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Ca2+/calmodulin-dependent protein kinase II (CaMKII) is a multifunctional kinase involved in vital cellular processes such as Ca2+ handling and cell fate regulation. In mammalian heart, two primary CaMKII isoforms, δB and δC, localize in nuclear and cytosolic compartments, respectively. Although previous studies have established an essential role of CaMKII-δC in cardiomyocyte apoptosis, the functional role of the more abundant isoform, CaMKII-δB, remains elusive.
Here we determined the potential role of CaMKII-δB in regulating cardiomyocyte viability and explored the underlying mechanism.
In cultured neonatal rat cardiomyocytes, the expression of CaMKII-δB and CaMKII-δC was inversely regulated in response to H2O2-induced oxidative stress with a profound reduction of the former and an increase of the later. Similarly, in vivo ischemia/repefusion (IR) led to an opposite regulation of these CaMKII isoforms in a rat myocardial IR model. Notably, overexpression of CaMKII-δB protected cardiomyocytes against oxidative stress-, hypoxia- and angiotensin II-induced apoptosis, whereas overexpression of its cytosolic counterpart promoted apoptosis. Using cDNA microarray, real time-PCR and Western blotting, we demonstrated that overexpression of CaMKII-δB but not CaMKII-δC elevated expression of heat shock protein 70 (HSP70) family members, including inducible HSP70 (iHSP70) and its homologous (Hst70). Moreover, overexpression of CaMKII-δB led to phosphorylation and activation of heat shock factor 1 (HSF1), the primary transcription factor responsible for HSP70 gene regulation. Importantly, gene silencing of iHSP70, but not Hst70, abolished CaMKII-δB-mediated protective effect, indicating that only iHSP70 was required for CaMKII-δB elicited anti-apoptotic signaling.
We conclude that cardiac CaMKII-δB and CaMKII-δC were inversely regulated in response to oxidative stress and IR injury, and that in contrast to CaMKII-δC, CaMKII-δB serves as a potent suppressor of cardiomyocyte apoptosis triggered by multiple death-inducing stimuli via phosphorylation of HSF1 and subsequent induction of iHSP70, marking both CaMKII-δ isoforms as promising therapeutic targets for the treatment of ischemic heart disease.
Ca2+/calmodulin-dependent protein kinase II (CaMKII) is a ubiquitous and multifunctional serine/threonine protein kinase family, which consists of 30 distinct members encoded by four different genes (α, β, δ, and γ). The δ isoform of CaMKII is predominantly expressed in the heart of many mammalian species, including rat, mouse and human 1-4. Two primary splicing variants of the δ isoform, CaMKII-δB and CaMKII-δC, have been cloned from rat heart 1. The only difference between these isoforms is the additional 11-amino acids conferring a nuclear targeting signal of CaMKII-δB 5. As a result, CaMKII-δB and CaMKII-δC localize to the nucleus and the cytoplasm, respectively, although they share many common biochemical properties 5.
CaMKII is activated upon the binding of Ca2+ and calmodulin complex 6, 7. In addition to the widely-accepted Ca2+/calmodulin-mediated activation, recent studies have demonstrated that CaMKII can be activated by reactive oxygen species (ROS)-induced oxidation in a Ca2+-independent fashion 8. Activation of CaMKII leads to phosphorylation of a penal of target proteins involved in multiple important physiological and pathological processes from Ca2+ handling, muscle contraction to memory encoding 9-11.
