Effects of Calcium, Inorganic Phosphate, and pH on Isometric Force in Single Skinned Cardiomyocytes From Donor and Failing Human Hearts
Background— During ischemia, the intracellular calcium and inorganic phosphate (Pi) concentrations rise and pH falls. We investigated the effects of these changes on force development in donor and failing human hearts to determine if altered contractile protein composition during heart failure changes the myocardial response to Ca2+, Pi, and pH.
Methods and Results— Isometric force was studied in mechanically isolated Triton-skinned single myocytes from left ventricular myocardium. Force declined with added Pi to 0.33±0.02 of the control force (pH 7.1, 0 mmol/L Pi) at 30 mmol/L Pi and increased with pH from 0.64±0.03 at pH 6.2 to 1.27±0.02 at pH 7.4. Force dependency on Pi and pH did not differ between donor and failing hearts. Incubation of myocytes in a Pi-containing activating solution caused a potentiation of force, which was larger at submaximal than at maximal [Ca2+]. Ca2+ sensitivity of force was similar in donor hearts and hearts with moderate cardiac disease, but in end-stage failing myocardium it was significantly increased. The degree of myosin light chain 2 phosphorylation was significantly decreased in end-stage failing compared with donor myocardium, resulting in an inverse correlation between Ca2+ responsiveness of force and myosin light chain 2 phosphorylation.
Conclusions— Our results indicate that contractile protein alterations in human end-stage heart failure alter Ca2+ responsiveness of force but do not affect the force-generating capacity of the cross-bridges or its Pi and pH dependence. In end-stage failing myocardium, the reduction in force by changes in pH and [Pi] at submaximal [Ca2+] may even be less than in donor hearts because of the increased Ca2+ responsiveness.
Received May 25, 2001; revision received June 29, 2001; accepted July 2, 2001.
Ischemia in cardiac muscle produces a rapid and pronounced fall of developed force. Studies on animal tissue have shown that the rapid fall in developed force during ischemia is mainly caused by the combined effects of changes in intracellular [Ca2+], inorganic phosphate (Pi) concentration, and pH on the contractile apparatus.1,2 In failing human hearts, changes have been observed in isoform composition3–6 and phosphorylation status7 of contractile proteins, which may alter the response to changes in Ca2+, Pi, and pH. However, the functional implications of contractile protein changes in human heart failure are largely unknown. In particular, data concerning the Pi and pH dependence of force development in human myocardium is limited,8 and studies on the Ca2+ responsiveness of force in nonfailing versus failing human hearts remain controversial.5–9
To examine whether differences in the response to ischemia develop gradually or abruptly with heart disease, a comparison was made between nonfailing donors and patients from different New York Heart Association (NYHA) classes. Moreover, this comparison yields information on the origin of changes in contractile properties with heart failure. Left ventricular biopsies were obtained during open heart surgery from patients with mitral or aortic valve disease (NYHA class I–III). Left ventricular tissue was also obtained from explanted end-stage failing hearts (NYHA class IV) and from donor hearts.
In the well-oxygenated heart, [Pi] is ≈2 mmol/L at pH 7, whereas during prolonged ischemia, [Pi] may increase to 30 mmol/L and, after an initial alkalization of ≈0.1 to 0.2 pH units, pH may drop to ≈6.10 Therefore, we investigated the effects of a broad range of [Pi] (0 to 30 mmol/L) and of pH (6.2 to 7.4) on isometric force development in mechanically isolated single Triton-skinned cardiomyocytes. To mimic the effects of prolonged ischemia, we studied the combined effects at 30 mmol/L Pi and pH 6.2. In addition, Ca2+ responsiveness of force and myosin light chain 2 (MLC-2) phosphorylation were studied in failing and nonfailing hearts. By removing all membranous structures, myofibrillar contractile properties can be measured under standardized conditions in which [Ca2+], [Pi], and pH can be varied independently, whereas MLC-2 phosphorylation is kept constant and diffusional limitations are negligible.
Left ventricular biopsy specimens were obtained from 12 patients undergoing valve replacement surgery (NYHA class I–III). Biopsy specimens were immediately immersed in cold relaxing solution (pH 7.0; in mmol/L: free Mg2+ 1, KCl 145, EGTA 2, ATP 4, imidazole 10). In addition, left ventricular samples were obtained during heart transplantation surgery from 3 explanted end-stage failing hearts (NYHA class IV) and from 4 nonfailing donor hearts. This tissue was transported in cardioplegic solution and on arrival in the laboratory, stored in liquid nitrogen. Characteristics of patients and donors are given in Table 1. Samples were obtained after informed consent and with approval of the local ethics committees. All procedures followed were in accordance with institutional guidelines.
