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(Circulation. 2000;102:1690.)
© 2000 American Heart Association, Inc.

Basic Science Reports

Cardiac-Specific Overexpression of Tumor Necrosis Factor- Causes Oxidative Stress and Contractile Dysfunction in Mouse Diaphragm

Xia Li, MD; Melanie R. Moody, MS; David Engel, MD; Stacey Walker, BS; Fred J. Clubb, Jr, DVM, PhD; Natarajan Sivasubramanian, PhD; Douglas L. Mann, MD; Michael B. Reid, PhD

From Baylor College of Medicine and Texas Heart Institute (F.J.C.), Houston, Tex.

Correspondence to Michael B. Reid, PhD, Pulmonary Medicine, Suite 520B, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030. E-mail reid{at}bcm.tmc.edu

	Abstract

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Background—We have developed a transgenic mouse with cardiac-restricted overexpression of tumor necrosis factor- (TNF-). These mice develop a heart failure phenotype characterized by left ventricular dysfunction and remodeling, pulmonary edema, and elevated levels of TNF- in the peripheral circulation from cardiac spillover. Given that TNF- causes atrophy and loss of function in respiratory muscle, we asked whether transgenic mice developed diaphragm dysfunction and whether contractile losses were caused by oxidative stress or tissue remodeling.

Methods and Results—Muscles excised from transgenic mice and littermate controls were studied in vitro with direct electrical stimulation. Cytosolic oxidant levels were measured with 2',7'-dichlorofluorescin diacetate; emissions of the oxidized product were detected by fluorescence microscopy. Force generation by the diaphragm of transgenic animals was 47% less than control (13.2±0.8 [±SEM] versus 25.1±0.6 N/cm²; P<0.001); this weakness was associated with greater intracellular oxidant levels (P<0.025) and was partially reversed by 30-minute incubation with the antioxidant N-acetylcysteine 10 mmol/L (P<0.01). Exogenous TNF- 500 µmol/L increased oxidant production in diaphragm of wild-type mice and caused weakness that was inhibited by N-acetylcysteine, suggesting that changes observed in the diaphragm of transgenic animals were mediated by TNF-. There were no differences in body or diaphragm weights between transgenic and control animals, nor was there evidence of muscle injury or apoptosis.

Conclusions—Elevated circulating levels of TNF- provoke contractile dysfunction in the diaphragm through an endocrine mechanism thought to be mediated by oxidative stress.

Key Words: heart failure • muscles • myocytes • free radicals • antioxidants • cytokines

	Introduction

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Respiratory muscle weakness is a clinical hallmark of congestive heart failure (CHF). As reviewed recently,^{1 2} diaphragm dysfunction may exceed loss of function in peripheral skeletal muscles. This influences the morbidity of CHF, in which respiratory muscle insufficiency is thought to exaggerate breathlessness and exercise intolerance. Despite a longstanding awareness of this problem, virtually nothing is understood about the mechanisms responsible for respiratory muscle weakness in CHF.

Sustained overexpression of tumor necrosis factor- (TNF-) appears to be one of several maladaptive mechanisms that contribute to the progression of heart failure.³ TNF- mRNA and protein are expressed in the failing human heart, but neither one is detectable in the healthy heart.^{4 5 6 7 8} TNF- has pleiotropic effects that may accelerate cardiac toxicity, acting to stimulate left ventricular remodeling,⁹ induce apoptosis of cardiac myocytes,¹⁰ and depress contractile function of the heart.^{11 12 13} Animal experiments have demonstrated that long-term infusion of TNF- leads to deleterious changes in left ventricular structure and function that mimic the phenotype characteristic of heart failure.⁹ Transgenic mice with cardiac-restricted overexpression of TNF- undergo similar changes, with progressive remodeling and dilation of the left ventricle and development of volume overload.^{14 15}

TNF- may also act at sites beyond the failing heart. Individuals with heart failure have elevated TNF- levels in the peripheral circulation,¹⁶ which have the potential to weaken respiratory muscles. Diaphragm force production is diminished by exposure to exogenous TNF-¹⁷ or by endotoxin-induced upregulation of TNF- within the muscle.¹⁸ Several mechanisms could mediate TNF-–induced weakness. TNF- stimulates the production of reactive oxygen species (ROS). Increased ROS activity can act directly on the diaphragm to inhibit contractile function.^{19 20} TNF- also can have catabolic effects on the diaphragm, stimulating loss of contractile protein and muscle atrophy.²¹ Despite the implications of these findings, TNF- has not been evaluated as a mediator of respiratory muscle weakness in the setting of chronic heart failure.

