Ca Spark Frequency Research Articles

CaMKII-dependent SR Ca leak contributes to doxorubicin-induced impaired Ca handling in isolated cardiac myocytes

Doxorubicin (DOX) is one of the most effective chemotherapeutic agents, but cardiotoxicity limits DOX therapy. Although the mechanisms are not entirely understood, reactive oxygen species (ROS) appear to be involved in DOX cardiotoxicity. Ca/calmodulin dependent protein kinase II (CaMKII) can be activated by ROS through oxidation and is known to contribute to myocardial dysfunction through Ca leakage from the sarcoplasmic reticulum (SR). We hypothesized that CaMKII contributes to DOX-induced defects in intracellular Ca ([Ca] i) handling. Cardiac myocytes were isolated from wild-type (WT) adult rat hearts and from mouse hearts lacking the predominant myocardial CaMKII isoform (CaMKIIδ −/−, KO) vs. WT. Isolated cardiomyocytes were investigated 30 min after DOX (10 μmol/L) superfusion, using epifluorescence and confocal microscopy. Intracellular ROS-generation ([ROS] i) and [Ca] i handling properties were assessed. In a subset of experiments, KN-93 or AIP (each 1 μmol/L) were used to inhibit CaMKII. Melatonin (Mel, 100 μmol/L) served as ROS-scavenger. Western blots were performed to determine the amount of CaMKII phosphorylation and oxidation. DOX increased [ROS] i and led to significant diastolic [Ca] i overload in rat myocytes. This was associated with reduced [Ca] i transients, a 5.8-fold increased diastolic SR Ca leak and diminished SR Ca content. ROS-scavenging partially rescued Ca handling. Western blots revealed increased CaMKII phosphorylation, but not CaMKII oxidation after DOX. Pharmacological CaMKII inhibition attenuated diastolic [Ca] i overload after DOX superfusion and led to partially restored [Ca] i transients and SR Ca content, presumably due to reduced Ca spark frequency. In line with this concept, isoform-specific CaMKIIδ-KO attenuated diastolic [Ca] i overload and Ca spark frequency. DOX exposure induces CaMKII-dependent SR Ca leakage, which partially contributes to impaired cellular [Ca] i homeostasis. Pharmacological and genetic CaMKII inhibition attenuated but did not completely abolish the effects of DOX on [Ca] i. In light of the clinical relevance of DOX, further investigations seem appropriate to determine if CaMKII inhibition could reduce DOX-induced cardiotoxicity.

Age-related regulation of excitation–contraction coupling in rat heart

Hearts from subjects with different ages have different Ca(2+) signaling. Release of Ca(2+) from intracellular stores in response to an action potential initiates cardiac contraction. Both depolarization-stimulated and spontaneous Ca(2+) releases, Ca(2+) transients and Ca(2+) sparks, demonstrate the main events of excitation-contraction coupling (ECC). Global increase in free Ca(2+) concentration ([Ca(2+)]( i )) consists of summation of Ca(2+) release events in cardiomyocytes. Since the Ca(2+) flux induced by Ca(2+) sparks reports a summation of ryanodine-sensitive Ca(2+) release channels (RyR2s)'s behavior in a spark cluster, evaluation of the properties of Ca(2+) sparks and Ca(2+) transients may provide insight into the role of RyR2s on altered heart function between 3-month-old (young adult) and 6-month-old (mature adult) rats. Basal [Ca(2+)]( i ) and Ca(2+) sparks frequency were significantly higher in mature adult rats compared to those of young adults. Moreover, amplitudes of Ca(2+) sparks and Ca(2+) transients were significantly smaller in mature adults than those of young adults with longer time courses. A smaller L-type Ca(2+) current density and decreased SR Ca(2+) load was observed in mature adult rats. In addition, RyR2s were markedly hyperphosphorylated, and phosphorylation levels of PKA and CaMKII were higher in heart from mature adults compared to those of young adults, whereas their SERCA protein levels were similar. Our data demonstrate that hearts from rats with different ages have different Ca(2+) signaling including hyperphosphorylation of RyR2s and higher basal [Ca(2+)]( i ) together with increased oxidized protein-thiols in mature adult rats compared to those of young adults, which play important roles in ECC. Finally, we report that ECC efficiency changes with age during maturation, partially related with an increased cellular oxidation level leading to reduced free protein-thiols in cardiomyocytes.

