This Article Abstract Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Simonini, A. Articles by Massie, B. M. Search for Related Content PubMed PubMed Citation Articles by Simonini, A. Articles by Massie, B. M.

(Circulation Research. 1996;79:128-136.)
© 1996 American Heart Association, Inc.

Articles

Heart Failure in Rats Causes Changes in Skeletal Muscle Morphology and Gene Expression That Are Not Explained by Reduced Activity

Americo Simonini, Carlin S. Long, Gary A. Dudley, Ping Yue, Jill McElhinny, Barry M. Massie

the Department of Medicine (C.S.L., B.M.M.), Cardiovascular Research Institute (A.S., C.S.L., P.Y., B.M.M.), and Clinical Pharmacology Division (A.S.) of the University of California, San Francisco; the Cardiology Section (B.M.M., C.S.L.) of the Department of Veterans Affairs Medical Center, San Francisco, Calif; and the Department of Exercise Science (J.M., G.A.D.), the University of Georgia, Athens.

Correspondence to Barry M. Massie, MD, Cardiology Section (111C), Veterans Affairs Medical Center, 4150 Clement St, San Francisco, CA 94121. E-mail massie.barry@sanfrancisco.va.gov.

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
In patients with congestive heart failure, skeletal muscle is characterized by a smaller proportion of slow-twitch oxidative fibers and reduced oxidative enzyme activity. However, whether these changes result from disuse or occur as a direct consequence of heart failure is unresolved. To address this issue, 18 rats with heart failure 8 weeks after left coronary artery ligation and 13 sham-operated control rats underwent quantification of locomotor activity by a photocell activation technique, measurements of hemodynamics and infarct size, histochemical and morphological analyses of the soleus and plantaris muscles, and Northern analyses of muscle contractile protein and oxidative enzyme mRNA expression. Although the rats with heart failure had elevated left ventricular end-diastolic pressures (24.1±2.6 mm Hg) and a mean infarct size of 35.1±4.1%, activity levels were similar to those found in the sham-operated rats (3849±304 versus 3526±130 counts per hour). With heart failure, there was a significant reduction of type I fibers in the soleus muscle and type IIa fibers in the plantaris muscle, with corresponding increases in intermediate staining of type IIab fibers in both muscles. This was associated with a 17% decrease in citrate synthase activity in both the soleus and plantaris muscles (26.2±1.6 versus 30.7±3.4 and 29.1±2.4 versus 35.7±3.4 µmol/L per minute per gram, respectively [P<.05]). In the soleus muscle, mRNA for both ß-myosin heavy chains and cytochrome C oxidase III (normalized to 18S RNA) was reduced (0.27±0.02 versus 0.65±0.02 and 0.23±0.04 versus 0.64±0.02 U), whereas the messages for IIx and IIb myosin heavy chains were increased. A similar decrease in messages for cytochrome oxidase and the primary myosin isoform was observed in the plantaris muscle. Both soleus ß-myosin heavy chain and cytochrome C oxidase expression show significant inverse relationships to left ventricular end-diastolic pressure and infarct size. In contrast, there was no relationship between either ß-myosin heavy chain or cytochrome C oxidase expression and locomotor activity. These results indicate that in rats heart failure produces changes in skeletal muscle gene expression at the pretranslational level that cannot be explained by inactivity.

Key Words: heart failure • skeletal muscle • fiber types • gene expression • locomotor activity

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Exercise intolerance is a hallmark of CHF and reflects a number of derangements that accompany this syndrome, including impaired cardiac reserve, alterations in the peripheral circulation, abnormalities of ventilation, and changes in skeletal muscle.^{1 2 3 4 5 6} Evidence for the last of these includes changes in muscle function and metabolism,^{3 5 6} and the severity of these abnormalities correlates closely with the degree of impairment of exercise capacity.^{5 6} Muscle biopsy findings in these patients show a reduced proportion of slow-twitch oxidative fibers and decreased oxidative enzyme activity,^{7 8 9 10} which could explain, at least in part, the increased susceptibility to fatigue, impaired oxidative metabolism, and enhanced anaerobic glycolysis during exercise.

Since these findings in many ways resemble those that occur with disuse,³ an important question has been whether the muscle changes play a role in the reduction of exercise tolerance or merely reflect the lower level of activity of heart-failure patients. Since longitudinal studies of exercise capacity in conjunction with measurements of muscle function, histology, and biochemistry have not been performed, this question remains unresolved. Therefore, the present study used an animal model of heart failure to determine (1) whether there are changes in skeletal muscle fiber-type composition and biochemistry, (2) whether these "phenotypic" changes result from changes in contractile protein and oxidative enzyme gene expression, (3) whether the magnitude of these changes is related to the severity of left ventricular dysfunction, and (4) whether the magnitude of the muscle changes is quantitatively related to the level of locomotor activity.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Experimental Model
The rat coronary ligation model was used to induce heart failure.¹¹ After acclimatization for 1 week in the animal facility, female Sprague-Dawley rats weighing 240 to 260 g were anesthetized with 2% isoflurane, intubated, and maintained on a Harvard rodent ventilator. A left thoracotomy was performed at the fifth intercostal space, and while the heart was exteriorized, the left coronary artery was ligated 1 to 2 mm from its origin with a 7-0 suture ligation of the proximal left coronary artery. In sham-operated control rats, the same procedure was used, but the suture was not tied. The heart was then repositioned, and the muscle and skin layer were closed with a purse-string suture. In the infarct group, operative mortality was 30%, and overall survival (n=24) at the time of study 8 weeks after surgery was 50%. All sham-operated rats (n=13) survived. After surgery, rats were housed in two-animal Plexiglas cages and received food and water ad libitum.

Activity Monitoring
Locomotor activity of age-matched heart-failure and sham-operated control animals was measured 8 weeks after surgery by photocell monitoring.¹² Each animal was placed into a novel open-field environment (Opto-Varimex Minor, Columbus Instruments), measuring 16.5 in by 16.5 in, equipped with 15 photocell beams in each direction located 4 cm off the cage floor. Interruptions of photocell beams were cumulatively computer-registered in 10-minute intervals.

