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(Circulation Research. 2001;88:1299.)
© 2001 American Heart Association, Inc.

Integrative Physiology

Contraction and Intracellular Ca²⁺ Handling in Isolated Skeletal Muscle of Rats With Congestive Heart Failure

Per Kristian Lunde, Anders J. Dahlstedt, Joseph D. Bruton, Jan Lännergren, Peter Thorén, Ole M. Sejersted, Håkan Westerblad

From the Institute for Experimental Medical Research (P.K.L., O.M.S.), University of Oslo, Ullevaal Hospital, Oslo, Norway, and the Department of Physiology and Pharmacology (A.J.D., J.D.B., J.L., P.T., H.W.), Karolinska Institutet, Stockholm, Sweden.

Correspondence to Håkan Westerblad, PhD, Department of Physiology and Pharmacology, Karolinska Institutet, 171 77 Stockholm, Sweden. E-mail Hakan.Westerblad{at}fyfa.ki.se

Abstract

Abstract—A decreased exercise tolerance is a common symptom in patients with congestive heart failure (CHF). This decrease has been suggested to be partly due to altered skeletal muscle function. Therefore, we have studied contractile function and cytoplasmic free Ca²⁺ concentration ([Ca²⁺]_i, measured with the fluorescent dye indo 1) in isolated muscles from rats in which CHF was induced by ligation of the left coronary artery. The results show no major changes of the contractile function and [Ca²⁺]_i handling in unfatigued intact fast-twitch fibers isolated from flexor digitorum brevis muscles of CHF rats, but these fibers were markedly more susceptible to damage during microdissection. Furthermore, CHF fibers displayed a marked increase of baseline [Ca²⁺]_i during fatigue. Isolated slow-twitch soleus muscles of CHF rats displayed slower twitch contraction and tetanic relaxation than did muscles from sham-operated rats; the slowing of relaxation became more pronounced during fatigue in CHF muscles. Immunoblot analyses of sarcoplasmic reticulum proteins and sarcolemma Na⁺,K⁺-ATPase showed no difference in flexor digitorum brevis muscles of sham-operated versus CHF rats. In conclusion, functional impairments can be observed in limb muscle isolated from rats with CHF. These impairments seem to mainly involve structures surrounding the muscle cells and sarcoplasmic reticulum Ca²⁺ pumps, the dysfunction of which becomes obvious during fatigue.

Key Words: heart failure • skeletal muscle • fatigue • intracellular Ca²⁺ handling

Decreased fatigue resistance and skeletal muscle weakness are important symptoms in humans with congestive heart failure (CHF). The reason for the decreased fatigue resistance is not clear. Often, there is no clear correlation between the degree of heart dysfunction and the decrease in exercise tolerance.¹ This suggests that there could be functional impairments within the skeletal muscles that are due to, for instance, alterations in the local environment with restricted local blood flow.² In addition, numerous studies have been focused on possible abnormalities in skeletal muscle cells, and significant changes have been found both at the mRNA and protein levels. Generally observed changes in skeletal muscle cells in CHF include a shift of myosin heavy chain distribution toward more fast-type myosin heavy chain,^{3 4 5} altered expression of sarcoplasmic reticulum (SR) Ca²⁺-ATPase (SERCA),^{6 7} and in later stages, decrements in mitochondrial enzymes and muscle cell atrophy.⁸ Functional studies of limb muscle function in CHF are more sparse. One study on bundles of muscle fibers from the fast-twitch extensor digitorum longus (EDL) muscles of rats showed marked dysfunction in CHF with 50% reductions in tetanic Ca²⁺ and force and markedly accelerated fatigue development, which was not due to muscle cell atrophy.⁹ There seems to be a discrepancy between these very dramatic functional changes and the relatively subtle changes of muscle protein levels observed in muscles from CHF subjects. In accordance, a more recent study showed more moderate and, in some instances, apparently opposite changes in contractile function and Ca²⁺ handling in skeletal muscle from rats with myocardial infarction.¹⁰ Having this in mind, we compared contractile function, intracellular Ca²⁺ handling, and fatigue resistance of intact, single, fast-twitch muscle fibers from CHF and sham-operated rats (CHF and sham fibers, respectively). The results show no major changes in the contractile function and Ca²⁺ handling of unfatigued fibers from CHF rats. The fatigue resistance of CHF fibers was similar to that of sham fibers, but the increase in baseline Ca²⁺ during fatigue was markedly larger in CHF fibers. Moreover, fibers from CHF rats were markedly more susceptible to damage during microdissection. We also studied contractions and fatigue in isolated slow-twitch soleus muscles. The most conspicuous result in soleus muscles from CHF rats was a pronounced slowing of relaxation and increase in baseline force during fatigue.

