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Transgene overexpression of B crystallin confers simultaneous protection against cardiomyocyte apoptosis and necrosis during myocardial ischemia and reperfusion

PARTHA S. RAY^*, JODY L. MARTIN, ERIC A. SWANSON, HAJIME OTANI^*, WOLFGANG H. DILLMANN and DIPAK K. DAS^*1

^* Cardiovascular Research Center, Department of Surgery, University of Connecticut Health Center, Farmington, Connecticut 06030-1110, USA; and
Department of Medicine, University of California at San Diego, San Diego, California, USA

¹Correspondence: Cardiovascular Division, Department of Surgery, 263 Farmington Ave., Farmington, CT 06030-1110, USA. E-mail : ddas{at}neuron.uchc.edu

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS Generation of {alpha}B... Northern and Western blot... Murine ex vivo isolated... Perfusion protocol for global... Measurement of malonaldehyde for... TUNEL assay for assessment... Measurement of infarct size Statistical analysis RESULTS DISCUSSION REFERENCES

 
We investigated whether enhanced expression of B crystallin, a stress-inducible molecular chaperone of the small heat shock family, can protect myocardial contractile apparatus against ischemia reperfusion (I/R) injury. Transgenic mice overexpressing B crystallin were generated using the 0.76 kb rat B crystallin cDNA cloned into a pCAGGS plasmid driven by a human cytomegalovirus expression system. Southern analysis confirmed transgene integration and Northern and Western blotting characterized expression (3.1-fold and 6.9-fold elevations in myocardial mRNA and protein levels, respectively). Extent of functional recovery over a 3 h reperfusion period following a 20 min ischemic period in transgenic and wild-type mouse hearts was assessed using an ex vivo work-performing heart preparation. The transgenic group displayed significantly higher values of DP at R45 min (29.14±1.9 mm Hg vs. 17.6±0.7 mm Hg), R60 min (31.56±1.7 mm Hg vs. 17.8±0.8 mm Hg), and R75 min (32.5±2.2 mm Hg vs. 16.9±0.9 mm Hg), and of dLVP/dt at R45 min (1740.2±111.5 mm Hg.s^-1 vs. 548.7±82.2 mm Hg.s^-1) and R60 min (1199.8±104.6 mm Hg.s^-1 vs. 466.9±61.1 mm Hg.s^-1). The transgenic group also displayed development of less oxidative stress, decreased extent of infarction, and attenuated cardiomyocyte apoptotic cell death. Transgene overexpression of B crystallin was therefore successful in diminishing the independent contributory effects of both necrosis and apoptosis on I/R-induced cell death.—Ray, P. S., Martin, J. L., Swanson, E. A., Otani, H., Dillmann, W. H., Das, D. K. Transgene overexpression of B crystallin confers simultaneous protection against cardiomyocyte apoptosis and necrosis during myocardial ischemia and reperfusion.

Key Words: B crystallin • transgenic • ischemia/reperfusion • oxidative stress

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS Generation of {alpha}B... Northern and Western blot... Murine ex vivo isolated... Perfusion protocol for global... Measurement of malonaldehyde for... TUNEL assay for assessment... Measurement of infarct size Statistical analysis RESULTS DISCUSSION REFERENCES

 
EVEN THOUGH TREMENDOUS progress has been made in our understanding of the pathophysiology of myocardial ischemia reperfusion (I/R) injury, the precise mechanisms underlying the progression of such cellular injury to ultimate cell death remain far from clear. Disturbances in calcium and lipid homeostasis, generation of reactive oxygen species, loss of high-energy phosphates, and the accumulation of detrimental catabolites have all been implicated and are still believed to play a crucial role in the pathogenesis of I/R injury (1 2 3) . A significant advance in the field of ischemia reperfusion research came with recent findings that demonstrated that necrosis and apoptosis are independent contributors to I/R-induced cell death (4) . Oxygen free radicals also play a significant role in I/R-induced cardiomyocyte apoptosis (5 , 6) .

The last decade has witnessed an explosive growth in research efforts to understand why cyclic episodes of short durations of ischemia, each followed by short durations of reperfusion, can protect the heart from the otherwise potentially lethal effects of a subsequent more severe ischemic insult (7 8 9) . This led to the development of the concept of ischemic preconditioning (IP). Subsequent research revealed that it is the myocardial adaptation to stress that comprises the fundamental basis of IP. Thus, a defined amount of any stress, including oxidative stress and heat shock, can protect the myocardium from suffering lethal ischemic injury if administered to the heart in the form of a preconditioning protocol prior to the ischemic duration of known lethal effect (10 , 11) . Several reports now indicate a role of heat shock protein (HSP) 70 in mediating such protective effects (12 13 14) . However, recent studies have challenged these findings with the observation that expression of HSP 70 may not be directly linked with the heat stress-mediated cardioprotection (14) .