To determine the appropriate period of activity monitoring, five rats were placed in the monitoring cages for a 74-hour period. Activity was first quantified in blocks of 2 hours, and this revealed significantly greater locomotor activity in the initial 2 hours compared with subsequent periods, representing a period of familiarization with the new environment. Then, excluding the first 2 hour period, blocks of 12 hours encompassing the daytime and nighttime periods were compared for the 3-day period. Nighttime activity was significantly higher than daytime activity, but there was no significant difference between the three successive nocturnal periods (3263±323, 3458±89, and 2890±223 counts per hour for the first, second, and third nocturnal periods, respectively). Therefore, animals were monitored for 14 hours beginning at 6:00 PM, and the final 12 hours were used to quantify locomotor activity.

Hemodynamic Measurements
On the day following activity measurements, rats were lightly anesthetized using ketamine (70 mg/kg) plus xylazine (10 mg/kg), and the right carotid artery was cannulated with PE-50 tubing. Under continuous pressure monitoring, the arterial catheter was advanced retrogradely into the left ventricle, and pressures were recorded. Only animals with LVEDPs of >15 mm Hg were included in the heart-failure group (n=18). Ligated animals with lower LVEDPs were analyzed separately as a small infarct group (n=6).

Muscle Harvesting
With animals still anesthetized, the hindlimbs were dissected, and the left soleus and plantaris muscles were excised and quickly frozen in precooled isopentane before storage in liquid nitrogen for subsequent histochemical analysis. The contralateral hindlimb muscles were then excised, snap-frozen in liquid nitrogen, and stored for RNA analysis. The hearts were excised, and the right and left ventricles were weighed separately. The left ventricles were preserved in 10% formalin, sectioned into four slices along the long axis, and then stained with Masson's trichrome for infarct size determination by planimetry.¹¹ Infarct size was expressed as the mean percentage of the circumference that was infarcted in the four slices.

mRNA Analysis
Total cellular RNA was isolated by the method of Chomczynski and Sacchi.¹³ RNA was quantified by absorbance at 260 nm, and its integrity was confirmed by examining the 28S and 18S rRNA bands in ethidium bromide-stained agarose gels. Total RNA (15 µg per lane) was separated by denaturing agarose gel electrophoresis, subjected to alkali pretreatment, transferred to nylon membranes, and cross-linked by ultraviolet irradiation. ß-MHC mRNA levels were detected by hybridization to a 40-base oligodeoxyribonucleotide probe (Oncogene Science, Inc) complementary to a unique 3' untranslated region of rat ß-MHC mRNA. Twenty-base oligodeoxyribonucleotides complementary to IIa, IIb, and IIx MHC mRNAs were synthesized according to published sequences^{14 15} at the Biomolecular Resource Center, University of California, San Francisco. All blots were also hybridized with a 784-bp Pst I fragment of the cDNA-encoding rat heart (mitochondrial-encoded) COX subunit III¹⁶ (provided by Anne F. Martin, PhD, of the University of Cincinnati). The cDNA oligodeoxynucleotides and 18S RNA probes were ³²P-radiolabeled by random priming, T4 kinase, and T7 RNA polymerase and hybridized by established protocols.¹⁷ After hybridization and washing, blots were exposed to Hyperfilm (Amersham) at -70°C for up to 2 days. Hybridizations were accomplished with one probe at a time, with subsequent stripping and reprobing of the same blot. The MHC signals from each sample were normalized to the signal obtained with an antisense riboprobe for 18S RNA (Ambion, Inc) using a scanning densitometry program (Scan Analysis, Biosoft).

Histochemical and Enzyme Analyses
The midbelly was cut from each soleus and plantaris muscle for sectioning after warming to -20°C. These samples were mounted on tongue blades using a medium of OCT compound and tragagunth gum, frozen in liquid nitrogen, and rewarmed to -20°C. Serial sections, 10 µm, were subsequently cut at -20°C and dried in room air for 1 hour. The end portions of each muscle were stored again at -70°C until they were analyzed for citrate synthase activity.

Cross sections were analyzed for fiber-type composition using a qualitative assay for actomyosin myofibrillar ATPase activity, as performed previously.¹⁸ Fibers were classified as type I (black staining), type IIa (light staining), type IIb (dark staining), and type IIab (intermediate staining) after acid preincubation at pH 4.35. On average, 348±21 (mean±SE) and 1692±124 fibers were used for fiber-type composition analyses in the soleus and plantaris muscles, respectively. Fiber cross-sectional area was determined for type I or type II fibers using cross sections assayed for actomyosin myofibrillar ATPase activity. Analyses were performed with a semiautomated image-analysis system using public domain software Image (NIH), as described elsewhere.¹⁹ Fibers in regions of a given cross section that were histologically abnormal because of either freeze damage or oblique sectioning were excluded from analyses. There were 141±8 and 186±10 representative fibers of each type measured in the soleus and plantaris muscles, respectively. Cross-sectional area was not assessed separately in fast-twitch subtypes, because the low percentage of type IIa and type IIb fibers precluded valid measures. These analyses provided data describing the percentage of type I, IIa, IIb, and IIab fibers in each muscle as well as average cross-sectional area of type I and type II fibers.

Muscle capillarity was assessed by combining stains for alkaline phosphatase and glycoprotein.^{20 21} The number of capillaries per square millimeter was determined by averaging the number of capillaries counted within three to five regions of a cross section of each muscle, as described previously.²²

Citrate synthase activity was determined in the combined end portions of each soleus or plantaris muscle as originally described by Srere²³ and carried out by Luginbuhl et al.¹⁸ Briefly, samples were thawed, blotted dry, weighed, minced, homogenized (175 mmol/L KCl, 10 mmol/L glutathione, and 2 mmol/L EDTA), and centrifuged at 700g. The supernatant was assayed spectrophotometrically using 5,5'-dithio-bis(2-nitrobenzoate). Assays were performed at 30°C in a Bausch and Lomb spectrophotometer (Spectronic 1001, Milton Roy Co) equipped with a thermoelectric flow cell and 1-cm light path. Each supernatant was also assayed for total protein using the Lowry method (Kit P5656, Sigma Chemical Co).

