INTRODUCTION

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Despite optimal medical therapy, patients with systolic ventricular dysfunction (VD) and chronic heart failure (CHF) often experience dyspnea and exercise intolerance (1). It is now established that alterations in peripheral and ventilatory muscles contribute to these symptoms (1). De Troyer and colleagues (2) were the first to note that cardiac disease was associated with compromised inspiratory muscle function. Meanwhile, several other groups demonstrated that inspiratory muscle weakness in CHF was more important than peripheral muscle weakness (3). This inspiratory muscle weakness may affect outcome variables in these patients. Indeed, inspiratory muscle weakness was shown to reduce life expectancy (6) and to precipitate respiratory failure in patients with COPD (7). We wanted to determine whether the diaphragm was affected differently from other inspiratory or peripheral muscles. To this end, a rat model was used in which systolic VD was induced by infarction after coronary ligation.

In addition, the present study was designed to evaluate whether altered insulin-like growth factor-I (IGF-I) expression was a possible mechanism for diaphragm weakness induced by VD. IGF-I plays an important role in skeletal muscle protein anabolism and growth (8). Reduced serum IGF-I levels have been described in patients with CHF (9). Moreover, diaphragmatic IGF-I expression was shown to be downregulated in steroid-induced myopathy (10). Therefore, we hypothesized that the skeletal muscle alterations seen in VD could be related to a decrease in either endocrine or autocrine IGF-I production.

The purpose of the present study was: (1) to evaluate whether in a model of VD the diaphragm was affected differently from other skeletal muscles; and (2) to determine whether altered autocrine or endocrine IGF-I production may be involved in causing this diaphragm dysfunction.

	METHODS

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Experiments were conducted on 37 male Wistar rats 14 to 20 wk of age and weighing 380 to 500 g. Twenty-two rats underwent a coronary occlusion leading to myocardial infarction and 15 age-matched rats received a sham operation and were used as controls. Two series of experiments were performed. In one series (17 rats with VD, 10 Co rats), body and tissue mass, in vitro contractile properties, histologic and histochemical analysis, IGF-I serum levels and expression were measured. In a second series hemodynamic measurements (VD = 5, Co = 5) were performed. Rats were studied 3 mo after the operation.

All sham-operated rats survived after surgery. In the infarction group, mortality was 36%. The study was approved by the Institutional Animal Care and Ethics Committee.

Experimental Protocol

For all experiments rats were anesthetized intraperitoneally with sodium pentobarbital (Nembutal [Sanofi, Brussels, Belgium]; 30 mg/kg for the coronary ligation operation, 60 mg/kg for the hemodynamic measurements and for the dissection). Except for hemodynamic measurements, rats were tracheotomized and a tracheal cannula (polyethylene tubing PE-200) was inserted. Subsequently, they were mechanically ventilated (Harvard pump respirator; Harvard Apparatus, Co., South Natick, MA), with an O₂-enriched gas mixture. During the coronary ligation operation, anesthesia was maintained with halothane (Fluothane; Zeneca, Destelbergen, Belgium), 1.5% in O₂, using a fluothane respirator (Fluotec 3; Cyprane, Heighley, UK).

Experimental Infarction in Rats

The technique for induction of myocardial infarction was similar to that previously described by Selye and coworkers (11). A left sided thoracotomy was performed and the left coronary artery was ligated 2 to 3 mm from its origin between the pulmonary conus and the left atrium with a 5-0 polypropylene suture. Co rats underwent a left-sided thoracotomy, but no suture was placed. After the operation 0.1 mg piritramide (Dipidolor; Janssen Pharmaceutica, Beerse, Belgium) was given intramuscularly as well as 22.5 mg cefuroxime (Zinacef; Glaxo, Brussels, Belgium).

Hemodynamic Measurements

The right carotid artery was cannulated with a micromanometer-tipped catheter (Millar Instruments, Houston, TX). The arterial catheter was advanced retrogradely into the left ventricle under continuous pressure monitoring. Left ventricle peak systolic (LVPSP) and end-diastolic (LVEDP) pressures were measured as well as systolic and diastolic aortic pressures.