Ec Coupling is Preserved in Ncx Knockout Mice Exposed to Metabolic Inhibitors

To determine whether ablating the cardiac sodium-calcium exchanger (NCX) prevents couplon loss during metabolic inhibition (MI), we recorded Ca transients or resting Ca spark frequency (CaSpF) in ventricular myocytes isolated from WT and ventricular-specific NCX knockout (KO) mice. In field stimulated myocytes loaded with 10 µM Fluo-3 AM, application of the mitochondrial and glycolytic inhibitors FCCP (50 nM) and 2-DG (10 mM) reduced Ca transients by 54.2 ± 3.3% in WT but only by 19.0 ± 6.0% in KO (p <0.05). Similarly, MI suppressed action potential evoked Ca sparks in patch clamped WT myocytes loaded with 1 mM fluo-3 salt by 60.0 ± 8.2% (p <0.05), but in KO by only 33.0 ± 3.4%. The reduction in ICa during MI was similar for both WT and KO and SR Ca stores remained at control levels in both cell types during MI. In patch clamped myocytes loaded with 1 mM K5Fluo-3 and held at −75 mV, MI reduced resting CaSpF in WT by 92.4 ± 6.7%, (p=0.008) but had no effect on CaSpF in KO Abolishing Na and Ca gradients by using the same “internal” solution for both the bath and pipette (current clamp mode, VRest = 0 mV), eliminated the KO's resistance to MI, causing CaSpF to decline to a similar extent in both WT and KO (59.0 ± 6.2% and 60.7 ± 10.9%, respectively). This finding suggests that elevated diadic cleft Ca, a major characteristic of KO myocytes (Pott et al, Biophys J, 92: 1431, 2007; Neco et al. Biophys J, 99: 755, 2010), maintains effective triggering of SR Ca release during MI despite reduced ICa.

Reactive Oxygen Species–Activated Ca/Calmodulin Kinase IIδ Is Required for Late I Na Augmentation Leading to Cellular Na and Ca Overload

In heart failure Ca/calmodulin kinase (CaMK)II expression and reactive oxygen species (ROS) are increased. Both ROS and CaMKII can increase late I(Na) leading to intracellular Na accumulation and arrhythmias. It has been shown that ROS can activate CaMKII via oxidation. We tested whether CaMKIIδ is required for ROS-dependent late I(Na) regulation and whether ROS-induced Ca released from the sarcoplasmic reticulum (SR) is involved. 40 μmol/L H(2)O(2) significantly increased CaMKII oxidation and autophosphorylation in permeabilized rabbit cardiomyocytes. Without free [Ca](i) (5 mmol/L BAPTA/1 mmol/L Br(2)-BAPTA) or after SR depletion (caffeine 10 mmol/L, thapsigargin 5 μmol/L), the H(2)O(2)-dependent CaMKII oxidation and autophosphorylation was abolished. H(2)O(2) significantly increased SR Ca spark frequency (confocal microscopy) but reduced SR Ca load. In wild-type (WT) mouse myocytes, H(2)O(2) increased late I(Na) (whole cell patch-clamp). This increase was abolished in CaMKIIδ(-/-) myocytes. H(2)O(2)-induced [Na](i) and [Ca](i) accumulation (SBFI [sodium-binding benzofuran isophthalate] and Indo-1 epifluorescence) was significantly slowed in CaMKIIδ(-/-) myocytes (versus WT). CaMKIIδ(-/-) myocytes developed significantly less H(2)O(2)-induced arrhythmias and were more resistant to hypercontracture. Opposite results (increased late I(Na), [Na](i) and [Ca](i) accumulation) were obtained by overexpression of CaMKIIδ in rabbit myocytes (adenoviral gene transfer) reversible with CaMKII inhibition (10 μmol/L KN93 or 0.1 μmol/L AIP [autocamtide 2-related inhibitory peptide]). Free [Ca](i) and a functional SR are required for ROS activation of CaMKII. ROS-activated CaMKIIδ enhances late I(Na), which may lead to cellular Na and Ca overload. This may be of relevance in hear failure, where enhanced ROS production meets increased CaMKII expression.

Ca2+ wave probability is determined by the balance between SERCA2-dependent Ca2+ reuptake and threshold SR Ca2+ content

In this manuscript, we determined the roles of the sarcoendoplasmic reticulum Ca(2+) ATPase 2 (SERCA2) and the ryanodine receptor (RyR) in Ca(2+) wave development during β-adrenergic stimulation. SERCA2 knockout mice (KO) were used 6 days after cardio-specific gene deletion, with left ventricular SERCA2a abundance reduced by 54 ± 9% compared with SERCA2(flox/flox) controls (FF) (P < 0.05). Ca(2+) waves occurred in fewer KO than FF myocytes (40 vs. 68%, P < 0.05), whereas the addition of isoproterenol (ISO) induced waves in an equal percentage of myocytes (82 vs. 64%). SERCA2-dependent Ca(2+) reuptake was slower in KO (-ISO, KO vs. FF: 15.4 ± 1.2 vs. 21.1 ± 1.4 s(-1), P < 0.05), but equal during ISO (+ISO, KO vs. FF: 21.9 ± 3.3 vs. 27.7 ± 2.7 s(-1)). Threshold SR Ca(2+) content for wave development was lower in KO (-ISO, KO vs. FF: 126.6 ± 10.3 vs. 159.3 ± 7.1 µmol/L, P < 0.05) and was increased by ISO only in FF (+ISO, KO vs. FF: 131.7 ± 8.7 vs. 205.5 ± 20.4 µmol/L, P < 0.05). During ISO, Ca(2+)/calmodulin-dependent kinase II (CaMKII)-dependent phosphorylation of RyR in KO was 217 ± 21% of FF (P < 0.05), and SR Ca(2+) leak indicated higher RyR open probability in KO. CaMKII inhibition decreased Ca(2+) spark frequency in KO by 44% (P < 0.05) but not in FF. Mathematical modelling predicted that increased Ca(2+) sensitivity of RyR in KO could account for increased Ca(2+) wave probability during ISO. In ventricular cardiomyocytes with reduced SERCA2 abundance, Ca(2+) wave development following β-adrenergic stimulation is potentiated. We suggest that this is caused by a CaMKII-dependent shift in the balance between SERCA2-dependent Ca(2+) reuptake and threshold SR Ca(2+) content.

