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Cardiovascular Research 2003 59(2):268-270; doi:10.1016/S0008-6363(03)00480-2
© 2003 by European Society of Cardiology
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Copyright © 2003, European Society of Cardiology

Ischemia/reperfusion-induced apoptosis: connecting nitric oxide and cell cycle regulators

Carla Pignatti and Claudio Stefanelli*

Department of Biochemistry "G. Moruzzi", University of Bologna, Via Irnerio, 48, 40126 Bologna, Italy

* Corresponding author. Tel.: +39-51-2091203; fax: +39-51-2091224. claudio.stefanelli{at}unibo.it

See article by Maejima et al. [1] (pages 308–320) in this issue.

Cardiomyocytes are able to divide throughout the fetal period of life and continue to increase in cell number up to the first 2-3 days after birth. Thereafter, they undergo terminal differentiation. Although in certain pathological conditions, such as myocardial infarction, cardiomyocyte proliferation has been observed also in the adult heart [2], postnatal cardiomyocytes are normally irreversibly withdrawn from the cell cycle, resulting in the loss of their regenerative capacity. In response to some stimuli, such as increased overload and ischemia, cardiac myocytes can grow by hypertrophy, which is accompanied by the upregulation of protein synthesis [3]. Hypertrophic and mitogenic stimuli share similar intracellular responses, such as multiple second messenger systems and induction of various immediate-early genes. Actually, serum stimulation of neonatal, terminally-differentiated cardiomyocytes, similarly to what happens in proliferating cells, upregulates some of the cell cycle regulators such as G1 cyclins and associated cyclin-dependent kinases (cdks), which are required for the induction of cardiac hypertrophy [4].

The cdk enzymes are members of a conserved family of serine/threonine kinases whose activity is dependent on the presence of activating subunits known as cyclins. The abundance of specific cyclins increases during phases of the cell cycle in which they are required, thus regulating cell cycle progression. Such a control system operates at the level of checkpoints that occur at the G1/S phase boundary, in the S phase, and during the G2/M phases. For example, cdk2 binds cyclin A or E at the transition G1/S of the cycle and the complex may phosphorylate specific proteins, thus allowing the cell cycle processes to continue. Whereas cyclin binding is required for cdk kinase activity, other proteins can inhibit the enzymatic activity of cyclin/cdk complexes and prevent cell cycle progression. The p21 family (p21cip1/waf1, p27kip1, and p57kip2) of these cdk inhibitors predominantly inhibits the cdks of the G1 to S phase transition. It is worth noting that these inhibitors can be transactivated by the "tumor suppressor" p53 protein.

In cardiomyocytes, the early cell cycle arrest is accompanied by the disappearance of most cyclins and cdks. However, adult cardiomyocytes continue to express some of the cell cycle regulators whose role is not fully understood [5,6]. In particular, some cdk inhibitors such as p21cip1/waf1 and p27kip1 are considered as key regulators of cell cycle arrest in postmitotic cardiomyocytes [5].

Cell cycle regulators may be also involved in apoptosis [7]. Actually, in proliferating cells many apoptotic stimuli induce cell cycle arrest before cell death, thereby affecting both cell cycle and apoptotic machinery. In hypoxic cardiomyocytes, Adachi and colleagues found an increased expression of cyclin A that was concomitant with activation of the associated kinase cdk2 [6]. In addition, the cdk inhibitor p21cip1/waf1 was decreased, possibly following cleavage by the apoptotic protease caspase-3. Notably, overexpression of cyclin A was sufficient to induce apoptosis in normoxic cardiomyocytes, highlighting a key role of cyclin A and the associated kinase activity in the signal transduction pathway leading to apoptosis in hypoxic cells. Moreover, in a model of simulated hypoxia, Hauck et al. [8] observed the activation of cdks 2 and 3 and identified the retinoblastoma protein as a substrate of the increased cyclin-associated kinase activity. The phosphorylation and inactivation of the retinoblastoma protein can result in activation of the transcription factor E2F-1, leading to transcriptional activation of E2F-responsive genes, including those for pro-apoptotic caspase-3 and -7 and cyclin A itself, as well as to downregulation of cdk inhibitors p21cip1/waf1 and p27kip1 [5]. These events suggest a role for cell cycle regulators in a positive feedback loop mediating apoptosis in hypoxic cells.

Nitric oxide (NO) is a signalling molecule and a pleiotropic mediator involved in a growing number of cell functions. NO can be formed by constitutive (eNOS) and by inducible (iNOS) isoforms of nitric oxide synthase and is relevant for cardiovascular pathophysiology since it plays a major role in vascular biology and heart failure [9]. NO has an important role in the regulation of cell death and cell survival, and it can be both pro-apoptotic and anti-apoptotic depending on its flow and concentration, the environmental redox state, and the nature of the cells and their differentiation and proliferative condition [10–12].

