SR Ca Load Research Articles

Ibrutinib impairs IGF-1-dependent activation of intracellular Ca handling in isolated mouse ventricular myocytes.

The Bruton tyrosine kinase (BTK) inhibitor Ibrutinib is associated with a higher incidence of cardiotoxic side effects including heart failure (HF). Ibrutinib is capable of inhibiting PI3K/Akt signaling in neonatal rat ventricular cardiomyocytes when stimulated with insulin-like growth factor 1 (IGF-1). We therefore hypothesized that Ibrutinib might disrupt IGF-1-mediated activation of intracellular Ca handling in adult mouse cardiomyocytes by inhibiting PI3K/Akt signaling. Isolated ventricular myocytes (C57BL6/J) were exposed to IGF-1 at 10 nmol/L in the presence or absence of Ibrutinib (1 µmol/L) or Acalabrutinib (10 µmol/L; cell culture for 24 ± 2 h). Intracellular Ca handling was measured by epifluorescence (Fura-2 AM) and confocal microscopy (Fluo-4 AM). Ruptured-patch whole-cell voltage-clamp was used to measure ICa. Levels of key cardiac Ca handling proteins were investigated by immunoblots. IGF-1 significantly increased Ca transient amplitudes by ∼83% as compared to vehicle treated control cells. This was associated with unaffected diastolic Ca, enhanced SR Ca loading and increased ICa. Co-treatment with Ibrutinib attenuated both the IGF-1-mediated increase in SR Ca content and in ICa. IGF-1 treated cardiomyocytes had significantly increased levels of pS473Akt/Akt and SERCA2a expression as compared to cells concomitantly treated with IGF-1 and Ibrutinib. SR Ca release (as assessed by Ca spark frequency) was unaffected by either treatment. In order to test for potential off-target effects, second generation BTK inhibitor Acalabrutinib with greater BTK selectivity and lower cardiovascular toxicity was tested for IGF1-mediated activation of intracellular Ca handling. Acalabrutinib induced similar effects on Ca handling in IGF-1 treated cultured myocytes as Ibrutinib in regard to decreased Ca transient amplitude and slowed Ca transient decay, hence implying a functional class effect of BTK inhibitors in cardiac myocytes. Inhibition of BTK by Ibrutinib impairs IGF-1-dependent activation of intracellular Ca handling in adult ventricular mouse myocytes in the face of disrupted Akt signaling and absent SERCA2a upregulation.

Impaired Right Ventricular Calcium Cycling Is an Early Risk Factor in R14del-Phospholamban Arrhythmias.

The inherited mutation (R14del) in the calcium regulatory protein phospholamban (PLN) is linked to malignant ventricular arrhythmia with poor prognosis starting at adolescence. However, the underlying early mechanisms that may serve as prognostic factors remain elusive. This study generated humanized mice in which the endogenous gene was replaced with either human wild type or R14del-PLN and addressed the early molecular and cellular pathogenic mechanisms. R14del-PLN mice exhibited stress-induced impairment of atrioventricular conduction, and prolongation of both ventricular activation and repolarization times in association with ventricular tachyarrhythmia, originating from the right ventricle (RV). Most of these distinct electrocardiographic features were remarkably similar to those in R14del-PLN patients. Studies in isolated cardiomyocytes revealed RV-specific calcium defects, including prolonged action potential duration, depressed calcium kinetics and contractile parameters, and elevated diastolic Ca-levels. Ca-sparks were also higher although SR Ca-load was reduced. Accordingly, stress conditions induced after contractions, and inclusion of the CaMKII inhibitor KN93 reversed this proarrhythmic parameter. Compensatory responses included altered expression of key genes associated with Ca-cycling. These data suggest that R14del-PLN cardiomyopathy originates with RV-specific impairment of Ca-cycling and point to the urgent need to improve risk stratification in asymptomatic carriers to prevent fatal arrhythmias and delay cardiomyopathy onset.

