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(Circulation Research. 2002;91:1015.)
© 2002 American Heart Association, Inc.

Cellular Biology

Abnormal Ca²⁺ Release, but Normal Ryanodine Receptors, in Canine and Human Heart Failure

Ming Tao Jiang, Andrew J. Lokuta, Emily F. Farrell, Matthew R. Wolff, Robert A. Haworth, Héctor H. Valdivia

From the Department of Physiology (M.T.J., A.J.L., E.F.F., H.H.V.), Medicine (M.R.W.), and Surgery (R.A.H.), University of Wisconsin Medical School, Madison, Wis. Present address for M.T.J. is the Department of Anesthesiology, Medical College of Wisconsin, Milwaukee, Wis.

Correspondence to Héctor H. Valdivia, MD, PhD, University of Wisconsin, 1300 University Ave, Madison, WI 53706. E-mail valdivia{at}physiology.wisc.edu

	Abstract

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Sarcoplasmic reticulum (SR) Ca²⁺ transport proteins, especially ryanodine receptors (RyR) and their accessory protein FKBP12.6, have been implicated as major players in the pathogenesis of heart failure (HF), but their role remain controversial. We used the tachycardia-induced canine model of HF and human failing hearts to investigate the density and major functional properties of RyRs, SERCA2a, and phospholamban (PLB), the main proteins regulating SR Ca²⁺ transport. Intracellular Ca²⁺ is likely to play a role in the contractile dysfunction of HF because the amplitude and kinetics of the [Ca²⁺]_i transient were reduced in HF. Ca²⁺ uptake assays showed 44±8% reduction of V_max in canine HF, and Western blots demonstrated that this reduction was due to decreased SERCA2a and PLB levels. Human HF showed a 30±5% reduction in SERCA2a, but PLB was unchanged. RyRs from canine and human HF displayed no major structural or functional differences compared with control. The P_o of RyRs was the same for control and HF over the range of pCa 7 to 4. Subconductance states, which predominate in FKBP12.6-stripped RyRs, were equally frequent in control and HF channels. An antibody that recognizes phosphorylated RyRs yields equal intensity for control and HF channels. Further, phosphorylation of RyRs by PKA did not appear to change the RyR/FKBP12.6 association, suggesting minor ß-adrenergic stimulation of Ca²⁺ release through this mechanism. These results support a role for SR in the pathogenesis of HF, with abnormal Ca²⁺ uptake, more than Ca²⁺ release, contributing to the depressed and slow Ca²⁺ transient characteristic of HF.

Key Words: heart failure • ryanodine receptor • sarcoplasmic reticulum • protein kinase A

	Introduction

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It is generally accepted that in ventricular cardiomyocytes, depolarization of the sarcolemma and T-tubules triggers contraction by the process of Ca²⁺-induced Ca²⁺ release (CICR).^1,2 Depolarization opens voltage-dependent Ca²⁺ channels/dihydropyridine receptors (DHPR), allowing a small flux of external Ca²⁺ (the inward Ca²⁺ current, I_Ca) that in mature cells is insufficient to elicit a full contraction. The incoming Ca²⁺, however, opens Ca²⁺ release channels/ryanodine receptors (RyR) of the sarcoplasmic reticulum (SR), producing a massive Ca²⁺ discharge and raising [Ca²⁺]_i to contracting levels. Synchronous and coordinated contraction of ventricular cells propels blood into arteries; diastolic refilling occurs when [Ca²⁺]_i is re-sequestered back into the SR by the Ca²⁺-ATPase and extruded from the cytosol by the Na⁺-Ca²⁺ exchanger, causing relaxation of the cell.

A prominent characteristic of several animal models of heart failure (HF) and of the failing human myocardium is a depression of the rate of contraction and relaxation of individual cardiomyocytes.^3–7 This contractile dysfunction is at least partly attributed to abnormal intracellular Ca²⁺ cycling, inasmuch as the peak and kinetics of the [Ca²⁺]_i transient appear to be depressed, too. However, despite extensive research, the exact molecular players and the mechanisms responsible for these alterations remain controversial. Although most studies agree that SR Ca²⁺ content is depressed in HF, some investigators attribute this decrease to abnormal density/function of Ca²⁺ uptake and extrusion mechanisms (ie, Ca²⁺-ATPase and Na⁺-Ca²⁺ exchanger)^6–8 and others to abnormal RyR function.^9,10

Proposing RyRs as central players in the contractile dysfunction of HF is sound and appealing for several reasons. First, RyRs are the main (if not the only) gate for Ca²⁺ release from the SR, and small variations in their activity may produce large fluctuations in intra-SR Ca²⁺. Second, RyRs are tightly bound to FKBP12.6, and disruption of the RyR/FKBP12.6 complex produces unstable channels with high activity and propensity to leak Ca²⁺ even at rest.¹¹ If PKA phosphorylation strips RyRs of FKBP12.6, as proposed recently,⁹ then the increased catecholamine levels found in HF would produce hyperphosphorylation of RyRs, potentially resulting in abnormal Ca²⁺ leak from the SR.

