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Cardiovascular Research Advance Access first published online on April 8, 2008
This version [Corrected Proof] published online on April 25, 2008
Cardiovascular Research, doi:10.1093/cvr/cvn091
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Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2008. For permissions please email: journals.permissions@oxfordjournals.org

Overexpression of heat shock protein 27 protects against ischaemia/reperfusion-induced cardiac dysfunction via stabilization of troponin I and T

Xi-Yuan Lu, Le Chen, Xiao-Long Cai and Huang-Tian Yang*

Laboratory of Molecular Cardiology, Institute of Health Sciences (IHS), Institutes for Biological Sciences (SIBS), Chinese Academy of Sciences (CAS) and Shanghai Jiao Tong University School of Medicine (SJTUSM), 225 Chong Qing Nan Rd, Build. No. 1 of Institute of Health Sciences, Rm. 613, Shanghai 200025, China

* Corresponding author. Tel: +86 21 63852593; fax: +86 21 63852593. E-mail address: htyang{at}sibs.ac.cn

Received 20 July 2007; revised 30 March 2008; accepted 2 April 2008

Time for primary review: 31 days


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Supplementary material
 Funding
 References
 
Aims: Heat shock protein 27 (Hsp27) renders cardioprotection from ischaemia/reperfusion (I/R) injury, but little is known about its role in myofilaments. We proposed that increased expression of Hsp27 may improve post-ischaemic contractile dysfunction by preventing I/R-induced cardiac troponin I (cTnI) and troponin T (cTnT) degradation.

Methods and results: Adenovirus-mediated Hsp27 overexpression improved contractile function in perfused rat hearts subjected to global no-flow I/R (30-min/30-min). Such improvement was further confirmed in Hsp27-overexpressing cardiomyocytes subjected to simulated I/R (20-min/30-min). Moreover, these cells showed restored myofilament response to Ca2+ but not intracellular Ca2+ transients. The protection correlated with attenuation of I/R-induced cTnI and cTnT degradation. Confocal microscopy revealed co-localization of Hsp27 with these proteins. Co-immunoprecipitation and pull-down assays further confirmed that Hsp27 interacted with the COOH-terminus of cTnI and the NH2-terminus of cTnT and that Hsp27 overexpression decreased the interaction between µ-calpain (a protease mediating proteolysis of cTnI and cTnT) and cTnI or cTnT under I/R.

Conclusion: The findings reveal a novel role of Hsp27 in the protection of cTnI and cTnT from I/R-induced degradation by preventing their proteolytic cleavage via interacting with these proteins. Such protection may result in restored post-ischaemic myofilament response to Ca2+ and improved post-ischaemic contractile function.

KEYWORDS Heat shock protein 27; Ischaemia/reperfusion; Contraction; Troponin I; Troponin T


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 Supplementary material
 Funding
 References
 
Myocardial dysfunction resulting from ischaemia/reperfusion (I/R) is a common clinical scenario in patients suffering from ischaemic heart disease. Such dysfunction is associated with prominent decreased myofilament Ca2+ responsiveness1–3 and appears to be related to specific proteolysis of myofilament proteins, especially cardiac troponin I (cTnI) and troponin T (cTnT).4–7 cTnI and cTnT are part of the cTn complex that, in concert with tropomyosin, regulates myocyte contraction in response to a rise of intracellular Ca2+ concentration ([Ca2+]i).8 The degradation of COOH-terminal cTnI and NH2-terminal region of cTnT in I/R by Ca2+-activated protease µ-calpain has been demonstrated to correlate with I/R-induced contractile dysfunction,4,5,9–11 whereas the protection against proteolysis of cTnI minimizes I/R-induced myocardial stunning (reversible post-ischaemic contractile dysfunction).10 Therefore, identification of therapeutic approaches and mechanisms that stabilize cTnI and cTnT during I/R are important for the improvement of ischaemic heart diseases associated with contractile dysfunction.

Heat shock protein 27 (Hsp27, Hsp25 in rodents) belongs to the family of small Hsps that bind denatured proteins following cell stress to prevent aggregation. During myocardial ischaemic injury or hypoxic stress, Hsp27 renders itself an excellent candidate for cardioprotection against apoptosis, infarction, and oxidative stress.12–15 Interestingly, Hsp27 has been shown to localize on the sarcomere in cardiomyocytes15 and I/R increases its myofilament translocation.16,17 Moreover, overexpressing a wild-type human Hsp27 (Hsp27wt) in a transgenic mouse model preserves contractile function of the Langendorff-perfusing heart with global I/R injury.14 We, therefore, hypothesized that the increasing of Hsp27 expression may protect the heart against post-ischaemic contractile dysfunction via stabilizing myofilaments, in particular cTnI and cTnT.