Chronic β-adrenergic stimulation reverses depressed Ca handling in mice overexpressing inhibitor-2 of protein phosphatase 1

RationaleA higher expression/activity of type 1 serine/threonine protein phosphatase 1 (PP1) may contribute to dephosphorylation of cardiac regulatory proteins triggering the development of heart failure. ObjectiveHere, we tested the putatively protective effects of PP1 inhibitor-2 (I2) overexpression using a heart failure model induced by chronic β-adrenergic stimulation. Methods and resultsTransgenic (TG) and wild-type (WT) mice were subjected to isoprenaline (ISO) or isotonic NaCl solution supplied via osmotic minipumps for 7 days. I2 overexpression was associated with a depressed PP1 activity. Basal contractility was unchanged in catheterized mice and isolated cardiomyocytes between TGNaCl and WTNaCl. TGISO mice exhibited more fibrosis and a higher expression of hypertrophy marker proteins as compared to WTISO. After acute administration of ISO, the contractile response was accompanied by a higher sensitivity in TGISO as compared to WTISO. In contrast to basal contractility, the peak amplitude of [Ca]i and SR Ca load were reduced in TGNaCl as compared to WTNaCl. These effects were normalized to WT levels after chronic ISO stimulation. Cardiomyocyte relaxation and [Ca]i decay kinetics were hastened in TGISO as compared to WTISO, which can be explained by a higher phospholamban phosphorylation at Ser16. Chronic catecholamine stimulation was followed by an enhanced expression of GSK3β, whereas the phosphorylation at Ser9 was lower in TG as compared to the corresponding WT group. This resulted in a higher I2 phosphorylation that may reactivate PP1. ConclusionOur findings suggest that the basal desensitization of β-adrenergic signaling and the depressed Ca handling in TG by inhibition of PP1 is restored by a GSK3β-dependent phosphorylation of I2.

Inhibition of CaMKII Attenuates Progressing Disruption of Ca(2+) Homeostasis Upon Left Ventricular Assist Device Implantation in Human Heart Failure.

In heart failure, left ventricular assist device (LVAD) implantation is performed to ensure sufficient cardiac output. Whereas some patients are subsequently weaned from LVAD support, other patients still need heart transplantation. To elucidate underlying mechanisms, we assessed the arrhythmogenic SR-Ca(2+) leak at the time of LVAD implantation (HF-Im) and heart transplantation (HF-Tx) and evaluated the effects of CaMKII-inhibition. Human left-ventricular cardiomyocytes were isolated, paced at 1 Hz for 10 beats to ensure SR-Ca(2+) loading and scanned for diastolic Ca(2+) sparks (confocal microscopy). In HF-Im, the high diastolic spark frequency (CaSpF) of 0.76 ± 0.12 × 100 μm(-1) × s(-1) could be reduced to 0.48 ± 0.10 × 100 μm(-1) × s(-1) by CaMKII inhibition (AIP, 1 μM). The amplitude of Ca(2+) sparks, width, and length was not significantly altered. In sum, CaMKII inhibition yielded a clear tendency toward a reduction of the SR-Ca(2+) leak (n cells/patients = 76/6 vs. 108/6, P = 0.08). In HF-Tx, we detected an even higher CaSpF of 1.00 ± 0.10 100 μm(-1) × s(-1) and a higher SR-Ca(2+) leak compared with HF-Im (increase by 81 ± 33%, n cells/patients = 156/7 vs. 130/7, P < 0.05), which fits to the further decreased LV function. Here, CaMKII inhibition likewise reduced CaSpF (0.35 ± 0.09 100 μm(-1) × s(-1,) P = 0.06) and significantly reduced spark duration (n sparks/patients = 58/3 vs. 159/3, P < 0.05). Conclusively, the SR-Ca(2+) leak was reduced by 69 ± 12% in HF-Tx upon CaMKII inhibition (n cells/patients = 53/3 vs. 91/3, P < 0.05). These data show that the SR-Ca(2+) leak correlates with the development of LV function after LVAD implantation and may represent an important pathomechanism. The fact that CaMKII inhibition reduces the SR-Ca(2+) leak in HF-Tx suggests that CaMKII inhibition may be a promising option to beneficially influence clinical course after LVAD implantation.

