The purpose of this investigation was to determine the effects of low extracellular calcium and calcium antagonists on skeletal muscle staircase and fatigue. Initial experiments revealed that, brief exposure (10 minutes) of single frog sartorius muscle to diltiazem, D-600(5 and 30 pM) and low calcium Ringer's solution (LCR, calcium replaced by magnesium and EGTA) had little effect on isometric twitches evoked every 30 seconds. However, when stimulated at 1 per second for 15 minutes, the calcium antagonists significantly decreased the magnitude and time course of the staircase, whereas LCR decreased only the time course. Each experimental condition significantly increased the rate of fatigue while diltiazem and 0-600 both increased the magnitude of fatigue. Following the stimulation period, caffeine (10 mM) elicited contractures from all muscles whereas high potassium (180 mM) elicited contractures from control muscles only. These results indicate that calcium channel antagonists depress the skeletal muscle staircase response. They also indicate that these compounds as well as LCR enhance the fatigue process. Extracellular calcium influx may therefore have some influence on skeletal muscle twitches during prolonged repetitive activity. Key words: diltiazem D-600 calcium channels extracellular calcium MUSCLE & NERVE 13:1118-1124 1990
EFFECTS OF LOW CALCIUM AND CALCIUM ANTAGONISTS ON SKELETAL MUSCLE STAIRCASE AND FATIGUE JAY H. WILLIAMS, PhD
Extracellular calcium influx via membranebound, voltage-gated channels plays several important roles in excitation and contraction of cardiac and smooth muscle including generation of the membrane action potential and control of crossbridge formation. '428 Organic compounds such as verapamil, diltiazem, and nifedipine, as well as the cations cobalt and manganese, block calcium entry and therefore inhibit the mechanical response of these tissues.15 Consequently, antagonism of calcium influx reduces myocardial contractility, causes vasodilation and is widely used in the treatment of cardiovascular disease manifestations such as angina pectoris, hypertension, and supraventricular tachycardia. l 5 In skeletal muscle, calcium is also an important
From the Muscular Function Laboratory, Division of Health, Physical Education and Recreation, Virginia Polytechnic Institute and State University, Blacksburg, Virginia. Address correspondence and reprint requests to J.H. Williams, PhD. HPER Division, Virginia Tech, Blacksburg. VA 24061 Accepted. for publication November 22, 1989. CCC 0148-639X/90/01201118- 07 $04.00 0 1990 John Wiley 8 Sons, Inc.
1118
Calcium and Muscle Fatigue
contraction regulator. Some time ago, Bianchi and Shanes8 and Curtis"' reported that during activation, a net uptake of calcium by frog sartorius muscle occ:urs. Although voltage-dependent calcium channels and calcium influx have been located in skeletal muscle,'^' current opinion holds that calcium stored intracellularly by the sarcoplasmic reticulum (SK) is the major source of that used in the contraction process.I2 This position is supported by findings that skelet.al muscle twitches and tetanic contractions are not depressed by removal of external calcium or by addition of calcium a n t a g o r i i ~ t s . " " ~ ~Several ~ , ~ ~ reports, however, suggest that under sotne conditions, potentiation of skeletal muscle contractions can be inhibited by inhibiting calcium influx. 'These include twitch potentiation induced by theophylline, l 3 nitrate ions,'9 epinephrine,32 and brief tetanic contractions19 and also contractures induced by high extracellular potassium.'6 Thus, it is unclear as to whether extracellular calcium influx plays any role in the regulation of skeletal muscle con tractions. During low frequency repetitive stimulation (eg, 1 to 5 Hz) of frog skeletal muscle, twitches are
MUSCLE & NERVE
December 1990
progressively potentiated during the initial riiinutes. This staircase response is then followed by a rapid decliric in tension (fatigue) to below control levels."" A t present, the mechanisms responsible for these two phenomena are not fully understood. l'hree pieces of circumstantial evidence indicate that the influx of extr-acellular calcium may sotilehow he involved. First, Bianchi arid Niiraya11"" show that during I H z stimulation, a net uptakc of calciuni occ~irsthat is temporally associated with the staircase response. As stirnulation continues, a net accumulation of calcium in the lumen of the transverse tubule (Tl') occurs that is associated with fatigue. Second, reports t.liat repetitive stimulation initially increases then decreases the cnlciuiii influx in cardiac cells. Third, Kolbeck and Speir'" show that diltiazeni, verapaniil, and zero-calcium incubation media eiihatice fatigue of the rat diaphragm. Whether changes iii intrdcxt racellular calcium cxchangc influence skeletal muscle twitches during repetitive stimulation or arc merely coincidental is questionable. I f ' these processes are causally linked then altering calcium influx via calcium antagonists or Iiy reduc:ed cxttacellular calcium cli.iring this type of activity should alter the time co~irseand magnitude of the staircase arid fatigue responses. Therefore, the purpose of' this investigatiori was to cxarnine the el'kcts of' low c.dl1iiuin .' and calcium charillel antagonists on skeletal muscle staircase a i d fhtigie. METHODS
For all experiments, a norrnal, phosphate buffered Kinger's solution (NR) containing the following (mA4): NaCI (1 15). KCI (2.5), CaCL, ( 1 4 , NaH,€'O.l (0.85), Na2HP0, (2.15), glucc.)se ( 5 ) , and d-tubocurarine (. 1 mg/mL), was used (pII 7 . 2 ) . Media were gassed prior to a r i d during use with room air. Stock drug solutioiis (500 FM) were prepared frcsh each day by dissolving either diltiazern (DIL'T) or K)-600 (D600) (Sigma Chemical Co.) in NK. Final drug concentrations were achieved by diluLing tlie stock with appropriate amounts of'N K. Low calcium Ringer's (LCR) was prepared by substituting 3 mM MgGl, and 1 mM EGI'A for CaCl,.
