Oxidative stress is caused by an imbalance between the production of reactive oxygen species (ROS) and the ability of an organism to eliminate these toxic intermediates. Although the Parkinson-susceptibility gene, Parkinson protein 7/DJ-1 (DJ-1), has been linked to the regulation of oxidative stress, the exact mechanism by which this occurs and its in vivo relevance have remained elusive. In the heart, oxidative stress is a major contributor to the development of heart failure (HF). Therefore, we hypothesized that DJ-1 inhibits the pathological consequences of ROS production in the heart, the organ with the highest oxidative burden. We report that DJ-1 is highly expressed in normal heart tissue but is markedly reduced in end-stage human HF. DJ-1-deficient mice subjected to oxidative stress by transaortic banding exhibited exaggerated cardiac hypertrophy and susceptibility to developing HF. This was accompanied by a Trp53 (p53)-dependent decrease in capillary density, an excessive oxidation of DNA, and increased cardiomyocyte apoptosis, key events in the development of HF. Impaired mitochondrial biogenesis and progressive respiratory chain deficiency were also evident in cardiomyocytes lacking DJ-1. Our results provide compelling in vivo evidence that DJ-1 is a unique and nonredundant antioxidant that functions independent of other antioxidative pathways in the cellular defense against ROS.cardiomyopathy | angiotensin II | JC-1

The tumor suppressor Trp53 (p53) inhibits cell growth after acute stress by regulating gene transcription. The mammalian genome contains hundreds of p53-binding sites. However, whether p53 participates in the regulation of cardiac tissue homeostasis under normal conditions is not known. To examine the physiologic role of p53 in adult cardiomyocytes in vivo, Cre-loxP–mediated conditional gene targeting in adult mice was used. Genome-wide transcriptome analyses of conditional heart-specific p53 knockout mice were performed. Genome-wide annotation and pathway analyses of >5,000 differentially expressed transcripts identified many p53-regulated gene clusters. Correlative analyses identified >20 gene sets containing more than 1,000 genes relevant to cardiac architecture and function. These transcriptomic changes orchestrate cardiac architecture, excitation-contraction coupling, mitochondrial biogenesis, and oxidative phosphorylation capacity. Interestingly, the gene expression signature in p53-deficient hearts confers resistance to acute biomechanical stress. The data presented here demonstrate a role for p53, a previously unrecognized master regulator of the cardiac transcriptome. The complex contributions of p53 define a biological paradigm for the p53 regulator network in the heart under physiological conditions.

Abstract-Statins are widely used clinical drugs that exert beneficial growth-suppressive effects in patients with cardiac hypertrophy. We investigated the role of the cell cycle inhibitor p21 CIP1/WAF1 (p21) in statin-dependent inhibition of hypertrophic growth in postmitotic cardiomyocytes. We demonstrate that lovastatin fails to inhibit cardiac hypertrophy to angiotensin II in p21 Ϫ/Ϫ mice and that reconstitution of p21 function by TAT.p21 protein transduction can rescue statin action in these otherwise normally developed animals. Lovastatin specifically recruits the forkhead box FoxO3a transcription factor to the p21 promoter, mediating transcriptional transactivation of the p21 gene as analyzed in isolated primary cardiomyocytes. Lovastatin also stimulates protein kinase B/Akt kinase activity, and Akt-dependent phosphorylation forces p21 in the cytoplasm, where it inhibits Rho-kinases contributing to the suppression of cardiomyocyte hypertrophy. Loss of p21 or FoxO3a by RNA interference causes a general inhibition of lovastatin signal transduction. These results suggest that p21 functions as FoxO3a downstream target to mediate an statin-derived anti-hypertrophic response. Taken together, our genetic and biochemical data delineate an essential function of p21 for statin-dependent inhibition of cardiac myocyte hypertrophy. (Circ Res. 2007;100:50-60.)

