Hypoxia-Inducible Factor-1α Mediates Increased Sympathoexcitation via Glutamatergic N-Methyl-d-Aspartate Receptors in the Paraventricular Nucleus of Rats With Chronic Heart FailureCLINICAL PERSPECTIVE by Neeru M. Sharma, Craig J. Cunningham, Hong Zheng, Xuefei Liu, and Kaushik P. Patel Circ Heart Fail Volume 9(11):e003423 November 3, 2016 Copyright © American Heart Association, Inc. All rights reserved.

Hypoxia-inducible factor 1α (HIF-1α) expression in the paraventricular nucleus (PVN) in chronic heart failure (CHF). Hypoxia-inducible factor 1α (HIF-1α) expression in the paraventricular nucleus (PVN) in chronic heart failure (CHF). A, Immunohistochemistry of PVN sections from sham and CHF rats. Representative pictures: lower magnification; Inset: higher magnification. B, Average values obtained by the reciprocal intensity method in each group are shown. Values are mean±SEM of analyses of 3 animals in each group. *P=0.004 vs sham. C, HIF-1α mRNA expression relative to rpl19 in punched PVN samples measured by real-time polymerase chain reaction in the PVN in CHF rats. Changes in HIF-1α mRNA are presented in folds with reference to the Sham group. Values are mean±SEM of quadruplicate analyses of 4 to 5 animals in each group. *P=0.014 vs sham. D, Western blot analysis of HIF-1α in the PVN punches from sham and CHF rats. Top: a representative Western blot; bottom: densitometry analyses of HIF-1α level normalized to tubulin. Values are mean±SEM of quadruplicate analyses of 4 to 5 animals in each group. *P=0.001 vs sham. Neeru M. Sharma et al. Circ Heart Fail. 2016;9:e003423 Copyright © American Heart Association, Inc. All rights reserved.

Hypoxia-induced hypoxia-inducible factor 1α (HIF-1α) expression in NG108 cells. Hypoxia-induced hypoxia-inducible factor 1α (HIF-1α) expression in NG108 cells. A, Western blot analysis was used to assess levels of HIF-1α protein after 0, 0.5, 1, 2, 4, 8, and 12h of exposure to hypoxia. Top: a representative Western blot; bottom: densitometry analyses of HIF-1α level normalized to GAPDH. *P=0.035 4 h vs 0 h control, $P=0.003 8 h vs 0 h control, &P=0.013 12 h vs 0 h control. B, Real-time polymerase chain reaction of HIF-1α relative to rpl19 after 0, 0.5, 1, 2, 4, 8, and 12h of exposure to hypoxia. C, HIF-1α protein expression after normoxia in hypoxia samples. Protein levels of HIF-1α were measured in NG108 cells after 12 h of hypoxia and after 12 h of hypoxia followed by 1 h of normoxia Top: a representative Western blot; bottom: densitometry analyses of HIF-1α level normalized to GAPDH. *P=0.002 compared with 12 h of hypoxia. Values were mean±SE from 4 independent experiments. Neeru M. Sharma et al. Circ Heart Fail. 2016;9:e003423 Copyright © American Heart Association, Inc. All rights reserved.

Hypoxia-induced N-methyl-d-aspartate-type 1 receptor (NMDA-NR1) expression in NG108 cells. Hypoxia-induced N-methyl-d-aspartate-type 1 receptor (NMDA-NR1) expression in NG108 cells. A, RT-PCR of NMDAR-NR1 relative to rpl19 after 0, 0.5, 1, 2, 4, 8, 12, and 24 hours of exposure to hypoxia. *P=0.051 4h vs. 0 h control, $P=0.011 8h vs. 0 h control, &P=0.023 12h vs. 0 h control, #P=0.001 24h vs. 0 h control. B, Western blot analysis was used to assess levels of NMDAR-NR1 present after 0, 0.5, 1, 2, 4, 8, 12, and 24 hours of exposure to hypoxia. Top: a representative western blot, bottom: densitometry analyses of NMDAR-NR1 level normalized to GAPDH. *P=0.003 12h vs. 0 h control. Values were mean+SE from four Independent experiments. C, Immunofluorescent staining for HIF-1α and NMDA-NR1 after 12 h of hypoxia exposure in NG108 cells. Representative images of NG108 cells before and after hypoxia exposure are presented. NMDA-NR1 (green), HIF-1α (red), and nuclei (blue). Neeru M. Sharma et al. Circ Heart Fail. 2016;9:e003423 Copyright © American Heart Association, Inc. All rights reserved.

