| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Pulmonary and Critical Care Division, Department of Medicine Medical College of Georgia, Augusta, Georgia
Submitted 11 October 2004 ; accepted in final form 15 April 2005
 |
ABSTRACT |
|---|
 TOP ABSTRACT METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
oxidative stress
The mechanism by which congestive heart failure alters skeletal muscle function remains unclear. Importantly, evidence indicates that inactivity alone cannot fully account for congestive heart failure-induced reductions in muscle function (25). Recent work indicates, however, that congestive heart failure is associated with an increase in generation of free radicals (i.e., molecules derived from superoxide anions) and other reactive oxygen species in some tissues (2, 32). Moreover, in several conditions (e.g., sepsis, wasting syndromes), heightened free radical formation in skeletal muscle has been recently linked to the development of muscle weakness and protein loss (4, 24).
On the basis of these previous findings, we postulated that cardiac dysfunction may result in heightened free radical formation in the respiratory muscles and that free radicals, in turn, may trigger alterations in respiratory muscle function. The purpose of the present study was to examine this issue by 1) comparing generation of reactive oxygen species for diaphragm muscle samples taken from control animals and animals after coronary ligation to induce cardiac dysfunction, 2) examining diaphragm muscles taken from control animals and animals with coronary ligation for evidence of free radical-mediated alterations in lipid and protein composition, and 3) examining the effect of administration of a free radical scavenger [polyethylene glycol-superoxide dismutase (PEG-SOD)] on diaphragm force generation in animals with cardiac dysfunction due to coronary ligation.
|
METHODS |
|---|
|
TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
300 g) were used for experimentation. Animals were given food and water ad libitum and housed in university animal facilities. The study was approved by the Medical College of Georgia Institutional Animal Care and Use Committee. Two groups of experiments were performed. In the first set of studies, two groups of animals were studied: 1) sham-operated control animals (n = 19) and 2) animals subjected to coronary ligation (n = 20). The coronary ligation surgical procedure is described below. Both sets of animals were killed at 6 wk after surgery. At the time of death, animals were anesthetized with face mask halothane, and groups of animals were used for the following assessments. 1) In four control and four coronary ligation animals, a Millar microtip catheter was placed via the left carotid artery to measure mean arterial pressure and left ventricular end diastolic pressures; catheters were then removed, and diaphragms were excised, frozen to –80°C, and used for assessment of 8-isoprostane and protein carbonyl levels. 2) In four control and four coronary ligation animals, diaphragms were excised, diaphragm strips dissected, and the diaphragm force-frequency relationship was assessed. 3) In three control and three coronary ligation animals, diaphragms were excised, mitochondria isolated, and mitochondrial respiration was assessed 4) In six control and seven coronary ligation animals, diaphragms were excised, mitochondria were isolated, and mitochondrial hydrogen peroxide (H2O2) generation was measured. And 5) in six control and six coronary ligation animals, hemidiaphragm preparations were created, and diaphragm superoxide generation was assessed by determining ethidium formation after infusion of hydroethidine.
In additional studies (n = 4), animals underwent coronary ligation as described below, and at 2 wk after surgery PEG-SOD administration (2,000 units·kg–1·day–1 ip) was started. At 6 wk after surgery (and 4 wk after beginning PEG-SOD administration), animals were anesthetized, diaphragms were removed, strips were dissected, and the diaphragm force-frequency relationship was assessed. Diaphragm weights and heart weights were recorded. The heart was then placed in 10% buffered formalin. After fixation, the left ventricle was cut into three transverse sections. Sections were mounted and projected onto a computer monitor. Image-analysis software (SigmaScan software, Richmond, CA) was used to outline the infarcted and noninfarcted portions of the left ventricular circumference. The ratio of left ventricular infarction to noninfarcted tissue was calculated from these measurements.
Coronary ligation technique. Ligation of the left descending coronary artery was performed to induce left ventricular infarction and cardiac dysfunction as previously described (1). Sham operated animals served as controls. For this procedure, the following protocol was followed: 1) initial face mask inhalational halothane anesthesia; 2) intubation with a small-animal laryngoscope and attachment to an anesthesia machine/small-animal ventilator; 3) a small incision in the left chest intercostal space (1 cm), with placement of a ligature about the left anterior descending coronary artery (or no ligature for shams); 4) closure of the chest; and 5) stoppage of anesthesia and removal of the endotracheal tube.
