ERK1/2 MAPK signaling in hypothalamic paraventricular nucleus contributes to sympathetic excitation in rats with heart failure after myocardial infarction

Abstract

Brain MAPK signaling pathways are activated in heart failure (HF) induced by myocardial infarction and contribute to augmented sympathetic nerve activity. We tested whether decreasing ERK1/2 (also known as p44/42 MAPK) signaling in the hypothalamic paraventricular nucleus (PVN), a forebrain source of presympathetic neurons, would reduce the upregulation of sympathoexcitatory mediators in the PVN and augmented sympathetic nerve activity in rats with HF. Sprague-Dawley rats underwent left anterior descending coronary artery ligation to induce HF, with left ventricular dysfunction confirmed by echocardiography. One week after coronary artery ligation or sham operation, small interfering (si)RNAs targeting ERK1/2 or a nontargeting control siRNA was microinjected bilaterally into the PVN. Experiments were conducted 5-7 days later. Confocal images revealed reduced phosphorylated ERK1/2 immunofluorescence in the PVN of HF rats treated with ERK1/2 siRNAs compared with HF rats treated with control siRNA. Western blot analysis confirmed significant reductions in both total and phosphorylated ERK1/2 in the PVN of HF rats treated with ERK1/2 siRNAs along with reduced expression of renin-angiotensin system components and inflammatory mediators. HF rats treated with ERK1/2 siRNAs also had reduced PVN neuronal excitation (fewer Fos-related antigen-like-immunoreactive neurons), lower plasma norepinephrine levels, and improved peripheral manifestations of HF compared with HF rats treated with control siRNAs. These results demonstrate that ERK1/2 signaling in the PVN plays a pivotal role in mediating sympathetic drive in HF induced by myocardial infarction and may be a novel target for therapeutic intervention.

Keywords: autonomic regulation; brain; extracellular signal-regulated kinases 1 and 2; mitogen-activated protein kinase; proinflammatory cytokines; renin-angiotensin system.

Fig. 1.

Protein levels of total and phosphorylated (p-)ERK1/2 in the paraventricular nucleus (PVN; A and B) and cerebral cortex (C and D) in normal rats without treatment, sham-operated (sham) rats treated with PVN microinjection of a nontargeting control (Con) small interfering (si)RNA, and heart failure (HF) rats treated with PVN microinjection of CON siRNA or ERK1/2 MAPK siRNAs. Representative Western blots are shown in A and C. Values were corrected by β-actin and are expressed as means ± SE; n = 4–7 rats/group. †P < 0.05 vs. normal or sham + CON siRNA; *P < 0.05, HF + ERK1/2 siRNAs vs. HF + CON siRNA.

Fig. 2.

Effects of microinjection of pooled ERK1/2 MAPK siRNAs on ERK1/2 activity in the PVN of HF rats. Representative confocal images from the PVN of HF rats that received bilateral microinjection of ERK1/2 siRNAs or control siRNA are shown. ERK1/2 siRNAs substantially reduced p-ERK1/2 immunofluorescence (right) compared with control siRNA (left). Middle: higher-power magnification of a section of the left image showing p-ERK1/2 expression (green) predominantly in neuronal (NeuN, red) elements. 3V, third ventricle.

Fig. 3.

Bilateral PVN microinjection of ERK1/2 MAPK siRNAs reduced expression of renin-angiotensin system and inflammatory mediators in the PVN in HF rats. A: representative Western blots. B–F: quantitative comparison of protein levels for angiotensin-converting enzyme (ACE; B), ANG II type 1 receptors (AT₁R; C), IL-1β (D), TNF-α (E), and cyclooxygenase (COX)-2 (F) in the PVN. Values were corrected by β-actin and are expressed as means ± SE; n = 7 rats/group. *P < 0.05 vs. HF + CON siRNA.

Fig. 4.

Effect of bilateral PVN microinjection of ERK1/2 MAPK siRNAs on PVN neuronal excitation and plasma norepinephrine (NE). A: representative sections from each group showing Fos-related antigen-like immunoreactivity (Fra-LI) among neurons in the PVN. Dark dots indicate single activated neurons. B: quantification of Fra-LI-positive neurons in subnuclear regions of the PVN in each treatment group (n = 4 rats/group). pm, posterior magnocellular PVN; mp, medial parvocellular PVN; vlp, ventrolateral parvocellular PVN; dp, dorsal parvocellular PVN. C: plasma levels of NE, a marker of sympathetic nerve activity, in each treatment group (n = 7 rats/group). Values are expressed as means ± SE. *P < 0.05 vs. HF + CON siRNA.

Fig. 5.

Effect of bilateral PVN microinjection of ERK1/2 MAPK siRNAs on peripheral manifestations of HF 2 wk after coronary artery ligation. A: body weight (BW). B: left ventricular (LV) weight-to-BW ratio. C: right ventricular (RV) weight-to-BW ratio. D: wet lung-to-BW ratio. Values are expressed as means ± SE; n = 7 rats/group. *P < 0.05 vs. HF + CON siRNA.