TRAF2, an Innate Immune Sensor, Reciprocally Regulates Mitophagy and Inflammation to Maintain Cardiac Myocyte Homeostasis

Abstract

Mitochondria are essential for cardiac myocyte function, but damaged mitochondria trigger cardiac myocyte death. Although mitophagy, a lysosomal degradative pathway to remove damaged mitochondria, is robustly active in cardiac myocytes in the unstressed heart, its mechanisms and physiological role remain poorly defined. We discovered a critical role for TRAF2, an innate immunity effector protein with E3 ubiquitin ligase activity, in facilitating physiological cardiac myocyte mitophagy in the adult heart, to prevent inflammation and cell death, and maintain myocardial homeostasis.

Keywords: AAV9, adeno-associated virus serotype 9; ER, endoplasmic reticulum; FS, fractional shortening; GFP, green fluorescent protein; IP, intraperitoneal; LV, left ventricular; MAM, mitochondria-associated membranes; MCM, MerCreMer; MEF, murine embryonic fibroblast; PINK1, PTEN-induced kinase 1; RFP, red fluorescent protein; TLR9, toll-like receptor 9; TRAF2; TRAF2, tumor necrosis factor receptor-associated factor-2; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling; cTnT, cardiac troponin T; cell death; inflammation; mitophagy.

Graphical abstract

Figure 1

TRAF2 Localizes to the Mitochondria in Human and Mouse Hearts (A and B) Immunoblots depicting TRAF2 expression in hearts from individuals evaluated as donors for cardiac transplantation, without evidence for cardiomyopathy (donor) or patients with end-stage ischemic cardiomyopathy (ICM) undergoing cardiac transplantation. The hearts were subjected to biochemical fractionation into mitochondria-enriched (B) and cytosolic (A) fractions, shown by segregation of VDAC, a mitochondrial protein and GAPDH, a cytosolic protein, respectively. (C and D) Quantitative assessment of TRAF2 expression in the respective biochemical fractions are shown in A and B. (E and F) Representative immunoblot and quantitation are shown depicting total TRAF2 expression in crude extracts from human hearts from patients with ischemic cardiomyopathy (ICM) or donors as control. (G and H) Representative immunoblot is shown depicting TRAF2 expression in hearts from male C57BL/6J mice (8 weeks old) subjected to ex vivo ischemia-reperfusion injury (IR) or sham (S) treatment as control. Expression of VDAC, FACL4, calreticulin, VAPB, and IRE1α is shown to evaluate cosegregation with mitochondria (mito, with VDAC), mitochondria-associated membranes (MAM, with FACL4), endoplasmic reticulum (ER, with calreticulin, VAPB, and IRE1α), and cytosol (cyto, with GAPDH). ∗P < 0.05 and ∗∗∗P < 0.001, by t-test. No statistically significant differences were observed between the groups for TRAF2 abundance in the cyto fraction (by t-test) or ER (by Mann-Whitney test).

Figure 2

TRAF2 Ablation in Adult Cardiac Myocytes Induces Cardiomyopathy With Damaged Mitochondria and Cell Death (A and B) Left ventricular (LV) endocardial fractional shortening (%FS (A) and end-diastolic diameter (B) in wild-type, Traf2 floxed (TRAF2 fl/fl), MerCreMer (MCM) mice, and mice homozygous for Traf2 floxed alleles carrying the MerCreMer transgene (to generate TRAF2-icKO), 14 days after tamoxifen treatment (20 mg/kg/d, IP 5 days per week for 3 weeks). n = 9 to 10 mice per group. ∗P < 0.05 and ∗∗∗P < 0.001 for comparison versus wild-type group; ###P < 0.001 for comparison versus Traf2 fl/fl group by post hoc test after 2-way ANOVA. (C and D) Representative images with hematoxylin and eosin (H and E) staining (C), and trichrome staining (D) to evaluate myocardial structure and fibrosis, respectively, in mice as treated in A and B. (E) Representative transmission electron microscopy images to evaluate myocardial ultrastructure in mice as in A. Arrows indicate swollen mitochondria with cristal rarefication. (F) Representative TUNEL-stained images to evaluate cardiac myocyte TUNEL positivity (green) in mice as in A. DAPI staining identifies nuclei. Staining for α-sarcomeric actin was performed to evaluate TUNEL staining in cardiac myocyte nuclei. %TUNEL-positive nuclei/total cardiac myocyte nuclei are depicted. n = 4 to 6/group. ∗∗P < 0.01 versus Traf2 fl/fl by post hoc test after 1-way ANOVA. ANOVA = analysis of variance; TAM = tamoxifen; TEM = transmission electron microscopy; TUNEL = terminal deoxynucleotidyl transferase dUTP nick end labeling; WT = wild type.

