Endothelial cells are required for the camp regulation of cardiac contractile proteins

Transcription

1 Proc. Natl. Acad. Sci. USA Vol. 9, pp , April 1993 Physiology Endothelial cells are required for the camp regulation of cardiac contractile proteins (endothelium/endothelial factors/actomyosin/atpase cardiac autoregulation/contractile proteins) GEORGE MCCLELLAN, ANDREA WEISBERG, LIN-ER LIN, DAVID ROSE, CLAUDIO RAMACIOTTI, AND SAUL WINEGRAD* Department of Physiology, School of Medicine, University of Pennsylvania, Philadelphia, PA Communicated by Robert E. Forster II, December 14, 1992 ABSTRACT The contractile proteins in mammalian cardiac muscle are regulated by a camp-dependent reaction that alters the activity of the actomyosin ATPase. The ATPase activity of cardiac actomyosin has also been shown to depend on factors released by small arteries in the myocardial tissue. Endothelial cells have been implicated in the regulation of the contractile force developed by isolated cardiac tissue. To determine whether endothelial cells are required for the campdependent regulation of the contractile proteins, the effect of camp on the actomyosin ATPase activity was measured in cryostatic sections of isolated, quickly frozen rat ventricular trabeculae. In half of the trabeculae, the endocardial endothelial cells had been damaged by a 1-sec exposure to.5% Triton X-1. In trabeculae with intact endothelial cells, camp increased actomyosin ATPase activity toward an apparently maximum value. In trabeculae with damaged endothelial cells, camp did not change actomyosin ATPase activity. The coronary venous effluent from an isolated heart has already been shown to modify the maximum isometric force developed by an isolated trabecula. The extent to which the force of the isolated trabecula is changed by the coronary venous effluent is dosely related to the degree to which camp has up-regulated the actomyosin ATPase activity in the isolated heart donating the coronary effluent: the greater the degree of up-regulation of ATPase activity, the greater the increase in force produced by the effluent. These results indicate that endothelial cells are required for the camp-dependent regulation of cardiac contractile proteins to function, and these results further suggest that the myocardium autoregulates by modulating the camp regulation of contractile proteins with endothelial-derived factors. Endothelial cells have been shown to play an important role in controlling the tone of vascular smooth muscle (1, 2). In response to multiple humoral agents and neurotransmitters, endothelial cells release substances that modify the contractile mechanism of smooth muscle and alter the level of force development. The most important of these substances appears to be nitric oxide (3), a powerful relaxant, and endothelin (4), which produces strong contraction. In addition to their effects on smooth muscle cells, these vasoregulatory substances also influence the rate of release of each other by endothelial cells. Besides responding to various chemical stimuli with the secretion of vasoregulatory substances, endothelial cells are also sensitive to local oxygen tension and to shear forces exerted by blood or artificial perfusate flowing in blood vessels (5-7). More recently, it has become clear that endothelial cells are also capable of modifying the contractile behavior of isolated cardiac tissue and isolated cardiac cells (7-1). The The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C solely to indicate this fact. first information implicating endothelial cells in the regulation of cardiac contractility came from studies comparing shape of the contraction of isolated papillary muscles or trabeculae before and after the endocardial endothelial layer had been damaged or destroyed by either rubbing the surface of the tissue bundle or treating the bundle very briefly with a very low concentration of detergent (8, 9). Disruption of the endocardial endothelium decreased the peak tension developed during a contraction and produced an earlier onset of relaxation without altering the rate of rise of force. Because most endothelial cells in the tissue bundle were in the endocardium, it was not clear from these observations whether the change in contraction was the result of the loss of a covering layer of cells or specifically of endothelial cells. Subsequent work strongly favors the latter hypothesis. Cultured cardiac endothelial cells produce a substance or substances that can reverse the effect of damage to the endocardial endothelium on contraction of the myocardial cells (1). The coronary venous effluent from an isolated perfused working heart contains substances that alter the contraction of an isolated trabecula by changing peak contractile tension and time to the onset of relaxation (7). Extending from the small arteries, even in the absence of perfusion of those arteries, gradients