A, Representative pictures of intracellular calcium ([Ca2+]i) as measured by Fura 2 in response to glutamate in hypoxic NG108 cells. A, Representative pictures of intracellular calcium ([Ca2+]i) as measured by Fura 2 in response to glutamate in hypoxic NG108 cells. B, A representative of time series of live cell confocal imaging of single cell from each group after glutamate stimulation. C, Cumulative data of intracellular calcium ([Ca2+]i) represented as arbitrary intensity units of control and hypoxia group at basal and after glutamate stimulation at 42 s, which is the time point showing maximum Fura 2 intensity in most of the neurons (n=20–22 cells from 3 coverslips in each group). *P=0.001 after glutamate stimulation compared with basal levels in control group. $P=0.001 after glutamate stimulation compared with basal levels in hypoxia group. #P=0.003 after glutamate stimulation hypoxic group vs control group. Neeru M. Sharma et al. Circ Heart Fail. 2016;9:e003423 Copyright © American Heart Association, Inc. All rights reserved.

(A) Chromatin immunoprecipitation (ChIP) assay of NG108 cells exposed to hypoxia using hypoxia-inducible factor 1α (HIF-1α) antibody and IgG as the control. (A) Chromatin immunoprecipitation (ChIP) assay of NG108 cells exposed to hypoxia using hypoxia-inducible factor 1α (HIF-1α) antibody and IgG as the control. Top: a representative agarose gel; bottom: densitometry analyses of N-methyl-d-aspartate-type 1 receptor (NMDA-NR1) promoter fragment levels normalized to input. Values were mean±SE from 3 independent experiments. *P=0.010 compared with 0 h control. B, HIF-1α and NMDA-NR1 expression after siRNA HIF-1α knockdown. Top: a representative Western blot; bottom: densitometry analyses of HIF-1α and level normalized to GAPDH. *P=0.030 HIF-1α, $P=0.036 NMDA-NR1 compared with scramble siRNA. Values were mean±SE from 4 independent experiments. Neeru M. Sharma et al. Circ Heart Fail. 2016;9:e003423 Copyright © American Heart Association, Inc. All rights reserved.

Renal sympathetic nerve activity (RSNA), mean arterial pressure (MAP), and heart rate (HR) responses to N-methyl-d-aspartate (NMDA) injected into the paraventricular nucleus (PVN). Renal sympathetic nerve activity (RSNA), mean arterial pressure (MAP), and heart rate (HR) responses to N-methyl-d-aspartate (NMDA) injected into the paraventricular nucleus (PVN). A, A segment of an original recording from an individual rat demonstrating the baseline parameters and peak changes in RSNA, integrated RSNA, MAP, and HR by administration of 200 pmol of NMDA into the PVN in the 4 groups: sham, CHF, sham+siRNA+HIF-1α, and CHF+HIF-1α. B, Mean data of baseline RSNA calculated as the percent of the maximum RSNA. *P=0.009 vs. sham. C, Mean data of changes in RSNA. *P=0.006 vs sham; #P=0.002 vs CHF. D, MAP *P=0.030 vs. sham; #P=0.040 vs CHF. E, HR *P=0.035 vs sham; #P=0.001 vs CHF after 200 pmol of NMDA into the PVN of the 4 groups. Values represent mean±SE of 5 to 6 animals in each group. Neeru M. Sharma et al. Circ Heart Fail. 2016;9:e003423 Copyright © American Heart Association, Inc. All rights reserved.

Proposed model for the transcriptional upregulation of N-methyl-d-aspartate-type 1 receptor (NMDA-NR1) by hypoxia-inducible factor 1 (HIF-1) in the paraventricular nucleus (PVN) in chronic heart failure (CHF). Proposed model for the transcriptional upregulation of N-methyl-d-aspartate-type 1 receptor (NMDA-NR1) by hypoxia-inducible factor 1 (HIF-1) in the paraventricular nucleus (PVN) in chronic heart failure (CHF). Hypoxia induces upregulation of HIF-1α, which binds to constitutively expressed HIF-1β to form the HIF-1 transcription complex. This transcription complex binds to the HRE1 and HRE2 located in the promoter of NMDA-NR1, leading to increased expression of NMDA-NR1 in the PVN during CHF causing an increase in sympathetic nerve activity. Neeru M. Sharma et al. Circ Heart Fail. 2016;9:e003423 Copyright © American Heart Association, Inc. All rights reserved.