Background: Studies in transgenic mice showed the key role of moloney murine leukemia virus 1 (Pim-1) in the control of cardiomyocyte function and viability. Objective: To investigate if Pim-1 is a therapeutic target for the cure of diabetic cardiomyopathy (DCM), a steadily increasing cause of non-ischemic heart failure. Methods and Results: Western blot analysis on hearts of streptozotocin-induced type-1 (T1D) mice showed a time-dependent reduction in Pim-1 (8-fold at 20 wks from T1D induction), a parallel decline in the Pim-1 activators STAT-3 (6-fold) and Akt (7-fold) and an increase of the Pim-1 direct inhibitor miR-1 (6-fold) (P 0.01 vs. age-matched non-diabetic (ND) mice for all comparisons). Moreover, diabetic hearts showed low levels of anti-apoptotic Bcl-2, high levels of pro-apoptotic Bad and increased caspase 3/7 activity (P<0.01 vs. ND for all comparisons). Studies on murine cardiomyocytes challenged with high glucose (HG) confirmed the in vivo expressional changes. In in vitro rescue studies, anti-miR-1 boosted Pim-1 and Bcl-2 expression and promoted cardiomyocytes survival under HG. Similarly, transfection with Pim-1 plasmid prevented cardiomyocyte apoptosis. Finally, at 4 wks from T1D induction, mice were randomly assigned to receive an i.v. injection of human (h) Pim-1 via cardiotropic serotype-9 adeno-associated virus (1X1010 or 5X1010 pfu, doses decided on pilot titration studies) or empty vector. Expression of hPim-1 was confirmed by Western blot and immunohistochemistry in cardiomyocytes and to a less extent in skeletal muscles (Fig.1a), but not in other organs. Echocardiography showed that hPim-1 gene therapy attenuates diastolic dysfunction and prevents the development of left ventricle dilatation and failure in T1D mice (Fig.1b). Conclusion: Down-regulation of pro-survival Pim-1 contributes in the pathogenesis of DCM. Systemic delivery of hPim-1 via cardiotropic AAV9 represents a novel and effective approach to treat DCM. © 2010 American Heart Association, Inc.