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Reduced levels of globular and asymmetric forms of acetylcholinesterase in rat left ventricle with pressure overload hypertrophy
Life Sciences,Vol. 55, No. 9, pp. 653-659, 1994 Copyright © 1994 Elsevier Science Ltd Printed in the USA. All tqghtsreserved 0024-32O5/94 $6.00 + .oo
Pergamon 007,4-3205(94)00176-6
REDUCED LEVELS OF GLOBULAR AND ASYMMETRIC FORMS OF ACETYLCHOLINESTERASE IN RAT LEFT VENTRICLE WITH PRESSURE OVERLOAD HYPERTROPHY
Cynthia Nyquist-Battie 1, Kelly E. Hagler I and Sandra Love2
1School of Biological Sciences and 2School of Pharmacy, University of Missouri, Kansas City, MO 64108 (Received in fmal form June 13, 1994)
Summary The aim of the present work was to determine the effect of abdominal aortic stenosis on molecular forms of acetylcholinesterase (ACHE) in rat heart. Pressure-overload, left ventricular hypertrophy was produced in male Sprague-Dawley rats by suprarenal abdominal aortic constriction. After two weeks the relative heart weight was increased over 20 % compared to shamsurgical controls, mostly due to left ventricular enlargement. Aortic constriction reduced AChE activity per wet weight and per unit protein by 2530% in the left ventricle and interventricular septum, but not in the other chambers. However, total AChE activity per chamber was normal in the left ventricle and interventricular septum, but was elevated in the atria. The molecular forms of AChE were separated in linear sucrose gradients and their specific activities were calculated from the resulting percent activities and total AChE activities. This data showed that although aortic constriction had no effect on ratios of the various forms, it did reduce the specific activities of globular and asymmetric forms in the left ventricle and interventricular septum. The reduced AChE activity suggests that slower rates of ACh hydrolysis occur in the left ventricle in pressure-overload hypertrophy. Key Words: acetylcholinesterase, heart, parasympathetic nervous system, cardiac hypertrophy
Parasympathetic and sympathetic nerve fibers are present in all chambers of mammalian heart (1). In addition to direct effects on heart, the parasympathetic system is antagonistic to the sympathetic system, acting both presynaptically by inhibiting norepinephrine (NE) release, and postsynaptically by inhibiting inotropic and chronotropic effects of NE on cardiac effector ceils (3). Cardiac hypertrophy alters both the sympathetic and parasympathetic effects on heart function (4, 5). Modified autonomic innervation, may be relevant for both the development of cardiac hypertrophy and the progression of cardiac hypertrophy to failure (6, 7, 8). For these reasons it is important to understand how cardiac hypertrophy alters autonomic neurotransmission. The effects of cardiac hypertrophy on neurotransmitter levels and enzymes responsible for transmitter synthesis have been Corresponding author: Cynthia Nyquist-Battie, School of Biological Sciences, Department of Cell Biology & Biophysics, University of Missouri-Kansas City, 2411 Holmes, Kansas City, MO 64108
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investigated (4, 5, 9-17). These previous studies showed that the nature of change in autonomic neural markers varies as a function of the cause and stage of cardiac hypertrophy (4). Cardiac hypertrophy does not always causes a simple dilution of neural markers, such as choline acetyltransferase (CHAT), due to the dispersion of nerves by the enlarging myocytes, but can produce their depletion (4) in part due to the loss of nerve fibers (18). In other studies, compensatory increases in CHAT, tyrosine hydroxylase, dopamine i3 hydroxylase, and catecholamine stores and turnover have been observed during hypertrophy, especially when hypertrophy develops gradually and is well-compensated (6, 10-12). These changes help to maintain normal autonomic function during cardiac enlargement. Ventricular hypertrophy may also alter cardiac function by reducing or increasing neurotransmitter inactivation. For example, cardiac hypertrophy reduces the ability of cardiac adrenergic nerves to take up monoamines, which is the main mechanism for NE inactivation (4, 19). Little is known regarding the effect of cardiac hypertrophy on acetylcholine (ACh) hydrolysis. In heart, ACh is not inactivated after release by a re-uptake system, but is inactivated by diffusion and hydrolysis (20). The efficiency of ACh hydrolysis is one factor that determines the duration of parasympathetic activation of heart (20). Acetylcholinesterase is the main enzyme responsible for this hydrolysis (21). Previously, globular and asymmetric forms of cardiac AChE have been identified (22-24). Globular (G) AChE forms are comprised of molecules which lack collagen