Transcutaneous Vagus Nerve Stimulation Ameliorates the Phenotype of Heart Failure With Preserved Ejection Fraction Through Its Anti-Inflammatory Effects

Abstract

Background: A systemic proinflammatory state plays a central role in the development of heart failure with preserved ejection fraction (HFpEF). Low-level transcutaneous vagus nerve stimulation (LLTS) suppresses inflammation in animals and humans, mediated by an α7nAchR (alpha7 nicotinic acetylcholine receptor)-dependent pathway. We examined the effects of LLTS on cardiac function, inflammation, and fibrosis in the presence of α7nAchR pharmacological blockade in a rat model of HFpEF.

Methods: Dahl salt-sensitive rats at 7 weeks of age were treated with high-salt diet for 6 weeks to induce HFpEF, followed by 4 weeks of (1) LLTS, (2) LLTS with the α7nAchR blocker methyllycaconitine, (3) sham, and (4) olmesartan. Blood pressure, cardiac function by echocardiography, heart rate variability, and serum cytokines were measured at 13 and 17 weeks of age. Cardiac fibrosis, inflammatory cell infiltration, and gene expression were determined at 17 weeks.

Results: LLTS attenuated the increase in blood pressure; improved cardiac function; decreased inflammatory cytokines, macrophage infiltration, and fibrosis; and improved survival compared with other groups. Methyllycaconitine attenuated these effects, whereas olmesartan did not improve cardiac function or fibrosis despite maintaining similar blood pressure as LLTS. Heart rate variability was similarly improved in the LLTS and LLTS plus methyllycaconitine groups but remained low in the other groups. LLTS reversed the dysregulated inflammatory signaling pathways in HFpEF hearts.

Conclusions: Neuromodulation with LLTS improved cardiac function in a rat model of HFpEF through its anti-inflammatory and antifibrotic effects. These results provide the basis for further clinical trials in humans.

Keywords: fibrosis; heart failure; heart rate; humans; inflammation.

Figure 1.

Study design, baseline data and effect of treatment on vital signs. A. Schematic of the study design, group assignment and endpoints. B. Changes in heart rate before and during LLTS at baseline (13 weeks), at different stimulation intensities (n=12). C. Representative ECG tracings from an animal at baseline and during active LLTS at 3mA, indicating a modest prolongation of the RR interval during active stimulation. Arrows indicate the stimulation artifact. D. Representative examples of echocardiographic images at 13 weeks from an animal treated with low salt diet compared to a high salt diet-treated animal, indicating development of left ventricular hypertrophy and diastolic dysfunction in the latter group. E. Systolic blood pressure (BP). F. Diastolic BP. G. Heart rate. H. Body weight. N=12–34 per group. LS = low salt. HS = high salt. Anti-HTN = antihypertensive (Olmesartan). MLA = methyllycaconitine. ECG = electrocardiogram. ECHO = echocardiogram. BP = blood pressure.

Figure 2.

Effect of treatment on echocardiographic parameters. A. Left ventricular (LV) ejection fraction. B. Interventricular septum (IVS) diameter. C. Ratio of the early to late mitral inflow Doppler velocity (E/A). D. Early diastolic mitral annulus Doppler velocity (e’). E. Ratio of the early mitral inflow Doppler velocity to the early diastolic mitral annulus velocity (E/e’). F. Circumferential strain (absolute values are plotted). N=10–24 per group. LS = low salt. HS = high salt. Anti-HTN = antihypertensive (Olmesartan). MLA = methyllycaconitine.

Figure 3.

Effect of treatment on serum inflammatory cytokines. A. Tumor necrosis factor (TNF)-α. B. Monocyte chemoattractant protein (MCP)-1. C. Osteopontin. D. Transforming growth factor (TGF)-β. N=12–24 per group. LS = low salt. HS = high salt. Anti-HTN = antihypertensive (Olmesartan). MLA = methyllycaconitine.

Figure 4.

Effect of treatment on heart rate variability. A. Frequency domain analysis using the FFT method (LF: low frequency. HF: high frequency. LF/HF: low frequency to high frequency ratio). B. Nonlinear analysis from Poincare plots [SD1: the standard deviation of the instantaneous beat-to-beat RR interval variability (minor axis of the ellipse), SD2: the standard deviation of the continuous long-term RR interval variability (major axis of the ellipse), SD1/SD2: the ratio of SD1 to SD2]. Logarithmic transformation was performed to satisfy the modeling assumptions. Box represents median values with interquartile intervals and whiskers represent 10^th and 90^th percentiles. N=10–24 per group. C. Symbolic dynamics analysis (0V: zero variation family; 1V: one variation family; 2LV: two like variations family; 2UV: two unlike variations family). N=8–12 per group. LS = low salt. HS = high salt. Anti-HTN = antihypertensive (Olmesartan). MLA = methyllycaconitine.

Figure 5.

Effect of treatment on heart weight, fibrosis and inflammatory infiltration. A. Heart weight normalized to tibia length (N=13–34 per group). B. Fibrosis area (as a percent of the left ventricular area) (N=12–24 per group). C. Inflammatory score (N=6 per group). D. Representative examples of histological images from animals from each group stained with Masson’s trichrome showing perivascular fibrosis. E. Representative examples of histological images from animals from each group stained with Masson’s trichrome showing interstitial fibrosis. F. Representative examples of histological images from animals from each group stained with hematoxylin and eosin. LS = low salt. HS = high salt. Anti-HTN = antihypertensive (Olmesartan). MLA = methyllycaconitine.

Figure 6.

Flow cytometry from left ventricular (LV) tissue isolated at endpoint. A. Effect of treatment on macrophages, granulocytes and CD4 T cells (N=8–12 per group). B. Representative examples of flow cytometry analysis of macrophage subsets in the hearts of animals from each group. LS = low salt. HS = high salt. Anti-HTN = antihypertensive (Olmesartan). MLA = methyllycaconitine.

Figure 7.

Effect of treatment on survival. Kaplan-Meier curve of survival according to treatment group. For this analysis, time 0 was considered when the mice were 13 weeks old. Survival was significantly better in the HS active group compared to all other HS groups. LS = low salt. HS = high salt. Anti-HTN = antihypertensive (Olmesartan). MLA = methyllycaconitine.

Figure 8.

Effect of treatment of left ventricular transcriptional profile. A. Venn diagram showing the number of transcripts that were differentially expressed in high salt sham vs. low salt hearts and were reversed by treatment with low level transcutaneous vagus nerve stimulation (LLTS). In the lower panel, Venn diagram of the number of transcripts that were differentially expressed in high salt sham vs. high salt active hearts and were reversed by treatment with methyllycaconitine (MLA). B. Heat map showing differentially expressed genes in each treatment group. C. Principal component analysis of changes in gene expression in hearts from different treatment groups. D. KEGG pathway analysis of the genes (n=656) that were normalized in the LLTS-treated hearts compared to sham-treated hearts. N=5–6 per group.