Activation of Histone Deacetylase 2 by Inducible Heat Shock Protein 70 in Cardiac Hypertrophy
Diverse cardiac diseases induce cardiac hypertrophy, which leads to dilatation and heart failure. We previously reported that hypertrophy can be blocked by class I histone deacetylase (HDAC) inhibitor, which prompted us to investigate the regulatory mechanism of class I HDACs. Cardiac hypertrophy was introduced by aortic banding, by infusion of isoproterenol or angiotensin II, or by swimming. Hypertrophic stimuli transiently elevated the activity of histone deacetylase-2 (Hdac2), a class I HDAC. In cardiomyocytes, forced expression of Hdac2 simulated hypertrophy in an Akt-dependent manner, whereas enzymatically inert Hdac2 H141A failed to do so. Hypertrophic stimuli induced the expression of heat shock protein (Hsp)70. The induced Hsp70 physically associated with and activated Hdac2. Hsp70 overexpression produced a hypertrophic phenotype, which was blocked either by siHdac2 or by a dominant negative Hsp70ΔABD. In Hsp70.1−/− mice, cardiac hypertrophy and Hdac2 activation were significantly blunted. Heat shock either to cardiomyocytes or to mice activated Hdac2 and induced hypertrophy. However, heat shock-induced Hdac2 activation was blunted in the cardiomyocytes isolated from Hsp70.1−/− mice. These results suggest that the induction of Hsp70 in response to diverse hypertrophic stresses and the ensuing activation of HDAC2 trigger cardiac hypertrophy, emphasizing HSP70/HDAC2 as a novel mechanism regulating hypertrophy.
- cardiac hypertrophy
- class I histone deacetylases
- histone deacetylase 2
- heat shock protein 70
- Hsp70.1−/− mice
Cardiac hypertrophy is a response, either adaptive or maladaptive, to pressure or volume overload, mutations, or loss of contractile mass. Hypertrophic growth accompanies many forms of heart disease, including ischemic diseases, myocardial infarction, hypertension, aortic stenosis, and valvular dysfunctions. Although the initial hypertrophic responses seem to be an adaptation to those stimuli, the sustained stress may lead to cardiomyopathy and heart failure, a major cause of human morbidity and mortality. However, few interventions have proven effective in blocking the hypertrophy or in preventing the transition to congestive heart failure.
Cardiomyocyte hypertrophy is characterized by an increase in individual myocyte size, enhanced protein synthesis, and heightened organization of the sarcomere,1 which are regulated by activation of heart-specific transcription factors such as GATA4, MEF2, and immediate early genes like c-jun and c-fos.2 The subsequent reactivation of the fetal gene program and repression of adult cardiac genes are closely related to the deterioration of heart function in hypertrophy.
Recently, modulation of gene transcription by altering chromatin structure, especially by adding or removing acetyl groups to histone tails, has been implicated in diverse human pathologies, including cardiac hypertrophy.3 Histone deacetylases (HDACs), which remove the acetyl group, repress downstream gene expression. Although HDACs are divided into 4 families, a major focus has been on class II HDACs (HDAC4, HDAC5, HDAC7, and HDAC9), which antagonize hypertrophy by repressing the fetal gene program4 by phosphorylation-dependent relocalization of those proteins from the cytoplasm to the nucleus.5 However, we and others showed that HDAC inhibitors can attenuate cardiac hypertrophy,6–8 which suggests that class I HDACs (HDAC1, HDAC2, HDAC3, and HDAC8), which are readily blocked by those inhibitors, are prohypertrophic.9 In a recent study using Hdac2−/− mice, Trivedi et al10 clearly showed that Hdac2 mediates hypertrophy by modulating inositol polyphosphate-5-phosphatase f (Inpp5f)/glycogen synthase kinase (GSK)3β signals, further implicating the opposite roles of both classes of HDACs.
Because class I and class II HDACs share the deacetylase domain with similar enzymatic action, these opposite actions of the two classes of HDACs raise critical questions as to the fundamental roles of HDAC activity. Previous studies of their actions were done either by means of chemical HDAC inhibitors7–9 or by utilizing a knockout strategy.4,10 Chemical inhibitors raise questions of selectivity, however. In addition, with the knockout, one has to consider the two factors of ‘existence’ and ‘function’ of the enzyme. Indeed, the enzymatic activity of the HDAC is not required in antihypertrophic processes, because MITR, a splice variant of HDAC9 that lacks a deacetylase domain, is still effective.4 Therefore, the functional role in hypertrophy of HDACs as enzymes, and not merely their presence, and how they are regulated should be investigated. We herewith report that diverse hypertrophic signals induce the enzymatic activation of HDAC2 by inducing heat shock protein (HSP) 70 in commencing cardiac hypertrophy.