Materials and Methods

Animals
Experiments were performed on a total of 48 male Wistar rats: 25 CHF rats and 23 sham-operated control (sham) rats. The primary surgery was performed on 3-month-old rats (weight 300 g). These were anesthetized with 2.5% halothane in 30% O₂/70% N₂O. CHF was produced by ligating the left coronary artery.¹¹ Sham rats were subjected to the same surgical procedures, but the coronary artery was not ligated. During the surgery, transmitters recording body temperature, heart rate, ECG, and gross locomotor activity were placed in the peritoneal cavity of some animals.¹²

The secondary surgery was performed after 6 weeks by using the same anesthetic method. Systolic aortic pressure and left ventricular end-diastolic pressure (LVEDP) were measured by a micromanometer-tipped catheter (SPR-407, Millar Instruments) inserted through the right carotid artery. Soleus and flexor digitorum brevis (FDB) muscles were dissected out and kept in standard Tyrode’s solution bubbled with 5% CO₂/95% O₂; 0.2% FCS was added to the solution to improve the survival of single fibers.¹³ Experiments were performed at room temperature (24°C). The present study was approved by the local ethics committee.

Experiments on Intact Single Fibers
Intact single fibers were dissected from FDB muscles by using dark-field illumination and x80 to x120 magnification.¹³ The isolated fiber was mounted between an Akers 801 force transducer (SensoNor) and an adjustable holder at the length giving maximum tetanic force. The fiber was continuously superfused by Tyrode’s solution, and electrical stimulation was achieved by brief supramaximum current pulses delivered via platinum plate electrodes lying parallel to the fiber.¹³

In the majority of fibers, the free myoplasmic Ca²⁺ concentration ([Ca²⁺]_i) was measured with the fluorescent Ca²⁺ indicator indo 1 (Molecular Probes). The pentapotassium salt of indo 1 was microinjected into fibers; this procedure avoids problems with the loading of organelles. The fluorescence of indo 1 was measured with a system consisting of a xenon lamp, a monochromator, and two photomultiplier tubes (PTI, Photo Med GmbH). Fluorescence signals were translated to [Ca²⁺]_i by using an intracellular calibration curve established in intact mouse muscle fibers.¹⁴ After the injection of indo 1, the fibers were allowed to rest for at least 60 minutes. Fibers that produced a force markedly lower than that before the injections were not used. The experimental protocol was started by first producing a single twitch and then, at 1-minute intervals, 350-ms tetani at 15 to 100 Hz. Fatigue was produced by applying 350-ms 70-Hz tetani at 1-second intervals until force was reduced to 40% of the control.

Experiments on Whole Soleus Muscles
Isolated soleus muscles were mounted at optimum length. After 30 minutes of rest, the muscle was electrically stimulated at 1-minute intervals with a single pulse or 2-second tetani at 10 to 70 Hz. Fatigue was produced by 700-ms 50-Hz tetani at 2-second intervals delivered until force was down to 40% of the control.

Immunoblot Analyses
Membrane proteins from FDB and soleus muscles (and also, for comparison, EDL muscles and pieces from the left ventricle of the heart) were isolated as previously described.¹⁵ A semiquantitative determination of different proteins was achieved by Western or slot-blot analysis with various amounts of protein to ensure that staining intensity was within the linear range of analysis.¹⁶ The primary antibodies and concentrations used were anti-SERCA1 antibody (MA3-912, Affinity BioReagents; 1:2500), anti-SERCA2 antibody (MA3-919, Affinity BioReagents; 1:1000), anti–Na⁺,K⁺-ATPase ₁-subunit antibody (MA3-929, Affinity BioReagents; 1:250), anti–Na⁺,K⁺-ATPase ₂-subunit antibody (No. 06-168, Upstate Biotechnology; 1:1000), anti–Na⁺,K⁺-ATPase ß₁-subunit antibody (No. 06-170, Upstate Biotechnology; 1:1000), and anti–ryanodine receptor antibody (MA3-925, Affinity BioReagents; 1:5000). Values are expressed in arbitrary units and normalized to the mean of sham muscles (100%).¹⁷

Statistical Analysis
Values are presented as mean±SE. Unpaired t tests were used to establish statistical differences between CHF and sham groups, and the significance level was set at P<0.05.