Determination of the Infarct Size

Infarct size was measured in all rats. The hearts were embedded in paraffin and sectioned transversely in 7-µm slices. The fraction of the left ventricular wall taken up by the infarction was determined microscopically and was expressed as a percentage of the total circumference as described previously (12).

In Vitro Contractile Properties

A laparotomy with transverse extension of incision was performed to remove the diaphragm. Two small strips of the diaphragm were dissected and placed at their optimal length (L_o), defined as the length at which peak twitch force was obtained. They were then stimulated as described in detail previously (13).

After a 15-min thermoequilibration period, the following measurements were successively performed: twitch force (P_t), its corresponding time to peak tension (TPT) and half-relaxation time (1/2 RT); tetanic force at 160 Hz (P_o); force-frequency curve; fatigue run at 25 Hz. Deterioration of the preparation because of successive stimulations was evaluated by measuring the tension developed at 160 Hz after each stimulus during the force-frequency protocol. This methodology has been described in detail elsewhere (13).

After completion of the experiment, bundle length, thickness, and width were measured at L_o using fine calipers. The bundle was dip dried and weighed. Cross-sectional area (CSA) was obtained by dividing weight by specific density (1.056) and muscle length at L_o. All forces were expressed per unit CSA (14). Twitch-to-tetanus ratio (P_t/ P_o) was also calculated for each muscle bundle.

The whole diaphragm (including the bundles), heart, liver, parasternal intercostals (including sternum and chondral parts of ribs), right scalenus medius, gastrocnemius, soleus, extensor digitorum longus, and plantaris were weighed after trimming and dipping dry.

Histologic and Histochemical Analysis

The gastrocnemius and diaphragm samples were cut with a cryostat kept at 20° C to obtain cross sections 10 µm thick as described elsewhere (13). Two serial sections of each muscle were stained for routine hematoxylin-eosin staining. The other serial sections were stained for myofibrillar adenosine triphosphatase (ATPase) after acid preincubation at pH 4.5 and 4.3. Muscle fibers were classified as type I, IIa, or type IIx/b fibers (15).

Fiber Size and Proportion

Morphometric examination was carried out microscopically (Leitz Laborhama S., Wetzlar, Germany) at magnification ×20 as described (13). For the diaphragm and gastrocnemius, CSAs were measured and corrected for the shortening occurring from optimal length to excised length by dividing CSAs with a correction factor. This correction factor was the ratio of optimal length to unstretched length and was established in our laboratory to be 1.53 and 1.04 for diaphragm and gastrocnemius, respectively (unpublished data). Finally, the residual interstitial space (RIS) was also evaluated by measuring the interstitial area between muscle fibers. RIS was expressed as a percentage of total fiber area.

IGF-I Serum Level Measurement

Blood was sampled from the thorax immediately after dissection of the heart. Serum was obtained by centrifugation at 3,000 rpm for 10 min at 4° C and was stored at 20° C.

IGF-I was measured in acid-ethanol-extracted sera by radioimmunoassay as described previously (16), using a guinea-pig polyclonal antiserum (Ciba-Geigy, Basel, Switzerland).

IGF-I mRNA Study-RNA Extraction and Analysis

Samples of diaphragm, gastrocnemius and liver were stored in liquid nitrogen. Total RNA was isolated using a modified guanidinium isothiocyanate-CsCl method (17). Quality and quantity of the RNA preparations were determined by measurement of absorbency at 260 and 280 nm and by Northern blot analysis. Samples of 20 µg of total RNA were separated by electrophoresis, prehybridized and hybridized by standard procedures using a probe labeled with -³²P-dCTP (17). A rat IGF-I 3'-cDNA probe (obtained from Dr. Derek Leroith, Betheseda, MD), excised from the pGEM3 vector with EcoRI and HindIII, was used for hybridization. Equal loading of the samples was checked by hybridization with an 18S ribosomal RNA probe. Quantification was performed by measuring the radioactivity level on the blot with a phosphorimager.

