INTRODUCTION

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Potassium (K⁺) channels regulate the resting membrane potential and action-potential duration of skeletal muscles. Blocking K⁺ channels pharmacologically with agents such as tetraethylammonium or the aminopyridines increases action-potential duration by slowing the rate of repolarization of the action potential, but generally has minimal effects on resting membrane potential (1). The net result is an augmentation of muscle force at low to intermediate contraction frequencies (e.g., by 25 to 160% for diaphragm twitch force with the aminopyridines) (1), which may persist over time during the course of repetitive stimulation (6, 7). The magnitude of the inotropic effect with some of the K⁺-channel blockers, and particularly 3,4-diaminopyridine (DAP) (7), is much greater than that reported for currently available inotropic agents, such as the methylxanthines and -adrenergic agonists (8), which have limited utility in the clinical management of diaphragm or limb-muscle weakness.

Muscle membranous ion conductances and their regulation may change with aging. Compared with young-adult muscle, aged limb muscle has substantially reduced macroscopic (whole-cell) chloride conductance (13). Furthermore, responses of chloride conductance to R-(+)-2-(p-chlorophenoxy)proprionic acid, high concentrations of taurine, 4--phorbol-12,13-dibutyrate, and cholera toxin differ in young-adult and old limb muscle (14, 15). Regarding muscle K⁺ conductance, there appears to be an increase in resting macroscopic (whole-cell) conductance with aging, although more modest in magnitude than the reduction in chloride conductance (13). However, changes with aging do not appear to be uniform among all types of K⁺ conductances, as aging has been associated with reductions in the density and open probability of adenosine triphosphate (ATP)-sensitive K⁺ channels in limb muscle (17).

An effective skeletal-muscle inotropic agent would be potentially useful in several groups of subjects, including those with: (1) specific neuromuscular diseases (e.g., muscular dystrophy); (2) systemic illnesses in which muscle weakness results from the direct effects of the illness (e.g., congestive heart failure [CHF], chronic obstructive pulmonary disease [COPD]); (3) muscle weakness resulting from the effects of bedrest during exacerbations of any illness; and (4) the frail elderly. The two middle groups comprise a high proportion of older people. Both the sick elderly with muscle weakness and the otherwise healthy but frail elderly would benefit from increased muscle strength, because muscle weakness in and of itself can reduce the ability to independently perform activities of daily living (ADL) and can increase the risk of falls. Diaphragm-muscle strength decreases with aging, and this weakness can be exacerbated by diseases with a high prevalence in the elderly (e.g., CHF, COPD). Respiratory-muscle weakness can induce and/or worsen respiratory failure in subjects with underlying diseases, and may contribute to the limitation of exercise capacity in the frail elderly. Because the elderly comprise a large group of potential candidates for skeletal-muscle inotropic therapy, it is important to determine whether inotropic agents affect aged muscle differently from young muscle.

The purpose of the present study was therefore to examine whether K⁺-channel blockade affects old muscle differently from young-adult muscle. Our specific hypothesis, based on the greater macroscopic K⁺ conductance of old than of young-adult muscle (16), was that K⁺-channel blockade would prolong action-potential duration and increase force to a greater extent in old than in young-adult muscle.

Studies directed at answering this question were performed with DAP, an aminopyridine. In concentrations producing their inotropic effects, the aminopyridines block K⁺ channels (18), but are not known to affect other ion channels or other regulatory proteins in skeletal muscle. These agents block many types of K⁺ channels, in particular delayed-rectifier K⁺ channels, but also others such as ATP-sensitive K⁺ channels. The positive inotropic effects of DAP in skeletal muscle are attributed predominantly to blockade of delayed-rectifier K⁺ channels (1), since specific blockers of either Ca²⁺-activated or ATP-sensitive K⁺ channels (e.g., apamin, charybdotoxin, glibenclamide) have very little effect on muscle force (especially in nonfatigued muscle) (22). By blocking delayed-rectifier K⁺ channels, the aminopyridines slow repolarization of the action potential, with resultant prolongation of the action-potential duration (1, 25, 26), and increase Ca²⁺ influx (26). The present study used DAP as the K⁺-channel blocker because this agent has greater inotropic effects than 4-aminopyridine (5) and is less toxic than 4-aminopyridine, allowing its use for the treatment of multiple sclerosis and Lambert-Eaton myasthenic syndrome in humans (27, 28).