in the actomyosin ATPase activity in cardiac cells have been shown by quantitative histochemistry (11). These gradients probably result from substances released by cells in the blood vessels that modulate activity of the contractile proteins in nearby myocardial cells. The mechanisms by which endothelial-derived factors modify cardiac contractility are not clear (12, 13). Endothelin increases contractility at nanomolar concentrations, but how it does this has not been defined. Evidence indicates that endothelin may increase the inward Ca2+ current, alter sensitivity of the contractile proteins to Ca2+, and change intracellular ph as a result of an effect on transmembrane ionic pumping. Other endothelial-derived cardioactive substances have not been identified, but two different studies have found that endothelin cannot account for the increase in contraction produced by either the culture medium for endothelial cells or the coronary venous effluent from isolated perfused hearts (1, 21). Regulation of cardiac contractility occurs primarily by either an increase in the concentration of activating Ca2+ ions or an alteration in the response of the contractile proteins to a given Ca2+ concentration. One demonstrated mechanism for regulating cardiac contractility involves a camp-sensitive alteration of the maximum Ca2+-activated force in permeabilized cardiac cells and the actomyosin ATPase activity in cryostatic sections of quickly frozen cardiac tissue (14-16). In the studies involving the measurement of force development in permeabilized cells, modification of contraction was *To whom reprint requests should be addressed at: Department of Physiology, School of Medicine, University of Pennsylvania, 37th and Hamilton Walk, Philadelphia, PA

2 2886 Physiology: McClellan et al. shown to involve two different reactions, one reaction that was camp sensitive and a second reaction that was initiated by exposing the tissue to a low detergent concentration. The following study was done to determine whether the role of endothelial-derived factors in modulating cardiac-cell contractility is related to the camp-sensitive mechanism for altering contractility. Measurement of the ATPase activity of actomyosin was used to monitor contractile function in the cardiac cells. METHODS Male rats obtained from Charles River Breeding Laboratories and weighing between 2 and 35 g were killed in accordance with accepted procedures (American Association for Accreditation of Laboratory Animal Care), and the hearts were removed immediately. The right ventricle was opened while the heart was bathed in Krebs' solution at room temperature. Ties were placed around the ends of up to three endocardial trabeculae or papillary muscles with crosssectional area between.1 and.2 mm2. The bundles of tissue were removed and attached between a force transducer and a mechanical ground at a sarcomere length of X2.2 Am. During the dissection and mounting, considerable care was taken to avoid damage to the endocardial endothelium from contact with dissection instruments and from contraction to a short length. The mounted bundles of tissue were superfused with Krebs' solution containing 118 mm NaCl, 4.7 mm KCl, 2.5 mm CaCl2, 1.2 mm MgSO4, 1.2 mm KH2PO4,.5 mm EDTA, 24.5 mm NaHCO3, and 11 mm glucose at 3 C and oxygenated with 95% 2/5% CO2 at a rate of.4 ml/min, which is sufficient to prevent any flow sensitivity of the contraction. The bundles were stimulated with platinum electrodes at.2 Hz until the shape of the isometric contraction stabilized; this normally required =12 min. Half of the tissue bundles were then exposed to.5% Triton X-1 in oxygenated Krebs' solution and then quickly washed copiously with a large volume of standard Krebs' solution before superfusion was reestablished. In the other half, superfusion with standard Krebs' solution was maintained throughout. Within 3 min all bundles of tissue were quickly frozen in isopentane cooled with liquid N2. The frozen tissue was sectioned generally within 1 day on a Hacker cryostat maintained below -2 C, and actin-activated ATPase activity of myosin was determined by an already published procedure that uses quantitative histochemistry (15). For examination of ATPase activity at least 8 serial transverse sections were cut. Sectioning began at the chordae for the papillary muscles and at one wall attachment for trabeculae; sections were 4-,m thick. The ATPase measurement involves trapping inorganic phosphate liberated by the enzymatic action and conversion to the opaque salt cobalt sulfide. The average optical density of, at least, 1 transverse sections was measured in each tissue for each protocol. For determination of the camp effect on actomyosin ATPase activity, 1,uM camp and 5 mm theophylline were added to the incubation media of sections cut serially to those used for control measurements of ATPase (15). Optical density was measured by digitizing