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The myotrophin/V-1 protein was originally found to be elevated in failing heart tissues and was described as an exogenously acting hypertrophy-inducing factor. However, several studies have proposed only intracellular functions for this protein. We investigated whether this protein is an exogenously acting hypertrophy-inducing trophin or an intracellular nuclear factor of kappa B (NFκB) regulatory protein. In the current report, immunofluorescence and cell fractionation studies showed that myotrophin is present only in the cytoplasm and is not actively released into the extracellular environment in response to hypertrophy-inducing stimuli. Moreover, in response to ischemia/reperfusion injury, an active release of myotrophin from adult rat myocardium was not observed. Furthermore, protein synthesis studies in rat neonatal myocytes indicated that exogenous myotrophin did not induce hypertrophy. On the other hand, myotrophin stimulates the generation of NFκB dimers in vitro and thus regulates the NFκB-mediated transcription in cardiac myocytes. Taken together, these studies suggest that myotrophin is a strictly cytosolic protein that regulates the NFκB-mediated transcriptional process.
The myotrophin/V-1 protein (Myo/V1) was first characterized in the mammalian heart,1 where it was called myotrophin,2–4 and in the rat cerebellum, where it was named V-1 protein.5 Myo/V1 is a 12-kD ankyrin repeat-containing protein6 that is found at elevated levels in tissues of failing human hearts3 and in the hearts of spontaneously hypertensive rats.2,4 A 2003 report7 indicated that Myo/V1 is also found at very low femtomolar levels in human plasma, both in healthy human subjects and in heart failure patients. However, in that study, the levels of this protein gradually decreased in human plasma during the progression of heart failure.7 Myo/V1 was found to be transiently up-regulated in the developing postnatal rat cerebellum5 and was later found ubiquitously expressed in all mammalian tissues.6–8 To date, however, the exact molecular function of Myo/V1 protein is still unknown.
Since its discovery, investigators have proposed various functions for the intracellular Myo/V1 protein.2,6,9–15 This protein was first described as a trophic growth-promoting protein (myotrophin) in rat neonatal myocytes, suggesting that it is an extracellular growth factor initiating cell surface signal transduction events that lead to cardiac hypertrophy.2,16 However, other reports indicated that, when added exogenously to cells, Myo/V1 does not exhibit this hypertrophy-inducing property in cardiac myocytes14 or in other mammalian cells.15 Later, another report from the same investigators11 suggested that Myo/V1 is a nuclear DNA binding protein capable of binding to NFκB-specific DNA binding sites. In contrast, our group9 proposed that Myo/V1 interacts with NFκB proteins only to generate different NFκB dimers in order to regulate gene transcription. Similarly, in other tissues, such as postnatal cerebellum, it was suggested that Myo/V1 might play an intracellular non-genomic transcriptional10 role in the granular cell differentiation process (NCBI-UniGene Mm. 4123).17 Furthermore, a “severe combined immunodeficiency complementing gene” (GenBank accession no: D78188), which rescued immune deficiency in SCID mouse cell lines, was also later found to code for Myo/V1 protein. Recently, a unique microRNA that targets Myo/V-1 mRNA in pancreatic β-cells has been implicated in insulin exocytosis.12,13 To sort out these conflicting functions of Myo/V1 protein, we examined first whether Myo/V1 acts as an extracellular signal in myocardial injury to induce hypertrophy. Later, we determined its cellular location, as well as its ability to localize outside of the cell during myocardial injury. We also analyzed the potential cytoplasmic and nuclear functions of Myo/V1 protein.
