Caffeine contractures, twitch characteristics and the threshold for Ca2+-induced Ca2+ release in skeletal muscle from horses with chronic intermittent rhabdomyolysis

Research in Veterinary Science 1993, 54, 110-117

Caffeine contractures, twitch characteristics and the threshold for Ca2+-induced Ca 2+ release in skeletal muscle from horses with chronic intermittent rhabdomyolysis J. BEECH, S. LINDBORG, Department of Clinical Studies, University of Pennsylvania, School

of Veterinary Medicine, New Bolton Center, Kennett Square, Pennsylvania 19348, USA, J. E. FLETCHER, F. LIZZO, L. TRIPOLITIS, Department of Anesthesiology, Hahnemann University, Philadelphia, 19102-1192, USA, K. BRAUND, Neuromuscular Laboratories, Scott Ritchey Research Program, College of Veterinary Medicine, Auburn University, Auburn, Alabama 36830, USA

Muscle from horses with intermittent exercise associated rhabdomyolysis was examined to determine if calcinm regulation was abnormal. In vitro studies on semimembranosus muscle fibre bundles showed the time to 50 per cent relaxation of caffeineinduced contractures was shorter and the electrically elicited twitch longer in horses with exercise associated rhabdomyolysis. Substitution of strontium for calcium eliminated the difference in caffeine contracture between the normal and rhabdomyolysis horses. The threshold of calciuminduced calcium release was lower than normal in terminal cisternae-containing fractions of muscle from horses with rhabdomyolysis. Thoroughbreds with rhabdomyolysis had a shorter time to peak twitch tension than standardbreds, and normal thoroughbreds had a shorter caffeine contracture than normal standardbreds. There was no difference in fibre typing between breeds or groups. Either no histological changes or low grade to moderate degenerative myopathy was seen in muscle from horses with rhabdomyolysis. These results suggest horses with intermittent exercise associated rhabdomyolysis have abnormal calcium regulation.

C H R O N I C intermittent exercise-associated rhabdomyolysis is an important cause of decreased performance and interrupted exercise programmes. It is characterised by signs of exercise-induced muscle stiffness and pain, decreased exercise tolerance and ability and abnormally increased serum concentrations ofcreatine kinase and aspartate aminotransferase in response to

exercise. Myoglobinuria may be seen in severe cases. The cause and pathogenesis often remain unknown and most studies have focused on histochemical and biochemical changes in the muscle as a result of an episode of rhabdomyolysis (Lindholm et al 1974, McEwen and Hulland 1986, Van Den Hoven et al 1986). A viral aetiology has been implicated (Harris 1990). There has been one report of a polysaccharide storage myopathy and a complex I mitochondrial enzyme deficiency associated with exertional rhabdomyolysis (Valberg et al 1990). In vitro muscle studies have been few. In vitro halothane contracture responses, typical of individuals with malignant hyperthermia, have been reported in skeletal muscle from several horses with chronic intermittent rhabdomyolysis (Waldron-Mease 1978, Hildebrand et a11987) and prolonged relaxation times of the electrically elicited twitch have been reported (Beech et al 1988). The contracture response to caffeine has been used to study excitation contraction coupling in muscle and increased sensitivity to caffeine has been demonstrated in malignant hyperthermia and some other human myopathies (Heiman-Patterson et al 1988a,b, Lehmann-Horn and Iaizzo 1990b). .Preliminary data showed that horses with chronic intermittent rhabdomyolysis had shorter caffeine contractures and a lower threshold for calcium induced calcium release in heavy sarcoplasmic reticulum fractions than control horses (Beech et al 1990). Calcium regulation has been shown to be abnormal in malignant hyperther-

vitromuscle muscle studiesin equine in equinerhabdomyolysis rhabdomyolysis In In vitro studies

