Ataxia-telangiectasia mutated gene product (ATM) is a 350 kDa Serine/Threonine kinase belonging to the family of Phosphatidylinositol-3 kinase like kinases. ATM functions as a key element in DNA Damage Response (DDR), a mechanism that maintains genomic integrity within the cells. ATM is activated after double stranded DNA damage and initiates signalling cascades that determine the process of decision-making of cell fate and involves the participation of multiple proteins. This vital protein acts first by sensing double stranded DNA breaks and second by transducing the signal and activating other downstream proteins of the repair pathway via its kinase function. This provides an important link between signals generated after DNA damage, the cell cycle pathway and apoptotic machinery. This function is crucial for mammalian cells which are constantly challenged by genotoxic agents from a variety of sources and therefore require a robust sensing and repair mechanism to maintain cell vitality. Cells lacking ATM are hypersensitive to cytotoxic insults, particularly genotoxic stress, induced through radiation or radiomimetic drugs. This thesis describes the discovery and characterisation of novel autoregulatory feedback loops of ATM kinase in human cells. Firstly, I have discovered that inhibition of ATM kinase activity causes induction of ATM protein expression followed by time dependent oscillations. This novel autoregulatory mechanism was demonstrated in cell cycle independent manner and both in the absence and presence of DNA damage. ATM promoter assay revealed that this autoregulation was governed at the transcriptional level. Furthermore, this autoregulatory induction of ATM was also accompanied by a transient upregulation of P53, pATR and E2F1 levels. Elucidation of the underlying trafficking mechanism of ATM during such autoregulation and in DDR also revealed a novel ATM sub-cellular trafficking mechanism which was dependent on its own kinase activity. This trafficking mechanism involved DNA damage induced Golgi to nuclear transport of phosphorylated ATM S-1981 to elicit DDR. This was found to be a conserved pathway required during the initiation of DDR and was demonstrated in multiple cell lines. Further studies into the sub-cellular transport machinery revealed the involvement of β-COPI coatomer protein in this mechanism of ATM trafficking, which was found to be autoregulated by ATM kinase, and required 387-388 ATM di-Lysine motif. The discovery of these functionally important autoregulatory mechanisms of ATM were further utilised to develop Luciferase reporter based biosensor of DNA damage and single cell fluorescence based ATM inhibition assay to screen for ATM inhibitors. Finally, following the discovery and characterisation of these functional spatio-temporal autoregulations of ATM, quantitative estimations of the kinetics of signalling cascades initiated by it during DDR and its overall outcome on cellular fate were determined to study ATM pathway systematically for employing a quantitative systems biology approach. These novel findings have immensely increased our understanding of ATM regulation and function. Elucidation of the mechanisms of novel autoregulations of ATM provide new dimensions through which DDR pathway could be manipulated, and as such could be utilised for achieving targeted cellular sensitivity in therapeutic intervention of cancer.

Background: Environmental and endogenous stresses induce genomic DNA damage. In order to combat cellular assaults and maintain genomic integrity, reparative processes including DNA damage repair (DDR) and autophagy are activated. A key protein involved in DDR is ataxia telangiectasia mutated kinase (ATM). Mutations in ATM gene cause a multi-systemic disease called ataxia telangiectasia. Approximately 1.4-2.0% of the population has heterozygous mutation in ATM gene, which associates with enhanced susceptibility to cancer and ischemic heart disease. Autophagy, a conserved catabolic process, functions to maintain genomic stability by the sequestration and removal of misfolded proteins and damaged organelles. Dysregulation of autophagy contributes to the pathogenesis of many diseases including heart disease. Previous work from our lab has demonstrated autophagic impairment in the myocardium of ATM deficient mice during an acute phase (4 hr) of myocardial infarction (MI). The objective of this study was to examine the role of ATM deficiency in autophagic impairment during a chronic phase (28 days) post-MI. Methods: Wildtype (WT) and ATM heterozygous knockout (hKO) mice underwent MI by the ligation of the left anterior descending artery. Expression and activity of proteins associated with autophagy were examined in the infarct left ventricular tissue 28 days post-MI using western blot analyses. The data were analyzed using ANOVA followed by Student-Newman-Keuls test. A p-value of Results: The ratio of microtubule-associated protein light chain 3 (LC3-II-to-LC3-I; an indicator of autophagic turnover) lower in hKO-sham vs WT-sham. MI led to significant decrease in this ratio in WT-MI vs WT-sham. Protein levels of p62 (an autophagic transport protein) remained unchanged among the four groups. Expression levels of beclin-1 (aids in the formation of the autophagophore) were similarly increased in both MI groups vs their sham controls. Levels of mature cathepsin D (a lysosomal protease involved in lysosomal degradation of misfolded proteins) were significantly higher in WT-MI vs WT-sham group. Interestingly, cathepsin D levels were significantly lower in hKO-MI vs WT-MI group. Activation of mTOR (a coordinator of autophagy, cell growth and metabolism) was significantly higher in hKO-MI, not in WT-MI, vs hKO-sham group. Activation of AMPK (a sensor and regulator of cellular energy homeostasis) was higher in WT-MI, not in hKO-MI, vs WT-sham. Conclusion: Thus, ATM deficiency alters autophagic response in the heart chronic post-MI.

