Stefanie Walther

Na+-dependent SR Ca2+ overload induces arrhythmogenic events in mouse cardiomyocytes with a human CPVT mutation

AIMS Mutations in the cardiac ryanodine receptor Ca(2+) release channel, RyR2, underlie catecholaminergic polymorphic ventricular tachycardia (CPVT), an inherited life-threatening arrhythmia. CPVT is triggered by spontaneous RyR2-mediated sarcoplasmic reticulum (SR) Ca(2+) release in response to SR Ca(2+) overload during beta-adrenergic stimulation. However, whether elevated SR Ca(2+) content--in the absence of protein kinase A activation--affects RyR2 function and arrhythmogenesis in CPVT remains elusive. METHODS AND RESULTS Isolated murine ventricular myocytes harbouring a human RyR2 mutation (RyR2(R4496C+/-)) associated with CPVT were investigated in the absence and presence of 1 micromol/L JTV-519 (RyR2 stabilizer) followed by 100 micromol/L ouabain intervention to increase cytosolic [Na(+)] and SR Ca(2+) load. Changes in membrane potential and intracellular [Ca(2+)] were monitored with whole-cell patch-clamping and confocal Ca(2+) imaging, respectively. At baseline, action potentials (APs), Ca(2+) transients, fractional SR Ca(2+) release, and SR Ca(2+) load were comparable in wild-type (WT) and RyR2(R4496C+/-) myocytes. Ouabain evoked significant increases in diastolic [Ca(2+)], peak systolic [Ca(2+)], fractional SR Ca(2+) release, and SR Ca(2+) content that were quantitatively similar in WT and RyR2(R4496C+/-) myocytes. Ouabain also induced arrhythmogenic events, i.e. spontaneous Ca(2+) waves, delayed afterdepolarizations and spontaneous APs, in both groups. However, the ouabain-induced increase in the frequency of arrhythmogenic events was dramatically larger in RyR2(R4496C+/-) when compared with WT myocytes. JTV-519 greatly reduced the frequency of ouabain-induced arrhythmogenic events. CONCLUSION The elevation of SR Ca(2+) load--in the absence of beta-adrenergic stimulation--is sufficient to increase the propensity for triggered arrhythmias in RyR2(R4496C+/-) cardiomyocytes. Stabilization of RyR2 by JTV-519 effectively reduces these triggered arrhythmias.

Endothelin-1 enhances nuclear Ca2+ transients in atrial myocytes through Ins(1,4,5)P3-dependent Ca2+ release from perinuclear Ca2+ stores.

Nuclear Ca2+ plays a key role in the regulation of gene expression. Inositol (1,4,5)-trisphosphate [Ins(1,4,5)P3)] might be an important regulator of nuclear Ca2+ but its contribution to nuclear Ca2+ signalling in adult cardiomyocytes remains elusive. We tested the hypothesis that endothelin-1 enhances nuclear Ca2+ concentration transients (CaTs) in rabbit atrial myocytes through Ins(1,4,5)P3-induced Ca2+ release from perinuclear stores. Cytoplasmic and nuclear CaTs were measured simultaneously in electrically stimulated atrial myocytes using confocal Ca2+ imaging. Nuclear CaTs were significantly slower than cytoplasmic CaTs, indicative of compartmentalisation of intracellular Ca2+ signalling. Endothelin-1 elicited a preferential (10 nM) or a selective (0.1 nM) increase in nuclear versus cytoplasmic CaTs. This effect was abolished by inhibition of endothelin-1 receptors, phospholipase C and Ins(1,4,5)P3 receptors. Fractional Ca2+ release from the sarcoplasmic reticulum and perinuclear stores was increased by endothelin-1 at an otherwise unaltered Ca2+ load. Comparable increases of cytoplasmic CaTs induced by β-adrenoceptor stimulation or elevation of extracellular Ca2+ could not mimic the endothelin-1 effects on nuclear CaTs, suggesting that endothelin-1 specifically modulates nuclear Ca2+ signalling. Thus, endothelin-1 enhances nuclear CaTs in atrial myocytes by increasing fractional Ca2+ release from perinuclear stores. This effect is mediated by the coupling of endothelin receptor A to PLC-Ins(1,4,5)P3 signalling and might contribute to excitation-transcription coupling.

