Coupled gating modifies the regulation of cardiac ryanodine receptors by luminal Ca2+

AbstractCardiac ryanodine receptors (RYR2s) infrequently exhibit coupled gating that is manifested by synchronous opening and closing. To better characterize this phenomenon, we investigated the regulation of coupled RYR2 channels by luminal Ca2+ focusing on effects that are likely mediated by the true luminal activation mechanism. By reconstituting an ion channel into a planar lipid bilayer and using substantially lower concentration of luminal Ba2+ (8mM, the virtual absence of Ca2+) and luminal Ca2+ (8mM), we show that response of coupled RYR2 channels to caffeine at a diastolic cytosolic Ca2+ (90nM) was affected by luminal Ca2+ in a similar manner as for the single RYR2 channel except the gating behavior. Whereas, the single RYR2 channel responded to luminal Ca2+ by prolongation in open and closed times, coupled RYR2 channels seemed to be resistant in this respect. In summary, we conclude that the class of Ca2+ sites located on the luminal face of coupled RYR2 channels that is responsible for the channel potentiation by luminal Ca2+ is functional and not structurally hindered by the channel coupling. Thus, the idea about non-functional luminal Ca2+ sites as a source of the apparent gating resistance of coupled RYR2 channels to luminal Ca2+ appears to be ruled out

Omecamtiv Mecarbil: A Myosin Motor Activator Agent with Promis- ing Clinical Performance and New in vitro Results

Background: Clinical treatment of heart failure is still suffering from limited efficacy and unfavorable side effects. The recently developed group of agents, the myosin motor activa- tors, act directly on cardiac myosin resulting in an increased force generation and prolongation of contraction. The lead molecule, omecamtiv mecarbil is now in human 3 stage. In addition to the promising clinical data published so far, there are new in vitro results indicating that the ef- fect of omecamtiv mecarbil on contractility is rate-dependent. Furthermore, omecamtiv mecarbil was shown to activate cardiac ryanodine receptors, an effect that may carry proarrhythmic risk. Methods: These new results, together with the controversial effects of the drug on cardiac oxy- gen consumption, are critically discussed in this review in light of the current literature on ome- camtiv mecarbil. Results: In therapeutically relevant concentrations the beneficial inotropic effect of the agent is not likely affected by these new results - in accordance with the good clinical data. At su- pratherapeutic concentrations, however, activation of cardiac ryanodine receptors may increase arrhythmia propensity, and the stronger effect on diastolic than systolic cell shortening, ob- served at higher pacing frequencies, may decrease or offset the inotropic effect of omecamtiv mecarbil. Conclusion: Further studies with definitely supratherapeutical concentrations of omecamtiv mecarbil should be designed to map the actual risk of these potentially harmful side-effects

PKA phosphorylation activates the calcium release channel (ryanodine receptor) in skeletal muscle: defective regulation in heart failure

The type 1 ryanodine receptor (RyR1) on the sarcoplasmic reticulum (SR) is the major calcium (Ca2+) release channel required for skeletal muscle excitation–contraction (EC) coupling. RyR1 function is modulated by proteins that bind to its large cytoplasmic scaffold domain, including the FK506 binding protein (FKBP12) and PKA. PKA is activated during sympathetic nervous system (SNS) stimulation. We show that PKA phosphorylation of RyR1 at Ser2843 activates the channel by releasing FKBP12. When FKB12 is bound to RyR1, it inhibits the channel by stabilizing its closed state. RyR1 in skeletal muscle from animals with heart failure (HF), a chronic hyperadrenergic state, were PKA hyperphosphorylated, depleted of FKBP12, and exhibited increased activity, suggesting that the channels are “leaky.” RyR1 PKA hyperphosphorylation correlated with impaired SR Ca2+ release and early fatigue in HF skeletal muscle. These findings identify a novel mechanism that regulates RyR1 function via PKA phosphorylation in response to SNS stimulation. PKA hyperphosphorylation of RyR1 may contribute to impaired skeletal muscle function in HF, suggesting that a generalized EC coupling myopathy may play a role in HF

