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(Circulation. 1997;95:910-916.)
© 1997 American Heart Association, Inc.

Articles

Chronic Congestive Heart Failure Elicits Adaptations of Endurance Exercise in Diaphragmatic Muscle

Boris Tikunov, PhD; Sanford Levine, MD; Donna Mancini, MD

the Division of Circulatory Physiology (D.M.), Columbia Presbyterian Medical Center, New York, NY, and the Philadelphia Veterans Administration Medical Center, Philadelphia, Pa.

	Abstract

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Background During rest and exercise, patients with heart failure hyperventilate; therefore, the diaphragm can be viewed as undergoing constant moderate-intensity exercise. Accordingly, we hypothesized that heart failure elicits adaptations in the diaphragm similar to those elicited by endurance exercise in the limb muscles of normal subjects.

Methods and Results Costal diaphragmatic biopsy samples were obtained from 7 normal subjects (age, 36±20 years) and 10 patients (age, 50±6 years; left ventricular ejection fraction, 18±8%) at the time of transplant or left ventricular assist-device placement. We measured the distribution of myosin heavy chain isoforms I, IIa, and IIb by SDS gel electrophoresis. We also measured the activities of the following enzymes: citrate synthase, a marker of oxidative metabolism; ß-hydroxyacyl-CoA dehydrogenase, a marker of lipolytic metabolism; and lactate dehydrogenase, a marker of glycolytic metabolism. In normal subjects, the distribution of myosin heavy chain isoforms I, IIa, and IIb was 43±2%, 40±2%, and 17±1%, respectively. In contrast, in heart failure subjects, the fiber distribution was 55±2%, 38±2%, and 7±2% for types I, IIa, and IIb, respectively. Therefore, in heart failure, myosin heavy chain I is increased (P<.0001) and myosin heavy chain IIb decreased from normal levels (P<.001). Additionally, citrate synthase activity (normal, 0.33±0.14; heart failure, 0.54±0.21 µmol·min^-1·mg protein^-1; P<.05) and ß-hydroxyacyl-CoA dehydrogenase activity (normal, 0.27±0.04; heart failure, 0.38±0.02 µmol·min^-1·mg protein^-1; P<.05) were greater in heart failure patients than in normal subjects, whereas lactate dehydrogenase activity was significantly less in heart failure patients than in normal subjects (normal, 11.6±4.6; heart failure,: 4.3±2.2 µmol·min^-1·mg protein^-1; P<.01).

Conclusions In the diaphragm in heart failure, there is a shift from fast to slow myosin heavy chain isoforms with an increase in oxidative capacity and a decrease in glycolytic capacity. These diaphragmatic muscle changes are consistent with those elicited by endurance training of the limb muscles in normal subjects.

Key Words: heart failure • muscles • diaphragm

	Introduction

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Exertional fatigue in patients with heart failure may result in part from intrinsic skeletal muscle changes. Many investigators have reported a variety of histochemical abnormalities in the limb musculature of these patients, including a decreased percentage of slow-twitch, oxidative, type I fibers; an increased percentage of fast-twitch, glycolytic, type IIb fibers; type II fiber atrophy; a decrease in oxidative enzymes; and mitochondrial abnormalities.^{1 2 3 4 5 6 7 8 9 10} All these changes suggest a shift from oxidative to glycolytic metabolism in these patients. The mechanism underlying these limb-muscle abnormalities is not known. Potential mechanisms include relative inactivity of the limb muscles in patients with heart failure, chronic underperfusion of limb muscles, and the effect of heart failure–related neurohumoral factors. It is unknown whether the heart failure state elicits a generalized myopathic process or if specific muscle changes occur.

In contrast to the limb muscles, little is known about adaptations elicited by heart failure in the respiratory muscles. The literature is largely limited to studies of the diaphragm in several animal models of heart failure. Recently, Supinski et al¹¹ showed a decrease in maximum transdiaphragmatic pressure with an increase in muscle fatigability in a canine heart failure model. Similarly, a decrease in maximum tetanic force has been observed during in vitro electric stimulation of diaphragmatic strips obtained from rabbits in heart failure.¹² Howell et al¹³ described decreases in maximum transdiaphragmatic pressure without any changes in fatigability in Yucatan minipigs with heart failure. Those authors also noted an increase in cross-sectional area of all fiber types and an increase in the proportion of fast-twitch fibers. There is only one preliminary report on changes elicited by heart failure in the diaphragm of patients undergoing cardiac transplantation. Lindsay et al¹⁴ reported the appearance of pathological "cores," tubular aggregates, and "bizarre myosin types." Reported fiber-type distribution and area were not different from those in control subjects undergoing cardiac surgery.

