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Carvedilol is one of the most effective beta-blockers for preventing ventricular tachyarrhythmias (VTs) in heart failure (HF), but the mechanisms underlying its favorable anti-arrhythmic benefits remain unclear. Spontaneous Ca2+ waves, also termed store-overload-induced Ca2+ release (SOICR), are known to evoke VTs in patients with HF. Here we show that carvedilol is the only beta-blocker that effectively suppresses SOICR by directly reducing the open duration of the cardiac ryanodine receptor (RyR2). This unique anti-SOICR activity of carvedilol combined with its beta-blocking activity likely contributes to its favorable anti-arrhythmic effect. To allow individual and optimal titration of these beneficial activities, we developed a novel SOICR-inhibiting, minimally-beta-blocking carvedilol analogue VK-II-86. We found that VK-II-86 alone prevented stress-induced VTs in RyR2 mutant mice, and was more effective when combined with a selective beta-blocker metoprolol or bisoprolol. Thus, SOICR inhibition combined with optimal beta-blockade presents a new, promising and potentially patient-tailorable anti-arrhythmic approach.
Ventricular tachyarrhythmias (VTs) are a leading cause of sudden death. Over the past 40 years, a variety of pharmacological therapies for VTs have been developed. Unfortunately, large clinical trials have recently demonstrated that most anti-arrhythmic drugs currently used have little or no survival benefit and, indeed, may even be harmful1-3. There is one exception. Beta-blockers generally reduce VTs and prevent sudden death. Particularly, carvedilol, a non-selective beta-blocker with anti-oxidant and alpha-blocking activities, is one of the most effective beta-blockers in reducing VTs and mortality in individuals with heart failure (HF)4-7. However, the mechanism underlying carvedilol’s favorable anti-arrhythmic effect remains unclear8, 9. The anti-oxidant and alpha-blocking activities of carvedilol have been suggested to contribute to its beneficial effects, but the benefits of anti-oxidants and alpha-blockers have not been corroborated by clinical studies10, 11. Thus, some other unknown carvedilol-specific action must be involved. Identifying this unknown action could have significant implications for the understanding and treatment of cardiac arrhythmias and the development of novel anti-arrhythmic therapies.
It is well known that excessive beta-adrenergic receptor (AR) stimulation can result in sarcoplasmic recticulum (SR) Ca2+ overload that can trigger spontaneous Ca2+ waves12-14, also known as store-overload-induced Ca2+ release (SOICR), mediated by the cardiac ryanodine receptor (RyR2)15, 16. This SOICR can evoke delayed afterdepolarizations (DADs) that can lead to triggered arrhythmias and sudden death17-23. Indeed, SOICR-evoked DADs are the cause of catecholaminergic polymorphic ventricular tachycardia (CPVT) associated with naturally occurring RyR2 mutations15, 16, 24-29. SOICR-evoked DADs are also a major cause of VTs and sudden death in HF12, 13, 30, 31. Beta-blockers suppress beta-AR mediated, stress-induced SR Ca2+ overload and its arrhythmogenicity. However, beta-blocker effectiveness is limited by drug intolerance and the existence of beta-AR independent causes of overload (e.g. Na+-dependent Ca2+ overload)2, 32, 33.
We propose that carvedilol, in addition to its beta-blocking action, directly suppresses SOICR itself. We tested this in two well-established SOICR cell models, HEK293 cells and cardiomyocytes expressing a SOICR-promoting, CPVT-causing RyR2 mutation (R4496C). Our data reveal that carvedilol is the only beta-blocker that effectively suppresses SOICR by directly altering the function of RyR2, independent of its beta- or alpha-blocking, or anti-oxidant activities. Our results also suggest that SOICR-inhibition combined with optimal beta-blockade is an effective and potentially patient-tailorable means to treat Ca2+-mediated VTs.
Of 14 beta-blockers tested, carvedilol was the only one that effectively inhibited SOICR (>95%). All the other beta-blockers had no impact on SOICR even at relatively high concentrations (30μM) (Fig.1).