Diverse cell morphology and intracellular calcium dynamics in pulmonary vein cardiomyocytes

Pulmonary veins (PVs) contain cardiomyocytes with a complex cellular morphology and high arrhythmogenesis. Ca(2+) regulation and Ca(2+) sparks play a pivotal role in the electrical activity of cardiomyocytes. The purpose of this study was to investigate whether the cell morphology can determine the PV electrical activity and Ca(2+) homeostasis. Through confocal microscopy with fluo-3 Ca(2+) fluorescence, Ca(2+) sparks and Ca(2+) transients were evaluated in isolated single rabbit left atria (LA) and PV cardiomyocytes according to the cell morphology (rod, rod-spindle and spindle/bifurcated). Twenty-two (20%) rod, 49 (43%) rod-spindle and 41 (37%) spindle/bifurcated cardiomyocytes were identified in the LA (n = 29) and PV (n = 83) cardiomyocytes. The PV cardiomyocytes with pacemaker activity had a higher incidence of spindle/bifurcated morphology than LA and PV cardiomyocytes without pacemaker activity. As compared to those in the rod or rod-spindle cardiomyocytes, spindle/bifurcated cardiomyocytes had a larger Ca(2+) transient amplitude and higher frequency of the Ca(2+) sparks with larger amplitude and longer duration. In contrast, rod-spindle and rod cardiomyocytes had similar Ca(2+) transients and Ca(2+) sparks. The cell length correlated well with the amplitude of the Ca(2+) transient and Ca(2+) spark duration with a linear regression. In conclusion, cell morphology and cell length play a potential role in the Ca(2+) homeostasis and Ca(2+) spark. The large Ca(2+) transients and high frequency of Ca(2+) sparks in spindle/bifurcated cardiomyocytes may cause a high arrhythmogenesis in the PV cardiomyocytes with pacemaker activity.

Limitations of FKBP12.6-directed treatment strategies for maladaptive cardiac remodeling and heart failure

Sarcoplasmic reticulum (SR) calcium (Ca) leak can be reduced by enhancing FKBP12.6 binding to SR Ca release channels (RyR2) and expression of a “sticky” FKBP12.6D37S mutant may correct reduced binding stoichiometry in RyR2 from failing hearts. Both calcium/calmodulin-dependent protein kinase IIδc (CaMKIIδc) and protein kinase A (PKA) are activated in heart failure and promote SR Ca leak at RyR2. It is possible that FKBP12.6 dissociation from RyR2 may promote remodeling and that interventions to reassociate FKBP12.6 with RyR2 reflect a future therapeutic strategy. We created transgenic (TG) mice expressing FKBP12.6D37S and tested their capacity to improve intracellular Ca handling and pathological remodeling in vivo. FKBP12.6D37S TG mice were cross-bred with CaMKIIδc TG mice, which are known to exhibit pronounced RyR2 dysfunction and heart failure. We observed a significant improvement of post-rest Ca transients and a higher SR Ca content in FKBP12.6D37S TG mice. In double-TG mice, a marked reduction of SR Ca spark frequency indicated reduced SR Ca leak but neither SR Ca transient amplitude, SR Ca content nor morphological or functional parameters improved in vivo. Likewise, FKBP12.6D37S TG mice subjected to increased afterload after aortic banding exhibited higher SR Ca load but did not exhibit any improvement in hypertrophic growth or functional decline. Enhancement of FKBP12.6-RyR2 binding markedly reduced RyR2 Ca leak in CaMKIIδc-induced heart failure and in pressure overload. Our data suggest that activation of CaMKIIδc and pressure overload confer significant resistance towards approaches aiming at FKBP12.6-RyR2 reconstitution in heart failure and maladaptive remodeling, although RyR2 Ca leak can be reduced.

