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Cardiovascular Research 2007 76(3):473-481; doi:10.1016/j.cardiores.2007.08.010
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Copyright © 2007, European Society of Cardiology

Inhibition of histone deacetylases triggers pharmacologic preconditioning effects against myocardial ischemic injury

Ting C. Zhaoa,*, Guangmao Chengb, Ling X. Zhangc, Yi T. Tsenga and James F. Padburya

aDepartment of Pediatrics, Women & Infants Hospital, The Warren Alpert Brown Medical School, Brown University, Providence, RI 02905, United States
bDepartment of Cardiology, Medical University of South Carolina, Charleston, SC 29403, United States
cDepartment of Oncology, Rhode Island Hospital, The Warren Alpert Brown Medical School, Brown University, Providence, RI 02903, United States

*Corresponding author. Cardiovascular Research, Department of Pediatrics, The Warren Alpert Brown Medical School at Brown University, 101 Dudley Street, Providence, RI 02905, United States. Tel.: +1 401 274 1122x8007; fax: +1 401 277 3617. Tzhao{at}wihri.org

Received 13 May 2007; revised 18 July 2007; accepted 6 August 2007


   Abstract
Top
 Abstract
1. Introduction
 2. Methods and materials
 3. Results
 4. Discussion
 Appendix A Supplementary data
 Acknowledgments
 References
 
Objectives Recent evidence has demonstrated the importance of histone deacetylases (HDAC) in the control of hypertrophic responses in the heart. However, it remains unknown whether inhibition of HDACs plays a role in myocardial ischemia and reperfusion (I/R) injury. We hypothesize that HDAC inhibition triggers preconditioning-like effects against I/R injury.

Methods and results Isolated mouse hearts were perfused with 3 cycles of 5-minute infusion and 5-minute washout of 50 nM of trichostatin A (TSA), a potent inhibitor of HDACs to mimic early pharmacologic preconditioning. This was followed by 30 min of ischemia and 30 min of reperfusion. In addition, mice were treated with saline or TSA (0.1 mg/kg, i.p.) to investigate delayed pharmacologic preconditioning. Twenty-four hours later, the hearts were subjected to I/R. Ventricular function and infarct size were measured, and HDAC 3, 4 and 5 were assessed by Western blot and immunofluorescence. HDAC and p38 mitogen-activated protein kinase activities were determined. TSA produced marked improvements in post-ischemic ventricular function recovery and a reduction in infarct size in both early and delayed preconditioning. Cardioprotection elicited by TSA was abrogated by SB203580, an inhibitor of p38. HDAC 3, 4 and 5 proteins were detected in mouse myocardium. TSA treatments resulted in a significant inhibition of HDAC activity. HDAC inhibition caused a dramatic increase in phosphorylation of p38 and p38 activity. Notably, HDAC inhibition also resulted in remarkable acetylation of p38 at lysine residues.

Conclusion These results suggest that inhibition of HDACs triggers pharmacologic preconditioning to protect the ischemic heart, which involves p38 activation.

KEYWORDS Trichostatin A; Histone deacetylase; Signal transduction; Ischemia


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods and materials
 3. Results
 4. Discussion
 Appendix A Supplementary data
 Acknowledgments
 References
 
Histone acetyltransferases (HAT) and histone deacetylases (HDAC) have garnered significant attention of late because these enzymes play a critical role in the regulation of a variety of cellular processes. Histone acetylation is mediated by histone acetyl transferase. The resulting modification in the structure of chromatin leads to nucleosomal relaxation and altered transcriptional activation. The reverse reaction is mediated by histone deacetylase, which induces deacetylation, chromatin condensation, and transcriptional repression [1–4]. Studies have shown that HAT and HDACs target non-histone protein, which may represent general regulatory mechanisms in biological signaling [5–7]. The biological roles of HDACs have been extensively explored in recent years [8].

