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Am J Physiol Heart Circ Physiol 289: H2519-H2525, 2005. First published August 12, 2005; doi:10.1152/ajpheart.00872.2004
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Effect of mutation of amino acids 246–251 (KRKHKK) in HSP72 on protein synthesis and recovery from hypoxic injury

M. R. Voss,4 S. Gupta,1 J. P. Stice,1 G. Baumgarten,5 L. Lu,1 J. M. Tristan,4 and A. A. Knowlton1,2,3,4

1Molecular and Cellular Cardiology and 2Department of Pharmacology and Toxicology, University of California, Davis and 3Department of Veterans Affairs Medical Center, Sacramento, California; 4Baylor College of Medicine, Houston, Texas; and 5Department of Anesthesiology and Intensive Care Medicine, University of Bonn, Bonn, Germany

Submitted 24 August 2004 ; accepted in final form 9 August 2005


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
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Heat shock protein (HSP)72, the inducible form of HSP70, protects cells against a variety of injuries, but underlying mechanisms are poorly defined. To investigate the protective effects of HSP72, multiple clones expressing wild-type (WT) HSP72 and two mutants with defective nucleolar and nuclear localization (M45 and 985A, respectively) were made with the tet-off system in C2C12 cells. Four different parameters of cell function/injury were examined after simulated ischemia: protein synthesis, polysome formation, DNA synthesis, and lactate dehydrogenase (LDH release). Overexpression of WT HSP72 was also compared to nontransfected C2C12 cells. As expected, overexpression of HSP72 protected against simulated ischemia and reoxygenation for all parameters. In contrast, both M45 and 985A showed abnormal protein synthesis and polysome formation, both after simulated ischemia and under control conditions. Total RNA was slightly reduced in M45 and 985A at baseline, but 1 h after hypoxia, RNA levels were protected in all clones but significantly decreased in nontransfected C2C12 cells. Clones expressing 985A had nuclear retention of mRNA, suggesting that HSP72 is needed for nuclear export of RNA. All clones, both WT and mutant, had protection of DNA synthesis compared to C2C12 cells, but 985A had greater release of LDH after injury than any other group. These results support a multifactoral protective effect of HSP72, some aspects dependent on nuclear localization with stress and some not. The protection of protein synthesis and polysome formation, and abnormalities in these with the mutants, support a role for HSP72 in these processes both in the normal cell and in injury.

heat shock proteins; necrosis; nuclear localization; nucleoli

HEAT SHOCK PROTEIN (HSP)72 s the major inducible form of HSP70, and it increases in response to heat, ischemia, hypoxia, and stretch among other stresses. Although HSP72 is widely recognized to protect the cell from injury, the exact cellular functions it protects remain incompletely defined. Membrane integrity, mitochondrial function, and indexes of apoptosis have been examined as possible points of protection by HSP72. These are primarily indexes of damage. Studies of transgenic mice overexpressing HSP72 have examined return of contractile function after ischemia, which is a global index of function (14, 20). However, investigations have not closely examined the role of HSP72 in the protection of normal cell functions. It is known that both in vivo and in vitro overexpression will protect cardiac myocytes from ischemia and that prevention of the endogenous increase in HSP72 will convert a mild injury to severe injury; however, the parameters of injury that have been studied have been rather general indexes of cell function and integrity, such as lactate dehydrogenase (LDH) release and propidium uptake. It is known that some, but not all, HSP72 translocates to the nucleus with stress. Previously we investigated (9) regulation of nuclear translocation of HSP72 and identified two mutants of HSP72's (inducible HSP70) nuclear localization signal, KRKHKK (amino acids 246–251): 985A (AAAHAA) and M45 (ARAHAK), which effect nuclear localization with stress. 985A has decreased nuclear accumulation after heat shock. M45 accumulates in the nucleus with stress but fails to move to the nucleolus. KRKHKK tagged to enhanced green fluorescent protein (EGFP) causes nucleolar concentration of EGFP. Despite loss of normal nuclear/nucleolar accumulation after stress, neither mutant affects viability immediately after heat shock.

