Role of PKCδ in Acute HFD-induced Increased Tonic GABA Current in Cardiac-projecting DMV Neurons

Abstract

High fat diet (HFD) is associated with reduced cardiac vagal motor output, a major contributor to progression of cardiovascular diseases. In cardiac projecting neurons in the dorsal motor nucleus of the vagus (CVNDMV), 15-day HFD exposure increases tonic GABAA receptor (GABAAR) current, which contributes to dampening cardiac parasympathetic output. However, the mechanism underlying this increase inhibition is unknown. In this study, we aimed to decipher the mechanism(s) underlying this acute HFD-induced tonic GABAAR current in CVNDMV. We hypothesized that increased activity of protein kinase C δ isoform (PKCδ) enhances tonic GABAAR current in CVNDMV after HFD since PKCδ activity is upregulated and this type of PKCδ activation promotes GABAAR membrane stabilization as well as increases tonic GABAAR current in neurons. Initial experiments using cardiac retrograde tracing paired with whole-cell patch-clamp electrophysiology in male C57BL/6Js confirmed that general inhibition of PKC activity with GFX (10 μM) eliminated HFD-induced increases in tonic GABAAR current in CVNDMV after 15D HFD (HFDGFX = 0.55 ± 0.34 pA/pF; N=3 cells / 2 mice vs NFDGFX = 0.87 ± 0.42 pA/pF; N=3 cells / 2 mice). To investigate the specific role of PKCδ, we first determined that PKCδ was expressed in all putative cholinergic neuronal subpopulations of DMV by mining a publicly available single nucleus RNAseq dataset (The Broad Institute Single Cell Portal) followed by confirmation that PKCδ was present in CVNDMV from normal mice (Ct value= 14.6 ± 0.74, N=8 cells/ 4 mice). PKCδ selective inhibition with rottlerin (10μM), similar to GFX, diminished the increase in tonic GABAAR current after 15D HFD (HFDno inhibitor vs rottlerin= 1.32 ± 0.14 vs 0.46 ± 0.06 pA/pF, P=0.0004; NFDrottlerin vs HFDrottlerin = 0.41 ±0.08 vs 0.46 ± 0.06 pA/pF, P=0.68; NNFD rottlerin = 3 cells /2 mice, NHFD rottlerin = 5 cells /3 mice). There was no effect of GFX or rottlerin on tonic current in NFD animals (NFDno inhibitor vs GFX: P=0.95; NFDno inhibitor vs rottlerin: P=0.42), suggesting that inhibition of PKCδ did not alter tonic current in normal conditions. Importantly, the impact of inhibition of PKCδ was specific to tonic GABAAR currents since rottlerin had no effect on phasic GABAAR neurotransmission (amplitude: P= 0.07; frequency: P=0.09; decay time: P=0.25). To begin elucidating mechanisms for an increase in PKCd activity, we examined single cell transcriptional expression in CVNDMV after 15D HFD and we revealed a strong trend for lower fold expression of PKCδ mRNA in HFD (2ΔΔCtNFD vs HFD = 1.41 ± 0.42 vs 0.51 ± 0.09, P=0.058, NNFD = 8 cells / 4 mice, NHFD = 8 cells / 4 mice). Altogether, these preliminary results suggest that PKCδ mediates HFD-induced increases in tonic GABAAR current. Given the differences between increase PKCδ functional expression and putative transcriptional downregulation, it is possible that HFD-induced increases of PKCδ activity occur at post-transcriptional level. Further experiments will be done to investigate PKCδ protein expression and its colocalization with GABAARs in CVNDMV. The outcome of this study, therefore, will provide understanding on mechanism to develop treatments targeting PKCδ for cardiovascular diseases associated with HFD. R01HL157366 NIHLB to CRB. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.