HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) All Versions of this Article: 291/1/H88    most recent 01372.2005v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Web of Science (3) Citing Articles via Google Scholar Google Scholar Articles by Sathish, V. Articles by Narayanan, N. Search for Related Content PubMed PubMed Citation Articles by Sathish, V. Articles by Narayanan, N.

Mechanistic basis of differences in Ca²⁺-handling properties of sarcoplasmic reticulum in right and left ventricles of normal rat myocardium

Department of Physiology and Pharmacology, University of Western Ontario, London, Ontario, Canada

Submitted 27 December 2005 ; accepted in final form 27 January 2006

	ABSTRACT

TOP ABSTRACT METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
This study investigated Ca²⁺-cycling properties of sarcoplasmic reticulum (SR) in right ventricle (RV) and left ventricle (LV) of normal rat myocardium. Intracellular Ca²⁺ transients and contractile function were monitored in freshly isolated myocytes from RV and LV. SR in RV displayed nearly fourfold lower rates of ATP-energized Ca²⁺ uptake in vitro than SR of LV. The Ca²⁺ concentration required for half-maximal activation of Ca²⁺ transport was nearly twofold higher in SR of RV. The lower Ca²⁺-sequestering activity of SR in RV was accompanied by a matching decrement in Ca²⁺-induced phosphoenzyme formation during the catalytic cycle of the Ca²⁺-pumping ATPase (SERCA2). Western immunoblot analysis showed that protein levels of Ca²⁺-ATPase and its inhibitor phospholamban (PLN) were only 15% lower in SR of RV than in SR of LV. Coimmunoprecipitation experiments revealed that PLN-bound, functionally inert Ca²⁺-ATPase molecules in SR of RV greatly exceed (>50%) that in SR of LV. Endogenous Ca²⁺/calmodulin-dependent protein kinase-mediated phosphorylation of SR substrates did not abolish the huge disparity in SR Ca²⁺ pump function between RV and LV. Intracellular Ca²⁺ transients, evoked by electrical field stimulation, were significantly prolonged in RV myocytes compared with LV myocytes, mainly because of slow decay of intracellular Ca²⁺ concentration. The slow decay of intracellular Ca²⁺ concentration in RV and consequent decrease in the speed of RV relaxation may promote temporal synchrony of the end of diastole in RV and LV. The preponderance of functionally silent SR Ca²⁺ pumps in RV reflects a higher diastolic reserve required to protect and maintain RV function in the face of a sudden rise in afterload or resistance in the pulmonary circulation.

calcium-adenosinetriphosphatase; phospholamban; phosphoenzyme; calcium transient; diastolic reserve

Cardiac adaptation to a variety of physiological stresses is inevitably linked to molecular remodeling of the sarcoplasmic reticulum (SR) Ca²⁺-cycling apparatus. The major Ca²⁺-cycling proteins in SR include the ryanodine receptor (RyR) Ca²⁺ release channel (CRC), which is responsible for Ca²⁺ release into cytosol on myocyte excitation to induce muscle contraction (5); sarco(endo)plasmic Ca²⁺-ATPase (SERCA2a), which actively sequesters Ca²⁺ back into SR lumen to promote muscle relaxation (18); the Ca²⁺-storage protein calsequestrin (27); and phospholamban (PLN), which regulates Ca²⁺-ATPase function (23, 34, 37). In its unphosphorylated state, PLN is thought to interact with Ca²⁺-ATPase, exerting an inhibitory effect manifested largely through a decrease in the enzyme's affinity for Ca²⁺; phosphorylation of PLN by cAMP-dependent protein kinase or calmodulin (CaM) kinase is thought to disrupt this interaction, resulting in enhanced affinity of the ATPase for Ca²⁺ and stimulation of Ca²⁺ pump activity (23, 34, 37). This long-standing view has been questioned recently, and it has been reported that Ca²⁺, but not phosphorylation of PLN, disrupts the interaction between Ca²⁺-ATPase and PLN (1). Besides PLN, CaM and CaM kinase are tightly associated with cardiac SR and have been implicated in modulation of Ca²⁺ uptake and release functions of SR through direct phosphorylation of Ca²⁺-ATPase (15, 31, 32, 43–46) and RyR CRC (14, 38, 42).

Increase in afterload of the systemic circulation is known to invoke tissue remodeling and altered Ca²⁺ homeostasis in LV. However, the impact of physiological load of RV on SR function, to our knowledge, has not been studied. As a first step in exploring the mechanisms of RV adaptation to stress, the present study evaluated potential intrinsic differences in SR Ca²⁺-cycling properties of RV and LV, given the huge difference in afterload encountered by these cardiac chambers in the normal physiological state. Although an earlier study documented differences in Ca²⁺ transport between regions of the dog heart (9), the mechanistic basis of the differences was not addressed. Here we combine biochemical analysis with functional studies of intracellular Ca²⁺ concentration ([Ca²⁺]_i) transients in freshly isolated myocytes to gain insights into the intrinsic Ca²⁺ cycling properties of RV and LV in normal rat myocardium. Our findings demonstrate striking chamber-specific differences, with a smaller fraction of the available SR Ca²⁺ pump units operative in RV than in LV, implying greater diastolic reserve in RV. We propose that this is of importance in preserving RV function in response to a sudden rise in afterload or impedance of blood flow created by the resistance in the pulmonary vasculature.

	METHODS

TOP ABSTRACT METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
Animals. Male Wistar rats weighing 250–300 g were purchased from Charles River (St. Constant, PQ, Canada). On arrival, the rats were housed individually in plastic cages in the Health Sciences Center animal care facility of this institution at 23°C on a 12:12-h light-dark cycle. The investigations were conducted under guidelines approved by the local Animal Care Committee in accordance with the standards of the Canadian Council on Animal Care. All animals had free access to food (Purina chow containing 20% protein) and water.

