Targeting of Cdc42‐Interacting Protein 4‐Calcineurin Signalosomes Improves Cardiac Structure and Function After Myocardial Infarction

Figures

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  Figure 1. Chronic ischemic cardiomyopathy is improved by CIP4 conditional gene targeting. A, 8 to 10‐wk‐old male and female CIP4 CKO CIP4 f/f ;Tg(Myh6‐cre/Esr1*) and control Tg(Myh6‐cre/Esr1*) (MCM) and CIP4 f/f mice were treated with tamoxifen‐laden chow for 1 wk to induce cre‐catalyzed recombination, followed by 1 wk on normal chow before being subjected to LCA PL or sham surgery. End point studies were performed 4 wk after induction of MI. B, Representative M‐mode echocardiography at end point for MI cohorts. C–E, LV diameter in diastole and systole and fractional shortening at end point. F and G, Gravimetric analysis at end point. For (C–G), n: MCM—Sham—6; CIP4 f/f —Sham—11; CIP4 CKO—Sham—6; MCM—MI—9; CIP4 f/f —MI—18; CIP4 CKO—MI—17. Data analyzed by 2‐way ANOVA (factors—genetic cohorts and surgery) and Tukey’s multiple comparisons test. H, Representative transverse cardiac sections stained with Masson’s Trichrome for MI cohorts. Scale bar—1 mm. I, Infarct size measured as % left ventricular circumference containing >50% transmural scar. n: MCM—4; CIP4 f/f —7; CIP4 CKO—5. J, Dysfunctional myocardium for MI cohorts is expressed as the ratio of the endocardial length for segments with…

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  Figure 2. Cardiac function after ischemia/reperfusion injury is improved by CIP4 conditional gene targeting. A, 7 to 8‐wk‐old male and female CIP4 CKO CIP4 f/f ;Tg(Myh6‐cre/Esr1*) and control Tg(Myh6‐cre/Esr1*) (MCM) and CIP4 f/f mice were treated with tamoxifen‐laden chow for 1 wk to induce cre‐catalyzed recombination, followed by 1 wk on normal chow before being subjected to transient ligation of the left coronary artery for 30 min and 24 h reperfusion. B–D, LV end‐systolic volume, end‐diastolic volume, and ejection fraction by 4‐dimensional imaging. n=13 for each cohort. Data analyzed by matched 2‐way ANOVA (factors—genetic cohorts and pre‐/post‐I/R) and Tukey’s multiple comparisons test. E and F, Area‐at‐risk and infarct size by histological Evans’ Blue and 2,3,5‐triphenyltetrazolium chloride staining. n=13 for each cohort. Data analyzed by 1‐way ANOVA and Tukey’s multiple comparisons test. AAR indicates area at risk; CIP4, Cdc42‐interacting protein 4; CKO, conditional knockout; I/R, ischemia/reperfusion; IS, infarct size; LV, left ventricular; MCM, MerCreMer transgene; and MI, myocardial infarction.

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  Figure 3. Calcineurin Aβ2 is cardioprotective during ischemia/reperfusion injury. A, A self‐complementary AAV9 vector was used to express a MIR30A CaNAβ2 small hairpin RNA minigene 29 under the direction of a cardiomyocyte‐specific chicken cardiac troponin T (TNNT2) promoter and a human calsequestrin (CASQ2) enhancer to deplete CaNAβ through the cardiomyocyte in vivo (Table S2). 30 , 31 Western blot of ventricular extracts for CaNAβ2 1 mo after injection of 6‐wk‐old male C57BL/6NJ mice with 1012 vg AAV. Equal loading shown by Ponceau total protein stain. Model drawing created in BioRender. Turcotte, M. (2025) https://BioRender.com/cvpyw1h. B, 1‐mo‐old male and female C57BL/6NJ mice were injected with 1012 vg AAV (~7×1013 vg/kg) 4 wk before being subjected to transient ligation of the left coronary artery for 30 min and 24 h reperfusion. C–E, LV end‐systolic volume, end‐diastolic volume, and ejection fraction by 4‐dimensional imaging. n=11 (shControl) and 14 (shCaNAβ2). F and G, Area at risk and infarct size by histological Evans’ Blue and 2,3,5‐triphenyltetrazolium chloride staining. n=7 (shControl) and 9 (shCaNAβ2). H, Representative bullseye plots for circumferential peak systolic strain…