Results

Body weight, body temperature, and gross locomotor activity were not significantly different between sham and CHF rats, whereas the heart rate was slightly lower in CHF rats (Table 1).¹⁸ Hemodynamic measurements of heart function were severely pathological in CHF rats: systolic aortic pressure was significantly lower and LVEDP was significantly higher in CHF rats than in sham rats. A recent study using echocardiography shows that selecting rats with LVEDP >15 mm Hg, as in the present study, ensures that the animals have a significant uncompensated heart failure.¹⁹ CHF rats also showed several clinical signs of severe heart failure, including pulmonary congestion, tachypnea, and pleural effusion (data not shown). Moreover, there was an extensive thin-walled scar in the left ventricle of all CHF rats. Finally, ECG records showed markedly pathological QRS complexes (broadened large Q waves) in four of five CHF rats, whereas ECG was normal in all three sham rats studied.

View this table: [in this window] [in a new window]   Table 1. Gross Characteristics and Heart Performance in Sham and CHF Rats

Experiments on Single FDB Muscle Fibers
One major difference was noted during the dissection of single muscle fibers: isolated fibers from CHF rats often did not respond to electrical stimulation, despite having a normal appearance in the dissection microscope, whereas fibers from sham rats were robust in this respect. To deal with this problem in CHF muscles, we adopted a procedure in which we dissected bundles of 3 intact fibers, and this bundle was transferred to the stimulation chamber. One fiber was then injected with indo 1, and when it had recovered from injection, as judged from the [Ca²⁺]_i transient and force response, the remaining fibers were cut open by a broken microelectrode; an all-or-none response to small increments of the stimulation strength verified activation of the injected fiber only. With this procedure, we had a success rate (number of fibers that could be used in experiments divided by the number of dissected fibers) of 20% in CHF muscles compared with 80% in sham muscles.

Mean data from twitch and tetanic contractions produced in unfatigued single muscle fibers did not show any significant differences between sham and CHF fibers, except for a higher twitch force in CHF fibers (Table 2). However, during 70-Hz (and also 100-Hz) tetanic stimulation, there was a trend for both [Ca²⁺]_i and force to be higher in CHF fibers than in sham fibers (Figure 1), but the difference was not significant (P>0.2). Resting [Ca²⁺]_i was not significantly different between the two groups: 43.6±10.4 nmol/L in sham fibers (n=9) and 45.7±10.8 nmol/L in CHF fibers (n=5).

View this table: [in this window] [in a new window]   Table 2. Contractile Properties of Unfatigued Sham and CHF Muscle

View larger version (12K): [in this window] [in a new window]   Figure 1. Original records of [Ca2+]i and force from 70-Hz tetani produced in unfatigued single FDB fibers of sham and CHF muscle. Period of stimulation is indicated below force records.

Force-[Ca²⁺]_i curves of unfatigued fibers were constructed by measuring mean force (F) and [Ca²⁺]_i during the final 100 ms of stimulation in tetani of different frequencies and fitting data points to the following equation: F=F_max[Ca²⁺]_i^N/(Ca₅₀^N+[Ca²⁺]_i^N), where F_max is the force at saturating [Ca²⁺]_i, Ca₅₀ is the [Ca²⁺]_i giving 50% of F_max, and N is a constant describing the steepness of the function. Neither of these parameters nor the frequency giving half-maximum force (obtained by linear interpolation) was significantly different between sham and CHF fibers (Table 2).

Original representative records of [Ca²⁺]_i and force obtained during fatiguing stimulation are shown in Figure 2, and mean data from all fibers studied are presented in Figure 3. The rate of force decline during fatigue was similar in CHF and sham fibers (Figure 3A). Although the mean tetanic [Ca²⁺]_i was higher in CHF fibers than in sham fibers throughout the fatigue runs (Figure 3B), this difference was not statistically significant at any time point. The fibers depicted in Figure 2 show a clear increase of baseline force in fatigue, and this increase was larger in the CHF fibers. This was a general finding, and at the end of fatiguing stimulation, baseline force (measured immediately before the onset of the last tetanic contractions) tended to be larger in CHF fibers (6.7±3.1% of the control tetanic force, n=7) than in sham fibers (2.4±0.7%, n=9), but the difference was not significant (P=0.16). The increase in baseline force in fatigue suggests a marked slowing of relaxation, and this was confirmed by measurements of the half-relaxation time in fatigue, which was significantly increased both in CHF and sham fibers. The half-relaxation time in fatigue was 260±71 ms in CHF fibers and 186±25 ms in sham fibers, which represents an increase by 175±63% and 99±31%, respectively. Thus, again the slowing of relaxation tended to be larger in CHF fibers, but the difference was not significant (P=0.25).