In this study, we used the tachycardia-induced canine model of HF and human failing myocardium to determine density and function of the major SR Ca²⁺ transport proteins. We found that, despite their great potential as regulators of SR Ca²⁺ content, RyRs remain largely unaffected by the pathophysiological mechanisms that occur in HF, and that an abnormal Ca²⁺ uptake, more than Ca²⁺ release, is likely to explain the blunted Ca²⁺ transient of HF.

	Materials and Methods

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Induction of Heart Failure
All animal studies were performed in accordance to the guidelines in the NIH Guide for the care and Use of Laboratory Animals DHHS publication No. [NIH] 85-23 (revised 1985), and approved by institutional review board. The protocol for induction of heart failure is detailed elsewhere.¹² A total of 18 control (sham-operated) and 14 HF dogs were used in this study. The studies involving human hearts were approved by the Institutional Review Committee of the University of Wisconsin and were conducted with informed consent from the patients. Human ventricular cells and myocardium (for isolation of cell organelles) were obtained from explanted hearts of 6 nonfailing (control) and 19 failing patients with ischemic or dilated cardiomyopathic failure (ICM and DCM, respectively). Echocardiographic and intracardiac pressure measurements in both canine and human failing hearts showed overt signs of congestive HF including LV dilation and systolic dysfunction.

Isolation of Ventricular Myocytes and Measurement of [Ca²⁺]_i Transients
Single ventricular myocytes were obtained by enzymatic digestion using the procedure of Haworth et al.¹³ Cells were suspended in Krebs-Henseleit HEPES medium and kept at room temperature until used, usually within 8 hours. For measurements of intracellular Ca²⁺, myocytes were perfused with a modified Tyrode solution containing (in mmol/L) NaCl 138, MgCl₂ 1, KCl 4.4, dextrose 11, CaCl₂ 1, HEPES 12, pH 7.4. Cells were incubated at room temperature in Tyrode solution with 5 µmol/L Fluo-4AM for 30 minutes. Confocal images (BioRad MR-1) were recorded with the scan line oriented along the long axis of the cell. Fluo-4 was excited at 488 nm, with emitted fluorescence measured at 515 nm. Ca²⁺ transients were reconstructed by stacking 512 consecutive line scans and performing a time-intensity plot using a software program running in IDL 5.4 (written by Dr Ana M. Gómez, INSERM U-390, Montpellier, France). [Ca²⁺]_i was calculated using a pseudoratio method assuming a K_d and resting [Ca²⁺]_i of 1.1 µmol/L and 150 nmol/L, respectively.

Preparation of SR-Enriched Microsomes
SR-enriched membranes were isolated from canine and human left ventricle by differential centrifugation as described in Lokuta et al.¹⁴ Ventricular tissue was flash-frozen in liquid N₂ immediately after explantation. In some experiments, NaF (20 mmol/L) and okadaic acid (1 µmol/L) were added to the homogenization medium to prevent protein dephosphorylation that might occur during homogenization.

⁴⁵Ca²⁺ Uptake Measurements
ATP-dependent, oxalate-facilitated ⁴⁵Ca²⁺ uptake by SR microsomes was determined by the rapid filtration technique, as described¹⁵ (see also the expanded Materials and Methods section, which can be found in the online data supplement available at http://www.circresaha.org).

