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. 2020 Oct 9;3(1):562.
doi: 10.1038/s42003-020-01289-2.

The E3 ubiquitin ligase HectD3 attenuates cardiac hypertrophy and inflammation in mice

Ashraf Yusuf Rangrez  1   2 , Ankush Borlepawar #  3   4 , Nesrin Schmiedel #  3   4 , Anushka Deshpande  3   4 , Anca Remes  3   4 , Manju Kumari  5 , Alexander Bernt  3   4 , Lynn Christen  3 , Andreas Helbig  6 , Andreas Jungmann  7 , Samuel Sossalla  8 , Andreas Tholey  6 , Oliver J Müller  3   4 , Derk Frank  3   4 , Norbert Frey  9   10
Affiliations

The E3 ubiquitin ligase HectD3 attenuates cardiac hypertrophy and inflammation in mice

Ashraf Yusuf Rangrez et al. Commun Biol. .

Abstract

Myocardial inflammation has recently been recognized as a distinct feature of cardiac hypertrophy and heart failure. HectD3, a HECT domain containing E3 ubiquitin ligase has previously been investigated in the host defense against infections as well as neuroinflammation; its cardiac function however is still unknown. Here we show that HectD3 simultaneously attenuates Calcineurin-NFAT driven cardiomyocyte hypertrophy and the pro-inflammatory actions of LPS/interferon-γ via its cardiac substrates SUMO2 and Stat1, respectively. AAV9-mediated overexpression of HectD3 in mice in vivo not only reduced cardiac SUMO2/Stat1 levels and pathological hypertrophy but also largely abolished macrophage infiltration and fibrosis induced by pressure overload. Taken together, we describe a novel cardioprotective mechanism involving the ubiquitin ligase HectD3, which links anti-hypertrophic and anti-inflammatory effects via dual regulation of SUMO2 and Stat1. In a broader perspective, these findings support the notion that cardiomyocyte growth and inflammation are more intertwined than previously anticipated.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. HectD3 interacts with and suppresses SUMO2-dependent activation of calcineurin-signaling and cardiomyocyte hypertrophy.
a Pictorial representation of functional domains of HectD3 and its interaction region with SUMO2. The SUMO2-binding region from Yeast-two hybrid assay was located in the APC10 domain of HectD3 which is also involved in the E3 ubiquitin ligation of its target proteins. b The SUMO2–HectD3 interaction was further confirmed by immunoprecipitation of the endogenous proteins using mouse heart lysate. SUMO2-interacting proteins were pulled-down using anti-SUMO2/3 antibody and were evaluated by immunoblotting using anti-HectD3 antibody. c Immunofluorescence microscopy suggests scattered localization of HectD3 in cytoplasm and perinuclear region where it co-localizes with SUMO2. d NFAT response element-driven Firefly luciferase reporter assay was performed in NRVCMs to study the effect of overexpression of HectD3 (HD3) in and/or SUMO2 (S2), with or without constitutively active calcineurin A (CnA). Box plot indicates the inhibitory effects of HectD3 on SUMO2-mediated or CnA-mediated activation of luciferase activity (n = 12 each). e Expression of fetal genes nppa and nppb determined by quantitative real-time PCR indicates downregulation of both the genes when HectD3 is overexpressed (n = 9 each). f Cell surface area measurement of NRVCM overexpressing SUMO2, HectD3, or both, indicates SUMO2 increases cellular hypertrophy, whereas, the presence of HectD3 attenuated this activation (n > 1500 each). g Immunoblots showing the expression of Calcineurin A (CnA), SUMO2/3, and HectD3 of cytosolic or nuclear protein extracted from neonatal rat cardiomyocytes expressing delta calcineurin A (δCnA) alone, with SUMO2, or with SUMO2 and HectD3. Histone H3 and GAPDH were used as markers for nuclear or cytoplasmic proteins, respectively. h Densitometry analysis of CnA indicates its reduced or increased expression in cytosol or nucleus, respectively, upon SUMO2 overexpression; the presence of HectD3 displayed no effect (n = 4 each). Densitometry analysis of δCnA however indicates its significantly reduced expression in both cytosol and nucleus in cardiomyocytes, where HectD3 was overexpressed together with SUMO2 and δCnA. Densitometry analysis of free SUMO2/3 indicates its significantly reduced expression in both cytosol and nucleus of cardiomyocytes where HectD3 was overexpressed together with SUMO2 and δCnA (n = 4 each). Statistical calculations were carried out by one-way ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001.
Fig. 2
Fig. 2. HectD3 regulates SUMO2-mediated sumoylation.
