Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: JOSD2 mediates isoprenaline-induced heart failure by deubiquitinating CaMKIIδ in cardiomyocytes

doi: 10.1007/s00018-023-05037-7

Figure Lengend Snippet: JOSD2 is upregulated in myocardial hypertrophy and fibrosis. A Wildtype mice were infused with saline (Sham) or ISO for 2 weeks. One lane represents one mouse. Representative western blot analysis for JOSD2 levels in WT-Saline or ISO-induced (WT-ISO) mouse heart tissues. GAPDH was used as the loading control. B Wildtype mice were subjected to MI surgery for 1 week. One lane represents one mouse. Representative western blot analysis for JOSD2 levels in WT-Sham or MI-induced (WT-MI) heart tissues. GAPDH was used as the loading control. C Representative western blot analysis for JOSD2 protein levels in neonatal rat ventricular myocytes (NRVMs), mouse peritoneal macrophages (MPMs) and primary fibroblasts (PFs). GAPDH was used as the loading control. D Immunofluorescence staining of mouse heart tissues for JOSD2 (red), sarcomeric alpha-actinin (green), and vimentin (green). Sections were counterstained with DAPI (blue). (scale bar = 25 μm). E Representative western blot analysis for JOSD2 levels with prolonged ISO stimulation time

Article Snippet: Small interfering RNA against JOSD2 and scrambled sequences were purchased from Genepharma (Shanghai, China).

Techniques: Saline, Western Blot, Immunofluorescence, Staining

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: JOSD2 mediates isoprenaline-induced heart failure by deubiquitinating CaMKIIδ in cardiomyocytes

doi: 10.1007/s00018-023-05037-7

Figure Lengend Snippet: JOSD2 knockout alleviates ISO-induced cardiac remodeling. Wildtype and JOSD2 knockout mice were infused with saline (Sham) or ISO for 2 weeks. A Representative M-mode echocardiographic images from mice in each group. B Representative images from harvested mouse heart tissues (scale bar: 2.5 mm). C-I Representative images and quantification of heart tissues from WT and JOSD2−/− mice stained with WGA (wheat germ agglutinin, C and D), H&E (E), Sirius Red (F and G) and Masson (H and I) in sections of hearts (scale bar: 100 μm). J mRNA levels of hypertrophy-associated and fibrosis-associated genes in heart tissues of mice. Data was normalized to Actb. K Western blot analysis of hypertrophy-associated β-MyHC, ANP and fibrosis-associated COL-1, TGF-β1 in heart tissues. L Densitometric quantification of blots in panel K, GAPDH was used as the loading control. M The content of ANP in plasma. All quantitative data is presented as Mean ± SEM; n = 6; ns = not significant; * = p < 0.05

Article Snippet: Small interfering RNA against JOSD2 and scrambled sequences were purchased from Genepharma (Shanghai, China).

Techniques: Knock-Out, Saline, Staining, Western Blot

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: JOSD2 mediates isoprenaline-induced heart failure by deubiquitinating CaMKIIδ in cardiomyocytes

doi: 10.1007/s00018-023-05037-7

Figure Lengend Snippet: Biometric and echocardiographic parameters in ISO-challenged mouse experiment

Article Snippet: Small interfering RNA against JOSD2 and scrambled sequences were purchased from Genepharma (Shanghai, China).

Techniques:

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: JOSD2 mediates isoprenaline-induced heart failure by deubiquitinating CaMKIIδ in cardiomyocytes

doi: 10.1007/s00018-023-05037-7

Figure Lengend Snippet: JOSD2 deficiency protects against myocardial injury induced by MI. Wildtype and JOSD2 knockout mice were subjected to MI surgery to model cardiac hypertrophy and fibrosis. Tissues from mice were analyzed after 1 week. A Representative M-mode echocardiographic images from mice in each group. B Representative images and quantification of heart tissues stained with WGA (B and C, scale bar: 50 μm), Sirius Red (D and E, scale bar: 100 μm), and TTC (F and G) in sections of hearts (scale bar: 1 mm). H-I The content of cTnT (H) and ANP (I) in plasma. J mRNA levels of hypertrophy-associated and fibrosis-associated genes in heart tissues of mice. Data was normalized to Actb. K Western blot analysis of hypertrophy-associated and fibrosis-associated proteins in heart tissues. L Densitometric quantification of blots in panel K, GAPDH was used as the loading control. All quantitative data is presented as Mean ± SEM; n = 6; ns = not significant; * = p < 0.05

Article Snippet: Small interfering RNA against JOSD2 and scrambled sequences were purchased from Genepharma (Shanghai, China).