The present study addresses this issue. We measured diaphragm function in transgenic mice that develop heart failure due to cardiac-specific overexpression of TNF-. These animals develop alterations in cardiac structure and function that mimic the human disease and exhibit circulating TNF- levels of 250 to 350 pg/mL. Our study was designed to test 3 hypotheses: (1) Cardiac-specific overexpression of TNF- causes contractile dysfunction of the diaphragm. This was tested by measuring isometric contractile properties and fatigue of diaphragm fiber bundles isolated from transgenic mice and littermate controls. (2) Oxidative stress contributes to loss of diaphragm function. This was tested by comparing oxidant levels in diaphragms of transgenic and control animals and by evaluating antioxidant effects on diaphragm function. (3) The diaphragm is weakened by structural changes: muscle atrophy, injury, or apoptosis. This was assessed by measuring muscle mass and by histological and ultrastructural analyses.

	Methods

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Experimental Animals
All procedures conformed to NIH guidelines and were approved by the animal protocol review committee of our institution. Mice were fed rat chow ad libitum and were maintained on a 12-hour light/dark cycle. We studied transgenic mice in which cardiac myocytes constitutively overexpress a transgene for secreted TNF-. The transgene (Figure 1) consists of a 5.5-kb fragment of the -myosin heavy chain promoter.²² Expression was targeted to cardiac myocytes by incorporation of a PstI-EcoRI 1.1-kb fragment of the murine TNF- gene²³ that encodes the complete coding region and a portion of the 3' untranslated region that contains 60% of the adenine-uridine–rich sequences. A 150-bp BglII-SalI fragment containing an SV40 polyadenylation signal is ligated to the 3' untranslated region to enhance mRNA stability. Individual fragments were assembled in a pBluescript vector plasmid, excised, and purified as a linear 6.8-kb DNA fragment. The purified fragment was microinjected into 1-cell C57BL6/JxICR hybrid mouse embryos, producing a founder line that exhibits cardiac-restricted expression of the TNF- transgene. Southern hybridization using a BglII-SalI -myosin heavy chain probe detected transgene expression in the myocardium but not the spleen, brain, lung, or liver. Transgenic animals develop left ventricular remodeling and dysfunction by 8 weeks of age.²⁴ Unlike other transgenic mouse lines that overexpress TNF- in the myocardium,^{14 15} serum TNF- levels are elevated in our mice (250 to 350 pg/mL) by 4 to 8 weeks of age. TNF- is not elevated in littermate controls (<2 pg/mL). Investigators were blinded to animal genotype until data collection was complete. Short-term TNF- effects were tested in muscles of adult ICR mice (Harlan, Indianapolis, Ind).

View larger version (8K): [in this window] [in a new window]   Figure 1. Schematic of TNF- transgene. Structure and synthesis are detailed in Methods.

Muscle Preparations
Animals were anesthetized by intraperitoneal injection (0.4 mL/kg) of ketamine 42.8 mg/mL, xylazine 8.6 mg/mL, and acepromazine 0.7 mg/mL. Under deep anesthesia, we excised the diaphragm, soleus, or extensor digitorum longus (EDL) muscle. For contractile studies, fiber bundles were isolated from the diaphragm; limb muscles were studied intact. For oxidant measurements, hemidiaphragms were isolated.

Ultrastructural Evaluation
Animals were euthanized by intraperitoneal injection (Euthasol; King Pharmaceuticals, Inc). The left ventricle was cannulated and perfused with chilled physiological saline, then with 2% glutaraldehyde. For transmission electron microscopy, 1-mm-thick sections were dehydrated through graded series of acetone and embedded in epoxy resin. Sections were cut (1 µmol/L), stained with toluidine blue, and examined for areas appropriate for thin sections. Sections in the silver-gray interference color area were placed on a copper grid, stained with uranyl acetate and lead citrate, and examined with a JEOL 1200 electron microscope (JEM-1200 EXII).

