Interspecies variation in myocardial physiology: the anomalous rat.

THE HEART OF THE ADULT RAT HAS LONG BEEN RECOGNIZED TO BE ANOMALOUS IN AT LEAST TWO RESPECTS: an absent or negative inotropic response to an increase in rate of electrical stimulation (negative staircase); resistance to digitalis glycosides. The heart of the neonatal rat (less than 2 weeks old), on the other hand, demonstrates a markedly positive staircase and a large increase in force upon application of glycoside. It is significant that the action potential of the neonate ventricle demonstrates a prolonged plateau component which progressively decreases with age. The shortening of the plateau correlates with the disappearance of the positive staircase and glycoside responses. Previous studies indicated that a major factor contributing to the prolonged plateau of the neonate was a high level of sodium (Na) conductance. Thus transmembranous Na movement associated with excitation is considerably greater in the neonatal heart as compared to the heart of the adult rat. The higher level of intracellular Na would produce increased activity of a proposed sodium-calcium (Na-Ca) carrier. This is believed to mediate the augmented influx of Ca which is responsible for positive staircase and glycoside responses. Ventricular muscle from the hearts of most mammalian species demonstrates a prolonged plateau with maintenance of a "slow" channel for Na. It appears that in the rat this channel closes with age. It follows that there would be a reduced tendency for the adult rat heart to accumulate intracellular Na, [Na](i), when excitation frequency is increased or the Na pump is inhibited by digitalis. Since elevation of [Na](i) is the stimulus for the proposed Na-Ca carrier, this system would not be activated, Ca influx would not increase and contractility would not be augmented.


Interspecies Variation in Myocardial
Physiology: the Anomalous Rat by Glenn A. Langer* The heart of the adult rat has long been recognized to be anomalous in at least two respects: an absent or negative inotropic response to an increase in rate of electrical stimulation (negative staircase); resistance to digitalis glycosides. The heart of the neonatal rat (less than 2 weeks old), on the other hand, demonstrates a markedly positive staircase and a large increase in force upon application of glycoside. It is significant that the action potential of the neonate ventricle demonstrates a prolonged plateau component which progressively decreases with age. The shortening of the plateau correlates with the disappearance of the positive staircase and glycoside responses. Previous studies indicated that a major factor contributing to the prolonged plateau of the neonate was a high level of sodium (Na) conductance. Thus transmembranous Na movement associated with excitation is considerably greater in the neonatal heart as compared to the heart of the adult rat. The higher level of intracellular Na would produce increased activity of a proposed sodium-calcium (Na-Ca) carrier. This is believed to mediate the augmented influx of Ca which is responsible for positive staircase and glycoside responses. Ventricular muscle from the hearts of most mammalian species demonstrates a prolonged plateau with maintenance of a "slow" channel for Na. It appears that in the rat this channel closes with age. It follows that there would be a reduced tendency for the adult rat heart to accumulate intracellular Na, [Na]i, when excitation frequency is increased or the Na pump is inhibited by digitalis. Since elevation of [Na]i is the stimulus for the proposed Na-Ca carrier, this system would not be activated, Ca influx would not increase and contractility would not be augmented.
In many biological investigations it is the mutant organism or the anomalous species which frequently provides additional insights into basic mechanisms of function. Definition of the basis for an anomaly may further define the normal condition. In the area of mammalian myocardial physiology the rat has provided just such an anomalous condition. The rat heart demonstrates anomalies with respect to its action potential (1, 2), forcefrequency response (1-3), ionic exchange parameters (2,(4)(5)(6) and response to digitalis glycosides (2,7,8). Investigation of these characteristics has provided further insight into fundamental properties of myocardial function not only in the rat but in other mammalian species.
I will first review the anomalies and then attempt to correlate them in such a manner as to indicate how the information has provided further definition of a model for control of myocardial force development. *