The potential cardioprotective role of the small heat shock protein family has begun to receive attention only recently and came with the observation that phosphorylation of MAPKAP kinase 2 leading to the phosphorylation of HSP 27 plays a crucial role in IP (15) . This observation is further strengthened by the fact that HSP 27 is involved in cytoskeletal stabilization (16) and that cytoskeletal injury plays a crucial role in the pathogenesis of I/R injury (17) . Consistent with these findings is a recent report demonstrating that enhanced expression of either hsp 27 or B crystallin in cardiomyocytes results in decreased cytosolic enzyme release after simulated ischemia (18) . B crystallin is a member of the small HSP family that shares many properties with HSP 27. Recent studies have demonstrated that B crystallin is the most abundantly expressed stress protein in the heart (19) , and its production is induced by agents that promote the disassembly of microtubules (20) . It has also been demonstrated that B crystallin helps to preserve microtubular integrity in the face of simulated ischemia in cardiomyocytes (21) .

The aim of our study was to assess whether the cardioprotective effects of enhanced expression of B crystallin against cellular ischemic injury as suggested by in vitro studies are sufficient to preserve myocardial contractile function and reduce infarct size when challenged by an ischemic insult. Furthermore, we also sought to determine whether B crystallin plays a role in attenuating apoptotic cell death associated with myocardial I/R injury. In the present study, transgenic mice overexpressing B crystallin were generated and characterized. An ex vivo isolated murine working heart model was used to assess postischemic recovery of contractile function. Coronary perfusate malonaldehyde levels were measured to assess the extent of oxidative stress and TUNEL assay to assess the extent of myocardial apoptotic cell death was performed. Results suggest that transgene overexpression of B crystallin exerted considerable cardioprotection against I/R injury as evidenced by its ability to significantly preserve postischemic contractile function and reduce associated oxidative stress. In addition, it reduced the extent of infarction associated with such injury, attenuated cardiomyocyte apoptotic cell death, and thereby diminished the independent contributory effects of both necrosis and apoptosis on I/R-induced cell death.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS Generation of {alpha}B... Northern and Western blot... Murine ex vivo isolated... Perfusion protocol for global... Measurement of malonaldehyde for... TUNEL assay for assessment... Measurement of infarct size Statistical analysis RESULTS DISCUSSION REFERENCES

 
All animals used in this study received humane care in compliance with the principles of laboratory animal care formulated by the National Society for Medical Research and Guide for the Care and Use of Laboratory Animals prepared by the National Academy of Sciences and published by the National Institutes of Health (publication no. NIH 85–23, revised 1985).

	Generation of B crystallin transgenic mice

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS Generation of {alpha}B... Northern and Western blot... Murine ex vivo isolated... Perfusion protocol for global... Measurement of malonaldehyde for... TUNEL assay for assessment... Measurement of infarct size Statistical analysis RESULTS DISCUSSION REFERENCES

 
For construction of transgenic mice, a rat B crystallin transgene was cloned into pCAGGS plasmid. Expression of rat B crystallin cDNA was driven by a human cytomegalovirus immediate-early enhancer linked to the chicken ß-actin promoter, followed by its first exon and intron (Fig. 1 ). The rat B crystallin with its own stop codon and poly(A) signal is followed by a rabbit ß-globin poly(A) sequence. The mRNA transcript of the transgene therefore consists of the first exon of chicken ß-actin, which is transcribed but untranslated, followed by the B crystallin transgene. To isolate the expression unit of the B crystallin transgene, the plasmid was cleaved with SalI and BglII. Standard techniques were used to generate the transgenic mice (23) . In brief, the pronucleus of eggs from superovulated C3H mice crossed with C57/black 6 male mice were injected with 1–2 pl of the purified DNA fragment at a concentration of 2 µg/ml. This amount is equivalent to 200 to 400 copies of the transgenes. The injected eggs were then transferred into the oviduct of pseudopregnant BalbC mice. Litters were delivered after 20 days of gestation.

View larger version (17K): [in this window] [in a new window]   Figure 1. Schematic depiction of the B crystallin transgene fragment used to generate B crystallin transgenic mice. The human cytomegalovirus immediate-early enhancer (CMVIE enhancer) is followed by the chicken ß-actin promoter with the first transcribed but untranslated exon, followed by the first chicken ß-actin intron. The rat B crystallin cDNA is followed by rabbit B globin poly(A) donor. Restriction enzyme sites and probes used for Southern and Northern hybridization are indicated.

To determine transgene integration, genomic DNA was extracted from the tails of 3-wk-old mice and subjected to Southern blot analysis. The tail DNA was digested with ApaI and PstI, resolved on an agarose gel, transferred to a nylon membrane, and hybridized to a ³²P-labeled transgene-specific probe that corresponds to the first chicken ß-actin intron (Fig. 1) . ApaI cuts in the chicken ß-actin intron and PstI cuts 600 bp into the B crystallin cDNA generating a 1.4 kb DNA fragment. The probe is presented by a 0.75 kb ApaI/XbaI digest releasing a fragment of the chicken ß-actin intron that specifically hybridizes to the transgene. From 44 founder animals, 4 transgene positive mice were identified. Two of the founders were bred into lines B1 and B2. The B1 mice had the highest expression levels and were used primarily for the reported studies.