In Situ DNA Ligation Assay
Excised diaphragm muscles were fixed in 10% zinc-buffered formalin and embedded in paraffin. Sections were cut (6 µmol/L) and mounted on glass slides. Double-stranded DNA breaks with single-base 3' overhangs were detected with the in situ DNA ligation assay of Didenko et al²⁵ with 2 modifications: (1) sections were washed with 4 changes of water >2 hours after proteinase K treatment, and (2) sections were washed twice in PBS at 65°C for 15 minutes after the ligation reaction. Double-stranded DNA breaks were stained with fluorescein (excitation, 495 nm; transmission, 525 nm). Sections were counterstained with DAPI (excitation/transmission, 310/353) to identify cell nuclei. Dye emissions were imaged by fluorescence microscopy. Images were compared to determine the fraction of total nuclei that was positive for ligase staining.

TNF- and TNF- Receptor Assays
Diaphragm samples were homogenized in ice-cold PBS, incubated with 1% Triton X-100 (10 µL/mL), rehomogenized, and centrifuged (8000 rpm for 15 to 20 minutes; 4°C). The supernatant was isolated and recentrifuged. Supernatant protein concentration was determined with a commercial assay (Pierce Chemical Co). Murine TNF-, TNF- type 1 receptor (TNFR1), and TNF- type 2 receptor (TNFR2) levels were quantified by immunoassay (R&D Systems) and normalized to protein content.

Contractile Protocols
Long-term effects of TNF- were tested in muscles from transgenic animals and age-matched littermate controls. Each muscle was studied at room temperature in Krebs-Ringer solution containing 0.025 mmol/L d-tubocurarine chloride bubbled with 95%/5% O₂/CO₂. The tendon was tied to a force transducer mounted on a micrometer by which muscle length was adjusted. Contraction was stimulated directly with platinum electrodes (4x37 mm) and supramaximal activation (13 V, 550 mA, 0.2-ms pulses). Transducer output was displayed on an oscilloscope from which contractile properties were recorded. Muscle length was adjusted to maximize twitch force (optimal length, L_o). Bath temperature then was increased to 37°C; in trials to assess antioxidant effects, N-acetylcysteine (NAC) 10 mmol/L was added to the bath medium at the time of temperature change. After 30 minutes of thermoequilibration at 37°C, twitch force, time to peak twitch force, and twitch half-relaxation time were recorded. Tetanic forces then were measured every 2 minutes at stimulation frequencies of 300 (maximal tetanic force), 15, 300, 30, 300, 40, 300, 50, 300, 80, 300, 120, 300, 160, 300, 200, and 300 Hz (300-ms stimulus trains). After 10 minutes of recovery, fatigue was induced by use of intermittent tetanic contractions (0.5 trains/s) stimulated at 30 Hz (diaphragm), 15 Hz (soleus), or 60 Hz (EDL) for 10 minutes.

Acute TNF- effects were assessed according to Wilcox et al.¹⁷ Diaphragm fiber bundles were mounted at L_o and incubated at 37°C in oxygenated Krebs-Ringer solution. The bath contained either buffer alone (control) or buffer containing (1) murine TNF- 500 µmol/L (Boehringer Mannheim Corp), (2) NAC 1 mmol/L, or (3) TNF- plus NAC. After 150 minutes of incubation, the force-frequency relationship was measured as described above.

After contractile studies, L_o was recorded. The muscle was trimmed of bone and connective tissue, blotted dry, and weighed. Forces were normalized for muscle cross section according to Close.²⁶

Intracellular Oxidant Assay
Oxidant levels were assessed with dichlorofluorescein (DCF) as described previously.^{27 28} In brief, paired hemidiaphragms from a donor animal were secured in Krebs-Ringer solution bubbled with 95% O₂/5% CO₂ and preloaded with 2',7'-dichlorofluorescin diacetate (Molecular Probes). Hemidiaphragms then were incubated separately for 50 minutes in buffer (control) or in buffer containing TNF- 500 µmol/L. During incubation, nonfluorescent dichlorofluorescin was oxidized to fluorescent DCF at a rate proportional to cytosolic oxidant levels. Accumulation of the reaction product was assessed by measurement of DCF emissions at 3 to 5 sites per hemidiaphragm with a fluorescence microscope (Labophot-2) with CCD camera (series 72; Dage-MTI Inc). Emission intensities from each hemidiaphragm were averaged to obtain a single gray-scale value reflecting mean DCF content.