To further assess whether the alpha-blocking and anti-oxidant activities of carvedilol contribute to its unique action, we tested the impact of alpha-blockers (prazosin and phentolamine) and anti-oxidants ((N-(2-mercaptopropionyl)-glycine (MPG) and α-tocopherol) on SOICR. None of these agents significantly altered SOICR (Fig.1b). Thus, carvedilol is the only beta-blocker that effectively suppresses SOICR, and this action is independent of its beta- and alpha-blocking and anti-oxidant activities.
To test the possibility that carvedilol suppresses SOICR by directly altering the function of RyR2, we assessed the impact of carvedilol on single native RyR2 channels from SR microsomes fused into lipid bilayers. Carvedilol (1 μM) significantly reduced RyR2 mean open time (OT) (P < 0.01) and open probability (Po) (P < 0.01), and increased the event frequency (P < 0.01). The mean closed time (CT) was not statistically altered by carvedilol (P= 0.10) (Fig.2a).
We also assessed the impact of carvedilol on single purified recombinant RyR2 channels. Carvedilol (1 μM) significantly reduced their mean OT (P < 0.01) and increased their event frequency (P< 0.05) (Fig.2b). Carvedilol also reduced intra-burst event duration, while increasing intra-burst event frequency (P<0.05) (Fig.S1). Thus, carvedilol consistently reduces the duration and increases the frequency of single RyR2 openings.
We next determined whether carvedilol is able to suppress SOICR in cardiomyocytes from mice harboring the SOICR-promoting, CPVT-causing RyR2 mutation, R4496C (Fig.S2). Line-scan confocal Ca2+ imaging showed that carvedilol (1 μM) significantly reduced the occurrence and frequency of SOICR (P < 0.05), whereas metoprolol (2 μM) and the vehicle DMSO had no significant effect (Fig.3a–c). The SR Ca2+ contents in myocytes treated with carvedilol, metoprolol, or DMSO were not significantly different (Fig.3d). We also employed epifluorescence single cell Ca2+ imaging to determine the impact of carvedilol on SOICR in a large number of myocytes. Consistently, carvedilol (1 μM) significantly decreased the occurrence and frequency of SOICR (P < 0.05). In contrast, metoprolol (2 μM) and the vehicle DMSO had no significant effect (Fig. 3e–g).
We also determined the impact of low concentrations of carvedilol on SOICR. Myocytes were incubated with 0.3 μM (or 122 ng/ml) carvedilol, which is similar to the peak plasma concentration of carvedilol (~100 ng/ml)34, 35, for 3 hours before the induction of SOICR. Myocytes treated with 0.3 μM carvedilol had a significantly reduced occurrence and frequency of SOICR compared to DMSO-treated cells (P < 0.01). The frequency and occurrence of SOICR in myocytes treated with 0.6μM metoprolol, 0.6 μM prazosin, or 6 μM α-tocopherol did not differ significantly from those in DMSO-treated cells (Fig.3h,i). Thus, carvedilol at clinically relevant concentrations suppresses SOICR in cardiomyocytes, independent of its beta- and alpha-blocking, and anti-oxidant activities.
To be able to individually and optimally titrate SOICR inhibition and beta-blockade, we set out to separate the anti-SOICR activity of carvedilol from its beta-blocking effect by generating SOICR-inhibiting carvedilol analogues with minimal beta-blockade. To this end, we have made a number of structural modifications to carvedilol (Fig.S3). Carvedilol and metoprolol markedly suppressed the enhanced heart rate in WT mice induced by isoproterenol (Iso), but one of the analogues VK-II-86 (like the vehicle DMSO) did not (Fig.4a,b). Treating the R4496C+/− mice with carvedilol or metoprolol for 5 days decreased the heart rate (P<0.01), but a 5-day VK-II-86 treatment did not (P=0.18) (Fig.4c).
Carvedilol, but not VK-II-86, abolished the Iso-induced PKA phosphorylation of RyR2 (Fig.S4). Radioligand binding studies also revealed that VK-II-86 exhibited >2,000-fold lower beta-AR binding affinity than carvedilol (Fig.S5, Supplementary Table 1). Thus, VK-II-86 lacks the normal beta-blocking activity of carvedilol and has little impact on heart rate.