Phosphorylation of SR Proteins by PKA
SR microsomes were phosphorylated at 37°C in medium containing (in mmol/L) 50 Tris-maleate (pH 6.8), 120 KCl, 2 MgCl₂, 10 NaF, 1 [-³²P]ATP (1000 to 3000 cpm/pmol), 1 µmol/L of okadaic acid, the catalytic subunit of PKA (40 U), and 2 µmol/L thapsigargin. Negative controls were prepared by adding 10 µmol/L peptide inhibitor of PKA (PKI, Sigma). The reaction (50 µL) was initiated by the addition of [-³²P]ATP and stopped after 3 minutes with 12.5 µL of sample buffer (4x) consisting of 0.25 mol/L Tris-HCl (pH 6.8), 8% SDS, 50% glycerol, and 0.4 mmol/L DTT. Aliquots (40 µg) of SR membranes were subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) in 4 to 20% polyacrylamide precast minigels (BioRad). Phosphorylated PLB and RyR were detected by autoradiography, and the gel slices containing both proteins were cut and counted by liquid scintillation spectrophotometry.

Western Blot Assays
SR microsomes or whole homogenates were dissolved in sample buffer at 37°C for 10 minutes and electrophoresed in SDS gels as described above for transfer onto nitrocellulose membranes, as described.¹⁶ Membranes were probed with primary antibodies against SERCA2a, PLB, and RyRs (Affinity Bioreagents Inc.), or against FKBP12 (polyclonal antibody that recognizes FKBP12.6 and FKBP12, kindly provided by Dr Angela Dulhunty, Australian National University, Canberra, Australia), or against phosphorylated RyR (P-S2809, Badrilla), diluted 1:2500 (FKBP12 and P-S2809) or 1:5000 (all others) in phosphate-buffered saline (PBS). After washing, membranes were incubated with secondary antibodies (IgG) conjugated to horseradish peroxidase (1:30 000 dilution in PBS). Protein-antibody reactions were detected by chemiluminescence using Kodak X-Omat films. The relative amount of proteins on the blots was determined by densitometric analysis using a HP 3c laser scanner and the program SigmaGel. Protein standards were included in each blot to normalize the densitometric data to a known amount of protein loaded.

[³H]Ryanodine Binding Assay
[³H]Ryanodine binding was performed as described.^14–17 The incubation medium contained 1 mol/L KCl, 20 mmol/L MOPS (pH 7.2), 60 to 120 µg of cardiac microsomes or 120 to 240 µg of homogenates, 10 µmmol/L CaCl₂ (total volume 100 µL), and [³H]ryanodine (0.5 to 20 nmol/L). The incubation lasted 90 minutes at 36°C. The Ca²⁺ dependence of [³H]ryanodine binding was determined in total homogenates in medium containing 20 mmol/L MOPS (pH 7.2), 150 mmol/L KCl, 3 mmol/L ß,-methylene-adenosine 5'-triphosphate (AMP-PCP, a nonhydrolyzable ATP analog), 1 mmol/L EGTA, 2.8 to 3 mmol MgCl₂ (final free [Mg²⁺] 0.55 mmol/L), and CaCl₂ to give a range of free [Ca²⁺] from pCa 7 to pCa 4.

Single-Channel Recording of RyRs and Activation by Fast Ca²⁺ Steps
RyRs in SR vesicles were incorporated into planar lipid bilayers and recorded as described.^14–17 The trans and the cis chamber (luminal and cytoplasmic side, respectively, 600-µL each) contained 300 mmol/L cesium methanesulfonate and 10 mmol/L HEPES-Na (pH 7.2). Rapid exchange of solutions at the cytosolic side of the channel (Figure 5) was achieved as described in the online data supplement.

View larger version (38K): [in this window] [in a new window]   Figure 5. Single-channel activity of dog control and HF RyRs. SR-embedded RyRs were fused to lipid bilayers and activated by rapid steps of Ca2+ (0.1100 µmol/L) applied to the cis (cytoplasmic) side of the channel. A, Two traces of representative channel activity from a control RyR or (B) from a HF RyR. Openings are represented as upward deflections of the baseline current. Holding potential=30 mV. C, Po of individual channels was calculated from ensemble currents generated using 24 and 28 traces from control and HF channels, respectively (n= 6 channels from each group). D, Time course and magnitude of the Ca2+ step used for activation of RyRs. See online data supplement for details.

Data Analysis
All data are presented as mean±SEM and differences are considered significant with P<0.05. Ca²⁺ transients and SR protein phosphorylation were compared with t tests. Ca²⁺ transport and its kinetic data (V_max, K_0.5, and Hill coefficients), effects of HF on P_o of RyRs at constant and transient [Ca²⁺] were analyzed with two-way ANOVA for repeated measurements, followed by post hoc analysis with t tests.