To test this hypothesis, we transfected adenoviruses carrying the Hsp27wt gene or green fluorescent protein (GFP) gene to adult rat hearts in vivo and to cultured cardiomyocytes in vitro. The roles of increasing Hsp27 expression in cardiac and myocyte contractile function, intracellular Ca2+ transients, and cTnI/cTnT degradation under I/R were investigated. In addition, potential protective mechanisms of Hsp27 against I/R-induced depression in contractility were analysed. Our findings demonstrated the cardioprotective effect of increasing Hsp27 expression by gene transfer in post-ischaemic contraction, and are the first to reveal the roles and protective mechanisms of Hsp27 in regulating myofilament response to Ca2+ and troponins under I/R.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 Supplementary material
 Funding
 References
 
Expanded methods for immunoblotting analysis, immunofluorescence analysis, and co-immunoprecipitation assay were provided in Supplementary material online.

2.1 Animals
Adult male Sprague-Dawley rats from 280 to 300 g (Shanghai Slac Laboratory Animal Co. Ltd.) were cared for in accordance with the Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85-23, Revised 1996). Animal procedures were approved by the Institutional Review Board at the Institute of Health Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, and Shanghai Jiao Tong University School of Medicine (Shanghai, China).

2.2 Construction of recombinant adenoviruses
Recombinant adenoviruses expressing human Hsp27wt (Ad.Hsp27wt) and GFP (Ad.GFP) were prepared as described previously18 by using pAdEasyTM vector system (Qbiogene, USA). Briefly, cDNA of Hsp27 from a plasmid SV2711, provided by Dr Eileen Hickey and Dr Lee Weber (University of Nevada), was subcloned into a shuttle vector pTrack-CMV (Qbiogene, USA). The pAdTrack-CMV-Hsp27 and pAdEasy-1 (Qbiogene, USA) expressing GFP gene were homologously recombinanted in bacteria BJ5183. The pAdEasy-1 and recombinant plasmid pAd.Hsp27 were propagated separately in HEK 293 cells. The propagated recombinant adenoviruses in the HEK 293 cells were purified and stored at –80°C in a solution containing (mmol/L): NaCl 135, KCl 5, MgCl2 1, Tris–HCl 10 (pH 7.4), and BSA 0.02% and glycerol 10%. The titers of stocks measured by plaque assays were 7 x 1010 pfu/mL for Ad.Hsp27wt and 3 x 1010 pfu/mL for AdGFP.

2.3 In vivo adenoviral gene delivery
The surgical procedures and adenoviral delivery were carried out as described.19 Briefly, the rats were anesthetized with sodium pentobarbital (60 mg/kg, ip) and a thoracotomy was performed. A 26 gauge needle containing 200 µL of diluted adenoviruses (3 x 1010 pfu/mL) or sterile saline was advanced from the apex of the left ventricle to the aortic root. The aorta and main pulmonary arteries were clamped for 10 s distal to the site of the injector and solution injected, and then the chest was closed. The hearts underwent haemodynamic studies at day 4 after the injection of the adenovirus.

2.4 Langendorff-perfused heart protocol
Vehicle control, Ad.GFP-, or Ad.Hsp27wt-infected hearts were removed from anesthetized rats and mounted on a Langendorff apparatus, perfused with a modified Krebs–Henseleit buffer containing (mmol/L) 118 NaCl, 4.7 KCl, 1.2 MgSO4, 1.8 CaCl2, 1.2 KH2PO4, 25 NaHCO3, 0.026 Na2EDTA, and 11.1 glucose. The buffer was saturated with 95% O2/5% CO2 (pH 7.4, 37°C) as previously described.20 A small fluid-filled balloon was inserted into the left ventricular (LV) cavity and coupled to a pressure transducer (Gould model P23 Db, AD Instrument, Castle Hill, Australia). The balloon was inflated until a LV end-diastolic pressure (LVEDP) reached 10 mmHg. Isolated hearts were stabilized for at least 15 min and then underwent 30 min of no-flow global ischaemia followed by 30 min of reperfusion (R30). Contractile performance of the left ventricle was evaluated on the basis of its developed pressure (LVDP), LVEDP, and maximum speeds of pressure development (+dP/dt) and pressure decay (–dP/dt) with PowerLab (AD Instrument, Australia).

2.5 Isolation, culture, and adenoviral infection of ventricular myocytes
Left ventricular myocytes were isolated from adult rat hearts using a standard enzymatic method as previously described.21,22 Briefly, the freshly isolated heart was successively perfused with nominally Ca2+-free Tyrode’s solution containing collagenase II (240 U/mL, Worthington Biochemical, USA) and protease (0.12 mg/mL, Sigma, USA) for 18–22 min. Finally, the cell suspension from left ventricles was rinsed with Tyrode’s solution followed by a gradual increase in the Ca2+ concentration up to 1.25 mM and >90% of isolated rod-shaped myocytes were Ca2+-tolerant. The isolated myocytes were then cultured with medium 199 (Sigma, USA) supplemented with L-carnitine (2 mmol/L), N-2-mercaptopropionyl glycine (5 mmol/L), taurine (5 mmol/L), insulin (0.1 µmol/L), 2.5% FBS (Gibico, USA), and penicillinstreptomycin (100 IU/mL). Adenoviral infection was performed as described previously.23 After 2 h of culture to achieve myocyte attachment, adenovirus-directed gene transfer was implemented by adding a minimal volume of the FBS-free medium 199 containing Ad.HSP27wt or Ad.GFP (a negative control) to the dish at a multiplicity of infection (MOI, the ratio of infectious virus particles to the number of cells being infected) of 100 for 2 h. All experiments were performed after 48 h of adenoviral infection.

