Molecular and Cellular Biochemistry 116: 139-145, 1992. © 1992 Kluwer Academic Publishers. Printed in the Netherlands.
Functional and metabolic effects of propionyl-L-carnitine in the isolated perfused hypertrophied rat heart Roberto Motterlini, Michele Samaja, Massimo Tarantola, Rosella Micheletti and Giuseppe Bianchi lstituto Scientifico San Raffaele, Dipartimento di Scienze e Tecnologie Biomediche dell'Universitgl di Milano, and Prassis Sigma Tau Institute, Milano, Italy
Abstract Aim of this study was to assess the effect of propionyl-L-carnitine (PLC), a naturally occurring derivative of L-carnitine, in cardiac hypertrophy induced by pressure overload in rats. The abdominal aorta was banded and the rats received one daily administration of PLC (50 mg/kg) or saline for four days. The hearts were excised 24 h after the last administration and were perfused retrogradely with oxygenated Krebs-Henseleit buffer containing 1.2mM palmitate bound to 3% (w/v) albumin, 2.5 IzM PLC and 25/~M L-carnitine. A saline-filled balloon was inserted into the left ventricle and the heart contractility was measured at three volumes of the balloon, corresponding to zero diastolic pressure and to increased volumes (110 and 220 ~1) over the zero volume. At the end of the perfusion, the hearts were freeze-clamped, weighed and analyzed for adenine nucleotide and phosphocreatine (PCr) content by HPLC methods. No differences in the myocardial performance were found at zero diastolic pressure. In contrast, at high intraventricular volume, the maximal rate of ventricular relaxation was increased in PLC-treated with respect to saline-treated controls (p < 0.05). In addition, the increase of the end-diastolic pressure at increasing balloon volume was more marked in controls than in the PLC-treated hearts (p < 0.02). These data correlate well with the measured higher level of total adenine nucleotides (p < 0.05) and ATP (p < 0.02) in the PLC-treated hearts, while PCr was the same in both groups. Parallel experiments performed in the absence of palmitate in the perfusing media failed to show any effect of PLC. We conclude that PLC improves the diastolic function by increasing the fraction of energy available from fatty acid oxidation in the form of ATP. (Mol Cell Biochem 116" 139-145, 1992)
Key words." isolated heart, propionyl-L-carnitine, palmitic acid, energetic metabolites
Introduction L-Carnitine is essential in the transport of long-chain fatty acids from cytosol into the mitochondria [1, 2]. The mitochondrial inner membrane is not permeable to long-chain fatty acids preventing the access of these substrates to the fatty acid [3-oxidation system, primary machinery for energy production in the cell. To over-
come this barrier, L-carnitine combines with fatty acylCoA in the cytosol and shuttles the fatty acid into the mitochondrion by a reaction catalyzed by L-carnitine acyltransferase. Thus, reduction in cell carnitine levels results in depressed mitochondrial oxidation of long chain fatty acids. The consequent decrease in rate of
Address for offprints. M. Samaja, Dipartimento di Scienze e Tecnologie Biomediche, via Olgettina 60, 20132 Milano, Italy
140 energy production is critical for those tissues whose function is heavily dependent on fatty acid oxidation, such as myocardium. Therefore, impaired fatty acid oxidation in the cardiomyopathies involving reduced myocardial level of L-carnitine [3-7], may further contribute to depress the cardiac function. Furthermore, the reduced utilization of long-chain fatty acids leads to the accumulation in the cytosol of potentially toxic substances that contribute to the pathogenesis of cardiac dysfunction [8, 9]. Propionyl-L-carnitine (PLC) is a naturally occurring derivative of L-carnitine to which the propionyl moiety seems to confer three potentially advantageous properties over L-carnitine, namely, a greater affinity for the cardiomyocyte sarcolemmal carrier [10], the ability to replenish mitochondria with intermediates of the citric acid cycle [11] and a greater positive inotropic activity demonstrated both in mechanical recovery after ischemia and in intact isolated hearts [10, 12-14]. Here, we tested the hypothesis that PLC improves the myocardial function secondary to specific metabolic adjustments. Aim of this study was: (1) to assess if free fatty acids are required for the cardiac action of PLC; (2) to study the relationship between changes in mechanical performance and high-energy phosphates content in the heart. We used the isolated perfused rat heart as experimental model to simulate the metabolic derangement due to carnitine deficiency. The hearts were made hypertrophic by pressure overload, a condition known to be associated with altered CoA and carnitine metabolism [15]. The suitability of this model to several biochemical and physiological measurements allows to relate metabolic changes to functional alterations. In addition, the response of the heart to PLC treatment is isolated from the response of the whole organism and can be studied in a greater detail.