In Situ Calibration of Nucleoplasmic versus Cytoplasmic Ca2+ Concentration in Adult Cardiomyocytes

Quantification of subcellularly resolved Ca²⁺ signals in cardiomyocytes is essential for understanding Ca²⁺ fluxes in excitation-contraction and excitation-transcription coupling. The properties of fluorescent indicators in intracellular compartments may differ, thus affecting the translation of Ca²⁺-dependent fluorescence changes into [Ca²⁺] changes. Therefore, we determined the in situ characteristics of a frequently used Ca²⁺ indicator, Fluo-4, and a ratiometric Ca²⁺ indicator, Asante Calcium Red, and evaluated their use for reporting and quantifying cytoplasmic and nucleoplasmic Ca²⁺ signals in isolated cardiomyocytes. Ca²⁺ calibration curves revealed significant differences in the apparent Ca²⁺ dissociation constants of Fluo-4 and Asante Calcium Red between cytoplasm and nucleoplasm. These parameters were used for transformation of fluorescence into nucleoplasmic and cytoplasmic [Ca²⁺]. Resting and diastolic [Ca²⁺] were always higher in the nucleoplasm. Systolic [Ca²⁺] was usually higher in the cytoplasm, but some cells (15%) exhibited higher systolic [Ca²⁺] in the nucleoplasm. Ca²⁺ store depletion or blockade of Ca²⁺ leak pathways eliminated the resting [Ca²⁺] gradient between nucleoplasm and cytoplasm, whereas inhibition of inositol 1,4,5-trisphosphate receptors by 2-APB reversed it. The results suggest the presence of significant nucleoplasmic-to-cytoplasmic [Ca²⁺] gradients in resting myocytes and during the cardiac cycle. Nucleoplasmic [Ca²⁺] in cardiomyocytes may be regulated via two mechanisms: diffusion from the cytoplasm and active Ca²⁺ release via inositol 1,4,5-trisphosphate receptors from perinuclear Ca²⁺ stores.

Subclinical abnormalities in sarcoplasmic reticulum Ca2+ release promote eccentric myocardial remodeling and pump failure death in response to pressure overload

OBJECTIVES This study sought to explore whether subclinical alterations of sarcoplasmic reticulum (SR) Ca(2+) release through cardiac ryanodine receptors (RyR2) aggravate cardiac remodeling in mice carrying a human RyR2(R4496C+/-) gain-of-function mutation in response to pressure overload. BACKGROUND RyR2 dysfunction causes increased diastolic SR Ca(2+) release associated with arrhythmias and contractile dysfunction in inherited and acquired cardiac diseases, such as catecholaminergic polymorphic ventricular tachycardia and heart failure (HF). METHODS Functional and structural properties of wild-type and catecholaminergic polymorphic ventricular tachycardia-associated RyR2(R4496C+/-) hearts were characterized under conditions of pressure overload induced by transverse aortic constriction (TAC). RESULTS Wild-type and RyR2(R4496C+/-) hearts had comparable structural and functional properties at baseline. After TAC, RyR2(R4496C+/-) hearts responded with eccentric hypertrophy, substantial fibrosis, ventricular dilation, and reduced fractional shortening, ultimately resulting in overt HF. RyR2(R4496C+/-)-TAC cardiomyocytes showed increased incidence of spontaneous SR Ca(2+) release events, reduced Ca(2+) transient peak amplitude, and SR Ca(2+) content as well as reduced SR Ca(2+)-ATPase 2a and increased Na(+)/Ca(2+)-exchanger protein expression. HF phenotype in RyR2(R4496C+/-)-TAC mice was associated with increased mortality due to pump failure but not tachyarrhythmic events. RyR2-stabilizer K201 markedly reduced Ca(2+) spark frequency in RyR2(R4496C+/-)-TAC cardiomyocytes. Mini-osmotic pump infusion of K201 prevented deleterious remodeling and improved survival in RyR2(R4496C+/-)-TAC mice. CONCLUSIONS The combination of subclinical congenital alteration of SR Ca(2+) release and pressure overload promoted eccentric remodeling and HF death in RyR2(R4496C+/-) mice, and pharmacological RyR2 stabilization prevented this deleterious interaction. These findings suggest potential clinical relevance for patients with acquired or inherited gain-of-function of RyR2-mediated SR Ca(2+) release.