a Immunoblot indicating the expression of SUMO2/3 and sumoylated proteins in neonatal rat cardiomyocytes (NRVCMs) overexpressing SUMO2, HectD3, both, or LacZ. b and c represents densitometry analysis of immunoblot a for free SUMO2/3 or sumoylated proteins, respectively (n = 4 each). Adenovirus-mediated expression of HectD3 in NRVCMs is found to downregulate SUMO2/3 and sumoylated proteins, both endogenous as well as overexpressed. NRVCMs expressing LacZ were used as a control condition. d Immunoblot indicating the expression of HectD3 and SUMO2/3-sumoylated proteins in cardiac protein lysates obtained from human patients suffering from hypertrophic cardiomyopathy (HCM). Non-failing (NF) heart samples were used as controls. e Immunoblot indicating the expression of SUMO2/3 and sumoylated proteins in neonatal rat cardiomyocytes (NRVCMs) overexpressing SUMO2 or both, HectD3 and SUMO2, in the absence or the presence of MG132, a proteasome inhibitor. f Box plot representing densitometry analysis of SUMO2/3-sumoylated proteins and HectD3 shown in image d (n = 4 (control), 5 (HCM)). HectD3 is found to be significantly downregulated, whereas, SUMO2/3-mediated protein sumoylation is significantly increased in HCM patient hearts. g Box plot representing densitometry analysis of free SUMO2/3 or sumoylated proteins shown in image e clearly shows that MG132 attenuates degradation of SUMO2/3 or sumoylated proteins by HectD3 (n = 3). Statistical calculations were carried out by one-way ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001.
Fig. 3
Fig. 3. HectD3 targets interferon response proteins and Stat1 signaling in NRVCMs.
a Heat map showing the differentially abundant proteins in NRVCMs after HectD3 overexpression quantified by LC–MS/MS. Protein lysate from NRVCMs overexpressing LacZ (n = 3 each). b A number of proteins involved in interferon response signaling, including Stat1, displayed significantly reduced levels during HectD3 overexpression. c Venn-diagram indicating the comparative analysis of differentially expressed proteins and mRNAs determined by mass spectrometry or RNA-sequencing, respectively. Overall, 13 molecules were significantly downregulated, both, at protein and RNA levels, which are shown in box plots b and d, respectively. e Immunoblot image displaying the expression of Stat1/p-Stat1 and Stat3/p-Stat3 in NRVCMs overexpressing LacZ (as a control) or HectD3. f Stat3 and p-Stat3 were not significantly altered, whereas, Stat1 and p-Stat1 were found to be significantly downregulated upon HectD3 overexpression as determined by densitometry analysis of image shown in e (n = 3 each). g Immunoblots indicating the input and IP samples showing Stat1, HectD3, and ubiquitin detected by anti-Stat1, anti-HectD3, and anti-HA antibodies, respectively. Images show that anti-HA beads could successfully pull down HectD3 as well as polyubiquitinated Stat1 only in the condition where HectD3 and ubiquitin were expressed with Stat1. Statistical calculations were carried out by two-tailed Student’s t-test. *p < 0.05, **p < 0.01, ***p < 0.001.
Fig. 4
Fig. 4. HectD3 inhibits LPS-/IFNγ-mediated activation of the inflammatory response in cardiomyocytes.
a Immunoblots displaying the expression of Stat1/p-Stat1 in control and HectD3 overexpressing NRVCMs in the absence or the presence of LPS. Densitometry analyses of a for Stat1/p-Stat1 are shown in b and c indicating that LPS significantly activated Stat1 by its phosphorylation, while HectD3 overexpression blunted this activation. (n = 3 each). d Immunoblots displaying the expression of Stat1/p-Stat1 in control and HectD3 overexpressing NRVCMs in the absence or the presence of IFNγ. Densitometry analyses of b for Stat1/p-Stat1 are shown in e and f indicating that IFNγ significantly activated Stat1 by its phosphorylation, while HectD3 overexpression blunted this activation (n = 3 each). g Transcript levels of several interferon responsive Stat1-signaling genes determined by quantitative real-time PCR revealing the same trend, i.e. upregulation upon LPS treatment in controls (LacZ) and reduction in upregulation upon HectD3 overexpression. (n = 6 each). Statistical calculations were carried out by two-way ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001.
Fig. 5
Fig. 5. HectD3 suppresses SUMO2-dependent activation of calcineurin-signaling and cardiac hypertrophy.
a Relative HectD3 expression in mouse hearts where HectD3-AAV was injected intravenously to find a suitable dose for further experiments. HectD3 was found to be significantly expressed only in mice injected with 1 × 1012 times vector/mouse genome (n = 5 each). b Immunoblots further confirms the upregulation of HectD3 in mouse hearts harvested from mice injected with HectD3 (1 × 1012 times vector/mouse genome) compared to the AAV-luciferase (control), which is presented as a box plot in c (n = 5 each). d Relative HectD3 expression in various tissues obtained from mice injected with AAV-HectD3 demonstrates significant upregulation of HectD3 only in the heart compared to other tissues (n = 5 each). e Box plot indicating the heart weight (wt):body wt ratio in mice that were injected with different doses of AAV-HectD3. Data indicate no significant effect of HectD3 overexpression (n = 5 each). f Schematic outline of the experimental workplan for AAV-mediated overexpression of HectD3 in mice, including transverse aortic constriction (TAC) and downstream experiments. g Box plot indicating the heart weight (wt):body wt ratios in mice that underwent TAC or sham operations compared to the respective control group where HectD3 or