Chemicals. Reagents for electrophoresis were obtained from Bio-Rad Laboratories (Mississauga, ON, Canada). [-³²P]ATP was purchased from Amersham (Oakville, ON, Canada), ⁴⁵CaCl₂ from New England Nuclear (Mississauga, ON, Canada); anti-PLN monoclonal antibody from Upstate Biotechnology (Lake Placid, NY), and fura-2 AM from Molecular Probes (Eugene, OR). SERCA2-specific antibody anti-87 (24) was a generous gift from Dr. A. K. Grover (McMaster University, Hamilton, ON, Canada). All other chemicals were obtained from Sigma (St. Louis, MO).

Preparation of SR membrane vesicles and muscle homogenate. SR membrane vesicles were isolated from RV and LV myocardium of rats according to the procedure described previously (22). Briefly, RV and LV free walls were isolated separately, minced, and homogenized (Polytron, Brinkman Instruments, Westbury, NY) with three 15-s bursts at 30-s intervals at setting 5.5 in 6 vol (based on tissue weight) of ice-cold buffer (10 mM NaHCO₃, pH 6.8). The homogenate was centrifuged at 1,000 g for 10 min at 4°C. The supernatant was decanted and kept in an ice slurry. The pellet was resuspended in 4 vol of ice-cold buffer and centrifuged as described above. The supernatant was decanted and combined with the first supernatant, and the pellet was discarded. The combined supernatant was centrifuged at 8,000 g for 20 min at 4°C. The supernatant was collected, and the pellet was discarded. KCl was added to the supernatant (44 mg/ml, 0.6 M final concentration), swirled until dissolved, left on ice for 25 min, and then centrifuged at 40,000 g for 1 h at 4°C. After isolation, the SR vesicles were suspended in 10 mM Tris-maleate (pH 6.8) containing 100 mM KCl and stored at –80°C after quick freezing in liquid nitrogen. Protein concentration was determined by the method of Lowry et al. (26) with bovine serum albumin used as the standard. The yield of SR membranes from RV and LV myocardium was similar (1.9 mg/g wet tissue). The relative purity of SR vesicles from RV and LV myocardium of rats did not differ as judged from essentially similar protein profiles revealed by SDS-PAGE.

In addition to SR membranes, RV and LV muscle homogenates were used in some experiments. The muscle tissue was homogenized (Polytron) with three 15-s bursts at 30-s intervals at setting 5.5 in 10 vol (based on tissue weight) of buffer (10 mM Tris·HCl-100 mM KCl, pH 6.8). The homogenates were filtered through four layers of cheese cloth.

Determination of Ca²⁺ uptake. ATP-dependent, oxalate-facilitated Ca²⁺ uptake by cardiac SR vesicles and muscle homogenates was determined using the Millipore filtration technique as described previously (29). The standard incubation medium for Ca²⁺ uptake (250 µl total volume) contained (in mM) 50 Tris-maleate (pH 6.8), 5 MgCl₂, 5 NaN₃, 120 KCl, 0.1 EGTA, 5 potassium oxalate, 5 ATP, 0.1 ⁴⁵CaCl₂ (8,000 cpm/nmol, 8.2 µM free Ca²⁺), and 0.025 ruthenium red and cardiac SR vesicles (7.5 µg protein) or muscle homogenate (10 µg protein). In experiments where Ca²⁺ concentration dependence was varied, EGTA concentration was held constant at 0.1 mM, and the amount of total ⁴⁵CaCl₂ added was varied to yield the desired free Ca²⁺ concentration according to the computer program of Fabiato (11). The Ca²⁺ uptake reaction was initiated by addition of SR to the assay components, which had been preincubated for 3 min at 37°C. The data on Ca²⁺ concentration dependence of Ca²⁺ uptake were analyzed by nonlinear regression analysis (SigmaPlot) and fitted to the following equation

where v is measured Ca²⁺ uptake activity at a given Ca²⁺ concentration, V_max is maximum activity, K_0.5 is Ca²⁺ concentration resulting in one-half V_max, and n is the Hill coefficient. To evaluate the effect of endogenous CaM kinase or exogenous PKA on Ca²⁺ uptake, assays were performed in the absence of CaM or PKA and in the presence of 2 µM CaM or PKA (catalytic subunit; 8 µg in 250 µl of incubation medium). The Ca²⁺ uptake reaction was initiated by addition of SR to the assay components, which had been preincubated for 3 min at 37°C.

Immunoblotting of SERCA2a and PLN. Western immunoblotting techniques were used for detection and estimation of the relative amounts of SERCA2a and PLN in SR of LV and RV of rat heart. The SR vesicles (25 µg protein/lane) were first subjected to SDS-PAGE in 10% (for SERCA2a) or 15% (for PLN) gels, and the fractionated proteins were transblotted to nitrocellulose membranes. The membranes were probed with antibodies specific for cardiac SERCA (anti-87, polyclonal, 1:1,000 dilution) and PLN (monoclonal, 0.5 µg/ml). A peroxidase-linked goat anti-rabbit IgG (for Ca²⁺-ATPase) or goat anti-mouse IgG (1:5,000 dilution) was used as the secondary antibody. Protein bands reactive with antibodies were visualized using the enhanced chemiluminescence detection system (Amersham). The images of the protein bands were optimized, captured, and analyzed by a video documentation system (ImageMaster, Pharmacia Biotech, San Francisco, CA).

Determination of phosphoenzyme formation and decomposition. The standard reaction medium (0.5 ml total volume) for measurement of phosphoenzyme (EP) formation/decomposition contained (in mM) 40 Tris·HCl (pH 7.0), 100 KCl, 5 MgCl₂, 5 NaN₃, 0.1 EGTA, 0.1 CaCl₂ (8.2 µM free Ca²⁺), and 0.002 [-³²P]ATP (12,000 cpm/pmol) and SR vesicles (50 µg protein). To determine EP formation (steady-state EP level), [-³²P]ATP was added to the reaction mixture, which had been preincubated for 3 min at 4°C. The reaction was quenched with 1 ml of stop solution (6% trichloroacetic acid-0.3 mM ATP-5 mM P_i) 15 s after addition of radioactive ATP. To determine EP decomposition, the steady-state level of ³²P-labeled EP was first obtained as described above. Then 5 µl of 12.5 mM nonradioactive ATP were added to the reaction mixture (124 µM final concentration); the reaction was acid quenched with stop solution at desired time intervals after addition of nonradioactive ATP. The acid-precipitated protein samples were subjected to acidic SDS-PAGE (33) and autoradiographed. The EP bands visualized on the autoradiograms were quantified by liquid scintillation counting of bands excised from the gel.