View larger version (55K): [in this window] [in a new window]   Figure 2. Original records of [Ca2+]i and force from representative fatigue runs produced in single FDB fibers of sham and CHF muscle.

View larger version (18K): [in this window] [in a new window]   Figure 3. A, Mean±SEM data of tetanic force during development of fatigue in single FDB muscle fibers of sham (, n=9) and CHF (•, n=7) muscle. Data shown are the 1st, 10th, and 20th fatiguing tetanus and the tetani by which 70% and 40% force was produced. B, Mean±SEM values of tetanic [Ca2+]i during fatigue measured at the same time points as in panel A. Open bars indicate sham (n=7); solid bars, CHF (n=5). C, Average [Ca2+]i tails obtained after 70-Hz tetani produced under control conditions (left) and at the end of fatiguing stimulation (right). Thin line indicates sham; thick line, CHF. Time axes start at the end of tetanic stimulation.

In Figure 3C, we show average [Ca²⁺]_i records obtained immediately after tetanic stimulation in control conditions and in fatigue. In control conditions, these "tails" of [Ca²⁺]_i were similar in CHF and sham fibers. However, in fatigue, the [Ca²⁺]_i tails were markedly slower in CHF fibers, and baseline [Ca²⁺]_i (measured as the mean over 100 ms obtained 1 second after the end of stimulation) was significantly (P<0.05) higher in CHF fibers (206±37 nmol/L, n=5) than in sham fibers (120±10 nmol/L, n=7). One minute after the end of fatiguing stimulation, baseline [Ca²⁺]_i had recovered substantially in both groups, and there was no longer any significant difference between CHF fibers (67±16 nmol/L) and sham fibers (44±11 nmol/L).

Recovery of tetanic force and [Ca²⁺]_i after fatigue was studied by producing tetani at regular intervals for 30 minutes. There was no significant difference between CHF and sham fibers at any time point, and at 30 minutes of recovery, tetanic force was 81±7% and 76±8% in CHF fibers (n=7) and sham fibers (n=8), respectively; corresponding values for tetanic [Ca²⁺]_i were 76±7% (n=5) and 92±6% (n=7).

Experiments on Soleus Muscles
Twitch contraction and tetanic relaxation in unfatigued soleus muscles were slower in CHF animals than in sham animals (Table 2). In addition, the twitch force was higher and the frequency required to produce 50% tetanic force was lower in CHF muscles than in sham muscles. Original force records from typical fatigue runs in sham and CHF muscles are shown in Figure 4, and mean data are presented in Figure 5. During fatiguing stimulation, peak tetanic force fell more slowly in CHF muscles than in sham muscles (Figure 5A), and the number of tetani required to bring peak force down to 40% of the original was significantly (P<0.05) higher in CHF muscles (103±4 tetani) than in sham muscles (76±7 tetani). A further difference between the two groups was that baseline force increased rapidly during fatigue in CHF muscles. Thus, at the end of fatiguing stimulation, it amounted to 24.1±2.2% of the original peak tetanic force, which was significantly (P<0.01) higher than the value in sham muscles (5.9±3.0%). In accordance, the half-relaxation time of the last fatiguing tetanus was markedly longer in CHF muscles (1896±163 ms) than in sham muscles (538±57 ms, P<0.001). During the 30-minute recovery period after fatiguing stimulation, peak tetanic force was generally lower in CHF muscles than in sham muscles (Figure 5B).

View larger version (35K): [in this window] [in a new window]   Figure 4. Original force records from typical fatigue runs in soleus muscles from sham and CHF rats.

View larger version (13K): [in this window] [in a new window]   Figure 5. Mean data of baseline and tetanic force during fatigue (A) and recovery (B) in sham ( and , n=10) and CHF ( and •, n=10) soleus muscles.