HSP72 accumulates in the nucleus and nucleoli with stress and/or injury (8, 22, 27). It is thought that HSP72 protects the ribosomal synthesis machinery (1, 2); however, others have found that protection of translation is independent of rRNA synthesis (26). HSP72 associates with translating ribosomes after heat stress and has been found to preferentially bind the 40S subunit (4). We were interested in the importance of nuclear localization of HSP72 for the recovery of cellular function after hypoxia and how this compared with a general index of damage. Therefore, we created a series of clones overexpressing HSP72 under control of the tet-off system. We determined the effect of overexpression of wild-type (WT) HSP72, compared with the two mutants with abnormal nuclear/nucleolar localization, on recovery of DNA synthesis, RNA levels, polysome formation, and protein synthesis after hypoxia. We report our findings here.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
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C2C12 cells (American Type Culture Collection) were grown in DMEM supplemented with 10% FBS, 50 U penicillin, and 50 µg/ml streptomycin. pRcCMVHSP70, without the Flag epitope tag, was cut with BamHI, and the cDNA for human HSP72 was then subcloned into the BamHI site of p10-3 (9). Orientation was verified by digesting with HindIII. The SacII/ClaI sites in HSP72 were used to subclone the mutated regions for 985A and M45 into p10-HSP72. Early-passage cells were transfected, using lipofectamine (Invitrogen) simultaneously with pTRE-tTA (tet-off) and p10-HSP72, p10-HSP72985A, or p10-HSP72M45. The tet-off system was the gift of Prof. H. Bujard, University of Heidelberg, Heidelberg, Germany (6). Tetracycline (2 µg/ml) was used to repress gene expression. G418 (400 µg/ml) was used to select for transfected cells. After 2 wk, cells were replated at a light density. Clones were identified and propagated. G418 (200 µg/ml) was used to maintain selective pressure. Clones were examined microscopically, and clones with grossly abnormal morphology were discarded. At least two clones were obtained for each construct.

Western blot analysis. Western blot analysis was carried out as previously described (17). Blots were developed with a chemiluminescent system (Pierce).

Protein synthesis. Protein synthesis was measured with a modification of the method of Morgan and colleagues (15, 16). Cells were placed in medium supplemented with 0.4 mM L-phenylalanine for 1 h to equilibrate the cellular pool of phenylalanine. As described originally by Morgan, this equilibrates the intracellular pool of phenylalanine with the extracellular pool; thus intracellular phenylalanine concentrations can be predicted and new protein synthesis calculated. Then cells were incubated with 10 µCi of [3H]phenylalanine for 3 h. Cells were washed with PBS supplemented with 10 mM phenylalanine and then collected in isolation detergent (0.9% NaCl, 4 mM Tris·HCl, 0.02% SDS, 1.5 mM PMSF, pH 7.4). An aliquot of each cell lysate was TCA precipitated in duplicate, and the protein was collected with a Millipore filter apparatus with GF/C filters. Filters were dried, placed in scintillation vials with fluor, and counted for 5 min. Total protein was measured on each lysate with a bicinchoninic acid (BCA) assay (Pierce) as previously described (17). New protein synthesis (phenylalanine incorporation) was expressed as a function of total protein with the following formula: µmol Phe·µg protein–1·h–1 = [cpm (sample)/(200 µl·BCA µg/µl)]/[cpm(media)/(5 µl·0.4 µmol/µl)]·h–1, where cpm is counts per minute.

Pilot studies were done to determine how long labeling needed to be done to reproducibly detect change and be in a linear range. As shown in Fig. 1, 3 h of labeling resulted in a significant labeling of protein in normal cells, such that a loss of synthesis could be reproducibly detected and was in a linear range for protein synthesis. Further experiments were done to determine that 10 h of hypoxia was needed to reproducibly impair protein synthesis in C2C12 cells (data not shown).



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  		Fig. 1. Protein synthesis is linear from 1 to 6 h of labeling. Incorporation is shown as µmol phenylalanine/µg protein. *P < 0.05 vs. 1 h; **P < 0.05 vs. 1 and 3 h.

 


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  		Fig. 3. Protein synthesis as measured by incorporation of [3H]phenylalanine. *P < 0.05 vs. WT control, no hypoxia (i.e., baseline).

 
LDH release. LDH release was measured as an index of cell injury as previously described (23).