Materials and Methods
The experimental protocols were approved by the Chonnam National University Medical School Research Institutional Animal Care and Use Committee. Details regarding animal models and drug administrations are described in the online data supplement, available at http://circres.aha.journals.org.
Cell Cultures, Constructs, siHdac2, and Transfection Study
Protocols for cell cultures of rat neonatal cardiomyocytes, H9c2 cells, and 293T cells, siHdac2, and constructs as well as the antibodies used are described in the online data supplement.
In Vivo Visualization of the Interaction of Hsp70 and Hdac2
To visualize the interaction, we utilized fluorescence protein fragment complementation methods as described (MBL International Corporation, Woburn, Mass).11
Protocols for HDAC assay, immunoprecipitation, Western blot analysis, fluorescent immunocytochemistry, cell size measurements, in vitro translation, GST pull-down assay, RT-PCR, and Northern blot are described in the online data supplement.
Hypertrophic Stimuli Activate Hdac2, a Class I HDAC
We first observed changes in Hdac2 activity in the mouse hearts treated with isoproterenol (ISP). Hdac2 activation was observed as early as the 3rd day of administration, when a substantial increase in the heart weight/body weight ratio (HW/BW) was not evident. This activation was blunted after 14 days, whereas the increase in HW/BW was well sustained even after 14 days (Figure 1A). Partial constriction of the ascending aorta by aortic banding (AB) increased the HW/BW. In this case as well, Hdac2 activation preceded the increase in HW/BW (Figure 1B). However, Hdac1, an alternate class I Hdac, was not activated by either ISP or AB (Figure IA and IB in the online data supplement). Chronic angiotensin (Ang) II infusion increased HW/BW and Hdac2 activity (Figure 1C and supplemental Figure IC). Interestingly, even 1 day after bolus injection, phenylephrine (PE) activated Hdac2 (Figure 1D and supplemental Figure ID). Forced swimming of mice induced physiological cardiac hypertrophy through AKT pathways (supplemental Figure IE).12 Swimming of BL6 female mice for 3 days was enough to activate Hdac2 (Figure 1E), although HW/BW was not increased (supplemental Figure IF). We examined the individual enzyme activities of class I HDACs (Figure 1F). ISP activated Hdac2 only.
Activation of Hdac2 in response to hypertrophic stimuli was further investigated in rat neonatal cardiomyocytes. PE significantly increased Hdac2 activity as early as 3 hours (Figure 2B). Significant induction of Natriuretic peptide precursor type A (Nppa) (encoding atrial natriuretic factor, ANF), a marker of hypertrophy, was observed at day 2 (Figure 2A). Transient transfection of Hdac2 activated −3003 Nppa promoter–luciferase (Figure 2C) but not Hdac1 or Hdac3 (Figure 2D).
The Hdac2-induced activation of Nppa promoter (Figure 2E) and Myh7 (myosin heavy polypeptide 7), known as β-myosin heavy chain (supplemental Figure IIB), was completely blocked by AKT dominant negative (supplemental Figure IIA) and Akti-1/2 (AKT inhibitor VIII, isozyme-selective Akti-1/2, Calbiochem, Figure 2E and supplemental Figure IIB and IIC). Replacement of His141 in Hdac2 with alanine caused a significant reduction in its activity (supplemental Figure III) without altering its binding capacities to other cofactors (data not shown).13 The enzymatically inert Hdac2 H141A could not activate the promoter-luciferase of either Myh7 (Figure 2F) or Nppa (data not shown). Transfection of wild type Hdac2 caused an increase in cell size and stress fiber formation (Figure 2G and 2H), whereas transfection of the mutant did not.
Hdac2 Regulation Mechanisms
The enzymatic activities of HDACs are regulated by several different mechanisms14: (1) HDAC protein amounts, (2) intracellular localization of HDAC protein, (3) phosphorylation status, and (4) association with other cofactors. Changes in protein amounts were ruled out (supplemental Figure IVA through IVC). Substantial Hdac2 activation took place mainly in the nucleus (supplemental Figure VA) because most of the Hdac2 was present in the nucleus (supplemental Figure VB). However, hypertrophy did not induce translocation of Hdac2 (supplemental Figure VB through VE). We also did not observe a significant increase in the phosphorylation of the endogenous Hdac2 (supplemental Figure VF).