tails, while asymmetric (A) AChE molecular forms are those that contain catalytic subunits covalently bound to a collagen-helical peptide, anchoring these forms to components of the extracellular matrix (25). In rat heart, all chambers contain monomeric (GO, dimeric (G2) and tetrameric (G4) globular forms of AChE (23-24). A12 ACHE, containing twelve catalytic subunits, was identified as the predominant A-form in adult rat heart (23-24). In the present study, the effect of aortic banding, which reduces the presynaptic cholinergic marker ChAT (13), on the activity of AChE molecular forms was determined. Globular and asymmetric forms of ACHE, including those that are externalized and thus physiologically active (17), were reduced per unit tissue in the hypertrophied left ventricle, suggesting that there may be a reduced ability to inactivate released ACh during pressure-overload cardiac hypertrophy. Methods Animal care and use conformed to the guidelines established by NIH and all protocols were approved by our institution's Animal Use Committee. Male Sprague - Dawley rats were obtained from Sasco, Inc. (Omaha, NE) and maintained with free access to Purina laboratory chow and water. Animals were fasted for 12 - 14 hours prior to surgery. Surgical induction of cardiac hypertrophy in rats, weighing approximately 220-230 g, was done under sodium pentobarbital and sterile conditions. To produce a constriction of approximately 0.8 mm, the abdominal aorta was isolated just above its superior mesenteric branch and a blunted 20 gauge needle was placed beside the aorta. Using a 3-0 silk ligature, the artery was tied against the needle, and then the needle was quickly slipped out. Shamoperated rats were subjected to all of the above steps except for aortic constriction. Each rat was administered penicillin, I.M. to prevent infection. Animals in both groups were sacrificed 14 days after surgery. Unless specified, all chemicals were obtained from Sigma Chemical Co. (St. Louis, MO) or Fisher Scientific (Springfield, N J). During sodium pentobarbital anesthesia, hearts were perfused through the left ventricle with 30 ml of phosphate-buffered saline, pH 7.4 to remove AChE contained in blood (22). After removal, the combined atria, the right and left ventricles and interventricular septum were separately homogenized at a 1/15 (w/v) dilution in 50 mM Tris-
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HC1, pH 7.3 with 1% (v/v) Triton X-100, 1 mM NaCI and 5 mM EDTA, as described previously (22). Homogenates were centrifuged at 24,000 g for 30 min at 4°C. The supernatant was assayed for AChE activity and subjected to velocity sedimentation analysis. Supernatant AChE activity was assayed as described previously (22), with incubation of triplicate samples in a 400/~1 final reaction volume, containing 50 mM potassium phosphate, pH 6.8 with 0.1% (v/v) Triton X-100 and 0.1 mM ISO-OMPA (tetraisopropyl pyrophosphoramide) to inhibit butyrylcholinesterase. After 20 min pre-incubation in the presence of ISO-OMPA, reactions were initiated by the addition of [3H]acetylcholine iodide (100 mCI/mmol, DuPont NEN Co., Boston, MA) at a final concentration of 0.1 mM. Assays conducted at 37°C, were terminated after 30 min by the addition of 600 #1 of 50 mM glycine, pH 1.25 with 1 M NaCI. The [3H]acetate product was quantified by liquid scintillation counting. For analysis of AChE molecular forms, aliquots (400/zl) of the supernatants were layered on linear 5-20 % (w/v) sucrose gradients containing the AChE homogenization buffer. Sedimentation was performed in a SW 40 rotor at 230,000 gmaxtO a ~o2 value of 1.09 x 10 ~2 rad2/s or for approximately 22 h at 4°C (24). Forty 300/xl fractions were collected from the top of each gradient. The enzymatic activity of each fraction was determined after preincubation for 20 min with 250/~1 of the 50 mM potassium phosphate assay buffer containing ISO-OMPA and Triton X-100. Reactions were initiated by the addition of [3H]acetylcholine iodide at a final concentration of 0.1 mM. Assays, performed at 37°C, were terminated after 90 min and were counted as per homogenate assays. The sedimentation coefficients of AChE molecular forms were estimated by comparison with those of bovine serum albumin (4.4S) and catalase (11.3 S), added to each gradient prior to centrifugation. The relative proportion of each AChE molecular form was calculated by comparing the enzymatic activities under each peak with that under the entire sedimentation profile (24). The activity of each molecular form was derived from supernatant AChE activity and the relative proportional data. Protein levels were determined by the method of Lowry et al. (26), using bovine serum albumin as the standard. Statistical significance was determined by unpaired, two-tailed