Development of Ca Alternans in Atrial Myocytes is Modulated by Action Potential Morphology

Cardiac alternans is defined as cyclic, beat-to-beat variations in contraction force, action potential duration (APD) and cytosolic Ca transient (CaT) amplitude at constant stimulation rate.The bidirectional coupling of beat-to-beat regulation of cytosolic [Ca]i and membrane voltage (Vm) is a key causative factor for the development of alternans. The majority of previous studies have emphasized the role of [Ca]i→Vm coupling and disturbances in [Ca]i regulation as the primary cause of APD alternans. In this study we investigated how alternation in AP morphology feeds back on intracellular Ca handling (Vm→[Ca]i coupling) in single rabbit atrial myocytes. From AP recordings during pacing induced simultaneous CaT and APD alternans two AP waveforms were selected that represent a typical short and long AP of APD alternans. These AP waveforms were used as command voltages in subsequent voltage clamp experiments (AP voltage clamp). At pacing frequencies below alternans induction, using a series of short or long APs voltage clamp pulses, APD affected CaT amplitude where short APs caused slightly smaller amplitude CaTs. At higher frequencies CaT alternans could be induced with AP voltage clamp protocols in the absence of APD alternans, however the pacing frequency threshold where CaT alternans could be induced decreased with increasing APD, i.e. a long APD was capable of triggering CaT alternans at lower frequencies than a short AP. When cells were stimulated with an APD alternans protocol (APs of alternating duration as command voltage) the CaT alternans threshold was significantly lower. The data identify a contribution of Vm→[Ca]i coupling to the development and severity of CaT alternans and demonstrate that Vm→[Ca]i coupling affects Ca handling through changes in diastolic SR Ca load and voltage-dependent regulation of L-type Ca channels and Na/Ca exchange.

Ip3 Receptor Mediated Ca Release in Atrial Cells from Normal and Failing Hearts

We studied excitation-contraction coupling (ECC) and inositol-1,4,5-triphosphate (IP3)-dependent Ca release in atrial cells from normal and heart failure (HF) rabbit hearts. Elementary Ca release events (Ca puffs) from IP3 receptors (IP3Rs) were detected in permeabilized myocytes after inhibition of ryanodine-receptor mediated Ca spark activity with tetracaine. Puff activity was induced by IP3 exposure and blocked by 2-APB, identifying their origin from IP3Rs. In normal myocytes puff activity was low and restricted to the cell periphery, whereas in HF myocytes their overall frequency was increased and puffs were more prominent in the cell center. Compared to normal cells, in HF atrial myocytes diastolic [Ca]i was increased, action potential (AP)-induced Ca transients (CaTs) were larger in amplitude (primarily due to enhanced Ca release from central non-junctional sarcoplasmic reticulum, SR), propagation of activation from cell periphery to the center was accelerated, and fractional SR Ca release was increased, whereas no difference in SR Ca load was observed. These differences were largely abolished by exposure to 2-APB, suggesting an increased basal IP3 activity in HF. Angiotensin II (AngII) increased diastolic [Ca]i in normal and HF myocytes whereas the effect was more pronounced in HF. The amplitude of AP-dependent CaTs was increased in the presence of AngII or after photolytical release of caged IP3 in normal cells, whereas the opposite effect was observed in HF. The frequency of spontaneous Ca waves was increased in HF, and returned to levels comparable to normal myocytes after 2-APB exposure. In conclusion, HF atrial myocytes revealed enhanced Ca release during ecc and more frequent spontaneous Ca waves, presumably resulting from increased basal IP3R-mediated Ca release. However, additional neurohumoral stimulation with AngII, as it might occur in-vivo in cardiac disease, negatively affected atrial Ca signaling during ECC.

