Nesfatin-1 as a novel cardiac peptide: identification, functional characterization, and protection against ischemia/reperfusion injury

Abstract

Nesfatin-1 is an anorexic nucleobindin-2 (NUCB2)-derived hypothalamic peptide. It controls feeding behavior, water intake, and glucose homeostasis. If intracerebrally administered, it induces hypertension, thus suggesting a role in central cardiovascular control. However, it is not known whether it is able to directly control heart performance. We aimed to verify the hypothesis that, as in the case of other hypothalamic satiety peptides, Nesfatin-1 acts as a peripheral cardiac modulator. By western blotting and QT-PCR, we identified the presence of both Nesfatin-1 protein and NUCB2 mRNA in rat cardiac extracts. On isolated and Langendorff-perfused rat heart preparations, we found that exogenous Nesfatin-1 depresses contractility and relaxation without affecting coronary motility. These effects did not involve Nitric oxide, but recruited the particulate guanylate cyclase (pGC) known as natriuretic peptide receptor A (NPR-A), protein kinase G (PKG) and extracellular signal-regulated kinases1/2 (ERK1/2). Co-immunoprecipitation and bioinformatic analyses supported an interaction between Nesfatin-1 and NPR-A. Lastly, we preliminarily observed, through post-conditioning experiments, that Nesfatin-1 protects against ischemia/reperfusion (I/R) injury by reducing infarct size, lactate dehydrogenase release, and postischemic contracture. This protection involves multiple prosurvival kinases such as PKCε, ERK1/2, signal transducer and activator of transcription 3, and mitochondrial K(ATP) channels. It also ameliorates contractility recovery. Our data indicate that: (1) the heart expresses Nesfatin-1, (2) Nesfatin-1 directly affects myocardial performance, possibly involving pGC-linked NPR-A, the pGC/PKG pathway, and ERK1/2, (3) the peptide protects the heart against I/R injury. Results pave the way to include Nesfatin-1 in the neuroendocrine modulators of the cardiac function, also encouraging the clarification of its clinical potential in the presence of nutrition-dependent physio-pathologic cardiovascular diseases.

Fig. 1

a Immunoblots of brain (lane 1), heart (lane 2), lung (lane 3), and stomach (lane 4) rat homogenates showing expression of Nesfatin-1 and β-actin as housekeeping protein, b real-time PCR of Nucleobindin 2 mRNA expression in heart, lung, and brain, c Nesfatin-1 ELISA assay of heart, plasma and lung. Changes were evaluated as mean ± SE of six experiments for each group. Significant difference from control values (one-way ANOVA), *p < 0.05

Fig. 2

Concentration–response curves of increasing concentrations (0.1 pmol/L–10 nmol/L) of Nesfatin-1 on LVP, +(LVdP/dt)_max, −(LVdP/dt)_max, T/−t, τ, HTR, and CP on the isolated and Langendorff-perfused rat heart. For abbreviations and basal values, see “Results”. Percentage changes were evaluated as mean ± SE of six experiments for each group. Significant difference from control values (one-way ANOVA), *p < 0.05

Fig. 3

a Effects of Nesfatin-1 before and after treatment with PTx (10 pmol/L), Anantin (100 nmol/L), PD (100 nmol/L), KT5823 (100 nmol/L), and EHNA (100 nmol/L) on LVP, +(LVdP/dt)_max, −(LVdP/dt)_max, in the isolated and Langendorff-perfused rat heart. b cGMP concentrations in control and Nesfatin-1-treated heart extracts. c Immunoblots of pERK1/2, ERK total, pAKT, and AKT total in control and Nesfatin-1-treated hearts (cardiac homogenates). d Nesfatin-1 co-immunoprecipitation with NPR-A. Percentage changes were evaluated as mean ± SE of six experiments for each group. Significant difference (one-way ANOVA) from control values of Nesfatin-1 versus Krebs–Henseleit solution (KHs), *p < 0.05. Comparison between groups treated with Nesfatin-1 alone versus antagonist, ^§ p < 0.05

Fig. 4

Local sequence alignment of the BNP and Nesfatin-1. The loop of BNP interacting with NPR-A (aa residues 5–16) has been aligned with Nesfatin-1 sequence by LALIGN software (http://www.ch.embnet.org/software/LALIGN_form.html). Hydrophobic amino acids which can interact with Pocket I and Pocket II of NPR-A are reported in bold and italic bold, respectively

Fig. 5

a Effects of Nesfatin-1 before and after treatment with L-NAME (10 μmol/L) on LVP on the isolated and Langendorff-perfused rat heart preparation, b immunoblots of peNOS, eNOS, pnNOS, and nNOS in control and Nesfatin-1 treated-hearts (cardiac homogenates), c biotin switch assay of S-nitrosylated proteins in the membrane fraction of homogenates from control and Nesfatin-1-treated hearts. Percentage changes were evaluated as mean ± SE of five experiments for each group. Significant difference from control values and from control values of Nesfatin-1 alone versus Krebs–Henseleit solution (KHs) (one-way ANOVA), *p < 0.05. Comparison between groups of Nesfatin-1 alone versus L-NAME, ^§ p < 0.05

Fig. 6

a Protocol groups. b LVP and LVEDP variations. Data are expressed as changes of LVP and LVEDP values (mmHg) from the stabilization to the end of the 120 min of reperfusion with respect to the baseline values for each group. Vertical lines indicate ischemic administration. Comparison between groups, ^§ p < 0.05. c Infarct size. The amount of necrotic tissue measured after 30-min global ischaemia and 120-min reperfusion is expressed as percentage of left ventricle (IS/LV %); *p < 0.05 with respect to I/R. Significant differences from control values of PostC or Nesfatin-1 PostC alone versus I/R (one-way ANOVA), *p < 0.05. d Nesfatin-1 ELISA measurement and real-time PCR of NUCB-2 of I/R and PostC hearts. Significant differences (one-way ANOVA), *p < 0.05. e Effects of Nesfatin-1 on LDH release. Values are expressed as mean ± SE of absolute data (U/wet wt, units per g of wet heart). Significant difference from control values (one-way ANOVA), *p < 0.05; changes were evaluated as mean ± SE of eight experiments for each group

Fig. 7

Cardiac performance. a LVP variations. Data are expressed as changes of LVP values (mmHg) at the end of 120-min reperfusion with respect to baseline values for each group. Significant differences from control values, *p < 0.05 with respect to I/R, for Nesfatin-1-Post (100 pmol/L), Nesfatin-1-Post (100 pmol/L) + epsilon-V1-2 (1 μmol/L), and Nesfatin-1-PostC (100 pmol/L) + 5HD (100 μmol/L). b LVEDP variations. Data are expressed as changes of LVEDP variations (mmHg) at the end of 120-min reperfusion with respect to baseline values for each group. Significant differences from control values, *p < 0.05 with respect to I/R, for Nesfatin-1-Post, Nesfatin-1-Post + epsilon-V1-2, and Nesfatin-1-Post + 5HD. c Infarct size. The amount of necrotic tissue measured after 30-min global ischaemia and 120-min reperfusion is expressed as percentage of the left ventricle (IS/LV %); *p < 0.05 with respect to I/R and each antagonist group

Fig. 8

Representative western blots and relative densitometric analysis of STAT3 and ERK1/2 phosphorylation in Sham, I/R, PostC, and Nesfatin-1-treated hearts. Percentage changes were evaluated as mean ± SE of four experiments for each group. Significant difference (one-way ANOVA) from control values, *p < 0.05