Short Communication: Angiotensin II Type 1 Receptor–Mediated Upregulation of Calcineurin Activity Underlies Impairment of Cardioprotective Signaling in Diabetic Hearts

Methods

Male Otsuka–Long–Evans–Tokushima fatty (OLETF) rats, which spontaneously develop obesity and type 2 diabetes, and their controls (Long–Evans–Tokushima–Otsuka [LETO] rats) were used. Preparation of myocardial infarction, immunoblotting, quantitative real-time RT-PCR, calcineurin activity assay, and determination of insulin sensitivity were performed by standard methods (see the expanded Methods section in the Online Data Supplement, available at http://circres.ahajournals.org).

Results

Compared with LETO rats, OLETF rats had greater body weight, higher levels of blood glucose and serum insulin, and lower M value, indicating phenotype of type 2 diabetes (Online Table I). Pretreatment with valsartan did not modify body weight in OLETF rats but reduced plasma insulin level by 32% and increased M value by 122%, suggesting an improvement in insulin sensitivity.

Blood pressure levels, heart rates, and risk area sizes were similar in the study groups except for baseline blood pressure in rats pretreated with AT₁ receptor blockers, which was 8 mm Hg lower than in untreated rats (Online Tables II and III). In LETO rats, EPO and DADLE reduced infarct size as a percentage of risk area (%I/R) from 48.4±1.3% in controls to 27.7±3.4% and 24.8±5.0%, respectively (Figure 1). This DADLE-induced protection was blocked by AG490. Infarct size in OLETF rats (60.4±1.6%) was larger than that in LETO rats, and EPO and DADLE failed to limit infarct size (53.9±5.3% and 55.0±2.2%). However, in OLETF rats pretreated with valsartan, EPO and DADLE were protective (39.4±4.9% and 38.9±3.4%), and this effect of valsartan on EPO-induced protection was mimicked by losartan (57.8±5.9% versus 31.2±7.5%, P<0.05). Valsartan alone did not limit infarct size in OLETF rats, and results were the same when the dose of valsartan was increased by 5-fold (10 mg/kg per day) in post hoc experiments (54.1±9.6%). In LETO rats, infarct size or protection by EPO was not modified by valsartan pretreatment (Figure 1). Administration of BIO to directly inhibit GSK-3β similarly reduced infarct size in LETO and OLETF rats (%I/R=26.8±2.5% and 27.1±3.8%, P<0.05 versus each control).

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Figure 1. Infarct size data in LETO (A) and OLETF (B) rats. *P<0.05 vs corresponding control (n=4 to ≈5). Val indicates valsartan; Los, losartan.

Protein levels of GRP78 and GRP94, ER stress chaperone proteins, in the myocardium were higher by 102% and by 114%, respectively, in OLETF than in LETO rats, and valsartan attenuated the elevation of these ER stress markers (Online Figure I). EPO infusion increased levels of phospho-Jak2 and phospho-Akt in LETO rats (Figure 2). EPO failed to induce Jak2 and Akt phosphorylation in OLETF rats, but responses of these kinases were preserved in OLETF rats pretreated with valsartan. There was no significant difference in the baseline level of Jak2 phosphorylation (Online Figure II) and EPO-induced ERK1/2 phosphorylation between LETO and OLETF rats (data not shown).

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Figure 2. Impaired phosphorylation of Jak2 and Akt in OLETF rats. Ratio of phosphorylated kinase to total kinase after EPO infusion is expressed as a percentage of its baseline value. A, Jak2. B, Akt. Val indicates valsartan (n=4 to ≈7). *P<0.05 vs baseline, #P<0.05 vs OLETF.

There was no significant difference between total SOCS3 levels or SOCS3 levels in the Jak2 immunoprecipitates in OLETF and LETO rats (Online Figure III). However, tissue calcineurin activity was higher in OLETF than in LETO rats (2.00±0.31 versus 0.82±0.06 nmol/mg per minute) (Figure 3A), although protein and mRNA levels of calcineurin were similar in the two groups (Online Figures IV and V). Pretreatment with valsartan for 2 weeks suppressed tissue calcineurin activity similar to pretreatment with FK506 or cyclosporine A (Figure 3A), and chronic treatment with valsartan or FK506 restored response of Jak2 to EPO (Figures 2A and 3⇓B). In contrast, Jak2 response was not recovered by acute infusion of FK506 for 30 minutes from 15 minutes before EPO infusion in isolated OLETF hearts. PTP1B and SHIP1 mRNA levels in the myocardium were similar in OLETF and LETO rats, although transforming growth factor-β1 mRNA level was elevated in OLETF rats, as reported previously (Online Figure V).⁴

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Figure 3. Upregulation of myocardial calcineurin activity in OLETF rats and its involvement in impaired Jak2 phosphorylation. A, Calcineurin activity. *P<0.05 vs LETO; #P<0.05 vs OLETF. Val indicates valsartan; FK, FK506; CsA, cyclosporine A. B, Effects of EPO on Jak2 phosphorylation. Levels of Jak2 phosphorylation are expressed as in Figure 2. Chronic indicates 7-day treatment with FK506; acute, acute treatment with FK506 in vitro after isolation of hearts (see text for details). *P<0.05 vs baseline; #P<0.05 vs OLETF (n=4 to ≈9).