Figure 3

TRAF2 Ablation in Adult Cardiac Myocytes Impairs Homeostatic Mitophagy (A) Representative immunoblot is shown depicting expression of PARKIN and mitochondrial proteins (VDAC, TOMM20, COXIV) in cardiac extracts from mice at day 35 after inducible adult-onset ablation of TRAF2 (TRAF2-icKO), 14 days after tamoxifen (TAM) treatment (20 mg/kg/d, intraperitoneal for 5 days per week for 3 weeks). n = 4/group. Protein levels were normalized to GAPDH. Upward arrow points to up-regulation in abundance of indicated protein indicated as a % change. ∗P < 0.05 for TRAF2-icKO versus Traf2 fl/fl groups by t-test; and. (B to D) Representative images (B) from hearts transduced with AAV9-mitoKeima in mice as in A; with the ratio of quantitation of mitoKeima emission in the red channel over green as an index of mitophagy (C). Total mitochondria were assessed by abundance of the green signal (arbitrary units, au) per unit area of transduced myocytes (D). ∗P < 0.05 and ∗∗P < 0.01 by t-test. (E-G) Representative images (E) for assessment of flux through macroautophagy in TRAF2-icKO versus Traf2 fl/fl mice carrying the RFP-GFP-LC3 reporter transgene, with quantitative assessment of the ratio of autolysosomes (punctate RFP signal) to autophagosomes (punctate RFP+GFP signal, in F); and total autophagosomes (RFP+GFP puncta) and autolysosomes (RFP puncta) per unit myocardial area in G. No statistically significant differences were observed between TRAF2-icKO versus Traf2 fl/fl mice by t-test in F and G. GFP = green fluorescent protein; RFP = red fluorescent protein.

Figure 4

TRAF2 Ablation in Adult Cardiac Myocytes Up-Regulates TLR9 and Mitochondrial DNA Sensing (A) Representative images demonstrating colocalization of PicoGreen with TLR9 in myocardial sections from mice with adult-onset inducible TRAF2 ablation (TRAF2-icKO) and Traf2 floxed control mice, without and with concomitant germline TLR9 ablation. Nuclei are stained blue (DAPI). Arrowheads demonstrate colocalization of PicoGreen with TLR9. Arrows point to cytosolic detection of DNA by PicoGreen in TRAF2-icKO-TLR9KO myocardium. (B and C) Immunoblot (B) and quantitation (C) depicting TLR9 expression in cardiac extracts from TRAF2-icKO and Traf2 floxed control mice modeled as in A. ∗P < 0.05 and ∗∗P < 0.01 by t-test. (D)Tlr9 transcript expression in cardiac extracts from TRAF2-icKO and Traf2 floxed control mice as in A. The P value depicted is by t-test. (E) In situ hybridization to detect Tlr9 transcript (red) and its colocalization with DESMIN (green) to identify cardiac myocytes. Arrowheads point to TLR9 transcripts in cardiac myocytes. Representative section from Traf2 fl/fl Tlr9-null mouse is shown as a control.

Figure 5

TLR9 Ablation Rescues Inflammatory Cell Infiltration and Cell Death in Mice With Inducible TRAF2 Ablation in Adult Cardiac Myocytes, in the Short Term (A) Representative images depicting CD68⁺ cells in myocardial section from mice with adult-onset inducible TRAF2 ablation (TRAF2-icKO) and Traf2 floxed control mice, without and with concomitant germline TLR9 ablation, 2 weeks after TRAF2 ablation. Nuclei are stained blue (DAPI). (B) Quantitation of CD68⁺ cells is shown as % of all DAPI-stained nuclei in mice as in A. P values are by post hoc test after 1-way ANOVA. (C and D) Representative TUNEL-stained images (C) with quantitative assessment (D) of cardiac myocyte TUNEL positivity (green; %TUNEL-positive nuclei/total cardiac myocyte [CM] nuclei) in mice as in A. DAPI staining identifies nuclei. Staining for α-sarcomeric actin was performed to evaluate TUNEL staining in cardiac myocyte nuclei. P values are by post hoc test after 1-way ANOVA. (E to H) Left ventricular endocardial fractional shortening (%FS, E), end-diastolic diameter (LVEDD, in millimeters, F), LV mass (in milligrams, G), and heart weight normalized to tibial length (in milligrams per millimeter, H) in mice modeled as in A. P values are by post hoc test after 1-way ANOVA. (I-L) Expression of Nppa(I), Nppb(J), Atp2a2(K), and Acta1(L) transcripts in mice modeled as in A. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001 by post hoc test after 1-way ANOVA for all panels except by Kruskal-Wallis test for H and J. Abbreviations as in Figure 2.

