Mechanical performance of myocardium from hibernating and nonhibernating mammals

Mechanical performance of myocardium from hibernating and nonhibernating mammals David E Smith, M.D. Bert Katzung, M.D., Ph.D.* San Francisco, Calif.

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t has long been recognized that one of the important obstacles to greater use of hypothermia in clinical medicine is the profound and sometimes catastrophic effect of low temperatures on cardiac function. Thus, the physiology of hibernation has been of great interest to investigators, since hibernators are capable of withstanding body temperatures close to 0°C. with no ill effects. It is now well established that entrance into hibernation is an active process in the intact animal and may involve complex autonomic and endocrine mechanisms preceding as well as during the period of lowered body temperature. For instance, heart rate and probably metabolic rate decrease before body temperature begins to drop. Thyroid, adrenals, gonads, and other endocrine glands undergo marked changes during hibernation.‘J Thus, it is of considerable interest to consider to what degree these extracardiac effects determine the ability of the cardiovascular system of hibernators to function at very low temperatures. It has been found that hearts of hibernating mammals and of poikilotherms

function efficiently at very low temperatures, both in vivo and in vitro, whereas those of nonhibernating species are severely compromised by temperatures below 20 to 25°C.3-7 Hibernators in the nonhibernating or active state have been utilized in some studies, specimens actually in hibernation were used in a few. Direct comparisons of myocardial performance in hibernating and in active specimens of a single species are hard to find in the literature. It was thought that such direct comparison of tissues might provide interesting information about the physiologic determinants of the ability to hibernate. Many comparative studies3-’ which have been carried out have utilized spontaneously contracting preparations. In such studies, the effects of rate (which is profoundly affected by temperature) on contractility complicate the direct effects of temperature on mechanical performance. It was thought that a study was needed in which the rate of contraction was controlled, and, for this reason, isolated electrically driven myocardial strips were utilized. The effect of rate was then studied

From the Department of Pharmacology. I:niversity of California Medical Center, San Francisco, Calif. Supported in part by grants from the American Heart Association (64-G-17). and National Institutes of Health (HE:07753), by a Medical Student Research Training Fellowship (STSGM4303) to D. B. Smith, and by TJnited States Public Health Service Grant FR-00122. ior computing services. Received for publication Aug. 3 1, 1965. *Address correspondence to: B. G. Katzung, Depnrtment of Pharn?acologp, 1210-S, University of California Medical Center, San Francisco, Calif.. 94122.

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as it controlled variable b> deterllGning the force-frequent!, relationships at several temperatures. Methods

1:ive species I\-ere studied. Hibernating species Ivere represented by ground squirrels (Citelhs lateralis and C. tereticudls) and the golden hamster (Mesocricetl~s czllreatlls). Nonhibernators included the rat (Sprague-I)awley and Long-Evans strains) and the guniea pig. The normal body temperature for each of these species is 36-38°C. ‘Fen to 15 animals of each of the five species I\-ere maintained in the animal quarters at an average temperature of 24°C. Ten to 15 other animals of each species were placed in individual cages in a cold room maintained at 5-7°C. Abundant \vood shavings were available and an excess of standard laborator), food pellets were placed in the cages of all the animals, except the ground squirrels. Food was removed from the cages of the ground squirrels within 72 hours after the). had been placed in the cold (this is thought to cause more prompt hibernation). All of the guinea pigs died within 72 hours. The rats survived for 7 to 14 days, but the number surviving 14 days WYEtoo small for useful study. All of the hamsters survived 14 days, and about SO per cent survived several months. Of the survivors, only about 50 per cent hibernated (using as criteria a body temperature within 5 to 8°C. of the environment and arousal frown the dormant state in response to moderate handling stimulation). All of the ground squirrels hibernated lvithin 1 week. Only 1 died, probably because of an infection. Isolated myocardial strips \vere prepared b>. decapitating or stunning and exsanguinating the animal and rapidly removing the heart to cold, oxygenated solution containing NaCl 154 tTl;\I.,/I,, KC1 5.6 mRI.,‘L., CaCl, 5.0 ,ll:\r.~I,., KaHCO.: 7.1 mRl./‘L., and dextrose 11 II~AI.;‘I,. ‘l‘hin strips (WC’ from each aninlal ) \\ere cut from the free \sall of tht~ right Vantricle anti \vere mounted in ;I teniperaturrcontrolled chatllberR c.ontaining the solut ion described, and gassed with 0.5 per cent oxygen and 5 per cent carbon dioxide. The preparations were equilibrated for

