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Am J Physiol Heart Circ Physiol 277: H474-H480, 1999;
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Vol. 277, Issue 2, H474-H480, August 1999

Human heart failure: cAMP stimulation of SR Ca2+-ATPase activity and phosphorylation level of phospholamban

Ulrich Schmidt1, Roger J. Hajjar2, Catherine S. Kim1, Djamel Lebeche1, Angelia A. Doye3, and Judith K. Gwathmey1

1 Integrated Physiology Research Laboratories, Cardiovascular Division, Department of Cardiovascular Medicine and Evans Department of Medicine, Boston University School of Medicine and the Whitaker Cardiovascular Institute, Boston 02118; 2 Massachusetts General Hospital, Boston 02114; and 3 The Institute for Cardiovascular Diseases and Muscle Research, Cambridge, Massachusetts 02138


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Failing human myocardium has been associated with decreased sarcoplasmic reticulum (SR) Ca2+-ATPase activity. There remains controversy as to whether the regulation of SR Ca2+-ATPase activity is altered in heart failure or whether decreased SR Ca2+-ATPase activity is due to changes in SR Ca2+-ATPase or phospholamban expression. We therefore investigated whether alterations in cAMP-dependent phosphorylation of phospholamban may be responsible for the reduced SR Ca2+-ATPase activity in human heart failure. Protein levels of phospholamban and SR Ca2+-ATPase, detected by Western blot, were unchanged in failing compared with nonfailing human myocardium. There was decreased responsiveness to the direct activation of the SR Ca2+-ATPase activity by either cAMP (0.01-100 µmol/l) or protein kinase A (1-30 µg) in failing myocardium. Using the backphosphorylation technique, we observed a decrease of the cAMP-dependent phosphorylation level of phospholamban by 20 ± 2%. It is concluded that the impaired SR function in human end-stage heart failure may be due, in part, to a reduced cAMP-dependent phosphorylation of phospholamban.

human myocardium; calcium ion; sarcoplasmic reticulum; calcium ion-adenosinetriphosphatase; phospholamban ; adenosine 3',5'-cyclic monophosphate


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

IN HUMAN FAILING MYOCARDIUM, the sarcoplasmic reticulum (SR) Ca2+ uptake rate measured from crude membranes is significantly reduced (11, 18, 30). Quantification of the steady-state levels of SR Ca2+-ATPase (SERCA2a) mRNA levels revealed marked reductions in both failing human hearts and in some experimental models of heart failure (32). Associated with a decrease in mRNA, a number of groups have also shown a decrease in SR Ca2+-ATPase activity and SR Ca2+ uptake in SR vesicles and membranes isolated from failing human hearts (5, 11, 15, 18, 27, 30, 32). Although some studies have failed to show differences in protein levels of SR Ca2+-ATPase in nonfailing and failing human myocardium (8, 30), others have reported that the protein levels of SR Ca2+-ATPase in failing human hearts are significantly reduced (11). It is unclear why these discrepancies exist among laboratories, but they may be due to inhomogeneities of human myocardium that are explanted from cardiac transplant candidates. Differences between mRNA levels and protein levels may also be related to mRNA processing and posttranslational modification. In addition, differences reported in SR Ca2+-ATPase activity may be due to structural changes of the Ca2+-ATPase in relation to calreticulin and calsequestrin within the SR.

Despite the discrepancy between protein levels and SR Ca2+-ATPase activity, decreased SR Ca2+-ATPase activity has been shown by a number of investigators to be associated with abnormal intracellular Ca2+ handling (9, 17). Phospholamban, which is an integral protein of the SR, strongly regulates SR Ca2+-ATPase activity and Ca2+ mobilization in the SR (2, 12-14, 18). In the unphosphorylated state, phospholamban inhibits SR Ca2+-ATPase activity by reducing the affinity for Ca2+. cAMP-dependent kinases phosphorylate phospholamban, thereby abolishing the inhibition of SR Ca2+-ATPase activity (2, 12-14, 18). Therefore, phospholamban affects both the maximal Ca2+ velocity and the apparent affinity of the pump for Ca2+. Because intracellular cAMP concentrations are decreased in human heart failure (16), we investigated whether the abnormal SR Ca2+-ATPase activity in failing human myocardium may be due to a reduced level of phosphorylation of phospholamban.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Study population. The investigation conforms with the principles outlined in the Declaration of Helsinki. This study was approved by the Subcommittee for Human Studies at Massachusetts General Hospital. Diseased myocardial tissues were obtained from 11 patients (two females, nine males) with end-stage heart failure (New York Heart Association class III or IV) undergoing cardiac transplantation at the Massachusetts General Hospital. Nonfailing myocardium was obtained from eight patients (three females, five males) who had neurological events: either cerebrovascular accidents or traumatic head injuries.