Since the identification of HDAC 1 in 1996, eighteen HDACs have been reported in mammals [8–10]. These mammalian HDACs can be grouped into three distinct classes. Class I HDACs consist of HDAC 1, 2, 3, and 8, which are ubiquitously expressed. Class II HDACs include HDAC 4, 5, 7 and 9. In contrast to class I, class II HDACs exhibit a tissue-specific pattern of expression; HDAC 4 and HDAC 5 are found at high levels in the heart, brain and skeletal muscle [11,12]. Class III HDACs were identified on the basis of sequence similarity with Sir, a yeast transcriptional repressor that requires the cofactor NAD+ for its deacetylase activity. Class III HDACs are not yet fully explored in mammalian systems and are insensitive to inhibition by the HDAC inhibitor trichostatin A [13,14]. Trichostatin A is a highly specific and potent HDAC inhibitor to Class I and Class II HDACs. HDAC inhibition was shown to markedly decrease infarct size and reduce ischemia-induced neurological deficit scores in focal cerebral ischemia model of rats [15]. Inhibition of HDACs in myocytes silences the fetal gene activation, renders myocytes insensitive to hypertrophic agonists and blocks cardiac hypertrophy induced by aortic banding [16–18]. It remains unclear whether inhibition of HDACs plays an important role in the regulation of myocardial ischemia and reperfusion (I/R) injury. Pharmacologic preconditioning has been shown to be a powerful way to achieve cardioprotective effects [19–21]. We investigate whether inhibition of HDACs with TSA could trigger a pharmacologic preconditioning-like effect to protect the heart against myocardial I/R injury. We and others have identified the role of p38 activation by preconditioning stimuli in the protection of myocardium against ischemic injury [19,22–26]. p38 signaling has been shown to mediate fetal hemoglobin induction following HDAC inhibition [27]. It remains unknown whether TSA-induced cardioprotection could be mediated through the activation of p38. We hypothesize that HDAC inhibition could elicit a preconditioning-like effect to protect the heart against myocardial I/R injury, which is dependent upon p38. By using established early and delayed pharmacologic preconditioning approaches [19,24,28], we investigated: 1) whether treatment with an HDAC inhibitor, TSA, triggers early and delayed preconditioning-like effects against myocardial I/R injury; 2) whether cardiac protection elicited by HDAC inhibition will be abrogated by pharmacologic inhibition of the p38 signaling pathway; 3) whether HDAC inhibition will result in an increase in p38 acetylation; 4) whether HDAC inhibition will increase p38 activity in pharmacologic preconditioned myocardium.


   2. Methods and materials
Top
 Abstract
 1. Introduction
 2. Methods and materials
3. Results
 4. Discussion
 Appendix A Supplementary data
 Acknowledgments
 References
 
2.1 Animals
Adult male outbred mice (ICR) were supplied by Harlan (Indianapolis, IN). Animal experiments were conducted to conform to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). The experimental procedures were carried out in accordance with the guidelines of the Institutional Animal Care and Use Committee.

2.2 Chemical supplies and antibodies
TSA and SB203580 were obtained from Calbiochem (San Diego, CA). SB203580 was dissolved in dimethylsulfoxide. The perfusion chemicals were purchased from Sigma (St. Louis, MO). Phospho-p38, p38 polyclonal antibodies, HDAC 3, 4, 5, polyclonal antibodies, and mouse acetylated-lysine monoclonal antibody were purchased from Cell Signaling (New England, MA). The anti-rabbit HRP antibody was purchased from Amersham (Hercules, CA). The goat anti-mouse HRP antibody was purchased from Invitrogen (Carlsbad, CA).

2.3 Langendorff isolated heart perfusion
The methodology of Langendorff isolated perfused mouse heart preparation and ventricular function measurement have been described previously [19,29]. Infarct size was measured as described in the literature [19,30,31].

2.4 Experimental protocol 1
Effect of TSA in early cardiac preconditioning. The early pharmacologic preconditioning protocol was performed as described by Burg [28]. The mice were randomly assigned to one of the following groups: 1) ischemic control (n=6), hearts were perfused with K–H buffer for 30 min; 2) TSA treatment (n=6), hearts were perfused with 3 cycles of 5-minute infusion and 5-minute washout of TSA (50 nM) in K–H buffer; 3) SB203580+TSA (n=5), hearts were perfused with TSA (50 nM) in combination with SB203580 (10 µM); 4) SB203580 (n=7), hearts were perfused with SB203580 alone (10 µM). All hearts were then subjected to 30 min of ischemia followed by 30 min of reperfusion. The in vitro dose of SB203580 (10 µM) has been demonstrated to inhibit p38 activity in preconditioned hearts [25].