	Northern and Western blot analysis

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS Generation of {alpha}B... Northern and Western blot... Murine ex vivo isolated... Perfusion protocol for global... Measurement of malonaldehyde for... TUNEL assay for assessment... Measurement of infarct size Statistical analysis RESULTS DISCUSSION REFERENCES

 
Isolation of RNA was performed as described by Chomezynski et al. (22) . For electrophoresis, Northern transfer, and hybridization, standard protocols were used (23) . As a loading standard, GAPDH mRNA was used. The Northern blots were densitometrically scanned and the ratio of B crystallin over GAPDH mRNA was expressed. For Western blot analysis, protein extracts were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrotransferred onto nitrocellulose using a semidry electrophoresis apparatus (Bio-Rad, Hercules, Calif.). The nitrocellulose membranes were reacted with polyclonal antibodies directed against B crystallin or rodent HSP25 (StressGen, Victoria, B.C., Canada). Protein loading was normalized by using monoclonal antibodies to constitutive HSP C70 (StressGen) and sarcomeric actin (Sigma, St. Louis, Mo.). Blots were then processed using the Enhanced Chemiluminescent kit (Amersham, Arlington Heights, Ill.). Protein and RNA results were quantitated by scanning the images. The images were then digitized and analyzed with the NIH image public domain image analysis software program.

	Murine ex vivo isolated work-performing heart model

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS Generation of {alpha}B... Northern and Western blot... Murine ex vivo isolated... Perfusion protocol for global... Measurement of malonaldehyde for... TUNEL assay for assessment... Measurement of infarct size Statistical analysis RESULTS DISCUSSION REFERENCES

 
Wild-type or transgenic B6C3 female mice (n=15) weighing 25–30 g were anesthetized with sodium pentobarbital (200 mg/kg b.w. intraperitoneal (i.p.) injection, Abbott Laboratories, North Chicago, Ill.) and anticoagulated with heparin sodium (500 U/kg b.w. i.p. injection, Elkins-Sinn Inc., Cherry Hill, N.J.). After ensuring adequate depth of anesthesia, a thoracotomy was performed, and the heart was excised and immediately immersed in ice-cold perfusion buffer. The aortic arch was quickly isolated and incised. The aorta was cannulated and retrograde perfusion in the Langendorff mode through the aortic cannula was initiated at a perfusion pressure of 60 mm Hg. The perfusion buffer used in this study consisted of a modified Krebs-Henseleit bicarbonate buffer [KHB: composed of (in mM) 118 NaCl, 4.7 KCl, 1.2 MgSO₄, 25 NaHCO₃, 10 glucose, and 1.7 CaCl₂, gassed with 95% O2:5% CO₂, filtered through a 5 µm filter to remove any particulate contaminants, pH 7.4] that was maintained at a constant temperature of 37°C and gassed continuously for the duration of the experiment.

Next, the pulmonary venous opening was located. The beveled sharp end of a PE-50 catheter was inserted through this opening and passed through the left-atrium, mitral valve and pushed out the apex so that its fluted end remained in the left ventricular lumen. This catheter was connected to a pressure transducer to monitor left ventricular pressure. The pulmonary venous opening was then cannulated with a short piece of PE-50 tubing and connected to the left atrial inflow line. Side arms of the aortic and left atrial cannulas were connected to pressure transducers to permit continuous monitoring of the respective chamber pressures. At this time, perfusion was switched to the working heart mode by stopping retrograde perfusion through the aortic cannula, opening the aortic outflow line and initiating antegrade perfusion through the left atrial inflow line, in that order. A bubble trap/pressure chamber containing an air cushion of 1.5 ml was located in the aortic output line to allow for adequate elastic recoil. The method followed was essentially the same as that described previously (24 , 25) except for a slight modification in that for our model we used a fixed preload of 15 mm Hg and a fixed afterload of 50 mm Hg maintained by hydrostatic columns. Left ventricular, aortic, and left atrial pressures (LVP, AOP, and LAP, respectively) were monitored, analyzed, and recorded in real time using the Digimed data acquisition and analysis system (Micromed, Louisville, Ky.). Heart rate (HR), left ventricular (DP) defined as the difference of the maximum, and minimum left ventricular pressures and maximum positive dLVP/dt were all derived or calculated from the continuously obtained left ventricular pressure signal.

	Perfusion protocol for global ischemia reperfusion

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS Generation of {alpha}B... Northern and Western blot... Murine ex vivo isolated... Perfusion protocol for global... Measurement of malonaldehyde for... TUNEL assay for assessment... Measurement of infarct size Statistical analysis RESULTS DISCUSSION REFERENCES

 
The preparation was allowed to attain steady-state values of functional parameters. After a 30 min stabilization period, baseline functional data were recorded and perfusate samples were collected. The hearts were then subjected to 20 min of global ischemia by arresting buffer flow through the left atrial cannula. Reperfusion for the first 10 min began in the retrograde Langendorff mode to allow for postischemic stabilization. Thereafter, perfusion was switched to the antegrade working heart mode as described above to allow for monitoring of functional parameters, which were recorded at 15, 30, 45, 60, 75, 90, 120, 150, and 180 min of reperfusion. Perfusate samples were also collected at the above time points and at 1, 3, 7, and 10 min of reperfusion to allow for assessment of oxidative stress in the immediate-early reperfusion period, when typically it is at its greatest.