After explantation, nonfailing and failing human hearts were placed in iced, oxygenated, pH-balanced Krebs-Henseleit solution and transported to the laboratory. Composition of Krebs-Henseleit solution was as follows (in mmol/l): 120 NaCl, 5.9 KCl, 2.6 CaCl2, 1.2 MgCl2, 1.2 NaH2PO4, 25 NaHCO3, 11.5 dextrose, 95% O2, and 5% CO2. The tissue was dissected from the hearts, immediately placed in liquid nitrogen, and stored at -80°C. This procedure did not change any enzymatic activity.

Three of the patients from the nonfailing group suffered cerebrovascular events, and five of the patients had traumatic head injuries. None of the patients was placed on cardiotonic drugs or required intra-aortic balloon placement. These patients had no evidence of heart failure at the time of explantation. The baseline clinical parameters of the patients with heart failure undergoing cardiac transplantation are listed in Table 1.

                              
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  		Table 1.   Clinical characteristics of patients with heart failure

Preparation of SR membranes. Left ventricular myocardial tissue, free of connective tissue, was chilled in ice-cold (4°C) homogenization buffer with the following composition (in mmol/l): 300 sucrose, 1 phenylmethylsulfonyl fluoride (PMSF), and 20 PIPES, pH 7.4. The tissue was motor-driven homogenized. The homogenate (membrane) was spun at 8,000 rpm (Beckman JA 20; Beckman, Munich, Germany) for 20 min. The supernatant was filtered through four layers of gauze, diluted with an equal volume of 400 mmol/l KCl, and placed on ice for 15 min. The suspension was then centrifuged at 37,000 rpm for 60 min at 4°C (Beckman TI 70). The final pellet was resuspended in a buffer solution containing (in mmol/l) 400 sucrose, 5 HEPES, and 5 Tris, pH 7.2. The total protein concentration was measured according to the method of Lowry et al. (16). To assess the similarity between the membrane preparations used, protein levels of the ryanodine receptors and calsequestrin in both failing and nonfailing groups were measured. There was no difference between the two groups (data not shown).

Western blot analysis. SDS-PAGE was performed on the isolated membranes under reducing conditions on a 7.5% separation gel with a 4% stacking gel in a Miniprotean II cell (Bio-Rad). Proteins were then transferred to a Hybond-enhanced chemiluminescence nitrocellulose for 2 h. The blots were blocked in 5% nonfat milk in Tris-buffered saline for 3 h at room temperature. For immunoreaction, the blot was incubated with 1:2,500 diluted monoclonal anti-SERCA2a antibody (Affinity BioReagents) or 1:2,500 diluted anti-cardiac phospholamban monoclonal IgG (UBI) for 90 min at room temperature. After being washed, the blots were incubated in a solution containing peroxidase-labeled goat anti-mouse IgG (dilution 1:1,000) for 90 min at room temperature. The blot was then incubated in a chemiluminescence system and exposed to an X-OMAT X-ray film (Fuji Films) for 1 min. The densities of the bands were evaluated using National Institutes of Health (NIH) Image. Normalization was performed by dividing densitometric units of each membrane preparation by the protein amounts in each of the preparations. Serial dilution of the membrane preparations revealed a linear relationship between the amounts of protein and the densities of the SERCA2a and phospholamban immunoreactive bands (data not shown).

SR Ca2+-ATPase activity. SR Ca2+-ATPase activity assays were carried out according to Chu et al. (3) based on a pyruvate/NADH-coupled reaction. With the use of a photometer (Beckman DU 640) adjusted at a wavelength of 340 nm, oxidation of NADH in the membranes was assessed at 37°C by the difference of the total absorbance and basal absorbance. The reaction was carried out in a volume of 1 ml, and all experiments were carried out in triplicate. The activity of the Ca2+-ATPase was calculated as absorbance/6.22 × protein × time in nanomoles ATP per milligram protein per minute. The measurements were repeated at different Ca2+ concentrations ([Ca2+]). The [Ca2+] used to stimulate SR Ca2+-ATPase activity were obtained using formulas from Fabiato (4). To verify that the SR Ca2+-ATPase activity we measured from the membrane preparations was SR related, we used the specific inhibitor cyclopiazonic acid (CPA) after maximally activating the SR Ca2+-ATPase with 10 µmol/l [Ca2+]. At CPA of 10 µmol/l, SR Ca2+-ATPase activity was decreased by >95% in all preparations. This was similar to all of our previous studies in which similar verifications were undertaken (10, 28, 30).

