Targeted inhibition of calcineurin attenuates cardiac hypertrophy in vivo

Abstract

The Ca(2+)-calmodulin-activated Ser/Thr protein phosphatase calcineurin and the downstream transcriptional effectors of calcineurin, nuclear factor of activated T cells, have been implicated in the hypertrophic response of the myocardium. Recently, the calcineurin inhibitory agents cyclosporine A and FK506 have been extensively used to evaluate the importance of this signaling pathway in rodent models of cardiac hypertrophy. However, pharmacologic approaches have rendered equivocal results necessitating more specific or genetic-based inhibitory strategies. In this regard, we have generated Tg mice expressing the calcineurin inhibitory domains of Cain/Cabin-1 and A-kinase anchoring protein 79 specifically in the heart. DeltaCain and DeltaA-kinase-anchoring protein Tg mice demonstrated reduced cardiac calcineurin activity and reduced hypertrophy in response to catecholamine infusion or pressure overload. In a second approach, adenoviral-mediated gene transfer of DeltaCain was performed in the adult rat myocardium to evaluate the effectiveness of an acute intervention and any potential species dependency. DeltaCain adenoviral gene transfer inhibited cardiac calcineurin activity and reduced hypertrophy in response to pressure overload without reducing aortic pressure. These results provide genetic evidence implicating calcineurin as an important mediator of the cardiac hypertrophic response in vivo.

Figure 1

(A) The noncompetitive calcineurin inhibitory domain from Cain contains a putative motif that is conserved in AKAP79 and FKBP12. (B) The 194-aa Flag-tagged Cain peptide (21 kDa) and the 299-aa Flag-tagged AKAP79 peptide (40 kDa) were placed under control of the 5.5-kb murine α-myosin heavy chain promoter to drive transgene expression in the mouse heart. (C) Western blot analysis by using anti-Flag antibody of heart extracts from ΔCain and ΔAKAP TG mice. Positive controls consisted of extracts from neonatal ventricular cardiomyocytes infected with adenovirus encoding each protein domain.

Figure 2

(A) Quantitation of heart-to-body weight ratios after isoinfusion (60 mg/kg/day) over 14 days demonstrates a significant attenuation of cardiac hypertrophy in ΔCain and ΔAKAP Tg mice containing either single- (+/0) or double- (+/+) copy transgene integrations. (B) Similarly, abdominal aortic banding-induced cardiac hypertrophy was significantly attenuated in both single- or double-copy ΔCain and ΔAKAP Tg mice over 14 days. (C and D) Isoproterenol infusion and aortic banding stimulated a significant increase in cardiac calcineurin activity as measured by RII peptide dephosphorylation in WT mice (n = 3, each), which was reduced in isoinfused ΔCain (n = 3) or aortic-banded ΔAKAP Tg mice (n = 3). *, P < 0.05 vs. nonTG-iso or banded; †, P < 0.05 vs. nonTG; and #, P < 0.05 vs. AKAP (+/0).

Figure 3

(A) Gross morphology from histological cross section of the hearts from the indicated groups. Aortic banding and isoinfusion induced a noticeable hypertrophy response that was blunted in ΔCain Tg mice. (B) Northern blots of atrial natriuretic factor, skeletal α-actin, and myosin light chain 2a showed increased cardiac mRNA levels in WT-banded mice compared with sham WT or sham Cain Tg mice. However, Cain-banded mice demonstrated a qualitative decrease in marker expression compared with WT-banded mice. Glyceraldehyde-3-phosphate dehydrogenase mRNA levels are shown as a loading control.

Figure 4

(A) Rat hearts were infected with adenoviruses expressing either β-galactosidase (Adβgal) or the ΔCain transgene (Adcain) by aortic-coronary perfusion and subjected to transverse aortic constriction for 7 days. Adcain-infected rats showed a significant attenuation of hypertrophy compared with Adβgal infection. (B) Adβgal- and Adcain-infected cohorts obtained a similar trans-aortic pressure gradient of ≈60 mmHg. (C) Measurement of RII-peptide dephosphorylation rates showed a significant increase in cardiac calcineurin activity after aortic banding in control rats, which was normalized in Adcain-infected and -banded rats. (D) Western blotting for the Flag epitope demonstrated significant Cain protein expression 7 days after in vivo infection. *, P < 0.05 vs. Adβgal-banded.