lo 00 rliirltllc+ \\irh 5~iiiiill,ktictli ;tt 0.7 per second :tnd l-c‘mper;itiir~ ;~t 36O. lsonletric tension \V;LS rlleasllred \\ ith f((‘.\ .i734 transducer triodes ;md recorded OII ink-Lvriting oscillographs. Stillluli \\ crc provided by Tektronix 161 l)ulse genetators using stij)ral~i;~silllnl voltage at 8 pulse duration of 10 msec. The relationship of stcad>~-state isometric tension to tcmperaturc \vas determined at the arbitraril>~ chosen rate of O..i stimuli per second. Ajfter equilibration at 36”C., the temperature \\as louvered at the rate of 1” pc‘r L to 3 Illinutes. Stimulus threshold voltage \\ as repeatedlv determined and the stimulus intensity \~as maintained at either ;tpproxinlately 1.5 tinles the threshold or 50 volts (the maximum available frown the stinlulator), whirhever \vas less. Recorded c.ontractile tension \\as measured :tt lo intervals from 36” down to the temperature at lvhich no contractions c,ould be elicited at the dcsignated rate (;rt slower rates some preparations continued to respond to slightly lower temperat urcsj. I n order to permit statistical treatnlent, thcb niaxinlum contractile tension for each preparation U-its determined alld set equal to 100 per cent. ,411of the other unensurements of tension \yere then converted to l)er cent of maxi60

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The force-freqllency relationship \\‘as determined at three temperatures: 35“, 25”, and l.i"C. St-imulus voltage was maintained as described in the preceding paragraph. !?tir~lulatiOtl \vas carried Out SUCcessively at frequencies of 0.1, 0.5, 1,2,3, etc., per second, until the preparation failed to respond to each stimulus. Each frequency ~1as maintained long enough for contractile tension to stabilize. The nlaximum contractile tension at each of the three temperatures was determined for each preparation, and isometric tension at each other frequencv \~as then expressed as per cent of this maximum. Results

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many other authors9 and suggests that, above 20-22”C., myocardial contractile force is not notably superior in hibernating species. Fig. 2 contrasts the performance of m)-ocardium from the hibernating species in the active and in the hibernating states. There were no significant differences between hibernating and nonhibernating ground squirrel myocardium and hibernating hamster tissue. Honever, the contractility of nonhibernating hamster myocardial tissue fell to levels significantly lower (p < .05) than the levels of contractility of the other tissues at ever! temperature below 20’. Another manifestation of the effect of temperature on myocardial performance is the tempetature at which peak contractile tension is registered. Since it had already been found that the several species differed significantly in performance at 101~ temperatures, it nas of interest to

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Fig. 1. Temperature-isometric force data for isolated right ventricular strips from nonhibernating species and hibernators in the awake state. Each curve represents the average of 4 to 10 preparations over the entire range of temperature. For each preparation the maximum contractility was set at 100 per cent, and the measurements at every other temperature were related to it. Vertical bars indicate the standard error (S.E.).

temperatures in hibernators. However, the apparent superiority of the myocardium of the rat to that of the hamster was unexpected. In order to rule out the possibility that the means computed using all of the data were biased by varying numbers of zero values (i.e., unresponsive muscles, recorded as producing zero tension), the data for the lower temperatmes were recalculated for each temperature using only data from the muscles still responding to stimulation with measurable contractions. However, there was no change in the relative performance of the four types of myocardium. It should also be noted that, in the temperature range of 36” to 22”, all of the preparations showed an increase in contractile tension with decreasing temperature. This confirms the previous reports of