At the end of the 3 h reperfusion period, hearts were removed from the apparatus and the atrial tissue was dissected away. The ventricles were either fixed in 10% buffered formalin or immersed in 1% triphenyl tetrazolium chloride (TTC) solution in phosphate buffer (Na₂HPO₄ 88 mM, NaH₂PO₄ 1.8 mM) at 37°C for 10 min. Hearts kept in formalin were later embedded in paraffin following standard procedures, and 3 µM thick transverse ventricular sections were obtained to perform TUNEL assays for the detection of apoptosis. Hearts immersed in TTC were stored at -70°C for later processing in infarct size calculations.

Six hearts from the transgenic group and seven hearts from the control group were excluded from the study on account of low baseline cardiac contractile performance after the 30 min stabilization period. Three hearts from the transgenic group and two hearts from the control group were also excluded as they did not maintain recordable contractile function until the end of the 3 h reperfusion period.

	Measurement of malonaldehyde for assessment of oxidative stress

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS Generation of {alpha}B... Northern and Western blot... Murine ex vivo isolated... Perfusion protocol for global... Measurement of malonaldehyde for... TUNEL assay for assessment... Measurement of infarct size Statistical analysis RESULTS DISCUSSION REFERENCES

 
Malonaldehyde (MDA) was assayed as described (26) to monitor the development of oxidative stress during ischemia reperfusion. Coronary perfusates were collected at the time of recording baseline functional parameters and thereafter at 1, 3, 5, 7, 15, 30, 60, 90, 120, 150, and 180 min into reperfusion for the measurement of MDA. MDA in the collected coronary perfusate samples was derivatized using 2,4-dinitrophenylhydrazine (DNPH); 1 ml of perfusate was added to 0.1 ml of DNPH reagent (310 mg DNPH in 100 ml 2N HCl, 1.56 mmol DNPH) in a 20 ml Teflon^TM lined screw-capped test tube, contents were vortexed, and 10 ml of pentane was added prior to intermittent rocking for 30 min. The aqueous phase was extracted three times with pentane, blown down with N₂, and reconstituted in 200 µl of acetonitrile. Aliquots of 25 µl in acetonitrile were injected onto a Beckman Ultrasphere C₁₈ (3 mm) column in a Waters HPLC (Waters Corp., Milford, Mass.). The products were eluted isocratically with a mobile phase containing acetonitrile-H₂ 0-CH₃COOH (34:66:0.1, v/v/v) and detected at three different wavelengths of 307 nm, 325 nm, and 356 nm. The peak for malonaldehyde was identified by co-chromatography with a DNPH derivative of the authentic standard, peak addition, comparison of the UV patterns of absorption at the three wavelengths, and gas chromatography-mass spectrometry. The amount of MDA was quantitated by performing peak area analysis using the Maxima software program (Waters) and expressed in pmol/ml.

	TUNEL assay for assessment of apoptotic cell death

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS Generation of {alpha}B... Northern and Western blot... Murine ex vivo isolated... Perfusion protocol for global... Measurement of malonaldehyde for... TUNEL assay for assessment... Measurement of infarct size Statistical analysis RESULTS DISCUSSION REFERENCES

 
Immunohistochemical detection of apoptotic cells was carried out using TUNEL in which residues of digoxigenin-labeled dUTP are catalytically incorporated into the DNA by terminal deoxynucleotidyl transferase11, an enzyme that catalyzes a template-independent addition of nucleotide triphosphate to the 3'-OH ends of double- or single-stranded DNA. The incorporated nucleotide was incubated with a sheep polyclonal anti-digoxigenin antibody, followed by a FITC-conjugated rabbit anti-sheep IgG as a secondary antibody as described by the manufacturer (Apop Tag Plus, Oncor Inc., Gaithersburg, Md). The sections (n=3) were washed in phosphate-buffered saline (PBS) three times, blocked with normal rabbit serum, and incubated with mouse monoclonal antibody recognizing cardiac myosin heavy chain (Biogenesis Ltd., Bournemouth, U.K.), followed by staining with TRIRC-conjugated rabbit anti-mouse IgG (200:1 dilution, Dako Japan; Tokyo, Japan). The fluorescence staining was viewed with a confocal laser microscope (Olympus Co., Tokyo, Japan).