Measurement of force generation. Diaphragm force generation was assessed as previously described (31). In brief, diaphragms were excised, they were placed in a dissecting dish, and diaphragm strips were excised. During strip dissection, care was taken to keep muscles intact from origin to insertion. Strips were then mounted vertically in an organ bath containing Krebs-Henselheit solution (22°C, 50 mg/l curare, pH 7.40, 135 mM NaCl, 5 mM KCl, 11.1 mM dextrose, 2.5 mM CaCl2, 1 mM MgSO4, 14.9 mM NaHCO3, 1 mM NaHPO4, and insulin 50 units/l, 95% O2-5% CO2). One end of each strip was tied to the base of the organ bath, and the other was attached to a Grass FT10 force transducer. Platinum-mesh field electrodes were used to deliver supramaximal currents using a constant-current amplifier driven by a Grass S48 stimulator. Muscle strip length was adjusted to optimal length for these studies. Strips were sequentially stimulated with trains of 1-, 10-, 20-, 50-, and 100-Hz stimuli (train duration 800 ms, 30 s between adjacent trains), and force was recorded. Cross-sectional area was calculated as muscle strip weight times muscle density (1.06) divided by muscle length. Specific muscle force was calculated as raw force divided by cross-sectional area.
Measurement of mitochondrial respiration.
Mitochondrial respiration was determined as previously described (6). After removal from the animal, diaphragms were rinsed in cold isolation buffer (180 mM KCl, 5 mM MOPS, and 2 mM EGTA, pH 7.25 at 4°C), blotted dry, weighed, and placed in fresh isolation buffer. After being minced finely with scissors, muscle pieces were homogenized for two, 7-s periods using a Polytron homogenizer set at one-half speed (15,000 rpm). The homogenate was filtered through two layers of cheesecloth into a clean centrifuge tube. The homogenate was centrifuged at 600 g for 7.5 min at 4°C; the resulting supernatant was then centrifuged at 5,000 g for 10 min at 4°C. The isolated mitochondrial pellet was washed five times with 1-ml volumes of isolation buffer, and then it was gently resuspended in isolation buffer to yield a final mitochondrial protein concentration of 20 mg/ml (Bio-Rad protein assay). To assess mitochondrial oxygen consumption, mitochondrial suspensions (final concentration 200 µg in 0.6 ml) in buffer (120 mM KCl, 5 mM KH2PO4, 5 mM MOPS, and 1 mM EDTA, pH 7.25, containing 10 mM pyruvate and 2.5 mM malate,) were placed in an Instech chamber (Plymouth Meeting, PA) containing a polaragraphic oxygen electrode. ADP (0.5 mM) was added to initiate state 3 respiration.
Measurement of diaphragm mitochondrial H2O2 formation. A standard assay was used to assess the formation of H2O2 by incubated mitochondrial suspensions (21); this assay measures the hydrogen peroxidase-mediated oxidation of p-hydroxyphenyl acetic acid (4-HPA) by tissue suspensions and uses oxidation rate as an index of H2O2 formation. Briefly, mitochondrial buffer containing 0.5 mM 4-HPA and 40 µg of mitochondria were added to an assay cuvette in an Aminco spectrophotofluorometer set at an excitation wavelength of 315 nm and an emission wavelength of 425 nm. After fluorescence signal zeroing, 12 units/ml of horseradish peroxidase were added to the reaction cuvette, and the increase in fluorescence over time was used as an index of H2O2 production. Standard concentrations of H2O2 were used for calibration of the fluorescent signal.
Effect of contraction on diaphragm free radical generation. Left hemidiaphragms with attached ribs and aorta were excised from control and coronary ligation rats and placed in an organ bath containing oxygenated (95% O2-5% CO2) Krebs-Henselheit solution. Hydroethidine was then infused into the diaphragm; after 20 min, diaphragms were removed, ethidium was extracted, and it was measured via spectrofluoroscopy (30). Studies were performed both with the diaphragm noncontracting ("resting") and with repetitive contractions performed (20-Hz trains of stimuli, 0.25/s train rate, for 20 min applied via field-stimulating electrodes placed in the bath about the muscle strip). Ethidium levels were taken as an index of superoxide generation, because superoxide reacts with hydroethidine to form ethidium and superoxide scavengers specifically inhibit ethidium formation by this experimental preparation (30).
