1Institute of Cardiovascular Sciences, St. Boniface General Hospital Research Centre, and Department of Physiology, Faculty of Medicine, University of Manitoba, Winnipeg, Manitoba, Canada R2H 2A6; and 2Department of General Medicine, Aoto Hospital, Jikei University School of Medicine, Tokyo 125-8506, Japan
Submitted 24 March 2004 ; accepted in final form 4 June 2004
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ABSTRACT |
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sarcoplasmic reticulum; cardiomyopathy; cAMP-dependent protein kinase; Ca2+/calmodulin-dependent protein kinase; sarco(endo)plasmic reticulum ATPase; phospholamban
It is now well established that the major player involved in regulating intracellular Ca2+ concentrations essential for cardiac contraction and relaxation is the sarcoplasmic reticulum (SR) (4). Irregularities in cardiac performance can be linked to alterations in the SR function because of its critical role in regulating intracellular Ca2+ (29, 34, 44). Although earlier reports (8, 42, 43) suggested that SR functions of Ca2+ uptake and release were compromised in cardiomyopathic hamster hearts, the major challenge lies in discovering the mechanisms by which this occurs. In this regard, one of the major SR Ca2+-cycling proteins, the cardiac sarco(endo)plasmic reticulum Ca2+-ATPase pump (SERCA2a) is responsible for pumping back 7592% of cytosolic Ca2+ into the SR to bring about cardiac relaxation (2, 3). The function of SERCA2a is in turn regulated by a low-molecular-weight protein, phospholamban (PLB) (15, 20). In its unphosphorylated form, PLB inhibits SERCA2a function, yet upon phosphorylation by regulatory proteins such as Ca2+/calmodulin-dependent protein kinase (CaMK) and cAMP-dependent protein kinase (PKA), this inhibition is relieved (15, 20). It is therefore feasible that impairment of SR Ca2+-cycling and regulatory proteins can underlie cardiac contractile dysfunction that occurs in the J2N-k cardiomyopathic hearts. Accordingly, we examined the SR function of the cardiac muscle in J2N-k and J2N-n hamsters.
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MATERIALS AND METHODS |
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Animal model. The animal model used in this study was the 36-wk cardiomyopathic J2N-k male hamster and was compared with the normal J2N-n male hamster. The hamsters were housed in humidity- and temperature-controlled rooms and allowed free access to water and standard chow.
Echocardiographic assessment of cardiac performance. Cardiac ultrasound studies were carried out using the SONOS 5500 ultrasonograph (Agilent Technologies). Transthoracic short-axis measurements were performed using a 12-MHz annular array ultrasound transducer, which recorded left ventricular diastolic and systolic measurements. The M-mode echocardiograms measured the following parameters: interventricular septum thickness (IVS), left ventricular internal dimension (LVID), and left ventricular posterior wall thickness (LVPW) at diastole and systole, as well as ejection fraction, percent fractional shortening, cardiac output, left ventricular mass, and heart rate. Echocardiographic assessment was carried out as the hamsters were anesthetized using isoflurane gas on 2 liters of oxygen.
General characterization of cardiomyopathic and control hamsters. Cardiomyopathic hamsters (J2N-k) and control hamsters (J2N-n) were anesthetized, and blood was collected for measuring creatine kinase activity using the IDEXX VetTest Chemistry Analyzer and dry chemistry slides from Ortho Diagnostics Laboratories (Westbrook, ME). Animals were anesthetized, and the heart muscle was isolated. Heart and body weights were determined, and wet and dry weights of the lungs and both kidneys were measured.
Isolation of SR vesicles. SR vesicles were obtained using a method described previously (2628, 40). Ventricular tissue was pulverized and homogenized in a buffer (10 ml/g tissue) containing (in mM) 10 NaHCO3, 5 NaN3, and 15 Tris·HCl (pH 6.8) with a Polytron homogenizer (Brinkmann, Westbury, NY). The homogenate was centrifuged for 20 min at 10,919 g. The resultant pellet was discarded, and the supernatant was further centrifuged for 45 min at 43,666 g (JA 20 rotor; Beckman). The supernatant obtained contained the cytosolic fraction and was aliquoted and frozen in liquid nitrogen before being stored at 80°C. The resultant pellet was resuspended in a buffer containing 0.6 M KCl and 20 mM Tris·HCl (pH 6.8) and centrifuged for 45 min at 43,666 g. The final pellet containing the SR fraction was suspended in a buffer containing 250 mM sucrose and 10 mM histidine (pH 7.0), aliquoted, and frozen in liquid nitrogen before being stored at 80°C. All buffers used for isolation contained a cocktail of protease inhibitors consisting of aprotinin, leupeptin, 4-(2-aminoethyl)benzenesulfonyl fluoride, and 0.1% phenylmethylsulfonyl fluoride to prevent any degradation of proteins during the isolation procedure.
