Peter P. Jones

Carvedilol and its new analogs suppress arrhythmogenic store overload-induced Ca2+ release

Carvedilol is one of the most effective beta blockers for preventing ventricular tachyarrhythmias in heart failure, but the mechanisms underlying its favorable antiarrhythmic benefits remain unclear. Spontaneous Ca2+ waves, also called store overload–induced Ca2+ release (SOICR), evoke ventricular tachyarrhythmias in individuals with heart failure. Here we show that carvedilol is the only beta blocker tested 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, probably contributes to its favorable antiarrhythmic effect. To enable optimal titration of carvedilols actions as a beta blocker and as a suppressor of SOICR separately, we developed a new SOICR-inhibiting, minimally beta-blocking carvedilol analog, VK-II-86. VK-II-86 prevented stress-induced ventricular tachyarrhythmias in RyR2-mutant mice and did so more effectively when combined with either of the selective beta blockers metoprolol or bisoprolol. Combining SOICR inhibition with optimal beta blockade has the potential to provide antiarrhythmic therapy that can be tailored to individual patients.

The ryanodine receptor store-sensing gate controls Ca2+ waves and Ca2+-triggered arrhythmias

Spontaneous Ca2+ release from intracellular stores is important for various physiological and pathological processes. In cardiac muscle cells, spontaneous store overload–induced Ca2+ release (SOICR) can result in Ca2+ waves, a major cause of ventricular tachyarrhythmias (VTs) and sudden death. The molecular mechanism underlying SOICR has been a mystery for decades. Here we show that a point mutation, E4872A, in the helix bundle crossing region (the proposed gate) of the cardiac ryanodine receptor (RyR2) completely abolishes luminal, but not cytosolic, Ca2+ activation of RyR2. The introduction of metal-binding histidines at this site converts RyR2 into a luminal Ni2+-gated channel. Mouse hearts harboring a heterozygous RyR2 mutation at this site (E4872Q) are resistant to SOICR and are completely protected against Ca2+-triggered VTs. These data show that the RyR2 gate directly senses luminal (store) Ca2+, explaining the regulation of RyR2 by luminal Ca2+, the initiation of Ca2+ waves and Ca2+-triggered arrhythmias. This newly identified store-sensing gate structure is conserved in all RyR and inositol 1,4,5-trisphosphate receptor isoforms.

Removal of FKBP12.6 Does Not Alter the Conductance and Activation of the Cardiac Ryanodine Receptor or the Susceptibility to Stress-induced Ventricular Arrhythmias

The 12.6-kDa FK506-binding protein (FKBP12.6) is considered to be a key regulator of the cardiac ryanodine receptor (RyR2), but its precise role in RyR2 function is complex and controversial. In the present study we investigated the impact of FKBP12.6 removal on the properties of the RyR2 channel and the propensity for spontaneous Ca2+ release and the occurrence of ventricular arrhythmias. Single channel recordings in lipid bilayers showed that FK506 treatment of recombinant RyR2 co-expressed with or without FKBP12.6 or native canine RyR2 did not induce long-lived subconductance states. [3H]Ryanodine binding studies revealed that coexpression with or without FKBP12.6 or treatment with or without FK506 did not alter the sensitivity of RyR2 to activation by Ca2+ or caffeine. Furthermore, single cell Ca2+ imaging analyses demonstrated that HEK293 cells co-expressing RyR2 and FKBP12.6 or expressing RyR2 alone displayed the same propensity for spontaneous Ca2+ release or store overload-induced Ca2+ release (SOICR). FK506 increased the amplitude and decreased the frequency of SOICR in HEK293 cells expressing RyR2 with or without FKBP12.6, indicating that the action of FK506 on SOICR is independent of FKBP12.6. As with recombinant RyR2, the conductance and ligand-gating properties of single RyR2 channels from FKBP12.6-null mice were indistinguishable from those of single wild type channels. Moreover, FKBP12.6-null mice did not exhibit enhanced susceptibility to stress-induced ventricular arrhythmias, in contrast to previous reports. Collectively, our results demonstrate that the loss of FKBP12.6 has no significant effect on the conduction and activation of RyR2 or the propensity for spontaneous Ca2+ release and stress-induced ventricular arrhythmias.

