Pim-1 regulates cardiomyocyte survival downstream of Akt

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© 2007 Nature Publishing Group http://www.nature.com/naturemedicine

Pim-1 regulates cardiomyocyte survival downstream of Akt John A Muraski1, Marcello Rota2, Yu Misao2, Jenna Fransioli1, Christopher Cottage1, Natalie Gude1, Grazia Esposito2, Francesca Delucchi2, Michael Arcarese2, Roberto Alvarez1, Sailay Siddiqi1, Gregory N Emmanuel1, Weitao Wu1, Kimberlee Fischer1, Joshua J Martindale1, Christopher C Glembotski1, Annarosa Leri2, Jan Kajstura2, Nancy Magnuson3, Anton Berns4, Remus M Beretta5, Steven R Houser5, Erik M Schaefer6, Piero Anversa2 & Mark A Sussman1 The serine-threonine kinases Pim-1 and Akt regulate cellular proliferation and survival. Although Akt is known to be a crucial signaling protein in the myocardium, the role of Pim-1 has been overlooked. Pim-1 expression in the myocardium of mice decreased during postnatal development, re-emerged after acute pathological injury in mice and was increased in failing hearts of both mice and humans. Cardioprotective stimuli associated with Akt activation induced Pim-1 expression, but compensatory increases in Akt abundance and phosphorylation after pathological injury by infarction or pressure overload did not protect the myocardium in Pim-1–deficient mice. Transgenic expression of Pim-1 in the myocardium protected mice from infarction injury, and Pim-1 expression inhibited cardiomyocyte apoptosis with concomitant increases in Bcl-2 and Bcl-XL protein levels, as well as in Bad phosphorylation levels. Relative to nontransgenic controls, calcium dynamics were significantly enhanced in Pim1–overexpressing transgenic hearts, associated with increased expression of SERCA2a, and were depressed in Pim-1–deficient hearts. Collectively, these data suggest that Pim-1 is a crucial facet of cardioprotection downstream of Akt.

Intracellular molecular signaling networks communicate through kinases that phosphorylate target substrates to regulate crucial aspects of growth and survival. Pim-1, a proto-oncogenic serine-threonine kinase, was originally discovered as the proviral integration site for Moloney murine leukemia virus1. Pim-1 expression can be induced by cytokines and growth factors including LIF, GM-CSF, EGF and most interleukins (reviewed in ref. 2), consistent with a role for Pim-1 in the proliferation and survival of hematopoietic cells. Pim-1 mediates proliferative signaling through phosphorylation of multiple target substrates, resulting in cell-cycle transition, and also mediates protective effects through phosphorylation of multiple targets including Bad at Ser112 (ref. 3). Recently, induction of Pim-1 expression has been linked to Akt activation in hematopoietic cells4. Like Pim-1, Akt is a serinethreonine kinase involved in cell proliferation and survival. After growth factor or cytokine receptor activation, Akt is phosphorylated, resulting in a conformational change that releases Akt from the membrane, allowing it to transit through the cytosol and eventually to the nucleus, where it affects the transcription of target genes5 and exerts cardioprotective activity6. The Akt-mediated induction of Pim-1 in nonmyocardial contexts after prolactin administration and

the direct binding of Akt to a prolactin response element on the Pim-1 promoter7,8 support a role for Akt in regulation of Pim-1 activity, but whether such a relationship also exists in the myocardium has not been assessed. This study was designed to investigate the potential cardioprotective role of Pim-1. RESULTS Developmental regulation of Pim-1 cardiac expression As previously described9, the 34-kDa isoform of Pim-1 is visualized as two bands corresponding to the phosphorylated, higher moiety and unphosporylated, lower moiety forms in whole-cell lysate immunoblots of mouse hearts (Supplementary Fig. 1a online). Pim-1 expression decreased with age in myocardial lysates from mice, with neonatal heart samples showing 6.3-fold more Pim-1 than 30-week-old mice (Fig. 1a). Postnatal expression levels declined, but remained significantly elevated until 8 weeks of age, when protein levels become comparable to 30-week-old hearts (Fig. 1a). Similarly, a 3.5-fold increase in Pim1 mRNA levels occurred in neonatal hearts, and they decreased significantly by 8 weeks of age (Supplementary Fig. 1b). As induction of Pim-1 in the hematopoietic system is linked to Akt activity4, we performed in vitro kinase assays on whole-heart lysates

1San Diego State University Heart Institute, San Diego State University, 5500 Campanile Drive, San Diego, California 92182, USA. 2Cardiovascular Research Institute, New York Medical College, Vosburgh Pavilion, Valhalla, New York 10595, USA. 3School of Molecular Biosciences, Washington State University, Pullman, Washington 99164, USA. 4Division of Molecular Genetics, The Netherlands Cancer Institute, 1066 CX Amsterdam, The Netherlands. 5Cardiovascular Research Center, Temple University School of Medicine, 3420 North Broad Street, Philadelphia, Pennsylvania 19140, USA. 6BioSource Cytokines & Signaling, Invitrogen Corporation 94 South Street, Hopkinton, Maryland 01748, USA. Correspondence should be addressed to M.A.S. ([email protected]).

Received 22 August; accepted 24 September; published online 25 November 2007; doi:10.1038/nm1671










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