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Studies were performed on diaphragm muscle of young adult (3 to 4 mo of age) and old (20 to 21 mo of age) male Fischer 344 rats. An age of 20 to 21 mo was chosen for the old animals, as in previous studies of aging in our laboratory (29), in order to avoid a possible survivorship bias that could be introduced by studying animals at even older ages. The mean weight of the young-adult rats was 320 ± 13 g (mean ± SE) and that of the old rats was 442 ± 14 g. The animals were anesthetized with intraperitoneal urethane (initial dose 1 g/kg, with additional smaller doses given as needed). The costal diaphragm was removed surgically with intact bony and tendinous origins and insertions, and placed in well-oxygenated physiologic solution during further dissection. In accord with what is done with other in vitro muscle studies in rodents, the animals were not tracheostomized prior to removal of the diaphragm (1, 2, 5, 10, 22, 31). For studies of resting membrane potential and action potentials, hemidiaphragms with an intact phrenic nerve supply were pinned to the bottom of a Sylgard-lined 35-mm Petri dish. For studies of contractility, small strips of muscle were cut from each hemidiaphragm and mounted vertically in a 25-ml double-jacketed tissue chamber. Both preparations had intact bony and tendinous origins and insertions during study. Muscle from a given animal underwent either electrophysiologic or contractility studies. All studies were performed at 37° C. This temperature was maintained with a Peltier device (Medical Systems, Greenvale, NY) surrounding the Petri dish for the electrophysiology studies, and by circulating warm water through the outer jacket of the tissue chamber for the contractility studies. The composition of the physiologic solutions was as follows, in mM: NaCl 135, KCl 5, CaCl₂ 2.5, MgSO₄ 1, NaH₂PO₄ 1, NaHCO₃ 15, and glucose 11, with the pH adjusted to 7.35 to 7.45 during aeration with 95% O₂/5% CO₂. For the electrophysiologic studies, well-aerated solution flowed constantly from a reservoir through the Petri dish; in order to minimize vibration, the solution was not aerated in the Petri dish. For the contractility studies, physiologic solution was aerated both in the reservoir and in the tissue bath. For both systems, the physiologic solution was verified to be well-oxygenated through use of a dissolved oxygen meter (World Precision Instruments, Sarasota, FL). Studies focused on a single DAP concentration of 0.3 mM, which was chosen on the basis of its producing a near-maximal inotropic effect in a previous study (7). For the contractility studies, an aliquot of DAP stock solution was added directly to the bath. An incubation time of 10 min was used, which in both a previous study of DAP (7) and the present study was found to be sufficient for force increases to reach a plateau. For the action-potential studies, a flow-through system was used in which physiologic solution without DAP was switched to physiologic solution with DAP. A longer incubation period of 15 min was therefore used to also allow for a 5-min wash-in time. The duration of the incubation was verified to be sufficient for the slowing of action-potential repolarization and the increase in action-potential area to reach a plateau. All chemicals were obtained from Sigma Chemical Co., St. Louis, MO.