Immunoblot Analyses
The contractile results and [Ca²⁺]_i measurements described above would indicate some changes in SERCA in CHF muscles. Therefore, immunoblot analyses of the expression of fast-type and slow-type SR Ca²⁺-ATPase (SERCA1 and SERCA2, respectively) were performed (Figure 6). As expected, SERCA1 was the predominant form in fast-twitch EDL and FDB muscles, whereas only traces of SERCA1 expression were seen in the soleus muscle, and none were seen in the heart. On the other hand, SERCA2 was expressed in slow-twitch soleus muscles and the heart (not shown) but not in the two fast-twitch muscles (only EDL shown). Furthermore, there was not a significant difference between SERCA1 expression in CHF and sham FDB muscles (Figure 6A) or between SERCA2 expression in CHF and sham soleus muscles (Figure 6B). Thus, the slowed force and [Ca²⁺]_i handling observed in CHF muscle cannot be explained by a reduced expression of SERCA. In FDB muscles, we also performed immunoblot analyses of several other proteins. Again, no significant difference between CHF and sham muscles was found regarding SR Ca²⁺-release channels (ie, ryanodine receptors; 98±9.0% versus 100±8.7%, respectively) or different isoforms and subunits of the sarcolemma Na⁺,K⁺-ATPase (₁, 94±13.2% versus 100±11.6%, respectively; ₂, 103±8.2% versus 100±7.3%, respectively; and ß₁, 99±3.2% versus 100±3.7%, respectively).

View larger version (20K): [in this window] [in a new window]   Figure 6. A, SERCA1 is expressed in fast-twitch (EDL and FDB) muscles, whereas the expression is small in slow-twitch (soleus) muscles and undetectable in the left ventricle of the heart (LV) (immunoblots). Bar graph shows mean data of SERCA1 expression in FDB from sham (n=11) and CHF (n=13) rats; note that there was no difference between the two groups. B, SERCA2 is expressed in slow-twitch soleus muscles but not in fast-twitch EDL muscles (immunoblots). Mean data showed no difference in SERCA2 expression between soleus muscles from sham (SH, n=11) and CHF (n=13) rats (bar graph).

Discussion

The major results of the present study are as follows: (1) single fibers from the hindlimb FDB muscle of CHF rats were more susceptible to damage during dissection; (2) in the unfatigued state, there were no differences or only moderate differences in contractile function and [Ca²⁺]_i handling between CHF and sham muscle; and (3) in the fatigued state, there was a marked reduction of the rate of [Ca²⁺]_i removal from the myoplasm in single fibers and a marked slowing of force relaxation of soleus muscles from CHF rats.

The present functional differences between CHF and sham muscles cannot be explained by inactivity in CHF rats, inasmuch as there was no difference in the locomotor activity pattern of the two groups, a finding that agrees with previous results obtained in rats with CHF.^{4 7 18} Rather, it appears likely that some systemic factors are involved in the development of skeletal muscle dysfunction in CHF, as recently reviewed by Lunde et al.²⁰ For instance, a major increase of tumor necrosis factor- has been observed in rats with CHF,⁵ and tumor necrosis factor- may (possibly together with other cytokines) induce myopathy and apoptosis.^{21 22}

The single fibers of FDB muscles had twitch contraction and half-relaxation times of 40 ms, and the stimulation frequency required to give half-maximal tetanic force was 20 Hz, compared with 90 to 150 ms and 10 Hz, respectively, in soleus muscles (Table 2). Thus, the single FDB muscle fibers would be fast-twitch fibers, whereas the soleus muscles would consist mainly of slow-twitch fibers, which fits with the expression pattern of SERCA isoforms.²³

The present study is the first in which force and [Ca²⁺]_i were measured in intact, single, skeletal muscle fibers of the rat. Single fibers were dissected from the hindlimb FDB muscle because the fibers of this muscle are short, which is advantageous for microdissection and dye injection. One disadvantage with the use of this muscle is that it contains almost exclusively fast-twitch fibers. In the present study, we observed larger differences between intact soleus muscles from CHF and sham rats than between the single fast-twitch FDB fibers of the two groups. Thus, it would have been interesting to measure force and [Ca²⁺]_i in single slow-twitch fibers from soleus muscles. However, soleus fibers are long, and their diameters are small, which make them less suitable for single-fiber experiments.

Increased Risk of Damage During Dissection of CHF Muscle Fibers
During the dissection of fibers, we observed a markedly increased fragility in muscles from CHF rats, and fibers that had a normal appearance in the dissection microscope often did not respond to electrical stimulation. The reason for this difference between CHF and sham muscles is not clear. One possibility is that the extracellular matrix is affected in skeletal muscles of CHF animals, and it may be hypothesized that this change precedes intrinsic changes in the muscle cells, as has been shown in failing hearts.²⁴ In line with this, it has recently been shown that in skeletal muscles from rats with CHF, apoptosis was first seen in interstitial cells, and it has been speculated that damage to endothelial cells diminishes the delivery of nutrients to the muscle cells, leading to secondary muscle cell damage.⁵ Interestingly, we observed larger changes in contractile function in the highly vascularized and oxygen-dependent soleus muscle, which, therefore, would be expected to be more vulnerable.

