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

Abstract

Background

CaN (calcineurin) promotes pathological cardiac remodeling but also cardioprotection in ischemia–reperfusion injury. CaN inhibitors are also immunosuppressants. This pleiotropy complicates targeting calcineurin in cardiovascular disease. CIP4/TRIP10 (Cdc42‐interacting protein 4) is a scaffold protein that binds the CaNAβ (calcineurin Aβ) N‐terminal polyproline domain and organizes a calcium and CaNAβ2 signaling compartment independent of contractile calcium. We showed that CIP4‐CaNAβ2 signalosomes promote pathological cardiac hypertrophy induced by pressure overload. It is unknown whether CIP4‐CaNAβ2 signalosomes contribute to cardioprotection and remodeling in ischemic disease.

Methods

CIP4 conditional knockout mice were studied following ischemia–reperfusion and permanent left coronary artery ligation that induce myocardial infarction. C57BL/6NJ mice were transduced with cardiotropic adeno‐associated virus expressing a CaNAβ2 small hairpin RNA to inhibit CaNAβ2 expression, a VIVIT peptide to inhibit CaN‐NFAT (nuclear factor of activated T cells) signaling, or a CaNAβ polyproline peptide to block CIP4‐CaNAβ2 binding and similarly studied by ischemia–reperfusion injury and left coronary artery ligation. CaNAβ polyproline‐dependent signaling was also studied in T cells.

Results

CIP4 conditional knockout and cardiomyocyte‐specific CaNAβ polyproline peptide expression improved cardiac function after ischemia–reperfusion injury and decreased infarct size and improved cardiac function after permanent left coronary artery ligation. In contrast, cardiomyocyte‐specific CaNAβ2 depletion and VIVIT expression worsened outcome after myocardial infarction. The polyproline peptide had no effect on T‐cell activation and cytokine expression in vitro.

Conclusions

CIP4‐CaNAβ2 signalosomes promote adverse cardiac remodeling and are not cardioprotective. Proof of concept is provided for the treatment of ischemic cardiomyopathy by a polyproline peptide gene therapy. Targeting these complexes may be beneficial in cardiovascular diseases, including ischemic cardiomyopathy and acute myocardial infarction.

Nonstandard Abbreviations and Acronyms

AAV

adeno‐associated virus

CaN

calcineurin

CaNAβ2

β2 isoform of the calcineurin A‐subunit

CIP4 CKO

CIP4 conditional knockout

CIP4

Cdc42‐interacting protein 4

GFP

green fluorescent protein

I/R

ischemia reperfusion

LCA PL

left coronary artery permanent ligation

MCM

MerCreMer transgene

NFAT

nuclear factor of activated T cells

PP

CaNAβ polyproline domain

SH3

Src homology 3

shCaNAβ2

CaNAβ2 small hairpin RNA

shControl

control small hairpin RNA

Clinical Perspective.

What Is New?

• Targeting CIP4 (Cdc42‐interacting protein 4), which is a scaffold protein for the phosphatase calcineurin, improved cardiac function in mice after acute myocardial infarction due to ischemia–reperfusion injury and in chronic ischemic cardiomyopathy.

What Are the Clinical Implications?

• This study establishes CIP4 signalosomes as a new drug target for the treatment of chronic pathological cardiac remodeling including after myocardial infarction.

• This study provides proof of concept for a new gene therapy approach to treating acute myocardial infarction and chronic ischemic cardiomyopathy.

Heart failure, the common end‐stage for cardiac disease, is a syndrome of major public health significance. The prevalence of heart failure among adult Americans is ~6.7 million, and 1 in 8 will die while in heart failure. ¹ Notably, coronary heart disease is the most common risk factor for heart failure. Although current guideline‐directed medical therapy for heart failure with reduced ejection fraction (EF) can significantly lower the risk of hospitalization and death, ² mortality remains high for all causes of heart failure, compelling the development of novel therapeutic strategies. ¹ Pathological cardiac remodeling underlies the development of heart failure. Although cardiomyocyte hypertrophy is the major intrinsic compensatory mechanism for chronic stress on the heart, in pathological conditions hypertrophy is accompanied by altered myocyte contractility and metabolism, the progressive loss of myocytes, myocardial inflammation, and interstitial fibrosis, together resulting in systolic and/or diastolic cardiac dysfunction. ³ This remodeling is regulated by a network of intracellular myocyte signaling pathways that represent potential candidate targets for drug development. ⁴

Diverse signaling enzymes have been identified as critical mediators of pathological cardiac remodeling. ⁴ One enzyme that is of longstanding interest is the Ca²⁺/calmodulin‐dependent phospho‐serine/threonine phosphatase calcineurin (CaN), also known as serine/threonine‐protein phosphatase 2B (PP2B) and protein phosphatase 3 (PPP3). ⁵ , ⁶ In mammals, there are 3 genes (PPP3C A‐C) encoding the CaN catalytic A‐subunit α, β and γ isoforms. ⁷ In addition, CaNAβ (calcineurin Aβ) is expressed as the alternatively spliced isoforms CaNAβ1 and CaNAβ2, the latter usually comprising the majority of CaNAβ in cells and sharing a C‐terminal domain structure similar to CaNAα. ⁸ Studies using mice with CaN Aα and Aβ genetic deletion have revealed isoform‐specific functions in different organs. ⁹ , ¹⁰ , ¹¹ , ¹² , ¹³ , ¹⁴ In particular, CaNAβ knockout attenuated the cardiac hypertrophy induced by pressure overload and angiotensin II and isoproterenol infusion. ⁹ The role of CaNAβ in cardiomyocyte hypertrophy is primarily through the action of CaNAβ2, whereas CaNAβ1 overexpression opposes pathological hypertrophy. ¹⁵ , ¹⁶ Although CaNAβ gene targeting results in the inhibition of pathological cardiac hypertrophy, CaNAβ knockout also increases myocardial loss after ischemia–reperfusion (I/R) injury. ¹⁷ In addition, CaN inhibitors are immunosuppressants, and CaNAβ gene targeting negatively affects T‐cell development and activation. ¹⁸ The roles for CaN in cardioprotection and the immune system have precluded the development of CaN‐targeting therapeutics for heart failure.

