348
Laboratory Animals (1991) 25, 348-353
The isolated, buffer-perfused ferret heart: A new model for the study of cardiac physiology and metabolism STEFAN
NEUBAUER
& JOANNE
S. INGWALL
NMR Laboratory for Physiological Chemistry, Department of Medicine, Harvard Medical School, Brigham and Women's Hospital, Boston, Massachusetts, USA Summary The isolated, buffer-perfused ferret heart is a new model for the study of cardiac physiology and metabolism. Compared to the more commonly used isolated heart preparation, the rat heart, the ferret has a lower rate-pressure product due to lower heart rate, a remarkably low coronary flow and almost complete oxygen extraction. The ferret heart remains in stable haemodynamic and metabolic conditions for a longer period of time than the rat heart. ATP contents of the two species are similar, but creatine phosphate content is higher in the ferret while NAD content is much lower.
Keywords: Isolated ferret spectroscopy
heart;
3/ P-NMR
The ferret is becoming a more widely-used animal for basic research in cardiology. This may be due in part to several special features of the ferret heart, such as remarkable mechanical and electrical stability during recovery from injury, in part to decreasing availability of dogs for laboratory studies, while ferrets are becoming more readily available from several breeding companies in larger numbers. The isolated, buffer- or blood-perfused ferret heart has been used for studies of normal and pathologic heart physiology and metabolism (for example, Morris et al., 1985; Allen et al., 1985; Moody et al., Correspondence to: Stefan Neubauer, MD, Medizinische Universitiitsklinik, Josef-Schneider-StraOe 2, 87 Wiirzburg, Germany.
Received 23 November 1989; accepted 22 April 1991
1985; Marban et al., 1986; Kusuoka et al., 1986, 1987; Neubauer et al., 1988). However, few physiologic and metabolic characteristics of the ferret heart have been determined. In the present report, we define several physiological and biochemical characteristics of the isolated, isovolumic, adult ferret heart perfused with buffer solution at constant pressure. Using 31 P-NMR spectroscopy as well as high-pressure liquid chromatography, we define coronary flow, oxygen extraction and consumption, high- and low-energy phosphate contents and intracellular pH. We compare our data with the most widely used model for isolated heart experiments, the rat heart, and describe similarities and distinctions of ferret and rat heart. These results should establish a framework for future studies of the isolated ferret heart. Methods Male, brown, castrated ferrets were obtained from Marshall Farms (North Rose, NY, USA) at an age of approximately 10 months with a body weight of 1500-2000 g. Ferrets were of the species European Polecat (mustela putorius furo). The designation for breed and genetic status was 'sable'. In our facility, the animals were housed in 70 X 70 X 50 cm aluminium cages (2 animals per cage) for a maximum period of one week before being used for experiments. They were fed commercial cat chow and had access to tap water, both supplied ad libitum. Artificial neon light was on from 600 to 2200 h. Room temperature was held at 22 °e, humidity was not controlled. For the experiments reported here, ferrets were anaesthetized by breathing chloroform in a
Downloaded from lan.sagepub.com at AMS/Girona Library on March 6, 2015
The isolated buffer-perfused ferret heart sealed cylinder (30 em diameter, height 45 em). A transverse laparotomy and a left and a right anterolateral thoracotomy were performed, and the heart was rapidly excised and immersed in ice-cold buffer. The aorta was dissected free, and mounted onto a cannula attached to a perfusion apparatus. Retrograde perfusion of the heart was started in the Langendorff mode at a constant temperature of 37°C and a constant coronary perfusion pressure of 100mmHg. This perfusion pressure was chosen because it yielded an optimal creatine phosphatel ATP ratio, indicative of maximum oxygen delivery. In preliminary experiments, this ratio was highest and was unchanged over a pressure range of 80 to 120mmHg, pressures which changed myocardial performance by 38070(Neubauer et al., 1988). Animals weighing 1500-2000 g had a mean heart weight of 9,9 ± O' 3 g. Before entering the heart, perfusate was passed through a membrane filter of pore size 5 j.tm (Gelman Sciences, Ann Arbor, MI, USA). A small vent made out of polyethylene tubing was pierced through the apex of the left ventricle to allow drainage of flow from Thebesian veinS'. For perfusion phosphate-free Krebs-Henseleit buffer was used containing (mM): NaCl (118), KCI-(4'7), CaClz (1' 75), MgS04 (1' 2), ethylenediaminetetraacetate tetrasodium (0' 5), NaHC03 (25·0) and glucose (11' 0). Equilibrating the buffer with 95% Oz, 5% COz yielded a pH of 7, 4. Coronary flow was measured by collecting coronary sinus effluent in a calibrated cylinder. Cardiac performance measurements A water-filled latex balloon was inserted into the left ventricle through an incision in the left atrial appendage, via the mitral valve, and secured by a ligature. The balloon was connected to a Statham P23Db pressure transducer (Gould Instruments) via a small-bore polyethylene tube for continuous measurement of left ventricular pressure and heart rate on a Hewlett-Packard 7754B recorder. Performance was estimated as the product of heart rate and left ventricular developed pressure (mmHg/min).