	Results

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Ca²⁺ Transients in Control and HF Myocytes
We used line-scan imaging to measure intracellular Ca²⁺ transients in Fluo 4–loaded control and HF canine ventricular myocytes. Cells were field-stimulated with a 10-ms, 30-V pulse applied at 1 Hz. Figure 1A shows stacked line-scan Ca²⁺ images in ventricular myocytes from control and HF dogs. Control cells showed a rapid and homogeneous increase in [Ca²⁺]_I, whereas the majority of HF cells displayed attenuated and slower [Ca²⁺]_i elevations. Figure 1B plots the integrated pixel intensity of the line-scan image versus time and represents the Ca²⁺ transient for each stimulation in the control (blue line) and HF (green line) cells. Both, the amplitude and the kinetics of the [Ca²⁺]_i transient were reduced in HF cells. Peak [Ca²⁺] was 0.91±0.012 µmol/L in control and 0.60±0.018 µmol/L in HF cells (n=34 and 32, respectively; P<0.05). Time to peak was 47.3±4.1 ms in control and 98.2±12 ms in HF cells. Time to 50% decay of the [Ca²⁺]_i transient was 625±36 ms in control and 861±64 ms in HF. Thus, HF cells display marked abnormalities in the [Ca²⁺]_i transient. These results are in agreement with previous studies that find that the contractile dysfunction of HF may not be solely attributed to defects of contractile proteins^3–8,18; abnormalities in mechanisms that regulate [Ca²⁺]_i may also contribute significantly to the depressed contraction and relaxation.

View larger version (31K): [in this window] [in a new window]   Figure 1. Ca2+ transients in canine control and HF cells. A, Line scan (2-ms) images were stacked horizontally to determine the time course of the [Ca2+]i transient in control and HF cells. Scan line was aligned along the longitudinal axis of the cell. Distance is therefore displayed on the y-axis (0.2 µm/pixel). B, Line scan images (Ca2+ transients) are presented as plots of integrated pixel intensity versus time. Cells of the HF group had Ca2+ transients with smaller amplitude and slower kinetics.

Alterations in SR Ca²⁺ Uptake and PKA Stimulation
Figure 2A shows ATP-supported and oxalate-facilitated ⁴⁵Ca²⁺ uptake by SR-enriched microsomes from control and HF dogs, in the absence and the presence of 4 µg/mL of the catalytic subunit of PKA. Compared with control, both the initial rate and the magnitude of Ca²⁺ uptake were lower in HF SR at various [Ca²⁺]. Figure 2B shows that PKA stimulated Ca²⁺ uptake in control and HF SR, especially at low [Ca²⁺], but the extent of stimulation was lower in the HF group (P<0.05 at 0.17 to 1.4 µmol/L Ca²⁺). Kinetic analysis indicated that V_max for ⁴⁵Ca²⁺ uptake was depressed by 45% (P<0.05) in HF compared with control, with no significant changes in K_0.5 for Ca²⁺ or n, the Hill coefficients. In control SR, PKA stimulated Ca²⁺ uptake, but did not significantly alter K_0.5 for Ca²⁺ or V_max. In HF SR, PKA reduced K_0.5 without changing significantly the V_max. These results suggest that the global Ca²⁺ alterations observed in isolated HF cells (Figure 1) may be due to reduced rate of SR Ca²⁺ transport.

View larger version (15K): [in this window] [in a new window]   Figure 2. Ca2+ uptake by control and HF SR. A, ATP-driven 45Ca2+ uptake into SR vesicles was measured for 3 minutes at 37°C in the absence and the presence of the catalytic subunit of PKA and the indicated [Ca2+]. Rate of 45Ca2+ uptake is lower in HF at all [Ca2+] (P<0.05 by two-way ANOVA), and PKA stimulated the rate of 45Ca2+ uptake at all [Ca2+] in both control and HF (P<0.05 to 0.001 with paired t test after ANOVA). ANOVA indicated the effect of PKA was more pronounced in control than in HF at 0.17, 0.32, and 1.4 µmol/L [Ca2+] (P<0.05). B, PKA-stimulated 45Ca2+ uptake (%). *Percent stimulation differs significantly between control and HF (P<0.05, ANOVA followed by t-test, n=8)

SR Ca²⁺ Pump and Phospholamban Density in HF
We compared the density of the SR Ca²⁺-ATPase (SERCA2a) and its regulatory protein, phospholamban (PLB), in control and HF. Figure 3A is a Western blot of SR-enriched microsomes from control and failing dog hearts probed with antibodies against SERCA2a and PLB. We first measured the intensity of 18 SERCA2a and 12 PLB bands from SR of control dogs and normalized each number to 100%; the intensity of the corresponding bands from the same number of HF samples was then compared against control. Figure 3B shows that SERCA2a and PLB were significantly reduced in HF (65±7% and 68±10% of control, respectively; P<0.05). Thus, a reduction in SERCA2a protein density and its regulator PLB in HF may be an underlying cause for the decreased ⁴⁵Ca²⁺ uptake in SR microsomes (Figure 2A) and conceivably, for the prolongation of the [Ca²⁺]_i transient observed in intact cells (Figure 1B).

