Correction of impaired calmodulin binding to RyR2 as a novel therapy for lethal arrhythmia in the pressure-overloaded heart failure.

BACKGROUND: Calmodulin (CaM) is a key modulator of the channel gating function of the ryanodine receptor (RyR). OBJECTIVE: The purpose of this study was to investigate the pathogenic role of RyR-bound CaM in diastolic Ca2+ leakage from the sarcoplasmic reticulum and arrhythmogenesis in pressure-overloaded heart failure. METHODS: Pressure overload was induced in 12-week-old mice by transverse aortic constriction (TAC) using a 27-gauge needle. RESULTS: TAC operation for 8 weeks produced a significant increase in left ventricular end-diastolic diameter and frequent occurrence of lethal arrhythmias after infusion of epinephrine and caffeine in TAC mice. The amount of RyR-bound CaM decreased significantly in TAC mice compared with sham mice. The apparent affinity of CaM binding to RyR decreased in pressure-overloaded cells compared with sham cells and untreated cells. High-affinity calmodulin (HA-CaM; ie, CaM whose binding affinity to RyR was significantly increased) restored a normal level of CaM-RyR binding properties in pressure-overloaded cells. HA-CaM corrected abnormally increased Ca2+ spark frequency in the pressure-overloaded cells to the level seen in the sham cells. The frequency of spontaneous Ca2+ transients in TAC cells during and after 1-5 Hz of field stimulation was 44%, whereas it was significantly attenuated by HA-CaM but not with CaM. CONCLUSION: Several disorders in the RyR channel function characteristic of pressure-overloaded cells (increased spontaneous Ca2+ leakage, delayed afterdepolarization, triggered activity, Ca2+ spark frequency, spontaneous Ca2+ transients) are caused by deteriorated CaM binding to RyR2. These disorders could be rectified by restoring normal CaM binding to RyR2.

Enhanced binding of calmodulin to RyR2 corrects arrhythmogenic channel disorder in CPVT-associated myocytes.

AIMS: Calmodulin (CaM) plays a key role in modulating channel gating in ryanodine receptor (RyR2). Here, we investigated (a) the pathogenic role of CaM in the channel disorder in CPVT and (b) the possibility of correcting the CPVT-linked channel disorder, using knock-in (KI) mouse model with CPVT-associated RyR2 mutation (R2474S). METHODS AND RESULTS: Transmembrane potentials were recorded in whole cell current mode before and after pacing (1-5 Hz) in isolated ventricular myocytes. CaM binding was assessed by incorporation of exogenous CaM fluorescently labeled with HiLyte Fluor(®) in saponin-permeabilized myocytes. In the presence of cAMP (1 µM) the apparent affinity of CaM binding to the RyR decreased in KI cells (Kd: 140-400 nM), but not in WT cells (Kd: 110-120 nM). Gly-Ser-His-CaM (GSH-CaM that has much higher RyR-binding than CaM) restored normal binding to the RyR of cAMP-treated KI cells (140 nM). Neither delayed afterdepolarization (DAD) nor triggered activity (TA) were observed in WT cells even at 5Hz pacing, whereas both DAD and TA were observed in 20% and 12% of KI cells, respectively. In response to 10nM isoproterenol, only DAD (but not TA) was observed in 11% of WT cells, whereas in KI cells the incidence of DAD and TA further increased to 60% and 38% of cells, respectively. Addition of GSH-CaM (100 nM) to KI cells decreased both DADs and TA (DAD: 38% of cells; TA: 10% of cells), whereas CaM (100 nM) had no appreciable effect. Addition of GSH-CaM to saponin-permeabilized KI cells decreased Ca(2+) spark frequency (+33% of WT cells), which otherwise markedly increased without GSH-CaM (+100% of WT cells), whereas CaM revealed much less effect on the Ca(2+) spark frequency (+76% of WT cells). Then, by incorporating CaM or GSH-CaM to intact cells (with protein delivery kit), we assessed the in situ effect of GSH-CaM (cytosolic [CaM]=~240 nM, cytosolic [GSH-CaM]=~230 nM) on the frequency of spontaneous Ca(2+) transient (sCaT, % of total cells). Addition of 10nM isoproterenol to KI cells increased sCaT after transient 5 Hz pacing (37%), whereas it was much more attenuated by GSH-CaM (9%) than by CaM (26%) (P<0.01 vs CaM). CONCLUSIONS: Several disorders in the RyR channel function characteristic of the CPVT-mutant cells (increased spontaneous Ca(2+) leak, delayed afterdepolarization, triggered activity, Ca(2+) spark frequency, spontaneous Ca(2+) transients) can be corrected to a normal function by increasing the affinity of CaM binding to the RyR.