Techniques: Knock-Out, Staining, Western Blot

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: JOSD2 mediates isoprenaline-induced heart failure by deubiquitinating CaMKIIδ in cardiomyocytes

doi: 10.1007/s00018-023-05037-7

Figure Lengend Snippet: Biometric and echocardiographic parameters in MI-induced mouse experiment

Article Snippet: Small interfering RNA against JOSD2 and scrambled sequences were purchased from Genepharma (Shanghai, China).

Techniques:

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: JOSD2 mediates isoprenaline-induced heart failure by deubiquitinating CaMKIIδ in cardiomyocytes

doi: 10.1007/s00018-023-05037-7

Figure Lengend Snippet: JOSD2 aggravates ISO-induced myocardial injury via activation of CaMKIIδ. WT mice were infected with AAV9 carrying JOSD2 cDNA (AAV9-JOSD2) or an empty vector (AAV9-NC) through tail vein. Four weeks post-injection, mice were infused with ISO. During the ISO-infused period, KN-93 was administered at 5 mg/kg/day. A Representative M-mode echocardiographic images from mice in each group. B Representative images from harvested heart tissues (scale bar: 2.5 mm). C–I Representative images and quantification of heart tissues stained with WGA (C–D), H&E (E), Sirius Red (F–G) and Masson (H–I) in sections of hearts (scale bar: 100 μm). J The content of ANP in plasma. K mRNA levels of hypertrophy-associated genes and fibrosis-associated genes in mouse heart tissues. Data was normalized to Actb. L Western blot analysis of hypertrophy-associated β-MyHC, ANP and fibrosis-associated COL-1, TGF-β1 in heart tissues. GAPDH was used as the loading control. M Western blot analysis of p-CaMKIIδ, CaMKIIδ in heart tissues. GAPDH was used as the loading control. All quantitative data is presented as Mean ± SEM; n = 6; ns = not significant; * = p < 0.05

Article Snippet: Small interfering RNA against JOSD2 and scrambled sequences were purchased from Genepharma (Shanghai, China).

Techniques: Activation Assay, Infection, Plasmid Preparation, Injection, Staining, Western Blot

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: JOSD2 mediates isoprenaline-induced heart failure by deubiquitinating CaMKIIδ in cardiomyocytes

doi: 10.1007/s00018-023-05037-7

Figure Lengend Snippet: JOSD2 regulates ISO-induced cardiomyocyte hypertrophy and fibrosis. NVRMs were used to model ISO effects in culture. Cells were transfected with siRNA against JOSD2 (panels A-D) or JOSD2 overexpressing vectors (panels E–H). Scrambled siRNA and empty vectors were used as control. Following transfections, cells were exposed to 10 μM ISO for 24 h. A Following ISO exposure, cells were stained with Phalloidin-TRITC to assess hypertrophic responses [scale bar = 100 μm]. B Mean cell area quantification of image in panel A. C Representative western blot for β-MyHC, ANP, COL-1, TGF-β1 and JOSD2 in NVRMs. GAPDH was used as the loading control. D mRNA levels of hypertrophy-associated and fibrosis-associated genes were measured in NVRMs. Data was normalized to Actb. E Hypertrophic response to ISO in cells overexpressing JOSD2. Representative Phalloidin-TRITC staining image [scale bar = 100 μm]. F Mean cell area quantification of the image in panel E. G Representative western blot for β-MyHC, ANP, COL-1, TGF-β1 and JOSD2 in NVRMs. GAPDH was used as a loading control. H mRNA levels of hypertrophy-associated genes and fibrosis-associated were measured in NVRMs. Data was normalized to Actb. All quantitative data is presented as Mean ± SEM; n = 3; ns = not significant; * = p < 0.05

Article Snippet: Small interfering RNA against JOSD2 and scrambled sequences were purchased from Genepharma (Shanghai, China).

