Single-nucleus RNA sequencing reveals Cntnap2+ astrocytes triggering a hypertensive effect in the rostral ventrolateral medulla by disrupting glutamate uptake

Figures

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  Fig. 1. Hemodynamic profiling and RVLM cellular composition in SHR and WKY rats. (A–D) Radiotelemetry, RSNA recording, ELISA, and immunofluorescence assay were employed to measure MAP, HR, sympathetic outflow, and RVLM neuronal excitability in SHRs and WKY rats at 13 weeks of age (n = 6). (E) UMAP revealed the different cell types in the RVLM on the basis of transcriptome data. (F) Number and percentage of every classified cell type, as determined by UMAP, in the combined six samples. (G) Sample types in the SHR and WKY groups as revealed by UMAP. (H) Expression of marker genes in the cell types identified through UMAP. (I) Box plot displaying the expression of gene markers specific to each cell type in diverse cell clusters. Mean ± SEM is utilized for data presentation. **p < 0.01, ***p < 0.001. ELISA: enzyme-linked immunosorbent assay; HR: heart rate; MAP: mean arterial pressure; RSNA: renal sympathetic nerve activity; RVLM: rostral ventrolateral medulla; SEM: standard error of the mean; SHR: spontaneously hypertensive rat; UMAP: uniform manifold approximation and projection; WKY: Wistar-Kyoto.

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  Fig. 2. Cntnap2 expression was markedly elevated in the astrocytes of the RVLM in SHRs. (A) Three different subtypes of astrocytes in UMAP. (B) Expression of marker genes in the subtypes identified through UMAP. (C) Box plot displaying the expression of gene markers specific to each cell type in the diverse subtypes. (D) Sample types in the SHR and WKY groups revealed by UMAP. (E) Enriched functional pathways based on the differentially expressed genes of the different subtypes. (F and G) RT-qPCR (n = 6) and Western blot (n = 6) were used to analyze the expression of Cntnap2 in the RVLM of SHRs and WKY rats. (H) Immunofluorescence was applied to examine the expression of Cntnap2 in the astrocytes of the RVLM in SHRs and WKY rats (n = 6). Mean ± SEM is utilized for data presentation. **p < 0.01, ***p < 0.001. RT-qPCR: reverse transcription quantitative polymerase chain reaction; RVLM: rostral ventrolateral medulla; SEM: standard error of the mean; SHR: spontaneously hypertensive rat; UMAP: uniform manifold approximation and projection; WKY: Wistar-Kyoto.

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  Fig. 3. Enhancing Cntnap2 in the astrocytes of the RVLM led to the development of hypertension in WKY rats. (A and B) Following the administration of the pLV-Cntnap2, the expression of Cntnap2 in the RVLM of WKY rats was assessed by using RT-qPCR (n = 6) and Western blot (n = 6). (C) Following the administration of the pLV-Cntnap2, the expression of Cntnap2 in the RVLM astrocytes of WKY rats was evaluated on the basis of immunofluorescence (n = 6). (D) MAP and HR in WKY rats after the overexpression of Cntnap2 in RVLM astrocytes was measured using radiotelemetry (n = 6). (E and F) Sympathetic outflow in WKY rats after the overexpression of Cntnap2 in RVLM astrocytes was evaluated through RSNA recording and ELISA (n = 6). (G) RVLM neuronal excitability in WKY rats after the overexpression of Cntnap2 in RVLM astrocytes was assessed by immunofluorescence (n = 6). Mean ± SEM is utilized for data presentation. **p < 0.01, ***p < 0.001, ns means nonsignificant. ELISA: enzyme-linked immunosorbent assay; HR: heart rate; MAP: mean arterial pressure; RSNA: renal sympathetic nerve activity; RT-qPCR: reverse transcription quantitative polymerase chain reaction; RVLM: rostral ventrolateral medulla;…

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  Fig. 4. Elevating Cntnap2 levels in primary astrocytes resulted in heightened primary neuronal excitability. (A and B) RT-qPCR (n = 6) and Western blot (n = 6) were employed to analyze the expression of Cntnap2 in primary astrocytes after pLV-Cntnap2 administration. (C and D) The primary neuronal excitability in cocultures of primary astrocytes/primary neurons was measured by using calcium imaging and immunofluorescence (n = 6). Mean ± SEM is utilized for data presentation. ***p < 0.001. RT-qPCR: reverse transcription quantitative polymerase chain reaction; SEM: standard error of the mean.

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  Fig. 5. Cntnap2 hindered the uptake of glutamate in astrocytes by inhibiting Eaat2. (A–C) RT-qPCR (n = 6), Western blot (n = 6), and immunofluorescence (n = 6) were used to assess the expression of Eaat2 in the RVLM astrocytes of SHRs and WKY rats. (D and E) After the delivery of the pLV-Cntnap2, the expression of Eaat2 in the RVLM of WKY rats was evaluated through RT-qPCR (n = 6) and Western blot (n = 6). (F and G) After 48 h of infecting primary astrocytes with the pLV-Cntnap2, the expression of Eaat2 was measured by using RT-qPCR (n = 6) and Western blot (n = 6). (H) Assessment of FITC-coupled glutamate uptake at different time intervals in diverse groups (n = 6). Mean ± SEM is utilized for data presentation. **p < 0.01, ***p < 0.001. FITC: fluorescein isothiocyanate; RT-qPCR: reverse transcription quantitative polymerase chain reaction; RVLM: rostral ventrolateral medulla; SEM: standard error of the mean; SHR: spontaneously hypertensive rat; WKY: Wistar-Kyoto.

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  Fig. 6. Cntnap2 was observed to elevate neuronal excitability by blocking Eaat2 in vitro. (A and B) Eaat2 expression in primary astrocytes postadministration of the pLV-Eaat2 was assessed by using RT-qPCR (n = 6) and Western blot (n = 6). (C and D) Primary neuronal excitability in cocultures of primary astrocytes and primary neurons was measured by using calcium imaging and immunofluorescence (n = 6). Mean ± SEM is utilized for data presentation. **p < 0.01, ***p < 0.001. RT-qPCR: reverse transcription quantitative polymerase chain reaction; SEM: standard error of the mean.

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  Fig. 7. Cntnap2/Eaat2 axis in RVLM astrocytes regulated BP, sympathetic nerve activity, and neuronal excitability in WKY rats. (A–C) Eaat2 expression in the RVLM astrocytes of WKY rats after the administration of the pLV-Eaat2 was evaluated by using RT-qPCR (n = 6), Western blot (n = 6), and immunofluorescence (n = 6). (D–G) MAP, HR, sympathetic outflow, and neuronal excitability in WKY rats following the overexpression of Cntnap2 and Eaat2 in RVLM astrocytes were measured by using radiotelemetry, RSNA recording, ELISA, and immunofluorescence (n = 6). Mean ± SEM is utilized for data presentation. **p < 0.01, ***p < 0.001, ns means nonsignificant. ELISA: enzyme-linked immunosorbent assay; HR: heart rate; MAP: mean arterial pressure; RSNA: renal sympathetic nerve activity; RT-qPCR: reverse transcription quantitative polymerase chain reaction; RVLM: rostral ventrolateral medulla; SEM: standard error of the mean; WKY: Wistar-Kyoto.