Spark Frequency Research Articles

Shear stress enhances Ca2 + sparks through Nox2-dependent mitochondrial reactive oxygen species generation in rat ventricular myocytes

Shear stress enhances diastolic and systolic Ca2+ concentration in ventricular myocytes. Here, using confocal Ca2+ imaging in rat ventricular myocytes, we assessed the effects of shear stress (~16dyn/cm2) on the frequency of spontaneous Ca2+ sparks and explored the mechanism underlying shear-mediated Ca2+ spark regulation. The frequency of Ca2+ sparks was immediately increased by shear stress (by ~80%), and increased further (by ~150%) during prolonged exposure (20s). The 2-D size and duration of individual sparks were increased by shear stimulation. Inhibition of nitric oxide synthase (NOS) only partially attenuated the prolonged shear-mediated enhancement in spark frequency. Pretreatment with antioxidants significantly attenuated the short- and long-term effects of shear on spark frequency. Microtubule or nicotinamide adenine dinucleotide phosphate oxidase 2 (Nox2) inhibition abolished the immediate shear-induced increase in spark frequency and suppressed the effects of prolonged exposure to shear stress by ~70%. Scavenging of mitochondrial reactive oxygen species (ROS) and mitochondrial uncoupling also abolished the effect of short-term shear on spark occurrence, and markedly reduced (by ~80%) the effects of prolonged shear. Mitochondrial ROS levels increased under shear; this was eliminated by blocking Nox2. Sarcoplasmic reticulum Ca2+ content was increased only by prolonged shear. Our data suggest that shear stress enhances ventricular spark frequency mainly via ROS generated from mitochondria through Nox2, and that NOS and higher sarcoplasmic reticulum Ca2+ concentrations may also contribute to the enhancement of Ca2+ sparks under shear stress. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.

Cardiac Specific IP3R Over-Expression: IP3ICR Contribution in Ca2+ Signaling

Inositol-1,4,5-trisphosphate (IP3) is a second messenger produced upon agonist binding to a G-protein coupled receptor (GPCR) and subsequently triggers SR-Ca2+ release through openings of IP3 receptors (IP3Rs). In cardiac muscle, IP3R type 2 (IP3R2) is the predominant isoform expressed both in ventricle and atrial tissue. Several studies have focused on the functional interaction between ryanodine receptors (RyRs) and IP3Rs in atrial myocytes. However, it is still unclear how IP3-induced Ca2+ release (IP3ICR) may contribute to excitation-contraction coupling in ventricle. Evidence suggests that IP3ICR modulates the RyR function by affecting its local Ca2+ environment. Under pathophysiological cellular remodeling conditions (e.g. atrial fibrillation and heart failure) a functional interplay of IP3R and RyR Ca2+ events may be significantly pronounced. Our aim in this study was to examine this interaction in a cardiac-specific IP3R2 overexpressing (TG) mouse model. Our experimental approach includes: characterization of the IP3 signaling pathway by various pharmacological interventions; electrophysiology under whole-cell configuration of the patch clamp technique in combination with rapid confocal Ca2+ imaging and complemented with molecular biology approaches (RT-PCR, Western Blot, immunostaining). Western blot results show an increase in IP3R2 protein expression in the TG model both in atria and ventricular tissue compared to its wild-type littermate (FVB). However, a similar distribution on the junctional SR was found, where IP3Rs co-localize with RyRs. Supporting the protein data, preliminary results show an increase in basal spark frequency (SpF) in TG atrial and ventricular myocytes compared to FVB. Upon IP3 stimulation FVB atrial and ventricular cardiomyocytes presented an increase in SpF. Furthermore, a larger increment was seen in TG atrial myocytes. In order to examine the contribution of the SR-Ca2+ leak mediated by IP3R2 a SR-Ca2+ leak protocol was established. However, even under control conditions a significant contribution of IP3ICR on the SR-Ca2+ leak was found. We conclude that overexpression of IP3R2 in the TG model affects Ca2+ handling significantly by functional interplay with the RyR2.