Isoform‐specific functions in some tissues may be attributable to differences in expression or substrate specificity. ¹³ , ¹⁹ However, differences such as these do not adequately explain why CaN isoforms have a nonredundant function in the adult heart, in which Aα and Aβ (mainly Aβ2) are normally present at similar protein levels and in which relevant substrates can be dephosphorylated with similar efficiency by both isoforms. ⁹ , ¹⁹ Specificity in enzyme function can be conferred by binding to multivalent scaffold proteins that localize the signaling enzyme within the cell and recruit relevant upstream activators and downstream effector substrates. Like other phosphatases, ²⁰ CaN is bound by scaffold proteins and localized to diverse intracellular compartments. ⁵ , ⁶ We discovered that in contrast to CaNAβ1, which is intracellularly localized via its different C‐terminal domain, ⁷ CaNAβ2 can be selectively localized by the CaNAβ‐specific N‐terminal polyproline domain to a compartment within the myocyte organized by the endosome‐associated scaffold protein CIP4 (Cdc42‐interacting protein 4, TRIP10). ¹⁶ CIP4 contains an N‐terminal F‐BAR (Fer‐CIP4 Homology‐Bin/Amphiphysin/Rvs) domain that binds membrane phospholipids and cytoskeletal proteins and confers CIP4 homo‐dimerization, an HR1 domain that binds active Rho family members (Cdc42 [cell division cycle 42], TC10, and TCL), and a C‐terminal SH3 (Src homology 3) domain that binds proteins involved in the control of the actin cytoskeleton and signaling. ²¹ The CIP4 SH3 domain binds the CaNAβ polyproline‐domain. ¹⁶

By imaging live ventricular myocytes expressing fluorescent biosensors, we found that CIP4‐bound CaNAβ2 was activated by G‐protein coupled receptor signaling, including angiotensin II, α‐ and β‐adrenergic receptors. ¹⁶ Remarkably, CIP4‐bound CaNAβ2 was not activated by pacing that induces myocyte contraction. Both CIP4 gene targeting and adeno‐associated virus (AAV)‐mediated CaNAβ polyproline peptide expression, which competitively inhibits CIP4‐CaNAβ2 binding, inhibited cardiac remodeling and improved cardiac function in response to pressure overload in mice, apparently independently of NFAT (nuclear factor of activated T cells) transcription factor regulation. ¹⁶ These results suggested that CIP4‐CaNAβ2 “signalosomes” might constitute a new target for intervention in pathological cardiac remodeling. As a major impediment to the development of CaN‐directed therapeutics are its roles in cardioprotection and immunity, we were interested whether CIP4‐CaNAβ2 signalosomes contribute to these functions, or, alternatively, whether CIP4‐CaNAβ2 might be safely targeted in ischemic cardiomyopathy and lack a role in T cells, in which case CIP4‐CaNAβ2 signalosomes might be acceptable drug targets for the treatment of heart failure. Here we report the effects of CIP4 cardiomyocyte‐specific knockout and CaNAβ polyproline anchoring disruption in adult mouse models of I/R injury and chronic ischemic cardiomyopathy. Inhibition of CIP4‐CaNAβ2 signalosomes is contrasted with inhibition of CaNAβ2 expression across intracellular compartments by RNA interference and with inhibition of CaN function by a VIVIT peptide that competes the allosteric binding of all CaNA isoforms by scaffold proteins and substrates like NFAT containing a PxIxIT short linear motif. ⁷ , ²² , ²³ , ²⁴ , ²⁵ In addition, we screen for a role of polyproline‐anchored CaNAβ in T‐cell activation in vitro. Results are presented that CIP4‐CaNAβ2 signalosomes comprise a novel compartment contributing to the deleterious effects of cardiomyocyte CaN signaling in ischemic heart disease that may be therapeutically tractable for the treatment and prevention of heart failure.

METHODS

Complete detailed methods are provided in the Supplemental Material. The data that support the findings of this study are available from the corresponding author upon reasonable request.

Animal Models

All experiments involving animals were approved by the Administrative Panel on Laboratory Animal Care Institutional Animal Care and Use Committee at Stanford University. The conditional “floxed” CIP4 (Trip10) mouse (Jackson Laboratory Strain #035591) was as previously described. ¹⁶ To induce gene knockout, CIP4 ^f/f ;Tg(Myh6‐cre/Esr1*) CIP4 conditional knockout (CKO) mice and control Tg(Myh6‐cre/Esr1*) MerCreMer transgene (MCM) and CIP4 ^f/f mice were fed tamoxifen‐laden chow (125 mg tamoxifen/kg chow, Harlan Teklad) for 1 week before study. For AAV studies, C57BL/6NJ mice (Jackson Laboratory Strain #005304) were injected intravenously with 10¹² viral genomes either 4 weeks before I/R injury or 2 days after permanent coronary artery ligation to induce myocardial infarction (MI). Echocardiography, strain analysis, and histological analyses were performed as described in the Supplemental Material (see also Figures S1 and S2).