An increased susceptibility to mechanical damage of CHF skeletal muscle suggests that these muscles are more easily damaged during eccentric contractions. To the best of our knowledge, this intriguing possibility has not been specifically investigated.

Minor Changes of Muscle Function in Unfatigued CHF Muscle
In muscles in the unfatigued state, we found only moderate differences in contractile function and Ca²⁺ handling between CHF and sham rats. In accordance, immunoblot analyses showed no difference between the two groups in the expression of SERCA isoforms, ryanodine receptors, or different subunits of sarcolemma Na⁺,K⁺-ATPase.

The small effects on contraction and [Ca²⁺]_i of unfatigued single fast-twitch fibers observed in the present study are markedly different from the results of a previous study (Perreault et al⁹ ) on fiber bundles from fast-twitch muscle of rats with CHF, in which major abnormalities were observed. CHF was induced by ligation of the left coronary artery in both studies; the degree of heart failure and infarct size are comparable; and in both cases, experiments were performed 6 weeks after inducing CHF. Thus, it is likely that the conflicting results are due to the different approaches used to assess muscle function. In the previous study, the Ca²⁺-sensitive photoprotein aequorin was loaded into bundles of muscle fibers by means of macroinjection into the interstitial space.⁹ This procedure inevitably must put a large stress on the muscle cells because a large amount of the protein is forced into the myoplasm. If the increased fragility during microdissection that we observed is taken into account, it is possible that recovery after the macroinjection of aequorin was markedly impaired preferentially in muscles from CHF rats. This would also explain the apparent conflict between the large effects on contractile function observed in the study of Perreault et al and the modest changes in contractile function and protein expression observed with similar models of CHF in the present and other^{7 10} studies. An alternative explanation might be that the isolated single fibers used in the present study are not representative of the whole muscle; eg, it could be that the fibers that contracted after dissection were the only fibers with almost unaltered properties. Although we cannot fully exclude this possibility, we consider it unlikely, because muscles from CHF rats contracted normally in early stages of dissection, and it was not until reaching the state of isolating a single cell from a bundle of 3 fibers that an increased fragility was observed.

Changes of Function in Fatigued CHF Muscle
In the single-fiber experiments, changes of tetanic force and [Ca²⁺]_i were not different between CHF and sham fibers at any time point during fatigue. The fatigue pattern observed in both groups of fibers, with an initial increase of tetanic [Ca²⁺]_i accompanied by a 10% to 20% force reduction followed by decreases of both tetanic [Ca²⁺]_i and force, is very similar to that previously observed in single mouse FDB muscle fibers, albeit the present rat fibers appear more fatigue resistant.²⁵ Nevertheless, CHF fibers tended to have higher tetanic [Ca²⁺]_i throughout fatiguing stimulation, and in the fatigued state, baseline [Ca²⁺]_i was significantly higher in CHF fibers than in sham fibers. This finding is consistent with a slower SR Ca²⁺ uptake in CHF fibers, which became manifest during fatigue.^{26 27}

In fatigued soleus muscles, force relaxation was markedly slower in CHF muscles than in sham muscles. In principle, a slowing of relaxation is a consequence of slowed crossbridge kinetics and/or slowed Ca²⁺ removal from the myoplasm, which mainly occurs by Ca²⁺ being taken up by the SR.²⁸ A major slowing of crossbridge kinetics would also be manifested as a marked reduction of the rate of force development at the start of tetanic contraction. In the present study, the rate of force development during fatigue was not markedly different in muscles from CHF and sham rats (data not shown). Thus, the pronounced slowing of relaxation in fatigued CHF soleus muscles was most likely due to an impaired Ca²⁺ uptake into the SR, which is also found in cardiomyocytes from rats with heart failure, in diaphragms from rabbits with heart failure, and probably in the present CHF single fibers.^{11 29}

Based on the above results, the following model can be proposed. Because there is only a modest or no difference between unfatigued CHF and sham muscles regarding speed of relaxation and Ca²⁺ removal from the myoplasm, no major difference in the number of SR Ca²⁺ pumps between the two groups is expected, which fits with the present immunoblot results and published data.⁷ However, during fatigue, a slowing of the rate of SR Ca²⁺ uptake becomes manifest, indicating that the Ca²⁺ pumps in CHF muscles are more sensitive to fatigue-induced changes. The situation would then be similar to that in aged muscle, in which the rate of SR Ca²⁺ pumping may be reduced and in which an increased sensitivity to posttranslation modifications of SR Ca²⁺ pumps has been observed.³⁰ Interestingly, it has been shown that the impairment of SR Ca²⁺ pump function in aged muscle predominantly occurs in slow-twitch soleus muscles, whereas fast-twitch gastrocnemius muscles are not affected.³¹ This agrees with the present results, in which the largest effects of CHF were seen in soleus muscles.

