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(Circulation Research. 1997;81:627-635.)
© 1997 American Heart Association, Inc.

Articles

Dilated Cardiomyopathy in Transgenic Mice With Cardiac-Specific Overexpression of Tumor Necrosis Factor-

Toru Kubota, Charles F. McTiernan, Carole S. Frye, Susan E. Slawson, Bonnie H. Lemster, Alan P. Koretsky, A. Jake Demetris, , Arthur M. Feldman

From the Cardiovascular Research Laboratories, the Division of Cardiology (T.K., C.F.M., C.S.F, B.H.L., A.M.F.) and the Division of Transplant Pathology (A.J.D.), University of Pittsburgh Medical Center, and the Biomedical Engineering Program and Pittsburgh NMR Center for Biomedical Research (S.E.S., A.P.K.), Carnegie Mellon University, Pittsburgh, Pa.

Correspondence to Arthur M. Feldman, MD, PhD, Division of Cardiology, University of Pittsburgh Medical Center, 200 Lothrop St, S 572 Scaife Hall, Pittsburgh, PA 15213. E-mail feldma{at}card2.cath.upmc.edu

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Abstract The failing human heart expresses tumor necrosis factor- (TNF-). However, its pathophysiological significance is not clear. We previously reported that robust overexpression of TNF- in the murine heart causes lethal myocarditis. In this study, we modified the transgene to reduce the production of TNF- by preserving the destabilizing sequence in TNF- cDNA. Expression was driven by the murine -myosin heavy chain promoter. Use of this modified construct allowed us to establish a murine transgenic line (TG). TG offspring were examined at 6, 12, and 24 weeks. All showed a significantly higher heart weight–to–body weight ratio. Northern blot analysis confirmed the expression of transgene in the heart, and enzyme-linked immunosorbent assay demonstrated the presence of TNF- protein. The TG heart demonstrated a mild, diffuse, lymphohistiocytic interstitial inflammatory infiltrate. Cardiomyocyte necrosis and apoptosis were present but not abundant. Magnetic resonance imaging showed that the TG heart was significantly dilated with reduced ejection fraction. Although the left ventricular dP/dt_max was not different at baseline, its responsiveness to isoproterenol was significantly blunted in TG. Atrial natriuretic factor was expressed in the TG ventricle. A group of TG died spontaneously, and subsequent autopsies revealed exceptional dilatation of the heart, increased lung weight, and pleural effusion, suggesting that they died of congestive heart failure. The cumulative mortality rate at 6 months was 23%. In conclusion, the mouse overexpressing TNF- recapitulated the phenotype of congestive heart failure. This provides a novel model to elucidate the role of this cytokine in the development of congestive heart failure.

Key Words: tumor necrosis factor- • heart failure • transgenic mouse

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Tumor necrosis factor- is a proinflammatory cytokine with pleiotropic biological effects.^{1 2} Elevated circulating levels of TNF- have been reported in patients with end-stage congestive heart failure,^{3 4 5 6 7 8 9 10} and recent studies suggest a relationship between the serum levels of TNF- and the severity of the disease.^{7 8 9 10} Previously, it was suggested that inflammatory cells within the cardiac interstitium or in the circulation were responsible for local cardiac expression and release of TNF-. However, recent studies report that the cardiomyocytes are also an abundant source of TNF- production.^{11 12} Indeed, failing human myocardium, but not nonfailing human hearts, express abundant quantities of TNF-.^{13 14 15 16} Furthermore, myocytes have TNF- receptors on their surfaces,¹⁷ and these receptors appear to be released into the circulation during heart failure.^{7 13} Several studies in vitro have demonstrated that TNF- exposure causes a substantial decrease in cardiac contractility^{18 19 20 21} and can recapitulate many of the phenotypic changes associated with the failing heart, including (1) upregulation of G_i,^{22 23} (2) a delay in the Ca²⁺ transient,^{19 24} and (3) induction of inducible nitric oxide synthase.^{14 16 21} However, it is unclear whether cardiac expression of TNF- is important for the development of the heart failure phenotype or whether it simply represents an epiphenomenon associated with end-stage congestive failure.