We next assessed the impact of VK-II-86 on SOICR and single RyR2 channels. Like carvedilol, VK-II-86 completely abolished SOICR in HEK293 cells (P<0.01) (Fig.4d, Fig.S4). VK-II-86 (1 μM) pre-treatment of cardiomyocytes for 30 min significantly reduced the occurrence and frequency of SOICR in both confocal (Fig.4e) and epifluorescence (Fig.4f, Fig.S4) imaged cells (P<0.01). Furthermore, pre-treatment with a lower concentration of VK-II-86 (0.3μM) for a longer period of time (3 hours) also significantly inhibited the occurrence (P<0.01) and frequency (P<0.05) of SOICR (Fig.4g).
Importantly, VK-II-86 also modified the function of both single native and purified recombinant RyR2 channels. Like carvedilol, it reduced the duration and increased the frequency of channel openings (Figs.4h, Fig.S6; P<0.01). It also reduced intra-burst event duration and increased intra-burst event frequency (Fig.S1). Thus, VK-II-86 is a minimally beta-blocking carvedilol analogue with anti-SOICR activity.
We determined whether the SOICR-inhibiting carvedilol analogues with minimal beta-blockade are effective in suppressing CPVT. We used the RyR2 R4496C+/− mice that are known to develop SOICR-driven CPVT24-26. In these mice (unlike their WT littermates), long-lasting VTs, including bidirectional VTs, can be evoked pharmacologically using caffeine and epinephrine (Fig.5a–d). Importantly, VK-II-86 suppressed CPVT in RyR2-R4496C+/− mice in a dose dependent manner (Fig.5e,f, Fig.S7). At 3.4 mg kg−1 day−1, VK-II-86 reduced the VT duration by 99.9% (P<0.01) without adversely lowering the heart rate (8%, P=0.08) (Figs.4c,,5f),5f), whereas carvedilol (1.6 mg kg−1 day−1) reduced the VT duration by 79% (P<0.01) and the heart rate by 36% (P<0.05)(Figs.4c,,5f5f).
We also determined the acute effect (post treatment) of VK-II-86 on VT in RyR2 R4496C+/+ homozygous mice (Fig. 5g). R4496C+/+ mice injected with the vehicle DMSO displayed VT for >30 min. VK-II-86 (3.4 mg kg−1) completely stopped the VT. Carvedilol (1.6 mg kg−1) also completely stopped the VT and appears to be more effective than VK-II-86 (1.7 mg kg−1) probably due to its beta-blocking activity, which would reduce the strength of the original epinephrine trigger.
We used metoprolol or bisoprolol as a selective beta-blocker and VK-II-86 as a SOICR inhibitor to test the premise that higher anti-arrhythmic efficacy can be achieved by both SOICR inhibition and beta-blockade. We found that treating R4496C+/− mice with VK-II-86 plus metoprolol or with VK-II-86 plus bisoprolol was much more effective in preventing CPVT than VK-II-86 (P<0.01), metoprolol (P<0.01), bisoporlol (P<0.05) (Fig.5h, Fig.S8), or propranolol (P<0.05) (Fig.S9) alone. These data demonstrate that the combination of SOICR inhibition and beta-blockade increases the anti-arrhythmic efficacy.
We also generated two additional carvedilol analogues (CS-I-34 and CS-I-59) (Fig.6a). Like VK-II-86, CS-I-34 and CS-I-59 had no effect on the Iso-enhanced heart rate (Fig.6b) or the non-stimulated heart rate (Fig.6c). CS-I-34 and CS-I-59 reduced the occurrence and frequency of SOICR in cardiomyocytes (P<0.01) (Fig.6d) and suppressed SOICR in HEK293 cells (Fig.6e). CS-I-34 and CS-I-59 also significantly reduced the duration of VT in R4496C+/− mice (Fig.6f; P<0.01). We also found that flecainide inhibited SOICR frequency and suppressed CPVT in RyR2- R4496C+/− mice (Fig. S10), consistent with those reported previously36.
Carvedilol is the beta-blocker of choice in preventing VTs and cardiovascular mortality in patients with HF5-7. Yet the mechanism underlying carvedilol’s anti-arrhythmic benefit is not well defined. In the present study, we tested the action of a number of known beta-blockers on SOICR, and surprisingly found that only carvedilol effectively inhibited SOICR. Since SOICR and SOICR-induced DADs are frequent in failing hearts12, 13, 30, 31, carvedilol’s unique anti-SOICR activity among beta-blockers likely contributes to its favorable anti-arrhythmic benefit.