2.6 Simulated I/R in isolated cardiomyocytes
To directly determine the roles of Hsp27 in the alternations of cell contraction and Ca2+ transients due to I/R, a cellular model of simulated I/R (20-min/30-min) in ventricular myocytes was used as previously described.22,24,25 Briefly, myocytes were equilibrated in modified Krebs–Henseleit solution at 35°C, pH 7.4. Then the solution was switched to ischaemic solution, containing (mmol/L): 123.0 NaCl, 8.0 KCl, 6.0 NaHCO3, 0.9 NaH2PO4, 0.5 MgSO4, 20.0 Na-lactate, 1.8 CaCl2, gassed with 95% N2/5% CO2, (pH 6.8) for 20 min followed by 30 min of reperfusion with modified Krebs–Henseleit solution. Left ventricular myocytes from the same heart were harvested for protein extraction at pre-ischaemia and 30 min of reperfusion.

2.7 Simultaneous measurements of cell shortening and Ca2+ transients
Cell shortening and intracellular Ca2+ transients were detected simultaneously by an IonOptix system (IonOptix, Milton, USA) as described.22,26 Cells were incubated with a Ca2+ indicator indo-1AM (5 µM, Molecular Probes, USA) at 25°C for 10 min. Loaded cells were electrically stimulated at 0.5 Hz, except during ischaemia. The ratio of emitted fluorescence at 405 and 485 nm was recorded as an indicator of cytosolic Ca2+ concentration. Simultaneously, cells were illuminated with red light (650–750 nm) through the bright-field path of the microscope and the cell shortening was detected by an optical edge-detector, collected using a charge-coupled device camera, and analysed using IonWizard 4.4 software in length mode (IonOptix, Milton, USA).

2.8 Measurement of extracellular Ca2+ concentration ([Ca2+]o)-shortening relation
Measurement of [Ca2+]o-shortening relation was performed as described previously.26 Changes in the peak cell shortening and Ca2+ transients induced by elevation of [Ca2+]o with or without I/R were simultaneously recorded by an IonOptix system as described in 2.7. [Ca2+]o was cumulatively elevated from 0.5, 1, 1.5, 2, 3, 4, 5, to 6 mmol/L.

2.9 Immunoblotting analysis
Left ventricles were homogenized and cells were lysed as described previously.4,27 Briefly, ~100 mg of freeze-clamped left ventricular tissues was homogenized at 4°C with a homogenizer in 10 vols of lysis buffer. Cell extracts were scraped into lysis buffer, followed by vigorous vortexing and cooling on ice for 15 min before 15 min of centrifugation at 12 000 g. Tissue homogenates, cell lysates, or immunoprecipitates were analysed by standard western blotting with antibodies against Hsp27 (1:3000), cTnI (1:500), cTnT (1:500), µ-calpain (1:400, Santa Cruz, USA), or actin (1:2500, Sigma, USA). The detailed method is in Supplementary material online.

2.10 Immunofluorescence analysis
The immunofluorescent experiments were performed as described previously.28 The paraformaldehyde-fixed myocytes were treated with 0.5% Triton X-100 in PBS for 15 min. The myocytes were then co-immunostained with antibodies anti-Hsp27 (1:100) and anti-cTnI (1:100) or anti-cTnT (1:100, Santa Cruz, USA). After washing with PBS, the cells were stained for 2 h with TRITC-conjugated anti-rabbit IgG, FITC-conjugated anti-mouse, or anti-goat IgG second antibodies (1:200, Jackson, USA), respectively. The stained cells were observed by laser-scanning confocal microscopy (Leica, Heidelberg, Germany). The detailed method is in Supplementary material online.

2.11 Co-immunoprecipitation assay
Cells were lysed with the lysis buffer described previously.27 Immunoprecipitations were performed as described29 by using 2 µg of rabbit anti-Hsp27, goat anti-cTnI, mouse anti-cTnT, rabbit anti-µ-calpain, and negative control antibodies, normal rabbit IgG, normal goat IgG, or normal mouse IgG (Santa Cruz, USA), respectively. Protein G-agarose beads (Santa Cruz, USA) were then added to isolate IgG antibodies. Immunoprecipitates were then separated from the supernatant by centrifugation at 5000 rpm and resolved on SDS–PAGE. The detailed method is in Supplementary material online.

