Pim-1 regulates cardiomyocyte survival downstream of Akt John A Muraski 1 , Marcello Rota 2 , Yu Misao 2 , Jenna Fransioli 1 , Christopher Cottage 1 , Natalie Gude 1 , Grazia Esposito 2 , Francesca Delucchi 2 , Michael Arcarese 2 , Roberto Alvarez 1 , Sailay Siddiqi 1 , Gregory N Emmanuel 1 , Weitao Wu 1 , Kimberlee Fischer 1 , Joshua J Martindale 1 , Christopher C Glembotski 1 , Annarosa Leri 2 , Jan Kajstura 2 , Nancy Magnuson 3 , Anton Berns 4 , Remus M Beretta 5 , Steven R Houser 5 , Erik M Schaefer 6 , Piero Anversa 2 & Mark A Sussman 1 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-X L protein levels, as well as in Bad phosphorylation levels. Relative to nontransgenic controls, calcium dynamics were significantly enhanced in Pim- 1–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 virus 1 . 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 protec- tive 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 cells 4 . Like Pim-1, Akt is a serine- threonine 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 genes 5 and exerts cardioprotective activity 6 . 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 promoter 7,8 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 described 9 , 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 immuno- blots of mouse hearts (Supplementary Fig. 1a online). Pim-1 expres- sion 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 signifi- cantly 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 activity 4 , we performed in vitro kinase assays on whole-heart lysates © 2007 Nature Publishing Group http://www.nature.com/naturemedicine Received 22 August; accepted 24 September; published online 25 November 2007; doi:10.1038/nm1671 1 San Diego State University Heart Institute, San Diego State University, 5500 Campanile Drive, San Diego, California 92182, USA. 2 Cardiovascular Research Institute, New York Medical College, Vosburgh Pavilion, Valhalla, New York 10595, USA. 3 School of Molecular Biosciences, Washington State University, Pullman, Washington 99164, USA. 4 Division of Molecular Genetics, The Netherlands Cancer Institute, 1066 CX Amsterdam, The Netherlands. 5 Cardiovascular Research Center, Temple University School of Medicine, 3420 North Broad Street, Philadelphia, Pennsylvania 19140, USA. 6 BioSource Cytokines & Signaling, Invitrogen Corporation 94 South Street, Hopkinton, Maryland 01748, USA. Correspondence should be addressed to M.A.S. (sussman@heart.sdsu.edu). NATURE MEDICINE ADVANCE ONLINE PUBLICATION 1 ARTICLES