Propranolol Prevents the Development of Heart Failure by Restoring FKBP12.6-Mediated Stabilization of Ryanodine Receptor
Background— In heart failure, protein kinase A-mediated hyperphosphorylation of ryanodine receptors (RyRs) in sarcoplasmic reticulum (SR) causes dissociation of FKBP12.6 from RyRs. This results in an abnormal Ca2+ leak through RyRs, possibly leading to cardiac dysfunction. In the present study, we assess whether β-blockers can correct this defect in RyR in tachycardia-induced heart failure and thereby improve cardiac function.
Methods and Results— SRs were isolated from dog left ventricular muscles (normal group, 4 weeks of rapid right ventricular pacing with or without propranolol [P(+) or P(−)]). End-diastolic and end-systolic diameters both increased less in P(+) than P(−), associated with a smaller decrease in fractional shortening in P(+). In SR from P(−), a prominent Ca2+ leak was observed, and FK506 (which dissociates FKBP12.6 from RyR) did not induce an additional Ca2+ leak. However, there was no appreciable Ca2+ leak in SR from P(+), although FK506 induced a Ca2+ leak as in normal SRs. In SR from P(+), an FK506-induced conformational change in RyR, which was virtually absent in SR from P(−), was observed as in normal SRs. Both the stoichiometry of FKBP12.6 versus RyR, assessed by [3H]FK506 and [3H]ryanodine binding assays, and the protein expression of FKBP12.6, assessed by Western blot analysis, were restored by propranolol toward the levels seen in normal SRs.
Conclusions— Low-dose propranolol corrects the defective interaction of FKBP12.6 with RyR (restoration of RyR conformational change and prevention of Ca2+ leak from RyR), apparently resulting in an attenuation of intracellular Ca2+ overload and hence preventing the development of left ventricular remodeling in heart failure.
Received October 31, 2001; revision received January 7, 2002; accepted January 7, 2002.
An abnormal regulation of intracellular Ca2+ by sarcoplasmic reticulum (SR) has been shown to be involved in the mechanism underlying the contractile and relaxation dysfunctions in heart failure. Several investigators have demonstrated that in cardiac hypertrophy or failure, Ca2+ uptake by the SR is decreased in conjunction with a decreased density of Ca2+ ATPase.1,2⇓
Within the last few years, an altered function of the SR Ca2+ release channel (ryanodine receptor [RyR]) has also been shown to contribute to cardiac dysfunction in heart failure.3–5⇓⇓ As described in our previous study,6 in a dog model of pacing-induced heart failure, a prominent abnormal Ca2+ leak occurs through the RyR. This is attributable to a partial loss of RyR-bound FKBP12.6 and the resultant conformational change in the RyR. Presumably, this abnormal Ca2+ leak causes an intracellular Ca2+ overload, which in turn leads to diastolic and systolic dysfunctions. We also found that in the failing heart, polylysine-induced Ca2+ release from SR vesicles was decreased, owing to an impaired gating function of the RyR,7 and this too is ascribable to a defective FKBP12.6-RyR interaction.8 Removal of FKBP12.6 from RyR causes uncoupled channel gating in the RyR, resulting in defective closure of these channels.9,10⇓ With regards to the mechanism responsible for the partial loss of FKBP12.6 from the RyR, Marx et al11 demonstrated that RyR hyperphosphorylation, mediated by protein kinase A (PKA), causes dissociation of FKBP12.6 from RyR, which in turn causes an increased sensitivity to Ca2+-induced activation and defective channel functions. These findings suggest that failing hearts lack FKBP-mediated channel regulation and that this is the major cause of the serious abnormality in their regulation of intracellular Ca2+ and their observed cardiac dysfunctions.
A common finding in patients with heart failure is that a hyperadrenergic state and elevated levels of circulating catecholamines are markers for increased risk of mortality.12 Moreover, clinical trials have shown that treatment with β-blockers restores cardiac function and reduces rate of mortality in patients with heart failure.13,14⇓ The experimental literature also suggests that the alterations in biology and contractility seen in the failing cardiac myocyte can be reversed by β-blockers.15 However, the actual mechanism responsible for these beneficial effects of β-blockers has not been fully elucidated. In the present study, we used a dog model of pacing-induced heart failure to investigate whether β-blockers would inhibit PKA-mediated RyR hyperphosphorylation, prevent the dissociation of FKBP12.6 from RyR, inhibit the abnormal Ca2+ leak through RyR, and thereby restore cardiac function.
