p53 regulates the cardiac transcriptome Tak W. Maka,1, Ludger Hauckb, Daniela Grotheb, and Filio Billiab,c,d,e,1 aCampbell Family Cancer Research Institute, Princess Margaret Hospital, Toronto, ON, Canada M5G 2M9; bToronto General Research Institute, Toronto, ON, Canada M5G 1L7; cDivision of Cardiology, University Health Network, Toronto, ON, Canada M5G 2C4; dHeart and Stroke Richard Lewar Centre of Excellence, University of Toronto, Toronto, ON, Canada M5S 1A8; and eInstitute of Medical Science, University of Toronto, Toronto, ON, Canada M5G 1A8 Contributed by Tak W. Mak, January 20, 2017 (sent for review October 10, 2016; reviewed by Kanaga Sabapathy and Karen H. Vousden) The tumor suppressor Trp53 (p53) inhibits cell growth after acute identifying the factors that effectively maintain cardiac tissue ho- stress by regulating gene transcription. The mammalian genome meostasis is of great scientific and clinical importance. contains hundreds of p53-binding sites. However, whether p53 Elevated p53 levels correlate with CM apoptosis and hypertro- participates in the regulation of cardiac tissue homeostasis under phy in end-stage human HF (15). The heart, as an obligate aerobic normal conditions is not known. To examine the physiologic role of organ, has the largest mitochondrial (mt) content to meet its high p53 in adult cardiomyocytes in vivo, Cre-loxP–mediated conditional energy demand (16). Reactive oxygen species (ROS), normal by- gene targeting in adult mice was used. Genome-wide transcriptome products of aerobic respiration, induce DNA damage (17), thereby analyses of conditional heart-specific p53 knockout mice were per- activating cellular defense systems against ROS through stabiliza- formed. Genome-wide annotation and pathway analyses of >5,000 tion of p53 (18). Therefore, we hypothesized that impairment of differentially expressed transcripts identified many p53-regulated p53 will have deleterious consequencesontheheart.Herewere- gene clusters. Correlative analyses identified >20 gene sets contain- port that p53 forms a critical hub in a comprehensive transcrip- ing more than 1,000 genes relevant to cardiac architecture and func- tional network. Our study suggests that p53 acts as a pleiotropic tion. These transcriptomic changes orchestrate cardiac architecture, regulator of cardiac structure and function. excitation-contraction coupling, mitochondrial biogenesis, and oxida- tive phosphorylation capacity. Interestingly, the gene expression sig- Results nature in p53-deficient hearts confers resistance to acute Cardiac-Specific Ablation of p53 Induces Age-Dependent Cardiac biomechanical stress. The data presented here demonstrate a role Hypertrophy and HF. We crossed transgenic mice expressing Cre for p53, a previously unrecognized master regulator of the cardiac recombinase flanked by mutated estrogen receptors (MerCreMer; transcriptome. The complex contributions of p53 define a biological mcm) with mice carrying loxP-flanked alleles of p53 to obtain fl/fl paradigm for the p53 regulator network in the heart under p53 ;mcm animals. The day of the last injection was arbitrarily physiological conditions. set to 0. Four consecutive daily i.p. Tamoxifen (Tam) injections induced genetic ablation of p53 with high recombination efficiency heart failure | tumor suppressor | cardiac hypertrophy | transcriptome | (Fig. 1 A–C). To determine the physiological consequence of p53 fl/fl cardiomyopathy ablation, cardiac morphology and function in p53 ;mcm mice were assessed in the presence and absence of Tam. fl/fl he tumor suppressor Trp53 (p53) is a transcription factor that Although p53 ;mcm mice were initially normal post-Tam, they MEDICAL SCIENCES Ttranslates growth and survival signals into specific gene ex- developed concentric hypertrophy by 6 mo. These mice showed pression patterns, regulating tumor-free survival of an organism significant increases in heart weight/body weight (HBW) ratios (1). In normal cells, p53 expression is kept at low levels by the E3 (Fig. 1D)(P < 0.001) and wall thickening (Fig. 1E). In contrast, fl/fl ubiquitin ligase Mdm2, which targets p53 for proteasomal degra- p53 ;mcm mice developed normally in the absence of Tam (Fig. 1 dation (2). In response to acute stress, Mdm2 is inactivated and D and E). The cross-sectional area of cardiomyocytes was 1.5-fold fl/fl increased p53 levels block cell division and induce apoptosis (3). greater in p53 ;mcm mice post-Tam (46 ± 3.4%; P < 0.001) Conversely, p53 can activate Mdm2 transcription, thereby forming compared with vehicle-injected controls (Fig. 1 F and G). The a negative feedback loop that curtails p53 activity (4). Cells with analysis of cardiac performance by echocardiography on older fl/fl mutated p53 proliferate aberrantly and generate outgrowths of p53 ;mcm mice post-Tam revealed significant decreases in frac- genetically unstable cells, leading to tumorigenesis as demonstrated tional shortening (31 ± 4.5%)incomparisonwithvehicle-injected by the early cancer predisposition of p53 knockout mice (p53KO) (5). Loss of Mdm2 in mice leads to death in