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Release of fatty acid-binding protein from isolated rat heart subjected to ischemia and reperfusion or to the calcium paradox
148
Biochimica et Biophysics Acta, 961 (1988) 148-152 Elsevier
BBA Report
BBA 50235
Release of fatty acid-binding protein from isolated rat heart subjected to ischemia and reperfusion or to the calcium paradox J.F.C. Glatz a, M. van Bilsen a, R.J.A. Paulussen b, J.H. Veerkamp b, G.J. van der Vusse a and R.S. Reneman a aDepartment
of Physiology,
Unioersity of Limburg, Maastricht
and ’ Department Nijmegen (The Netherlands)
of Biochemistry,
University of Nijmegen,
(Received 4 December 1987) (Revised manuscript received 25 April 1988)
Key words: Fatty acid binding protein; Ischemia; Calcium paradox; (Rat heart)
The release of cardiac fatty acid-binding protein (cFABP) and of fatty acids from isolated rat hearts was measured during both reperfusion following 60 min of ischemia and the calcium paradox (readmission of Ca*+ after a period of Ca *+-free perfusion). Total cFABP release was much more pronouned after Ca*+ readmission (over 50% of tissue content) than during post-ischemic reperfusion (on average, 3% of tissue content), but in both cases, it closely paralleled the release of lactate dehydrogenase. Only minor amounts of long-chain fatty acids, if any, were released from the heart. These observations are challenging the idea that cFABP plays a fatty acid-buffering role under the pathophysiological conditions studied.
Myocardial ischemia, when exceeding 30-45 min, results in significantly increased levels of long-chain fatty acids [l], substances known to be toxic for the heart when present at high concentration [2,3]. Accumulation of fatty acids to similar levels is also found during the calcium paradox, i.e., during readmission of Ca2+-containing fluids after a period of Ca 2+-free perfusion [4]. Neither the precise origin nor the intracellular fate of the accumulated fatty acids is known. Previously, we have postulated that they may be bound to the cardiac fatty acid-binding protein (cFABP) of 15 kDa, which is abundantly present in the cytosolic
Abbreviations: cFABP, cardiac fatty acid-binding protein; LDH, lactate dehydrogenase (EC 1.1.1.27). Correspondence: J.F.C. Glatz, Department of Physiology, University of Limburg, P.O. Box 616, 6200 MD Maastricht, The Netherlands. 00052760/88/$03.50
compartment, as it comprises 4-8% of the cytosolic protein mass [5]. The exact mode of action and physiological significance of this and other FABP types are still unclear [6,7]. Protecting the cardiac structures against deleteriously high levels of fatty acids could be an important function of cFABP. To study the possible role of cFABP in sequestrating fatty acids, we measured the release of cFABP and of fatty acids from isolated rat hearts during both reperfusion following 60 mm of ischemia and the calcium paradox. In each case, the release of cFABP paralleled that of lactate dehydrogenase but, interestingly, only minor amounts of fatty acids, if any, were released from the heart. Hearts were excised from either-anaesthetized male Lewis rats of 240-300 g, and cannulated and perfused at 37 o C, either as isolated working hearts (ischemia/ reperfusion experiments) or according to the Langendorff technique (calcium paradox
0 1988 Eisevier Science Publishers B.V. (Biomedical Division)
149