Statistical Analysis

Statistics were calculated using Graphpad Prism 10. n refers to the number of individual mice. All data are expressed as mean±SEM. For 2‐group comparisons, 2‐tailed t test was performed. For comparisons of multiple groups, data sets were analyzed by 1‐way ANOVA followed by Tukey’s post hoc testing. Two‐way ANOVA (with interactions considered throughout) was used for experiments with 2‐factor design followed by Tukey’s multiple comparisons (multiple treatment groups) or uncorrected Fisher’s least significant difference (2 treatment groups) tests, with matching as appropriate.

RESULTS

Inhibition of Postmyocardial Infarction Remodeling by CIP4 Gene Targeting

We previously described the CIP4 CKO CIP4 ^f/f ;Tg(Myh6‐cre/Esr1*) mouse, in which cardiomyocyte‐specific deletion of CIP4 Exons 2 and 3 results from tamoxifen activation of a MerCreMer estrogen receptor–cre recombinase fusion protein (MCM) expressed in cardiac myocytes. ¹⁶ , ²⁶ CIP4 CKO prevented the development of pathological cardiac remodeling and heart failure in response to chronic pressure overload, apparently in association with the inhibition of CaNAβ2 signaling. ¹⁶ To test whether CIP4‐CaNAβ has a broader role in the regulation of pathological remodeling, we now considered whether CIP4 CKO would be similarly beneficial in the chronic ischemic cardiomyopathy associated with MI induced by left coronary artery permanent ligation (LCA PL). ²⁷ The 8 to 10‐week‐old CIP4 CKO mice were fed tamoxifen‐laden chow for 1 week and then subjected 1 week later to LCA PL or sham survival surgery (Figure 1A). Tamoxifen‐treated control cohorts included MCM and CIP4 ^f/f mice to account for effects of MCM expression and CIP4 gene loxP site insertion, respectively. Consistent with our previously published results, ¹⁶ no significant differences were observed by M‐mode echocardiography between CIP4 CKO, MCM, and CIP4 ^f/f mice 4 weeks after sham operation (Figure 1B through 1E), demonstrating that CIP4 gene deletion has little effect on physiological cardiac function. 4 weeks after MI, MCM, and CIP4 ^f/f mice were found to have left ventricular (LV) dilatation with 25% and 29% increased end‐diastolic LV diameter, respectively (Figure 1B and 1C). Systolic function was impaired with LV end‐systolic diameter increased 60% and 62% and fractional shortening less in value by 20% and 18%, respectively for the MCM and CIP4 ^f/f cohorts (Figure 1D and 1E). Surprisingly, CIP4 CKO mice did not have significantly increased LV end‐diastolic diameter and exhibited less systolic dysfunction than the control cohorts, including a lesser increase in end‐systolic LV diameter (24%) and a decrease in fractional shortening of 9%. The relatively preserved systolic function post MI of the CIP4 CKO mice was confirmed by strain analysis using B‐mode parasternal long axis images (Figure S3A through S3C; Videos S1 through S6). Both segmental and global longitudinal strain were improved by CIP4 CKO following MI when compared with the 2 control cohorts.

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 decreased longitudinal peak systolic strain (see Table S1 for control values) to the overall endocardial length in parasternal long axis echocardiographic images (Figure S1). n: MCM—9; CIP4 ^f/f —12; CIP4 CKO—10. Data in (I and J) analyzed by 1‐way ANOVA and Tukey’s multiple comparisons test. CIP4 indicates Cdc42‐interacting protein 4; CKO, conditional knockout; LCA PL, left coronary artery permanent ligation; LV, left ventricular; MCM, MerCreMer transgene; and MI, myocardial infarction.

Gravimetric analysis confirmed that CIP4 attenuated post‐MI cardiac remodeling. Cardiac hypertrophy (increased biventricular weight indexed to tibial length) was less for the CIP4 CKO mice than MCM and CIP4 ^f/f cohorts (Figure 1F). In addition, indexed wet lung weight, a marker for heart failure, was increased only for the 2 control cohorts and not for CIP4 CKO mice (Figure 1G). Notably, the histological measurement of infarct size at end point showed that CIP4 CKO reduced infarct size by 35% to 38% compared with control mice (Figure 1H and 1I). This result was corroborated by the measurement of fractional dysfunctional myocardium by segmental longitudinal strain analysis (Figure 1J). Finally, as a control, western blot of ventricular extracts confirmed that as previously published ¹⁶ tamoxifen‐induced CIP4 CKO reduced the expression of the myocyte‐specific isoform CIP4h >90% (Figure S3D). Consistent with earlier results that CIP4h binds a small pool of CaNAβ2 in the cardiomyocyte, ¹⁶ CaNAβ2 levels were not affected by CIP4 CKO nor by MI. CIP4h was also not changed in expression by MI. Expression of the ubiquitously expressed isoform CIP4a, which is primarily expressed in nonmyocyte cardiac cell types, ¹⁶ was as expected not altered in expression by CIP4 CKO, but, interestingly, was increased in expression 50% following MI (P=0.008 for pooled MCM and CIP4 ^f/f data). Together, these results showed that CIP4 CKO preserved cardiac structure and function in a model of chronic ischemic cardiomyopathy, suggesting that like in chronic pressure overload ¹⁶ CIP4 targeting inhibits pathological cardiac remodeling after MI.

