Specialized Proresolving Mediators Protect Against Experimental Autoimmune Myocarditis by Modulating Ca2+ Handling and NRF2 Activation

Abstract

Specialized proresolving mediators and, in particular, 5(S), (6)R, 7-trihydroxyheptanoic acid methyl ester (BML-111) emerge as new therapeutic tools to prevent cardiac dysfunction and deleterious cardiac damage associated with myocarditis progression. The cardioprotective role of BML-111 is mainly caused by the prevention of increased oxidative stress and nuclear factor erythroid-derived 2-like 2 (NRF2) down-regulation induced by myocarditis. At the molecular level, BML-111 activates NRF2 signaling, which prevents sarcoplasmic reticulum-adenosine triphosphatase 2A down-regulation and Ca²⁺ mishandling, and attenuates the cardiac dysfunction and tissue damage induced by myocarditis.

Keywords: 8OHdG, 8-hydroxy-2'-deoxyguanosine; BML-111, 5(S), (6)R, 7-trihydroxyheptanoic acid methyl ester; Ctrl, control; Cys, cysteine; EAM, experimental autoimmune myocarditis; EC, excitation-contraction; Epi, 15-epi-lipoxin A4; LXA4, lipoxin A4; Lut, luteolin; NRF2; NRF2, nuclear factor erythroid-derived 2-like 2; SCR, spontaneous diastolic Ca2+ release; SERCA2A; SERCA2A, sarcoplasmic reticulum–adenosine triphosphatase 2A; SPM, specialized proresolving mediator; SR, sarcoplasmic reticulum; Veh, vehicle; calcium handling; mRNA, messenger RNA; myocarditis; pro-resolving mediators.

Graphical abstract

Figure 1

BML-111 Administration Prevents Cardiac Dysfunction and Systolic Ca²⁺ Dysregulation in EAM-induced Mice (A) Experimental design. (B) Representative hematoxylin and eosin–stained slides of hearts from control + vehicle, (Ctrl+Veh), control + 5(S), (6)R, 7-trihydroxyheptanoic acid methyl ester (BML-111) (Crtl+ BML), experimental autoimmune myocarditis (EAM) + vehicle (EAM+Veh), and EAM + BML-111 (EAM+BML)-treated mice. Original magnification ×20. (C) Individual and mean values of ejection fraction (EF) in Ctrl+Veh (N = 7), Ctrl+BML (N = 8), EAM+Veh (N = 9), and EAM+BML (N = 10) mice. (D, left) Representative traces of calcium influx through L-type channels (I_CaL) obtained from −60 to +60 mV acquired in a cardiomyocyte from a vehicle-treated mouse. (Right) Mean density values of I_CaL recorded at all voltages tested in cardiomyocytes from Ctrl+Veh (N = 16, N = 4), Ctrl+BML (N = 18, N = 3), EAM+Veh (N = 12, N = 5), and EAM+BML (N = 18, N = 4) groups. (E) Representative line-scan confocal images and the corresponding profiles of Ca²⁺ transients from 1 cell in each experimental group. (F) Individual and mean values of peak fluorescence Ca²⁺ transients (left), the decay time constant (Tau) (center), and cell shortening (right) obtained in cardiomyocytes from Ctrl+Veh (N = 21, N = 5), Ctrl+BML (N = 28, N = 4), EAM+Veh (N = 26, N = 4), and EAM+BML (N = 26, N = 4) mice. Data show individual values and mean ± SD. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001 versus Ctrl+Veh; and ##P < 0.01 and ###P < 0.001 versus EAM+Veh.

Figure 2

BML-111 Treatment Normalizes SR-Ca²⁺ Load, Atp2a2 Down-Regulation, and Diastolic Ca²⁺ Release in EAM-Induced Mice (A, left) Individual and mean values of cardiac levels of Atp2a2 from Ctrl+Veh (N = 11), Ctrl+ BML (N = 7), EAM+Veh (N = 8), and EAM+BML (N = 10) mice. (Right) Relative quantification of sarcoplasmic reticulum–adenosine triphosphatase 2A (SERCA2A) by proteomics; Zq values are log₂ ratios expressed in SD units (see methods). (B, left) Representative line-scan images of caffeine-induced Ca²⁺ transients from all experimental groups. (Right) Individual and mean values of the amplitude of caffeine-induced Ca²⁺ transients (F/F0) in cardiomyocytes from Ctrl+Veh (N = 13, N = 5), Ctrl+BML (N = 22, N = 4), EAM+Veh (N = 23, N = 3), and EAM+BML (N = 18, N = 3) mice. (C, left) Representative 3-dimensional line-scan confocal images of spontaneous diastolic Ca²⁺ release (SCR) recordings (spontaneous Ca²⁺ transients release [upper] and Ca²⁺ waves [lower]) from EAM+Veh cardiomyocytes. (Right) SCR occurrence in cardiomyocytes from Ctrl+Veh (N = 14, N = 5), Ctrl+BML (N = 19, N = 5), EAM+Veh (N = 21, N = 3), and EAM+BML (N = 24, N = 4) mice. Data show individual values and mean ± SD. ∗P < 0.05 and ∗∗∗P < 0.001 versus EAM+Veh; and #P < 0.05 and ##P < 0.01 versus EAM+Veh. Abbreviations as in Figure 1.