cAMP- and Ca2+/calmodulin-dependent protein kinases mediate inotropic, lusitropic and arrhythmogenic effects of urocortin 2 in mouse ventricular myocytes

BACKGROUND AND PURPOSE Urocortin 2 is beneficial in heart failure, but the underlying cellular mechanisms are not completely understood. Here we have characterized the functional effects of urocortin 2 on mouse cardiomyocytes and elucidated the underlying signalling pathways and mechanisms.

Angiotensin II and myosin light-chain phosphorylation contribute to the stretch-induced slow force response in human atrial myocardium

AIMS Stretch is an important regulator of atrial function. The functional effects of stretch on human atrium, however, are poorly understood. Thus, we characterized the stretch-induced force response in human atrium and evaluated the underlying cellular mechanisms. METHODS AND RESULTS Isometric twitch force of human atrial trabeculae (n = 252) was recorded (37 degrees C, 1 Hz stimulation) following stretch from 88 (L88) to 98% (L98) of optimal length. [Na(+)](i) and pH(i) were measured using SBFI and BCECF epifluorescence, respectively. Stretch induced a biphasic force increase: an immediate increase [first-phase, Frank-Starling mechanism (FSM)] to approximately 190% of force at L88 followed by an additional slower increase [5-10 min; slow force response (SFR)] to approximately 120% of the FSM. FSM and SFR were unaffected by gender, age, ejection fraction, and pre-medication with major cardiovascular drugs. There was a positive correlation between the amplitude of the FSM and the SFR. [Na(+)](i) rose by approximately 1 mmol/L and pH(i) remained unchanged during the SFR. Inhibition of Na(+)/H(+)-exchange (3 microM HOE642), Na(+)/Ca(2+)-exchange (5 microM KB-R7943), or stretch-activated channels (0.5 microM GsMtx-4 and 80 microM streptomycin) did not reduce the SFR. Inhibition of angiotensin-II (AngII) receptors (5 microM saralasin and 0.5 microM PD123319) or pre-application of 0.5 microM AngII, however, reduced the SFR by approximately 40-60%. Moreover, stretch increased phosphorylation of myosin light chain 2 (MLC2a) and inhibition of MLC kinase (10 microM ML-7 and 5 microM wortmannin) decreased the SFR by approximately 40-85%. CONCLUSION Stretch elicits a SFR in human atrium. The atrial SFR is mediated by stretch-induced release and autocrine/paracrine actions of AngII and increased myofilament Ca(2+) responsiveness via phosphorylation of MLC2a by MLC kinase.

Increased Heart Failure Development After Pressure Overload-Induced Hypertrophy in Mice with a RyR2-R4496C+/- Knock-In Mutation