Improvement in Cardiac Function After I/R Injury Following CIP4 Gene Targeting

CaNAβ promotes pathological cardiac remodeling but is also cardioprotective. ⁹ , ¹⁷ Therefore, we next considered whether despite CIP4 CKO’s benefit in chronic remodeling, CIP4 CKO would be detrimental in acute MI induced by I/R injury. As measured by 4‐dimensional echocardiography and corroborated by strain measurements (Figure 2A through 2D; Figure S4; Videos S7 through S15), injury induced by LCA ligation for 30 minutes and 24 hours reperfusion resulted in systolic dysfunction with minimal ventricular dilatation (increased end‐diastolic volume) for the three CIP4 CKO, MCM, and CIP4 ^f/f cohorts, including increased end‐systolic volume and decreased ejection fraction (EF). Notably, after I/R injury, EF was 7% and 13% higher for the CIP4 CKO cohort than for the MCM and CIP4 ^f/f controls, respectively. This correlated with improvement in global endocardial circumferential strain for the CIP4 CKO cohort when compared with the control cohorts (CIP4 CKO: −23.8%±1.1% versus CIP4 ^f/f : −16.3%±0.6%, P<0.0001; versus MCM: −21.0%±0.9%, P=0.054; Figure S4H).

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.

To directly test for a cardioprotective role for CIP4‐CaNAβ signalosomes, infarct size was measured by Evans’ Blue and TTC histological staining (Figure S2). Importantly, although area at risk was similar for all 3 cohorts, infarct size was not increased by CIP4 CKO (Figure 2E and 2F). In addition, CIP4 CKO showed decreased infarct size compared with CIP4 ^f/f controls cohort, and a trending decrease relative to the MCM controls when measured histologically or by segmental strain analysis (Figure 2E and 2F; Figure S4D and S4I). Taken together, these results suggest that in contrast to CaNAβ knockout, ¹⁷ CIP4 CKO is not deleterious during I/R injury, and, moreover, is functionally beneficial in both acute and chronic MI.

Cardioprotection Conferred by Cardiomyocyte CaNAβ2

It has been reported that global CaNAβ gene knockout worsened I/R injury, ¹⁷ whereas transgenic cardiomyocyte‐specific CaNAβ1 overexpression did not improve cardiac function in acute MI due to I/R injury despite inhibiting late remodeling after MI. ¹⁵ , ²⁸ These results suggested that CaNAβ2 is the primary CaNAβ mediator of cardioprotection in I/R injury. However, even though CIP4‐associated CaN activity is dependent upon CaNAβ2 expression (and polyproline‐dependent CIP4 anchoring), ¹⁶ CIP4 CKO was not deleterious in I/R injury (Figure 2). To test directly whether cardiomyocyte CaNAβ2 serves a role in cardioprotection, we assessed the effect on I/R injury of cardiomyocyte‐specific CaNAβ2 inhibited expression. Serotype 9 AAV9 were generated to express in cardiomyocytes CaNAβ2 (shCaNAβ2) or control (shControl) MIR30A small hairpin RNA. shCaNAβ2 expression reduced ventricular CaNAβ2 expression 83% in naïve mice (P=0.014; Figure 3A).

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 analysis of parasternal short axis images. Sectors labeled with bolded text indicate regions with decreased strain. I, Dysfunctional myocardium as assessed by circumferential peak systolic strain analysis and expressed as % LV volume. n=9 (shControl) and 12 (shCaNAβ2). C–E and I, Data analyzed by matched 2‐way ANOVA (factors—genetic cohorts and pre−/post‐IR) and uncorrected Fisher’s least significant difference test. F and G, Data analyzed by 2‐tailed t tests. AAR indicates area at risk; AAV, adeno‐associated virus; CaNAβ2, calcineurin Aβ; CIP4, Cdc42‐interacting protein 4; I/R, ischemia/reperfusion; IS, infarct size; ITR, inverted terminal repeat (Δ—deleted); IV, intravenous; LV, left ventricular; pA ‐ SV40 polyadenylation signal; and WPRE, woodchuck hepatitis virus posttranscriptional regulatory element.

AAV9‐transduced wild‐type C57BL/6NJ mice were subjected to I/R injury (Figure 3B). LV EF was decreased after I/R injury 12% for the shCaNAβ2 cohort compared with 5% for the shControl control (Figure 3C through 3E; Videos S16 and S17). The worse systolic dysfunction of the infarcted shCaNAβ2 cohort was corroborated by measurement of LV global and segmental circumferential and longitudinal strain (Figure S5). Histological assay showed that shCaNAβ2 increased infarct size 78% over shControl mice (Figure 3F and 3G). Measurement of dysfunctional myocardium by segmental analysis of either circumferential and longitudinal strain was consistent with increased injured myocardium in the presence of CaNAβ2 small hairpin RNA (Figure 3H and 3I; Figure S5D). In sum, these results suggest that in contrast to CIP4, CaNAβ2 expression is important for cardioprotection during I/R injury.