349
NMR measurements The perfused hearts were placed into a 30 mm NMR sample tube and inserted into a custom probe which was seated in the bore of a superconducting wide-bore (89 mm) 8·4 Tesla magnet (Oxford Instruments) as previously described (Neubauer et al., 1988). A Nicolet 1280 computer was used in the pulsed Fourier transform mode to generate 3Ip_NMR spectra from a Nicolet NT-360 spectrometer operating at 145·75 MHz. Before inserting hearts into the probe, an 18-channel Oxford Instrumentation Shim Supply was used to homogenize the magnetic field. A sample of phosphoric acid (500 mM), occupying a volume and position similar to that of the beating heart, was inserted into the probe, and the signal intensity was maximized by minimizing the linewidth of phosphorus (typically about 40 Hz). A temperature of 37°C was maintained by means of a variable temperature unit attached to the NMR-probe. Spectra were accumulated over two minute periods, averaging data from 44 free induction decays which were obtained using a pulse time of 60 j.tS,a pulse angle of 60° and an interpulse delay of 2·60 s. The resonance areas corresponding to ATP, creatine phosphate, inorganic phosphate, mono phosphate esters and NAD, which are proportional to the number of phosphorus atoms of the respective compound, were measured using the Nicolet Integration Program. Saturation factors were determined by comparing the spectra obtained for a pulse angle of 60° and an interpulse delay of 2·6 s to spectra obtained using a pulse angle of 90° and an interpulse delay of 12s (fully relaxed spectra): 1· 24 (creatine phosphate), 1· 00 (y-, a- and {3-P resonance of ATP). In each heart, the area of the [{3-P] ATP resonance of the first spectrum obtained under control conditions was arbitrarily set to 100% and used as the reference value for all resonances in the set of 3IP-NMR spectra obtained for the protocol. The ATP concentration of oxygenated, buffer-perfused ferret heart was measured by high pressure liquid chromatography (see below) and was found to be 38,6 ± 2' 6 nmol/mg protein (n == 4). Cytosolic
Downloaded from lan.sagepub.com at AMS/Girona Library on March 6, 2015
350
Neubauer
A TP concentration (in mM) was calculated from this value by using a mg protein/mg wet weight ratio of O· 176 and assuming that 50% of the wet weight of isolated hearts is intracellular water. Cytosolic ATP concentration in normoxic hearts was 13 . 6 mM; the cytosolic concentrations of the other metabolites during the protocol were calculated by multiplying the ratio of resonance area of metabolite to the [,,-P] -ATP area by 13,6 mM. Intracellular pH (pHi) was measured by comparing the chemical shift between inorganic phosphate and creatine phosphate with values obtained from a standard curve (Ingwall, 1982).
Experimental protocol All hearts (n = 6) were given a ca. 20 min stabilization period where end-diastolic pressure was set to 10 mmHg. One 31P-NMR spectrum was then recorded. A 20 min period without accumulation of data followed, after which a second spectrum was recorded. Haemodynamic measurements were taken at the beginning and at the end of the protocol. Hearts were then freeze-clamped for subsequent high pressure liquid chromatography analysis. Applying the criteria that hearts showing more than a 10070 change in any physiologic or metabolic parameter during the protocol would have been discarded, we found that all hearts maintained stability.