111111 tractureandand twitchstudies studiesa section a sectionof ofaligned aligned mia, witha lower a lowerthreshold thresholdforfor calcium-induced tracture mia, with calcium-induced twitch musclefibres fibresapproximately approximately 2.52.5cmcmto to3.53.5 calciumrelease release heavy sarcoplasmicreticulum reticulum muscle calcium in in heavy sarcoplasmic cmcm fractionsfrom frommuscle muscle frommalignant malignanthypertherhyperther- long long× 2x cm 2 cm x 0.5 was placedin in oxygenated fractions from × 0.5 cmcm was placed oxygenated mia-susceptiblepigs pigs (Nelson1983, 1983,Ohnishi et al modified modified Krebs Krebsbuffer buffersolution solution(Fletcher (Fletcherandand mia-susceptible (Nelson Ohnishi et al 1983, Fletcher et al 1991). As the authors’ pre- Rosenberg Rosenberg1985) 1985) at roomtemperature temperatureforfor 1983, Fletcher et al 1991). As the authors' preat room thethe dis-disliminary findings findings indicated indicated that thathorses horseswith with section sectionof of tibre bundles. A 3 to 5 g section was liminary fibre bundles. A 3 to 5 g section was placed in cold Krebs bufferandand kept before chronicintermittent intermittent rhabdomyolysis rhabdomyolysis hadhad abnor- placed chronic abnorin cold Krebs buffer kept onon iceice before heavysarcoplasmic sarcoplasmicreticulum reticulumfraction fractionisolation. isolation. mal in vitromuscle muscle function,thisthis study’s purpose heavy mal in vitro function, study's purpose was to determineif these if these findingswere were consistent A Asection section was keptrefrigerated refrigeratedforforlessless than24 24 was to determine findings consistent was kept than a larger populationof of horses to determine hours hoursbefore beforefreezing freezingin in isopentaneforfor histology in ainlarger population horses andand to determine isopentane histology whetherthere therewaswasa difference a differencebetween betweenthorthor- andandhistochemistry. histochemistry.Biopsies Biopsies were processedforfor whether were processed oughbredsandand standardbreds.Because Because of pheny- in vitro in vitrostudies studies within90 90 minutesof of collectionas as oughbreds standardbreds. ofphenywithin minutes collection toin’sprophylactic prophylacticefficacy efficacyforforchronic chronicintermitintermit- previously previouslydescribed described(Beech (Beech 1988). toin's et et al al 1988). tent rhabdomyolysis in horses in horses (Beech et al 1988) tent rhabdomyolysis (Beech et al 1988) they wished to determine whether the drug also Muscle twitch, caffeine and halothane contracthey wished to determine whether the drug also twitch, caffeine and halothane contracaffectedthethecaffeine caffeinecontracture contracture response responseif ifit it Muscle affected ture studies ture studies addedduring duringin in vitrotesting. testing. waswas added vitro Fibrebundles bundleswere were dissected placed Fibre dissected andand placed in in a 5a 5 ml tissue bath containing modified Krebs’ solution ml tissue bath containing modified Krebs' solution Materialsandand methods Materials methods bubbledwith with cent oxygen : 5 per cent carbon bubbled 95 95 perper cent oxygen : 5 per cent