Measurement of Ca2+ uptake. SR Ca2+ uptake was measured using a procedure described previously (2628, 40). The reaction mixture contained (in mM) 50 Tris-maleate (pH 6.8), 5 NaN3, 5 ATP, 5 MgCl2, 120 KCl, 5 potassium oxalate, 0.1 EGTA, 0.1 45CaCl2 (20 mCi/l), and 25 µM ruthenium red. The initial free Ca2+ concentration in this medium, determined using the program of Fabiato (6), was 8.2 µM. Ruthenium red was added as an inhibitor of the Ca2+ release channel. The reaction was initiated by adding SR vesicles (20 µg protein) to the reaction mixture at 37°C and was terminated after 1 min by filtration. The filters were washed, dried, and counted in a beta scintillation counter. SR Ca2+ uptake at 1 min was found to be in the linear range (see Fig. 2, inset).
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Western blot analysis. The protein content of SR Ca2+-cycling proteins SERCA2a and PLB and its phosphorylated forms, serine-16 PLB and threonine-17 PLB, were determined by Western blot analysis as described previously (27, 28, 40). SR samples (20 µg) were separated by SDS-polyacrylamide gel electrophoresis on 10% (for SERCA2a) and 15% (for PLB, serine-16 PLB, and threonine-17 PLB) gels and transferred to polyvinylidene difluoride membranes. A linear relationship in the reactivity of bands and amounts of PLB and SERCA2a was demonstrated (see Fig. 3, A and B, insets). Membranes were probed with a monoclonal anti-SERCA2a antibody obtained from Affinity Bioreagents (Golden, CO), monoclonal anti-PLB polyclonal antibody obtained from Upstate Biotechnology, polyclonal anti-serine-16 PLB antibody obtained from Santa Cruz Biotechnology (Santa Cruz, CA), and polyclonal anti-threonine-17 PLB antibody obtained from Badrilla (Leeds, UK). Appropriate secondary antibodies were used, and the antibody-antigen complexes in all membranes were detected using the ECL kit (Amersham Life Science, Oakville, ON, Canada). An imaging densitometer (model GS-800; Bio-Rad, Hercules, CA) was used to scan the protein bands, and data were quantified using Quantity One 4.4.0 software (Bio-Rad).
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Measurement of phosphatase activity. Determination of the phosphatase activity of the SR was based on a technique established previously (2628). Briefly, the phosphatase activity was determined using a serine/threonine assay kit (Upstate Biotechnology). This assay is based on the principle of dephosphorylation of the synthetic phosphopeptide KRpTIRR. The reaction was initiated by adding 30 µg of SR to microtiter wells in the presence or absence of the synthetic substrate (200 µm) and incubating for 30 min. The reaction was terminated by adding malachite green solution, and the absorbance was read after 15 min at 660 nm to determine the amount of inorganic phosphate released. This assay was performed in both the presence and absence of the exogenous substrate.
Statistical analysis. Results are expressed as means ± SE and were statistically evaluated using one-way analysis of variance. A level of P < 0.05 was considered the threshold for statistical significance between the control and various experimental groups.
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RESULTS |
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Because the SR plays a crucial role in cardiac contractility, its function was examined in control and cardiomyopathic hamsters. Western blot analysis of calsequestrin (CQS), an exclusive protein of the SR, was performed to determine purity of SR preparations isolated from control and cardiomyopathic hearts. CQS levels were increased two- to threefold in the SR fraction compared with homogenates (Fig. 1), indicating a two- to threefold purification of the SR. Figure 2 shows a decrease in cardiac SR Ca2+ uptake in cardiomyopathic hamsters compared with controls. Because a reduction in cardiac SR Ca2+ uptake in cardiomyopathic hamsters could be a direct consequence of a decrease in the expression of SERCA2a or inhibition of SERCA2a by PLB, the protein content of SERCA2a and PLB was also determined. Figure 3A shows that whereas the level of PLB protein was not altered, the level of SERCA2a protein content was, in fact, reduced (Fig. 3B), with a concomitant increase in the ratio of PLB-to-SERCA2a levels (Fig. 2C) in the cardiomyopathic hamster. Because the depression in the protein levels of SERCA2a in cardiomyopathic hamsters may be a direct result of the reduction in its message levels, we examined the mRNA levels of SERCA2a in control and cardiomyopathic hamsters. Figure 4 shows that SERCA2a mRNA levels were significantly decreased in cardiomyopathic hamsters compared with controls.
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DISCUSSION |
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Previous cardiomyopathic hamster studies have reported abnormalities in SR function; however, the underlying mechanisms are not fully understood (8, 42, 44). In this study, the decrease in heart function was associated with a reduction in SR Ca2+ uptake of the cardiomyopathic heart. Analysis of the protein content of the Ca2+-cycling proteins responsible for SR Ca2+ uptake revealed a decrease in protein levels of SERCA2a, with no change in PLB cardiomyopathic hamster heart. The ratio of PLB-to-SERCA2a protein levels was increased and would therefore result in increased inhibition of SERCA2a by PLB in the cardiomyopathic hamster heart. A reduction in SERCA2a protein content could be directly linked to a decrease in the mRNA levels of SERCA2a. These results could possibly be used to explain the reduction of SR Ca2+ uptake in the cardiomyopathic heart.