2.5 Experimental protocol 2
Effect of TSA in delayed cardiac preconditioning. In vivo delayed pharmacologic preconditioning was conducted as previously described [19,24]. Briefly, mice were randomized to the following four groups: 1) Vehicle (n=5), mice were injected with saline (0.1 ml, i.p.); 2) TSA (n=6), mice were injected with TSA (0.1 mg/kg, i.p.), 3) SB203580+TSA (n=5), the same as group 2 with SB203580 (0.1 mg/kg, i.p.) given 30 min prior to administration of TSA; 4) SB203580 (n=6), mice were administered SB203580 only (0.1 mg/kg, i.p.). Twenty-four hours later, the hearts were removed and subjected to 30 min of ischemia followed by 30 min of reperfusion. The in vivo dose of SB203580 (0.1 mg/kg, i.p.) has been shown to block cardiac protection by p38 activation in our previous study [19].

2.6 Experimental protocol 3
A subset of animals in Experimental protocol 1 and Experimental protocol 2 without sustained ischemia and reperfusion was used solely for the purpose of measuring p38 phosphorylation, acetylation, HDAC activity and p38 activity. For early preconditioning, samples were collected immediately after the third cycle of administration of TSA and 5 min washout. For delayed pharmacologic preconditioning, animals were treated with TSA for 30 min and heart tissues were collected. The hearts were frozen in liquid nitrogen, and cardiac lysates were extracted as previously described [19].

2.7 HDAC activity assay in myocardium
Measurement of HDAC activity in cardiac tissue was conducted using the colorimetric HDAC activity assay kit (BioVision Research, Mountain View, CA, USA).

2.8 Western blot analysis and immunoprecipitation
Western blots were performed as per the manufacturer's instructions (Cell Signaling Technology). Proteins (50 µg/lane) were separated by SDS-PAGE (12% SDS for phospho-p38, p38, acetylated-lysine and HDAC 3; 6% SDS for HDAC 4 and 5). The blots were incubated with the respective primary antibody, washed and visualized by incubation with anti-rabbit HRP secondary antibody and ECL Chemiluminescence Detection Reagent (Amersham Pharmacia Biotech). For the measurement of the acetylation of p38, cardiac tissue lysates (200 µg) were immunoprecipitated with an antibody (1 µg) specific for phospho-p38 or total p38 and gently agitated for 2 h, followed by additional incubation with EZview red protein A affinity gel (Sigma, Saint Louis). Rabbit IgG was used as a negative control for immunoprecipitation. Immunoblotting was performed as described above.

2.9 p38 activity measurement
p38 activity was measured according to manufacture's instructions using the p38 kinase assay kit from Cell signaling (New England, MA).

2.10 Statistics
All data are expressed as mean±SE. Differences among the groups were analyzed by one-way analysis of variance (ANOVA), followed by post hoc Bonferroni comparison or Student's unpaired t test for two groups. A probability of p<0.05 was considered to represent a significant difference.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods and materials
 3. Results
4. Discussion
 Appendix A Supplementary data
 Acknowledgments
 References
 
3.1 Baseline ventricular function
Baseline functional parameters including LVSP, LVEDP, heart rate, LV-dP/dt max and LV-dP/dt min, were recorded before ischemia. There were no differences in baseline ventricular function among groups, which is detailed in the online supplementary data Table 1a and Table 1b.


Figure 6
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  		Table 1a Baseline ventricular function in early pharmacologic preconditioning following HDACs inhibition

 

Figure 7
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  		Table 1b Baseline ventricular function in delayed pharmacologic preconditioning following HDACs inhibition.

