Mitochondrial dysfunction is associated with a pro-apoptotic cellular environment in senescent cardiac muscle
Introduction
The aging process is characterized by a progressive impairment in biochemical and physiologic capacity, inevitably resulting in increased morbidity and mortality. Aging causes a decline in cardiac function, due to decrements in stroke volume, ejection fraction, and cardiac output (Lakatta, 1993). Remodeling of the aging heart involves a significant loss of cardiac myocytes (∼30%), reactive hypertrophy of the remaining cells, and increased connective tissue (Anversa et al., 1986, Anversa et al., 1990, Olivetti et al., 1991, Wanagat et al., 2002). The reduction in the number of cardiomyocytes undoubtedly contributes to the decline in cardiac functional capacity in aged animals. Apoptosis, also termed programmed cell death, represents an important process mediating the loss of cardiac myocytes with advanced age (Kajstura et al., 1996, Lee et al., 2002, Phaneuf and Leeuwenburgh, 2002). Moreover, previous evidence indicates that apoptotic signaling increases protein degradation and impairs contractile function, independent of cell loss (Ruetten et al., 2001, Du et al., 2004). Thus, understanding the mechanisms involved in the aging-associated apoptotic process in cardiac myocytes is important for the development of future therapeutic and clinical approaches to combat this phenomenon.
Progressive impairment of mitochondrial function is a hallmark of aging (Wallace, 2005, Hiona and Leeuwenburgh, 2008). Mitochondria also play a key role in regulating apoptosis. There are numerous death-inducing signals, such as reactive oxygen species (ROS), which can trigger mitochondria to release apoptogenic proteins such as cytochrome c and AIF into the cytosol (Phaneuf and Leeuwenburgh, 2002, Primeau et al., 2002). Indeed, one of the many intriguing aspects of apoptosis regulation by mitochondria is their spatial and functional relationships with a number of pro-apoptotic proteins within the cell. For example, as part of the electron transport chain, cytochrome c is integral to the proper functioning of mitochondria in the synthesis of ATP. However, upon an apoptotic stress, cytochrome c is released from the organelle and proceeds to activate the cytosolic assembly of the apoptosome which in turn results in DNA fragmentation and cell death (Gustafsson and Gottlieb, 2008). Members of the Bcl-2 family of proteins also influence apoptotic susceptibility according to their subcellular localization and translocation with respect to the mitochondria. Certain pro- and anti-apoptotic members reside predominantly in the mitochondria, whereas other members such as Bax reside in the cytosol of healthy cells. In a pro-apoptotic cellular environment, Bax translocates to the outer mitochondrial membrane (Gross et al., 1998, Saikumar et al., 1998, Murphy et al., 1999). After translocation of Bax to the organelle, mitochondria lose their membrane potential (ΔP) and release apoptogenic proteins. In addition, Bax promotes fragmentation of the mitochondrial network by interacting with the mitochondrial fission machinery (Karbowski et al., 2002, Arnoult et al., 2005b, Neuspiel et al., 2005, Karbowski et al., 2006, Sheridan et al., 2008).
Mammalian cells maintain mitochondrial morphology by balancing the opposing processes of mitochondrial fusion and fission. Dynamin-related protein 1 (Drp 1) is partly responsible for mitochondrial membrane constriction and fission of organelle reticula, while Optic atrophy type 1 protein (Opa 1) seems to be involved in the regulation of mitochondrial cristae morphology and is essential for the fusion of mitochondria. There is evidence that multiple components of the mitochondrial morphogenesis machinery, including Drp 1 and Opa 1, can positively and negatively regulate apoptosis (Frank et al., 2001, Olichon et al., 2003, Lee et al., 2004, Arnoult et al., 2005a, Arnoult et al., 2005b, Frezza et al., 2006, Estaquier and Arnoult, 2007). For example, Drp 1 is an apoptogenic protein that when stimulated, translocates from the cytosol to the mitochondria to participate in the release of pro-apoptotic factors housed within the organelle (Lee et al., 2004, Arnoult et al., 2005b, Estaquier and Arnoult, 2007). Furthermore, Opa 1 is thought to play an active role in the release of cytochrome c during apoptosis (Olichon et al., 2003, Arnoult et al., 2005a, Frezza et al., 2006). The influence of these proteins on the mitochondrially mediated apoptotic pathway of the myocardium during advanced stages of aging is currently unknown.