luciferase (Luc) was overexpressed using equivalent AAV9 particles. Data indicates that TAC significantly increased the heart weight (wt):body wt ratio in control mice, whereas, AAV9-mediated overexpression of HectD3 reduced the ratio (n = 8 (AAV-Luc sham), 9 (AAV-HectD3 sham), 13 (AAV-Luc TAC), 14 (AAV-HectD3 TAC)). h Similarly, increased HectD3 expression reduced the cardiomyocyte cell surface area in mice that underwent TAC compared to the respective Luc mice group (n > 150 each). i Fractional shortening (%) as a measure of contractile function was also moderately improved in TAC operated mice when HectD3 was overexpressed. Expression of fetal genes determined by quantitative real-time PCR indicates upregulation of nppa j, nppb k, rcan1.4 l, β-myosin heavy chain (myh7) m, and downregulation of α-myosin heavy chain (myh6) n after TAC; HectD3 overexpression however reduced this up-/down-regulation (n = 8 (AAV-Luc sham), 9 (AAV-HectD3 sham), 13 (AAV-Luc TAC), 14 (AAV-HectD3 TAC)). Similarly, TAC upregulated fibrosis markers Collagen 1a (col1a) o, and 3a (col3a) p which was again attenuated by HectD3 overexpression (n = 8 (AAV-Luc sham), 9 (AAV-HectD3 sham), 13 (AAV-Luc TAC), 14 (AAV-HectD3 TAC)). Statistical calculations were carried out by two-tailed Student’s t-test or two-way ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001.
Fig. 6
Fig. 6. HectD3 attenuates Stat1-mediated inflammatory response in vivo.
a Schematic outline of the experimental work-plan for AAV-mediated overexpression of HectD3 in mice, including TAC and downstream experiments. b Immunoblots indicating proteins levels of Stat1/p-Stat1 and Stat3/p-Stat3 in mouse hearts where HectD3 was overexpressed via AAV-mediated gene transfer. AAV-luciferase injected mice were used as a control group. c Densitometry analysis of images shown in b is presented as a box plot that shows a strong reduction in the phosphorylation of both Stat1 and Stat3 by HectD3 overexpression (n = 5 each). d Immunoblots indicating the protein levels of Stat1/p-Stat1 and Stat3/p-Stat3 after TAC or sham surgery in mouse hearts where HectD3 was overexpressed via AAV-mediated gene transfer. AAV-luciferase injected mice were used as control group. e Densitometry analysis of images shown in d are presented as box plots for Stat1, p-Stat1, Stat3, and p-Stat3 showing that the phosphorylation of Stat1 was increased, whereas phosphorylation of Stat3 was reduced after TAC in control mice. HectD3 overexpression strongly reduced the phosphorylation of Stat1, yet maintained the phosphorylation levels of Stat3 comparable to control sham mice after TAC. f Transcript levels of several inflammatory markers and downstream targets (Bst2, Ccl2, Ccl5, Ifit1, Ifit3, IFNγ, Stat1, and Stat3) of interferon-signaling and Stat1-signaling are detected by quantitative real-time PCR in mouse hearts after TAC in mouse hearts where HectD3 was overexpressed by AAV-mediated gene transfer. g Representative immunofluorescence microscopy images of cardiac tissue sections of mouse hearts obtained from luciferase (control) or HectD3 injected mice that underwent TAC operations or Angiotensin-II (AngII) infusion (with respective sham or PBS controls). Tissue sections were stained with F4/80 as a marker of macrophage and counterstained with DAPI for nuclear staining. HectD3 overexpression reduced the infiltration of inflammatory macrophages to the mouse hearts after TAC compared to the respective control mice. h Immunofluorescence intensity analysis of F4/80 positive cells are presented in the form of a box plot indicating a dramatic increase in the macrophages in TAC operated or AngII-treated control mice. HectD3 overexpression however significantly reduced macrophage numbers (n = 6 each). i Representative images of RAW 264.7 cells after chemotaxis assay in modified Boyden chambers containing polycarbonate membranes. j Optical density measurements are presented in the form of a box plot showing accelerated migration of RAW 264.7 cells when treated with LPS. However, cells treated with culture supernatant from NRVCMs overexpressing HectD3 reduced this migration. Contrasting results were obtained with HectD3 knockdown condition (n = 3 each). Statistical calculations were carried out by two-way ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001.
Fig. 7
Fig. 7. Schematic representation of HectD3-mediated dual inhibitory regulation of cardiac hypertrophy and inflammation.
Right half panel indicate that HectD3 targets SUMO2 for UPS-mediated degradation, subsequently attenuating nuclear localization of constitutively active calcineurin A (δCnA) and activation of CnA-NFAT hypertrophic signaling in vitro and in vivo. On the other hand, left half panel demonstrates that HectD3 interacts with and targets interferon response factor Stat1 thereby downregulating pro-inflammatory Stat1-target genes, consequently resulting in reduced secretion of chemoattractants, and infiltration of pro-inflammatory macrophages to the heart.
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