 
3.2 HDAC inhibition improves post-ischemic ventricular function, which depends on p38
In early preconditioning, TSA treatment improved the recovery of LVDP as compared with the control group (p<0.05 Fig. 1 A). Similarly, TSA treatment markedly increased recovery of LVEDP (Fig. 1 B). Both post-ischemic LV-dP/dt max and LV-dP/dt min recoveries were significantly improved in groups infused with TSA as compared with the control group (p<0.05, Fig. 1 C and D). The rate pressure product remarkably increased in the TSA group versus the control group (p<0.05, Fig. 1 E). Heart rate did not change significantly among groups (Fig. 1 F). The improvements in LVEDP, LV-dP/dt max, LV-dP/dt min and RPP following TSA treatment, however, were blocked by SB203580, a p38 inhibitor. There were no significant differences in functional recoveries between SB203580 alone, control, or the SB203580+TSA groups. To further support the selective role of SB203580 for p38 in our study, we infused SB203580 at the lower concentration (1 µmol/L), which has been shown to selectively inhibit p38 in isolated perfused hearts [32]. We found that the co-infusion of SB203580 at 1 µmol/L with TSA still abrogated the functional improvement induced by TSA. However, ventricular functional improvement elicited by TSA was not blocked by co-infusion of the c-Jun N-terminal kinase (JNK) inhibitor VIII (10 µmol/L) in the presence of TSA (see the online supplementary data Fig. S1 and Fig. S2).


Figure 1
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  		Fig. 1 The effects of TSA on ventricular function in early preconditioning. Values represent mean±SE (n=5–7). *p<0.05.

 

Figure 2
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  		Supplementary Fig. S1 The effects of SB 203580 (1 µmol/L) in the presence of TSA (50 nmol/L) on ventricular function in early preconditioning. Values represent mean±SE (n=5). TSA indicates trichostatin A. *p<0.05.

 

Figure 3
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  		Supplementary Fig. S2 The effects of JNK inhibitor VIII in the presence of TSA (50 nmol/L) on ventricular function in early preconditioning. Values represent mean±SE (n=5). TSA indicates trichostatin A. *p<0.05.

 
In delayed preconditioning, there was a significant improvement in the recovery of LVDP following TSA treatment (p<0.05 versus the vehicle group, Fig. 2 A). Post-ischemic LVEDP markedly improved in the TSA-treated group as compared with the vehicle group (p<0.05 Fig. 2 B). TSA-induced improvement in LVEDP was abolished by SB203580 (p<0.05, Fig. 2 B). The recovery of post-ischemic LV-dP/dt max and LV-dP/dtmin was also dramatically increased following TSA treatment (p<0.05, Fig. 2 C and D). The improvement in post-ischemic LV-dP/dtmax and LV-dP/dtmin induced by TSA treatment was mitigated with pretreatment with SB203580 (p<0.05, Fig. 2 C and D). In addition, TSA treatment significantly augmented the recovery of RPP (Fig. 2 E), which was abrogated by pretreatment with SB203580 (p<0.05 versus the TSA-treated group). The recoveries of heart rate did not vary among groups (Fig. 2 F). There were no significant differences in ventricular functional recoveries between SB203580 alone, vehicle, or the SB203580+TSA groups.


Figure 2
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  		Fig. 2 The effects of TSA on ventricular function in delayed preconditioning. Values represent mean±SE (n=5–7). *p<0.05.

 
3.3 HDAC inhibition reduces infarct size
In early preconditioning, myocardial infarct size was 28±2% of the risk zone in the control group (Fig. 3). Infusion of TSA before ischemia significantly reduced the infarct size to 11±1%. Simultaneous perfusion of SB203580 with TSA abolished the reduction of infarct size, as indicated by an increase in infarct size to 26±2% (p<0.05 versus the TSA group). The infarct size in the SB203580 alone group was 29±2%, which was not different from that of the SB203580+TSA group. Furthermore, SB203580 at the concentration of 1 µmol/L completely blocked the infarct-limiting effect of TSA whereas co-infusion of JNK inhibitor VIII to inhibit JNK activity failed to abrogate the infarct-limiting effect of TSA (see the online supplementary data Fig. S3).


Figure 3
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  		Fig. 3 Infarct size was examined in early preconditioning. The bar diagram shows infarct size expressed as percentage of area at risk. Values represent mean±SE. TSA indicates trichostatin A. *p<0.05.

 
In delayed preconditioning, TSA treatment significantly reduced myocardial infarct size (9±2% versus 31±2% in the vehicle group, Fig. 4). Pre-treatment of SB203580 eliminated TSA-induced reduction in infarct size after delayed pharmacologic preconditioning (33±6%, p<0.05 versus TSA group). The infarct size in the SB203580 group was 36±2%, which was not different from that of the SB203580+TSA group.


Figure 4
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  		Fig. 4 Infarct size was examined in delayed preconditioning. The bar diagram shows infarct size expressed as percentage of area at risk. Values represent mean±SE. TSA indicates trichostatin A. *p<0.05.