View larger version (44K): [in this window] [in a new window]   Figure 3. SERCA2a and phospholamban density in canine HF. A, Western blots of SERCA2a and PLB in control and HF samples. In this and the remaining figures, each C and HF indicate a different control and HF sample, respectively. B, Cumulative data from 8 to 10 separate samples for each group. Density of SERCA2a and PLB were both depressed in HF (P<0.05) with no apparent alteration in stoichiometry. Data are presented as mean±SE, n=10.

Density and Function of RyRs in Canine HF
We first conducted Western blot analysis and [³H]ryanodine binding experiments to measure the density of RyRs in control and HF samples. Neither Western blots (Figure 4A) nor saturation binding experiments with [³H]ryanodine (Figure 4B) yielded different density values for control and HF samples. The intensity of the RyR band was 1.0±0.10 in control and 1.07±0.11 in HF (n=14 samples each). B_max, the maximal density of [³H]ryanodine binding sites, was 0.37±0.07 and 0.39±0.09 pmol/mg protein in control and HF microsomes, respectively (n=13).

View larger version (16K): [in this window] [in a new window]   Figure 4. Density and Ca2+-sensitivity of RyR in control and failing hearts. A, Western blots of RyR in control and HF canine SR. RyRs were probed with a specific antibody as described in Materials and Methods section. B, Equilibrium [3H]ryanodine binding curves show no difference between control and HF SR. C, Ca2+ dependence of [3H]ryanodine binding in control and HF. SR-enriched microsomes were incubated with 7 nmol/L [3H]ryanodine and the indicated [Ca2+] at 37°C for 90 minutes. Ca2+-dependent binding was similar in control and HF samples. Thus, both Bmax and K0.5 for Ca2+ were identical (n=8 dogs of each group).

We also examined whether Ca²⁺-dependent activation of [³H]ryanodine binding differs between control and HF. Binding was conducted in the presence of Mg²⁺ and AMP-PCP (a nonhydrolyzable analogue of ATP), two cytosolic ligands of RyRs that modulate the RyR response to Ca²⁺. Figure 4C shows that Ca²⁺ was equally effective in activating RyRs from control and HF homogenates. Both data sets could be fitted with the same line. For the range of [Ca²⁺] tested, the activation curve was sigmoidal with a half-maximal effective Ca²⁺ concentration (K_0.5) =1.8 µmol/L. Thus, the Ca²⁺ sensitivity of RyRs does not appear altered in HF (see also Figure 5).

We reconstituted SR microsomes into planar lipid bilayers to directly determine the basal activity and the Ca²⁺ sensitivity of RyRs from control and failing hearts (Figure 5). Single RyRs were stimulated by fast and calibrated elevations of cis (cytosolic) [Ca²⁺]. Ca²⁺ was elevated from 0.1 to 100 µmol/L with a rapid ( 15 ms) microinjection system that exchanged the solution in the vicinity of the RyR (0.5 µL) without altering substantially the composition of the bath solution (600 µL). The Ca²⁺ step produced a sudden increase of channel activity (Figures 5A and 5B), but then the activity decayed even though [Ca²⁺] remained elevated (Figure 5D). The ensemble current generated by summing sweeps of single channel currents (Figure 5C) revealed that the probability of the channel being open, P_o, was high immediately after the injection, and then slowly decayed to a new steady-state 2 seconds after the injection (RyR adaptation).^17,19 An exponential fit of the ensemble current showed that the time constant of activation (_on=20 ms, not shown), the peak P_o (0.94±0.08), and the time constant of decay (_decay=1.4±0.21 seconds) were identical in control and HF RyRs. Thus, the basal activity and the capacity of RyRs to respond to rapid and sustained Ca²⁺ stimuli appears intact in RyRs from canine failing hearts.