RNA studies: total RNA. Because 98% of cellular RNA is ribosomal, measurement of total RNA will reflect rRNA levels. RNA was isolated from the clones 1 h after hypoxia and from controls with RNAzol as previously described (17). In a second set of experiments, cells were separated into nuclear and cytoplasmic fractions as previously described (11). Total RNA was isolated and measured, and the nuclear-to-cytoplasmic ratio (N/C) was calculated. mRNA was then isolated from each fraction (Oligotex Direct mRNA Mini Kit; Qiagen) and measured. In a third set of experiments, RNA distribution between the nucleus and cytoplasm was examined by immunofluorescence microscopy. Cells were grown on coverslips, fixed in ice-cold methanol, and then stained with a probe for RNA (SYTO RNA select green fluorescent stain; Molecular Probes), incubated with LAP-1 antibody (1:100; Affinity BioReagents, Golden, CO), and developed with affinity-purified anti-mouse-IgG-Texas red (1:500; Abcam, Cambridge, MA). LAP-1 antibody allowed visualization of the nuclear membrane. Cells were examined by confocal microscopy (Zeiss), images were collected, and "stack" analysis was done at 0.4-µm intervals to visualize different layers of the cell.

DNA synthesis. Bromodeoxyuridine (BrdU) incorporation was determined with a kit (Boehringer Mannheim). Methods were as described by the manufacturer, except that cells were counterstained with methyl green at completion of the protocol. Cells were scored as BrdU negative or positive with an Olympus B60X without knowledge of treatment group.

Polysome analysis. Cells were collected 1 h after hypoxia. Controls and time 0 cells were collected 48 h after discontinuation of antibiotics in experiments done in conjunction with hypoxia experiments. Polysomes were isolated by the method of Davies and Abe (5) with slight modifications. Cells were homogenized in 5 vol of homogenization buffer [0.25 M sucrose, 5 mM mercaptoethanol, 500 µg/ml heparin in 1x HKM buffer (HKM buffer 5x: 0.25 M HEPES pH 7.4, 0.5 M potassium acetate, and 25 mM magnesium acetate)]. The homogenate was centrifuged at 12,000 g for 10 min at 4°C. The supernatant (200 µl) was layered on 15–60% sucrose gradient in 1x HKM buffer and centrifuged at 250,000 g for 4.5 h in a Beckman SW-40 rotor. The free polysome pellets were dissolved in HKM buffer and stored at –80°C until use or immediately diluted in the same buffer to a concentration of 150 A260 units/ml. Samples were collected with a Bio-Rad Econosystem chromatography system with a gradient monitor scanning at 254 nm.

Simulated ischemia. Cells were placed in ischemic buffer (in mM: 137 NaCl, 12 KCl, 0.49 MgCl2, 0.9 CaCl·2H2O, 4 HEPES, and 20 Na lactate, pH 6.2) and placed in an anaerobic chamber as previously described for 10 h (7). These conditions more closely simulate in vivo ischemia than hypoxia alone. In pilot studies it was determined that this treatment was sufficient to injure nontransfected C2C12 cells and to reduce protein synthesis. We were interested in whether the mutant constructs would be able to protect cellular functions against injury compared with WT HSP72. Nontransfected C2C12 cells were also studied for comparison. Cells were studied at time 0, i.e., baseline, no hypoxia, and 3, 6, and 24 h after simulated ischemia. For all experiments all antibiotics were stopped 48 h before cells were studied. Pilot studies were done to determine the time point for tetracycline withdrawal before study to have good expression levels of the constructs. As the experiments were performed over a period of 24 h, the antibiotics were discontinued in a staggered fashion so that all samples at time of analysis had been off antibiotics for the same number of hours.

Statistics. Results were compared by ANOVA followed by a Dunnett's test. For normalized data (BrdU) an ANOVA on ranks was used. Data were compared with untreated WT HSP72, because the effect of the mutations on the protective effects of HSP72 was the primary end point of these studies. Results are expressed as means ± SE.