Incubation Media.
Sartoi ius nruscles were taken from srriall (4to 6 ( i n , 20 to 30 g ) niale glassfrogs (Rnno l'zpzenr) with silk thirads (5-0) tied to the distal and proximal tendons. Single muscles were inouiited vertically, under tension ( 2 g) in a tem-
Muscle Preparation.
Calcium and Muscle Fatigue
peratiire-cnntt.olled (25"C, 25 mI,) muscle charnbcr. Muscles were first allowed to equi1ibrat.e in N R for 60 minutes wliilr twit.clies werc evoked cvery 2 minutes. Only if' twitches obtained during the final 20 niinirtes o f equilibration were stable was the tissuc used for experirneiitatioti. Twitches were e1et:trically evoked by passing square wave pulses ( 2 rrisec) of supramaxirnal (1.5 x maximal voltage) current across platinurn ring electrodes p1at:ed at eitlier end of the muscle. Stiriiuli were delivered b y a stirnulus isolation unit (SIU-5) wliich was, iri turn, driven hy a Grass S-48 stirnulator.
Twitches were registered by an isornetric: force transducer (Harvard). Amplified signals were displayed on a strip-chart recorder (Kipp & %onen ED-8) and also digitized (MetraRyte DAS-16, 12 bit resolution, 1000 Hz) and recorded by mici-ocoml~uter(IBM 1%-XT). 'l'witches sampled b y thc microcomputer system were analyzed, off-line, for peak tension (PT), time t.o peak tension (TTPT), 1ialf-rclax;ition time (HRT), and the peak rate of tension increase (+dY/dt) atid dcctease (-dP/tlt).
Tension Recording.
Initially, the effects of DIL'I', DGOO and LCR o n single twitches were examined. Muscles were incubated for 10-niinute periods in allernating NR and DILT, 1)600 (5 and 30 pM), 01- I.CR solutions while twitchcs were evoked, recorded, and analyzed every 30 seconds. 'To examine the ef'fkcls of the 3 experimental conditions 011 musclc staii-case and fatigue, 1 inusclc of each pair was iiicuhaLed in N K and the other in either DILT (SO FM), D(i00 (30 kM), o r I X K in the following nianner. M~isclcswere initially incubatcd in NR for 5 ~niiiutesand twitches evoked every 30 scconds. They wcre thcri incubated for 5 minutes i n h e niedia to be used during the repetitive stirnulation bout with twitches again elicited every 30 seconds. After- these preincubations, muscles were stimulatecl at 1 H7 for 15 minutes. From the cotnputer and strip-c:hart recordings, temporal changes in P T were examined for initial twitch PI' (€''rO), the higlicst P'T reached during the s~;rirc:ascresponse (P'r,,,), the time a t which 1"11,, occurred (TTI"l1,,),final twitch PT (PT,,) and thc half-time (t,,2) of the c1ct:lirie in I'T from I'T",. Immediately al'ter thc fatigue bout, muscles were exposed the same incubation media coiit.airiing cither high potassium (180 riiM) or cal'feine (10 r M ) concentrations to elicit a contracturc. Experimental Protocols.
MUSCLE & NERVE
December 1990
1119
Table I. The effects of the four experimental conditions on single muscle twitches. ~
~~
PT
TTPT (msec)
HRT (msec)
+dPldt (Nlsec)
-dP/dt (Nlsec)
124.2 (17.6) 144.1 (11.8) 153.9 (12.7) 139.2 (11.8) 141.1 (12.7) 126.4 (13.7)
32 2 (2 2)
36 5 (2 0) 38 0 (2 0) 38 9 (1 8) 36 9 (2 1 ) 37 5 (2 0) 37 7 (2 2)
2.33 (.25)
1.16 (.06) 1.10 (.07) 1.08 (.09) 1.13(.09) 1.12 (.09) 1.02 (.08)
Condition
NR
DILT (5 pM) DlLT (30 pM) 0600 (5 pM) D600 (30 pM) LCR
31 9 (2 3) 31 8 (2 1 ) 32 4 (2 5) 32 8 (7 4) 30 9 (2 2)
Values are means (StM) See text for definitions (N
=
8 for
each condition)
dicated that within a single pair, responses under NR conditions were very similar. During repetitive stimulation, all muscles exhibited initial positive staircase responses during the first ti minutes of' stimulation. This response was followed by an exponential decline in P T through the remainder of the stimulation period. Incubation of' muscles in DILT and D600 significantly depressed the magnitude (PT,,,/Y'To) of the staircase response by 10 to 20% ( P < .05) (Table 2). LCR, however, only tended to depress the staircase (.05 < f < . l o ) . In 7 of the 10 muscle pairs exposed to LCR, P'r,,,/Ply0 was noticeably decreased while in the other pairs the data were equivocal. T h e calcium antagonists, as well as LCK, significantly reduced the time course (TT'PT,,) of the staircase by approximately 35%. All three experimental conditions also signific:antly increased the rate (t,&) of fatigue with D600 exerting the greater effect on tlPL. After the 15-
Analyses of variance, adjusted for paired muscles, were used to determine differences in the dependent variables between each incubation condition ( P < .05). RESULTS Twitches. Mean responses of single twitches to DILT, U600, and LCR are presented in Table 1 . As can be seen, these conditions had no sigriificant effects on the magnitude and time course of the twitch although the higher concentration of DI1.T tended to increase both P'1'and HKT ( . l o < P < .15).