With respect to isoform-specific functions in the heart, most studies over the past decade have been focused on the role of CaMKII-δC in regulating cardiac excitation-contraction coupling 12-14 and maladaptive cardiac remodeling 15. In addition, recent studies have shown that CaMKII-δC is a common intermediate of cardiac cell apoptosis induced by diverse death-inducing stimuli, including excessive β1-adrenergic receptor (β1-AR) stimulation, H2O2, intracellular high Ca2+, and acidosis 16-18. Despite the higher abundance of CaMKII-δB, it has received far less attention than its cytosolic counterpart. The functional role of CaMKII-δB in the heart remains largely unknown, although emerging evidence suggests that overexpression of CaMKII-δB induces cardiac hypertrophy 19, 20. In the current study, we demonstrate that the expression of the major cardiac CaMKII isoforms are inversely regulated in response to oxidative stress and ischemia/reperfusion (IR) injury, and that enhanced CaMKII-δB signaling potently protects cardiomyocytes against oxidative stress-, hypoxia-, and angiotensin II (Ang II)-induced apoptosis, while activation of CaMKII-δC is apoptotic. The anti-apoptotic effect is mediated by CaMKII-δB-induced phosphorylation of an important transcriptional factor, heat shock factor 1 (HSF1), which positively regulates the expression of inducible heat shock protein70 (iHSP70, also known as HSP72). These findings not only define a novel cardiac protective function of CaMKII-δB, but also imply that CaMKII-δB downregulation or malfunction may be a crucial pathogenic element and a potential therapeutic target for various forms of heart disease, particularly ischemic heart disease.
Unless otherwise indicated, all chemicals were purchased from Sigma-Aldrich. An expanded Methods section is available in the online data supplement at http://circres.ahajournals.org.
Separation of cytosolic and nuclear protein was achieved with a Nuclear/Cytosolic Fractionation Kit (Biovision Research Products, CA, USA) following the manufacturer's instructions. Western blot to assay the abundance of CaMKII-δ in cytosolic and nuclear extracts was performed with an anti-CaMKII-δ antibody (Santa Cruz Biotechnology, Inc., sc-5392), as previously described 17. Confocal immunocytochemical imaging was performed as previously described 16. See the online data supplement for further details.
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay or DNA laddering/Hoechst staining was used as cell viability or apoptosis index, respectively, as described previously 21. See the online data supplement for further details.
Cardiomyocytes were transfected with siRNA targeted specifically to iHSP70 or Hst70 by Lipofectamine™ RNAiMAX (Invitrogen) following the manufacturer's instructions. See the online data supplement for further details.
Data are expressed as the mean ± S.E.. Statistical comparisons used one-way analysis of variance, followed by Bonferroni's procedure for multiple-group comparisons. P<0.05 was considered statistically significant.
To determine the potential functional role of CaMKII-δB in regulating cardiomyocyte viability, we first examined the expression of the major cardiac CaMKII isoforms, CaMKII-δB and CaMKII-δC, in response to oxidative stress. H2O2 treatment elevated CaMKII-δC gene expression in a concentration- and time-dependent manner (Figure 1 A&B). In contrast, CaMKII-δB gene expression was profoundly suppressed in response to H2O2 treatment (Figure 1 A&B). To determine whether these CaMKII isoforms were also oppositely regulated at their protein levels by oxidative stress, we fractionalized cardiomyocyte proteins and detected CaMKII-δB and CaMKII-δC abundance by assaying CaMKII-δ in the nuclear and the cytosolic fractions, respectively, using the same antibody reacting with both CaMKII-δ isoforms. CaMKII-δB protein abundance was rapidly and profoundly reduced by half at 4 h after H2O2 (200 μM) treatment and reached the minimal level at 24 h after treatment, whereas the protein level of CaMKII-δC was significantly elevated after H2O2 exposure (Figure 1C).
To investigate whether CaMKII-δB and CaMKII-δC were oppositely regulated in vivo during cardiac IR injury, we utilized a rat myocardial IR model. The experimental protocol and cardiac damage readouts, including myocardial infarction and lactate dehydrogenase (LDH) release, were illustrated in Online Figure I (online data supplements). Importantly, IR led to a marked increase in CaMKII-δC and a decrease in CaMKII-δB expression at both mRNA and protein levels (Figure 1 D-E). These_results indicate there is an inverse regulation of the expression of CaMKII-δB and CaMKII-δC by oxidative stress or IR injury in in vivo as well as in cultured cardiomyocytes, highlighting that these CaMKII isoforms may elicit distinctly different, even opposing functional roles in regulating cardiomyocyte viability.
