High Ca Content of Pacemaker Tissues in the Frog Heart

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1 Short Communication Japanese Journal of Physiology, 34, ,1984 High Ca Content of Pacemaker Tissues in the Frog Heart Yasuichiro FUKUDA Department of Physiology II, School of Medicine, Chiba University, Chiba, Chiba, 280 Japan Summary Cation contents of various cardiac regions were determined in the isolated perfused bullfrog heart. The Ca content was much higher in the pacemaker tissues (sinus venosus and atrio-ventricular ring muscle) than in the atrial or ventricular muscles even with extracellular Ca deficiency. A possible relationship between stored Ca and spontaneous pacemaker activity is discussed. Key Words: frog heart, Ca content, pacemaker activity. Ion concentration differences between the intra- and extracellular compartments underlie the cardiac resting potential, excitability, and contractility. The present experiment on the frog heart aims to determine whether the cation (Na, K, and Ca) contents of pacemaker tissues (sinus venosus and atrio-ventricular (AV) ring muscle) differ from that of atrial or ventricular muscles. Although intracellular ion electrodes have been developed recently, determination of tissue ion contents by `classical' chemical analysis may be of value when contents of several ions are simultaneously measured in various tissues. Since most of the total Ca in the tissue is bound or sequestered in the intracellular organelles (SPERELAKIs, 1979; WINEGRAD, 1979), the tissue Ca content represents, though indirectly, the size of `stored' Ca. The bullfrog heart was excised and perfused with the Yagi-Hartung cannula with Ringer solution aerated with air. The preparations were spontaneously active. Normal Ringer solution contained the following composition in mmol/l: NaCI 111.0, KC1 1.9, CaCl2 1.1, NaHC03 2.4, NaH2P , and glucose 5.6. After perfusing for 2 hr, the heart was dissected and divided into the sinus venosus, atrium, AV ring muscle, and ventricle. All extraneous and cut end tissues were removed. The total Na, K, and Ca contents were analyzed by flame spectrophotometry (FUKUDA, 1975), and the extracellular fluid (ECF) space was measured by inulin method (FUKUDA, 1975). The cation contents of tissues were corrected for the inulin space (tissue cation content). In some experiments the heart was Received for publication July 4,

2 1118 Y. FUKUDA initially perfused with normal solution for 2 hr and then perfused with nominally Ca-free solution for 1 hr under hydrostatic pressure difference of about 10 cm H2O. In a separate experiment, the spontaneous pacemaker activities of the sinus venosus or AV ring muscle were recorded by intracellular microelectrodes while the excised heart was continuously superfused with Ca-free solution or solution containing 2 mmol/l MnC12. The inulin space of the bullfrog sinus venosus, atrium, AV ring muscle, and ventricle were respectively 0.71±0.03 (n=20) 0.45±0.02 (n=32), 0.54±0.04 (n= 20), and 0.26±0.01 (n=32) ml/g tissue wet weight (mean±s.e.). Due to large ECF volume and high ECF [Na+], the tissue Na content values showed a wide variation in each heart region except for the ventricle (Fig. 1). Since some of the intracellular Na may be sequestered in the sarcoplasmic reticulum (SPERE- LAKIS, 1979), the intracellular free [Na+] will be less than those shown in Fig. 1. Although the ventricle contained less tissue K than that of the AV ring muscle, there was no significant difference between the tissue K content of the sinus venosus and atrium or ventricle. It seemed, therefore, unlikely that the Na and K contents of pacemaker tissues differ clearly from those of working cardiac muscles when the contents were corrected for the ECF spaces. The result is completely Fig. 1. Cation distribution in various regions of the bullfrog heart. Left, total and tissue Na content; Center, total and tissue K content; Right, total and tissue Ca content; S, sinus venosus; A, atrium; AV, atrio-ventricular ring muscle; V, ventricle. Each cloumn represents mean value + S.E. Numbers in parentheses are number of observations. Tissue cation contents corrected for inulin spaces were expressed as mmol (corrected amount)/kg corrected wet weight. The tissue Na content in various heart regions were not significantly different from each other. The tissue K content of the ventricle was lower than that of AV ring muscle (p<0.02, t-test). The tissue Ca content of four heart regions were significantly different from each other (p <0.005 or lower, t-test). In the right, column indicated as tissue (Ca-free) represents tissue Ca content determined after perfusing with Ca-free solution for 1 hr. Japanese Journal of Physiology

3 Ca AND CARDIAC PACEMAKER 1119 compatible with measurements by DANIELSON (1964) on the toad heart, though WALKER and LADLE (1973) described that intracellular K activities measured with K electrode are lower in the frog sinus venosus than in the atrium or ventricle. The total and tissue Ca contents, on the other hand, differed markedly between different heart regions. The sinus venosus and AV ring muscle contained a much higher amount of tissue Ca than the atrial and ventricular muscles. The Ca content difference between the sinus venosus and AV ring muscle was also significant. As shown previously (FUKUDA, 1975), the atrium contained a higher Ca than the ventricle. Ca wash-out study showed that about 94% and 79 % of tissue Ca remained in the sinus venosus and the AV ring muscle respectively, whereas more than 40 % of tissue Ca was washed out in the atrium and ventricle (Fig. 1 right). Thus a large part of tissue Ca in the pacemaker tissues could not easily be released into the bulk ECF space in a Ca deficient condition. The present experiment demonstrated that the frog pacemaker tissues are highly capable of accumulating Ca, although it could show neither instantaneous movements nor function of stored Ca. It is uncertain at present whether muscle fibers of the pacemaker tissues themselves possess a strong affinity for Ca uptake. Since the intracellular concentration of Ca2+ is an order of 10 mol/l, most of the tissue Ca will be bound elsewhere in the tissue including intracellular organelles (mitochondria and sarcoplasmic reticulum), membranes, connective tissues, and even intercellular matrices. No previous studies, however, have given direct evidence for the existence of large intracellular organelles storing sufficient Ca in the pacemaker tissues. An interesting observation concerning Ca storage in tissues is that the basement membrane contains plenty of negatively charged free or Fig. 2. Effect of perfusing with Ca-free solution or Mn on pacemaker potentials of the bullfrog sinus venosus. A, effect of Ca-free solution; B, effect of 2 mmol/l MnCl2 in normal Ca Ringer solution. Vol. 34, No. 6, 1984