Discussion

Earlier studies have demonstrated impairment of PI3K-Akt and ERK signaling in the myocardium of rat models of type 1 and type 2 diabetes mellitus.^1–3 However, little is known about dysfunction of signaling mechanisms upstream of PI3K and ERK. Thus, we focused on alterations by diabetes in the Jak2-mediated signaling pathway, which is crucial for both cytokine receptors and G protein–coupled receptors to transmit cytoprotective signaling.^5–8 EPO and DADLE failed to activate Jak2 in OLETF rats, whereas direct inhibition of GSK-3β by BIO limited infarct size in OLETF and LETO rats, indicating disrupted signaling upstream of Jak2 and preserved protective mechanisms downstream of GSK-3β in diabetic myocardium. AT₁ receptor blockers restored both Jak2-PI3K-Akt signaling and myocardial response to EPO and DADLE in OLETF rats, although neither infarct size nor EPO-induced protection was modified by AT₁ blockade in LETO rats. These results indicate that AT₁ receptor activation is responsible for impaired Jak2 activation and interruption of cytoprotective signaling from the EPO and δ-opioid receptors in diabetic hearts.

We first assumed that SOCS3, a negative regulator of Jak2, is responsible for AT₁ receptor–mediated inhibition of Jak2 phosphorylation in the diabetic myocardium because SOCS3 has been shown to be upregulated by AT₁ receptor activation in noncardiac cells.⁹ However, this possibility was not supported by the results that level of SOCS3 bound to Jak2 was similar in OLETF and LETO rats. A significant difference was not detected also for expression of SHP-1 and PTP1B, protein phosphatases involving in Jak2 dephosphorylation.¹⁰

The hypothesis that calcineurin is responsible for lack of EPO-induced phosphorylation of Jak2 in OLETF rats was based on a report of upregulated calcineurin in the diabetic kidney¹¹ and a reported function of calcineurin as a Tyr protein phosphatase.¹² This hypothesis was supported by the findings that calcineurin activity was elevated by more than 2-fold in the myocardium of OLETF rats and that inhibition of calcineurin restored the response of Jak2 to EPO receptor activation (Figure 3). Furthermore, blockade of the AT₁ receptor normalized calcineurin activity, indicating that AT₁ receptor mediates upregulation of calcineurin in OLETF rats. However, direct dephosphorylation of Jak2 by calcineurin is unlikely to be a mechanism by which calcineurin inhibited increase in phospho-Jak2 level after EPO receptor activation, because acute FK506 infusion could not restore response of Jak2 in OLETF rats (Figure 3B).

How calcineurin activity is upregulated by the AT₁ receptor in the diabetic myocardium remains unclear. In contrast to reports on calcineurin in the renal cortex of diabetic rats,¹¹ protein and mRNA levels of calcineurin were not elevated in OLETF myocardium (Online Figures IV and V). Because tissue calcineurin activity was assayed less than condition of sufficiently high levels of Ca²⁺ and calmodulin, the difference between levels of calcineurin activity in the diabetic myocardium and nondiabetic myocardium would be attributable to difference in modulatory factors other than Ca²⁺ and calmodulin. Candidates for such factors include RCAN1 (regulator of calcineurin 1)¹³ and heat shock protein-90.¹⁴

Relationships among ER stress, Jak2 signaling, and antiinfarct tolerance of the myocardium in the diabetic heart are complex. Our previous studies have shown that PI3K-Akt and ERK pathways downstream of the EPO receptor have complementary roles in cardioprotection.^6,7 However, in diabetic hearts, both PI3K-Akt-GSK-3β signaling and ERK- GSK-3β signaling are impaired.^2,3 We recently found that suppression of ER stress restored ERK-GSK-3β signaling without recovery of Jak2-PI3K-Akt signaling.³ Suppression of ER stress by a chemical chaperone reduced infarct size in OLETF rats to the level of infarct size in LETO rats without change in heart weight,³ indicating that modest ventricular hypertrophy in OLETF rats is not a major cause of the infarct size difference. In the present study, blockade of the AT₁ receptor prevented both elevation of ER stress markers and loss of Jak2 phosphorylation response in diabetic hearts. Taken together, these observations indicate that blockade of the AT₁ receptor can be more proximal therapy compared with an ER stress modulator for preservation of myocardial response to Jak2-mediated cardioprotective interventions (Online Figure VI).