The maintenance of normal heart function requires proper control of protein turnover. The ubiquitin-proteasome system is a principal regulator of protein degradation. Mdm2 is the main E3 ubiquitin ligase for p53 in mitotic cells thereby regulating cellular growth, DNA repair, oxidative stress and apoptosis. However, which of these Mdm2-related activities are preserved in differentiated cardiomyocytes has yet to be determined. We sought to elucidate the role of Mdm2 in the control of normal heart function. We observed markedly reduced Mdm2 mRNA levels accompanied by highly elevated p53 protein expression in the hearts of wild type mice subjected to myocardial infarction or trans-aortic banding. Accordingly, we generated conditional cardiac-specific Mdm2 gene knockout (Mdm2f/f;mcm) mice. In adulthood, Mdm2f/f;mcm mice developed spontaneous cardiac hypertrophy, left ventricular dysfunction with early mortality post-tamoxifen. A decreased polyubiquitination of myocardial p53 was observed, leading to its stabilization and activation, in the absence of acute stress. In addition, transcriptomic analysis of Mdm2-deficient hearts revealed that there is an induction of E2f1 and c-Myc mRNA levels with reduced expression of the Pgc-1a/Ppara/Esrrb/g axis and Pink1. This was associated with a significant degree of cardiomyocyte apoptosis, and an inhibition of redox homeostasis and mitochondrial bioenergetics. All these processes are early, Mdm2-associated events and contribute to the development of pathological hypertrophy. Our genetic and biochemical data support a role for Mdm2 in cardiac growth control through the regulation of p53, the Pgc-1 family of transcriptional coactivators and the pivotal antioxidant Pink1.

Defining the roadblocks responsible for cell cycle arrest in adult cardiomyocytes lies at the core of developing cardiac regenerative therapies. p53 and Mdm2 are crucial mediators of cell cycle arrest in proliferative cell types, however, little is known about their function in regulating homeostasis and proliferation in terminally differentiated cell types, like cardiomyocytes. To explore this, we generated a cardiac-specific conditional deletion of p53 and Mdm2 (DKO) in adult mice. Herein we describe the development of a dilated cardiomyopathy, in the absence of cardiac hypertrophy. In addition, DKO hearts exhibited a significant increase in cardiomyocyte proliferation. Further evaluation showed that proliferation was mediated by a significant increase in Cdk2 and cyclin E with downregulation of p21 and p27. Comparison of miRNA expression profiles from DKO mouse hearts and controls revealed 11 miRNAs that were downregulated in the DKO hearts and enriched for mRNA targets involved in cell cycle regulation. Knockdown of these miRNAs in neonatal rat cardiomyocytes significantly increased cytokinesis with an upregulation in the expression of crucial cell cycle regulators. These results illustrate the importance of the cooperative activities of p53 and Mdm2 in a network of miRNAs that function to impose a barrier against aberrant cardiomyocyte cell cycle re-entry to maintain cardiac homeostasis.

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Cardiac homeostasis requires proper control of protein turnover. Protein degradation is principally controlled by the Ubiquitin-Proteasome System. Mule is an E3 ubiquitin ligase that regulates cellular growth, DNA repair and apoptosis to maintain normal tissue architecture. However, Mule’s function in the heart has yet to be described. In a screen, we found reduced Mule expression in left ventricular samples from end-stage heart failure patients. Consequently, we generated conditional cardiac-specific Mule knockout (Mule fl/fl(y);mcm) mice. Mule ablation in adult Mule fl/fl(y);mcm mice prevented myocardial c-Myc polyubiquitination, leading to c-Myc accumulation and subsequent reduced expression of Pgc-1α, Pink1, and mitochondrial complex proteins. Furthermore, these mice developed spontaneous cardiac hypertrophy, left ventricular dysfunction, and early mortality. Co-deletion of Mule and c-Myc rescued this phenotype. Our data supports an indispensable role for Mule in cardiac homeostasis through the regulation of mitochondrial function via maintenance of Pgc-1α and Pink1 expression and persistent negative regulation of c-Myc.