NOD1 Activation Induces Cardiac Dysfunction and Modulates Cardiac Fibrosis and Cardiomyocyte Apoptosis

<div><p>The innate immune system is responsible for the initial response of an organism to potentially harmful stressors, pathogens or tissue injury, and accordingly plays an essential role in the pathogenesis of many inflammatory processes, including some cardiovascular diseases. Toll like receptors (TLR) and nucleotide-binding oligomerization domain-like receptors (NLRs) are pattern recognition receptors that play an important role in the induction of innate immune and inflammatory responses. There is a line of evidence supporting that activation of TLRs contributes to the development and progression of cardiovascular diseases but less is known regarding the role of NLRs. Here we demonstrate the presence of the NLR member NOD1 (nucleotide-binding oligomerization domain containing 1) in the murine heart. Activation of NOD1 with the specific agonist C12-iEDAP, but not with the inactive analogue iE-Lys, induces a time- and dose-dependent cardiac dysfunction that occurs concomitantly with cardiac fibrosis and apoptosis. The administration of iEDAP promotes the activation of the NF-κB and TGF-β pathways and induces apoptosis in whole hearts. At the cellular level, both native cardiomyocytes and cardiac fibroblasts expressed NOD1. The NLR activation in cardiomyocytes was associated with NF-κB activation and induction of apoptosis. NOD1 stimulation in fibroblasts was linked to NF-κB activation and to increased expression of pro-fibrotic mediators. The down-regulation of NOD1 by specific siRNAs blunted the effect of iEDAP on the pro-fibrotic TGF-β pathway and cell apoptosis. In conclusion, our report uncovers a new pro-inflammatory target that is expressed in the heart, NOD1. The specific activation of this NLR induces cardiac dysfunction and modulates cardiac fibrosis and cardiomyocyte apoptosis, pathological processes involved in several cardiac diseases such as heart failure.</p> </div

CICR gain is increased in CPVT mice due to reduced <i>I_Ca</i> at low voltages.

<p><i>A</i>. Representative examples of Ca²⁺ entry and release fluxes simultaneously recorded in WT (black traces) and CPVT (red traces) myocytes. Beneath the images is the corresponding profile of fluorescence, expressed as F/F₀, where F is fluorescence and F₀ is diastolic fluorescence, after background correction. <i>B</i>. Voltage-dependent Ca²⁺ induced-Ca²⁺ release gain (CICR-gain) decreased monotonically, giving rise to an L-shaped in CPVT (filled symbols, n = 14) and WT (open symbols, n = 16) cells. <i>C & D</i>. Voltage dependence of peak [Ca²⁺]_i transients (C) and peak of <i>I_Ca</i> density (D) displayed bell-shaped, graded function with the membrane potential. * P<0.05 and ** P<0.005.</p

CPVT cells demonstrated rightward and leftward shifts in the voltage-dependent activation and inactivation of <i>I_Ca</i>, respectively.

<p><i>A</i>. Activation kinetics of <i>I_Ca</i> over the whole voltage range were not significantly different between WT (open symbols) and CPVT (filled symbols) cells. <i>B</i>. The time course of inactivation of <i>I_Ca</i>, which encompass slow and fast components, were similar in WT and CPVT cells at all voltages studied. <i>C.</i> Superimposed voltage-dependence of <i>I_Ca</i> activation and inactivation. <i>I_Ca</i> activationis shifted to more positive values in CPVT <i>vs</i> WT cells, whereas inactivation of <i>I_Ca</i> is shifted to more hyperpolarized potential in CPVT cells compared with WT cells. <i>D</i>. Voltage dependence of <i>I_Ca</i> window current () display a bell-shaped voltage-dependence, however, the peak of is reduced in CPVT cells (continuous line) compared to WT cells (dashed line). * P<0.05 and ** P<0.005.</p

Parameters of Boltzmann fittings of activation (d∞) and inactivation (f∞) curves (in mV).

<p>*p<0.05 vs WT;</p>#<p>p<0.05 vs control WT;</p>†<p>p<0.05 vs control CPVT.</p

Increased Ca²⁺ spark occurrence limits <i>I_Ca</i> window current ().

