Pflfigers Archiv
Pfltigers Arch. 380, 259--266 (1979)
EuropeanJournal of Phys~bgy 9 by Springer-Verlag 1979
Functional and Metabolic Features of an Isolated Perfused Guinea Pig Heart Performing Pressure-Volume Work R. Bringer, O. Sommer, G. Walter, H. Stiegler, and E. Gerlach Physiologisches Institut der Universitfit M/inchen, Pettenkoferstral3e 12, D-8000 Mfinchen2, Federal Republic of Germany
Abstract. Cardiac performance and some parameters of glycolytic and oxidative metabolism were analyzed in
isolated perfused guinea pig hearts performing pressure-volume work. Perfusion medium was an oxygenated Krebs-Henseleit bicarbonate buffer (pH 7.4) which contained glucose and physiological concentrations of pyruvate and insulin. The pressure-flow relationship in the coronary vascular bed indicated autoregulation of coronary flow. Left ventricular function was influenced by aortic pressure (Pa) and venous filling pressure (Pv) in accordance with the Frank-Starling principle, i.e. stroke work increased as a function of Pa or P, to a certain maximum and then decreased. Myocardial oxygen consumption (MVO2), on the other hand, was linearly correlated with P~ and Pv, respectively, over the entire pressure range. Efficiency of the left ventricle, therefore, increased to an optimum (16 ~o) and decreased at higher pressures. Myocardial contents of glycogen, ATP and creatine phosphate were not markedly influenced by a change in P~ or Pv. L-Noradrenaline (0.08 gM, NA) stimulated stroke work and MVOz at a all Pv tested; efficiencies reached physiologic values (21%) at high volume loads. The increased MVOz was associated with an acceleration ofpyruvate decarboxylation and lactate release up to 10- and 15-fold, respectively, at elevated but physiological NA concentrations (0.2 ~tM). Our results demonstrate that the isolated perfused working guinea pig heart compares favourably with the non-failing Starling heart-lung preparation and hearts in situ, as far as coronary function, left ventricular performance and oxidative metabolism are concerned. Key words: Flow-autoregulation - Ventricular func-
tion - External heart work - Myocardial oxygen consumption - Efficiency - Pyruvate decarboxylation - Noradrenaline.
Introduction
Isolated hearts perfused according to Langendorff [20] exhibit satisfactory functional and metabolic stability provided they are supplied with suitable substrates [8,9]. The Langendorff heart, however, does not perform pressure-volume work. This disadvantage was overcome by perfusing hearts via the left atrium [23, 25] and allowing the left ventricle to eject perfusion fluid into the aorta. In studies with isolated perfused hearts it is essential to know whether the coronary system is capable of flow-autoregulation [4,9,31] and whether the relationships between aortic or filling pressure and external work o1 oxygen consumption, respectively, are comparable to those in the blood-perfused Starling heart-lung preparation [14, 17,33] and hearts in situ [6,28]. In the hemoglobin-free perfused working rat heart coronary function seems to be impaired [24,25] and oxygen consumption does not correlate with the external work performed [25]. These problems prompted us to study function and energetics as well as some metabolic parameters in an isolated perfused working heart preparation supplied with glucose and pyruvate as substrates in the presence of insulin.