carbon dioxide and brought to and maintained at 37°C dioxide and brought to and maintained at 37°C Subject selectionandand biopsy Subject selection biopsy as described (Fletcher Rosenberg 1985). Testing as described (Fletcher andand Rosenberg 1985). Testing Semimembranosus muscle muscle samples samples were were waswas Semimembranosus completedwithin withinfour fourto tosixsixhours hoursof ofthethe completed obtainedfrom fromcontrol controlhorses, horses, fromhorses horses biopsy. biopsy. The muscle was adjusted for the optimum obtained andandfrom The muscle was adjusted for the optimum withchronic chronicintermittent intermittent rhabdomyolysis. rhabdomyolysis. Ages Ages length length maximumtwitch twitch tension stimulated with forfor maximum tension andand stimulated rangedfrom fromtwotwo years each group.The The supramaximally supramaximallyat 0-2 at 0.2 Strips exhibitinga twitch a twitch ranged to to 14 14 years forfor each group. Hz.Hz. Strips exhibiting breedandandsexsex distribution of of horses each greater greater than0.50.5 g tensionwere were consideredviable viable breed distribution thethe horses in in each than g tension considered groupforforeach each studyareare presentedin inTable Table1 1forfor testing were then equilibratedforfor group study presented testing andand were then equilibrated 30 30 to to 45 45 minutes. The following parameters were determined except studies in whichgroups groupscontained containedfive five minutes. except forfor studies in which The following parameters were determined twitch:time time cent cent fewerhorses. horses. or or fewer forfor thethe twitch: to to 50 50 perper cent andand 100100 perper cent peaktension, tension,andand time cent Thetechnique techniqueforforbiopsy biopsyhashas beendescribed described peak The been time to to 50 50 perper cent andand 90 90 perper cent relaxation.TheThe times cent previously(Beech (Beech 1988). Briefly,a section a section cent previously et et al al 1988). Briefly, relaxation. times to to50 50 perper cent andand 90 90 per cent relaxation were measured from the time of aligned fibres from the belly of the semimemof aligned fibres from the belly of the semimem- per cent relaxation were measured from the time peaktension tensiondevelopment. development.TheThemuscle muscle strips branosusmuscle musclewaswas asepticallyremoved removedafter after of of peak strips branosus aseptically were subsequentlyexposed exposed a bolus injectionof of anaesthetisingthethe skin andsubcutaneous subcutaneoustissue. tissue. were subsequently to to a bolus injection anaesthetising skin and 8 mMcaffeine. caffeine.A A0.50.5 g twitchmagnitude magnitudewaswas The horse was lightlysedated. sedated.ForForin in vitroconcon- 8 mM g twitch a a The horse was lightly vitro TABLE1: Sex 1: Sex breeddistribution distribution of horsesin of horses the in the studies TABLE andand breed studies Study Study Muscletwitch twitch Muscle Normal group Normal group CIR group cm group Caffeinecontracture contracture Caffeine Normal group Normal group CIR group cm group TWX TCICR Normal group Normal group group C~RCIR group Histology Histology Normal group Normal group CIR group cla group

