CARDIAC A N D SKELETAL MUSCLE E N Z Y M E LEVELS IN HYPERTENSIVE A N D AGEING RATS
PETER JOHNSON* and JANET L. HAMMER Department of Chemistry and College of Osteopathic Medicine, Ohio University, Athens, OH 45701,
U.S.A. (Tel. 614 593-1744)
(Received 8 June 1992; accepted 20 July 1992)
Abstract--1. The activities of glycolytic, fatty acid oxidation and citric acid cycle enzymes were measured in hypertensive and ageing rat cardiac and skeletal muscles.
2. Lactate dehydrogenase and fl-hydroxyacyl-CoA dehydrogenase were significantly decreased in hypertensive, but not senescent, cardiac muscle.
3. Total phosphorylase activity was significantly increased in senescent, but not hypertensive, cardiac muscle.
4. In ageing rat cardiac and skeletal muscles, calpain II titers increased significantly with age, but in normotensive and hypertensive muscles, the titers showed no significant difference.
INTRODUCTION
Muscle is a tissue which is capable of undergoing significant biochemical and physiological change in response to a variety of external and internal stimuli. During normal maturation and senescence, signifi- cant changes in muscle enzymology and in the con- tractile apparatus have been observed in different skeletal (Essen-Gustavsson and Borges, 1986; Orlan- der et al., 1978; Costell et al., 1989; Kline and Bechtel, 1990) and cardiac muscles (Dunaway et al., 1986; Dowell, 1987; Karttunen and Vornanen, 1987; Lakatta, 1987a; De Tata et al., 1988), and such changes may explain differences which occur in muscle performance during development.
In addition to changes in muscle related to devel- opment, changes also occur in response to patterns of use or disuse (Kainulainen et al., 1990; Lacaille et al., 1990; Rusko et al., 1991), in hypoxia (Pastoris et al., 1991) and in diseases such as muscular dystrophy (Johnson and Hammer, 1988). In experimentally- induced or spontaneous hypertension, a number of enzymatic changes have also been observed to occur in skeletal (Nagaoka et al., 1987; Ben Bachir-Lamrini et al., 1990), vascular (Uehara et al., 1988) and cardiac muscle (Lakatta, 1987b; Anand-Srivastava, 1988; Ilieva et al., 1989; Lakatta, 1990; Smith et al., 1990). In the case of alterations in the cardiac myosin isoenzyme pattern in hypertensive myocardium, these differences have been implicated in changes in the contractile response in this tissue (Pauletto et al., 1988; Lakatta, 1990).
Because of the significance of the energy-product- ing metabolic pathways in the contractile process in
*To whom correspondence should be addressed. Abbreviations: ELISA, enzyme-linked immunosorbent
assay.
muscle, we have investigated the activity levels of key marker enzymes of glycogenolysis, glycolysis, fatty acid degradation and the citric acid cycle in hyperten- sive animals in order to determine if energy pro- duction mechanisms in muscle tissue show significant changes in hypertension. In addition, because of suggestions that rates of protein turnover may be altered in hypertensive muscle (Lakatta, 1987b), we have also examined the level of muscle calpain II in hypertensive muscle, as this enzyme may be involved in intracellular protein turnover (Croall and De- Martino, 1991). In view of previous comparisons of the ageing process with hypertension (Lakatta, 1987b, 1990), we have performed similar analyses on ageing skeletal and cardiac muscles in order to deter- mine if there are in fact enzymatic similarities be- tween the ageing process and the changes induced by hypertension.
MATERIALS AND METHODS
Animals
Normotensive (WKY) and spontaneously hyper- tensive (SHR) male Wistar rats (Rattus rattus) ap- proximately eight months of age were purchased from Taconic Farms, NY. For studies on ageing rats, male Fischer 344/NHsd BR rats of 3, 12 and 27 months of age were purchased from Harlan Sprague Dawley, IN.
Materials
Substrates and purified enzymes for use in the enzyme assay protocols were purchased from Sigma, St Louis, MO. Falcon Microtest III assay plates for ELISA were purchased from Fisher Scientific, and the GAM-HRP secondary antibody used for ELISA was obtained from Organo Teknika, PA.