An important question is how does carvedilol suppress arrhythmogenic SOICR. Using single channel recordings in lipid bilayers, we demonstrate that carvedilol directly modifies the gating properties of RyR2 by reducing the duration and increasing the frequency of channel openings. Interestingly, reducing the duration of RyR2 channel openings has also been shown to be the underlying mechanism by which flecainide, a Na channel blocker, suppresses SOICR and prevents SOICR-evoked CPVT in both mice and humans36-38. Thus, it appears that reducing RyR2 open duration is not only common to both drugs (carvedilol and flecainide) but also an effective means to suppress SOICR and SOICR-evoked VTs. However, flecainide is contraindicated in patients with previous myocardial infarction or significant left ventricular dysfunction because of the risk of VTs39. Carvedilol has also been reported to suppress SR ‘Ca2+ leak’ by reducing the levels of RyR2 phosphorylation or oxidation via its beta-blocking and anti-oxidant activities 40, 41. These modulation pathways of SR Ca2+ leak, however, are not carvedilol specific. In contrast, we show here that carvedilol’s unique anti-SOICR activity arises from a direct action on the RyR2 channel and is independent of the drug’s beta-blocking and anti-oxidant activities. Thus, it is mechanistically different from those described by Reiken et al. and Mochizuki et al.40, 41.
The peak plasma concentration of carvedilol at therapeutic doses is ~100 ng/mL with more than 98% of this bound to plasma proteins34, 35. Despite its high plasma protein association, carvedilol has a large volume of distribution (132L)34, indicating that it accumulates (over time) in extravascular tissues like cardiac muscle42. This is likely due to its high degree of lipophilicity8, 43. In line with this view, the concentrations of carvedilol found in the heart are 5-7 fold higher than that in the plasma44, 45. We also found that lower carvedilol concentrations were needed to suppress SOICR at longer drug exposure times. Thus, carvedilol at therapeutic doses may accumulate in cardiac cells to a level that is able to modify the function of RyR2.
Since carvedilol possesses both the beta-blocking and anti-SOICR activities, it would suppress both the stress-induced SR Ca2+ overload that triggers SOICR and the SOICR itself. This dual action of carvedilol likely contributes to its favorable anti-arrhythmic benefits. However, the concentrations of carvedilol required to suppress SOICR (0.3-1 μM) are much higher than those for beta-blockade (~1 nM)46. Therefore, strong SOICR inhibition would require high doses of carvedilol, which could produce excessive beta-blockade and the accompanying adverse effects such as bradycardia47. In other words, carvedilol’s anti-SOICR benefit is dose limited by the drugs beta-blocking activity. We were successful at pharmacologically separate carvedilol’s beta-blocking and anti-SOICR activities, so that the benefits of beta-blocking and SOICR inhibition could be individually and optimally titrated. We synthesized a number of carvedilol analogues with structural changes in regions thought to be essential for beta-blockade. Three carvedilol analogues, VK-II-86, CS-I-34, and CS-I-59 exhibited minimal beta-blocking activity, but retained the ability to suppress SOICR. These novel agents prevented CPVT in mice without causing bradycardia. Further, we showed that VK-II-86 when combined with selective beta-blockers (metoprolol or bisoprolol) is more effective than VK-II-86 or metoprolol or bisoprolol alone. Thus, the combination of agents (our new SOICR inhibitor and a selective beta-blocker) represents a novel and more effective approach for preventing SOICR-evoked VTs.
Carvedilol is a pleiotropic agent with diverse actions5, 6, 48-51. Thus, it is important to note that our novel carvedilol analogues, even though they lack beta-blocking activity, may have other actions besides the anti-SOICR activity demonstrated here. Given the significant role of SOICR in arrhythmogenesis, however, it is very likely that their anti-SOICR activity will play a key role in their anti-arrhythmic action.
Stable, inducible HEK293 cells expressing a CPVT-causing RyR2 mutant, R4496C, display robust SOICR, but parental HEK293 cells do not15, 16. These RyR2-R4496C cells were used to assess the impact of beta-blockers and other reagents on SCR using single-cell Ca2+ imaging as described previously15, 16.