Stimulation of SR Ca2+-ATPase by cAMP and protein kinase A. The membranes were preincubated with cAMP (0.01-100 µmol/l) or protein kinase A (PKA; catalytic subunit; 1-30 µg) for 5 min at 37°C. Soluble cAMP levels and membrane-bound cAMP are in the range of 30-80 pmol/mg protein (31). Therefore, we used concentrations of cAMP in the range of 0.01-100 µmol/l. These experiments were performed at a [Ca2+] of 600 nmol/l.

Backphosphorylation. Membranes were maintained in a buffer containing (in mmol/l) 300 sucrose, 1 PMSF, 20 PIPES, and 4.9 NaN3. Membranes (40 µg) were phosphorylated in a mixture containing 40 mmol/l histidine hydrochloride (pH 6.8), 100 mmol/l NaCl, 10 mmol/l MgCl2, 1 mmol/l EGTA, 15 mmol/l NaF, 10 µg catalytic subunit of PKA, and 50 µmol/l [32P]ATP in a volume of 50 µl. The membranes were preincubated for 2 min at 30°C. The reaction was started by the addition of [32P]ATP. The reaction was stopped after 3 min by the addition of an ice-cold stop solution containing 50 mmol/l H3PO4 (pH 6.8), 30 mmol/l EDTA, 7% (wt/vol) SDS, and 6% (wt/vol) mercaptoethanol. The myocardial membranes were applied to a 7.5% SDS-polyacrylamide gel under reducing conditions. The gels were stained with Coomassie blue, dried, and exposed to an X-ray film. An image analysis program (NIH) was used to detect the bands. The phosphorylation of phospholamban was maximal after 1 min and remained stable up to 10 min (Fig. 1).


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  		Fig. 1.   Time dependency of incorporation of 32P into phospholamban.

Chemicals and reagents. All chemicals and reagents were of the highest analytical grade and were obtained from Sigma (St. Louis, MO). The SERCA2a antibody was obtained from Affinity Bioreagents, and the phospholamban antibody was obtained from Upstate Biotechnology.

Statistical analysis. Data are represented as means ± SE. The distributions of the continuous variables were checked for normality. Univariate Pearson and Spearman correlation coefficients and accompanying significant P values were generated for the normal and nonnormal continuous variables, respectively. Student's t-test was used to compare the means of continuous variables between groups. A two-sided P value of <0.05 was considered significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

SR Ca2+-ATPase activity in failing ventricles. To investigate whether the SR Ca2+-ATPase activity differs between the failing and nonfailing group, we examined SR Ca2+-ATPase activity at a [Ca2+] of 600 nmol/l. As shown in Fig. 2, SR Ca2+-ATPase activity was decreased in failing when compared with nonfailing myocardium.


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  		Fig. 2.   Sarcoplasmic reticulum (SR) ATPase activity to increasing levels of Ca2+ in membranes from human nonfailing (open bar, n = 7) and failing (filled bar, n = 11) myocardium measured using an NADH-coupled assay. * Significantly different from values on nonfailing myocardium.

Levels of phospholamban and SERCA2a in failing and nonfailing SR membranes. To evaluate whether the abnormalities in SR Ca2+-ATPase activity are due to changes in SERCA2a or phospholamban expression, we quantitated the levels of both phospholamban and SERCA2a in membrane preparations from nonfailing and failing human myocardium. As shown in Fig. 3, there was no significant difference in either phospholamban or SERCA2a proteins in the failing group when compared with the nonfailing group. Also, within the failing group, the protein expression of SERCA2a and phospholamban did not differ between ischemic and dilated cardiomyopathic hearts (data not shown).


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  		Fig. 3.   Protein levels of phospholamban and SR Ca2+-ATPase (SERCA2a) in failing (n = 8) and nonfailing (n = 8) SR membranes.

Stimulation of SR Ca2+-ATPase activity by cAMP. Increasing concentrations of cAMP increased SR Ca2+ activity in both failing and nonfailing myocardium (Fig. 4). cAMP-induced SR Ca2+-ATPase activity was significantly higher in nonfailing myocardium compared with failing myocardium.


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  		Fig. 4.   Response of SR Ca2+ activity to increasing levels of cAMP in membranes from human nonfailing (n = 7) and failing (n = 10) myocardium measured using an NADH-coupled assay. * Significantly different from values on nonfailing myocardium.