t-tests. Values are given as means + standard error of the mean. Results Aortic constriction of two weeks duration had no effect on body weight, as shown in Table 1. In contrast, total heart weight and relative heart weight (heart weight per body weight x 103) were increased over 20%. Increased heart weight was the result of significant left ventricular enlargement, as well as increased atrial and interventricular septal weights. Aortic constriction significantly (p < 0.01) reduced AChE activity (per wet weight and per mg protein) in the left ventricle and interventricular septum. For example, AChE activity in left ventricle was reduced 31%: 178 + 2 (control) versus 122 -t- 8 (aortic banded) pmol/min/mg wet weight. In the interventricular septum, the values were 284 _ 14 (control) and 209 + 3 (aortic-banded), a reduction of 26 %. In the other chambers, AChE activity was not altered by aortic banding. In the right ventricle, the values were 219 + 7 (control) and 226 -I- 5 (aortic banded). In the atria, the values were 390 + 10 (control) and 397 + 9 (aortic-banded). Total AChE activity per chamber (nanomoles/chamber) was normal in the left ventricle after aortic banding (sham: 92.9 + 3; aortic banded: 90.3 + 4). Similarly, the right ventricle and interventricular septum of aortic-banded rats exhibited normal levels of AChE activity per chamber (data not shown). The atria of aortic-banded rats showed elevated chamber activity for AChE (sham: 53.8 + 2; aortic-banded: 73 + 5, p < 0.05).
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The percent activities, obtained from the sucrose gradient separation of the various AChE molecular forms, were not affected by aortic-banding (data not shown). The specific activities of AChE molecular forms, isolated from all chambers of aortic- and sham-banded rats, were calculated from the percent activities and total AChE activities as described in "Experimental Procedures" and the results are shown in Table 2. In the left ventricle and interventricular septum of aortic banded rats, globular and asymmetric AChE forms were reduced between 30-35 % compared to controls. In atria and right ventricle, no reductions in AChE molecular form specific activities were evident after aortic constriction. TABLE I Effect of Abdominal Aortic Constriction of Two Weeks Duration on Body, Heart and Relative Heart Weight. Control Body weight (g) 312 ___ 14 Heart weight (g) 1.03 5:0.05 Left ventricle weight (g) 0.56 5:0.02 Atria weight (g) 0.13 5:0.013 0.017" Right ventricle weight (g) 0.16 + 0.019 Intraventricular Septum weight (g) 0.14 ± 0.02 Relative heart weight 3.33 5:0.14
Aortic-banded 311 5 : 2 6 1.42 ± 0.05** 0.75 5: 0.02* 0.1 9 -t021 ± ~019 0.25 ± 0.01"* 4.16 ± 0.06**
Values are means + SEM for n=5-6 animals. Animals were killed two weeks after surgery. Statistical significance was determined by unpaired, two-tailed t-tests with * =p < 0.05 and • * = p < 0.01. Relative heart weight represents heart weight/body weight x 103. Discussion The present study extends our knowledge of parasympathetic control during ventricular hypertrophy by showing that left ventricular enlargement, produced by aortic constriction above both renal arteries, causes a loss of globular and asymmetric AChE activities per unit weight but not per chamber. The decreased AChE activity resulted from similar reductions in the activities of both globular and asymmetric forms. In contrast to skeletal muscle where most AChE is produced by muscle fibers (25), cardiac nerve endings contain a significant proportion of total AChE (23, 27). In cardiac nerve endings, G 4 AChE is the predominate form, G~ and G2 AChE are secondary forms and Aa2 is a minor form (23). In contrast to nerve endings, cardiomyocytes contain less (34 AChE relative to the other G-forms (23). Additionally, AI~ AChE is a larger component of the AChE pool in cardiomyocytes (27). Since AChE is associated with both cardiomyocytes and nerve endings, one can explain the loss of left ventricular AChE activity per unit weight in conjunction with normal AChE activity per chamber in several ways. A lack of a compensatory increase in AChE production by the enlarged cardiac myocytes may have occurred. The significant reduction of A12 ACHE, a form associated with cardiomyocytes but a minor component in nerve endings, would suggest that this may be the case. Reduced levels of AChE mRNA leading to reduced AChE production may be present in the enlarged myocytes similar to the reduction in Ca-2+ATPase mRNA previously reported in left ventricular hypertrophy (28). The reduced AChE specific activity in the left ventricle may
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TABLE II Effect of Abdominal Aortic-Constriction of Two Weeks Duration on Acetylcholinesterase Molecular Forms Activity.