The mechanisms governing the signal transduction pathway that links NO to apoptosis are largely unknown but are beginning to be elucidated. Increasing evidence indicates that the targets of NO include molecules involved in signal transduction and DNA repair as well as proteins implicated in the apoptotic process such as caspases, p53, bax, and cdk inhibitors. The cross-talk between cell destructive and protective signal pathways determines the impact of NO in cell death and survival [13].

NO represents both an autocrine and paracrine modulator in protecting the myocardium from ischemia/reperfusion-induced injury. Furthermore, an increasing body of evidence supports a role for NO as a trigger of ischemic preconditioning, highlighting the cardioprotective action of NO [14,15]. Actually, whereas the exact physiological role of NO in many forms of cardiomyocyte apoptosis remains controversial, the protective action of NO against ischemia/reperfusion-induced myocardial apoptosis [16–18], an important mode of cell loss observed in a number of situations including angina, myocardial infarction, and cardiac surgery, is well established.

Despite the large body of literature, the molecular mechanisms involved in the anti-apoptotic effect of NO in postischemic cardiomyocytes are poorly understood. Increased production of NO can result in direct inhibition of caspase activity due to S-nitrosation of an essential cysteine residue of caspase-3 and other caspases [16,19]. NO can also regulate signal transduction leading to activation of survival pathways such as those linked to nuclear factor {kappa}B. It appears likely that NO, which exerts many effects on cellular biochemistry, can affect the apoptotic machinery at more than one site.

In this issue of Cardiovascular Research, Maejima and colleagues [1] add a piece to the puzzle of the molecular mechanisms involved in the induction of apoptosis in cardiomyocytes exposed to ischemia/reperfusion. The investigators, using cultured neonatal rat cardiomyocytes, show that NO can modulate the changes in cell cycle-related proteins that accompany apoptosis. In their paper, Maejima et al. extend their previous studies in hypoxic cells [6] to ischemia/reperfusion conditions and confirm an important role of cyclin A-associated kinase activity in the development of apoptosis. The increase of this activity during ischemia/reperfusion was caused by accumulation of the activating cyclin A and downregulation of the inhibiting p21cip1/waf1. According to previous reports [20], Maejima et al. also show that ischemia/reperfusion increases endogenous NO synthesis. The newly synthesized NO, whose formation appears to be linked to the reperfusion step, is cardioprotective and attenuates the induction of apoptosis. The authors then asked whether NO could affect the changes in cell cycle-related proteins that they had observed. By blocking endogenous NO formation by pharmacological inhibition or increasing NO levels by use of a donor molecule, Maejima and colleagues provide data showing that the NO produced by cardiomyocytes during reperfusion both decreases levels of the cyclin A protein and upregulates p21cip1/waf1. This results in attenuation of the cyclin A-associated kinase activity stimulated by ischemia/reperfusion and in protection against apoptosis.

These findings are relevant for understanding the mechanisms that underlie the induction of apoptosis in the ischemic/reperfused heart and the protective role of NO and raise some questions that can be answered in upcoming research. First, what is the source of NO in this experimental system? Several data from the literature indicate a role for both the constitutive [21] and the inducible [17] isoform of NOS in cardioprotection against ischemia/reperfusion. If iNOS is involved, it is likely that NO formation is triggered by addition of serum during the reperfusion of ischemic cardiomyocytes. It is worth noting that the events leading to activation of cyclin A-associated kinase probably have an earlier origin, i.e. during ischemia, since they can be observed in hypoxic cells [6,8]. But what are these events triggered by hypoxia that lead to cdk activation in cardiomyocytes? Further, what are the mechanisms responsible for the effects of NO on cell cycle regulator proteins in cardiomyocytes? As regards p21cip1/waf1, in smooth muscle cells NO can both increase the expression [22] as well as inhibit the degradation [23] of the p21cip1/waf1 protein. However, p21cip1/waf1 is also cleaved by caspase-3 [24] that is inhibited by NO [16,19], and this mechanism might contribute to increase the stability of the protein. Lastly, an intriguing question concerns the possible role (if any) of p53 in the action of NO. The p53 protein could well be involved in apoptosis of hypoxic cardiomyocytes [25] and is closely implicated in cell cycle regulation, apoptosis, hypoxia, and NO-dependent pathways [26].

In conclusion, the paper by Maejima et al. is the first to show that NO can play a role in protecting cardiomyocytes by modulating cell cycle regulator proteins. This study underlines an emerging role of cell cycle regulator proteins in the induction of cardiomyocyte apoptosis [5,6,8] and indicates a new direction for future research in order to find new therapeutic targets for the control of cardiac apoptosis.


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