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

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 (CVN DMV ), 15-day HFD exposure increases tonic GABA A receptor (GABA A R) 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 GABA A R current in CVN DMV . We hypothesized that increased activity of protein kinase C δ isoform (PKCδ) enhances tonic GABA A R current in CVN DMV after HFD since PKCδ activity is upregulated and this type of PKCδ activation promotes GABA A R membrane stabilization as well as increases tonic GABA A R 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 GABA A R current in CVN DMV after 15D HFD (HFD GFX = 0.55 ± 0.34 pA/pF; N=3 cells / 2 mice vs NFD GFX = 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 CVN DMV 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 GABA A R current after 15D HFD (HFD no inhibitor vs rottlerin = 1.32 ± 0.14 vs 0.46 ± 0.06 pA/pF, P=0.0004; NFD rottlerin vs HFD rottlerin = 0.41 ±0.08 vs 0.46 ± 0.06 pA/pF, P=0.68; N NFD rottlerin = 3 cells /2 mice, N HFD rottlerin = 5 cells /3 mice). There was no effect of GFX or rottlerin on tonic current in NFD animals (NFD no inhibitor vs GFX : P=0.95; NFD no 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 GABA A R currents since rottlerin had no effect on phasic GABA A R 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 CVN DMV after 15D HFD and we revealed a strong trend for lower fold expression of PKCδ mRNA in HFD (2 ΔΔCt NFD vs HFD = 1.41 ± 0.42 vs 0.51 ± 0.09, P=0.058, N NFD = 8 cells / 4 mice, N HFD = 8 cells / 4 mice). Altogether, these preliminary results suggest that PKCδ mediates HFD-induced increases in tonic GABA A R 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 GABA A Rs in CVN DMV . 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.