Left Ventricle Control Aortic-banded Intraventricular Septum Control Aortic-banded Right Ventricle Control Aortic-banded Atria Control Aortic-banded
GI + G2
G4
AI2
79 + 1 56 + 3**
85 + 1 57 + 4**
14 + 1 9 + 1"*
110 + 3 83 + 5*
168 + 1 99 + 3**
19 ___ 2 13 ___ 1"
82 + 3 78 + 2
127 ___ 4 118 + 3
17 ___ 2 15 -1- 1
150 + 11 164 + 9
221 + 17 211 + 13
27 + 2 26 + 2
Values are means + SEM, n=5-6 and represent AChE activities expressed as pmol/min/mg wet weight. Statistical significance between treated and control animals was determined by unpaired, two-tailed t-test (* = p < .03; ** = p < .001). Since G t and G2 AChE activities were not well separated during density gradient centrifugation, their activities were pooled for calculations. G1, G2 and G4 refer to globular monomeric, dimeric and tetrameric ACHE, respectively. A~2 refers to the asymmetric form of AChE with a collagen-like tail and twelve catalytic subunits. also be caused by cardiac myocyte enlargement diluting the number of AChE-containing nerve fibers per unit volume. This hypothesis is supported by the earlier finding of reduced ChAT activity, a marker of cholinergic nerve fibers, in left ventricle during hemodynamic overload (13). In contrast to the left ventricle, the atria, which also exhibited a significant weight increase, were able to maintain per mg activity by increasing total AChE activity per chamber. In skeletal muscle, A12 AChE is the physiologically active A-form and G4 AChE is the major physiologically active G- form (25). We have shown that these forms are the predominate extracellular forms in cardiac muscle, and therefore consider them to be the active forms involved in ACh hydrolysis (17). Interestingly, these physiologically active forms of AChE were reduced in our study. Hypothetically, decreased local levels of extracellular AChE would cause a reduced rate of ACh hydrolysis effectively increasing parasympathetic stimulation. In cardiac hypertrophy, decreased ACh hydrolysis may offset the effects of reduced ACh production, suggested by lowered ChAT activity (13), on parasympathetic tone. The normalization of reduced parasympathetic tone would be beneficial to cardiac function during hypertrophy, because reduced parasympathetic tone is associated with increased mortality under certain conditions (29).
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Acknowled~,ements This research was supported by a grant from the National Institutes of Health (HL41389) and the Lettie B. MclIvain Frederic Fund. The animal research protocol adhered to the "Guide for the Care and Use of Laboratory Animals" prepared by the Committee on Care and Use of Laboratory Animals of the Institute of Laboratory Animal Resources, National Research Council (NIH publication 86-23, revised 1985). References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24.
C.B. HIGGENS, S.F. VATNER and E. BRAUNWALD Pharmacol. Rev. 25 119155 (1973). W.C. RANDALL Neural Regulation of Heart, W.C. Randall (Ed.), 13-41, Oxford Univ. Press, New York (1977). M.N. LEVY Fed. Proc. 43 2598-2602 (1984). J.F. SPANN Functional Changes in Pathologic Hypertrophy, R. Zak (Ed.) 421-466, Raven Press, New York (1984). P.G. SCHMID, D.D. LUND and R. ROSKOSKI, JR. Disturbances in Neurogenic Control of the Circulation, 33-48, Am. Physiol. Society, Bethesda, MD (1981). P.K. GANGULY, S.L. LEE, R.E. BEAMISH and N.S. DHALLA Am. Heart. J. 118 520-525 (1989). H.E. MORGAN and K.M. BAKER Circulation 83 13-25 (1991). C.V. LEIER, P.F. BINKLEY and R.J. CODY Circulation 82-I 68-76 (1990). L.J. HELLER and J.R. PROHASKA J. Mol. Cell. Cardioi. 16 987-993 (1984). K. LINDPAINTNER, D.D. LUND and P.G. SCHMID Circ. Res. 61 55-62 (1987a). K. LINDPAINTNER, D.D. LUND and P.G. SCHMID J. Cardiovasc. Pharmac. 10 (Suppl. 12), $211-$220 (1987b). D.D. LUND, P.G. SCHMID, J.A. DAVIS, F.M. SHRABI and R. ROSKOSKI Life Sci. 32 2257-2264 (1983). R. ROSKOSKI, JR., P.G. SCHMID, H.E. MAYER and F.M. ABBOUD Circ. Res. 36 547-552 (1975). P.G. SCHMID, D.D. LUND, J.A. DAVID, C.A. WHITEIS, R.K. BHATNAGAR and R. ROSKOSKI, JR. Am. J. Physiol. 