For electrophysiologic studies, the phrenic nerve was placed in a suction electrode (A-M Systems, Everett, WA) and stimulated electrically with a pulse width of 0.2 ms and supramaximal voltage. Intracellular recordings were made with glass microelectrodes fabricated with a Flaming-Brown micropipette puller (Sutter Instruments, Novato, CA). The electrodes had resistances of 5 to 15 M when filled with 3 M KCl. Resting membrane potential and action potentials were recorded with an Axoclamp 2B amplifier (Axon Instruments, Foster City, CA). After determination of resting membrane potential, the phrenic nerve was stimulated at a low frequency (2 Hz), and the resulting action potential(s) was recorded. Repetitive muscle contraction frequently dislodged the electrode from the cell, so that it was not feasible to study the same muscle fiber before and after DAP. Therefore, muscle fibers were sampled before and after addition of drug, with care taken not to sample from a given fiber more than once under a given condition. Preliminary studies indicated that resting-membrane-potential and action-potential characteristics remained stable for at least 1 h in the absence of addition of drug. Electrophysiologic signals were digitized, collected on-line (Axotape software; Axon Instruments, Foster City, CA), and stored on the hard drive of a computer for later analysis. Data analysis utilized a combination of the manually driven cursors provided with the Axotape software and the semiautomated analysis capabilities of the Strathclyde Electrophysiology Software Whole-Cell Program (distributed by Dagan, Minneapolis, MN). Small segments of data containing the action potentials were exported from Axotape to the Whole-Cell Program for the latter method of analysis. Action-potential properties were characterized as follows: height (difference between resting membrane potential and the peak positive voltage), overshoot (amount by which voltage exceeded 0 mV at the peak of the action potential), area (the integral of membrane potential during the action potential measured relative to resting membrane potential), 10 to 90% rise time (the time required for the depolarizing phase to go from 10 to 90% of action-potential height), maximal rate of rise (most rapid rate of depolarization), 50% decay time (T 50%, the time required for the action potential to repolarize 50% of the way back to resting membrane potential), and 80% decay time (T 80%, the time required for the action potential to repolarize 80% of the way back to resting membrane potential).

For studies of muscle force, the muscle strips were stimulated with a pulse width of 1 ms and supramaximal voltage delivered via platinum electrodes placed on either side of the muscle. Force was measured with a high-sensitivity isometric transducer (Kent Scientific Corporation/Radnotti Glass Technology, Monrovia, CA), digitized, collected on-line (Axotape software), and stored on the hard drive of a computer for later analysis. With this technique, twitch forces of ~ 0.5 kg/cm² are obtained routinely in rat diaphragm (6). Twitch force was measured during 0.1-Hz stimulation for a 3-min baseline period before addition of drug (or no drug); muscle strips in which force varied by more than 5% during the baseline period were rejected for analysis. The (rare) strips with poor baseline stability were rejected in the present and previous studies (6, 7, 32) because it is difficult to accurately quantify the effects of a drug in a muscle strip whose baseline force is unstable. Studies of muscle fatigue used trains of 20-Hz stimuli, with a train duration of 0.33 s delivered once per second, for a total duration of 2 min. Data analysis was performed with manually driven cursors provided with the data-acquisition program, to determine peak force and isometric-twitch kinetics. The force of contraction of a muscle strip is determined to a large extent by the size of the strip. To factor out the effects of variability in muscle-strip size, force was normalized relative to that found during a baseline period, as is commonly done in studies of muscle contractility and particularly fatigue (22, 33). The approach used in the present study and our previous studies (6, 7, 32) was to normalize force relative to the value during the predrug, prefatigue baseline period (the 3-min period during which muscle strips were stimulated at a frequency of 0.1 Hz prior to the addition of drug or no drug). Force during the baseline period was assigned a value of 100, and all subsequent force measurements were normalized relative to this value. This method of normalization is identical to that used by Wickendon and colleagues (22) in their study of effects on muscle force of agents that affect ATP-sensitive K⁺ channels. Rate of muscle contraction and relaxation during twitches were quantified by the isometric contraction time (time between the onset of contraction and peak force) and the half-relaxation time (time required for peak force to decay by 50%). During the 20-Hz trains used to test fatigue resistance, contraction time was measured from the first twitch of the train, and half-relaxation time was measured from the last twitch of the train.