Single
Repetitive stimulation. Teniporal patterns of the changes in PT for each iricubation condition are 3hown in Figures 1, 2,arid 3 . Although the time course oi individual changes in PT were somewhat variable between muscle pairs, pilot data in-
0
1
2
3
2.41 (.21) 2.46 (25) 2.41 (.22) 2.42 (.21) 2.28 (.24)
4
5
6
7
8
9 1 0 1 1 1 2 1 3 1 4 1 5
STIMULATION TIME (min) FIGURE 1. Responses of single sartorius muscles to NR and DlLT during repetitive stimulation. Data were obtained from paired sartorii in which one muscle was exposed to NR and the other to DILT. Values are means t SEM and normalized with respect to PT,.
1120
Calcium and Muscle Fatigue
MUSCLE & NERVE
December 1990
0
1.8
0
0
1
2
4
3
5
6
7
6
9
-0 CONTROL - 0-600
1 0 1 1 1 2 1 3 1 4 1 ~
STIMULATION TIME (min) FIGURE 2. Responses of single sartorius muscles to NR and D600 during repetitive stimulation.
DISCUSSION
minute stimulation period, the calcium antagonists, h i t not LCR, significantly increased the magnitude of fatigue (PT,,/PT,,). Iri all cases, DILI' rendered the muscle niechanically inactive after approximately 1 1 Ininutes. Uiitler all conditions, caf-feine exposure immediately following thc stimulation period, resulted in contracture (data not shown). Although peak responses to caffeine w e r e off scale for thc recording configuvation used, ~ l i cinitial tinic courses were very similar. Only in NK muscles did high potassium elicit a measurable contracture. In LCR muscles, a sIriall potassium coritracture occurred but was minimally detect.ahle.
1.8
\
5
0.8 -
a
0.6 0.4
-
0.2
-
Roth l)(iO0 and DlLT have been shown to inarkcdl y in hibit calcium influx in skeletal muscle."~20 Likewise, incubation media in which calciurn has been replaced by inagnesiuni and EGTA also inhibit. calcium entry. As evident from recent results and those of other^,^^'"^'."^ DILI+, D600, and LCR appear to have little influence on individual muscle t.witches. During repetitive siirnulation, howevcr, thc calcium antagonists depressed the staircase response and all 3 conditions accelerated fatigue. It is possible that the effects of the three experitncntal condit.ions on repet.itively evoked twitches
0 - - T I -
0.0L'
'
'
'
'
'
'
'
'
1
2
3
4
5
6
7
B
9
0
I
'
-
0
'
'
CONTROL LOW co2+
'
'
1 0 1 1 1 2 1 3 1 4 1 5
STIMULATION TIME (rnin) FIGURE 3. Responses of single sartorius muscles to NR and LCR during repetitive stimulation
Calcium and Muscle Fatigue
MUSCLE & NERVE
December 1990
1121
Table 2. The effects of the 3 experimental conditions on the parameters of skeletal muscle fatigue. Condition
PT,IPTo
TTPT,,, (sec)
PT, JPT,
ti/,
(set)
~
~
NR DlLT
1 70 ( 098) 1 49 ( 057)*
292.6 (22.7) 192.0 (7.29)*
0 27 ( 041) 0.00 (.OOO)"
230.6 (23.9) 139.6 (6.78)*
NR
1 73 ( 082) 140 (loo)*
281.8 (21.4) 186.2 (10.5)"
0.31 (052) 0.08 (010)*
225.7 (25.6) 80.5 (1 1.O)*
1 6 2 (130) 1 56 ( 090)
241.7 (19.8) 121.6 (20.6)'
0.30 (059) 0.15 ( 093)
217.3 (19.8) 297.6 (24.2)'
D600 NR
LCR
Values are means (SEMJ. See text for definitions. r P < .05 vs NR)
could have been due to mechanisms other than inhibited calcium influx. However, it is unlikely that the electrical activity of the muscle was influenced since DILT, D600, and LCR do not normally alter skeletal muscle resting or action potential^.^,'^ Additionally, the calcium channel antagonists are thought to act only on rnembrane bound calcium channels and not to inhibit calcium release channels located in the SKS2 At present the mechanisms which mediate the staircase response are no1 fully understood. Some suggest that this phenomenon niay be due to rnyosin phosphorylatior~,'i altered intracellular calcium exchange." Bianchi and Narayai~"."~~ note that the increasc calcium uptake by the r n usclc during the initial moments of' repetitive stimulation (1 Hz) may be linked to the staircase response. 'The present results add additional support to this notion that when calcium influx was blocked, the staircase magnitude was reduced. Further, Gamboa-Aldeco et all9 have recently shown that removal of exter-nal calcium or addition of nifedipine significantly rcduced posttetanic twitch potentiation, a phenomenon similar to the staircase response. Exactly how increased extracellular calcium influx might mediate twitch potentiation during the staircase response is not known. Clearly the amount of calcium entering the muscle is far too small to markedly increase tension via direct elevation of niyoplasmic calcium. Williams and Barnes34 suggest that extracellular calcium rriost likely acts indirectly by: ( 1 ) increasing "calciuminduced" SK calcium release, (2) increasing resting myoplasrnic calcium concentration, and/or ( 3 ) increasing SR calcium load arid release. In either case, the amount of calcium present in the myoplasm during the twitch would be increased secondary to enhanced influx. In any event, the staircase response seems to be partially dependent on extracellular calcium influx. It should be noted,