Ibandronate and Ventricular Arrhythmia Risk

Bisphosphonates, including ibandronate, are used in the prevention and treatment of osteoporosis. We report a case of suspected ibandronate-associated arrhythmia, following a single dose of ibandronate in a 55-year-old female. ECG at presentation revealed frequent ectopy and QT/QTc interval prolongation; at follow-up 9 months later the QT/QTc intervals were normalized. Proarrhythmic potential of ibandronate was assessed with a combination of in vivo and in vitro approaches in canines and canine ventricular myocytes. We observed late onset in vivo repolarization instability after ibandronate treatment. Myocytes superfused with ibandronate exhibited action potential duration (APD) prolongation and variability, increased early afterdepolarizations (EADs) and reduced Ito (P<0.05), with no change in IKr . Ibandronate-induced APD changes and EADs were prevented by inhibition of intracellular calcium cycling. Ibandronate increased sarcoplasmic reticulum calcium load; during washout there was an increase in calcium spark frequency and spontaneous calcium waves. Computational modeling was used to examine the observed effects of ibandronate. While reductions in Ito alone had modest effects on APD, when combined with altered RyR inactivation kinetics, the model predicted effects on APD and SR Ca(2+) load consistent with observed experimental results. Ibandronate may increase the susceptibility to ventricular ectopy and arrhythmias. Collectively these data suggest that reduced Ito combined with abnormal RyR calcium handling may result in a previously unrecognized form of drug-induced proarrhythmia.

Redox modification of ryanodine receptors by mitochondria‐derived reactive oxygen species contributes to aberrant Ca2+ handling in ageing rabbit hearts

Ageing is associated with a blunted response to sympathetic stimulation and an increased risk of arrhythmia and sudden cardiac death. Aberrant calcium (Ca(2+)) handling is an important contributor to the electrical and contractile dysfunction associated with ageing. Yet, the specific molecular mechanisms underlying abnormal Ca(2+) handling in ageing heart remain poorly understood. In this study, we used ventricular myocytes isolated from young (5-9 months) and old (4-6 years) rabbit hearts to test the hypothesis that changes in Ca(2+) homeostasis are caused by post-translational modification of ryanodine receptors (RyRs) by mitochondria-derived reactive oxygen species (ROS) generated in the ageing heart. Changes in parameters of Ca(2+) handling were determined by measuring cytosolic and intra-sarcoplasmic reticulum (SR) Ca(2+) dynamics in intact and permeabilized ventricular myocytes using confocal microscopy. We also measured age-related changes in ROS production and mitochondria membrane potential using a ROS-sensitive dye and a mitochondrial voltage-sensitive fluorescent indicator, respectively. In permeablized myocytes, ageing did not change SERCA activity and spark frequency but decreased spark amplitude and SR Ca(2+) load suggesting increased RyR activity. Treatment with the antioxidant dithiothreitol reduced RyR-mediated SR Ca(2+) leak in permeabilized myocytes from old rabbit hearts to the level comparable to young. Moreover, myocytes from old rabbits had more depolarized mitochondria membrane potential and increased rate of ROS production. Under β-adrenergic stimulation, Ca(2+) transient amplitude, SR Ca(2+) load, and latency of pro-arrhythmic spontaneous Ca(2+) waves (SCWs) were decreased while RyR-mediated SR Ca(2+) leak was increased in cardiomyocytes from old rabbits. Additionally, with β-adrenergic stimulation, scavenging of mitochondrial ROS in myocytes from old rabbit hearts restored redox status of RyRs, which reduced SR Ca(2+) leak, ablated most SCWs, and increased latency to levels comparable to young. These data indicate that an age-associated increase of ROS production by mitochondria leads to the thiol-oxidation of RyRs, which underlies the hyperactivity of RyRs and thereby shortened refractoriness of Ca(2+) release in cardiomyocytes from the ageing heart. This mechanism probably plays an important role in the increased incidence of arrhythmia and sudden death in the ageing population.

Age‐associated attenuation of autophagy underlies ryanodine receptor hyperactivity

Aging is associated with reduced autophagy and altered mitochondrial function; yet, the link between these processes and altered calcium (Ca) homeostasis remains unknown. We used ventricular myocytes from young (5–9 months) and old (4–5 years) rabbit hearts to test the hypothesis that reduced autophagy leads to dysfunctional mitochondria that produces ROS, leading to oxidation of ryanodine receptors (RyRs) and changes in Ca homeostasis. Markers of autophagy were measured via western blotting and β‐galactosidase staining. Age‐associated changes in ROS production, mitochondrial membrane potential, and Ca handling were determined in myocytes using confocal microscopy. Specific markers confirmed reduced autophagy. In aged myocytes, ROS production was increased and mitochondrial membrane was depolarized; both parameters were exacerbated with beta‐adrenergic agonist isoproterenol. Also with isoproterenol, aged myocytes showed decreased Ca transient amplitude, SR Ca load, and latency of spontaneous Ca waves, which were reversed with the mitochondria‐specific antioxidant mito‐TEMPO. These data suggest that age‐related attenuation of autophagy may lead to mitochondrial dysfunction that underlies increased thiol‐oxidation of RyRs. This process could explain the reduced RyR refractoriness leading to spontaneous Ca release in senescent cardiomyocytes making aged hearts more prone to arrhythmia.