All values presented are means ± SE. Sample sizes are indicated in the figure legends. Statistical comparison of the effects of DAP on young-adult versus old muscle was done with analysis of variance (ANOVA), which was followed by the Newman-Keuls test when analysis of variance indicated significant differences. Statistical analysis of fatigue data utilized ANOVA for repeated measures, followed by the Newman-Keuls test when indicated. The criterion for statistical significance was p < 0.05.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Resting membrane potential did not differ between young-adult and old muscle, and was not affected by DAP (Figure 1). Action-potential height, overshoot, area, rate of rise, and rate of decay also did not differ between young-adult and old muscle (Figures 1 and 2). DAP did not affect action-potential height, overshoot, or rate of rise of either young-adult or old muscle. However, DAP slowed the action-potential repolarization of both young-adult and old muscle (Figure 2). In young-adult muscle, the T 50% increased by ~ 181% (p < 0.05) and the T 80% increased by ~ 471% (p < 0.05), whereas in old muscle the T 50% increased by ~ 95% (p < 0.05) and the T 80% increased by ~ 190% (p < 0.05). As a result of the slowing of action-potential repolarization, DAP increased the action-potential area of young-adult muscle by ~ 245% (p < 0.05) and that of old muscle by ~ 86% (p < 0.05). In the absence of DAP, action-potential area was not significantly different in young-adult and old muscle, whereas in the presence of DAP action-potential area was ~ 44% greater in young-adult than in old muscle (p < 0.05) (Figure 1).

View larger version (16K): [in this window] [in a new window]   Figure 1.   Resting membrane potential and action-potential height, overshoot, and area of young-adult and old diaphragm muscle in the absence and presence of DAP (0.3 mM). Values are mean ± SE; error bars are not indicated when error was smaller than the symbol size. Significant differences are indicated: *significant difference for no drug versus DAP; +significant difference for young-adult versus old muscle. Sample sizes were as follows: young-adult muscle, 15 fibers before DAP and 15 after DAP; old muscle, 15 fibers before DAP and 15 fibers after DAP.

View larger version (15K): [in this window] [in a new window]   Figure 2.   Action potential 10 to 90% rise time and maximum rate of rise, and times for action-potential to decay by 50 and 80%, of young-adult and old diaphragm muscle in the absence and presence of DAP (0.3 mM). Values are mean ± SE; error bars are not indicated when error was smaller than the symbol size. Significant differences are indicated: *significant difference for no drug versus DAP. Sample sizes are the same as for Figure 1.

To determine the functional consequences of the greater effects of DAP in increasing the action-potential area of young-adult as compared with old muscle, we conducted studies of muscle force during twitches and short tetani. DAP increased twitch force and 20-Hz tetanic force of both young-adult and old muscle (Figure 3). In the absence of DAP, force was similar for young-adult and old muscle, whereas in the presence of DAP force was ~ 15% greater for young-adult than for old muscle during twitches (p < 0.05) and ~ 19% greater for young-adult than for old muscle during 20-Hz stimulation (p < 0.05). The isometric-twitch-contraction times of both young-adult and old muscle were prolonged by DAP (Figure 3). Twitch contraction time did not differ for young-adult muscle and old muscle in the absence of DAP, but was ~ 14% longer for young-adult than for old muscle with DAP (p < 0.05). Isometric half-relaxation time was prolonged by DAP only for young-adult muscle (p < 0.05), so that in the presence of DAP this time was ~ 48% longer for young-adult than for old muscle (p < 0.05) (Figure 3).

View larger version (19K): [in this window] [in a new window]   Figure 3.   Isometric twitch force, force during 20-Hz stimulation, twitch contraction time, and twitch half-relaxation time of young-adult and old diaphragm muscle in the absence and presence of DAP (0.3 mM). Values are mean ± SE; error bars are not indicated when error was smaller than the symbol size. Significant differences are indicated: *significant difference for no drug versus DAP; +significant difference for young-adult versus old muscle. Sample sizes are as follows: young-adult muscle, five muscle strips without DAP and six muscle strips with DAP; old muscle, five muscle strips without DAP and six muscle strips with DAP.