Elevated spontaneous Ca2+ release from the sarcoplasmic reticulum (SR) Ca2+ release channel (ryanodine receptor-RyR2) due to a gain-of-function of the RyR2 defect contributes to contractile dysfunction and arrhythmias in heart failure (HF). However, whether increased diastolic SR Ca2+ release can promote HF is yet unclear.RyR2R4496C+/- mice associated with an increased SR Ca2+ leak and wild-type (WT) littermates underwent surgery with (TAC) or without (Sham) transverse aortic constriction. Transthoracic echocardiography was performed 1 and 3 weeks post-surgery. Hearts and lungs were dissected, weighed and normalized to tibia length. Gross morphology and collagen content were examined in paraffin-embedded heart slices stained with hematoxylin/eosin and picrosirius red, respectively. Arrhythmias were monitored (24h) with radiotelemetry. Data are presented as mean±S.E.M.RyR2R4496C+/--Sham hearts exhibited no gross baseline changes in ventricular structure and function and fibrosis compared to WT-Sham. 1 week after TAC, WT-TAC mice showed concentric left ventricular (LV) hypertrophy with preserved ejection fraction. In contrast, RyR2R4496C+/--TAC mice exhibited eccentric hypertrophy and significant deterioration of phenotypic changes associated with the transition to HF, such as chamber dilation (LV end-diastolic diameter;WT:-1.4±3%,RyR2R4496C+/-:19.7±3.6%,P<0.05) and reduced ejection fraction (WT:61.7±3.2%,RyR2R4496C+/-:39.4±2.8%,n=14,P<0.05). 3 weeks post-TAC, relative heart weight was increased in RyR2R4496C+/- mice vs. WT-TAC (WT:9.4±0.3mg/mm,RyR2R4496C+/-:11.3±0.7mg/mm,n=13-23,P<0.05). The HF phenotype in RyR2R4496C+/--TAC mice further aggravated 3 weeks after TAC, ultimately resulting in increased relative lung weight (WT:17±8%,RyR2R4496C+/-:101±21%,n=16-20,P<0.05). Hypertrophy continued to increase in RyR2R4496C+/--TAC mice, while it saturated in WT-TAC mice (LV mass;WT:65.2±8.1%,RyR2R4496C+/-:119±13.3%,n=14-21,P<0.05). Strikingly, RyR2R4496C+/--TAC mice did not die for a primary arrhythmic death, but with a progressive electrical deterioration resembling an electromechanical dissociation (mortality rate:60% 4 weeks post-TAC,P<0.05).In summary, HF development was facilitated in RyR2-R4496C+/- mice after pressure overload-induced hypertrophy. Thus, spontaneous SR Ca2+ leak per se may be causally related to the development of HF.

In Situ Calibration of Cytoplasmic and Nucleoplasmic Calcium Concentration in Adult Rat and Mouse Cardiac Myocytes

Quantifying subcellularly resolved Ca2+ signals in cardiac myocytes is essential for understanding Ca2+ fluxes in excitation-contraction and excitation-transcription coupling. Translation of changes in Ca2+-dependent fluorescence into changes in [Ca2+] relies on the indicators behavior in situ, but properties of fluorescent indicators in different intracellular compartments may differ. Thus, we determined the in situ calibration of a frequently used Ca2+ indicator, Fluo-4, and evaluated its use in reporting cytoplasmic and nucleoplasmic Ca2+ signals in isolated cardiac myocytes.Calibration solutions were made by mixing known quantities of EGTA and CaEGTA solutions and the free [Ca2+] was confirmed with a Ca2+-sensitive electrode. Solutions contained metabolic inhibitors and cyclopiazonic-acid (5μM) to block active Ca2+ transport and the Ca2+ ionophore A-23187 (10μM) to allow equilibration of [Ca2+] between bath solution and cell interior. Ventricular rat and mouse myocytes were loaded with Fluo-4/AM (8μM, 20min). Fluo-4 fluorescence (excitation/emission: 488/>505nm) was recorded using a Nipkow dual disc-based confocal microscope.Concentration-response curves were obtained and a significant difference in the apparent Ca2+ binding affinities (Kd) of Fluo-4 between cytoplasmic (993±56nM; 1026±65nM) and nucleoplasmic (1211±73nM; 1251±71nM) compartments was observed for both mouse and rat cells, respectively (both n=15, P<0.01). The established curves were used to transform raw Fluo-4 fluorescence signals during electrically stimulated [Ca2+] transients (1Hz, room temperature) into nucleoplasmic and cytoplasmic [Ca2+]. There was a significant difference in diastolic (121±24nM vs 149±35nM; 99±17nM vs 121±26nM) and systolic (420±148nM vs 364±102nM; 787±172nM vs 491±157nM) [Ca2+] between cytoplasmic and nucleoplasmic compartments in mouse and rat cells, respectively (both n=15; P<0.01).The results reveal that, in cardiac myocytes, the Ca2+-dependent fluorescent properties of Fluo-4 differ between cytoplasm and nucleoplasm and that significant differences between cytoplasmic and nucleoplasmic [Ca2+] exist during diastole as well as systole.