Background Increased sympathetic outflow is a major contributor to the progression of chronic heart failure (CHF). Potentiation of glutamatergic tone has been causally related to the sympathoexcitation in CHF. Specifically, an increase in the N-methyl-D-aspartate-type1 receptor (NMDA-NR1) expression within the paraventricular nucleus (PVN) is critically linked to the increased sympathoexcitation during CHF. However, the molecular mechanism(s) for the up-regulation of NMDA-NR1 remains unexplored. We hypothesized that hypoxia via HIF-1α might contribute to the augmentation of the NMDA-NR1 mediated sympathoexcitatory responses from the PVN in CHF. Methods and Results Immunohistochemistry staining, mRNA, and protein for hypoxia-inducible-factor 1α (HIF-1α) were up-regulated within the PVN of left coronary artery ligated CHF rats. In neuronal cell line (NG108-15) in vitro, hypoxia caused a significant increase in mRNA as well as protein for HIF-1α (2-fold) with the concomitant increase in NMDA-NR1 mRNA, protein levels, as well as glutamate-induced Ca+ influx. ChIP assay identified HIF-1α binding to NMDA-NR1 promoter during hypoxia. Silencing of HIF-1α in NG108 cells leads to a significant decrease in expression of NMDA-NR1 suggesting that expression of HIF-1α is necessary for the upregulation of NMDA-NR1. Consistent with these observations, HIF-1α silencing within the PVN abrogated the increased basal sympathetic tone as well as sympathoexcitatory responses to microinjection of NMDA in the PVN of rats with CHF. Conclusions These results uncover a critical role for HIF-1 in the up-regulation of NMDA-NR1 to mediate sympathoexcitation in CHF. We conclude that subtle hypoxia within the PVN may act as a metabolic cue to modulate sympathoexcitation during CHF.

Increased sympathetic outflow is a common feature of chronic heart failure (HF). Potentiation of glutamatergic tone (increase in N‐methyl‐D‐aspartate (NMDA)‐type1 (NR1) receptors expression) within the paraventricular nucleus (PVN) has been related to the increased sympathetic drive in HF. However, the underlying molecular mechanism(s) for the upregulation of NR1 remains unclear. Immunohistochemistry of the PVN of rats with HF (coronary artery ligation‐induced) revealed upregulation of Hypoxia‐Inducible‐Factor 1α (HIF‐1α) compared to Sham. We hypothesized that increased HIF‐1α may contributes to the augmentation of the NR1 expression in the PVN of rats with HF. Initially, using an in vitro neuronal cell line (NG108–15), we found 2‐fold increase in HIF‐1α expression after 12h of hypoxia treatment in chambers. Concomitantly, hypoxia also showed significant increases in NR1 mRNA at 4h (6.5 fold) and 8h (7.2 fold) and protein levels after 12h (40%). Silencing of HIF‐1α in NG108 neurons led to decrease in expression of NR1. Taking this in vitro data to the whole animal studies, HIF‐1α knockdown in the PVN of rats with HF ameliorated the increased sympathoexcitation to NMDA (57.4±5.3 vs. 27.0±1.3). Taken together, these results suggest a contributory role for HIF‐1α in the regulation of NR1 expression, providing a novel mechanistic insight for the modulation of sympathoexcitation induced by the PVN in HF.Supported by NIHHLS2222.

Increased sympathetic outflow is a prominent feature of chronic heart failure (HF) associated with higher mortality. Multiple studies from our lab have reported that higher glutamatergic tone (increase in N‐methyl‐D‐aspartate (NMDA)‐type 1 (NR1) receptors expression) within the paraventricular nucleus (PVN) increase the sympathetic drive in HF. Further, HF (coronary artery ligation‐induced) augmented the expression of hypoxia‐inducible‐factor (HIF)‐1α both at mRNA (9 fold) and protein (2.5 fold) levels in the PVN which, led us to hypothesized that increased levels of HIF‐1α may contribute to exaggerated sympathetic drive via increasing NR1 expression in the PVN of rats with HF. Neuronal cell line (NG108‐15) exposed to hypoxia (2% O₂, 5% CO₂, 93% N₂) showed 2‐fold increase in HIF‐1α (12h) expression with concomitant increases in NR1 at transcription and protein levels after 4h (6.5 fold) and 12h (40%) respectively. Live cell imaging also showed marked increase in intracellular calcium in response to glutamate under hypoxic conditions. Mechanistically, hypoxia promotes the direct binding of HIF‐1α (3.2 fold) at NR1 promoter in neurons exposed to hypoxia as demonstrated by chromatin immunoprecipitation analysis. Furthermore, silencing of HIF‐1α by using HIF‐1α ‐targeted small interfering RNA in neurons led to 2.9 fold decrease in the expression of NR1. Validating our in vitro data, HIF‐1α knockdown by HIF‐1α‐siRNA in the PVN of rats with HF ameliorated the increased sympathoexcitation to NMDA (57±5% vs. 27±1% of basal value). Taken together, our study suggests a contribution of HIF‐1α in the regulation of NR1 expression within the PVN in HF, and provides a novel mechanism for the modulation of sympathoexcitation in HF. Supported by NIH HL62222. Grant Funding Source: NIH HL62222.