Summary The mammalian heart has a remarkable regenerative capacity for a short period of time after birth, after which the majority of cardiomyocytes permanently exit cell cycle. We sought to determine the primary post-natal event that results in cardiomyocyte cell-cycle arrest. We hypothesized that transition to the oxygen rich postnatal environment is the upstream signal that results in cell cycle arrest of cardiomyocytes. Here we show that reactive oxygen species (ROS), oxidative DNA damage, and DNA damage response (DDR) markers significantly increase in the heart during the first postnatal week. Intriguingly, postnatal hypoxemia, ROS scavenging, or inhibition of DDR all prolong the postnatal proliferative window of cardiomyocytes, while hyperoxemia and ROS generators shorten it. These findings uncover a previously unrecognized protective mechanism that mediates cardiomyocyte cell cycle arrest in exchange for utilization of oxygen dependent aerobic metabolism. Reduction of mitochondrial-dependent oxidative stress should be important component of cardiomyocyte proliferation-based therapeutic approaches.

The adult mammalian heart is incapable of regeneration following cardiomyocyte loss, which underpins the lasting and severe effects of cardiomyopathy. Recently, it has become clear that the mammalian heart is not a post-mitotic organ. For example, the neonatal heart is capable of regenerating lost myocardium, and the adult heart is capable of modest self-renewal. In both of these scenarios, cardiomyocyte renewal occurs via the proliferation of pre-existing cardiomyocytes, and is regulated by aerobic-respiration-mediated oxidative DNA damage. Therefore, we reasoned that inhibiting aerobic respiration by inducing systemic hypoxaemia would alleviate oxidative DNA damage, thereby inducing cardiomyocyte proliferation in adult mammals. Here we report that, in mice, gradual exposure to severe systemic hypoxaemia, in which inspired oxygen is gradually decreased by 1% and maintained at 7% for 2 weeks, results in inhibition of oxidative metabolism, decreased reactive oxygen species production and oxidative DNA damage, and reactivation of cardiomyocyte mitosis. Notably, we find that exposure to hypoxaemia 1 week after induction of myocardial infarction induces a robust regenerative response with decreased myocardial fibrosis and improvement of left ventricular systolic function. Genetic fate-mapping analysis confirms that the newly formed myocardium is derived from pre-existing cardiomyocytes. These results demonstrate that the endogenous regenerative properties of the adult mammalian heart can be reactivated by exposure to gradual systemic hypoxaemia, and highlight the potential therapeutic role of hypoxia in regenerative medicine.

In the last few years, the methodical development of the 2D-electrophoresis has resulted in an increasing use of proteomic methods in the field of biological and pharmacological research. The shift from genome sequencing to large-scale analysis of proteomes required high throughput methods for the identification of proteins and provoked the development of mass spectrometry of protein fragments, which facilitated the automation of sample preparation and sample processing. So far, because of its low speed and the high chemical costs, the classical amino terminal sequencing of proteins for their identification has been often displaced by mass spectrometry. The book is clearly arranged and starts with a compact overview of mass spectrometry for protein identification including basic construction of mass spectrometers and ionization techniques. The special focus of this book is tandem mass spectrometry, but additional emphasis is also placed on the fragmentation chemistry of tryptic peptides. The proposed algorithm for the interpretation of product ion spectra was applied and discussed using five sample mass spectra, which assist a newcomer to retrace all steps of interpretation. The following chapters deal with the experimental and technical details including gel electrophoresis, digestion of proteins, mass spectrometric analysis of tryptic digests using capillary liquid chromatography and protein identification by database searching. The suggested protocols, which were used in the laboratory of the authors, are comprehensible and can be quickly used. Additionally, the use of tandem mass spectrometry for the sequence analysis of novel proteins and for the characterization of post-translationally modified proteins is presented in the final two chapters. Concerning a de-novo-sequencing of proteins, the limitations of producing new sequences are pointed out and strategies to enhance and confirm the interpretation of product spectra, such as fragmentation of product ions, synthetic peptides and peptide derivatization, were critically discussed. Finally, the book completely covers the procedures for analyzing protein sequences by mass spectrometry. Therefore, it is worth reading for researchers working in the field of mass spectrometry of proteins as well as for newcomers who want to learn the basics of mass spectrometry and plan to use these techniques in their own laboratories.