A variety of pulmonary function abnormalities have been described in patients in heart failure.^{15 16 17} The lungs of patients with heart failure exhibit a restrictive impairment on conventional pulmonary function tests that is accompanied by decreases in dynamic compliance and increases in airway resistance; all of these abnormalities result in an increased work of breathing.^{18 19} Previously, we demonstrated that the tension-time index of the diaphragm in heart failure patients, a measurement that reflects energy expenditure, was greater than normal both at rest and during exercise in these patients.²⁰ Thus, the diaphragm in heart failure patients can be viewed as undergoing constant moderate-intensity exercise. Therefore, we hypothesized that the diaphragm in heart failure patients would exhibit adaptations similar to those noted in the limb muscles of normal subjects who have undergone endurance training. An alternate hypothesis was that skeletal muscle histochemical abnormalities similar to those seen in the limb would be observed if there was a generalizable myopathic process in heart failure. We performed the present study to test these hypotheses.

	Methods

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Patient Population
The average age of the 2 women and 8 men who participated in the study was 50±6 years (range, 38 to 57 years). Left ventricular ejection fraction was 18±8%. The origin of heart failure was dilated cardiomyopathy in 7 patients and coronary artery disease in the remaining 3. Mean duration of illness was 27±3 months (range, 1 to 76 months). Peak VO₂ was severely reduced at 4.9±3.3 mL·kg^-1·min^-1. Six of the 10 patients were assigned an assumed peak VO₂ of 3.5 mL·kg^-1·min^-1 (ie, equivalent to resting level of one metabolic unit) as the specimens were obtained at the time of left ventricular assist-device placement. All patients were currently nonsmokers, whereas 5 of the patients had a past history of smoking. All patients had received therapy with digoxin, high-dose diuretics, and ACE inhibition. Only 3 patients were not receiving parenteral inotropic support at the time of study. Pulmonary vascular resistance averaged 3.7±1.9 Wood units.

Seven "brain-dead" organ donors (four women, three men) with an average age of 36±20 years (range, 17 to 64 years) served as control subjects. These subjects tended to be younger than the patients, but the difference in age between the two groups did not achieve statistical significance. Only two subjects had a past history of smoking. All subjects were intubated and mechanically ventilated. The average duration of immobilization (ie, the time from admission to harvest) for the control subjects was 27±9 hours. The majority of patients were victims of head trauma sustained during motor vehicle accidents. Only two of the control subjects were receiving moderate to high-dose inotropic support (Table 1).

View this table: [in this window] [in a new window]   Table 1. Clinical Characteristics of Control Subjects

The protocol was approved by the Human Studies Committee at Columbia Presbyterian Hospital. Written informed consent was obtained from all heart failure subjects, whereas approval for the control diaphragmatic biopsies was obtained from the donor families by the regional procurement agency.

Protocol
Diaphragmatic biopsies were performed under direct vision at the time of cardiac transplantation and/or insertion of left ventricular assist device in patients with heart failure. Diaphragmatic biopsies in control subjects were obtained from donors at the time of harvest. All needle biopsies were procured from the left anterior quadrant of the costal diaphragm.

One specimen was frozen immediately in liquid nitrogen for biochemical analysis. The other specimen was placed in embedding medium and frozen in isopentane cooled by liquid nitrogen for histochemical analysis.

Biochemical Measurements
Two types of biochemical measurements were performed: proportions of various MHC isoforms and assays for activities of three enzymes of intermediary metabolism—CS, BOAC, and LDH.