Changes in Human Heart Failure
We also tested for changes in density of SR Ca²⁺ transport proteins in human HF. Myocardial homogenates and SR microsomes were obtained from freshly explanted hearts of 12 ICM and 7 DCM patients and compared with the same from 6 nonfailing (control) individuals whose heart could not be matched for transplantation. As with the canine model above, samples from control and HF groups were run side-by-side for a direct comparison of protein expression. Western blots revealed that RyR expression tended to be lower in HF, but the difference did not reach statistical significance (control=1.0±0.089, n=5, and HF 0.87±0.082, n=19; P<0.2) (Figure 6A). Unlike the canine model of HF, there was no difference in PLB expression, either for the high molecular weight form (H) (1.0±0.085 and 0.97±0.13 for control and HF, respectively) or the low molecular weight form (L) (1.0±0.080 and 0.96±0.076 for control and HF, respectively). However, like human HF, canine HF showed a significant reduction of SERCA2a (1.0±0.067 in control versus 0.70±0.050 in HF; P<0.002) (Figure 6B).

View larger version (69K): [in this window] [in a new window]   Figure 6. Density of SR Ca2+ transport proteins in human control and HF. A, Western blots of RyRs, SERCA2a, and the high (H) and low (L) molecular weights of PLB from ventricular homogenates. B, Pooled data from 8 control and 12 HF samples. Intensity values of control samples were normalized to 1.0.

Human RyRs from control and HF SR were also quantified with [³H]ryanodine binding assays and reconstituted in lipid bilayers to assess their gating kinetics and P_o. Like in the canine model of HF, neither density nor function of RyRs were different in human HF. B_max was 0.34±0.05 (control n=5), 0.315±0.089 (ICM n=12), and 0.359±0.033 (DCM n=7) (not shown). Figures 7A and 7B show that, when reconstituted in lipid bilayers, both groups of RyRs had the same level of activity in the presence of steady concentrations of Ca²⁺; furthermore, neither population of RyR channels showed a significant frequency of subconducting states, a characteristic that would have been found if FKBP12.6 were dissociated from RyRs.^11,20 The current-voltage relationship for the full-conducting level (>96% of all events) was linear and had a slope of 750 pS for both types of channels (Figure 7E). Although there were some detectable subconducting states (Figures 7A and 7B, arrow in the expanded traces), they were also present in control RyRs and were not frequent enough to skew the Gaussian distribution of open events in the current histograms (Figures 7C and 7D). Instead, subconducting states were abundant when control RyRs were subjected to trypsin digestion (Figure 7F). Addition of trypsin (0.3 U/mL) to the cis (cytoplasmic) side of the channel first abolished the fast, full conducting openings, increased mean open time, and induced fractional openings before causing an almost complete blockade of channel activity. Hence, subconducting states appear to be relatively rare occurrences in RyR gating unless channels are subjected to strenuous conditions, eg, limited proteolysis or FKBP12.6 detachment.

View larger version (51K): [in this window] [in a new window]   Figure 7. Single-channel activity of human RyRs. A and B, Two-second traces of representative single channel activity from control and HF RyRs. Activating [Ca2+] was nominally free and measured at 5 µmol/L. The last trace is an expansion of the indicated segment above, chosen to display the different subconductance states observed (arrows). Scale bar=500 ms (x-axis) and 30 pA (y-axis). C and D, Current histograms constructed with data from 14 control and 16 HF channels from 4 different preparations each. Arrows point to subconducting states. E, Current-voltage relation for human control and HF channels. Points could be fitted with the same line, which had a slope conductance=750 pS (n=4). F, Control canine RyR exhibits subconducting states (middle) before blockade of channel activity by trypsin (bottom). Recording conditions as in previous figures. Holding potential=30 mV.