Cardiac myocytes from 2-day-old Sprague-Dawley rat pups (Charles River Laboratories, Inc.; Wilmington, Mass) were isolated as described previously.2 Cells were treated with rat recombinant Myo/V1 (0, 10, 20, and 500 ng; ~1–40 nM) for 24 hours in the presence of L-[2,3,4,5,6-3H]-phenylalanine (specific activity, 132 Ci/mmol; Amersham Biosciences, part of GE Healthcare; Piscataway, NJ). Phenylephrine (100 μM) was used as a positive control. [3H]-Phenylalanine (5 μCi/mL) was added to the cardiac myocyte culture medium, which contained unlabeled phenylalanine (0.36 mM). At the end of the treatment, cells were rinsed 3 times with ice-cold phosphate-buffered saline (PBS) and treated with 1 mL of 10% trichloroacetic acid (TCA) for 60 min at 4 °C to precipitate proteins. The precipitate was collected and washed 3 times with 95% ethanol, and the radioactivity was determined. Separately, an aliquot of each cell sample was used to measure the DNA content by the Hoechst dye method.18 [3H]-Phenylalanine incorporation results were normalized to nanograms of DNA/well to correct for cell number.
Rat neonatal myocytes were grown on glass cover slips, washed with 1 × PBS, and fixed in methanol/acetone solution (1:1) for 30 min at 4 °C. Myo/V1 was detected with anti-Myo/V1 antibody (polyclonal antibody raised against the full-length recombinant Myo/V1 protein; 1:500 dilution) followed by fluorescein isothiocyanate (FITC)-conjugated anti-rabbit IgG antibody (1:100 dilution), and confocal images were captured as described previously.9 Paraffin sections of normal adult feline myocardium were processed similarly.
To determine whether Myo/V1 was secreted, rat neonatal myocytes were treated with a diluent medium or phenylephrine (100 μM) for 24 hours, and the serum-free conditioned medium was collected for Myo/V1 analysis. Similarly, neonatal myocytes were infected with recombinant adenovirus that expresses Myo/V1 (AdMyo/V1) for 48 hours, and the conditioned medium was collected. AdMyo/V1 was constructed by recombinant DNA methods, as reported earlier.9 Soluble cytosolic extracts were also prepared from the myocyte samples by homogenizing the cells in 10 mM MES buffer (pH, 6.1), and the total proteins were concentrated by TCA precipitation as described before. Fifty μg of bovine serum albumin (BSA) was added as a carrier protein to the serum-free conditioned medium of unstimulated cells, phenylephrine-treated cells, and AdMyo/V1-infected cells, and the total proteins were TCA precipitated. Protein concentration was measured by bicinchoninic acid (BCA) method (Pierce Biotechnology, Inc.; Rockford, Ill), and equivalent amounts (20 μg) of cytosolic proteins and all of the proteins precipitated from conditioned medium were fractionated in 10% Tris-tricine SDS-PAGE, then Western transferred and immunoblotted to identify Myo/V1 protein. Myo/V1 from postnatal rat heart cytosolic extracts was used as a control to identify Myo/V1 protein.
As previously described,19 mice were injected intraperitoneally with heparin (10,000 U/kg; Sigma-Aldrich Co.; St. Louis, Mo) 30 min before the experiment. Later, after anesthesia with tribromoethanol (Avertin), mouse hearts were excised and placed in ice-cold Krebs-Henseleit (KH) buffer solution. A short perfusion cannula was inserted into the aortic root, and retrograde perfusion was initiated immediately at a constant perfusion pressure of 80 mmHg with KH buffer (pH, 7.4) containing the following (in mM): NaCl, 118; NaHCO3, 24; KCl, 4.7; KH2PO4, 1.2; MgSO4, 1.2; CaCl2, 2.2; and glucose, 10. The buffer was oxygenated with 95% oxygen and 5% carbon dioxide at 37 °C before use.