Fluo-3 was obtained from Molecular Probes, and SAED was from Pierce. [3H]ryanodine, [3H]dihydro-FK506, and [γ-32P]ATP were purchased from Dupont NEN. Anti-FKBP12 (C-19) antibody, which cross-reacts with FKBP12.6,16 was purchased from Santa Cruz Biotechnology. Anti-RyR antibody and Anti-SERCA2 antibody were obtained from Oncogene Research Products and Affinity Bioreagents Inc, respectively. Human recombinant FKBP12.6 was produced in our laboratory. FK506 was provided by Fujisawa Pharmaceutical Co Ltd (Osaka, Japan).
Production of Pacing-Induced Heart Failure
In beagle dogs weighing 10 to 14 kg (KITAYAMA LABES Co, Ltd, Nagano, Japan), we induced heart failure by 28 days of rapid right ventricular (RV) pacing at 250 bpm using an externally programmable miniature pacemaker (Medtronic Inc), as described previously.7 Then, under anesthesia, we chronically implanted a 5-F micromanometer in the left ventricle (LV) via the apex for the measurement of LV pressure, and we placed a pair of crystals (5 MHz, 2 mm in diameter) on the endocardium of the anterior and posterior walls perpendicular to the long axis of the LV, midway between the apex and the base of the heart. After allowing a recovery period of 1 week, we measured LV pressure and recorded two-dimensional echocardiograms at the level of the head of the papillary muscle in the conscious state, ≈1 hour after the termination of rapid RV pacing. The care of the animals and the protocols used were in accordance with guidelines set by the Animal Ethics Committee of Yamaguchi University School of Medicine.
To determine the dose of propranolol to be used for chronic administration, we evaluated the concentration-dependent effect of propranolol on hemodynamic parameters in normal conscious dogs (Figure 1). Propranolol was continuously infused for 1 day, starting at a rate of 0.05 mg/kg per day and increasing incrementally to 2 mg/kg per day. A recovery period of 1 day was allowed before each incremental increase. Propranolol (2 mg/kg per day) was found significantly to decrease maximal inotropic response induced by isoproterenol (0.80 μg · kg−1 · min−1 IV) (Figure 1). In our first dog, we continuously infused this dose of propranolol simultaneously with the initiation of RV pacing. However, after 1 day of this infusion, the dog died of severe cardiac dysfunction. Therefore, in the next dog, we continuously infused 0.5 mg/kg per day of propranolol, by which means a 17% decrease in the inotropic response had been obtained in normal dogs (Figure 1). However, after infusion for 3 days with pacing, that dog also died from the same cause. We then decided to try a much lower dose, 0.05 mg/kg per day, for chronic administration in the following experiments. At this dose, neither the baseline max dP/dt nor the isoproterenol-induced inotropic response was decreased, although baseline heart rate was decreased significantly (by 15%) in normal conscious dogs (Figure 1). Thus, a negative chronotropic effect, but not a negative inotropic effect, was elicited at this dose.
To obtain LV diastolic pressure-diameter relationship over a wide range of diastolic pressures, phenylephrine (2 to 10 μg · kg−1 · min−1) was infused intravenously for 10 minutes to increase LV pressure in 4 conscious dogs (4 dogs, propranolol −; 4 dogs, propranolol +).
Preparation of SR Vesicles
Ca2+ Uptake and Leak Assays
We first incubated SR vesicles (0.2 mg/mL) in 0.5 mL of solution containing 0.15 mol/L potassium gluconate, 1 mmol/L MgCl2, 0.2 mmol/L EGTA-calcium buffer (free [Ca2+], 0.3 μmol/L), 10 mmol/L NaN3, and 20 mmol/L MOPS, pH 6.8. Ca2+ uptake was initiated by the addition of 0.5 mmol/L ATP into the cuvette. After the Ca2+ uptake had reached a plateau, we added 1 μmol/L thapsigargin to inhibit SR Ca2+-ATPase activity with or without FK506 (30 μmol/L) and monitored the resultant Ca2+ leak. We monitored the time course of Ca2+ uptake spectrophotometrically (F-2000, Hitachi) using fluo-3 as a Ca2+ indicator (excitation 480 nm, emission 530 nm), as described previously.6 The magnitude of the Ca2+ leak was taken as the value obtained 60 seconds after the addition of thapsigargin, and it was expressed as a percentage of the preceding Ca2+ uptake.