Methods Materials PLC was synthesized at Sigma Tau Chemical Drug Dept. (batch 2649). Solutions for animal injection were prepared immediately before use dissolving PLC in saline and buffering pH to 7.4. The heart peffusion buffer contained l l 6 m M NaC1, 4.7raM KC1, 1.2mM KH2PO4, 0.5mM EDTA, 1.2mM Na2SO4, 28.5mM NaHCO3, 3 mM CaC12, 1.2 mM MgC12and 16.6 mM glucose. Half of the hearts were perfused with 1.2 mM palmitate bound to
3% (w/v) bovine albumin added to the medium. All reagents were provided from Sigma Chemical (St. Louis, MO) and were analytical grade. In addition, 2.5/xM PLC and 25/xM L-carnitine were also present in the perfusion buffer.
Animals Twenty male Wistar Kyoto rats (Charles River, Italy) weighing 200-250 g were used in this preliminary study. The animals were fed a standard diet (Altromin MT, Bolzano, Italy) containing approximately 55 nmol/g total L-carnitine. Cardiac hypertrophy was induced by banding the abdominal aorta above the renal arteries. The abdominal cavity was opened and a silver band (0.7 mm internal diameter) was fitted around the aorta. After 4 weeks recovery, the animals were randomly assigned to treatment consisting in one daily administration through an intraarterial catheter of either PLC (50 mg/kg) or saline (control group) for four days. The studies were performed conforming to the guiding principles of the National Society for Medical Research.
Heart perfusion Heart perfusion experiments were performed 24 h after the fourth administration of PLC or saline. Rats were anaesthetized with i.p. heparinized sodium thiopental (100mg/kg). The chest was opened, the aorta was mounted onto a cannula and the perfusion was started retrogradely with the described buffer, pH 7.4, 37° C. The coronary flow rate was controlled by a peristaltic pump. The coronary sinus return was collected through an outflow cannula in the pulmonary artery. A latex balloon was introduced into the left ventricle through the left auricular appendage and connected to a pressure transducer to monitor end-diastolic pressure (EDP), left-ventricular developed pressure (LVDP), heart rate (HR), maximal rate of heart contraction (+ dP/dtmax) and relaxation (-dP/dtmax). The spontaneously contracting hearts were stabilized for 30 min at coronary flow rate = 15 ml/min, with the volume of the ventricular balloon adjusted to achieve EDP -- 0.5 + 0.5 Torr (V0). At the end of the stabilization, the baseline values for the myocardial function were recorded and the intraventricular balloon was filled with accurately measured volumes of saline using a
141 glass gas-tight 1 ml syringe (Hamilton Co, Reno, Nevada) and a micrometer. The selected volumes of the balloon were V0 + ll0I~L (Vl~0) or V0 + 220~1 (V220). The hearts were then allowed to stabilize and a complete series of measurements was taken again.
Metabolic measurements
The 02 uptake (VO2) was evaluated measuring the pO 2 in the arterial inflow and in the coronary sinus return (YSI mod. 5300 Oxygen Monitor, Yellow Springs Inc., OH). In the palmitate experiments, the myocardial content of high-energy phosphate compounds was also determined at the end of the perfusion. The hearts were freeze-clamped in liquid nitrogen, weighed and transferred to a tube containing 2 ml of 0.5 M cold perchloric acid. The content was homogenized using an OMNI 1000 (OMNI Int'l, Waterbury, Connecticut) operating at 20,000 RPM. After 15 min, the suspension was centrifuged, 0.5 ml of the supernatant was neutralized with 0.4 ml of 0.5 M K O H and 0.1 ml of 1M KH2PO4, centrifuged again 30min later, and filtered through 0.22/~m pore size membrane (Nihon Millipore, Japan). ATP, ADP, AMP, creatine and creatine phosphate in myocardial extract were analyzed by HPLC. The equipment (Kontron Instruments, Milano, Italy) was composed of two rood. 420 pumps and a mod. 432 UV/Vis detector set at 210 nm. The 3/~m Supelcosil LC18 column (Supelco, Bellefonte, Pennsylvania) was equilibrated with 0.1 M KHzPO 4 and 5 mM tetrabutylammonium sulphate, the sample (20tzl) was injected and eluted using a composed gradient with a buffer containing 0.1M KH2PO4, 4 m M tetrabutyl-ammonium sulphate and 90% (v/v) CH3CN. The analysis was completed in 25 min and data were analyzed with the Kontron's dedicated software.