CBP(B) 104/I--E 63
64 PETER JOHNSON and JANET L. HAMMER
Muscle extract preparation
Immediately after euthanasia of animals by as- phyxiation with CO2 or diethyl ether, two individual skeletal muscles (longissimus dorsi and quadriceps femoris) and the mixed cardiac muscle from the outer wall of the left ventricle were excised. Muscle samples were immediately cooled to and stored at - 7 0 ° until further use, and portions of approximately 100 mg were removed from the frozen sample and pulverized in a stainless steel macerator bathed in liquid nitrogen (Pette and Reichmann, 1982). The frozen muscle powder macerate was then suspended in 1.5 ml of 5 m M EDTA, 0.1 M sodium phosphate, pH7.2 , homogenized in a 5 ml capacity Thomas glass-teflon hand homogenizer, and the supernatant fluid col- lected after centrifugation (Reichmann et al., 1983). Protein concentrations of supernatant samples were measured using the Bio-Rad Bradford assay kit.
Enzyme assays
Using suitably-sized aliquots of the muscle super- natant preparations to give measurable activity rates in each assay, rates were measured on a Gilford Response spectrophotometer using the enzyme kin- etics program of the spectrophotometer. For these studies, the activities of hexokinase, phosphofructok- inase, lactate dehydrogenase and fl-hydroxyacyl-CoA dehydrogenase were determined by the protocols of Reichmann et aL (1983). Total glycogen phosphoryl- ase (i.e. phosphorylase a plus phosphorylase b) was assayed by the method of Simoneau et al. (1985) and citrate synthase by the method of Shepherd and Garland (1969). Activity rates in each procedure were converted into units of enzyme activity as defined in the original method by the construction of standard curves using commercially-purified preparations of each enzyme. Enzyme activities in muscle prep- arations were then expressed as units of enzyme activity per mg of supernatant protein.
Calpain H titer measurement
Determination of calpain II levels in muscle ex- tracts was performed by the ELISA procedure as described by Hussain et al. (1987), using a mono- clonal antibody which recognizes an epitope on the large subunit of the enzyme (Johnson and Hammer,
1988). In the ELISA, muscle extract samples were diluted to a protein concentration of 40 #g/ml, and 50 #1 aliquots were added to the assay plate wells. Because of the inherent variability of ELISA ab- sorbance readings between different assay plates for the same sample, complete sample sets of duplicates of each of the six samples of each individual muscle type in an ageing or normotensive/hypertensive com- parison were analyzed on the same 96-well plate. Following incubation and wash procedures, plates were read in a Dynatech Minireader II spectrometer and results were expressed in arbitrary units of optical density.
Statistical analysis
Six animals in each age group, six normotensive and six hypertensive animals were used in the studies. Mean values + SD were calculated from the results obtained for each muscle group of a particular age or normotensive/hypertensive designation, and Stu- dent's t test was used to evaluate significant difference between data sets based on the confidence level (P value) calculated for the difference. Values were calculated to four significant figures, but for simplic- ity are displayed to three significant figures in the Tables.
RESULTS
Enzyme activity levels in normotensive and hyperten- sive muscles
The results of enzyme activity measurements on the two skeletal muscles and on the cardiac left ventricu- lar muscle are shown in Table 1. These studies show that, in the longissimus dorsi, no significant differ- ences between activity levels in normotensive and hypertensive muscles were found, whereas in the quadriceps femoris muscle, phosphofructokinase ac- tivity in hypertensive animals was almost twice that in normotensive animals and the P value indicated that the difference was highly significant. In the cardiac muscle, the activities of lactate dehydrogen- ase and /~-hydroxyacyl-CoA dehydrogenase both showed statistically significant decreases in hyperten- sive muscle to about 70% of the activity levels found in the normotensive muscle.
Table 1. Enzyme activity levels in skeletal and cardiac muscles of normotensive (WKY) and hypertensive (SHR) rats Phosphofructose Lactate Hydroxyacyl Citrate
Muscle Rat Phosphorylase Hexokinase k inase dehydrogenase dehydrogenase synthase
Enzyme activity levels + SD are expressed as units per mg of supernatant protein as defined in Materials and Methods. P values for significant difference between activity levels in the same muscle type for normotensive and hypertensive animals are shown between the values compared. NSD indicates no significant difference between the values (P/> 0.1 or larger).