Myocyte isolation. Myocytes were isolated as previously described (10). Briefly, hearts were mounted on a Langendorff apparatus and perfused with Ca²⁺-free buffer containing (in mM) 120 NaCl, 5.4 KCl, 1 MgCl₂, 0.33 NaH₂PO₄, 10 HEPES, and 10 glucose at pH 7.4. After a brief equilibration period, 1.16 mg/ml type II collagenase (Worthington Biochemical, Lakewood, NJ) and 0.1 mg/ml protease type XIV were added to the buffer, and the heart was perfused for 9 min in a recirculating manner. Collagenase was washed out with buffer containing 0.2 mM Ca²⁺, and the heart was anatomically separated into LV and RV and diced with scissors. After incubation at 37°C, tissues were filtered through a nylon mesh and allowed to settle. The cells were exposed to a series of sedimentation and resuspension steps in buffer containing increasing concentrations of Ca²⁺ (0.2–1.0 mM). Cell yield was assessed microscopically, and the density was diluted to 10⁵ cells/ml; 50–60% of LV and RV cells were healthy and rod-shaped.

Measurement of [Ca²⁺]_i. Fura-2 was used to measure Ca²⁺ transients in single myocytes. Cells were loaded by incubation with 1 µM fura-2 AM for 30 min at 35°C and then allowed to settle onto a glass coverslip that comprised the bottom of a perfusion chamber (0.75 ml vol). The chamber was mounted on a Nikon inverted microscope and continuously perfused with bathing solution at 2–3 ml/min at room temperature. Cells were considered viable if they demonstrated a characteristic rod shape without blebbing and contracted reversibly after electrical pacing at frequencies of 0.25–1.0 Hz using a 2.5-ms-duration pulse with a pair of platinum electrodes. Cells were illuminated with alternating 345- and 380-nm light using a Deltascan system (Photon Technology International), with the 510-nm emission detected using a photometer, as previously described (35). [Ca²⁺]_i was calibrated according to the methods of Grynkiewicz et al. (12), with [Ca²⁺]_i = [K_d(R – R_min)/(R_max – R)]Sf₂/Sb₂, where R_min and R_max are ratios of fluorescence intensity at 345 nm to fluorescence intensity at 380 nm with Ca²⁺-free and saturated conditions, respectively, and Sf₂/Sb₂ is the ratio of fluorescence of Ca²⁺-free to Ca²⁺-bound indicator measured at 380 nm. We used a dissociation constant (K_d) of 225 nM for binding of Ca²⁺ to fura-2 (12) and a viscosity factor of 0.6. Data were corrected for background fluorescence. Calculation of [Ca²⁺]_i involves a number of assumptions, and factors such as inhomogeneity of Ca²⁺ within cells introduce uncertainty in the values. However, the time course of decay of the [Ca²⁺]_i transient is not influenced by the calibration.

Contraction was quantified from digital recordings of cells (Coolsnap CCD, ImageMaster 5, Photon Technology International) stimulated at 0.5 Hz as described above. Myocyte length and fractional shortening were measured along the central axis of each cell.

Measurement of CaM kinase II-mediated SR protein phosphorylation. Phosphorylation of SR proteins by endogenous CaM kinase II was determined as described previously (43). The phosphorylation assay medium (50 µl total volume) contained (in mM) 50 HEPES (pH 7.4), 10 MgCl₂, 0.1 CaCl₂, 0.1 EGTA, 0.001 calmodulin, and 0.8 [-³²P]ATP (specific activity 200–300 cpm/pmol) and SR (25 µg of protein). The phosphorylation reaction was initiated by addition of [-³²P]ATP after preincubation of the rest of assay components for 3 min at 37°C. The Ca²⁺/CaM dependence of phosphorylation was monitored in parallel assays without Ca²⁺ (in the presence of 1 mM EGTA) and CaM in the assay system. Reactions were terminated after 2 min by addition of 15 µl of SDS sample buffer, and the samples were subjected to SDS-PAGE in 4–18% gradient gel, stained with Coomassie brilliant blue, dried, and autoradiographed. Phosphorylation was quantified by liquid scintillation counting after excision of the radioactive bands from the gels as described previously (21, 45).

Coimmunoprecipitation of SERCA2a and PLN. The coimmunoprecipitation protocols were similar to those described by Asahi et al. (1). For quantification of a stable PLN-SERCA interaction, 1 mg/ml SR protein in 0.25 M sucrose, 10 mM Tris·HCl (pH 7.5), 20 µM CaCl₂, 3 mM 2-mercaptoethanol, and 150 mM KCl were mixed with an equal volume of solubilizing buffer [40 mM HEPES-NaOH (pH 7.5), 300 mM NaCl, 2 mM EDTA, 4 mM phenylmethylsulfonyl fluoride, and 1% Tween 20]. The samples were vortexed for 30 s and centrifuged in a Beckman microcentrifuge for 30 min at 16,000 g. The supernatants were rotated with BSA-treated protein G-Sepharose for 30 min and centrifuged to remove protein bound nonspecifically to protein G-Sepharose. The supernatants were then mixed with a protein G-Sepharose-PLN monoclonal antibody complex. The samples were rotated for 30 min at 4°C and centrifuged. The pellets were washed three times with a buffer composed of 20 mM HEPES (pH 7.5), 150 mM NaCl, 1 mM EDTA, and 0.5% Tween 20. The samples were loaded on 8% polyacrylamide gels, and the proteins were separated by standard SDS-PAGE protocols and transferred to nitrocellulose membranes. After blocking with a skim milk suspension, the membranes were probed with antibody specific for cardiac SERCA (anti-87, polyclonal), washed in Tris·HCl, pH 7.5, 100 mM KCl, 0.1% Tween 20 (TBS-0.1% Tween), and treated with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody. Membranes were washed in TBS-0.1% Tween 20, and the signals were detected with an enhanced chemiluminescence detection system (Amersham). The images of protein bands were optimized, captured, and analyzed by a video documentation system (ImageMaster).