Statistics
Data are expressed as mean±SEM. Differences among individual variables were tested by Student’s t test for normally distributed data or the Mann-Whitney rank-sum test for nonnormally distributed data.²⁹ Two-way repeated-measures ANOVA was used to test parameters measured repeatedly over time.³⁰ Comparisons were considered significant at P<0.05.

	Results

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Animal Characteristics and Muscle Weights
Transgenic mice were not different from littermate controls in appearance, spontaneous activity (ambulation, grooming behavior, response to touch, etc), or body weight (Table 1). Nor were diaphragm or limb muscle weights different (Table 1). In contrast, the hearts of transgenic animals were 24% heavier than control, reflecting cardiac hypertrophy.²⁹

View this table: [in this window] [in a new window]   Table 1. Whole-Body and Individual Muscle Weights (g)

Diaphragm Morphology and Apoptosis
We detected no evidence of diaphragm injury in transgenic animals at the ultrastructural level (Figure 2). Myofibrillar organization was maintained, mitochondria exhibited clearly defined cristae, and membrane structure was preserved at the sarcolemma and triad regions. There was little evidence of diaphragmatic apoptosis by in situ DNA ligase assay (data not shown). Few apoptotic myonuclei were observed in muscles of transgenic or control animals, and there was no detectable difference between groups. This contrasts with the myocardium of transgenic animals, in which apoptotic changes are prominent.

View larger version (132K): [in this window] [in a new window]   Figure 2. Diaphragm ultrastructure. Electron micrographs of diaphragm samples from transgenic (A) and littermate control (B) animals; bars=2 µm and 500 nm (inset).

TNF- and TNF- Receptors in Diaphragm
TNF- content was not elevated in the diaphragm of transgenic mice. Tissue levels averaged 6.5±1.9 (SEM) pg TNF-/mg protein versus 12.2±3.0 in diaphragm of littermate controls (n=6 per group). Nor were TNF- receptor populations different. TNFR1 levels averaged 185±19 pg/mg protein in diaphragm of transgenic animals and 187±11 in control muscle; TNFR2 levels were 189±14 and 183±21 pg/mg protein, respectively (n=6 per group).

Diaphragm Dysfunction in Transgenic Animals
The diaphragm of transgenic animals was grossly dysfunctional (Table 2). Forces developed during twitch and maximal tetanic contractions were 50% of the corresponding control values. Twitch timing also was shortened, with 10% decrements in both time to peak force and half-relaxation time. Weakness was evident over the entire range of diaphragm activation; absolute forces were diminished at all stimulus frequencies (Figure 3). Normalized for maximal force, the relative forces of twitch and submaximal tetanic contractions were not different between groups (Table 2), and relative force-frequency relationships were indistinguishable (data not shown). During the fatigue protocol, force production by control diaphragm fell progressively (Figure 4). The muscles of transgenic animals generated less force at the onset of stimulation. This disparity persisted throughout the fatigue trial. Contractile function was not altered in either limb muscle that we examined. The absolute force-frequency relationships of soleus and EDL muscles (Figure 5) were identical between groups. Soleus and EDL of transgenic animals also had normal twitch properties, maximal forces, and fatigue characteristics (data not shown).

View this table: [in this window] [in a new window]   Table 2. Contractile Properties of Diaphragm Fiber Bundles

View larger version (16K): [in this window] [in a new window]   Figure 3. Diaphragm force-frequency characteristics. Forces developed by muscles of transgenic (•) and littermate control () animals at stimulus frequencies of 0.1 (twitch) to 300 Hz. Values are mean±SEM; n=6/group; *P<0.001.

View larger version (19K): [in this window] [in a new window]   Figure 4. Diaphragm fatigue. Forces developed by muscles of transgenic (•) and littermate control () animals during repetitive contractions. Values are mean±SEM; n=6/group; *P<0.001.