Regulation of myocyte contraction via neuronal nitric oxide synthase: role of ryanodine receptor S‐nitrosylation

The sarcoplasmic reticulum (SR) Ca(2+) release channel (ryanodine receptor, RyR2) has been proposed to be an end target of neuronal nitric oxide synthase (NOS1) signalling. The purpose of this study is to investigate the mechanism of NOS1 modulation of RyR2 activity and the corresponding effect on myocyte function. Myocytes were isolated from NOS1 knockout (NOS1(/)) and wild-type mice. NOS1(/) myocytes displayed a decreased fractional SR Ca(2+) release, NOS1 knockout also led to reduced RyR2 S-nitrosylation levels. RyR2 channels from NOS1(/) hearts had decreased RyR2 open probability. Additionally, knockout of NOS1 led to a decrease in [(3)H]ryanodine binding, Ca(2+) spark frequency (CaSpF) and a rightward shift in the SR Ca(2+) leak/load relationship. Similar effects were observed with acute inhibition of NOS1. These data are indicative of decreased RyR2 activity in myocytes with NOS1 knockout or acute inhibition. Interestingly, the NO donor and nitrosylating agent SNAP reversed the depressed RyR2 open probability, the reduced CaSpF, and caused a leftward shift in the leak/load relationship in NOS1(/) myocytes. SNAP also normalized Ca(2+) transient and cell shortening amplitudes and SR fractional release in myocytes with NOS1 knockout or acute inhibition. Furthermore, SNAP was able to normalize the RyR2 S-nitrosylation levels. These data suggest that NOS1 signalling increases RyR2 activity via S-nitrosylation, which contributes to the NOS1-induced positive inotropic effect. Thus, RyR2 is an important end target of NOS1.

Prolonged mechanical unloading affects cardiomyocyte excitation‐contraction coupling, transverse‐tubule structure, and the cell surface

Prolonged mechanical unloading (UN) of the heart is associated with detrimental changes to the structure and function of cardiomyocytes. The mechanisms underlying these changes are unknown. In this study, we report the influence of UN on excitation-contraction coupling, Ca2+-induced Ca2+ release (CICR) in particular, and transverse (t)-tubule structure. UN was induced in male Lewis rat hearts by heterotopic abdominal heart transplantation. Left ventricular cardiomyocytes were isolated from the transplanted hearts after 4 wk and studied using whole-cell patch clamping, confocal microscopy, and scanning ion conductance microscopy (SICM). Recipient hearts were used as control (C). UN reduced the volume of cardiomyocytes by 56.5% compared with C (UN, n=90; C, n=59; P<0.001). The variance of time-to-peak of the Ca2+ transients was significantly increased in unloaded cardiomyocytes (UN 227.4±24.9 ms2, n=42 vs. C 157.8±18.0 ms2, n=40; P<0.05). UN did not alter the action potential morphology or whole-cell L-type Ca2+ current compared with C, but caused a significantly higher Ca2+ spark frequency (UN 3.718±0.85 events/100 μm/s, n=47 vs. C 0.908±0.186 events/100 μm/s, n=45; P<0.05). Confocal studies showed irregular distribution of the t tubules (power of the normal t-tubule frequency: UN 8.13±1.12×105, n=57 vs. C 20.60± 3.174×105, n=56; P<0.001) and SICM studies revealed a profound disruption to the openings of the t tubules and the cell surface in unloaded cardiomyocytes. We show that UN leads to a functional uncoupling of the CICR process and identify disruption of the t-tubule-sarcoplasmic reticulum interaction as a possible mechanism.—Ibrahim, M., Al Masri, A., Navaratnarajah, M., Siedlecka, U., Soppa, G. K., Moshkov, A., Abou Al-Saud, S., Gorelik, J., Yacoub, M. H., Terracciano, C. M. N. Prolonged mechanical unloading affects cardiomyocyte excitation-contraction coupling, transverse-tubule structure, and the cell surface.

Ca SR Leak is Modulated by CaMKII Phosphorylation on RyR2-S2814

CaMKII has been shown to increase cardiac SR Ca leak through RyRs. RyR2-S2814 has been suggested as the phosphorylation site responsible for SR Ca leak triggering cardiac arrhythmias in heart failure. Here we test the requirement of S2814 for these effects, in knock-in mice expressing only RyR2-S2814A or -S2814D. Ca spark frequency (CaSpF) and SR load were studied in intact and permeabilized cardiomyocytes using confocal microscopy. At baseline CaSpF is higher in S2814D vs. WT (8.7±0.4 (n=9) vs 6.44±0.3, n=8, p<0.01) without altered SR Ca load. Activation of endogenous CaMKII (1.2 μM Ca-calmodulin) in WT increases CaSpF as described by Guo et al (Circ. Res, 2006), but only to the level seen in S2814D at baseline. Moreover, CaMKII did not further increase CaSpF in S2814D myocytes. In RyR2-S2814A CaMKII activation produces a very small CaSpF increase vs WT (19 vs 60%), and that response in S2814A is secondary to increased SR Ca load (by 15%, n=10, p<0.01). In intact myocytes (as above), basal CaSpF was highest in S2814D vs. WT and S2814A (which were similar). Baseline Ca transients were not different among the groups. The cAMP activated GTP exchange factor Epac may activate CaMKII to induce SR Ca leak (Pereira et al, J Physiol, 2007). In WT mice the Epac activator 8-CPT enhanced CaSpF and decreased both SR Ca load and Ca transient amplitude (consistent with a primary effect on SR Ca leak). However, 8-CPT had no effect on any of these parameters in either S2814A or S2814D myocytes. These data indicate that RyR2-S2814 is the critical RyR2 site responsible for CaMKII-dependent enhancement of SR Ca leak and potential arrhythmogenesis in heart failure, and confirm that Epac signaling can work through this same pathway.