Data analysis. Statistical analysis was performed using the SigmaPlot scientific graph program (Systat) and Student's t-test for unpaired data. P < 0.05 was taken as the level of significance. Results were averaged and are expressed as means ± SE of experiments using separate preparations; n denotes the number of independent determinations using separate SR/myocyte preparations.

	RESULTS

TOP ABSTRACT METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
SR Ca²⁺ sequestration function differs in RV and LV. ATP-dependent, oxalate-facilitated Ca²⁺ uptake by SR vesicles is commonly used to measure Ca²⁺ pump function of SR in vitro. The rates of ATP-driven Ca²⁺ uptake into cardiac SR vesicles from RV and LV of normal rat myocardium are presented in Fig. 1A. The rate of Ca²⁺ uptake, measured in the presence of saturating free Ca²⁺ (8.2 µM, see below), was strikingly lower (4-fold) in RV than in LV. Experiments using unfractionated muscle homogenates also showed a similar difference in the Ca²⁺-sequestering activity between RV and LV (Fig. 1B). Furthermore, the huge disparity in Ca²⁺ uptake rates of SR in RV and LV could be observed at a wide range of Ca²⁺ concentrations (0.01–8.2 µM; Fig. 2). Analysis of the kinetic parameters revealed a significant difference in the maximum velocity of Ca²⁺ pumping (V_max = 176 ± 13 and 45 ± 3 nmol Ca²⁺·mg protein^–1·min^–1 SR in LV and RV, respectively, P < 0.05) and the apparent affinity of the Ca²⁺ pump for Ca²⁺ (K_0.5 = 0.54 ± 0.03 and 0.90 ± 0.1 µM for SR in LV and RV, respectively, P < 0.05) between SR of RV and SR of LV. These findings demonstrate a striking chamber-specific difference between RV and LV with respect to SR Ca²⁺ pump function.

View larger version (10K): [in this window] [in a new window]   Fig. 1. Time course of ATP-energized Ca2+ uptake in vitro by sarcoplasmic reticulum (SR) isolated from right ventricle (RV) and left ventricle (LV) of rat myocardium (A) and by tissue homogenates of RV and LV (B). Reaction was carried out under standard assay conditions (see METHODS); concentration of free Ca2+ in the assay was 8.2 µM. Values are means ± SE of 5 experiments using separate SR preparations. *P < 0.05 vs. LV.

View larger version (12K): [in this window] [in a new window]   Fig. 2. Ca2+ concentration dependence of ATP-driven Ca2+ uptake by SR from RV and LV of rat myocardium. Ca2+ uptake reaction was carried out under standard assay conditions (see METHODS); concentration of free Ca2+ in the assay was varied as indicated. Values are means ± SE of 4 experiments using separate SR preparations. Kinetic parameters of Ca2+ transport derived from these data are summarized in RESULTS.

View larger version (17K): [in this window] [in a new window]   Fig. 3. Relative amounts of Ca2+-ATPase (SERCA2a isoform) and phospholamban (PLN) in RV and LV of rat myocardium as determined by Western immunoblot. Bottom: typical immunoblots of SERCA2a and PLN obtained using 3 separate SR preparations each from RV and LV. Identical amount of SR (25 µg protein) was loaded in each lane. A and B: amounts of SERCA2a and PLN as determined by computer-assisted scanning densitometry of immunoblots. C: ratio of SERCA2a to PLN. Values are means ± SE of 6 experiments using separate SR preparations. AU, arbitrary units. *P < 0.05 vs. LV.

View larger version (18K): [in this window] [in a new window]   Fig. 4. Formation and decomposition of phosphoenzyme intermediate (EP) in catalytic cycle of SR Ca2+-ATPase in RV and LV of rat myocardium. Top: EP formation (steady-state EP level) and EP decomposition determined as described in METHODS. Bottom: typical autoradiograms depicting steady-state EP level (A) and rate of EP decomposition (B) by Ca2+-ATPase. EP levels were quantified by liquid scintillation counting of Ca2+-ATPase band excised from gels. Values are means ± SE of 3 experiments using separate SR preparations. *P < 0.05 vs. LV.

View larger version (26K): [in this window] [in a new window]   Fig. 5. Intracellular Ca2+ transients in myocytes from RV and LV of rat myocardium. Cytosolic free Ca2+ concentration ([Ca2+]i) was measured by microspectrofluorometry of fura-2 AM-loaded myocytes. A: typical Ca2+ transients recorded during field stimulation at a frequency of 0.5 Hz. Representative traces for LV and RV are aligned and superimposed to illustrate more rapid recovery in LV myocytes. B and C: Ca2+ transients illustrate kinetics of LV and RV myocytes with data expanded from A. First-order best-fit exponential curves (dashed lines) are drawn through data in both transients. D: average values for time constant of decay of [Ca2+]i showing more rapid decline in LV myocytes over entire range of frequencies studied. E: basal and peak rise of intracellular Ca2+ level of RV and LV myocytes. Values are means ± SE in 19 cells from 5 different preparations. *P < 0.05 vs. LV.

View larger version (67K): [in this window] [in a new window]   Fig. 6. Contractile properties of myocytes isolated from RV and LV of rat myocardium. A: series of video frames illustrating digital images of a representative cardiac RV myocyte during 1 cycle [diastolic, systolic, and recovery (diastolic)] when field stimulation was applied at 0.5 Hz. B and C: mechanical indexes of myocyte shortening, including peak systolic and diastolic cell length and fractional shortening, determined as fractional difference in length between systolic and diastolic state. Values are means ± SE of 40 cells from 5 separate preparations. *P < 0.05 vs. LV.