Recently, we reported that robust cardiac-specific overexpression of TNF- in transgenic mice was lethal.²⁵ The transgene construct used in those experiments used the murine -MHC promoter fused to the coding sequence for murine TNF- to achieve cardiac-specific overexpression of TNF-. However, to ensure robust expression, we had deleted the AU-rich destabilizing sequence in the 3' untranslated region of the TNF- gene.^{26 27} When that construct was used, no transgenic mice survived past 11 days of age, and their hearts demonstrated a florid interstitial infiltrate with edema. However, we hypothesized that a more modest overexpression of TNF- might abrogate the lethality associated with robust expression and thus improve survival and provide a mouse model of congestive heart failure. Therefore, we modified the transgene to reduce the extent of TNF- overproduction by including the AU-rich destabilizing sequence. This modification of the transgene construct resulted in improved survival and the development of progeny that display the heart failure phenotype.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Construction of the Transgene
A transgene construct was made containing the murine -MHC promoter with cDNA to murine TNF-. To generate the plasmid vector, we first used a PCR-based approach to amplify a portion of the pGL2-Basic plasmid (Promega), which has a multiple cloning site, a firefly luciferase gene, and the SV40 T antigen intron and polyadenylation signals (SVpA). Amplification primers 5'-ggg aag ctt gat ctt tgt gaa gga acc-3' (nucleotides 1892 to 1909, sense strand) and 5'-ctt tat gtt ttt ggc gtc ttc ca-3' (complement to nucleotides 77 to 99) were used to amplify a region of pGL2-Basic that excluded the luciferase coding sequences and introduced a HindIII site at the 3' end of the amplified DNA. After HindIII digestion, the amplified plasmid was self-ligated to generate a pGL-SVpA vector. Finally, a 5.5-kb Sac I–Sal I fragment of the murine -MHC promoter (generously provided by Dr Jeffrey Robins, University of Cincinnati [Ohio])^{28 29} was isolated and ligated into Sac I–Xho I–digested pGL-SVpA to generate pGL-MHC-SVpA.

RAW 264.7 cells treated with lipopolysaccharide for 6 hours³⁰ were used to generate cDNA to murine TNF-. The cDNA was amplified with a sense primer (5'-gga gaa cag aaa ctc cag aac atc c-3') corresponding to TNF- cDNA nucleotides 34 to 58³¹ and an antisense primer (5'-ggg gat ccc caa gcg atc ttt att tct ctc-3') containing a BamHI site not found in the murine sequence, followed by nucleotides complementary to TNF- cDNA bases 1608 to 1627.³¹ TNF- cDNA was cloned then into the pCR 2.1 vector using a TA Cloning Kit (Invitrogen) to create plasmid pCR-TNF. The identity of the TNF- coding sequence^{31 32} was confirmed by sequence analysis (Sequenase Version 2.0 DNA Sequence Kit, USB). The pCR-TNF was digested first at the vector EcoRV site. Then HindIII linkers were added, and the pCR-TNF was digested with HindIII to create a clonable end. BamHI was added subsequently to cleave at the BamHI site introduced in the initial PCR amplification immediately after TNF- cDNA base 1627. The 1624-bp fragment (containing 7 nt of the HindIII linker, 18 bp of the pCR 2.1 vector, TNF- cDNA nucleotides 34 to 1627, and 5 nt from the PCR-introduced BamHI site) of pCR-TNF was cloned into an 8.4-kb fragment of pGL-MHC-SVpA after a HindIII/partial BamHI digestion. This digestion removed SVpA from pGL-MHC-SVpA. BamHI digestion of pGL-MHC-TNF produced a linear 7.1-kb fragment (MHC-TNF) used for microinjection into mouse embryos.

Generation and Identification of Transgenic Mice
FVB mice were used for the generation of transgenic animals. Microinjection of the transgene was accomplished in the Transgenic Mouse Facility, Children's Hospital of Pittsburgh (Pa) by Dr Maria T. Sáenz Robles and Suzanne Bertera. After offspring were born, the tails were biopsied at the age of 10 days. Genomic DNA was isolated using a proteinase K method previously described.³³ Transgenic mice were identified by PCR with a sense primer (5'-cca cat tct tca gga ttc tct-3') specific to the -MHC promoter exon 2²⁸ and an antisense primer (5'-cag cct tgt ccc ttg aag aga-3') specific to the TNF- cDNA nucleotides 579 to 599,³¹ as described previously.²⁵

Southern Blot Analysis
The integrity and copy number of the inserted transgene was examined by Southern blot analysis. After complete digestion by BamHI, genomic DNA (10 µg/lane) was electrophoretically separated in a 0.5% agarose gel and transferred onto a nylon membrane (Zeta-Probe GT, Bio-Rad Laboratories). A 1.1-kb EcoRI fragment of the transgene, a part of mouse TNF- cDNA, was used as a hybridization probe. The probe was radiolabeled to at least 1x10⁸ cpm/µg using a Random Primed DNA Labeling Kit (Boehringer-Mannheim). After prehybridization with 7% SDS, 500 mmol/L NaP, 1 mmol/L EDTA, 1% bovine serum albumin, and 100 µg/mL sheared salmon sperm DNA, the membrane was hybridized with the probe overnight at 65°C and washed two times with 5% SDS, 40 mmol/L NaP, and 1 mmol/L EDTA at 65°C and three times with 1% SDS, 40 mmol/L NaP, and 1 mmol/L EDTA at 65°C. Radioactive images from hybridizations were obtained with a PhosphorImager (Molecular Dynamics) and quantified with ImageQuant software (Molecular Dynamics). The expected size of the inserted transgene after BamHI digestion was 7.1 kb. The copy number was calculated as the ratio of the transgene to the endogenous TNF- (two copies in each animal).