2.12 Plasmid construction for expressing wild-type and truncated cTnI and cTnT
Mouse wild-type cTnI and COOH-terminal deletion mutant cTnI1–199 were amplified by reverse transcriptase–polymerase chain reaction (RT–PCR) as described previously.30,31 The common forward primer for cTnI and cTnI1–199 was 5'-CTGGAGATCACCATGGCTGATGAAAGCAGC-3', and the reserve primers for cTnI and cTnI1–199 were 5'-GTGGGATCCAAGGGCTCAGCCCTCAAACTTTTT C-3') and 5'-CTTTTTCTTGCGGGATCCCATGCCTCACAGTGCATCGATATTG-3', respectively. Mouse wild-type cTnT and NH2-terminal deletion mutant cTnT72–291 were amplified by RT–PCR using a common reserve primer 5'-CCGAAGCTTTCATCATTTCCAACGCCCGGTGACTTTG-3' and forward primers 5'-AATCATATGTCTGACGCCGAGGAGGTGGTG-3' for wild-type cTnT or 5'-AGCCCCATATGCTCTTCATGCCCAACTT-3' for cTnT72–291. Total RNA (0.5 mg) from myocytes was converted to cDNA by using Superscript II reverse transcriptase (Invitrogen) in a final volume of 20 mL, and 0.4 mL of this was used for each PCR. PCR was performed using Taq DNA polymerase (Promega) in a Mastercycler gradient (Eppendorf, Hamburg, Germany) at 95°C for 5 min followed by 35 cycles, each cycle consisted of denaturation at 95°C for 45 s, annealing at 55°C for 40 s, and extension at 72°C for 45 s. After a completion of the last cycle, there was an autoextension for 5 min at 72°C. The PCR productions were then subcloned into a pET-28a cloning vector carrying a NH2-terminal His (histidine) Tag (Novagen, USA). The final constructs pET-28a-cTnI, pET-28a-cTnT, pET-28a-cTnI{Delta}C (cTnI1–199, missing 12 residues), and pET-28a-cTnT{Delta}N (cTnT72–291, missing 71 residues) were verified by sequencing. GST-HSP27pGEX-4T-2 was provided by Prof. Madhavi J. Rane (University of Louisville).

2.13 His pull-down assay
Glutathione S-transferase (GST) and histidine (His) proteins were expressed and prepared according to the manufacturer’s instructions from Novagen (USA). The prepared GST and His tagged fusion protein plasmids, pET-28a-cTnI, pET-28a-cTnT, pET-28a-cTn{Delta}C (cTnI1–199), pET-28a-cTnT{Delta}N (cTnT72–291), and GST-HSP27pGEX-4T-2 were transformed into E. coli (BL21) and incubated with 0.2 mM isopropylb-D-1-thiogalactopyranoside (IPTG, Sigma, USA) to express the proteins. The fusion proteins were purified from bacterial lysates with GSH-Sepharose 4B beads and His-bind Resins (Novagen, USA). For His-pull-down assays, 0.5 µg of GST-Hsp27 fusion protein were incubated with 0.5 µg of His fusion proteins (His-cTnI, His-cTnT, His-cTnI{Delta}C, or His-cTnI{Delta}N) conjugated with His-bind resins, respectively, in 250 µL of TBS-N (20 mmol/L Tris–HCl, pH 7.6, 200 mmol/L NaCl, and 0.1% Nonidet P-40) at 4°C for 2 h. After His-pull-down, the samples were processed for immunoblotting analysis with anti-Hsp27 antibody as previously mentioned in 2.9.

2.14 Statistical analysis
Data are presented as mean±SEM. Statistical analysis was performed using two-tailed Student t-test for unpaired data and ANOVA for multiple comparisons. P < 0.05 was considered statistically significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 Supplementary material
 Funding
 References
 
3.1 Recovery of post-ischaemic function in Hsp27 overexpressing hearts
To evaluate whether overexpressing of Hsp27wt was capable of preserving contractile function during I/R, we examined post-ischaemic haemodynamic changes in isolated perfused-hearts with global no-flow I/R (30-min/30-min). Immunoblotting analysis showed a ~3.5-fold increase in Hsp27 level in Ad.Hsp27wt-infected hearts related to vehicle or Ad.GFP hearts 4 days post-infection (Figure 1A). No significant differences were observed in LVDP, LVEDP, and ±dP/dt between vehicle and Ad.GFP controls during pre-ischaemic and I/R phases (data not shown). Baseline functional values before initiation of I/R were also similar between Ad.GFP and Ad.Hsp27wt groups (Figure 1B). After 30 min of reperfusion, LVDP, +dP/dt, and –dP/dt recovered by 20.7 ± 5.2%, 19.1 ± 3.5%, and 18.5 ± 5.1%, respectively, in Ad.GFP hearts, but the LVDP, +dP/dt, and –dP/dt were significantly improved to 43.3 ± 5.2%, 30.7 ± 4.7%, and 35.4 ± 5.7%, respectively, in Ad.Hsp27wt hearts (Figure 1B, P < 0.01 each). Concomitantly, increased LVEDP by I/R in the Ad.GFP hearts (68 ± 2.5 mmHg) was markedly decreased in the Ad.Hsp27wt hearts (48.9 ± 2.3 mmHg, P < 0.01).


