Oxidation Directly Activates CaMKII
CaMKII is activated by Ca2+/CaM, but autophosphorylation at T287 sustains catalytic activity after dissociation of Ca2+/CaM (Figure 1A) because the negatively charged phosphate prevents reassociation of the catalytic domain and autoinhibitory region (Hudmon and Schulman, 2002). CaMKII activity may also be enhanced by pro-oxidant conditions (Zhu et al., 2007); we therefore hypothesized that oxidation of the regulatory domain in the vicinity of T287 could sustain CaMKII catalytic activity by an analogous mechanism. Exposure of purified CaMKII to H2O2 in the absence of any pretreatment yielded no discernable CaMKII activity (Figure 1B). However, exposure to H2O2 after pretreatment with Ca2+/CaM yielded persistent CaMKII activation even in the presence of EGTA. These data suggest that Ca2+/CaM binding exposed a key segment of CaMKII for oxidation, and that oxidation interfered with the interaction of the autoinhibitory and catalytic domains. Activation of wild-type (WT) CaMKII by H2O2 was dose dependent (Figure 1C). The concentration of EGTA used was sufficient to block CaMKII activity without the addition of H2O2 (Figure 1B), suggesting that activity observed in the pro-oxidant condition was independent of sustained Ca2+/CaM binding.
Pretreatment with Ca2+/CaM was also necessary for autophosphorylation-dependent CaMKII activation, indicating that autophosphorylation and oxidation of CaMKII occur by parallel mechanisms. CaMKII bearing a T287A substitution underwent normal Ca2+/CaM-dependent activation but did not maintain persistent Ca2+-independent activity in the presence of ATP (Figure 1D). However, the T287A mutant was activated by H2O2 (Figure 1C), and the extent of this activation was statistically indistinguishable at all but the highest concentration of H2O2 tested (1 mM).Weinterpret these observations as evidence that activation of CaMKII by ROS and autophosphorylation occur by a similar mechanism, but by independent modifications to nearby sites. Activation of the kinase by either mechanism requires the enzyme to be initially ‘‘opened’’ by Ca2+/CaM to allow access to the autoinhibitory domain for oxidation or autophosphorylation (Figures 1A and 1E). Either of these modifications can prevent subsequent interaction of the autoinhibitory region with the catalytic domain, providing for sustained Ca2+-independent activation of CaMKII. Consistent with these ideas, direct measurements of intrinsic fluorescence revealed that autophosphorylation and oxidation of CaMKII independently induce similar conformational changes in CaMKII (Figure S1 available online).
Proteomic analysis of the synthetic peptide that contains the 281/282 methionine residues was used to probe for oxidative modification upon treatment with H2O2. We observed a clear decrease in the unoxidized form coupled with an increase in the various oxidized forms of this peptide based on the chromatographic traces and on the change in the number of observed spectra (Figure S2). In addition to the synthetic peptide, we analyzed the peptide containing the 281/282 methionine residues after treatment of the whole protein with H2O2 followed by trypsin cleavage. We were able to determine the relative change in oxidation of this peptide upon hydrogen peroxide treatment (Tables S1 and S2). The MS/MS spectra of the oxidized forms of the peptide were identical to those from the synthetic peptide, verifying that the oxidized peptide was correctly identified.
Given these observations and the recognized susceptibility of methionine residues to oxidation (Hoshi and Heinemann, 2001), we made methionine to valine mutations for the paired residues (M281/282V) andfor anothermethionine(M308V) intheCaM-binding region. Thesemutants were exposed to H2O2 and assayed for activity in the presence of EGTA (Figure 1C). The H2O2-dependent activation of CaMKII was preserved in the M308V mutant. However, oxidation-dependent CaMKII activity was completely abolished in the M281/282V and M281/282/308V mutants. Our data, obtained in cell-free assay conditions, point to direct oxidation of the M281/282 pair as the primary H2O2-dependent activation pathway for CaMKII. Importantly, all the methionine to valine mutants showed a normal activity response to autophosphorylation (Figure 1D), further supporting the concept that Ca2+-autonomous CaMKII activation by ROS or T287 autophosphorylation are independent events. While the paired methionine motif is conserved in the b, g, andd isoformsofCaMKII, the neuronal a isoform substitutes a cysteine residue for the first methionine of the pair (position 280 in CaMKIIa). The side chain of cysteine is also susceptible to oxidation. We generated a M281C mutant of CaMKIId to mimic the substitution in CaMKIIa. Additionally, we generated and purified CaMKIIa. Both the M281C CaMKIId mutant and the purified CaMKIIa were activated by H2O2, indicating that the cysteine substitution seen in CaMKIIa also supports ROS-dependent activation (Figure 1C). To further elucidate the role of M281 and M282 in ROS-dependent activation, these sites were individually mutated (Figure S3). The M282V mutation completely ablated oxidation-dependentactivation, while the M281V mutationpartially reduced activation by 65%, indicating that a single oxidation event within the regulatory domain is insufficient to activate CaMKII.
