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1Department of Pharmacology and Cell Biophysics, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267; and 2Department of Life Science and National Research Laboratory of Proteolysis, Kwangju Institute of Science and Technology, Kwangju 500-712, South Korea
Submitted 22 December 2003 ; accepted in final form 4 June 2004
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ABSTRACT |
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sarcoplasmic reticulum; calcium cycling; contractility
A newly discovered histidine-rich, Ca-binding protein (HRC) has been shown to increase SR Ca storage capacity in neonatal rat myocytes (14). This protein appears to be an additional component of the SR Ca release structure, composed of the RyR, calsequestrin (CSQ), triadin (TRI), and junctin (JNT) (18, 33). HRC binds Ca (3, 11, 25) and has been proposed to be either in the lumen of the SR (10, 15, 18, 30, 31) or on the cytoplasmic surface of the SR (5, 6, 27, 28). There is a single isoform of HRC, identified in rabbit skeletal, cardiac, and arteriolar smooth muscles (10, 24, 26, 28). The deduced amino acid sequence of HRC reveals structural features that are analogous to CSQ, suggesting that HRC may play a similar role to CSQ during excitation-contraction coupling, as a Ca storage protein (18). In addition, biochemical studies in skeletal muscle have shown that HRC associates with TRI in a Ca-dependent manner, implicating HRC to be involved in regulation of Ca release from the SR by an interaction with TRI (18, 27, 28).
In this study, we assessed the protein levels of HRC in animal models of heart failure and in human heart failure and identified significant decreases compared with nonfailing hearts. To gain insight into the functional significance of altered HRC expression levels, we infected adult rat cardiac ventricular myocytes with a recombinant adenovirus that contains the full-length mouse HRC cDNA (mHRC). For the first time, we demonstrate that overexpression of HRC in adult rat cardiomyocytes results in increased SR Ca storage capacity, but this Ca is not available for release, resulting in depressed myocyte Ca kinetics and mechanics. Thus HRC may be an integral regulatory protein in the cardiac muscle Ca-cycling cascade.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONGRANTSREFERENCES |
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q-overexpressing mouse hearts (7)]. Human tissues were obtained from four nonfailing donor and five failing left ventricles (LVs). Once in the laboratory, the tissue was cut in pieces, frozen in liquid N2, and stored at –80°C. All tissue was homogenized in a buffer (pH 7.0) containing 50 mM potassium phosphate buffer, 10 mM NaF, 1 mM EDTA, 0.3 M sucrose, 0.3 mM PMSF, 0.5 mM DTT, and a cocktail inhibitor protease inhibitor tablet (Roche). According to the established linear range, animal (10 µg) and human (20 µg) homogenates were then subjected to SDS-PAGE and immunoblotted as described below. This investigation conformed with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Pub. No. 85-23, Revised 1996) and with the principles outlined in the Declaration of Helsinki (32). Adult rat ventricular myocyte isolation and culture. Hearts from adult male Sprague-Dawley rats (8 wk old) were perfused with modified Krebs-Henseleit buffer (KHB; 118 mM NaCl, 4.8 mM KCl, 25 mM HEPES, 1.25 mM K2HPO4, 1.25 mM MgSO4, 11 mM glucose, 5 mM taurine, and 10 mM butanedione monoxime; pH 7.4) for 5 min. Subsequently, hearts were perfused with an enzyme solution [KHB containing 0.7 mg/ml collagenase type II (263 U/mg), 0.2 mg/ml hyaluronidase, 0.1% BSA, and 25 µM Ca] for 10 min. Then, 25 µM Ca was added to the perfusion buffer, and hearts were perfused for 5 min. Subsequently, the Ca concentration in the perfusion buffer was raised to 100 µM, and perfusion continued for 5 min. Finally, LV tissue was excised, minced, pipette dissociated, and filtered through a 240-µm screen. Cells were harvested and resuspended in 1 mM Ca-KHB + 1% BSA. After brief centrifugation, the cells were resuspended in 1.8 mM Ca-KHB + 1% BSA, centrifuged briefly again, and resuspended in ACCT medium (DMEM containing 2 mg/ml BSA, 2 mM L-carnitine, 5 mM creatine, 5 mM taurine, 100 IU/ml penicillin, and 100 µg/ml streptomycin). Cells were then counted and plated on laminin-coated glass coverslips or dishes.