 
3.4 HDACs proteins and HDAC activity in myocardium
As shown in Fig. 5 A, Western blot demonstrated the expression of HDAC 3, HDAC 4 and HDAC 5 in normal non-preconditioned hearts, which evidences the presence of HDAC 3, 4, 5 in adult mouse myocardium. This is consistent with the observations that HDAC 4 and HDAC 5 are mainly found in the heart, and HDAC3 is ubiquitously expressed [11,12]. In addition, immunofluorescent staining indicated that HDAC 3 and HDAC 4 showed significant aggregation in the cytoplasm whereas HDAC 5 appeared in a speckled pattern (see the online supplementary data Fig. S4). There was a basal level of HDAC activity in the normal heart, but HDAC activity was significantly inhibited by treatments of TSA in both early (Fig. 5 B) and delayed preconditioned (Fig. 5 C) hearts.


Figure 5
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  		Fig. 5 The Western blot shows HDAC 3, 4 and 5 (A). HDAC activities were measured in both early (B) and delayed (C) preconditioned hearts. Values are mean±SE. *p<0.05 vs the control or vehicle group.

 
3.5 HDAC inhibition activates p38 phosphorylation and enhances p38 activity
Next, we examined the impact of TSA on p38 phosphorylation in early preconditioned hearts. TSA treatment was associated with a significant increase in p38 phosphorylation (Fig. 6 A), indicating an augmentation of p38 phosphorylation following an increase of hyperacetylation. To determine the relationship between acetylation and p38 phosphorylation, cardiac tissue lysates were immunoprecipitated with an antibody specific for phospho-p38 or p38 and immunoblotted with an antibody against acetylated-lysine. There was a significant increase in the acetylation in phospho-p38 but not total p38 (Fig. 6 B). There was no detectable acetylation of IgG as a negative control (not shown).


Figure 6
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  		Fig. 6 The effect of TSA on p38 phosphorylation and acetylation in early pharmacologic preconditioning. (A) Western blot shows the effect of TSA on p38 phosphorylation. The bar graph shows the results of densitometric measurement of phospho-p38. Values are mean±SE. (n=3), *p<0.05 vs the control group. (B) Cardiac tissue lysates were immuoprecipitated with either phospho-p38 or total p38, and immunoblotted with anti-acetylated lysine antibody.

 
In delayed preconditioning, TSA treatment significantly increased p38 phosphorylation compared to the vehicle group (Fig. 7 A). An immunoprecipitation experiment further demonstrated that TSA treatment increased the acetylation of phospho-p38 (Fig. 7 B). These studies demonstrate that phospho-p38 was hyperacetylated after HDAC inhibition in both early and delayed preconditioning.


Figure 7
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  		Fig. 7 The effect of TSA on p38 phosphorylation and acetylation in delayed pharmacologic preconditioning. (A) Western blot shows effect of TSA on p38 phosphorylation. The bar graph shows the results of densitometric measurement of phospho-p38. Values are mean±SE (n=3). *p<0.05 vs the vehicle group. (B): cardiac tissue lysates were immuoprecipitated with either phospho-p38 or total p38, and immunoblotted with anti-acetylated lysine antibody.

 
We next examined whether p38 activity could be increased following HDAC inhibition in preconditioned hearts. As shown in Fig. 8, the basal level of p38 activity was detected in the control and vehicle groups, but treatment of hearts with TSA significantly increased p38 activity, as indicated by increases in phospho-ATF-2 in both early (Fig. 8 A) and delayed preconditioned (Fig. 8 B) hearts.


Figure 8
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  		Fig. 8 p38 activity following HDAC inhibition in both early (A) and delayed (B) pharmacologic preconditioning. Western blot shows phospho-ATF-2 levels. The bar graph shows the results of densitometric measurement of p38 activity. *p<0.05 vs control or vehicle group. Values represent means±SE (n=3).