RyR Phosphorylation and FK506-Binding Protein in Heart Failure
Marx et al⁹ postulated that FKBP12.6 dissociates from RyRs on phosphorylation, and that this reaction is increased in HF. We first determined whether PKA phosphorylation of RyRs causes dissociation of FKBP12.6. Figure 8A, top panel, is an autoradiogram showing that the phosphorylation protocol using the catalytic subunit of PKA effectively phosphorylates control canine RyRs. An 450-kDa band corresponding to RyRs incorporates [³²P-]ATP in a saturable and time-dependent manner. Phosphorylation is prevented by including 10 µmol/L of PKI peptide in the phosphorylation cocktail (not shown), indicating that the reaction is specific for PKA. Using this protocol, we back-phosphorylated control and HF canine RyRs side by side (Figure 8A, bottom panel) to determine their level of phosphorylation in vivo. Although some control and HF samples markedly deviated from the average back-phosphorylation level, the pooled data from n=12 control and 10 HF samples (Figure 8D) yielded no significant difference. Figure 8B is a Western blot of control dog and human ventricular homogenates probed with a polyclonal antibody that recognizes the FKBP12.6 and FKBP12 isoforms. The homogenate was incubated with the cocktail of Figure 8A in the absence and the presence (+PKA) of PKA and immediately fractionated in soluble (Cyt) and membranous (Membr) components to test whether phosphorylation dissociates either immunophilin from membrane-associated proteins (RyRs). Figure 8B shows that the distribution pattern of both, FKBP12.6 and FKBP12, is independent of RyR phosphorylation. Although the FKBP12.6/FKBP12 ratio differs between dog and human cardiomyocytes, in agreement with previous reports,²¹ neither immunophilin dissociates from membrane-bound proteins after phosphorylation. Lastly, we used an antibody that recognizes the phosphorylated form of RyRs only (phosphorylated serine 2809) for a direct assessment of phosphorylation levels in HF. In rat hearts perfused with isoproterenol (100 nmol/L for 1 minute) before homogenization, the intensity of the RyR band is 4-fold higher than in hearts without isoproterenol treatment (Figure 8C), indicating specificity of the antibody. Control and HF RyRs from dog homogenates (Figure 8C) show variable levels of native phosphorylation, but again, the pooled data from n=8 control and 10 HF samples (Figure 8D) yields no significant difference. Thus, there was no structural or functional evidence that phosphorylation removes FK506-binding protein from RyRs and that this process is exaggerated in HF.

View larger version (49K): [in this window] [in a new window]   Figure 8. Phosphorylation of RyRs and FKBP12.x sedimentation. A, Autoradiograms showing the time course of PKA phosphorylation of RyRs, conducted as described in Materials and Methods section, and back-phosphorylation of 4 control and 4 HF canine SR. B, Western blots of FKBP12.6 and FKBP12, recognized by the same antibody and separated based on gel retention. Ventricular homogenates of control dogs and humans were phosphorylated with PKA as in (A) and immediately spun at 12 000g for 10 minutes. Aliquots of supernatant (Cyt) and pellet (Membr) (80 µg each) were run in SDS-PAGE, transferred to nitrocellulose membranes and blotted with FKBP12.x antibody. C, Top, Western blots of homogenates (60 µg/lane) of rat hearts stimulated without (-) or with isoproterenol immediately before homogenization. Homogenates were probed with the P-S2809 antibody that recognizes the phosphorylated form of the RyR only. Bottom, Four control and 4 HF RyRs from dog SR run side-by-side. D, Pooled results from back-phosphorylation and Western blots experiments. Data from n=8 control samples was normalized to 1.0; HF samples (n=10) were then compared against control.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
There is agreement that the depressed cardiac performance characteristic of HF is likely to result, at least in part, from abnormal [Ca²⁺]_i cycling, but a more controversial issue is the level of involvement of specific molecules transporters of Ca²⁺. Although there is substantial evidence suggesting that upregulation of the sarcolemmal Na⁺-Ca²⁺ exchanger may depress the [Ca²⁺]_i transient in human failing cells and in animal models of HF, the involvement of the SR has yet to be defined,²¹ especially in regard to specific Ca²⁺ transporters, ie, RyRs, SERCA2a, and PLB. In the present study, we used the fast-paced canine model of HF and human failing myocardium to carry out a detailed analysis of the density and main function of SR Ca²⁺ transporters. Previous studies have measured density of SERCA2a and PLB in canine HF,³ Na⁺-Ca²⁺ exchanger function in human failing cells,⁷ and RyR activity in both canine and human HF.⁹ Our results do not invalidate previous conclusions; however, if we accept that the depressed SR Ca²⁺ load observed in HF partly underlies the blunted [Ca²⁺]_i transient, they are only congruent with the idea that abnormal Ca²⁺ uptake, not Ca²⁺ release, contributes to the contractile dysfunction of HF.