Figure 1
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  		Figure 1 Hsp27 overexpression improves post-ischaemic cardiac performance in isolated perfused hearts subjected to no-flow global I/R (30-min/30-min). (A) Schematic representation of adenoviruses encoding GFP (Ad.GFP) and Hsp27wt (Ad.Hsp27wt, left panel); right panel, immunoblotting analysis for Hsp27 protein level at 4 days after the adenoviral infection. Actin was used as a control. (B) Left ventricular (LV) performance during I/R in Ad.GFP and Ad.Hsp27wt hearts. LVDP, LV developed pressures; LVEDP, LV end-diastolic pressure; and +dP/dt and –dP/dt, maximum speed of LV pressure development and decline. After 30 min of reperfusion, LVDP, +dP/dt and –dP/dt recovered by 20.7 ± 5.2%, 19.1 ± 3.5%, and 18.5 ± 5.1%, respectively, in Ad.GFP hearts. LVDP, +dP/dt, and –dP/dt were significantly improved to 43.3 ± 5.2%, 30.7 ± 4.7%, and 35.4 ± 5.7%, respectively, after I/R in Ad.Hsp27wt hearts. There was also a concomitant improvement in LVEDP (n = 4). *P < 0.05, **P < 0.01 vs. corresponding Ad.GFP values.

 
3.2 Cell contraction and Ca2+ transients in hsp27 overexpressing myocytes
To explore cellular mechanisms underlying Hsp27-mediated protection on the contractile function during I/R, we next measured cell contractions and intracellular Ca2+ transients simultaneously in control, Ad.GFP- or Ad.Hsp27wt-infected cardiomyocytes under simulated I/R. Hsp27 protein levels were at a 6-fold increase in Ad.HSP27wt-infected cells relative to non-infected or Ad.GFP control cells after 48 h of infection (Figure 2A). No significant differences in cell shortening and Ca2+ transients were observed between control and Ad.GFP cells with or without I/R (data not shown). I/R (20-min/30-min) significantly decreased amplitude, rise, and decay rates of cell shortening and Ca2+ transients in Ad.GFP cells (Figure 2B–H). Decreases in the amplitude of cell shortening were significantly larger than that of Ca2+ transients (deceased by 56.4 ± 3.4% in cell shortening vs. 25.4 ± 3.4% in Ca2+ transients at R30, P < 0.01, Figure 2C and D). HSP27 overexpression did not affect the pre- or post-ischaemic dynamics of Ca2+ transients (Figure 2B, D, F, and H), but it significantly improved recovery of cell-shortening characterized as the amplitude and ±dL/dtmax at R20 and R30 (Figure 2B, C, E, and G). These data reveal that Hsp27 directly improves post-ischaemic cell contraction in a mechanism not related to the alteration of [Ca2+]i.


Figure 2
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  		Figure 2 Protein expression of Hsp27 and dynamics of cell contractions and intracellular Ca2+ transients in left ventricular myocytes subjected to simulated I/R (20-min/30-min) after 48 h of Ad.GFP and Ad.Hsp27wt infection. (A) Immunoblotting analysis for Hsp27 and actin expression in myocytes with or without Ad.GFP and Ad.Hsp27wt infecton. Actin was used as a control. (B–H) Representative traces (B) and the averaged dynamic changes of amplitudes (C and D), maximum speed of cell shortening (–dL/dtmax, E) and re-lengthening (+dL/dtmax, F); maximum speed of rise (+d[Ca2+]i/dtmax, G) and decay (–d[Ca2+]i/dtmax, H) of Ca2+ transients recorded in myocytes at pre-ischaemia (Pre) and 10, 20, and 30 min of reperfusion (R). Number of myocytes indicated in parentheses. *P < 0.05, **P < 0.01, and ***P < 0.01 vs. pre-ischaemic values. #P < 0.05, ##P < 0.01, ###P < 0.001 vs. the Ad.GFP values.

 
3.3 Roles of Hsp27 in regulation of myofilament Ca2+ responsiveness
Because myofilament Ca2+ activation is the other key regulator of myocardial contractility, we next assessed the relationships between the amplitude of Ca2+ transients and cell shortening induced by elevation of [Ca2+]o, an indirect measurement of myofilament Ca2+ sensitivity26 in Ad.GFP- and Ad.Hsp27wt-transfected cardiomyocytes. Elevation of [Ca2+]o resulted in a similar concentration-dependent increase of the Ca2+ transients and cell shortening between Ad.GFP and Ad.Hsp27wt cells without I/R (Figure 3A). This relationship was significantly shifted downward by simulated I/R in GFP control cells. However, the relationship was shifted to the left in Ad.Hsp27wt cells (Figure 3B), indicating that Hsp27 can restore I/R-suppressed myofilament Ca2+ responsiveness.


Figure 3
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  		Figure 3 Relationships between the amplitudes of cell shortening and intracellular Ca2+ transients induced by elevation of [Ca2+]o in Ad.GFP- and Ad.Hsp27wt-transfected cardiomyocytes at control (without I/R, A) or 30 min of reperfusion following 20 min of simulated ischaemia (B). Concentrations of [Ca2+]o: 0.5, 1, 1.5, 2, 3, 4, 5, and 6 mmol/L. Number of myocytes indicated in parentheses.