1122
Calcium and Muscle Fatigue
however, that since this response was not completely abolished by DILI', D600, or LCR, other mechanisms such as those noted previously are also probably involved. Like the staircase response, the niechanisms which cause skeletal muscle fatigue are also n o t fully understood although corisidcrable evidence favors the notion that is most probably due to excitation-contraction (E-c) coupling failure." Gonzalez-Scrratos et al." show that calcium content of the SK terminal cisterriae is not reduced during fatigue. T h e present results and those of others clearly demonst.rate that high caffeine and potassium concentrations will elicit contractures of similar magnitude in both rested arid fatigued 11111scle.~"~~ Thus SR calcium content seems adequate enough to support contraction but is somehow unable to be released during fatigue. Failure of E-C coupling could result from either a failure of the 'I'T action pot.entia1 or a reduced stimulus for calcium release." With re ard to the first rnechanisrn, Bianchi arid Narayan5- ar-
i
gue that the net efflux of calcium into the I'T lumen during fatigue leads to elevation of T I calcium concentration. This, in turn, could elevate the mechanical threshold and decrease the cfficiency of E-C coupling. In this investigation, blocking calcium entry (or re-entry) by 1)ILT and D600 would liavc led t o a more rapid accumulation of T'l' calcium, an early elcvation of' the mechanical threshold and hence an early onset of fatigue. However, if' calciuin accutnulation in the T I ' contributed to fatigue then LCR should have reduced rather than increased the rate of fatigue sincc low initial 'I'T calcium concentrations woulcl have clelayed calcium acc:Lir-nulatiorl. Failure of' the ' I T action potential could a l s o occur as ;I rcsult of changes in sodiurri ant1 potassium concentration in the r ~ ~ r 1 7 . ' '~' l o w c v e r since , low con(:cntralions 01' calciuni ;intagorlists do not alter wnductance of' sodium rind pot
MUSCLE 8, NERVE
December 1990
in fatigue obser-vcti in this iiivcstigution ;ire probably cl iic t o factors other than altered socliuiiiipotassiurn stasis. Kcgiirdiiig tlie scc.ond mcchaiiism, the precisca Iiophysical 01- biochcinical processes wliich stimulate SK calciuin release a i r uriknown. Some suggest that calciuin ious t hetiiselvcs evoke calcium release. ' p , 1 7 Recent cviclerice suggests t ~ i a t a though a "calriii in-induced" cdciiini release rnechanism may riot 1.x the primary triechanisiri of SK calciurn release, il may play some role in this p ~ o c c s s . ~PerIiaps ~" this "seconttary" means of releasing SR calcium liecomes increasingly iniportant dLir-iii(>-repetitive stimulatioii leading to fa? Ligue. Duriii g do p(dariza t io Ii (IIci 11111 coi i I cl er i t e I' thc: cell and dircc:tly stiinulate or facilitate tlir: release 01' SR calciuni." Also, c;ilcium influx c.ould act iiitlirectly by cnteririg the niiisclc and r-estoriiig ;in internal "trigger-" c&:iuiii 111 either case, inhiliiting citkiiirn influx Ijy l)ILrl', I)(iOO, antl I.CR coiild i.cduce thc siir~iiilusfor SR cal-
ciuiii releasc: aiid accclrrate fatigue. l ' h c present r-esults t1ierc"oi.e suggest that extracellular calcium iriflux is somehow involved in the fittipie process. 1'o sii mrnai-ize, the calci u r n antagonists, tli Itiazem, and M O O depress thc slaircase response iuid enliarice fatigue of' frog skcletal riiiisclc whcr-eas reduced external calcium has no effect of the staircase but increases tlic: rate 01' f'atiguc. 'Iliese i-esults support the notion that cxtracellular calcium inllux m a y have sonic iriflucnce on skeletal muscle staircase arid f'atiguc. They also fiirtlier support the Iiotion that culcium influx has soiiic Iole iri the process of skeletal muscle excitatioiiconttxtioii coupling. l ' h e notioil that ciilcium chanricl antagonisis depi-ess contr'actioiis during rcpetitive activity might also h a v e irriplicatioris for individLials using these compo~i tids for treatniciit of i1iyoc;trdial disease rnmifestations such ;is hypcrtension and those who x c enganged in a physically active lifestyle.
