Cardiovascular Research, 1977, 11, 172-176
Lactate dehydrogenase isoenzymes in the human hypertrophic heart N . W . REVIS', R . Y .T H O M S O N , A N D A. J. V. C A M E R O N From the Cardiac Department, Western Infirmary, Glasgow. and Department of Biochemistry, University of Glasgow, Glasgow
Lactate dehydrogenase isoenzymes have been measured in human hearts at autopsy. As compared with normal hearts, hypertrophic hearts a n d ischaemic hypertrophic hearts showed a shift in the isoenzyme distribution. This shift appeared to be more closely associated with an increase in the relative proportion of fibrous tissue than with changes in the myocardial cells.
SUMMARY
Lactate dehydrogenase catalyses the interconversion of lactate and pyruvate. There are 5 isoenzymes, formed as the 2 different subunits, H and M, combined in tetramers. Early studies suggested that H subunits predominate in tissues (eg heart) which oxidise lactate via pyruvate to COa and H20, whereas M subunits predominate in tissues (eg skeletal muscle) which convert glucose to lactate via pyruvate (Dawson et al., 1964). The finding of Goodfriend et al. (1966) that exposure of heart cells in culture to low oxygen tension provokes an increase in M subunits would support the idea that the pattern of lactate dehydrogenase isoenzymes is related to the metabolic activity of the tissue. It has long been believed that during cardiac hypertrophy a decrease in ratio of capillaries to myofibrils leads to hypoxia in the myocardium as a result of the increase in the diffusion distance from the capillary lumen to the centre of the hypertrophied area (Warn, 1940; Woods, 1961). This might be expected to result in an isoenzyme pattern shift. Such a shift was sought by Bello and Messer (1968) in hearts from patients with ischaemic heart disease and myocardial hypertrophy. They found an increase in M subunits only if both conditions were present; neither hypertrophy nor ischaemia by itself produced a significant shift. It was concluded that these changes were associated with increased anaerobic glycolysis as a result of marked cardiac hypoxia. The present experiments were designed to reinvestigate this question, in particular by looking at hearts in which hypertrophy was more marked than in those examined by Bello and Messer and
by determining whether any change in isoenzyme pattern reflected changes in the myocardial cells or in other cell types. Methods
Subjects were selected on the following criteria. The controls were patients who had died accidentally or by violence. The heart weight (range 290 to 330g) and coronary arteries were normal. Patients in the second group all had marked cardiac hypertrophy (range 570 to 800 g) but normal coronary arteries. The patients in the third group showed marked ischaemia and moderate hypertrophy (range 420 to 540 g). The term marked ischaemia was defined by severe coronary atheroma or occlusion with recent or old infarction. At autopsy, which was performed within 7 to 24 h of death, hearts were dissected out, rinsed with ice-cold 0.25 mol sucrose blotted and weighed, the left and right ventricular wall thickness recorded. The left ventricular free wall was then cut into small slices and homogenated in ice-cold 0.1 mol phosphate buffer pH 7.4 using an ultra-turrax homogeniser. The homogenate was centrifuged at 20 OOO g for 30 min. The supernatant was decanted, diluted 1 :50, and 10 plitre aliquots were used to determine the lactate dehydrogenase (LDH) isoenzymes. The LDH isoenzymes were separated electrophoretically and stained according to the method of Dietz and Lubrano (1967). Helu cells (human embryonic lung cells which have been replaced by predominately fibroblasts) of the fourth passage were suspended in ice-cold 'Address for correspondence and reprints: N. W. Revis, 0.25 mol sucrose (approximately and centriUniversity of Tennesste Biomedical Graduate School, Biology Division, PO Box Y,Oak Ridge, Tennessee 37830, fuged at 300g for 10 min and the supernatant decanted. This process was repeated 3 times. After USA. 172
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Lactate dehydrogenase in cardiac hypertrophy