Materials and Methods Na-pyruvate, lactate dehydrogenase (E.C. 1.1.1.27), amyloglucosidase (E.C. 3.2.1.3), glucose-6-phosphate dehydrogenase (E.C. 1.1A.49), NAD, NADH and NADP were purchased from Boehringer, Mannheim. Insulin and L-noradrenaline were obtained from Sigma Chemic, Mfinchen, l-l~C-pyruvate (specific activity 14.4mCi/mmol) from Amersham-Buchler, Braunschweig. All other chemicals from E. Merck, Darmstadt, and C. Roth, Karlsruhe, were of highest available purity. The Isolated Working Heart Preparation. In an initial adaptation period (20 rain), hearts isolated from fed guinea pigs (body weight 350-450 g) were perfused via the aorta according to Langendorff [20,9]. Veins were ligated close to the surface of the atria. The 1eft atrium was cannulated via an incision of the left auricle. A cathether placed into the puhnonary artery drained the coronary effluent
0031-6768/79/0380/0259/$01.60
Pflfigers Arch. 380 (1979)
260 Overflow
F Inflow Reservoir
]re
Aortic Pressur6
Fig. 1. Schematic illustration of the device for non-recirculating perfusion of isolated hearts performing left ventricular pressurevolume work. The heart preparation was placed in a moist chamber (dotted rectangle). Arrows indicate direction of flow during left ventricular ejection. Aortic and atrial pressures were measured at right angles to the direction of flow by means of rigid tubes communicating with the enlarged fluid chambers (volumes approximately 5 ml) in the aortic and atrial cannulae, respectively. Aortic flow in diastole was maintained as a result of decompression of the aortic "Windkessel". For further details see Methods. RA = right atrium; LA = left atrium perfusate which was collected in Erlenmeyer flasks (volume 250 ml) for determination of metabolites. In order to obtain a heart preparation capable of performing pressure-volume work, perfusion via the aorta was replaced by perfusion via the left atrium (Fig. 1). Oxygenated perfusion medium flowed from a reservoir (approximately 21) through an adjustable resistance which allowed perfusion of the heart at various filling pressures. The left ventricle ejected the perfusion fluid via an aortic cannula into an adjustable overflow system in which the aortic pressure and thus coronary perfusion pressure could be varied. The right ventride did not perform mechanical work, since the pulmonary drainage pressure was kept 4 - 6 c m H20 below the atmospheric pressure. Inflow and outflow elasticity chambers (airfilled "Windkessel", volume 15 and 9 ml, respectively) were attached to the left atrial and aortic cannula, respectively, as indicated in Fig. 1. Perfusion medium was a non-recirculating modified (1.25 mM Ca 2 +) Krebs-Henseleit bicarbonate buffer (pH 7.4, 38~C) saturated with 95 % 0 2 - 5 ~ C O 2 [19]. If not otherwise stated the arterial perfnsion fluid contained 0.2mM pyruvate, 5mM glucose and 40 pU/I insulin. Hemodynamic parameters were measured with strain gages (P 23 BB, P 23 Db, Statham) and electromagnetic flow transducers (M 4000, Statham) located in the perfusion system as shown in Fig. 1. Atrial filling pressure, aortic pressure, coronary flow and aortic flow were continuously recorded (Dynograph, type R411, Beckman Instruments). Mean pressure and flow were obtained by electroni-
cally averaging the phasic input signals. Spontaneous heart rate was monitored using a cardiotachometer (Beckman Instruments) which received phasic pressure signals. Pressure-volume work was the product [cardiac output] x [pressure gradient across the left ventricle]; the pressure gradient was calculated as the difference [mean aortic pressure] - [mean left atrial filling pressure]. Acceleration work during ejection was determined using the formula for kinetic energy and was the product 1/2 x [ejected volume] x [mean velocity of flow] a [14,17, 33]. The sum [pressure-volume work per heart beat] + [acceleration work during systole] was the stroke work, i.e. the total external work performed by the left ventricle during ejection. The kinetic energy was neglected at low filling pressures (_< 10cm H20), since it was less than 3 % of stroke work under these conditions. The inflow resistance was altered in order to change the filling pressure and, thus, the volume load of the left ventricle. In such experiments mean aortic pressure or the pressure gradient across