	Measurement of infarct size

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS Generation of {alpha}B... Northern and Western blot... Murine ex vivo isolated... Perfusion protocol for global... Measurement of malonaldehyde for... TUNEL assay for assessment... Measurement of infarct size Statistical analysis RESULTS DISCUSSION REFERENCES

 
Hearts to be used for infarct size calculations (n=3) were taken upon termination of the experiment, immersed in 1% triphenyl tetrazolium solution in PBS (Na₂HPO₄ 88 mM, NaH₂PO₄ 1.8 mM) for 10 min at 37°C, and stored at -70°C for later processing. Frozen hearts (including only ventricular tissue) were sliced transversely in a plane perpendicular to the apico-basal axis into 0.5 mm thick sections, blotted dry, placed between microscope slides, and scanned on a Hewlett-Packard Scanjet 5p single-pass flat bed scanner (Hewlett-Packard, Palo Alto, Calif.). Using the NIH Image 1.6.1 image processing software, each digitized image was subjected to equivalent degrees of background subtraction, brightness, and contrast enhancement for improved clarity and distinctness. Risk as well as infarct zones of each slice were traced and the respective areas were calculated in terms of pixels. The weight of each slice was then recorded to facilitate the expression of total and infarct masses of each slice in grams in order to remove the introduction of any errors due to nonuniformity of heart slice thickness. The individual risk masses and infarct masses of each slice were summed to obtain the risk and infarct masses for the whole heart. Infarct size was expressed as a percentage of the area at risk for any one heart.

	Statistical analysis

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS Generation of {alpha}B... Northern and Western blot... Murine ex vivo isolated... Perfusion protocol for global... Measurement of malonaldehyde for... TUNEL assay for assessment... Measurement of infarct size Statistical analysis RESULTS DISCUSSION REFERENCES

 
The values for myocardial functional parameters, MDA, risk, and infarct volumes, and infarct sizes were all expressed as the mean ± standard error of mean (SE). Differences between data were analyzed for significance by performing a Student’s t test. The results were considered significant if P<0.05.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS Generation of {alpha}B... Northern and Western blot... Murine ex vivo isolated... Perfusion protocol for global... Measurement of malonaldehyde for... TUNEL assay for assessment... Measurement of infarct size Statistical analysis RESULTS DISCUSSION REFERENCES

 
Characterization of transgenic mice
Southern blot analysis of mouse tail DNA with a transgene specific probe identified members of the B 1 line as shown in Fig. 2 . In the hearts of heterozygous B1 mice, B crystallin mRNA levels were 3.1-fold elevated above the level in control mice. There was a 6.9-fold increase at the protein level. Endogenous B crystallin expression in the rodent heart is relatively high, with published protein concentration estimates ranging up to 0.5% of total protein (27) . The Western blot of the mouse heart extracts (Fig. 3 ) demonstrates a basal level of closer to 0.1% of total protein in our studies. The level of transgene expression in the heart was quite high, especially in lane 1. In addition, significant protein expression occurred in skeletal muscle: the increase in protein levels was even more dramatic than that observed in the heart. A 4.8-fold increase was seen at the RNA level and a 7.9-fold increase at the protein level (Fig. 4 ). Significant increases in protein expression also occurred in the brain. In other organs like liver, kidney, and spleen, no B crystallin derived transgene expression could be identified. The level of HSP25 mRNA or protein was not significantly altered in the B crystallin transgenic mice in the heart or in the other organs where transgene expression occurred.

View larger version (55K): [in this window] [in a new window]   Figure 2. Southern blot of mouse tail DNA. Mouse tail DNA was digested with ApaI and PstI and hybridized with the transgene-specific probe that corresponds to the first chicken B actin intron generated by ApaI-XbaI digestion of the transgene. The (-) lane represents DNA from transgene negative littermates and the (+) lane contains DNA from a transgene positive B crystallin mouse. The 3rd lane contains a ApaI-PstI digest of the plasmid containing the B crystallin transgene.

View larger version (33K): [in this window] [in a new window]   Figure 3. Western blot analysis. Homogenates of mouse ventricles and skeletal muscle were electrophoresed, transferred, and probed with antibodies against actin and B crystallin. 100 micrograms of protein were loaded for each sample. The + or - signs indicate animals that tested positive or negative by Southern blot analysis. Recombinant B crystallin was loaded in the first lane for comparative purposes. In the heart and skeletal muscle of transgene positive mice, a significant increase in the amount of B crystallin occurs.

View larger version (27K): [in this window] [in a new window]   Figure 4. Northern blot. RNA samples were isolated from mouse heart ventricle and skeletal muscle and subsequently hybridized to 32P-labeled probes corresponding to B crystallin and GAPDH used to demonstrate loading levels. + or - indicates transgene positive or transgene negative animals as identified by Southern blots. In transgene positive animals, significant increases in the amount of B crystallin occurred in skeletal muscle and heart.

Recovery of myocardial contractile performance
There were no statistically significant differences between the control and transgenic groups with regard to baseline cardiac function. Evidence of improved postischemic recovery of myocardial contractile recovery, however, was clearly apparent in the transgenic group as early as R15, although statistically significant differences could only be reported from R30 onward.