the third wash the cells were suspended in ice-cold 0.1 mol phosphate buffer pH 7.4 and homogenised using a Dounce (B) homogeniser by 10 up and down strokes. The homogenate was centrifuged at 20 OOO g for 30 min and the supernatant was used to measure the LDH isoenzymes as previously described. After electrophoresis and staining, gels were scanned with a Joyce Lobel densitometer with integrator attachment and a 595 nm filter. The percentage of each isoenzyme was determined directly from the integrator marks. The relative amounts of H and M subunits were calculated as described by Appella and Markert (1961) and Cahn et al. (1962). Total LDH activity was assayed by measuring the disappearance of NADH at 340 nm as described by Wrbblewski and LaDue (1955) in the same supernatant that was used for the separation of the LDH isoenzymes. The incubation mixture contained 0.1 mol phosphate buffer pH 7.5, NADH 20 mmol, and low (0.4mmol) or high (4.0mmol) concentration of sodium pyruvate. At 7, 10, 15, 20, and 30 h of death the percentage of H and M subunits and total lactate dehydrogenase activity in the supernatants were not significantly different within the 3 groups, although differences were recorded between the various groups. After 30 h the M subunits were decreased by 30% within the 3 groups while total LDH activity remained normal. Hydroxyproline was used as a measure of fibrous tissue. This amino acid is found exclusively in collagen in which it occurs in a constant proportion of 13.4% by weight (Neuman and Logan, 1950). The concentration of collagen and thus fibrous tissue in the myocardium can therefore be estimated by determination of hydroxyproline. The ventricles were removed and weighed and dried at 105°C to a constant weight. The dry tissue was hydrolysed in 6N HCI for a period of 16 h in sealed test-tubes at a temperature of 105°C; 3 cm3 of 6N HCI were used for each 100 mg of tissue. Hydroxyproline was determined in an aliquot after neutralisation according to the method of Stegemann (1958) as described by Woessner (1961). For histology the left ventricular free wall was divided into 3 parts, anterior, lateral, and posterior. Following fixation and dehydration 6 pm sections from each part were stained for collagen using Masson trichrome stain and counterstained with Haemalum. After staining, sections were examined under a binocular Watson-Barnett microscope (magnification x 100) to determine the relative proportions of muscle cells, fibrous tissue (collagen and fibroblasts), and other cellular elements (blood cells and endothelial cells) in each section. This was
done by inserting a grid into 1 of the eyepieces of the microscope. Twentyfive fields were examined in each section and in each field the number of grid intersections that fell upon muscle cells, fibrous tissue, and other cellular elements were recorded.
Results The heart weight and left and right ventricular wall thickness in the ‘marked hypertrophy’ group are all far greater than in the ‘marked ischaemia/moderate hypertrophy’ group (Table 1). Table 2 shows the LDH isoenzyme pattern in each group. The markedly hypertrophic hearts show significantly less of the isoenzyme I than the controls. Similar changes were found in the ischaemia/ moderate hypertrophy group. Fig. 1 shows the relative proportions of M subunits. In both the ‘marked hypertrophy’ and the ‘marked ischaemia/moderate hypertrophy’ groups the proportions of M subunits are significantly greater than the controls. The increase, however, is not proportional to the increase in heart weight. It is significantly greater in hypertrophy’ ‘marked ischaemia / moderate (P< 0.0005) than in ‘marked hypertrophy’ group. These conclusions were confirmed by observing the extent to which LDH activity is inhibited in each case by pyruvate. It has been shown that relatively high concentrations of pyruvate inhibit the H subunits of LDH (Dawson et al., 1964). This inhibition has been shown by studies in vitro to be directly proportional to the percentage of H subunits present in the enzyme (Plagemann et al., 1960). Table 3 shows that both the marked hypertrophy and
Table 1 Heart weight and ventricular wall thickness in patient groups studied Patient groups
Controls
Marked Marked hypertrophy ischaemial moderate hypertrophy
__
-_ Number of cases 19
10
14
Heart weight (g)
320 i40
669 f 80
*
477t
1.5 10.05
2.8f
*0.10
2.1* 10.07
0.21t
0.18.