Ankyrin-B reduction enhances Ca spark-mediated SR Ca release promoting cardiac myocyte arrhythmic activity

Ankyrin-B (AnkB) loss-of-function may cause ventricular arrhythmias and sudden cardiac death in humans. Cardiac myocytes from AnkB heterozygous mice (AnkB(+/-)) show reduced expression and altered localization of Na/Ca exchanger (NCX) and Na/K-ATPase (NKA), key players in regulating [Na](i) and [Ca](i). Here we investigate how AnkB reduction affects cardiac [Na](i), [Ca](i) and SR Ca release. We found reduced NCX and NKA transport function but unaltered [Na](i) and diastolic [Ca](i) in myocytes from AnkB(+/-) vs. wild-type (WT) mice. Ca transients, SR Ca content and fractional SR Ca release were larger in AnkB(+/-) myocytes. The frequency of spontaneous, diastolic Ca sparks (CaSpF) was significantly higher in intact myocytes from AnkB(+/-) vs. WT myocytes (with and without isoproterenol), even when normalized for SR Ca load. However, total ryanodine receptor (RyR)-mediated SR Ca leak (tetracaine-sensitive) was not different between groups. Thus, in AnkB(+/-) mice SR Ca leak is biased towards more Ca sparks (vs. smaller release events), suggesting more coordinated openings of RyRs in a cluster. This is due to local cytosolic RyR regulation, rather than intrinsic RyR differences, since CaSpF was similar in saponin-permeabilized myocytes from WT and AnkB(+/-) mice. The more coordinated RyRs openings resulted in an increased propensity of pro-arrhythmic Ca waves in AnkB(+/-) myocytes. In conclusion, AnkB reduction alters cardiac Na and Ca transport and enhances the coupled RyR openings, resulting in more frequent Ca sparks and waves although the total SR Ca leak is unaffected. This could enhance the propensity for triggered arrhythmias in AnkB(+/-) mice.

Effect of Ryanodine Receptor Refractoriness on the Induction of Calcium Alternans

A numerical model of the rabbit ventricular cardiomyocyte is developed, that properly reproduces calcium alternans at high pacing frequency and experimental postrest-potentiation. We show that calcium alternans may appear due to the alternation in the number of RyR2 ready to open before each beat and not, despite the fact they are present, to oscillations in the SR Ca load. This mechanism is present when activation rates of RyR2 are low and is also favored by a slowing of the RyR2 recovery time from inactivation. Generally, changes in RyR2 refractoriness lead to alternans via two different mechanisms, (the already established) SR Ca2+ load and (a new one) recovery form inactivation. Drugs or mutations which target RyR2 refractoriness should be arrythmogenic via the activation of one of those mechanisms if they decrease the activation or inactivation rate.

Synchronization of Local Calcium Releases by Beta-Adrenergic Stimulation in Cardiac Pacemaker Cells