Differential Effects of VIVIT and polyproline CaN Anchoring Disruptor Peptides After I/R Injury

Previously published live cell imaging results suggest that CIP4‐bound CaNAβ2 comprises a small pool of CaNAβ in the cardiomyocyte that does not regulate NFAT transcription factors. ¹⁶ We, therefore, considered that CIP4‐bound CaNAβ2 might be unrelated to cardioprotective CaNAβ‐dependent NFAT signaling elsewhere in the myocyte. ¹⁷ CIP4 binds the N‐terminal polyproline domain of CaNAβ, and by proximity ligation assay and live cell imaging studies, we previously showed that CIP4 binds CaNAβ2 but not CaNAα in cardiomyocytes. ¹⁶ Notably, we demonstrated that expression of a PP‐GFP (polyproline‐green fluorescent protein) fusion peptide by an AAV9 vector engineered to confer cardiomyocyte‐specific expression blocked CIP4‐CaNAβ2 binding in cardiomyocytes in vivo. ¹⁶ In addition, like CIP4 CKO, PP‐GFP expression improved pathological cardiac remodeling in response to chronic pressure overload. ¹⁶ To test whether CIP4‐bound CaNAβ2 contributes to cardioprotection, adult wild‐type C57BL/6N mice were administered the same AAV9.PP‐GFP vector or AAV9.GFP control (Figure 4A; Figure S6A). The mice were studied 1 month later by I/R injury (Figure 4B). For comparison, a VIVIT‐GFP fusion protein was also expressed using AAV9. The VIVIT peptide is based upon a PxIxIT short linear motif present within scaffold proteins and substrates like NFAT that bind a conserved allosteric docking site on all CaN catalytic A‐subunits. ⁷ Expression of the VIVIT peptide has been shown to inhibit NFAT activation and myocyte survival in vitro. ¹⁷ , ²³ , ²⁴

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 analysis of parasternal short axis images post I/R. Sectors labeled with bolded text indicate regions with decreased strain. H, Dysfunctional myocardium as assessed by circumferential peak systolic strain analysis and expressed as % LV volume. n: GFP—12; VIVIT‐GFP—12; PP‐GFP—10. C–E and H, Data analyzed by matched 2‐way ANOVA (factors—AAV cohorts and pre−/post I/R) and Tukey’s multiple comparisons test. AAR indicates area at risk; AAV, adeno‐associated virus; CaNAβ2, calcineurin Aβ; CIP4, Cdc42‐interacting protein 4; EGFP, enhanced green fluorescent protein; I/R, ischemia/reperfusion; IS, infarct size; ITR, inverted terminal repeat; IV, intravenous; LV, left ventricular; PP‐GFP, polyproline‐green fluorescent protein; and SV40 pA, SV40 polyadenylation signal.

Compared with GFP, VIVIT‐GFP expression induced mild systolic dysfunction (EF: 47%±1% versus 53%±2%, P=0.0034) 1 month after AAV9 administration (Figure 4C through 4E), whereas PP‐GFP expression had no significant effect in uninjured mice. Following I/R injury, VIVIT‐GFP expression resulted in a 13% further decrease in EF, whereas EF decreased 10% for control GFP‐treated mice (Videos S18 through S20). In contrast, PP‐GFP expression preserved both end‐systolic volume and EF. The worsening and general preservation of systolic function by VIVIT‐GFP and PP‐GFP expression, respectively, was corroborated by measurement of global and segmental circumferential and longitudinal peak strain (Figure S6). VIVIT‐GFP worsened infarct size measured histologically 67%, like shCaNAβ2, whereas infarct size was not different for PP‐GFP‐ and GFP‐treated mice (Figure 4F). In contrast, the extent of dysfunctional myocardium assessed by segmental circumferential and longitudinal peak strain analysis was dramatically less following I/R injury for the PP‐GFP cohort, whereas VIVIT‐GFP expression resulted in increased dysfunction, consistent with histological findings (Figure 4G and 4H; Figure S6E). Taken together, these results suggest that while VIVIT‐GFP exacerbated I/R injury, PP‐GFP preserved cardiac function despite the absence of histologically detectable cardioprotection.

Inhibition of Remodeling in Chronic Ischemic Cardiomyopathy by Polyproline Anchoring Disruption

As PP‐GFP expression and CIP4 CKO improved systolic function after I/R injury, we next tested whether like CIP4 CKO, PP‐GFP might also be beneficial in chronic ischemic cardiomyopathy. Wild‐type male C57BL/6N mice were subjected to LCA PL or sham operation and randomized 2 days later by EF for treatment with AAV9.PP‐GFP, AAV9.VIVIT‐GFP, or AAV9.GFP control (Figure 5A and 5B). At end point 8 weeks after survival surgery, the sham‐operated cohorts were similar in cardiac structure and function (Figure 5C through 5E; Videos S21 through S23). During the 8 weeks post‐MI, the GFP‐treated infarcted cohort showed a steady decline in systolic function, evident as a 5.2% drop in EF (P=0.04; Video S24). The VIVIT‐GFP treated MI cohort exhibited a more profound phenotype, with a 14% drop in EF and a 47 and 48 μL increase in end‐systolic volume and end‐diastolic volume, respectively (P<0.0001 for each parameter; Video S25). In contrast, the PP‐GFP treated MI cohort exhibited no LV dilatation and, notably, an 11% increase in EF (P<0.0001) to 44.4%±0.8% at end point that correlated with improvements in stroke volume and cardiac output (Figure S7A through S7E; Video S26). Segmental and global circumferential and longitudinal strain analysis confirmed these differences in end point systolic function (Figure S7F through S7L).