Increased Ca Sparks and Waves Frequency and Unchanged [Na]i in Hearts from Ankyrin-B Heterozygous Mice

The interplay between Na/Ca exchanger (NCX) and Na/K-ATPase (NKA) is essential in regulating cardiac [Na]i, [Ca]i and contractility. The membrane targeting and stability of both NCX and NKA require direct interaction with cytoskeletal protein ankyrin B (AnkB). Humans with AnkB loss-of-function mutations and AnkB heterozygous mice (AnkB+/-) display a complex cardiac phenotype that includes ventricular arrhythmias and sudden death. Cardiac myocytes from AnkB+/- mice show reduced NCX and NKA expression at the T-tubules, larger cellular and SR Ca load and increased frequency of delayed afterdepolarizations (DADs). How the coordinated loss of NCX and NKA affects SR Ca release and [Na]i is poorly understood. We measured Ca sparks and waves in myocytes from AnkB+/- and wild-type (WT) mice with and without 1 μM isoproterenol. Under control conditions, Ca transients, SR Ca load and Ca sparks frequency (CaSpF), even normalized to SR Ca content, were higher in AnkB+/- mice at all frequencies. With isoproterenol, Ca transient amplitude and SR Ca content were similar in myocytes from WT and AnkB+/- mice, but CaSpF was still elevated in AnkB+/- mice. Ca waves occurred preponderantly in AnkB+/- mice. In some cases, Ca waves and sparks evolved into synchronous after-Ca transients that are indicative of DADs. This data suggest that SR Ca release is enhanced in AnkB+/- mice. We measured [Na]i to determine whether the increased CaSpF in AnkB+/- mice is due to higher [Na]i, which may elevate diastolic Ca. [Na]i was slightly, but not significantly, lower in AnkB+/- vs. WT mice (11.4±1.1 vs. 13.2±1.7 mM in WT). Isoproterenol lowered [Na]i by a comparable amount in both AnkB+/- and WT mice (to 8.1±0.9 mM and 11.1±1.5 mM). Thus, altered [Na]i does not explain the increased Ca sparks frequency in AnkB+/- mice.

Refractoriness of Ryanodine Receptors During Calcium Alternans in Rabbit Atrial Myocytes

Electro-mechanical and Ca alternans is a known pro-arrhythmic factor. At the cellular level Ca alternans appears as cytosolic Ca transients of alternating amplitude at regular beating frequency. Direct intra-sarcoplasmic reticulum (SR) [Ca] measurements with the low affinity Ca indicator fluo-5N entrapped in the SR revealed that alternans in diastolic SR content are not a prerequisite for cytosolic Ca alternans, and thus SR Ca content is not the sole determinant of alternans. The goal of this study was to determine whether alternans of the kinetics of recovery from inactivation of ryanodine receptors and refractoriness of release represent a key factor underlying cytosolic Ca alternans. Alternans was induced by electrical pacing (1.6 to 2.5 Hz). After Ca alternans was established, pacing was stopped and the occurrence of spontaneous Ca waves and Ca sparks was quantified. The time interval from cessation of stimulation to the appearance of the first Ca waves was significantly shorter and the frequency of Ca sparks was higher after the small Ca transient compared to the large transient. Application of 0.1 mM caffeine or 10 μM isoproterenol rescued Ca alternans and shortened the rest interval until appearance of Ca waves. Photolysis of caged Ca (DM-nitrophen) to produce photolytically triggered Ca release (PTCR) from the SR was used to probe the refractoriness of SR Ca release during alternans. During the decay phase of the Ca transient PTCR was significantly less during the large Ca transient. During the rising phase of the Ca transient PTCR was greater during the large Ca transient, and was capable of inducing a phase reversal of Ca alternans. We conclude that alternating ryanodine receptor inactivation recovery intervals, together with alternations in SR Ca load, represent key determinants of Ca alternans. (VMS and CL contributed equally).