A previous study of young-adult rat diaphragm found that the inotropic effects of DAP could be maintained over time during repetitive contractions (7). To determine whether there may be differences between young-adult and old muscle in the modulatory effects of DAP on force over time, muscles were subjected to repetitive stimulation with 20-Hz trains. In the absence of DAP, force was similar for young-adult and old muscle throughout the course of repetitive stimulation (Figure 4). DAP had two effects on the force of young-adult and old muscle during the course of repetitive 20-Hz stimulation: a large degree of positive inotropism, and a modest acceleration of the rate at which force declined over time. Force of both young-adult and old diaphragm was approximately threefold higher in DAP-treated than in untreated muscle at the onset of repetitive stimulation, and remained signficantly higher with than without DAP for the first 30 to 40 s of the fatigue run (Figure 4). However, because of the mildly accelerated rate of force loss in DAP-treated muscle, force was no longer significantly different in DAP-treated than in untreated muscle during the second half of the fatigue run. Despite the higher rate of force decline with than without DAP, however, muscle force was never lower in the presence of DAP than in the absence of DAP for either young-adult or old muscle. In the presence of DAP, diaphragm force was significantly greater for young-adult than for old muscle during a short portion of the period of repetitive stimulation (40 to 50 s into the fatigue run). During repetitive stimulation, DAP increased contraction and half-relaxation times of both young-adult and old muscle (Figure 5), with these effects most prominent during the early portion of the period of repetitive stimulation for contraction time and the middle of the period of repetitive stimulation for half-relaxation time. However, effects of DAP on isometric contraction and half-relaxation times during fatigue were not affected by age.

View larger version (17K): [in this window] [in a new window]   Figure 4.   Fatigue during intermittent 20-Hz stimulation of young-adult and old muscle in the absence and presence of DAP (0.3 mM). Values are mean ± SE; error bars are not indicated when error was smaller than the symbol size. Significant differences are indicated: *significant difference for no drug versus DAP; +significant difference for young-adult versus old muscle. Sample sizes are the same as for Figure 3.

View larger version (21K): [in this window] [in a new window]   Figure 5.   Contraction and half-relaxation times during intermittent 20-Hz stimulation of young-adult and old muscle in the absence and presence of DAP (0.3 mM). Values are mean ± SE; error bars are not indicated when error was smaller than the symbol size. Significant differences are indicated: *significant difference for no drug versus DAP. Sample sizes are the same as for Figure 3.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Limited data are available regarding changes in skeletal-muscle K⁺ conductance with aging or the functional consequences thereof. De Luca and colleagues found variable increases in macroscopic (whole-cell) K⁺ conductance with aging in rat limb muscle (13). In their earlier studies of Wistar rats, an 8-48% increase in K⁺ conductance with aging from 6 to 9 to 29 mo was not statistically significant, whereas in subsequent studies of a larger number of fibers from inbred Wistar Kyoto rats, Deluca and colleagues found a significant increase in K⁺ conductance, from 316 to 422 µS/cm², with aging from 6 to 9 to 23 mo. Of interest is that this latter increase could be reduced by glibenclamide, suggesting that ATP-sensitive K⁺ channels contributed to the increased overall K⁺ conductance (14). This contrasts with the data of Tricarico and coworkers (17), who performed patch-clamp recordings of ATP-sensitive K⁺ channels in rat-toe muscles. They found that the open probability of 0.196 for channels from 7- to 11-mo-old rats decreased to 0.057 and 0.041 for channels from 20- to 23- and 25- to 26-mo-old rats, respectively. Furthermore, there was an apparent reduction in membranous ATP-sensitive K⁺-channel density, but no change in single-channel conductance, with aging.

Many types of K⁺ channels are found in mammalian skeletal muscle, including inward-rectifier, delayed-rectifier, ATP-sensitive, and large- and small-conductance Ca²⁺-activated K⁺ channels (17, 22, 34). The voltage-sensitive inward- and delayed-rectifier channels regulate resting membrane potential and action-potential duration, respectively, and hence muscle-force production. The role of the other K⁺ channels in regulating muscle contractile performance under physiologic conditions is less clear. Specifically, agents that block several types of K⁺ channels (e.g., tetraethylammonium or the aminopyridines) prolong action-potential duration and augment force (1, 25), whereas specific blockers of ATP-sensitive K⁺ channels (e.g., glibenclamide) or Ca²⁺-activated K⁺ channels (e.g., apamin or charybdotoxin) have little effect on the force of nonfatigued muscle (22). Thus, the action-potential-prolonging and inotropic effects of the aminopyridines are attributed predominantly to their blockade of delayed-rectifier K⁺ channels. Previous studies of K⁺ conductance in aging skeletal muscle have focused on resting conductance (which is regulated predominantly by inward-rectifier K⁺ channels, with possible contributions from ATP-sensitive or Ca²⁺-activated K⁺ channels) (13), or specifically on conductance through ATP-sensitive K⁺ channels (17). In contrast, the present study, by examining effects of DAP on action-potential repolarization, most likely focussed on changes in delayed-rectifier K⁺ channels with aging. The differences among studies in the direction of change in K⁺-channel conductances with aging can therefore be explained by heterogeneous effects of aging on various types of K⁺ channels.

