Parsing the Roles of the Transcription Factors GATA-4 and GATA-6 in the Adult Cardiac Hypertrophic Response Jop H. van Berlo 1 , Bruce J. Aronow 1 , Jeffery D. Molkentin 1,2 * 1 From the Department of Pediatrics, Cincinnati Children’s Hospital Medical Center, University of Cincinnati, Ohio, United States of America, 2 Howard Hughes Medical Institute, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio, United States of America Abstract The transcriptional code that programs cardiac hypertrophy involves the zinc finger-containing DNA binding factors GATA-4 and GATA-6, both of which are required to mount a hypertrophic response of the adult heart. Here we performed conditional gene deletion of Gata4 or Gata6 in the mouse heart in conjunction with reciprocal gene replacement using a transgene encoding either GATA-4 or GATA-6 in the heart as a means of parsing dosage effects of GATA-4 and GATA-6 versus unique functional roles. We determined that GATA-4 and GATA-6 play a redundant and dosage-sensitive role in programming the hypertrophic growth response of the heart following pressure overload stimulation. However, non- redundant functions were identified in allowing the heart to compensate and resist heart failure after pressure overload stimulation, as neither Gata4 nor Gata6 deletion was fully rescued by expression of the reciprocal transgene. For example, only Gata4 heart-specific deletion blocked the neoangiogenic response to pressure overload stimulation. Gene expression profiling from hearts of these gene-deleted mice showed both overlapping and unique transcriptional codes, which is presented. These results indicate that GATA-4 and GATA-6 play a dosage-dependent and redundant role in programming cardiac hypertrophy, but that each has a more complex role in maintaining cardiac homeostasis and resistance to heart failure following injury that cannot be compensated by the other. Citation: van Berlo JH, Aronow BJ, Molkentin JD (2013) Parsing the Roles of the Transcription Factors GATA-4 and GATA-6 in the Adult Cardiac Hypertrophic Response. PLoS ONE 8(12): e84591. doi:10.1371/journal.pone.0084591 Editor: Sakthivel Sadayappan, Loyola University Chicago, United States of America Received October 2, 2013; Accepted November 24, 2013; Published December , 2013 Copyright: ß 2013 van Berlo et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by grants from the National Institutes of Health (J.D.M., J.H.v.B.) and the Howard Hughes Medical Institute (J.D.M.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: Jeff.Molkentin@cchmc.org Introduction The adult heart typically undergoes a process of hypertrophic growth in response to injury or disease stimulation that promotes neurohumoral activation and/or increases in wall stress [1]. The heart responds to disease or stress by activating signaling and transcriptional pathways that ultimately result in the activation of pro-growth and adaptive genes in an attempt to compensate for an injury event or disruption of homeostasis [1]. The zinc-finger containing transcription factors GATA-4 and GATA-6 are each expressed in the heart where they play a prominent role in myocyte lineage commitment and differentiation during embryo- genesis [2,3] but each is also induced and re-employed in the adult heart following injury where they participate in mediating the hypertrophic growth of individual myocytes [3]. We have shown previously that heart-specific deletion of Gata4 in the adult mouse renders the heart less able to hypertrophy with agonist or pressure overload stimulation, as well as more likely to succumb to heart failure, even with aging [4]. Loss of Gata4 from the heart also negatively impacted neoangiogenesis following stress stimulation, further defining its role in homeostasis and prevention of heart failure [5]. By comparison, cardiac-specific deletion of Gata6 similarly resulted in a defective hypertrophic response with pressure overload stimulation, as well as a greater propensity towards heart failure [6]. During development Gata4 and Gata6 seem to be completely redundant in programming cardiomyocyte commitment, as deletion of either gene alone still permitted myocyte formation, yet deletion of both together resulted in a complete loss of the lineage [7]. Moreover, Gata4 +/2 Gata6 +/2 (double heterozygous) mice are embryonic lethal, similar to homozygous null mutations in either gene alone, again suggesting functional redundancy and that the total dosage of the four Gata4/ 6 alleles is most critical with respect to their function [8]. Here we attempted to investigate if GATA-4 and GATA-6 had unique functionality in the adult heart during the hypertrophic response and/or in maintaining proper homeostatic gene expression that underlies cardiac ‘‘well-being’’ by using a reciprocal gene replacement strategy. Materials and Methods Animal Models and Methods Gata4-loxP (fl) and Gata6-loxP (fl) mice were each described previously, as were transgenic mice expressing a tetracycline transactivator (tTA) driven inducible and cardiac-specific a- myosin heavy chain (aMHC) promoter or deletion with the bMHC-promoter driven, cre-expressing transgene [4,6]. Pressure overload induced by transverse aortic constriction (TAC) was performed as described previously, as well as assessment of cardiac ventricular performance by echocardiography [4–6]. Assessment of capillary density in the heart with isolectin B4 on frozen histological sections was performed as described previously [5]. Western blotting from cardiac nuclear protein extracts was performed as described previously [9]. mRNA collection from PLOS ONE | www.plosone.org 1 December 2013 | Volume 8 | Issue 12 | e84591 31