experiments), as previously described [8-lo]. The standard perfusion medium was a modified Tyrode buffer, containing 28.6 mM NaHCO,, 130 mM NaCl, 5.6 mM KCl, 1.2 mM NaH,PO,, 1.0 mM MgCl,, 1.3 mM (calcium paradox) or 2.2 mM (ischemia,/ reperfusion) CaCl, , supplemented with 11 mM glucose (calcium paradox) or 11 mM glucose and 5 mM pyruvate (ischemia/ reperfusion) and gassed with O,/CO, (95 : 5). The pH of the medium was 7.4. Aortic and left ventricular pressure were measured with catheters connected to external pressure transducers. Aortic flow was measured el~troma~etically. After 30 min of normoxic perfusion at a left atria1 filling pressure (preload) of 1 kPa and a diastolic aortic pressure (afterload) of 8 kPa, the isolated working hearts were subjected to 60 min of global no-flow ischemia at 37 * C, followed by a reperfusion period of 30 min. Reperfusion resulted in a variable recovery of cardiac function. At the end of the reperfusion period, the left ventricular developed pressure (systolic minus diastolic left ventricular pressure) and aortic flow had returned to, on average, 50 and 34% of their pre-ischemic values, respectively. These data are in agreement with previous findings in our laboratory [S]. Coronary perfusate was collected during the last 15 min of the pre-ischemic control period and during 30 min of reperfusion, and sampled in 5 or 10 mill aliquots. In the calcium paradox experiments, the hearts were perfused with standard perfusate for 20 min (perfusion pressure, 8 kPa). Thereafter, perfusion was continued with a Ca2+-free solution for 10 min, and finally the hearts were perfused again with the Ca24-containing perfusate, also for 10 min. Effluent was collected during the last 5 min of the initial perfusion period, the first and second 5 min of the Cazf-free period and the first and second 5 min after reintroduction of Ca2+. All effluent samples were stored at - 80 OC until analysis. The content of cFABP in the coronary effluent samples was measured with an enzyme-linked immunosorbent assay, using rabbit anti-rat cFABP antiserum [6], as will be described elsewhere. The detection limit was 0.3 pmol (5 ng)/ml, and the recovery of pure cFABP added to the effluent samples amounted to 92 rf: 13% (mean f S.D. of
three determinations). Molecular weight of cFABP was taken as 14992 [ll]. Lactate dehydrogenase activity in the coronary effluent was measured according to the method of Bergmeyer and Bernt [12]. Citrate synthase (EC 4.1.3.7) activity was assayed in supematants of sonicated effluent samples according to the method of Shepherd and Garland [13]. Lipids were determined in 5 ml of the perfusate samples as earlier described [14,15]. In brief, after extraction with c~oroform/meth~ol (2 : 1, v/v), phospholipids were separated from neutral lipids by stepwise elution from a silica gel column. Triacylglycerols and fatty acids were further separated by thin-layer chromatography [14]. Phospholipids were quantitated by measuring the phosphorus content according to Bartlett 1161; triacylglycerols and fatty acids were determined by gas-liquid chromato~aphy [ 151. During normoxic control perfusion, either with Ca2+-containing or Ca 2+-free buffer, the release of cFABP into the coronary perfusate was negligible (Fig. 1). However, variable but significant amounts of cFABP were released during reperfusion following a 60 min period of ischemia. The maximal release was measured between 5 and 10 min after the start of reperfusion (Fig. 1). Readmission of Ca2+ after 10 min of Ca2+-free perfusion resulted in a marked release of cFABP (Fig. 1). The maximal depletion occurred during the first 5 min after Ca*+ reintroduction and was about 30-times as high as that observed during post-ischemic reperfusion. For comparison, the release of the cytosolic marker enzyme lactate dehydrogenase (LDH), the activity of which is commonly assayed in coronary perfusates to assess the extent of myocardial cell damage [17], was also measured. In the ischemia/ reperfusion and the calcium paradox experiments, the general pattern of release of LDH was similar to that of cFABP (Fig. 1). Furthermore, the 21-fold higher total amount of LDH released after induction of the calcium paradox as compared with the ischemia experiments corresponds with the 19-fold higher total release of cFABP under these circumstances (Table I). From each individual experiment, it also appeared that both proteins were continuously released at a fixed ratio (data not shown).