243 H175-H180 (1982). M.J. SOLE, A.B. KAMBLE and M.N. HUSSAIN Circ. Res. 41 814-817 ( 1 9 ) . H. TSUBOI, O.OSAMU, K. OGAWA, I. TAKAYUK1, H. HASHIMOTO, K. OKUMURA and T. SATAKE J. Hypertens. 5 323-330 (1987). C.NYQUIST-BATTIE, K.E. HAGLER, L. WlNDBERG and J.V. THOTTASSERY Neurochem. Int. 22 143-151 (1993). F. BORCHARD Normal Path. Anat. 33 1-68 (1978). J.E. FISCHER, W.D. HORST and I.J. KOPIN Nature Lond. 207 951-953. (1965). R. LINDMAR, K. LOFFELHOLZ and W. WEIDE Naunyn-Schmeideberg's Arch. Pharmacol. 318 295-300 (1982). R. STANLEY, J. CONATSER and W.D. DETTBARN Biochem. Pharmacol. 27 2409-2411 (1978). C. NYQUIST-BATIIE, C. HODGES-SAVOLA and H.L. FERNANDEZ J. Molec. Cell. Cardio. 1___~9935-943 (1987). C. NYQUIST-BATI'IE, R.J. DOWELL and H.L. FERNANDEZ J. Molec. Cell. Cardiol. 21 987-994 (1989). C. NYQUIST-BATTIE and K. TRANS-SALTZMAN Circ. Res. 65 55-62 (1989).
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25. 26. 27. 28. 29.
AChE in Pressure Overload Hypertrophy
J. MASSOULIE and S. BON. Ann. Rev. Neurosci. 5 57-106 (1982). O.H. LOWRY, N.J. ROSEBROUGH, A.L. FARR and R. RANDALL J. Biol. Chem. 193 265-275 (1951). C. NYQUIST-BA'Iq'IE, K.E. HAGLER and W.R. MILLINGTON J. Mol. Cell. Cardiol. 25 1111-1118 (1993). A.M. FELDMAN, E.O. WEINBERG, P.E. RAY and B.H. LONELL Circ. Res. 73 184-192 (1993). J.M. WHARTON,R.E. COLEMAN and H.C. STRAUSS Trends Cardiovasc. Med. 2 65-71 (1992).
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Pergamon 007,4-3205(94)00176-6
REDUCED LEVELS OF GLOBULAR AND ASYMMETRIC FORMS OF ACETYLCHOLINESTERASE IN RAT LEFT VENTRICLE WITH PRESSURE OVERLOAD HYPERTROPHY
Cynthia Nyquist-Battie 1, Kelly E. Hagler I and Sandra Love2
1School of Biological Sciences and 2School of Pharmacy, University of Missouri, Kansas City, MO 64108 (Received in fmal form June 13, 1994)
Summary The aim of the present work was to determine the effect of abdominal aortic stenosis on molecular forms of acetylcholinesterase (ACHE) in rat heart. Pressure-overload, left ventricular hypertrophy was produced in male Sprague-Dawley rats by suprarenal abdominal aortic constriction. After two weeks the relative heart weight was increased over 20 % compared to shamsurgical controls, mostly due to left ventricular enlargement. Aortic constriction reduced AChE activity per wet weight and per unit protein by 2530% in the left ventricle and interventricular septum, but not in the other chambers. However, total AChE activity per chamber was normal in the left ventricle and interventricular septum, but was elevated in the atria. The molecular forms of AChE were separated in linear sucrose gradients and their specific activities were calculated from the resulting percent activities and total AChE activities. This data showed that although aortic constriction had no effect on ratios of the various forms, it did reduce the specific activities of globular and asymmetric forms in the left ventricle and interventricular septum. The reduced AChE activity suggests that slower rates of ACh hydrolysis occur in the left ventricle in pressure-overload hypertrophy. Key Words: acetylcholinesterase, heart, parasympathetic nervous system, cardiac hypertrophy
Parasympathetic and sympathetic nerve fibers are present in all chambers of mammalian heart (1). In addition to direct effects on heart, the parasympathetic system is antagonistic to the sympathetic system, acting both presynaptically by inhibiting norepinephrine (NE) release, and postsynaptically by inhibiting inotropic and chronotropic effects of NE on cardiac effector ceils (3). Cardiac hypertrophy alters both the sympathetic and parasympathetic effects on heart function (4, 5). Modified autonomic innervation, may be relevant for both the development of cardiac hypertrophy and the progression of cardiac hypertrophy to failure (6, 7, 8). For these reasons it is important to understand how cardiac hypertrophy alters autonomic neurotransmission. The effects of cardiac hypertrophy on neurotransmitter levels and enzymes responsible for transmitter synthesis have been Corresponding author: Cynthia Nyquist-Battie, School of Biological Sciences, Department of Cell Biology & Biophysics, University of Missouri-Kansas City, 2411 Holmes, Kansas City, MO 64108