Tissue levels of 8-isoprostane and protein carbonyl measurements.
Measurement of muscle 8-isoprostane levels (i.e., 8-isoprostaglandin F2
) were performed using a competitive phase enzyme immunoassay (17). For this determination, we pulverized muscle samples (20–50 mg) under liquid nitrogen. Subsequently, this powder was homogenized in methanol (20 ml/g of tissue), an aliquot of this homogenate (1 ml) was mixed with 5 ml of 0.1 M potassium phosphate buffer (pH 7.4), and the mixture was passed through a Sep-Pak C18 filter (prewashed with methanol and water). After the C18 filter was washed with additional methanol-phosphate buffer and with ultrapure water, samples were eluted from the filter using a 99% ethyl acetate-1% methanol mixture. The ethyl acetate was subsequently evaporated under a stream of nitrogen passed through a heating system. The residue remaining after solvent evaporation was redissolved in buffer, placed in assay wells coated with mouse monoclonal rabbit antibody, and mixed with rabbit antiserum to 8-isoprostane and 8-isoprostane linked to anticholinesterase. After incubation, wells were washed and Ellman's reagent [containing acetylthiocholine and 5,5'-dithiobis(2-nitrobenzoic acid)] added to wells. A spectrophotometric plate reader set at wavelength of 412 nm was used to measure the 5-thio-2-nitrobenzoic acid formed by reaction of Ellman's reagent with acetylcholinesterase. Known concentrations of 8-isoprostane were used to construct a standard curve, and tissue 8-isoprostane concentrations were calculated by reference to this curve.
For determination of muscle protein carbonyl side group content (23), frozen muscle samples were pulverized under liquid nitrogen, and powdered tissue was homogenized in 0.05 M K2HPO4 and 5 mM EDTA, pH 7.4. Homogenates were precipitated with trichloroacetic acid and centrifuged, and one portion of pellet was resuspended using 2 N HCl containing 0.1% 2,4-dinitrophenylhydrazine (DNP). A second pellet portion was resuspended in HCl without DNP. Samples were incubated at room temperature for 60 min, and proteins were reprecipitated with trichloroacetic acid. The precipitate was washed with an ethanol-ethyl acetate mixture (1:1 vol/vol) and dissolved in 6 M guanidine-HCl. The absorbance of the DNP-derived sample, minus the absorbance of the nonderived protein, was taken as an index of protein carbonyl group content, using a molar extinction coefficient of 21,000.
Statistical analysis. Unpaired t-tests (Sigma-Stat Software) were used to compare parameters across experimental groups for the first group of experiments. ANOVA was used for comparison of forces generated between control, coronary ligation, and coronary ligation + PEG-SOD-treated groups, and Tukey's test was used to determine differences between individual groups following ANOVA. A P value of <0.05 was taken as indicating statistical significance. Data are presented as means ± SE.
|
RESULTS |
|---|
|
TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
|
|
|
|
|
|
|
|
DISCUSSION |
|---|
|
TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
Because of the difficulties and expense of administering PEG-SOD over several weeks to rats, we did not include a group of control, sham-operated animals treated with PEG-SOD alone in the present study. Our laboratory has administered PEG-SOD to control animals previously, however (see Refs. 7, 24, and 29), and found that PEG-SOD alone had no effect on the force-frequency curve of normal, control animals in all of these previous experiments.
Muscle function in congestive heart failure. Exercise intolerance is almost always present in patients with congestive heart failure. Traditionally, exercise limitation in these patients has been attributed to inadequate cardiac output with a resultant reduction in blood flow and oxygen delivery to working muscle. Recent findings, however, indicate that congestive heart failure elicits alterations in the intrinsic work capacity of muscle and that these peripheral maladaptive responses may be largely responsible for the reduced exercise capacity seen in this syndrome (9, 16, 20, 33). Specifically, studies have shown that exercise tolerance does not immediately improve after cardiac transplantation or after administration of therapies that improve cardiac function despite the fact that cardiac output increases abruptly in response to these interventions (26). In addition, direct measures of muscle strength and endurance have been found to be reduced in heart failure patients, leading to the concept that heart failure induces a myopathic state, and it is this resultant myopathy that is responsible for many of the symptoms seen in this syndrome (9, 16, 20).