150
ISCHEMIA+
REPERFUSION
CALCIUM PARADOX
407
20-
-15
0
60
70
60
90 min
min
o-dI -5
0
10
20 min
min
Fig. 1. Time-course of the release of LDH and cFABP from isolated rat hearts subjected to ischemia and reperfusion and to the calcium paradox. Results are expressed as meansfS.D. of four and five experiments, respectively. Note the 20-fold difference in scale between each of the left and right panels. I, ischemic period.
As the rat hearts (average wet weight of 1.0 g) contained about 100 nmol (1500 pg) of cFABP [l&19] and 355 + 60 U of LDH (mean _t S.D. for eight animals), the relative release of their total tissue content was, on average, 2.9% for cFABP
TABLE
I
TOTAL RELEASE OF LDH, cFABP AND FATTY ACIDS FROM RAT HEART AS INDUCED BY REPERFUSION FOLLOWING 60 min OF ISCHEMIA OR BY THE CALCIUM PARADOX Values represent the amounts released during a 30 min reperfusion period in the ischernia/reperfusion experiments and a 10 min Ca*+ readmission period in the calcium paradox experiment. Data are given as means* S.D. of the number of experiments indicated within parentheses. n.m., not measurable (below the detection limit of 3 nmol fatty acids per 5 ml effluent). Experiment type
Total release per heart LDH (units)
Ischemia/reperfusion Calcium paradox [5]
[4]
cFABP
fatty acids
(nmol)
(nmol)
12.2k 9.9 2.9k 4.4 n.m. 263 +48 55.7 f 13.6 4+ 5
and 3.4% for LDH during post-ischemic reperfusion, and 56% for cFABP and 74% for LDH during Ca2+ -repletion. Because of the marked interindividual variation, these values are mutually not significantly different. The mitochondrial matrix enzyme citrate synthase could not be detected in any of the effluent samples (data not shown), indicating that neither mitochondria nor their matrix proteins were released from the heart upon cell injury. Only minor amounts of fatty acids could be detected in the perfusate samples collected during the calcium paradox experiments after readmission of Ca *+ (Table I). The a mounts observed were close to the detection limit of our assay of 3 nmol fatty acids per 5 ml sample [14,15]. Fatty acids were not found in the effluent collected during post-ischemic reperfusion. Triacylglycerols and phospholipids were not found in any of the effluent samples. Upon tissue injury, cytosolic proteins will be released from damaged cells into the circulation, The total amount of proteins released is generally
151
a good reflection of the severity of the injury. Therefore, the release of activity of the intracellular enzyme LDH is often taken as a measure of myocardial tissue damage [17]. In the present study, we observed that the release of the 15 kDa cFABP from rat heart closely parallels that of the much larger (138 kDa) LDH, after both modest (reperfusion following ischemia) and considerable cell injury (induction of calcium paradox). In the latter case, more than half of the total tissue content of these cytosolic proteins could be detected in the coronary perfusate. Since proteins from the mitochondrial matrix, such as citrate synthase, were not released, the similarity between the release of LDH and cFABP strongly suggests that in rat heart, all cFABP is present in the cytosolic compartment, and not partly within ~t~hon~a, as was proposed by Fournier et al. [20] on the basis of immunocytochemical analysis. Our findings indicate that myocardial tissue damage can be estimated from the release of cFABP. In the myocardium, endogenous long-chain fatty acid levels significantly increase during prolonged ischemia [1,15]. After 60 min of ischemia, the mean total (non-esterified) fatty acid content of rat heart had increased from 40 to 220 nmol/g wet weight [l]. Theoretically, these fatty acids