Autophosphorylation at T287 dramatically increases the binding affinity of CaMKII for CaM, a phenomenon known as ‘‘CaM trapping’’ (Meyer et al., 1992). In the absence of ATP the Ca2+/CaM/CaMKII complex was very rapidly dissociated following addition of EGTA, independent of the redox state, as measured by fluorescence anisotropy of dansylated CaM (Figure S4A). CaMKII exposure to H2O2 for 10 min induced Ca2+/CaM-independent activity (as in Figure 1B) but also failed to induce CaM trapping (not shown). These observations indicate that under normal experimental conditions, oxidation of CaMKII is not sufficient to induce CaMtrapping. Dissociation ofCa2+/CaMfrom autophosporylated CaMKII and CaM was significantly slower than from nonphosphorylated enzyme, consistent withCaMtrapping. However, pretreatment withH2O2 prior to EGTA had no significant effect on the dissociation kinetics. Thus, oxidation of CaM or CaMKII does not prevent or enhance CaM trapping by autophosphorylated CaMKII. CaM trapping is reduced by phosphorylation of T306/307 (Colbran, 1993), so we investigated whether oxidation of M308 might prevent CaM trapping by a parallel mechanism. We did observe a significant slowing of dissociation of the CaM/CaMKII complex after H2O2 treatment of the M308 mutant (Figures S4B and S4C). These data suggest that the absence of CaM trapping during oxidation is partly due to M308.
It seemed possible that conditions capable of oxidizing methionine residues would also oxidize unprotected cysteine residues. Although mutation of methionine residues at 281 and 282 was sufficient to completely ablate ROS-dependent activation of CaMKII, we created a C290V mutant to determine whether this cysteine residue within the CaMKII regulatory domain could also play a role. Both the Ca2+/CaM-dependent activity and the ROS-dependent activity of the C290V mutant were indistinguishable from that with WT CaMKII (Figure S4D). Our finding that oxidation of paired amino acids (M281/282 in CaMKIId) was required for activation by H2O2 supports a view that oxidation of a lone residue is insufficient to confer Ca2+/CaM-autonomous CaMKII activity. In order to comprehensively test the potential role of all accessible cysteines in contributing to oxidation-dependent CaMKII activity we measured CaMKII activity responses to H2O2 in the presence of iodoacetic acid, a reagent that blocks oxidation of unprotected cysteine residues (Zangerle et al., 1992). Cysteine-protected CaMKIId showed equivalent H2O2 activity responses compared to CaMKIId without iodoacetic acid (Figure S2D). We used an established colorometric assay to quantify the available cysteine residues and verify that cysteine protection by iodoacetic acid was effective. Our results confirmed that most or all of the 11 cysteines in CaMKIId were accessible to the Ellman’s reagent after Ca2+/
CaM binding, while treatment with iodoacetic acid blocked the accessibility of cysteine residues to biochemical modification (data not shown). Taken together, these findings demonstrate that oxidative activation of CaMKIId is independent of cysteines.
Oxidation of CaMKII Occurs In Vivo
We developed an immune serum against oxidized M281/282 to detect ROS effects on CaMKII in vivo. We validated the fidelity of the antiserum using purified CaMKII protein by immunoblotting against WT CaMKII and the M281/282V mutant in control conditions and after treatment with H2O2 or Ca2+/CaM/ATP. Blots were also assayed with a phospho- and site-specific antibody against T287 (p-287). WT CaMKII exposed to H2O2 after pretreatment with Ca2+/CaM showed significant reactivity to our oxidized M281/282 antiserum, but untreated and T287-phosphorylated CaMKII samples were not recognized by our antiserum (Figure 2A). The
M281/282Vmutant hadminimal reactivity to our antiserumamong the three treatments. These findings demonstrated that phospho-T287 and oxidized M281/282 were immunologically distinct sites. We performed additional immunoblots in which oxidized CaMKII was probed with the antiserum along with increasing concentrations of the peptide antigen (Figure 2B). Band intensity decreased with increasing peptide concentration, indicating that the immune serum was specific for oxidized CaMKII.