Oxygen consumption Because of the necessity to use short, metallic lines of tubing impermeable to oxygen for oxygen consumption measurements, these measurements cannot be made simultaneously with NMR measurements. Accordingly, additional hearts (n = 4) were subjected to the protocol described above. Oxygen tension was measured in the perfusion medium at the level of the aortic cannula and in the coronary effluent in the right ventricle with a Clark-type electrode (Yellow Springs Instrument Co., Yellow Springs, Ohio, USA). Oxygen consumption was calculated according to the formula (Neely et al., 1967): (perfusate p02 difference across the heart) x (solubility of 02/mmHg) x (coronary flow)/(dry weight in grams).
& Ingwall
High pressure liquid chromatography Freeze-clamped tissue was powdered in a stainless steel percussion mortar cooled in liquid nitrogen. The powder was homogenized in 0,4 N perchloric acid at O°C and aliquots of the homogenate were removed for protein determination according to Lowry et al. (1951). The homogenate was neutralized and centrifugated for 5 min. The supernatant was used for the determination of purine (A TP, ADP, AMP, GTP, GDP), pyrimidine (UTP, CTP) and pyridine (NAD) nucleotide contents by high pressure liquid chromatography as previously described (Lange et al., 1984). Briefly, ATP, GTP, UTP, CTP, GDP and ADP were separated by applying aliquots of the supernatant to a Partisil SAX column. Nucelotides were eluted isocratically using 0·16 M K2HP04 + 0·1 M KCI, pH 6, 5, at room temperature at a flow rate of I' 4 ml/min (t.. = 254 nm). AMP, inosine, hypoxanthine, xanthine and NAD were separated by using a CwJ.tBondapak column. Elution conditions were 0·025 M NH4H2P04, pH 4' 3, 2· 0 ml/min, at room temperature (t.. + 254 nm). Metabolite concentrations for HPLC results are expressed as nmol/mg protein. The adenylate energy charge was calculated as (A TP + I/2ADP)/(ATP + ADP + AMP).
Statistical analysis Data from rat and ferret hearts were compared using the unpaired t-test (Zar, 1974). Calculations were aided by the Stat View 512 + Professional, Graphic, Statistics Utility (BrainPower Inc., Calabasas, CA, USA). All data are presented as meaniSE. Results and discussion
Stability of the isolated ferret heart preparation The isolated adult ferret heart exhibits excellent physiologic and metabolic stability, which is greater than in the isolated rat heart, where instability occurs after - 90 min of buffer perfursion (Bittl & Ingwall, 1985). In the ferret, all physiologic and metabolic parameters were stable for at least 130 min. Here, the standard errors for the parameters over this time, expressed
Downloaded from lan.sagepub.com at AMS/Girona Library on March 6, 2015
The
isolated
buffer-perfused
ferret
351
heart
as the percentage of the means, were creatine phosphate, 2·2070; inorganic phosphate, 2'7%; rate-pressure product, 1·1 %; and coronary flow, O' 9%. In rat, standard errors for 90 min of perfusion were creatine phosphate, 1'2%; inorganic phosphate, 3, 2%; rate-pressure product, 2'5%; and coronary flow, 2·4%. After that time, instability occurred in both models.
Cardiac performance Data from Table 1 show that the spontaneously beating, isolated buffer-perfused ferret heart has a heart rate of 152 ± 4/min, while heart rate of the rat model is almost twice as high. Left ventricular developed pressure is similar in both species, and, therefore, the rate-pressure product is only about half as high (58%) in ferret than it is in rat. Coronary flow was higher in ferret than in rat. However, coronary flow/g wet weight was as low as 3, 5 mUmin x g, almost an order of magnitude lower than coronary flow values we have measured for beating, buffer-perfused hearts of other species, such as rat (16'8± 1'0; n==6) or rabbit (12' 8 ± 1· 1; n = 6). Thus, the buffer-perfused ferret heart has an extraordinarily small demand for coronary flow. However, oxygen consumption is not reduced in proportion, amounting to 55% of oxygen consumption in rat, which corresponds Table 1. Cardiac extraction
LVDP
HR RPP
CF CF/g wet wt AVD-O, MV02
performance and oxygen consumption of the isolated ferret and rat heart
well with the lower workload in ferret. This relativ~ly high oxygen consumption in the presence of low coronary flow is achieved by an almost complete oxygen extraction from coronary perfusate, which does not occur in rat (51 ± 1%): 02 saturation of perfusate entering the coronary arteries was 90%, perfusate leaving the heart via the coronary sinus was saturated to 6% only, and thus, oxygen extraction was 84%. CrP
A
y
P'
MPT.~J
I
~'GPC PPM
and
Ferret
Rat
(n=6)
(n = 6)
119±5 152±4 I7'9±0'9 35±3 3'5±0'3
118±5 260± 830·7 ± 2·020± I16·8 ± 1·0-
± 1-
84± 1
51
18'5±3'0
33·6±2·7-
L VDP, left ventricular developed pressure (mmHg); HR, heart rate (min-'); RPP, rate-pressure product (103 mmHg/ min); CF, coronary flow (ml/min); CF/g wet wt, coronary flow per gram wet weight (ml/min x g); SO, ven, coronary sinus O2 saturation (070); AVD-02, 'arteriovenous' 02 difference or O,-extraction of the heart (0J0, 'arterial' S02 was always 90%); MV02, 02-consumption Vtmol/g dry weight x min); - P< O· 05 ferret versus rat. In all hearts, end-diastolic pressure was set to 10 mmHg by adjusting the volume of the left ventricular balloon.