   RESULTS
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All three groups of clones showed much higher expression of HSP72, as would be expected, compared with nontransfected C2C12 cells, as shown in Fig. 2. The tet-off system was selected for these experiments because we previously found that these cells will not maintain overexpression of HSP72 (A. A. Knowlton, unpublished observation). Treatment with penicillin and streptomycin had no effect on HSP72 expression in the C2C12 cells, although streptomycin has been thought to possibly induce HSP72. For all studies, multiple clones were used for each construct, and no significant variation was observed among the clones. WT and C2C12 cells had identical basal levels of protein synthesis (8.9 ± 0.5 and 8.9 ± 1.6 µM phenylalanine incorporation·µg protein–1·h–1, respectively). Both mutants showed reduced baseline protein synthesis as shown in Fig. 3 (5.9 ± 0.5 and 5.7 ± 0.4 µM phenylalanine incorporation·µg protein–1·h–1). Thus expression of either of the two mutant constructs in the absence of injury depressed protein synthesis. After simulated ischemia all groups had virtually the same low level of protein synthesis, but WT showed more rapid recovery. C2C12 cells did not have recovery of protein synthesis until 24 h. Both mutants returned to basal synthesis rates but remained significantly depressed compared with WT. There was no difference among clones of any of the constructs.



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  		Fig. 2. Representative Western blot showing expression of heat shock protein (HSP)72 in transfected and nontransfected C2C12 cells. A: C2C12 cells off penicillin-streptomycin for 48 h. Each lane represents a different set of cells. B: C2C12 cells treated with penicillin-streptomycin. Each lane represents a different clone. C: wild-type (WT) HSP72 clone. D: 985A clone. E: M45 clone A. F: M45 clone B. All were loaded at 50 µg protein/lane.

 
At baseline, DNA synthesis was the same for all groups. After simulated ischemia both mutants and WT showed no loss of DNA synthesis as measured by BrdU incorporation. As shown in Fig. 4, although levels of BrdU incorporation decreased at 3 h this was not significant. In contrast, C2C12 cells had significant depression of BrdU incorporation, and this did not recover until 24 h. Again, there was no difference among individual clones for any construct.



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  		Fig. 4. DNA synthesis as measured by bromodeoxyuridine (BrdU) incorporation, expressed as % of BrdU-positive cells. *P < 0.05 vs. WT baseline.

 
Polysome formation. Overexpression of WT HSP72 protected polysomes from hypoxia-reoxygenation compared with the nontransfected C2C12 cells (Fig. 5). Both M45 and 985A showed abnormal polysome distribution even under normal conditions, with few polysomes present, and had near total loss of polysomes after hypoxia-reoxygenation (see Fig. 5). Thus inhibition of protein synthesis was associated with loss of polysome formation.



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  		Fig. 5. A: polysome formation under normoxic conditions. B: after hypoxia. Clones as marked. Left to right is large to small; thus more ribosomes are present in polysomes on left (i.e., better formation of polysomes with multiple ribosomes). Clones expressing the 2 mutants show abnormal polysome formation, with little (M45) or no (985A) polysome formation even under control conditions. 80S is final peak on right. Each tracing is representative example from at least 3, and all clones were studied.

 
Total RNA. Because 98% of RNA in cell is ribosomal, total levels of RNA will reflect levels of rRNA. Total RNA was measured both in control cells and 1 h after hypoxia. As shown in Fig. 6A, at baseline there was less RNA in both mutants. After hypoxia, cells overexpressing WT HSP72 and mutants maintained RNA levels, whereas a significant drop in RNA occurred in the nontransfected C2C12 cells. Thus both the mutant constructs and WT protected RNA levels after hypoxia, although RNA levels for M45 and 985A were lower at baseline than in WT.



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  		Fig. 6. A: total RNA with and without hypoxia. Samples were collected 1 h after hypoxia. C, control, no hypoxia; H, hypoxia. *P < 0.05 vs. WT baseline, no hypoxia. B: nuclear-to-cytoplasmic ratio (N/C) of total RNA; n = 7–9/group. C: N/C for mRNA; n = 4–6/group. *P < 0.05 vs. all others.

 
HSP72 is a chaperone, and it folds and refolds molecules as they cross membranes. One possible mechanism for depressed protein synthesis and loss of polysome formation in the mutant clones was that nuclear HSP72 was needed for export of RNA. Total RNA N/C were calculated after isolating total RNA from nuclear and cytoplasmic fractions. As shown in Fig. 6B, 985A tended to have higher amounts of nuclear RNA, but this did not reach significance. In contrast, when mRNA was isolated from total RNA, mRNA N/C clearly showed retention of mRNA in the nucleus in 985A-expressing cells (Fig. 6C). The N/C for 985A was twice that of WT HSP72. Finally, confocal microscopy was used to examine the distribution of RNA in the clones. Anti-LAP-1 was used as a marker of the nuclear membrane. As shown in Fig. 7B, M45 had more prominent nucleoli than the other groups. At times, a large amount of RNA appeared to be present in the nucleus of M45, but this was not consistent. 985A had smaller nucleoli than in the other clones (Fig. 7C). Stack analysis (0.4 µm) showed no additional abnormalities (data not shown). Thus 985A, with abnormal nuclear localization, had retention of mRNA in its nuclei and small nucleoli on microscopy. In contrast, M45 had large nucleoli, but the distribution of RNA was otherwise normal.