Sympathetic heart rate acceleration is a complex orchestration of intercellular, subcellular and molecular events. According to a modern concepts, rhythmic local Ca2+releases (LCRs), generated by a subcellular “Ca-clock”, interact with a “membrane-voltage-clock”, forming a coupled-clock system that produces rhythmic action potentials (AP). We imaged LCRs using a fast-2D-camera with simultaneous AP recording via perforated-patch-clamping to explore how β-AR stimulation modulates the phase and synchronization of the ensemble of individual LCRs within a given diastole to accelerate spontaneous beating rate. In single rabbit SA-node cells (36oC) isoproterenol (100nM): (i) synchronized LCR occurrence [the standard deviation of individual LCR periods (time from Ca2+transient peak to LCR occurrence) decreased (−30%; baseline,71.5ms vs. ISO,50ms)], (ii) shifted the LCR phases to earlier time-point, (iii) shifted “Ca-nadir”, i.e. time-point when AP-induced Ca2+-transient decay ceases and [Ca2+] begins to increase due to the occurrence of LCRs (time from maximum-diastolic-potential to Ca-nadir: baseline, 103ms vs. ISO,86ms); (iv) accelerated diastolic depolarization (baseline,0.09mV/ms vs. ISO,0.15mV/ms); (v) shortened spontaneous cycle length (baseline,420ms vs. ISO,345ms). We previously had demonstrated a close relationship between the decay of the global AP-induced Ca2+ transient and the LCR phase (within a given cycle) and the AP cycle length; we now found that the decays of each LOCAL Ca2+Transient (LCaT) induced by the prior AP vary in different subcellular locations/neighborhoods and that isoproterenol not only accelerated LCaT-decays (average time constant τ: baseline, 104 vs. ISO,87ms) but also synchronized LCaT-decays among neighborhoods (τ's SD/mean: baseline,40.5% vs. ISO,33.7%). More synchronous LCaT-decay mirrors more synchronous local SR Ca2+-pumping and therefore more synchronous achievement of the threshold SR Ca load required for spontaneous LCR occurrence. Thus, earlier and more synchronized LCR occurrence under β-AR stimulation insures an earlier and larger Na+/Ca2+-exchanger current that ultimately accelerates diastolic depolarization rate.

Dual role of junctin in the regulation of ryanodine receptors and calcium release in cardiac ventricular myocytes

Junctin, a 26 kDa intra-sarcoplasmic reticulum (SR) protein, forms a quaternary complex with triadin, calsequestrin and the ryanodine receptor (RyR) at the junctional SR membrane. The physiological role for junctin in the luminal regulation of RyR Ca(2+) release remains unresolved, but it appears to be essential for proper cardiac function since ablation of junctin results in increased ventricular automaticity. Given that the junctin levels are severely reduced in human failing hearts, we performed an in-depth study of the mechanisms affecting intracellular Ca(2+) homeostasis in junctin-deficient cardiomyocytes. In concurrence with sparks, JCN-KO cardiomyocytes display increased Ca(2+) transient amplitude, resulting from increased SR [Ca(2+)] ([Ca(2+)](SR)). Junctin ablation appears to affect how RyRs 'sense' SR Ca(2+) load, resulting in decreased diastolic SR Ca(2+) leak despite an elevated [Ca(2+)](SR). Surprisingly, the β-adrenergic enhancement of [Ca(2+)](SR) reverses the decrease in RyR activity and leads to spontaneous Ca(2+) release, evidenced by the development of spontaneous aftercontractions. Single channel recordings of RyRs from WT and JCN-KO cardiac SR indicate that the absence of junctin produces a dual effect on the normally linear response of RyRs to luminal [Ca(2+)]: at low luminal [Ca(2+)] (<1 mmol l(-1)), junctin-devoid RyR channels are less responsive to luminal [Ca(2+)]; conversely, high luminal [Ca(2+)] turns them hypersensitive to this form of channel modulation. Thus, junctin produces complex effects on Ca(2+) sparks, transients, and leak, but the luminal [Ca(2+)]-dependent dual response of junctin-devoid RyRs demonstrates that junctin normally acts as an activator of RyR channels at low luminal [Ca(2+)], and as an inhibitor at high luminal [Ca(2+)]. Because the crossover occurs at a [Ca(2+)](SR) that is close to that present in resting cells, it is possible that the activator-inhibitor role of junctin may be exerted under periods of prevalent parasympathetic and sympathetic activity, respectively.

