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 -NG-nitro arginine methyl ester 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 the accumulation of Spp1 -expressing CCR2 (C-C chemokine receptor type 2) ⁺ cardiac resident macrophages (CRM). Furthermore, treatment with tVNS reduced the number of CCR2 ⁺ CRM and the expression of Spp1 while also inducing the expression of Igf1 in TLF ⁺ (Timd4 ⁺ [T-cell immunoglobulin and mucin domain containing 4 ⁺ ]/Lyve1 ⁺ [Lymphatic vessel endothelial hyaluronan receptor 1 ⁺ ]/Folr2 ⁺ [Folate receptor 2 ⁺ ]) and MHC2 ⁺ (Major histocompatibility complex 2 ⁺ ) 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/α7nAChR (α7 nicotinic acetylcholine receptor) signaling, either via pharmacological inhibition of α7nAChR or choline acetyltransferase deletion in CD4 ⁺ (cluster of differentiation) T cells. CONCLUSIONS: Collectively, our data indicate that tVNS improves HFpEF by reducing Spp1 expressing CCR2 ⁺ CRM and inducing expression of proreparative Igf1 in TLF ⁺ /MHC2 ⁺ CRM. These effects are mediated through cholinergic signaling, highlighting a neuroimmune pathway in HFpEF.

Abnormal cardiac metabolism precedes and contributes to structural changes in heart failure. Low-level tragus stimulation (LLTS) can attenuate structural remodeling in heart failure with preserved ejection fraction (HFpEF). The role of LLTS on cardiac metabolism is not known. Dahl salt-sensitive rats of 7 weeks of age were randomized into three groups: low salt (0.3% NaCl) diet (control group; n = 6), high salt diet (8% NaCl) with either LLTS (active group; n = 8), or sham stimulation (sham group; n = 5). Both active and sham groups received the high salt diet for 10 weeks with active LLTS or sham stimulation (20 Hz, 2 mA, 0.2 ms) for 30 min daily for the last 4 weeks. At the endpoint, left ventricular tissue was used for RNA sequencing and transcriptomic analysis. The Ingenuity Pathway Analysis tool (IPA) was used to identify canonical metabolic pathways and upstream regulators. Principal component analysis demonstrated overlapping expression of important metabolic genes between the LLTS, and control groups compared to the sham group. Canonical metabolic pathway analysis showed downregulation of the oxidative phosphorylation (Z-score: −4.707, control vs. sham) in HFpEF and LLTS improved the oxidative phosphorylation (Z-score = −2.309, active vs. sham). HFpEF was associated with the abnormalities of metabolic upstream regulators, including PPARGC1α, insulin receptor signaling, PPARα, PPARδ, PPARGC1β, the fatty acid transporter SLC27A2, and lysine-specific demethylase 5A (KDM5A). LLTS attenuated abnormal insulin receptor and KDM5A signaling. HFpEF is associated with abnormal cardiac metabolism. LLTS, by modulating the functioning of crucial upstream regulators, improves cardiac metabolism and mitochondrial oxidative phosphorylation.

Background: Postural orthostatic tachycardia syndrome (POTS) is increasingly recognized after SARS‐CoV‐2 infection. We compared autonomic phenotype, functional impact, and circulating immune markers in POTS, postacute sequelae of COVID‐19 (PASC), and healthy controls. Methods: In this cross‐sectional study (August 2021 to December 2022), we recruited patients with POTS (n=24) or PASC (n=24) and healthy controls (n=19). Participants underwent 10‐minute active stand testing, 24‐hour Holter monitoring, serum cytokine, and adrenergic autoantibody assays (cell‐based activation). Patient‐reported outcome measures were collected via secure electronic link. Results: The study included 67 participants (mean age, 32.4±8.9 years; 71% women). Compared with controls and participants with PASC, participants with POTS demonstrated significantly higher orthostatic tachycardia (active stand heart rate change from supine to standing, 46.3±14.0 bpm versus 12.7±6.6 bpm in controls and 34.6±13.7 bpm in PASC; P <0.001; POTS versus PASC P =0.005). Among participants with PASC, 62.5% met formal POTS criteria. Cytokine analysis revealed lower interleukin‐2 and higher interleukin‐8 levels in POTS versus controls, with POTS cases showing elevated tumor necrosis factor‐α compared with those without POTS. Autoantibody activation measures did not differ significantly among groups, and multivariate biomarker models lacked predictive utility for POTS diagnosis. Patient‐reported outcome measures indicated greater fatigue, orthostatic intolerance, and autonomic symptom burden and reduced health‐related quality of life in POTS and PASC groups compared with controls. Conclusions: POTS and PASC exhibit overlapping autonomic symptomology and profound functional impairment. Alterations in cytokines suggest a possible cytokine‐linked or compartmentalized immune contribution, but cytokine and autoantibody measures alone do not predict POTS. Routine autonomic assessment in persistent postviral illness and larger longitudinal, multimodal studies are warranted. Registration: Australian New Zealand Clinical Trial Registry; ACTRN: 12621000476831.