A hand-made balloon was inserted into the left ventricle (LV) through the mitral valve via an incision in the left atrium and connected to an ML844 pressure transducer (ADInstruments, Inc.; Colorado Springs, Colo). The balloon was inflated with saline to adjust enddiastolic pressure to 7 to 10 mmHg. After 30 min stabilization time, the hearts were subjected to 30 min of no perfusion pressure (0 mmHg) to generate acute ischemic injury, followed by 30 min of reperfusion (80 mmHg). Control hearts were perfused for 90 min at 80 mmHg. Functional data were recorded at 1 kHz on a data acquisition system (PowerLab/4SP, ADInstruments). Left ventricular function was measured by LV-developed pressure. The effusates (2 mL) were collected at 3-min intervals at the indicated times after ischemic injury, and creatine kinase and lactate release were measured to confirm the ischemic injury. Total creatine kinase activity was measured in the effusates using reagents from Diagnostic Chemicals Ltd. (Charlottetown, PEI, Canada) and used as an indicator of ischemic injury and as a positive control for protein released during ischemic injury. For quantifying Myo/V1 protein, 50 μg BSA was added as a carrier protein to the pooled effusates, and the total proteins were precipitated with 10% TCA. The TCA precipitate was washed with 90% ethanol, dissolved in SDS-sample buffer, and incubated at 37 °C for 30 min. Later, the proteins were fractionated in 10% Tris-tricine SDS-PAGE, and Western transferred and immunoblotted for Myo/V1 protein as described previously.9
The potential ability of Myo/V1 to bind to DNA containing a κB site and the effect of Myo/V1 on the native NFκB proteins in unstimulated myocytes were determined using NFκB gel-shift assays6,9,20; the substrate was the conventional κB-Igκ/HIV oligonucleotide (5′-AGTTGAGGGGACTTTCCCAGGC-3′ from Santa Cruz Biotechnology, Inc.; Santa Cruz, Calif). Recombinant Myo/V1 protein was purified from Escherichia coli as described previously.11 κB-DNA-protein binding reactions with increasing concentrations (1, 5, 10, and 20 μg) of recombinant Myo/V1, but without any nuclear extracts, were carried out as described previously.6,9 Nuclear extracts from unstimulated rat neonatal myocytes were prepared using NE-PER reagents (Pierce Biotechnology, Inc.) according to the manufacturer's protocol. κB-DNA-protein binding reactions with increasing concentrations of recombinant Myo/V1 plus myocyte nuclear extracts were carried out, also as described previously, and the κB-DNA complexes were fractionated in 4% PAGE and autoradiographed.6,9
The effect of Myo/V1 on the NFκB-mediated transcription process was measured in unstimulated myocytes using an NFκB luciferase reporter assay as described previously.9 The expression plasmids pκB-tk-luc, pcDNA3-AM1.1-Myo/V1, and pRSV-RelA(p65) were constructed earlier by recombinant DNA methods.9 Rat neonatal myocytes were plated at a density of 0.5 × 106 and transfected 1 day after cell isolation. The FuGENE™ 6 Transfection Reagent (Roche Diagnostics Corporation; Indianapolis, Ind) was used according to the manufacturer's protocol. Two sets of transfection experiments were performed: 1) pκB-tk-luc reporter plasmid (0.5 μg) was transfected in both the presence and the absence of pcDNA3-AM1.1-Myo/V1 plasmid (0.5 μg), which overexpresses Myo/V1 protein; and 2) pκB-tk-luc reporter and pcDNA3-AM1.1-Myo/V1 expression plasmids were transfected in the presence of pRSV-RelA(p65) expression plasmid (0.5 μg), which expresses the NFκB p65 protein. As a control, pcDNA3 vector was used in place of Myo/V1 expression vector. Forty-eight hours later, cells were harvested, lysed, and assayed for luciferase activity. Protein concentration was measured using the BCA method. To normalize for transfection efficiency differences, pSV-β-galactosidase plasmid (0.5 μg) was included in all the transfection samples. Luciferase activity was normalized for the protein content of each extract and with β-galactosidase activity. Transfection experiments were repeated 4 times.
All values are reported as mean ± the standard error of the mean unless otherwise indicated. Comparison of the groups was done by unpaired Student's t-test. All statistical analysis was performed with SigmaStat (SPSS Inc.; Chicago, Ill). Significance was accepted at P ≤0.05.