CGMP reduces the sarcoplasmic reticulum Ca2+ loading in airway smooth muscle cells: a putative mechanism in the regulation of Ca2+ by cGMP

Ca(2+) and cGMP have opposite roles in many physiological processes likely due to a complex negative feedback regulation between them. Examples of opposite functions induced by Ca(2+) and cGMP are smooth muscle contraction and relaxation, respectively. A main Ca(2+) storage involved in contraction is sarcoplasmic reticulum (SR); nevertheless, the role of cGMP in the regulation of SR-Ca(2+) has not been completely understood. To evaluate this role, intracellular Ca(2+) concentration ([Ca(2+)]i) was determinated by a ratiometric method in isolated myocytes from bovine trachea incubated with Fura-2/AM. The release of Ca(2+) from SR induced by caffeine was transient, whereas caffeine withdrawal was followed by a [Ca(2+)]i undershoot. Caffeine-induced Ca(2+) transient peak and [Ca(2+)]i undershoot after caffeine were reproducible in the same cell. Dibutyryl cGMP (db-cGMP) blocked the [Ca(2+)]i undershoot and reduced the subsequent caffeine peak (SR-Ca(2+) loading). Both, the opening of SR channels with ryanodine (10 μM) and the blockade of SR-Ca(2+) ATPase with cyclopiazonic acid inhibited the [Ca(2+)]i undershoot as well as the SR-Ca(2+) loading. The addition of db-cGMP to ryanodine (10 μM) incubated cells partially restored the SR-Ca(2+) loading. Cyclic GMP enhanced [Ca(2+)]i undershoot induced by the blockade of ryanodine channels with 50 μM ryanodine. In conclusion, the reduction of SR-Ca(2+) content in airway smooth muscle induced by cGMP can be explained by the combination of SR-Ca(2+) loading and the simultaneous release of SR-Ca(2+). The reduction of SR-Ca(2+) content induced by cGMP might be a putative mechanism limiting releasable Ca(2+) in response to a particular stimulus.

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.

Suppression of Cardiac L-Type Ca Channel Activity Causes Cardiac Hypertrophy

Increased L type Ca channel (LTCC) activity was proposed to induce Ca dependent hypertrophy and heart failure by activating CaMK and calcineurin (CaN) signaling. Previous experiments showed that upregulation of LTCC activity induced profound cardiomyopathy. However, the enhanced Ca influx did not activate CaN or hypertrophy; but led to necrotic myocyte death. We hypothesized that reducing LTCC by genetic deletion of 50% of Cav1.2 would be cardioprotective like β-adrenergic receptor or LTCC blockers. Surprisingly, this deletion enhanced disease, CaN activation and hypertrophy. These myocytes with reduced Ca current exhibited significantly increased diastolic Ca. To further address these surprising findings we analyzed the Ca dynamics of mice with a standard null allele crossed with a cardiac specific flox allele (α1cf/-Cre), which led to an even greater deletion of the α1c protein in the heart. Here we measured Ca handling dynamics in α1cf/-Cre mice. Twitch amplitudes were significantly reduced (50%) and relaxation time was prolonged (16%). SR Ca load was reduced significantly (∼25%). These findings may be explained by increased diastolic SR Ca leak that we measured in myocytes from α1cf/-Cre mice. Thus, the deletion of the α1c protein in mouse cardiac myocytes leads to an increased basal [Ca]i by increasing Ca leak from the SR. Interestingly, the CaMKII inhibitor KN93 completely reversed the effects of the reduced LTCC: twitch amplitude and relaxation as well as SR Ca load were rescued. These results suggest that resting [Ca]i and the gain in ECC are increased to maintain contractility. This increase might drive hypertrophy by activating CaN and CaMKII.

Dantrolene Restores Altered RyR2-Mediated Ca Signaling in Heart Failure

In heart failure (HF) arrhythmogenic Ca release and chronic [Ca]SR depletion arise due to altered function of ryanodine receptors. Dantrolene, a therapeautic agent used to treat malignant hyperthermia associated with mutations of the type 1 ryanodine receptor (RyR1), is purported to be without effects on the cardiac type 2 ryanodine receptor (RyR2). However, recent investigations suggest that dantrolene may correct abnormal RyR2-mediated calcium release associated with HF. In this investigation, we tested if dantrolene exerts anti-arrhythmic effects on heart failure ventricular myocytes by examining the intra-SR Ca threshold for arrhythmogenic Ca waves. Using the low-affinity calcium indicator fluo-5N entrapped in the SR, direct measurement of [Ca]SR showed that in normal rabbit myocytes dantrolene (1 microM) had no effect on SR Ca content, the amplitude of action potential induced intra-SR Ca depletions, or on the threshold for spontaneous Ca wave initiation (i.e., the SR Ca content at which spontaneous waves initiate). Furthermore, in field stimulated (0.5 Hz and 1.0 Hz) normal cardiomyocytes loaded with indo-1, dantrolene treatment had no effect on Ca transient amplitude, SR Ca load, or post-rest decay of SR Ca content. In cardiomyocytes from failing rabbit hearts, SR Ca content and the wave initiation threshold were decreased compared to normal myocytes. Interestingly, treatment of HF cardiomyocytes with dantrolene restored the SR Ca content and increased the wave initiation threshold. Together, these data suggest that dantrolene may exert anti-arrhythmic effects in heart failure cardiomyocytes by increasing the intra-SR Ca threshold at which spontaneous Ca waves occur.