could to a large extent be bound by the 100 nmol of cFABP, which is assumed to have one [6] or two 1211 fatty acid-binding sites per protein molecule. During subsequent reperfusion, only a few nmol of cFABP, and no measurable amounts of fatty acids, were released into the perfusate. Hence, these observations do not allow a conclusion on the role of cFABP under these circumstances. In calcium paradox experiments, similar changes in endogenous fatty acid levels occur. The fatty acid content of Ca2+-depleted hearts of about 50 nmol/g wet weight further increases to about 270 nmol/g wet weight during 10 min of Ca*+ suppletion [4]. In this time interval, only about 4 nmol of fatty acids could be detected in the effluent, whereas more than half of the tissue content (or about 56 nmol) of cFABP was released. This observation indicates that fatty acids were either accumulating at cellular sites not accessible to cFABP or stripped from cFABP prior to its release from the tissue, e.g., by binding to mem-
branous structures. Alternatively, cFABP could be released from cells prior to the occurrence of the mass accumulation of fatty acids. Therefore, the proposed protective function of cFABP in sequestrating fatty acids accumulating in injured myocardial cells seems questionable. In summary, cFABP was shown to be released from myocardial tissue upon cellular injury at a rate and to an extent similar to that of LDH. The corrolary of this finding is that the plasma level of cFABP might be used to estimate myocardial infarct size in patients. Upon severe injury of myocardial cells during the calcium paradox, the release of cFABP was not associated with a release of fatty acids, despite elevated tissue levels. This finding does not sustain a role of cFABP in the binding of accumulating intracellular fatty acids under the pathophysiolo~cal circ~st~ces presently studied. Acknowledgements We would like to thank Lucienne de Boer for her help in preparing the manusc~pt. This study was supported by grant No. 900-516-091 from Medigon/ZWO. References Van der Vusse, G.J., Prinzen, F.W., Van Bilsen, M., Engels, W. and Reneman, R.S. (1987) Basic Res. Cardiol. 82 Suppl. 1,157-167. Katz, A.M. and Messineo, F.C. (1981) Circ. Res. 48, l-16. Piper, H.M., Sezer, O., Schwartz, P., Huetter, J.F. and Spieekermann, P.G. (1983) B&him. Biophys. Acta 732, 193-203. Van der Vusse, G.J., Van B&en, M. and Reneman, R.S. (1987) J. Mol. Cell Cardiol. 19, S. 100 (abstr.). Glatz, J.F.C., Paulussen, R.J.A. and Veerkamp, J.H. (1985) Chem. Phys. Lipids 38, 115-129. Veerkamp, J.H. and Paulussen, R.J.A. (1987) B&hem. Sot. Trans. 15, 331-336. Sweetser, D.A., Heuckeroth, R.O. and Gordon, J.I. (1987) Annu. Rev. Nutr. 7, 337-359. Snoeckx, L.H.E.H., Van der Vusse, G.J., Coumans, W.A., Willemsen, P.H.M., Van der Nagel, T. and Reneman, R.S. (1986) Cardiovasc. Res. 20, 67-75. Van B&en, M., Engels, W., Willemsen, P.H.M., Coumans, W.A., Van der Vusse, G.J. and Reneman, R.S. (1987) Progr. Appl. Microcirc. 12, 236-243. 10 Engels, W., Van B&en, M., Van der Vusse, G.J., Willemsen, P.H.M., Coumans, W.A., Kamps, M.A.F., Endert, J. and Reneman, R.S. (1987) Basic Res. Cardiol. 82 suppl. 1, 245-251.
152 11 Sacchettini, J.C., Said, B., Schulz, H. and Gordon, J.I. (1986) J. Biol. Chem. 261, 8218-8223. 12 Bergmeyer, H.-U. and Bemt, E. (1974) in Methods of Enzymatic Analysis (Bergmeyer, H.-U., ed.), Vol. 1, 3rd Edn., pp. 607-612, Verlag Chemie, Weinheim. 13 Shepherd, D. and Garland, P.B. (1969) Methods Enzymol. 13,11-13. 14 Roemen, T.H.M. and Van der Vusse, G.J. (1985) J. Chromatogr. 344, 304-308. 15 Van der Vusse, G.J., Roemen. T.H.M., Prinzen, F.W., Coumans, W.A. and Reneman, R.S. (1982) Circ. Res. 50, 538-546.