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investigated (4, 5, 9-17). These previous studies showed that the nature of change in autonomic neural markers varies as a function of the cause and stage of cardiac hypertrophy (4). Cardiac hypertrophy does not always causes a simple dilution of neural markers, such as choline acetyltransferase (CHAT), due to the dispersion of nerves by the enlarging myocytes, but can produce their depletion (4) in part due to the loss of nerve fibers (18). In other studies, compensatory increases in CHAT, tyrosine hydroxylase, dopamine i3 hydroxylase, and catecholamine stores and turnover have been observed during hypertrophy, especially when hypertrophy develops gradually and is well-compensated (6, 10-12). These changes help to maintain normal autonomic function during cardiac enlargement. Ventricular hypertrophy may also alter cardiac function by reducing or increasing neurotransmitter inactivation. For example, cardiac hypertrophy reduces the ability of cardiac adrenergic nerves to take up monoamines, which is the main mechanism for NE inactivation (4, 19). Little is known regarding the effect of cardiac hypertrophy on acetylcholine (ACh) hydrolysis. In heart, ACh is not inactivated after release by a re-uptake system, but is inactivated by diffusion and hydrolysis (20). The efficiency of ACh hydrolysis is one factor that determines the duration of parasympathetic activation of heart (20). Acetylcholinesterase is the main enzyme responsible for this hydrolysis (21). Previously, globular and asymmetric forms of cardiac AChE have been identified (22-24). Globular (G) AChE forms are comprised of molecules which lack collagen tails, while asymmetric (A) AChE molecular forms are those that contain catalytic subunits covalently bound to a collagen-helical peptide, anchoring these forms to components of the extracellular matrix (25). In rat heart, all chambers contain monomeric (GO, dimeric (G2) and tetrameric (G4) globular forms of AChE (23-24). A12 ACHE, containing twelve catalytic subunits, was identified as the predominant A-form in adult rat heart (23-24). In the present study, the effect of aortic banding, which reduces the presynaptic cholinergic marker ChAT (13), on the activity of AChE molecular forms was determined. Globular and asymmetric forms of ACHE, including those that are externalized and thus physiologically active (17), were reduced per unit tissue in the hypertrophied left ventricle, suggesting that there may be a reduced ability to inactivate released ACh during pressure-overload cardiac hypertrophy. Methods Animal care and use conformed to the guidelines established by NIH and all protocols were approved by our institution's Animal Use Committee. Male Sprague - Dawley rats were obtained from Sasco, Inc. (Omaha, NE) and maintained with free access to Purina laboratory chow and water. Animals were fasted for 12 - 14 hours prior to surgery. Surgical induction of cardiac hypertrophy in rats, weighing approximately 220-230 g, was done under sodium pentobarbital and sterile conditions. To produce a constriction of approximately 0.8 mm, the abdominal aorta was isolated just above its superior mesenteric branch and a blunted 20 gauge needle was placed beside the aorta. Using a 3-0 silk ligature, the artery was tied against the needle, and then the needle was quickly slipped out. Shamoperated rats were subjected to all of the above steps except for aortic constriction. Each rat was administered penicillin, I.M. to prevent infection. Animals in both groups were sacrificed 14 days after surgery. Unless specified, all chemicals were obtained from Sigma Chemical Co. (St. Louis, MO) or Fisher Scientific (Springfield, N J). During sodium pentobarbital anesthesia, hearts were perfused through the left ventricle with 30 ml of phosphate-buffered saline, pH 7.4 to remove AChE contained in blood (22). After removal, the combined atria, the right and left ventricles and interventricular septum were separately homogenized at a 1/15 (w/v) dilution in 50 mM Tris-