Fig. 1. 3IP_NMR spectra. Panel A: 2 min spectrum from a ferret heart. 44 acquisitions, pulse angle 60°, interpulse delay 2'60 s. Panel B: 5 min spectrum from a rat heart. 128 acquisitions, pulse angle 45°, interpulse delay 2.15 s. MPE, monophosphate esters, Pi, inorganic phosphate; GPC, glycerophosphoryl choline; CrP, creatine phosphate; y., Ciand ,J-phosphorus atom of ATP; NAD, nicotinamide adenine dinucleotid.
Downloaded from lan.sagepub.com at AMS/Girona Library on March 6, 2015
Neubauer & Ingwall
352
Table 2. High- and low-energy intracellular pH measured
A TP (mM) (calculated) CrP (mM) Pi (mM) pH;
phosphate concentrations and, by 31P_NMR spectroscopy
Ferret
Rat
(n =6)
(n =6)
13,6 23·9 ±4-1 4·1 ±0·8 7'IS±0'01
13·3 ±O'3* 3-7 ±O'4 7-14±O'OI
II ·2
CrP, creatine phosphate; Pi, inorganic phosphate; *P
Laboratory Animals (1991) 25, 348-353
The isolated, buffer-perfused ferret heart: A new model for the study of cardiac physiology and metabolism STEFAN
NEUBAUER
& JOANNE
S. INGWALL
NMR Laboratory for Physiological Chemistry, Department of Medicine, Harvard Medical School, Brigham and Women's Hospital, Boston, Massachusetts, USA Summary The isolated, buffer-perfused ferret heart is a new model for the study of cardiac physiology and metabolism. Compared to the more commonly used isolated heart preparation, the rat heart, the ferret has a lower rate-pressure product due to lower heart rate, a remarkably low coronary flow and almost complete oxygen extraction. The ferret heart remains in stable haemodynamic and metabolic conditions for a longer period of time than the rat heart. ATP contents of the two species are similar, but creatine phosphate content is higher in the ferret while NAD content is much lower.
Keywords: Isolated ferret spectroscopy
heart;
3/ P-NMR
The ferret is becoming a more widely-used animal for basic research in cardiology. This may be due in part to several special features of the ferret heart, such as remarkable mechanical and electrical stability during recovery from injury, in part to decreasing availability of dogs for laboratory studies, while ferrets are becoming more readily available from several breeding companies in larger numbers. The isolated, buffer- or blood-perfused ferret heart has been used for studies of normal and pathologic heart physiology and metabolism (for example, Morris et al., 1985; Allen et al., 1985; Moody et al., Correspondence to: Stefan Neubauer, MD, Medizinische Universitiitsklinik, Josef-Schneider-StraOe 2, 87 Wiirzburg, Germany.