 
3.4 Roles of Hsp27 in cTnI and cTnT degradation
We then investigated whether Hsp27 prevents I/R-induced degradation of cTnI and cTnT. Such degradation has been demonstrated to affect the Ca2+ response and thus has been proposed to play a key role in ischaemic contractile dysfunction.4,5,7,9 Immunoblotting analysis showed strong bands at 31 kDa (full-length cTnI) and 39 kDa (full-length cTnT) in Ad.GFP and Ad.Hsp27w heart or myocyte lysis, and a ~27 kDa additional faint band (fragmented cTnI) in myocytes (Figure 4). I/R increased fragmented cTnI and cTnT (~27 kDa) bands in both Ad.GFP myocytes and hearts. Such increases were significantly reduced by overexpressing of Hsp27 (Figure 4).


Figure 4
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  		Figure 4 Immunoblot analysis of cTnI (A) and cTnT (B) degradation products at control (without I/R) and 30 min of reperfusion (R30) in Ad.GFP- and Ad.Hsp27wt-infected hearts and cardiomyocytes. Upper panel in (A) and (B), representative immunoblots in Ad.GFP- and Ad.Hsp27wt-infected hearts (left) and cardiomyocytes (right); low panel in (A) and (B), averaged fractional intensity (%) of the fragmented cTnI or cTnT over the total immunoreactivity per lane (fragmented/(fragmented+unfragmented cTnI/cTnT) at R30. I/R increased degradation of cTnI and cTnT seen in the Ad.GFP group was reduced by Hsp27 overexpression in both hearts and cardiomyocytes (n = 4 each). **P < 0.01 and ***P < 0.001 vs. corresponding Ad.GFP group.

 
3.5 Cellular localization and interaction of Hsp27 with cTnI and cTnT in vivo and in vitro
To explore whether the protection of Hsp27 on cTnI and cTnT is related to a possible intermolecular interaction between them, we visualized the specific immunofluorescence of Hsp27 and cTnI or cTnT using confocal immunocytochemical imaging. Hsp27 appeared to be present in a striated pattern in sarcomere regions, co-localizing with these two proteins (Figure 5A).


Figure 5
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  		Figure 5 Co-localization and interaction of Hsp27 with cTnI or cTnT in rat left ventricular myocytes. (A) Representative images of dual immunostaining by using antibodies for Hsp27 (red; b, b', e, e') and cTnI (green; a, a') or cTnT (green; d, d'). Nuclei were labelled with Hochest 33342 (blue). Scale bars, 8 µm. (B) Interaction of Hsp27 with cTnI or cTnT at pre-ischaemia (left) and 30 min of reperfusion (right) in myocytes after 48 h of Ad.Hsp27wt infection. The lysates from myocytes were subjected to immunoprecipitation (IP). The whole cell lysates were incubated with agarose beads coated with normal rabbit IgG, normal goat IgG, and normal mouse IgG (as negative controls), and anti-Hsp27, anti-cTnI (upper panel), or anti-cTnT antibodies (low panel) followed by immunoblotting with indicated antibodies. Ten percent of lysates used for IP reactions were included as a positive control (input). The interaction of Hsp27 with cTnI or cTnT in the immunoprecipitates was analysed by immunoblotting with anti-Hsp27 and anti-cTnI or anti-cTnT antibodies. Similar results were obtained in at least three independent experiments.

 
To confirm whether Hsp27 can interact with cTnI or cTnT in vivo, we performed co-immunoprecipitation experiments using lysates from myocytes overexpressing Hsp27wt and harvested at pre-ischaemia and 30 min of reperfusion. cTnI and cTnT were detected in the immunoprecipitates with anti-Hsp27 and Hsp27 was also detected in cTnI or cTnT immunoprecipitates at pre-ischaemia and I/R (Figure 5B), suggesting an interaction of Hsp27 with cTnI and cTnT in vivo. We then determined whether Hsp27 can directly interact with cTnI or cTnT. His-pull-down assays were performed with GST fusion protein Hsp27 and His fusion proteins cTnI and cTnT. As shown in Figure 6B, immobilized His-cTnI or His-cTnT, but not His alone, was able to pull down GST-Hsp27, indicating a direct interaction of Hsp27 with cTnI and cTnT. Next, we determined interacting domains of cTnI and cTnT with Hsp27 by His-pull down experiment. Mutant cTnI with COOH-terminal deletion cTnI1-199 (His-cTnI{Delta}C) or mutant cTnT with NH2-terminal deletion cTnT72-291 (His-cTnT{Delta}N, Figure 6A) were expressed as His fusion proteins. Immobilized His-cTnI{Delta}C or His-cTnT{Delta}N failed to pull down GST-Hsp27 (Figure 6B). Thus, cTnI COOH-terminus and cTnT NH2-terminus are required for the interaction with Hsp27.