First, the biochemistry biopsy specimens were weighed, minced with scissors, and homogenized on ice in 5 vol of 100 mmol/L Tris-HCl (pH 8.0) with 5% BSA. Portions of these homogenates were used to determine the above-noted enzyme activities.^{21 22} Other portions of these homogenates were centrifuged at 4000g for 15 minutes at 4°C, and the pellet was used for preparation of myofibrils according to the method of Solaro et al.²³ ß-Mercaptoethanol (4 mmol/L) was included in all solutions. Last, myofibrils were diluted with 50% glycerol and stored at -20°C. The concentrations of proteins were determined by Bradford's reaction with the use of a Bio-Rad Standard Protein Plus kit.

SDS-Gel Electrophoresis for MHCs
The MHC isoforms²⁴ were quantified by modification of the method of Talmadge and Roy.²⁵ First, samples were prepared by dilution of myofibrils with SDS reducing buffer and then heated at 95°C for 4 to 6 minutes. SDS-gel electrophoresis was performed²⁵ with the following modifications: (1) glycerol concentration in the separating gel was increased from 30% to 40%, and (2) the gels were initially run at 50 V for 1 hour, then at 150 V for 2 hours, and then at 220 V for 40 hours at 8°C. Protein (0.1 to 0.2 mg) was loaded for each lane. A Bio-Rad Protean IIxi cell of 16x20-cm configuration and 20-well combs of 0.75-mm thickness were used in these experiments. Subsequently, the gels were fixed and stained with silver by use of Bio-Rad Silver Stain Plus kits or with Coomassie R-250 followed by destaining with 40% methanol and 10% acetic acid. Thereafter, the bands were analyzed by scanning the gels with Pharmacia LKB Ultrascan densitometer. All quantitated measures of MHC refer to the silver stains, as do the illustrative gels.

Western Blotting
For Western blot analysis, proteins were transferred from the SDS gels onto polyvinylidenefluoride membranes in 10 mmol/L 3-(cyclohexylamino)-1-propanesulfonic acid (Sigma) and then treated with 5% methanol for 16 to 18 hours at 4°C with the use of a Bio-Rad Trans Blot Cell. The procedure that we used was similar to the protocol of Hughes et al.²⁶ The supernatants of monoclonal antibodies were obtained from the developmental studies Hybridoma Bank maintained by the Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, Md, and the Department of Biological Sciences, University of Iowa, Iowa City, under contract N01-HD-6-2915 from the NICHD.

Myosin ATPase Staining
Serial cross sections of 1-µm thickness were obtained from the biopsies by use of a cryostat. Muscle fiber types were identified by use of the standard myosin-ATPase staining procedure of Brook and Kaiser, as adjusted for human skeletal muscles.²² We preincubated our samples at pH 4.6 because according to the classification of Staron and Hikida,²⁷ all three primary human fiber types (ie, types I, IIa, and IIb) can be identified at this pH. Additionally, oil red O staining for neutral lipids, periodic acid–Schiff staining for glycogen, and trichrome staining for collagen were also performed.

Statistical Analysis
Data from patients with heart failure and normal subjects were compared with the use of Student's grouped t tests. Additionally, the relations between variables were examined by linear regression analysis. A value of P<.05 was considered significant. Data are expressed as mean±SD.

	Results

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Enzymatic Analysis
BOAC activity was significantly higher in heart failure than in normal subjects (normal, 0.27±0.04; heart failure, 0.38±0.02 µmol·min^-1·mg protein^-1; P<.05). CS was also significantly greater in heart failure than in normal diaphragms (normal, 0.33±0.14; heart failure, 0.54±0.21 µmol·min^-1·mg protein^-1; P<.05). In contrast, LDH activity was significantly decreased in heart failure compared with normal diaphragms (normal, 11.6±4.6; heart failure, 4.3±2.2 µmol·min^-1·mg protein^-1; P<.01). Calculated activities ratios of LDH/CS in heart failure subjects were decreased >4-fold compared with normal subjects (normal, 0.36±0.1; heart failure, 0.08±0.03; P<.001). Thus, oxidative enzymatic activity was significantly increased and glycolytic enzymatic activity decreased in heart failure subjects (Fig 1).

View larger version (12K): [in this window] [in a new window]   Figure 1. Enzymatic activity in normal and heart failure (CHF) subjects. Values are mean±SEM, *P<.05; **P<.001.

