681
Journal of Physiology (1992), 456, pp. 681-687 With 2 figures Printed in Great Britain
ROLE OF BASAL RELEASE OF NITRIC OXIDE ON CORONARY FLOW AND MECHANICAL PERFORMANCE OF THE ISOLATED RAT HEART
By M. AMRANI, J. O'SHEA, N. J. ALLEN, S. E. HARDING, J. JAYAKUMAR, J. R. PEPPER, S. MONCADA* AND M. H. YACOUB From the Departments of Cardiothoracic Surgery and Cardiac Medicine, National Heart and Lung Institute, Dovehouse Street, London SW3 6LY and the *Wellcome Research Laboratories, Beckenham, Kent
(Received 9 January 1992) SUMMARY
1. The role of nitric oxide (NO) in the regulation of coronary flow and mechanical function was studied in isolated working rat hearts. 2. The inhibition of basal release of NO by L-N"--monomethylarginine (L-NMMA; 500 AvM), a specific inhibitor of its synthesis, induced a reduction of coronary flow to 3941 % (± 3 4) of its basal level. 3. Associated with this was a drop of cardiac output to 58-10 % (± 542) of control values. 4. This mechanical dysfunction, which was more pronounced in hypertrophied hearts, appears to be related to ischaemia, as it was prevented by simultaneously administered glyceryl trinitrate. Furthermore, L-NMMA did not alter the contractile activity of isolated cardiac myocytes, thereby excluding a direct toxic effect. 5. These findings provide evidence that NO plays a crucial role in the maintenance of basal coronary flow and appears to be essential for sustaining mechanical activity.
INTRODUCTION
The endothelium plays a major role in regulating coronary vasomotor tone through the production and metabolism of various vasodilating and vasoconstricting agents (Vanhoutte & Houston, 1985). Since the discovery of endothelium-derived relaxing factor (EDRF) (Furchgott & Zawadzki, 1980), it became evident that endothelial cells can strongly modulate the responsiveness of the vascular smooth muscle. Recent studies showed that EDRF and nitric oxide (NO) have similar properties and that NO is released from vascular endothelial cells, thus suggesting that EDRF is NO (Palmer, Ferrige & Moncada, 1987). Nitric oxide is formed from the terminal guanidino nitrogen of the amino acid L-arginine, (Palmer, Ashton & Moncada, 1988a). An arginine analogue i.e. L-N-monomethylarginine (L-NMMA) has been demonstrated specifically to inhibit the formation of NO from L-arginine, resulting in attenuation of endothelium-dependent relaxation (Palmer, Rees, Ashton & Moncada, MS 1026
M. AMRANI AND OTHERS 1988b). This effectof L-NMMA can be reversed by L-arginine. In addition, NO has been shown to be released continuously under basal conditions in isolated hearts (Amezcua, Dusting, Palmer & Moncada, 1988; Kelm & Schrader, 1988; Amezcua, Palmer, de Souza & Moncada, 1989). This endogenous secretion is quantitatively sufficient to influence the coronary tone and its inhibition by L-NMMA increases coronary perfusion pressure in a dose-dependent manner (Kelm & Schrader, 1988; Amezcua et al. 1989). A reduced release of NO may be associated with some pathological conditions (Chester, O'Neil, Moncada, Tadjkarimi & Yacoub, 1990). The extent of this NO-induced vasodilator tone and its physiological importance in maintaining adequate flow to sustain mechanical function in normal and hypertrophied heart has not been addressed. The present study was designed to assess the effect of a maximal inhibition of NO on cardiac mechanical performance in the 682
normal and hypertrophic isolated rat heart. METHODS
Langendorff heart preparation Male Wistar WKY rats and their spontaneously hypertensive (SHR) strain with hypertrophied heart (Olac Harlan Ltd, Bicester, Oxon), weighing between 300 and 350 g, were used in all experiments. Throughout the procedure animals were anaesthetized with halothane mixed with 95 % 02-5% CO2. The groin was dissected, the femoral vein exposed and heparin (200 IU) was then excised and injected. One minute later, a bilateral thoracotomy was performed. The heart was and Langendorff into cold fluid. The aorta cannulated was (10 'C) perfusion immediately placed the left atrium was perfusion was initiated for a 3 min wash-out period. During this time cannulated and the pulmonary artery excised to ensure the ejection of coronary effluent. In some experiments the heart was converted to a working mode preparation for 20 or 60 min. The isolated working rat heart model used in this study has already been described in detail by preparation, Neely, Leibermeister, Battersby & Morgan (1967). Briefly, in this left heart 25 NaHCO2, oxygenated Krebs-Henseleit bicarbonate buffer (consisting of (mM): 118-5 NaCl, and gassed 4.75 KCl, 1'19 MgSO4, 1118 KH2PO4 and 2 5 pH 7 4, containing 