Figure 6
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  		Figure 6 Interaction of Hsp27 with cTnI or cTnT in vitro and interacting regions of cTnI/ cTnT (A and B). (A) Schematic representation of His-tagged wild-type cTnI (His-cTnI), His-tagged mutant cTnI with the C-terminal deletion (His-cTnI{Delta}C, 1-199), His-tagged wild-type cTnT (His-cTnT), and His-tagged mutant cTnT with the N-terminal deletion (His-cTnT{Delta}N, 72-291) fusion proteins. (B) Determination of interaction and interacting regions between Hsp27 and cTnI or cTnT by His pull-down assay. Purified GST-Hsp27 fusion protein was incubated with His fusion proteins of wild-type and mutant cTnI or cTnT. Proteins pulled down by His-bind resins were analysed by immunoblotting with anti-Hsp27 antibody. Original lysates were used for a positive immunoblotting control (input) and His was used as a negative control. (C) Interaction of µ-calpain with cTnI or cTnT in cardiomycoytes. The lysates, from simulated I/R (20-min/30-min) myocytes after 48 h transfection of Ad.GFP and Ad.Hsp27wt, were subjected to immunoprecipitations. The cell lysates were incubated with agarose beads coated with normal goat IgG or mouse IgG as negative control, and anti-cTnI (left) or anti-cTnT (right) antibodies. The interaction of µ-calpain with cTnI or cTnT in the immunoprecipitates was analysed by immunoblotting with anti-µ-calpain with anti-cTnI (left panel) or anti-cTnT (right panel) antibodies. Similar results were obtained in at least three independent experiments.

 
To further explore mechanisms underlying the protection of Hsp27 against cTnI and cTnT degradation, we examine whether Hsp27 affects the interaction between the protease µ-calpain and cTnI or cTnT. µ-Calpain is a calcium-activated protease that appears to play a key role in myocardial dysfunction following I/R by degrading contractile proteins, such as cTnI and cTnT.7,10 Co-immunoprecipitation experiments were performed using lysates harvested from Ad.GFP and Ad.Hsp27wt myocytes subjected to simulated I/R. Immunoblotting with anti-cTnI or anti-cTnT antibody revealed the same amount of cTnI or cTnT in the immunoprecipitates (Figure 6C, bottom bands). However, the amount of µ-calpain pulled down with anti-cTnI or anti-cTnT was reduced in Hsp27 overexpressing cells compared with that in GFP cells (Figure 6C, upper bands). Thus, Hsp27 probably prevents cTnI and cTnT degradation via reducing the interaction of µ-calpain with cTnI and cTnT.


   4. Discussion
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 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
Supplementary material
 Funding
 References
 
Our results demonstrate that (i) increasing Hsp27 expression by gene transfer in vivo improves recovery of post-ischaemic contractile function; (ii) such protection is associated with the improvement of cell contractions and myofilament Ca2+ responsiveness but not the [Ca2+]i; (iii) the improvement correlates with the prevention of I/R-induced cTnI and cTnT degradation; and (iv) Hsp27 can interact with cTnI at COOH-terminus and cTnT at NH2-terminus, which may prevent protease µ-calpain from degrading these proteins under I/R. These results confirm and extend previous findings indicating that Hsp27 plays an important role in regulation of contractile function during I/R and provide new insight into the mechanisms mediating the protective effects of Hsp27 in myofilament regulatory proteins.

4.1 Overexpression of Hsp27 by gene transfer improves post-ischaemic contractile function
I/R is recognized to induce contractile dysfunction. Preservation of myocardial function after I/R is crucial and depends on endogenous adaptive responses. Hsp27 is one particularly interesting small Hsp as it can protect cardiomyocytes against thermal, hypoxic, or I/R-induced cell death12,15,27 and heart infarction.13 We demonstrated that Hsp27 overexpression by in vivo gene delivery to both isolated myocytes and whole hearts improves post-ischaemic recovery of contractile function. This is consistent with the observation from transgenic mice that Hsp27 overexpression improves post-ischaemic contractile dysfunction in Langendorff perfused hearts.14 Our results suggest that increasing Hsp27 expression in the heart by gene delivery is a novel biological strategy to minimize myocardial contractile dysfunction and improve cell viability during I/R.

4.2 Hsp27 prevents I/R-induced depression in myofilament Ca2+ responsiveness
Cardiac contractility is regulated by alterations of [Ca2+]i, myofilament response to Ca2+, or both.2,3,32,33 In I/R myocytes, intracellular Ca2+ amplitude decreases significantly. Because the activator Ca2+ is mainly released via ryanodine receptors (RyRs) from sarcoplasmic reticulum (SR) and is taken up by SR Ca2+-ATPase (SERCA2),34 the inhibition could be explained by the decreased expression and function of RyRs and SERCA2 due to I/R.22,35,36 In addition, inhibited activity of Na+/Ca2+ exchange during ischaemia may also contribute to the decreased Ca2+ amplitude.22 Concomitantly, I/R myocytes show a significant decrease in the myofilament Ca2+ responsiveness, characterized by a more marked decrease in the cell shortening than that in the Ca2+ transients. Our observation of unchanged dynamics of intracellular Ca2+ transients in both pre-ischaemic and post-ischaemic phases by increasing Hsp27 expression suggests that protection of hsp27 against post-ischaemic contractile dysfunction may be associated with the regulation of the myofilament response to Ca2+ 2,16,33,37 rather than affect [Ca2+]i regulated by those Ca2+ handing proteins.