a video image of the section formed by a compound microscope with a x 25 objective having a.4 numerical aperature and a video camera. Sensitivity of the density measurement was limited to 1/256 by the number of gray levels of a pixel. Noncellular regions of the tissue section could be excluded from the measurement. For determining the relation between ATPase activity of actomyosin and effect of coronary venous effluent on contractility of an isolated trabecula, a cascade system was used (7). Isolated, perfused working hearts were prepared by a Proc. Natl. Acad Sci. USA 9 (1993) small modification (17) of the Neely-Morgan (18) preparation with hearts from male rats weighing between 25 and 35 g. The aorta, pulmonary artery, left atrium, and right atrium were cannulated to control left-atrial filling pressure and to measure cardiac output, coronary sinus flow, total coronary flow, and systolic and diastolic pressures. The perfusion medium was the same as the bicarbonate Krebs' solution used for superfusion of isolated trabeculae, except that the perfusion medium was maintained at 37 C. Forty minutes was allowed for recovery from dissection and stabilization. At appropriate times after the contractile activity of an endocardial trabecula isolated from the heart of another rat had stabilized, the coronary venous effluent was collected from the pulmonary artery of the perfused heart for 1.5 min, reoxygenated with 95%2/5%CO2, brought to 3 C, and used to replace the Krebs' solution bathing the trabeculae. Coronary venous effluent flow varied between 15 and 25 ml/min. The change in the wave form of the contraction produced by superfusion with coronary venous effluent was determined from computer-averaged wave forms for five consecutive contractions. The details of this procedure have already been described (7). After the samples of medium perfusing the isolated heart had been used to superfuse the isolated trabecula, the isolated heart was quickly frozen in isopentane cooled with liquid N2. Four-micrometer sections were cut through the ventricles with a Hacker cryostat. At least 8 serial sections were cut, generally starting with the epicardial surface, but as a result of the orientation of the tissue with respect to the knife edge, most sections included the full thickness of the ventricular wall. At least 1 sections consisting of every fourth or every eighth section in the series were used for measurement of the control or the camp-stimulated actomyosin ATPase activity. The details of this procedure have been published (11). Values reported are the averages of values from the 1 sections. Data were analyzed by unpaired and, where appropriate, paired Student's t test. Statistical significance was accepted at P <.5. RESULTS Isolated Cardiac Trabeculae. Peak force and shape of the contraction were measured in a population of isolated endocardial trabeculae taken from the right ventricle of rat hearts. To get a broad sampling of forces normalized for cross-sectional area, trabeculae with different thicknesses were chosen. This was done because the normalized force produced either by maximum Ca2+ activation in permeabilized cells or by electrical stimulation in intact cells has been demonstrated to decline exponentially with increased thickness of the tissue bundle (19). Trabeculae were assigned randomly to the two groups before the cross-sectional area was accurately measured on the cryostatic sections. Approximately half of the trabeculae were treated for 1 sec with.5% Triton X-1 in Krebs' solution to damage the endothelial cells (8), and then both trabeculae populations were quickly frozen for measurement of actomyosin ATPase activity. Damage of the endocardial endothelium by.5% Triton X-1 caused a 14 ± 3% decline in peak force of contraction and a decrease in the time to 5%o relaxation by 18 ± 4% (n = 21). Response of the actomyosin ATPase activity to camp of the bundles with intact and with damaged endocardial endothelium is shown in Table 1. The response of ATPase activity to camp exposure in tissue sections of the bundles with intact endothelial cells differed markedly from that in tissue samples with damaged endothelial cells. In the sections from the control cells, actomyosin ATPase activity significantly increased, whereas no significant increase occurred in