11 1 mm glucose with 96 % 02-5% CO2 at 37 entered the cannulated left atrium, passed into the left ventricle, from which it was spontaneously ejected via an aortic cannula against a hydrostatic pressure of 100 cmH2O. Electrical pacing was not used. Total cardiopulmonary bypass with maintained perfusion may be simulated by clamping the left atrial cannula and introducing perfusion coronary fluid at 37°C from a reservoir 100 cm above the heart. Three reservoirs were used, each containing either single Krebs buffer or single Krebs solution with the addition of L-NMMA (with or without is essentially that glyceryl trinitrate, GTN), L-arginine or L-lysine. This preparation, which described by Langendorff (1895), continued to beat but did not perform external work. Control values for cardiac output (CO; derived from the sum of aortic and coronary flows), peak aortic pressure (PAP), and electronically differentiated rate of aortic pressure rise (dP/dT) were
°C,
recorded
CaCl2),
(Chart recorder RS 3400, Gould, Ilford, Essex). Any heart that did not reach a steady
level of function with a cardiac output greater than 65 ml/min was rejected). Experimental procedure Normal and hypertrophied hearts (n = 4 for each) were subjected to a Langendorff perfusion,
Krebs solution for 20 min then with different concentrations of L-NMMA for the same initiallyatwith the end of which coronary flow was measured. Values are expressed as a percentage of the period,level obtained during Krebs perfusion. basal In another group of hearts (n = 6), a 20 min working control period using single buffer perfusion was initiated. The cardiac performance parameters recorded during this period were taken as 100 % reference. This group of hearts was then subjected to three further consecutive periods of 20 min working mode during which each heart was perfused with Krebs solution containing 500,UM L-NMMA, 1 mM L-lysine, 1 mM L-arginine respectively. The same protocol was repeated with hypertrophied hearts (n = 6).
NITRIC OXIDE AND CARDIAC FUNCTION
683
In another group of normal hearts (n = 6) the 20 min control period was followed by a further 20 min period during which hearts were perfused with a mixture of 500 UM L-NMMA plus GTN at a concentration of 15 mg/l. The concentration of GTN was chosen after we had demonstrated it to reverse the effect of 500 /M L-NMMA on coronary flow. At the end of each working period, hearts were perfused with the next solution in the Langendorff mode for 2 min during which the device was emptied of the previous solution and refilled with the following one. In those groups the total time of perfusion was 89 min. The perfusion fluid was collected for lactate assessment after a 20 min period of either Langendorff or working mode under either Krebs buffer or L-NMMA. In another experiment, designed to assess the influence of longer periods of L-NMMA infusion, normal hearts (n = 6) were initially perfused for 20 min (control period) with single Krebs buffer, then with L-NMMA for 60 min and finally with L-arginine for 20 min. The total time of perfusion was 104 min. Cardiac myocytes Cardiac myocytes were isolated from rat hearts perfused in the Langendorff mode, followed by collagenase and hyaluronidase, as previously described (Harding, Vescovo, Kirby, Jones, Gurden & Poole-Wilson, 1988). Myocytes were perfused on the stage of an inverted microscope with warmed (32 'C) Krebs-Henseleit solution containing 2 mm calcium, and electrically stimulated to beat at 0 5 Hz. The image of the cell was scanned with a camera every millisecond and displayed on a video monitor. The length change and the velocities of contraction and relaxation during each beat were obtained using an edge-detection device, and were continuously monitored using a chart recorder. The change in cell length with each beat was normalized for cell length, to give percentage shortening values. The velocity was expressed as the percentage of total length change that occurred during one 20 ms scan. Cells were selected by various criteria designed to exclude myocytes that had been damaged during the isolation procedure (Harding et al. 1988). Cumulative concentration-response curves to L-NMMA were performed, with the lower concentrations being in contact with the cells for 5 min, and the highest (1 mm) for 10 min. This time is sufficient for responses to be obtained for the inotropes so far tested, which include catecholamines, endothelin, calcium, forskolin and dibutyryl cyclic AMP (Harding et al. 1988).
Lactate assay Lactate level was assessed by a standard colometric assay (Sigma diagnostics, Procedure No. 735, Sigma, Poole, Dorset).