Figure 6

TLR9 Ablation Does Not Rescue Mitochondrial Abnormalities and Does Not Prevent Cardiomyopathy With Inducible Cardiac Myocyte TRAF2 Ablation With Longer-Term Follow-Up (A) Representative transmission electron microscopy images to evaluate myocardial ultrastructure in mice as with adult-onset inducible TRAF2 ablation (TRAF2-icKO) and Traf2 floxed control mice, without and with concomitant germline TLR9 ablation, 2 weeks after TRAF2 ablation. (B to E) Mice with adult-onset inducible TRAF2 ablation or Traf2 floxed alleles in TLR9-null (knockout [KO]) background (TRAF2-icKO TLR9KO and TRAF2 fl/fl TLR9KO) were followed by echocardiographic evaluation at 36 weeks after tamoxifen (TAM) treatment to induce TRAF2 ablation. Left ventricular end-diastolic diameter (LVEDD, mm (B), endocardial fractional shortening (%FS) (C), mass (LVM, in milligrams (D), and ratio of the chamber diameter to wall thickness (r/h) (E) was assessed as shown. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001 by t-test.

Figure 7

Restoration of TRAF2, but Not Its E3 Ligase–Deficient Mutant, Rescues Cardiomyopathy and Mitochondrial Abnormalities With Inducible Cardiac Myocyte TRAF2 Ablation (A) Schematic depicting experimental strategy for tamoxifen-inducible TRAF2 ablation in mice homozygous for floxed Traf2 alleles carrying the Myh6-MerCreMer transgene, followed by AAV9-cTnT-TRAF2 or TRAF2Rm particles at 5.0 × 10¹¹ viral particles per mouse. (B) Left ventricular endocardial fractional shortening (%FS) in TRAF2icKO mice or TRAF2 floxed mice transduced with AAV9 particles coding for TRAF2 or TRAF2Rm, driven by a cardiac troponin T promoter as shown in A. ∗P < 0.05 by post hoc test after 1-way ANOVA. (C-G) Representative immunoblot (C) with quantitation of TRAF2 expression (D) and abundance of mitochondrial proteins (VDAC, E; TOMM20, F; COXIV, G); all normalized to actin) in cardiac extracts from mice modeled as in A. ∗∗P < 0.01 and ∗∗∗P < 0.001 by post hoc test after 1-way ANOVA. (H) Representative transmission electron microscopy images to evaluate myocardial ultrastructure in mice as in A. Scale bar = 500 nm. (I and J) Representative images (I) are shown demonstrating mitophagy with mitoQC reporter expression with quantitative assessment (J), 4 weeks after injections of AAV9-cTnT-TRAF2 or empty viral particles (AAV9-cTnT-null) in 8-week-old mitoQC reporter mice. ∗P < 0.05 by t-test. Abbreviations as in Figure 2.

Figure 8

TRAF2 Ablation Induced Cell Death, Which Can Be Rescued by Parkin-Induced Mitophagy (A) Murine embryonic fibroblasts (MEFs) carrying floxed Traf2 alleles were modeled for TRAF2 ablation with adenoviral Cre (or LacZ as control, each at a multiplicity of infection [MOI] = 100) treatment for 72 hours as in Figure S11 and subjected to transmission electron microscopic (TEM) analysis. Representative TEM images are shown demonstrating mitochondrial abnormalities noted after Traf2 ablation as compared with control mice. (B and C) Representative images (B) and quantitative analyses of mitoKeima emission in the red channel over green as an index of mitophagy (C), in Traf2-null MEFs (modeled as in A) expressing mitoKeima transduced with adenoviral Parkin or LacZ (MOI = 100 for 48 hours) as control. Adenoviral particles coding for LacZ were added to equalize the number of viral particles per treatment. ∗P < 0.05 by post hoc test after 1-way ANOVA. (D) Cell death in Traf2 null MEFs (modeled as in A) and transduced with adenoviral Parkin or LacZ (MOI = 100 for 72 hours) as control. Adenoviral particles coding for LacZ were added to equalize the number of viral particles per treatment. ∗∗∗P < 0.001 by post hoc test after 1-way ANOVA. (E) Schematic depicting role of TRAF2-induced mitophagy in preventing inflammation and cell death in cardiac myocyte homeostasis. TRAF2 localizes to the mitochondria and promotes mitophagy via K63-ubiquitination activity in unstressed cardiac myocytes (left). Ablation of TRAF2 results in mitochondrial DNA leak, and up-regulates TLR9 abundance to provoke inflammation via mitochondria DNA sensing. The resultant inflammation and accumulation of damaged mitochondria provoke cardiac myocyte cell death and cardiomyopathy (middle). Concomitant ablation of TLR9 in the setting of TRAF2 deficiency prevents inflammation and cell death, and rescues left ventricular hypertrophy and function in the short term; but does not prevent accumulation of damaged mitochondria and cell death inexorably resulting in cardiomyopathy during follow up (right).