Statistical Analysis
Fiber cross-sectional area data were analyzed using a three-way ANOVA (group by fiber type by muscle) for independent measures. Fiber-type percentage data for the soleus or plantaris muscle were analyzed using a two-way ANOVA (group by fiber type) for independent measures. Citrate synthase and capillary data were also compared between groups using two-way analysis, but the variables were group and muscle. A least-squares means test was used to compare means when significant interaction was found. Comparisons of weights, hemodynamic measurements, and quantitative mRNA variables between groups were made using a one-way ANOVA. Correlations were determined using linear regression and Pearson's product correlations. The use of linear regression was verified by the adequate r values and by examining the residuals, which showed a random pattern. Data are presented as mean±SE.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Hemodynamics and Organ Weights
Hemodynamics, weight, and locomotor activity data are shown in Table 1. The heart-failure group consisted of the 18 infarcted rats with LVEDPs of 15 mm Hg (mean, 24.1±2.6 mm Hg). Their mean left ventricular infarction size was 35.1±4.9%. The sham-operated group consisted of 13 animals with a mean LVEDP of 1.7±0.5 mm Hg. Right ventricular weight, normalized to body weight, was increased in heart-failure rats compared with sham-operated rats (0.94±0.04 versus 0.57±0.04 mg/g [P<.001]), suggesting chronically elevated pulmonary artery pressures in the heart-failure group. Left ventricular weight was also significantly increased (3.10±0.09 versus 2.89±0.08 mg/g [P<.006]). The soleus and plantaris muscles were smaller in the heart-failure rats than in the sham-operated rats (0.40±0.01 versus 0.48±0.02 mg/g [P<.001] and 1.05±0.07 versus 1.29±0.06 mg/g [P<.02], respectively).

View this table: [in this window] [in a new window]   Table 1. Weights, Hemodynamics, and Locomotor Activity Measurements

The small infarction group (n=6) had, by definition, LVEDPs of <15 mm Hg; their infarctions constituted 7.8±1.9% of the left ventricle. They had ventricular and muscular weights similar to those of the sham-operated control group.

Activity
Locomotor activities were quantified from the cumulative photocell counts triggered by each rat during the 12-hour nocturnal period in an Opto-Varimex box. Representative activity-time plots are shown in Fig 1. After the first hour, in which all animals displayed increased activity triggered by exposure to the new environment, activity levels were relatively constant over the monitoring period. The mean values for nocturnal activity were not different between heart-failure rats and sham-operated rats (3849±304 versus 3526±130 counts per hour), whether analyzed by parametric or nonparametric tests. The activity varied more in the heart-failure animals than in the sham-operated animals, as reflected in the large standard deviation, but this difference in the variances was not statistically significant (P=.37), and the values were normally distributed in both groups. Correlations between activity and the subsequently measured LVEDP, infarct size, and right ventricular weight were all poor (r>.16 for each relationship), indicating that the level of activity was not related to the severity of heart failure.

View larger version (36K): [in this window] [in a new window]   Figure 1. Plots of locomotor activity measurements of a typical heart-failure rat (top) compared with a control sham-operated rat (bottom) during the nocturnal cycle. Each point represents the cumulative photocell activation counts over a 10-minute interval. The first hour (or six intervals) of relative hyperactivity in both animals represents a "discovery" period after the animals are placed in the monitoring boxes. There was no significant difference in mean overnight photocell counts between the two groups.

RNA Analysis
Representative Northern blots of soleus and plantaris mRNAs are shown in Figs 2 through 4. The content of soleus mRNA encoding ß-MHC in heart-failure rats was 40% of that in sham-operated rats when the messages were normalized to 18S ribosomal RNA signals (0.27±0.02 versus 0.65±0.02 arbitrary units [P<.001]). COX III mRNA levels were also significantly reduced in the heart-failure animals (0.23±0.04 versus 0.64±0.02 arbitrary units [P<.001]). As is evident from Fig 4, neither IIb nor IIx MHC mRNA was detectable in the soleus muscle of the sham-operated rats. In contrast, among the heart-failure group, IIb MHC mRNA was detected in 7 of 11 soleus RNA samples analyzed (P=.003, ² test), whereas message for IIx was present in 8 of 11 samples (P=.002, ² test). MHC type IIa mRNA was not found in the soleus muscles of either group. When compared with sham-operated rats, the plantaris muscles of the heart-failure rats demonstrated a decrease in the predominant MHC mRNA species (types IIa and IIb) as well as a reduction in COX mRNA.

View larger version (43K): [in this window] [in a new window]   Figure 2. Representative Northern blot of soleus RNA extracts. In this and the following two figures, signals shown represent sequential probing of the same Northern blot with individual RNA-specific probes as indicated. Samples were standardized to the 18S (pT7 18S) rRNA to ensure equal loading. Levels of mRNAs encoding ß-MHC (the predominant MHC species in the soleus) and COX III (a mitochondrial oxidative enzyme) were reduced in heart-failure (CHF) rats (seven right lanes) compared with control rats (five left lanes).

View larger version (96K): [in this window] [in a new window]   Figure 3. Northern blot of plantaris muscle RNA extracts. Messages for IIa and IIb MHC (the predominant species in the plantaris) and for COX III were reduced in heart-failure (CHF) rats (three right lanes) compared with control (Con) rats.

View larger version (39K): [in this window] [in a new window]   Figure 4. Representative Northern blots demonstrating the changes in soleus MHC isoform mRNA levels. Whereas heart failure (right five lanes) induces a decrease in soleus type I myosin (ß-MHC) expression, it also results in a de novo expression of type II isoforms (IIb, IIx).