Subcellular compartmentalization is also an essential aspect of signal transduction for the human Src homology and collagen (Shc) protein of 66-kDa (p66Shc). p66Shc regulates life span in mammals and is a critical component of the apoptotic response to oxidative stress (Migliaccio et al., 1999, Giorgio et al., 2005, Orsini et al., 2006). Under basal conditions, a fraction of p66Shc localizes to the mitochondria (Nemoto et al., 2006, Orsini et al., 2004). However, oxidative stress triggers a mitochondrial accumulation of the protein, and once imported into the organelle, p66Shc causes alterations in mitochondrial Ca2+ handling and three-dimensional structure, thus inducing apoptosis (Pinton et al., 2007). Interestingly, Ventura et al. (2004) demonstrated the specific and selective localization of p46Shc to the mitochondrial matrix which suggests that p46Shc may exert a nonredundant function in signal transduction pathways involving mitochondria. A number of studies have demonstrated an association between p66Shc and cardiomyocyte apoptosis under conditions of cardiac stress (Cesselli et al., 2001, Graiani et al., 2005, Bianchi et al., 2006, Obreztchikova et al., 2006, Rota et al., 2006, Malhotra et al., 2009). However, no investigations have examined how advanced aging affects the mechanisms of mitochondrially mediated apoptotic signaling involving the Shc proteins, as well as the mitochondrial morphology machinery, with the specific focus of identifying the subcellular localization of these apoptogenic factors. Therefore, the purpose of the present study was to assess the impact aging had on the compartmentalization of apoptogenic factors active in mitochondrially mediated cell death signaling in cardiac muscle. We hypothesized that the myocardium from aged animals would present with mitochondrial dysfunction, and that decrements in organelle function would be associated with pro-apoptotic signaling involving the mitochondrial compartment.
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Animals
Male Fischer 344 Brown Norway rats aged 6 (young) and 36 (senescent) months (mo) were studied. The animals (n = 15/group) were obtained from the National Institute of Aging stock located at Harlan Animal Colonies (Indianapolis, Indiana). They were housed two to a cage in a temperature-controlled room (20–21 °C) with a 12-h light–dark cycle and given food and water ad libitum. The Principles of Laboratory Animal Care (NIH publication No. 86-23, revised 1985) were followed and the York University
Cardiovascular physiology in young and senescent animals
The effects of age on the HW/BW ratio, MAP, (HW/BW)/MAP, and HR are summarized in Table 1. A significant, but modest 12% cardiac hypertrophy was observed in the senescent animals, as indicated by the HW/BW. This increase in heart size occurred in the absence of a proportional increase in MAP. In fact, there was a significantly lower MAP in the senescent (−19%), compared to the young animals. HR was also depressed (−20%; P < 0.05) in the aged animals (Table 1). These cardiovascular characteristics
Discussion
The aging-induced decline in cardiac function is associated with the pathological remodeling of the heart. The reduction in the number of cardiomyocytes undoubtedly contributes to the decline in cardiac functional capacity in aged animals. Apoptotic cell death represents an important process regulating the loss of cardiac myocytes with advanced age (Kajstura et al., 1996, Lee et al., 2002, Phaneuf and Leeuwenburgh, 2002, Gustafsson and Gottlieb, 2008). Progressive impairment of mitochondrial
Acknowledgements
We are grateful to Elmira Raeifar, Hafez Khalili, Julianna H. Huang, as well as to Drs. Anna-Maria Joseph and Béatrice Chabi for their dedicated technical expertise. This work was supported by the Canadian Institutes of Health Research (CIHR). During the course of this investigation, Vladimir Ljubicic was a recipient of a Doctoral Research Award from the Heart and Stroke Foundation of Canada. Keir J. Menzies is a CIHR Doctoral scholar. David A. Hood holds the Canada Research Chair in Cell
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