Clinical variables including ejection fraction, age, duration of illness, pulmonary vascular resistance, VO₂, and New York Heart Association class were correlated with enzymatic activity. The only statistically significant correlations were between left ventricular ejection fraction and LDH activity, which were positively correlated (r=.84; P<.01), and between ejection fraction and CS activity, which were negatively correlated (r=-.75; P<.05). These correlations suggest a shift to oxidation metabolism in the diaphragm as the disease progresses. However, these observations may be spurious because the range of ejection fraction for this gravely ill group of patients may have been falsely elevated owing to a combination of multiple inotropic therapies or the presence of mitral regurgitation.

Histochemical Analysis
Standard histological stains of muscle specimens from patients and control subjects revealed no evidence of inflammation or fiber necrosis and no accumulation of intracellular fat, glycogen, or collagen.

ATPase staining was difficult. When our histochemical methodology and gray-scale digitization are used, preincubation at pH 4.6 causes the three primary fiber types (ie, I, IIa, and IIb) to have the following appearances: I, dark; IIa, white; and IIb, intermediate (or moderately dark). Fig 2 shows all three fiber types in the control diaphragm, whereas the heart failure diaphragm contained only types I and IIa. Due to tissue distortion associated with freezing, we did not report cross-sectional area of fiber types.

View larger version (99K): [in this window] [in a new window]   Figure 2. Representative myosin-ATPase–stained cross sections from costal diaphragm in normal subject (a) and patient with heart failure (b) at preincubation at pH 4.6, with type I fibers staining dark, type IIa white, and type IIb intermediate (or moderately dark).

MHC Composition
The content of MHC isoforms in normal and heart failure subjects are shown in the scatterplots in Fig 3. All three panels show a relatively narrow range for the three MHC isoforms in each of the subject groups. The proportions of MHC types I, IIa, and IIb for the control group were 43±2%, 40±2%, and 17±1%, respectively, whereas those for the heart failure group were 55±4%, 38±2%, and 7±2%, respectively. Type I MHC was significantly increased and type IIb MHC isoforms were significantly reduced in these patients (both P<.01).

View larger version (14K): [in this window] [in a new window]   Figure 3. Content of MHC isoforms in normal (NL) and heart failure subjects (HF). *P<.001;**P<.0001.

Fig 4 shows the electrophoretic separation of MHCs in three normal and three heart failure subjects. Three distinct bands can be seen representing MHC isoforms IIb, IIa, and I from top to bottom of each gel. The striking finding is the almost total depletion of type IIb MHC isoforms in the heart failure subjects.

View larger version (35K): [in this window] [in a new window]   Figure 4. Electrophoretic separation of MHC isoforms in three normal (NL) and three heart failure subjects (HF).

Confirmation of MHC bands were made by immunoblotting techniques (Western blots) with the use of the monoclonal antibodies of Hughes et al²⁶ (Table 2 and Fig 5). To verify that the bands developed on the SDS gels corresponded to different MHCs and not to other proteins with a molecular weight of 200 kD, we used antibody A4.1025, which recognizes all MHC isoforms expressed in adult human skeletal muscle. A high degree of identity between the immunoblots and SDS gels in both normal and heart failure diaphragms was observed (Fig 5). These bands were identified with the use of antibodies specifically directed at the different MHC isoforms. Antibodies A4.951 and A4.840, specific for MHC type I, reacted only with the lower band on the SDS gels in both normal and heart failure gels. Therefore, this lower band can be definitively attributed to the MHC type I isoform. Antibody N2.261, which recognizes both MHC types I and IIa, reacted with the lower (ie, MHC type I) and intermediate bands on SDS gels in both groups. Only the intermediate band reacted positively with antibody N1.551, which is specific for MHC type IIa. Thus, the intermediate band can be identified as the MHC type IIa isoform. Antibodies A4.74 and A4.1519 are specific for all fast MHC (IIa and IIb) isoforms. These antibodies recognized MHC IIa (ie, intermediate band) in normal and heart failure gels. However, the positive reaction with the upper band was apparent only for the normal subjects; minimal or no reaction was noted in heart failure subjects. These results suggest that (1) the upper band on the SDS gels should correspond to the fast MHC isoform, other than MHC IIa, and (2) qualitatively, this isoform is markedly reduced or completely eliminated in heart failure. Because only three MHC isoforms are known to be expressed in human diaphragm (ie, MHC types I, IIa, and IIb), this isoform should be fast MHC IIb.