The peak LVP (maximum systolic left ventricular pressure) recovered most dramatically in the transgenic group (Fig. 5 , top), being most prominent at R30 (57.22±3.99 mm Hg vs. 32.68±2.04 mm Hg in control group P<0.05), R45 (69.44±2.67 mm Hg vs. 29.45±2.36 mm in control group, P<0.001), and R60 (60.3±3.19 mm Hg vs. 26.0±1.17 mm Hg in control group P<0.05). This represented the percent of recoveries of 66.92%, 81.22%, and 70.53% at time points of R30, R45, and R60 compared to baseline peak LVP in the transgenic group whereas in the control group the corresponding percent recoveries were 41.88%, 37.74%, and 33.32% of baseline peak LVP at the same time points. The transgenic group continued to have consistently higher values of peak LVP until termination of the experiment, being significantly different when compared to values of the control group even at R180. Mean aortic pressure (Fig. 6 , bottom) closely followed the above trend, being significantly higher in the transgenic group than in the control group at time points of R30, R60, and R75 (29.48±1.54 mm Hg vs. 19.73±1.65 mm Hg, 41.78±2.67 mm Hg vs. 17.38±1.32 mm Hg, and 36.48±3.44 mm Hg vs. 16.43±1.18 mm Hg, respectively). Heart rate (Fig. 7 , top) was preserved closer to baseline values in the transgenic group for the entire experiment. This was especially apparent at R60 (306.6±11.71 beats/min vs. 203.83±4.60 beats/min in the control group).

View larger version (52K): [in this window] [in a new window]   Figure 5. Recovery of peak left ventricular pressure (top) and left ventricular developed pressure (bottom) during a 180 min reperfusion period after 20 min of global ischemia in isolated working mouse hearts. and indicate performance in control and B crystallin transgenic mouse hearts, respectively. *P<0.05, **P<0.001.

View larger version (48K): [in this window] [in a new window]   Figure 6. Recovery of positive dLVP/dt (top) and mean aortic pressure (bottom) during 180 min reperfusion period after 20 min of global ischemia in isolated working mouse hearts. and indicate performance in control and B crystallin transgenic mouse hearts, respectively. *P<0.05.

View larger version (49K): [in this window] [in a new window]   Figure 7. Recovery of heart rate (top) and amount of malonaldehyde (MDA) formation (bottom) during 180 min reperfusion period after 20 min of global ischemia in isolated working mouse hearts. and indicate values for control and B crystallin transgenic mouse hearts, respectively. *P<0.05 MDA formation was most prominent in the control group after 7 min of reperfusion.

The trends observed with respect to left ventricular developed pressure (Fig. 5 , bottom) and positive dLVP/dt (Fig. 6 , top) were, however, different. The transgenic group did display considerable postischemic recovery of these parameters up until the R60 time point, but was unable to maintain such recovery thereafter. Significantly different values for DP were observed at R45 (29.14±1.90 mm Hg transgenic vs. 17.6±0.66 mm Hg control), R60 (31.56±1.65 mm Hg transgenic vs. 17.75±0.84 mm Hg control), and R75 (32.5±2.21 mm Hg transgenic vs. 16.95±0.88 mm Hg control). Significant differences in values of positive dLVP/dt were demonstrable at R45 (1740.18±111.51 mm Hg/s transgenic vs. 548.73±82.23 mm Hg/s control) and at R60 (1199.82±104.57 mm Hg/s transgenic vs. 466.85±61.11 mm Hg/s control).

MDA formation
Production of MDA is an index of the occurrence of lipid peroxidation and the development of oxidative stress. Coronary perfusate MDA levels were found to be significantly reduced in the transgenic group as compared to the control group (Fig. 7 , bottom), the difference being especially apparent at time points of R1 (45.57±7.05 pmol/ml vs. 72.79±8.96 pmol/ml), R7 (43.39±4.17 pmol/ml vs. 161.67±23.11 pmol/ml), and R10 (39.03±4.56 pmol/ml vs. 84.36±5.82 pmol/ml).

Infarct size
Infarct size (percent infarct of total area at risk) was noticeably reduced in the transgenic group as compared to the control group (35.41±2.95% vs. 18.59±0.89%) as shown in Fig. 8 .

View larger version (53K): [in this window] [in a new window]   Figure 8. Representative scanned images of mouse heart ventricular sections stained with triphenyl tetrazolium chloride (TTC) to demonstrate extent of myocardial infarction after 20 min of global ischemia and 180 min of reperfusion. Tissue stained positive appears red and represents viable myocardium. Myocardium that does not stain positive appears yellow and represents infarct. Infarct size is expressed as percent infarct mass of risk mass calculated in grams. and indicate values for control and B crystallin transgenic mouse hearts, respectively.*P<0.05

Apoptotic cell death
Control hearts displayed massive apoptosis as detected with TUNEL in both cardiomyocytes and noncardiomyocytes. As stated in Materials and Methods, we used double antibody to specifically stain cardiomyocytes. Thus, in conjunction with TUNEL, we used monoclonal antibody recognizing cardiac myosin heavy chain, followed by staining with TRIRC-conjugated rabbit anti-mouse IgG. The results shown in Fig. 9A , C demonstrate increased apoptotic cardiomyocytes in the wild-type ischemic reperfused hearts. Apoptotic cardiomyocytes were rarely observed in B crystallin transgenic mouse hearts (Fig. 9B , D ).