Cardiomyocytes were mechanically isolated and mounted in the experimental setup as described previously.6 Before mechanical isolation, tissue from end-stage failing and donor hearts was defrosted in relaxing solution. A single cardiomyocyte was attached between a force transducer and a piezoelectric motor (Figure 1). To dissolve all membranous structures, the cardiomyocyte was immersed for 45 seconds in relaxing solution containing 0.3% Triton X-100.
Isometric force measurements were performed at 15°C and a sarcomere length of 2.2 μm. After the first control activation (0 Pi, pH 7.1), resting sarcomere length was readjusted to 2.2 μm if necessary. The second control measurement was used to calculate maximal isometric tension (ie, force divided by the cross-sectional area). The next 5 measurements were carried out in solutions with different amounts of Pi (1, 2, 5, 15, and 30 mmol/L) in random order (Pi series), followed by a control measurement. Thereafter, 4 activations in solutions with different pH values (6.2, 6.5, 6.8, 7.4) were performed, which were also followed by a control measurement. To test if the effects of Pi and pH interact, force was measured in a solution with high Pi (30 mmol/L) and pH 6.2.
The composition of relaxing and activating solutions used during force measurements was calculated2 as described by Fabiato.11 Force values obtained in solutions at different pH and/or with Pi were normalized to the interpolated control values.
Maximal control force was increased after the series of activations at different [Pi]. In a number of myocytes, force was measured at different submaximal [Ca2+] before and after the Pi series to investigate the effect of Pi incubation on Ca2+ responsiveness. The pCa, that is, −log10 [Ca2+], of the relaxing and activating solution (pH 7.1, 0 Pi) were, respectively, 9 and 4.5. Solutions with lower free [Ca2+] were obtained by mixing of the activating and relaxing solutions.
Two-Dimensional Gel Electrophoresis
Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE)12 was performed on trichloroacetic acid-treated donor and end-stage failing hearts to quantify differences in the phosphorylation status of MLC-2. Moreover, to investigate if alterations in MLC-2 phosphorylation were involved in the potentiation of force, cardiomyocytes (∼2 mg wet wt) were incubated in 1 mL of activating solution with and without Pi (30 mmol/L) for 10 minutes at room temperature. Samples (600 μg dry weight) were loaded on immobiline strips with a pH gradient of 4.5 to 5.5 (Amersham Pharmacia Biotech). In the second dimension, proteins were separated by SDS-PAGE.6 Gels were stained with Coomassie blue, scanned and analyzed using Image Quant (Molecular Dynamics).
Force-pCa relations were fit to a modified Hill equation:
F (Ca2+)/F0=[Ca2+]nH/(Ca50nH+[Ca2+]nH), where F is steady-state force. F0 denotes the steady force at saturating [Ca2+], nH reflects the steepness of the relation, and Ca50 (or pCa50) represents the midpoint of the relation.
Values are given as mean±SEM of n experiments. Mean values for donor, NYHA class I–III, and NYHA class IV samples were compared by 1-way ANOVA. When ANOVA yielded a significant overall effect (P<0.05), Bonferroni post hoc tests were performed to compare pairs of group means. Paired Student’s t tests were performed for the combined experiments on single cardiomyocytes at high Pi and low pH as well as for the experiments concerning maximal force and Ca2+ sensitivity before and after Pi series.
Maximal isometric tension was higher, although not significantly, in NYHA class I–III hearts (46.7±8.3 kN/m2; n=12) compared with donor and end-stage failing myocardium (26.4±3.5, n=4, and 21.9±3.7 kN/m2, n=3respectively), which had been stored in liquid nitrogen. Passive tension amounted to 1.7±0.4, 3.3±0.7, and 1.4±0.2 kN/m2 in donor, NYHA class I–III, and end-stage failing hearts. In Figure 2, recordings of force development during a control experiment (0 Pi, pH 7.1), with 15 mmol/L Pi, at pH 6.2 and with 30 mmol/L Pi at pH 6.2 are shown.
Effects of Pi
Figure 2 illustrates that force decreased in the presence of 15 mmol/L Pi to ≈30% of the control force. The dependency of force on Pi derived from the complete set of experiments is summarized in Figure 3A. At 30 mmol/L Pi, relative force development was significantly different among the 3 groups (P<0.05). A linear dependence of force on the logarithmic of [Pi] was found (Figure 3B). For donor, NYHA class I–III, and end-stage failing hearts, the slopes amounted to 0.35±0.03, 0.36±0.02, and 0.31±0.02, respectively. The differences between slopes were not significant. Hence, the relations between force and [Pi] were similar in donor and failing hearts.