Adenovirus-mediated gene transfer. Recombinant adenovirus HRC was generated by using the Adeno-X expression system kit as described before (14). Two hours after isolation and culture, myocytes seeded on coverslips or dishes were infected with either mHRC adenovirus (AdHRC) or green fluorescent protein adenovirus (AdGFP) in diluted media, at a multiplicity of infection of 800, for 2 h before the addition of a suitable volume of culture media. Twenty-four hours later, the myocytes were washed in 1 mM Ca-KHB and harvested for quantitative immunoblotting, immunostaining, or use in the experiments outlined below.
Quantitative immunoblotting. Cultured myocytes were then harvested and lysed for 20 min at 4°C in lysis buffer [20 mM Tris·HCl (pH 7.5), 150 mM NaCl, 1% NP-40, 10% glycerol, 0.4 mM sodium orthovanadate, 10 mM sodium pyrophosphate, 10 mM sodium fluoride, 0.5 mM DTT, and 2 µl/ml protease inhibitor cocktail]. The supernatants obtained after centrifugation at 8,000 g for 10 min were then used in subsequent experiments. Protein loading (6–30 µg) varied for each protein; however, a linear range was performed on each immunoblot to ensure that protein loading was within the linear range of detection. Protein samples were electrophoresed on 6% (RyR), 8% (TRI, HRC, CSQ), or 12% (JNT, PLB, SERCA, CSQ) SDS-polyacrylamide gels under reducing conditions and then transferred to nitrocellulose membranes (various transfer conditions per respective protein). Membranes were blocked with 5% nonfat milk in Tris-buffered saline for 1 h at room temperature and then incubated overnight at 4°C or at room temperature for 2–3 h with the respective primary antibody: RyR (Affinity Bioregents); TRI (Affinity Bioreagents); HRC (polyclonal rabbit anti-mouse HRC antibody, generous gift from Dr. Woo Jin Park); JNT (polyclonal rabbit anti-canine JNT antibody, generous gift from Dr. Larry Jones); PLB (Affinity Bioreagents); SERCA (homemade polyclonal rabbit anti-SERCA antibody); and CSQ (Affinity Bioreagents). Blots were then washed in Tris-buffered saline, incubated with horseradish peroxidase-conjugated secondary antibodies (Amersham Biosciences) for 1 h at room temperature, washed again, and subsequently developed using the ECL chemiluminescence detection kit (Amersham Biosciences) according to the manufacturer's instructions. Protein loading was normalized to endogenous CSQ levels, as CSQ levels were not altered in HRC-overexpressing myocytes.
Immunostaining.
Twenty-four hours after cardiomyocytes (grown on coverslips) were infected with either AdGFP or AdHRC, the cells were washed with chilled PBS, fixed for 15 min in a 4% formaldehyde solution, immersed in 0.5% Triton X-100 solution at room temperature for 15 min, washed twice in chilled PBS (5-min washes), and blocked in 5% BSA solution (in PBS) at 37°C for 30 min. Subsequently, the cells were incubated with diluted polyclonal rabbit anti-mHRC antibody (1:500 in blocking solution) for 1 h at room temperature, washed four times with chilled PBS (5-min washes), incubated with Alexa Fluor 488-conjugated goat -rabbit IgG (Molecular Probes) secondary antibody (1:500 in blocking solution) for 1 h at room temperature, and washed again four times with chilled PBS. Finally, the myocyte-covered coverslip was inverted onto a slide containing 10 µl of Vectashield mouting media (VECTA), and immunofluorescence was analyzed.
Contractile parameter measurements. Myocytes that adhered to the coverslips were bathed in temperature (37°C)-equilibrated KHB containing 1 mM Ca for 20 min. The myocyte suspension was then placed in a Plexiglas chamber, which was positioned on the stage of an inverted epifluorescence microscope (Nikon Diaphot 200). Myocyte contraction was field stimulated by a Grass S5 stimulator (0.5 Hz, square waves), and contractions were videotaped and digitized on a computer. A video edge motion detector (Crescent Electronics) was used to measure myocyte length and cell shortening, from which the percent fractional shortening (%FS) {[(resting cell length – maximal cell shortening length)/resting cell length]x100} and maximal rates of contraction and relaxation (±dL/dt) were calculated (4). To investigate the response of adult rat cardiomyocytes ovexpressing HRC to isoproterenol, a maximal concentration of isoproterenol (100 nM) was added to the KHB (including 1 mM Ca) in the Plexiglas chamber, and the above-mentioned measurements were repeated. All data were analyzed using software from Felix 1.1 Software (Photon Technology) and Ionwizard.