Enzymes in hypertension and ageing
Table 2. Enzyme activity levels in the left ventricular cardiac muscle of ageing rats Age Phosphofructose Lactate Hydroxyacyl Citrate (mo) Phosphorylase Hexokinase kinase dehydrogenase dehydrogenase synthase
Enzyme activity levels + SD are expressed as units per mg of supernatant protein as defined in Materials and Methods. P values for significant difference between activity levels are shown between the values compared. NSD indicates no significant difference between the values (P >/0.1).
Enzyme activity levels in ageing muscles
As shown in Table 2, there were no significant differences detectable in the activity levels of the enzymes in the left ventricular muscle during matu- ration. In senescence, only glycogen phosphorylase appeared to undergo a significant change in activity, with the activity rising to approximately twice the level found in the mature animals.
Because previous studies (Orlander et al., 1978; Essen-Gustavsson and Borges, 1986) had determined the skeletal muscle levels of all the enzymes under consideration in this study with the exception of glycogen phosphorylase, only glycogen phosphoryl- ase assays were performed on ageing skeletal muscles. These analyses (Table 3) indicate that the activity levels in both muscles showed no statistically signifi- cant changes from 3 to 27 months of age.
Calpain H titers in ageing and hypertensive muscles
The results of the immunological assays of calpain II titers in both skeletal and cardiac muscles are shown in Table 4. A consistent feature observed in the studies on ageing muscles was the statistically significant increases in the calpain titers between 3 and 27 months of age, with the increased titers in the senescent muscles being approximately twice the titers in the muscles from the 3-month-old rats. In contrast to these results, the calpain titers of the two skeletal muscles and the cardiac muscle showed no significant differences between hypertensive animals and normotensive controls.
DISCUSSION
Previous reports have established that there are significant morphological and physiological changes that occur in cardiac muscle as a result of hyperten-
Table 3. Total glycogen phosphorylase activity levels in skeletal muscles of ageing rats
Muscle Age (mo) Longlssimus dorsi Quadriceps femoris
Total (phosphorylase a plus phosphorylase b) glycogen phosphoryl- ase activities _+ SD are expressed as units per mg of supernatant protein as defined in Materials and Methods. No significant differences between enzyme activity levels (P > 0.1) were found in muscles of different ages.
sion (Ben Bachir-Lamrini et al., 1990; Lakatta, 1990; Johnson and Hammer, 1992). Other studies have also reported changes in hypertensive myocardium in the levels of enzymes involved in energy production, including changes in creatine kinase isoenzyme ex- pression (Pauletto et al., 1988; Smith et al., 1990), decreases in adenylate cyclase (Anand-Srivastava, 1988) and glycogen phosphorylase a (Ilieva et al., 1989), and increases in calmodulin activator (Huang et al., 1988), lactate dehydrogenase and citrate syn- thase (Smith et al., 1990).
The present studies indicate that in the hyperten- sive myocardium, changes in enzyme activities related to carbohydrate and fatty acid oxidation are quanti- tatively minor, with only lactate dehydrogenase and fl-hydroxyacyl-CoA dehydrogenase showing signifi- cant differences (decreases of less than 30%) between the normotensive and hypertensive states. The de- crease of 29% in lactate dehydrogenase activity in hypertensive myocardium contrasts with a previous report of an elevation in lactate dehydrogenase activity in a rat model for renal hypertension (Smith et al., 1990). The difference in these results may be explained because the previous studies were per- formed using surgically- and drug-induced hyperten- sive states. In contrast to earlier studies which reported changes in glycogen phosphorylase a and citrate synthase activity in hypertensive myocardium (Ilieva et al., 1989; Smith et al., 1990), our results indicate that changes in citrate synthase and total phosphorylase activity (phosphorylase a plus phos- phorylase b) do not appear to be statistically signifi- cant. These differences may in part be explained because the previous studies on phosphorylase only measured phosphorylase a activity, and in the case of citrate synthase, the results were not obtained from SHR rats. Our results therefore suggest that, in the spontaneously hypertensive myocardium, glycogen, glucose, lactate and fatty acid utilization are probably not significantly decreased, and flux through the citric acid cycle, as judged by the level of citrate synthase activity, is comparable to the normotensive state.