To determine the effects of Myo/V1 on cardiac myocyte growth, we measured the amount of de novo protein synthesis by [3H]-phenylalanine incorporation into neonatal myocyte proteins in the presence and absence of recombinant Myo/V1. Phenylephrine stimulation was used as a positive control. As shown in Figure 1, stimulation with 100 μM phenylephrine led to a significant increase (P=0.03) of protein synthesis by 30.2% ± 7.1%. However, a 24-hour treatment with increasing concentrations (10, 20, and 500 ng; ~1–40 nM) of recombinant Myo/V1 resulted in no significant increase (P=0.23) in protein synthesis. Although nonsignificant, the decrease in protein synthesis was unexpected and was not caused by toxicity of recombinant Myo/V1, since the DNA content and cell number were maintained between samples (data not shown). A similar decrease in protein synthesis has been reported to occur in Myo/V1 protein at 0.4 nM.8 Therefore, these studies suggest that exogenously added Myo/V1 does not induce neonatal cardiac myocyte growth as measured by protein synthesis and cell number.
Since protein synthesis experiments did not support an exogenous trophic role for Myo/V1 protein, we hypothesized that the cellular location of Myo/V1 might provide clues about the function of Myo/V1. Therefore, we sought to determine the cellular location of Myo/V1 in rat neonatal myocytes and non-myocytes and in adult feline myocardium by indirect immunofluorescence with use of confocal microscopy. Under basal conditions (Figs. 2A and 2B), Myo/V1 was observed predominantly in the cytoplasm in a widely diffuse pattern surrounding the nucleus. Phase contrast light fluorescence microscopy of adult feline myocardium (Fig. 2C) further confirmed that Myo/V1 was present mostly in the cytosol and in the perinuclear region. The absence of cell-surface fluorescence in these cells emphasized that Myo/V1 was not present on the plasma membrane.
To further clarify the localization of Myo/V1, we performed a Western blot analysis of conditioned medium and cytosolic extracts from neonatal cardiac myocytes under different physiological conditions. As shown in Figure 3, Myo/V1 was not detected in the conditioned medium from neonatal myocytes under basal conditions (lane NM-CM). Even cells treated with phenylephrine (lane NMP-CM), an agonist that induces myocyte hypertrophy, or cells infected with AdMyo/V1 (lane AdMyo-CM), which overexpresses Myo/V1, did not secrete Myo/V1 into the medium. On the contrary, we observed Myo/V1 only in the soluble cytosolic fractions (lanes NM-Cyt, NMP-Cyt, and AdMyo-Cyt) of these samples. In addition, Myo/V1 was not detected in the insoluble membrane fractions (data not shown) in these samples.
To further confirm these results in vivo, we tested whether Myo/V1 is released from the mammalian heart in response to ischemic injury. For this study, we subjected adult mouse hearts to ischemia/reperfusion injury using a Langendorff System (ADInstruments) and collected the effusates at different times after ischemic injury. The total proteins from these effusates were concentrated by TCA precipitation and analyzed for Myo/V1 by Western blot analysis. As shown in Figure 4A, none of the effusates from “no-flow” ischemic injury contained Myo/ V1 protein, while the same fractions did exhibit creatine kinase activity (Fig. 4B). In summary, cellular localization (Fig. 3) and ischemic/reperfusion injury studies (Fig. 4A) indicate that Myo/V1 is neither secreted nor present in the extracellular location under basal or injury conditions to the mammalian heart.
A previous report has also indicated that Myo/V1 has the capability to bind directly to DNA containing a κB consensus site and thus exert its functions.11 However, our earlier studies showed that Myo/V1 interacts only with NFκB proteins.6,9 Therefore, in this study, we reexamined whether purified recombinant Myo/V1 protein has the ability to bind to a κB consensus-DNA binding site directly. Recombinant Myo/V1 was purified from E. coli (Fig. 5A), and its ability to bind to κB site-containing DNA was tested in a gel-shift assay at different concentrations (Fig. 5B). Although Myo/V1 did not bind to κB site-containing DNA at all concentrations that we tested (lanes 2–5 in Fig. 5B with 1, 5, 10, and 20 μg of purified recombinant Myo/V1), the nuclear extracts from a positive control heart tissue (MHCsTNF),21 which overexpresses TNFα, exhibited κB-site binding activity (lane 1 in Fig. 5B). These data indicate that Myo/V1 is not a κB-DNA binding protein.