Action Potential
As compared to most other mammalian species, the action potential (AP) recorded from ventricular cells of the adult rat is considerably shortened (1,2,9) with total duration in the range of 100 msec. The AP demonstrates an essential absence of the plateau (phase 2) component and this accounts for the major part of the shortening and the fact that the effective refractory period is less than 50 msec in duration (9). It is interesting that the short AP develops as the rat ages. Prenatally and during the early neonatal period the duration is much increased (2, 10, 1H). At 1 day of age a prominent plateau is present with a total AP duration of approximately 300 msec. There is then a progressive loss of the plateau through the next 3-4 weeks such that the adult configuration is achieved (Fig. 1).
It is likely that the changes in the plateau are secondary to changes in both sodium and potassium conductance (9Na and gK). Diminution in both delayed and anomalous rectification for K would shorten the plateau as would loss of a slow conducting Na channel. There is experimental evidence for the latter (2). If the Na concentration in the per- fusion medium is reduced to one-h (choline chloride substituted to maintair ity) for the 1 day old ventricle the j amplitude and duration decrease marke4 This suggests the presence of a significward Na current during the early neor with progressive closing of the slow-chz rat ages.
It should be noted that the configurn AP in the early neonatal rat is similar tc in adults of other mammalian species. 1 heart AP becomes anomalous with age. question, in a teleological sense, what t] from the progression. The adult rat has of 400-500 beats/min. A short plateau wi dent short refractory period obviously p high heart rates to be established. As perhaps, is the closure of the slow Na c tem. In other mammalian species with and prominent plateau, the slow Na cha approximately 75% of the Na which i tracellularly with each AP (12). At high this large influx places a large load on th of the cell. In the adult rat heart most ( channel Na influx is eliminated, leav pump to handle the influx associated wi component (phase 0) of the AP which is on the fast Na channel. Therefore a la nent of the energy required for active N is eliminated.

Force-Frequency Respons
In most mammalian species an increa lation frequency induces an increase in force development known as the Bowditch staircase phenomenon (13). Within similar frequency range 6 DAY the rat heart shows a decline in contractile force with increasing stimulation frequency (1-4).
As indicated with the AP, in the neonate as compared to the adult, the force-frequency response differs markedly in the young as compared to the old rat. A markedly positive staircase is present ADULT during the first week of life which then declines by three weeks to give the negative response typical of 50D% Noa] the adult rat (Fig. 2). This temporal sequence of mechanical function matches the sequence of the electrophysiological changes so that as the AP shortens the positive staircase declines and be-.ction potential comes negative. )te the progres-Again, the staircase response of the neonatal rat of the plateau. is the response found in the hearts from adults of y and 26 days. other species. Therefore the force-frequency remplitude oftheel sponse, as does the AP progression, becomes produced with anomalous with age.
Lalf normal Ionic Exchange n isosmolal-AP plateau Upon an increase in stimulation frequency most Py ( Fig. 1) mammalian species demonstrate a net loss of K ant slow in-from the heart (14)(15)(16). In contrast, the adult rat iatal period heart demonstrates little or no loss of cellular K.
innel as the Instead the increase in frequency is associated with an increase in the steady-state rate of cellular K ation of the exchange, i.e., increased turnover rate without a ) that found change in the quantity of K within the cell. This Thus the rat increased steady-state exchange with higher stimu-One might lation frequencies is consistent with the absence of he rat gains a plateau component of the action potential and, a heart rate more specifically, the absence of an anomalously ith its atten-rectifying channel for K. Though the studies have ermits such not been done, it would be expected that the early important, neonatal heart would demonstrate considerably less hannel sys-increase in steady-state K exchange upon increase longer APs in stimulation frequency. This expectation is based nnel admits on the probable presence of anomalous rectification influxes in-in the neonatal heart which acts to reduce gK below lheart rate, resting level during the plateau phase and cancel, so ie Na pump to speak, the augmented outward K current which f this slow develops during repolarization. This combination ing the Na acts to maintain steady-state K exchange unaltered th the spike over a large range of stimulation frequency. The th hespedent absence of a net loss of K will be discussed further rdependent below.
,a transport There are other interventions to which the rat responds anomalously in terms of its ionic exchange properties. One is the response of myocardial K ;%0 e exchange to respiratory acidosis. Upon induction of a respiratory acidosis the beating rabbit ventricle vse in stimudemonstrates a net increase in its K content. This is  contrasted to the absence of increased uptake in the quiescent, nonelectrically stimulated rabbit heart and in the stimulated rat heart-i.e., the rabbit's response is made ratlike by the absence of membrane depolarization. The response of K exchange to magnesium is also dissimilar in the rabbit and the rat (6,17). In the rabbit, Mg did not affect ventricular K exchange under normal physiological perfusion conditions, whereas K exchange rate in the rat ventricle was markedly depressed by Mg. In order that an effect of Mg be demonstrated in the rabbit, K efflux had first to be increased by administration of a digitalis compound. Mg would then abolish the glycosideinduced net K loss. In this case, then, the rabbit was made ratlike by prior administration of digitalis.
Mg also affects force development very differently in the rat and rabbit. Perfusion of the rabbit ventricle with 20mM Mg decreases the rate of tension development (dPldt) to 84% of control whereas the same concentration reduces dPldt of the rat ventricle to 15% of control (6,17). Obviously the rat demonstrates a great deal more sensitivity to the excitation-contraction uncoupling effects of Mg.