Histochemistry
As shown in Table 2, a significant difference was found between the fiber-type distribution in the heart-failure and control rats. The percentage of type I fibers was lower (89.8±1.8 versus 94.8±1.1 [P=.004]) in the soleus muscles of heart-failure rats compared with control rats. This muscle also showed a higher percentage of type IIab fibers in heart-failure rats (5.5±1.0 versus 1.7±1.6 [P<.03]). In the plantaris muscle, there were fewer type IIa (8.3±0.4 versus 14.1±1.4 [P<.025]) and more type IIab (79.5±1.6 versus 72.0±3.2 [P<.004]) fibers in heart-failure animals than in control animals. There was no significant difference between the groups with regard to fiber cross-sectional area in either muscle.

View this table: [in this window] [in a new window]   Table 2. Soleus and Plantaris Muscle Fiber-Type Composition in Heart-Failure and Control Rats

Citrate synthase activity was reduced by a mean of 17% in the heart-failure animals in both the soleus and plantaris muscles (26.2±1.6 versus 30.7±3.4 and 29.1±2.4 versus 35.7±3.4 µmol/L per minute per gram, respectively [P=.048]). There were no differences between the two groups in total protein or capillary density (Table 3).

View this table: [in this window] [in a new window]   Table 3. Soleus and Plantaris Muscle Citrate Synthase Activity, Total Protein Content, and Capillarity in Heart-Failure and Control Rats

Correlation of Heart-Failure Severity and Activity to Changes in Gene Expression
Fig 5 shows the relationships between the message for ß-MHC in the soleus muscle, normalized for 18S mRNA, and both the severity of heart failure, as quantified by LVEDP, and the level of locomotor activity. There was a significant inverse relationship between ß-MHC expression and end-diastolic pressure (r=-.83 for all animals [P<.001] and r=-.66 for infarct animals only [P=.01]) but no relationship with activity. Fig 6 shows the relationship between soleus COX message and the same indexes of heart failure and activity. Again, there was a significant inverse correlation with LVEDP (r=-.86 for all animals and r=-.76 for infarct animals only [both P<.001]) but not with activity. Very similar correlations were present between ß-MHC or COX expression and either infarct size (shown in Fig 7) or the right ventricular weight-to-body weight ratio.

View larger version (13K): [in this window] [in a new window]   Figure 5. Correlation of soleus ß-MHC expression (normalized to the 18S RNA signal) and LVEDP. There was an inverse correlation between type I myosin expression and the degree of heart failure (r=-.83 [P<.001] for all animals, r=-.66 [P<.01] for infarcted rats only). When correlated with activity, no relationship between ß-MHC and locomotion was found. MI indicates myocardial infarction; HF, heart failure.

View larger version (13K): [in this window] [in a new window]   Figure 6. Correlation of soleus COX III expression (normalized to the 18S RNA signal) and LVEDP. As with ß-MHC expression, there was a negative correlation between COX III expression and LVEDP (r=-.66 [P<.001] for all rats, r=-.76 [P<.001] for infarcted rats) and no relationship with activity. MI indicates myocardial infarction; HF, heart failure.

View larger version (12K): [in this window] [in a new window]   Figure 7. Correlations of soleus ß-MHC and COX mRNA (normalized to 18S mRNA) and left ventricular infarct size, showing significant inverse relationships (r=-.83 [P<.001] and r=-.87 [P<.001], respectively, for all animals; r=-.68 [P=.16] and r=-.78 [P=.003], respectively, for infarcted rats).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
A growing body of data indicate that patients with CHF often exhibit profound changes in skeletal muscle function, metabolism, and morphology.^{5 6 7 8 9 10} Many of these changes are inversely related to exercise capacity in these patients and have been thought to play a role in their exercise intolerance. The present study was designed to address the question of whether abnormalities of skeletal muscle occur as a consequence of heart failure itself or reflect a pattern of disuse secondary to inactivity. To accomplish this, we determined muscle fiber-type distribution, oxidative enzyme activity, and the expression of skeletal muscle contractile proteins and oxidative enzymes in a rat model of heart failure and then related these measurements to quantitative indexes of both the severity of heart failure and locomotor activity.

The principal findings of the present study were that rats with heart failure induced by coronary artery ligation exhibit (1) atrophy of both the soleus and plantaris muscles, (2) a decrease in the percentage of slow twitch-type fibers and an increase in transitional fast twitch-type IIab fibers in the normally slow-twitch soleus muscle, (3) a decrease in the activity of citrate synthase, a mitochondrial oxidative enzyme, (4) changes in mRNA encoding for contractile proteins, including a decrease in ß-MHC message with emergence of type IIb and IIx MHC, not characteristic of the soleus, and decrease in messages for the predominant MHC species within the plantaris, and (5) a decrease in message for the mitochondrial oxidative enzyme COX in both muscles. Most important, the observed changes in the soleus and plantaris muscles appeared to occur independently of locomotor activity but were strongly correlated to the severity of heart failure and the size of the myocardial infarction. The implications of these findings are discussed below.

Skeletal Muscle in CHF
Indexes of muscle endurance and strength are reduced in heart-failure patients, and quantitative relationships between these indexes and peak VO₂ have been demonstrated.^{5 24} Studies of muscle metabolism have also shown consistent abnormalities in CHF patients, such as rapid depletion of high-energy phosphates and development of intracellular acidosis with exercise^{3 4} ; these too are related to the severity of symptoms and exercise impairment.⁶ A variety of abnormalities of skeletal muscle morphology and biochemistry have been described previously.^{3 7 8 9 10} The most consistent of these have been a reduction in the proportion of type I fibers, atrophy of type II fibers, a decrease in the activity of a variety of oxidative enzymes, and changes in mitochondrial ultrastructure consistent with impaired oxidative capacity. The magnitude and consistency of these findings has led to the suggestion that heart failure may induce a form of myopathy,⁹ although as previously discussed, many of the changes could also be explained by disuse.

The myocardial infarction model used in the present study mimics many of the changes in skeletal muscle previously reported in patients with heart failure. As in patients, both the soleus and plantaris muscles were atrophied in rats with heart failure, and in the normally slow-twitch soleus, there was a decrease in the proportion of type I fibers and an increase in fast-twitch type II fibers, most prominently the type IIab variety. These changes in muscle phenotype corresponded to changes in expression of the several myosin isoforms. There was also a decrease in muscle oxidative enzyme activity and in the mRNA for COX. Both of these changes in gene expression were quantitatively related to the severity of heart failure.