Received 23 November 1989; accepted 22 April 1991
1985; Marban et al., 1986; Kusuoka et al., 1986, 1987; Neubauer et al., 1988). However, few physiologic and metabolic characteristics of the ferret heart have been determined. In the present report, we define several physiological and biochemical characteristics of the isolated, isovolumic, adult ferret heart perfused with buffer solution at constant pressure. Using 31 P-NMR spectroscopy as well as high-pressure liquid chromatography, we define coronary flow, oxygen extraction and consumption, high- and low-energy phosphate contents and intracellular pH. We compare our data with the most widely used model for isolated heart experiments, the rat heart, and describe similarities and distinctions of ferret and rat heart. These results should establish a framework for future studies of the isolated ferret heart. Methods Male, brown, castrated ferrets were obtained from Marshall Farms (North Rose, NY, USA) at an age of approximately 10 months with a body weight of 1500-2000 g. Ferrets were of the species European Polecat (mustela putorius furo). The designation for breed and genetic status was 'sable'. In our facility, the animals were housed in 70 X 70 X 50 cm aluminium cages (2 animals per cage) for a maximum period of one week before being used for experiments. They were fed commercial cat chow and had access to tap water, both supplied ad libitum. Artificial neon light was on from 600 to 2200 h. Room temperature was held at 22 °e, humidity was not controlled. For the experiments reported here, ferrets were anaesthetized by breathing chloroform in a
Downloaded from lan.sagepub.com at AMS/Girona Library on March 6, 2015
The isolated buffer-perfused ferret heart sealed cylinder (30 em diameter, height 45 em). A transverse laparotomy and a left and a right anterolateral thoracotomy were performed, and the heart was rapidly excised and immersed in ice-cold buffer. The aorta was dissected free, and mounted onto a cannula attached to a perfusion apparatus. Retrograde perfusion of the heart was started in the Langendorff mode at a constant temperature of 37°C and a constant coronary perfusion pressure of 100mmHg. This perfusion pressure was chosen because it yielded an optimal creatine phosphatel ATP ratio, indicative of maximum oxygen delivery. In preliminary experiments, this ratio was highest and was unchanged over a pressure range of 80 to 120mmHg, pressures which changed myocardial performance by 38070(Neubauer et al., 1988). Animals weighing 1500-2000 g had a mean heart weight of 9,9 ± O' 3 g. Before entering the heart, perfusate was passed through a membrane filter of pore size 5 j.tm (Gelman Sciences, Ann Arbor, MI, USA). A small vent made out of polyethylene tubing was pierced through the apex of the left ventricle to allow drainage of flow from Thebesian veinS'. For perfusion phosphate-free Krebs-Henseleit buffer was used containing (mM): NaCl (118), KCI-(4'7), CaClz (1' 75), MgS04 (1' 2), ethylenediaminetetraacetate tetrasodium (0' 5), NaHC03 (25·0) and glucose (11' 0). Equilibrating the buffer with 95% Oz, 5% COz yielded a pH of 7, 4. Coronary flow was measured by collecting coronary sinus effluent in a calibrated cylinder. Cardiac performance measurements A water-filled latex balloon was inserted into the left ventricle through an incision in the left atrial appendage, via the mitral valve, and secured by a ligature. The balloon was connected to a Statham P23Db pressure transducer (Gould Instruments) via a small-bore polyethylene tube for continuous measurement of left ventricular pressure and heart rate on a Hewlett-Packard 7754B recorder. Performance was estimated as the product of heart rate and left ventricular developed pressure (mmHg/min).
349
NMR measurements The perfused hearts were placed into a 30 mm NMR sample tube and inserted into a custom probe which was seated in the bore of a superconducting wide-bore (89 mm) 8·4 Tesla magnet (Oxford Instruments) as previously described (Neubauer et al., 1988). A Nicolet 1280 computer was used in the pulsed Fourier transform mode to generate 3Ip_NMR spectra from a Nicolet NT-360 spectrometer operating at 145·75 MHz. Before inserting hearts into the probe, an 18-channel Oxford Instrumentation Shim Supply was used to homogenize the magnetic field. A sample of phosphoric acid (500 mM), occupying a volume and position similar to that of the beating heart, was inserted into the probe, and the signal intensity was maximized by minimizing the linewidth of phosphorus (typically about 40 Hz). A temperature