Targeting NLRP3 signaling reduces myocarditis-induced arrhythmogenesis and cardiac remodeling

Abstract

Background: Myocarditis substantially increases the risk of ventricular arrhythmia. Approximately 30% of all ventricular arrhythmia cases in patients with myocarditis originate from the right ventricular outflow tract (RVOT). However, the role of NLRP3 signaling in RVOT arrhythmogenesis remains unclear.

Methods: Rats with myosin peptide-induced myocarditis (experimental group) were treated with an NLRP3 inhibitor (MCC950; 10 mg/kg, daily for 14 days) or left untreated. Then, they were subjected to electrocardiography and echocardiography. Ventricular tissue samples were collected from each rat's RVOT, right ventricular apex (RVA), and left ventricle (LV) and examined through conventional microelectrode and histopathologic analyses. In addition, whole-cell patch-clamp recording, confocal fluorescence microscopy, and Western blotting were performed to evaluate ionic currents, intracellular Ca²⁺ transients, and Ca²⁺-modulated protein expression in individual myocytes isolated from the RVOTs.

Results: The LV ejection fraction was lower and premature ventricular contraction frequency was higher in the experimental group than in the control group (rats not exposed to myosin peptide). Myocarditis increased the infiltration of inflammatory cells into cardiac tissue and upregulated the expression of NLRP3; these observations were more prominent in the RVOT and RVA than in the LV. Furthermore, experimental rats treated with MCC950 (treatment group) improved their LV ejection fraction and reduced the frequency of premature ventricular contraction. Histopathological analysis revealed higher incidence of abnormal automaticity and pacing-induced ventricular tachycardia in the RVOTs of the experimental group than in those of the control and treatment groups. However, the incidences of these conditions in the RVA and LV were similar across the groups. The RVOT myocytes of the experimental group exhibited lower Ca²⁺ levels in the sarcoplasmic reticulum, smaller intracellular Ca²⁺ transients, lower L-type Ca²⁺ currents, larger late Na⁺ currents_, larger Na⁺-Ca²⁺ exchanger currents, higher reactive oxygen species levels, and higher Ca²⁺/calmodulin-dependent protein kinase II levels than did those of the control and treatment groups.

Conclusion: Myocarditis may increase the rate of RVOT arrhythmogenesis, possibly through electrical and structural remodeling. These changes may be mitigated by inhibiting NLRP3 signaling.

Keywords: Myocarditis; NLRP3; Right ventricular outflow tract; Ventricular tachycardia.

Fig. 1

Study design and in vivo electrophysiology. A Experimental protocol. B Echocardiographic parameters of the control (healthy rats; n = 7), experimental (rats with myocarditis; n = 9), and treatment (MCC950-treated rats with myocarditis; n = 8) groups. C ECG readings before and after myosin peptide injection; rats with myocarditis had abnormal ECG morphologies with QRS fragmentation. D Representative electrocardiographic tracings and average data of the control (n = 7), experimental (n = 7), and treatment (n = 7) groups on days 1 (baseline) and 21 (after treatment). E ECG tracing revealing spontaneous premature ventricular contractions in a rat with myocarditis. F ECG tracing indicating caffeine (120 mg/kg)-induced ventricular tachycardia in a rat with myocarditis. ECG, electrocardiography

Fig. 2

Results of hematoxylin–eosin and immunohistochemical staining performed to measure NLRP3 expression. Representative sections (day 21) of the RVOTs, RVAs, and LVs of the control (healthy rats), experimental (rats with myocarditis), and treatment (MCC950-treated rats with myocarditis) groups after hematoxylin–eosin staining and immunohistochemical staining for CD45 and NLRP3 (magnification, 400 ×). A Average counts of inflammatory cells (lymphocytes and macrophages) in the control (n = 3), experimental (n = 4), and treatment (n = 3) groups. Macrophages (red arrow) and lymphocytes (yellow arrow) were observed in addition to interstitial space edema (blue arrow) and cardiomyocyte necrosis (green arrow). B Relative expression of CD45 in the RVOTs of the control group was compared with that in different heart regions of the experimental and treatment groups. Average CD45 immunostaining results of the control (n = 4), experimental (n = 4), and treatment (n = 4) groups. C Relative expression of NLRP3 in the RVOTs of the control group was compared with that in the experimental and treatment groups. Average NLRP3 immunostaining results of the control (n = 4), experimental (n = 4), and MCC950-treated (n = 4) groups. RVOT, right ventricle outflow tract; RVA, right ventricular apex; LV, left ventricle