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  		Fig. 7. Confocal images showing distribution of RNA (green, left) and LAP-1 (Texas red, center), a protein found in nuclear membrane. Merged images are shown at right. HSP72 clones as indicated. Magnification x630.

 
Release of LDH occurred in C2C12 cells and in 985A after simulated ischemia and to a lesser extent in M45 or WT, as shown in Fig. 8. Interestingly, 985A LDH release began earlier than in C2C12 cells and had not recovered by 24 h, whereas in C2C12 cells LDH release peaked at 6 h and returned to normal by 24 h.



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  		Fig. 8. Lactate dehydrogenase release after hypoxia and reoxygenation. *P < 0.05 vs. WT baseline, no hypoxia.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
HSP72 is known to concentrate in the nucleus and nucleolus with cellular injury (8, 22, 27). It is thought that HSP72 protects the ribosomal synthesis machinery (1,2), but others have found that protection of translation is independent of rRNA synthesis (25). We showed previously (9) that 985A reduced nuclear accumulation after heat shock. M45 has normal accumulation in the nucleus but fails to appear in the nucleoli. EGFP tagged with KRKHKK, when transiently transfected into Cos cells, localizes into the nucleoli (9). Both mutant proteins bind ATP, demonstrating preservation of function (9). The current study takes these observations further by dissecting the ability of these mutants to protect cellular functions after an injury.

Protein synthesis. Both mutants had depressed baseline protein synthesis but recovered to their baseline after hypoxia within the same time frame as WT HSP72. WT HSP72 had significant protection of protein synthesis compared to C2C12 cells, supporting the function of HSP72 in protecting protein synthesis. On the other hand, in the absence of injury, there was no difference in polysome formation or protein synthesis between WT, cells overexpressing HSP72, and nontransfected C2C12 cells. This is not unexpected, because HSP72 protects, but does not enhance basal cell function.

Total RNA content and distribution. Total RNA levels at baseline were quite similar among the four groups, although the mutant clones had slightly lower levels, a change that was statistically significant. Distinct differences were apparent in the distribution of mRNA, which was clearly concentrated in the nucleus for 985A compared with the other groups. Confocal studies demonstrated that M45-expressing cells had abnormally large nucleoli, whereas those of 985A-expressing cells were small compared with WT HSP72-expressing cells. This suggests that HSP72's localization to the nucleus and nucleoli is important for formation of nucleoli and export of mRNA.

The constitutive form of HSP70 (HSC70) is more highly expressed than the inducible HSP72 in nonstressed cells (10), and it is thought that HSC70 rather than HSP72 functions in the normal cell as a nuclear transport chaperone; however, HSC70 and HSP72 have very similar sequences, such that they may be interchangeable in some settings. It has been observed that overexpression of HSP72 suppresses expression of HSC70 (18, 24). Thus one possible explanation of the findings is that the overexpressed HSP72 inhibits the expression of HSC70 and then replaces it in key functions in the normal, noninjured cell. Thus some of the effects of mutant HSP72 on nuclear export of RNA may reflect substitution of HSP72 for the suppressed HSC70, rather than reflecting a primary function of HSP72.