Hsp70 Interacts With Hdac2
HDAC activity may be regulated by interacting with other proteins,14 and HSPs have been reported as one of the regulators of class I HDACs.15 In heart biology, HSPs, as molecular chaperons, are induced by various stresses to protect cardiomyocytes. Thus, we postulated that hypertrophic stimuli-induced HSPs may work as regulators of HDAC2.
First, we investigated whether Hsps physically interact with Hdac2 and found that Hdac2 successfully pulled down Hsp70, but not Hsp56 or Hsc70 (heat shock cognate) (constitutive form; Figure 3A) in 293T cells. Inversely, Hsp70 also recruited Hdac2 (Figure 3B) in H9c2 cells. Likewise, in the cell-free system, recombinant GST-Hsp70 protein successfully pulled down the eluted His-Hdac2 (Figure 3C).
We next sought the Hdac2-interacting domain of Hsp70. Hsp70 has two conserved domains: ATP-binding domain (ABD) and peptide-binding domain (PBD) (Figure 3D).16 His-tagged chimeric proteins of full or truncated Hsp70s were induced from bacteria and in vitro translated Hdac2 was applied. Hdac2 failed to associate with the mutants lacking either part of the PBD (Hsp70ΔC) or the NH2-terminal region (Hsp70ΔN) (Figure 3E).
Hsp70 and Hdac2 were colocalized in the nucleus of H9c2 cells (Figure 3F). Direct in vivo association of both proteins was visualized with fluorescent protein fragment complementation methods using the CoralHue Fluo-chase system. In this system, fluorescence can be emitted only when divided fluorophore fragments closely approach by the interaction of two target molecules of interest.11 Mammalian expression vectors to make chimeric proteins of both mKGN-Hdac2 and mKGC-Hsp70 were cloned (online data supplement). Green fluorescence was detected in the nucleus of H9c2 cells (Figure 3G) or HeLa cells (data not shown), when both constructs were simultaneously transfected.
Hsp70 Activates Hdac2
Next, we investigated whether Hsp70 can activate Hdac2. Transfection of Hsp70 successfully activated Hdac2 in cardiomyocytes (Figure 4A) and in H9c2 cells, whereas other Hsps failed (Figure 4B), although those proteins were correctly expressed (data not shown). Hsp70 did not activate Hdac1 (supplemental Figure VI). The recombinant GST-Hdac2 protein did not elicit the intrinsic Hdac2 activity (Figure 4C). Previously, it was reported that all HDACs except HDAC8 generated from bacteria are enzymatically inactive and that cellular components are required in the Hdac assay.17 We also measured the enzymatic activity of the recombinant proteins when H9c2 cell lysates were applied. Interestingly, Hdac2 activity was further increased when recombinant Hsp70 was added (Figure 4C).
The domain mapping study with Hsp70 gave us the idea that the Hsp70ΔABD that can bind to Hdac2 may work in a dominant negative fashion. When pCMV-Hsp70ΔABD was cotransfected, the Hdac2 recruited by Hsp70 full was significantly reduced (Figure 4D). Next, Hdac2 activity was measured in the presence of Hsp70ΔABD in cardiomyocytes. Hsp70ΔABD itself did not activate Hdac2 (Figure 3E). However, transfection of Hsp70ΔABD dose-dependently blocked Hsp70 full-induced activation of Hdac2 (Figure 4F). Hsp70ΔN or Hsp70ΔC, both of which failed to interact with Hdac2 (Figure 3E), could not activate Hdac2 (Figure 4E), further suggesting that binding of intact Hsp70 is required for the activation of Hdac2.
Hsp70 Induces Cardiac Hypertrophy
Hsps are induced by hypertrophic stimuli.18 Thus, we postulated that induction of Hsp in response to hypertrophic stresses and subsequent activation of Hdac2 may induce cardiac hypertrophy. First, we studied which HSPs are upregulated by hypertrophic signals in mice. At the 5th day of ISP-infusion, Hsp70 transcript was dramatically increased, whereas Hsp27 (also known as Hspb2), Hsp40 (Dnajb1), Hsp56 (Fkbp4), Hsp90α/β, and Hsc70 (Hspa8) were not changed (Figure 5A). The increase in Hsp70 protein was also observed in AB or swimming models (supplemental Figure VIIA and VIIB). Hypertrophic stresses such as pressure overload have been reported to elevate Hsp70 expression within 1 hour after the stimuli.19 Likewise, in our experiments, administration of ISP or PE to mice dramatically increased the expression of Hsp70 as early as 3 hours, implying that Hsp70 precedes eventual heart weight increase (Figure 5B and supplemental Figure VIIC). The increase in Hsp70 was attenuated at 14 days (Figure 5A).