Identification of the active region responsible for the anti-thrombotic activity of anopheline anti-platelet protein from a malaria vector mosquito.

We previously identified an anti-platelet protein, anopheline anti-platelet protein (AAPP), from the salivary gland of female Anopheles stephensi (a mosquito vector of human malaria). AAPP specifically blocks platelet adhesion to collagen by binding directly to collagen and subsequently causing platelet aggregation. The aim of this study was to identify the active region of AAPP responsible for the anti-thrombotic activity because we hypothesized that AAPP could be used as a candidate anti-platelet drug. Various truncated forms of AAPP were produced using an Escherichia coli expression system. Each protein was examined for binding activities to soluble/fibrillar collagen and anti-thrombotic activity using a plate assay and platelet/whole blood aggregation study, respectively. Among the truncated forms examined, only a protein encoded by exon 3-4 (rAAPPex3-4) effectively bound to soluble/fibrillar collagen in a concentration-dependent and saturable manner. The EC50 values of full-length AAPP and rAAPPex3-4 for soluble collagen binding were 35 nM and 36 nM, respectively. In contrast to soluble collagen, there was a difference in binding affinity to fibrillar collagen between full-length AAPP and rAAPPex3-4, with EC50 values of 31 nM and 51 nM, respectively. rAAPPex3-4 also inhibited aggregation of platelets/whole blood, and the IC50 values of full-length AAPP and rAAPPex3-4 for platelet aggregation were 35 nM and 93 nM, respectively. These results indicated that the essential moiety of AAPP for collagen binding and anti-thrombotic activity was in the region encoded by exon 3-4, which is highly conserved among the counterpart regions of other mosquito species.

Enhanced binding of calmodulin to the ryanodine receptor corrects contractile dysfunction in failing hearts.

AIMS: The channel function of the cardiac ryanodine receptor (RyR2) is modulated by calmodulin (CaM). However, the involvement of CaM in aberrant Ca(2+) release in diseased hearts remains unclear. Here, we investigated the pathogenic role of defective CaM binding to the RyR2 in the channel dysfunction associated with heart failure. METHODS AND RESULTS: The involvement of CaM in aberrant Ca(2+) release was assessed in normal and pacing-induced failing canine hearts. The apparent affinity of CaM for RyR2 was considerably lower in failing sarcoplasmic reticulum (SR) compared with normal SR. Thus, the amount of CaM bound to RyR2 was markedly decreased in failing myocytes. Expression of the CaM isoform Gly-Ser-His-CaM (GSH-CaM), which has much higher binding affinity than wild-type CaM for RyR1, restored normal CaM binding to RyR2 in both SR and myocytes of failing hearts. The Ca(2+) spark frequency (SpF) was markedly higher and the SR Ca(2+) content was lower in failing myocytes compared with normal myocytes. The incorporation of GSH-CaM into the failing myocytes corrected the aberrant SpF and SR Ca(2+) content to normal levels. CONCLUSION: Reduced CaM binding to RyR2 seems to play a critical role in the pathogenesis of aberrant Ca(2+) release in failing hearts. Correction of the reduced CaM binding to RyR2 stabilizes the RyR2 channel function and thereby restores normal Ca(2+) handling and contractile function to failing hearts.

Anopheline anti-platelet protein from a malaria vector mosquito has anti-thrombotic effects in vivo without compromising hemostasis.

INTRODUCTION: The saliva of blood-feeding animals (e.g., mosquitoes, ticks, bats) has pharmacological activities that facilitate efficient blood-sucking. We previously identified a unique anti-platelet protein, anopheline anti-platelet protein (AAPP), from the salivary gland of female Anopheles stephensi (human malaria vector mosquito). AAPP specifically blocks platelet adhesion to collagen by binding directly to collagen and subsequently aggregating platelets. To examine the potential of AAPP as a therapeutic agent, we investigated the in vivo anti-thrombotic effects of AAPP. MATERIALS AND METHODS: Effects of AAPP on whole blood/platelet aggregation in mice were examined. AAPP was also challenged in an established model of pulmonary thromboembolism in mice. We simultaneously investigated the side-effects of the protein (prolongation of bleeding time and coagulation time). Aspirin was used as a positive control for comparison of anti-thrombotic effects. RESULTS AND CONCLUSIONS: AAPP inhibited whole blood aggregation induced by collagen at 10mg/kg body weight. AAPP prevented pulmonary death at a lower dose (3mg/kg) without prolongation of bleeding time compared with aspirin (100mg/kg) that compromised hemostasis. AAPP and aspirin did not affect coagulation time. These results indicate that AAPP has great potential as a new anti-platelet agent with a better risk/benefit ratio than that seen with aspirin (the most widely used anti-platelet agent).

