A novel sympathetic neuronal GABAergic signalling system regulates NE release to prevent ventricular arrhythmias after acute myocardial infarction

Figures

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  Figure 1. GABAergic signalling system and P2X7Rs expressed SCG neurons. A: TH‐positive (green) neurons in rat SCG slices were positively stained for the GABA synthetic enzyme GAD65/67 (red in the first panel). GABAAR subunits (GABAARβ2) were located on both the cell body and axons (red colour in the second panel) of the SCG TH‐positive neurons (green colour). The distribution pattern of the P2X7Rs (red colour in the third panel) is the same as that of GABAARβ2, which is located on TH‐positive neurons (green colour). B: Fura‐Red AM staining of cultured SCG neurons for testing the intracellular Ca2+ concentration ([Ca2+]i). A higher fluorescence intensity indicates a lower [Ca2+]i. The representative pictures show the 0‐ and 20‐minute results. ATP increased [Ca2+]i significantly, but muscimol blocked the increase in Ca2+. The GABAAR antagonist SR95531 blocked the inhibitory effects of GABA on the ATP‐induced [Ca2+]i elevation. C: Statistical analysis of [Ca2+]i after treatment with ATP (n = 3), ATP + muscimol (n = 4), or SR95531 + ATP+muscimol (n = 5). At 20 minutes, [Ca2+]i in the ATP + muscimol group was significantly lower than that in the ATP group (P < 0.05), and [Ca2+]i in the SR95531…

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  Figure 2. The SCG GABAergic signalling system was involved in MI. The protein band of GABAARβ2 was smaller in the MI rat SCG than in the sham surgery rat SCG, but the administration of GABA attenuated the change in GABAARβ2 levels (A). Compared with MI alone (n = 6), pretreatment with GABA (n = 4) before the MI procedure significantly reduced the rat HR (B, a and c), and the arrhythmias were relatively benign in the pretreatment group compared with those in the MI group. However, the infarct area of the heart in the MI and MI + GABA groups was not markedly different (B, a and b). “*” P < 0.05 compared with the sham surgery group. “#” P < 0.05 compared with the MI group. One‐way ANOVA was used to compare the different groups

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  Figure 3. Exogenous GABA reduced peripheral sympathetic activity and the incidence of VAs. In MI rats, the plasma and cardiac NE levels were significantly higher than those in sham surgery rats (*, P < 0.05, A and B). GABA reduced both plasma and cardiac NE levels in the MI + GABA group compared with MI alone (#, P < 0.05, A and B). However, NE levels in the MI + GABA group were still higher than those in the sham surgery group (*, P < 0.05, A and B). C, Recordings of typical inducible VAs with premature ventricular beats (PVC) (a), sustained ventricular tachycardia (b) and sustained ventricular fibrillation (c). D, The percentage of inducible total VAs/stimulation times was 1.5 times lower in the MI + GABA group than in the MI group (#, P < 0.05), but those in both the MI and MI + GABA groups were higher than those in the sham surgery group (*, P < 0.05). E, Statistical analysis of inducible VAs in the three groups. The inducible quotient of VAs was 3‐4 times higher in the MI group than in the sham surgery group (*, P < 0.05) and was 2 times lower in the MI + GABA group than in the MI group (#, P < 0.05). However, the inducible quotient of VAs in the MI + GABA group was still 2 times…

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  Figure 4. Knockdown of SCG GABAARβ2 increased RSNA and HR. Transduction efficiency after targeted injection of GABAARβ2 shRNA in the SCG was tested by qPCR and Western blotting. A, SCG slices showed a positive intracellular infection after 3 days of treatment with vehicle and shRNA vectors (both were GFP‐tagged). B, and C, GABAARβ2 was downregulated at both the protein and mRNA levels. D, Typical recordings of the RSNA in the vehicle and shRNA groups. Baseline RSNA was significantly increased in the GABAARβ2 shRNA group (*, P < 0.05). F, Heart rate (HR) was significantly higher in the GABAARβ2 shRNA group than in the vehicle group on day 3 (*, P < 0.05, E). The HR remained at a high level until day 7. RSNA, renal sympathetic nerve activity. SCG, superior cervical ganglion. The scale bar in each graph of A is 160 μm. “*” P < 0.05 compared with the vehicle group. One‐way ANOVA was used to compare the different groups

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  Figure 5. SCG GABAARβ2 knockdown induced sympathoexcitoxicity in MI rats. A, The survival rate of the SCG GABAARβ2 knockdown (shRNA) group was only 30%, and the survival rate of the MI group was approximately 70% at 7 days post MI. SCG‐targeted injection of vehicle and shRNA did not affect the survival rate. B, Plasma NE was higher in the MI + vehicle and MI + shRNA groups than in the control + vehicle group, and NE levels were highest in the MI + shRNA group. C, Baseline RSNA was higher in the shRNA only, MI + vehicle and MI + shRNA groups than in the control + vehicle group, and baseline RSNA was highest in the MI + shRNA group. D and E, The inducible quotient of ventricular arrhythmias (VAs) and inducible VAs were both higher in the MI groups (n = 6). The MI + shRNA group (n = 4) showed an even higher inducible quotient of VAs and inducible VAs than the MI + vehicle group. “*,” P < 0.05 compared with the control + vehicle group; “#,” P < 0.05 compared with the MI + vehicle group. Nonparametric testing was used to compare the different groups

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  Figure 6. The schematic pathway of the SCG GABAergic signalling system involved in sympathetic activity and arrhythmias. SCG sympathetic neurons were positive for both P2X7Rs and GABAARs. GABA blocked the ATP‐induced [Ca2+]i elevation in vitro. Our results likely show another receptor‐receptor interaction model that is commonly observed in central neuronal synaptic functions. By inhibiting P2X7Rs, GABA blocks NE release from sympathetic neurons both inside and outside of cardiac tissues. By regulating sympathetic activity, GABA can attenuate arrhythmias in post‐MI rats, indicating that the GABAergic system could be a potential therapeutic target to protect the heart from arrhythmias post MI