Background: The cytoplasmatic pattern recognition receptor, NOD2 (nucleotide-binding oligomerization domain 2), belongs to the innate immune system and is among others responsible for the recognition of single-stranded RNA. With Coxsackievirus B3 (CVB3) being a single-stranded RNA virus, and the recent evidence that the NOD2 target, NLRP3 (NOD-like receptor family, pyrin domain containing 3) is of importance in the pathogenesis of CVB3-induced myocarditis, we aimed to unravel the role of NOD2 in CVB3-induced myocarditis. Methods and Results: Endomyocardial biopsy NOD2 mRNA expression was higher in CVB3-positive patients compared with patients with myocarditis but without evidence of persistent CVB3 infection. Left ventricular NOD2 mRNA expression was also induced in CVB3-induced myocarditis versus healthy control mice. NOD2 knockdown(−/−) mice were rescued from the detrimental CVB3-mediated effects as shown by a reduced cardiac inflammation (less cardiac infiltrates and suppression of proinflammatory cytokines), cardiac fibrosis, apoptosis, lower CAR (Coxsackievirus and adenovirus receptor) expression and CVB3 copy number, and an improved left ventricular function in NOD2−/− CVB3 mice compared with wild-type CVB3 mice. In agreement, NOD2−/− decreased the CVB3-induced inflammatory response, CVB3 copy number, and apoptosis in vitro. NOD2−/− was further associated with a reduction in CVB3-induced NLRP3 expression and activity as evidenced by lower ASC (apoptosis-associated speck-like protein containing a CARD) expression, caspase 1 activity, or IL-1&bgr; (interleukin-1&bgr;) protein expression under in vivo and in vitro CVB3 conditions. Conclusions: NOD2 is an important mediator in the viral uptake and inflammatory response during the pathogenesis of CVB3 myocarditis.

Introduction: Nucleotide binding oligomerization domain 2 (NOD2) is a cytoplasmatic pattern recognition receptor belonging to the NOD-like receptor family, which is part of the innate immune system. Hypothesis: Since NOD2 recognises ssRNA and Coxsackievirus B3 (CVB3) is a ssRNA virus, we hypothesised that NOD2 regulates cardiac inflammatory signaling in CVB3-induced myocarditis. Methods: Gene expression was analyzed on endomyocardial biopsies (EMBs) taken from ejection fraction- and age-matched CVB3+ (n=10) and CVB3- control (n=7) patients and on EMBs from CVB3+patients who spontaneously eliminated CVB3 (n=6). NOD2 -/- mice and wild-type (wt) C57BL/6 mice were i.p. infected with 5 x 105 p.f.u. of CVB3. Seven days after infection, mice were hemodynamically characterized, and left ventricles (LV) were isolated. Results: NOD2 mRNA expression was 3.7-fold (p<0.05) increased in CVB3+ vs CVB3- control patients. Interestingly, NOD2 and Nrlp3 mRNA expression significantly dropped in EMBs of CVB3+ patients who spontaneously eliminated CVB3. In addition, LV NOD2 mRNA expession was increased in CVB3 vs wt mice. NOD2-/- CVB3 mice showed an improved LV function compared to CVB3 wt mice. In parallel, infiltration of CD4-, CD8-, CD11b- and CD68-positive cells was less pronounced in CVB3-infected NOD2-/- mice compared to CVB3 wt mice. Concomitantly, LV mRNA levels for TNF-alpha, IL-1ß, IFN-gamma, IFN-ß, TLR4, and MyD88 were 3.9-fold, 2.9-fold, 5.3-fold, 8.0-fold, 1.4-fold and 2.0-fold reduced in CVB3 NOD2-/- vs CVB3 wt mice, respectively (p<0.05). LV viral copy number was lower in CVB3 NOD2-/- vs CVB3 wt mice. CVB3-infected NOD2-/- mice exhibited less pronounced cardiac fibrosis, as indicated by lower collagen I and III mRNA expression and a 2.7-fold (p<0.0005) lower collagen I/III protein ratio. NOD2 knockdown in HL-1 cardiomyocytes was associated with a decreased inflammatory response, a 1.4-fold (p<0.05) lower CVB3 copy number, and a 3.4-fold (p<0.005) reduced TLR4 protein expression and underlying signaling. Conclusions: NOD2 knock down improves LV function and attenuates pathophysiological key mechanisms in acute CVB3-induced myocarditis mice. Modulation of NOD2 might represent a promising therapeutic strategy to treat viral myocarditis.

Sildenafil inhibits cyclic GMP-specific phosphodiesterase type-5A (PDE5A) and can prevent cardiac hypertrophy and left ventricular (LV) dysfunction in mice subjected to severe pressure-overload. The pathophysiological role of sildenafil in adverse remodeling in the hypertensive heart after chronic renin–angiotensin aldosterone system stimulation is unknown. Therefore, we studied the efficacy of the PDE5A inhibitor sildenafil for treating advanced cardiac hypertrophy and LV remodeling due to angiotensin (Ang)II-induced heart failure (HF) in vivo. C57BL6/J mice were subjected to AngII-induced cardiac hypertrophy for 3 weeks and cardiac dysfunction, cardiac inflammatory stress response, adverse remodeling as well as apoptosis were documented. Mice were subsequently treated with sildenafil (100 mg/kg/day) or placebo with delay of 5 days for treating AngII infusion-induced adverse events. Compared to controls, AngII infusion resulted in impaired systolic (dP/dt max −46 %, SV −16 %, SW −43 %, E a +51 %, EF −37 %, CO −36 %; p < 0.05) and diastolic (dP/dt min −36 %, LV end diastolic pressure +73 %, Tau +21 %, stiffness constant β +74 %; p < 0.05) LV function. This was associated with a significant increase in cardiac hypertrophy and fibrosis. Increased inflammatory response was also indicated by an increase in immune cell infiltration and apoptosis. Treatment with sildenafil led to a significant improvement in systolic and diastolic LV performance. This effect was associated with less LV hypertrophy, remodeling, cardiac inflammation and apoptosis. PDE5A inhibition with sildenafil may provide a new treatment strategy for cardiac hypertrophy and adverse remodeling in the hypertensive heart.