Pathological Analysis
Mice were euthanized under methoxyflurane anesthesia before pathological evaluation. All surgical procedures were performed according to the protocols approved by the Institutional Animal Care and Use Committee, University of Pittsburgh (Pa). After measurement of body weight, the heart was excised and rinsed in ice-cold 30 mmol/L KCl. Tissues were either snap-frozen in liquid nitrogen for RNA and protein analysis or immediately immersion-fixed in 10% neutral buffered formalin for staining with either hematoxylin and eosin or Masson's trichrome. Apoptotic cells were assessed using a commercially available TUNEL kit (ApopTag, Oncor).³⁴

Northern Blot Analysis
Total RNA was extracted from frozen tissues by using an acid guanidinium thiocyanate–phenol–chloroform method.³⁵ The concentration of RNA in each sample was assessed spectrophotometrically. RNA (5 to 10 µg) was electrophoresed in formaldehyde-agarose gels and transferred to nitrocellulose (Optitran, Schleicher & Schuell). Hybridization probes were prepared from the following sources: (1) mouse TNF- cDNA, a 1.1-kb EcoRI fragment of the transgene, (2) rat atrial natriuretic factor cDNA,³⁶ and (3) a 24-mer oligonucleotide complementary to rat 18S ribosomal RNA.³⁷ cDNA probes were radiolabeled to at least 1x10⁸ cpm/µg using a Random Primed DNA Labeling Kit (Boehringer-Mannheim). Radiolabeled cDNA probes were hybridized overnight at 42°C in 50% formamide, 10% dextran sulfate, 5x SSC, 25 mmol/L sodium phosphate, 5x Denhardt's, and 0.2% SDS. The membranes received a final wash in 0.2x SSC/0.1% SDS at 60°C. Radioactive images from hybridizations were obtained with a PhosphorImager (Molecular Dynamics) and quantified with ImageQuant software (Molecular Dynamics). Then, the membranes were stripped and rehybridized with the 18S oligonucleotide. The 18S rRNA oligomer probe (labeled using T4 polynucleotide kinase) was combined with a 400-fold mass excess of unlabeled 18S oligonucleotide and hybridized overnight at 50°C in 6x SSC, 0.1% SDS, 0.05% sodium pyrophosphate, and 1x Denhardt's solution; the membranes were washed at room temperature in 3x SSC/0.1% SDS. Then, the membranes were reexposed and quantified, and the results of the cDNA hybridization were normalized to that of the 18S probe to correct for differences in RNA mass and efficiency of transfer.

RT-PCR
First-strand cDNA was synthesized by reverse transcription of 1 µg of total RNA by using oligo-dT primers according to manufacturer's instructions (SuperScript II, GIBCO-BRL). Primers specific to the -MHC promoter exon 1²⁸ (5'-tca gag att tct cca acc cag-3') and complementary to the TNF- cDNA 579 to 599³¹ (5'-cag cct tgt ccc ttg aag aga-3') were used to detect the transgene transcripts as described previously.²⁵

ELISA
Tissue levels of TNF- protein were assessed using a commercially available ELISA kit for mouse TNF- (Factor-Test-X, Genzyme) as reported previously.^{13 25 38} Frozen tissues (5 to 20 mg) were homogenized in 300 to 500 µL ice-cold phosphate-buffered saline containing 1 mmol/L phenylmethylsulfonyl fluoride protease inhibitor (Sigma Chemical Co). After brief centrifugation, samples were kept on ice for the duration of the assay. Total protein levels were quantified using a commercially available assay (Bio-Rad Protein Assay, Bio-Rad Laboratories) with bovine serum albumin (Sigma) as a standard (0 to 2 mg/mL). Tissue samples were standardized to 20 µg total protein in 100 µL diluent buffer for each immunoassay. Mouse TNF- provided by the manufacturer was used as a standard (0 to 2240 pg/mL). All assays were done in duplicate. Results were analyzed spectrophotometrically at a wavelength of 450 nm with a microtiter plate reader. TNF- values are reported as pg/mL of standardized sample (equivalent to pg/200 µg total protein).