REFERENCES I . Almeis W, Fink R, I';ilade P 1': Calcium depletion in frog muscle ciibules: 'The decline of calciuni current under maintained depolariz;itiori. 1 Physiol (Luizdj 19813 12:177'10 1. 2. Almets W, Palade P7': Slow calciurn antl potaszium currents am^ frog rriemhrane: Measurements with viiaclinegap tcchniqirc. j ~ h y s i o (I.imd) l 1
EFFECTS OF LOW CALCIUM AND CALCIUM ANTAGONISTS ON SKELETAL MUSCLE STAIRCASE AND FATIGUE JAY H. WILLIAMS, PhD
Extracellular calcium influx via membranebound, voltage-gated channels plays several important roles in excitation and contraction of cardiac and smooth muscle including generation of the membrane action potential and control of crossbridge formation. '428 Organic compounds such as verapamil, diltiazem, and nifedipine, as well as the cations cobalt and manganese, block calcium entry and therefore inhibit the mechanical response of these tissues.15 Consequently, antagonism of calcium influx reduces myocardial contractility, causes vasodilation and is widely used in the treatment of cardiovascular disease manifestations such as angina pectoris, hypertension, and supraventricular tachycardia. l 5 In skeletal muscle, calcium is also an important
From the Muscular Function Laboratory, Division of Health, Physical Education and Recreation, Virginia Polytechnic Institute and State University, Blacksburg, Virginia. Address correspondence and reprint requests to J.H. Williams, PhD. HPER Division, Virginia Tech, Blacksburg. VA 24061 Accepted. for publication November 22, 1989. CCC 0148-639X/90/01201118- 07 $04.00 0 1990 John Wiley 8 Sons, Inc.
1118
Calcium and Muscle Fatigue
contraction regulator. Some time ago, Bianchi and Shanes8 and Curtis"' reported that during activation, a net uptake of calcium by frog sartorius muscle occ:urs. Although voltage-dependent calcium channels and calcium influx have been located in skeletal muscle,'^' current opinion holds that calcium stored intracellularly by the sarcoplasmic reticulum (SK) is the major source of that used in the contraction process.I2 This position is supported by findings that skelet.al muscle twitches and tetanic contractions are not depressed by removal of external calcium or by addition of calcium a n t a g o r i i ~ t s . " " ~ ~Several ~ , ~ ~ reports, however, suggest that under sotne conditions, potentiation of skeletal muscle contractions can be inhibited by inhibiting calcium influx. 'These include twitch potentiation induced by theophylline, l 3 nitrate ions,'9 epinephrine,32 and brief tetanic contractions19 and also contractures induced by high extracellular potassium.'6 Thus, it is unclear as to whether extracellular calcium influx plays any role in the regulation of skeletal muscle con tractions. During low frequency repetitive stimulation (eg, 1 to 5 Hz) of frog skeletal muscle, twitches are
MUSCLE & NERVE
December 1990
progressively potentiated during the initial riiinutes. This staircase response is then followed by a rapid decliric in tension (fatigue) to below control levels."" A t present, the mechanisms responsible for these two phenomena are not fully understood. l'hree pieces of circumstantial evidence indicate that the influx of extr-acellular calcium may sotilehow he involved. First, Bianchi arid Niiraya11"" show that during I H z stimulation, a net uptakc of calciuni occ~irsthat is temporally associated with the staircase response. As stirnulation continues, a net accumulation of calcium in the lumen of the transverse tubule (Tl') occurs that is associated with fatigue. Second, reports t.liat repetitive stimulation initially increases then decreases the cnlciuiii influx in cardiac cells. Third, Kolbeck and Speir'" show that diltiazeni, verapaniil, and zero-calcium incubation media eiihatice fatigue of the rat diaphragm. Whether changes iii intrdcxt racellular calcium cxchangc influence skeletal muscle twitches during repetitive stimulation or arc merely coincidental is questionable. I f ' these processes are causally linked then altering calcium influx via calcium antagonists or Iiy reduc:ed cxttacellular calcium cli.iring this type of activity should alter the time co~irseand magnitude of the staircase arid fatigue responses. Therefore, the purpose of' this investigatiori was to cxarnine the el'kcts of' low c.dl1iiuin .' and calcium charillel antagonists on skeletal muscle staircase a i d fhtigie. METHODS
For all experiments, a norrnal, phosphate buffered Kinger's solution (NR) containing the following (mA4): NaCI (1 15). KCI (2.5), CaCL, ( 1 4 , NaH,€'O.l (0.85), Na2HP0, (2.15), glucc.)se ( 5 ) , and d-tubocurarine (. 1 mg/mL), was used (pII 7 . 2 ) . Media were gassed prior to a r i d during use with room air. Stock drug solutioiis (500 FM) were prepared frcsh each day by dissolving either diltiazern (DIL'T) or K)-600 (D600) (Sigma Chemical Co.) in NK. Final drug concentrations were achieved by diluLing tlie stock with appropriate amounts of'N K. Low calcium Ringer's (LCR) was prepared by substituting 3 mM MgGl, and 1 mM EGI'A for CaCl,.