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 length by gravimetric measure. n: GFP Sham—3; VIVIT‐GFP Sham—5; PP‐GFP Sham—5; GFP MI—7; VIVIT‐GFP MI—10; PP‐GFP MI—7. H, Representative transverse cardiac sections stained with Masson’s Trichrome for infarcted cohorts. Scale bar—1 mm. I, Infarct size measured as % left ventricular circumference containing >50% transmural scar. n: GFP—5; VIVIT‐GFP—8; PP‐GFP MI—6. Data analyzed by 1‐way ANOVA and Tukey’s multiple comparisons test. J, Dysfunctional myocardium as assessed by circumferential peak systolic strain analysis and expressed as % LV volume. n: GFP Sham—3; VIVIT‐GFP Sham—5; PP‐GFP Sham—5; GFP MI—7; VIVIT‐GFP MI—9; PP‐GFP MI—7. C–G and J, Data analyzed by 2‐way ANOVA and Tukey’s multiple comparisons test. Factors were AAV cohorts and time after surgery (matched, C–E) or AAV cohorts and surgical condition (F,G, and J). AAV indicates adeno‐associated virus; CaNAβ2, calcineurin Aβ; CIP4, Cdc42‐interacting protein 4; EGFP, enhanced green fluorescent protein; ITR, inverted terminal repeat; IV, intravenous; LCA, left coronary artery; LV, left ventricular; MI, myocardial infarction; NFAT, nuclear factor of activated T cells; PL, permanent ligation; PP‐GFP, polyproline‐green fluorescent protein; and SV40 pA, SV40 polyadenylation signal.

Histological assessment of infarct size showed that VIVIT‐GFP increased 20%, whereas PP‐GFP decreased 32% infarct size when compared with GFP control (Figure 5H and 5I). Measurement of fractional dysfunctional myocardium by segmental circumferential and longitudinal strain analysis corroborated these results (Figure 5J; Figure S7I). As a control for the differential action of the VIVIT‐GFP and PP‐GFP peptides, ventricular extracts were assayed by western blot for dephosphorylation of NFATc2 transcription factor (Figure S7M). As expected, VIVIT‐GFP, but not PP‐GFP inhibited NFATc2 dephosphorylation at end point after LCA PL when compared with GFP control, ¹⁶ , ²⁴ consistent with the VIVIT‐inhibited cardioprotection by CaN‐NFAT signaling. ¹⁷ In sum, expression of VIVIT‐GFP, which inhibits CaN‐NFAT signaling, worsened cardiac function and structure after MI due to LCA PL. In contrast, PP‐GFP anchoring disruption, which inhibits the formation of CIP4‐CaNAβ2 signalosomes but does not inhibit NFAT activation, markedly improved cardiac function and structure in chronic ischemic cardiomyopathy. These results support the hypothesis that in contrast to the broad‐based targeting of CaNAβ2, selective inhibition of CIP4‐CaNAβ2 signalosomes is beneficial in ischemic heart disease.

Lack of Effect by CaNAβ polyproline Anchoring Disruption on T Cells In Vitro

CaNAβ gene targeting has been reported to inhibit T‐cell development and function in young mice, overlapping in phenotype with cyclosporin A‐induced immunosuppression. ¹⁸ Although the experiments presented above are based upon cardiomyocyte‐specific AAV expression of PP‐GFP, an alternative approach could involve developing a small chemical inhibitor that disrupts CIP4‐CaNAβ binding. In order to screen for a function of CaNAβ in T cells that might be inhibited by a systemic therapy targeting polyproline‐anchored CaNAβ, we transduced primary mouse CD4⁺ T cells, which express CIP4, ³² with lentivirus that expresses PP‐GFP or GFP control. As the degree of T‐cell receptor stimulation dictates T‐cell activation outcomes and T cells are central to cardiac repair and ischemic heart failure, ³³ , ³⁴ T cells were stimulated by high and low doses of αCD3 and αCD28 activating antibodies. T cells were transduced with similar efficacy as shown by flow cytometry for GFP expression (40%±17% and 36%±17% [mean±SD] of the GFP and PP‐GFP lentivirus‐transduced cells treated with the high dose of activating antibodies, respectively; Figure S8). Viability was also not different between PP‐GFP and GFP transduced T cells (Figure 6B). Importantly, surface expression of early (CD69) and late (CD25) markers of T‐cell activation were increased in GFP‐expressing cells in an αCD3 and αCD28 dose‐dependent manner, regardless of expression of the polyproline anchoring disruptor peptide (Figure 6C through 6E). Likewise, mRNA levels of cytokines associated with T‐cell activation and known to be regulated by NFAT, such as IL‐2 (interleukin‐2), IFNγ (interferon‐gamma), IL‐17, and TNFα (tumor necrosis factor α), ³⁵ , ³⁶ , ³⁷ were comparably elevated in both groups regardless of GFP or PP‐GFP expression (Figure 6F). These data demonstrate that polyproline anchoring of CaNAβ does not serve a significant role in T‐cell activation and, moreover, suggest that a heart failure drug targeting CIP4‐CaNAβ signalosomes would not be associated with immunosuppression.

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, tumor necrosis factor α.

DISCUSSION

CaNAβ has been established by gene knockout as an important mediator of both pathological cardiac hypertrophy and cardiomyocyte survival, as well as serving important roles in the immune system and other tissues. ⁵ , ⁶ , ³⁸ This pleiotropy has been a roadblock to the development of CaN‐targeted therapeutics for heart failure, particularly in the context of chronic ischemic cardiomyopathy where this a significant risk of recurrent myocardial infarction. ¹ We have shown that the endosomal scaffold protein CIP4 binds CaNAβ via its polyproline domain, constituting an independent Ca²⁺ and CaNAβ2 signaling compartment that promotes myocyte hypertrophy and pathological cardiac remodeling in response to chronic pressure overload. ¹⁶ The results presented herein demonstrate that in contrast to generalized CaNAβ2 depletion or inhibition of CaN interactions with the large family of PxIxIT motif‐containing substrates that include NFAT transcription factors, ⁷ CIP4 gene targeting and polyproline‐anchoring disruption are not deleterious and, instead, can be functionally beneficial during I/R injury (Figure S9). Notably, both cardiomyocyte‐specific CIP4 knockout and PP‐GFP expression improved cardiac structure and function in chronic ischemic cardiomyopathy. CIP4‐CaNAβ2 signalosomes may, therefore, constitute a favorable target for intervention in both acute MI and chronic pathological cardiac remodeling under diverse pathophysiological conditions.