Fig. 3

Morphology of AP and incidence of premature ventricular contractions and ventricular tachycardia. A Superimposed traces depicting APs in the RVOTs of the control (healthy rats), experimental (rats with myocarditis), and treatment (MCC950-treated rats with myocarditis) groups (n = 8). APA and APD at repolarizations of 20%, 50%, and 90% (APD₂₀, APD₅₀, and APD₉₀) are indicated. B Superimposed traces depict APs in the RVAs of the control, experimental, and treatment groups (n = 8). APA, APD₂₀, APD₅₀, and APD₉₀ are indicated. C Superimposed traces depict APs in the LVs of the control, experimental, and treatment groups (n = 8). APA, APD₂₀, APD₅₀, and APD₉₀ are indicated. D Premature ventricular contractions and ventricular tachycardia in the RVOTs, RVAs, and LVs of the control (n = 6), experimental (n = 10), and treatment (n = 7) groups. AP, action potential; APA, action potential amplitude; APD, action potential duration; RVOT, right ventricle outflow tract; RVA, right ventricular apex; LV, left ventricle

Fig. 4

Estimates of I_Na-Late, I_Ca-L, and NCX current in rat RVOTs. A Tracings and I–V relationship of NCX current in the RVOT cardiomyocytes of the control (healthy rats; n = 10), experimental (rats with myocarditis; n = 10), and treatment (MCC950-treated rats with myocarditis; n = 9) groups. B Tracings and I–V relationship of I_Ca-L in the RVOT cardiomyocytes of the control (n = 14), experimental (n = 16), and treatment (n = 12) groups C Current tracings and average data of I_Na-Late in the RVOT cardiomyocytes of the control (n = 12), experimental (n = 12), and treatment (n = 8) groups. I_Na-Late, late Na⁺ current; I_Ca-L, L-type Ca²⁺ current; NCX, Na⁺–Ca²⁺ exchanger; RVOT, right ventricle outflow tract

Fig. 5

ROS, intracellular Ca²⁺, SR Ca²⁺, Ca²⁺ leak, and cytosolic Na⁺ levels in rat RVOTs. A Tracings and average levels of [Ca²⁺]_i transients and fractional SR Ca²⁺ release in the RVOT cardiomyocytes of the control (n = 30), experimental (rats with myocarditis; n = 28), and treatment (MCC950-treated rats with myocarditis; n = 30) groups. B Tracings and average SR Ca²⁺ levels in the RVOT cardiomyocytes of the control (n = 12), experimental (n = 12), and treatment (n = 12) groups. C Tracings and average Ca²⁺ leak levels in the RVOT cardiomyocytes of the control (n = 19), experimental (n = 14), and treatment (n = 15) groups. D Average levels of cytosolic ROS in the RVOT cardiomyocytes of the control (n = 30), experimental (n = 31), and treatment (n = 25) groups. E Average levels of mitochondrial ROS in the RVOT cardiomyocytes of the control (n = 33), experimental (n = 30), and treatment (n = 33) groups. F Average levels of cytosolic Na⁺ in the RVOT cardiomyocytes of the control (n = 30), experimental (n = 30), and treatment (n = 29) groups. ROS, reactive oxygen species; SR, sarcoplasmic reticulum; RVOT, right ventricle outflow tract

Fig. 6

Effects of the NLRP3/CaMKII axis on Ca²⁺ regulatory proteins in rat RVOTs. Representative Western blots and summary data for sarcoplasmic/endoplasmic reticulum Ca²⁺ ATPase 2a, CaMKII phosphorylated at Thr286, RyR2, total phospholamban, RYR phosphorylated at Ser2808, phospholamban phosphorylated at Thr17, protein kinase A, Cav1.2, NLRP3, nuclear factor-κB, and interleukin-1β expression in the RVOT tissues of the control (healthy rats; n = 7), experimental (rats with myocarditis; n = 7), and treatment (MCC950-treated rats with myocarditis; n = 7) groups. Glyceraldehyde-3-phosphate dehydrogenase served as a loading control. CaMKII, Ca2 + /calmodulin-dependent protein kinase II; RYR, ryanodine receptor; RVOT, right ventricle outflow tract

Fig. 7

Potential mechanisms underlying the role of NLRP3 signaling in myocarditis. MCC950 treatment may reverse myocarditis-induced Na⁺–Ca²⁺ dysregulation by mitigating the alterations in levels of ROS, CMKII, RyR2, and ionic channels in cardiomyocytes. I_Na-Late: late Na⁺ current; NCX, Na⁺–Ca²⁺ exchanger; ROS, reactive oxygen species; RyR2, ryanodine receptor 2; RyR2-pS2808, RyR2 phosphorylated at serine 2808; SR, sarcoplasmic reticulum; SERCA, sarcoplasmic reticulum ATPase