Phosphorylation of AKT and the following inactivation by phosphorylating antihypertrophic GSK3β are key markers of the physiological hypertrophy resulting from increased oxygen demand, such as exercise (supplemental Figure IE).12 Activation of AKT/GSK3β signal cascades are also involved in pathological hypertrophy, especially at the early phase.20 In our experimental model using ISP or AB, both of which are the typical inducers of pathological hypertrophy, Akt/Gsk3β phosphorylation was transiently upregulated after ISP infusion (Figure 5C) or AB (supplemental Figure VIID), when dramatic increase in Hsp70 and activation of Hdac2 take place.
We next studied whether hypertrophy is associated with the increase in binding of Hsp70 and Hdac2. AB induced Hsp70, which results in its increased binding to Hdac2, compared with sham-operated mice (Figure 5D). Likewise, in PE-treated cardiomyocytes, greater amounts of Hsp70 were recruited by Hdac2 than in those without PE treatment (Figure 5E).
On the basis of the activation of Hdac2 by direct association with Hsp70 and the increase in Hsp70 in the early phase of cardiac hypertrophy, we postulated that HSP70 may play a role as a prohypertrophic mediator. We tested this by checking whether Hsp70 induces hypertrophic markers. Transfection of Hsp70 dose-dependently increased promoter activity of Nppa (Figure 5F) and Myh7 (Figure 5G). Interestingly, this Hsp70-induced activation of the Nppa promoter was completely abolished by transfection of siHdac2, suggesting that Hsp70-induced events are Hdac2-dependent. We also studied whether Hsp70ΔABD, a dominant negative Hsp70 mutant that failed to activate Hdac2, blocks Hsp70-induced activation of Nppa promoter. First, the effect of Hsp70ΔABD itself on the promoter activity was examined. Like Hsp70ΔN and Hsp70ΔC, both of which failed to interact with Hdac2 (Figure 3E), Hsp70ΔABD did not induce promoter activation (supplemental Figure VIII). Addition of dominant negative Hsp70ΔABD completely abolished Hsp70 full-induced −3003 Nppa promoter activity (Figure 5H). Electroporation of Hsp70 caused stress fiber formation (Figure 5I) and an increase in cell size (Figure 5J), clearly demonstrating that Hsp70 induces hypertrophic phenotypes.
ISP-Induced Hypertrophy Is Blunted in Hsp70.1−/− Mice
We further confirmed that Hsp70 activates Hdac2 in response to hypertrophic stimuli by utilizing Hsp70.1−/− mice.21 In contrast with Hsp70.1+/+ mice, the increase in Hdac2 activity was completely blocked in Hsp70.1−/− mice (Figure 6A). In addition, although ISP could still elevate HW/BW, the increase in HW/BW was significantly blunted in Hsp70.1 mutant mice (Figure 6B and supplemental Table I). Ang II–induced cardiac hypertrophy was also significantly attenuated in the null mice (data not shown). Echocardiogram analysis showed that ISP-induced thickening of ventricular walls was significantly blunted (Figure 6C and see supplemental Table II) in the null mice. We next investigated the responses of hypertrophy markers in Hsp70.1 mutant mice. The increase in either ANF or α-tubulin, an alternate hypertrophy marker,22 was significantly blunted in Hsp70.1−/− mice (Figure 6D).
Our observations that HSP70/HDAC2 induces activation of AKT signaling pathways led us to investigate if Akt/Gsk3β signal activation is impaired in Hsp70.1−/− mice as was previously reported in Hdac2−/− mice.10 Our results showed that the phosphorylation of Akt (Figure 6E) and Gsk3β (Figure 6F) in the ISP-treated Hsp70−/− heart was significantly blunted.
Heat Shock Induces Cardiac Hypertrophy In Vivo and In Vitro
Given that Hsp70 is induced by heat shock, the question arises whether heat shock can activate Hdac2 and induce hypertrophy. One-hour heat shock to cardiomyocytes, which induces Hsp70, was sufficient to activate Hdac2 (Figure 7A). To rule out the possible involvement of Hsps other than Hsp70, heat shock was introduced to cardiomyocytes isolated from Hsp70.1−/− mice. Heat shock failed to activate Hdac2 in Hsp70.1-null cardiomyocytes, indicating that heat shock-induced activation of Hdac2 is mediated by Hsp70 (Figure 7B). Heat shock for 1 hour elevated Nppa promoter-luciferase activity (Figure 7C). Likewise, heat shock significantly increased the individual cell size (Figure 7D).