Detailed experimental procedures for the generation of the RyR2-R4496C mutant mice are available in the Supplementary Methods.
Ca2+ waves in freshly isolated ventricular myocytes were measured using single-cell Ca2+ imaging as described previously52.
Laser scanning confocal imaging of Ca2+ dynamics was performed as described previously53.
Detailed experimental procedures and characterization data of novel compounds are described in the Supplementary Methods.
All animal studies were approved by the Institutional Animal Care and Use Committees at the University of Calgary, and performed in accordance with NIH guidelines. Adult R4496C mutant mice and wild type control littermates (2-4 months old) were used for all experiments.
The effects of drugs on ventricular tachycardias provoked by epinephrine and caffeine were studied in anesthetized R4496C mutant mice. Briefly, mice were lightly anesthetized with isoflurane vapor (0.5-1%). Anesthetized mice were placed on a heating pad (28 °C) and subcutaneous needle electrodes were applied to the left, right upper limb and right lower limb for ECG recording (BIOPAC MP System, Goleta, CA). Baseline ECG was recorded for 5 minutes, followed by the intraperitoneal injection of epinephrine (1.6 mg/kg) and caffeine (120 mg/kg). ECG was then continuously recorded for another 30 minutes. ECG recordings were analyzed using the AcqKnowledge 3.9.1 program (BIOPAC, Goleta, CA).
Epinephrine (1.6 mg/kg)/caffeine (120 mg/kg) challenge was performed twice in the same R4496C+/− mutant mouse at an interval of 7 days. All animals went through first ECG test before any drug treatments to obtain the baseline. These animals were then randomized into different groups and treated with various drugs by intraperitoneal injection for 5 days. The final dose of drug was administered 30 minutes before the second ECG test. The percentage of time that the mice were in ventricular tachycardia within the 30 min recording period in both the first and second ECG tests was determined and compared. To evaluate the acute effect of different drugs on CPVT, the R4496C+/+ mutant mice, continuously monitored with ECG recordings, were intraperitoneally injected with epinephrine (1.6 mg/kg) and caffeine (120 mg/kg) to induce ventricular tachycardia. One dose of each drug tested was then intraperitoneally injected into the mice 12 minutes after the injection of epinephrine and caffeine. ECG was continuously recorded for additional 21 minutes.
All values shown are mean ± SEM unless indicated otherwise. To test for differences between groups, we used Student’s t test (2-tailed) or one-way ANOVA with post hoc test. Statistical analyses were performed using the SPSS V.15.0 (SPSS, Chicago, IL). A P value <0.05 was considered to be statistically significant.
Supported by grants from the Canadian Institutes of Health Research to S.R.W.C., US National Institutes of Health to S.R.W.C., L-S.S., M.F., and A.M.F., the Heart and Stroke Foundation of Alberta to A.M.G. and H.J.D., the Intramural Research Program of the National Institute on Aging to D.Y. and H.C.. D.J., W.C., X.T. had Alberta Innovates-Health Solutions (AIHS) Studentship Awards, C.D.S.: AIHS Fellowship Awards, P.P.J.: AIHS and Heart and Stroke Foundation Fellowship Awards, H.J.D. and S.R.W.C.: AIHS Scientist Awards. We want to thank the King family and the Libin Cardiovascular Institute of Alberta for their generous donations and Drs. S. Pogwizd and S. Priori for critical reading of the manuscript.
Extended, detailed experimental procedures are available in the Supplementary Information.
AUTHOR CONTRIBUTIONS Q.Z., J.X., D.J., R.W., K.V., A.W., C.S., W.Z., D.Y., J.C., A.M.G., H.J.D., H.C., A.M.F., L-S.S., M.F., T.G.B., S.R.W.C., designed research; Q.Z., J.X., D.J., R.W., K.V., A.W., C.S., C.X., W.C., J.Z., W.Z., X.T., P.J., X.Z., A.G., H.C., L.Z., D.Y., X.L., performed research; Q.Z., J.X., D.J., C.X., W.C., J.Z., W.Z., X.T., P.J., X.Z., A.G., H.Y., L.Z., D.Y., analyzed data; and Q.Z., R.W., C.S., W.Z., M.F., T.G.B., S.R.W.C., wrote the paper.
The authors declare no competing financial interests.