Absence of the inhibitory G-protein Galphai2 predisposes to ventricular cardiac arrhythmia

Abstract

Background: We explored the role that inhibitory heterotrimeric G-proteins play in ventricular arrhythmia.

Methods and results: Mice with global genetic deletion of Galpha(i2) [Galpha(i2) (-/-)] were studied and found, based on telemetry, to have a prolonged QT interval on surface ECG when awake. In vivo electrophysiology studies revealed that the Galpha(i2) (-/-) mice have a reduced ventricular effective refractory period and a predisposition to ventricular tachycardia when challenged with programmed electrical stimulation. Neither control nor combined global deletion of Galpha(i1) and Galpha(i3) mice showed these abnormalities. There was no evidence for structural heart disease at this time point in the Galpha(i2) (-/-) mice as assessed by cardiac histology and echocardiography. The absence of Galpha(i2) thus leads to a primary electrical abnormality, and we explored the basis for this finding. With patch clamping, single isolated ventricular cells showed that Galpha(i2) (-/-) mice had a prolonged ventricular action potential duration (APD) but steeper action potential shortening as the diastolic interval was reduced in restitution studies. Gene expression studies showed increased expression of L-type Ca(2+) channel subunits, and patch clamping revealed an increase in these currents in Galpha(i2) (-/-) mice. There were no changes in K(+) currents.

Conclusions: The absence of inhibitory G-protein signaling mediated through Galpha(i2) is a substrate for ventricular arrhythmias.

Figure 1. Single lead II surface ECG characteristics recorded from conscious freely moving mice under basal conditions using implantable telemetry

A. Evidence of unprovoked spontaneous ventricular ectopic activity was observed in 2/6 Gα_i2 (−/−) mice. No ventricular ectopics were observed in any littermate controls or in mice with the combined knockout of Gα_i1 and Gα_i3. The morphology of ectopic beats (highlighted by an asterix) suggests a ventricular origin. The complexes (highlighted by a +) are either ventricular in origin or are aberrantly conducted sinus beats. B. Representative signal averaged ECG recording from a Gα_i2 (−/−) (TOP) mouse showing evidence of QT prolongation compared with control (BOTTOM). Signal recorded and averaged from 900 successive sinus beats per mouse.

Figure 2. In vivo murine cardiac EP data and VT induced by programmed electrical stimulation

A. The measurement of VERP and this was significantly reduced in Gα_i2 (−/−) mice. B. Example of programmed electrical stimulation with one extrastimulus (S2) demonstrating VT induction. Control (TOP) shows non sustained VT induction at very short S1–S2 coupling. Gα_i2 (−/−) (Bottom) mice develop sustained VT using a similar induction protocol.

Figure 3. Echocardiography in wild type and Gα_i2 (−/−) mice

A. Pulsed-wave Doppler in the aortic arch. Velocity-time integral represents the area under each envelope (used to calculate stroke volume). Peak blood flow velocity is denoted by the orange cross (x). B. Parasternal short axis view of the left ventricle during M-mode. Dimensions of the anterior wall (yellow bars), left ventricle (green bars) and posterior wall (red bars) during diastole (1) and systole (2).

Figure 4. The action potential duration and its’ rate dependence in control and Gα_i2 (−/−) mice

A. Ventricular action potentials in control and Gα_i2 (−/−) mice. Representative traces of an action potential measured after stimulation of the cells by a 5 ms pulse after pacing at 1 Hz for 60 seconds. B. An illustration of the protocol for restitution with a 1000 millisecond test interval. C. Restitution curve under basal conditions (■ = wild type mice ▲ = Gα_i2 (−/−) mice). D. Restitution curve in the presence of 10 μmol\L isoprenaline (■ = wild type mice ▲ = Gα_i2 (−/−) mice). E. Regression analysis of the linear portion of the restitution curve under basal conditions (■ = wild type mice ▲ = Gα_i2 (−/−) mice).

Figure 5. Electrophysiological properties in control and Gα_i2 (−/−) mice

A. Quantitative real-time PCR data comparing transcript expression between control and Gα_i2 (−/−) mice (four mice in each group with each assay performed in triplicate). The data are expressed as ΔΔC_t i.e. −(ΔC_t(Gα_i2 (−/−) − ΔC_t(control)). ΔC_t is measured relative to the house keeping gene GAPDH. Data are shown as mean±SEM. *=p<0.05 and NS=not significant. ΔΔC_t of 1 is equivalent to two fold increase and 2 a four fold increase etc. B. Representative traces of calcium current in calcium solutions (see methods) C. Mean current-voltage relationships and a bar graph of current density. The Gα_i2 (−/−) mice show asignificant increase in basal current density (Table 3). D. In Ba²⁺ containing solutions a similar increase was observed in basal and isoprenaline induced currents. E. Representative recordings of outward K⁺ currents in control and Gα_i2 (−/−) mice. The mean current parameters are summarised in Table 3 but there were no significant differences between the two groups.