View larger version (162K): [in this window] [in a new window]   Figure 9. Double immunofluorescent staining used to demonstrate cardiac myosin heavy chain (red) and apoptotic nuclei (yellow) using TUNEL in mouse hearts subjected to 20 min of global ischemia and 180 min of reperfusion. A, C) Images of control hearts; B, D) images of B crystallin transgenic hearts. Original magnification 600x.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS Generation of {alpha}B... Northern and Western blot... Murine ex vivo isolated... Perfusion protocol for global... Measurement of malonaldehyde for... TUNEL assay for assessment... Measurement of infarct size Statistical analysis RESULTS DISCUSSION REFERENCES

 
The results of our study documented that transgenic mice overexpressing B crystallin are resistant to myocardial ischemic reperfusion injury. Using the isolated working mouse heart preparation, we were able to show that the mouse hearts overexpressing B crystallin had improved postischemic recovery of left ventricular function compared to wild-type mouse hearts. These observations were further supported by results that revealed that transgenic mouse hearts had reduced infarct size and lower incidence of cardiomyocyte apoptosis compared to wild-type hearts.

The crystallins (A and B) are predominantly present in the vertebrate lens and are members of the small heat shock protein family (24) . Whereas A crystallin is predominantly expressed in lens, B crystallin mRNA and protein have been found to occur in high levels in heart, lung, skeletal muscle, and kidney (28) . In heart, B crystallin is bound to myocardial cytoskeletal proteins (29) , suggesting its role in cytoskeletal stabilization. Although the actual physiological function of B crystallin remains unclear, this small heat shock protein (25–27 kDa family) is known to function as a molecular chaperone in protein biosynthesis to facilitate protein folding and translocation (30) , and undergoes posttranslational modifications (autokinase activity) (31) . B crystallin also has a role in development and differentiation of striated muscle lineages (32) .

B crystallin expression can be induced in response to stress stimuli such as heat shock and oxidative stress (33 , 34) . Evidence is rapidly accumulating to support the protective effects of B crystallin against noxious stresses like hyperthermia and environmental stress. For example, ectopic expression of B crystallin has been found to render NIH 3T3 cells thermoresistant (35) as well as glioma cells through stabilization of cytoskeletal structure (36) . In ischemic rats, B crystallin was found to bind with the myofibrils, suggesting its cardioprotective role (29) . Recovery from exercise was associated with specific transient changes in the expression of B crystallin, which suggests that this heat shock protein may have specific roles in the remodeling process evoked by repeated bouts of contractile activity (37) . B crystallin and HSP 25 have been found to be associated with sarcomeric structures directly after heat shock, indicating a cytoskeletal protective function (38) .

Recent studies point to the involvement of B crystallin in mediating cellular protection against myocardial ischemia reperfusion injury. Ischemia was found to cause a rapid redistribution of B crystallin from the cytosolic pool to intercalated disks and Z lines of the myofibrils, which suggests its role in the protection of myocardial contractile apparatus against ischemia reperfusion injury (39) . This study showed that ischemia-induced phosphorylation of cardiac B crystallin is accompanied by a complete shift of this protein from the soluble to insoluble fraction, followed by its translocation to intercalated disks and myofibrillar Z lines, two cytoskeletal structures known to become destabilized during prolonged ischemia. An increased expression of B crystallin through an adenovirus vector system protected the adult cardiomyocytes against injury mediated by simulated ischemia (18) . In another related study, the same investigators found that microtubular integrity was significantly better preserved after ischemia in cells overexpressing B crystallin (40) . The results of the present study demonstrating improved postischemic ventricular contractile function are consistent with these previous reports.

Our study further documented that hearts of the transgenic mice overexpressing B crystallin had significantly reduced infarct size. Recent studies indicate that necrosis and apoptosis are two independent contributors of myocardial infarction (4) . Reperfusion of ischemic myocardium has been found to contain a significant number of apoptotic cardiomyocytes (41) . However, after 180 min of reperfusion it is unlikely that apoptotic cells will be TTC positive (red stain indicating viability). Therefore, it is a realistic contention that TTC will not stain either necrotic or apoptotic cells at 180 min reperfusion. Therefore, since there are two contributors to TTC nonstaining, infarct size (TTC nonstained area) should be greater in the control group. If an experimental intervention reduces only necrotic cell death, infarct size in the experimental group should be less than that observed in the control group, but not by as much as in a group where the experimental intervention reduces both contributors to TTC nonstaining: necrosis and apoptosis. This explains why there was a large difference in the observed infarct sizes between the two groups in our study and supports our contention that overexpression of B crystalline confers simultaneous protection against both cardiomyocyte apoptosis and necrosis.

The ex vivo isolated work-performing heart model was used primarily to detect differences in cardiac contractile function and cannot be expected to reveal hemodynamic data comparable to that which might be obtained from an in vivo model. The main advantage of the ex vivo model is that it affords the investigator a means to study effects on cardiac contractile function independent of innate neurohumoral protective/compensatory mechanisms that stay intact in an in vivo system. Only after studies using an ex vivo model can one conclude that cardioprotective effects, if any, are primarily due to the experimental intervention under consideration. If only the in vivo system is used to assess such cardioprotection (even if enhanced postischemic ventricular contractile function is observed in such a study), it cannot reasonably be concluded that such cardioprotection is due solely to the experimental intervention and has no contribution from the aforementioned innate protective/compensatory mechanisms. For this reason we used the ex vivo model. Furthermore, there is deterioration over time in the contractile function of such an ex vivo heart preparation (regardless of whether we use mouse or rat heart) even under control conditions in the absence of an ischemic insult. It is therefore the accepted standard that cardioprotection in the form of enhanced contractile function, if any, can be reliably detected only in the early to midreperfusion times when using such a model.