Regulation of Sarcoplasmic Reticulum Calcium Leak by Cytosolic Calcium in Rabbit Ventricular Myocytes

Diastolic sarcoplasmic reticulum (SR) Ca leak determines Ca content and, therefore, the amplitude of action potential-induced global Ca transients in ventricular myocytes. However, the pathways and properties of SR Ca leak have been poorly described. Here, we studied the effects of cytosolic [Ca] ([Ca]i) on SR Ca leak in permeabilized rabbit ventricular myocytes. Using confocal microscopy we simultaneously measured intra-SR free Ca ([Ca]SR) with fluo-5N and cytosolic Ca sparks with rhod-2, and monitored SR Ca leak as the change in [Ca]SR over time after complete SERCA inhibition with thapsigargin (10 μM). Increasing [Ca]i from 150 to 250 nM significantly increased SR Ca leak (by ∼30%) over the entire range of [Ca]SR. This increase in SR Ca leak associated with an increase in Ca spark frequency. Further increasing [Ca]i to 350 nM led to rapid [Ca]SR depletion due to the occurrence of spontaneous Ca waves. In contrast, lowering [Ca]i to 50 nM markedly decreased SR Ca leak rate (by ∼60%) and nearly abolished Ca spark activity. When the ryanodine receptor (RyR) was completely inhibited with ruthenium red (50 μM), changes in [Ca]i between 50 and 350 nM did not produce any significant effect on SR Ca leak, showing that changes of [Ca]i over a physiological range alter SR Ca leak solely by regulating RyR activity. However, decreasing [Ca]i to a lower, non-physiologically level (5 nM) activated additional SR Ca leak pathway(s) that were insensitive to RyR or SR Ca ATPase inhibition. In summary, [Ca]i plays an important role in regulating SR Ca leak by activating RyR and preventing Ca leak through unspecified pathways.

Increased Ca2+ leak and spatiotemporal coherence of Ca2+ release in cardiomyocytes during β‐adrenergic stimulation

beta-Adrenergic receptor (beta-AR) stimulation of cardiac muscle has been proposed to enhance Ca(2+) release from the sarcoplasmic reticulum (SR) through ryanodine receptors (RyRs). However, the anticipated increase in RyR Ca(2+) sensitivity has proven difficult to study in intact cardiomyocytes, due to accompanying alterations in SR Ca(2+) content, inward Ca(2+) current (I(Ca)) and diastolic cytosolic Ca(2+) concentration ([Ca(2+)](i)). Here, we studied whole-cell Ca(2+) release and spontaneous Ca(2+) leak (Ca(2+) sparks) in guinea-pig ventricular myocytes with confocal Ca(2+) imaging before and during beta-AR stimulation by isoproterenol (Iso), but under otherwise nearly identical experimental conditions. The extent of SR Ca(2+) loading was controlled under whole-cell voltage-clamp conditions. UV flash-induced uncaging of Ca(2+) from DM-nitrophen was employed as an invariant trigger for whole-cell Ca(2+) release. At matched SR Ca(2+) content, we found that Iso enhanced the spatiotemporal coherence of whole-cell Ca(2+) release, evident from spatially intercorrelated release and accelerated release kinetics that resulted in moderately (20%) increased release amplitude. This may arise from higher RyR Ca(2+) sensitivity, and was also reflected in spontaneous SR Ca(2+) leak. At comparable SR Ca(2+) content and cytosolic [Ca(2+)](i), we observed an approximately 4-fold increase in Ca(2+) spark frequency in Iso that also appeared in quiescent cells within 2 min without increased SR Ca(2+) content. This was likely to have been mediated by Ca(2+)/calmodulin-dependent protein kinase (CaMKII), rather than cAMP dependent protein kinase (PKA). We conclude that Iso increases the propensity of RyRs to open, both in response to rapid elevations of [Ca(2+)](i) and at diastolic [Ca(2+)](i). While this could be beneficial in enhancing and synchronizing systolic whole-cell SR Ca(2+) release, the same behaviour could also be proarrhythmogenic during diastole.

Dantrolene, a Therapeutic Agent for Malignant Hyperthermia, Inhibits Catecholaminergic Polymorphic Ventricular Tachycardia in a RyR2R2474S/+ Knock-In Mouse Model

Dantrolene, a specific agent for the treatment of malignant hyperthermia, was found to inhibit Ca(2+) leak through not only the skeletal ryanodine receptor (RyR1), but also the cardiac ryanodine receptor (RyR2) by correcting the defective inter-domain interaction between N-terminal (1-619 amino acid) and central (2,000-2,500 amino acid) domains of RyRs. Here, the in vivo anti-arrhythmic effect of dantrolene in a human catecholaminergic polymorphic ventricular tachycardia (CPVT)-associated RyR2(R2474S/+) knock-in (KI) mouse model was investigated. ECG was monitored in KI mice (n=6) and wild-type (WT) mice (n=6), before and after an injection of epinephrine (1.0mg/kg) or on exercise using a treadmill. In all KI (but not WT) mice, bi-directional ventricular tachycardia (VT) was induced after an injection of epinephrine or on exercise. Pre-treatment with dantrolene (for 7-10 days) significantly inhibited the inducible VT (P<0.01). In KI cardiomyocytes, Ca(2+) spark frequency (SpF; s(-1)·100µm(-1): 5.8±0.3, P<0.01) was much more increased after the addition of isoproterenol than in WT cardiomyocytes (SpF: 3.6±0.2). The increase in SpF seen in KI cardiomyocytes was attenuated by 1.0µmol/L dantrolene (SpF: 3.6±0.5, P<0.01). Dantrolene prevents CPVT, presumably by inhibiting Ca(2+) leak through the RyR2.