Chemicals L-NMMA (supplied by Wellcome Laboratories, Beckenham, Kent), L-arginine, L-lysine (Sigma Chemical Company, Poole, Dorset) and GTN (David Bull, Mulgrave, Victoria, Australia) were each diluted in Krebs solution. In this study L-lysine was used as control instead of D-arginine which has frequently been used (Palmer et al. 1988a, b). D-Arginine is not taken up by the amino acid uptake mechanism and therefore cannot be considered as a good control.
Expression of results The values of cardiac performance parameters were expressed as percentages of the basal levels obtained after 20 min perfusion with Krebs buffer. Lactate concentration was expressed in mg/dl. Differences between groups were determined using Student's t test and significance was assumed when the P value was 0 05 or less. Values are given as means + S.E.M.
RESULTS
Dose-response of coronary flow to L-NMMA At concentrations between 10 and 100 AM L-NMMA the coronary flow dropped to 88-5 % (± 3-18) and to 49 3 % (± 6 66) respectively of the basal level. Concentrations above 500 IUM produced no further significant decrease. A concentration of 500 UM
M. AMRANI AND OTHERS
684
TABLE 1. Dose-response of coronary flow to L-NMMA L-NMMA concentration (aM) 10 50 100 500 1000 Normal 885 + 3418 67-3 + 310 49-3 + 6-6 39-1 + 3-4 36-0 + 3-0 coronary flow SHR 92-3+t12 74-5+ 3 8 54-5+ 2-0 37-4+ 2-7 36-8+2 1 coronary flow Each point represents the mean + S.E.M. of four normal or hypertrophied (SHR) hearts subjected to 20 min Langendorff perfusion, initially with Krebs buffer then L-NMMA. Results are expressed as the percentage of basal value obtained with Krebs buffer.
TABLE 2. Functional parameters of normal and hypertrophic hearts Normal (20 min) Co PAP dP/dT
Co PAP
dP/dT
L-NMMA 58410±5-42 68-43+W277 75&23+6-56 L-NMMA 47'90+4-58 85-60+ 180 6710+5-40
L-Lysine 63-50+11410 91-50+ 194 79-78+8-64
P
L-Lysine 010001 52-60 + 10-70 01001 83-40+3'32 0'005 70-50+7-71
P
P
Journal of Physiology (1992), 456, pp. 681-687 With 2 figures Printed in Great Britain
ROLE OF BASAL RELEASE OF NITRIC OXIDE ON CORONARY FLOW AND MECHANICAL PERFORMANCE OF THE ISOLATED RAT HEART
By M. AMRANI, J. O'SHEA, N. J. ALLEN, S. E. HARDING, J. JAYAKUMAR, J. R. PEPPER, S. MONCADA* AND M. H. YACOUB From the Departments of Cardiothoracic Surgery and Cardiac Medicine, National Heart and Lung Institute, Dovehouse Street, London SW3 6LY and the *Wellcome Research Laboratories, Beckenham, Kent
(Received 9 January 1992) SUMMARY
1. The role of nitric oxide (NO) in the regulation of coronary flow and mechanical function was studied in isolated working rat hearts. 2. The inhibition of basal release of NO by L-N"--monomethylarginine (L-NMMA; 500 AvM), a specific inhibitor of its synthesis, induced a reduction of coronary flow to 3941 % (± 3 4) of its basal level. 3. Associated with this was a drop of cardiac output to 58-10 % (± 542) of control values. 4. This mechanical dysfunction, which was more pronounced in hypertrophied hearts, appears to be related to ischaemia, as it was prevented by simultaneously administered glyceryl trinitrate. Furthermore, L-NMMA did not alter the contractile activity of isolated cardiac myocytes, thereby excluding a direct toxic effect. 5. These findings provide evidence that NO plays a crucial role in the maintenance of basal coronary flow and appears to be essential for sustaining mechanical activity.