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HC1, pH 7.3 with 1% (v/v) Triton X-100, 1 mM NaCI and 5 mM EDTA, as described previously (22). Homogenates were centrifuged at 24,000 g for 30 min at 4°C. The supernatant was assayed for AChE activity and subjected to velocity sedimentation analysis. Supernatant AChE activity was assayed as described previously (22), with incubation of triplicate samples in a 400/~1 final reaction volume, containing 50 mM potassium phosphate, pH 6.8 with 0.1% (v/v) Triton X-100 and 0.1 mM ISO-OMPA (tetraisopropyl pyrophosphoramide) to inhibit butyrylcholinesterase. After 20 min pre-incubation in the presence of ISO-OMPA, reactions were initiated by the addition of [3H]acetylcholine iodide (100 mCI/mmol, DuPont NEN Co., Boston, MA) at a final concentration of 0.1 mM. Assays conducted at 37°C, were terminated after 30 min by the addition of 600 #1 of 50 mM glycine, pH 1.25 with 1 M NaCI. The [3H]acetate product was quantified by liquid scintillation counting. For analysis of AChE molecular forms, aliquots (400/zl) of the supernatants were layered on linear 5-20 % (w/v) sucrose gradients containing the AChE homogenization buffer. Sedimentation was performed in a SW 40 rotor at 230,000 gmaxtO a ~o2 value of 1.09 x 10 ~2 rad2/s or for approximately 22 h at 4°C (24). Forty 300/xl fractions were collected from the top of each gradient. The enzymatic activity of each fraction was determined after preincubation for 20 min with 250/~1 of the 50 mM potassium phosphate assay buffer containing ISO-OMPA and Triton X-100. Reactions were initiated by the addition of [3H]acetylcholine iodide at a final concentration of 0.1 mM. Assays, performed at 37°C, were terminated after 90 min and were counted as per homogenate assays. The sedimentation coefficients of AChE molecular forms were estimated by comparison with those of bovine serum albumin (4.4S) and catalase (11.3 S), added to each gradient prior to centrifugation. The relative proportion of each AChE molecular form was calculated by comparing the enzymatic activities under each peak with that under the entire sedimentation profile (24). The activity of each molecular form was derived from supernatant AChE activity and the relative proportional data. Protein levels were determined by the method of Lowry et al. (26), using bovine serum albumin as the standard. Statistical significance was determined by unpaired, two-tailed t-tests. Values are given as means + standard error of the mean. Results Aortic constriction of two weeks duration had no effect on body weight, as shown in Table 1. In contrast, total heart weight and relative heart weight (heart weight per body weight x 103) were increased over 20%. Increased heart weight was the result of significant left ventricular enlargement, as well as increased atrial and interventricular septal weights. Aortic constriction significantly (p < 0.01) reduced AChE activity (per wet weight and per mg protein) in the left ventricle and interventricular septum. For example, AChE activity in left ventricle was reduced 31%: 178 + 2 (control) versus 122 -t- 8 (aortic banded) pmol/min/mg wet weight. In the interventricular septum, the values were 284 _ 14 (control) and 209 + 3 (aortic-banded), a reduction of 26 %. In the other chambers, AChE activity was not altered by aortic banding. In the right ventricle, the values were 219 + 7 (control) and 226 -I- 5 (aortic banded). In the atria, the values were 390 + 10 (control) and 397 + 9 (aortic-banded). Total AChE activity per chamber (nanomoles/chamber) was normal in the left ventricle after aortic banding (sham: 92.9 + 3; aortic banded: 90.3 + 4). Similarly, the right ventricle and interventricular septum of aortic-banded rats exhibited normal levels of AChE activity per chamber (data not shown). The atria of aortic-banded rats showed elevated chamber activity for AChE (sham: 53.8 + 2; aortic-banded: 73 + 5, p < 0.05).