MRI
Female transgenic mice and wild-type control mice at the ages of 9 and 24 weeks were studied. Images were acquired on a Bruker Avance 7T spectrometer using a home-built single-loop radiofrequency coil. To anesthetize the mice, sodium pentobarbital was applied intraperitoneally in two doses: the first dose was 0.06 mg/g body wt, and 20 minutes later, a second dose of 0.04 mg/g body wt was applied. After sedation, the mouse was placed prone on the coil, with the heart positioned above the coil center. Image acquisition was gated to both the respiratory and cardiac cycles. A pilot coronal image was obtained from which the long axis of the heart was identified. Eight oblique transverse slices, with a slice thickness of 1 mm and an interslice gap of 0.2 mm, provided short-axis images through the heart, covering the entire left ventricle. The short-axis images were acquired with a spin echo imaging sequence with the following parameters: echo time, 11.3 milliseconds; field of view, 1.5 cm; matrix size, 128x128; number of averages, 4; and repetition time, 1 second. The acquisition order of the multislice set was rotated so that every slice was obtained at eight different time points (24 milliseconds apart), allowing for images to be obtained from end diastole, through systole, and into mid diastole (the average cardiac cycle was 275 milliseconds). Diffusion gradients were used to crush the signal from flowing blood. The left ventricular volume was estimated by tracing the intraventricular area in several consecutive short-axis images where each slice was 1.2 mm thick. The first image acquired after the R wave was chosen as end diastole. End systole was taken as the image when the left ventricular opening was minimal. In several animals, the heart was weighed after the study and compared with the MRI-estimated ventricular mass, where the specific gravity of myocardium was 1.055 g/cm³.

Left Ventricular Pressure Measurement
Five 12-week-old and seven 24-week-old transgenic mice and five 12-week-old and five 24-week-old wild-type control mice were studied. All mice were female, except for three transgenic and two wild-type mice at 12 weeks of age. After anesthetization with 2.5% Avertin (18 µL/g body wt IP, Aldrich Chemical Co), mice were placed in a supine position and surgically intubated with a modified 20-gauge needle connected to a volume-cycled rodent ventilator (Harvard Apparatus) with a tidal volume of 0.2 to 0.4 mL and respiratory rate of 110/min. The chest was opened, and a 1.4F micromanometer catheter (Millar Instruments) was inserted into the left ventricle through the apex. Left ventricular pressure was recorded at the baseline and 2 minutes after low and high doses of isoproterenol injection (0.1 and 1 µg IP). All data were digitized at 1 kHz and stored on a customized personal computer for subsequent analysis.

Statistical Analysis
All statistical analysis was performed using SPSS software (SPSS Inc). The results are presented as mean±SD, unless mentioned otherwise. Student's t test was used to compare each variable between the transgenic mice and the age-matched control mice. A Mann-Whitney rank-sum test was used to compare the mice that had died spontaneously with the mice that were intentionally killed for study, because those that died spontaneously had a significantly larger variance as a group. ANOVA, with or without repeated measures, was used when more than two groups were compared. The post hoc multiple comparisons were made by the Student-Newman-Keuls test. Mortality rate was estimated by Kaplan-Meier survival analysis. Differences were considered significant at a value of P<.05.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Generation of Transgenic Mice
The transgene construct was microinjected into the pronuclei of one-cell mouse embryos, after which the embryos were surgically reimplanted into pseudopregnant mice. Screening of the offspring by PCR identified three potential founders harboring the TNF- transgene, all of which were grossly indistinguishable from nontransgenic littermates. The Southern blot analysis of the founders is shown in Fig 1. The probe detected both transgene and endogenous TNF-. The estimated copy number of transgene in each founder is as follows: founder 1, 39.8±2.6 (mean±SEM); founder 2, 22.3±1.2; and founder 3, 21.0±1.3. One mouse (founder 1) was male but produced no offspring despite mating with several females and was killed at the age of 39 weeks to examine the pathological changes in the heart, which showed a diffuse lymphohistiocytic inflammatory infiltrate in the interstitium. A second mouse (founder 2) was female and delivered 79 offspring in six litters. However, only one of them, which died at 3 days of age, carried the transgene. This founder died spontaneously at 44 weeks of age, and a subsequent autopsy demonstrated pathology consistent with heart failure, including ventricular dilatation and pulmonary edema. The third mouse (founder 3) was also female and produced a number of offspring harboring the TNF- transgene. This founder also developed subsequent congestive heart failure at 50 weeks of age and died with a profoundly dilated heart. The TNF- transgene appeared to be X-linked, since only female mice harbored the transgene when transgenic males were outcrossed with wild-type females. Furthermore, when transgenic females were outcrossed with wild-type males, both males and females harbored the transgene.