Potentiation of Force After Pi Series
Usually force development declined slightly during a series of force measurements (eg, at different [Ca2+]).6 However, after activation of myocytes during a Pi series, control force was larger than before the Pi series. The potentiating effect on maximal force amounted to 3±1%, 6±2%, and 5±4% in donor (n=10), NYHA class I–III (n=27), and NYHA class IV (n=7) myocytes, respectively. The potentiating effect would have been larger if the decline in force development during the series of measurements would have been taken into account. Maximal force after a series of measurements at different [Ca2+] had declined to 95±1%, 98±2%, and 97±1% in donor, NYHA class I–III, and end-stage failing cardiomyocytes, respectively. From these values, we estimate that the actual potentiation of maximal force ranged between 5% and 10%. Ca2+-activated force development in the presence of Pi was required, as potentiation did not occur after incubations for a similar period in relaxing solution containing 30 mmol/L Pi.
Force was also measured at submaximal [Ca2+] before and after the Pi series. The average force-pCa relations obtained in donor, NYHA class I–III, and end-stage failing hearts before Pi are shown in Figure 4A. It can be noted that Ca2+-responsiveness did not differ between donor and NYHA class I–III myocardium (pCa50, 5.56±0.04 and 5.60±0.05, respectively) but that it was substantially increased in end-stage failing hearts (pCa50, 5.88±0.05) (P<0.05 among groups). The steepness of the force-pCa relations did not significantly differ among the 3 groups.
At submaximal [Ca2+], Pi increased force to a larger extent than at maximal activation. This is reflected in an increase in the mid-point of the force-pCa relations, on average by 0.05±0.01 pCa units (Figure 4B). It can be seen that the relative potentiation of force amounted to a factor of about 2 at pCa 6. In Table 2, the pCa50 and nH values before and after Pi are summarized.
In the well-oxygenated heart, [Pi] is ∼2 mmol/L. To investigate if this concentration is sufficient to potentiate force, an additional set of experiments was performed in donor cardiomyocytes. In these experiments, control activations were followed by a Pi series with 2 mmol/L Pi. In accordance with the previous Pi series, 5 measurements were performed in activating solution with 2 mmol/L Pi followed by a control activation. These activations resulted in a minor, nonsignificant increase in maximal force by 2±3% (n=4). To investigate if the force potentiation is time-dependent, cardiomyocytes (n=3) were incubated for 3 successive periods of 5 minutes in activating solution with 2 mmol/L Pi. After each incubation period, maximal control force was determined. After the first incubation period, maximal force had increased by 9±1% (P<0.05). Force did not increase further after the second incubation period, whereas after the third incubation period it had decreased to 97±2%.
Effect of pH
Figure 2 indicates that force at pH 6.2 decreased to ≈50% of the control value. The results for normalized force as a function of pH are summarized in Figure 3C. Relative force increased fairly linearly with pH. No significant differences were found between the pH dependency of force in donor and failing myocardium.
Combined Effects of Pi and pH
During ischemia, pH and [Pi] change concomitantly. Figure 2 shows that force was markedly reduced to ≈15% in the presence of 30 mmol/L Pi at pH 6.2. For each set of paired measurements the product of normalized forces at 30 mmol/L Pi (pH 7.1) and at pH 6.2 (0 Pi) was calculated and compared with the value found for the combined intervention (30 mmol/L Pi, pH 6.2). Values are given in Table 3. The products of force at pH 6.2 and force with 30 mmol/L Pi did not differ significantly from the values obtained at pH 6.2 with 30 mmol/L Pi. The average value obtained for the product of isometric force at pH 6.2 and force with 30 mmol/L Pi was significantly different among the 3 groups (P<0.05).
Myosin Light Chain 2 Phosphorylation
The MLC-2 in the human ventricle consists of 2 isoforms (MLC-2 and MLC-2*), which may both be phosphorylated (MLC-2P and MLC-2*P). Figure 5 shows 2D gels from donor myocytes incubated in activating solution without (A) and with 30 mmol/L Pi (B). It can be seen that the phosphorylation level of MLC-2 was not altered by incubation of myocytes in Pi-containing activating solution. The distribution of unphosphorylated and phosphorylated MLC-2 isoforms is given in Table 4. Our values of MLC-2 isoform expression evidenced by the ratio between MLC-2 and MLC-2* did not differ between donor and failing hearts and amounted on average to 70/30, which is similar to the values found by Morano.12 Troponin T (TnT) phosphorylation was also visible on these gels. The monophosphorylated TnT, in percentage of total TnT, amounted to 63.5±0.3% and 69.8±3.5% without and with 30 mmol/L Pi, respectively.