View larger version (21K): [in this window] [in a new window]   Fig. 7. Effect of activation of endogenous calmodulin (CaM) kinase II (A) or exogenous PKA (B) on Ca2+ uptake activity of SR isolated from RV and LV of rat myocardium. Ca2+ uptake reaction was carried out under standard assay conditions at subsaturating (0.6 µM) or saturating (8.2 µM) free Ca2+ concentration without CaM (–CaM) or PKA (–PKA) and with calmodulin (+CaM) or PKA (+PKA). When present in the assay, CaM concentration was 2 µM, and PKA (catalytic subunit) concentration was 8 µg in 250 µl of incubation medium. Reaction was initiated by addition of SR to the rest of the assay components preincubated for 3 min at 37°C. Values are means ± SE of 4 separate cardiac SR preparations. A: *P < 0.05 vs. –CaM. B: *P < 0.05 vs. –PKA.

View larger version (22K): [in this window] [in a new window]   Fig. 8. Comparison of endogenous CaM kinase II-mediated protein phosphorylation in SR from RV and LV of rat myocardium. A: Coomassie blue-stained SDS-polyacrylamide gel depicting typical protein profiles of SR from LV and RV subjected to incubation in phosphorylation reaction medium in the absence (–) and presence (+) of Ca2+ and CaM (left) and autoradiogram of the same gel depicting phosphorylation of phospholamban (PLN: H, high molecular weight form; L, low molecular weight form), Ca2+-ATPase and ryanodine receptor (RyR) due to activation of endogenous CaM kinase II (right). B: quantitative data on phosphorylation of RyR, Ca2+-ATPase and PLN as determined by liquid scintillation counting of radioactive bands excised from gels (see METHODS). Data for PLN include 32P incorporation into low- and high-molecular-weight forms of this protein. Values are means ± SE of 6 separate SR preparations. *P < 0.05 vs. LV.

View larger version (15K): [in this window] [in a new window]   Fig. 9. Physical interaction between Ca2+-ATPase (SERCA2a) and PLN in SR membranes isolated from RV and LV of rat myocardium. SR membranes were solubilized with Tween 20, and supernatants were rotated with BSA-treated protein G-Sepharose and centrifuged to remove proteins bound nonspecifically to protein G-Sepharose. Supernatants were then mixed with a protein G-Sepharose-PLN monoclonal antibody complex, and samples were rotated for 30 min to facilitate coimmunoprecipitation of Ca2+-ATPase with PLN. Immunoprecipitates were fractionated by SDS-PAGE and transferred to nitrocellulose membranes, and coimmunoprecipitated Ca2+-ATPase was detected by Western blotting with SERCA2 antibody (anti-87, polyclonal) and quantified (see METHODS). Western blots were obtained using 2 separate SR preparations each from RV and LV. Values are means ± SE of 4 experiments using separate SR preparations. *P < 0.05 vs. LV.

	DISCUSSION

TOP ABSTRACT METHODS RESULTS DISCUSSION GRANTS REFERENCES

A number of experimental approaches were utilized to characterize the mechanisms underlying the difference in SR Ca²⁺ pump activity between RV and LV and discern its physiological relevance. When protein levels were assessed by Western blotting, small differences in SERCA2 Ca²⁺ pump density and PLN levels were apparent, but these could not account for the large difference in SR Ca²⁺ pump activity. Moreover, protein levels do not necessarily reflect the functional status of the Ca²⁺ pump units. Measurement of the steady-state level of the EP intermediate in the catalytic and ion transport cycle of the Ca²⁺-ATPase revealed a nearly fourfold lower EP level in SR from RV than in SR from LV. Because the SERCA2 content in RV and LV differed only marginally (14%), this finding implies that <50% of the SR Ca²⁺ pump units in RV are catalytically active. Therefore, we conclude that the large disparity in SR Ca²⁺ sequestration between RV and LV stems mainly from a preponderance of functionally inert SR Ca²⁺ pump units in RV compared with LV. The decomposition rate of preformed EP and the EP turnover estimated from V_max-to-EP ratios did not differ significantly between RV and LV, suggesting an apparently similar Ca²⁺-cycling rate by the active SR Ca²⁺ pump units in RV and LV.

Our observations also provide important insights into the mechanism underlying the suppression of function of a large portion of the SR Ca²⁺ pump units in RV. We have found that the K_0.5 for Ca²⁺ activation of Ca²⁺ transport is almost twofold higher in SR from RV than in SR from LV. Such a decrease in Ca²⁺ affinity is a characteristic feature of the PLN-bound state of the Ca²⁺-ATPase (34). Furthermore, protein coimmunoprecipitation experiments revealed that the relative amount of stable PLN-bound Ca²⁺-ATPase molecules is significantly greater in SR from RV than in SR from LV. These findings suggest that the suppression of function of a larger proportion of the SR Ca²⁺ pump units in RV may result from their apparently stable association with PLN. According to the generally held view of PLN regulation, unphosphorylated PLN interacts with the Ca²⁺-ATPase, exerting an inhibitory effect manifested mainly through a decrease in the enzymes's affinity for Ca²⁺; phosphorylation of PLN by cAMP-dependent protein kinase or CaM kinase is thought to disrupt this interaction, resulting in enhanced affinity of the ATPase for Ca²⁺ and stimulation of Ca²⁺ pump activity (4, 23, 34, 37). This long-standing view has been questioned recently, and it has been reported that Ca²⁺, but not phosphorylation of PLN, disrupts the physical interaction between Ca²⁺-ATPase and PLN (1). Recent work from our laboratory demonstrated that 1) PLN-bound Ca²⁺-ATPase is catalytically nonproductive and 2) a Ca²⁺/CaM-dependent process disrupts the physical interaction between Ca²⁺-ATPase and PLN, resulting in restoration of Ca²⁺-ATPase function (30). In the present study, we have found that activation of endogenous SR CaM kinase by Ca²⁺/CaM, which results in phosphorylation of PLN and Ca²⁺-ATPase (Fig. 8) (15, 39, 43), does not attenuate the disparity in Ca²⁺ sequestration between SR from RV and SR from LV. Exogenous PKA, which phosphorylates PLN (34), also fails to overcome the disparity in SR Ca²⁺ pump function between RV and LV. Therefore, it appears that a major portion of Ca²⁺ pump units in SR from RV remains stably associated with PLN; these PLN-bound Ca²⁺ pump units are functionally inert and resistant to recruitment by conventional Ca²⁺/CaM- or PKA-mediated processes. The existence of a pool of functionally silent "reserve SR Ca²⁺ pumps" in RV might be of considerable physiological significance, inasmuch as these Ca²⁺ pumps can be called into action to protect and maintain RV function when a sudden rise in afterload or resistance is encountered by the pulmonary circulation. Future studies are needed to address whether induction of experimental pulmonary hypertension will result in recruitment of the reserve SR Ca²⁺ pumps in RV.