There are several possible explanations for greater effects of DAP on young-adult than on old muscle. One is that K⁺-channel density is lower, or that the open probability of these channels is lower, in old than in young-adult muscle, so that blocking these channels will have a smaller effect in old and young-adult muscle. Another is that baseline channel density and open probability do not differ between young-adult and old muscle, but that the K⁺ channels of old muscles are less sensitive than those of young-adult muscle to inhibition by DAP. In our study, young-adult and old muscle did not differ in action-potential properties in the absence of DAP. This would favor differential K⁺-channel sensitivity to DAP in young-adult versus old muscle as the mechanism responsible for the present findings. However, differential K⁺-channel density or open probability in young-adult as compared with old muscle, if accompanied by other changes in ionic conductances with aging, could also account for the findings. Action-potential repolarization results from the following factors: (1) inactivation of previously opened Na⁺ channels; (2) activation of previously closed K⁺ channels (predominantly delayed-rectifier channels); and (3) the effects of baseline membranous K⁺ and Cl conductances (through the channels that regulate resting membrane potential in resting musclewhich for K⁺ are mainly the inward-rectifier K⁺ channels). With multiple regulatory mechanisms contributing to the rapidity of action-potential prolongation, an alteration in one mechanism could be compensated for by a change in another (e.g., baseline chloride conductance, which changes considerably with aging) to maintain near-normality of action-potential repolarization.

The greater DAP-induced augmentation of action-potential area for young-adult than for old muscle was associated with a greater augmentation of muscle force for young-adult than for old muscle. Lin-Shiau and coworkers (2) have found a linear relationship between prolongations of action-potential duration and increases in diaphragm force in response to various K⁺-channel blockers (4-aminopyridine, tetraethylammonium, uranyl nitrate) administered singly or in combination. These data support the notion that increases in contractile force are the direct consequence of delayed action-potential repolarization, as has also been concluded by several other investigators (1, 3, 4). Delayed action-potential repolarization would be expected to lengthen contraction times, and indeed this was found in the present and previous studies (5). The present data suggest that the greater force increase of young-adult than of old muscle was due in part to a greater lengthening of contraction time. That DAP prolonged the half-relaxation time of fatigued muscle is consistent with previous findings for both DAP and 4-aminopyridine in young-adult muscle (6, 7). Slowing of rates of contraction and relaxation has several consequences. First, for a given level of peak force, the total force- time integral will be increased. Second, during stimulation at subtetanic frequencies, there will be greater fusion of contractions, which can increase peak force.

During repetitive stimulation, DAP produced substantial positive inotropism and a modest acceleration of the rate of decline of force in both young-adult and old muscle, consistent with previous data on young-adult muscle (7). The beneficial effects of the inotropism outweighed the detrimental effects of the more rapid rate of decline of force, in that force with DAP was either signficantly higher or tended to be higher, but was never lower, than force without DAP. Whether DAP would have a beneficial or detrimental effect on force over time in diseased muscle (e.g., dystrophic muscle) cannot be predicted from the present data.

The magnitude of the changes in rate of repolarization of the action potential and twitch height noted in the present study differ to some extent from values reported in other mammalian muscle in response to the aminopyridines. Delbono and Kotsias (1) found that 2.5 mM DAP increased the twitch force of rat diaphragm by 59%, and prolonged the action-potential duration by 100% (from 0.9 to 1.8 ms, measured at 40 mV) as the result of a 50% reduction in the rate of action-potential repolarization (the rate of repolarization decreased from 156 to 77 V/s). Lin-Shiau (2) found that 0.3 mM 4-aminopyridine increased the twitch force of diaphragm muscle by 25%, and prolonged the action-potential duration by ~ 20% (from 0.51 to 0.62 ms). In rat diaphragm we have found that 0.3 mM 4-aminopyridine increased force by 47 to 64% and prolonged contraction time by 20 to 32% (5, 6), whereas 0.3 mM DAP increased force by 158% and prolonged contraction time by 29% (7). Quantitative differences between findings in the present and previous studies can be attributed to several factors, including the use of DAP versus 4-aminopyridine, differing concentrations of drug used, differences in the animal model (age, gender, species, and strain), and different methods of quantifying the action-potential repolarization rate.