K201 (JTV519) suppresses spontaneous Ca2+ release and [3H]ryanodine binding to RyR2 irrespective of FKBP12.6 association

K201 (JTV519), a benzothiazepine derivative, has been shown to possess anti-arrhythmic and cardioprotective properties, but the mechanism of its action is both complex and controversial. It is believed to stabilize the closed state of the RyR2 (cardiac ryanodine receptor) by increasing its affinity for the FKBP12.6 (12.6 kDa FK506 binding protein) [Wehrens, Lehnart, Reiken, Deng, Vest, Cervantes, Coromilas, Landry and Marks (2004) Science 304, 292-296]. In the present study, we investigated the effect of K201 on spontaneous Ca2+ release induced by Ca2+ overload in rat ventricular myocytes and in HEK-293 cells (human embryonic kidney cells) expressing RyR2 and the role of FKBP12.6 in the action of K201. We found that K201 abolished spontaneous Ca2+ release in cardiac myocytes in a concentration-dependent manner. Treating ventricular myocytes with FK506 to dissociate FKBP12.6 from RyR2 did not affect the suppression of spontaneous Ca2+ release by K201. Similarly, K201 was able to suppress spontaneous Ca2+ release in FK506-treated HEK-293 cells co-expressing RyR2 and FKBP12.6. Furthermore, K201 suppressed spontaneous Ca2+ release in HEK-293 cells expressing RyR2 alone and in cells co-expressing RyR2 and FKBP12.6 with the same potency. In addition, K201 inhibited [3H]ryanodine binding to RyR2-wt (wild-type) and an RyR2 mutant linked to ventricular tachycardia and sudden death, N4104K, in the absence of FKBP12.6. These observations demonstrate that FKBP12.6 is not involved in the inhibitory action of K201 on spontaneous Ca2+ release. Our results also suggest that suppression of spontaneous Ca2+ release and the activity of RyR2 contributes, at least in part, to the anti-arrhythmic properties of K201.

Endoplasmic reticulum Ca2+ measurements reveal that the cardiac ryanodine receptor mutations linked to cardiac arrhythmia and sudden death alter the threshold for store-overload-induced Ca2+ release

A number of RyR2 (cardiac ryanodine receptor) mutations linked to ventricular arrhythmia and sudden death are located within the last C-terminal approximately 500 amino acid residues, which is believed to constitute the ion-conducting pore and gating domain of the channel. We have previously shown that mutations located near the C-terminal end of the predicted TM (transmembrane) segment 10, the inner pore helix, can either increase or decrease the propensity for SOICR (store-overload-induced Ca2+ release), also known as spontaneous Ca2+ release. In the present study, we have characterized an RyR2 mutation, V4653F, located in the loop between the predicted TM 6 and TM 7a, using an ER (endoplasmic reticulum)-targeted Ca2+-indicator protein (D1ER). We directly demonstrated that SOICR occurs at a reduced luminal Ca2+ threshold in HEK-293 cells (human embryonic kidney cells) expressing the V4653F mutant as compared with cells expressing the RyR2 wild-type. Single-channel analyses revealed that the V4653F mutation increased the sensitivity of RyR2 to activation by luminal Ca2+. In contrast with previous reports, the V4653 mutation did not alter FKBP12.6 (FK506-binding protein 12.6 kDa; F506 is an immunosuppressant macrolide)-RyR2 interaction. Luminal Ca2+ measurements also showed that the mutations R176Q/T2504M, S2246L and Q4201R, located in different regions of the channel, reduced the threshold for SOICR, whereas the A4860G mutation, located within the inner pore helix, increased the SOICR threshold. We conclude that the cytosolic loop between TM 6 and TM 7a plays an important role in determining the SOICR threshold and that the alteration of the threshold for SOICR is a common mechanism for RyR2-associated ventricular arrhythmia.

Phosphodiesterase 4D Regulates Baseline Sarcoplasmic Reticulum Ca2+ Release and Cardiac Contractility, Independently of L-Type Ca2+ Current

Rationale: Baseline contractility of mouse hearts is modulated in a phosphatidylinositol 3-kinase-&ggr;–dependent manner by type 4 phosphodiesterases (PDE4), which regulate cAMP levels within microdomains containing the sarcoplasmic reticulum (SR) calcium ATPase type 2a (SERCA2a). Objective: The goal of this study was to determine whether PDE4D regulates basal cardiac contractility. Methods and Results: At 10 to 12 weeks of age, baseline cardiac contractility in PDE4D-deficient (PDE4D−/−) mice was elevated mice in vivo and in Langendorff perfused hearts, whereas isolated PDE4D−/− cardiomyocytes showed increased whole-cell Ca2+ transient amplitudes and SR Ca2+content but unchanged L-type calcium current, compared with littermate controls (WT). The protein kinase A inhibitor Rp-adenosine-3′,5′ cyclic monophosphorothioate (Rp-cAMP) lowered whole-cell Ca2+ transient amplitudes and SR Ca2+ content in PDE4D−/− cardiomyocytes to WT levels. The PDE4 inhibitor rolipram had no effect on cardiac contractility, whole-cell Ca2+ transients, or SR Ca2+ content in PDE4D−/− preparations but increased these parameters in WT myocardium to levels indistinguishable from those in PDE4D−/−. The functional changes in PDE4D−/− myocardium were associated with increased PLN phosphorylation but not cardiac ryanodine receptor phosphorylation. Rolipram increased PLN phosphorylation in WT cardiomyocytes to levels indistinguishable from those in PDE4D−/− cardiomyocytes. In murine and failing human hearts, PDE4D coimmunoprecipitated with SERCA2a but not with cardiac ryanodine receptor. Conclusions: PDE4D regulates basal cAMP levels in SR microdomains containing SERCA2a-PLN, but not L-type Ca2+ channels or ryanodine receptor. Because whole-cell Ca2+ transient amplitudes are reduced in failing human myocardium, these observations may have therapeutic implications for patients with heart failure.