Figure 3

Comparative Proteomics Analysis Reveals Coordinated Protein Abundance Changes in the Heart Proteome of EAM-Induced Mice After BML-111 Treatment (A) Functional categories significantly altered (∗false discovery rate <0.05) in the EAM+Veh, Ctrl+BML, and EAM+BML groups with respect to Ctrl+Veh (control). The Systems Biology Triangle algorithm was used to detect coordinated protein changes by BML-111 treatment. Proteins were functionally annotated using Gene Ontology, Kyoto Encyclopedia of Genes and Genomes, and Reactome terms retrieved from the Uniprot repository. Zc values are log₂ ratios of categories expressed in units of SD. Supplemental Table 4 displays Zq values for the protein components of the functional categories that were significantly altered. (B) Relative quantification of the proteins making up the “response to oxidative stress” category showing a highly interconnected network. Protein-protein associations were taken from the STRING database.(C) Redox proteomics analysis using the filter-aided stable isotope labeling of oxidized cysteine (Cys) method detects a significant increase in reversible Cys oxidation levels in the heart proteome in the comparisons EAM+Veh versus Ctrl+Veh and EAM+BML versus Veh+BML groups taking as reference the global behavior of all the peptides from the proteome, which follows the expected null hypothesis. The increase in oxidation level was significantly lower when comparing the modified Cys-containing peptide abundance changes from untreated animals (EAM+Veh vs Ctrl+Veh) with the corresponding BML-treated animals (EAM+BML vs Ctrl+BML). The cumulative distribution of Zpq values for the total and the modified Cys-containing peptides is shown. ATP = adenosine triphosphate; MHC = major histocompatibility complex; TCA = tricarboxylic acid; other abbreviations as in Figure 1.

Figure 4

Luteolin Administration Blunts the Cardiac Antioxidative Effects and NRF2 Activation Produced by BML-111 in EAM-Induced Mice (A) Experimental design. (B) Representative cardiac 8-hydroxy-2'-deoxyguanosine immunofluorescence staining in all experimental groups. (C) Individual and mean values of fluorescence expressed in arbitrary units (AUs) from Ctrl+Veh (N = 8), Ctrl+BML (N = 5), EAM+Veh (N = 9), and EAM+BML (N = 8) mice in the absence of luteolin and from Ctrl+Veh (N = 9), Ctrl+BML (N = 7), EAM+Veh (N = 8), and EAM+BML (N = 10) mice cotreated with luteolin. (D, upper) Representative Western blots of SERCA2A and laminin in nuclear heart lysates from all experimental groups. (Lower) Scatter plots summarizing the data in Ctrl+Veh (N = 4), Ctrl+BML (N = 6), EAM+Veh (N = 5), and EAM+BML (N = 5) cardiac samples from mice without luteolin or in Ctrl+Veh (N = 5), Ctrl+BML (N = 4), EAM+Veh (N = 6), and EAM+BML (N = 5) cardiac samples from mice cotreated with luteolin. Data show individual values and mean ± SD. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001 versus Ctrl+Veh; and $P < 0.05 and $$P < 0.01 versus EAM+Veh without luteolin. Abbreviations as in Figure 1.