To test the above hypothesis, we investigated whether activation of the nuclear isoform, CaMKII-δB, regulates cardiac cell viability and, if so, whether the effect of CaMKII-δB differs from that of CaMKII-δC. Using adenoviral gene transfer, we overexpressed either CaMKII-δB or CaMKII-δC in cultured neonatal or adult rat cardiomyocytes in the presence or absence of H2O2 (200 μM). Overexpressed CaMKII-δB was enriched in the nuclear compartment while CaMKII-δC in the cytosol in neonatal cardiomyocytes (Figure 2A), as is the case in adult rodent cardiac myocytes 16. Expression levels and phosphorylation status of both CaMKII isoforms were elevated in a titer-dependent manner in neonatal myocytes infected with Adv-CaMKII-δB or Adv-CaMKII-δC for 24 h (Figure 2B). H2O2 exposure triggered robust cardiomyocyte apoptosis, as manifested by DNA laddering (Figure 2 C&D) and an increase in Hoechst staining-positive cells (Figure 2E) compared with non-treated cells. Remarkably, overexpression of CaMKII-δB, but not CaMKII-δC or β-gal, protected both neonatal and adult rat cardiac myocytes from H2O2-mediated apoptosis (Figure 2 C&D) as well as necrosis (Online Figure II, online data supplements). H2O2-induced DNA fragmentation assayed by DNA laddering (Figure 2 C&D) and apoptotic nuclear morphological changes visualized by Hoechst staining (Figure 2E) were markedly blunted in cells infected by Adv-CaMKII-δB. In contrast, overexpression CaMKII-δC promoted apoptosis in cultured neonatal cardiac myocytes even in the absence of H2O2 treatment, and overtly enhanced apoptosis induced by H2O2 (100 μM) (Figure 2 E). CaMKII-δC-induced apoptosis was abolished by overexpression of CaMKII-δB (Online Figure III, online data supplements).
Next, we determined whether CaMKII-δB can protect cardiomyocytes from other death-inducing stimuli, in addition to oxidative stress. Overexpression of CaMKII-δB abolished hypoxia- and Ang II-induced myocyte apoptosis (Figure 2 F-G, respectively). These results provided the first documentation that activation of the most abundant cardiac CaMKII isoform, CaMKII-δB, potently protects cardiomyocytes against apoptosis triggered by multiple death-inducing stimuli, whereas its cytosolic counterpart promotes apoptosis.
To delineate the mechanism underlying the protective effect of CaMKII-δB, we examined possible differential gene expression profiles with cDNA microarray analysis in neonatal cardiomyocytes infected with Adv-CaMKII-δB, Adv-CaMKII-δC, or Adv-β-gal (all at 20 m.o.i for 36 h). The expression levels of 174 genes were significantly altered in neonatal cardiomyocytes infected with Adv-CaMKII-δB relative to those in cells infected with Adv-β-gal (>1.5-fold or < 0.5-fold, n = 3). Among them, 17 genes were specifically upregulated or downregulated by overexpression of CaMKII-δB but not CaMKII-δC (>1.5 fold or <0.5 fold, n = 3) (Online Table I and Online Figure IV, online data supplements), although the majority of the 174 genes were similarly regulated by both CaMKII isoforms. Importantly, among the 17 differentially regulated genes, we identified an important cell protective factor, iHSP70 (also known as HSP72), which was selectively upregulated in response to overexpression of CaMKII-δB but not CaMKII-δC. The cDNA microarray analysis revealed a 1.7-fold increase in iHSP70 gene expression in cells infected with Adv-CaMKII-δB but not Adv-CaMKII-δC relative to those infected by Adv-β-gal (Figure 3A). Using real-time PCR, we validated the CaMKII-δB-dependent elevation of iHSP70 gene expression at either 36 h or 48 h after adenoviral infection (Figure 3B). Likewise, iHSP70 protein abundance was also augmented by 2.4-folds only in cells infected with Adv-CaMKII-δB (m.o.i. 20 for 48 h) (Figure 3C).