In this project, we took advantage of recent advances in 4‐dimensional echocardiography and strain analysis to corroborate histological results following I/R injury and chronic MI. MI due to I/R injury is traditionally measured by Evans’ Blue and TTC histological staining, which relies on colorimetric differences to quantify perfused, viable, and nonviable myocardium. ³⁹ Chronic MI can be quantified in Masson’s Trichrome stained fixed tissue by the measurement of blue scar tissue. As infarcted tissue is hypocontractile, MI can also be identified as regions exhibiting reduced longitudinal or circumferential strain using high‐resolution echocardiography. ⁴⁰ , ⁴¹ Dysfunctional myocardium measured as % endocardial length in parasternal long axis views or % volume using parasternal short axis images correlated with histological measurement of infarct size both in acute and chronic MI, with the notable exception of the experiment involving PP‐GFP expression and I/R injury. Although VIVIT‐GFP expression resulted in worse I/R injury than either PP‐GFP or GFP regardless of quantification method, PP‐GFP resulted in no difference in I/R injury when measured histologically but significantly improved I/R injury when measured by strain. It is possible that the different results reflect a relative lack of sensitivity in the histological measurement of infarction post I/R. Alternatively, the strain measurements used in this study are not designed to distinguish stunned and nonviable myocardium. ⁴² It is possible that the improvement in systolic function following treatment with AAV9.PP‐GFP, whether measured by end‐systolic volume, EF, or strain, reflects decreased stunning relative to the VIVIT‐GFP and GFP cohorts as opposed to improved myocardial survival.

Parenthetically, we note that despite the similarity in phenotype of MCM and CIP4 ^f/f controls in uninjured and sham‐operated mice, as well as in chronic MI following LCA PL, MCM control mice had decreased infarct size and improved cardiac function after I/R injury when compared with CIP4 ^f/f control littermates (Figures 1 and 2). Tamoxifen‐treated MCM mice have been reported to exhibit transient systolic dysfunction, which is absent at the low dose of tamoxifen used here. ⁴³ In contrast, the present results suggest that a low dose of tamoxifen in conjunction with the MCM transgene may induce a preconditioning‐like phenomenon, underscoring the need for appropriate controls in genetic studies.

It is well established that CaN phosphatase activity is important for cardiac myocyte survival and hypertrophy via activation of NFAT transcription factors. ⁶ Early on, expression of a constitutive active truncated CaNAα was shown to decrease cardiomyocyte cell death following I/R injury in mice. ⁴⁴ This improvement in cardiac survival was in contrast to its induction of pathological cardiac hypertrophy and heart failure in uninjured transgenic mice. ⁴⁵ Conversely, via decreased NFAT activation, infarct size and myocyte apoptosis following I/R injury were found to be increased in global, constitutive CaNAβ knockout mice, ¹⁷ which also exhibited decreased pressure overload‐induced pathological hypertrophy. ⁹ Notably, transgenic expression of the minor CaNAβ isoform CaNAβ1, that is localized by palmitoylation to the plasma membrane and Golgi by its unique C‐terminus, ⁴⁶ improved cardiac function and reduced scar size after MI induced by permanent coronary artery ligation. ¹⁵ CaNAβ1, however, activated AKT (protein kinase B), ATF4, and serine and 1‐carbon metabolism, but not NFAT, and was antihypertrophic. ¹⁵ , ⁴⁷ We show here that like CaN isoform‐nonspecific VIVIT‐mediated inhibition, depletion by RNA interference of CaNAβ2 exacerbated I/R injury in a myocyte autonomous manner (Figures 3 and 4). Taken together, these results support a role for CaN in cardioprotection, and, moreover, suggest that loss of cardiomyocyte CaNAβ2 is a major contributor to the worsened phenotype in I/R injury conferred by global CaNAβ gene targeting.

Paradoxically, CaN signaling can also be proapoptotic. ⁴⁸ By binding the CaNAβ polyproline domain, CIP4 binds mainly, if not exclusively, CaNAβ2 in the adult cardiomyocyte. ¹⁶ Consistent with the association of CaN with diverse scaffold proteins, ⁵ , ⁷ results previously obtained by live cell imaging suggest that CIP4‐associated CaNAβ2 comprises a small, localized pool of CaN within the myocyte. ¹⁶ Although CIP4‐CaNAβ2 signalosomes are prohypertrophic, CIP4‐CaNAβ2 activity has not been associated with NFAT activation. ¹⁶ Neither PP‐GFP nor CIP4 CKO was found to increase infarct size after I/R injury. These results contrast with those for VIVIT‐GFP in I/R injury, which inhibits NFAT activation. ²⁴ Instead, both PP‐GFP and CIP4 CKO improved cardiac structure and function long term after MI. Although we have defined the physiological relevance of CIP4‐CaNAβ2 signalosomes, how CIP4‐bound CaNAβ2 promotes adverse remodeling remains unclear. Future research will focus on identifying which CaN substrates contribute to adverse CIP4 signaling in that subcellular compartment.