Role of Disuse
Many of the changes in skeletal muscle in patients with CHF, such as muscle atrophy, fiber atrophy, and decreased oxidative enzyme activity, are similar to those observed with disuse.^{3 4} Furthermore, the ability to reverse some of the functional and metabolic changes with systemic or local muscle training^{25 26} and the gradual time course of improvement in exercise tolerance with therapy are consistent with deconditioning as a mechanism. On the other hand, some findings in heart failure are not typical of disuse, such as the relatively well-preserved muscle strength in spite of significant atrophy, the disproportionate atrophy of type II relative to type I fibers, and the rapid and extreme local muscular fatigue in patients that is disproportionate to the degree of reduced mitochondrial content.^{27 28 29} Because many changes in muscle are nonspecific and because heart failure in the clinical setting is usually accompanied by some decrease in activity, the contribution of deconditioning to the muscle findings is uncertain.

The additional assessments in the present study provide a further basis for comparing changes in heart failure with those expected from disuse. Skeletal muscle disuse has been extensively studied with regard to its effect on mitochondrial enzyme activity, protein synthesis, and gene expression. Both slow and fast muscles of the rat hindlimb subjected to hindlimb suspension, microgravity, immobilization, or denervation have exhibited changes in the content of mRNAs encoding for both the structural and metabolic proteins needed to sustain normal contractile activity, including ß-actin, MHC isoforms, COX, and citrate synthase. Investigations using tail-suspended rats have shown deconditioning to result in a similar shift from type I (slow) to type II (fast) fibers as found in muscle biopsies from heart-failure patients,³⁰ but the data on MHC mRNA levels in disuse have been variable. Whereas Thomason et al³¹ demonstrated decreases in the content and synthetic rate of myofibrillar proteins without a decrease in ß-MHC mRNA, Diffee et al³² reported concordant reductions in ß-MHC protein and mRNA levels similar those measured in our heart-failure group.

Impaired skeletal muscle metabolism secondary to reduced mitochondrial enzyme content is also a characteristic common to both heart failure and disuse. The expression and protein synthetic rates of COX, a multiunit mitochondrial component of the electron-transport chain, have been shown to respond to alterations in contractile activity (training and detraining).^{33 34} Further evidence for the role of pretranslational mechanisms in muscle atrophy was demonstrated in the rat model by the decrease of both COX mRNA and ß-actin after limb immobilization.³⁵ The association between muscle contractile activity and mitochondrial and sarcomeric gene transcription has been the rationale for suggesting disuse as a primary mechanism in the development of skeletal muscle abnormalities in patients with CHF.³⁶

Locomotor Activity
Perhaps the most important new information provided by the present study is the quantitative measurements of locomotor activity. Laboratory rats maintained on a 12/12-hour light-dark cycle and given continuous access to food and water consume over 75% of their food and water and exhibit 90% of their locomotor activity at night.³⁷ This nocturnal activity in rats corresponds to an elevation in skeletal muscle metabolism and limb muscle blood flow, resulting in an increase in cardiac output.³⁸ In the present investigation, measurements of locomotor activity made at night, when foraging and exploratory activities were presumably at their highest, did not distinguish the heart-failure group from the sham-operated group. This finding is in agreement with a previous study (Teerlink and Clozel³⁹ ) that employed telemetric monitoring of activity in rats with similar degrees of heart failure after coronary ligation (infarct size, 39±2% of the left ventricle compared with 35±5% in the present study). In the study of Teerlink and Clozel, no difference in activity was observed between heart-failure rats and sham-operated rats when 7-day periods of continuous monitoring were compared over an 8-week period after infarction.

Since the soleus muscle, which exhibited the greatest histochemical and gene expression changes, is a postural hindlimb muscle that is involved in these forms of activity, it very likely that this activity assessment provided an accurate measure of soleus activity. The lack of intergroup differences in activity strongly suggests that mechanisms other than disuse are involved in the genesis of the muscle changes. Furthermore, the lack of a relationship between changes in gene expression and the activity level within the heart-failure rats despite the larger variation in locomotor activity in this group argues against inactivity as the responsible mechanism. However, since few of these rats had low activity levels, these data do not exclude the possibility that disuse could cause still greater abnormalities in skeletal muscle.

Significance
As important as the lack of relationship between the measured level of activity and the severity of the muscle abnormalities is their strong correlation with indexes of heart-failure severity. What possible mechanisms may explain this relationship? The striking plasticity of muscle phenotype is well characterized and is illustrated by the heterogeneity of contractile protein isoforms and their modulation by a variety of stimuli.⁴⁰ In addition to training and disuse, altered loading, electrical stimulation, and hormone administration can induce changes of the magnitude associated with heart failure.^{14 41 42} CHF is a multidimensional syndrome characterized by a number of abnormalities that could affect skeletal muscle. Potential candidates include the altered neurohormonal milieu, impaired muscle blood flow, changes in the release of endothelial regulating factors, and cytokine activation, all of which are known to induce changes in sarcomeric growth and gene expression in skeletal or cardiac muscle.^{43 44 45}

The changes in sarcomeric and mitochondrial gene expression in heart-failure animals observed in the present study could represent the effect of factors that influence either transcriptional or posttranscriptional mechanisms (ie, mRNA stability, rate and/or efficiency of mRNA translation, or protein half-life). The Northern and histological data from the soleus and plantaris muscles suggest that the altered phenotype associated with impaired muscle function and metabolism observed in patients is most likely due, at least in part, to a transcriptional mechanism.

In addition to the need to define the mechanism responsible for the skeletal muscle abnormalities more precisely, several further questions arise. Is this a specific form of myopathy, or do similar changes occur in other chronic disease conditions? Are these changes responsible for the functional and metabolic abnormalities in heart-failure patients, and to what extent do they contribute to their reduction in exercise tolerance?