To determine whether the elevation in iHSP70 expression is causatively linked to CaMKII-δB-induced anti-apoptotic effect, we utilized siRNA-mediated iHSP70 gene silencing. siRNAs (including siRNA1 and siRNA2) effectively reduced the expression of iHSP70 at mRNA (Figure 4A) and protein levels (Figure 4B). Notably, iHSP70 gene silencing substantially abolished CaMKII-δB-mediated anti-apoptotic effect, as assayed by DNA laddering in cardiomyocytes treated with either siRNA1 or siRNA2 of iHSP70, regardless of CaMKII-δB overexpression (Figure 4C). Moreover, MTT assay further revealed that overexpression of CaMKII-δB could not protect cardiomyocytes from H2O2-induced cell death in the presence of iHSP70 siRNA1 or siRNA2 (Figure 4D). These results provide strong evidence to indicate that upregulation of iHSP70 is essentially involved in CaMKII-δB-elicited anti-apoptotic signaling.
Interestingly, another closely related member of the HSP70 family, namely Hst70 in rats (but called HSP70.2 in mouse), was also significantly increased by 1.8-fold in cells overexpressing CaMKII-δB but not in those overexpressing CaMKII-δC (Figure 3A and Online Table I, online data supplements). However, gene silencing of Hst70 had no detectable effect on CaMKII-δB-induced protection. Specifically, two out of three tested siRNA sequences (Hst70 siRNA1 and 3) suppressed Hst70 mRNA level by 60-70 % (Figure 5A), but could not abolish the protective effect of CaMKII-δB assayed by DNA laddering (Figure 5B) and MTT (Figure 5C), indicating that Hst70, unlike HSP72, is not involved in CaMKII-δB-mediated anti-apoptotic signaling.
To investigate the mechanism underlying CaMKII-δB-mediated upregulation of iHSP70, we analyzed the expression and phosphorylation status of HSF1, the transcription factor primarily responsible for the regulation of iHSP70 expression. Immunofluorescent imaging revealed that CaMKII-δB and HSF1 were co-localized at the nuclear compartment in cardiomyocytes infected with Adv-CaMKII-δB (Figure 6A). Using a site-specific antibody reacting with phosphorylated HSF1 at Ser230, which has been shown to positively contribute to the transcriptional activity of HSF1 22, we found that overexpression of CaMKII-δB but not CaMKII-δC or β-gal elevated phosphorylation level of HSF1 at Ser230 relative to the control group (Figure 6B). In contrast, overexpression of CaMKII-δB had no detectable effect on phosphorylation of HSF1 at Ser303, a constitutively phosphorylated residue, indicating that CaMKII-δB selectively phosphorylates HSF1 at Ser230 (Figure 6B). It is noteworthy that H2O2 (200 μM for 15 min) treatment transiently increased CaMKII-δB mediated phosphorylation of HSF1-Ser230 (Figure 6C) and HSF1 nuclear translocation (Online Figure V, online data supplements).