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The percent activities, obtained from the sucrose gradient separation of the various AChE molecular forms, were not affected by aortic-banding (data not shown). The specific activities of AChE molecular forms, isolated from all chambers of aortic- and sham-banded rats, were calculated from the percent activities and total AChE activities as described in "Experimental Procedures" and the results are shown in Table 2. In the left ventricle and interventricular septum of aortic banded rats, globular and asymmetric AChE forms were reduced between 30-35 % compared to controls. In atria and right ventricle, no reductions in AChE molecular form specific activities were evident after aortic constriction. TABLE I Effect of Abdominal Aortic Constriction of Two Weeks Duration on Body, Heart and Relative Heart Weight. Control Body weight (g) 312 ___ 14 Heart weight (g) 1.03 5:0.05 Left ventricle weight (g) 0.56 5:0.02 Atria weight (g) 0.13 5:0.013 0.017" Right ventricle weight (g) 0.16 + 0.019 Intraventricular Septum weight (g) 0.14 ± 0.02 Relative heart weight 3.33 5:0.14
Aortic-banded 311 5 : 2 6 1.42 ± 0.05** 0.75 5: 0.02* 0.1 9 -t021 ± ~019 0.25 ± 0.01"* 4.16 ± 0.06**
Values are means + SEM for n=5-6 animals. Animals were killed two weeks after surgery. Statistical significance was determined by unpaired, two-tailed t-tests with * =p < 0.05 and • * = p < 0.01. Relative heart weight represents heart weight/body weight x 103. Discussion The present study extends our knowledge of parasympathetic control during ventricular hypertrophy by showing that left ventricular enlargement, produced by aortic constriction above both renal arteries, causes a loss of globular and asymmetric AChE activities per unit weight but not per chamber. The decreased AChE activity resulted from similar reductions in the activities of both globular and asymmetric forms. In contrast to skeletal muscle where most AChE is produced by muscle fibers (25), cardiac nerve endings contain a significant proportion of total AChE (23, 27). In cardiac nerve endings, G 4 AChE is the predominate form, G~ and G2 AChE are secondary forms and Aa2 is a minor form (23). In contrast to nerve endings, cardiomyocytes contain less (34 AChE relative to the other G-forms (23). Additionally, AI~ AChE is a larger component of the AChE pool in cardiomyocytes (27). Since AChE is associated with both cardiomyocytes and nerve endings, one can explain the loss of left ventricular AChE activity per unit weight in conjunction with normal AChE activity per chamber in several ways. A lack of a compensatory increase in AChE production by the enlarged cardiac myocytes may have occurred. The significant reduction of A12 ACHE, a form associated with cardiomyocytes but a minor component in nerve endings, would suggest that this may be the case. Reduced levels of AChE mRNA leading to reduced AChE production may be present in the enlarged myocytes similar to the reduction in Ca-2+ATPase mRNA previously reported in left ventricular hypertrophy (28). The reduced AChE specific activity in the left ventricle may
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TABLE II Effect of Abdominal Aortic-Constriction of Two Weeks Duration on Acetylcholinesterase Molecular Forms Activity.
Left Ventricle Control Aortic-banded Intraventricular Septum Control Aortic-banded Right Ventricle Control Aortic-banded Atria Control Aortic-banded
GI + G2
G4
AI2
79 + 1 56 + 3**
85 + 1 57 + 4**
14 + 1 9 + 1"*
110 + 3 83 + 5*
168 + 1 99 + 3**
19 ___ 2 13 ___ 1"
82 + 3 78 + 2
127 ___ 4 118 + 3
17 ___ 2 15 -1- 1
150 + 11 164 + 9
221 + 17 211 + 13
27 + 2 26 + 2
Values are means + SEM, n=5-6 and represent AChE activities expressed as pmol/min/mg wet weight. Statistical significance between treated and control animals was determined by unpaired, two-tailed t-test (* = p < .03; ** = p < .001). Since G t and G2 AChE activities were not well separated during density gradient centrifugation, their activities were pooled for calculations. G1, G2 and G4 refer to globular monomeric, dimeric and tetrameric ACHE, respectively. A~2 refers to the asymmetric form of AChE with a collagen-like tail and twelve catalytic subunits. also be caused by cardiac myocyte enlargement diluting the number of AChE-containing nerve fibers per unit volume. This hypothesis is supported by the earlier finding of reduced ChAT activity, a marker of cholinergic nerve fibers, in left ventricle during hemodynamic overload (13). In contrast to the left ventricle, the atria, which also exhibited a significant weight increase, were able to maintain per mg activity by increasing total AChE activity per chamber. In skeletal muscle, A12 AChE is the physiologically active A-form and G4 AChE is the major physiologically active G- form (25). We have shown that these forms are the predominate extracellular forms in cardiac muscle, and therefore consider them to be the active forms involved in ACh hydrolysis (17). Interestingly, these physiologically active forms of AChE were reduced in our study. Hypothetically, decreased local levels of extracellular AChE would cause a reduced rate of ACh hydrolysis effectively increasing parasympathetic stimulation. In cardiac hypertrophy, decreased ACh hydrolysis may offset the effects of reduced ACh production, suggested by lowered ChAT activity (13), on parasympathetic tone. The normalization of reduced parasympathetic tone would be beneficial to cardiac function during hypertrophy, because reduced parasympathetic tone is associated with increased mortality under certain conditions (29).
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Acknowled~,ements This research was supported by a grant from the National Institutes of Health (HL41389) and the Lettie B. MclIvain Frederic Fund. The animal research protocol adhered to the "Guide for the Care and Use of Laboratory Animals" prepared by the Committee on Care and Use of Laboratory Animals of the Institute of Laboratory Animal Resources, National Research Council (NIH publication 86-23, revised 1985). References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24.
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