Abstract

Heart failure with preserved ejection fraction (HFpEF) is characterized by an increased sympathetic drive and decreased left ventricle compliance. We recently described that HFpEF rats displayed chronic neuronal activation in the rostral ventrolateral medulla (RVLM), a major region involved in the regulation of sympathetic outflow. Importantly, reactive oxygen species, inflammation and angiotensin II (AngII) have been suggested to partially mediate sympathoexcitation in cardiovascular diseases. Recently, endoplasmic reticulum (ER) stress has been point out as a major corner stone in the AngII-mediated ROS production. Currently, there is no evidence showing the plausible mechanisms underpinning chronic central sympathetic neuron hyper-activation in HFpEF. Accordingly, we aimed to determine whether changes in ER stress, O2− radical formation, AngII type 1 receptor (AT1R), Cu/ZnSOD, gp91phox (NOX2), NF-kB p65 subunit and inflammatory cytokine expression were associated with neuronal hyper-activation in the RVLM of HFpEF rats. Adult male Sprague-Dawley rats underwent volume overload to induce HFpEF. Cardiac function was determined by pressure-volume loops. RVLM micropunches were obtained from control and HF rats for the study of neuronal activation and for protein and mRNA expression using immunoblot and qRT-PCR, respectively. DHE staining was used to quantify O2− radical formation by confocal microscopy. Compared to Sham, HFpEF rats display (HFpEF vs. Sham): normal EF (75.9±3.4 vs. 72.1±2.3%, P<.05), increased end diastolic pressure (EDP) (5.6±0.1 vs. 3.8±0.3 mmHg, P<.05) and EDP-volume relationship (0.007±0.001 vs. 0.003±0.001 1/μl, P<.05) and overt signs of cardiac hypertrophy (heart to body weight ratio, 6.1±0.3 vs. 4.0±0.5 mg/g; P<.05). HFpEF rats displayed RVLM neuronal activation (FosB expression) compared to Sham (252.0±56.5 vs. 100.0±3.9%, P<.05) and increased production of superoxide radical (12.3±1.6 vs. 3.7±0.6 AU, P<.05). In addition, compared to Sham rats, HFpEF rats showed an augmented expression of AT1R (276.2±48.1 vs. 100.0±20.3%, P<.05) and p65 expression (279.9±48.1 vs. 100.0±33.9%, P<.05). Also, HFpEF rats displayed a shift in the expression of anti-oxidant and pro-oxidant enzymes that favors RVLM oxidative stress (Cu/ZnSOD decreases; NOX2 increases). Furthermore, ER stress markers were significantly increased in HFpEF compared to sham. Indeed, we found that CHOP and XBP1 mRNAs were increased by ~60% and ~90% respectively, in the RVLM from HFpEF rats compared to Sham rats. Finally, we found an increased de novo synthesis of the proinflammatory cytokines TNF-α and IL-1β in the RVLM from HFpEF animals compared to sham. Our data show for the first time that neuronal activation in the RVLM of HFpEF rats is associated with ER stress. In addition, we found that AT1R and NF-kB p65 protein expression are both up-regulated in the RVLM of HFpEF rats suggesting that activation of the AngII signaling pathways and/or inflammatory signaling cascade in the RVLM may play a role in the maintenance of sympathetic neuron hyper-activation in HFpEF through an ER stress-dependent mechanism.