Atrial Spceific Pitx2 Insufficiencyincreases the Frequency of Calcium Sparks, Waves, and After-Depolarizations in Mouse Atrial Myocytes

The transcription factor Pitx2 has been proposed as a molecular link between single nucleotide polymorphisms on chromosome 4q25 and increased risk of atrial fibrillation in carriers of the risk variant. Since atrial fibrillation has been associated with calcium handling disturbances in isolated atrial myocytes, we here tested the hypothesis that Pitx2 insufficiency alters the calcium homeostasis in atrial myocytes.To test this hypothesis, we used right atrial myocytes from a transgenic mouse model with inducible atrial specific Pitx2 deletion. Spontaneous calcium release was detected with confocal calcium imaging and resulting ion currents or membrane depolarizations were measured with patch-clamp technique in myocytes from wild-type (Pitx2+/+) and heterozygous Pitx2+/- mice.Calcium imaging revealed that the frequency of calcium sparks (2.1±0.7 vs 0.2±0.1 events/cell/s, p<0.05) and waves (3.2±1.2 vs. 0 events/min, p<0.05) were significantly higher in Pitx2+/- mice. This was also true for the frequency of transient inward currants activated by calcium waves (2.8±0.5 vs 1.2±0.5 events/min, p<0.05). The higher frequency was of spontaneous calcium release was likely due to a higher caffeine releasable sarcoplasmic reticulum calcium load in the Pitx2+/- mice (23.8±5.8 vs 14.4±1.9 amol/pF, p<0.05). Moreover, only myocytes from Pitx2+/- had spontaneous action potentials at a resting potential of −80 mV (0.5±0.4/min). At −60 mV the frequency of spontaneous action potentials was 5.3±1.8/min for Pitx2+/- and 0.4±1.2/min for Pitx2+/+ mice. Importantly, spontaneous after-potentials were also recorded in myocytes subjected to field stimulation, but only in myocytes from Pitx2+/- mice. These results were not unique to right atrial myocytes as the calcium spark frequency was also 3.9 fold higher in left atrial myocytes from Pitx2+/- mice (p<0.001).Together, these results demonstrate that Pitx2 insufficiency promote both spontaneous calcium release and spontaneous action potentials. Both of these features are hallmarks of myocytes from patients with atrial fibrillation, suggesting that Pitx2-mediated modulation of intracellular calcium handling plays an important role in electrophysiological processes associated with atrial fibrillation.

Calmodulin Regulation of Ryanodine Receptors (RyR2) Depends on RyR2 Phosphorylation at Residues S2808 and S2814

Introduction: Calmodulin (CaM) is a calcium-binding protein that regulates target proteins directly or via their regulatory kinases. CaM binds RyR2 and forms part of the RyR2 macromolecular complex that includes Protein Kinase A (PKA), CaM-dependent kinase II (CaMKII) and the phosphatases, PP1 and PP2A. In the complex, CaM influences SR Ca2+ release either by directly binding RyR2 (inhibition) or by phosphorylation of the RyR2 at S2814 through CaMKII (activation). CaM binding to RyR2 is critical for normal cardiac function and loss of CaM inhibiting efficacy leads to pathological activation of RyR2 that produce spontaneous Ca2+ releases from the SR. These in themselves can trigger miss-timed APs, cardiac arrhythmias and sudden death. Several recent studies have reported that CaM binding affinity to RyR2 is reduced by oxidation of both CaM and RyR2. Here we test the hypothesis that the action of CaM on the activity of RyR2 also depends on the phosphorylation state of the channel. Methods and Results: CaM effect on Ca2+ sparks (and hence RyR2 activity) was tested on permeabilized cardiomyocytes from transgenic mice where CaMKII or PKA phosphorylation sites in RyR2 were ablated (S2814A and S2808A, respectively) or where CaMKII phosphorylation site in RyR2 was fully active (S2814D). RyR2 phosphorylation was confirmed by Western Blot. CaM only inhibited Ca2+ sparks frequency in cardiomyocytes with high phosphorylation levels of RyR2 in CaMKII site (S2814D), in PKA site (S2814A+cAMP or S2808A+cAMP) or in both residues (RyR2 S2814D+cAMP). In contrast, CaM did not have an inhibitory effect in cardiomyocytes with low phosphorylation levels of RyR2 (S2814A and S2808A). Conclusion: Our results show that CaM inhibitory efficacy is dependent on RyR2 phosphorylation in CaMKII and PKA sites S2814 and S2808 in mouse ventricular myocytes.