In addition to investigations of the myocardium in hypertension, previous studies have also reported changes in vascular and skeletal muscles in hyperten- sion in terms of changes in phospholipase activity (Uehara et al., 1988), Na +, K ÷ transport (Nagaoka et al., 1987) and ant±oxidant levels (Johnson and Hammer, 1992). However, our findings indicate that
66 PETER JOHNSON and JANET L. HAM,,mR
Table 4. Calpain titers in skeletal and cardiac muscles of ageing, normotensive (WKY) and hypertensive (SHR) rats
L. dorsi 30.2_+4.4 P <0.05 39.0+8.6 P <0.01 53.3+6.4 61.7_+3.1 NSD 60.8_+13.1 Q. femoris 21.8 ___ 4.2 P < 0.05 30.8 ± 7.5 P < 0.05 40.2 ± 5.7 47.8 ± 3.5 NSD 52.2 -+ 7.4 Left 27.2 + 5.7 40.7 + 5.0 51.8 + 5.7 54.2 + 7.2 57.2 + 8.3 ventricle - P < 0.001 - P < 0.01 - - NSD -
Calpain titers were determined by ELISA on 2/zg aliquots of protein per ELISA well, and the results are expressed as mean absorbances × 102 + SD for an ageing or normotensive/hypertensive comparison of a muscle type read on the same ELISA plate. P values for significant difference between two data sets are shown below and between each data set. NSD indicates that there was no statistically significant difference between two data sets (P i> 0.1).
in the cases of the two skeletal muscles investigated, there were no significant changes in hypertension in the activities of the enzymes under investigation, with the exception of the significant increase in phospho- fructokinase activity in the quadriceps femoris to approximately twice the normotensive level. These findings suggest that, as in the myocardium, energy production in skeletal muscle probably is not dimin- ished as a consequence of hypertension.
In both skeletal and cardiac muscle, the titers of calpain II were found to be unaffected as a conse- quence of hypertension, which would suggest that there is no elevation in rates of proteolysis of the in si tu substrates of this enzyme (Croall and DeMartino, 1991). In this context, it is interesting to note that hypertension is generally accompanied by myocardial hypertrophy (Lakatta, 1987b; Smith et al., 1990), a feature which also suggests that levels of intracellular protein degradation in the hypertensive myocardium are probably not elevated.
Because of previous suggestions that the hyperten- sive state and the ageing process in cardiac muscle may be similar (Lakatta, 1987b, 1990), enzyme ac- tivity level comparisons were made between hyper- tensive and ageing muscle enzyme activities. Previous studies on ageing rat cardiac muscle have reported both activity increases and decreases in phosphofruc- tokinase (De Tata et al., 1988; Dunaway et al., 1986), a decrease in citrate synthase (Kainulainen and Komulainen, 1989), and an increase in lactate dehy- drogenase (De Tara et al., 1988; Kainulainen and Komulainen, 1989). In our studies, comparisons between enzyme levels in hypertensive and senescent myocardium in comparison to their respective normotensive and mature adult control groups re- vealed that in both senescence and hypertension, the activities of hexokinase, phosphofructokinase and citrate synthase were unaltered. In contrast, different enzymes showed significant activity changes in senes- cence and hypertension, with an increase in glycogen phosphorylase activity in senescence (compared to no change in hypertension) and with decreases in lactate dehydrogenase and fl-hydroxyacyl-dehydrogenase in hypertensive animals (compared to no change in senescent myocardium).
Although there are differences in the results ob- tained in different laboratories for cardiac enzyme activity level changes in ageing and hypertension,
comparison of these results suggests that only in the case of hexokinase activity, which shows no change in ageing or hypertension, is there full agreement in the data in terms of a similar activity level of one of the enzymes in ageing or hypertension relative to the control level. These results therefore suggest that, in terms of the enzymology of the energy-producing metabolic pathways, the changes in senescent and hypertensive myocardium do not have striking similarities.