Regulatory mechanisms involved in modulating heart function on a beat-to-beat basis include the precisely balanced complementary reactions of phosphorylation/dephosphorylation (30). In particular, the two major signaling pathways that activate CaMK and PKA, regulating SR function, were investigated in this study. Under normal physiological conditions, enhanced SR Ca2+ uptake is achieved primarily by the phosphorylation of PLB by CaMK and PKA (15, 18, 20). The underlying inhibitory effect of PLB on SERCA2a is relieved as a result of the phosphorylation of PLB by PKA and CaMK (5, 13, 15, 18, 20, 33), which in turn increases the affinity of SERCA2a for Ca2+, as well as Vmax for SR Ca2+ transport. This study shows a significant decrease in PLB phosphorylation at serine-16 by PKA. This alteration was of a specific nature, because PLB phosphorylation at threonine-17 by CaMK was unaffected in the cardiomyopathic heart. Because PLB protein content was unaltered in the cardiomyopathic hearts, reduced PLB phosphorylation by PKA could be directly due to a significant decrease in the SR-associated PKA activity. The alteration in PKA activity was not exclusively restricted to the SR-associated PKA pool, because the cytosolic PKA activity was also significantly reduced in the cardiomyopathic heart. The unaltered status of PLB phosphorylation by CaMK in cardiomyopathic hearts was consistent with no changes in the SR-associated CaMK activity. Thus a reduction in PLB phosphorylation by PKA could also contribute to the impairment of SR Ca2+ transport in cardiomyopathic hearts by increased inhibition of PLB on SERCA2a.
Because phosphorylation and dephosphorylation are complementary regulatory mechanisms, increased SR-associated phosphatase activity observed in the cardiomyopathic heart could potentially contribute to decreased PLB phosphorylation. It has been reported that the protein phosphatase endogenous to the SR is a type I phosphatase (37) that functions to dephosphorylate PLB at both the CaMK and PKA sites (16). By dephosphorylating PLB, protein phosphatase 1 inhibits the activity of SERCA2a and ultimately prolongs the duration of contraction, as Ca2+ uptake into the SR is reduced. Earlier studies reported mechanisms of SR dysfunction in dilated cardiomyopathic human hearts; these include a reduction in expression of SERCA2a (21), reduced PLB phosphorylation at serine-16 (35), and increased phosphatase activity (22). Our results on SR dysfunction in cardiomyopathic hamster hearts were consistent with those of cardiomyopathic human hearts. Nonetheless, it must be noted that in the human heart studies, dilated cardiomyopathy was of unknown nature (idiopathic). Incidentally, our results specifically reveal mechanisms underlying cardiac contractile dysfunction in inherited genetic disorders such as X-linked DCM, as well as Duchenne and Becker muscular dystrophies, as a direct consequence of the deletion of the -sarcoglycan gene. Earlier studies on different cardiomyopathic models reported abnormalities in SR Ca2+ uptake and release, as well as defects in the mechanisms responsible for these functions. With regard to mechanisms underlying SR Ca2+ release in cardiomyopathic hamsters, alterations in ryanodine receptor (RyR) binding and RyR mRNA levels (17) were observed in BIO 14.6 cardiomyopathic hamsters, whereas RyR binding and RyR protein levels were abnormal in UM-X7.1 cardiomyopathic hamsters (42). With respect to the mechanisms responsible for SR Ca2+ uptake in the cardiomyopathic hamsters, reduced SERCA2a and PLB protein levels were observed in BIO 14.6 cardiomyopathic hamsters (11). SERCA2a mRNA levels were also found to be reduced in BIO 53.58 cardiomyopathic hamster hearts (1). Conclusively, our study is the first to date that highlights novel mechanisms such as an increased ratio of PLB-to-SERCA2a protein levels, reduced PLB phosphorylation by PKA, decreased PKA activity, and increased SR-associated protein phosphatase activity in cardiomyopathic hamster hearts.
In summary, our results indicate that cardiac contractile dysfunction occurred in the J2N-k cardiomyopathic hamster and that this phenomenon could be associated with the impairment of SR function. SR dysfunction occurred as the result of a marked inhibition of SERCA2a, caused by the increase in PLB-to-SERCA2a ratio, as well as by reduced levels of phosphorylation of PLB by PKA (a result of decreased PKA activity and increased phosphatase activity) in the cardiomyopathic hamster heart.
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GRANTS |
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FOOTNOTES |
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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. Section 1734 solely to indicate this fact.
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