Inhibition of Rac1 reduces store overload-induced calcium release and protects against ventricular arrhythmia

Abstract

Rac1 is a small GTPase and plays key roles in multiple cellular processes including the production of reactive oxygen species (ROS). However, whether Rac1 activation during myocardial ischaemia and reperfusion (I/R) contributes to arrhythmogenesis is not fully understood. We aimed to study the effects of Rac1 inhibition on store overload-induced Ca(2+) release (SOICR) and ventricular arrhythmia during myocardial I/R. Adult Rac1(f/f) and cardiac-specific Rac1 knockdown (Rac1(ckd) ) mice were subjected to myocardial I/R and their electrocardiograms (ECGs) were monitored for ventricular arrhythmia. Myocardial Rac1 activity was increased and ventricular arrhythmia was induced during I/R in Rac1(f/f) mice. Remarkably, I/R-induced ventricular arrhythmia was significantly decreased in Rac1(ckd) compared to Rac1(f/f) mice. Furthermore, treatment with Rac1 inhibitor NSC23766 decreased I/R-induced ventricular arrhythmia. Ca(2+) imaging analysis showed that in response to a 6 mM external Ca(2+) concentration challenge, SOICR was induced with characteristic spontaneous intracellular Ca(2+) waves in Rac1(f/f) cardiomyocytes. Notably, SOICR was diminished by pharmacological and genetic inhibition of Rac1 in adult cardiomyocytes. Moreover, I/R-induced ROS production and ryanodine receptor 2 (RyR2) oxidation were significantly inhibited in the myocardium of Rac1(ckd) mice. We conclude that Rac1 activation induces ventricular arrhythmia during myocardial I/R. Inhibition of Rac1 suppresses SOICR and protects against ventricular arrhythmia. Blockade of Rac1 activation may represent a new paradigm for the treatment of cardiac arrhythmia in ischaemic heart disease.

Keywords: Rac1; arrhythmia; ischaemia and reperfusion; reactive oxygen species; store overload-induced calcium release.

Figure 1

Myocardial Rac1 activity after I/R. (A) Representative Western blots of Rac1‐GTP with α‐actinin as loading control in Rac1^f/f and Rac1^ckd hearts after myocardial I/R. (B) Quantification of Rac1 activity using Rac1‐GTP to total Rac1 protein ratios. (C) Quantification of total Rac1 to α‐actinin protein ratios in the heart. (D) Representative Western blots of total Rac1 and α‐actinin proteins in isolated cardiomyocytes from adult Rac1^f/f and Rac1^ckd mice. (E) Quantification of total Rac1 to α‐actinin protein ratios in the isolated cardiomyocytes. Data are mean ± SEM from three to four mice per group. * P < 0.05 versus Rac1^f/f sham in B or Rac1^f/f in E, †P < 0.05 versus respective Rac1^f/f.

Figure 2

Inhibition of Rac1 decreases cardiac arrhythmia during 45 min. of ischaemia followed by 1 hr of reperfusion (I/R). (A) Representative ECG tracings showing premature ventricular complex (PVC) and ventricular tachycardia (VT). (B) Total number of PVC. (C) Total incidence of VT. (D) Representative sections of Evans Blue and triphenyltetrazolium chloride stained hearts showing area at risk (blue) and infarct area (white) respectively. (E) Quantification of infarct area to area at risk. Data are mean ± SEM. n = 7–13 mice per group in B and C, n = 6 mice per group in E. **P < 0.01 versus Rac1^f/f.

Figure 3

Effects of Rac1 inhibition decreases store overload‐induced Ca²⁺ release (SOICR) of adult ventricular myocytes. (A–C) Representative Fura‐2 ratios of Rac1^f/f, NSC23766‐treated Rac1^f/f and Rac1^ckd myocytes in response to increasing extracellular Ca²⁺ concentrations (1, 3 and 6 mM) followed by caffeine (10 mM) stimulation (arrows). The boxed areas are enlarged. The threshold of SOICR was increased after Rac1 inhibition with NSC23766 and in Rac1^ckd myocytes. (D–F) Quantification of frequency, amplitude and occurrence of Ca²⁺ transients in Rac1^f/f, NSC23766‐treated Rac1^f/f (Rac1^f/f +NSC) and Rac1^ckd cardiomyocytes in response to 6 mM extracellular Ca²⁺. (G) sarcoplasmic reticulum (SR) Ca²⁺ content assessed by caffeine stimulation. Data are mean ± SEM. Numbers in bars indicate myocyte numbers from three mice per group, **P < 0.01, ***P < 0.001 versus Rac1^f/f.

Figure 4

Rac1 inhibition reduces cardiomyocyte Ca²⁺ transients and sarcoplasmic reticulum (SR) Ca²⁺ load. (A–D) Representative Fura‐2 ratios in Rac1^f/f and Rac1^ckd myocytes, which were perfused at 1 and 6 mM extracellular Ca²⁺ concentrations and paced at 0.5 Hz for 20 sec. followed by treatment with caffeine. (E) Ca²⁺ transient amplitude in Rac1^f/f and Rac1^ckd myocytes during pacing. (F) SR Ca²⁺ load assessed by caffeine (10 mM) in Rac1^f/f and Rac1^ckd myocytes. Data are mean ± SEM of 10–16 myocytes from 3 mice per group, *P < 0.05, **P < 0.01 versus corresponding Rac1^f/f.

Figure 5

Rac1 inhibition decreases myocardial superoxide generation and oxidation of RyR2 after I/R. (A) Representative dihydroethidium (DHE) fluorescence staining (red) of heart sections after 45 min. of ischaemia followed by 1 hr of reperfusion (I/R). (B) Quantification of the red fluorescence intensity after I/R in Rac1^ckd and Rac1^f/f hearts. (C) Representative blots of RyR2 oxidation after 45 min. of ischaemia followed by 15 min. of reperfusion (I/R) using monobromobimane (mBB). (D) Quantification of free thiols of RyR2 in Rac1^f/f and Rac1^ckd hearts after I/R. Data are mean ± SEM. N = 3–5 mice per group, *P < 0.05 versus Rac1^f/f sham, †P < 0.05 versus Rac1^f/f I/R, ‡P < 0.05 versus Rac1^ckd sham.