To investigate whether stimulation by cAMP restores the SR ATPase activity as a function of [Ca2+] in failing hearts, ATPase activity was measured in the presence of 10 µmol/l cAMP in failing and nonfailing membrane preparations. As shown in Fig. 5, cAMP induced a shift upward of the ATPase-[Ca2+] relationship in both failing and nonfailing membrane preparations. The Ca2+ responsiveness of SR Ca2+-ATPase in cAMP-stimulated preparations from nonfailing hearts was greater than in samples from failing hearts up to 1 µmol/l [Ca2+]. At higher [Ca2+], SR Ca2+-ATPase activity was similar between cAMP-stimulated nonfailing and failing human myocardium. The Ca2+ responsiveness was restored in cAMP-stimulated failing myocardium to levels seen in non-cAMP-stimulated samples from nonfailing hearts up to 1 µmol/l [Ca2+]. At higher [Ca2+], SR Ca2+-ATPase activities in cAMP-stimulated preparations from failing hearts were higher than values obtained in non-cAMP-stimulated preparations from nonfailing hearts.


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  		Fig. 5.   Effect of 10 µmol/l cAMP on SR ATPase-Ca2+ concentration ([Ca2+]) relationships in membranes from human nonfailing (n = 6) and failing (n = 6) myocardium (dashed lines post-cAMP). , Nonfailing + 10 µM cAMP; , nonfailing; open circle , failing + 10 µM cAMP; , failing. * Significantly different from values on nonfailing myocardium.

Stimulation of SR Ca2+-ATPase activity by PKA. The SR Ca2+-ATPase activity was stimulated by increasing concentrations of PKA in both failing and nonfailing myocardium (Fig. 6). Membrane preparations from nonfailing myocardium demonstrated a significantly higher ATPase activity when compared with failing myocardium. A higher concentration of PKA was required to initiate stimulation of SR Ca2+-ATPase activity in failing human hearts (~10 µg).


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  		Fig. 6.   Response of SR Ca2+ activity to increasing levels of protein kinase A (PKA) in membranes from human nonfailing (n = 6) and failing (n = 9) myocardium measured using an NADH-coupled assay. * Significantly different from values on nonfailing myocardium.

Backphosphorylation of phospholamban. To examine whether the phosphorylation of phospholamban is altered in human heart failure, we measured the incorporation of 32P in the presence of ATP and cAMP protein kinase in failing and nonfailing SR membranes. There was increased 32P incorporation into SR membranes from failing hearts compared with nonfailing hearts (5.8 ± 0.6 vs. 4.1 ± 0.6 pmol 32P/mg, P < 0.05, n = 8).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Failing human myocardium has been demonstrated to have impaired intracellular Ca2+ mobilization that results in elevated diastolic [Ca2+] and slowed diastolic relaxation. This impaired SR Ca2+ uptake could be due to 1) a decreased expression of SERCA2a in failing myocardium or 2) a change in the regulatory relationship between phospholamban and SR Ca2+-ATPase activity. The present study was designed to provide insight into the mechanism for the reduced SR Ca2+ uptake reported in failing human myocardium.

The mRNA and protein expression of the SR Ca2+-ATPase has long been the main focus of a number of research groups (10, 19, 27, 30, 32). A decrease in the steady-state mRNA levels of SR Ca2+-ATPase in failing human myocardium was first reported by Mercadier et al. (19) and confirmed by others (11, 30, 32). Contradictory results have been obtained as to whether the protein expression of SR Ca2+-ATPase is decreased in failing human myocardium, thereby explaining an observed decrease in SR Ca2+-ATPase activity. Hasenfuss et al. (11) observed a decrease in the protein expression of the SR Ca2+-ATPase in human end-stage heart failure. This is in contrast to our findings and those of others (7, 29) who did not observe a change in SERCA2a protein expression yet reported a decrease in SR Ca2+-ATPase activity. SR Ca2+ uptake is, however, not only determined by the protein level of SERCA2a but also by the interaction of phospholamban and SERCA2a.

Luo et al. (17) demonstrated a positive correlation between the relative levels of phospholamban in the mouse heart and the affinity of the SR Ca2+ pump for Ca2+. Furthermore, this group has elegantly shown that the increase in velocity of relaxation and contraction in myocytes from phospholamban-deficient mice depends on the level of phospholamban in these hearts (12-14, 17-18). We, like Movsesian et al. (22, 23), found no change in phospholamban protein expression in samples from failing human hearts.