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establish the degree to which this was due to differences in peak temperature rather than differences in slope. A second temperature variable of interest was the temperature (arrest temperature) at which the isolated tissue no longer responded to maximal stimuli delivered at the standard rate (0.5 per second). Table I shows the results of these computations. By both measures, the ventricular tissue of the nonhibernating hamster was less tolerant of low temperatures than that of the nonhibernating ground squirrel and preparations from hibernating individuals of both species. The apparent superiority of rat myocardium to that of the nonhibernating hamster is again noted in the significantly lower arrest temperatures found for the rat. Since it is well known that heart rate does not remain constant as body temperature changes, it was important to evaluate the influence of frequency of contraction on contractile performance at representative temperatures. The force-frequency curves obtained using animals in the nonhibernating state are shown in Fig. 3. The curves obtained for the “cold-resistant” species, rat and ground squirrel, were remarkably stable for the 20” temperature range studied. In comparison, the guinea pig and hamster preparations showed more marked reduction in highfrequency. performance as temperature UYIS lowered, This progressive reduction of the optimal frequency (contraction rate producing maximal contractility) and max-

imum follow frequency (highest stimulation frequency at which the muscle responds to every stimulus) has been previously reported by Kruta and Stejskalova.‘” Changes ,in stimulus Ihreshold. 1)uring determination of the force-temperature data, the temperature at which the stitnulus threshold (as estimated from the stimulus voltage required) began to increase was noted. In all preparations, threshold remained approximately constant from 36” down to 20” or less and then began to rise steeply. The mean temperatures at which this steep rise began were: guinea pig, 17”; rat, 1.5’; hamster, 13”; and ground squirrel, 9Y‘. Data for the latter two species are for tissues from nonhibernating individuals. Discussion

The data presented strongly support the hypothesis that certain intrinsic properties of the myocardium of hibernating species permit efficient function at low tetnperatures. However, these properties do not seem to be absent in all nonhibernating species, since \ve have shown that isolated rat myocardium is capable of responding to stimuli down to temperatures well below the lethal temperature (16”C.)s for the intact animal. Furthermore, since isolated nlyocardium was used in this study, it. iippears that integrated endocrine and,,‘ot central nervous systeni alterations are no1 required for effective c;lrdiac function in all hibernators (a conclusion reached b!z4dolph)“. ‘l‘hus, Ion-temperature per-

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formance of myocardium from awake and active ground squirrels did not differ detectably from that of tissue from hibernating squirrels. In contrast, myocardium from hibernating hamsters was markedly more tolerant of cold than was tissue from active hamsters. This suggests that the biochemical milieu necessary for function

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at low temperatures is always present in ground squirrel heart but requires special adaptive changes in the caseof the hamster. It is of interest that some author@ have been able to demonstrate significant changes in metabolism in hamster tissues during a period of exposure to cold preceding hibernation. These authors’2 and others2J3

It is knowi that critical temperatures for survival of intact animals fairly closely approximate ihe arrest temperatures of spontaneously. beating isolated tnyocardial preparations.“,” ‘Thus, it is paradoxical that electricall~~ driven rat myocardiutn should appear to have a tolerance of cold superior to that of the nonhibernating hamster, since the heart in the intact hamster, isolated spontaneously beating hamster heart, and isolated hamster atria have all been shown to function at lo\+.er temperatures than do rat preparations.” c.i However, in none of the studies cited was contractility measured. Furthermore, since the measurements of contractilit)made in this study were relative, the mechanical superiority of rat myocardium demonstrated in Ipig. 1 may be onI>. relative and not absolute. Since the force-frequenqr relationship of hamster heart deteriorated more with decreasing temperature, the lower arrest tetnperature (Table 1) found for rats than for hamsters in this stud) a tnanifestatioti of the Ina> represent greater temperature stability of the forcefrequency relation in rat heart (Fig. 31, and the fact that the arrest temperature \vas deternlined using a fixed rate (0.; per second) higher than the normal spantaneous rate for any species at these tentthat peratures. A4dolphtt has remarked isolation of the heart affects its performance more in the hamster than in the rat or the mouse, and Lyman and Blinks7 noted an “inherent delicac)” of the hamster heart, and the fact that tissue cultures front infant hamster and rat heart were not detectahl>. different in their response to decreased temperature. Finall>., it should he noted that the differences in mechanical performance of myocardial strips from the species studied were largely quantitative. That is, increments in contractile force were observed in all preparations xvhen the temperature \vas lowered from 36” to 22”, and derremerits were observed in all cases belobv 13 to 18°C. Nevertheless, the ability of hibernating hamster ventricle and both Iiit~ertl;~ting and nonhibernating ground