Ataxia telangiectasia mutated kinase (ATM) is activated in response to DNA damage. We have previously shown that ATM plays a critical role in myocyte apoptosis and cardiac remodeling after myocardial infarction (MI). Here, we tested the hypothesis that ATM deficiency results in autophagic impairment in the heart early during MI. MI was induced in wild-type (WT) and ATM heterozygous knockout (hKO) mice by ligation of the left anterior descending artery. Structural and biochemical parameters of the heart were measured 4 h after left anterior descending artery ligation. M-mode echocardiography revealed that MI worsens heart function, as evidenced by reduced percent ejection fraction and fractional shortening in both groups. However, MI-induced increase in left ventricular end-diastolic and end-systolic diameters and volumes were significantly lower in hKO hearts. ATM deficiency resulted in autophagic impairment during MI, as evidenced by decreased microtubule-associated protein light chain 3-II increased p62, decreased cathepsin D protein levels, and increased aggresome accumulation. ERK1/2 activation was only observed in WT-MI hearts. Activation of Akt and AMP-activated protein kinase (AMPK) was lower, whereas activation of glycogen synthase kinase (GSK)-3β and mammalian target of rapamycin (mTOR) was higher in hKO-MI hearts. Inhibition of ATM using KU-55933 resulted in autophagic impairment in cardiac fibroblasts, as evidenced by decreased light chain 3-II protein levels and formation of acidic vesicular organelles. This impairment was associated with decreased activation of Akt and AMPK but enhanced activation of GSK-3 β and mTOR in KU-55933-treated fibroblasts. Thus, ATM deficiency results in autophagic impairment in the heart during MI and cardiac fibroblasts. This autophagic impairment may occur via the activation of GSK-3 β and mTOR and inactivation of Akt and AMPK. NEW & NOTEWORTHY Ataxia telangiectasia mutated kinase (ATM) plays a critical role in myocyte apoptosis and cardiac remodeling after myocardial infarction (MI). Here, we provide evidence that ATM deficiency results in autophagic impairment during MI. Further investigation of the role of ATM in autophagy post-MI may provide novel therapeutic targets for patients with ataxia telangiectasia suffering from heart disease.

Ataxia-telangiectasia mutated kinase (ATM) is a serine/threonine kinase. Mutations in the ATM gene cause a rare autosomal multisystemic disease known as Ataxia-telangiectasia (AT). Individuals with mutations in both copies of the ATM gene suffer from increased susceptibility to ionizing radiation, predisposition to cancer, insulin resistance, immune deficiency, and premature aging. Patients with one mutated allele make-up ~1.4 to 2% of the general population. These individuals are spared from most of the symptoms of the disease. However, they are predisposed to developing cancer or ischemic heart disease, and die 7-8 years earlier than the non-carriers. DNA double-strand breaks activate ATM, and active ATM is known to phosphorylate an extensive array of proteins involved in cell cycle arrest, DNA repair, and apoptosis. The importance of ATM in the regulation of DNA damage response signaling is fairly well-established. This review summarizes the role of ATM in the heart, specifically in cardiac remodeling following β-adrenergic receptor stimulation and myocardial infarction.