Techniques: Transfection, Staining, Western Blot

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: JOSD2 mediates isoprenaline-induced heart failure by deubiquitinating CaMKIIδ in cardiomyocytes

doi: 10.1007/s00018-023-05037-7

Figure Lengend Snippet: JOSD2 interacts CaMKIIδ directly and promotes CaMKIIδ activation. A-B MS/MS spectrum of the peptide showing FTDEYQLFEELGK and ESTESSNTTIEDEDVK from CaMKIIδ. Single-letter abbreviations: K, Lys; V, Val; I, Ile; G, Gly; Q, Gln; L, Leu; N, Asn; E, Glu; T, Thr; S, Ser; F, Phe; D, Asp; Y, Tyr. m/z, mass/charge ratio. C The complex of JOSD2 and CaMKIIδ in NRVMs were detected by Coimmunoprecipitation. NRVMs were transfected with Flag-tagged JOSD2. Lysates from cells were immunoprecipitated with an anti-Flag antibody. CaMKIIδ levels were detected by immunoblotting. IgG was used as a control for IP. D In HEK-293 T cells which express a few endogenous CaMKII, Flag-tagged JOSD2 and GFP-tagged CaMKIIδ were transfected. Lysates from cells were immunoprecipitated with anti-Flag antibody. GFP levels were detected by immunoblotting. IgG was used as a control for IP. E Representative western blot for CaMKIIδ and p-CaMKIIδ in NRVMs with gradient levels of JOSD2. F–G JOSD2 was silenced or overexpressed in NRVMs. Representative western blotting for CaMKIIδ and p-CaMKIIδ in NRVMs. GAPDH was used as a loading control. (H-I) Representative western blotting for CaMKIIδ and p-CaMKIIδ in heart tissues from ISO- and MI-induced mice. J–K Confocal images (J)and representative recordings (K) of SR Ca2+ determined by the Fluo-4 AM fluorescence signal in cardiomyocytes transfected with si-JOSD2 or si-NC before ISO stimulation for 24 h. n = 3 cells in each group. [Scale bar, 50 μm]. L–M Confocal images (L) and representative recordings (M) of SR Ca2+ determined by the Fluo-4 AM fluorescence signal in cardiomyocytes transfected with Flag-JOSD2 or empty vector (EV) before ISO stimulation for 24 h. n = 6 cells in each group. [Scale bar, 50 μm]

Article Snippet: Small interfering RNA against JOSD2 and scrambled sequences were purchased from Genepharma (Shanghai, China).

Techniques: Activation Assay, Tandem Mass Spectroscopy, Transfection, Immunoprecipitation, Western Blot, Fluorescence, Plasmid Preparation

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: JOSD2 mediates isoprenaline-induced heart failure by deubiquitinating CaMKIIδ in cardiomyocytes

doi: 10.1007/s00018-023-05037-7

Figure Lengend Snippet: JOSD2 directly removes ubiquitin from CaMKIIδ at residue K227. A HEK-293 T cells were transfected with GFP-CaMKIIδ, Flag-JOSD2, Myc-Ub (WT), Myc-Ub(K48) and Myc-Ub(K63). Cells were then exposed to MG132 for 6 h. Ubiquitinated CaMKIIδ was detected by immunoblotting. B A schematic illustration of the construct with mutated ubiquitination site of CaMKIIδ. (C) HEK-293 T cells were transfected with Flag-JOSD2, Myc-Ub, GFP-CaMKIIδ (WT), GFP-CaMKIIδK(K138R), GFP-CaMKIIδ(K227R) and GFP-CaMKIIδ(K268R). Cells were then exposed to MG132 for 6 h. Ubiquitinated CaMKIIδ was detected by immunoblotting. D A schematic illustration of the construct with the mutated active site of JOSD2. E HEK-293 T cells were transfected with GFP-CaMKIIδ, Myc-Ub, Flag-JOSD2(WT) and Flag-JOSD2(C24A). Cells were then exposed to MG132 for 6 h. Ubiquitinated CaMKIIδ was detected by immunoblotting

Article Snippet: Small interfering RNA against JOSD2 and scrambled sequences were purchased from Genepharma (Shanghai, China).

Techniques: Residue, Transfection, Western Blot, Construct

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: JOSD2 mediates isoprenaline-induced heart failure by deubiquitinating CaMKIIδ in cardiomyocytes

doi: 10.1007/s00018-023-05037-7

Figure Lengend Snippet: Biometric and echocardiographic parameters in ISO-challenged mice with the treatment of AAV9 (Empty Vector, JOSD2) with or without KN-93

Article Snippet: Small interfering RNA against JOSD2 and scrambled sequences were purchased from Genepharma (Shanghai, China).

Techniques: Plasmid Preparation