[3H]dihydro-FK506 and [3H]ryanodine Binding Assays
We performed [3H]dihydro-FK506 and [3H]ryanodine binding assays as described previously.6–8⇓⇓ We determined the density of high affinity [3H]ryanodine binding sites in SR vesicles by Scatchard analysis of [3H]ryanodine binding isotherms, as described previously.6–8⇓⇓
Site-Directed Fluorescent Labeling of the RyR
We performed specific fluorescent labeling of RyR in SR vesicles using the cleavable hetero-bifunctional cross-linking reagent sulfosuccinimidyl 3-((2-(7-azido-4-methylcoumarin-3-acetamido) ethyl) dithio)propionate (SAED), with polylysine as a site-specific carrier, as described previously.6,18,19⇓⇓ We monitored the time course of the FK506-induced changes in the fluorescence intensity (arbitrary units) of the RyR-bound methylcoumarin-acetate (MCA) probe (excitation 335 nm, emission 450 nm) under the same conditions as those used for the Ca2+ leak assay (except that there was no fluo-3 in the reaction solution). For this monitoring, we used a fluorescence spectrophotometer (F-2000; Hitachi).
We performed immunoblot analyses for FKBP12.6 and SR Ca2+-ATPase as previously described.6 By using the method by Marx et al,11 we achieved coimmunoprecipitation of FKBP12.6 from SR using anti-RyR antibody followed by immunoblotting with anti-FKBP12 antibody. PKA-mediated phosphorylation of immunoprecipitated RyR was assessed with back-phosphorylation using [γ-32P]ATP, as previously described.11
Intragroup comparisons were made by paired t test. Intergroup analysis was performed by ANOVA with a post hoc Schiff’s test. Data are expressed as mean±SD. We accepted P<0.05 as statistically significant.
In the propranolol-treated dogs with chronic RV pacing, both systolic and diastolic functions were preserved, and none of these dogs developed heart failure (Table 1 and Figure 2A). The representative diastolic pressure-diameter relationship obtained during phenylephrine infusion shown in Figure 2B revealed that in the propranolol-untreated dog, the diastolic pressure-diameter relationship curve shifted to the right after a 4-week period of pacing, indicating the development of LV remodeling. In contrast, there was a much less pronounced shift in the propranolol-treated dog (Figure 2B). The plasma contents of norepinephrine and atrial natriuretic peptide and angiotensin II were higher in dogs with rapid chronic RV pacing than in normal dogs. Chronic administration of propranolol during pacing significantly reduced these levels (Figure 2C). These data indicate that our propranolol-treated dogs showed no signs of heart failure despite chronic RV pacing.
Addition of 1 μmol/L thapsigargin to normal SR vesicles produced a small Ca2+ leak, whereas addition of 30 μmol/L FK506 together with 1 μmol/L thapsigargin produced a much more pronounced leak (Figure 3). In contrast, in failing (propranolol-untreated) SR vesicles, addition of thapsigargin alone produced a prominent Ca2+ leak, but addition of FK506 produced no additional increase. In SR vesicles from paced, propranolol-treated dogs, a spontaneous Ca2+ leak was not observed, and FK506 had the same effect as in normal SR (that is, it greatly increased the Ca2+ leak).
In normal SR vesicles, the addition of FK506 after Ca2+ uptake had plateaued induced an increase in MCA fluorescence at a faster rate than Ca2+ leak seen in the same SR vesicles, but it produced virtually no increase in MCA fluorescence in failing (propranolol-untreated) SR vesicles (Figure 4). In propranolol-treated SR vesicles, FK506 induced an increase in the MCA fluorescence intensity, just as it did in normal SR. As we proposed previously,6 MCA fluorescence changes reflect the time course of conformational changes in RyR produced by the FK506-induced dissociation of FKBP12.6 from RyR. Therefore, the reappearance of FK506-induced MCA fluorescence change in propranolol-treated failing SR suggests that the conformational state of RyR was restored in these vesicles.
In the propranolol-untreated SR vesicles, RyR was PKA-hyperphosphorylated, whereas it was reversed in the propranolol-treated SR vesicles, returning the channel phosphorylation to the levels seen in normal hearts (Figure 5A). The amount of the RyR-associated FKBP12.6 was in fact decreased by the chronic RV pacing, but the decrease was prevented by propranolol treatment (Figure 5B). Moreover, in the propranolol-treated vesicles, the Bmax value obtained for [3H]dihydro-FK506 binding was significantly larger than that obtained for the propranolol-untreated vesicles, although it was still less than in the normal vesicles (Figure 5C and Table 2). Because RyR contains a single high-affinity ryanodine-binding site per tetramer, the ratio of the Bmax for [3H]dihydro-FK506 binding to that of the high-affinity [3H]ryanodine in SR vesicles provides an approximate estimate of the molar ratio of the FKBP bound per Ca2+ release channel tetramer.20 This molar ratio was larger for the propranolol-treated failing vesicles (2.38±0.46) than for their propranolol-untreated counterparts (1.14±0.16) and approached the value obtained for normal SR vesicles (3.62±0.62) (Table 2).