Previous studies from our laboratory have suggested that Myo/V1 splits the activated p50-p65 heterodimers into monomeric subunits and converts them to generate p50-p50 and p65-p65 homodimers.9 These observations raised the possibility that Myo/V1 might also be directly involved in generating NFκB dimers from inactive NFκB subunits. Therefore, we tested this possibility in unstimulated rat neonatal myocyte nuclear extracts using recombinant Myo/V1 protein. Recombinant Myo/V1 was added to nuclear extracts of unstimulated neonatal myocytes at various concentrations (0–100 ng), and its ability to generate NFκB dimers capable of binding to κB-DNA was studied. Figure 5C shows a representative result of a gel-shift assay. In the absence of recombinant Myo/V1 protein, the nuclear extracts of neonatal myocytes did not exhibit any κB-DNA binding activity under basal conditions, which indicated the absence of activated NFκB dimers (lane 1 in Fig. 5C). However, when additional recombinant Myo/V1 protein was added to the reaction, both p50-p50 and p65-p65 homodimers started to form (lane 2 in Fig. 5C), and its levels were correlated with the increasing concentrations of Myo/V1 (lanes 2–7 in Fig. 5C). p50 and p65 antibody supershifts (lanes 8 and 9 in Fig. 5C) further confirmed the nature of these κB-DNA-protein complexes as respective NFκB homodimers. Therefore, these studies suggest that Myo/V1 acts as a chaperone and contributes to NFκB subunit dimerization in neonatal cardiac myocytes.
In order to identify the functional relevance of Myo/V1 with respect to the generation of NFκB dimers, we first studied the role of Myo/V1 on the endogenous NFκB-mediated transcription process at the basal level. Neonatal myocytes were co-transfected with pκB-tk-luc and pcDNA-AM1.1-Myo/V1 expression vectors (Fig. 6A). Forty-eight hours later, luciferase reporter enzyme activity was measured. The results (Fig. 6A) showed that overexpression of Myo/V1 significantly (P ≤0.05) reduced the κB luciferase reporter activity from its basal levels. To further confirm these results, we studied the effect of Myo/V1 on the p65-stimulated NFκB transcription process (Fig. 6B). Neonatal myocytes were co-transfected with pκB-tk-luc and pRSV-RelA(p65) vectors in the presence and absence of pcDNA-AM1.1-Myo/V1 vector plasmid, and luciferase reporter enzyme activity was measured as described previously.9 As shown in Figure 6B, Myo/V1-overexpressing cells exhibited a significant (50%) inhibition (P ≤0.05) on p65-stimulated luciferase reporter activity. These results suggest that “inhibitory NFκB dimers” are activated during Myo/V1 overexpression in myocytes. Since p50-p50 homodimers are known for transcriptional down-regulation,22–29 and since Myo/V1 can generate p50-p50 homodimers both in vitro and in vivo,9 this current observation (Fig. 6) is probably due to the generation of these homodimers in vivo.
Myo/V1 was first identified as a unique protein to be up-regulated in failing rodent2,4 and human3 heart tissues. In contrast to previous reports, which suggested that Myo/V1 is an exogenously acting “hypertrophy-inducing trophic” protein (myotrophin) in rat neonatal myocytes,2,16 the results of the present study suggest that Myo/V1 is a predominantly cytosolic protein that could regulate the NFκB-mediated transcription process.