Incubation Media.
Sartoi ius nruscles were taken from srriall (4to 6 ( i n , 20 to 30 g ) niale glassfrogs (Rnno l'zpzenr) with silk thirads (5-0) tied to the distal and proximal tendons. Single muscles were inouiited vertically, under tension ( 2 g) in a tem-
Muscle Preparation.
Calcium and Muscle Fatigue
peratiire-cnntt.olled (25"C, 25 mI,) muscle charnbcr. Muscles were first allowed to equi1ibrat.e in N R for 60 minutes wliilr twit.clies werc evoked cvery 2 minutes. Only if' twitches obtained during the final 20 niinirtes o f equilibration were stable was the tissuc used for experirneiitatioti. Twitches were e1et:trically evoked by passing square wave pulses ( 2 rrisec) of supramaxirnal (1.5 x maximal voltage) current across platinurn ring electrodes p1at:ed at eitlier end of the muscle. Stiriiuli were delivered b y a stirnulus isolation unit (SIU-5) wliich was, iri turn, driven hy a Grass S-48 stirnulator.
Twitches were registered by an isornetric: force transducer (Harvard). Amplified signals were displayed on a strip-chart recorder (Kipp & %onen ED-8) and also digitized (MetraRyte DAS-16, 12 bit resolution, 1000 Hz) and recorded by mici-ocoml~uter(IBM 1%-XT). 'l'witches sampled b y thc microcomputer system were analyzed, off-line, for peak tension (PT), time t.o peak tension (TTPT), 1ialf-rclax;ition time (HRT), and the peak rate of tension increase (+dY/dt) atid dcctease (-dP/tlt).
Tension Recording.
Initially, the effects of DIL'I', DGOO and LCR o n single twitches were examined. Muscles were incubated for 10-niinute periods in allernating NR and DILT, 1)600 (5 and 30 pM), 01- I.CR solutions while twitchcs were evoked, recorded, and analyzed every 30 seconds. 'To examine the ef'fkcls of the 3 experimental conditions 011 musclc staii-case and fatigue, 1 inusclc of each pair was iiicuhaLed in N K and the other in either DILT (SO FM), D(i00 (30 kM), o r I X K in the following nianner. M~isclcswere initially incubatcd in NR for 5 ~niiiutesand twitches evoked every 30 scconds. They wcre thcri incubated for 5 minutes i n h e niedia to be used during the repetitive stirnulation bout with twitches again elicited every 30 seconds. After- these preincubations, muscles were stimulatecl at 1 H7 for 15 minutes. From the cotnputer and strip-c:hart recordings, temporal changes in P T were examined for initial twitch PI' (€''rO), the higlicst P'T reached during the s~;rirc:ascresponse (P'r,,,), the time a t which 1"11,, occurred (TTI"l1,,),final twitch PT (PT,,) and thc half-time (t,,2) of the c1ct:lirie in I'T from I'T",. Immediately al'ter thc fatigue bout, muscles were exposed the same incubation media coiit.airiing cither high potassium (180 riiM) or cal'feine (10 r M ) concentrations to elicit a contracturc. Experimental Protocols.
MUSCLE & NERVE
December 1990
1119
Table I. The effects of the four experimental conditions on single muscle twitches. ~
~~
PT
TTPT (msec)
HRT (msec)
+dPldt (Nlsec)
-dP/dt (Nlsec)
124.2 (17.6) 144.1 (11.8) 153.9 (12.7) 139.2 (11.8) 141.1 (12.7) 126.4 (13.7)
32 2 (2 2)
36 5 (2 0) 38 0 (2 0) 38 9 (1 8) 36 9 (2 1 ) 37 5 (2 0) 37 7 (2 2)
2.33 (.25)
1.16 (.06) 1.10 (.07) 1.08 (.09) 1.13(.09) 1.12 (.09) 1.02 (.08)
Condition
NR
DILT (5 pM) DlLT (30 pM) 0600 (5 pM) D600 (30 pM) LCR
31 9 (2 3) 31 8 (2 1 ) 32 4 (2 5) 32 8 (7 4) 30 9 (2 2)
Values are means (StM) See text for definitions (N
=
8 for
each condition)
dicated that within a single pair, responses under NR conditions were very similar. During repetitive stimulation, all muscles exhibited initial positive staircase responses during the first ti minutes of' stimulation. This response was followed by an exponential decline in P T through the remainder of the stimulation period. Incubation of' muscles in DILT and D600 significantly depressed the magnitude (PT,,,/Y'To) of the staircase response by 10 to 20% ( P < .05) (Table 2). LCR, however, only tended to depress the staircase (.05 < f < . l o ) . In 7 of the 10 muscle pairs exposed to LCR, P'r,,,/Ply0 was noticeably decreased while in the other pairs the data were equivocal. T h e calcium antagonists, as well as LCK, significantly reduced the time course (TT'PT,,) of the staircase by approximately 35%. All three experimental conditions also signific:antly increased the rate (t,&) of fatigue with D600 exerting the greater effect on tlPL. After the 15-
Analyses of variance, adjusted for paired muscles, were used to determine differences in the dependent variables between each incubation condition ( P < .05). RESULTS Twitches. Mean responses of single twitches to DILT, U600, and LCR are presented in Table 1 . As can be seen, these conditions had no sigriificant effects on the magnitude and time course of the twitch although the higher concentration of DI1.T tended to increase both P'1'and HKT ( . l o < P < .15).