To determine the role of CaMKII oxidation in apoptosis, mice were treated with saline, AngII, or isoproterenol (Iso) for 1 week, and transverse heart sections from these mice were probed for the production of oxidized CaMKII in vivo. WT mice treated with AngII produced more oxidized CaMKII than those treated with saline or Iso (Figure 2C). Total CaMKII immunoreactivity remained constant regardless of treatment. Conversely, mice lacking a critical subunit of NADPH oxidase (p47-/-) did not show increased levels of oxidized CaMKII in response to AngII. The p47-/- mice did not assemble the ROS-producing complex NADPH oxidase (Munzel and Keaney, 2001), the main source of ROS due to AngII stimulation in many cell types (Lyle and Griendling, 2006). Heart sections from WT mice showed increased staining for T287-phosphorylated CaMKII after AngII treatment, while p47-/- mice were unaffected (Figure S5). Other studies have suggested that protein phosphatase activity is decreased by pro-oxidant conditions (Howe et al., 2004), indicating the possibility of coordinate activation of CaMKII both by direct oxidation at the Met281/282 sites and by phosphatase inactivation leading to increased phosphorylation at the T287 site.
We also homogenized hearts from mice treated with saline, AngII, or Iso, and whole heart lysates were analyzed by immunoblot for oxidized CaMKII. While total CaMKII was not significantly different among the three treatment groups, heart lysates from mice treated with AngII showed significantly increased oxidized CaMKII levels (Figure 2D). Taken together, these findings demonstrate that oxidation of CaMKII occurs in vivo, and that elevated levels of AngII increase CaMKII oxidation at M281/282 compared to saline or Iso.
AngII Triggers ROS Production and CaMKII-Dependent Apoptosis in Cardiomyocytes
Given our results, we hypothesized that cells deficient in ROS production or CaMKII activity would be resistant to AngII-mediated apoptosis. We treated cardiomyocytes from mice that express an inhibitory peptide against CaMKII (AC3-I, Zhang et al., 2005) with 100 nM AngII for 24 hr in parallel with isolated cardiomyocytes fromWTand p47-/- mice. AngII caused a significant increase in the percent of TUNEL-positive nuclei inWT cells but had no significant effect in p47-/- or AC3-I cardiomyocytes (Figure 3A). Activity assays for caspase-3, a downstream target enzyme in the CaMKII apoptotic signaling pathway in heart, recapitulated the results from the TUNEL assay (Figure 3B). Importantly, direct addition of ROS in the form of H2O2 caused significant apoptosis in p47-/- cells, demonstrating that their resistance to AngII-induced apoptosis is a result of impaired ability to produce ROS rather than a lack of sensitivity to ROS. The apoptotic effect of H2O2 in AC3-I cells was blunted by more than half compared to WT or p47-/- cells (Figure 3A), indicating the critical importance of CaMKII activation to ROS- and Iso-dependent apoptosis.
In order to validate the connection between AngII and ROS in our experimental model, we treated isolated cardiomyocytes from WT and p47-/- mice with 100 nM AngII and monitored production of ROS by imaging DHE, a fluorescent reporter for superoxide and hydrogen peroxide (Figure 3C). We also incubated WT cardiomyocytes with fura-2 AM, a cell-permeant calcium indicator, to observe changes in intracellular Ca2+ ([Ca2+]i). Treatment with AngII caused a significant increase in ROS production in WT but not in p47-/- cardiomyocytes (Figure 3D). On the other hand, increases in [Ca2+]i were significantly less after AngII compared to Iso treatment (Figures 3E and 3F) for cardiomyocytes from both WT and p47-/- mice. These data show that AngII signaling predominantly increases ROS, while Iso predominantly increases [Ca2+]i under our experimental conditions.
CaMKII Knockdown Prevents AngII- and Iso-Induced Apoptosis
In order to further test the role of CaMKII and specifically define the effects of M281/282 on myocardial apoptosis, we used a knockdown and replacement strategy in cultured neonatal cardiomyocytes. Rat cardiomyocytes were cultured and treated with shRNA-encoding lentivirus against rat CaMKIId. After 48 hr CaMKII expression was significantly reduced, as measured by immunoblot and activity assays (Figure 4A). Cells were then transduced with lentivirus encoding shRNA-resistant WT or M281/282V mutant CaMKII. Control cells were transduced with GFP-encoding lentivirus. After 48 hr, cells transduced with CaMKII rescue constructs showed significant recovery of CaMKII expression compared to control cells. Addition of Ca2+/ CaM to lysates from cells transduced with either CaMKII-encoding virus had similar total activity to native cells. However, H2O2-induced activity was only rescued in cells expressing the WT CaMKII construct. These cellular studies support our earlier finding with molecular CaMKII (Figure 1) by showing that oxidation of M281/282 is critical for ROS-triggered CaMKII activity. In addition, this strategy created cardiomyocytes that express ROS-resistant CaMKII, providing a system for investigating ROS- and CaMKII-dependent apoptosis.