Regulation of Cardiac Pacemaker Activity by PDE4 Isoforms

Background Numerous epidemiological and clinical studies have revealed a positive correlation between heart rate (HR) and cardiovascular morbimortality. The autonomic nervous system is the major extracardiac determinant of HR. During sympathetic stimulation, the activation of β-adrenergic receptors (βAR) induces an increase in cAMP levels, leading to a positive chronotropic effect. Among the 5 cAMP-PDE families expressed in the heart, PDE4 is critical for controlling excitation-contraction coupling (ECC) during βAR stimulation in atrial and ventricular cells. PDE4 may also be important for automaticity. 3 genes encode for cardiac PDE4s: pde4a, pde4b and pde4d. Their respective contribution to the regulation of pacemaker activity remains ill-defined. Methods The total enzymatic PDE activity was determined in mouse sinoatrial node (SAN) tissue as the cAMP hydrolytic activity measured in the absence of PDE inhibitor and the fraction corresponding to PDE4 activity was assessed by including the PDE4 inhibitor Ro-20-1724 (10 µM). The in vitro pacemaker activity was assessed by measuring the spontaneous Ca2+ transients in Fluo4-loaded-SAN intact tissue. Images were obtained using confocal microscopy. Results Ro-20-1724 increased the beating rate of intact mouse SAN and increased PKA-phosphorylation levels of key ECC actors (ryanodine receptor, phospholamban). PDE4 enzymatic activity was found to account for 60% of the total cAMP-PDE activity in SAN. The 3 isoforms PDE4A, 4B and 4D were found to be expressed in mouse SAN. In PDE4D-, but not in PDE4B-deficient mice, Ca2+ homeostasis was altered in control conditions and after βAR stimulation. Indeed, ablation of PDE4D induced a decreased beating rate and an increased Ca2+ spark frequency in control and βAR-stimulated conditions. Conclusion Our preliminary results reveal that PDE4 controls pacemaker function in mice and that PDE4D ablation strongly perturbs normal SAN activity.

Mechanism of Sinoatrial Node Dysfunction in a RyR2R420Q Mouse Model Ofcatecholaminergic Polymorphic Ventricular Tachycardia

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a genetic disease characterized by stress-induced syncope and/or sudden death in young individuals with structurally normal heart. More than 150 mutations located in the cardiac Ca2+ release channel (type-2 ryanodine receptor, RyR2) gene are related to CPVT. Besides ventricular tachycardia (VT) under stress, sinoatrial node (SAN) dysfunction is frequently observed in CPVT patients. However, the cellular mechanisms remain underexplored. We created a KI mice model bearing a mutation in the N-terminal portion of the RyR2 found in a CPVT family, RyR2(R420Q). ECGs were recorded in KI and WT littermates in resting condition and after epinephrine/caffeine (2/120 mg/kg) challenge (i.p. injection). All KI mice (n=7) showed sustained bidirectional VT, validating the model. Moreover, resting heart rhythm was slower in KI mice during rest period (525.3±9.0 bpm n=4 and 498.5±4.9 bpm n=3, P<0.05), denoting alteration in SAN function. To examine the mechanisms of this dysfunction, SANs were dissected from WT and KI mice, loaded with the cell-permeant Ca2+ dye, Fluo-4 AM, and viewed by confocal microscopy using a white light laser fitted to 500 nm and resonant scanning. The cycle length of spontaneous [Ca2+]i transients was significantly longer in KI mice (696±2ms in 70 WT cells vs. 994±5ms in 48 KI cells, P<0.0001). [Ca2+]i transient amplitudes were reduced in KI cells (peak F/F0: 3.2±0.2, n= 43, p<0.05) compared to WT cells (peak F/F0: 3.7±0.1, n= 67), but with similar kinetics. Ca2+ sparks frequency between spontaneous beats was similar between KI and WT but the time to peak (22.8±0.4ms in 938 WT Ca2+ sparks vs. 41.2±0.7ms in 1617 KI Ca2+ sparks, P<0.0001) and duration were prolonged in KI mice (duration at half maximum: 34.1±0.5ms, n=1135 WT; vs. 57.8±0.6ms n=2237 KI, P<0.0001). In conclusion, our data show that RYR2R420Q SAN mutation: 1) induce CPVT phenotype in mice, 2) decreases the SAN rhythm, 3) promotes more [Ca2+]i leak during diastole. These alterations may contribute to supraventricular arrhythmia in CPVT.

Glycosylated Chromogranin A in Heart Failure: Implications for Processing and Cardiomyocyte Calcium Homeostasis.

Chromogranin A (CgA) levels have previously been found to predict mortality in heart failure (HF), but currently no information is available regarding CgA processing in HF and whether the CgA fragment catestatin (CST) may directly influence cardiomyocyte function. CgA processing was characterized in postinfarction HF mice and in patients with acute HF, and the functional role of CST was explored in experimental models. Myocardial biopsies from HF, but not sham-operated mice, demonstrated high molecular weight CgA bands. Deglycosylation treatment attenuated high molecular weight bands, induced a mobility shift, and increased shorter CgA fragments. Adjusting for established risk indices and biomarkers, circulating CgA levels were found to be associated with mortality in patients with acute HF, but not in patients with acute exacerbation of chronic obstructive pulmonary disease. Low CgA-to-CST conversion was also associated with increased mortality in acute HF, thus, supporting functional relevance of impaired CgA processing in cardiovascular disease. CST was identified as a direct inhibitor of CaMKIIδ (Ca2+/calmodulin-dependent protein kinase IIδ) activity, and CST reduced CaMKIIδ-dependent phosphorylation of phospholamban and the ryanodine receptor 2. In line with CaMKIIδ inhibition, CST reduced Ca2+ spark and wave frequency, reduced Ca2+ spark dimensions, increased sarcoplasmic reticulum Ca2+ content, and augmented the magnitude and kinetics of cardiomyocyte Ca2+ transients and contractions. CgA-to-CST conversion in HF is impaired because of hyperglycosylation, which is associated with clinical outcomes in acute HF. The mechanism for increased mortality may be dysregulated cardiomyocyte Ca2+ handling because of reduced CaMKIIδ inhibition.