The results of CaNAβ and CIP4 knockout and PP‐GFP expression in chronic disease models, including the results shown here for chronic ischemic cardiomyopathy, support a role for CaNAβ2 in pathological cardiac remodeling, ⁹ , ¹⁶ whether through CIP4 or in other subcellular compartments (Figure S9). In contrast to the beneficial effects of VIVIT peptide in pressure overload hypertrophy, ²⁵ the worse outcome following VIVIT‐GFP in the chronic MI model presumably reflects the unique importance of CaNAβ2‐mediated cardioprotection in ischemic disease. Likewise, the exacerbation of I/R injury by shCaNAβ2 stands in stark contrast to the lack of increased infarct size and the improved cardiac function for CIP4 CKO and PP‐GFP‐expressing mice in the I/R model. A limitation of this study is that PP‐GFP expression was used to probe the function of CIP4‐associated CaNAβ. PP‐GFP would be expected to compete CaNAβ binding to other scaffolds that bind the polyproline domain, as well the binding to CIP4 of other CIP4 SH3 domain binding partners. ¹⁶ Although we hypothesize that CIP4‐bound CaNAβ2 does not contribute to cardioprotection like CaNAβ2 in other intracellular compartments, it is also possible that CaNAβ2 is simply not associated with CIP4 during I/R and that PP‐GFP blockade of binding of other protein partners to the CIP4 SH3 domain confers the beneficial phenotype in that model. Regardless, that neither CIP4 CKO nor PP‐GFP promoted myocardial loss after I/R injury supports the safety of targeting CIP4‐CaNAβ2 signalosomes complexes in pathological cardiac remodeling and chronic cardiac disease.

Current therapy for acute MI is generally focused on coronary reperfusion, ⁴⁹ and targeting of CIP4‐CaNAβ2 signalosomes, which might enhance myocardial function, could be advantageous in this setting. In addition, together with earlier findings in the pressure overload model, ¹⁶ results obtained followed permanent coronary artery ligation constitute proof‐of‐concept that targeting of CIP4‐CaNAβ2 signalosomes can be efficacious for the prevention of heart failure arising from diverse causes. A cardiac‐specific AAV gene therapy could be effective for the prevention or treatment of heart failure, but due to the latency of AAV therapies, a small molecule inhibiting the CIP4‐CaNAβ protein–protein interaction would presumably be preferable for the treatment of acute MI. This approach would represent a distinct therapeutic strategy compared with currently available CaN inhibitors such as the immunosuppressants cyclosporin A and FK506 (tacrolimus). Although instrumental for the widespread deployment of solid organ transplant therapies, ³⁸ CaN inhibitors currently in clinical use have a variety of off‐target effects, including prominent renal toxicity. ⁵⁰ Interestingly, CaN inhibitor‐associated nephrotoxicity has been associated with inhibition of renal CaNAα. ⁵¹ CaNAβ is also important for T‐cell development and function, such that CaNAβ knockout mice have defective allograft rejection. ¹⁸ Young CaNAβ knock‐out mice have reduced CD3+ T cells, as well as CD4+ and CD8+ cells. ¹⁸ , ⁵² In addition, T cells isolated from CaNAβ knock‐out mice have reduced proliferation in response to αCD3 and αCD28 and inhibited IL‐2 expression in response to phorbol ester and ionomycin. ¹⁰ , ¹⁸ Our results demonstrate that in vitro T‐cell activation and viability are similar regardless of polyproline‐anchored CaNAβ. Although further studies will be necessary regarding potential off‐target effects of polyproline anchoring disruption therapies, the lack of effect of PP‐GFP on T‐cell activation suggest that systemic targeting of polyproline‐anchored CaNAβ using a small chemical inhibitor would not be immunosuppressive. This would be necessary for therapeutic translation, as T cells play a critical role in the acute response to cardiac ischemia. ⁵³

Like polyproline‐anchored CaNAβ, CIP4 may itself be an acceptable drug target. We have found that CIP4 cardiomyocyte‐specific knockout in the healthy adult mouse induces no obvious phenotype. ¹⁶ In addition, CIP4 global knockout mice exhibit no obvious anatomical, behavioral, growth, or reproductive pathology, with the exception of a mild thrombocytopenia. ⁵⁴ , ⁵⁵ Interestingly, CIP4 targeting is potentially beneficial for the treatment of diabetes, and decreased postprandial serum glucose levels and increased adipocyte glucose uptake and insulin sensitivity were observed for CIP4 knock‐out mice, apparently due to decreased glucose transporter 4 internalization. ⁵⁴ CIP4 knockout mice also exhibit normal T‐ and B‐cell development and generally normal immune responses, with defects specifically in T‐cell dependent IgG and IgE antibody responses and contact hypersensitivity. ³²

CONCLUSIONS

Given the improvement in cardiac structure and function in ischemic and pressure overload cardiomyopathy conferred by CIP4‐CaNAβ targeting in mice, ¹⁶ further research is warranted into the translational potential of targeting CIP4‐CaNAβ signalosomes in cardiovascular disease, including by a CaNAβ polyproline peptide gene therapy.

Sources of Funding

This work was supported by National Institutes of Health Grants R01 HL158052 (Dr Kapiloff), R01 HL166547 (Dr Dodge‐Kafka), R01 HL144477 and R01 HL165725 (Dr Alcaide), and F30 HL162200 (Dr Bayer), and the National Heart, Lung, and Blood Institute Gene Therapy Resource Program.

Disclosures

None.

Supporting information

Tables S1–S3

Supplemental Methods

Major Resources Table

Figures S1–S9

References 56–62

Videos S1–S26