This is why, in the late reperfusion period in an ex vivo isolated working heart model, the functional parameters measured do not necessarily parallel myocyte cell viability at 180 min reperfusion. We do not know whether there is greater apoptotic cell death occurring in the control hearts above and beyond the 40% infarction in control. Even if it is occurring in control hearts and is being prevented in the B crystallin transgenic hearts, this increased cell viability cannot be expected to be reflected in functional measurements so late in the reperfusion period in an ex vivo isolated working heart model due to the deficiencies in the model outlined above. The only way to confirm this is in an in vivo model, which can potentially constitute the subject of future studies.

Cardiomyocyte apoptosis appears to occur in a variety of mammalian species including mouse, rat, rabbit, and pig (42 43 44) . Reactive oxygen species appear to play a crucial role in cardiomyocytes apoptosis, because antioxidants such as superoxide dismutase or ebselen can reduce the number of apoptotic cells significantly (45) . In another related study, when subjected to transgenic mice overexpressing GSHPx-1 were found contain minimal number of apoptotic cardiomyocytes whereas the knockout mice devoid of any copy of GSHPx-1 contained significantly higher number of apoptotic cells compared to the wild-type mouse hearts (46) . The results of the present study showed very few apoptotic cardiomyocytes and nonmyocyte cells in transgenic mouse hearts subjected to ischemia/reperfusion compared with the wild-type mouse hearts.

Although molecular characterization of the B crystallin transgenic mice suggested expression primarily in parenchymal tissues such as cardiac myocytes and not diffusely in other tissues, the findings of the TUNEL assay suggested that such overexpression was successful in preventing apoptosis in both cardiac myocytes and nonmyocytes. Possible reasons might include the general reduction in reactive oxygen species/oxidative stress as evidenced by the reduced formation of MDA (thereby reducing the occurrence of apoptosis in both myocytes and nonmyocytes equally) or the reduced secretion from myocytes of a potential paracrine proapoptotic mediator. Although the reduction in oxidative is probably potentially responsible for the observation of reduced apoptotic cell death, the possibility of reducing the release of a potential paracrine proapoptotic mediator from myocytes cannot be ruled out. Nevertheless, we used double antibody to specifically stain cardiomyocytes. Thus, in conjunction with TUNEL, we used monoclonal antibody recognizing cardiac myosin heavy chain, followed by staining with TRIRC-conjugated rabbit anti-mouse IgG. Thus, our results correctly reflect increase in apoptotic cardiomyocytes in the control ischemic reperfused hearts. We have certainly identified apoptotic cardiomyocytes and nonmyocytes in the control ischemic reperfused myocardium, but our interpretation is based on the results of apoptotic myocytes.

In concert, reduced amount of MDA formation was found in the transgenic mouse hearts overexpressing B crystallin subjected to ischemia and reperfusion. A large number of studies support that reactive oxygen species are generated during ischemia and reperfusion leading to the development of oxidative stress (47) . Our HR demonstrated that B crystallin overexpression in hearts reduces the formation of oxygen-derived free radicals during ischemia and reperfusion as evidenced by reduced MDA formation, a presumptive marker for oxidative stress (48) .

MDA levels were elevated in the hearts of wild-type mice subjected to ischemia reperfusion, the elevation being most marked in the early reperfusion period, which is consistent with what is generally observed in the working heart model (for both mouse and rat heart). Our data clearly demonstrate that MDA levels in the hearts of wild-type mice stay significantly elevated above baseline levels even after 180 min of reperfusion. Thus, the MDA findings suggest more than just short term protection/stunning of the myocardium since, in the B crystallin group, MDA levels were maintained at or near baseline levels throughout the course of the experiment.

In summary, our results demonstrated that transgenic mouse hearts overexpressing B crystallin are resistant to ischemic reperfusion injury compared to those from wild-type mice as evidenced by improved postischemic ventricular contractile function and reduced infarct size. Out results also showed for the first time that B crystallin possesses an antiapoptotic role because overexpression of this protein in the hearts prevented both myocytes and nonmyocytes apoptosis. Reduction of oxidative stress in the ischemic reperfused myocardium suggests that B crystallin may reduce cardiomyocyte apoptosis by virtue of its ability to reduce oxidative stress.

	ACKNOWLEDGMENTS

 
This study was supported in part by NIH HL 34360, HL 22559, HL 56803, and HL 49434.

Received for publication March 27, 2000. Revision received July 14, 2000.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS Generation of {alpha}B... Northern and Western blot... Murine ex vivo isolated... Perfusion protocol for global... Measurement of malonaldehyde for... TUNEL assay for assessment... Measurement of infarct size Statistical analysis RESULTS DISCUSSION REFERENCES

 

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