View larger version (12K): [in this window] [in a new window]   Figure 5. Limb muscle force-frequency characteristics. Forces developed by soleus (top) and EDL (bottom) of transgenic (•) and littermate control () animals at frequencies of 0.1 (twitch) to 300 Hz. Values are mean±SEM; n=5 (soleus) or 4 (EDL) per group.

Oxidants as Mediators
Oxidant levels were clearly elevated in the diaphragm of transgenic animals. DCF oxidation was accelerated in 5 of 6 paired comparisons (Figure 6). Exaggerated oxidant levels apparently contributed to contractile dysfunction; 43% of the force deficit observed in diaphragms of transgenic animals could be abolished by 30 minutes of exposure to NAC, a thiol donor with antioxidant properties. NAC did not increase force production by control muscle (Figure 7).

View larger version (15K): [in this window] [in a new window]   Figure 6. Diaphragm oxidant levels. DCF emissions measured from individual muscles of transgenic animals (•) and littermate controls (); lines define paired comparisons; n=6/group; *P<0.025.

View larger version (16K): [in this window] [in a new window]   Figure 7. NAC effect on transgenic diaphragm. Force produced by diaphragm of transgenic animals after 30 minutes of incubation with NAC 10 mmol/L (). Values are mean±SEM; n=6; dashed lines transcribed from Figure 2; *P<0.01 vs untreated transgenic muscle.

Direct Effects of TNF-
TNF- exposure caused a progressive decline in force production by diaphragm of wild-type mice. As shown in Figure 8, TNF- depressed force at all stimulation frequencies and increased diaphragm oxidant content. Addition of NAC to the bathing medium markedly attenuated the drop in force caused by TNF-, suggesting that weakness is caused by increased intracellular oxidants.

View larger version (12K): [in this window] [in a new window]   Figure 8. TNF- effects on wild-type diaphragm. A, Mean forces (±SEM) produced by diaphragm incubated with (•) or without () TNF- 500 µmol/L; n=6/group; *P<0.001. B, Paired comparisons of DCF emissions from hemidiaphragms incubated with (•) or without () TNF- 500 µmol/L; n=6/group; *P<0.05. C, Mean forces (±SEM) produced by diaphragm incubated with TNF- plus NAC 10 mmol/L; dashed lines transcribed from A; n=6; *P<0.001 vs TNF- alone.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
This study demonstrates that cardiac overexpression of TNF- stimulates oxidant production by diaphragm muscle fibers, causing profound weakness. Loss of diaphragm function is partially reversible by antioxidant treatment and is not accompanied by muscle atrophy, overt injury, or apoptosis. The diaphragm appeared to be particularly susceptible to TNF-, because limb muscles from the same animals functioned normally. The long-term effects of TNF- overexpression were duplicated by acute exposure of wild-type diaphragm to exogenous TNF-, suggesting that long-term changes are a direct response to the cytokine in vivo. These observations identify a novel, oxidant-mediated mechanism whereby TNF- can cause respiratory muscle weakness. This finding is especially relevant to individuals with CHF, in whom elevated TNF- levels and ventilatory insufficiency are common.

Transgenic Animals
The mice used in this study were from a stable line of transgenic animals that overexpress TNF- in the cardiac compartment.²⁴ In brief, cardiac myocytes of these animals overexpress a targeted TNF- transgene (25 copies per cell). Consistent with previous reports by other investigators,^{14 15} our TNF- transgenic mice develop left ventricular dysfunction and left ventricular remodeling by 8 weeks of age. In contrast to previously reported transgenic lines, our mice have elevated circulating levels of TNF- as early as 4 weeks of age. This may relate to shortening of the transgene 3' untranslated region, which confers increased message stability. Transgene expression appears to be limited to the myocardium. TNF- mRNA is not detectable in noncardiac tissues, nor are TNF- protein levels increased in diaphragm.