16 Bartlett, G.R. (1959) J. Biol. Chem. 234, 466-468. 17 Waldenstroem, A.P., Hjalmarson, A.C., Jodal, M. and Waldenstroem, J. (1977) Acta Med. Stand. 201, 525-532. 18 Paulussen, R.J.A., Jansen, G.P.M. and Veerkamp, J.H. (1986) B&him. Biophys. Acta 877, 342-349. 19 Crisman, T.S., Claffey, K.P., Saouaf, R., Hanspal, J. and Brecher, P. (1987) J. Mol. Cell. Cardiol. 19, 423-431. 20 Foumier, N.C. and Rahim, M. (1985) Biochemistry 24, 2387-2396. 21 Offner, G.D., Troxler, R.F. and Brecher, P. (1986) J. Biol. Chem. 261. 5584-5586.
Biochimica et Biophysics Acta, 961 (1988) 148-152 Elsevier
BBA Report
BBA 50235
Release of fatty acid-binding protein from isolated rat heart subjected to ischemia and reperfusion or to the calcium paradox J.F.C. Glatz a, M. van Bilsen a, R.J.A. Paulussen b, J.H. Veerkamp b, G.J. van der Vusse a and R.S. Reneman a aDepartment
of Physiology,
Unioersity of Limburg, Maastricht
and ’ Department Nijmegen (The Netherlands)
of Biochemistry,
University of Nijmegen,
(Received 4 December 1987) (Revised manuscript received 25 April 1988)
Key words: Fatty acid binding protein; Ischemia; Calcium paradox; (Rat heart)
The release of cardiac fatty acid-binding protein (cFABP) and of fatty acids from isolated rat hearts was measured during both reperfusion following 60 min of ischemia and the calcium paradox (readmission of Ca*+ after a period of Ca *+-free perfusion). Total cFABP release was much more pronouned after Ca*+ readmission (over 50% of tissue content) than during post-ischemic reperfusion (on average, 3% of tissue content), but in both cases, it closely paralleled the release of lactate dehydrogenase. Only minor amounts of long-chain fatty acids, if any, were released from the heart. These observations are challenging the idea that cFABP plays a fatty acid-buffering role under the pathophysiological conditions studied.
Myocardial ischemia, when exceeding 30-45 min, results in significantly increased levels of long-chain fatty acids [l], substances known to be toxic for the heart when present at high concentration [2,3]. Accumulation of fatty acids to similar levels is also found during the calcium paradox, i.e., during readmission of Ca2+-containing fluids after a period of Ca 2+-free perfusion [4]. Neither the precise origin nor the intracellular fate of the accumulated fatty acids is known. Previously, we have postulated that they may be bound to the cardiac fatty acid-binding protein (cFABP) of 15 kDa, which is abundantly present in the cytosolic
Abbreviations: cFABP, cardiac fatty acid-binding protein; LDH, lactate dehydrogenase (EC 1.1.1.27). Correspondence: J.F.C. Glatz, Department of Physiology, University of Limburg, P.O. Box 616, 6200 MD Maastricht, The Netherlands. 00052760/88/$03.50
compartment, as it comprises 4-8% of the cytosolic protein mass [5]. The exact mode of action and physiological significance of this and other FABP types are still unclear [6,7]. Protecting the cardiac structures against deleteriously high levels of fatty acids could be an important function of cFABP. To study the possible role of cFABP in sequestrating fatty acids, we measured the release of cFABP and of fatty acids from isolated rat hearts during both reperfusion following 60 mm of ischemia and the calcium paradox. In each case, the release of cFABP paralleled that of lactate dehydrogenase but, interestingly, only minor amounts of fatty acids, if any, were released from the heart. Hearts were excised from either-anaesthetized male Lewis rats of 240-300 g, and cannulated and perfused at 37 o C, either as isolated working hearts (ischemia/ reperfusion experiments) or according to the Langendorff technique (calcium paradox
0 1988 Eisevier Science Publishers B.V. (Biomedical Division)
149