View larger version (52K): [in this window] [in a new window]   Figure 1. Southern blot analysis of three potential founders and a wild-type control mouse.

After the screening at 10 days, 15 of 176 transgenic mice were found to have died spontaneously. This was in marked contrast to an absence of death in wild-type littermates (n=195). As seen in Fig 2, the survival rate for the transgenic mice was substantially different from that of littermate control mice, with the mean time to death being 297±17 (mean±SEM) days (P<.005). The cumulative mortality rate at 6 months was 23%. Although there were no statistical differences in mortality between transgenic males and females, the generations of mice had significant effects on mean survival time (F0, 351 days; F1, 209±25 days; F2, 160±2 days; and F3, 86±2 days; P<.05). Subsequent autopsies suggested that at least 11 of the 15 deaths were associated with congestive heart failure.

View larger version (11K): [in this window] [in a new window]   Figure 2. Survival function curves from transgenic mice (TG, n=176) and wild-type littermates (WT, n=195). *P<.005 vs WT.

Expression of TNF- Transgene and Protein in the Heart
A group of transgenic mice and wild-type control mice was killed for analysis at the ages of 6, 12, and 24 weeks. Transgene expression was assessed using Northern blot analysis of total RNA from various tissues, including heart, lung, and liver. Robust expression of the transgene was found in the heart; however, no transgene expression was noted in either the liver or the lung (Fig 3). RT-PCR detected multiple bands for the TNF- transgene, consistent with our earlier observation of the presence of multiple splice variations of the TNF- transgene.²⁵ Fig 4 shows cardiac TNF- levels by ELISA in transgenic mice and wild-type control mice at each age. TNF- levels were significantly higher in the transgenic mice than in littermate control mice (P<.001). However, age did not appear to influence TNF- levels, since there was virtually no difference in levels at 6, 12, and 24 weeks. Similarly, gender had no effects on TNF- levels: 12-week-old males (269±70 pg/mL, n=3) and females (361±138 pg/mL, n=6) had similar levels of TNF-. In addition, TNF- levels in mice that died spontaneously (243±94 pg/mL) were similar to those in the transgenic mice that were killed for study. However, we cannot exclude the possibility that proteolysis decreased the levels of TNF- in the carcasses. Cardiac levels of TNF- in transgenic mice of the present study were approximately an order of magnitude less than those found in the heart of a mouse expressing the TNF- transgene in which the 3' destabilizing region had been deleted (1483 pg/mL).²⁵ Immunoreactive TNF- levels in either lung, liver, or sera were not different between 12-week-old transgenic mice and wild-type control mice (Table 1), consistent with the Northern blot analysis demonstrating that the transgene transcripts were limited to the heart.

View larger version (50K): [in this window] [in a new window]   Figure 3. Northern blot analysis for the transgene transcripts. A, Total RNA from ventricles in 12-week-old transgenic mice (TG) and age-matched control mice (WT); B, Total RNA from ventricle, lung, and liver in a 12-week-old TG.

View larger version (13K): [in this window] [in a new window]   Figure 4. TNF- protein levels in the ventricle by ELISA. Transgenic mice (TG) and wild-type control mice (WT) were analyzed at the ages of 6, 12, and 24 weeks. Values are expressed as mean+SD. *P<.001 vs WT.

View this table: [in this window] [in a new window]   Table 1. Tissue TNF- Protein Levels

Gross Pathology and Histology
Gross pathological observation of the hearts harboring the transgene suggested the presence of cardiac enlargement. This finding was confirmed by the presence of an elevation in heart weight–to–body weight ratios as early as 6 weeks of age (Table 2). An elevation in heart weight was also recognized in these animals.

View this table: [in this window] [in a new window]   Table 2. Body, Heart, and Lung Weight by Age

The histology of the transgenic animals was easily distinguishable from age-matched control animals at all ages (6, 12, and 24 weeks), and an evolution of histopathological changes developed over time. Representative images are presented in Fig 5. Changes in the interstitium were obvious by routine light microscopy. There was an increase and hypertrophy of interstitial cells, which appeared to consist mostly of histiocytes, fewer lymphocytes, and hypertrophied interstitial connective tissue cells. Apoptotic cell death in this population was evident both by routine light microscopy and by the TUNEL assay. There was also mild interstitial edema, an apparent increase in the production of matrix material, and focal interstitial fibrosis. The fibrosis in some animals was more pronounced near the endocardium. The interstitial changes were associated with mild myocyte hypertrophy and focal myocyte degeneration in the form of rare areas of single-cell dropout and occasional myocyte multinucleation. Apoptotic myocytes were present but seen rarely.

