β-adrenergic stimulation. Using whole cell patch-clamp and confocal Ca2+ imaging of cardiomyocytes derived from TAB hearts (TCMs) we found that membrane-permeable SK enhancers NS309 and CyPPA (10 μM) attenuated frequency of spontaneous Ca2+ waves and delayed afterdepolarizations. Furthermore, mSK inhibition enhanced (UCL-1684, 1 μM); while activation reduced mitochondrial ROS production in TCMs measured with MitoSOX. Protein oxidation assays demonstrated that increased oxidation of ryanodine receptors (RyRs) in TCMs was reversed by SK enhancers. Experiments in permeabilized TCMs showed that SK enhancers restored SR Ca2+ content, suggestive of substantial improvement in RyR function.

Conclusion: These data suggest that enhancement of mSK channels in hypertrophic rat hearts protects from Ca2+-dependent arrhythmia and suggest that the protection is mediated via decreased mitochondrial ROS and subsequent decreased oxidation of reactive cysteines in RyR, which ultimately leads to stabilization of RyR-mediated Ca2+ release.

Keywords: Cardiac hypertrophy; Reactive oxygen species; Ryanodine receptor; Small conductance Ca2+-activated K+ channels; Ventricular arrhythmia.

Figure 1

Pharmacological enhancement of SK channels attenuates Ca²⁺-dependent arrhythmia in ex vivo optically mapped hypertrophic hearts. (A) Representative ECGs at baseline and in the presence of 50 nmol/L isoproterenol, membrane-permeable SK enhancer NS309 (2 μmol/L) and cell impermeable SK inhibitor apamin (10 nmol/L). (B) Representative traces of action potentials and corresponding pooled data, *^#P < 0.05, vs Sham and TAB + NS309 + ISO, paired, and unpaired Student’s t-test where appropriate. (C) Representative activation maps in Sham and TAB hearts. (D) Pulled data for conduction velocities and conduction velocity heterogeneity, not significant at P < 0.05, unpaired Student’s t-test and One-way ANOVA where appropriate. (E) Representative Ca²⁺traces in hearts paced at 150 ms cycle length before pause. Red arrows indicate spontaneous Ca²⁺release. (F, G) Amplitude and rate of rise of spontaneous Ca²⁺release during pause. *^#P < 0.05, vs. Sham + ISO and TAB + NS309 + ISO, respectively, One-way ANOVA.

Figure 2

Pharmacological enhancement of SK channels decreases while inhibition increases arrhythmic potential in isolated ventricular myocytes from hypertrophic (TAB) rat hearts. (A) Representative simultaneous recording of Ca²⁺ transients and Vm in hypertrophic myocyte exposed to 100 nmol/L ISO before and 10 min after application of SK ehancer CyPPA (10 μmol/L) or SK inhibitor UCL-1684 (1 μmol/L); (B, C) Number of ISO-treated hypertrophic cells field-stimulated at 0.5 Hz for 1 min exhibiting Ca²⁺ waves and wave frequency, respectively. Cell numbers are shown in the columns, *P < 0.05 vs. TAB, Exact Fisher test and hierarchical linear model where appropriate. Number of TAB animals used was 7.

Figure 3

Expression and subcellular distribution of SK isoforms in ventricular myocytes from Sham and TAB rat hearts. (A) Identification of SK1, SK2, and SK3 using specific shRNAs expressed in cultured rat ventricular myocytes for 48 h. (B) SK channels distribution in membrane and mitochondrial fractions. Cyt C and MCU were used as the markers of mitochondria fraction. Pan-Cadherin and α1c and α2 subunits of L-type Ca²⁺channels were used as the markers membrane fraction. (C) Pooled data for normalized optical density (OD, %) of SK1-3 Western Blot analysis in membrane and mitochondrial fractions from isolated myocytes from 5 to 7 Sham and TAB hearts, *P < 0.05, Student’s t-test. Surface membrane SKs (pSK2 and SK3) were normalized to Pan-Cadherin and mSKs SK1 and mSK2 were normalized to MCU. (D, E) Representative western blots and pooled data for OD of SK1-3 in unfractionated myocytes. GAPDH was used as loading control. *P < 0.05, n = 7–12, Student’s t-test.