<p><i>A</i>. Analyze of the patch clamped-Ca²⁺ sparks revealed that the average of Ca²⁺ sparks frequencies in CPVT cells (filled circles, n = 10) were significantly higher compared to WT cells (open circles, n = 3). * P<0.05. <i>Right insets</i>. Representative examples of line-scan images of Ca²⁺ sparks elicited by depolarizing step to −48 mV. <i>B & C</i>. BAPTA dialysates (B) and thapsigargin-treatment (C) eliminates the difference in between CPVT and WT cells. <i>D</i>. In presence of Ryanodol in the perfusion solution, is reduced in WT cells compared to control condition, shown as light gray line.</p

Cardiac parameters collected after M-mode ultrasound evaluation of mice.

<p>Data are means ± SE. HW, heart weight, BW, body weight, HR heart rate, EF, left-ventricle ejection fraction, FS, fractional shortening, LVESD, left-ventricle end-systolic diameter; LVEDD, left-ventricle end-diastolic diameter; SV, systolic volume; DV, diastolic volume;</p>**<p><i>p</i><0.01,</p>***<p><i>p</i><0.001 Vehicle (Veh.) <i>vs.</i> iE (50, 150 or 200 µg iEDAP treated mice for two weeks).</p

Calmodulin inhibition reduced in WT cells.

<p><i>A & B</i>. CaM antagonists, W7 (<i>A</i>) or CALP2 (<i>B</i>) reduced in WT cells (dashed line) without affecting it in CPVT cells (continuous line). <i>C</i>. Comparison of the Ca²⁺ sparks occurrence at rest in myocytes from WT (open bars) and CPVT (closed bars) cells in control conditions and after incubation with W7 (left hatched bars) or CALP2 (right hatched bars). * P<0.05 vs WT. <i>D & E</i>. Representative immunoblots and quantification of CaM protein levels in cardiac heart lysates (<i>D</i>, normalized to the corresponding actin level and normalized to respective controls) and detected in the membrane fraction (<i>E</i>, normalized to the corresponding Ca_v1.2 level and normalized to respective controls) from WT (open bars, n = 4) and CPVT (closed bars, n = 4) mice.</p

Murine cardiac tissue expresses NOD1. Specific stimulation of NOD1 induces NF-κB pathway activation.

<p>(A) Histograms show NOD1 mRNA levels in different mouse tissues. (B) NOD1 protein levels in mouse hearts were analyzed by western-blot. Animals received i.p. 150 µg of iEDAP (iE)/day, a selective agonist of NOD1, or vehicle (Veh.). After 2 weeks of treatment an up-regulation of P-RIP2/RIP2, P-IKK/IKK, P-IκBα/IκBα protein ratio (B), higher p65 binding to κB motifs determined by ELISA (C), and NOS2 and COX2 (D) protein levels were observed in iE treated hearts. NOS2 and COX2 protein values were normalized with GAPDH. Representative blots are shown in the left panels and right panels illustrate the histograms representing the mean (band ratio)±SEM values <i>vs.</i> Veh. (100%); n = 4–6 animals. *p<0.05, ***p<0.001 <i>vs.</i> vehicle.</p

Selective activation of NOD1 stimulates NF-κB and TGF-β pathways in cardiac fibroblasts.

<p>(A) Confocal microscopy images of cardiac fibroblasts isolated from mouse hearts stained for NOD1. (B) Isolated murine cardiac fibroblasts were incubated for 15 to 60 min, 24 h or 48 h with 40 µg/ml iE. iE-treatment induces an up-regulation of P-RIP2/RIP2, P-IKK/IKK, P-IκBα/IκBα (15–60 min), NOS2 (24 h) and COX2 (48 h) protein levels. (C) Treatment of fibroblasts with iE for 72 h promoted an increase of TRβ2, P-Smad/Smad, PAI-1 and FGF-2 protein levels. GAPDH was used to normalize all target protein levels. Data are illustrated in histograms as mean±SEM <i>vs.</i> vehicle (100%; n = 3–5 samples).*p<0.05, **p<0.01 and ***p<0.001 <i>vs.</i> vehicle.</p

Effect of erythrodiol and uvaol on the fibrotic effect of angiotensin II in cardiac myofibroblasts.

<p>Time course of angiotensin II (Ang II; 1 µM)-stimulated collagen I protein production (A). Effect of erythrodiol (ERY; 5 µM) or uvaol (UVA; 5 µM) on CTGF (B), collagen I (C) and galectin 3 (D) protein expression in angiotensin II-treated cardiac myofibroblasts for 12 hours. Representative immunoblots of 4 experiments. Values are mean ± SEM of four assays. *p<0.05 <i>vs</i> vehicle. †p<0.05 vs angiotensin II. Quantification of band intensities was measured by densitometry and normalized to respective α-tubulin.</p