Intracellular Ca transient and SR Ca load measurements. To obtain intracellular Ca signals, myocytes that adhered to coverslips were incubated with the AM form of fura-2 (2 µM) for 20 min at room temperature and then resuspended in KHB containing 1 mM Ca. After four washes, the coverslip was mounted in a Plexiglas chamber containing 2 ml of fresh KHB (including 1 mM Ca), which was field stimulated to contract at 0.5 Hz between platinum wire electrodes. The fura-2 fluorescence was determined at room temperature using a Delta Scan dual-beam spectrofluorophotometer (Photon Technology) operating at an emission wavelength of 510 nm with excitation wavelengths of 340 and 380 nm. Baseline, amplitude (estimated by the 340-to-380-nm ratio), the time for 50% decay of the Ca signal, and the fast time constant of decay of the Ca signal were acquired. To assess SR Ca load, caffeine-induced Ca release was initiated by rapid application of 10 mM caffeine (in Ca-free KHB). All data were analyzed using software from Felix 1.1 Software (Photon Technology) and Ionwizard.
Statistics. All results are expressed as means ± SE. ANOVA, followed by a Bonferroni multiple-comparison post test, was used to compare animal models of heart failure. Student's paired t-test was used to compare data between human donor and heart failure samples and between AdGFP and AdHRC groups in the isolated myocyte studies. Furthermore, animal-based data (not cell based) was used for statistical comparisons. In all analyses, P < 0.05 was considered statistically significant.
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RESULTS |
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q overexpression (7). HRC levels were significantly decreased (
40–50%) in both animal models of heart failure relative to wild-type (WT) mice (Fig. 1A). It is interesting to note that compared with WT mouse hearts, there were no alterations in the expression levels of HRC in CSQ-overexpressing mouse hearts (data not shown), which develop hypertrophy but do not transition to heart failure (29). We also examined the levels of HRC in biopsy samples from human nonfailing (n = 4) and failing (n = 5) hearts with dilated cardiomyopathy and observed that HRC levels were significantly reduced (17%) in failing hearts (Fig. 1B). This suggests that a perturbation in the levels of HRC may have a potential role in heart failure.
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130 kDa, which is much greater than its expected molecular mass (79,860 Da), consistent with previous observations (14). Immunostaining with the anti-mouse HRC antibody in adult rat ventricular myocytes revealed a consistent labeling pattern across the length and depth of myocytes infected with either AdGFP or AdHRC (data not shown). The staining pattern was more pronounced in the AdHRC-expressing myocytes compared with the controls, confirming the increased expression of HRC in AdHRC-infected myocytes. Immunostaining with anti-CSQ was also performed in parallel to determine a pattern for SR protein staining in adult rat ventricular myocytes (data not shown). The anti-CSQ staining pattern was similar to the anti-mHRC pattern in AdGFP and AdHRC-infected myocytes, indicating that HRC was localized to the same compartment as CSQ.
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-adrenergic responses, adult rat cardiomyocytes were stimulated with a maximum concentration of isoproterenol (100 nM). The contractile and Ca kinetic parameters of infected myocytes are shown in Fig. 6. Isoproterenol stimulation resulted in enhancement of mechanics and Ca kinetic parameters in both AdGFP- and AdHRC-infected cardiac myocytes. However, the depressed basal contractile and Ca kinetic parameters in the HRC-infected myocytes remained significantly depressed even after maximal isoproterenol stimulation (Fig. 6, A and B).
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DISCUSSION |
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TOPABSTRACTEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONGRANTSREFERENCES |
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The SR internal Ca store of cardiac cells has a limited capacity; thus replenishment of Ca from the cytosol is necessary during each cycle of excitation-contraction coupling, and any perturbation in SR Ca cycling has been suggested to have detrimental effects on contractile function (22). HRC overexpression leads to an increased SR Ca load; however, because this SR Ca is bound by the excess amount of HRC, the quantal release of SR Ca is severely depressed, and this is associated with attenuation of single-cell Ca transients and cardiac contractility (Figs. 3 and 4). As a result, the diminished cytosolic Ca levels during contraction would lead to an attenuation of SERCA Ca transport rates and diminished rates of relaxation. Strikingly, this study suggests that a modest increase in expression of HRC (1.7-fold) is associated with a more dramatic phenotype than a 20-fold overexpression of CSQ (29). Importantly, even though the SR Ca pools are unresponsive to electrical stimulation in HRC-overexpressing myocytes, they are released upon caffeine application, similar to findings in CSQ-overexpressing myocytes (13, 29). This may suggest a defect in the RyR release of Ca, possibly due to an alteration in the sensitivity of the Ca regulatory site on the RyR, associated with the increased SR Ca-buffering capacity in myocytes overexpressing HRC. Thus the present study provides the first evidence that HRC is a regulator of SR Ca release. Future studies in lipid bilayer preparations from models with altered HRC expression levels will provide information on the effects of HRC on RyR function.
