Transcriptional response of the heart to vagus nerve stimulation

Abstract

Heart failure is a major clinical problem, with treatments involving medication, devices, and emerging neuromodulation therapies such as vagus nerve stimulation (VNS). Considering the ongoing interest in using VNS to treat cardiovascular disease it is important to understand the genetic and molecular changes developing in the heart in response to this form of autonomic neuromodulation. This experimental animal (rat) study investigated the immediate transcriptional response of the ventricular myocardium to selective stimulation of vagal efferent activity using an optogenetic approach. Vagal preganglionic neurons in the dorsal motor nucleus of the vagus nerve were genetically targeted to express light-sensitive chimeric channelrhodopsin variant ChIEF, and stimulated using light. RNA sequencing of left ventricular myocardium identified 294 differentially expressed genes (DEGs, false discovery rate <0.05). Qiagen Ingenuity Pathway Analysis (IPA) highlighted 118 canonical pathways that were significantly modulated by vagal activity, of which 14 had a z-score of ≥2/≤-2, including EIF-2, IL-2, Integrin, and NFAT-regulated cardiac hypertrophy. IPA revealed the effect of efferent vagus stimulation on protein synthesis, autophagy, fibrosis, autonomic signalling, inflammation, and hypertrophy. IPA further predicted that the identified DEGs were the targets of 50 upstream regulators, including transcription factors (e.g., MYC, NRF1) and microRNAs (e.g., miR-335-3p, miR-338-3p). These data demonstrate that the vagus nerve has a major impact on myocardial expression of genes involved in regulation of key biological pathways. The transcriptional response of the ventricular myocardium induced by stimulation of vagal efferents is consistent with the beneficial effect of maintained/increased vagal activity on the heart.

Keywords: RNA sequencing; autonomic nervous system; heart; transcriptome; vagus nerve..

Figure 1. Genetic targeting of DVMN vagal preganglionic neurons to express light sensitive channel ChIEF for selective stimulation of vagal efferent activity using light.

a) The rat brain drawn in sagittal and coronal projections illustrating the anatomical location of the DVMN targeted with a lentiviral vector (LVV) to express ChIEFtdTomato under the control of the PRSx8 promoter; b) Schematic drawing of the experimental design in anaesthetized rats instrumented for stimulation of the DVMN neurons expressing ChIEF by application of 445 nm laser light followed by RNA-seq of the left ventricular myocardium; c) Photomicrograph of a coronal section of the rat dorsal brainstem taken at low magnification illustrating a representative example of ChIEFtdTomato expression in the caudal regions of the DVMN. Arrows point at projecting axons of the transduced DVMN neurons, forming the efferent vagus nerve. XII, hypoglossal nucleus. CC, central canal. Scale bar = 200 μm; d) Representative recordings illustrating changes in arterial blood pressure (ABP) and heart rate (HR) during 4 hours of DVMN stimulation (optoVNS; 5 Hz) in an isoflurane-anesthetised rat; e) Mean (±SEM) values of mean arterial blood pressure (MAP) and HR at baseline (0) and at 1, 2, 3 and 4 hours of optoVNS or sham stimulation in isoflurane-anesthetised rats. Stimulation of DVMN neurons at 5 Hz had no effect on ABP and HR.

Figure 2. Analysis of differentially expressed genes (DEGs) in the left ventricular myocardium in response to stimulation of vagal efferent activity.

a) Principal component (PC) plot of PC1 vs. PC2 of control (Sham 1–5) and experimental (optoVNS 1–6) samples; b) Volcano plot of differential expression data of all identified genes. Grey: P > 0.05; Red: upregulated, P < 0.05; blue: downregulated, P < 0.05; c) Heat map of top 294 DEGs with a false discovery rate of < 0.05. Scale bar: z-score; d) The list of top 10 most upregulated DEGs; e) The list of top 10 most downregulated DEGs; FDR, false discovery rate. FC, fold change.

Figure 3. Canonical pathways modulated by vagal efferent activity.

a) Canonical pathways which were significantly enriched in response to optoVNS (P < 0.05, z-score ≥ 2/≤ -2); b) Additional canonical pathways of interest (P < 0.05, z-score ≥ 1/≤ - 1). Data labels: number of overlapping DEGs from the dataset (full listing of genes is given in Supplementary Table 2); c) Chord diagram of 6 most significantly enriched pathways, and their corresponding DEGs; d) Chord diagram of 2 cardiac hypertrophy canonical pathways and their corresponding DEGs (red: upregulated, blue: downregulated).

Figure 4. Upstream regulators modulated by vagal efferent activity.

Upstream regulators predicted to be activated (positive z-score) or inhibited (negative z-score) in response to optoVNS (criteria: P value of overlap < 0.05, absolute z-score ≥ 2; uppercase: transcription factors; mir: microRNA; miR: mature microRNA). Data labels: number of overlapping DEGs (full listing of genes is given in Supplementary Table 3).