n

Thoroughbred Thoroughbred CM CM F F M M

n

16 16 41 41

5 4

5 4

10 IO 18 18

2 3

;

8 8

8 8 34 34

8 8

Standardbred Standa~bred CM CM F F M M

Other Other CM CM F

4 4 10 10

2 2 5 5

0 0 4 4

0 0

0 0

0 0

0 0

F

5 5 17 17

0 0 1 1

2 6

2 6

0 0 0 0

4 3

4 3

0 0 3 3

0 0 1 1

2 2

2 2

0 0

0 0

1 1 1 1

2 5

2 5

0 0 0 0

2 0

2 0

0 0 1 1

0 0 0 0

3 3 1 1

0 0

0 0

1 1 7 7

3 9

3 9

0 0 0 0

3 7

3 7

0 0 5 5

0 0 2 2

1 1 3 3

0 0 1 1

CIR Chronicintermittent intermittentrhabdomyolysis rhabdomyolysis cm Chronic TCICR Thresholdfor calcium for calciuminduced inducedcalcium calciumrelease releasein the in the sarcoplasmic reticulum reticulum TCICR Threshold sarcoplasmic CM Castratedmale, male, F Female,M Male M Male CMCastrated F Female,

J. Beech, J. E. Fletcher, F. Lizzo, L. Tripolitis, K. G. Braund, S. Lindborg

112

strontium (Sr2+) for calcium (Ca 2+) in the bathing medium for one hour were evaluated on caffeine contractures. These contractures were compared with at least one paired contracture to caffeine of another muscle fibre bundle from the same horse in a Ca2+-containing medium on the same day. Paired fibre bundles were used because repeated exposures to caffeine do not give consistent results on cut fibre bundles. Caffeine contracture in a calcium-free medium was evaluated in 11 horses. In addition to evaluating differences between the groups of horses and the effects of changing the in vitro medium, results were also analysed to determine whether standardbreds differed from thoroughbreds. For the evaluation of peak tension generated in the CaZ+-free, CaZ+-containing and SrZ+-containing medium, and for comparing standardbreds to thoroughbreds results were analysed by one way ANOVA with repeated measures and a Sheffe test. Results of all other tests were statistically analysed using one- or two-tail t tests as appropriate. Muscle from four normal horses and four horses with chronic intermittent rhabdomyolysis were tested for in vitro halothane response. Strips considered viable for halothane contracture testing for malignant hyperthermia susceptibility were those exhibiting a twitch greater than 0-9 g to direct stimulation. The contracture test was considered positive for malignant hyperthermia susceptibility using estabfished criteria (Allen et al 1990).

A

I

Caffeine (8 ma) lg 1 min B

Threshold for calcium-induced calcium release !

t Caffeine (8 mM) FIG I :The contractu re response to caffeine of isolated fibre bundles from a horse with chronic intermittent exercise associated rhabdomyolysis in (a) a calcium containing medium and (b) a medium in which strontium was substituted for calcium. The twitch heights were allowed to go off scale after caffeine addition to present the increase in resting tension more clearly

prerequisite for caffeine contracture testing. Strips showing less than a 0-4 g rise in peak tension following the addition of caffeine to the bath were not included in the caffeine contracture study. In some cases the effects of phenytoin (10 kdVI)addition to the bath for one hour, or substitution of

A heavy sarcoplasmic reticulum fraction was isolated by differential centrifugation (8000 to 12000 g) of skeletal muscle homogenates (Nelson 1983) from eight normal horses on 16 separate occasions and from eight horses with chronic intermittent rhabdomyolysis. The threshold for CaE+-induced Ca 2÷ release was determined using a pyrophosphate method to increase assay sensitivity (Palade 1987), as previously described (Fletcher et al 1990a, 1991). Arsenazo III was used as the Ca 2÷ indicator and the absorbance monitored on a dual wavelength spectrophotometer (Aminco DW-2) at 650 and 700 nm. Ca2+ was added in 10 laM increments to 1.5 ml incubation buffer maintained at 37°C. Protein was determined by standard methodology (Markwell et al 1978). The threshold for Ca2+-induced Ca2+ release was expressed in ~maolCa Hmol/mg protein.

In vitro muscle studies in equine rhabdomyolysis Histology Histopathology was done on biopsies from seven normal horses and 34 horses with chronic intermittent rhabdomyolysis. Histochemistry and muscle fibre typing was done on muscle from five normal horses and 32 horses with chronic intermittent exertional rhabdomyolysis. Serial sections of all muscle samples were cut in transverse and longitudinal planes at 8 ~rn, using a cryostat microtome maintained at -20°C. Sections were stained with haematoxylin and eosin, modified Gomori trichrome, reduced nicotinamide adenine dinucleotide tetrazolium reductase (NADH-TR), and myosin adenosine triphosphatase (ATPase) at pH 10.2 and 4.3 (Dubowitz and Brooke 1973). Fibre types were more clearly differentiated at a pH of 10-2 than 4.3. Percentages of type I and type II fibres were determined from a random count of 1000 fibres from two transverse muscle sections stained with ATPase at pH 10-2. Results