Further differences between muscle enzyme activity levels in hypertension and senescence were also found from comparisons of the skeletal muscle enzyme levels in hypertension with changes in enzyme activity levels in senescent skeletal muscle. In both ageing (Orlander et al., 1978; Essen-Gustavsson and Borges, 1986) and hypertension (this study), levels of glycogen phosphorylase, hexokinase and lactate dehydrogenase remain unchanged, whereas the levels of fl-hydroxyacyl-CoA dehydrogenase and citrate synthase are altered during ageing but not in hyper- tension. In the case of skeletal muscle phosphofruc- tokinase, activity increases in ageing have been reported (Orlander et al., 1978) and in the present studies a significant increase in phosphofructokinase activity in quadriceps femoris, but not in longissimus dorsi, was observed. These comparisons suggest that although there are some similarities in skeletal muscle enzyme levels in ageing and hypertension, significant differences do exist in ageing and hyper- tension with respect to changes in other enzyme activity levels.
Our analyses of calpain II titers in hypertensive and ageing muscles also indicate that there are clear differences between these two states, as calpain II levels were significantly increased in cardiac and skeletal muscle during development and senescence, whereas no significant changes in calpain II titer were seen in the same muscles in a comparison of normo- tensive and hypertensive animals.
In summary, we conclude that although hyperten- sion results in some changes in muscle enzyme levels related to energy production, these changes probably do not have major effects on the energy production mechanisms in hypertensive muscle. A further con- clusion is that the enzyme activity changes observed in hypertensive muscles are different in some respects from those observed in ageing muscle, indicating that
Enzymes in hypertension and ageing 67
biochemical changes accompanying hypertension and ageing differ quite significantly.
Acknowledgement--The authors thank Dr Huzoor Akbar for generous provision of WKY and SHR rats.
REFERENCES
Anand-Srivastava M. B. (1988) Altered responsiveness of adenylate cyclase to adenosine and other agents in the myocardial sarcolemma and aorta of spontaneously- hypertensive rats. Biochem. Pharmacol. 37, 3017-3022.
Ben Bachir-Lamrini L., Sempore B., Mayet M. H. and Favier R. J. (1990) Evidence of a slow-to-fast fiber type transition in skeletal muscle from spontaneously hyper- tensive rats. Am. J. Physiol, 258, R352-R357.
Costell M., O'Connor J. E. and Grisolia S. (1989) Age- dependent decrease of carnitine content in muscle of mice and humans. Biochem. Biophys. Res. Commun. 161, 1135-1143.
Croall D. E. and DeMartino G. N. (1991) Calcium- activated neutral protease (calpain) system: Structure, function and regulation. Physiol. Rev. 71, 813-847.
De Tata V., Gori Z. and Bergamini E. (1988) Changes in the transmural distribution of glucose-metabolizing enzymes across the left and right ventricular wall of rat heart during growth and ageing. Arch. Gerontol. Geriatr. 7, 23-30.
Dowell R. T. (1987) Phosphorylcreatine shuttle enzymes during perinatal heart development. Biochem. Med. Metab. Biol. 37, 374-384.
Dunaway G. A., Kasten T. P. and Kolm P. (1986) Alter- ation of 6-phosphofructo-l-kinase isozyme pools during heart development and aging. J. biol. Chem. 261, 17,170-17,173.
Essen-Gustavsson B. and Borges O. (1986) Histochemical and metabolic characteristics of human skeletal muscle in relation to age. Acta Physiol. Scand. 126, 107-114.
Hussain H., Dudley G. A. and Johnson P. (1987) Effects of denervation on calpain and calpastatin in hamster skeletal muscle. Exp. Neurol. 97, 635-643.
Huang S. L., Wen Y. I., Kupranycz D. B., Pang S. C., Schlager G., Hamet P. and Tremblay J. (1988) Abnormal- ity of calmodulin activity in hypertension. Evidence of the presence of an activator. J. clin. Invest. 82, 276-281.
Ilieva T., Petkova I., Popova N., Kiprov D. and Tsoncheva A. (1989) Comparative studies on catecholamine content and glycogen phosphorylase activity in the myocardium of spontaneously hypertensive and normotensive rats. Cor Vasa 31, 55-63.
