Targeted nonviral gene-based inhibition of Gα(i/o)-mediated vagal signaling in the posterior left atrium decreases vagal-induced atrial fibrillation

Abstract

Background: Pharmacologic and ablative therapies for atrial fibrillation (AF) have suboptimal efficacy. Newer gene-based approaches that target specific mechanisms underlying AF are likely to be more efficacious in treating AF. Parasympathetic signaling appears to be an important contributor to AF substrate.

Objective: The purpose of this study was to develop a nonviral gene-based strategy to selectively inhibit vagal signaling in the left atrium and thereby suppress vagal-induced AF.

Methods: In eight dogs, plasmid DNA vectors (minigenes) expressing Gα(i) C-terminal peptide (Gα(i)ctp) was injected in the posterior left atrium either alone or in combination with minigene expressing Gα(o)ctp, followed by electroporation. In five control dogs, minigene expressing scrambled peptide (Gα(R)ctp) was injected. Vagal- and carbachol-induced left atrial effective refractory periods (ERPs), AF inducibility, and Gα(i/o)ctp expression were assessed 3 days following minigene delivery.

Results: Vagal stimulation- and carbachol-induced effective refractory period shortening and AF inducibility were significantly attenuated in atria receiving a Gα(i2)ctp-expressing minigene and were nearly eliminated in atria receiving both Gα(i2)ctp- and Gα(o1)ctp-expressing minigenes.

Conclusion: Inhibition of both G(i) and G(o) proteins is necessary to abrogate vagal-induced AF in the left atrium and can be achieved via constitutive expression of Gα(i/o)ctps expressed by nonviral plasmid vectors delivered to the posterior left atrium.

Figure 1

Attenuation by minigene-expressing Gα_i/octp of vagal stimulation (VS)- and carbachol (CCh)-induced effective refractory period (ERP) shortening. A: i: Compared to Gα_Rcpt, vagal-induced ERP shortening in the posterior left atrium (PLA) is attenuated in Gα_i2ctp dogs and is nearly eliminated in Gα_i2ctp+ Gα_o1ctp dogs. In Gα_i2ctp+ Gα_o1ctp dogs, vagal-induced ERP shortening was also significantly attenuated in the pulmonary vein (PV) (ii) and left atrial appendage (LAA) (iii). B: ERP shortening in the PLA in response to CCh. In Gα_Rcpt dogs, significant ERP shortening was noted in response to each CCh concentration (3, 10, 30 μM). In contrast, in Gα_i2ctp dogs, ERP shortening was noted only at 30 μM CCh. In Gα_i2ctp+ Gα_o1ctp dogs, there was no significant ERP shortening at any dose of CCh. *P <.05 vs baseline ERP at terminal study.

Figure 2

Decrease in vagal stimulation (VS)- and carbachol CCh-induced atrial fibrillation (AF) by minigene-expressing Gα_i/o peptide. A: Decrease in VS-induced AF in Gα_i2ctp and Gα_i2ctp+ Gα_o1ctp dogs compared to Gα_Rcpt. Both the AF inducibility index (i) and mean AF duration (ii) showed a progressive decrease in Gα_i2ctp and Gα_i2ctp+ Gα_o1ctp dogs, respectively. B: Decrease in CCh-induced AF in Gα_i2ctp dogs and in Gα_i2ctp+ Gα_o1ctp dogs compared to minigene-expressing Gα_Rcpt. Both the AF inducibility index (i) and mean AF duration (ii) showed a progressive decrease in Gα_i2ctp and Gα_i2ctp+ Gα_o1ctp dogs, respectively.

Figure 3

Attenuation by minigene-expressing Gα_i2ctp of vagal stimulation (VS)-induced changes in atrial fibrillation (AF) dominant frequency (DF). A: VS led to a significant increase in AF DF in the posterior left atrium (PLA) and pulmonary vein (PV) of Gα_Rcpt but not in Gα_i2ctp. No significant change in DF was noted in the left atrial appendage (LAA) in either Gα_i2ctp or Gα_Rcpt dogs. B: Representative examples of AF electrograms recorded from the PV, PLA, and LAA and their corresponding power spectra. Electrograms recorded from the Gα_i2atp group show a modest increase in DF with VS compared to baseline. The Gα_Rp group showed a much larger increase in DF.

Figure 4

Verification of Gα_x peptide transgene expression in the left atrium. A: Results of polymerase chain reaction on posterior left atrium (PLA), pulmonary vein (PV), and left atrial appendage (LAA) tissue. Transgene expression (for both Gα_i2ctp and Gα_Rcpt minigenes) was noted in the PLA. There was minimal expression in the adjoining PV and no expression in the LAA. B: Results of Western blotting for FLAG-tagged Gα_i2ctp. FLAG-tagged peptide was detected in the PLA but not in the LAA. Calsequestrin Q is the loading control for each lane. C: Example of immunohistochemistry for FLAG-tagged Gα_i2ctp. FLAG-tagged peptide (heavy brown stain) was detected in PLA myocytes (i) and in nerve bundles in the PLA (ii) but not in the LAA (iii).