The Cardiac Ryanodine Receptor Provides a Suitable Pathway for the Rapid Transport of Zinc (Zn²⁺)

The sarcoplasmic reticulum (SR) in cardiac muscle is suggested to act as a dynamic storage for Zn2+ release and reuptake, albeit it is primarily implicated in the Ca2+ signaling required for the cardiac cycle. A large Ca2+ release from the SR is mediated by the cardiac ryanodine receptor (RYR2), and while this has a prominent conductance for Ca2+ in vivo, it also conducts other divalent cations in vitro. Since Zn2+ and permeant Mg2+ have similar physical properties, we tested if the RYR2 channel also conducts Zn2+. Using the method of planar lipid membranes, we evidenced that the RYR2 channel is permeable to Zn2+ with a considerable conductance of 81.1 ± 2.4 pS, which was significantly lower than the values for Ca2+ (127.5 ± 1.8 pS) and Mg2+ (95.3 ± 1.4 pS), obtained under the same asymmetric conditions. Despite similar physical properties, the intrinsic Zn2+ permeability (PCa/PZn = 2.65 ± 0.19) was found to be ~2.3-fold lower than that of Mg2+ (PCa/PMg = 1.146 ± 0.071). Further, we assessed whether the channel itself could be a direct target of the Zn2+ current, having the Zn2+ finger extended into the cytosolic vestibular portion of the permeation pathway. We attempted to displace Zn2+ from the RYR2 Zn2+ finger to induce its structural defects, which are associated with RYR2 dysfunction. Zn2+ chelators were added to the channel cytosolic side or strongly competing cadmium cations (Cd2+) were allowed to permeate the RYR2 channel. Only the Cd2+ current was able to cause the decay of channel activity, presumably as a result of Zn2+ to Cd2+ replacement. Our findings suggest that the RYR2 channel can provide a suitable pathway for rapid Zn2+ escape from the cardiac SR; thus, the channel may play a role in local and/or global Zn2+ signaling in cardiomyocytes

Luminal addition of non-permeant Eu3+ interferes with luminal Ca2+ regulation of the cardiac ryanodine receptor

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Omecamtiv mecarbil activates ryanodine receptors from canine cardiac but not skeletal muscle

Due to the limited results achieved in the clinical treatment of heart failure, a new inotropic strategy of myosin motor activation has been developed. The lead molecule of myosin activator agents is omecamtiv mecarbil, which binds directly to the heavy chain of the cardiac β-myosin and enhances cardiac contractility by lengthening the lifetime of the acto-myosin complex and increasing the number of the active force-generating cross-bridges. In the absence of relevant data, the effect of omecamtiv mecarbil on canine cardiac ryanodine receptors (RyR 2) has been investigated in the present study by measuring the electrical activity of single RyR 2 channels incorporated into planar lipid bilayer. When applying 100 nM Ca2+ concentration on the cis side ([Ca2+]cis) omecamtiv mecarbil (1–10 µM) significantly increased the open probability and opening frequency of RyR 2, while the mean closed time was reduced. Mean open time was increased moderately by 10 µM omecamtiv mecarbil. When [Ca2+]cis was elevated to 322 and 735 nM, the effect of omecamtiv mecarbil on open probability was evident only at higher (3–10 µM) concentrations. All effects of omecamtiv mecarbil were fully reversible upon washout. Omecamtiv mecarbil (up to 10 µM) had no effect on the open probability of RyR 1, isolated from either canine or rabbit skeletal muscles. It is concluded that the direct stimulatory action of omecamtiv mecarbil on RyR 2 has to be taken into account when discussing the mechanism of action or the potential side effects of the compound

Omecamtiv mecarbil activates ryanodine receptors from canine cardiac but not skeletal muscle

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