Polysomes and rRNA. Ribosome assembly and polysome formation are necessary for protein synthesis. Both mutants showed abnormal polysome formation under normal conditions as well as after hypoxic injury. This supports the idea that ribosome formation, even under normal, baseline conditions, is abnormal in the presence of the mutants. Temperature-sensitive mutants of an Escherichia coli homolog of HSP70 impair ribosome assembly (1). Similarly, HSP72 is associated with the ribosome in the thermotolerant cell that has undergone a brief heat shock (2). Polysomes disassociate under conditions of ischemia and other similar injury (12, 19, 21). In the current experiments, overexpression of WT HSP72 was associated with preservation of polysomes and better recovery of protein synthesis than in the nontransfected C2C12 cells. The mutant clones had abnormal basal protein synthesis and returned to this level of depressed protein synthesis as quickly as WT clones. Others have reported that recovery of protein synthesis after heat shock is independent of ribosomal RNA synthesis based on the simultaneous recovery of RNA and protein synthesis after heat shock (25). These investigators did not evaluate total RNA levels. In the current study, total RNA levels were markedly decreased in the nontransfected C2C12 cells 1 h after hypoxia but were unchanged in WT HSP72 and the mutants. A small decrease in M45 total RNA was observed at baseline compared with WT HSP72 levels. The lack of nuclear or nucleolar localization had only a small effect on total RNA levels. This suggests that HSP72 protects the assembled ribosome in the cytosol. This is in contrast to the role of HSC70 in protein synthesis, which is to enable proper folding of nascent polypeptides (3).

It is also possible that HSP72 protects factors involved in the assembly of the translation machinery. Phosphorylation of eukaryotic initiation factor-2{alpha}, and thus inactivation, has been shown to be dependent on HSP90 and HSC70, but activation of this factor appears to be dependent on HSP70 (13). HSP72 has been found to be associated with the ribosomal subunits after heat shock in HepG2 cells, whereas HSC70 was associated with newly synthesized polypeptides (2–4). The protection of polysome formation by overexpression of WT HSP72 supports association of HSP72 with the ribosome rather than having an effect on rRNA synthesis.

Withdrawal of tetracycline was timed so that all experimental time points occurred at 48 h after tetracycline withdrawal. Thus nonhypoxic cells (time 0) had induced levels of the constructs. Even in this resting state both mutants prevented normal protein synthesis, which was seen with all of the mutant clones. In contrast, WT and nontransfected C2C12 cells had similar protein synthesis rates before hypoxia.

DNA synthesis. None of the clones had impaired DNA synthesis, but C2C12 cells showed significant depression of DNA synthesis after simulated ischemia, and this did not recover until 24 h. This occurred even though both mutants have impaired nuclear localization. These findings support other protective functions of HSP72 independent of nuclear localization of the protein. Overall protection of cellular functions outside the nucleus might be expected to result in more rapid resumption of DNA synthesis and cell division.

General cellular injury. The protective effect against membrane damage (release of LDH after hypoxia-reoxygenation) of the mutants differed. As expected, overexpression of WT HSP72 protected against injury, as manifested by decreased LDH release compared with control, nontransfected C2C12 cells; however, significant differences were observed between 985A and M45. Overexpression of 985A, which does not localize in the nucleus with stress but does bind to ATP [as we reported previously (9)], showed greater LDH release than nontransfected, control C2C12 cells. On the other hand, M45 provided protection similar to WT HSP72 against release of LDH with hypoxia-reoxygenation. This suggests that nuclear localization of HSP72 provides protection against injury independent of localization to the nucleoli.

The current studies provide further insight into the functions of HSP72. Complete loss of nuclear localization (mutant 985A) was associated with increased necrotic injury after hypoxia and reoxygenation (LDH release). Loss of nucleolar localization, but with preservation of nuclear localization (mutant M45), was associated with protection against necrosis but abnormal protein synthesis and polysome formation, which was also seen with mutant 985A. Abnormal protein synthesis and polysome formation is most likely secondary to inhibition of transport of mRNA to the cytosol for 985A; however, other factors cannot be completely excluded. Both WT and mutant constructs protected DNA synthesis compared with nontransfected C2C12 cells. These results suggest that HSP72 protects the translational machinery during injury and may contribute to its function under normal conditions, based on the abnormal polysome formation and protein synthesis depression seen with all the mutant clones.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Institutes of Health Grants AG-19327 (A. A. Knowlton) and HL-58515 (A. A. Knowlton) and by a Department of Veterans Affairs Merit Award (A. A. Knowlton).


   FOOTNOTES
 

Address for reprint requests and other correspondence: A. A. Knowlton, Molecular and Cellular Cardiology, Genomics and Biomedical Sciences Facility, Rm. 6317, Univ. of California, Davis, 451 East Health Sciences Way, Davis, CA 95616 (e-mail: aaknowlton{at}ucdavis.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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