Effect of Low-Level Tragus Stimulation on Cardiac Metabolism in Heart Failure with Preserved Ejection Fraction: A Transcriptomics-Based Analysis

Abstract

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.

Keywords: heart failure; metabolic regulation; metabolism; tragus stimulation.

Figure 1

IPA comparison analysis of metabolic and signaling pathways.

Figure 2

Principal component analysis of differentially expressed genes related to enriched metabolic (A), metabolic signaling (B), and oxidative phosphorylation pathways (C). On the first two principal components, each of the three groups clusters together with overlap between control (LS) and active (LLTS) groups. Sham (HS sham) group clusters further away from control and LLTS. HS: high salt; LLTS: low-level tragus stimulation; LS: low salt.

Figure 3

IPA prediction of oxidative phosphorylation pathway in response to LLTS. Comparison between LS vs. HS sham (A) and HS sham group compared to HS active (B). IPA analysis predicted the downregulation of the oxidative phosphorylation pathway in the HFpEF (HS sham group) compared to the control group, suggesting a potential impairment of mitochondrial function in HFpEF. In contrast, an improvement in the oxidative Phosphorylation pathway was observed in the HS active group following LLTS compared to the HS sham group, indicating a potential beneficial effect of LLTS on mitochondrial function in HFpEF. The predictions were based on transcriptomic analysis data and visualized using interactive diagrams generated by Ingenuity Pathway Analysis (IPA) software. Color coding in the figure represents predicted changes in functional status (activation/inhibition) and quantity of different in different pathways and molecules, respectively. HS: high salt; LLTS: Low-level tragus stimulation; LS: low salt.

Figure 4

IPA prediction of acetyl-CoA and propionyl-CoA generation (LS vs. HS sham). Acetyl-CoA is generated from FA, ketone bodies, and amino acids. IPA predicted reduced generation of acetyl-CoA from all sources and propionyl-CoA from valine and isoleucine. Abbreviations: ACAA2—acetyl-coenzyme A acyltransferase 2; ACAT1—acetyl-CoA acetyltransferase 1; ACSL1—acyl-CoA synthetase long-chain family member 1; ALDH6A1—aldehyde dehydrogenase 6 family, member A1; BCAT2—branched-chain amino acid transaminase 2; BCKDH—branched-chain α-ketoacid dehydrogenase complex; ECHS1—enoyl-CoA hydratase, short chain 1; FA—fatty acid; GCDH—glutaryl-CoA dehydrogenase; HADH—hydroxyacyl-coenzyme A dehydrogenase; HIBADH—β-hydroxybutyrate dehydrogenase; HSD17B8—hydroxysteroid 17-beta dehydrogenase 8. Decreased measurement indicates Ingenuity Pathway Analysis predicted reduced concentration of metabolic product based on altered gene expression.

Figure 5

Experimental protocol: HS active indicates the HFpEF group receiving LLTS.