Data Analysis

Statistical analysis was performed using the SAS Statistical package (SAS Institute, Cary, NC). Data from the two diaphragm strips obtained from each rat were averaged. Differences between means of rats with VD and Co rats controls were assessed using the Student's t test. In addition, for the fatigue run, two-way analysis of variance with repeated measures was used for assessment of differences as a function of time. Correlations were determined with Spearman's ranks product correlation. Statistical significance was set at a p value < 0.05. Data are expressed as mean ± standard deviation (SD).

	RESULTS

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Quantification of Infarcts and Hemodynamics

In the Co rats there were no signs of myocardial infarction as determined by histologic examination. In rats with coronary ligation, the average infarct size was 44 ± 20% of the left ventricular circumference in the series of rats in which hemodynamics were measured. In the series of rats in which the other measurements were done the infarct size was 32 ± 10%. Differences in infarct size between the two groups were not statistically significant.

LVPSP was significantly decreased by 19% in the rats with experimental VD compared with the control rats (106 ± 17 in VD versus 131 ± 10 mm Hg in Co; p < 0.05). However, LVEDP was not significantly different (pooled values of 2 ± 3 mm Hg). Both the systolic (100 ± 14 in VD versus 128 ± 16 mm Hg in Co; p < 0.05) as well as the diastolic aortic pressures (78 ± 16 in VD versus 110 ± 14 mm Hg in Co; p < 0.05) were significantly decreased after coronary ligation.

Body, Muscle, and Organ Weight

Body weight was not different between the two groups at the time of operation (375 ± 24 in VD versus 366 ± 23 g in Co rats) nor at the time of the study (442 ± 36 in VD versus 450 ± 27 g in Co rats).

In VD rats, heart weight was increased by 27% compared with that in control rats (1.4 ± 0.2 in VD versus 1.1 ± 0.1 g in Co rats; p < 0.005). Lung weight tended to be increased by 30% in VD rats (1.8 ± 0.5 g in VD and 1.4 ± 0.1 g in Co), but this increase was only borderline significant (p = 0.05). Liver weight was similar in the two groups (14.2 ± 1.9 g in VD and 14.5 ± 1.4 g in Co).

Diaphragm weight was significantly reduced by 17% in VD rats (622 ± 52 mg in VD versus 749 ± 54 mg in Co; p < 0.0005). For control and infarcted animals taken together, there was a significant inverse correlation between diaphragm weight and infarct size (r = 0.74; p < 0.001). For the infarcted animals only, the same tendency was present (r = 0.74; p = 0.06). VD did not affect the mass of the other respiratory or peripheral muscles examined.

Diaphragm Contractile Properties

P_t and P_o were similar in the two groups (pooled values of 506 ± 75 and 2,251 ± 365 g/cm², respectively). Similarly, TPT and 1/2 RT were identical (pooled values of 23 ± 8 and 22 ± 6 ms, respectively).

During the force-frequency curve, diaphragm force was comparable in the two groups at all frequencies, whether force was expressed as a percentage of the interposed 160 Hz stimulations (Figure 1) or in absolute terms. The decrease in interposed 160 Hz stimulations during the force-frequency protocol was 5 ± 6% and 8 ± 4% in VD and Co animals, respectively. This difference was not statistically significant.

View larger version (13K): [in this window] [in a new window]   Figure 1.   Force-frequency curve of the diaphragm in rats with VD (closed circles) and sham-operated rats (open circles). Force is expressed as a percentage of maximal tetanic force (Po).

During the low frequency fatigue run, no differences in force decline were observed between the two groups. Force decline at the end of the fatigue protocol averaged 57 ± 7 and 57 ± 8% in VD and Co rats, respectively (Figure 2).