First, as shown in Figure 1, we did not observe an increase in protein synthesis in Myo/V1-stimulated neonatal cardiac myocytes, despite using a broad range of concentrations of MyoV1. In contrast, there was a 30.2% ± 7.1% increase in protein synthesis in phenylephrine-stimulated cardiac myocytes, which is consistent with previous reports in the literature.30 These results are also consistent with a previous report, which suggested that Myo/V1 is not a hypertrophy-inducing protein14 when exogenously added to the cardiac myocytes.
Second, as shown in Figures 2–4, our results suggest that Myo/V1 is an intracellular protein that is not released into the extracellular space in vitro, nor is it released by the heart after a transient episode of ischemia/reperfusion-induced injury ex vivo. These findings support the earlier observation that this protein was originally isolated only from the heart tissues.2 While a 2003 report7 suggested that Myo/V1 is found at very low femtomolar levels in human plasma (2.402 fmol/mL or 28.82 pg/mL) of heart failure patients, this protein is also found in the plasma of healthy human subjects at similar levels (2.268 fmol/mL or 27.21 pg/mL). Since Myo/V1 has been shown to induce hypertrophy in myocytes only at nanomolar levels,2 we speculate whether the very low increase of plasma Myo/V1 (0.13 fmol/mL [from 2.27 to 2.40 fmol/mL] or ~1.6 pg/mL [from ~27.2 to ~28.8 pg/mL]) in heart failure patients7 would have elicited any substantial hypertrophic effects on the myocardium. Moreover, that published study7 showed that plasma Myo/V1 levels decreased from already-low levels during the progression of heart failure. Those authors and an associated editorial commentary31 suggested that human plasma Myo/V1 might have come from “dead or damaged cells” or from skeletal muscle rather than from the myocardium. Similarly, our studies did not demonstrate that Myo/V1 is actively secreted into the extracellular space from the myocardium or myocytes (Fig. 2). Although cardiac hypertrophy was observed in the Myo/V1 transgenic mice,32 peripheral levels of Myo/V1 protein were not detected in these animals. Therefore, we conclude that the pathological effects observed in these transgenic animals32 are the consequence of higher levels of intracellular Myo/V1 rather than its extracellular effects. Moreover, the primary6 and 3-dimensional structural analysis9 (Fig. 7) of Myo/V1 did not reveal any significant homology to known extracellular growth factors, cytokines, or hormones.
In general, several types of proteins exist in the peripheral circulation of mammals. Proteins such as growth factors and cytokines are actively secreted into the peripheral circulation by live cells from the tissues and exert their biological effects through their specific receptors at picomolar levels.33–35 In addition, intracellular pro-teins (for example, troponin, creatine kinase, heat shock factors, and chaperone proteins) are passively released into the peripheral circulation from dead (necrotic) cells and thus exist at very low levels in the circulation.36–39 Reports indicate that some of these protein's36–40 exhibit their in vitro biological effects in higher concentrations at nano- and micromolar levels, and no cell surface receptors have yet been clearly identified for these proteins. It is possible that these proteins are either taken up by endocytosis in cell-culture conditions or that they chaperone the extracellular domains of cell surface receptors in a nonspecific manner and thus generate their biological effects, rather than through specific ligand-receptor interactions. Because Myo/V1 is a ubiquitously expressed intracellular cytosolic protein in all tissues6 and since the in vitro trophic activity observed on cardiac myocytes was inconsistent,14,15 we conclude that it may belong to a group of intracellular proteins that are passively released from a dying cell. Previous reports that have shown ubiquitous intracellular proteins generating cytokine-like effects extracellularly are currently being questioned for their physiological relevance.41 Therefore, caution should be exercised,41,42 especially regarding the extracellular physiological relevance, when ubiquitous intracellular proteins are identified in the periphery and if their in vitro biological effects are demonstrated to occur at nano- or micromolar levels.