After 4 weeks’ rapid RV pacing, both the SR Ca2+ uptake and the amount of SR Ca2+-ATPase were decreased, and in each case the extent of the decrease was similar in the propranolol-untreated and -treated groups (Figure 6).
Evidence has accumulated as to the beneficial effect of β-blockers on the prognosis of patients with heart failure. Indeed, recent long-term survival studies have proved that β-blockers decrease both mortality and morbidity in such patients.13,14⇓ The mechanisms proposed to explain the benefits of β-blocker therapy include a reduction in sympathetic nervous activity, restoration of the β1-receptor population with improved contractile performance, enhanced myocardial relaxation, and an improved cardiac efficiency with an associated reduction in heart rate.21 However, the basis of the adrenergic cascade-related myocyte abnormalities remains largely unclear.
The important new finding made in this study is that in tachycardia-induced canine heart failure, low-dose propranolol, which had only a negative chronotropic but not a negative inotropic effect in normal dogs, restored channel regulation in RyRs and thereby improved cardiac function. In previous studies, β-blockers enhanced the SR Ca2+-ATPase expression in association with increased peak of intracellular Ca2+ transient in human end-stage heart failure,22 and it increased the activity of SR Ca2+-ATPase in a turkey model of furazolidone-induced dilated cardiomyopathy.23 In the present study, treatment with propranolol had no effect on the protein expression of SR Ca2+-ATPase or on Ca2+ uptake function. This discrepancy might be attributable to the different experimental dose of β-blocker used, the dose used in our study being much lower than doses used in the other studies. The regulation of SR Ca2+-ATPase expression might be different depending on the magnitude of the β antagonism.
Recently we found that the development of the heart failure induced by chronic RV pacing can be completely prevented by restoring FKBP12.6-mediated stabilization of RyR using the cardioprotective reagent JTV519 (unpublished data, 2001). Taken together with the recent finding that in heart failure, PKA-mediated hyperphosphorylation of RyR leads to a defective FKBP12.6-mediated channel regulation of RyR,11 our results make it very likely that the mechanism by which propranolol improves cardiac function and prevents LV remodeling, without a change in Ca2+ uptake function, depends on an inhibition of Ca2+ leak through RyR that results from a defective interaction of FKBP12.6 and RyR. The present finding that treatment with propranolol reversed the phosphorylation of RyR in conjunction with a reassociation of FKBP12.6 back to RyR supports the above view. Recently, Reiken et al24 reported that the β1-selective blocker metoprolol reversed PKA-mediated hyperphosphorylation of RyR2, restored the stoichiometry of RyR2 macromolecular complex, and normalized single-channel function in a canine model of heart failure. We confirmed the beneficial effect of β-blockers on RyR channel function using Ca2+ leak and site-directed fluorescent assays. This effect seems to be exerted as a class effect of β-blockers, because similar results were drawn regardless of the selectivity of the β-blocker (β1 selective or nonselective).
In heart failure, the contractile dysfunction that develops within myocytes during the process of LV remodeling is likely to involve other factors besides alterations in the excitation-contraction coupling process,25 ie, progressive loss of myofilaments from cardiac myocytes26 or alterations in cytoskeletal proteins, as well as desensitization of β-adrenergic signaling.27 Resensitization of the β-adrenergic signaling cascade could in part explain the beneficial effects of some β-blockers, such as metoprolol, in heart failure.28
Jeyakumar et al29 have demonstrated that the canine heart is exceptional with respect to the interaction of FKBP with RyR. Although in canine hearts only FKBP12.6 is bound to RyR2, this may be different in other species, including human hearts. Therefore, the findings in the present study may not necessarily be converted to human hearts.
In conclusion, in a canine model of heart failure, low-dose chronic propranolol treatment corrected the defective interaction of FKBP12.6 with RyR (restoration of RyR conformational change and prevention of Ca2+ leak from RyR), and this apparently resulted in an alteration of intracellular Ca2+ overload and a consequent prevention of the development of LV remodeling. The present results may provide a molecular basis for the common clinical observation that use of β-blockers improves prognosis among patients with heart failure.
This work was supported by a grant-in-aid for scientific research from the Ministry of Education in Japan (grants No. 13877107 and 13670717).