Mg2+ Inhibits Cardiac SR Calcium Release and has Biphasic Effects on Calmodulin Binding to RyR2

Mg2+ is a potent inhibitor of RyR channel gating, as demonstrated in lipid bilayer and SR vesicles. Less is known about Mg2+ effects on RyR2 function in the cardiac myocyte environment or how it might alter RyR2 interaction with accessory proteins like calmodulin (CaM), which by itself binds to, and inhibits RyR2 opening. We used permeabilized mouse ventricular cardiomyocytes to study the effects of Mg2+ on spontaneous Ca2+ release and CaM affinity for RyR2. We recorded Ca sparks at three [Mg2+]: 0.1 mM (low), 1 mM (physiological), and 3 mM (high). We found that in the presence of endogenous WT-CaM, with increasing [Mg2+], Ca spark frequency (CaSpF) decreased monotonically, and was accompanied by an increase in Ca spark amplitude and SR Ca2+ load. To test whether Mg2+ also modulates RyR2 interaction with accessory proteins, we measured binding affinity of WT-CaM and two CPVT-linked CaM mutants (N54I and N98S). The apparent affinity of WT-CaM for RyR2 in myocytes was highest at 1 mM Mg2+ and decreased at both 0.1 mM and 3 mM Mg2+. The same pattern was found for N54I- and N98S-CaM. We conclude that in cardiomyocytes, Mg2+ decreases CaSpF and increases SR Ca2+ load. As [Mg2+] increases in the physiologically range (0.1 - 1 mM), part of the Ca spark inhibition may be due to enhanced CaM binding to RyR2. In contrast, pathophysiologically high [Mg2+] (3 mM) may (1) limit RyR2-CaM binding (and thus CaM inhibition of RyR2) and (2) still directly inhibit RyR2 opening.

The effect of PKA-mediated phosphorylation of ryanodine receptor on SR Ca2 + leak in ventricular myocytes

Functional impact of cardiac ryanodine receptor (type 2 RyR or RyR2) phosphorylation by protein kinase A (PKA) remains highly controversial. In this study, we characterized a functional link between PKA-mediated RyR2 phosphorylation level and sarcoplasmic reticulum (SR) Ca2+ release and leak in permeabilized rabbit ventricular myocytes. Changes in cytosolic [Ca2+] and intra-SR [Ca2+]SR were measured with Fluo-4 and Fluo-5N, respectively. Changes in RyR2 phosphorylation at two PKA sites, serine-2031 and -2809, were measured with phospho-specific antibodies. cAMP (10μM) increased Ca2+ spark frequency approximately two-fold. This effect was associated with an increase in SR Ca2+ load from 0.84 to 1.24mM. PKA inhibitory peptide (PKI; 10μM) abolished the cAMP-dependent increase of SR Ca2+ load and spark frequency. When SERCA was completely blocked by thapsigargin, cAMP did not affect RyR2-mediated Ca2+ leak. The lack of a cAMP effect on RyR2 function can be explained by almost maximal phosphorylation of RyR2 at serine-2809 after sarcolemma permeabilization. This high RyR2 phosphorylation level is likely the consequence of a balance shift between protein kinase and phosphatase activity after permeabilization. When RyR2 phosphorylation at serine-2809 was reduced to its “basal” level (i.e. RyR2 phosphorylation level in intact myocytes) using kinase inhibitor staurosporine, SR Ca2+ leak was significantly reduced. Surprisingly, further dephosphorylation of RyR2 with protein phosphatase 1 (PP1) markedly increased SR Ca2+ leak. At the same time, phosphorylation of RyR2 at serine 2031 did not significantly change under identical experimental conditions. These results suggest that RyR2 phosphorylation by PKA has a complex effect on SR Ca2+ leak in ventricular myocytes. At an intermediate level of RyR2 phosphorylation SR Ca2+ leak is minimal. However, complete dephosphorylation and maximal phosphorylation of RyR2 increases SR Ca2+ leak.