Next, we extended our hypothesis to the animal model by examining whether heat shock to live mice can induce cardiac hypertrophy as well as activation of Hdac2. First, we treated CD1 male mice with single heat shock (30 minutes at 42°C) and measured Hdac2 activity as well as HW/BW; we did not observe any significant changes (data not shown). On the contrary, repeated heat shock for 5 consecutive days dramatically activated Hdac2 (Figure 7E), accompanied by an increase in HW/BW (Figure 7F). The inductions of ANF and Hsp70 were confirmed by Western blot (Figure 7G).
A model for the HSP70/HDAC2-mediated cardiac hypertrophy signaling pathway is shown in Figure 8. In summary, we found that diverse hypertrophic stimuli activate HDAC2, a class I HDAC, in the early phase of cardiac hypertrophy, and the induction of HSP70 in response to exogenous hypertrophic stimuli is a key regulatory mechanism to activate HDAC2. Our present observations highlight new directions. First, we clearly showed that HDAC2 activation mediates cardiac hypertrophy in response to exogenous stresses. Our work extends prior studies10 by identifying new aspects of HDAC2 function initiating cardiac hypertrophy. Second, we elucidated a key mechanism bridging HSPs to cardiac hypertrophy. Because HSPs possess a protective function against exogenous stresses including heat or ischemia by inhibiting apoptotic signaling pathways,23 it is plausible that the heart may react to protect itself from those stresses, especially in the early adaptive phase, by increasing the antiapoptotic cascades such as AKT signaling,24 possibly via an HSP70/HDAC2 pathway.
Enzymatic Activation of Hdac2 Is Required for Cardiac Hypertrophy
Although Trivedi et al10 observed that Hdac2 knockout mice are resistant to exogenous hypertrophic stresses, the implications of HDAC2 in the development of cardiac hypertrophy are in debate, because other recent work25 showed that Hdac2 knockout mice are still vulnerable to hypertrophy. However, these studies with knockout mice may reflect the presence of the enzymes rather than their enzymatic activities. Moreover, a sustained, lifetime loss of the enzyme may not account for the acute enzymatic responses to the exogenous hypertrophic stimuli. Indeed, deletion of one HDAC could cause compensatory activation of redundant HDACs, which may affect the function of individual enzymes.26 In this study, we measured the enzymatic activities of HDACs induced by hypertrophic stimuli, which circumvents either long-term effects of loss of enzyme or compensatory mechanisms of other HDACs in knockout mice, and clearly demonstrated that diverse hypertrophic stimuli induce enzymatic activation of HDAC2. It is noteworthy that activation of HDAC2 preceded substantial hypertrophic events, implying that activation of HDAC2 is not a result but a cause of hypertrophy.
Hsp70 Regulates Hdac2 Enzymatic Activity
We found no evidence that alterations in either protein amounts or localization are involved in the mechanism of Hdac2 regulation of cardiac hypertrophy. Likewise, in our experimental model, we did not observe a dramatic increase in the phosphorylation level of the endogenous Hdac2. We next investigated HSP as a regulator of HDAC2 in response to cardiac hypertrophic stresses. We showed that Hsp70 activated Hdac2 by direct association. In addition, we showed that binding capacity of Hsp70 to Hdac2 as well as a functioning domain is required for the activation.
Hsp70 Triggers Cardiac Hypertrophy
In the present study, Hsp70 expression and its association with Hdac2 was transiently increased in the early phase of cardiac hypertrophy, which coincided well with the activation of Hdac2. In addition, we showed that forced expression of Hsp70 exaggerated hypertrophy in cardiomyocytes. This finding was further supported by the in vivo animal model: impairment of Hdac2 activation accompanied the attenuation of the ISP-induced increase in HW/BW in Hsp70.1−/− mice.