View larger version (116K): [in this window] [in a new window]   Figure 5. Hematoxylin and eosin staining (A, D, and G; magnification x5), trichrome staining (B, E, and H; magnification x20), and TUNEL assay (C, F, and I; magnification x200) of the heart. Panels A, B, and C represent a 24-week-old wild-type control mouse; panels D, E, and F, a 24-week-old transgenic animal; and panels G, H, and I, a mouse that died spontaneously at the age of 20 weeks. Each section was taken at the midventricular level, which was confirmed by the presence of papillary muscle in the cavity and the semicircular shape of right ventricular free wall. In the heart harboring the transgene, eccentric hypertrophy (D), patchy interstitial fibrosis (E), and interstitial inflammation (F) were observed. The large arrow in panel F indicates myocyte multinucleation, and small arrows show apoptosis in the interstitial cells. The mouse that died spontaneously developed marked biventricular dilatation (G). The arrows in panels E and H represent patchy interstitial fibrosis; the arrow in panel I shows an apoptotic cardiomyocyte.

Autopsies were also performed with 16 transgenic mice, including two founders, which died spontaneously during the study period. Both ventricles and atria were enlarged extremely in these animals, and lung weight was increased also (Table 2). Pleural effusions with or without ascites were present in nine mice, and organized thrombi were observed in six of 16 atria. Exceptional dilatation of the heart accompanied by increased lung weight and pleural effusion suggested that most of these animals (at least 12 of 16) died because of congestive heart failure.

The histopathological changes in the interstitium were similar qualitatively to those seen in the transgenic mice that were killed for study, but in several of the mice, there was a noticeable increase in the lymphocytic component of the interstitial cells. This was associated with more clearly recognizable myocyte injury, myocyte and interstitial cell multinucleation, and rare but definite myocyte apoptosis. The interstitial fibrosis was relatively severe in some of the animals, but overall, interstitial fibrosis was not an overriding feature. The endocardium and pericardium were slightly fibrotic, but there was no evidence of significant pathological change in the intramyocardial branches of the coronary arteries.

Cardiac Function
Because of the small size and rapid heart rate of the transgenic mouse heart, functional analysis of transgenic myocardium has been challenging. In particular, sensitive measures of in vivo cardiac function have been technically difficult. However, recent advances in MRI technology have provided the capability to evaluate cardiac mass and function in the anesthetized mouse. To verify the accuracy of MRI volume measurement, we compared ventricular mass estimated by MR (VM_MR) with that by gravimetry (VM_G) in seven transgenic and five wild-type animals (Fig 6). The least-squares regression analysis yields the following line: VM_MR=1.02xVM_G-3.06 (r=.983, P<.0001). The errors between the two methods were 0.28±2.70 mg, which indicated the high accuracy and sensitivity of MRI volume measurement. These results agree with those reported in our previous study.³⁹ Representative MR images from a 24-week-old transgenic mouse and an age-matched wild-type control mouse are shown in Fig 7. The coronal view image of the transgenic mouse (Fig 7A) clearly demonstrated that the heart was dilated and globoid. Left ventricular volume was estimated by tracing the intraventricular area in five consecutive short-axis images, where each slice was 1.2 mm thick. The end-diastolic and end-systolic volumes were 0.104 and 0.052 mL, respectively, in the transgenic mouse (ejection fraction, 50%) and 0.064 and 0.018 mL, respectively, in the wild-type control mouse (ejection fraction, 72%). Cumulative data, presented in Table 3, demonstrate a progressive increase in end-diastolic and end-systolic volumes with age in mice overexpressing TNF-. As a result, left ventricular ejection fraction was decreased significantly at 24 weeks of age (P<.01).

View larger version (21K): [in this window] [in a new window]   Figure 6. Relationship between actual ventricular mass and that estimated by MRI.

View larger version (95K): [in this window] [in a new window]   Figure 7. Representative MR images from a 24-week-old transgenic mouse (A, B, and C) and an age-matched wild-type control mouse (D, E, and F). Panels A and D illustrate coronal views of the mice; panels B and E, midventricular short-axis views of the heart in end diastole; and panels C and F, midventricular short-axis views of the heart in end systole. Bars=2.5 mm.