Single
Repetitive stimulation. Teniporal patterns of the changes in PT for each iricubation condition are 3hown in Figures 1, 2,arid 3 . Although the time course oi individual changes in PT were somewhat variable between muscle pairs, pilot data in-
0
1
2
3
2.41 (.21) 2.46 (25) 2.41 (.22) 2.42 (.21) 2.28 (.24)
4
5
6
7
8
9 1 0 1 1 1 2 1 3 1 4 1 5
STIMULATION TIME (min) FIGURE 1. Responses of single sartorius muscles to NR and DlLT during repetitive stimulation. Data were obtained from paired sartorii in which one muscle was exposed to NR and the other to DILT. Values are means t SEM and normalized with respect to PT,.
1120
Calcium and Muscle Fatigue
MUSCLE & NERVE
December 1990
0
1.8
0
0
1
2
4
3
5
6
7
6
9
-0 CONTROL - 0-600
1 0 1 1 1 2 1 3 1 4 1 ~
STIMULATION TIME (min) FIGURE 2. Responses of single sartorius muscles to NR and D600 during repetitive stimulation.
DISCUSSION
minute stimulation period, the calcium antagonists, h i t not LCR, significantly increased the magnitude of fatigue (PT,,/PT,,). Iri all cases, DILI' rendered the muscle niechanically inactive after approximately 1 1 Ininutes. Uiitler all conditions, caf-feine exposure immediately following thc stimulation period, resulted in contracture (data not shown). Although peak responses to caffeine w e r e off scale for thc recording configuvation used, ~ l i cinitial tinic courses were very similar. Only in NK muscles did high potassium elicit a measurable contracture. In LCR muscles, a sIriall potassium coritracture occurred but was minimally detect.ahle.
1.8
\
5
0.8 -
a
0.6 0.4
-
0.2
-
Roth l)(iO0 and DlLT have been shown to inarkcdl y in hibit calcium influx in skeletal muscle."~20 Likewise, incubation media in which calciurn has been replaced by inagnesiuni and EGTA also inhibit. calcium entry. As evident from recent results and those of other^,^^'"^'."^ DILI+, D600, and LCR appear to have little influence on individual muscle t.witches. During repetitive siirnulation, howevcr, thc calcium antagonists depressed the staircase response and all 3 conditions accelerated fatigue. It is possible that the effects of the three experitncntal condit.ions on repet.itively evoked twitches
0 - - T I -
0.0L'
'
'
'
'
'
'
'
'
1
2
3
4
5
6
7
B
9
0
I
'
-
0
'
'
CONTROL LOW co2+
'
'
1 0 1 1 1 2 1 3 1 4 1 5
STIMULATION TIME (rnin) FIGURE 3. Responses of single sartorius muscles to NR and LCR during repetitive stimulation
Calcium and Muscle Fatigue
MUSCLE & NERVE
December 1990
1121
Table 2. The effects of the 3 experimental conditions on the parameters of skeletal muscle fatigue. Condition
PT,IPTo
TTPT,,, (sec)
PT, JPT,
ti/,
(set)
~
~
NR DlLT
1 70 ( 098) 1 49 ( 057)*
292.6 (22.7) 192.0 (7.29)*
0 27 ( 041) 0.00 (.OOO)"
230.6 (23.9) 139.6 (6.78)*
NR
1 73 ( 082) 140 (loo)*
281.8 (21.4) 186.2 (10.5)"
0.31 (052) 0.08 (010)*
225.7 (25.6) 80.5 (1 1.O)*
1 6 2 (130) 1 56 ( 090)
241.7 (19.8) 121.6 (20.6)'
0.30 (059) 0.15 ( 093)
217.3 (19.8) 297.6 (24.2)'
D600 NR
LCR
Values are means (SEMJ. See text for definitions. r P < .05 vs NR)
could have been due to mechanisms other than inhibited calcium influx. However, it is unlikely that the electrical activity of the muscle was influenced since DILT, D600, and LCR do not normally alter skeletal muscle resting or action potential^.^,'^ Additionally, the calcium channel antagonists are thought to act only on rnembrane bound calcium channels and not to inhibit calcium release channels located in the SKS2 At present the mechanisms which mediate the staircase response are no1 fully understood. Some suggest that this phenomenon niay be due to rnyosin phosphorylatior~,'i altered intracellular calcium exchange." Bianchi and Narayai~"."~~ note that the increasc calcium uptake by the r n usclc during the initial moments of' repetitive stimulation (1 Hz) may be linked to the staircase response. 'The present results add additional support to this notion that when calcium influx was blocked, the staircase magnitude was reduced. Further, Gamboa-Aldeco et all9 have recently shown that removal of exter-nal calcium or addition of nifedipine significantly rcduced posttetanic twitch potentiation, a phenomenon similar to the staircase response. Exactly how increased extracellular calcium influx might mediate twitch potentiation during the staircase response is not known. Clearly the amount of calcium entering the muscle is far too small to markedly increase tension via direct elevation of niyoplasmic calcium. Williams and Barnes34 suggest that extracellular calcium rriost likely acts indirectly by: ( 1 ) increasing "calciuminduced" SK calcium release, (2) increasing resting myoplasrnic calcium concentration, and/or ( 3 ) increasing SR calcium load arid release. In either case, the amount of calcium present in the myoplasm during the twitch would be increased secondary to enhanced influx. In any event, the staircase response seems to be partially dependent on extracellular calcium influx. It should be noted,