Some work also suggests that diaphragm function may be depressed in congestive heart failure (13, 28). Reports have suggested that some patients with severe heart failure have reduced diaphragm strength (13). In addition, we demonstrated a significant reduction in diaphragm force-generating capacity in dogs with heart failure induced using a rapid ventricular pacing model (28). Maximal diaphragm force generation was reduced by 24% in the pacing model, and diaphragms of heart failure animals fatigued twice as fast compared with controls.
Moreover, previous work indicates that the severity of the peripheral and diaphragm muscle dysfunction engendered by heart failure is related to the magnitude of the cardiac dysfunction that is present. Some studies, in which lesser degrees of cardiac infarction were induced, have reported little or no reduction in muscle force generation. For example, Stassijns et al. (27) found only a small, nonsignificant reduction in low-frequency force diaphragm generation in response to coronary ligation that induced a 44% left ventricular infarction size (27). In the present study, a somewhat greater infarction size was produced (51 ± 3%), and larger reductions in low-frequency force generation were observed. Our animals had trace edema, had mild ascites, and seemed to ambulate relatively easily in their cages. In contrast, in our laboratory's previous study using a pacing model to induce heart failure, animals had severe heart failure (manifested as severe ascites, a marked reduction in cardiac output, and severe peripheral edema) and had a marked reduction in both low- and high-frequency force generation (28). The level of cardiac dysfunction examined in the present study may, however, be a better approximation of the severity of heart failure present in patients at the point they would normally seek medical attention.
Although force generation by diaphragm muscle strips was reduced in our animals, diaphragm mitochondrial capacity was not different from control animals. This latter observation differs from the report of Garnier et al. (11), who found significant reductions in skeletal muscle mitochondrial function in severe congestive heart failure. The difference between the present results and this previous report may, as indicated above, be related to differences in the severity of heart failure examined in the two reports.
Diaphragm free radical generation. Our interest in examining the effects of cardiac dysfunction on free radical generation in skeletal muscle derives from recent reports by our laboratory and others examining the effects of sepsis on limb and respiratory skeletal muscle (4, 24). Animal models have consistently demonstrated significant sepsis-induced reductions in diaphragm muscle force generation (4, 24). Although a number of processes appear to interact to reduce diaphragm force generation in sepsis, oxygen-derived free radicals appear to play a major role in the genesis of this condition, with a number of studies demonstrating that administration of free radical scavengers blocks sepsis-induced reductions in muscle force generation (19, 24). In addition, markers of free radical-mediated protein and lipid peroxidation rise in skeletal muscle during sepsis, and mitochondria isolated from septic muscles generate higher levels of free radicals than muscles taken from control animals (18).
The findings of the present study parallel, in many respects, observations made on diaphragms from septic animals. We found that administration of PEG-SOD, a free radical scavenger, improved low-frequency force generation for the coronary ligated animal group, mirroring the known effect of PEG-SOD to improve diaphragm function in septic animals (24). We also observed an increase in mitochondrial generation of free radicals for diaphragms from coronary-ligated animals, comparable to previous studies reporting an increase in diaphragm mitochondrial free radical generation in animal models of infection (18). The markers of free radical-mediated tissue protein oxidation and lipid peroxidation found to increase in response to coronary ligation in the present study, i.e., protein carbonyls and 8-isoprostane levels, have also been reported to increase in response to sepsis in previous work (18).
The similarity of the observations made in the present study to findings regarding the effects of sepsis on the diaphragm raises the possibility that similar upstream stimuli are responsible for evoking free radical generation in these two conditions. In both heart failure and sepsis, cytokine levels are elevated (e.g., TNF-, IL-1
, endothelin, etc.), and cytokines have been shown to elicit increases in tissue free radical generation in smooth muscle, insulin-producing -cells, and cardiac tissue (8, 10, 12, 14). It is therefore reasonable to speculate that the enhanced diaphragm free radical generation observed in the present study may be cytokine induced. This possibility is supported by recent work by Li et al. (15).