View this table: [in this window] [in a new window]   Table 3. Left Ventricular Volume and Ejection Fraction

Left ventricular pressure was measured in 12 transgenic and 10 wild-type mice at 12 or 24 weeks of age. Because neither age nor gender had a significant effect on hemodynamic parameters, results are summarized as a whole. There were no significant differences in heart rate or systolic blood pressure between the transgenic mice (342±37 bpm, 56±9 mm Hg) and the wild-type control mice (364±49 bpm, 66±15 mm Hg). After 0.1 and 1 µg isoproterenol injection, heart rate increased in the transgenic mice as well as in the control mice (113±16% versus 111±15% and 137±20% versus 135±20%). Blood pressure did not change significantly after isoproterenol injections in either group. We used left ventricular dP/dt_max as an index of contractility. As shown in Fig 8, although left ventricular dP/dt_max was not significantly different at baseline, its responsiveness to isoproterenol was blunted in the transgenic animals (P<.05). In 8 mice in which we had performed MRI on the day preceding the pressure measurement, there was a weak but significant correlation between the dP/dt_max at baseline and the ejection fraction estimated by MRI (r=.73, P<.05).

View larger version (16K): [in this window] [in a new window]   Figure 8. Left ventricular dP/dtmax before and after isoproterenol injections in transgenic mice (TG) and wild-type control mice (WT). Values are mean±SD. *P<.05 vs WT.

Expression of Atrial Natriuretic Factor mRNA
To assess whether the hearts overexpressing TNF- activated the fetal gene program consistent with heart failure, we evaluated the levels of mRNA encoding atrial natriuretic factor in the ventricular myocardium. As seen in Fig 9, hearts with TNF- overexpression demonstrated reexpression of atrial natriuretic factor in the ventricle, whereas no atrial natriuretic factor expression was obvious in hearts from wild-type littermate control mice.

View larger version (44K): [in this window] [in a new window]   Figure 9. Northern blot analysis of atrial natriuretic factor (ANF) mRNA in ventricles from 12-week-old transgenic mice (TG) and age-matched wild-type control mice (WT).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
That end-stage congestive heart failure is associated with a marked increase in circulating TNF- levels has been reported by numerous investigators.^{3 4 5 6 7 8 9 10} Furthermore, there appears to be a direct relationship between disease severity and circulating levels of TNF-.^{7 8 9 10} Additionally, recent studies have demonstrated that although the nonfailing heart does not express TNF-, the failing myocardium expresses abundant amounts of this proinflammatory cytokine.^{13 14 16} Whereas initial reports suggested that TNF- was expressed by inflammatory cells within the myocardium, it is now clear that myocytes themselves can express TNF- at times of stress.^{11 12} However, the precise role of TNF- in the pathophysiology of congestive failure and/or the transition from compensated to decompensated heart failure remains unclear. Similarly, studies have not defined whether these changes in cardiac TNF- expression are pathophysiologically important or simply an epiphenomenon associated with end-stage heart failure.

In the present study, we demonstrate that chronic overexpression of TNF- results in the development of a dilated cardiomyopathy that shows (1) ventricular hypertrophy, (2) ventricular dilatation, (3) interstitial infiltrates, (4) interstitial fibrosis, (5) rare myocyte apoptosis, (6) a diminished ejection fraction, (7) attenuation of ß₁-adrenergic responsiveness, and (8) expression of atrial natriuretic factor in the ventricle. Additionally, mice overexpressing the TNF- transgene had a marked increase in mortality. More than half of the mice that died spontaneously presented with exceptional dilatation of the heart, increased lung weight, and pleural effusion, suggesting that they died of congestive heart failure.

In an earlier report,²⁵ we demonstrated that robust overexpression of TNF- in a mouse resulted in the development of a lethal myocarditis before the completion of weaning. By contrast, the mice in the present report demonstrated levels of TNF- that were approximately an order of magnitude less than those reported in the mouse with lethal myocarditis. It is unlikely that this disparity could be attributed to the analysis of tissue samples, for the tissues in the earlier study were obtained from carcasses, whereas those in the present study were obtained from fresh specimens. Myocardial TNF- levels from the carcasses of mice that died spontaneously in the present study were similar to those in the animals that were randomly killed for study. Rather, it would appear that the destabilizing region in the 3' tail of the TNF- gene plays an important part in regulating TNF- expression.

Although only a single transgenic line was established in the present study, three potential founders were produced, and two of them (founders 2 and 3) died of congestive heart failure. The gross pathology and histology were indistinguishable between these two animals. Furthermore, the third potential founder (founder 1), which was killed before the development of heart failure, also demonstrated mild diffuse myocarditis with a lymphohistiocytic infiltrate. Additionally, at least two potential founders harboring the more robust construct developed a similar though lethal phenotype.²⁵ Thus, five founders overexpressing TNF- constructs demonstrated similar cardiac phenotypes. Therefore, it is unlikely that identical insertion sites could have been a causative factor for the development of myocarditis and congestive heart failure in these animals.