experiments), as previously described [8-lo]. The standard perfusion medium was a modified Tyrode buffer, containing 28.6 mM NaHCO,, 130 mM NaCl, 5.6 mM KCl, 1.2 mM NaH,PO,, 1.0 mM MgCl,, 1.3 mM (calcium paradox) or 2.2 mM (ischemia,/ reperfusion) CaCl, , supplemented with 11 mM glucose (calcium paradox) or 11 mM glucose and 5 mM pyruvate (ischemia/ reperfusion) and gassed with O,/CO, (95 : 5). The pH of the medium was 7.4. Aortic and left ventricular pressure were measured with catheters connected to external pressure transducers. Aortic flow was measured el~troma~etically. After 30 min of normoxic perfusion at a left atria1 filling pressure (preload) of 1 kPa and a diastolic aortic pressure (afterload) of 8 kPa, the isolated working hearts were subjected to 60 min of global no-flow ischemia at 37 * C, followed by a reperfusion period of 30 min. Reperfusion resulted in a variable recovery of cardiac function. At the end of the reperfusion period, the left ventricular developed pressure (systolic minus diastolic left ventricular pressure) and aortic flow had returned to, on average, 50 and 34% of their pre-ischemic values, respectively. These data are in agreement with previous findings in our laboratory [S]. Coronary perfusate was collected during the last 15 min of the pre-ischemic control period and during 30 min of reperfusion, and sampled in 5 or 10 mill aliquots. In the calcium paradox experiments, the hearts were perfused with standard perfusate for 20 min (perfusion pressure, 8 kPa). Thereafter, perfusion was continued with a Ca2+-free solution for 10 min, and finally the hearts were perfused again with the Ca24-containing perfusate, also for 10 min. Effluent was collected during the last 5 min of the initial perfusion period, the first and second 5 min of the Cazf-free period and the first and second 5 min after reintroduction of Ca2+. All effluent samples were stored at - 80 OC until analysis. The content of cFABP in the coronary effluent samples was measured with an enzyme-linked immunosorbent assay, using rabbit anti-rat cFABP antiserum [6], as will be described elsewhere. The detection limit was 0.3 pmol (5 ng)/ml, and the recovery of pure cFABP added to the effluent samples amounted to 92 rf: 13% (mean f S.D. of
three determinations). Molecular weight of cFABP was taken as 14992 [ll]. Lactate dehydrogenase activity in the coronary effluent was measured according to the method of Bergmeyer and Bernt [12]. Citrate synthase (EC 4.1.3.7) activity was assayed in supematants of sonicated effluent samples according to the method of Shepherd and Garland [13]. Lipids were determined in 5 ml of the perfusate samples as earlier described [14,15]. In brief, after extraction with c~oroform/meth~ol (2 : 1, v/v), phospholipids were separated from neutral lipids by stepwise elution from a silica gel column. Triacylglycerols and fatty acids were further separated by thin-layer chromatography [14]. Phospholipids were quantitated by measuring the phosphorus content according to Bartlett 1161; triacylglycerols and fatty acids were determined by gas-liquid chromato~aphy [ 151. During normoxic control perfusion, either with Ca2+-containing or Ca 2+-free buffer, the release of cFABP into the coronary perfusate was negligible (Fig. 1). However, variable but significant amounts of cFABP were released during reperfusion following a 60 min period of ischemia. The maximal release was measured between 5 and 10 min after the start of reperfusion (Fig. 1). Readmission of Ca2+ after 10 min of Ca2+-free perfusion resulted in a marked release of cFABP (Fig. 1). The maximal depletion occurred during the first 5 min after Ca*+ reintroduction and was about 30-times as high as that observed during post-ischemic reperfusion. For comparison, the release of the cytosolic marker enzyme lactate dehydrogenase (LDH), the activity of which is commonly assayed in coronary perfusates to assess the extent of myocardial cell damage [17], was also measured. In the ischemia/ reperfusion and the calcium paradox experiments, the general pattern of release of LDH was similar to that of cFABP (Fig. 1). Furthermore, the 21-fold higher total amount of LDH released after induction of the calcium paradox as compared with the ischemia experiments corresponds with the 19-fold higher total release of cFABP under these circumstances (Table I). From each individual experiment, it also appeared that both proteins were continuously released at a fixed ratio (data not shown).