Statistics All values are expressed as mean + SEM. Between group comparison was by two-tailed unpaired Student's t-test. The significance was defined as a probability equal or less than 0.05.
Results The described aortic constriction procedure induced a
marked increase of the heart size with respect to normal (50%), indicating the presence of cardiac hypertrophy. As described earlier [1@ this condition was associated with a reduction of left ventricle total carnitine content, that was decreased by about 40% with respect to control sham-operated animals. Two series of heart perfusion experiments were performed. In the first series, the hearts were perfused with Krebs-Henseleit buffer with no palmitate added, and the two groups (PLC-treated and controls, n = 5 for each) were compared at V0 and Vn0- In the second series, ( n = 10) buffer contained 1.2raM palmitate bound to 3% (w/v) albumin, and measurements were made at three volumes of the balloon (0, 110, and 220 pA). Baseline data taken at V0 for the two series are shown in Table 1. In both series, no differences were detected at V0 between the controls and the PLC-treated hearts for any of the parameters evaluated including the double product LVDP x HR, index of the heart systolic function. Coronary pressure as well as contractility indexes were different in the two series of experiments probably because of the effects of fatty acidbound albumin [17]. Figure 1 shows the diastolic function (EDP and -dP/dtma×) monitored at Vl10 in all the groups. No effect of PLC treatment was detected in the absence of palmitate. In contrast, in the presence of palmitate, EDP increased less and the maximal rate of relaxation ( - dP/dtmax) was higher (p < 0.05 for both) in the PLCtreated hearts than in controls. The improvement of the diastolic function was not associated to higher systolic performance as shown by the values of LVDP x H R and + dP/dtmax. In the second series of the experiments carried out in the presence of palmitate, more marked differences were detected between PLC-treated and control hearts when balloon volume was increased by 220 ~1 over V0 (V220): EDP was 53.2_+ 5.0 vs 82.2_+ 8.4 Torr (p
Functional and metabolic effects of propionyl-L-carnitine in the isolated perfused hypertrophied rat heart Roberto Motterlini, Michele Samaja, Massimo Tarantola, Rosella Micheletti and Giuseppe Bianchi lstituto Scientifico San Raffaele, Dipartimento di Scienze e Tecnologie Biomediche dell'Universitgl di Milano, and Prassis Sigma Tau Institute, Milano, Italy
Abstract Aim of this study was to assess the effect of propionyl-L-carnitine (PLC), a naturally occurring derivative of L-carnitine, in cardiac hypertrophy induced by pressure overload in rats. The abdominal aorta was banded and the rats received one daily administration of PLC (50 mg/kg) or saline for four days. The hearts were excised 24 h after the last administration and were perfused retrogradely with oxygenated Krebs-Henseleit buffer containing 1.2mM palmitate bound to 3% (w/v) albumin, 2.5 IzM PLC and 25/~M L-carnitine. A saline-filled balloon was inserted into the left ventricle and the heart contractility was measured at three volumes of the balloon, corresponding to zero diastolic pressure and to increased volumes (110 and 220 ~1) over the zero volume. At the end of the perfusion, the hearts were freeze-clamped, weighed and analyzed for adenine nucleotide and phosphocreatine (PCr) content by HPLC methods. No differences in the myocardial performance were found at zero diastolic pressure. In contrast, at high intraventricular volume, the maximal rate of ventricular relaxation was increased in PLC-treated with respect to saline-treated controls (p < 0.05). In addition, the increase of the end-diastolic pressure at increasing balloon volume was more marked in controls than in the PLC-treated hearts (p < 0.02). These data correlate well with the measured higher level of total adenine nucleotides (p < 0.05) and ATP (p < 0.02) in the PLC-treated hearts, while PCr was the same in both groups. Parallel experiments performed in the absence of palmitate in the perfusing media failed to show any effect of PLC. We conclude that PLC improves the diastolic function by increasing the fraction of energy available from fatty acid oxidation in the form of ATP. (Mol Cell Biochem 116" 139-145, 1992)