Limitations
Although conducting these experiments in an animal model permitted a collection of data that is not available in clinical studies, the issue always arises as to whether these results can be extrapolated to humans. In this regard, the rat infarct model is characterized by many of the hemodynamic and neurohormonal changes typical of heart failure in humans, and studies of interventions with this model have proven predictive of subsequent clinical trials.⁴⁶ This model is also characterized by impaired exercise tolerance, abnormal muscle function and metabolism, and reduced oxidative enzyme content.^{47 48}

All mRNA signals were scanned, and densitometries were calculated well within the linear range to avoid the risk of oversaturation. Although many of the mRNAs analyzed appeared to be reduced, the increase in type II MHC species in the soleus provides evidence that heart failure is associated with altered transcriptional regulation and not merely a global reduction in gene expression. Furthermore, mRNA changes were also associated with alterations in the muscle phenotype, as evidenced by the histochemical and biochemical data.

The measurements of locomotor activity were performed for a 12-hour period. Although the period was relatively brief, several hours are generally considered more than sufficient to assess rodent activity.^{49 50} Furthermore, in our pilot studies, no differences in locomotor activity were observed during 3 consecutive days of monitoring, and as noted above, heart-failure rats monitored continuously by telemetry over 8 weeks also did not exhibit reduced activity.³⁹

Conclusion
Exercise intolerance is a major manifestation of CHF, and skeletal muscle abnormalities appear to contribute to this limitation. However, whether the muscle changes are primary or secondary to reduced activity levels has been unresolved. The present study shows for the first time that heart failure is associated with changes in gene expression that could explain many of the morphological and biochemical changes in skeletal muscle and, possibly, the functional and metabolic abnormalities. Most important, these changes are strongly related to the severity of heart failure but not to quantitative measurements of locomotor activity. Taken together, these findings suggest that the skeletal muscle abnormalities in CHF are mediated, at least in part, by transcriptional mechanisms not attributable to disuse.

	Selected Abbreviations and Acronyms

 

CHF = congestive heart failure COX = cytochrome C oxidase LVEDP = left ventricular end-diastolic pressure MHC = myosin heavy chain

	Acknowledgments

 
This study was supported by grant 5T32-GM07456 (Dr Simonini) from the National Institutes of Health, by the Department of Veterans Affairs Research Service (Drs Long, Massie, and Dudley), and by Grant-in-Aid 92-41 from the American Heart Association, California Affiliate, Inc (Dr Simonini). We would like to thank Paul C. Simpson, MD, for his assistance and guidance and the staff of the Laboratory of Neurology and Psychiatry, Department of Psychiatry, University of California, San Francisco (Paul Berger, MD, Bryan Tolliver, Malcolm Reid, and Kevin Souzer) for their assistance in obtaining locomotor analyses.