Cardiomyocytes treated with shRNAs/CaMKIId-encoding lentivirus were exposed to saline, AngII, or Iso as above (Figure 4B). The apoptotic response to AngII and Iso was significantly attenuated in CaMKII knockdown cells compared to myocytes without shRNA. Moreover, expression of shRNA-resistant WT CaMKII fully rescued apoptotic responses to both agonists (Figure 4C). In contrast, expression of the ROS-resistant M281/282V CaMKII mutant rescued the apoptotic response to Iso but, importantly, failed to rescue the apoptotic response to AngII after 24 hr (Figure 4C). Cells expressing the M281/282V CaMKII remained susceptible to Iso-induced apoptosis (Figures 4B and 4C), indicating that elimination of these residues does not affect activation of CaMKII by catecholamine stimulation. These cellular studies are performed in a complex biological environment compared to studies with isolated CaMKII but nevertheless support the concept that direct oxidation of CaMKII by AngII is sufficient to confer enhanced CaMKII activity and trigger apoptosis.
ROS Production and CaMKII Activity Are Critical for AngII-Mediated Cardiac Apoptosis In Vivo
Intracellular ROS levels increase dramatically in models of structural heart disease (Hare, 2001), particularly those initiated by AngII (Tojo et al., 2002). Stimulation by AngII leads to activation of the NADPH oxidase complex, increasing intracellular superoxide and hydrogen peroxide levels. To establish an in vivo context for our previous findings and to test the role of CaMKII in AngII-stimulated cardiac apoptosis, p47-/-, AC3-I, and WT mice were treated with saline, AngII, or Iso for 1 week. Transverse heart sections from these mice were stained for evidence of apoptosis. After 1 week WT mice treated with either AngII or Iso showed significant cardiac apoptosis, as determined by TUNEL staining of heart sections (Figure 5). The p47-/- mice had no significant increase in cardiac apoptosis after treatment with AngII, most likely because these mice were unable to produce ROS in response to AngII stimulation (Figure 3D). However, the p47-/- mice showed a preserved apoptotic response to Iso, suggesting that Iso-induced apoptosis occurs independently of oxidative stress generated by NADPH oxidase in vivo under these conditions. In contrast, the AC3-I mice with CaMKII inhibition were resistant to apoptosis induced by either AngII or Iso, indicating that CaMKII is a necessary signal element for apoptosis initiated by AngII or Iso in vivo.
Increased CaMKII Oxidation, Apoptosis, Cardiac Dysfunction, and Death in MsrA-/- Mice
Methionine oxidation is specifically reversed by MsrA (Weissbach et al., 2002), so we hypothesized that MsrA-/- mice would show enhanced vulnerability to AngII-mediated CaMKII oxidation and apoptosis. In order to test this idea we implanted MsrA-/- and WT control mice with AngII- or saline-eluting osmotic minipumps. Hearts from MsrA-/- mice treated with AngII in vivo showed significantly more CaMKII oxidation (Figures 6A and 6B) and increased TUNEL staining (Figure 6C) compared to saline-treated MsrA-/- mice and to saline- or AngII-treated control hearts. The increased CaMKII oxidation by AngII in MsrA-/- hearts showed that CaMKII oxidation is dynamically regulated by MsrA in myocardium in vivo and suggested that MsrA-/- mice would be more vulnerable to severe myocardial stress due to increased methionine oxidation. Myocardial infarction is the most common cause of sudden cardiac death and heart failure in patients, and p47-/- (Doerries et al., 2007) and AC3-I mice (Zhang et al., 2005) are protected from left ventricular dilation and dysfunction after myocardial infarction surgery, suggesting that ROS activation of CaMKII may be important in myocardial infarction. In order to test if CaMKII oxidation and apoptosis were regulated by NADPH oxidase and MsrA in the setting of myocardial infarction, we subjected MsrA-/-, p47-/-, and WT mice to myocardial infarction surgery. MsrA-/- mice showed significantly more CaMKII oxidation (Figures 7A and 7B) and myocardial apoptosis (Figure 7C) compared to WT and p47-/- mice. These data indicate that CaMKII oxidation is dynamically regulated by NADPH oxidase and MsrA in the setting of myocardial infarction. We performed myocardial infarction surgery on a larger cohort of MsrA-/- and WT control mice to determine if increased CaMKII oxidation and apoptosis in MsrA-/- mice translated into poorer functional outcomes.