Intravascular pressure augments cerebral arterial constriction by inducing voltage-insensitive Ca2+ waves

This study examined whether elevated intravascular pressure stimulates asynchronous Ca2+ waves in cerebral arterial smooth muscle cells and if their generation contributes to myogenic tone development. The endothelium was removed from rat cerebral arteries, which were then mounted in an arteriograph, pressurized (20–100 mmHg) and examined under a variety of experimental conditions. Diameter and membrane potential (VM) were monitored using conventional techniques; Ca2+ wave generation and myosin light chain (MLC20)/MYPT1 (myosin phosphatase targeting subunit) phosphorylation were assessed by confocal microscopy and Western blot analysis, respectively. Elevating intravascular pressure increased the proportion of smooth muscle cells firing asynchronous Ca2+ waves as well as event frequency. Ca2+ wave augmentation occurred primarily at lower intravascular pressures (<60 mmHg) and ryanodine, a plant alkaloid that depletes the sarcoplasmic reticulum (SR) of Ca2+, eliminated these events. Ca2+ wave generation was voltage insensitive as Ca2+ channel blockade and perturbations in extracellular [K+] had little effect on measured parameters. Ryanodine‐induced inhibition of Ca2+ waves attenuated myogenic tone and MLC20 phosphorylation without altering arterial VM. Thapsigargin, an SR Ca2+‐ATPase inhibitor also attenuated Ca2+ waves, pressure‐induced constriction and MLC20 phosphorylation. The SR‐driven component of the myogenic response was proportionally greater at lower intravascular pressures and subsequent MYPT1 phosphorylation measures revealed that SR Ca2+ waves facilitated pressure‐induced MLC20 phosphorylation through mechanisms that include myosin light chain phosphatase inhibition. Cumulatively, our findings show that mechanical stimuli augment Ca2+ wave generation in arterial smooth muscle and that these transient events facilitate tone development particularly at lower intravascular pressures by providing a proportion of the Ca2+ required to directly control MLC20 phosphorylation.

Reduced Threshold for Luminal Ca2+ Activation of RyR1 Underlies a Causal Mechanism of Porcine Malignant Hyperthermia

Naturally occurring mutations in the skeletal muscle Ca2+ release channel/ryanodine receptor RyR1 are linked to malignant hyperthermia (MH), a life-threatening complication of general anesthesia. Although it has long been recognized that MH results from uncontrolled or spontaneous Ca2+ release from the sarcoplasmic reticulum, how MH RyR1 mutations render the sarcoplasmic reticulum susceptible to volatile anesthetic-induced spontaneous Ca2+ release is unclear. Here we investigated the impact of the porcine MH mutation, R615C, the human equivalent of which also causes MH, on the intrinsic properties of the RyR1 channel and the propensity for spontaneous Ca2+ release during store Ca2+ overload, a process we refer to as store overload-induced Ca2+ release (SOICR). Single channel analyses revealed that the R615C mutation markedly enhanced the luminal Ca2+ activation of RyR1. Moreover, HEK293 cells expressing the R615C mutant displayed a reduced threshold for SOICR compared with cells expressing wild type RyR1. Furthermore, the MH-triggering agent, halothane, potentiated the response of RyR1 to luminal Ca2+ and SOICR. Conversely, dantrolene, an effective treatment for MH, suppressed SOICR in HEK293 cells expressing the R615C mutant, but not in cells expressing an RyR2 mutant. These data suggest that the R615C mutation confers MH susceptibility by reducing the threshold for luminal Ca2+ activation and SOICR, whereas volatile anesthetics trigger MH by further reducing the threshold, and dantrolene suppresses MH by increasing the SOICR threshold. Together, our data support a view in which altered luminal Ca2+ regulation of RyR1 represents a primary causal mechanism of MH.