As can be noted from Table 4, the level of MLC-2 phosphorylation was lower in failing cardiomyocytes. Two-dimensional PAGE analysis revealed a significant difference in MLC-2 phosphorylation between donor and end-stage failing hearts (Table 4). A significant inverse correlation was found between Ca2+-responsiveness of force and percentage of MLC-2 phosphorylation (Figure 5C).
Although Ca2+ sensitivity of force was increased in end-stage failing myocardium, we did not observe a difference in the response to Pi and pH between donor and failing hearts. A serendipitous finding was the potentiating effect of Pi preincubation on maximal force and its Ca2+ sensitivity, which has never been observed in previous studies on multicellular preparations.2,13 MLC-2 phosphorylation was significantly decreased in end-stage failing compared with donor hearts, resulting in an inverse correlation between Ca2+ responsiveness of force and percentage of MLC-2 phosphorylation.
Effect of Pi
Force gradually decreased, on average, to 33% of the control value in donor and failing myocardium when [Pi] was increased to 30 mmol/L. The overall relations between force and [Pi] were similar in donor and failing hearts. ANOVA revealed a significant difference in relative force at 30 mmol/L Pi, but no significant differences were present between pairs of group means. Relative force at 30 mmol/L was almost identical in donor and end-stage failing myocardium (Table 3). Therefore, our results indicate that Pi dependency of force in nonfailing and failing myocardium is very similar. The logarithmic relations between relative force and [Pi] reveal that force in human ventricular tissue decreases by ≈34% per decade. Kentish13 and Ebus et al2 reported a decline of ≈45% per decade in rat hearts. Hence, human myocardium appears less sensitive to Pi than rat myocardium. The basis of the metabolic effects on force resides in the kinetic scheme for the actomyosin ATPase activity. During muscle contraction, a cyclic interaction exists between myosin and actin, the forming and breaking of cross-bridges, which is driven by the energy available from hydrolysis of ATP into ADP and Pi. Depression of isometric force by Pi can be explained by mass action of Pi, which promotes the transition of attached cross-bridges from the force producing state to the nonforce or low-force state.14 The free energy available from ATP hydrolysis is reduced when [Pi] is increased,1 which may also result in a reduction of force.2,15
After activation of myocytes in Pi-containing activating solutions, force was increased at saturating [Ca2+] and almost doubled at pCa 6. This effect is opposite to the reduction in force by adding Pi during maximal activation (Figure 3A). In addition, in the presence of Pi, a reduction in Ca2+ responsiveness of the contractile apparatus was found,16 whereas Pi preincubation enhanced Ca2+ sensitivity. The potentiating effect of Pi was both concentration and time dependent and was not present after incubation of myocytes in a Pi-containing relaxing solution, which indicates that Ca2+ was required. Prolonged incubation of cardiomyocytes in activating solution with 2 mmol/L Pi increased maximal force (9±1%), which indicates that the potentiating effect of Pi may already be fully developed under normal physiological conditions. It should be noted that the effect of Pi on force might have influenced the force-Pi and force-pH relations (Figure 3) because of its concentration and time dependence. However, an increase in force by ≈10% will have only a minor effect on the relative force values obtained with Pi and at various pH values. The potentiating effect of Pi on force development has not been reported in previous studies that were performed on thicker multicellular preparations. It may have been obscured by Pi accumulation in these preparations, which occurs during contraction, or by the force decline that occurs during repeated contraction.2 Accumulation of Pi in our single-cell preparations was negligibly small.
Phosphorylation of MLC-2 has been shown to increase Ca2+ sensitivity of force by 0.1 pCa units but not maximal force development.12 A kinetic effect of Pi on MLC-2 phosphorylation might possibly explain the observed increase in Ca2+-sensitivity after activation of myocytes in Pi-containing solutions. However, our 2D PAGE results indicated no significant alterations in the MLC-2 phosphorylation after incubation of cardiomyocytes in a Pi-containing activating solution. Therefore, the mechanism behind the potentiating effect of Pi remains unclear.