Figure 5

Luteolin Blocks the Improvement in Cardiac Function and Ca²⁺ Dynamics Produced by BML-111 in EAM-Induced Mice (A) Experimental design. (B) Plots illustrate individual and mean values of EF in Ctrl+Veh (N = 10), Ctrl+ BML (N = 10), EAM+Veh (N = 7), and EAM+BML (N = 10) mice treated with luteolin. (C-E) Individual and mean values of peak fluorescence Ca²⁺ transients (C), decay time constant (D), and cell shortening (E) in cardiomyocytes from Ctrl+Veh (N = 36, N = 4), Ctrl+BML (N = 25, N = 4), EAM+Veh (N = 26, N = 4), and EAM+BML (N = 37, N = 4) from luteolin-treated mice. Data show individual values and mean ± SD. (F) Individual and mean values of the amplitude of caffeine-induced Ca²⁺ transients (F/F0) in cardiomyocytes from Ctrl+Veh (N = 10, N = 4), Ctrl+BML (N = 12, N = 4), EAM+Veh (N = 16, N = 4), and EAM+BML (N = 24, N = 4) mice treated with luteolin. (G) Graph shows SCR occurrence in cardiomyocytes from Ctrl+Veh (N = 10, N = 4), Ctrl+BML (N = 12, N = 4), EAM+Veh (N = 16, N = 4), and EAM+BML (N = 24, N = 4) mice treated with luteolin. Data show individual values and mean ± SD. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001 versus Ctrl+Veh; and $P < 0.05, $$P < 0.01, and $$$P < 0.001 versus Ctrl+BML. SR = sarcoplasmic reticulum; other abbreviations as in Figures 1 and 2.

Figure 6

Genetic Deletion of Nrf2 Tempers the Elevated Systolic Ca²⁺ Release and SR-Ca²⁺ Load and Uptake Induced by Epi in Isolated Cardiomyocytes Cardiomyocytes from wild-type (WT) and Nrf2 knockout (Nrf2^−/−) mice were incubated for 1 hour with Veh or 250 nmol/L 15-epi-lipoxin A4 (Epi). (A) Representative line-scan confocal images and the corresponding profiles of Ca²⁺ transients from 1 cell in each experimental group. (B) Individual and mean values of peak fluorescence Ca²⁺ transients (left), decay time constant (Tau) (center), and the rate of Ca²⁺ uptake (right) in WT+Veh (N = 25, N = 4); WT+Epi (N = 36, N = 4), Nrf2^−/−+Veh (N = 22, N = 3) and Nrf2^−/−+Epi (N = 24, N = 3) cardiomyocytes. (C) Individual and mean values of the amplitude of the caffeine-induced Ca²⁺ transients (F/F0) obtained in WT+Veh (N = 26, N = 4), WT+Epi (N = 36, N = 4), Nrf2^−/−+Veh (N = 15, N = 3), and Nrf2^−/−+Epi (N = 15, N = 3) cells. Data show individual values and mean ± SD. ∗∗P < 0.01 versus WT+Veh. (D, upper) Representative Western blots of SERCA2A and vinculin from cytosolic heart lysates from WT mice treated with Veh (Ctrl) or BML-111 (BML) for 6 hours. (Lower) Scatter plots summarizing the data in Ctrl (N = 4) and BML-111–treated (N = 5) mice expressed as arbitrary units. Data show individual values and mean ± SD. ∗P < 0.01 versus Ctrl. Abbreviations as in Figures 1, 2, and 5.

Figure 7

Correlation Between NRF2 and SERCA2A2 Expression in Human Ventricular Cells and in Myocardium From Patients With Myocarditis (A) AC16 cells were transduced with a lentivirus carrying a short hairpin RNA (shRNA) against a scramble sequence (shCTRL) or against NRF2 (shNRF2). (Left) Representative Western blots of the indicated proteins were determined 7 days post-transduction. Vinculin was used as loading control. (Right) Densitometry quantification of SERCA2A protein levels normalized to vinculin levels. Data are mean ± SD (N = 4). ∗∗∗P < 0.001 versus shCTRL transduced cells. (B) AC16 cells were transduced with a lentivirus expressing green fluorescent protein (GFP) or a constitutive active version of NRF2 (NRF2-ΔETGE). (Left) Representative images showing efficient transduction 4 days post-lentivirus delivery. (Center) Representative Western blots of the indicated proteins (NRF2, HMOX1, NQO1, GFP, and SERCA2A) were determined 4 days post-transduction. Vinculin was used as a loading control. (Right) Densitometry quantification of SERCA2A protein levels normalized to vinculin levels. Data are mean ± SD (N = 4). ∗P < 0.05 and ∗∗∗P < 0.001 versus GFP transduced cells. (C, left) Representative hematoxylin and eosin–stained slides of healthy myocardium (N = 5) and myocardium from patients with myocarditis (N = 8). Original magnification ×10 (right) and ×20 (left). (D, E) Individual and mean values of messenger RNA levels of NFE2L2(D) and ATP2A2(E) normalized to human 36B4. (F) Pearson correlation analysis of ATP2A2 and NFE2L2 messenger RNA levels in human samples. A linear regression of the data is shown. Data show individual values and mean ± SD.∗P < 0.01 and ∗∗P < 0.01 versus healthy group. FI = fold induction; other abbreviations as in Figures 1 and 2.