Alterations in diaphragm force generation. There are several potential processes that could account for the observed effect of coronary ligation to shift the diaphragm force-frequency curve so that force generation in response to low-frequency stimulation is depressed. One possibility is that coronary ligation elicited an alteration in the contribution of different fiber types to force generation (e.g., by an alteration in the cross-sectional area of selected fiber groups) so that type I fibers made a smaller contribution to force generation. Other possibilities include an effect of coronary ligation to alter sarcoplasmic reticulum calcium flux or to alter the characteristics of the contractile protein force-pCa relationship. Although the present data are insufficient to determine which of these possibilities may account for the observed reduction in low-frequency force in our experiments, it is of interest that free radicals have effects on muscle that could produce any or all of these alterations. Free radicals have been reported to influence caspase activation, protein synthesis, and proteolytic pathway activation, i.e., cellular pathways that influence fiber size and contractile protein content (4). Several free radical species can alter sarcoplasmic reticulum calcium flux, and H2O2, in particular, has been shown to impair excitation-contraction coupling by reducing sarcoplasmic reticulum calcium release (3). Free radical species can also directly alter contractile protein function, with both hydroxyl radicals and peroxynitrite shown to alter contractile protein calcium sensitivity in skinned-fiber studies (5). Our observation that PEG-SOD, a specific superoxide scavenger, prevented reductions in low-frequency diaphragm force generation supports the possibility that free radicals, by one or more of the above mechanisms, contribute to diaphragm dysfunction resulting from coronary ligation.
Alternatively, it is also possible that PEG-SOD administration may have influenced superoxide-sensitive signaling mechanisms in white blood cells and muscles so as to either alter either the levels of cytokines produced in heart failure or modify muscle cellular responses to cytokine levels at intramuscular sites well "upstream" of the contractile proteins and sarcoplasmic reticulum. Future experiments using highly targeted free radical scavengers (e.g., examination of the responses of organ- and organelle-specific inducible SOD-overexpressing transgenic animals) will be necessary to sort out the importance of direct effects of superoxide (e.g., on the contractile proteins) and indirect effects (i.e., influencing systemic cytokine levels) on skeletal muscle function in heart failure.
Regardless of mechanism, however, the observed coronary ligation-induced reduction in low-frequency diaphragm force generation may be clinically significant. Diaphragm motor units are usually activated over the same frequency range for which diaphragm force generation was impaired in the present study. As a result, maintenance of normal levels of diaphragm force generation should require substantially higher than usual motor unit firing frequencies in the presence of this level of diaphragm dysfunction. In addition, the development of a concurrent illness that increases the respiratory workload (e.g., pulmonary edema) could further increase diaphragm force requirements, resulting in an increase in motor unit firing frequencies to nonsustainable levels. As a result, the pattern of diaphragm weakness observed in this study would be expected to predispose to the development of respiratory failure.
|
GRANTS |
|---|
|
TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
|
FOOTNOTES |
|---|
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
|
REFERENCES |
|---|
|
TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
This article has been cited by other articles:
|
|
 |
|
  J. D. Bruton, N. Place, T. Yamada, J. P. Silva, F. H. Andrade, A. J. Dahlstedt, S.-J. Zhang, A. Katz, N.-G. Larsson, and H. Westerblad Reactive oxygen species and fatigue-induced prolonged low-frequency force depression in skeletal muscle fibres of rats, mice and SOD2 overexpressing mice J. Physiol., January 1, 2008; 586(1): 175 - 184. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. W. H. van Hees, H. F. M. van der Heijden, C. A. C. Ottenheijm, L. M. A. Heunks, C. J. C. Pigmans, F. W. A. Verheugt, R. M. H. J. Brouwer, and P. N. R. Dekhuijzen Diaphragm single-fiber weakness and loss of myosin in congestive heart failure rats Am J Physiol Heart Circ Physiol, July 1, 2007; 293(1): H819 - H828. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Yamada, T. Mishima, M. Sakamoto, M. Sugiyama, S. Matsunaga, and M. Wada Myofibrillar protein oxidation and contractile dysfunction in hyperthyroid rat diaphragm J Appl Physiol, May 1, 2007; 102(5): 1850 - 1855. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. J. Biesiadecki and R. J. Solaro When Hearts Fail So Does Skeletal Muscle: Breaking a Vicious Cycle Circ. Res., June 23, 2006; 98(12): 1456 - 1458. [Full Text] [PDF]
|
|
|
|
|
|
P. K. Lunde, O. M. Sejersted, H.-M. S. Thorud, T. Tonnessen, U. L. Henriksen, G. Christensen, H. Westerblad, and J. Bruton Effects of Congestive Heart Failure on Ca2+ Handling in Skeletal Muscle During Fatigue Circ. Res., June 23, 2006; 98(12): 1514 - 1519. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Visit Other APS Journals Online |
