Fig 2: ISG15 deficiency attenuates LV systolic dysfunction in mice after TAC.(A and B) Representative M-mode echocardiographs (A) and LV mass (B) in WT and Isg15–/– mice 8 weeks after sham or TAC surgery. (C–F) Ejection fraction (C), fractional shortening (D), cardiac output (E), and stroke volume (F) in WT and Isg15–/– mice 8 weeks after sham or TAC. WT sham, n = 15; WT TAC, n = 12; Isg15–/– sham, n = 13; Isg15–/– TAC, n = 14. Values are mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by 1-way ANOVA followed by Tukey’s post hoc test (skew-distributed data in B–F were log-transformed before statistical comparison).

Fig 3: ISG15 is upregulated in human NICM.(A) ISG15 in human LV samples from patients with nonfailing hearts (NF), dilated cardiomyopathy (DCM), or ischemic cardiomyopathy (ICM) (28). Differential expression determined by linear model ANOVA; P value (pval) adjusted for Benjamini-Hochberg FDR ≤ 0.05 (28). RPKM, reads per kilobase of transcript, per Mmllion mapped reads. (B) qRT-PCR in control human heart tissue (n = 3) and tissue from patients with end-stage heart failure due to NICM (n = 6). (C) RNAscope in situ hybridization for ISG15 and immunofluorescence for troponin I in human LV tissue. The arrows mark ISG15 RNAscope puncta in troponin I+ cardiomyocytes. Scale bars: 20 μm. (D) Immunoblotting for ISG15 and quantification of ISG15 protein conjugates in human control (n = 3) and NICM (n = 6) heart tissue. Values are mean ± SD. *P < 0.05 by unpaired 2-tailed Mann-Whitney test.

Fig 4: CCR2+ cardiac macrophages induce a cardiomyocyte IFN response.(A) qRT-PCR for IFN response genes (Isg15, Irf7, Ifit1, Ifit3, Ifi2712a, Ifitm3, Oasl2, Lgals3bp, Bst2, Gvin1) in mouse cardiomyocytes in medium conditioned by CCR2+ cardiac macrophages isolated from Ccr2gfp/+ mouse hearts 1 week after TAC, or under control conditions. Control, n = 4; CCR2+ cardiac macrophage–conditioned medium, n = 8. (B) Culture medium IFN-β concentration in bone marrow–derived macrophages (BMDMs) from Ccr2gfp/+ mice or Isg15–/– mice incubated with LPS (1 μg/mL), poly(I:C) (500 ng/mL), or STING agonist-4 (5 μmol/L) for 24 hours (n = 6 per condition). (C and D) Immunoblotting (C; n = 6 per condition) and qRT-PCR (D; n = 5 per condition) for ISG15 in mouse cardiomyocytes exposed to CD4+ T cell–conditioned medium for 24 hours. Values are mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by unpaired 2-tailed Mann-Whitney test (A), 1-way ANOVA followed by Tukey’s post hoc test (B), or unpaired 2-tailed Student’s t test (C and D).

Fig 5: ISG15 induction impairs cardiomyocyte protein turnover.(A) Immunoblotting for filamin-C in the soluble and insoluble fractions of WT and Isg15–/– mouse hearts 8 weeks after sham or TAC surgery (n = 6 per group). (B) Immunoblotting for ISG15, ISG15-conjugated proteins, p62, and LC3 in human cardiomyocytes transfected with siRNA directed against ISG15 for 6 hours before incubation with 500 IU/mL IFN-β for 48 hours. Values are mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by 1-way ANOVA followed by Tukey’s post hoc test.