Sensitized signalling between L-type Ca2+ channels and ryanodine receptors in the absence or inhibition of FKBP12.6 in cardiomyocytes.

The heart contraction is controlled by the Ca2+-induced Ca2+ release (CICR) between L-type Ca2+ channels and ryanodine receptors (RyRs). The FK506-binding protein FKBP12.6 binds to RyR subunits, but its role in stabilizing RyR function has been debated for long. Recent reports of high-resolution RyR structure show that the HD2 domain that binds to the SPRY2 domain of neighbouring subunit in FKBP-bound RyR1 is detached and invisible in FKBP-null RyR2. The present study was to test the consequence of FKBP12.6 absence on the in situ activation of RyR2. Using whole-cell patch-clamp combined with confocal imaging, we applied a near threshold depolarization to activate a very small fraction of LCCs, which in turn activated RyR Ca2+ sparks stochastically. FKBP12.6-knockout and FK506/rapamycin treatments increased spark frequency and LCC-RyR coupling fidelity without altering LCC open probability. Neither FK506 nor rapamycin further altered LCC-RyR coupling fidelity in FKBP12.6-knockout cells. In loose-seal patch-clamp experiments, the LCC-RyR signalling kinetics, indexed by the delay for a LCC sparklet to trigger a RyR spark, was accelerated after FKBP12.6 knockout and FK506/rapamycin treatments. These results demonstrated that RyRs became more sensitive to Ca2+ triggers without FKBP12.6. Isoproterenol (1 μM) further accelerated the LCC-RyR signalling in FKBP12.6-knockout cells. The synergistic sensitization of RyRs by catecholaminergic signalling and FKBP12.6 dysfunction destabilized the CICR system, leading to chaotic Ca2+ waves and ventricular arrhythmias. FKBP12.6 keeps the RyRs from over-sensitization, stabilizes the potentially regenerative CICR system, and thus may suppress the life-threatening arrhythmogenesis.

Abstract 11235: Improvement of Defective Channel Function on RyR2 Mediated by CaMKII Activation Suppresses Arrhythmogenesis in TnT-related Hypertrophic Cardiomyopathy

Backgrounds: In Familial Hypertrophic Cardiomyopathy (FHC) caused by cardiac troponin T (cTnT) mutations, Ca 2+ sensitivity in myofilaments is increased, results in leading to sudden cardiac death. The activation of calcium/calmodulin-dependent protein kinase II (CaMKII) is known to be one of the key elements for the alteration of calcium handling in heart failure, presumably mediated through defective channel function of cardiac ryanodine receptor (RyR2). However, how cTnT mutations lead to lethal arrhythmias and abnormal calcium handling through defective RyR2 remains elusive. Here, we investigated the pathogenic role of CaMKII on aberrant Ca 2+ release through RyR2 for arrhythmogenesis in FHC hearts and therapeutic effects of dantrolene, which was found to correct inter-domain interactions between N-terminal and central domain of RyR2 in FHC-related cTnT-mutated transgenic mouse (TG; cTnT-delta160E). TG have been reported myocardial disarray in histology and increased Ca 2+ sensitivity in myofilaments. Methods and results: On confocal Ca 2+ imaging by fluo4-AM exposed to isoproterenol (ISO; 10 nmol/L), the Ca 2+ spark frequency (SpF:s-1·100μm-1) was much higher in TG cardiomyocytes (ISO-TG: n=11: 7.8±0.6; p&lt;0.05) than in non-TG (n=8: 3.4±0.4). It was largely reversed by CaMKII inhibitor {KN-93(1μM), n=7: 5.2±0.5; p&lt;0.05}, but not by PKA inhibitor (PKI){H-89(1μM), n=7: 7.3±0.4}. The significant increase of spontaneous Ca 2+ transient (sCaT), which was measured after sequential pacing at 5 Hz was appeared in ISO-TG (42.1%; p&lt;0.05 vs ISO-treated non-TG 14.0%), whereas it was again attenuated by CaMKII inhibitor (15.8%), but not by PKI (33.0%). These abnormal events in ISO-TG were reproduced by adding EGTA-AM into ISO-treated non-TG, suggesting that increased Ca 2+ buffering capacity, causing an increase in diastolic [Ca 2+ ], predisposes to aberrant Ca 2+ release events. Moreover, after the treatment of dantrolene (1μM), ISO-induced SpF (n=6: 5.2±0.3; p&lt;0.05 vs ISO-TG) and sCaT (25.9%) were decreased in TG. Conclusions: CaMKII activation may play a role in the development of abnormal calcium handling in FHC-linked cTnT mutated hearts, possibly through defective inter-domain interaction between N-terminal and central domain of RyR2.