3 Table 1. Effect of camp on ATPase activity of trabeculae with intact or damaged endothelial cells ATPase activity Endothelium n Control +camp* P Intact ± Damaged 21 1 ± 6 98 ± *Expressed in units relative to control values. sections of cells from the trabeculae with damaged endothelial cells. The nature of the difference in response of actomyosin ATPase to camp is more apparent in a plot of the relation between ATPase activity with camp as a function of the ATPase activity level without camp in serial sections of the same tissue. This plot shows how much the ATPase activity of a given trabeculae was altered by camp exposure (Figs. 1 and 2). In the control trabeculae, the shape of the relation is logarithmic with apparent saturation. The relative increase in ATPase activity produced by camp is greater where the control value is lower. In the trabeculae with damaged endothelium, the slope of the relation is one, indicating that at all values of actomyosin ATPase activity, camp has no effect on the ATPase activity. Isolated Perfused Heart. In a previous study (7) it had been shown that superfusion of a trabecula with the reoxygenated coronary venous effluent collected from an isolated perfused heart could alter contractility of the isolated trabecula. The change in contractility was an increase or a decrease, depending upon the relative oxygen tension of the coronary effluent before it was reoxygenated; an increase was associated with higher oxygen tension. On the basis of these observations (7) and others (11), it was suggested that the effluent contained separate factors produced by endothelial cells that could either raise or lower contractility. The change actually seen was a function of the relative concentrations of the two different types of cardioactive factors. To determine whether the ability of the coronary venous effluent to alter contractility of the trabecula was related to the level of regulated activity of the ATPase of actomyosin in the cardiac cells in the perfused heart, the following study was done. The coronary venous effluent was applied, after reoxygenation, to the isolated trabecula, and the effect on the peak force developed during a contraction of the trabecula was measured. Both (i) actomyosin ATPase activity of the perfused heart and (ii) change in the peak tension developed by the + Physiology: McClellan et al..3 zt % acto ATPaSe FIG. 1. Relation between actomyosin ATPase (acto ATPase) activity without and with 1 AM camp during measurement of enzymatic activity in cryostatic sections of intact trabeculae and papillary muscles. Each point represents the value for one trabecula or papillary muscle. The line is the best fit and represents the equation y = log x. The correlation coefficient equals.86. The x intercept of the function is significantly greater than zero (P <.1). o.2 - Proc. Natl. Acad. Sci. USA 9 (1993) 2887 i * /m U acto ATPase FIG. 2. Relation between actomyosin ATPase (acto ATPase) activity without and with 1,uM camp during measurement of enzymatic activity in cryostatic sections of trabeculae and papillary muscles in which endocardial endothelium was damaged by 1-sec exposure to.5% Triton X-1 in Krebs' solution. Each point represents the value for one trabecula or papillary muscle. The line indicates the least-squares linear regression, the equation being y = x. The value for the intercept is not significantly different from the origin (P =.67), and the slope is not significantly different from 1 (P <.1). The correlation coefficient equals.9 and P <.5. isolated trabeculae during superfusion with reoxygenated coronary venous effluent have been shown to be functions of venous P2 (7, 11). The perfused heart was then quickly frozen, and actomyosin ATPase activity with and without camp was measured in serial sections. The percentage change in ATPase activity in response to camp was used as a measure of the relative level of camp regulation of actomyosin ATPase activity in the perfused heart. This value was compared with the relative change in peak tension developed during a contraction of the trabecula as a result of superfusion with the reoxygenated coronary venous effluent collected from the same perfused heart. The results shown in Fig. 3 indicate that the regulatory state of actomyosin ATPase in the isolated perfused heart is closely related to the ability of its coronary effluent to alter the contraction of an isolated trabecula: the smaller the response of ATPase activity in the isolated heart to camp, whether measured by percentage change or absolute increment in ATPase activity, the higher the levels of camp up-regulation existing before exposure to 1,uM camp and the greater the effect of the effluent on contraction of the trabecula. In other words, the greater the degree of upregulation of contractile proteins in the perfused heart, the greater is the ability of the coronary effluent to up-regulate the contraction of other cardiac tissue. DISCUSSION Previous studies have shown that the ATPase activity of actomyosin in cardiac cells can be changed by a campdependent reaction (15). In permeabilized heart cells, maximum Ca2+-activated force, as well, is increased, but in this preparation a second reaction or event that is activated by a low concentration of detergent is required (14, 16). Work reported here indicates that the presence of undamaged endocardial endothelial cells is required for camp to alter the ATPase activity of actomyosin. With a damaged endocardial endothelium, camp has no effect on ATPase activity, regardless of the original level of the ATPase activity. The validity of this conclusion depends heavily on the ability of the 1-sec exposure to.5% Triton X-1 to specifically damage the endocardial endothelium without damaging the underlying myocardial cells. Several laboratories have