of 37°C was maintained by means of a variable temperature unit attached to the NMR-probe. Spectra were accumulated over two minute periods, averaging data from 44 free induction decays which were obtained using a pulse time of 60 j.tS,a pulse angle of 60° and an interpulse delay of 2·60 s. The resonance areas corresponding to ATP, creatine phosphate, inorganic phosphate, mono phosphate esters and NAD, which are proportional to the number of phosphorus atoms of the respective compound, were measured using the Nicolet Integration Program. Saturation factors were determined by comparing the spectra obtained for a pulse angle of 60° and an interpulse delay of 2·6 s to spectra obtained using a pulse angle of 90° and an interpulse delay of 12s (fully relaxed spectra): 1· 24 (creatine phosphate), 1· 00 (y-, a- and {3-P resonance of ATP). In each heart, the area of the [{3-P] ATP resonance of the first spectrum obtained under control conditions was arbitrarily set to 100% and used as the reference value for all resonances in the set of 3IP-NMR spectra obtained for the protocol. The ATP concentration of oxygenated, buffer-perfused ferret heart was measured by high pressure liquid chromatography (see below) and was found to be 38,6 ± 2' 6 nmol/mg protein (n == 4). Cytosolic
Downloaded from lan.sagepub.com at AMS/Girona Library on March 6, 2015
350
Neubauer
A TP concentration (in mM) was calculated from this value by using a mg protein/mg wet weight ratio of O· 176 and assuming that 50% of the wet weight of isolated hearts is intracellular water. Cytosolic ATP concentration in normoxic hearts was 13 . 6 mM; the cytosolic concentrations of the other metabolites during the protocol were calculated by multiplying the ratio of resonance area of metabolite to the [,,-P] -ATP area by 13,6 mM. Intracellular pH (pHi) was measured by comparing the chemical shift between inorganic phosphate and creatine phosphate with values obtained from a standard curve (Ingwall, 1982).
Experimental protocol All hearts (n = 6) were given a ca. 20 min stabilization period where end-diastolic pressure was set to 10 mmHg. One 31P-NMR spectrum was then recorded. A 20 min period without accumulation of data followed, after which a second spectrum was recorded. Haemodynamic measurements were taken at the beginning and at the end of the protocol. Hearts were then freeze-clamped for subsequent high pressure liquid chromatography analysis. Applying the criteria that hearts showing more than a 10070 change in any physiologic or metabolic parameter during the protocol would have been discarded, we found that all hearts maintained stability.
Oxygen consumption Because of the necessity to use short, metallic lines of tubing impermeable to oxygen for oxygen consumption measurements, these measurements cannot be made simultaneously with NMR measurements. Accordingly, additional hearts (n = 4) were subjected to the protocol described above. Oxygen tension was measured in the perfusion medium at the level of the aortic cannula and in the coronary effluent in the right ventricle with a Clark-type electrode (Yellow Springs Instrument Co., Yellow Springs, Ohio, USA). Oxygen consumption was calculated according to the formula (Neely et al., 1967): (perfusate p02 difference across the heart) x (solubility of 02/mmHg) x (coronary flow)/(dry weight in grams).
& Ingwall
High pressure liquid chromatography Freeze-clamped tissue was powdered in a stainless steel percussion mortar cooled in liquid nitrogen. The powder was homogenized in 0,4 N perchloric acid at O°C and aliquots of the homogenate were removed for protein determination according to Lowry et al. (1951). The homogenate was neutralized and centrifugated for 5 min. The supernatant was used for the determination of purine (A TP, ADP, AMP, GTP, GDP), pyrimidine (UTP, CTP) and pyridine (NAD) nucleotide contents by high pressure liquid chromatography as previously described (Lange et al., 1984). Briefly, ATP, GTP, UTP, CTP, GDP and ADP were separated by applying aliquots of the supernatant to a Partisil SAX column. Nucelotides were eluted isocratically using 0·16 M K2HP04 + 0·1 M KCI, pH 6, 5, at room temperature at a flow rate of I' 4 ml/min (t.. = 254 nm). AMP, inosine, hypoxanthine, xanthine and NAD were separated by using a CwJ.tBondapak column. Elution conditions were 0·025 M NH4H2P04, pH 4' 3, 2· 0 ml/min, at room temperature (t.. + 254 nm). Metabolite concentrations for HPLC results are expressed as nmol/mg protein. The adenylate energy charge was calculated as (A TP + I/2ADP)/(ATP + ADP + AMP).