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods and materials
 3. Results
 4. Discussion
Appendix A Supplementary data
 Acknowledgments
 References
 
4.1 Salient findings
In this study, we demonstrated that 1) the HDAC inhibitor, TSA, triggers both early and delayed pharmacologic preconditioning-like effects to increase resistance to myocardial I/R injury, as indicated by an improvement in the recovery of ventricular function and the reduction of myocardial necrosis; 2) the TSA-induced cardioprotection in both early and delayed preconditioning was mitigated by SB203580, suggesting an essential role for p38 in the cardioprotection elicited by HDAC inhibition; 3) HDAC inhibition following TSA treatment resulted in a dramatic increase in phosphorylated p38 and acetylation of phospho-p38 in the myocardium. 4). Inhibition of HDAC activity by TSA led to remarkable increases in p38 activity in both early and delayed pharmacologically preconditioned hearts. To the best of our knowledge, this is the first investigation demonstrating that HDAC inhibition induces preconditioning-like effects to generate a cardioprotective effect against I/R injury in mice. These results suggest the importance of inhibition of histone deacetylases to achieve cardioprotection against ischemic injury.

4.2 HDAC inhibition and cardioprotection
It has been demonstrated that class II HDACs are abundantly expressed in the heart [11,12]. In this study, we confirmed expression of HDAC 3, HDAC 4 and HDAC 5 in adult mouse myocardium although we have not detected the other HDACs. There is a basal level HDAC activity in the myocardium, which was significantly inhibited by TSA. TSA was originally isolated from Streptomyces hygroscopicus and acts as a potent inhibitor of HDAC with a Ki value of 3.4 nM in in vitro experiments using purified HDAC from mouse mammary tumor cells [33]. The deacetylase-TSA crystal structure reveals that TSA binds the deacetylase catalytic core to form a tubular pocket, a zinc-binding site and two Asp-His charge-relay system to inhibit the histone deacetylase [34]. We demonstrated a significant suppression of HDAC activity following TSA treatments in both early and delayed preconditioning, which suggests an effective inhibition of histone deacetylases in the myocardium. Preconditioning stimuli have been shown to provide effective protection to the heart against ischemic injury [19–21]. In this study, we explored whether TSA triggers early preconditioning-like effect. Previous work suggests that three cycles of 5-minute infusion of the pharmacologic reagent separated by 5-minute washout could effectively induce early preconditioning [28]. On this basis, hearts were perfused with TSA, which consisted of 3 cycles of 5-minute infusion of TSA and 5-minute washout prior to ischemia. TSA, at a concentration of 50 nM, was used to inhibit HDAC activity in culture myocytes [16]. Infusion of TSA itself did not change ventricular function in non-ischemic hearts in this study. However, when the perfused heart was subjected to I/R in vitro, TSA treatment resulted in an improvement in the recovery of ventricular function. In addition, the extent of infarct size was decreased by TSA. In vivo administration of pharmacologic reagents has been shown to produce delayed cardioprotection from mouse to human in studies in the preconditioning field [19,24,35]. To elucidate whether TSA treatment also provides delayed protection against myocardial I/R injury, mice were treated with TSA (0.1 mg/kg) 24 h before ischemic insult. As found in early preconditioning in vitro, TSA led to an improvement in cardiac performance and a decrease in infarct size. Although evidence demonstrates the role of HDACs in the regulation of the development of cardiac hypertrophy and fetal gene expression [16–18,36,37], the significance of HDAC inhibition on ischemic myocardium has not been reported. Our findings have shown that HDAC inhibition following TSA treatment elicits a preconditioning-like effect against myocardial I/R injury.

4.3 HDAC inhibition-induced cardioprotection via p38 activation
The p38 family of mitogen-activated protein kinases has been shown to play an important role in mediating stress-induced signaling in mammalian cells [38]. Our previous studies show that activation of p38 protects the heart against I/R injury [19]. In line with our observation, activation of p38 with preconditioning stimuli or over-expression of MKK3/MKK6 has been reported to protect the heart against myocardial I/R injury [19,22–26,32]. Mutation of p38 β isoform also resulted in increased myocardial injury [39]. However, during lethal ischemia, ischemia and reperfusion as well as post-conditioning, p38 inhibition has been shown to result in cardiac protection [40–42]. Transgenic mice expressing a cardiac dominant p38 {alpha} attenuated cardiac I/R injury. Disruption of a single copy of p38 {alpha} in mice was reported to be less susceptible to myocardial I/R injury [43,44]. These conflicts remain unresolved. These discrepancies may have to do with the strength of p38 activation and activation of different isoforms, vastly different stress conditions, and animal species [45–47]. In this study, cardioprotection induced by HDAC inhibition in both early and delayed preconditioning was abrogated by inhibition of p38 with SB203580. This was extended by our observation that TSA-induced cardioprotection was also diminished by the low concentration of SB203580 (1 µmol/L), but not by the inhibition of JNK in early preconditioning. Again, our results suggest that HDAC inhibition-mediated protection is dependent on activation of p38.