Received December 26, 1995; accepted April 3, 1996.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Mason DT, Zelis R, Longhurst J, Lee G. Cardiocirculatory responses to muscular exercise in congestive heart failure. Prog Cardiovasc Dis. 1977;19:475-489.[Medline] [Order article via Infotrieve]
2. Weber KT, Kinasewitz GT, Janicki JS, Fishman AP. Oxygen utilization and ventilation during exercise in patients with chronic cardiac failure. Circulation. 1982;65:1213-1223.[Abstract/Free Full Text]
3. Minotti JR, Christoph I, Massie BM. Skeletal muscle function, morphology, and metabolism in patients with congestive heart failure. Chest. 1992;101(suppl):333S-339S.
4. Wilson JR, Mancini DM. Factors contributing to the exercise limitation of heart failure. J Am Coll Cardiol. 1993;22(suppl A):93A-98A.
5. Minotti JR, Christoph I, Oka R, Wiener MW, Wells L, Massie BM. Impaired skeletal muscle function in patients with congestive heart failure: relationship to systemic exercise performance. J Clin Invest. 1991;88:2077-2082.
6. Massie BM, Conway M, Yonge R, Frostick S, Sleight P, Ledingham J, Radda G, Rajagopalan B. Skeletal muscle metabolism in patients with congestive heart failure: relation to clinical severity and blood flow. Circulation. 1987;76:1009-1019.[Abstract/Free Full Text]
7. Mancini DM, Coyle E, Coggan A, Beltz A, Ferraro N, Montain S, Wilson JR. Contribution of intrinsic skeletal muscle changes to 31P NMR skeletal muscle abnormalities in patients with chronic heart failure. Circulation. 1989;80:1338-1346.[Abstract/Free Full Text]
8. Sullivan MJ, Green HJ, Cobb FR. Skeletal muscle biochemistry and histology in ambulatory patients with chronic heart failure. Circulation. 1990;80:1338-1346.
9. Drexler H, Reide U, Just H. Alterations of skeletal muscle in chronic heart failure. Circulation. 1992;85:1751-1759.[Abstract/Free Full Text]
10. Massie BM, Simonini A, Sahgal P, Wells L, Dudley GA. Relationship of systemic and local muscle exercise capacity to skeletal muscle characteristics in patients with congestive heart failure. J Am Coll Cardiol. 1996;27:140-146.[Abstract]
11. Pfeffer MA, Pfeffer JM, Fishbein MC, Fletcher PJ, Spadaro J, Kloner RA, Braunwald E. Myocardial infarct size and ventricular function in rats. Circ Res. 1979;44:503-512.[Abstract/Free Full Text]
12. Tyler TD, Tessel RE. A new device for the simultaneous measurement of locomotor and stereotypic frequency in mice. Psychopharmacology. 1979;64:285-90.[Medline] [Order article via Infotrieve]
13. Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987;162:156-159.[Medline] [Order article via Infotrieve]
14. Swoap SJ, Haddad F, Caiozzo VJ, Herrick RE, McCue SA, Baldwin KM. Interaction of thyroid hormone and functional overload on skeletal muscle isomyosin expression. J Appl Physiol. 1994;77:621-629.[Abstract/Free Full Text]
15. Adams GR, Haddad F, Baldwin KM. Interaction of chronic creatine depletion and muscle unloading: effects on postural and locomotor muscles. J Appl Physiol. 1994;77:1198-1205.[Abstract/Free Full Text]
16. Grosskopf R, Feldmann H. Analysis of a DNA segment from rat liver mitochondria containing the genes for cytochrome oxidase subunits I, II, and III, ATPase subunit 6, and several tRNA genes. Curr Genet. 1981;4:151-158.
17. Maniatas T, Fritsch EF, Sambrook J. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Publications; 1982.
18. Luginbuhl AJ, Dudley GA, Staron RS. Fiber type changes in rat skeletal muscle after intense interval training. Histochemistry. 1984;81:55-58.[Medline] [Order article via Infotrieve]
19. Ploutz LL, Tesch PA, Biro RL, Dudley GA. Effects of resistance training on muscle use during exercise. J Appl Physiol. 1994;76:1675-1681.[Abstract/Free Full Text]
20. Seligman AM, Heymann H, Barrnett RJ. The histochemical demonstration of alkaline phosphatase activity with indoxyl phosphate. J Histochem Cytochem. 1954;2:441-442.
21. Tranzer JP, Pearse AGE. Cytochemical demonstration of ubiquinones in animal tissues. Nature. 1963;199:1063-1065.[Medline] [Order article via Infotrieve]
22. Hather BM, Mason CE, Dudley GA. Histochemical demonstration of skeletal muscle fibre types and capillaries on the same transverse section. Clin Physiol. 1991;11:127-134.[Medline] [Order article via Infotrieve]
23. Srere PA. Citrate synthase. Methods Enzymol. 1969;230:946-950.
24. Minotti JR, Pillay P, Oka R, Wells L, Christoph I, Massie BM. Skeletal muscle size: relationship to muscle function in heart failure. J Appl Physiol. 1993;75:373-381.[Abstract/Free Full Text]
25. Minotti JR, Johnson EC, Hudson TL, Zuroske G, Murata G, Fukushima E, Cagle TG, Chick TW, Massie BM, Icenogle MV. Skeletal muscle response to exercise training in congestive heart failure. J Clin Invest. 1990;86:751-758.
26. Stratton JR, Dunn F, Adamopoulos S, Kemp GJ, Coats AJ, Rajagopalan B. Training partially reverses skeletal muscle metabolic abnormalities during exercise in heart failure. J Appl Physiol. 1994;76:1575-1582.[Abstract/Free Full Text]
27. Hather BM, Adams GR, Tesch PA, Dudley GA. Skeletal muscle responses to lower limb suspension in humans. J Appl Physiol. 1992;72:1943-1948.
28. Hikida RS, Gollnick PD, Dudley GA, Convertino VA, Buchanan P. Structural and metabolic characteristics of human skeletal muscle following 30 days of simulated microgravity. Aviat Space Environ Med. 1989;60:664-670.[Medline] [Order article via Infotrieve]
29. Berg HE, Dudley GA, Hather B, Tesch PA. Work capacity and morphologic characteristics of the human quadriceps in response to unloading. Clin Physiol. 1993;13:337-347.[Medline] [Order article via Infotrieve]
30. Thomason DB, Herrick RE, Surdyka D, Baldwin KW. Time course of soleus myosin expression during hindlimb suspension and recovery. J Appl Physiol. 1987;63:130-137.[Abstract/Free Full Text]
31. Thomason DB, Biggs RB, Booth FW. Protein metabolism and ß-myosin heavy-chain mRNA in unweighted soleus muscle. Am J Physiol. 1989;257:R300-R305.[Abstract/Free Full Text]
32. Diffee GM, Haddad F, Herrick RE, Baldwin KM. Control of myosin heavy chain expression: interaction of hypothyroidism and hindlimb suspension. Am J Physiol. 1991;261:C1099-C1106.[Abstract/Free Full Text]
33. Booth FW. Changes in skeletal muscle gene expression consequent to altered weight bearing. Am J Physiol. 1992;262:R329-R332.[Abstract/Free Full Text]
34. Williams RS. Regulation of nuclear and mitochondrial gene expression by contractile activity in skeletal muscle. J Biol Chem. 1986;261:376-380.[Abstract/Free Full Text]
35. Babij P, Booth FW. Alpha-actin and cytochrome C mRNAs in atrophied adult rat skeletal muscle. Am J Physiol. 1988;254:C651-C656.[Abstract/Free Full Text]
36. Williams RS, Harlan W. Adaptation of skeletal muscle to increased contractile activity. J Biol Chem. 1987;262:2764-2767.[Abstract/Free Full Text]
37. Delp MD, Manning RO, Bruckner JV, RB Armstrong RB. Distribution of cardiac output during diurnal changes in activity in rats. Am J Physiol. 1991;261:H1487-H1493.[Abstract/Free Full Text]
38. Smith TL, Coleman KA, Stanek KA, Murphy WR. Hemodynamic monitoring for 24 hours in unanesthetized rats. Am J Physiol. 1987;253:H1335-H1341.[Abstract/Free Full Text]
39. Teerlink JR, Clozel J-P. Hemodynamic variability and circadian rhythm in rats with heart failure: role of locomotor activity. Am J Physiol. 1993;264:H2111-H2118.[Abstract/Free Full Text]
40. Mahdavi V, Izumo S, Nadal-Ginard B. Developmental and hormonal regulation of sarcomeric myosin heavy chain gene family. Circ Res. 1987;60:804-814.[Abstract/Free Full Text]
41. Schiaffino S, Reggiani C. Myosin isoforms in mammalian skeletal muscle. J Appl Physiol. 1994;77:493-501.[Abstract/Free Full Text]
42. Periasamy M, Gregory P, Martin BJ, Stirewalt WS. Regulation of myosin heavy-chain gene expression during skeletal muscle hypertrophy. Biochem J. 1989;257:691-698.[Medline] [Order article via Infotrieve]
43. Klein I, Ojamaa K, Samarel AM, Welikson R, Hong C. Hemodynamic regulation of myosin heavy chain gene expression: studies in the transplanted heart. J Clin Invest. 1992;89:68-73.
44. Nadal-Ginard B, Mahdavi V. Molecular basis of cardiac performance: plasticity of the myocardium generated through protein isoform switches. J Clin Invest. 1989;84:1693-1700.
45. Waspe LE, Ordahl CP, Simpson PC. The cardiac beta-myosin heavy chain isogene is induced selectively in alpha 1-adrenergic receptor-stimulated hypertrophy of cultured rat heart myocytes. J Clin Invest. 1990;85:1206-1214.
46. Pfeffer JM, Pfeffer MA, Braunwald E. Influence of chronic captopril therapy on the infarcted left ventricle of the rat. Circ Res. 1985;57:84-95.[Abstract/Free Full Text]
47. Musch TI, Moore RI, Leathers DJ, Bruno A, Zelis R. Endurance training in rats with chronic heart failure induced by myocardial infarction. Circulation. 1986;74:431-441.[Abstract/Free Full Text]
48. Arnolda L, Brosnan J, Rajagopalan B, Radda GK. Skeletal muscle metabolism in heart failure in rats. Am J Physiol. 1991;261:H434-H442.[Abstract/Free Full Text]
49. Ossenkopp KP, Macrae LK, Teskey GC. Automated multivariate measurement of spontaneous motor activity in mice: time course and reliabilities of the behavioral measures. Pharmacol Biochem Behav. 1987;27:565-568.[Medline] [Order article via Infotrieve]
50. Minematsu S, Hiruta M, Taki M, Fujii Y, Aburada M. Automatic monitoring system for the measurement of body weight, food and water consumption and spontaneous activity of a mouse. J Toxicol Sci. 1991;16:61-73.[Medline] [Order article via Infotrieve]