During the present type of isometric contractions, peak tetanic force fell more slowly in CHF muscles than in sham muscles. This might be taken as an index of an increased fatigue resistance in CHF muscles, which would be opposite the reduced endurance observed in patients with CHF. However, the marked slowing of relaxation in fatigue would cause a decreased exercise tolerance during normal types of locomotion, which involve alternating movements, because slowed relaxation of antagonist muscles will impede the intended movements.³²

Conclusions
Functional impairments can be observed in skeletal muscle isolated from rats with CHF. In fast-twitch muscle, the impairment appears to mainly involve structures surrounding the muscle cells, but an impaired SR Ca²⁺ pumping also seems to become manifest during fatigue. In slow-twitch muscle, SR Ca²⁺ pumping appears to be impaired, and this becomes especially clear during fatigue.

Acknowledgments

The present study was supported by grants from the Swedish Medical Research Council (Project 10842), the Swedish National Center for Sports Research, Funds at the Karolinska Institutet, Sigurd and Elsa Goljes Memorial Foundation, The Ullevaal Hospital fund, Anders Jahre’s fund for the Promotion of Science, and The Research Council of Norway.

Footnotes

Original received November 13, 2000; resubmission received March 30, 2001; revised resubmission received April 24, 2001; accepted April 24, 2001.