Subcellular Mechanisms of Early Impaired Calcium Homeostasis with Chronic Beta1-Adrenergic Stimulation in Mice

Chronic beta-adrenergic stimulation leads to heart failure (HF). In mice overexpressing beta1-adrenoceptors (TG), increased diastolic Ca load in cardiomyocytes at early age is pivotal for the development of HF. The mechanisms underlying intracellular Ca dysregulation are unclear. We examined cytosolic Ca transients (Fluo4-AM, field stimulation), Na-Ca-exchanger (NCX) function and protein expression, cytosolic Na (SBFI) and T-tubular structures (Di8-ANEPPS) in cardiomyocytes from young (8-16 wks) TG mice and wildtype (WT) littermates.Results: Systolic [Ca] amplitude was unchanged, time to peak [Ca] (140±5 vs. 127±3 ms) and [Ca] decay (time constant, tau, 223±16 vs. 182±9 ms) were significantly prolonged in TG vs. WT. Diastolic Ca leak from the SR (quantified as tetracaine-sensitive change in diastolic [Ca] or diastolic Ca spark frequency) was unchanged. However, cytosolic Ca removal by NCX during coffein application was significantly slower (tau, 3683±337 in TG vs. 2304±272 ms in WT), indicating reduced forward mode NCX activity. NCX protein expression was unchanged. Preliminary results indicate increased cytosolic [Na] in young TG. Furthermore, confocal line scans revealed delayed (> 15 ms until half-maximal) systolic Ca release in 24.7±2.6 (TG) vs. 4.6±1.4% (WT) of the intracellular regions (n=32 and 31 cells, resp., p<0.01). The extent of dyssynchronous Ca release correlated with time to peak systolic [Ca] (R=0.51, P<0.001) and was associated with a lower density and increased irregularity of T-tubules in TG (22.8±1.6% of cell volume in TG vs. 26.1±2.5% in WT). In summary, in early HF remodeling with chronic beta1-adrenergic stimulation, slowed cytosolic Ca clearance isnot related to increased diastolic SR Ca leak but associated with decreased NCX forward mode activity, which may be related to increased cytosolic [Na]. Reduced T-tubule density with dyssynchronous, slowed systolic Ca release additionally contribute to increased cytosolic Ca load.

Defective Interaction of Calmodulin With RyR2 Plays a Key Role in the Pathogenesis of Leaky Channel in Failing Hearts

Background: We previously reported that interaction between N-terminal1-600 and central domains2000-2500 of ryanodine receptor (RyR2) is defective (i.e. domain unzipping) in failing hearts. Here, we investigated the pathogenic role of calmodulin (CaM), one of the accessory proteins in RyR2, on Ca2+ release in pacing-induced failing hearts. Methods & Results: Cardiomyocytes were isolated from dog LV muscles {normal; 4-weeks rapid RV pacing (heart failure) }. To assess CaM binding to RyR2, sarcoplasmic reticulum (SR) was mixed with CaM-SANPAH conjugate (16 nM-1μM), followed by UV photolysis. Then, the RyR2-bound CaM was detected by Western blotting using anti-CaM antibody. The affinity of CaM binding to RyR2 was lower in failing SR than normal SR. In normal SR, the decreased CaM binding seen in failing SR was reproduced by addition of FK506, which was found to induce domain unzipping mimicking failing SR. In normal cardiomyocytes, Ca2+ spark frequency (CaSF) was markedly increased by adding FK506, but this increase was inhibited by addition of exogenous CaM. In failing cardiomyocytes, CaSF was higher than normal at baseline, but it was again reduced by exogenous CaM. Conclusion: The defective inter-domain interaction within RyR2 seems to increase spontaneous Ca2+ release events in failing SR, via the reduced CaM-binding to RyR2.

Calcium/Calmodulin-Dependent Protein Kinase II Contributes to Cardiac Arrhythmogenesis in Heart Failure