Because we hypothesized there is a tight balance between the levels of proteins involved in SR Ca uptake, storage, and release and that selective overexpression of one protein in the complex may interrupt their tight balance and cause alterations of other proteins, we investigated the levels of SERCA, PLB, RyR, TRI, JNT, and CSQ. We discovered that overexpression of HRC did not affect the levels of the proteins regulating SR Ca uptake (SERCA and PLB) but was associated with upregulation of TRI and JNT, two junctional SR proteins that have been suggested to associate into a stable quaternary complex at the SR junctional membrane, regulating SR Ca release. Interestingly, the levels of the other two proteins, RyR and CSQ, postulated to be part of the quaternary complex with TRI and JNT, remained unaltered. In light of the novel finding that TRI and JNT levels are upregulated in HRC-overepressing adult rat ventricular myocytes, it is important to speculate what effect upregulation of these proteins may have on increased SR Ca load and the resulting attenuated Ca transients and decreased contractile parameters observed in these myocytes. Indeed, it is possible that overexpression of HRC alone may alter the conformation of the SR quaternary release complex and, therefore, cause inhibition of RyR channel function. However, a recent study has shown that cardiac myocytes isolated from TRI-1-ovexpressing mice (5-fold) are characterized by delayed rates of Ca transient decay as well as decreased rates of cell shortening and relengthening, suggesting that upregulation of TRI-1 impairs myocardial contractility (16). In addition, cardiac myocytes isolated from JNT-overexpressing mice (24- to 29-fold) exhibit lower basal intracellular Ca concentrations (12), whereas myocytes isolated from JNT-overexpressing mice (10-fold) show impaired relaxation parameters, although caffeine-induced Ca release was attenuated compared with WT mice (17). Thus it is more likely that perturbation in the levels of TRI, JNT, and HRC coordinately contribute to the resulting abnormal Ca cycling and impaired contractility observed in AdHRC-infected adult rat myocytes.
Recently, HRC has been suggested to be an additional component of the SR quaternary structure involved in Ca release (18) and may play a similar role to CSQ in SR Ca storage (14, 18). Furthermore, an interaction between HRC and TRI has been reported (18, 27, 28), although the apparent cross-talk between the players in the SR Ca release complex has not been elucidated. Interestingly, overexpression of JNT was associated with downregulation of TRI protein levels (12, 17) and vice verca (16), suggesting an important compensatory cross-talk, because the overall structures and interacting characteristics of JNT and TRI are similar (12, 32). Furthermore, in CSQ-overexpressing mouse myocytes, TRI and JNT levels were reduced by 50%, relative to control (13). Thus this is the first evidence suggesting that perturbation in the level of one of the proteins in the stable quaternary complex at the SR junctional membrane, HRC, results in upregulation of both TRI and JNT and that this is associated with increased SR Ca load, decreased SR fractional Ca release, and impaired contractility in adult rat ventricular myocytes. These data, taken together, suggest that proper formation of a quaternary molecular complex between RyR, TRI, JNT, CSQ, and HRC is imperative for efficient SR Ca release and, subsequently, for normal cardiac function. Furthermore, this is the first data implicating HRC in regulation of SR Ca release, apart from its role in SR Ca storage.
Indeed, Ca cycling in the cardiac myocyte is a complex process, and further elucidation of the proteins involved in Ca uptake, storage, and release is necessary to better understand the basic physiology and pathophysiology of the heart. However, our findings indicate that HRC may be a regulatory protein in the cardiac muscle SR Ca-release cascade and that perturbation in HRC levels could be a potential mechanism underlying cardiac dysfunction. Given the role of HRC in increasing the SR Ca load that is unavailable for release, it is possible that HRC levels are downregulated in heart failure as a compensatory mechanism to increase the amount of Ca released from the SR, in an attempt to enhance Ca cycling and cardiac function. However, it is also likely that HRC levels may be critical for proper SR Ca cycling and any alterations, such as decreases in human and experimental heart failure or increases such as in our rat cardiac myocytes, result in abnormal Ca cycling and cardiac dysfunction. Further investigation in HRC-overexpressing and HRC knockout mice is required to more extensively address HRC's additional binding partners in cardiac SR as well as its physiological and pathophysiological role in SR Ca cycling.
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ACKNOWLEDGMENTS |
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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.
* G.-C. Fan and K. N. Gregory contributed equally to this work. 
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) in AdHRC-infected myocytes compared with AdGFP-infected myocytes. n = 6 hearts for each group and 15–25 myocytes/heart, with 98 (AdGFP) and 123 (AdHRC) total myocytes/group. Values are means ± SE. *P < 0.05.