Abstract 11917: Muscarinic Stimulation Facilitates SR Ca +2 Release by Increasing PKG Phosphorylation of RyR2 at Ser-2808 and Decreasing CaMKII Phosphorylation at Ser-2814

Autonomic regulation of SR Ca +2 release is critical for modulating cardiac performance to match physiologic demand. During heart failure (HF), autonomic balanced shifts to a dominant sympathetic state to increase fractional SR Ca +2 release. Although intended to be compensatory, this increase in sympathetic input causes increases in the frequency of proarrhythmic spontaneous SR Ca +2 release events. Interestingly, parasympathetic augmentation was recently shown to alleviate HF phenotypes by shifting autonomic balance away from sympathetic dominance. While the effects of sympathetic stimulation and mechanism behind their action on cardiomyocyte Ca +2 handling are relatively well established in health and disease, the modes of action and mechanisms of parasympathetic modulation of SR Ca +2 release are poorly defined. To address this question, we investigated the effects of the cholinergic agonist, carbachol (CCH), on Ca +2 handling (Fluo-4 FF AM) and protein phosphorylation levels in cardiomyocytes using a combination of pharmacological approaches and genetic mouse models. In settings of reduced SR Ca +2 load, exposure to the parasympathetic agonist CCH increased fractional Ca +2 release without affecting Ca +2 spark frequency in cardiomyocytes. Immunoblot measurements showed that this “efficient” mode of excitation-contraction (EC) coupling involves reciprocal changes in the phosphorylation state of RyR2. Specifically, CCH treatment of cardiomyocytes increased Ser-2808 phosphorylation, mediated by muscarinic receptor subtype 2 and activation of PKG. Conversely, CCH treatment was associated with dephosphorylation at Ser-2814 through activation of muscarinic receptor subtype 3 and decreased ROS-dependent activation of CaMKII. We further demonstrate that parasympathetic stimulation improved Ca +2 cycling efficiency in cardiomyocytes isolated from failing hearts by restoring altered RyR2 phosphorylation balance. These results suggest that parasympathetic stimulation initiates a novel mode of EC coupling in cardiomyocytes that enhances the efficiency of cardiac SR Ca +2 store mobilization during systolic Ca +2 release, while inhibiting pathologic diastolic Ca +2 leak.

Treatment of catecholaminergic polymorphic ventricular tachycardia in mice using novel RyR2-modifying drugs

RationaleCatecholaminergic polymorphic ventricular tachycardia (CPVT) is a potentially lethal arrhythmic disorder caused by mutations in the type-2 ryanodine receptor (RyR2). Mutant RyR2 cause abnormal Ca2+ leak from the sarcoplasmic reticulum (SR), which is associated with the development of arrhythmias. ObjectiveTo determine whether derivatives of tetracaine, a local anesthetic drug with known RyR2 inhibiting action, could prevent CPVT induction by suppression of RyR2-mediated SR Ca2+ leak. Methods and resultsConfocal microscopy was used to assess the effects of tetracaine and 9 derivatives (EL1–EL9) on spontaneous Ca2+ sparks in ventricular myocytes isolated from RyR2-R176Q/+ mice with CPVT. Whereas each derivative suppressed the Ca2+ spark frequency, derivative EL9 was most effective at the screening dose of 500nmol/L. At this high dose, the Ca2+ transient amplitude was not affected in myocytes from WT or R176Q/+ mice. The IC50 of EL9 was determined to be 13nmol/L, which is about 400× time lower than known RyR2 stabilizer K201. EL9 prevented the induction of ventricular tachycardia observed in placebo-treated R176Q/+ mice, without affecting heart rate or cardiac contractility. ConclusionsTetracaine derivatives represent a novel class of RyR2 stabilizing drugs that could be used for the treatment of the potentially fatal disorder catecholaminergic polymorphic ventricular tachycardia.

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

Calmodulin (CaM) is a key modulator of the channel gating function of the ryanodine receptor (RyR). 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. Pressure overload was induced in 12-week-old mice by transverse aortic constriction (TAC) using a 27-gauge needle. 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. 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.

SPEG (Striated Muscle Preferentially Expressed Protein Kinase) Is Essential for Cardiac Function by Regulating Junctional Membrane Complex Activity.