Dissociation of calmodulin from cardiac ryanodine receptor causes aberrant Ca(2+) release in heart failure.

AIMS: Calmodulin (CaM) is well known to modulate the channel function of the cardiac ryanodine receptor (RyR2). However, the possible role of CaM on the aberrant Ca(2+) release in diseased hearts remains unclear. In this study, we investigated the state of RyR2-bound CaM and channel dysfunctions in pacing-induced failing hearts. METHODS AND RESULTS: The characteristics of CaM binding to RyR2 and the role of CaM on the aberrant Ca(2+) release were assessed in normal and failing canine hearts. The affinity of CaM binding to RyR2 was lower in failing sarcoplasmic reticulum (SR) than in normal SR. Addition of FK506, which dissociates FKBP12.6 from RyR2, to normal SR reduced the CaM-binding affinity. Dantrolene restored a normal level of the CaM-binding affinity in either FK506-treated (normal) SR or failing SR, suggesting that the defective inter-domain interaction between the N-terminal domain and the central domain of RyR2 (the therapeutic target of dantrolene) is involved in the reduction of the CaM-binding affinity in failing hearts. In saponin-permeabilized cardiomyocytes, the frequency of spontaneous Ca(2+) sparks was much more increased in failing cardiomyocytes than in normal cardiomyocytes, whereas the addition of a high concentration of CaM attenuated the aberrant increase of Ca(2+) sparks. CONCLUSION: The defective inter-domain interaction between N-terminal and central domains within RyR2 reduces the binding affinity of CaM to RyR2, thereby causing the spontaneous Ca(2+) release events in failing hearts. Correction of the defective CaM binding may be a new strategy to protect against the aberrant Ca(2+) release in heart failure.

Dantrolene, a therapeutic agent for malignant hyperthermia, markedly improves the function of failing cardiomyocytes by stabilizing interdomain interactions within the ryanodine receptor.

OBJECTIVES: We sought to investigate the effect of dantrolene, a drug generally used to treat malignant hyperthermia, on the Ca2+ release and cardiomyocyte function in failing hearts. BACKGROUND: The N-terminal (N: 1-600) and central (C: 2000-2500) domains of the ryanodine receptor (RyR) harbor many mutations associated with malignant hyperthermia in skeletal muscle RyR (RyR1) and polymorphic ventricular tachycardia in cardiac RyR (RyR2). There is strong evidence that interdomain interaction between these regions plays an important role in the mechanism of channel regulation. METHODS: Sarcoplasmic reticulum vesicles and cardiomyocytes were isolated from the left ventricular muscles of dogs (normal or rapid ventricular pacing for 4 weeks), for Ca2+ leak, transient, and spark assays. To assess the zipped or unzipped state of the interacting domains, the RyR was labeled fluorescently with methylcoumarin acetate in a site-directed manner. We used a quartz-crystal microbalance technique to identify the dantrolene binding site within the RyR2. RESULTS: Dantrolene specifically bound to domain 601-620 in RyR2. In the sarcoplasmic reticulum isolated from pacing-induced failing dog hearts, the defective interdomain interaction (domain unzipping) had already occurred, causing spontaneous Ca2+ leak. Dantrolene suppressed both domain unzipping and the Ca2+ leak, demonstrating identical drug concentration-dependence (IC50 = 0.3 micromol/l). In failing cardiomyocytes, both diastolic Ca2+ sparks and delayed afterdepolarization were observed frequently, but 1 micromol/l dantrolene inhibited both events. CONCLUSIONS: Dantrolene corrects defective interdomain interactions within RyR2 in failing hearts, inhibits spontaneous Ca2+ leak, and in turn improves cardiomyocyte function in failing hearts. Thus, dantrolene may have a potential to treat heart failure, specifically targeting the RyR2.

Alteration of methamphetamine-induced striatal dopamine release in mint-1 knockout mice.

Mint-1, which is also called as X11 or mammalian Lin10, protein has been implicated in the synaptic vesicle exocytosis and the targeting and localization of synaptic membrane proteins. Here, we established mint-1 gene knockout (mint-1 KO) mice and investigated vesicular and transporter-mediated dopamine (DA) release evoked by high K(+) and methamphetamine (METH), respectively. Compared with wild-type control, high K(+)-evoked striatal DA release was attenuated, but not significantly, in the KO mice as measured by microdialysis method. The METH-induced DA release was significantly attenuated in the KO mice. In addition, METH-induced stereotypy was also significantly attenuated in the KO mice. Mint-1 KO mice showed more sensitive and prominent behavioral response to an approaching object as compared with wild-type mice. These results suggest that mint-1 protein is involved in transporter-mediated DA release induced by METH.