Triad3A-Mediated K48-Linked ubiquitination and degradation of TLR9 impairs mitochondrial bioenergetics and exacerbates diabetic cardiomyopathy

Abstract

Introduction: Targeted protein degradation represents a promising therapeutic approach, while diabetic cardiomyopathy (DCM) arises as a consequence of aberrant insulin secretion and impaired glucose and lipid metabolism in the heart..

Objectives: Considering that the Toll-like receptor 9 (TLR9) signaling pathway plays a pivotal role in regulating energy metabolism, safeguarding cardiomyocytes, and influencing glucose uptake, the primary objective of this study was to investigate the impact of TLR9 on diabetic cardiomyopathy (DCM) and elucidate its underlying mechanism.

Methods: Mouse model of DCM was established using intraperitoneal injection of STZ, and mice were transfected with adeno-associated virus serotype 9-TLR9 (AAV9-TLR9) to assess the role of TLR9 in DCM. To explore the mechanism of TLR9 in regulating DCM disease progression, we conducted interactome analysis and employed multiple molecular approaches.

Results: Our study revealed a significant correlation between TLR9 expression and mouse DCM. TLR9 overexpression markedly mitigated cardiac dysfunction, myocardial fibrosis, oxidative stress, and apoptosis in DCM, while inflammation levels remained relatively unaffected. Mechanistically, TLR9 overexpression positively modulated mitochondrial bioenergetics and activated the AMPK-PGC1a signaling pathway. Furthermore, we identified Triad3A as an interacting protein that facilitated TLR9's proteasomal degradation through K48-linked ubiquitination. Inhibiting Triad3A expression improved cardiac function and pathological changes in DCM by enhancing TLR9 activity.

Conclusions: The findings of this study highlight the critical role of TLR9 in maintaining cardiac function and mitigating pathological alterations in diabetic cardiomyopathy. Triad3A-mediated regulation of TLR9 expression and function has significant implications for understanding the pathogenesis of DCM. Targeting TLR9 and its interactions with Triad3A may hold promise for the development of novel therapeutic strategies for diabetic cardiomyopathy. Further research is warranted to fully explore the therapeutic potential of TLR9 modulation in the context of cardiovascular diseases.

Keywords: Diabetic cardiomyopathy; Mitochondrial bioenergetics; Toll-like receptor 9; Triad3A; Ubiquitination.

Graphical abstract

Fig. 1

Decreased TLR9 levels in HG-stimulated cardiomyocytes and diabetic mice heart. A. TLR9 level in NRCMs stimulated by high glucose (HG) at different time periods (n = 6); *p < 0.05 vs the PBS group; B. Representative images of immunofluorescence of TLR9 (red), and α-actinin (green) in HG-treated NRCMs (n = 6); C. Representative western blot analysis of TLR9 in cardiac tissues from healthy and diabetic mice (n = 6); D. Immunohistochemical staining of TLR9 in heart tissues from healthy and diabetic mice (n = 6). *p < 0.05 vs the Veh group. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 2

TLR9 overexpression attenuates cardiac dysfunction and fibrosis in diabetic mice. A. Blood glucose of mice at duration of diabetes (n = 10); B-C. hemodynamic measurements. Peak derivative of pressure overtime (+dP/dt) is the maximal value of the instantaneous first derivative of left ventricular pressure; minimum peak derivative of pressure over time (-dP/dt) is the minimum value of the instantaneous first derivative of left ventricular pressure (n = 10); D-I. Representative B-model and M−model echocardiography in mice and cardiac function presented as ejection fraction (EF), fractional shortening (FS), cardiac output, left ventricular diastolic diameter (LVIDd) and E/A ratio (n = 10); J-K. Representative images of Sirius red staining and quantification of fibrotic area in cardiac tissues (n = 6); L-N. Immunoblotting blot analysis of Col-Ⅰ and α-sma protein levels in cardiac tissues; O. The mRNA levels of ctgf, col 1,col 3 in the cardiac tissues (n = 6). Significance was assessed by one-way ANOVA and Tukey’s post hoc test. Data are shown as the mean ± SEM. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 3

TLR9 overexpression alleviates diabetes-induced oxidative injury and apoptosis. A-B. Representative images of DHE staining in the cardiac tissues and quantification of the corresponding fluorescence intensity (n = 6); C. 3-NT levels in cardiac tissues (n = 6); D. MDA levels in cardiac tissues (n = 6); E. 4-HNE levels in cardiac tissues (n = 6); F. SOD activity in cardiac tissues (n = 6); G-H. Representative images and quantification (n = 6, 10 + fields per heart) of 4-HNE Immunohistochemistry staining in diabetic hearts; I-K. Western blot analysis of p67phox and SOD2 in diabetic mice hearts (n = 6); L-M. TUNEL assay by double staining with DAPI (blue) and TUNEL (green) detected apoptotic cells in mouse hearts. The quantification of TUNEL positive nuclei is shown (n = 6); N-P. Representative western blot and analysis of Bcl-2, Bax in diabetic hearts (n = 6). Significance was assessed by one-way ANOVA and Tukey’s post hoc test. Data are shown as the mean ± SEM. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 4