Herzinsuffizienz

Heart failure (HF) is a disease with an increasing prevalence and results in both reduced quality of life and decreased lifespan for patients. Despite improved therapy mortality remains very high. HF is induced by events that lead to reduced function of the heart, e.g. myocardial infarction and increased chronic afterload through arterial hypertension. For compensation to occur, neurohumoral mechanisms temporarily maintain cardiac function. Over time this results in left ventricular remodelling and, by means of a vicious circle, compensated HF becomes symptomatic HF. The myocardium of patients with HF is characterised by a dysfunction in excitation-contraction coupling (ECC), which causes reduced cell contractility due to reduced Ca(2+) transients and SR Ca(2+) load. The Ca(2+)/calmodulin-dependent protein kinase IIδ (CaMKIIδ) plays an important role in the onset of HF. CaMKIIδ phosphorylates several functional key proteins, leads to reduced SR Ca(2+) load and Ca(2+)-transients in HF, acts as an arrhythmogenic protein by increasing late I(Na), and contributes to diastolic dysfunction by accumulation of intracellular Ca(2+). CaMKIIδ also plays an important role in atrial fibrillation. Interestingly, with regard to increased cardiac load, CaMKIIδ is activated in increased afterload but not in preload. The important role of CaMKIIδ in HF implies new therapeutic options to improve HF therapy in the future.

Spontaneous Ca waves in ventricular myocytes from failing hearts depend on Ca2+-calmodulin-dependent protein kinase II

Increased cardiac ryanodine receptor (RyR)-dependent diastolic SR Ca leak is present in heart failure and in conditions when adrenergic tone is high. Increasing Ca leak from the SR could result in spontaneous Ca wave (SCaW) formation. SCaWs activate the inward Na/Ca exchanger (NCX) current causing a delayed afterdepolarization (DAD), potentially leading to arrhythmia. Here we examine SCaWs in ventricular myocytes isolated from failing and healthy rabbit hearts. Myocytes from healthy hearts did not exhibit SCaWs under baseline conditions versus 43% of those exposed to isoproterenol (ISO). This ISO-induced increase in activity was reversed by inhibition of Ca-calmodulin-dependent protein kinase II (CaMKII) by KN93. Inhibition of cAMP-dependent protein kinase (PKA) by H89 had no observed effect. Of myocytes treated with forskolin 50% showed SCaW activity, attributable to a large increase in SR Ca load ([Ca]SRT) versus control. At similar [Ca]SRT (121μM) myocytes treated with ISO plus KN93 had significantly fewer SCaWs versus those treated with ISO or ISO plus H89 (0.2±0.28 vs. 1.1±0.28 and 1.29±0.39 SCaWs cell−1, respectively). In myocytes isolated from failing hearts ISO induced an increase in the percentage of cells generating SCaWs vs. baseline (74% vs. 11%) with no increase in [Ca]SRT. Inhibiting CaMKII reversed this effect (14%). At similar [Ca]SRT (71µM) myocytes treated with ISO or ISO plus H89 had significantly more SCaWs per cell vs. untreated (2.5±0.5; 1.6±0.7 vs. 0.36±0.3, respectively). Treatment with ISO plus KN93 completely abolished this effect. The evidence suggests the ISO-dependent increase in SCaW activity in both healthy and failing myocytes is CaMKII-dependent, implicating CaMKII in arrhythmogenesis.

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.