The Ca²⁺ transients in freshly isolated myocytes in response to electrical filed stimulation revealed characteristic differences between RV and LV myocytes. The most striking among these was a clearly prolonged Ca²⁺ transient in RV myocytes compared with LV myocytes, mainly due to a slow rate of Ca²⁺ decay. In rat ventricular myocytes, Ca²⁺ sequestration by the SR is thought to account for removal of 90% of Ca²⁺ from the cytosol; thus the rate of Ca²⁺ decay largely reflects the rate of SR Ca²⁺ uptake (2, 6). Therefore, the slowed intracellular Ca²⁺ clearing (longer time constant of Ca²⁺ decay) in RV myocytes is consistent with our findings demonstrating slow rates of Ca²⁺ sequestration by SR from RV compared with SR from LV. Slowing the decay of the Ca²⁺ transient will also reduce the speed of relaxation. Because the peak systolic pressure developed in RV is markedly lower than that developed in LV, the slow rate of RV relaxation may promote temporal synchrony of the end of diastole in the two ventricular chambers. There was no significant difference between LV and RV peak systolic and diastolic cell length. However, when fractional shortening was determined as the difference between systolic and diastolic length, the degree of shortening was greater in LV than in RV myocytes. Because the SR Ca²⁺ pump activity is a major determinant of SR Ca²⁺ load (and, hence, the amount of Ca²⁺ available for release) (20, 36, 41), the greater SR Ca²⁺ pump activity in LV is consistent with more shortening in LV myocytes.

In conclusion, our findings demonstrate strikingly slower rates of Ca²⁺ sequestration by SR from RV than by SR from LV, despite similar levels of SR Ca²⁺ pump (Ca²⁺-ATPase) units in RV and LV. A major fraction of available Ca²⁺ pump units in RV are functionally inert because of their apparently stable association with the Ca²⁺ pump inhibitor protein PLN. Intracellular Ca²⁺ transients, evoked by electrical field stimulation, are prolonged in RV myocytes compared with LV myocytes, mainly because of slow [Ca²⁺]_i decay due to the lower SR Ca²⁺ pump activity in the RV myocytes. Because of the vast difference in peak systolic pressure between RV and LV, the slow [Ca²⁺]_i decay in RV and the consequent decrease in the speed of RV relaxation may promote temporal synchrony of the end of diastole in RV and LV. The PLN-bound, functionally silent SR Ca²⁺ pumps in RV apparently reflect the higher diastolic reserve required to protect and maintain RV function in the face of a sudden rise in afterload or resistance in the pulmonary circulation. An interesting species-specific difference in diastolic reserve has been demonstrated recently in a study that compared the Ca²⁺-cycling properties of rabbit and horse cardiomyocytes. The density of SR Ca²⁺ pump units was similar in rabbit and horse cardiomyocytes, although the resting heart rate is much lower in the horse (25). The horse, however, has the ability to increase heart rate 10-fold during strenuous exercise (25).

	GRANTS

TOP ABSTRACT METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
This work was supported by Canadian Institutes of Health Research Grants MOP-9553 (to N. Narayanan) and MOP-10019 (to S. M. Sims). M. Karmazyn holds a Canada Research Chair in Experimental Cardiology. V. Sathish is a postdoctoral fellow supported by Heart and Stroke Foundation of Ontario Program Grant in Heart Failure.

	FOOTNOTES

 

Address for reprint requests and other correspondence: N. Narayanan, Dept. of Physiology and Pharmacology, Medical Sciences Bldg., Univ. of Western Ontario, London, ON, Canada N6A 5C1 (e-mail address: njanoor.narayanan{at}schulich.uwo.ca)

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.

	REFERENCES

TOP ABSTRACT METHODS RESULTS DISCUSSION GRANTS REFERENCES

 