Caffeine and halothane and twitch studies The caffeine contracture in a Ca2+-containing medium of muscle from horses with chronic intermittent rhabdomyolysis had shorter times to 50 per cent relaxation than did normal horses (Table 2). Two female half sibling thoroughbreds with chronic exercise-associated rhabdomyolysis from the same stable that had greatly prolonged times to 50 per cent relaxation (2372 and 1440 seconds) were deleted from this statistical comparison and not included in the table. When Sr2+ was substituted for Ca 2÷ in the bath, time to peak tension of caffeine-induced contractures was shortened in both groups, and time to 50 per cent relaxation was no longer significantly different between the groups (Table 2). There was no difference between the two groups in percentage decrease in time to peak tension and peak tension was not changed by substituting Sr2+ for Ca 2÷ (see Fig 1 for this effect on a caffeine contracture from a horse with intermittent exertional rhabdomyolysis). However, no strips having under 0.4 g contracture were included in this part of the study. In six normal horses and five with rhabdomyolysis incubation in vitro for one hour with phenytoin (10 ~M) had no

113

effect on caffeine contractures with either Sr2+ or Ca 2÷ in the medium (data not shown). In 11 biopsies from normal and chronic intermittent rhabdomyolysis horses caffeine induced a mean peak tension of 0.1 + 0.1 g in a Ca2+free medium which was significantly lower than the values in Table 2 when the medium contained Ca 2+ (P<0-0001) or Sr2+ (P<0.02). Caffeine contracture data with Ca 2÷in the bath was analysed to determine whether standardbreds differed from thoroughbreds (Table 3). The standardbreds in the control group had significantly longer times to peak tension and lower peak tension than the control thoroughbreds, but these parameters were not significantly different between the two breeds in the chronic intermittent rhabdomyolysis group. In neither group was there a breed difference in time to 50 per cent relaxation. The contracture response to halothane exposure was evaluated in four horses within each group and was negative for malignant hyperthermia susceptibility. Analysis of twitch data using a two-tailed t test (Table 4) showed chronic intermittent rhabdomyolysis horses had a significantly longer time to 100 per cent peak tension but time to 50 per cent peak tension did not differ from the control group. Both time to 50 per cent and 90 per cent relaxation were significantlylonger in the chronic intermittent rhabdomyolysis group. The peak tension did not differ (data not shown) within each group. When standardbreds were compared with thoroughbreds there were no differences except that thoroughbreds with chronic intermittent rhabdomyolysis had a shorter time to 100 per cent peak tension than standardbreds with chronic intermittent rhabdomyolysis; thoroughbreds also had a longer time to 50 per cent relaxation than control thoroughbreds. No other comparisons were statistically significant. Threshold for CaZ+-induced Ca 2+ release The threshold for CaZ+-induced Ca 2+ release in sarcoplasmic reticulum fractions from eight horses with chronic intermittent rhabdomyolysis (6.7 _+1.0 ~tmol Ca2+/mg protein; mean + SD) was significantly (P<0-004) lower than that of eight control horses (10-8 _+2-7 pmol Ca2+/mg protein). There was no relation between the threshold for Ca2+_induced Ca2+ release and time to peak tension of the caffeine contracture.

114

J. Beech, J. 17,.Fletcher, F. Lizzo, L. Tripolitis, K. G. Braund, S. Lindborg

TABLE 2: Caffeine contractures in normal horses and those with chronic intermittent exercise induced rhabdomyolysis (OR). Time to peak tension (TTP), peak tension (P) and time to 50 per cent relaxation (TTR50 per cent) of the contracture induced by a bolus administration of caffeine (8 mM) Normal horses

ClR horses

(~+ SD)

Ca 2÷ medium Sr 2÷ medium

(~+SD)

TrP (S)

P g tension

TrR 50% (s)

TTP (s)

P g tension

TrR 50% (s)

142 + 53(10)a 82+22(10)

1"22 + 0'53 1-35+0.78

582 + 222* 129+_ 58

126 + 35(18) b 81 _+28(18)

1 "37 -+ 0'77 1.41 _+0,87

278 -+ 184(15) 107+ 51(17)