Key words." isolated heart, propionyl-L-carnitine, palmitic acid, energetic metabolites
Introduction L-Carnitine is essential in the transport of long-chain fatty acids from cytosol into the mitochondria [1, 2]. The mitochondrial inner membrane is not permeable to long-chain fatty acids preventing the access of these substrates to the fatty acid [3-oxidation system, primary machinery for energy production in the cell. To over-
come this barrier, L-carnitine combines with fatty acylCoA in the cytosol and shuttles the fatty acid into the mitochondrion by a reaction catalyzed by L-carnitine acyltransferase. Thus, reduction in cell carnitine levels results in depressed mitochondrial oxidation of long chain fatty acids. The consequent decrease in rate of
Address for offprints. M. Samaja, Dipartimento di Scienze e Tecnologie Biomediche, via Olgettina 60, 20132 Milano, Italy
140 energy production is critical for those tissues whose function is heavily dependent on fatty acid oxidation, such as myocardium. Therefore, impaired fatty acid oxidation in the cardiomyopathies involving reduced myocardial level of L-carnitine [3-7], may further contribute to depress the cardiac function. Furthermore, the reduced utilization of long-chain fatty acids leads to the accumulation in the cytosol of potentially toxic substances that contribute to the pathogenesis of cardiac dysfunction [8, 9]. Propionyl-L-carnitine (PLC) is a naturally occurring derivative of L-carnitine to which the propionyl moiety seems to confer three potentially advantageous properties over L-carnitine, namely, a greater affinity for the cardiomyocyte sarcolemmal carrier [10], the ability to replenish mitochondria with intermediates of the citric acid cycle [11] and a greater positive inotropic activity demonstrated both in mechanical recovery after ischemia and in intact isolated hearts [10, 12-14]. Here, we tested the hypothesis that PLC improves the myocardial function secondary to specific metabolic adjustments. Aim of this study was: (1) to assess if free fatty acids are required for the cardiac action of PLC; (2) to study the relationship between changes in mechanical performance and high-energy phosphates content in the heart. We used the isolated perfused rat heart as experimental model to simulate the metabolic derangement due to carnitine deficiency. The hearts were made hypertrophic by pressure overload, a condition known to be associated with altered CoA and carnitine metabolism [15]. The suitability of this model to several biochemical and physiological measurements allows to relate metabolic changes to functional alterations. In addition, the response of the heart to PLC treatment is isolated from the response of the whole organism and can be studied in a greater detail.
Methods Materials PLC was synthesized at Sigma Tau Chemical Drug Dept. (batch 2649). Solutions for animal injection were prepared immediately before use dissolving PLC in saline and buffering pH to 7.4. The heart peffusion buffer contained l l 6 m M NaC1, 4.7raM KC1, 1.2mM KH2PO4, 0.5mM EDTA, 1.2mM Na2SO4, 28.5mM NaHCO3, 3 mM CaC12, 1.2 mM MgC12and 16.6 mM glucose. Half of the hearts were perfused with 1.2 mM palmitate bound to
3% (w/v) bovine albumin added to the medium. All reagents were provided from Sigma Chemical (St. Louis, MO) and were analytical grade. In addition, 2.5/xM PLC and 25/xM L-carnitine were also present in the perfusion buffer.
Animals Twenty male Wistar Kyoto rats (Charles River, Italy) weighing 200-250 g were used in this preliminary study. The animals were fed a standard diet (Altromin MT, Bolzano, Italy) containing approximately 55 nmol/g total L-carnitine. Cardiac hypertrophy was induced by banding the abdominal aorta above the renal arteries. The abdominal cavity was opened and a silver band (0.7 mm internal diameter) was fitted around the aorta. After 4 weeks recovery, the animals were randomly assigned to treatment consisting in one daily administration through an intraarterial catheter of either PLC (50 mg/kg) or saline (control group) for four days. The studies were performed conforming to the guiding principles of the National Society for Medical Research.