Transgenic progeny demonstrated phenotypic heterogeneity, since survival was considerably different among different mice and since TNF- levels did not appear to reflect prognosis. However, this finding was not surprising, since clinical heterogeneity is a hallmark in patients with heart failure as well as in patients with genetically linked heart muscle disease.^{40 41} It has been proposed that hypertrophy, myocyte loss, and interstitial fibrosis are early components of the transition from compensated to decompensated myocardium.⁴² Indeed, the development of cardiac dilatation in the transgenic mouse occurs over a period of 6 months, and hypertrophy and fibrosis appear to precede dilatation and diminished pump function.

It might also be of interest to compare cardiac TNF- protein levels in our transgenic mice with those reported in human heart failure. There are two reports that used ELISA to demonstrate the presence of TNF- protein in the failing human heart: one study¹³ reported 200 pg/g protein, and the other¹⁵ reported 4 pg/mg protein. In contrast, TNF- in our transgenic mice was 300 pg/mL, which is 1500 pg/mg when normalized for the amount of protein loaded in each well (20 µg/100 µL). There appears to be more TNF- in our transgenic hearts than in the failing human heart. However, such comparisons may be problematic, since the values were obtained by different methods. Furthermore, nonspecific background activity from tissue homogenates limits our ability to detect low levels of TNF- by ELISA and may result in an overestimation of absolute TNF- levels.

The measurement of cardiac mass and function in the mouse is technically challenging because of the small size of the heart and its rapid rate. Transthoracic echocardiography has been used successfully to evaluate the transgenic mouse heart.^{43 44 45 46} In the present study, we have taken advantage of the recent adaptation of MRI technology to noninvasively assess cardiac structure and function in the mouse. MRI is able to provide simultaneous measures of morphology and function by gating the image acquisition to both the electrocardiogram and the respiratory signal, thereby abrogating motion artifacts and ensuring that images are obtained at specific points through the cardiac cycle. Furthermore, multiple image slices in MRI can be obtained in the same amount of time required for a single slice. This permits acquisition of eight short-axis slices at many different points through the cardiac cycle in a decreased amount of time. A previous study has demonstrated the ability of MRI to detect relatively small changes in cardiac mass secondary to catecholamine induced hypertrophy.³⁹ In the present study, we demonstrate the usefulness of MRI in evaluating left ventricular hypertrophy, cavity dilatation, and function in the TNF- transgenic mice compared with wild-type control mice.

Although the present study strongly suggests an important pathophysiological role for TNF- in the pathogenesis of end-stage heart failure, we cannot exclude the possibility that other pathways are of complementary importance in the development of the heart failure phenotype. For example, chronic overexpression of the ß₁-adrenergic receptor⁴⁷ as well as overexpression of G_s⁴⁸ can effect phenotypic changes associated with congestive heart failure. Furthermore, this mouse model does not provide the opportunity to evaluate the mechanisms that initiate the expression of TNF- after myocardial damage. In addition, tissue TNF- levels observed in the transgenic mice appear higher than those reported in the failing human heart. However, the TNF- transgenic mice develop a primary phenotype that is consistent with congestive heart failure, displaying both cardiac dilatation and left ventricular dysfunction. Therefore, it should provide a novel tool with which to analyze physiological and biochemical changes that accompany the transition from compensated to decompensated myocardium and the development of end-stage heart failure. Additionally, the opportunity to modify or rescue the heart failure phenotype by directed crossbreeding with mice harboring selected gene mutations will facilitate an understanding of the mechanisms that compensate or progress cardiac dysfunction. Similarly, the TNF- overexpression model may provide a unique platform for preclinical evaluation of gene transfer or pharmacological therapy.

	Selected Abbreviations and Acronyms

 

MHC = myosin heavy chain MR = magnetic resonance MRI = MR imaging PCR = polymerase chain reaction RT = reverse transcriptase SV = simian virus TNF- = tumor necrosis factor- TUNEL = terminal deoxynucleotidyl transferase–mediated dUTP-biotin nick end labeling

	Acknowledgments

 
Dr Kubota is the recipient of a Japan Heart Foundation & Bayer Yakuhin Research Grant Abroad. The MRI work was supported by National Institutes of Health grant RR-03631 and Research Career Development Award HL-02847 and the National Science Foundation Graduate Research Traineeship Program (BIR-9256343).

Received May 13, 1997; accepted July 25, 1997.

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