Left ventricular wall Thickness (cm)
Right ventricular wall 0.14 Thickness (cm) 10.01
*0.04
* 50
*0.03
Mean +SEM. *P
Lactate dehydrogenase isoenzymes in the human hypertrophic heart N . W . REVIS', R . Y .T H O M S O N , A N D A. J. V. C A M E R O N From the Cardiac Department, Western Infirmary, Glasgow. and Department of Biochemistry, University of Glasgow, Glasgow
Lactate dehydrogenase isoenzymes have been measured in human hearts at autopsy. As compared with normal hearts, hypertrophic hearts a n d ischaemic hypertrophic hearts showed a shift in the isoenzyme distribution. This shift appeared to be more closely associated with an increase in the relative proportion of fibrous tissue than with changes in the myocardial cells.
SUMMARY
Lactate dehydrogenase catalyses the interconversion of lactate and pyruvate. There are 5 isoenzymes, formed as the 2 different subunits, H and M, combined in tetramers. Early studies suggested that H subunits predominate in tissues (eg heart) which oxidise lactate via pyruvate to COa and H20, whereas M subunits predominate in tissues (eg skeletal muscle) which convert glucose to lactate via pyruvate (Dawson et al., 1964). The finding of Goodfriend et al. (1966) that exposure of heart cells in culture to low oxygen tension provokes an increase in M subunits would support the idea that the pattern of lactate dehydrogenase isoenzymes is related to the metabolic activity of the tissue. It has long been believed that during cardiac hypertrophy a decrease in ratio of capillaries to myofibrils leads to hypoxia in the myocardium as a result of the increase in the diffusion distance from the capillary lumen to the centre of the hypertrophied area (Warn, 1940; Woods, 1961). This might be expected to result in an isoenzyme pattern shift. Such a shift was sought by Bello and Messer (1968) in hearts from patients with ischaemic heart disease and myocardial hypertrophy. They found an increase in M subunits only if both conditions were present; neither hypertrophy nor ischaemia by itself produced a significant shift. It was concluded that these changes were associated with increased anaerobic glycolysis as a result of marked cardiac hypoxia. The present experiments were designed to reinvestigate this question, in particular by looking at hearts in which hypertrophy was more marked than in those examined by Bello and Messer and
by determining whether any change in isoenzyme pattern reflected changes in the myocardial cells or in other cell types. Methods
Subjects were selected on the following criteria. The controls were patients who had died accidentally or by violence. The heart weight (range 290 to 330g) and coronary arteries were normal. Patients in the second group all had marked cardiac hypertrophy (range 570 to 800 g) but normal coronary arteries. The patients in the third group showed marked ischaemia and moderate hypertrophy (range 420 to 540 g). The term marked ischaemia was defined by severe coronary atheroma or occlusion with recent or old infarction. At autopsy, which was performed within 7 to 24 h of death, hearts were dissected out, rinsed with ice-cold 0.25 mol sucrose blotted and weighed, the left and right ventricular wall thickness recorded. The left ventricular free wall was then cut into small slices and homogenated in ice-cold 0.1 mol phosphate buffer pH 7.4 using an ultra-turrax homogeniser. The homogenate was centrifuged at 20 OOO g for 30 min. The supernatant was decanted, diluted 1 :50, and 10 plitre aliquots were used to determine the lactate dehydrogenase (LDH) isoenzymes. The LDH isoenzymes were separated electrophoretically and stained according to the method of Dietz and Lubrano (1967). Helu cells (human embryonic lung cells which have been replaced by predominately fibroblasts) of the fourth passage were suspended in ice-cold 'Address for correspondence and reprints: N. W. Revis, 0.25 mol sucrose (approximately and centriUniversity of Tennesste Biomedical Graduate School, Biology Division, PO Box Y,Oak Ridge, Tennessee 37830, fuged at 300g for 10 min and the supernatant decanted. This process was repeated 3 times. After USA. 172
173
Lactate dehydrogenase in cardiac hypertrophy