Summary

The tnechanical perfortnancc of electrit~tlly driven right ventricular tnl\.ocardiuru frotn guinea pig, rat, hatnster, and ground squirrel was studied in vitro at tentperatures frotn 1” to 36°C’. Tissue front hibernating specitttens (ground squirrels and hatnsters) detnonstrnted signific-nntlv better perfornnance (less diminution in ~isontetric tension, and lo\Ver arrest temperatures) at low temperature than did myocardiurn front nonhibernating species (guinea pig and rat). However, tnpocatdium from nonhibernating hamsters was detnonstrably lesstolerantof low temperatures than \V;LStissue front hibernating specitmens,which suggests that ;I significant adaptation had taken place in individuals entering hibernation. In contrast, no significant difference between hibernating :ind ttotthibernating ground squirrel ntyocardiunt was demonstrated. ‘The tn~~ocardixl force-frequent>. relationship nxs found to be cluite sensitive to tetnperature itt guinea pigs and hamsters but relativeI>. resistant in t-:tts and ground squirrels.

Lyman, C. P., ;md Chatiield, 1’. 0.: Physiolog? of hibernation in nwnmals, I’hysiol. Rev. 35:403, 1955. Kayser, C.: The physiology of natural hihernation, Sew York, 1961, I’ergamon Press, 1’. 29. Adolph, fi:. F.: Some differences in responses to low temper:\tures between xam-blooded and cold-blooded vertebrates, Am. J. Physiol. 166:92, 1951. Hegnauer. A. H., md D’,2rmto, 1-I. E.: Oxygen consumption and cardiac output in the hypothermic doz. .Ain. 1. I’hvsiol. 178:138, 1954. Marshall, J’: hf., at& Wiliis, J. S.: The effects of ternper;iturc on the membrane potentials in isolated atria of the ground squirrel, Citellus tridecemlineatus, J. I’hysiol. 164:61, 1962. Hirvcmen. I,.: Temperature range of the sponI~IIC’~W+ xctivit>, of the isolated hedgehog, hamster, xncl r,lt attrick, rkta Physiol. Scandinal.. 36:38, 1956. Lyman, C. P., and Blinks, 1.). C.: The effect of temperature on the isolated hearts of closely related hibcrll;~tors ;tlld nonhibernators, J. Cell. & Cott~p. Physiol. M:53. 1959. li:~t~wg. 13. G: Intt:gl-.rtetl tn~wlt~ chamher-

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transducer system, J. Appl. Physiol. 19:800, 1964. Blipks, J. R., and Kochweser, J.: Physical factors in the analysis of the actions of drugs on myocardial contractility, Pharmacol. Rev. 15531, 1963. Kruta, V., and Stejskalova, J.: Allure de la contractilite et frequence optimale du myocarde auriculaire chez quelques mammiferes. Arch. Int. Physiol. 68:152, 1960. Adolph, E. F.: Responses to hypothermia in several species of infant mammals, Am. J. Physiol. 166:75, 1951.

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Denyes, A., and Hasset, J.: A study of the metabolism of liver, diaphragm and kidney in cold-exposed and hibernating hamsters, in Mammalian Hibernation, Proceedings of the First International Symposium, Bull. Mus. Comp. Zoology, 124:437, 1960. Kalabukhov, N. I.: Comparative ecology of hibernating mammals, in Mammalian Hibernation, Proceedings of the First International Symposium, Bull. Mus. Comp. Zoology 124:45, 1960.