em- Significance bryogenesis through p53-induced apoptosis, which is prevented by p53 codeletion (6). These findings provide genetic evidence The tumor suppressor Trp53 (p53) is a gene that regulates the that Mdm2 exerts a physiologically critical role in regulating p53 expression of many genes. However, the role of p53 in the heart in vivo. has not been well characterized. This work documents the im- Adult mammalian cardiomyocytes (CM) are differentiated portant role for p53 in the heart as a master regulator of the postmitotic cells that lack significant proliferative potential cardiac transcriptome. The contribution of p53 to the mainte- through their inability to reactivate the cell cycle (7). This is caused nance of cardiac tissue homeostasis is complex under by the lack of G1 cyclin/cyclin-dependent kinases (Cdk), crucial physiological conditions. positive cell-cycle modulators, and high levels of cell-cycle inhibi- tors (8), such as the retinoblastoma proteins pRb/p130 (9) and the Author contributions: L.H. and F.B. designed research; L.H., D.G., and F.B. performed re- Cdk inhibitors (Cdki) p21/p27 (10). The limited mitotic capacity of search; T.W.M. and D.G. contributed new reagents/analytic tools; T.W.M., D.G., and F.B. analyzed data; and T.W.M., L.H., and F.B. wrote the paper. mature CM (11) renders the adult mammalian heart functionally Reviewers: K.S., National Cancer Centre; and K.H.V., Cancer Research United Kingdom unable to repair itself after ischemic injury (12). Instead, surviving Beatson Institute. CM undergo hypertrophy to compensate for the ensuing hemo- The authors declare no conflict of interest. dynamic stress manifested as cell enlargement, myofibrillar disar- Freely available online through the PNAS open access option. ray, and re-expression of fetal genes (13). This process becomes 1To whom correspondence may be addressed. Email: [email protected] or tmak@ maladaptive with time, leading to the development of heart failure uhnres.utoronto.ca. (HF) with significant morbidity and mortality (14). The molecular This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. mechanisms underlying HF remain poorly understood. As such, 1073/pnas.1621436114/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1621436114 PNAS | February 28, 2017 | vol. 114 | no. 9 | 2331–2336 Downloaded by guest on September 29, 2021 fl/fl cardiac contractility (Pln, Ryc2, Serca2a) in older p53 ;mcm mice post-Tam (Fig. 1J). Finally, sarcomere organization profoundly influences cardiac function with abnormalities in this structure commonly observed in human HF. Key contractile proteins fl/fl (Tpm1, Tnnc1, Tnnt1, Ttn) were down-regulated in older p53 ; mcm mice post-Tam (Fig. 1J), demonstrating that p53 is important for proper expression of the contractile apparatus. Beyond these genes, we also found key regulators of fatty acid metabolism and mitochondrial respiration to be transcriptionally down-regulated in older p53KO, including Esrrβ/γ,Pparα/γ, Pgc-1a, Tfam, and Tfb1/2m (Fig. 1J). These data strongly suggest that p53 acts in concert with other transcriptional components that are necessary for the regulation of physiological hypertrophy. p53-Deficient Hearts Are Resistant to Pressure Overload. We then examined the responses of p53 loss to mechanical stress in younger fl/fl mice by subjecting 10-wk-old Tam-treated p53 ;mcm mice to transaortic banding (TAB). After 3 wk of TAB, HBW in vehicle- fl/fl injected p53 ;mcm mice increased by 2.3-fold, compared with fl/fl Tam-treated sham p53 ;mcm animals (P < 0.001) (Fig. 2A). Vehicle-treated hearts subjected to TAB exhibited significantly greater dilatation and increases in left ventricular wall thickness fl/fl than Tam-injected p53 ;mcm mice (Fig. 2B). To confirm that higher HBW in control hearts after TAB represents cardiomyocyte hypertrophy, we determined that the width of cardiomyocytes from fl/fl hearts of vehicle-treated mice was 2.1-fold greater than in p53 ; mcm mice injected with Tam post-TAB (P < 0.001) (Fig. 2 C and D). This was accompanied with significantly higher levels of ANP, BNP, and β-MHC compared with p53-deficient hearts (Fig. 2E). Importantly, TAB treatment reduced fractional shortening in ve- fl/fl fl/fl hicle-injected p53 ;mcm mice, as compared with p53 ;mcm animals in the presence of Tam (P < 0.001) (Fig. 2F). fl/fl Fig. 1. Heart-specific ablation of p53 triggers age-dependent concentric hy- Next, we investigated the insensitivity of p53 ;mcm mice to pertrophy with cardiac dysfunction. (A)ImmunoblotanalysisofLVextracts TAB by immunoblotting of left ventricular lysates (Fig. 2 G–I). (60 μg total protein/lane) of p53fl/fl;mcm mice at various time points after Tam Mef2a, Pln, and Serca2 were markedly down-regulated in vehicle- using anti-p53 antibodies. Animals were 13 wk old. For normalization, West- treated p53KO, but not in Tam-treated p53KO (Fig. 2G). ern blots were probed with anti-nucleophosmin (Npm1). Immunoblots were repeated once with similar results. (B) PCR of DNA isolated from LV and liver samples of wild-type, vehicle-injected control p53fl/fl;mcm mice 7 d post-Tam.