View this table: [in this window] [in a new window]   Table 2. Comparison of Western Blots of SDS Gels: Normal Versus Heart Failure Subjects

View larger version (29K): [in this window] [in a new window]   Figure 5. SDS gel electrophoresis and immunoblotting of MHC from the costal diaphragm in a normal subject and a heart failure subject (CHF). The central panels are the SDS gels of a normal and a heart failure subject. Panels a, b, c, and d correspond to the immunoblots with antibodies specific for all MHC isoforms (MHC I, MHC IIa, and MHC IIb).

Linear correlations for age and percent of MHC subtypes were performed for each group and the entire study population. No significant correlations were observed for the population as a whole (all r<.4; P=NS). The only significant correlations observed for each group were between age and MHC IIb. For the normal subjects, there was a significant positive correlation (r=.9; P<.01), whereas in the heart failure subjects, there was a significant negative correlation between age and percent of MHC IIb (r=–.68; P<.02). Thus, the heart failure patients had less type IIb with aging rather than the normal increased percentage.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
In this study, we demonstrated diaphragmatic changes in patients with heart failure consistent with an increase in oxidative metabolism. This is in contrast to the histochemical changes observed in the limb muscles of these patients. These findings are important in that they suggest that the muscle changes observed in heart failure are organ specific.

Enzymatic activities have not been previously reported in the diaphragm of patients with heart failure. In the present study, we demonstrated a significant increase in the activity of oxidative enzymes CS and BOAC and a significant reduction in glycolytic enzyme activity, ie, LDH, compared with control subjects. The ratio of LDH/CS has been used as an index of oxidative metabolism, ie, the lower the ratio, the greater the oxidative potential of the muscle.²⁸ In patients with heart failure, this ratio was significantly lower than in normal subjects. Although LDH is not the rate-limiting enzyme for glycolysis, studies on single muscle fibers have demonstrated that with chronic stimulation of muscle at low frequencies, changes in LDH activity parallel those of glycolytic enzymes such as phosphofructokinase.²⁹ Therefore, once again our findings suggest a shift to oxidative metabolism.

We also observed that the magnitude of the oxidative enzyme changes varied inversely with the ejection fraction, whereas the anaerobic enzyme adaptations varied directly. This also suggests that there is a shift to oxidative metabolism with increasing disease severity.

This is the first report to quantify diaphragm MHC isomers in normal or diseased subjects. The myosin molecule is a hexamer composed of three pairs of different types of polypeptide chains: two identical heavy chains and four light chains. In human diaphragmatic muscle, three major fiber types (I, IIa, and IIb) can be expressed: MHC I, MHC IIa, and MHC IIb. These MHC isoforms have been shown to be a major determinant of the following properties in muscle fibers^{30 31 32 33 34} : speed of isometric contraction, time to peak tension, and the rate of ATP utilization. Muscle fibers exhibiting greater proportions of slow myosin isoforms (MHC I) and lesser proportions of fast myosin isoforms (MHC IIa or MHC IIb) are known to be less fatigable than those muscle fibers exhibiting the reverse composition.

In the present study, we demonstrated a highly significant increase in the slow MHC I isomer and a significant decrease in the fast MHC IIb isomer in patients with heart failure. These MHC adaptations manifested by the heart failure diaphragm have the following physiological consequences: (1) a decrease in the maximum speed of contraction; (2) a decrease in the rate of ATP utilization per contraction; and (3) a decrease in fatigability. These findings, along with the observed changes in enzymatic activity, suggest a shift to oxidative metabolism in the diaphragm in heart failure. The pathophysiological consequence of the increased oxidative enzyme activities is increased ATP generation via aerobic oxidative pathways; this, coupled with a decrease in the rate of ATP consumption by myofibrillar proteins, should decrease the energy cost of contraction in the heart failure diaphragm.