Junctional membrane complexes (JMCs) in myocytes are critical microdomains, in which excitation-contraction coupling occurs. Structural and functional disruption of JMCs underlies contractile dysfunction in failing hearts. However, the role of newly identified JMC protein SPEG (striated muscle preferentially expressed protein kinase) remains unclear. To determine the role of SPEG in healthy and failing adult hearts. Proteomic analysis of immunoprecipitated JMC proteins ryanodine receptor type 2 and junctophilin-2 (JPH2) followed by mass spectrometry identified the serine-threonine kinase SPEG as the only novel binding partner for both proteins. Real-time polymerase chain reaction revealed the downregulation of SPEG mRNA levels in failing human hearts. A novel cardiac myocyte-specific Speg conditional knockout (MCM-Spegfl/fl) model revealed that adult-onset SPEG deficiency results in heart failure (HF). Calcium (Ca2+) and transverse-tubule imaging of ventricular myocytes from MCM-Spegfl/fl mice post HF revealed both increased sarcoplasmic reticulum Ca2+ spark frequency and disrupted JMC integrity. Additional studies revealed that transverse-tubule disruption precedes the development of HF development in MCM-Spegfl/fl mice. Although total JPH2 levels were unaltered, JPH2 phosphorylation levels were found to be reduced in MCM-Spegfl/fl mice, suggesting that loss of SPEG phosphorylation of JPH2 led to transverse-tubule disruption, a precursor of HF development in SPEG-deficient mice. The novel JMC protein SPEG is downregulated in human failing hearts. Acute loss of SPEG in mouse hearts causes JPH2 dephosphorylation and transverse-tubule loss associated with downstream Ca2+ mishandling leading to HF. Our study suggests that SPEG could be a novel target for the treatment of HF.

High throughput nanoparticle generation utilizing high-frequency spark discharges via rapid spark plasma removal

ABSTRACTScaling up the production capacity of spark discharge generated nanoparticles is crucial for their industrial application. Among various options, high-frequency spark discharge generation is a promising route as the mass production rate of nanoparticles should simply scale linearly with the spark frequency. However, reports of spark discharge generators operating above 1 kHz are scarce in the literature, as spark discharges at higher frequencies have been observed to be irregular, leading to premature spark discharges that result in a significantly lower mass production rate. In this study, we present a wire-to-plate electrode configuration that suppresses premature spark discharges during high-frequency operations above 1 kHz, and investigate the factors that contribute to the occurrence of premature spark discharges by comparing the performance of the wire-to-plate electrode configuration to those of rod-to-rod and wire-to-rod configurations. We identified that spark duration should be minimized...

Junctophilin-2 gene therapy rescues heart failure by normalizing RyR2-mediated Ca2 + release

BackgroundJunctophilin-2 (JPH2) is the primary structural protein for the coupling of transverse (T)-tubule associated cardiac L-type Ca channels and type-2 ryanodine receptors on the sarcoplasmic reticulum within junctional membrane complexes (JMCs) in cardiomyocytes. Effective signaling between these channels ensures adequate Ca-induced Ca release required for normal cardiac contractility. Disruption of JMC subcellular domains, a common feature of failing hearts, has been attributed to JPH2 downregulation. Here, we tested the hypothesis that adeno-associated virus type 9 (AAV9) mediated overexpression of JPH2 could halt the development of heart failure in a mouse model of transverse aortic constriction (TAC). Methods and resultsFollowing TAC, a progressive decrease in ejection fraction was paralleled by a progressive decrease of cardiac JPH2 levels. AAV9-mediated expression of JPH2 rescued cardiac contractility in mice subjected to TAC. AAV9-JPH2 also preserved T-tubule structure. Moreover, the Ca2+ spark frequency was reduced and the Ca2+ transient amplitude was increased in AAV9-JPH2 mice following TAC, consistent with JPH2-mediated normalization of SR Ca2+ handling. ConclusionsThis study demonstrates that AAV9-mediated JPH2 gene therapy maintained cardiac function in mice with early stage heart failure. Moreover, restoration of JPH2 levels prevented loss of T-tubules and suppressed abnormal SR Ca2+ leak associated with contractile failure following TAC. These findings suggest that targeting JPH2 might be an attractive therapeutic approach for treating pathological cardiac remodeling during heart failure.

Observation of quasi-periodic solar radio bursts associated with propagating fast-mode waves