4 2888 Physiology: McClellan et al. C Sa 8 C % change acto ATPase with camp IntX 5- * m mu.* I a a acto ATPase increment FIG. 3. Change in force vs. change in ATPase: Relation between change in actomyosin ATPase (acto ATPase) activity produced by 1,AM camp with cryostatic sections of isolated perfused hearts and relative change in peak isometric force developed by isolated trabecula when bathed with the coronary venous effluent from perfused hearts. Each point represents the value for a different perfused heart and trabecula. (Upper) Abcissa represents percentage change in ATPase activity. (Lower) Abcissa represents increment in ATPase activity produced by camp. In both graphs the line is the leastsquares linear regression. The correlation coefficient is.93 in Upper and.9 in Lower. In both cases P <.5. already used this procedure with the same results (8-1). There is a decline in peak force and an earlier onset of relaxation without a change in the rate of rise of tension. One laboratory has shown that these changes can be completely reversed by a factor present in the culture medium of cardiac endothelial cells (1), and another laboratory has shown a reversal of a major portion of the change by raising the Ca2+ concentration in the medium to very high levels (8). These observations would seem to indicate that the basic ability of the contractile proteins to generate force has not been damaged by the very brief exposure to the detergent. Ultrastructural studies with transmission electron microscopy show the absence of or damage to essentially all endocardial endothelial cells, but they do not reveal any structural changes in the cardiac myocytes. Intracellular electrical recordings do not show any significant change in the resting potential or the action potential. One would expect that if the cardiac myocytes were directly affected by the detergent, the function of the sarcolemma would be the first and most sensitive indicator of the action. The absence of significant change in the electrical properties of the sarcolemma argues strongly against any direct effect of the detergent on the myocardial cells. In isolated trabeculae, most of the endothelial cells, with the exception of those in the capillaries, are present in the endocardium. The remainder are in the blood vessels within the tissue. The thicker the tissue, the greater the relative proportion of noncapillary endothelial cells present in the tissue (11). For trabeculae of the thicknesses used in this Proc. Natl. Acad. Sci. USA 9 (1993) study, the endocardium contains 75-95% of the total noncapillary population of endothelial cells. Therefore, one can assume the treatment with detergent is affecting most noncapillary endothelial cells in the tissue. The effect of camp on the actomyosin ATPase of trabeculae can be determined by measuring ATPase activity with and without camp in alternate serial sections of each trabecula, and therefore the measurement has a high degree of sensitivity. Damage to the endocardial endothelium clearly totally inhibits the effect of camp. In the trabeculae with intact endothelial cells, the higher the control ATPase activity, the smaller the relative increment in ATPase activity with camp, and at the highest control values of ATPase activity no increase was observed. This apparent saturation could be explained were the differences in control ATPase activity due primarily to different degrees of existing camp upregulation. One explanation for this difference among trabeculae of different thickness could be different levels of function of the endothelial cells. Such an effect of thickness on endothelial cell function in superfused trabeculae has been predicted by a model based on the gradients of ATPase activity found in transverse sections of trabeculae ofdifferent thicknesses (7, 19). If the difference in control ATPase activity was due solely to differences in degree of camp up-regulation, then the values for camp-stimulated ATPase activity should all be the same. If the differences in control ATPase activity were due to damage to the contractile proteins, one would expect the same percentage increase from camp, as all undamaged contractile protein would respond similarly in the different preparations, unless the nature of the damage to the contractile proteins prevented their up-regulation without eliminating their control ATPase activity. An earlier study showed that the coronary venous effluent from an isolated perfused heart altered the contractility of an isolated trabecula superfused with the effluent (7). The conclusion was that endothelial cells