Cardiac myocyte apoptosis plays a pivotal role in the development of a variety forms of heart disease, including ischemic heart disease, congestive heart failure, and acute myocardial infarction 23, 24. ROS has been defined as a major contributor in triggering cardiomyocyte apoptosis during myocardial ischemia/reperfusion injury, dilated cardiomyopathy and the progression of heart failure 25. Recent studies have established a link between oxidative stress and activation of CaMKII and defined an essential role of CaMKII signaling in oxidative stress-triggered cardiomyocyte apoptosis 8, 17. However, isoform-specific expression regulation and function of cardiac CaMKII-δB and CaMKII-δC in response to oxidative stress, IR injury, or hypoxia have not been well characterized until now. Given their distinct intracellular distributions, these cardiac CaMKII isoforms may be differentially regulated and exhibit different actions. Indeed, in this study, we have revealed following important differences between these CaMKII-δ isoforms in their responses to oxidative stress or IR injury and their functional roles in regulating cardiac muscle cell viability. First, the expression profile of CaMKII-δB and CaMKII-δC is inversely regulated in cardiomyocytes subjected to oxidative stress with a downregulation of CaMKII-δB and an upregulation of CaMKII-δC. Similar opposite regulation of these cardiac CaMKII-δ isoforms is induced by myocardial IR injury in vivo. Second, overexpression of CaMKII-δB markedly attenuates H2O2-, hypoxia-, and AngII-induced apoptotic cell death, whereas overexpression of CaMKII-δC promotes cardiomyocyte apoptosis. Moreover, overexpression of CaMKII-δB, but not CaMKII-δC, selectively elevates phosphorylation of HSF1 at Ser230, resulting in increased expression of the major inducible HSP70, iHSP70, that is obligated to CaMKII-δB-induced cardiomyocyte protection. These present findings support the perception that the nuclear-located isoform, CaMKII-δB, constitutes an important cardiac protective factor against multiple insulting stimuli-induced myocardial damage, and that a reduction in this beneficial isoform in conjunction with a simultaneous increase in the detrimental isoform, CaMKII-δC, is a potential cause factor of various cardiac disorders, including cardiac myocyte apoptosis, IR injury, cardiac arrhythmia, and heart failure 26-29.
Although the present findings underscore a potentially important anti-apoptotic effect of the nuclear-located isoform, CaMKII-dB, it is noteworthy that overexpression of CaMKII-δB activates hypertrophic gene expression and produces hypertrophy in cultured cardiomyocytes 30. Similarly, transgenic mice with cardiac-specific overexpression of the nuclear-targeted cardiac CaMKII-isoform develop cardiac hypertrophy, cardiomyocyte enlargement and increases in hypertrophic gene expression 20. Thus, exaggerated upregulation of either cardiac CaMKII isoform or an imbalance between these two major isoforms might cause pathological consequences in the heart.
Differences between these CaMKII-δ isoforms are manifested by the distinct phenotypes of transgenic mouse models with cardiac-specific overexpression of CaMKII-δB or CaMKII-δC. Although both transgenic models can develop cardiac hypertrophy, CaMKII-δC transgenic mice display much more accelerated cardiac functional deterioration and shortened life span as compared to CaMKII-δB transgenic mice even with a matched increase in their total cellular CaMKII activity 15, 20. Additionally, transgenic overexpression of the cytosolic isoform, CaMKII-δC, leads to hyper-phosphorylation of substrates involved in cardiac excitation-contraction coupling such as ryanodine receptor (RyR) and phospholamban (PLB), resulting in increased SR Ca2+ sparks and decreased SR Ca2+ load 12, 15, whereas none of these parameters are changed in age-matched transgenic mice with the nuclear targeted CaMKII-δB 20. The isoform-specific phenotypes of CaMKII-δB and CaMKII-δC transgenic mouse models can be explained, at least in part, by our previous notion that overexpression of CaMKII-δC, instead of CaMKII-δB, exaggerates the adult mouse cardiomyocyte apoptotic death induced by β1-AR stimulation and relays apoptotic signal for multiple death-inducing stimuli 16, 17. Here, we have further demonstrated that CaMKII-δB, opposite to CaMKII-δC, protects cardiomyocytes against apoptosis induced by multiple insulting stimuli such as oxidative stress, hypoxia and AngII stimulation. It is also noteworthy that in response to oxidative stress and IR injury, expression of CaMKII-δB and CaMKII-δC are inversely regulated at gene and protein levels. The downregulation of the protective factor, CaMKII-δB, as well as the upregulation of the deleterious factor, CaMKII-δC, are expected to contribute to oxidative stress- or IR-induced cell injury and cell death. Taken together, there are distinct differences between these cardiac CaMKII isoforms in their function and regulation under various physiological and pathological circumstances.