Third, consistent with previous findings from our laboratory,9 we have shown here that Myo/V1 generates NFκB dimers in nuclear extracts of unstimulated neonatal myocytes (Fig. 5C) and that overexpression of Myo/V1 down-regulates the NFκB-mediated transcription process (Fig. 6), possibly by generating p50-p50 homodimers.9,22–29 Although a 2002 report suggested that Myo/V1 could also be a κB-DNA binding protein,11 results from our study (Fig. 5B) do not support this conclusion. Moreover, we have not observed any DNA binding domain in the Myo/V1 protein (Fig. 7) similar to rel DNA binding domains of NFκB proteins.
In summary, Myo/V1 is an evolutionarily conserved protein that is ubiquitously expressed at low basal levels in every mammalian organ and cell type.6,8 Although the exact intracellular function of Myo/V1 is not known, 3-dimensional comparative structural analysis9 shows that Myo/V1 possesses unique ankyrin repeats that are capable of interacting with the rel domain of NFκB proteins. Myo/V1 resembles a truncated form of IκBα protein without the signal response domain (SRD), nuclear localization signal masking domain (NLSM), and PEST degradation domain (Fig. 7). The generation of p50 and p65 homodimers by the mere addition of excess Myo/V1 protein (Fig. 5C) points out a previously unidentified chaperone mechanism for regulating NFκB functions and raises the interesting possibility that Myo/V1 may play an important role in regulating stress responses in adult mammalian myocardium. Ongoing studies are being conducted in our laboratory to examine this possibility.
Address for reprints: Natarajan Sivasubramanian, PhD,Winters Center for Heart Failure Research, Baylor College of Medicine, 1102 Bates Street, FC 0430.03, Houston, TX 77030. E-mail: ude.mcb@stan
This research was supported by Grants-in-Aid (0050786Y & 0355095Y)from the American Heart Association, Texas affiliate, and an RO1 grant from the National Institutes of Health (HL 072024) to N. Sivasubramanian. P. Knuefermann was supported by the Deutsche Forschungsgemeinschaft (KN521/1-1 and 1-2).
Drs. Baumgarten and Knuefermann are now at the Department of Anesthesiology and Intensive Care Medicine, University of Bonn, Bonn, Germany; Dr. Shi is at Texas Tech University Health Sciences Center, Lubbock, Texas; Dr. Chen is at the Division of Pulmonary and Critical Care Medicine, University of Washington School of Medicine, Seattle, Washington; and Dr. Sakata is at the Department of Internal Medicine and Therapeutics, Osaka University Graduate School of Medicine, Yamadaoka, Suita, Japan.
The authors have provided a key to abbreviations at the end of the paper.
Key to Abbreviations: AdMyo/V1 = recombinant adenovirus expressing Myo/V1; BCA = bicinchoninic acid; BSA = bovine serum albumin; CaCl2 = calcium chloride; FITC = fluorescein isothiocynate; IκBα = inhibitor of kappa B-α; IgG = immunoglobulin G; κB-Igκ/HIV = oligonucleotide containing a κB site from immunoglobulin κ chain gene or human immunodeficiency viral genome; KCl = potassium chloride; KH2PO4 = potassium phosphate (monobasic); MES = 2-morpholinoethanesulfonic acid; MgSO4= magnesium sulfate; MHCsTNF = transgenic mice with cardiac-restricted overexpression of tumor necrosis factor; Myo/V1 = myotrophin or V-1 protein; Myotrophin = a protein found in mammalian heart; NaCl = sodium chloride; NaHCO3 = sodium bicarbonate; NE-PER reagents = nuclear and cytoplasmic extraction reagents kit; NFκB = nuclear factor of kappa B; NLSM = nuclear localization signal masking domain; PAGE = polyacrylamide gel electrophoresis; PBS = phosphate-buffered saline; PEST = proline, glutamic acide, serine, threonine degradation domain; SCID = severe combined immunodeficiency disease; SDS = sodium dodecyl sulfate; SRD = signal response domain; TCA = trichloroacetic acid; V-1 = a protein analogous to myotrophin but found in rat cerebellum