Johnson P. and Hammer J. L. (1988) Calpain and calpas- tatin levels in dystrophic hamster skeletal muscles. Int. J. Biochem. 20, 1227-1230.
Johnson P. and Hammer J. L. (1992) Histidine dipeptide levels in ageing and hypertensive rat skeletal and cardiac muscles. Comp. Biochem. Physiol. 103B, 981-984.
Kainulainen H., Komulainen J., Leinonen A., Rusko H. and Vihko V. (1990) Regional differences of substrate
oxidation capacity in rat hearts: effects of extra load and endurance training. Basic Res. Cardiol. 85, 630-4539,
Karttunen P. and Vornanen M. (1987) Sarcolemmal ATPase activities of the rat heart ventricle---dependence on age and sodium ion. Comp. Biochem. Physiol. 86B, 815-820.
Kline K. H. and Bechtel P. J. (1990) Changes in the metabolic profile of equine muscle from birth through 1 yr of age. J. appl. Physiol. 68, 1399-1404.
Lacaille M., Mercier C. and Simard C. (1990) Effects of electrical stimulation on the activity of hexokinase in the medial gastrocnemius muscle of aged rats after hindlimb suspension. Comp. Biochem. Physiol. 96A, 469-471.
Lakatta E. G. (1987a) Cardiac muscle changes in senes- cence. Annu. Rev. Physiol. 49, 519-531.
Lakatta E. G. (1987b) Do hypertension and aging have a similar effect on the myocardium? Circulation 75,169-177.
Lakatta E. G. (1990) Similar myocardial effects of aging and hypertension. Eur. Heart J. 11, G29-G38.
Nagaoka R., Yamashita S., Maruyama T. and Akaike N, (1987) Active sodium-potassium transports in skeletal muscles of deoxycorticosterone hypertensive rats. Brain Res. 5, 283-291.
Orlander J., Kiessling K.-H., Larsson L., Karlsson J. and Aniansson A. (1978) Skeletal muscle metabolism and ultrastructure in relation to age in sedentary men. Acta Physiol. Scand. 1114, 249-261.
Pastoris O., Vercesi L. and Dossena M. (1991) Effects of hypoxia and pharmacological treatment on enzyme ac- tivities in skeletal muscle of rats of different ages. Exp. Gerontol. 26, 77-87.
Pauletto P., Piccolo D., Scannapieco G., Vescovo G., Dalla Libera L. and Dal Palu C. (1988) Left ventricular hyper- trophy in hypertension. Changes in isomyosins and cre- atine-kinase isoenzymes. Am. J. Med. 84, 122-124,
Pette D. and Reichmann H. (1982) A method for quantitat- ive extraction of enzymes and metabolites from tissue samples in the milligram range. J. Histochem. Cytochem. 30, 401-402.
Reichmann H., Srihari T. and Pette D. (1983) Ipsi- and contralateral fibre transformations by cross-reinner- vation. A principle of symmetry. Pflugers Arch, 397, 202-208.
Rusko H., Bosco C., Komulainen J., Leinonen A. and Vihko V. (1991) Muscle enzyme adaptations to added load during training and nontraining hours in rats. J. appl. Physiol. 70, 764-769,
Shepherd D, and Garland P. B. (1969) Citrate synthase from rat liver. Meth. Enzymol. 13, 11-16.
Simoneau J, A., Lortie G., Boulay M. R., Thibault M. C. and Bouchard C. (1985) Skeletal muscle histochemical and biochemical characteristics in sedentary male and female subjects. Can. J. Physiol, Pharmacol. 63, 30-35.
Smith S. H., Kramer M. F., Reis I., Bishop S. P. and Ingwall J. S. (1990) Regional changes in creatine kinase and myocyte size in hypertensive and nonhypertensive cardiac hypertrophy. Circ. Res. 67, 1334-1344.
Uehara Y., Ishii M., Ishimitsu T. and Sugimoto T. (1988) Enhanced phospholipase C activity in the vascular wall of spontaneously hypertensive rats, Hypertension 11, 28-33.
