Review articleCaMKII oxidative activation and the pathogenesis of cardiac disease
Introduction
Oxidative stress plays an important role in the development of cardiac disease [1]. It is unclear, however, how increased oxidative stress is “translated” into deleterious disease phenotypes. One molecule that has recently been implicated to be a sensor of oxidative stress in the heart and lung is the Ca2 +/calmodulin dependent protein kinase II (CaMKII). CaMKII is activated in numerous cardiac diseases, and contributes to the development of heart failure, and arrhythmias [2]. These pathways have intersected with the discovery of direct regulation of CaMKII activity by cellular reactive oxygen species (ROS) by direct oxidation of the enzyme's regulatory domain [3].
CaMKII functions as a homo- or hetero-multimer consisting of 12 subunits, each consisting of three conserved domains: an amino-terminal catalytic domain, a central autoregulatory domain, and a carboxy-terminal association domain. The catalytic domain contains the ATP and substrate binding pockets, providing the catalytic activity of the protein. The autoregulatory domain contains an inhibitory pseudosubstrate sequence, several sites for post-translational modification (Fig. 1), and the calmodulin-binding region. The association domain is responsible for oligomerization of the subunits to form the holoenzyme, and also contains variable regions that are alternatively spliced to form different splice variants of CaMKII [4].
CaMKII activity is autoinhibited by its pseudosubstrate region, which resides in the autoregulatory domain. This region binds the catalytic domain and sterically blocks the substrate and ATP binding pockets [5], [6]. Activation of CaMKII occurs upon binding of calcium-activated calmodulin (Ca2 +/CaM) to the autoregulatory domain. The binding of Ca2 +/CaM displaces the pseudosubstrate region, allowing the substrate and ATP access to the catalytic domain. Sustained activation of the kinase in the presence of ATP results in autophosphorylation across subunits at Thr287 [7]. This phosphorylation event leads to a 1000-fold increase in affinity for CaM, and prevents the reassociation of the catalytic domain resulting in the persistence of enzyme activity even in the absence of Ca2 +/CaM [8]. Activation of CaMKII subunits by Ca2 +/CaM, and subsequent intra-subunit phosphorylation also stimulate subunit exchange between holoenzymes. This exchange of active subunits leads to further activation of inactive holoenzyme via phosphorylation of neighboring subunits even in the absence of Ca2 +/CaM binding [9]. These studies illustrate the complexities of CaMKII activation and persistent, or autonomous, activity following autophosphorylation.
More recently, our group identified an alternative mechanism for CaMKII to remain active in the absence of Ca2 +/CaM. CaMKII can be oxidized at methionines 281 and 282 in the presence of ROS. Initial binding of Ca2 +/CaM is required to displace the pseudosubstrate region from the catalytic domain before the enzyme can maintain its activated state in the absence of Ca2 +/CaM. Mutation of the methionine residues to non-oxidizable valines renders the enzyme insensitive to persistent activity in the presence of ROS. Development of antiserum that specifically recognizes oxidized CaMKII has allowed us to validate that CaMKII is oxidized in vivo [3]. Oxidation of CaMKII at its paired methionine residues appears to act as a sensor of cellular ROS, and not a signal for oxidative damage and subsequent protein degradation. Increased oxidation of CaMKII, determined by immunoblotting, does not correlate with a decrease in the total amount of CaMKII protein present [10], [11], [12], but with an increase in kinase activity [3].
Oxidation of CaMKII occurs via ROS produced from a variety of sources including NADPH oxidase, and mitochondria. Elimination of either of these pathways via genetic knockout, or targeted ROS scavenging, results in a reduction of CaMKII oxidation [3], [13]. Additionally, oxidation of CaMKII is a reversible event occurring via enzymatic reduction of the methionine residues by methionine sulfoxide reductase A (MsrA) [3]. Chronic CaMKII activation, as well as subsequent dysfunction, occurs when the balance of autonomously active versus auto-inhibited enzyme is shifted towards increased activity, and its downstream targets become phosphorylated inappropriately. In the case of oxidation, this occurs when the anti-oxidant systems in the heart cannot “keep up” with the oxidation of CaMKII by cellular ROS. This concept is demonstrated in mice with altered MsrA activity. MsrA knockout enhances CaMKII oxidation, cell death, and post-myocardial infarction (MI) mortality in mice [3], while MsrA transgenic over-expression reduces CaMKII oxidation and prevents pathological consequences of aldosterone [10] and angiotensin II [14] in myocardium.