HSP belong to an important family of endogenous protective proteins that rapidly respond to a wide variety of stress stimuli, such as heat shock, hypoxia, ischemia, increased ROS, and inflammation. Among various members of HSP family, HSP70 has been implicated as cardiac protective 31-33. The HSP70 family consists of, at least, eight highly homologous members, including constitutively expressed (or cognate) and highly inducible proteins that differ from each other by the intracellular localization and expression pattern 34. In particular, Hst70 (encoded by Hspa2 gene) is expressed at high levels in testis and at considerably lower or undetectable levels in other tissues 35, 36. In contrast, iHSP70 (also named HSP72, encoded by Hspa1b gene) is expressed at low or undetectable levels in most unstressed normal cells and tissues, but its expression is rapidly induced by multiple physical and chemical stresses, and therefore it is often called the major stress-inducible HSP70. Nevertheless, both iHSP70 and Hst70 can be induced under diverse cell stress conditions 37, 38, and have been implicated in promoting cell survival 39, 40.
In searching for potential mechanisms responsible for the protective effect of CaMKII-δB, we have found that both iHSP70 and Hst70 are specifically upregulated by overexpression of CaMKII-δB but not its cytosolic counterpart. Importantly, gene silencing of iHSP70 blocks the protective effect of CaMKII-δB, establishing an essential role of iHSP70 in relaying CaMKII-δB anti-apoptotic signaling. In contrast, gene silencing of the closely related HSP70 family member, Hst70, has no detectable impact on CaMKII-δB-mediated protection. Thus, iHSP70 but not its homologous Hst70 plays an essential role in delivering CaMKII-δB activated anti-apoptotic signaling.
HSF1 is the transcription factor responsible for stress-induced expression of HSP70. In unstressed cells, HSF1 is present in both the cytoplasm and nucleus in a monomeric form that has no DNA binding activity. In response to heat shock and other stress stimuli, HSF1 assembles into a trimer, accumulates within the nucleus and binds with the proximal promoter heat shock element (HSE) on the HSP70 gene to induce the transcription of HSP70 41-43. Previous studies have shown that hyper-phosphorylation of HSF1 is required for the transcriptional competence of the factor 44-46. There are two kinds of phosphorylation sites on HSF1, i.e. inducible residue (Ser 230), which positively contributes to the transcriptional competence of HSF1 22, and constitutive residues (Ser 303, Ser 307 and Ser363), which repress the transcriptional activity of HSF1 47, 48.
In the present study, we have demonstrated that CaMKII-δB and HSF1 are co-localized in the nuclear compartment and that phosphorylation of HSF1 at Ser230, but not a constitutive residue Ser303, is clearly elevated in myocytes infected with Adv-CaMKII-δB in the presence or absence of H2O2 treatment (Online Figure V, online data supplements). Our data indicate that CaMKII-δB specifically phosphorylates the inducible residue of HSF1-Ser230, and subsequently enhances its transcriptional activity, leading to increased expression of iHSP70 which is obligated to CaMKII-δB-mediated cardioprotection.
In summary, we have demonstrated, for the first time, oxidative stress and IR injury leads to a downregulation of CaMKII-δB and an upregulation of CaMKII-δC, and that the two major cardiac CaMKII isoforms exhibit opposing effects on cardiomyocyte viability with CaMKII-δB anti-apoptotic and CaMKII-δC pro-apoptotic (Figure 7). Phosphorylation of HSF1 and subsequent induction of iHSP70 are essentially involved in CaMKII-δB-mediated cell survival signaling (Figure 7). These findings mark both cardiac CaMKII isoforms as promising therapeutic targets for preventing or alleviating cardiomyocyte loss caused by myocardial infarction, IR injury, heart failure, and many other etiologies.
We would like to thank LP Wei, CY Li, ZY Peng, WJ Xie, HQ Fang, H Huang, and HL Zhang for their excellent technical support.
Sources of Funding
This work was supported by Peking University 985 Project (Wei Peng, Yan Zhang, Ming Zheng, Heping Cheng, Chun-Mei Cao, and Rui-Ping Xiao) and 973 Program (2007CB512100) (Chun-Mei Cao, Ming Zheng and Heping Cheng) and, in part, by the Intramural Research Program of the NIH, National Institute on Aging (Weizhong Zhu and Rui-Ping Xiao).