150
ISCHEMIA+
REPERFUSION
CALCIUM PARADOX
407
20-
-15
0
60
70
60
90 min
min
o-dI -5
0
10
20 min
min
Fig. 1. Time-course of the release of LDH and cFABP from isolated rat hearts subjected to ischemia and reperfusion and to the calcium paradox. Results are expressed as meansfS.D. of four and five experiments, respectively. Note the 20-fold difference in scale between each of the left and right panels. I, ischemic period.
As the rat hearts (average wet weight of 1.0 g) contained about 100 nmol (1500 pg) of cFABP [l&19] and 355 + 60 U of LDH (mean _t S.D. for eight animals), the relative release of their total tissue content was, on average, 2.9% for cFABP
TABLE
I
TOTAL RELEASE OF LDH, cFABP AND FATTY ACIDS FROM RAT HEART AS INDUCED BY REPERFUSION FOLLOWING 60 min OF ISCHEMIA OR BY THE CALCIUM PARADOX Values represent the amounts released during a 30 min reperfusion period in the ischernia/reperfusion experiments and a 10 min Ca*+ readmission period in the calcium paradox experiment. Data are given as means* S.D. of the number of experiments indicated within parentheses. n.m., not measurable (below the detection limit of 3 nmol fatty acids per 5 ml effluent). Experiment type
Total release per heart LDH (units)
Ischemia/reperfusion Calcium paradox [5]
[4]
cFABP
fatty acids
(nmol)
(nmol)
12.2k 9.9 2.9k 4.4 n.m. 263 +48 55.7 f 13.6 4+ 5
and 3.4% for LDH during post-ischemic reperfusion, and 56% for cFABP and 74% for LDH during Ca2+ -repletion. Because of the marked interindividual variation, these values are mutually not significantly different. The mitochondrial matrix enzyme citrate synthase could not be detected in any of the effluent samples (data not shown), indicating that neither mitochondria nor their matrix proteins were released from the heart upon cell injury. Only minor amounts of fatty acids could be detected in the perfusate samples collected during the calcium paradox experiments after readmission of Ca *+ (Table I). The a mounts observed were close to the detection limit of our assay of 3 nmol fatty acids per 5 ml sample [14,15]. Fatty acids were not found in the effluent collected during post-ischemic reperfusion. Triacylglycerols and phospholipids were not found in any of the effluent samples. Upon tissue injury, cytosolic proteins will be released from damaged cells into the circulation, The total amount of proteins released is generally
151
a good reflection of the severity of the injury. Therefore, the release of activity of the intracellular enzyme LDH is often taken as a measure of myocardial tissue damage [17]. In the present study, we observed that the release of the 15 kDa cFABP from rat heart closely parallels that of the much larger (138 kDa) LDH, after both modest (reperfusion following ischemia) and considerable cell injury (induction of calcium paradox). In the latter case, more than half of the total tissue content of these cytosolic proteins could be detected in the coronary perfusate. Since proteins from the mitochondrial matrix, such as citrate synthase, were not released, the similarity between the release of LDH and cFABP strongly suggests that in rat heart, all cFABP is present in the cytosolic compartment, and not partly within ~t~hon~a, as was proposed by Fournier et al. [20] on the basis of immunocytochemical analysis. Our findings indicate that myocardial tissue damage can be estimated from the release of cFABP. In the myocardium, endogenous long-chain fatty acid levels