Previous investigations have suggested a role for histone hyperacetylation interacting with p38 in regulation of fetal hemoglobin induction [27]. However, it remains unclear whether HDAC inhibition will regulate activation of p38 to transduce signaling pathway. We showed that TSA treatment resulted in increased p38 phosphorylation and activity, suggesting p38 activation is the target of HDAC inhibition. Recent studies using this HDAC inhibitor suggested a role for non-histone acetylation [5–7]. One possible mechanism by which TSA activates p38 could be through acetylation, which augments stress signal transduction. In addition, it has been shown that acetylation can be observed within minutes, implying that acetylation is immediate [48]. Interestingly, our results indicate that phosphorylated p38 was acetylated following TSA treatment, suggesting an association of p38 acetylation with p38 activation following HDAC inhibition. However, it remains to be elucidated as to how p38 acetylation mediates p38 phosphorylation following HDAC inhibition. Future work is needed to examine the molecular mechanism(s) by which p38 acetylation regulates its activation using in vitro analysis approach. In addition, it is likely that a transcriptional response regulates the development of delayed preconditioning following HDAC inhibition. Our data indicates that p38 was activated following HDAC inhibition in both early and delayed preconditioning, meaning that p38 activation may be a common mechanism by which TSA is beneficial in both the early and delayed preconditioning. However, whether or not p38 activation following HDAC inhibition mediates distinct downstream signaling pathways for early and delayed preconditioning is a subject matter which merits further investigation.

In summary, we are the first to demonstrate a crucial role for HDAC inhibition in protecting the heart against I/R injury in both early and delayed pharmacologic preconditioning. Selective inhibition of p38 abrogated cardiac protection following TSA treatments, indicating the essential role of p38 in mediating protection elicited by HDAC inhibition. HDAC inhibition resulted in remarkable increases in phosphorylated p38 as well as acetylation of phospho-p38 at lysine residues. Further, HDAC inhibition caused a dramatic increase in p38 activity in pharmacologically preconditioned hearts, further supporting that HDAC inhibition with p38 activation is critical to achieve cardioprotection. Our findings shed new light on a novel signaling pathway in myocardial I/R injury and hold great promise for clinical implication.

Time for primary review 19 days


   Appendix A Supplementary data
Top
 Abstract
 1. Introduction
 2. Methods and materials
 3. Results
 4. Discussion
 Appendix A Supplementary data
Acknowledgments
 References
 
Online supplement.

Appendix A. Supplementary data
Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.cardiores.2007.08.010.


Figure 4
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  		Supplementary Fig. S3 The effects of SB203580 (1 µmol/L, A) or JNK inhibitor VIII (B) in the presence of TSA (50 nmol/L) on infarct size in early preconditioning. Values represent mean±SE (n=5). TSA indicates trichostatin A. *p<0.05.

 


Figure 5
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  		Supplementary Fig. S4 Immunofluorescent detection shows the HDAC 3 (a), HDAC 4 (b), HDAC 5 (c) and negative control (d). Myocytes were stained with anti-sarcomeric actinin (red), HDACs were stained with anti-HDAC 3, 4 and 5, respectively (green). Nuclei were stained with 4'6-diamidino-2-phenylindole (DAPI, blue). Images show an overlay of myocytes, HDACs, and nuclei. Bar=50 µm. HDACs primary antibodies were omitted in the negative control.

 


   Acknowledgments
Top
 Abstract
 1. Introduction
 2. Methods and materials
 3. Results
 4. Discussion
 Appendix A Supplementary data
 Acknowledgments
References
 
The work was supported by National American Heart Association-Scientist Development Grant 0735458N, Rhode Island Foundation 20052634 and National Institutes of Health Grant NIH 1P20RR01872802.


   References
Top
 Abstract
 1. Introduction
 2. Methods and materials
 3. Results
 4. Discussion
 Appendix A Supplementary data
 Acknowledgments
 References
 

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