were adding cardioregulatory substances to the coronary perfusate, which then altered contractility of the isolated trabecula. Here we have shown that the amplitude of change in the contractility of the trabeculae produced by the coronary venous effluent is closely and directly related to the level of camp regulation of the contractile proteins. In other words, the effluent tends to impose on the trabeculae the same regulatory state of the contractile proteins that existed in the heart contributing the effluent. In view of this correlation, it is reasonable to conclude that the regulatory state of the contractile proteins in the perfused heart is modulated by factors in the fluid perfusing the heart. These factors appear to be produced by the endothelial cells in the small arteries of the heart. Two aspects of the data showing the response of ATPase to camp merit further comment: (i) the much greater apparent likelihood of finding a very low ATPase activity in a trabecula with undamaged endothelial cells and (ii) the decrease in ATPase activity produced by camp in perfused hearts where the coronary effluent causes the largest increase in force. Both of these observations are probably the result of the fact that the endothelial cells release both up- and down-regulating factors (7). The very low ATPase observed in some trabeculae with undamaged endocardial endothelial cells may be from down-regulating factors released by the endothelial cells in the endocardium. These effects would not be seen when the endothelial cells had been damaged. It is not clear at this time why down-regulating factors should be predominant in some preparations. Evidence for a downregulating factor producing a decrease in ATPase activity from camp has already been presented (2). To explain how camp produces a negative effect in a heart with a high level of contractility, we propose that the changes in ATPase activity are from two different camp-sensitive phosphory-

5 Physiology: McClellan et al. lations accompanying, respectively, the up- and downregulating endothelial factors. In the presence of both factors the up-regulating camp reaction occurs preferentially. The down-regulating effect is seen in the presence of both types of endothelial factors when the up-regulating effect is near saturation and the camp concentration is raised to a high level. In summary, it appears that one of the major mechanisms by which heart contractility is controlled is through modulation of a camp-dependent mechanism for regulating the ATPase activity of the cardiac contractile proteins by factors released from endothelial cells. Although the manner in which camp alters the contractile proteins to effect this regulation is unclear, preliminary data (data not shown) suggest that this mechanism may involve phosphorylation of a protein in each of the thick and thin filaments. This work was supported by grants from the National Institutes of Health (HL161 and HL33294) and from the Mary Smith Charitable Trust. D.R. was a Howard Hughes Medical Institute Medical Student Research Training Fellow. 1. Furchgott, R. F. & Zawadski, J. (198) Nature (London) 288, Furchgott, R. F. & Vanhoutte, P. M. (1989) FASEB J. 3, Palmer, R. M., Ferrige, A. G. & Moncada, S. (1987) Nature (London) 327, Yanagisawa, M., Kurihara, H., Tombe, S., Kobayashi, M., Mitsui, Y., Goto, K. & Masaki, T. (1988) Nature (London) 332, Rubanyi, G. & Vanhoutte, P. (1985) J. Physiol. (London) 364, Proc. Natl. Acad. Sci. USA 9 (1993) Yoshizumi, M., Kurihara, H., Sugiyama, T., Takaku, F., Yanagisawa, M., Masaki, T. & Yazaki, Y. (1989) Biochem. Biophys. Res. Commun. 162, Ramaciotti, C., Sharkey, A., McClellan, G. & Winegrad, S. (1992) Proc. Natl. Acad. Sci. USA 89, Brutsaert, D., Meulemans, A. L., Sipido, K. & Sys, S. V. (1988) Circ. Res. 62, Brutsaert, D. (1989) Annu. Rev. Physiol. 51, Smith, J. A., Shah, A. M. & Henderson, H. J. (1991) J. Physiol. (London) 439, McClellan, G., Weisberg, A., Kato, N., Ramaciotti, C., Sharkey, A. & Winegrad, S. (1992) Circ. Res. 7, Kelly, R., Eid, H., Kramer, B., O'Neill, M., Liang, B., Reers, M. & Smith, T. (199) J. Clin. Invest. 86, Ishikawa, T., Yanagisawa, M., Kimura, S., Goto, K. & Masaki, T. (1988) Am. J. Physiol. 255, H97-H McClellan, G. & Winegrad, S. (198) J. Gen. Physiol. 75, Winegrad, S., Weisberg, A., Lin, L.-E. & McClellan, G. (1986) Circ. Res. 58, Winegrad, S., McClellan, G., Horowits, R., Tucker, M., Lin, L.-E. & Weisberg, A. (1983) Fed. Proc. Fed. Am. Soc. Exp. Biol. 42, Kato, N. S., Weisberg, A. & Winegrad, S. (1991) Circ. Res. 68, Neely, J., Liebermeister, H., Bathersby, E. & Morgan, H. (1967) Am. J. Physiol. 212, Lin, L.-E., McClellan, G., Weisberg, A. & Winegrad, S. (1991) J. Physiol. (London) 441, McClellan, G., Weisberg, A. & Winegrad, S. (199) Biophys. J. 57, 336a (abstr.). 21. Ramaciotti, C., McClellan, G., Sharkey, A., Rose, D., Weisberg, A. & Winegrad, S. (1993) Circ. Res., in press.