Day
Digitalis Glycosides It is well known that the adult rat heart is remarkably resistent to the action of the digitalis glycosides (7,8). Doses of glycosides which are markedly toxic to most mammalian hearts produce little or no effect in the rat. The resistance to the drug has been proposed to be due to its more rapid dissociation from binding sites in the rat heart as compared to the dissociation from hearts of other species (3,4,18).
As with AP and force-frequency the neonatal rat myocardium presents a different response which is much less anomalous (2). Papillary muscle from a rat 5 days old demonstrated an increase of 160o in dPldt upon perfusion with 5 x 10-5M ouabain. A muscle from rats 22 days old demonstrated an increase in dPldt of only 12% given the same dose of glycoside (Fig. 2). It is notable that the sensitivity of the target enzyme of the glycoside, Na-K ATPase, was exactly the same in the hearts from rats 5 days old and from adult rats (personal communication, K. D. Philipson). This indicates that the different response cannot be attributed to a change in the characteristics of the enzymes with age. The reason for the progressive resistance to glycoside must be related to another system which is operative in the control of contractile force.
The anomalies of the rat heart discussed above have provided important clues leading to the further definition of the systems important to control of contractility in the mammalian heart.

Model for Myocardial Contractile Control
Details of a model (Fig. 3) for the ionic control of myocardial contractility have been developed in a number of recent publications (19,20), but a brief summary is necessary here in order to understand how the anomalous rat is the exception that may lead to proving the rule or, in this case, the model.
Mammalian hearts, including the rat, are dependent upon a rapidly exchangeable source of calcium (Ca) for the support of contractile force. It remains uncertain as to whether the major portion of Ca involved in E-C coupling moves across the sarcolemma with each beat or whether a portion is released from internal sarcotubular sites by the mechanism of Ca-induced Ca release (21). It is possible that a combination of the two mechanisms is operative, but it remains, nevertheless, certain that modulation of the amount of Ca which crosses the sarcolemma upon excitation is of critical importance in control of the contractile response. This Ca is probably derived from sites at the surface of the cell within the surface-coat/external lamina com- COAT LIPID UNIT MEMBRANE FIGURE 3. Model for Ca movement. Ca is bound to negatively charged sites in the external lamina of the membrane. These sites are in rapid equilibrium with Ca in the interstitium. The external lamina is proposed to supply the Ca that moves across the sarcolemma via two routes: (1) through a pore system formed by integral proteins embedded in the lipid membrane; movement through this system would be electrogenic; (2) with a carrier (coupled to outward Na movement) such that movement via this system is electroneutral. Reproduced with permission of International Review of Physiology, Copyright, 1976. University Park Press, Baltimore.
plex (22). This complex contains large amounts of glycoprotein and glycolipid and carries a large quantity of negatively charged sites which are believed to represent the loci for Ca binding. Although extensive interspecies studies have not been done it is likely that the surface coat/external lamina complex functions similarly in mammalians, including the rat. It should be noted however, that the rat's sensitivity to uncoupling by Mg (see above) may indicate some differences in the structure of the surface complex. The next component of the model is concerned with the movement of Ca from its binding sites across the sarcolemmal complex. Evidence now indicates that a major portion of this movement occurs via a carrier system which couples the movement of one Ca ion inward to the movement of two Na ions outward (23). The rate at which the carrier moves or the number of carriers which are activated (the models are equivalent) depends upon the concentration of a component of intracellular sodium [Na]i. An increase in [Na]i causes increased carrier activity which results in greater Na movement outward and greater Ca movement inward. The latter would be expected to bring additional Ca to the myofilaments and result in a positively inotropic response (Fig. 3).
If such a Na-Ca carrier is operative, any intervention which results in increased [Na]i would result in increased contractile force. In most mammalian hearts, except the adult rat, increased frequency of stimulation and administration of digitalis clearly produce a significant increase in [Na]i and a significant increase in contractile force. The response fits and is predicted by the Na-Ca carrier system. As I have discussed, the adult rat does not demonstrate a positive inotropy upon an increase in stimulation frequency or when given digitalis.
Neither does the heart show a gain in [Na]i (as indicated by absence of a net K loss) with the above interventions and the reason for this may be found in the configuration of its action potential. As shown (Fig. 1), the plateau is progressively lost with age. A component of the plateau was shown to be dependent upon a slow Na channel which seemed to be present and operative in the young neonatal rat (and in other adult mammalians) but lost in the adult. The closure of this Na channel would be expected to greatly reduce the Na influx with each excitation. It would follow that there would be a reduced tendency for the adult rat to accumulate [Na]i when the excitation frequency is increased or the Na pump is inhibited by digitalis. Since elevation of [Na]i is the stimulus for the Na-Ca carrier activation, this activation would not occur, Ca influx would not increase and contractility would not be augmented. The sequence as described for the neonate to adult is consistent with progressive closure of the slow Na channel, accounts for the anomalous behavior described and adds support to the model proposed for the control of Ca movement in the mammalian heart.