significantly increase during prolonged ischemia [1,15]. After 60 min of ischemia, the mean total (non-esterified) fatty acid content of rat heart had increased from 40 to 220 nmol/g wet weight [l]. Theoretically, these fatty acids could to a large extent be bound by the 100 nmol of cFABP, which is assumed to have one [6] or two 1211 fatty acid-binding sites per protein molecule. During subsequent reperfusion, only a few nmol of cFABP, and no measurable amounts of fatty acids, were released into the perfusate. Hence, these observations do not allow a conclusion on the role of cFABP under these circumstances. In calcium paradox experiments, similar changes in endogenous fatty acid levels occur. The fatty acid content of Ca2+-depleted hearts of about 50 nmol/g wet weight further increases to about 270 nmol/g wet weight during 10 min of Ca*+ suppletion [4]. In this time interval, only about 4 nmol of fatty acids could be detected in the effluent, whereas more than half of the tissue content (or about 56 nmol) of cFABP was released. This observation indicates that fatty acids were either accumulating at cellular sites not accessible to cFABP or stripped from cFABP prior to its release from the tissue, e.g., by binding to mem-
branous structures. Alternatively, cFABP could be released from cells prior to the occurrence of the mass accumulation of fatty acids. Therefore, the proposed protective function of cFABP in sequestrating fatty acids accumulating in injured myocardial cells seems questionable. In summary, cFABP was shown to be released from myocardial tissue upon cellular injury at a rate and to an extent similar to that of LDH. The corrolary of this finding is that the plasma level of cFABP might be used to estimate myocardial infarct size in patients. Upon severe injury of myocardial cells during the calcium paradox, the release of cFABP was not associated with a release of fatty acids, despite elevated tissue levels. This finding does not sustain a role of cFABP in the binding of accumulating intracellular fatty acids under the pathophysiolo~cal circ~st~ces presently studied. Acknowledgements We would like to thank Lucienne de Boer for her help in preparing the manusc~pt. This study was supported by grant No. 900-516-091 from Medigon/ZWO. References Van der Vusse, G.J., Prinzen, F.W., Van Bilsen, M., Engels, W. and Reneman, R.S. (1987) Basic Res. Cardiol. 82 Suppl. 1,157-167. Katz, A.M. and Messineo, F.C. (1981) Circ. Res. 48, l-16. Piper, H.M., Sezer, O., Schwartz, P., Huetter, J.F. and Spieekermann, P.G. (1983) B&him. Biophys. Acta 732, 193-203. Van der Vusse, G.J., Van B&en, M. and Reneman, R.S. (1987) J. Mol. Cell Cardiol. 19, S. 100 (abstr.). Glatz, J.F.C., Paulussen, R.J.A. and Veerkamp, J.H. (1985) Chem. Phys. Lipids 38, 115-129. Veerkamp, J.H. and Paulussen, R.J.A. (1987) B&hem. Sot. Trans. 15, 331-336. Sweetser, D.A., Heuckeroth, R.O. and Gordon, J.I. (1987) Annu. Rev. Nutr. 7, 337-359. Snoeckx, L.H.E.H., Van der Vusse, G.J., Coumans, W.A., Willemsen, P.H.M., Van der Nagel, T. and Reneman, R.S. (1986) Cardiovasc. Res. 20, 67-75. Van B&en, M., Engels, W., Willemsen, P.H.M., Coumans, W.A., Van der Vusse, G.J. and Reneman, R.S. (1987) Progr. Appl. Microcirc. 12, 236-243. 10 Engels, W., Van B&en, M., Van der Vusse, G.J., Willemsen, P.H.M., Coumans, W.A., Kamps, M.A.F., Endert, J. and Reneman, R.S. (1987) Basic Res. Cardiol. 82 suppl. 1, 245-251.
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