the left ventricle was held constant by appropriately adjusting the hydrostatic height of the overflow system. Conversely, the hydrostatic pressure in the overflow system was varied while holding the filling pressure constant, thus imposing various pressure loads on the ventricle. The different work loads were changed stepwise, each load lasting for 6 - 8 min. Efficiency was calculated as the ratio [external left ventricular work performed]/[energy equivalent of left ventricular oxygen uptake]. Measured oxygen consumption (M'~O2) had thus to be corrected for the contribution of the right ventricle. Since this ventricle did not perform mechanical work, its oxygen uptake could be assessed by extrapolation of total MVO z to an aortic pressure equal to atmospheric pressure and by multiplication of the obtained value (1.05 gmol x rain- 1 x g- 1, as derived from the data in Table 1) by the weight portion of the right ventricle (24.5 % of the weight of both ventricles [1]). Similarly, the external work performed by the left ventricle was estimated from external heart work corrected for ventricular weight divided by the weight portion of the left ventricle (75.5 ~ of the weight of both ventricles [1]). The energy equivalent of the oxygen consumed, i.e. the change in free energy per atom oxygen that was reduced in the respiratory chain, was assumed as 52.7 kcal [21] (0.441 J per gmol oxygen), since with glucose and pyruvate as main substrates for the energy yielding metabolism mitochondrial respiration depends predominantly upon the oxidation of mitochondrial NADH. Analytical Procedures. Experiments designed to study the influence of altered work on myocardial energy state were terminated by stopfreezing the hearts between almninum blocks precooled in liquid nitrogen. Extraction of ventricular myocardium as well as separation and determination of high energy phosphate compounds were performed as described elsewhere [15,16]. Glycogen was determined as glucose in heart homogenates incubate d with amyloglucosidase [3]. Oxygen content was measured in the inflow and effluent perfusate using a teflon shielded platinum electrode (Dr. Schwab, Mtinchen) which was calibrated with air. Lactate and pyruvate were measured enzymatically [3]. 14CO2 production from pyruvate-l-l*C was used as a measure of oxidative decarboxylation of pyruvate [30]. The coronary effluent perfusate and the aortic output were collected for periods of one minute. The perfusate was acidified and carbon dioxide was trapped in phenethylamine (Roth, Karlsruhe). The radioactivity was mea~ sured in a liquid scintillation spectrometer (Model 3380, Packard) using toluol/ethanol/2-methoxy-ethanol (5.5/0.5/2.6) containing POPOP (0.4raM) and PPO (21 raM). Metabolic rates were calculated from arterio-venous concentration differences of metabolites and the coronary flow rate. If not otherwise stated, hemodynamic and metabolic rates presented in figures and tables are expressed per gram wet weight of ventricular tissue.
R. Bringer et aI. : Function and Energetics of Isolated Working Hearts
261
Results
8
A. Autoregutation of Coronary Flow
W F-
Changes in coronary perfusion pressure - caused by sudden alterations in the hydrostatic pressure of the overflow system - were followed by typical autoregulatory flow responses. A reduction in perfusion pressure was associated with an initial decrease of flow followed by an increase towards the control value. Conversely, an elevation of perfusion pressure resulted in an initial increase in flow which subsequently declined towards the flow value measured before the change in perfusion pressure. Steady state pressureflow relationships, shown in Fig. 2, panel A, demonstrate that coronary flow rate did not increase linearly with the perfusion pressure; instead, the pressure-flow relationship was similar to that in the heart in situ [4]. Furthermore, the pressure-flow curve exhibited a sigmold shape indicating that autoregulation occurred in the mean aortic pressure range between 40 and 80 mmHg. In Fig. 2, panel B, data are given for the concentration ratios of lactate and pyruvate in the coronary effluent perfusate. With perfusion pressures above 40mmHg the ratios were low and constant. However, when aortic pressure fell below 30 mmHg, the ratios increased due to an inadequate myocardial oxygen supply.