An increase in cAMP levels activates the cAMP-dependent protein kinase, which phosphorylates phospholamban (31, 33, 34). In human failing myocardium, cAMP levels have been reported to be decreased (6). Böhm et al. (2) reported a cAMP concentration of 46.7 ± 8.3 pmol/mg protein in the soluble fraction of nonfailing myocardium vs. 29.6 ± 6.0 pmol/mg protein in failing tissue, which may contribute to our observed decrease in the phosphorylation state of phospholamban. They also reported that cAMP-dependent phosphorylation of phospholamban was unchanged in failing compared with nonfailing human myocardium. Anti-phospholamban antibody, which stimulates Ca2+ uptake on maximal Ca2+ sequestration, revealed no difference between failing and nonfailing myocardium (21). We observed an unchanged maximal effect of cAMP to stimulate SR Ca2+-ATPase in membranes from failing myocardium. Addition of 10 µmol/l cAMP restored SR Ca2+-ATPase activity over physiological [Ca2+]. Our findings, therefore, show no change in the potential for phospholamban to be phosphorylated. The earlier reports by Movsesian et al. (21-23) and Böhm et al. (2) support our findings that the potential of phospholamban to be phosphorylated is not altered in failing myocardium and suggest a normal functional relationship between SR Ca2+-ATPase activity and phospholamban.

In this study, we report a decreased basal phosphorylation level of phospholamban in failing human myocardium compared with nonfailing myocardium. Consistent with our observation, Bartel et al. (1) recorded a decrease in the isoproterenol-induced phosphorylation of phospholamban. Böhm et al. (2) found no change in the membrane-bound PKA activity between failing and nonfailing myocardium. We have demonstrated that the reduced basal phosphorylation level of phospholamban is of functional consequence in the regulation of SR Ca2+-ATPase activity and Ca2+ mobilization. Under basal conditions, SR Ca2+-ATPase activity was significantly reduced in failing tissue, and maximal stimulation with PKA did not restore this parameter to levels seen in similarly stimulated preparations from nonfailing hearts. The concentration-response relationship for PKA and cAMP-stimulated SR Ca2+-ATPase activity was shifted to higher concentrations in failing myocardium. These data indicate a decreased sensitivity to cAMP and PKA stimulation in failing human hearts.

Enhanced diastolic intracellular [Ca2+] and decreased SR Ca2+-ATPase activity have been implicated in the impaired relaxation in failing human myocardium (8, 20). An increase of the Ca2+ sequestration by the SR should therefore improve relaxation in failing human myocardium. A slight enhancement of the intracellular cAMP concentration with isoproterenol (26) or forskolin (24) is also able to restore the negative force frequency relationship reported in human failing myocardium. In the presence of isoproterenol or forskolin, the phosphorylation state may become similar to that seen in nonfailing myocardium, resulting in a positive force-frequency relationship. Although the stimulation of the SR by cAMP in failing myocardium did not reach levels seen in nonfailing myocardium, the relative change was similar. This indicates that cAMP did restore the SR Ca2+-ATPase activity to levels seen in nonstimulated, nonfailing myocardium and supports the hypothesis that a small enhancement of intracellular cAMP levels in failing myocardium may be of benefit for Ca2+ sequestration in failing hearts.

It is concluded that a decreased phosphorylation level of phospholamban may be, in part, responsible for the lower SR Ca2+-ATPase activity in the absence of a change in SERCA2a protein expression reported in end-stage human heart failure. Stimulation of SR Ca2+-ATPase by cAMP-dependent substances may therefore be beneficial for myocardial function in end-stage heart failure.


    ACKNOWLEDGEMENTS

This work was supported in part by National Heart, Lung, and Blood Institute (NHLBI) Grants R01-HL-49574, R55-HL-52249, and R43-HL-60323, National Science Foundation Grant IBN-9419915 (to J. K. Gwathmey), and NHLBI Grant K08-HL-03561 (to R. J. Hajjar).


    FOOTNOTES

Gwathmey, Inc., is thanked for technical support of this project. The National Disease Research Interchange is acknowledged for support of this project.

Address for reqprint requests and other correspondence: J. K. Gwathmey, Integrated Physiology Laboratories, Boston Univ. School of Medicine, 763 Bldg., E Concord Ave., Cambridge, MA 02138 (E-mail: gwathmey{at}tiac.net).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Received 20 July 1998; accepted in final form 31 March 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Heart Circ Physiol 277(2):H474-H480
0002-9513/99 $5.00 Copyright © 1999 the American Physiological Society


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