The relationship of MHC proportions to the histochemical fiber-type proportions of a muscle is somewhat complex. MHCs contain the sites for ATP hydrolysis. Thus, there is a correlation between myofibrillar ATPase on the basis of histochemical separation of muscle fiber types and MHC content. Assuming that each of the major fiber types in human muscle (ie, I, IIa, and IIb) contains predominantly a single MHC isoform, a high degree of correlation between the relative percentages of different MHC isoforms and the percent contribution of each of the major fiber types to the cross-sectional area of the muscle (ie, the quotient of the cross-sectional area occupied by a given fiber type divided by the total cross-sectional area of all of the fiber types multiplied by 100) is expected. However, human muscle contains a continuum of intermediate fiber populations that coexpress more than one MHC isoform; this fact causes the correlation between percent MHC and percent fiber-type contribution to be highly dependent on the histochemical method used for fiber typing.²⁷

For example, the percentage of MHC IIb that we noted in our normal diaphragmatic samples (17±1%) is close to the contribution of IIb fibers noted by Lieberman et al³⁵ in their study of normal diaphragm. The normal fiber distribution in the study by Lieberman et al was 55% type I, 21% type IIa, and 24% type IIb. In contrast, in our normal samples, the percentage of MHC I isomer (43%) was lower and that of MHC IIa isomer (40%) was higher than the percent contribution of the corresponding fiber types. One possible explanation for this discrepancy is that some type I and IIa fibers are coexpressing both MHC I and IIa isomers. Those fibers with coexpression have been referred to as C fibers. Under certain circumstances, C fibers may account for >15% of the fibers in human skeletal muscle. These type C muscle fibers would not have been identified by the techniques used by Lieberman et al or other investigators.^{35 36 37 38} C fibers that expressed more MHC I and less MHC IIa (ie, type I C) would have been classified as fiber type I, whereas those C fibers containing less MHC I and more MHC IIa (ie, type IIa C) would have been classified as IIa fibers. Unfortunately, we were unable to complete typing of diaphragmatic fibers in our subjects because of technical problems and thus cannot draw direct comparisons with our samples.

In some heart failure subjects for whom ATPase staining was possible, we observed an increased number of type I and decreased number of type IIb fibers (Fig 4). These observations are consistent with our MHC isoform data. One prior report¹⁴ described no difference in fiber subgroups in the diaphragm of 8 heart failure patients and 12 subjects with normal left ventricular function at the time of cardiac surgery. Comparison of this preliminary report with our data is difficult. The composition of the control groups was clearly different. However, it is unlikely that the intense catecholamine discharge associated with brain death could alter muscle type within 48 hours.

The heart failure–induced changes in enzymatic activities also suggest a shift in fiber-type composition. Single-fiber analyses^{39 40} indicate that the activities of the main oxidative enzymes in human skeletal muscle range from highest to lowest in the following fiber-type order: I>IIa>IIb. In contrast, in these same studies, glycolytic activity decreased in the same fiber-type order, ie, I<IIa<IIb. Therefore, our heart failure–induced enzyme adaptations can also be explained by an increase in the relative contribution of the slow-twitch, MHC I–rich fiber types and a decrease in that of fast-twitch, MHC IIa/IIb fibers.

Histochemical and enzymatic abnormalities in limb muscles of patients with heart failure are well described and include muscular atrophy, shift in fiber-type composition from slow- to fast-twitch fiber types, reduced oxidative capacity, and accumulation of fat.^{1 2 3 4 5 6 7 8 9 10} The skeletal muscle histochemical changes are similar in patients with heart failure of different disease origins.⁶ In a preliminary report, Vescovo et al⁴¹ isolated MHC from muscle biopsies from the lower limb of patients with heart failure. They reported a significant reduction in MHC I and an increase in MHC IIb compared with patients with disuse atrophy.⁴¹ Heart failure–induced changes in the diaphragm are therefore opposite to those of the limb muscles. One possible explanation for these different responses is that skeletal muscle adapts to heart failure in a muscle-specific manner. The increase in work of the diaphragm at rest and during exercise can be viewed as constant moderate-intensity exercise of this muscle. Long-term functional overuse results in muscle fatigue–resistant adaptations, ie, training. The shift of MHC composition toward slow isoforms and an increase in oxidative enzyme capacity in limb muscle are well known consequences of endurance exercise in experimental animals and humans. Similar shifts in MHC composition and enzymatic activity have been described in the rodent diaphragm after endurance or resistive training.^{42 43 44 45 46 47} Thus, the respiratory muscles that are subjected to long-term increases in the work of breathing will respond with endurance training–like adaptations. In contrast, the physical inactivity of heart failure patients may result in changes in the limb muscle characteristic of deconditioning.