B. Relationships Between Myocardial Oxygen Consumption and Cardiac Pelformance Under the Influence of Different Workloads and Noradrenaline Function of the left ventricle was altered by changing the aortic pressure (P,) at constant left atrial filling pressure (Pv) (pressure load) or vice versa (volume load). Figure 3 illustrates the changes in stroke volume and stroke work as well as those in oxygen consumption ( M ~ / O 2 ) caused by stepwise increments in P, (panels A, C) or Pv (panels B, D), respectively. With Pv
Pfltigers Arch. 380, 259--266 (1979)
EuropeanJournal of Phys~bgy 9 by Springer-Verlag 1979
Functional and Metabolic Features of an Isolated Perfused Guinea Pig Heart Performing Pressure-Volume Work R. Bringer, O. Sommer, G. Walter, H. Stiegler, and E. Gerlach Physiologisches Institut der Universitfit M/inchen, Pettenkoferstral3e 12, D-8000 Mfinchen2, Federal Republic of Germany
Abstract. Cardiac performance and some parameters of glycolytic and oxidative metabolism were analyzed in
isolated perfused guinea pig hearts performing pressure-volume work. Perfusion medium was an oxygenated Krebs-Henseleit bicarbonate buffer (pH 7.4) which contained glucose and physiological concentrations of pyruvate and insulin. The pressure-flow relationship in the coronary vascular bed indicated autoregulation of coronary flow. Left ventricular function was influenced by aortic pressure (Pa) and venous filling pressure (Pv) in accordance with the Frank-Starling principle, i.e. stroke work increased as a function of Pa or P, to a certain maximum and then decreased. Myocardial oxygen consumption (MVO2), on the other hand, was linearly correlated with P~ and Pv, respectively, over the entire pressure range. Efficiency of the left ventricle, therefore, increased to an optimum (16 ~o) and decreased at higher pressures. Myocardial contents of glycogen, ATP and creatine phosphate were not markedly influenced by a change in P~ or Pv. L-Noradrenaline (0.08 gM, NA) stimulated stroke work and MVOz at a all Pv tested; efficiencies reached physiologic values (21%) at high volume loads. The increased MVOz was associated with an acceleration ofpyruvate decarboxylation and lactate release up to 10- and 15-fold, respectively, at elevated but physiological NA concentrations (0.2 ~tM). Our results demonstrate that the isolated perfused working guinea pig heart compares favourably with the non-failing Starling heart-lung preparation and hearts in situ, as far as coronary function, left ventricular performance and oxidative metabolism are concerned. Key words: Flow-autoregulation - Ventricular func-
tion - External heart work - Myocardial oxygen consumption - Efficiency - Pyruvate decarboxylation - Noradrenaline.
Introduction
Isolated hearts perfused according to Langendorff [20] exhibit satisfactory functional and metabolic stability provided they are supplied with suitable substrates [8,9]. The Langendorff heart, however, does not perform pressure-volume work. This disadvantage was overcome by perfusing hearts via the left atrium [23, 25] and allowing the left ventricle to eject perfusion fluid into the aorta. In studies with isolated perfused hearts it is essential to know whether the coronary system is capable of flow-autoregulation [4,9,31] and whether the relationships between aortic or filling pressure and external work o1 oxygen consumption, respectively, are comparable to those in the blood-perfused Starling heart-lung preparation [14, 17,33] and hearts in situ [6,28]. In the hemoglobin-free perfused working rat heart coronary function seems to be impaired [24,25] and oxygen consumption does not correlate with the external work performed [25]. These problems prompted us to study function and energetics as well as some metabolic parameters in an isolated perfused working heart preparation supplied with glucose and pyruvate as substrates in the presence of insulin.