Numbers in brackets in first column indicate number of horses tested which is constant along rows of parameters unless marked as different by (n) Superscript letters indicate a significant difference (P<0.05) in the Ca 2+ versus Sr2+ medium *Indicates a significant difference between normal and ciR horses (P<0.01)

TABLE 3: Caffeine contractures in standardbreds compared to thoroughbreds in the normal and in the chronic intermittent exercise induced rhabdomyolysis (CIR) group. Times to peak tension (TrP), peak tension (P) and times to 50 per cent relaxation (rrR 50 per cent) following bolus administration of 8 mM caffeine

Thoroughbred Standardbred

TI-P (S)

Normal horses P g tension

TrR 50% (s)

TTP (s)

cln horses P g tension

TTR 50% (s)

95 + 11(4)* 176 + 58(4)

1.7 _+0.5* 0.9 + 0-2

455 + 263* 566 + 42

117 + 38(9) 138 _+32(6)

1.6 + 0.8 0.9 _+0.4

260 + 196 378 _+263

*Indicates figures in same column are significantly different (P<0.05) by the two-tailed t test Caffeine contractures were in a calcium containing medium without additives (Values given as x +so)

Histology and histochemistry Muscle samples from the seven control horses studied showed no lesions and a type II fibre distribution predominated in the five examined (Table 5). Of the 34 chronic intermittent rhabdomyolysis horses evaluated, seven had no histological changes, one had a vacuolar myopathy, and 26 had low grade to moderate degenerate myopathy characterised by occasional internalised nuclei, focal necrosis, fibre splitting, and sometimes fibrosis (minimal) and fatty infiltration. There was variable atrophy and hypertrophy of fibres. When necrosis was seen, it was usually focal or multifocal. The microscopic lesions in this muscle did not appear to correlate with the frequency or severity of clinical bouts of rhabdomyolysis. Type II fibre distribution predominated in all horses. Fibre typing did not differ (P>0.05; two-tailed group t test) between the control group and horses with rhabdomyolysis (Table 5). Within the exertional rhabdomyolysis group there was no difference in fibre type distribution among thoroughbreds, standardbreds and horses of mixed breeds. Within the normal group, fibre typing was done on only four thoroughbreds; there was no difference between these

and the thoroughbreds with exertional rhabdomyolysis. Fibre types and in vitro muscle test parameters were examined to determine if the fibre type influenced the latter. Nineteen of the horses with rhabdomyolysis had caffeine contractures and muscle fibre typing performed and fibre typing and twitch studies were done on 21 horses. No statistical correlations were found between fibre type and time to 50 per cent relaxation of the caffeine contracture or for twitch parameters (time to 100 per cent peak tension and times to 50 per cent and 90 per cent relaxation). Although percentage type I fibres was directly correlated with time to 100 per cent peak tension of the caffeine contracture (P=0-011) the r value (0-567) suggested very little predictive value (data not shown). Discussion

Caffeine-induced contractures of intact fibre bundles or chemically skinned fibres have been studied in only a few muscle disorders. A low threshold of contracture to caffeine has been reported in human patients with various myopathic or neurogenic disorders (Takagi et a11983, Lehmann-Horn and Iaizzo 1990a,b). Prolonged

115

In vitro muscle studies in equine rhabdomyolysis TABLE 4: In vitro muscle twitch responses. Times to peak tension ('rrP 50 and 100 per cent) and times to relaxation (TrR 50 and 90 per cent) of the electrically elicited twitch