Effect of pH
The effects of changes in pH were studied in the range from 6.2 to 7.4, which encompasses the values that are known to occur during ischemia. Control force gradually decreased to ≈65% at pH 6.2. Again, rat myocardium appeared to be more sensitive than human, since control force decreased to 54% at pH 6.2 in rat hearts.2
It is not well established how changes in pH affect force development. Kentish13 has found that pH may influence cross-bridge cycling at several transitions. This complicates the explanation of the observed pH dependence. We did not observe a difference in pH dependency of force between donor and failing myocardium, indicating that during the development of heart failure the sensitivity of the heart to changes in pH remains the same.
Combined Effects of Pi and pH
The combined effects of 30 mmol/L Pi and pH 6.2 were studied to mimic the major metabolic alterations during ischemia. A synergistic interaction between Pi and pH on force was reported by Nosek et al17 in skeletal muscle, although this was not supported by others.18 It has been shown that the effects of Pi and pH on isometric force are independent in cardiac muscle.2,13,19 Our results are in agreement with these studies because the product of the relative reductions in force by 30 mmol/L Pi and pH 6.2 separately did not significantly differ from the combined effect found under simulated ischemic conditions (30 mmol/L Pi, pH 6.2).
Donor Versus Failing Myocardium
In accordance with previous studies on human hearts, no difference was observed between maximal isometric tension of donor and end-stage failing myocardium.5,7,8 Maximal tension was lower but not significant in cardiomyocytes from frozen tissue (donor and NYHA class IV) compared with myocytes from fresh biopsy specimens obtained during valve replacement surgery (NYHA class I–III). A reduction in maximal tension might be due to the difference in storage of the tissue. However, Ca2+ sensitivity of force was similar in donor and NYHA class I–III myocardium. This provides the first evidence that Ca2+ responsiveness remains unaltered until end-stage heart failure develops. Because the shift in Ca2+ sensitivity in end-stage failing myocardium coincides with previous observations,5–7 we are confident that differences in isolation procedures do not interfere with the conclusions presented in this study. Surprisingly, a decrease in maximal tension was observed in cardiomyocytes from failing rat hearts,20,21 whereas Ca2+-responsiveness was decreased20 or remained unaltered.21 Hence, in this respect, there exist important differences between studies on rats and humans.
The degree of MLC-2 phosphorylation was significantly lower in end-stage failing compared with donor hearts. MLC-2 may be phosphorylated by Ca2+ calmodulin-dependent myosin light chain kinase and protein kinase C and dephosphorylated by light chain phosphatase. Previously, enhanced type 1 phosphatase activity was found in failing human hearts,22 which may explain the decreased phosphorylation level of MLC-2. A significant inverse correlation was found between pCa50 and percentage of phosphorylated MLC-2, that is, MLC-2 phosphorylation is associated with a decrease in Ca2+ responsiveness. However, Morano12 has shown that phosphorylation of MLC-2 increases Ca2+ responsiveness. Previously, expression of atrial light chain 1,5 phosphorylation of troponin I,7 and reexpression of fetal troponin T6 have been related with increased Ca2+ sensitivity in human heart failure. It is therefore likely that the increased Ca2+ responsiveness is the resultant of a coordinated pattern of contractile protein changes in end-stage heart failure. In this respect, MLC-2 dephosphorylation may represent a potential compensatory mechanism to oppose the detrimental effects of increased Ca2+ sensitivity and impaired Ca2+ handling on diastolic function in human heart failure.
Recently, Hajjar et al8 reported a reduction in maximal force in the presence of 15 mmol/L Pi, which was larger in nonfailing than in failing hearts. In addition, they did not find a difference in Ca2+ responsiveness at pH 7.1. In contrast, in our study, force dependency on Pi and pH did not differ between donor hearts and hearts with mild to severe cardiac disease, whereas Ca2+ sensitivity of force was significantly increased in end-stage failing hearts at pH 7.1. The experiments by Hajjar et al8 were performed on multicellular preparations. Our observations on single cardiomyocytes, in which diffusion distances are minimized and interference of extracellular components is excluded, indicate that changes in contractile protein composition that occur in human heart failure alter Ca2+ responsiveness but do not affect the Pi and pH dependency of the force-generating capacity of cross-bridges.
In conclusion, our results on human cardiomyocytes indicate that the Pi and pH dependence of maximum force is not altered by heart failure. In addition, we have demonstrated that Ca2+ responsiveness of force correlated inversely with MLC-2 phosphorylation and that it only changes during the late stages of heart failure. Finally, force development at submaximal [Ca2+] may even be less depressed under ischemic conditions in end-stage failing myocardium than in donor hearts because of the increased Ca2+ responsiveness.
This study was supported by the Netherlands Heart Foundation (grant 99.155).
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