Transgenic (TG) Ca/calmodulin-dependent protein kinase II (CaMKII)delta(C) mice have heart failure and isoproterenol (ISO)-inducible arrhythmias. We hypothesized that CaMKII contributes to arrhythmias and underlying cellular events and that inhibition of CaMKII reduces cardiac arrhythmogenesis in vitro and in vivo. Under baseline conditions, isolated cardiac myocytes from TG mice showed an increased incidence of early afterdepolarizations compared with wild-type myocytes (P<0.05). CaMKII inhibition (AIP) completely abolished these afterdepolarizations in TG cells (P<0.05). Increasing intracellular Ca stores using ISO (10(-8) M) induced a larger amount of delayed afterdepolarizations and spontaneous action potentials in TG compared with wild-type cells (P<0.05). This seems to be due to an increased sarcoplasmic reticulum (SR) Ca leak because diastolic [Ca](i) rose clearly on ISO in TG but not in wild-type cells (+20+/-5% versus +3+/-4% at 10(-6) M ISO, P<0.05). In parallel, SR Ca leak assessed by spontaneous SR Ca release events showed an increased Ca spark frequency (3.9+/-0.5 versus 2.0+/-0.4 sparks per 100 microm(-1).s(-1), P<0.05). However, CaMKII inhibition (either pharmacologically using KN-93 or genetically using an isoform-specific CaMKIIdelta-knockout mouse model) significantly reduced SR Ca spark frequency, although this rather increased SR Ca content. In parallel, ISO increased the incidence of early (54% versus 4%, P<0.05) and late (86% versus 43%, P<0.05) nonstimulated events in TG versus wild-type myocytes, but CaMKII inhibition (KN-93 and KO) reduced these proarrhythmogenic events (P<0.05). In addition, CaMKII inhibition in TG mice (KN-93) clearly reduced ISO-induced arrhythmias in vivo (P<0.05). We conclude that CaMKII contributes to cardiac arrhythmogenesis in TG CaMKIIdelta(C) mice having heart failure and suggest the increased SR Ca leak as an important mechanism. Moreover, CaMKII inhibition reduces cardiac arrhythmias in vitro and in vivo and may therefore indicate a potential role for future antiarrhythmic therapies warranting further studies.

Dissociation of FKBP12.6 from ryanodine receptor type 2 is regulated by cyclic ADP-ribose but not β-adrenergic stimulation in mouse cardiomyocytes

Beta-adrenergic augmentation of Ca(2+) sparks and cardiac contractility has been functionally linked to phosphorylation-dependent dissociation of FK506 binding protein 12.6 (FKBP12.6) regulatory proteins from ryanodine receptors subtype 2 (RYR2). We used FKBP12.6 null mice to test the extent to which the dissociation of FKBP12.6 affects Ca(2+) sparks and mediates the inotropic action of isoproterenol (ISO), and to investigate the underlying mechanisms of cyclic ADP-ribose (cADPR) regulation of Ca(2+) sparks. Ca(2+) sparks and contractility were measured in cardiomyocytes and papillary muscle segments from FKBP12.6 null mice, and western blot analysis was carried out on sarcoplasmic reticulum microsomes prepared from mouse heart. Exposure to ISO resulted in a three- and two-fold increase in Ca(2+) spark frequency in wild-type (WT) and FKBP12.6 knockout (KO) myocytes, respectively, and Ca(2+) spark kinetics were also significantly altered in both types of cells. The effects of ISO on Ca(2+) spark properties in KO cells were inhibited by pre-treatment with thapsigargin or phospholamban inhibitory antibody, 2D12. Moreover, twitch force magnitude and the rate of force development were not significantly different in papillary muscles from WT and KO mice. Unlike beta-adrenergic stimulation, cADPR stimulation increased Ca(2+) spark frequency (2.8-fold) and altered spark kinetics only in WT but not in KO mice. The effect of cADPR on spark properties was not entirely blocked by pre-treatment with thapsigargin or 2D12. In voltage-clamped cells, cADPR increased the peak Ca(2+) of the spark without altering the decay time. We also noticed that basal Ca(2+) spark properties in KO mice were markedly altered compared with those in WT mice. Our data demonstrate that dissociation of FKBP12.6 from the RYR2 complex does not play a significant role in beta-adrenergic-stimulated Ca(2+) release in heart cells, whereas this mechanism does underlie the action of cADPR.

Does Deregulation Of Calcium Handling Precede Or Follow Alterations Of Cardiac Function During Progression To Heart Failure?

In heart failure (HF), abnormalities of in vivo cardiac function are closely correlated with reduced sarcoplasmic reticulum (SR) Ca release and myocyte contractility, supporting the concept that HF is a result of deranged Ca cycling. However, most studies addressing the role and mechanisms of altered Ca handling in HF have been performed at advanced stages of HF providing little information as to whether these deficiencies are causes or consequences of HF.In the present study we compared the time course of development of alterations in myocytes Ca handling, using a canine tachypacing model of chronic HF. In vivo function of left ventricle was assessed at several time points (1, 4, and >8 mo) with parallel studies in single ventricular myocytes of Ca handling utilizing Ca imaging in intact, permeabilized, and patch-clamped cells.LV fractional shortening progressively decreased by 50, 65, and 75 % of control values at 1, 4, and >8 month of tachypacing, respectively. The frequency of Ca sparks and the rate of SR Ca leak increased progressively with the duration of tachypacing, while diastolic [Ca]SR decreased with the duration of tachypacing. Na/Ca exchange activity was significantly augmented at 1 mo, and did not change thereafter. The SERCA-mediated Ca uptake and the density of peak Ca current were not changed up to >8 mo of tachypacing. Progressive decreases in the amplitude of depolarization-induced Ca transients and single-cell contractions were observed only starting at the 4th month of tachypacing.These results suggest that diminished SR Ca release follows rather than precedes deterioration of in vivo cardiac function, thus is not likely to be a cause of HF.