References

1. Wilson JR. Exercise intolerance in heart failure: importance of skeletal muscle. Circulation. 1995;91:559–561.
2. Kindig CA, Musch TI, Basaraba RJ, Poole DC. Impaired capillary hemodynamics in skeletal muscle of rats in chronic heart failure. J Appl Physiol. 1999;87:652–660.
3. Mancini DM, Coyle E, Coggan A, Beltz J, Ferraro N, Montain S, Wilson JR. Contribution of intrinsic skeletal muscle changes to ³¹P NMR skeletal muscle metabolic abnormalities in patients with chronic heart failure. Circulation. 1989;80:1338–1346.
4. Simonini A, Long CS, Dudley GA, Yue P, McElhinny J, Massie BM. Heart failure in rats causes changes in skeletal muscle morphology and gene expression that are not explained by reduced activity. Circ Res. 1996;79:128–136.
5. Vescovo G, Zennaro R, Sandri M, Carraro U, Leprotti C, Ceconi C, Ambrosio GB, Dalla Libera L. Apoptosis of skeletal muscle myofibers and interstitial cells in experimental heart failure. J Mol Cell Cardiol. 1998;30:2449–2459.
6. Peters DG, Mitchell HL, McCune SA, Park S, Williams JH, Kandarian SC. Skeletal muscle sarcoplasmic reticulum Ca²⁺-ATPase gene expression in congestive heart failure. Circ Res. 1997;81:703–710.
7. Simonini A, Chang K, Yue P, Long CS, Massie BM. Expression of skeletal muscle sarcoplasmic reticulum calcium-ATPase is reduced in rats with postinfarction heart failure. Heart. 1999;81:303–307.
8. Delp MD, Duan C, Mattson JP, Musch TI. Changes in skeletal muscle biochemistry and histology relative to fiber type in rats with heart failure. J Appl Physiol. 1997;83:1291–1299.
9. Perreault CL, Gonzalez-Serratos H, Litwin SE, Sun X, Franzini-Armstrong C, Morgan JP. Alterations in contractility and intracellular Ca²⁺ transients in isolated bundles of skeletal muscle fibers from rats with chronic heart failure. Circ Res. 1993;73:405–412.
10. Williams JH, Ward CW. Changes in skeletal muscle sarcoplasmic reticulum function and force production following myocardial infarction in rats. Exp Physiol. 1998;83:85–94.
11. Holt E, Tønnessen T, Lunde PK, Semb SO, Wasserstrom JA, Sejersted OM, Christensen G. Mechanisms of cardiomyocyte dysfunction in heart failure following myocardial infarction in rats. J Mol Cell Cardiol. 1998;30:1581–1593.
12. Johansson C, Thoren P. The effects of triiodothyronine (T₃) on heart rate, temperature and ECG measured with telemetry in freely moving mice. Acta Physiol Scand. 1997;160:133–138.
13. Lännergren J, Westerblad H. The temperature dependence of isometric contractions of single, intact fibres dissected from a mouse foot muscle. J Physiol. 1987;390:285–293.
14. Andrade FH, Reid MB, Allen DG, Westerblad H. Effect of hydrogen peroxide and dithiothreitol on contractile function of single skeletal muscle fibres from the mouse. J Physiol. 1998;509:565–575.
15. Ploug T, Wojtaszewski J, Kristiansen S, Hespel P, Galbo H, Richter EA. Glucose transport and transporters in muscle giant vesicles: differential effects of insulin and contractions. Am J Physiol. 1993;264:E270–E278.
16. Johansson C, Lännergren J, Lunde PK, Vennström B, Thorén P, Westerblad H. Isometric force and endurance in soleus muscle of thyroid hormone receptor-₁- or -ß-deficient mice. Am J Physiol. 2000;278:R598–R603.
17. Semb SO, Lunde PK, Holt E, Tonnessen T, Christensen G, Sejersted OM. Reduced myocardial Na⁺, K⁺-pump capacity in congestive heart failure following myocardial infarction in rats. J Mol Cell Cardiol. 1998;30:1311–1328.
18. Witte K, Hu K, Swiatek J, Mussig C, Ertl G, Lemmer B. Experimental heart failure in rats: effects on cardiovascular circadian rhythms and on myocardial ß-adrenergic signaling. Cardiovasc Res. 2000;47:350–358.
19. Sjaastad I, Sejersted OM, Ilebekk A, Bjornerheim R. Echocardiographic criteria for detection of postinfarction congestive heart failure in rats. J Appl Physiol. 2000;89:1445–1454.
20. Lunde PK, Sjaastad I, Schiøtz Thorud H-M, Sejersted OM. Skeletal muscle disorders in heart failure. Acta Physiol Scand. 2001;171:277–294.
21. McMurray J, Abdullah I, Dargie HJ, Shapiro D. Increased concentrations of tumour necrosis factor in "cachectic" patients with severe chronic heart failure. Br Heart J. 1991;66:356–358.
22. Krown KA, Page MT, Nguyen C, Zechner D, Gutierrez V, Comstock KL, Glembotski CC, Quintana PJ, Sabbadini RA. Tumor necrosis factor -induced apoptosis in cardiac myocytes: involvement of the sphingolipid signaling cascade in cardiac cell death. J Clin Invest. 1996;98:2854–2865.
23. Ranatunga KW. Temperature-dependence of shortening velocity and rate of isometric tension development in rat skeletal muscle. J Physiol. 1982;329:465–483.
24. Boluyt MO, O’Neill L, Meredith AL, Bing OH, Brooks WW, Conrad CH, Crow MT, Lakatta EG. Alterations in cardiac gene expression during the transition from stable hypertrophy to heart failure: marked upregulation of genes encoding extracellular matrix components. Circ Res. 1994;75:23–32.
25. Lännergren J, Westerblad H. Force decline due to fatigue and intracellular acidification in isolated fibres from mouse skeletal muscle. J Physiol. 1991;434:307–322.
26. Westerblad H, Allen DG. Mechanisms underlying changes of tetanic [Ca²⁺]_i and force in skeletal muscle. Acta Physiol Scand. 1996;156:407–416.
27. Westerblad H, Allen DG. Changes of myoplasmic calcium concentration during fatigue in single mouse muscle fibers. J Gen Physiol. 1991;98:615–635.
28. Westerblad H, Lännergren J, Allen DG. Slowed relaxation in fatigued skeletal muscle fibers of Xenopus and mouse: contribution of [Ca²⁺]_i and cross-bridges. J Gen Physiol. 1997;109:385–399.
29. MacFarlane NG, Darnley GM, Smith GL. Cellular basis for contractile dysfunction in the diaphragm from a rabbit infarct model of heart failure. Am J Physiol. 2000;278:C739–C746.
30. Chen B, Jones TE, Bigelow DJ. The nucleotide-binding site of the sarcoplasmic reticulum Ca-ATPase is conformationally altered in aged skeletal muscle. Biochemistry. 1999;38:14887–14896.
31. Narayanan N, Jones DL, Xu A, Yu JC. Effects of aging on sarcoplasmic reticulum function and contraction duration in skeletal muscles of the rat. Am J Physiol. 1996;271:C1032–C1040.
32. Allen DG, Lännergren J, Westerblad H. Muscle cell function during prolonged activity: cellular mechanisms of fatigue. Exp Physiol. 1995;80:497–527.

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