4 1120 Y. FUKUDA protein-bound mucopolysaccharide material with specific affinity for Ca (FLECKEN- STEIN, 1981; LANGER, 1973). Some of tissue Ca may be stored in such extramuscle fiber matrices. It is also assumed that cardiac sarcolemmal membrane plays an important role as a Ca accumulating system from which Ca enters the interior of the myocardial fiber through the slow channels (FLECKENSTEIN, 1981). Such a superficial Ca pool and subsequent influx of Ca2+ are augmented by adrenergic catecholamines even with extracellular Ca deficiency (FLECKENSTEIN, 1981). A high Ca storage in the pacemaker tissues may be relevant to their dense autonomic innervations (RUSKA, 1965). During perfusing with Ca-free Ringer, the pacemaker potentials recorded from the ventral part of the sinus venosus close to the sino-atrial ring muscle were reduced in magnitude, frequency, and rate of rise of slow diastolic depolarization (Fig. 2). However, even at 60 min (or longer period) of Ca washout, the sinus venosus could still initiate regular spontaneous activity with prolonged action potential (Fig. 2A). The result indicates that free Ca2+ in the ECF may not be a crucially important ion for maintaining spontaneous action potentials, but this conflicts with the fact that Mn2+ which effectively blocks slow inward current carried mainly by Ca2+ (or Nat) quickly abolishes the pacemaker activity (Fig. 2B). Similar effects on pacemaker potentials of Ca-free Ringer and of Mn2+ were seen in the AV ring muscle. One possibility is that other ions except for Ca2+ carry the current for pacemaker depolarization. Thus slow inward current by Na+ or the deactivation of an outward current carried by K+ may be essential for diastolic depolarization (BROWN, 1982; YANAGIHARA and IRISAWA, 1980). There is, however, another explanation that Ca2+ release from the highly stored site(s) such as membrane surface or intercellular (extra-muscle fiber) matrices into the cell interior contributes to the spontaneous depolarization. Many Ca2+ entry blockers and Mn2+ have been shown to rapidly suppress the spontaneous pacemaker activity indicating that Ca2+ is a major cation for carrying inward current in the pacemaker tissues (BROOKS and LU, 1972; FLECKENSTEIN, 1981; KOHLHARDT and FLECKENSTEIN, 1976; WIT and CRANEFIELD, 1974). In conclusion, high Ca contents of pacemaker tissues were found in the frog heart. However., site(s) of Ca storage and a relationship between stored Ca and spontaneous pacemaker activity could not be demonstrated. REFERENCES BROOKS, C. McC. and Lu, H.-H. (1972). The Sinoatrial Pacemaker of the Heart. Thomas, Springfield. BROWN, H. F. (1982). Electrophysiology of the sinoatrial node. Physiol. Rev., 62: DANIELSON, Bo G. (1964). The distribution of some electrolytes in the heart. Acta Physiol. Scand. (Suppl.), 62: 236. FLECKENSTEIN, A. (1981) Fundamental actions of calcium antagonists on myocardial and cardiac pacemaker cell membranes. In: New Perspectives of Calcium Antagonists, ed. by WEISS, G. B. Am. Physiol. Soc., Bethesda, Maryland, pp Japanese Journal of Physiology

5 Ca AND CARDIAC PACEMAKER 1121 FUKUDA, Y. (1975) Difference in calcium content of atrial and ventricular muscle. Jpn. J. Physiol., 25: KOHLHARDT, M. and FLECKENSTEIN, A. (1976) The slow membrane channel as the predominant mediator of excitation process of the sinoatrial pacemaker cell. Basic Res. Cardiol., 71: LANGER, G. A. (1973) Excitation-contraction coupling. Anna. Rev. Physiol., 35: RUSKA, H. (1965) Electron microscopy of the heart. In: Electrophysiology of the Heart, ed. by TACCARDI, B. and MARCHETTI, G. Pergamon Press Ltd., Oxford, London, pp SPERELAKIS, N. (1979) Origin of the cardiac resting potential. In: Handbook of Physiology, Section 2, The Cardiovascular System, Vol. 1, The Heart, ed. by BERNE, R. M., SPERELAKIS, N., and GEIGER, S. K. Am. Physiol. Soc., Bethesda, Maryland, pp WALKER, J. L. and LADLE, R. 0. (1973) Frog heart intracellular potassium activities measured with potassium microelectrodes. Am. J. Physiol., 225: WINEGRAD, S. (1979) Electromechanical coupling in heart muscle. In: Handbook of Physiology, Section 2, The Cardiovascular System, Vol. 1, The Heart, ed. by BERNE, R. M., SPERELAKIS, N., and GEIGER, S. K. Am. Physiol. Soc., Bethesda, Maryland, pp WIT, A. L. and CRANEFIELD, P. F. (1974) Effect of verapamil on the sinoatrial and atrioventricular nodes of the rabbit and the mechanism by which it arrests reentrant atrioventricular nodal tachycardia. Circ. Res., 35, YANAGIHARA, K. and IRISAwA, H. (1980) Potassium current during the pacemaker depolarization in rabbit sinoatrial node cell. Pflugers Arch., 388: Vol. 34, No. 6, 1984