Increased CaMKII expression and activity have been associated with several cardiac diseases. Overexpression of CaMKII in the heart leads to deranged calcium homeostasis and heart failure [15], [16], and arrhythmias [17]. CaMKII activity and expression are also elevated in cardiac injury models, including defined above, use abbreviation MI only (MI) [18], [19] and ischemia–reperfusion (I/R) injury [20], [21]. Oxidation of CaMKII has been directly measured or implicated in all of these conditions, suggesting a critical role for oxidative activation of CaMKII in the pathogenesis of cardiac disease.
Section snippets
Myocardial injury and inflammation
Inflammation is an innate immune response that is activated early in the wound-healing process, and involves a cascade of signaling events that recruit immune cells to the injury in order to “clean up” the injured tissue. Inflammation occurs following cardiac injury, such as MI or I/R, in order to repair the damaged myocardium. Oxidative stress and ROS production are associated with the inflammatory response, and play a critical role in the pathogenesis of cardiac disease following an injury
Neurohumoral signaling
Pathologic activation of neurohumoral signaling pathways, including β-adrenergic receptor, angiotensin II, and aldosterone, contribute to heart failure [27]. For example, elevated angiotensin II and aldosterone are seen in patients with heart failure [28], particularly heart failure related to MI [29], [30]. CaMKII is activated downstream of these signaling pathways, and is a critical mediator of the detrimental effects. These pathways are also associated with increases in ROS production and
Arrhythmia
Arrhythmia causes most cardiac-related sudden death. Arrhythmias are abnormal changes in heartbeats, and can arise from changes in ion channel properties, loss of normal intracellular Ca2 + homeostasis, or arise as the result of tissue remodeling. Both ROS and CaMKII have been shown to play a role in the development of a variety of arrhythmias [2], [35]. A recent study demonstrated that ROS-induced arrhythmias in isolated myocytes are blocked by inhibiting CaMKII activity [36], suggesting that
Diabetes
Diabetes is an important risk factor in the development of heart disease. Diabetes is known to cause increased oxidative stress, which may contribute to increased disease complications in these patients [50]. We recently reported that CaMKII plays a critical role in the increase in mortality in MI in the setting of diabetes. Oxidized CaMKII was elevated in the right atrium of diabetic patients compared to non-diabetic patients with MI as well as in the right atrium of diabetic mice. In this
Mitochondria
Mitochondria play an integral role in the heart in both calcium- and ROS-mediated signaling. ROS is produced in mitochondria as a byproduct of electron transport and ATP production, and is increased when oxygen consumption becomes uncoupled from ATP production [52]. Mitochondria become uncoupled following dissipation of the negative membrane potential across the inner mitochondrial membrane. Uncoupling can occur after excessive calcium uptake into the mitochondria resulting in the opening of
Conclusions
CaMKII is a critical sensor of oxidant stress in the heart, integrating calcium signals and ROS signals in several subcellular locations (Fig. 2). The effects of enhancing CaMKII activity via oxidation appear to be complex and widespread in cardiomyocytes. Global anti-oxidant therapies for patients have been largely unsuccessful [57], [58], suggesting that improved understanding of the mechanisms by which ROS-mediated signaling leads to cardiac disease will provide more specific, local targets
Funding
This work was funded in part by the US National Institutes of Health (R01HL70250, R01HL079031, R01HL113001, and R01HL096652) and a grant (08CVD01) from the Fondation Leducq as part of the Alliance for CaMKII Signaling in Heart.
Disclosures
M.E. Anderson is a named inventor on intellectual property claiming to treat myocardial infarction by CaMKII inhibition and is a cofounder of Allosteros Therapeutics, a biotech company aiming to develop enzyme-based therapies.
Acknowledgments
We are grateful for the artistic contributions of Shawn Roach.
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