1. Asahi M, McKenna E, Kurzydlowski K, Tada M, and MacLennan DH. Physical interactions between phospholamban and sarco(endo)plasmic reticulum Ca²⁺-ATPases are dissociated by elevated Ca²⁺, but not by phospholamban phosphorylation, vanadate, or thapsigargin, and are enhanced by ATP. J Biol Chem 275: 15034–15038, 2000.[Abstract/Free Full Text]
2. Bassani JW, Bassani RA, and Bers DM. Relaxation in rabbit and rat cardiac cells: species-dependent differences in cellular mechanisms. J Physiol 476: 279–293, 1994.[Abstract/Free Full Text]
3. Bers DM.Excitation-Contraction Coupling and Cardiac Contractile Force. Dordrecht, The Netherlands: Kluwer Academic, 1991.
4. Colyer J. Control of the calcium pump of cardiac sarcoplasmic reticulum. A specific role for the pentameric structure of phospholamban? Cardiovasc Res 27: 1766–1771, 1993.[Free Full Text]
5. Coronado R, Morrissette J, Sukhareva M, and Vaughan DM. Structure and function of ryanodine receptors. Am J Physiol Cell Physiol 266: C1485–C1504, 1994.[Abstract/Free Full Text]
6. Curl CL, Wendt IR, and Kotsanas G. Effects of gender on intracellular [Ca²⁺] in rat cardiac myocytes. Pflügers Arch 441: 709–716, 2001.[CrossRef][Web of Science][Medline]
7. Dell'Italia LJ. The right ventricle: anatomy, physiology, and clinical importance. Curr Probl Cardiol 16: 653–720, 1991.[Medline]
8. Dhalla NS, Golfman L, Liu X, Sasaki H, Elimban V, and Rupp H. Subcellular remodeling and heart dysfunction in cardiac hypertrophy due to pressure overload. Ann NY Acad Sci 874: 100–110, 1999.[CrossRef][Web of Science][Medline]
9. Dhalla NS, Sulakhe PV, Lee SL, Singal PK, Varley KG, and Yates JC. Subcellular Ca²⁺ transport in different areas of dog heart. Can J Physiol Pharmacol 58: 360–367, 1980.[Medline]
10. Ebihara Y and Karmazyn M. Inhibition of - but not ₁-mediated adrenergic responses in isolated hearts and cardiomyocytes by nitric oxide and 8-bromo cyclic GMP. Cardiovasc Res 32: 622–629, 1996.[CrossRef][Web of Science][Medline]
11. Fabiato A. Computer programs for calculating total from specified free or free from specified total ionic concentrations in aqueous solutions containing multiple metals and ligands. Methods Enzymol 157: 378–417, 1988.[Web of Science][Medline]
12. Grynkiewicz G, Poenie M, and Tsien RY. A new generation of Ca²⁺ indicators with greatly improved fluorescence properties. J Biol Chem 260: 3440–3450, 1985.[Abstract/Free Full Text]
13. Gupta RC, Yang XP, Mishra S, and Sabbah HN. Assessment of sarcoplasmic reticulum Ca²⁺-uptake during the development of left ventricular hypertrophy. Biochem Pharmacol 65: 933–939, 2003.[Medline]
14. Hain J, Onoue H, Mayrleitner M, Fleischer S, and Schindler H. Phosphorylation modulates the function of the calcium release channel of sarcoplasmic reticulum from cardiac muscle. J Biol Chem 270: 2074–2081, 1995.[Abstract/Free Full Text]
15. Hawkins C, Xu A, and Narayanan N. Sarcoplasmic reticulum calcium pump in cardiac and slow-twitch skeletal muscle but not fast-twitch skeletal muscle undergoes phosphorylation by endogenous and exogenous Ca²⁺/calmodulin-dependent protein kinase. Characterization of optimal conditions for calcium pump phosphorylation. J Biol Chem 269: 31198–31206, 1994.[Abstract/Free Full Text]
16. Headley JM and Von Rueden KT. The right ventricle: significant anatomy, physiology, and interventricular considerations. J Cardiovasc Nurs 6: 1–11, 1991.[Medline]
17. Ikemoto N. Structure and function of the calcium pump protein of sarcoplasmic reticulum. Annu Rev Physiol 44: 297–317, 1982.[CrossRef][Web of Science][Medline]
18. Inesi G. Mechanism of calcium transport. Annu Rev Physiol 47: 573–601, 1985.[CrossRef][Web of Science][Medline]
19. Inesi G, Sumbilla C, and Kirtley ME. Relationships of molecular structure and function in Ca²⁺-transport ATPase. Physiol Rev 70: 749–760, 1990.[Free Full Text]
20. Janczewski AM, Spurgeon HA, Stern MD, and Lakatta EG. Effects of sarcoplasmic reticulum Ca²⁺ load on the gain function of Ca²⁺ release by Ca²⁺ current in cardiac cells. Am J Physiol Heart Circ Physiol 268: H916–H920, 1995.[Abstract/Free Full Text]
21. Jiang MT, Moffat MP, and Narayanan N. Age-related alterations in the phosphorylation of sarcoplasmic reticulum and myofibrillar proteins and diminished contractile response to isoproterenol in intact rat ventricle. Circ Res 72: 102–111, 1993.[Abstract/Free Full Text]
22. Jones DL and Narayanan N. Defibrillation depresses heart sarcoplasmic reticulum calcium pump: a mechanism of postshock dysfunction. Am J Physiol Heart Circ Physiol 274: H98–H105, 1998.[Abstract/Free Full Text]
23. Kadambi VJ and Kranias EG. Phospholamban: a protein coming of age. Biochem Biophys Res Commun 239: 1–5, 1997.[CrossRef][Web of Science][Medline]
24. Khan I, Tabb T, Garfield RE, Jones LR, Fomin VP, Samson SE, and Grover AK. Expression of the internal calcium pump in pregnant rat uterus. Cell Calcium 14: 111–117, 1993.[CrossRef][Web of Science][Medline]
25. Loughrey CM, Smith GL, and MacEachern KE. Comparison of Ca²⁺ release and uptake characteristics of the sarcoplasmic reticulum in isolated horse and rabbit cardiomyocytes. Am J Physiol Heart Circ Physiol 287: H1149–H1159, 2004.[Abstract/Free Full Text]
26. Lowry OH, Rosebrough NJ, Farr AL, and Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 193: 265–275, 1951.[Free Full Text]
27. Lytton J and MacLennan DH. Sarcoplasmic reticulum. In: The Heart and Cardiovascular System (2nd ed.), edited by Fozzard HA, Haber E, Jennings RB, Katz AM, and Morgan HE. New York: Raven, 1992, p. 1203–1221.
28. MacLennan DH. Molecular tools to elucidate problems in excitation-contraction coupling. Biophys J 58: 1355–1365, 1990.[Web of Science][Medline]
29. Narayanan N, Newland M, and Neudorf D. Inhibition of sarcoplasmic reticulum calcium pump by cytosolic protein(s) endogenous to heart and slow skeletal muscle but not fast skeletal muscle. Biochim Biophys Acta 735: 53–66, 1983.[Medline]
30. Narayanan N and Xu A. Calmodulin triggers Ca²⁺ pump function in native cardiac sarcoplasmic reticulum by disrupting Ca²⁺-ATPase-phospholamban interaction (Abstract). J Mol Cell Cardiol 37: 226, 2004.
31. Netticadan T, Temsah R, Osada M, and Dhalla NS. Status of Ca²⁺/calmodulin protein kinase phosphorylation of cardiac SR proteins in ischemia-reperfusion. Am J Physiol Cell Physiol 277: C384–C391, 1999.[Abstract/Free Full Text]
32. Netticadan T, Temsah RM, Kawabata K, and Dhalla NS. Sarcoplasmic reticulum Ca²⁺/calmodulin-dependent protein kinase is altered in heart failure. Circ Res 86: 596–605, 2000.[Abstract/Free Full Text]
33. Sarkadi B, Enyedi A, Foldes-Papp Z, and Gardos G. Molecular characterization of the in situ red cell membrane calcium pump by limited proteolysis. J Biol Chem 261: 9552–9557, 1986.[Abstract/Free Full Text]
34. Simmerman HK and Jones LR. Phospholamban: protein structure, mechanism of action, and role in cardiac function. Physiol Rev 78: 921–947, 1998.[Abstract/Free Full Text]
35. Sims SM, Jiao Y, and Zheng ZG. Intracellular calcium stores in isolated tracheal smooth muscle cells. Am J Physiol Lung Cell Mol Physiol 271: L300–L309, 1996.[Abstract/Free Full Text]
36. Stern MD. Theory of excitation-contraction coupling in cardiac muscle. Biophys J 63: 497–517, 1992.[Web of Science][Medline]
37. Tada M and Katz AM. Phosphorylation of the sarcoplasmic reticulum and sarcolemma. Annu Rev Physiol 44: 401–423, 1982.[CrossRef][Web of Science][Medline]
38. Takasago T, Imagawa T, Furukawa K, Ogurusu T, and Shigekawa M. Regulation of the cardiac ryanodine receptor by protein kinase-dependent phosphorylation. J Biochem (Tokyo) 109: 163–170, 1991.[Abstract/Free Full Text]
39. Toyofuku T, Curotto Kurzydlowski K, Narayanan N, and MacLennan DH. Identification of Ser³⁸ as the site in cardiac sarcoplasmic reticulum Ca²⁺-ATPase that is phosphorylated by Ca²⁺/calmodulin-dependent protein kinase. J Biol Chem 269: 26492–26496, 1994.[Abstract/Free Full Text]
40. Vatner DE, Sato N, Kiuchi K, Shannon RP, and Vatner SF. Decrease in myocardial ryanodine receptors and altered excitation-contraction coupling early in the development of heart failure. Circulation 90: 1423–1430, 1994.[Abstract/Free Full Text]
41. Wier WG, Egan TM, Lopez-Lopez JR, and Balke CW. Local control of excitation-contraction coupling in rat heart cells. J Physiol 474: 463–471, 1994.[Abstract/Free Full Text]
42. Witcher DR, Kovacs RJ, Schulman H, Cefali DC, and Jones LR. Unique phosphorylation site on the cardiac ryanodine receptor regulates calcium channel activity. J Biol Chem 266: 11144–11152, 1991.[Abstract/Free Full Text]
43. Xu A, Hawkins C, and Narayanan N. Phosphorylation and activation of the Ca²⁺-pumping ATPase of cardiac sarcoplasmic reticulum by Ca²⁺/calmodulin-dependent protein kinase. J Biol Chem 268: 8394–8397, 1993.[Abstract/Free Full Text]
44. Xu A and Narayanan N. Ca²⁺/calmodulin-dependent phosphorylation of the Ca²⁺-ATPase, uncoupled from phospholamban, stimulates Ca²⁺-pumping in native cardiac sarcoplasmic reticulum. Biochem Biophys Res Commun 258: 66–72, 1999.[CrossRef][Web of Science][Medline]
45. Xu A and Narayanan N. Effects of aging on sarcoplasmic reticulum Ca²⁺-cycling proteins and their phosphorylation in rat myocardium. Am J Physiol Heart Circ Physiol 275: H2087–H2094, 1998.[Abstract/Free Full Text]
46. Xu A and Narayanan N. Reversible inhibition of the calcium-pumping ATPase in native cardiac sarcoplasmic reticulum by a calmodulin-binding peptide. Evidence for calmodulin-dependent regulation of the V_max of calcium transport. J Biol Chem 275: 4407–4416, 2000.[Abstract/Free Full Text]