View larger version (13K): [in this window] [in a new window]   Figure 2.   Low-frequency fatigue run of the diaphragm in rats with VD (closed circles) and sham-operated rats (open circles). Force is expressed as a percentage of initial force.

Histology and Histochemistry

Hematoxylin-eosin staining showed a normal muscular pattern of the diaphragm and gastrocnemius in the two groups. No myopathic alterations were present in VD rats in contrast to data obtained in cardiomyopathic Syrian hamsters before (18).

ATPase staining of the diaphragm revealed a significant atrophy of type I and type IIx/b fibers in VD rats, cross-sectional areas being reduced by 13 and 16%, respectively (p < 0.05 for both). CSA of type IIa fibers was not significantly decreased. The fiber proportions remained unchanged. In the gastrocnemius no significant alterations in fiber size or proportions were observed.

Both in diaphragm and gastrocnemius no significant alterations were seen in the residual interstitial space, expressed as a percentage of the total CSA. This averaged 11.2 ± 3.6% versus 9.8 ± 3.7% in diaphragm; 2.9 ± 2.2% versus 3.4 ± 3.08% in the internal part of the gastrocnemius, and 3.6 ± 2.8% versus 4.6 ± 2.2% in the external part of the gastrocnemius, in VD and Co rats, respectively.

Serum IGF-I Levels

The IGF-I serum levels were not significantly different between the two groups. They averaged 596 ± 77 ng/ml in VD animals compared with 630 ± 102 ng/ml in Co animals.

IGF-I mRNA Study

After hybridization with a rat cDNA probe for IGF-I, three bands were detected on Northern blotting of the liver, diaphragm (Figure 3), and gastrocnemius. The apparent sizes of these transcripts were 7 to 7.8, 1.6 to 2.1, and 0.8 to 1.2 kilobases, respectively. Because the largest band was the most prominent and was clearly distinct from the background, this band was used for phosphorimager quantification of IGF-I mRNA. These data were subsequently normalized to the data obtained after hybridization with an 18S rRNA probe. IGF-I expression was increased by 55% in the diaphragm of VD rats (24,022 ± 5,974 arbitrary units [AU] in VD versus 15,469 ± 1,455 AU in Co; p < 0.05). In the liver and gastrocnemius, IGF-I expression was comparable in the two groups.

View larger version (81K): [in this window] [in a new window]   Figure 3.   Northern blot autoradiography of the diaphragm in representative animals (VD = animals after coronary ligation; sham = sham-operated rats) after hybridization with -32P-IGF-I 3' cDNA (upper panel ) and 18S probe (bottom panel ). Location of ribosomal RNA (18S and 28S) is indicated.

	DISCUSSION

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The present study demonstrated that VD induced by coronary ligation in rats was associated with: (1) selective diaphragm atrophy, which was related to the infarct size; (2) a decrease in diaphragm type I and type IIx/b fiber cross-sectional area, without associated myopathic changes; (3) a specific increase in IGF-I expression in the diaphragm, which was not observed in liver or gastrocnemius.

Validation of the Model

The coronary ligation model has been widely used and accepted as a model for chronic heart failure. This model leads to heart failure, provided that the loss of myocardium is large enough (12). Because in our study infarctions were moderate (32 and 44% of the left ventricular circumference) and hemodynamic measurements revealed no signs of congestive heart failure, it was a model of VD and not of congestive heart failure. This is in keeping with previous studies in which only large infarctions (i.e., more than 47% of left ventricular circumference) produced important congestion (12). The slight increase in heart and lung weight observed, however, signals that limited congestion was present in our experiments. Muscle alterations may also be seen with smaller infarcts. Indeed, in rats with smaller infarctions, histologic alterations and pretranscriptional changes in myosin heavy chain expression in peripheral muscles have been described (19).