It is possible that changes with aging could differ from those described here in muscle from female animals, or those in other skeletal muscles than diaphragm. Among a group of twelve previous studies examining the effects of aminopyridines on skeletal muscle, three used exclusively male animals (5), one used exclusively female animals (25), one used both male and female animals but without commenting about differences between genders (2), and seven did not specify the gender of the animals (1, 3, 4, 18). All of the studies concurred that the aminopyridines block K⁺ channels, prolong action-potential duration through the slowing of action-potential repolarization, and increase the twitch force of skeletal muscle. On the basis of this limited information, there do not appear to be major qualitative differences between male and female muscle, although quantitative differences could certainly exist. Regarding the issue of differences among skeletal muscles, the 12 studies described previously examined diaphragm, sternohyoid, flexor digitorum longus, semitendinosus, cutaneous pectoris, and toe muscles (1, 18, 25). All concurred that the aminopyridines block K⁺ channels, prolong action-potential duration through the slowing of action-potential repolarization, and increase the twitch force of skeletal muscle. Khan and Edman (3) found that 4-aminopyridine augmented twitch force and slowed contraction time in frog semitendinosus muscle and rat toe muscle, but only in frog semitendinosus muscle was the relaxation rate slowed. Whether the latter difference between frog semitendinosus muscle and rat toe muscle was due to variability among muscles or variability among species is unclear. More recently, we found that the increase in force in response to 4-aminopyridine was similar for the sternohyoid (55%) and diaphragm muscles (64%), whereas the increase in force in response to another K⁺-channel blocker, tetraethylammonium, was significantly greater for the sternohyoid (33%) than for the diaphragm (7%) (5). This raises the possibility that there may be differences among muscles in the degree to which aging alters electrophysiologic and contractile responses to K⁺-channel blockers.

The present study was subject to the inherent limitations of in vitro approaches. Particularly relevant is that in vivo, alterations in blood flow induced by K⁺ release may influence muscle contractile performance, so that altering K⁺-channel conductance may modulate muscle fatigue by more than one mechanism. However, there are also a number of major advantages of in vitro over in vivo testing. First, it is technically not feasible to record diaphragm resting membrane potential and action potentials in vivo, due to muscle movement dislodging the electrodes from their intracellular location. Therefore, use of an in vivo model would not have provided insight into the cellular electrophysiologic mechanisms by which DAP altered the contractile force of young-adult versus old muscle. Second, the purpose of this study was to examine changes with aging that occur at the level of the muscle rather than at the level of the entire animal. Aging affects cardiovascular function, and furthermore, K⁺-channel blockers can affect vascular smooth muscle. Had this study utilized an in vivo approach, it would have been unclear whether different responses to DAP in old versus young-adult muscle were due to differences in cardiovascular and blood-flow effects or to differences in direct effects on the muscle.

In conclusion, the results of the present study agree with those of previous work indicating changes in skeletal-muscle K⁺ channels with aging (13). In addition, the results support the notion that effects of aging may differ among various membranous K⁺ conductances (13). Functionally, the inotropic effect of DAP was greater in young-adult than in old muscle, as was the degree of DAP-induced slowing of isometric contraction time. The difference between young-adult and old muscle in the inotropic effect (181% versus 144% increase) of DAP was modest in relation to the degree of inotropism. The increase in twitch force noted in old animals was much greater than increases in twitch force generally noted with currently available inotropic agents, such as the methylxanthines and -adrenergic agonists (8). Hence if DAP or DAP-like agents are used as a therapeutic approach for muscle weakness, their use should not be precluded in the elderly.