Normal cm Normal STB Normal TB CIRSTB CIR TB

n

TrP 100%

16 41 6 10 18 22

67 _+6 a 7 4 + 10 71 + 5 64 + 6 79 + 11d 70 _+7

TrP 50% TYR 50% Time (ms); mean _+SD 29 31 30 29 33 30

_+2 _+5 + 1 _+2 +5 --+4

41 ± 6 b 47_+8 47 -+ 5 38 _+3 48 + 5 47 _+9

TIR 90%

88 _+ 15c 9 9 + 17 100 + 19 80 + 6 106 _+ 17 93 _+ 15

aSignificant difference between normal and ClR horses (P<0"01) bSignificant difference between normal and cm horses (P<0.005) CSignificant difference between normal and CIR horses (P<0'02) dSignificant difference between CiR STB and crR TB (P<0"009) TB Thoroughbred STB Standardbred cm Chronic intermittent exercise induced rhabdomyolysis

caffeine contractures have been reported for human patients with McArdle's disease (Gruener et al 1968). In the present study the caffeine contracture was shorter in horses with chronic intermittent rhabdomyolysis and in both groups substitution of Ca 2÷ with Sr2+ in the bath shortened the caffeine contracture. The pathogenesis of the abnormal caffeine contractures is unclear as it could be non-specific or associated with altered Ca2+ regulation in the myoplasm. The same area of the same muscle was used in all horses in this study, the ratio of type I:type II fibres in the biopsied muscle did not differ between the two groups or between thoroughbreds and standardbreds, and there was no relationship between fibre types and caffeine contractures and twitches between the two groups. The reason for the very prolonged caffeine contractures in two half sibling female thoroughbreds with chronic intermittent rhabdomyolysis from the same stable is unknown; whether it reflects a genetic disorder is speculative. As the biopsy technique and laboratory methodology were constant these would seem unlikely influences and, reportedly, neither horse was on medication or an unusual training regimen. Although the reason for the different contracture times is unknown, the concentration of Ca2+available and the efflux and uptake rates of Ca2+ from or into the sarcoplasmic reticulum are likely factors. The time to peak tension would correlate with net Ca 2÷ release from the sarcoplasmic reticulum and the relaxation time with net Ca 2÷ uptake. The shorter time to 50 per cent relaxation of the caffeine contractures of the chronic intermittent rhabdomyolysis horses suggests Ca 2÷ uptake in the sarcoplasmic reticulum is more rapid in their muscle than in control horses. The absence

of a difference in time to peak tension between the two groups could indicate that altered Ca 2+ uptake specifically, not Ca 2÷ release is affected. However, the findings of a low threshold for Ca 2÷induced Ca 2+ release would suggest that release is also affected. It is unclear whether Ca2+-induced Ca2+ release shares the same pathway as caffeineinduced Ca 2+release (Sorenson et al 1986), making cause-effect comparisons difficult. The dramatic decline in caffeine contracture response of equine muscle in a Ca 2÷ free medium differs from reports in the literature on other species showing caffeine contractures are decreased by only 50 per cent in the absence of external Ca 2÷ (Fletcher et al 1990b). In normal pigs, low Ca2+ depressed caffeine-induced twitch potentiation by about 25 per cent (Williams et al 1991). The present findings could suggest that in the horse intracellular Ca2+ may become depleted or alternatively cannot function as the sole source of Ca z÷ for caffeine-induced contractures. TABLE 5: Percentage fibre type distribution in biopsies of

semimembranosus muscle from control horses and those with chronic intermittent exertional rhabdomyolysis Breed and group

n

Percentage fibre type (~ _+so) Type I Type II

CIR

STB TB Other Total Normal STB TB Total

14 14 4 32

11 + 2 10+4 10 + 4 10 + 4

89+2 90+4 90 + 4 90 _+3

1 4 5

15 7+3 9_+4

85 93+3 91 + 4

ClR Chronic intermittent exertional rhabdomyolysis STB Standardbred TB Thoroughbred