Materials and Methods Na-pyruvate, lactate dehydrogenase (E.C. 1.1.1.27), amyloglucosidase (E.C. 3.2.1.3), glucose-6-phosphate dehydrogenase (E.C. 1.1A.49), NAD, NADH and NADP were purchased from Boehringer, Mannheim. Insulin and L-noradrenaline were obtained from Sigma Chemic, Mfinchen, l-l~C-pyruvate (specific activity 14.4mCi/mmol) from Amersham-Buchler, Braunschweig. All other chemicals from E. Merck, Darmstadt, and C. Roth, Karlsruhe, were of highest available purity. The Isolated Working Heart Preparation. In an initial adaptation period (20 rain), hearts isolated from fed guinea pigs (body weight 350-450 g) were perfused via the aorta according to Langendorff [20,9]. Veins were ligated close to the surface of the atria. The 1eft atrium was cannulated via an incision of the left auricle. A cathether placed into the puhnonary artery drained the coronary effluent
0031-6768/79/0380/0259/$01.60
Pflfigers Arch. 380 (1979)
260 Overflow
F Inflow Reservoir
]re
Aortic Pressur6
Fig. 1. Schematic illustration of the device for non-recirculating perfusion of isolated hearts performing left ventricular pressurevolume work. The heart preparation was placed in a moist chamber (dotted rectangle). Arrows indicate direction of flow during left ventricular ejection. Aortic and atrial pressures were measured at right angles to the direction of flow by means of rigid tubes communicating with the enlarged fluid chambers (volumes approximately 5 ml) in the aortic and atrial cannulae, respectively. Aortic flow in diastole was maintained as a result of decompression of the aortic "Windkessel". For further details see Methods. RA = right atrium; LA = left atrium perfusate which was collected in Erlenmeyer flasks (volume 250 ml) for determination of metabolites. In order to obtain a heart preparation capable of performing pressure-volume work, perfusion via the aorta was replaced by perfusion via the left atrium (Fig. 1). Oxygenated perfusion medium flowed from a reservoir (approximately 21) through an adjustable resistance which allowed perfusion of the heart at various filling pressures. The left ventricle ejected the perfusion fluid via an aortic cannula into an adjustable overflow system in which the aortic pressure and thus coronary perfusion pressure could be varied. The right ventride did not perform mechanical work, since the pulmonary drainage pressure was kept 4 - 6 c m H20 below the atmospheric pressure. Inflow and outflow elasticity chambers (airfilled "Windkessel", volume 15 and 9 ml, respectively) were attached to the left atrial and aortic cannula, respectively, as indicated in Fig. 1. Perfusion medium was a non-recirculating modified (1.25 mM Ca 2 +) Krebs-Henseleit bicarbonate buffer (pH 7.4, 38~C) saturated with 95 % 0 2 - 5 ~ C O 2 [19]. If not otherwise stated the arterial perfnsion fluid contained 0.2mM pyruvate, 5mM glucose and 40 pU/I insulin. Hemodynamic parameters were measured with strain gages (P 23 BB, P 23 Db, Statham) and electromagnetic flow transducers (M 4000, Statham) located in the perfusion system as shown in Fig. 1. Atrial filling pressure, aortic pressure, coronary flow and aortic flow were continuously recorded (Dynograph, type R411, Beckman Instruments). Mean pressure and flow were obtained by electroni-
cally averaging the phasic input signals. Spontaneous heart rate was monitored using a cardiotachometer (Beckman Instruments) which received phasic pressure signals. Pressure-volume work was the product [cardiac output] x [pressure gradient across the left ventricle]; the pressure gradient was calculated as the difference [mean aortic pressure] - [mean left atrial filling pressure]. Acceleration work during ejection was determined using the formula for kinetic energy and was the product 1/2 x [ejected volume] x [mean velocity of flow] a [14,17, 33]. The sum [pressure-volume work per heart beat] + [acceleration work during systole] was the stroke work, i.e. the total external work performed by the left ventricle during ejection. The kinetic energy was neglected at low filling pressures (_< 10cm H20), since it was less than 3 % of stroke work under these conditions. The inflow resistance was altered in order to change the filling pressure and, thus, the volume load of the left ventricle. In such experiments mean aortic pressure or the pressure gradient across the left ventricle was held constant by appropriately adjusting the hydrostatic height of the overflow system. Conversely, the hydrostatic pressure in the overflow system was varied while holding the filling pressure constant, thus imposing various pressure loads