INTRODUCTION
The endothelium plays a major role in regulating coronary vasomotor tone through the production and metabolism of various vasodilating and vasoconstricting agents (Vanhoutte & Houston, 1985). Since the discovery of endothelium-derived relaxing factor (EDRF) (Furchgott & Zawadzki, 1980), it became evident that endothelial cells can strongly modulate the responsiveness of the vascular smooth muscle. Recent studies showed that EDRF and nitric oxide (NO) have similar properties and that NO is released from vascular endothelial cells, thus suggesting that EDRF is NO (Palmer, Ferrige & Moncada, 1987). Nitric oxide is formed from the terminal guanidino nitrogen of the amino acid L-arginine, (Palmer, Ashton & Moncada, 1988a). An arginine analogue i.e. L-N-monomethylarginine (L-NMMA) has been demonstrated specifically to inhibit the formation of NO from L-arginine, resulting in attenuation of endothelium-dependent relaxation (Palmer, Rees, Ashton & Moncada, MS 1026
M. AMRANI AND OTHERS 1988b). This effectof L-NMMA can be reversed by L-arginine. In addition, NO has been shown to be released continuously under basal conditions in isolated hearts (Amezcua, Dusting, Palmer & Moncada, 1988; Kelm & Schrader, 1988; Amezcua, Palmer, de Souza & Moncada, 1989). This endogenous secretion is quantitatively sufficient to influence the coronary tone and its inhibition by L-NMMA increases coronary perfusion pressure in a dose-dependent manner (Kelm & Schrader, 1988; Amezcua et al. 1989). A reduced release of NO may be associated with some pathological conditions (Chester, O'Neil, Moncada, Tadjkarimi & Yacoub, 1990). The extent of this NO-induced vasodilator tone and its physiological importance in maintaining adequate flow to sustain mechanical function in normal and hypertrophied heart has not been addressed. The present study was designed to assess the effect of a maximal inhibition of NO on cardiac mechanical performance in the 682
normal and hypertrophic isolated rat heart. METHODS
Langendorff heart preparation Male Wistar WKY rats and their spontaneously hypertensive (SHR) strain with hypertrophied heart (Olac Harlan Ltd, Bicester, Oxon), weighing between 300 and 350 g, were used in all experiments. Throughout the procedure animals were anaesthetized with halothane mixed with 95 % 02-5% CO2. The groin was dissected, the femoral vein exposed and heparin (200 IU) was then excised and injected. One minute later, a bilateral thoracotomy was performed. The heart was and Langendorff into cold fluid. The aorta cannulated was (10 'C) perfusion immediately placed the left atrium was perfusion was initiated for a 3 min wash-out period. During this time cannulated and the pulmonary artery excised to ensure the ejection of coronary effluent. In some experiments the heart was converted to a working mode preparation for 20 or 60 min. The isolated working rat heart model used in this study has already been described in detail by preparation, Neely, Leibermeister, Battersby & Morgan (1967). Briefly, in this left heart 25 NaHCO2, oxygenated Krebs-Henseleit bicarbonate buffer (consisting of (mM): 118-5 NaCl, and gassed 4.75 KCl, 1'19 MgSO4, 1118 KH2PO4 and 2 5 pH 7 4, containing 11 1 mm glucose with 96 % 02-5% CO2 at 37 entered the cannulated left atrium, passed into the left ventricle, from which it was spontaneously ejected via an aortic cannula against a hydrostatic pressure of 100 cmH2O. Electrical pacing was not used. Total cardiopulmonary bypass with maintained perfusion may be simulated by clamping the left atrial cannula and introducing perfusion coronary fluid at 37°C from a reservoir 100 cm above the heart. Three reservoirs were used, each containing either single Krebs buffer or single Krebs solution with the addition of L-NMMA (with or without is essentially that glyceryl trinitrate, GTN), L-arginine or L-lysine. This preparation, which described by Langendorff (1895), continued to beat but did not perform external work. Control values for cardiac output (CO; derived from the sum of aortic and coronary flows), peak aortic pressure (PAP), and electronically differentiated rate of aortic pressure rise (dP/dT) were
°C,
recorded
CaCl2),
(Chart recorder RS 3400, Gould, Ilford, Essex). Any heart that did not reach a steady
level of function with a cardiac output greater than 65 ml/min was rejected). Experimental procedure Normal and hypertrophied hearts (n = 4 for each) were subjected to a Langendorff perfusion,
Krebs solution for 20 min then with different concentrations of L-NMMA for the same initiallyatwith the end of which coronary flow was measured. Values are expressed as a percentage of the period,level obtained during Krebs perfusion. basal In another group of hearts (n = 6), a 20 min working control period using single buffer perfusion was initiated. The cardiac performance parameters recorded during this period were taken as 100 % reference. This group of hearts was then subjected to three further consecutive periods of 20 min working mode during which each heart was perfused with Krebs solution containing 500,UM L-NMMA, 1 mM L-lysine, 1 mM L-arginine respectively. The same protocol was repeated with hypertrophied hearts (n = 6).