Circulating TNF- appears to exert endocrine effects on organs beyond the heart. For example, liver and lung weights are increased in transgenic animals. However, limb skeletal muscles appear to remain largely unaffected by TNF- overexpression. We examined 2 muscles that represent opposite extremes of adaptation. Soleus is a slow-twitch, aerobic muscle adapted for maintaining posture; EDL is a fast-twitch, glycolytic muscle adapted for sprinting. Both had normal contractile properties. Muscle weights also were unaltered for soleus, EDL, and gastrocnemius, the latter being a mixed-fiber muscle with intermediate metabolic properties.

Diaphragm Weakness
In contrast to limb muscles, the diaphragm was profoundly weakened in transgenic animals. This deficit did not result from increased TNF- levels in the diaphragm of transgenic animals, nor were TNF- receptor populations altered. Rather, contractile dysfunction appears to reflect an endocrine action of circulating TNF-. In support of this interpretation, short-term exposure to exogenous TNF- produced weakness of similar magnitude in diaphragm of wild-type animals. The latter experiment establishes that TNF- can directly inhibit contraction of murine diaphragm and confirms a prior report by Wilcox and coworkers¹⁷ in which TNF- impaired the function of hamster diaphragm.

Unlike the heart, diaphragm dysfunction was not associated with structural damage. Tissue weight was not diminished. Intracellular structures appeared normal, and there was no evidence of apoptosis. These findings suggest loss of function at a more subtle level, ie, 1 regulatory proteins within diaphragm myofibers. Hopkins³¹ reported that TNF- disrupts sodium and magnesium fluxes across the sarcolemma in the short term, thereby altering intracellular calcium regulation. Muscle contraction also can be inhibited at later steps in the process of excitation-contraction coupling. Existing data cannot rule out possible actions of TNF- on the sarcoplasmic reticulum or myofilaments.

Oxidants as TNF- Mediators
Our data indicated that oxidant production by diaphragm myofibers is accelerated, both in muscles of transgenic animals and in wild-type muscle treated with exogenous TNF-. The capacity of TNF- to accelerate oxidant production had not been directly demonstrated in skeletal muscle fibers heretofore. But the response was expected. TNF- stimulates production of ROS in a variety of nonmuscle cell types,^{32 33} and mitochondria-derived ROS play a critical role in TNF- signal transduction within skeletal muscle cells.^{34 35} Cyclooxygenase activity represents an additional source of ROS³⁶ that has been implicated in TNF-–induced weakness of hamster diaphragm.¹⁷ The present data are consistent with the effects of ROS, which depress force both in vivo¹⁹ and in vitro.²⁰ More importantly, ROS-induced contractile depression can be reversed in the short term by antioxidants that function as thiol donors.²⁰ Oxidative modification of target proteins is a plausible mechanism for TNF-–induced weakness. Force may be decreased via reversible oxidative modification of regulatory proteins of the sarcolemma, sarcoplasmic reticulum, or myofilaments.³⁷

Note that the DCF assay is sensitive to nitric oxide (NO) derivatives as well as ROS.²⁸ Skeletal muscle fibers constitutively express both type I and type III NO synthases, muscles generate endogenous NO, and NO depresses force.³⁸ However, the evidence indicates that TNF- alone does not stimulate NO production by muscle,^{35 39 40} suggesting that NO is unlikely to mediate the responses observed in this study.

Conclusions
We conclude that TNF- overexpression by cardiac myocytes can compromise the diaphragm. This suggests an endocrine mechanism whereby the failing myocardium could stimulate oxidant production and loss of function in the respiratory muscles. The observation that contractile depression was partially reversible by NAC further suggests that antioxidants might benefit patients with CHF. If respiratory muscle strength can be improved, the sensation of dyspnea should be less intense and ventilatory failure is less likely.

	Acknowledgments

 
This research was supported by National Institutes of Health grants HL-45721, HL-59878, HL-58081, HL-61543, and P50-HL-O6H. We are grateful for the technical assistance of Dorellyn Lee Jackson, Ralph Nichols, Barbara Molinari, Blake Deady, and Pamela Potts.

	Footnotes

 
Guest Editor for this article was Ishwarlah Jialah, MD, PhD, FRCPath, DABCC, FACN, Southwestern Medical School, Dallas, Tex.

Received July 16, 1999; revision received April 26, 2000; accepted May 9, 2000.

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