This article has been cited by other articles:

				T. Urashima, M. Zhao, R. Wagner, G. Fajardo, S. Farahani, T. Quertermous, and D. Bernstein Molecular and physiological characterization of RV remodeling in a murine model of pulmonary stenosis Am J Physiol Heart Circ Physiol, September 1, 2008; 295(3): H1351 - H1368. [Abstract] [Full Text] [PDF]

				V. Sathish, F. Leblebici, S. N. Kip, M. A. Thompson, C. M. Pabelick, Y. S. Prakash, and G. C. Sieck Regulation of sarcoplasmic reticulum Ca2+ reuptake in porcine airway smooth muscle Am J Physiol Lung Cell Mol Physiol, April 1, 2008; 294(4): L787 - L796. [Abstract] [Full Text] [PDF]

				G. H. Borchert, M. Giggey, F. Kolar, T. M. Wong, P. H. Backx, and P. V. Escriba 2-Hydroxyoleic acid affects cardiomyocyte [Ca2+]i transient and contractility in a region-dependent manner Am J Physiol Heart Circ Physiol, April 1, 2008; 294(4): H1948 - H1955. [Abstract] [Full Text] [PDF]

				N. Geoghegan-Morphet, D. Burger, X. Lu, V. Sathish, T. Peng, S. M. Sims, and Q. Feng Role of neuronal nitric oxide synthase in lipopolysaccharide-induced tumor necrosis factor-alpha expression in neonatal mouse cardiomyocytes Cardiovasc Res, July 15, 2007; 75(2): 408 - 416. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: 291/1/H88    most recent 01372.2005v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Web of Science (3) Citing Articles via Google Scholar Google Scholar Articles by Sathish, V. Articles by Narayanan, N. Search for Related Content PubMed PubMed Citation Articles by Sathish, V. Articles by Narayanan, N.