Figure 4

Pharmacological enhancers and inhibitors of SK channels modulate ΔΨm in Sham myocytes. (A) Representative recording of changes in mitochondrial membrane potential in response to increase in intracellular Ca²⁺from 50 nmol/L to 1 μmol/L followed by application of 1 μmol/L UCL-1684, a selective SK inhibitor. Myocytes from healthy hearts were permeabilized with saponin and immobilized with 10 μmol/L Cytochalasin D. TMRE signal was normalized to minimum fluorescence obtained by application of 50 μmol/L FCCP and presented as a % of baseline. (B) Pooled data for normalized TMRE signal, *^#P < 0.05 vs. 50 nmol/L [Ca²⁺]cyt and 1 μmol/L [Ca²⁺]cyt, respectively, hierarchical linear modelling. Numbers of cells are presented in columns; number of Sham animals was 3. (C) Representative recordings of ΔΨm in permeabilized cells exposed to GW542573X (selective SK1 enhancer, black) and CyPPA (selective SK2 and SK3 enhancer, red); (D) Pooled data for C, *P < 0.05 vs. baseline, hierarchical linear modelling. Numbers of cells are presented in columns; number of Sham animals was 5.

Figure 5

Pharmacological inhibition of SK channels increases while enhancement decreases ΔΨm in TAB myocytes. (A) Representative recording of changes in ΔΨm in response to SK channels block by 1 μmol/L UCL-1684 in permeabilized Sham (black) and TAB (grey) myocytes. (B) Pooled data for A, *P < 0.05, hierarchical linear model. Numbers of cells are presented in columns, number of Sham animals was 4, and number of TABs was 5. (C) Representative recordings of ΔΨm in permeabilized cells exposed to 100 μmol/L GW542573X (selective SK1 enhancer, black), CyPPA (selective SK2 and SK3 enhancer, red), and NS309 (enhancer of SK1, SK2, and SK3, blue). (D) Pooled data for C, *P < 0.05 vs. baseline, hierarchical linear model. Numbers of cells are presented in columns; number of TABs was 6.

Figure 6

Reversal of RyR oxidation in TAB myocytes by SK-enhancers via attenuation of mitochondrial ROS. (A) CyPPA reduced thiol-oxidation of RyR in TAB ventricular myocytes. Cells were incubated with 10 μmol/L CyPPA for 10 min before lysis. ROS scavenger dithiothreitol (10 mmol/L) and oxidant 2,2′-dithiodipyridine (200 μmol/L) were used to obtain minimal and maximal oxidation for normalization. Top: Representative fluorescence images of oxidation-sensing dye mBB. Bottom: RyR western for normalization. (B) Pooled data for A. Additional group of cells was treated with 10 μmol/L NS309 for 10 min in the same way as CyPPA, *P < 0.05 vs. Sham and ^#P < 0.05 vs. TAB, n = 4 hearts for both Sham and TAB, one-way ANOVA. (C) SK modulators alter rate of ROS production measured with MitoSOX in CMs. Cell numbers are shown in the columns. *P < 0.05 vs. Sham and ^#P < 0.05 vs. TAB, hierarchical linear model. Number of Sham animals was 3; number of Tab animals was 4.

Figure 7

SK enhancement stabilizes RyRs in TAB myocytes. Pre-incubation of TAB myocytes with 10 μmol/L CyPPA or 10 μmol/L NS309 for 10 min normalizes SR Ca²⁺content in permeabilized TAB myocytes. (A, B) Representative line scan images and pooled data for spark frequency, respectively. (C, D) Representative traces of caffeine-induced Ca²⁺transients (20 mmol/L) and pooled data for caffeine-induced Ca²⁺transients amplitude, respectively. Ca²⁺sparks and Ca²⁺transients were measured in saponin-permeabilized myocytes using Fluo-4. Cell numbers are shown in the columns, *^#P < 0.05 vs. Sham and TAB respectively, hierarchical linear model. Number of Sham animals was 4; number of TAB animals was 6.

Figure 8

Schematic presentation of proposed cellular mechanisms of SK-mediated protection from Ca²⁺-dependent ventricular arrhythmia. (i) Functional upregulation of SK2 and 3 in the plasma membrane limits disease-related prolongation of APD and reduces Ca²⁺-dependent EADs and DADs countering I_NCX; (ii) Functional upregulation of mitochondria-targeted SK2 attenuates disease-related increase in mito-ROS. Pharmacological enhancement of SK2 reduces mito-ROS resulting in normalization of the oxidative state of RyR, stabilization of RyR-mediated Ca²⁺release and abolishment of DAD-driving SCWs.