Radio emission observations from the Learmonth and Bruny Island radio spectrographs are analysed to determine the nature of a train of discrete, periodic radio \lq sparks\rq (finite-bandwidth, short-duration isolated radio features) which precede a type II burst. We analyse extreme ultraviolet (EUV) imaging from SDO/AIA at multiple wavelengths and identify a series of quasi-periodic rapidly-propagating enhancements, which we interpret as a fast wave train, and link these to the detected radio features. The speeds and positions of the periodic rapidly propagating fast waves and the coronal mass ejection (CME) were recorded using running-difference images and time-distance analysis. From the frequency of the radio sparks the local electron density at the emission location was estimated for each. Using an empirical model for the scaling of density in the corona, the calculated electron density was used to obtain the height above the surface at which the emission occurs, and the propagation velocity of the emission location. The period of the radio sparks, $\delta t_\mathrm{r}$=1.78$\pm$0.04 min, matches the period of the fast wave train observed at 171 $\AA$, $\delta t_\mathrm{EUV}$=1.7 $\pm$ 0.2 min. The inferred speed of the emission location of the radio sparks, 630 km s$^{-1}$, is comparable to the measured speed of the CME leading edge, 500 km s$^{-1}$, and the speeds derived from the drifting of the type II lanes. The calculated height of the radio emission (obtained from the density) matches the observed location of the CME leading edge. From the above evidence we propose that the radio sparks are caused by the quasi-periodic fast waves, and the emission is generated as they catch up and interact with the leading edge of the CME.

Recombinant Atrial Natriuretic Peptide Prevents Aberrant Ca2+ Leakage through the Ryanodine Receptor by Suppressing Mitochondrial Reactive Oxygen Species Production Induced by Isoproterenol in Failing Cardiomyocytes.

Catecholamines induce intracellular reactive oxygen species (ROS), thus enhancing diastolic Ca2+ leakage through the ryanodine receptor during heart failure (HF). However, little is known regarding the effect of atrial natriuretic peptide (ANP) on ROS generation and Ca2+ handling in failing cardiomyocytes. The aim of the present study was to clarify the mechanism by which an exogenous ANP exerts cardioprotective effects during HF. Cardiomyocytes were isolated from the left ventricles of a canine tachycardia-induced HF model and sham-operated vehicle controls. The degree of mitochondrial oxidized DNA was evaluated by double immunohistochemical (IHC) staining using an anti-VDAC antibody for the mitochondria and an anti-8-hydroxy-2′-deoxyguanosine antibody for oxidized DNA. The effect of ANP on ROS was investigated using 2,7-dichlorofluorescin diacetate, diastolic Ca2+ sparks assessed by confocal microscopy using Fluo 4-AM, and the survival rate of myocytes after 48 h. The double IHC study revealed that isoproterenol (ISO) markedly increased oxidized DNA in the mitochondria in HF and that the ISO-induced DNA damage was markedly inhibited by the co-presence of ANP. ROS production and Ca2+ spark frequency (CaSF) were increased in HF compared to normal controls, and were further increased in the presence of ISO. Notably, ANP significantly suppressed both ISO-induced ROS and CaSF without changing sarcoplasmic reticulum Ca2+ content in HF (p<0.01, respectively). The survival rate after 48 h in HF was significantly decreased in the presence of ISO compared with baseline (p<0.01), whereas it was significantly improved by the co-presence of ANP (p<0.01). Together, our results suggest that ANP strongly suppresses ISO-induced mitochondrial ROS generation, which might correct aberrant diastolic Ca2+ sparks, eventually contributing to the improvement of cardiomyocyte survival in HF.

Antiarrhythmic effects of dantrolene in human diseased cardiomyocytes

Cardiac type 2 ryanodine receptors (RyR2s) play a pivotal role in cellular electrophysiology and contractility. Increased RyR2-mediated diastolic sarcoplasmic reticulum (SR) Ca2+ release is linked to heart failure (HF) and arrhythmias. Dantrolene, a drug used for the treatment of malignant hyperthermia, is known to stabilize RyRs in skeletal muscle. The purpose of this study was to investigate the effects of dantrolene on arrhythmogenic triggers and contractile function in human atrial fibrillation (AF) and HF cardiomyocytes (CM). Human CM were isolated from either patients with HF (ventricular) or patients with AF (atrial), and Ca2+ imaging, patch-clamp, or muscle strip experiments were performed. After exposure to dantrolene, human atrial AF and left ventricular HF CM showed significant reductions in proarrhythmic SR Ca2+ spark frequency and diastolic SR Ca2+ leak. Moreover, dantrolene decreased the frequency of Ca2+ waves and spontaneous Ca2+ transients in HF CM. Patch-clamp experiments revealed that dantrolene significantly suppressed delayed afterdepolarizations in HF and AF CM. Importantly, dantrolene had no effect on action potential duration in AF or in HF CM. In addition, dantrolene had neutral effects on contractile force of human isometrically twitching ventricular HF trabeculae. Our study showed that dantrolene beneficially influenced disrupted SR Ca2+ homeostasis in human HF and AF CM. Cellular arrhythmogenic triggers were potently suppressed by dantrolene, whereas action potential duration and contractility were not affected. As a clinically approved drug for the treatment of malignant hyperthermia, dantrolene may be a potential antiarrhythmic drug for patients with rhythm disorders and merits further clinical investigation.