116

J. Beech, J. E. Fletcher, F. Lizzo, L. Tripolitis, K. G. Braund, S. Lindborg

Substitution of Sr2+ for Ca 2+ in the external medium resulted in a shortened caffeine contracture with similar peak tension in all horses. Studies in other species have demonstrated that Sr2+ can substitute for Ca 2+ in caffeine-induced contractures (Uhrik and Zacharova 1988, Fletcher et al 1990b) but effects on the duration of the contracture have not been reported. The mechanism responsible for shortening the caffeine contracture has not been demonstrated but could be due to the more rapid movement of Sr2+ or movement of a greater amount per unit time across the sarcoplasmic reticulum (Mermier and Hasselbach 1976). In isolated sarcoplasmic reticulum vesicles, Sr2÷ uptake is more rapid than that of Ca2+ and when ATPis depleted both uptake and release of Sr2+ are much faster (Mermier and Hasselbach 1976). In this small number of horses tested for malignant hyperthermia susceptibility, none tested positive. Positive in vitro responses have been reported in biopsies of equine omobrachialis muscle (predominantly type II fibre composition); some of these animals developed malignant hyperthermia-like episodes when exposed to halothane, and several had exercise induced myopathy (Hildebrand et al 1987). Three of four horses with postoperative myositis were reported to have a positive response to halothane when fibre bundles of the semimembranosus muscle were tested (Waldron-Mease and Rosenberg 1979) and increased sensitivity to halothane and caffeine was reported for gracilis muscle from three horses with exercise associated myositis (Rosenberg and Waldron-Mease 1977). The true incidence of in vitro susceptibility in horses is unknown. Clinical manifestations of malignant hyperthermia in horses have been infrequently reported (Hildebrand et al 1987, Klein 1975). This study showed muscle from horses with chronic intermittent rhabdomyolysis had longer times to 50 per cent and 90 per cent relaxation following an electrically stimulated twitch (Table 4). In an earlier study on a smaller population of horses, prolonged times to 50 per cent and 90 per cent relaxation were also reported for muscle twitch in horses with chronic intermittent rhabdomyolysis (Beech et al 1988). In that study the relaxation time was measured from the stimulus point. When converted to times measured from the point of peak tension those values are similar to these in the present study. The current study

also showed a longer time to 100 per cent peak tension in horses with chronic intermittent rhabdomyolysis. The horses with chronic intermittent exercise associated rhabdomyolysis had variable histological changes in the muscle. This is not unexpected given the variable severity of signs of the horses and time period between an episode of rhabdomyolysis and the biopsy. Quantitative variability in lesions has been reported previously in a group of horses with histories of exertional rhabdomyolysis although qualitatively their lesions were similar both among the horses in that study and to horses in this study (McEwen and Hulland 1986). Type II fibres are reported to be selectively but not exclusively affected acutely after a bout of rhabdomyolysis (Lindholm et a11974, McEwen and Hulland 1986). The absence of selective fibre involvement in the horses is not unexpected as they were not biopsied immediately after exercise or a bout of rhabdomyolysis. In this study no lesions were found on light microscopy that were likely to explain the differences in in vitro responses between the groups. The severity of histological lesions did not appear to correlate with the historical clinical severity. This could be due to both the variable period between the biopsy and bouts of rhabdomyolysis, variable involvement of different muscles as previously reported (McEwen and Hulland 1986) and other factors involved in muscle cramping and pain. Although training and exercise cannot be eliminated as possible influences on in vitro function, exercise was variable for the horses with chronic intermittent rhabdomyolysis; frequently there was a period of rest before their biopsy and several of the normal horses had been strenuously exercising before being biopsied. The failure to detect differences between normal horses and those with chronic intermittent rhabdomyolysis within each of the two predominant breed groups was probably due to the smaller numbers of horses combined with the wide variability of data. The wide variation between horses may reflect biological variability and also the degree of clinical involvement in the horses with rhabdomyolysis. These results show that muscle function itself is affected in horses with chronic intermittent rhabdomyolysis and that several different sites in the muscle may be abnormal. The shortened caffeine contracture and lowered threshold for Ca2÷-induced

In vitro muscle studies in equine rhabdomyolysis

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