This article has been cited by other articles:

				J. M. Lawler, H.-B. Kwak, W. Song, and J. L. Parker Exercise training reverses downregulation of HSP70 and antioxidant enzymes in porcine skeletal muscle after chronic coronary artery occlusion Am J Physiol Regulatory Integrative Comp Physiol, December 1, 2006; 291(6): R1756 - R1763. [Abstract] [Full Text] [PDF]

				S. Jelic and T. H. Le Jemtel Diagnostic Usefulness of B-Type Natriuretic Peptide and Functional Consequences of Muscle Alterations in COPD and Chronic Heart Failure. Chest, October 1, 2006; 130(4): 1220 - 1230. [Abstract] [Full Text] [PDF]

				J. Zoll, L. Monassier, A. Garnier, B. N'Guessan, B. Mettauer, V. Veksler, F. Piquard, R. Ventura-Clapier, and B. Geny ACE inhibition prevents myocardial infarction-induced skeletal muscle mitochondrial dysfunction J Appl Physiol, August 1, 2006; 101(2): 385 - 391. [Abstract] [Full Text] [PDF]

				D. J. Barr, H. J. Green, D. S. Lounsbury, J. W. E. Rush, and J. Ouyang Na+-K+-ATPase properties in rat heart and skeletal muscle 3 mo after coronary artery ligation J Appl Physiol, August 1, 2005; 99(2): 656 - 664. [Abstract] [Full Text] [PDF]

				A. Linke, V. Adams, P. C. Schulze, S. Erbs, S. Gielen, E. Fiehn, S. Mobius-Winkler, A. Schubert, G. Schuler, and R. Hambrecht Antioxidative Effects of Exercise Training in Patients With Chronic Heart Failure: Increase in Radical Scavenger Enzyme Activity in Skeletal Muscle Circulation, April 12, 2005; 111(14): 1763 - 1770. [Abstract] [Full Text] [PDF]

				J. L. Hagen, D. J. Krause, D. J. Baker, M. H. Fu, M. A. Tarnopolsky, and R. T. Hepple Skeletal Muscle Aging in F344BN F1-Hybrid Rats: I. Mitochondrial Dysfunction Contributes to the Age-Associated Reduction in VO2max J. Gerontol. A Biol. Sci. Med. Sci., November 1, 2004; 59(11): 1099 - 1110. [Abstract] [Full Text] [PDF]

				P. McDonough, B. J. Behnke, T. I. Musch, and D. C. Poole Effects of chronic heart failure in rats on the recovery of microvascular PO2 after contractions in muscles of opposing fibre type Exp Physiol, July 1, 2004; 89(4): 473 - 485. [Abstract] [Full Text] [PDF]

				R. Ventura-Clapier, A. Garnier, and V. Veksler Energy metabolism in heart failure J. Physiol., February 15, 2004; 555(1): 1 - 13. [Abstract] [Full Text] [PDF]

				R. T. Hepple, K. D. Ross, and A. B. Rempfer Fiber Atrophy and Hypertrophy in Skeletal Muscles of Late Middle-Aged Fischer 344 x Brown Norway F1-Hybrid Rats J. Gerontol. A Biol. Sci. Med. Sci., February 1, 2004; 59(2): B108 - 117. [Abstract] [Full Text] [PDF]

				S. Gielen, V. Adams, S. Mobius-Winkler, A. Linke, S. Erbs, J. Yu, W. Kempf, A. Schubert, G. Schuler, and R. Hambrecht Anti-inflammatory effects of exercise training in the skeletal muscle of patients with chronic heart failure J. Am. Coll. Cardiol., September 3, 2003; 42(5): 861 - 868. [Abstract] [Full Text] [PDF]

				T. E. Richardson, C. A. Kindig, T. I. Musch, and D. C. Poole Effects of chronic heart failure on skeletal muscle capillary hemodynamics at rest and during contractions J Appl Physiol, September 1, 2003; 95(3): 1055 - 1062. [Abstract] [Full Text] [PDF]

				B. Helwig, K. M. Schreurs, J. Hansen, K. S. Hageman, M. G. Zbreski, R. M. McAllister, K. E. Mitchell, and T. I. Musch Training-induced changes in skeletal muscle Na+-K+ pump number and isoform expression in rats with chronic heart failure J Appl Physiol, June 1, 2003; 94(6): 2225 - 2236. [Abstract] [Full Text] [PDF]