↵*These authors contributed equally to this study.
- ↵Cory CR, McCutcheon LJ, O’Grady M, et al. Compensatory downregulation of myocardial Ca channel in SR from dogs with heart failure. Am J Physiol. 1993; 264: H926–H937.
- ↵Marks AR. Intracellular calcium-release channels: regulators of cell life and death. Am J Physiol. 1997; 272: 597–605.
- ↵Hasenfuss G. Alterations of calcium-regulatory proteins in heart failure. Cardiovasc Res. 1998; 37: 279–289.
- ↵Marks AR. Cardiac intracellular calcium release channels: role in heart failure. Circ Res. 2000; 87: 8–11.
- ↵Yano M, Ono K, Ohkusa T, et al. Altered stoichiometry of FKBP12.6 versus ryanodine receptor as a cause of abnormal Ca2+ leak through ryanodine receptor in heart failure. Circulation. 2000; 102: 2131–2136.
- ↵Yamamoto T, Yano M, Kohno M, et al. Abnormal Ca2+ release from cardiac sarcoplasmic reticulum in tachycardia-induced heart failure. Cardiovasc Res. 1999; 44: 146–155.
- ↵Ono K, Yano M, Ohkusa T, et al. Altered interaction of FKBP12.6 with ryanodine receptor as a cause of abnormal Ca2+ release in heart failure. Cardiovasc Res. 2000; 48: 323–331.
- ↵Marx SO, Gaburjakova J, Gaburjakova M, et al. Coupled gating between cardiac calcium release channels (ryanodine receptors). Circ Res. 2001; 88: 1151–1158.
- ↵Marx SO, Ondrias K, Marks AR. Coupled gating between individual skeletal muscle Ca2+ release channels (ryanodine receptors). Science. 1998; 281: 818–821.
- ↵Tsutsui H, Spinale FG, Nagatsu M, et al. Effects of chronic β-adrenergic blockade on the left ventricular and cardiocyte abnormalities of chronic canine mitral regurgitation. J Clin Invest. 1994; 93: 2639–2648.
- ↵Prestle J, Janssen PM, Janssen AP, et al. Overexpression of FK506-binding protein FKBP12.6 in cardiomyocytes reduces ryanodine receptor-mediated Ca2+ leak from the sarcoplasmic reticulum and increases contractility. Circ Res. 2001; 88: 188–194.
- ↵El-Hayek R, Yano M, Ikemoto N. A conformational change in the junctional foot protein is involved in the regulation of Ca2+ release from sarcoplasmic reticulum. J Biol Chem. 1995; 270: 15634–15638.
- ↵Yano M, El-Hayek R, Ikemoto N. Conformational changes in the junctional foot protein/Ca2+ release channel mediated depolarization-induced Ca2+ release from sarcoplasmic reticulum. J Biol Chem. 1995; 270: 3017–3021.
- ↵Timerman AP, Ogunbumni E, Freund E, et al. The calcium release channel of sarcoplasmic reticulum is modulated by FK-506-binding protein: dissociation and reconstitution of FKBP-12 to the calcium release channel of skeletal muscle sarcoplasmic reticulum. J Biol Chem. 1993; 268: 22992–22999.
- ↵Kubo H, Margulies KB, Piacentino VIII, et al. Patients with end-stage congestive heart failure treated with β-adrenergic receptor antagonists have improved ventricular myocyte calcium regulatory protein abundance. Circulation. 2001; 104: 1012–1018.
- ↵Judith K, Catherine S, Roger J, et al. Cellular and molecular remodeling in a heart failure model treated with the β-blocker carteolol. Am J Physiol. 1999; 276: 1678–1690.
- ↵Reiken S, Gaburjakova M, Gaburjakova J, et al. β-Adrenergic receptor blockers restore cardiac calcium release channel (ryanodine receptor) structure and function in heart failure. Circulation. 2001; 104: 2843–2848.
- ↵Beuckelmann DJ, Nabauer M, Erdmann E. Intracellular calcium handling in isolated ventricular myocytes from patients with terminal heart failure. Circulation. 1992; 85: 1046–1055.
- ↵Schaper J, Froede R, Hein ST, et al. Impairment of the myocardial ultrastructure and changes of the cytoskeleton in dilated cardiomyopathy. Circulation. 1991; 83: 504–514.
- ↵Gilbert EM, Abraham WT, Olsen S, et al. Comparative hemodynamic, LV functional, and antiadrenergic effects of chronic treatment with metoprolol vs carvedilol in the failing heart. Circulation. 1996; 94: 2817–2825.