Diaphragm Is the Only Muscle Atrophied of All Muscles Examined in Our Model

The diaphragm was the only skeletal muscle atrophied of all muscles examined in the present study, suggesting that it is one of the most vulnerable muscles even to moderate degrees of VD. Some studies of heart failure in humans also suggest that the diaphragm is more affected than the limb muscles (3- 5), but to the best of our knowledge no clear demonstration of this is available in animal models. It is possible that moderate VD only affects the diaphragm, whereas severe heart failure also affects other inspiratory muscles and peripheral muscles. For the parasternals, a lack of detectable muscle atrophy might be related to the fact that muscle weight also included the sternum and costal cartilages. The absence of an effect on the scalenus medius might be due to differences in biochemical composition compared with the diaphragm. The scalenus medius is a predominantly glycolytic muscle, whereas the diaphragm is highly oxidative. The lack of alterations in the scalenus is in line with the observation that in a rat model of heart failure glycolytic metabolism was well preserved, whereas oxidative metabolism was decreased (20).

Contractile Properties and Histology

In the present study, contractile properties normalized to CSA remained unaltered. Because atrophy was present, a global reduction in diaphragm force was likely to be present. The observed muscle atrophy was not associated with alterations in contractile properties or fatiguability. This demonstrates the absence of myopathy or could be due to the fact that alterations were masked by the concomitant decrease in the CSA of both type I and type IIx/b canceling out each other's effect. These data are in contrast with the data obtained by Supinski and coworkers (21). They found in dogs with CHF induced by ventricular pacing, an increase in diaphragm fatiguability in vitro. Conversely, heart failure in Yucatan minipigs induced by supraventricular pacing, did not cause changes in diaphragm fatiguability (22). The latter is in line with the present study. Similarly, in a dog model of ventricular pacing, heart failure induced an atrophy of the gracilis muscles without any alteration in its contractile properties (23). Discordant results between different animal models may be related either to species differences or to the fact that the degree of VD was different in the above-mentioned studies. Hemodynamics are, however, difficult to compare since different variables have been measured and values are not necessarily comparable between different animal models.

The observed reduction in diaphragm weight can be explained by the type I and type IIx/b atrophy. Reduction in residual interstitial space appeared not to contribute to the diaphragm atrophy since it was comparable in VD and control animals. Only a few prior studies have addressed fiber composition or fiber surface area in the diaphragm in CHF in humans and in animals (5, 22, 24). Heart failure, induced by supraventricular tachycardia in Yucatan minipigs, caused a generalized fiber atrophy in the diaphragm (22), which was more or less in line with our findings. Other studies have yielded variable results, going from no fiber atrophy (5) to an atrophy pattern dependent upon age (24). Differences in age of the examined subjects as well as differences in the degree or etiology of heart failure, or species differences, are possible explanations for the variation in results.

IGF-I in Our Model

Serum IGF-I was significantly reduced in patients with myocardial infarction, suggesting that IGF-I metabolism may be altered (9). In the present study, however, no differences in IGF-I serum levels in VD rats were detected, which may be due to the fact that VD induced in our model was moderate. Along the same lines, expression of IGF-I in the liver was not significantly altered, confirming the unaltered IGF-I serum levels. In our model, diaphragm IGF-I mRNA was clearly increased. In other models of atrophy such as after denervation of the gastrocnemius (25), or after ischemia in the extensor digitorum of rats (26), a significant increase in IGF-I mRNA was also found. However, diaphragm atrophy in steroid-induced myopathy was associated with a decrease in IGF-I mRNA (10). In a malnutrition model, in which diaphragm atrophy was also present, IGF-I expression remained unchanged (10). The fact that different models of atrophy elicit different IGF-I patterns suggests that the underlying mechanism is not a simple and exclusive one but is modulated by different and still unknown factors. Determining IGF-I expression might possibly help in distinguishing different origins of muscle atrophy in the future.

The nature of the observed increase in IGF-I mRNA in the diaphragm cannot be elucidated from the present study. However, it may constitute a compensatory mechanism to minimize the atrophy induced by VD or to accelerate the regeneration process. Alternatively, the increased work load of the diaphragm secondary to the VD (27) may also induce an increase in IGF-I mRNA.