Transcutaneous vagal nerve stimulation improves heart failure with preserved ejection fraction by modulating cardiac resident macrophage function

Background: We previously showed in a rat model of heart failure with preserved ejection fraction (HFpEF) that transcutaneous vagus nerve stimulation (tVNS) reduced cardiac fibrosis and inflammation. However, macrophage-mediated mechanisms through which tVNS rescues cardiac function remain poorly understood. Methods: We induced HFpEF in 8-week-old mice by a combination of a high-fat diet and L-NAME for 5 weeks followed by 4 weeks of tVNS or sham stimulation. At this time, we analyzed cardiac function by echocardiography and immune cell numbers by single cell RNA sequencing and flow cytometry. Results: Our data demonstrate that HFpEF mice exhibited diastolic dysfunction, left ventricular hypertrophy and fibrosis, consistent with HFpEF, and that tVNS significantly improved HFpEF severity. Analysis of merged single cell RNA sequencing data from control, HFpEF + sham, and HFpEF + tVNS mice showed that HFpEF was associated with accumulation of Spp1 expressing CCR2+ cardiac resident macrophages (CRM). Further, treatment with tVNS reduced the number of CCR2+ CRM and the expression of Spp1, while also inducing the expression of Igf1 in Timd4+/Lyve1+/Folr2+ (TLF+) and MHC2+ CRM. Global deletion of Spp1 or blockade of CCR2+ CRM recruitment improved HFpEF, whereas TLF+/MHC2+ specific deletion of Igf1 reversed the protective effect of tVNS on HFpEF. The benefits of tVNS were also abolished in the setting of disrupted acetylcholine (ACh)/α7 neuronal ACh receptor (α7nAChR) signaling, either via pharmacological inhibition of α7nAChR or choline acetyltransferase deletion in CD4⁺ T cells. Conclusion: Collectively, our data indicate that tVNS improves HFpEF by reducing Spp1 expressing CCR2+ CRM and inducing expression of pro-reparative Igf1 in TLF+/MHC2+ CRM. These effects are mediated through cholinergic signaling, highlighting a neuroimmune pathway in HFpEF.