TLR9 reinstates mitochondrial function and reduces energy substrates in the Mouse Model of Diabetes. A. Representative transmission electron microscopic images of mitochondria in mice treated with vehicle or STZ (magnification × 3000 [3 K], scale bar 5 μm; ×12,000 [12 K], scale bar 1 μm); B-E. Quantitative analysis of mitochondrial size, mitochondrial cristae number, cristae area and the proportion of mitochondria with disorganised cristae. (n = 12); F-G. Activity of pyruvate dehydrogenase (PDH) and citrate synthase (CS) were analyzed in heart tissues from indicated mice (n = 8); H, Oxygen consumption rate (OCR) in AMCMs isolated from indicated mice (n = 6); I, Extracellular flux analyzer traces of a glycolysis stress measured as the extracellular acidification rate (ECAR) in AMCMs isolated from indicated mice (n = 6); J-N. Measurements of ATP, ADP, AMP, ADP/ATP ratio and AMP/ATP ratio in cardiac tissues (n = 6). Significance was assessed by one-way ANOVA and Tukey’s post hoc test. Data are shown as the mean ± SEM.

Fig. 5

TLR9 promotes PGC-1α activation and Nrf2 activation and nuclear translocation in vivo A-H. Western blot analysis (left panel) and densitometric quantification (right panel) of phosphorylated AMPKα2, total AMPKα2, PGC-1α, Nrf2, HO1, Nox2, Nox4 in cardiac tissues of each group (n = 6); I-J. Representative images and quantification (n = 6, 10 + fields per heart) of PGC-1α in mice hearts (n = 6); K. Representative images of immunofluorescence of Nrf2 (red) and α-actinin (green) in cardiac tissues. Significance was assessed by one-way ANOVA and Tukey’s post hoc test. Data are shown as the mean ± SEM. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 6

Triad3A interacts with TLR9 and promotes K48-linked ubiquitination A. The mRNA of TLR9 in cardiac tissues from healthy and diabetic mice (n = 6); B. Representative western blot analysis of Triad3A in cardiac tissues from healthy and diabetic mice (n = 6); C. Immunoblots of the ubiquitinated tlr9 after Triad3A overexpression; D. Immunoblots of the ubiquitinated TLR9 in NRCMs treated with or without HG; E. Immunoblots of the ubiquitinated TLR9 in myocardial cells treated with or without STZ; F. Coimmunoprecipitation (coIP) assays were performed to examine the interaction of Triad3A and TLR9 in 293 T cells transfected with the indicated plasmids; G. GST pull-down assays showing the interaction Triad3A and TLR9 in 293 T cells transfected with the indicated plasmids. H-I. In screening for potential lysine ubiquitination types, the ubiquitination of Myc-TLR9 in response to Triad3A overexpression was examined in NRCMs transfected with the wild-type (WT) and mutated Myc-Ub plasmids and treated with HG. J. Immunoblotting analysis of TLR9 in NRCMs infected with HA-Triad3A and treated with HG (20 μM) for 24 h and cycloheximide (CHX, 50 μM) for the indicated time points. K, Immunoblotting analysis of TLR9 in NRCMs after Triad3A overexpression for 24 h and treatment with HG for 24 h, dimethyl sulfoxide (DMSO), MG132 (50 μM) or chloroquine (CQ, 50 μM) for 6 h.

Fig. 7

Triad3A silence inhibits STZ-induced diabetic heart injury and energy metabolism disorders through TLR9. A. hemodynamic measurements. Peak derivative of pressure overtime (+dP/dt) is the maximal value of the instantaneous first derivative of left ventricular pressure (n = 10); B-C. Echocardiographic assessment for each group (n = 10); D. left ventricular diastolic diameter (n = 10); E. E/A ratio(n = 10); F. Representative images of Sirius red staining, DHE staining, and TUNEL staining in cardiac tissues. G. Quantification of fibrotic area (n = 10); H. Quantification of the DHE fluorescence intensity (n = 10); I. Quantification of the TUNEL positive nuclei (n = 10); J-N. Measurements of ATP, ADP, AMP, ADP/ATP ratio, and AMP/ATP ratio in cardiac tissues. O-Q. Western blot analysis (left panel) and densitometric quantification (right panel) of phosphorylated AMPKα2, total AMPKα2, PGC-1α in cardiac tissues (n = 6). Significance was assessed by one-way ANOVA and Tukey’s post hoc test. Data are shown as the mean ± SEM. R. Graphical Abstract: Triad3A elevation induced by STZ or HG stimulation leads to degradation of TLR9 K48-linked proteasomal ubiquitination, which impairs mitochondrial function and reduces the AMP/ATP ratio and inhibits the activation of AMPK and PGC-1, which in turn leads to oxidative stress, apoptosis and fibrosis and other pathological changes. STZ: Streptozotocin; HG: High glucose; TLR9: Toll-like receptor 9; AMP: Adenosine monophosphate; ATP: Adenosine triphosphate; AMPK: Adenosine 5‘-monophosphate (AMP)-activated protein kinase; PGC-1α: Peroxisome-proliferator-activated receptorγcoactivator-1α. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)