Recently, it was reported that RyRs play a fundamental role in the abnormal Ca²⁺ cycling that occurs in HF.⁹ According to this study, PKA phosphorylation of RyRs dissociates FKBP12.6; as catecholamine levels are increased in HF,²² PKA would produce hyperphosphorylation of RyRs and dissociation of FKBP12.6, increasing the channel’s sensitivity to Ca²⁺ and causing diastolic SR Ca²⁺ leak.⁹ Upregulation of Na⁺-Ca²⁺ exchange^6–8 may then extrude the Ca²⁺ leak. Thus, the overall effect of RyR hyperactivity would be a depressed SR Ca²⁺ load, which alone could explain the reduced amplitude and kinetics of the [Ca²⁺]_i transient in HF. As intelligible as it sounds, we failed to detect key observations that support this scheme. Ca²⁺ sensitivity of RyRs was not different between control and HF groups (Figures 4C and 5). Subconductance states, predominant in FKBP12.6-stripped RyRs,^11,20 were equally frequent in control and HF channels (Figures 5 and 7). Phosphorylation of RyRs by PKA did not appear to dissociate FKBP12.6 (or FKBP12) from SR-embedded RyRs (Figure 8). Finally, the effects of rapamycin on single channels and [³H]ryanodine binding were the same for control and HF RyRs (not shown), suggesting that FKBP12.6 remains associated to both groups of channels. These crucial differences cannot be explained on variability in models of HF, as Marx et al⁹ and the present study both used the tachycardia canine model and failing human myocardium. However, a potential source of discrepancy is that Marx et al⁹ used solubilized and immunoprecipitated RyRs for phosphorylation and single channel recordings, whereas we used SR-embedded RyRs. It is possible that immunoprecipitation concentrates proteins such as phosphatases, kinases, or proteases and favor reactions that would not normally occur in more intact preparations. It may be argued also that we failed to observe hyperphosphorylation of RyRs and the resultant subconductance states because the SR microsomes might have been dephosphorylated during isolation, but the presence or the absence of phosphatase inhibitors (NaF and okadaic acid) in the isolation media did not change our results (not shown). Also, if abnormal dissociation of phosphatases would have occurred in HF, as proposed,⁹ we would not have detected the same level of RyR-associated FKBP12.6 in control and HF SR.

ß-Adrenergic receptor (ß-AR) activation produces important inotropic and chronotropic effects on cardiac contraction via activation of GTP-binding protein (G_s). G_s activates adenylate cyclase and increases cAMP levels which, in turn, activate PKA. PKA phosphorylation of sarcolemmal proteins (particularly DHPR) and SR proteins increase SR Ca²⁺ uptake and release, and myofibrils (troponin I and protein C) decrease their Ca²⁺ sensitivity to relax at a faster rate.²³ PLB and the RyR are the main proteins of the SR phosphorylated by PKA. It is conceivable then, that the PKA-induced increase of Ca²⁺ uptake and release result from phosphorylation of PLB and RyR, respectively. However, sustained stimulation of RyRs causes transient increases in Ca²⁺ release only,²⁴ because the resultant decrease in SR Ca²⁺ load exerts a negative feedback on RyRs, thus attenuating their activity despite persistent presence of the activator. Furthermore, a recent study determined that phosphorylation of RyRs is functionally inconsequential for Ca²⁺ release if SR Ca²⁺ levels remain constant.²⁵ From this perspective, it is improbable that phosphorylation of RyRs alone may account for the sustained anomalies of Ca²⁺ cycling and the persistently reduced levels of SR Ca²⁺ observed in HF. Although there is consensus that the etiology of the contractile dysfunction of HF is multifactorial, phosphorylation of RyRs as pivotal cause of depressed SR Ca²⁺ load is unlikely given the self-correcting mechanisms on Ca²⁺ release that are triggered by Ca²⁺ inside the SR.

In summary, we found that the abnormal [Ca²⁺]_i transients underlying contractile dysfunction in HF are at least partly due to depressed SR function. Because RyRs appear structurally and functionally normal in HF, we conclude that downregulation of SERCA2a function in HF directly leads to depressed SR Ca²⁺ load and indirectly to reduced Ca²⁺ release. Our results suggest that therapeutic interventions directed at increasing SR Ca²⁺ uptake may prove more beneficial than those than nonselectively increase e-c coupling gain.

	Acknowledgments

 
This work was supported by grants from the NIH HL-55438 and PO1-HL47053 (to H.H.V.) and HL-61534 (to R.A.H., M.R.W., and H.H.V.) and by AHA grant 0060443Z (to M.T.J.). We wish to thank Larry Whitesell for technical support in preparing the canine heart failure model.

Received April 22, 2002; revision received October 15, 2002; accepted October 15, 2002.

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