Another hypothesis for the observed diaphragmatic changes is chronic underperfusion. Because blood flow per gram of muscle is much greater for the diaphragm than for other skeletal muscles, it is unlikely that the histochemical changes observed result from chronic ischemia, although this hypothesis cannot be excluded. Moreover, Musch⁴⁸ demonstrated in a rat model that in contrast to the intercostals and transversus abdominis muscles, the diaphragm in chronic heart failure increases blood flow during exercise. Therefore, it is unlikely that the observed changes result from chronic ischemia. If this were the mechanism for the muscle changes, then it would be anticipated that both the limb and respiratory muscle changes would parallel each other, which they do not. Similarly, it is unlikely that the muscle changes are the result of a generalizable cytokine effect on muscle function.

Study Limitations
Our control group can be criticized because these subjects had been exposed to high-dose catecholamines and the neurohormonal consequences of brain death. We believe the control subjects were adequate because it is unlikely that histochemical changes would occur in such a short time period. Changes in MHCs occur over a 2-week period. Similarly, enzymatic changes occur over days and not hours.

Diaphragmatic tissue was procured from the same regional segment of the diaphragm in all subjects; thus, it is unlikely that the observed differences were due to variations in muscle regionality. Moreover, previous investigators did not describe differences in the fiber-type distribution in different costal regions of the diaphragm.^{35 44}

Significant alterations of skeletal muscle morphological and biochemical properties can occur with aging. Although there was no statistical difference between the age of the normal and heart failure subjects, the normal subjects in our study tended to be younger. A recent study of the MHC isomer composition in the diaphragm of rats did not detect any change in the percent of MHC I or IIa with aging. However, the contribution of MHC IIb was higher in the older animals.⁴⁹ These changes are analogous to what we observed in our normal subjects. The effect of aging on the MHC composition of the diaphragm has not been described in humans, although there appears to be an increase in the coexistence of MHC isoforms in single fibers in the limb muscles with aging.^{50 51}

Clinical Implications
Exertional fatigue and dyspnea in patients with heart failure can result from abnormal resting and exercise hemodynamic responses, endothelial and vascular smooth muscle dysfunction, and intrinsic skeletal muscle changes. Alteration of the neurohormonal levels in heart failure result in elevations in catecholamines, angiotensin, atrial natriuretic factor, tumor necrosis factor, and other cytokines. These circulating substances may affect muscle and/or vascular function. Our findings suggest that the muscle changes observed in heart failure are not a result of circulating neurohormones or chronic underperfusion but result from local factors. Patients with heart failure do not exhibit a generalized myopathy but have organ-specific changes.

In conclusion, in chronic heart failure, the ventilatory muscles exhibit adaptations similar to those elicited by long-term endurance training, probably as a consequence of the increased work of breathing in heart failure subjects. This is in contrast to the limb skeletal muscle, in which a shift from oxidative to glycolytic metabolism is observed. The changes in the skeletal muscle in heart failure are not generalized but are organ specific.

	Selected Abbreviations and Acronyms

 

BOAC = ß-hydroxyacyl-CoA dehydrogenase CS = citrate synthase LDH = lactate dehydrogenase MHC = myosin heavy chain

	Acknowledgments

 
This study was supported by a Grant-in-Aid from the New York City Affiliate of the American Heart Association and by the Division of Research Resources, General Clinical Research Centers Program, NIH (No. 5-M01-RR00645). We would like to thank Dr Stefano Ravalli for his help in performing and interpreting the histochemical stains.

	Footnotes

 
Reprint requests to Donna M. Mancini, MD, Division of Circulatory Physiology, Department of Medicine, Columbia Presbyterian Medical Center, 622 W 168th St, New York, NY 10032.

Received June 27, 1996; revision received August 26, 1996; accepted September 30, 1996.

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Top Abstract Introduction Methods Results Discussion References

 

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