In recent studies we demonstrated that systemic levels of protein-bound nitrotyrosine (NO 2 Tyr) and myeloperoxidase (MPO), a protein that catalyzes generation of nitrating oxidants, serve as independent predictors of atherosclerotic risk, burden, and incident cardiac events. We now show both that apolipoprotein A-I (apoA-I), the primary protein constituent of HDL, is a selective target for MPO-catalyzed nitration and chlorination in vivo and that MPO-catalyzed oxidation of HDL and apoA-I results in selective inhibition in ABCA1-dependent cholesterol efflux from macrophages. Dramatic selective enrichment in NO 2 Tyr and chlorotyrosine (ClTyr) content within apoA-I recovered from serum and human atherosclerotic lesions is noted, and analysis of serum from sequential subjects demonstrates that the NO 2 Tyr and ClTyr contents of apoA-I are markedly higher in individuals with cardiovascular disease (CVD). Analysis of circulating HDL further reveals that higher NO 2 Tyr and ClTyr contents of the lipoprotein are each significantly associated with diminished ABCA1-dependent cholesterol efflux capacity of the lipoprotein. MPO as a likely mechanism for oxidative modification of apoA-I in vivo is apparently facilitated by MPO binding to apoA-I, as revealed by cross-immunoprecipitation studies in plasma, recovery of MPO within HDL-like particles isolated from human atheroma, and identification of a probable contact site between the apoA-I moiety of HDL and MPO. To our knowledge, the present results provide the first direct evidence for apoA-I as a selective target for MPO-catalyzed oxidative modification in human atheroma. They also suggest a potential mechanism for MPO-dependent generation of a proatherogenic dysfunctional form of HDL in vivo.that promote oxidative damage, cell injury, and conversion of LDL, the major carrier of cholesterol in plasma, into an atherogenic form (9, 14). Protein-bound nitrotyrosine (NO 2 Tyr), a posttranslational modification specific for protein oxidation by , is markedly enriched within human atheroma (8, 21). Further, recent clinical studies demonstrate that systemic levels of protein-bound NO 2 Tyr serve as an independent predictor of atherosclerotic risk and burden in subjects and are modulated by known CVD risk-reducing therapies such as statins (10,22). Few studies to date have focused on defining the molecular targets of nitration in subjects with CVD, the attendant functional alterations, and the enzymatic participants in nitration.One potential enzymatic source for generation of NO-derived oxidants within human atheroma is the heme protein myeloperoxidase (MPO). MPO utilizes hydrogen peroxide (H 2 O 2 ) and a variety of low-molecular weight organic and inorganic substances as substrates to form reactive oxidant species capable of promoting protein halogenation, nitration, and oxidative cross-linking (4, 5). For example, MPO directly utilizes both NO (23) and the NO metabolite nitrite (NO 2 − ) as substrates in vitro (17-19, 24) and participates

In recent studies we demonstrated that systemic levels of protein-bound nitrotyrosine (NO(2)Tyr) and myeloperoxidase (MPO), a protein that catalyzes generation of nitrating oxidants, serve as independent predictors of atherosclerotic risk, burden, and incident cardiac events. We now show both that apolipoprotein A-I (apoA-I), the primary protein constituent of HDL, is a selective target for MPO-catalyzed nitration and chlorination in vivo and that MPO-catalyzed oxidation of HDL and apoA-I results in selective inhibition in ABCA1-dependent cholesterol efflux from macrophages. Dramatic selective enrichment in NO(2)Tyr and chlorotyrosine (ClTyr) content within apoA-I recovered from serum and human atherosclerotic lesions is noted, and analysis of serum from sequential subjects demonstrates that the NO(2)Tyr and ClTyr contents of apoA-I are markedly higher in individuals with cardiovascular disease (CVD). Analysis of circulating HDL further reveals that higher NO(2)Tyr and ClTyr contents of the lipoprotein are each significantly associated with diminished ABCA1-dependent cholesterol efflux capacity of the lipoprotein. MPO as a likely mechanism for oxidative modification of apoA-I in vivo is apparently facilitated by MPO binding to apoA-I, as revealed by cross-immunoprecipitation studies in plasma, recovery of MPO within HDL-like particles isolated from human atheroma, and identification of a probable contact site between the apoA-I moiety of HDL and MPO. To our knowledge, the present results provide the first direct evidence for apoA-I as a selective target for MPO-catalyzed oxidative modification in human atheroma. They also suggest a potential mechanism for MPO-dependent generation of a proatherogenic dysfunctional form of HDL in vivo.