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  Figure 4. CaNAβ polyproline and VIVIT calcineurin anchoring disruptor peptides improve and worsen outcome after ischemia/reperfusion injury, respectively. A, (top) PP‐GFP blocks CIP4‐CaNAβ binding, whereas VIVIT‐GFP inhibits CaN‐NFAT signaling. CIP4‐CaNAβ signaling is not associated with NFAT activation. 16 (bottom) AAV9 vectors were used to express VIVIT‐GFP, PP‐GFP, and GFP control fusion proteins under the direction of a cardiomyocyte‐specific chicken cardiac troponin T (TNNT2) promoter 31 (Table S3). Created in BioRender. Turcotte, M. (2025) https://BioRender.com/up7ibgy. B, 1‐mo‐old male and female C57BL/6NJ mice were injected with 1012 vg AAV (~7×1013 vg/kg) 4 wk before being subjected to transient ligation of the left coronary artery for 30 min and 24 h reperfusion. C–E, LV end‐systolic volume, end‐diastolic volume and ejection fraction by 4‐dimensional imaging. n: GFP—12; VIVIT‐GFP—13; PP‐GFP—15. F, Area‐at‐risk and infarct size by histological Evans’ Blue and 2,3,5‐triphenyltetrazolium chloride staining. n: GFP—8; VIVIT‐GFP—10; PP‐GFP—11. Data analyzed by 1‐way ANOVA and Tukey’s multiple comparisons test. G, Representative bullseye plots for circumferential peak systolic strain…

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  Figure 5. CaNAβ polyproline and VIVIT calcineurin anchoring disruptor peptides improve and worsen outcome in chronic ischemic cardiomyopathy, respectively. A, (top) PP‐GFP blocks CIP4‐CaNAβ binding, whereas VIVIT‐GFP inhibits CaN‐NFAT signaling. CIP4‐CaNAβ signaling is not associated with NFAT activation. 16 (bottom) AAV9 vectors were used to express VIVIT‐GFP, PP‐GFP, and GFP control fusion proteins under the direction of a cardiomyocyte‐specific chicken cardiac troponin T (TNNT2) promoter. 31 Created in BioRender. Turcotte, M. (2025) https://BioRender.com/up7ibgy. B, 9 to 11‐wk‐old male C57BL/6NJ mice were subjected to LAD PL, randomized 2 d later by ejection fraction acquired by 4‐dimensional echocardiography, and the following day injected with 1012 vg AAV (~4×1013 vg/kg). C–E, LV end‐systolic volume, end‐diastolic volume and ejection fraction by serial 4‐dimensional echocardiography. n: GFP Sham—3; VIVIT‐GFP Sham—5; PP‐GFP Sham—4; GFP MI—6; VIVIT‐GFP MI—9; PP‐GFP MI—7. *P‐values for GFP MI vs PP‐GFP MI; † P‐values for GFP MI vs VIVIT‐GFP MI; ‡ P‐values for PP‐GFP MI vs VIVIT‐GFP MI; § P‐values for VIVIT‐GFP Sham vs PP‐GFP Sham. F and G, Heart and wet lung weight indexed to tibial…

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  Figure 6. polyproline anchoring disruption does not affect T‐cell activation. A, Naïve CD4+ T cells were isolated from wild‐type C57B6/J mice and stimulated with 1 or 5 μg/mL αCD3 and αCD28 before transduction with lentivirus that express PP‐GFP or GFP control and FACS analysis and RNA isolation. Drawing created in BioRender. Bayer, A. (2025) https://BioRender.com/d4i12pc. B, Cell viability by 7AAD stain. C, Representative FACS analysis for CD69 and CD25 MFI for 5 μg/mL αCD3/CD28‐stimulated group. D and E, Quantification of CD69 and CD25 MFI. CD69 and CD25 MFI for unstimulated, naïve cells were 66±11 and 124±14 (mean±SEM, not shown), respectively. F, Quantitative reverse transcription polymerase chain reaction for fold gene expression relative to naïve cells. Data analyzed by matched 2‐way ANOVA (factors—B, D, and E: lentivirus and antibody dose, F: lentivirus and genes) and uncorrected Fisher’s least significant difference tests. n=3 independent T‐cell preparations. 7AAD 7‐amino‐actinomycin D; CaNAβ2, calcineurin Aβ; FACS, fluorescence‐activated cell sorting; IFNγ, interferon‐gamma; IL, interleukin; MFI, mean fluorescence intensity; PP‐GFP, polyproline‐green fluorescent protein; and TNF,…