Statistical analysis Data from rat and ferret hearts were compared using the unpaired t-test (Zar, 1974). Calculations were aided by the Stat View 512 + Professional, Graphic, Statistics Utility (BrainPower Inc., Calabasas, CA, USA). All data are presented as meaniSE. Results and discussion
Stability of the isolated ferret heart preparation The isolated adult ferret heart exhibits excellent physiologic and metabolic stability, which is greater than in the isolated rat heart, where instability occurs after - 90 min of buffer perfursion (Bittl & Ingwall, 1985). In the ferret, all physiologic and metabolic parameters were stable for at least 130 min. Here, the standard errors for the parameters over this time, expressed
Downloaded from lan.sagepub.com at AMS/Girona Library on March 6, 2015
The
isolated
buffer-perfused
ferret
351
heart
as the percentage of the means, were creatine phosphate, 2·2070; inorganic phosphate, 2'7%; rate-pressure product, 1·1 %; and coronary flow, O' 9%. In rat, standard errors for 90 min of perfusion were creatine phosphate, 1'2%; inorganic phosphate, 3, 2%; rate-pressure product, 2'5%; and coronary flow, 2·4%. After that time, instability occurred in both models.
Cardiac performance Data from Table 1 show that the spontaneously beating, isolated buffer-perfused ferret heart has a heart rate of 152 ± 4/min, while heart rate of the rat model is almost twice as high. Left ventricular developed pressure is similar in both species, and, therefore, the rate-pressure product is only about half as high (58%) in ferret than it is in rat. Coronary flow was higher in ferret than in rat. However, coronary flow/g wet weight was as low as 3, 5 mUmin x g, almost an order of magnitude lower than coronary flow values we have measured for beating, buffer-perfused hearts of other species, such as rat (16'8± 1'0; n==6) or rabbit (12' 8 ± 1· 1; n = 6). Thus, the buffer-perfused ferret heart has an extraordinarily small demand for coronary flow. However, oxygen consumption is not reduced in proportion, amounting to 55% of oxygen consumption in rat, which corresponds Table 1. Cardiac extraction
LVDP
HR RPP
CF CF/g wet wt AVD-O, MV02
performance and oxygen consumption of the isolated ferret and rat heart
well with the lower workload in ferret. This relativ~ly high oxygen consumption in the presence of low coronary flow is achieved by an almost complete oxygen extraction from coronary perfusate, which does not occur in rat (51 ± 1%): 02 saturation of perfusate entering the coronary arteries was 90%, perfusate leaving the heart via the coronary sinus was saturated to 6% only, and thus, oxygen extraction was 84%. CrP
A
y
P'
MPT.~J
I
~'GPC PPM
and
Ferret
Rat
(n=6)
(n = 6)
119±5 152±4 I7'9±0'9 35±3 3'5±0'3
118±5 260± 830·7 ± 2·020± I16·8 ± 1·0-
± 1-
84± 1
51
18'5±3'0
33·6±2·7-
L VDP, left ventricular developed pressure (mmHg); HR, heart rate (min-'); RPP, rate-pressure product (103 mmHg/ min); CF, coronary flow (ml/min); CF/g wet wt, coronary flow per gram wet weight (ml/min x g); SO, ven, coronary sinus O2 saturation (070); AVD-02, 'arteriovenous' 02 difference or O,-extraction of the heart (0J0, 'arterial' S02 was always 90%); MV02, 02-consumption Vtmol/g dry weight x min); - P< O· 05 ferret versus rat. In all hearts, end-diastolic pressure was set to 10 mmHg by adjusting the volume of the left ventricular balloon.
Fig. 1. 3IP_NMR spectra. Panel A: 2 min spectrum from a ferret heart. 44 acquisitions, pulse angle 60°, interpulse delay 2'60 s. Panel B: 5 min spectrum from a rat heart. 128 acquisitions, pulse angle 45°, interpulse delay 2.15 s. MPE, monophosphate esters, Pi, inorganic phosphate; GPC, glycerophosphoryl choline; CrP, creatine phosphate; y., Ciand ,J-phosphorus atom of ATP; NAD, nicotinamide adenine dinucleotid.
Downloaded from lan.sagepub.com at AMS/Girona Library on March 6, 2015
Neubauer & Ingwall
352
Table 2. High- and low-energy intracellular pH measured
A TP (mM) (calculated) CrP (mM) Pi (mM) pH;
phosphate concentrations and, by 31P_NMR spectroscopy
Ferret
Rat
(n =6)
(n =6)
13,6 23·9 ±4-1 4·1 ±0·8 7'IS±0'01
13·3 ±O'3* 3-7 ±O'4 7-14±O'OI
II ·2
CrP, creatine phosphate; Pi, inorganic phosphate; *P
