Abnormal Ca2+ Release, but Normal Ryanodine Receptors, in Canine and Human Heart Failure
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 Vmax 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 Po 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 β-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.
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, ICa) 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
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 Kd 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 [γ-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 [γ-32P]ATP and stopped after 3 minutes with 12.5 μL of sample buffer (4×) 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 β,γ-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.
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 (Vmax, K0.5, and Hill coefficients), effects of HF on Po of RyRs at constant and transient [Ca2+] were analyzed with two-way ANOVA for repeated measurements, followed by post hoc analysis with t tests.
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 proteins3–8,18⇓⇓⇓⇓⇓⇓; abnormalities in mechanisms that regulate [Ca2+]i may also contribute significantly to the depressed contraction and relaxation.
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 Vmax for 45Ca2+ uptake was depressed by 45% (P<0.05) in HF compared with control, with no significant changes in K0.5 for Ca2+ or n, the Hill coefficients. In control SR, PKA stimulated Ca2+ uptake, but did not significantly alter K0.5 for Ca2+ or Vmax. In HF SR, PKA reduced K0.5 without changing significantly the Vmax. 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.
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).
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). Bmax, 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).
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 (K0.5) =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 (τ ≈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, Po, 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 (τon=20 ms, not shown), the peak Po (0.94±0.08), and the time constant of decay (τdecay=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).
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 Po. Like in the canine model of HF, neither density nor function of RyRs were different in human HF. Bmax 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.
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-γ]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.
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+ exchange6–8⇓⇓ 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.
β-Adrenergic receptor (β-AR) activation produces important inotropic and chronotropic effects on cardiac contraction via activation of GTP-binding protein (Gs). Gs 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.
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.
Original received April 22, 2002; revision received October 15, 2002; accepted October 15, 2002.
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