the third wash the cells were suspended in ice-cold 0.1 mol phosphate buffer pH 7.4 and homogenised using a Dounce (B) homogeniser by 10 up and down strokes. The homogenate was centrifuged at 20 OOO g for 30 min and the supernatant was used to measure the LDH isoenzymes as previously described. After electrophoresis and staining, gels were scanned with a Joyce Lobel densitometer with integrator attachment and a 595 nm filter. The percentage of each isoenzyme was determined directly from the integrator marks. The relative amounts of H and M subunits were calculated as described by Appella and Markert (1961) and Cahn et al. (1962). Total LDH activity was assayed by measuring the disappearance of NADH at 340 nm as described by Wrbblewski and LaDue (1955) in the same supernatant that was used for the separation of the LDH isoenzymes. The incubation mixture contained 0.1 mol phosphate buffer pH 7.5, NADH 20 mmol, and low (0.4mmol) or high (4.0mmol) concentration of sodium pyruvate. At 7, 10, 15, 20, and 30 h of death the percentage of H and M subunits and total lactate dehydrogenase activity in the supernatants were not significantly different within the 3 groups, although differences were recorded between the various groups. After 30 h the M subunits were decreased by 30% within the 3 groups while total LDH activity remained normal. Hydroxyproline was used as a measure of fibrous tissue. This amino acid is found exclusively in collagen in which it occurs in a constant proportion of 13.4% by weight (Neuman and Logan, 1950). The concentration of collagen and thus fibrous tissue in the myocardium can therefore be estimated by determination of hydroxyproline. The ventricles were removed and weighed and dried at 105°C to a constant weight. The dry tissue was hydrolysed in 6N HCI for a period of 16 h in sealed test-tubes at a temperature of 105°C; 3 cm3 of 6N HCI were used for each 100 mg of tissue. Hydroxyproline was determined in an aliquot after neutralisation according to the method of Stegemann (1958) as described by Woessner (1961). For histology the left ventricular free wall was divided into 3 parts, anterior, lateral, and posterior. Following fixation and dehydration 6 pm sections from each part were stained for collagen using Masson trichrome stain and counterstained with Haemalum. After staining, sections were examined under a binocular Watson-Barnett microscope (magnification x 100) to determine the relative proportions of muscle cells, fibrous tissue (collagen and fibroblasts), and other cellular elements (blood cells and endothelial cells) in each section. This was
done by inserting a grid into 1 of the eyepieces of the microscope. Twentyfive fields were examined in each section and in each field the number of grid intersections that fell upon muscle cells, fibrous tissue, and other cellular elements were recorded.
Results The heart weight and left and right ventricular wall thickness in the ‘marked hypertrophy’ group are all far greater than in the ‘marked ischaemia/moderate hypertrophy’ group (Table 1). Table 2 shows the LDH isoenzyme pattern in each group. The markedly hypertrophic hearts show significantly less of the isoenzyme I than the controls. Similar changes were found in the ischaemia/ moderate hypertrophy group. Fig. 1 shows the relative proportions of M subunits. In both the ‘marked hypertrophy’ and the ‘marked ischaemia/moderate hypertrophy’ groups the proportions of M subunits are significantly greater than the controls. The increase, however, is not proportional to the increase in heart weight. It is significantly greater in hypertrophy’ ‘marked ischaemia / moderate (P< 0.0005) than in ‘marked hypertrophy’ group. These conclusions were confirmed by observing the extent to which LDH activity is inhibited in each case by pyruvate. It has been shown that relatively high concentrations of pyruvate inhibit the H subunits of LDH (Dawson et al., 1964). This inhibition has been shown by studies in vitro to be directly proportional to the percentage of H subunits present in the enzyme (Plagemann et al., 1960). Table 3 shows that both the marked hypertrophy and
Table 1 Heart weight and ventricular wall thickness in patient groups studied Patient groups
Controls
Marked Marked hypertrophy ischaemial moderate hypertrophy
__
-_ Number of cases 19
10
14
Heart weight (g)
320 i40
669 f 80
*
477t
1.5 10.05
2.8f
*0.10
2.1* 10.07
0.21t
0.18.
Left ventricular wall Thickness (cm)
Right ventricular wall 0.14 Thickness (cm) 10.01
*0.04
* 50
*0.03
Mean +SEM. *P