These results are in contrast to a previous report that hearts from Hsp70 knockout mice are enlarged,27 which could be interpreted as showing that Hsp70 may function as an antihypertrophic regulator. Indeed, we also observed that basal levels of some hypertrophic indicators, such as HW/BW or ANF expression, were slightly increased in our 5- to 6-week-old null-mice, although not significantly so. It is noteworthy, however, that in the previous report,27 hypertrophy was apparent when the mutant mice grew older than 20 weeks. Considering that heat shock factor (HSF)/HSP70 maintains physiological hypertrophy and that declines of HSF/HSP70 trigger transition to pathological hypertrophy when the stresses are sustained for a long time,28 it is quite likely that HSP70 may work to prevent maladaptive hypertrophy by maintaining physiological hypertrophy. Thus, the cardiac hypertrophy induced by the sustained loss of HSP70 in the older knockout mice might be maladaptive and pathological. On the contrary, our experiments focused on seeking the initial triggering signals in the early phase. In agreement with previous reports,19,29 we observed that HSP70 is dramatically increased even before the hypertrophic phenotype appears. Moreover, we clearly showed that forced expression of Hsp70 in cardiomyocytes provoked hypertrophy and that response to hypertrophic stimuli was significantly blunted in young Hsp70 knockout mice.
Hsp70 protein is produced by the duplicated genes Hsp70.1 (Hspa1b) and Hsp70.3 (Hspa1a), which differ in only one amino acid.30 Thus, one may argue that half of the protein is theoretically present even in Hsp70.1−/− mice. However, it is noteworthy that Hsp70 is inducible but not constitutive protein. Moreover, the two genes respond differently to exogenous stimuli; for example, osmotic stress induces Hsp70.1 but not Hsp70.3.31,32 Indeed, in our preliminary study, we also observed that hypertrophic stimuli preferentially induce the transcription of Hsp70.1 rather than that of Hsp70.3 (data not shown). Thus, the amount of Hsp70 proteins induced by hypertrophic stimuli in Hsp70.1−/− mice must be much less than 50%, which may explain why Hdac2 activation and hypertrophy was significantly blunted in Hsp70.1−/− mice in our study.
The stresses used to induce pathological hypertrophy, such as adrenergic stimuli or AB, can cause the phosphorylation of AKT and GSK3β,20 both of which are key markers of physiological hypertrophy.33 In our experimental models, phosphorylation of Akt and Gsk3β was transiently increased in the early phase of hypertrophy induced by ISP and by AB, as well as by swimming. In addition, Hdac2-induced activation of Nppa or Myh7 promoter was blocked by either Akti-1/2 or dominant negative Akt. These results suggest the possible involvement of the AKT signal in the relaying of HDAC2 to hypertrophic phenotypes, consistent with the previous report that Akt-phosphorylation was decreased in Hdac2-knockout mice but was increased in Hdac2-transgenics.10
Heat Shock Induces Cardiac Hypertrophy
One striking finding of this study is the induction of cardiac hypertrophy by heat shock. First, in the cardiomyocyte model, heat shock induced Hdac2 activation, ANF-expression, and enlargement of the cells. Interestingly, the heat shock-induced cardiac hypertrophy was reproduced in the animal model; repeated exposure to heat shock activated Hdac2 and increased HW/BW, suggesting that heat shock is a prohypertrophic stimulus.
In summary, we herewith propose that induction of HSP70 and subsequent activation of HDAC2 are important triggering signals of cardiac hypertrophy. It is noteworthy, however, that the HSP70/HDAC2 axis is not the only pathway in hypertrophy signaling. For example, the importance of neurohormonal signals mediated by G-protein and calcium-dependent signaling pathways in cardiac hypertrophy is well established. Nevertheless, the signal cascades proposed in the present work are worthy of further scrutiny, because they may provide novel targets for overcoming or averting cardiac disorders.
We are grateful to Prof Jonathan A. Epstein (University of Pennsylvania) for helpful suggestions, Prof Emeritus Young Johng Kook (Chonnam National University Medical School) for reviewing the manuscript, and Ji Myung Kim and Prof Woo Jin Park (Gwangju Institute of Science and Technology) for technical support. Mammalian expression vectors of HSPs were supplied by N.S. Kim (21C Frontier Human Gene Bank), which is supported by the 21C Frontier Functional Human Genome Project, Korea.
Sources of Funding
This work was supported by Molecular and Cellular BioDiscovery Research Program Grant 2006M10601000081-06N0100-08110 from the Ministry of Science and Technology (South Korea) and by the Korea Science and Engineering Foundation through the Medical Research Center for Gene Regulation grant R13-2002-013-03002-0 at Chonnam National University. H.J.K. and H.J. were supported in part by the Brain Korea 21 Project through Chonnam National University Medical School. H.J.K. was also supported by the Chonnam National University Specialization Grant.
↵*Both authors contributed equally to this work.
Original received January 11, 2008; revision received September 25, 2008; accepted September 30, 2008.
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