A novel finding here is that Hsp27 overexpression restores I/R-suppressed myofilament Ca2+ responsiveness. Earlier studies suggest that reduced myofilament Ca2+ responses account in part for I/R-induced contractile depression.3,33 This view is supported by the recent observation that specific enhancement of myofilament response to Ca2+ is able to attenuate I/R-induced contractile dysfunction.38 Our observations further confirmed that Hsp27 overexpression-attenuated deleterious effects of I/R on contractile function is associated with a specific improvement of myofilament response to Ca2+. The findings provide new insights into the mechanism of Hsp27-conferred protection on the contractile function and support the view that specific improvement of post-ischaemic myofilament Ca2+ responsiveness may be an important therapeutic approach for ischaemic heart diseases.38

4.3 Hsp27 prevents I/R-induced cTnI and cTnT degradation
One proposed mechanism for depressed contractile function and myofilament response to Ca2+ under I/R involves the specific degradation of myofilament proteins.2 Several studies have demonstrated that the dysfunction is correlated with partial proteolysis of cTnI and/or cTnT under mild or severe I/R in rat hearts4,9 or human left ventricular tissues.39 Increases in the severity of ischaemia result in an increase in the extent of cTnI degradation.4 We consistently detected cTnI and cTnT degradation in I/R myocytes as well as in global no-flow I/R hearts. Interestingly, we found for the first time that Hsp27 overexpression significantly attenuates I/R-induced degradation of cTnI and cTnT. Such degradation could be caused by the Ca2+-activated protease µ-calpain.5,7,40 I/R-induced proteolytic cleavage of the COOH-terminal of cTnI is associated with diminished maximum force-generating capacity9 and impairs diastolic function in the human myocardium.41 The restricted proteolytic truncation of cTnT72–291 during I/R appears to participate in the thin filament regulatory function7 and truncated cTnT77–289 shows a decrease of MgATPase activity in relaxed and maximal Ca2+-activated states, indicating that the NH2-terminal region of cTnT is essential for maximal activity of cardiac myofilaments.11 Recently, the variable region in the NH2-terminus of cTnT was found to contribute to the Ca2+ sensitivity of force development.42 This is supported by our results showing an association of cTnI and cTnT degradation under I/R with decreases of myofilament response to Ca2+ and contractility. Thus, degradation of the COOH-terminal of cTnI and the NH2-terminal of cTnT appears to contribute to the depressed myofilament Ca2+ responsiveness and contractile dysfunction. Furthermore, Hsp27 overexpression-prevented degradation of cTnI and cTnT under I/R is associated with the improvement of the post-ischaemic myofilament response to Ca2+ and contractility. Thus, prevention of degradation of cTnI and cTnT may constitute therapeutic targets for ischaemic heart diseases.10,38,43 Whether the recovery of post-ischaemic myofilament response to Ca2+ is also caused by the protection of Hsp27 on the other targets needs to be explored.

4.4 Mechanisms of Hsp27-mediated protection against I/R-induced cTnI and cTnT degradation
Another novel finding in the present study is the establishment of an interaction between Hsp27 and cTnI/cTnT at both pre-ischaemic and post-ischaemic status. Moreover, we demonstrated that the cTnI residues 200–211 at the COOH-terminus and the cTnT residues 1–71 at the NH2-terminus are necessary for this interaction. Such interaction may competitively prevent µ-calpain from binding to the cleavage region, and thereby protects against I/R-induced proteolytic cleavage of cTnI and cTnT. This is supported by our observation that Hsp27 overexpression attenuates the interaction between endogenous µ-calpain and cTnI or cTnT. Indeed, calpain activity is increased during myocardial ischaemia10 and proteolytic cleavage of the COOH-terminal of cTnI and the NH2-terminal of cTnT occurs under myocardial I/R.4,5,7,9 Ischaemia- or heat-shock-induced translocation of Hsp27 to Z bands or sarcomere in rodent hearts16,17 appears to be involved in the stabilization of myofilaments via interaction with their protease cleavage regions. Our findings provide new mechanisms underlying the cardioprotection of Hsp27 in modulating myofilaments. Further studies are required to elucidate whether the protection of Hsp27 on cTnT and cTnI is also related to enhance refolding or decreased misfolding of cTnT or cTnI.

In conclusion, we demonstrate here that Hsp27 gene transfer improves post-ischaemic recovery of contractile function in both isolated myocytes and hearts subjected to I/R without affecting baseline contraction. This protective effect is, at least partially, mediated by the recovery of myofilament response to Ca2+ during I/R, and more specifically, by prevention of I/R-induced cTnI and cTnT degradation via its interaction with the COOH-terminus of cTnI and the NH2-terminus of cTnT. Therefore, Hsp27 gene delivery may provide a potential therapeutic approach for ischaemic heart diseases.


   Supplementary material
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Supplementary material
Funding
 References
 
Supplementary material is available at Cardiovascular Research online.


   Funding
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Supplementary material
 Funding
References
 
This study was supported by Grants from National Natural Sciences Foundation of China (30393133, 30600210), and the Major State Basic Research Development Program of China (2006CB504106, 2007CB512100).


   Acknowledgements
 
We are grateful to Eileen Hickey and Lee Weber (University of Nevada) and Madhavi J. Rane (University of Louisville) for offering us plasmids.

Conflict of interest: none declared.


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Supplementary material
 Funding
 References
 

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