on the ventricle. The different work loads were changed stepwise, each load lasting for 6 - 8 min. Efficiency was calculated as the ratio [external left ventricular work performed]/[energy equivalent of left ventricular oxygen uptake]. Measured oxygen consumption (M'~O2) had thus to be corrected for the contribution of the right ventricle. Since this ventricle did not perform mechanical work, its oxygen uptake could be assessed by extrapolation of total MVO z to an aortic pressure equal to atmospheric pressure and by multiplication of the obtained value (1.05 gmol x rain- 1 x g- 1, as derived from the data in Table 1) by the weight portion of the right ventricle (24.5 % of the weight of both ventricles [1]). Similarly, the external work performed by the left ventricle was estimated from external heart work corrected for ventricular weight divided by the weight portion of the left ventricle (75.5 ~ of the weight of both ventricles [1]). The energy equivalent of the oxygen consumed, i.e. the change in free energy per atom oxygen that was reduced in the respiratory chain, was assumed as 52.7 kcal [21] (0.441 J per gmol oxygen), since with glucose and pyruvate as main substrates for the energy yielding metabolism mitochondrial respiration depends predominantly upon the oxidation of mitochondrial NADH. Analytical Procedures. Experiments designed to study the influence of altered work on myocardial energy state were terminated by stopfreezing the hearts between almninum blocks precooled in liquid nitrogen. Extraction of ventricular myocardium as well as separation and determination of high energy phosphate compounds were performed as described elsewhere [15,16]. Glycogen was determined as glucose in heart homogenates incubate d with amyloglucosidase [3]. Oxygen content was measured in the inflow and effluent perfusate using a teflon shielded platinum electrode (Dr. Schwab, Mtinchen) which was calibrated with air. Lactate and pyruvate were measured enzymatically [3]. 14CO2 production from pyruvate-l-l*C was used as a measure of oxidative decarboxylation of pyruvate [30]. The coronary effluent perfusate and the aortic output were collected for periods of one minute. The perfusate was acidified and carbon dioxide was trapped in phenethylamine (Roth, Karlsruhe). The radioactivity was mea~ sured in a liquid scintillation spectrometer (Model 3380, Packard) using toluol/ethanol/2-methoxy-ethanol (5.5/0.5/2.6) containing POPOP (0.4raM) and PPO (21 raM). Metabolic rates were calculated from arterio-venous concentration differences of metabolites and the coronary flow rate. If not otherwise stated, hemodynamic and metabolic rates presented in figures and tables are expressed per gram wet weight of ventricular tissue.
R. Bringer et aI. : Function and Energetics of Isolated Working Hearts
261
Results
8
A. Autoregutation of Coronary Flow
W F-
Changes in coronary perfusion pressure - caused by sudden alterations in the hydrostatic pressure of the overflow system - were followed by typical autoregulatory flow responses. A reduction in perfusion pressure was associated with an initial decrease of flow followed by an increase towards the control value. Conversely, an elevation of perfusion pressure resulted in an initial increase in flow which subsequently declined towards the flow value measured before the change in perfusion pressure. Steady state pressureflow relationships, shown in Fig. 2, panel A, demonstrate that coronary flow rate did not increase linearly with the perfusion pressure; instead, the pressure-flow relationship was similar to that in the heart in situ [4]. Furthermore, the pressure-flow curve exhibited a sigmold shape indicating that autoregulation occurred in the mean aortic pressure range between 40 and 80 mmHg. In Fig. 2, panel B, data are given for the concentration ratios of lactate and pyruvate in the coronary effluent perfusate. With perfusion pressures above 40mmHg the ratios were low and constant. However, when aortic pressure fell below 30 mmHg, the ratios increased due to an inadequate myocardial oxygen supply.
B. Relationships Between Myocardial Oxygen Consumption and Cardiac Pelformance Under the Influence of Different Workloads and Noradrenaline Function of the left ventricle was altered by changing the aortic pressure (P,) at constant left atrial filling pressure (Pv) (pressure load) or vice versa (volume load). Figure 3 illustrates the changes in stroke volume and stroke work as well as those in oxygen consumption ( M ~ / O 2 ) caused by stepwise increments in P, (panels A, C) or Pv (panels B, D), respectively. With Pv