1122
Calcium and Muscle Fatigue
however, that since this response was not completely abolished by DILI', D600, or LCR, other mechanisms such as those noted previously are also probably involved. Like the staircase response, the niechanisms which cause skeletal muscle fatigue are also n o t fully understood although corisidcrable evidence favors the notion that is most probably due to excitation-contraction (E-c) coupling failure." Gonzalez-Scrratos et al." show that calcium content of the SK terminal cisterriae is not reduced during fatigue. T h e present results and those of others clearly demonst.rate that high caffeine and potassium concentrations will elicit contractures of similar magnitude in both rested arid fatigued 11111scle.~"~~ Thus SR calcium content seems adequate enough to support contraction but is somehow unable to be released during fatigue. Failure of E-C coupling could result from either a failure of the 'I'T action pot.entia1 or a reduced stimulus for calcium release." With re ard to the first rnechanisrn, Bianchi arid Narayan5- ar-
i
gue that the net efflux of calcium into the I'T lumen during fatigue leads to elevation of T I calcium concentration. This, in turn, could elevate the mechanical threshold and decrease the cfficiency of E-C coupling. In this investigation, blocking calcium entry (or re-entry) by 1)ILT and D600 would liavc led t o a more rapid accumulation of T'l' calcium, an early elcvation of' the mechanical threshold and hence an early onset of fatigue. However, if' calciuin accutnulation in the T I ' contributed to fatigue then LCR should have reduced rather than increased the rate of fatigue sincc low initial 'I'T calcium concentrations woulcl have clelayed calcium acc:Lir-nulatiorl. Failure of' the ' I T action potential could a l s o occur as ;I rcsult of changes in sodiurri ant1 potassium concentration in the r ~ ~ r 1 7 . ' '~' l o w c v e r since , low con(:cntralions 01' calciuni ;intagorlists do not alter wnductance of' sodium rind pot
MUSCLE 8, NERVE
December 1990
in fatigue obser-vcti in this iiivcstigution ;ire probably cl iic t o factors other than altered socliuiiiipotassiurn stasis. Kcgiirdiiig tlie scc.ond mcchaiiism, the precisca Iiophysical 01- biochcinical processes wliich stimulate SK calciuin release a i r uriknown. Some suggest that calciuin ious t hetiiselvcs evoke calcium release. ' p , 1 7 Recent cviclerice suggests t ~ i a t a though a "calriii in-induced" cdciiini release rnechanism may riot 1.x the primary triechanisiri of SK calciurn release, il may play some role in this p ~ o c c s s . ~PerIiaps ~" this "seconttary" means of releasing SR calcium liecomes increasingly iniportant dLir-iii(>-repetitive stimulatioii leading to fa? Ligue. Duriii g do p(dariza t io Ii (IIci 11111 coi i I cl er i t e I' thc: cell and dircc:tly stiinulate or facilitate tlir: release 01' SR calciuni." Also, c;ilcium influx c.ould act iiitlirectly by cnteririg the niiisclc and r-estoriiig ;in internal "trigger-" c&:iuiii 111 either case, inhiliiting citkiiirn influx Ijy l)ILrl', I)(iOO, antl I.CR coiild i.cduce thc siir~iiilusfor SR cal-
ciuiii releasc: aiid accclrrate fatigue. l ' h c present r-esults t1ierc"oi.e suggest that extracellular calcium iriflux is somehow involved in the fittipie process. 1'o sii mrnai-ize, the calci u r n antagonists, tli Itiazem, and M O O depress thc slaircase response iuid enliarice fatigue of' frog skcletal riiiisclc whcr-eas reduced external calcium has no effect of the staircase but increases tlic: rate 01' f'atiguc. 'Iliese i-esults support the notion that cxtracellular calcium inllux m a y have sonic iriflucnce on skeletal muscle staircase arid f'atiguc. They also fiirtlier support the Iiotion that culcium influx has soiiic Iole iri the process of skeletal muscle excitatioiiconttxtioii coupling. l ' h e notioil that ciilcium chanricl antagonisis depi-ess contr'actioiis during rcpetitive activity might also h a v e irriplicatioris for individLials using these compo~i tids for treatniciit of i1iyoc;trdial disease rnmifestations such ;is hypcrtension and those who x c enganged in a physically active lifestyle.
REFERENCES I . Almeis W, Fink R, I';ilade P 1': Calcium depletion in frog muscle ciibules: 'The decline of calciuni current under maintained depolariz;itiori. 1 Physiol (Luizdj 19813 12:177'10 1. 2. Almets W, Palade P7': Slow calciurn antl potaszium currents am^ frog rriemhrane: Measurements with viiaclinegap tcchniqirc. j ~ h y s i o (I.imd) l 1