Heart perfusion Heart perfusion experiments were performed 24 h after the fourth administration of PLC or saline. Rats were anaesthetized with i.p. heparinized sodium thiopental (100mg/kg). The chest was opened, the aorta was mounted onto a cannula and the perfusion was started retrogradely with the described buffer, pH 7.4, 37° C. The coronary flow rate was controlled by a peristaltic pump. The coronary sinus return was collected through an outflow cannula in the pulmonary artery. A latex balloon was introduced into the left ventricle through the left auricular appendage and connected to a pressure transducer to monitor end-diastolic pressure (EDP), left-ventricular developed pressure (LVDP), heart rate (HR), maximal rate of heart contraction (+ dP/dtmax) and relaxation (-dP/dtmax). The spontaneously contracting hearts were stabilized for 30 min at coronary flow rate = 15 ml/min, with the volume of the ventricular balloon adjusted to achieve EDP -- 0.5 + 0.5 Torr (V0). At the end of the stabilization, the baseline values for the myocardial function were recorded and the intraventricular balloon was filled with accurately measured volumes of saline using a
141 glass gas-tight 1 ml syringe (Hamilton Co, Reno, Nevada) and a micrometer. The selected volumes of the balloon were V0 + ll0I~L (Vl~0) or V0 + 220~1 (V220). The hearts were then allowed to stabilize and a complete series of measurements was taken again.
Metabolic measurements
The 02 uptake (VO2) was evaluated measuring the pO 2 in the arterial inflow and in the coronary sinus return (YSI mod. 5300 Oxygen Monitor, Yellow Springs Inc., OH). In the palmitate experiments, the myocardial content of high-energy phosphate compounds was also determined at the end of the perfusion. The hearts were freeze-clamped in liquid nitrogen, weighed and transferred to a tube containing 2 ml of 0.5 M cold perchloric acid. The content was homogenized using an OMNI 1000 (OMNI Int'l, Waterbury, Connecticut) operating at 20,000 RPM. After 15 min, the suspension was centrifuged, 0.5 ml of the supernatant was neutralized with 0.4 ml of 0.5 M K O H and 0.1 ml of 1M KH2PO4, centrifuged again 30min later, and filtered through 0.22/~m pore size membrane (Nihon Millipore, Japan). ATP, ADP, AMP, creatine and creatine phosphate in myocardial extract were analyzed by HPLC. The equipment (Kontron Instruments, Milano, Italy) was composed of two rood. 420 pumps and a mod. 432 UV/Vis detector set at 210 nm. The 3/~m Supelcosil LC18 column (Supelco, Bellefonte, Pennsylvania) was equilibrated with 0.1 M KHzPO 4 and 5 mM tetrabutylammonium sulphate, the sample (20tzl) was injected and eluted using a composed gradient with a buffer containing 0.1M KH2PO4, 4 m M tetrabutyl-ammonium sulphate and 90% (v/v) CH3CN. The analysis was completed in 25 min and data were analyzed with the Kontron's dedicated software.
Statistics All values are expressed as mean + SEM. Between group comparison was by two-tailed unpaired Student's t-test. The significance was defined as a probability equal or less than 0.05.
Results The described aortic constriction procedure induced a
marked increase of the heart size with respect to normal (50%), indicating the presence of cardiac hypertrophy. As described earlier [1@ this condition was associated with a reduction of left ventricle total carnitine content, that was decreased by about 40% with respect to control sham-operated animals. Two series of heart perfusion experiments were performed. In the first series, the hearts were perfused with Krebs-Henseleit buffer with no palmitate added, and the two groups (PLC-treated and controls, n = 5 for each) were compared at V0 and Vn0- In the second series, ( n = 10) buffer contained 1.2raM palmitate bound to 3% (w/v) albumin, and measurements were made at three volumes of the balloon (0, 110, and 220 pA). Baseline data taken at V0 for the two series are shown in Table 1. In both series, no differences were detected at V0 between the controls and the PLC-treated hearts for any of the parameters evaluated including the double product LVDP x HR, index of the heart systolic function. Coronary pressure as well as contractility indexes were different in the two series of experiments probably because of the effects of fatty acidbound albumin [17]. Figure 1 shows the diastolic function (EDP and -dP/dtma×) monitored at Vl10 in all the groups. No effect of PLC treatment was detected in the absence of palmitate. In contrast, in the presence of palmitate, EDP increased less and the maximal rate of relaxation ( - dP/dtmax) was higher (p < 0.05 for both) in the PLCtreated hearts than in controls. The improvement of the diastolic function was not associated to higher systolic performance as shown by the values of LVDP x H R and + dP/dtmax. In the second series of the experiments carried out in the presence of palmitate, more marked differences were detected between PLC-treated and control hearts when balloon volume was increased by 220 ~1 over V0 (V220): EDP was 53.2_+ 5.0 vs 82.2_+ 8.4 Torr (p