NITRIC OXIDE AND CARDIAC FUNCTION
683
In another group of normal hearts (n = 6) the 20 min control period was followed by a further 20 min period during which hearts were perfused with a mixture of 500 UM L-NMMA plus GTN at a concentration of 15 mg/l. The concentration of GTN was chosen after we had demonstrated it to reverse the effect of 500 /M L-NMMA on coronary flow. At the end of each working period, hearts were perfused with the next solution in the Langendorff mode for 2 min during which the device was emptied of the previous solution and refilled with the following one. In those groups the total time of perfusion was 89 min. The perfusion fluid was collected for lactate assessment after a 20 min period of either Langendorff or working mode under either Krebs buffer or L-NMMA. In another experiment, designed to assess the influence of longer periods of L-NMMA infusion, normal hearts (n = 6) were initially perfused for 20 min (control period) with single Krebs buffer, then with L-NMMA for 60 min and finally with L-arginine for 20 min. The total time of perfusion was 104 min. Cardiac myocytes Cardiac myocytes were isolated from rat hearts perfused in the Langendorff mode, followed by collagenase and hyaluronidase, as previously described (Harding, Vescovo, Kirby, Jones, Gurden & Poole-Wilson, 1988). Myocytes were perfused on the stage of an inverted microscope with warmed (32 'C) Krebs-Henseleit solution containing 2 mm calcium, and electrically stimulated to beat at 0 5 Hz. The image of the cell was scanned with a camera every millisecond and displayed on a video monitor. The length change and the velocities of contraction and relaxation during each beat were obtained using an edge-detection device, and were continuously monitored using a chart recorder. The change in cell length with each beat was normalized for cell length, to give percentage shortening values. The velocity was expressed as the percentage of total length change that occurred during one 20 ms scan. Cells were selected by various criteria designed to exclude myocytes that had been damaged during the isolation procedure (Harding et al. 1988). Cumulative concentration-response curves to L-NMMA were performed, with the lower concentrations being in contact with the cells for 5 min, and the highest (1 mm) for 10 min. This time is sufficient for responses to be obtained for the inotropes so far tested, which include catecholamines, endothelin, calcium, forskolin and dibutyryl cyclic AMP (Harding et al. 1988).
Lactate assay Lactate level was assessed by a standard colometric assay (Sigma diagnostics, Procedure No. 735, Sigma, Poole, Dorset).
Chemicals L-NMMA (supplied by Wellcome Laboratories, Beckenham, Kent), L-arginine, L-lysine (Sigma Chemical Company, Poole, Dorset) and GTN (David Bull, Mulgrave, Victoria, Australia) were each diluted in Krebs solution. In this study L-lysine was used as control instead of D-arginine which has frequently been used (Palmer et al. 1988a, b). D-Arginine is not taken up by the amino acid uptake mechanism and therefore cannot be considered as a good control.
Expression of results The values of cardiac performance parameters were expressed as percentages of the basal levels obtained after 20 min perfusion with Krebs buffer. Lactate concentration was expressed in mg/dl. Differences between groups were determined using Student's t test and significance was assumed when the P value was 0 05 or less. Values are given as means + S.E.M.
RESULTS
Dose-response of coronary flow to L-NMMA At concentrations between 10 and 100 AM L-NMMA the coronary flow dropped to 88-5 % (± 3-18) and to 49 3 % (± 6 66) respectively of the basal level. Concentrations above 500 IUM produced no further significant decrease. A concentration of 500 UM
M. AMRANI AND OTHERS
684
TABLE 1. Dose-response of coronary flow to L-NMMA L-NMMA concentration (aM) 10 50 100 500 1000 Normal 885 + 3418 67-3 + 310 49-3 + 6-6 39-1 + 3-4 36-0 + 3-0 coronary flow SHR 92-3+t12 74-5+ 3 8 54-5+ 2-0 37-4+ 2-7 36-8+2 1 coronary flow Each point represents the mean + S.E.M. of four normal or hypertrophied (SHR) hearts subjected to 20 min Langendorff perfusion, initially with Krebs buffer then L-NMMA. Results are expressed as the percentage of basal value obtained with Krebs buffer.
TABLE 2. Functional parameters of normal and hypertrophic hearts Normal (20 min) Co PAP dP/dT
Co PAP
dP/dT
L-NMMA 58410±5-42 68-43+W277 75&23+6-56 L-NMMA 47'90+4-58 85-60+ 180 6710+5-40
L-Lysine 63-50+11410 91-50+ 194 79-78+8-64
P
L-Lysine 010001 52-60 + 10-70 01001 83-40+3'32 0'005 70-50+7-71
P
P
