Isolated Cardiac Ryanodine Receptor Function Varies Between Mammals.

Concerted robust opening of cardiac ryanodine receptors' (RyR2) Ca2+ release 1oplasmic reticulum (SR) is fundamental for normal systolic cardiac function. During diastole, infrequent spontaneous RyR2 openings mediate the SR Ca2+ leak that normally constrains SR Ca2+ load. Abnormal large diastolic RyR2-mediated Ca2+ leak events can cause delayed after depolarizations (DADs) and arrhythmias. The RyR2-associated mechanisms underlying these processes are being extensively studied at multiple levels utilizing various model animals. Since there are well-described species-specific differences in cardiac intracellular Ca2+ handing in situ, we tested whether or not single RyR2 function in vitro retains this species specificity. We isolated RyR2-rich heavy SR microsomes from mouse, rat, rabbit, and human ventricular muscle and quantified RyR2 function using identical solutions and methods. The single RyR2 cytosolic Ca2+ sensitivity was similar across these species. However, there were significant species differences in single RyR2 mean open times in both systole and diastole-like solutions. In diastole-like solutions, single rat/mouse RyR2 open probability and frequency of long openings (> 6 ms) were similar, but these values were significantly greater than those of either single rabbit or human RyR2s. We propose these in vitro single RyR2 functional differences across species stem from the species-specific RyR2 regulatory environment present in the source tissue. Our results show the single rabbit RyR2 functional attributes, particularly in diastole-like conditions, replicate those of single human RyR2 best among the species tested.

Inositol 1,4,5-trisphosphate receptor - reactive oxygen signaling domain regulates excitation-contraction coupling in atrial myocytes.

The inositol 1,4,5-trisphosphate receptor (InsP3R) is up-regulated in patients with atrial fibrillation (AF) and InsP3-induced Ca2+ release (IICR) is linked to pro-arrhythmic spontaneous Ca2+ release events. Nevertheless, knowledge of the physiological relevance and regulation of InsP3Rs in atrial muscle is still limited. We hypothesize that InsP3R and NADPH oxidase 2 (NOX2) form a functional signaling domain where NOX2 derived reactive oxygen species (ROS) regulate InsP3R agonist affinity and thereby Ca2+ release. To quantitate the contribution of IICR to atrial excitation-contraction coupling (ECC) atrial myocytes (AMs) were isolated from wild type and NOX2 deficient (Nox2-/-) mice and changes in the cytoplasmic Ca2+ concentration ([Ca2+]i; fluo-4/AM, indo-1) or ROS (2',7'-dichlorofluorescein, DCF) were monitored by fluorescence microscopy. Superfusion of AMs with Angiotensin II (AngII: 1 µmol/L) significantly increased diastolic [Ca2+]i (F/F0, Ctrl: 1.00 ± 0.01, AngII: 1.20 ± 0.03; n = 7; p < 0.05), the field stimulation induced Ca2+ transient (CaT) amplitude (ΔF/F0, Ctrl: 2.00 ± 0.17, AngII: 2.39 ± 0.22, n = 7; p < 0.05), and let to the occurrence of spontaneous increases in [Ca2+]i. These changes in [Ca2+]i were suppressed by the InsP3R blocker 2-aminoethoxydiphenyl-borate (2-APB; 1 µmol/L). Concomitantly, AngII induced an increase in ROS production that was sensitive to the NOX2 specific inhibitor gp91ds-tat (1 µmol/L). In NOX2-/- AMs, AngII failed to increase diastolic [Ca2+]i, CaT amplitude, and the frequency of spontaneous Ca2+ increases. Furthermore, the enhancement of CaTs by exposure to membrane permeant InsP3 was abolished by NOX inhibition with apocynin (1 µM). AngII induced IICR in Nox2-/- AMs could be restored by addition of exogenous ROS (tert-butyl hydroperoxide, tBHP: 5 µmol/L). In saponin permeabilized AMs InsP3 (5 µmol/L) induced Ca2+ sparks that increased in frequency in the presence of ROS (InsP3: 9.65 ± 1.44 sparks*s-1*(100µm)-1; InsP3 + tBHP: 10.77 ± 1.5 sparks*s-1*(100µm)-1; n = 5; p < 0.05). The combined effect of InsP3 + tBHP was entirely suppressed by 2-APB and Xestospongine C (XeC). Changes in IICR due to InsP3R glutathionylation induced by diamide could be reversed by the reducing agent dithiothreitol (DTT: 1 mmol/L) and prevented by pretreatment with 2-APB, supporting that the ROS-dependent post-translational modification of the InsP3R plays a role in the regulation of ECC. Our data demonstrate that in AMs the InsP3R is under dual control of agonist induced InsP3 and ROS formation and suggest that InsP3 and NOX2-derived ROS co-regulate atrial IICR and ECC in a defined InsP3R/NOX2 signaling domain.

JNK2, a Newly-Identified SERCA2 Enhancer, Augments an Arrhythmic [Ca2+]SR Leak-Load Relationship.

RATIONALE: We recently discovered pivotal contributions of stress kinase JNK2 (c-Jun N-terminal kinase isoform 2) in increased risk of atrial fibrillation through enhanced diastolic sarcoplasmic reticulum (SR) calcium (Ca2+) leak via RyR2 (ryanodine receptor isoform 2). However, the role of JNK2 in the function of the SERCA2 (SR Ca2+-ATPase), essential in maintaining SR Ca2+ content cycling during each heartbeat, is completely unknown. OBJECTIVE: To test the hypothesis that JNK2 increases SERCA2 activity SR Ca2+ content and exacerbates an arrhythmic SR Ca2+ content leak-load relationship. METHODS AND RESULTS: We used confocal Ca2+ imaging in myocytes and HEK-RyR2 (ryanodine receptor isoform 2-expressing human embryonic kidney 293 cells) cells, biochemistry, dual Ca2+/voltage optical mapping in intact hearts from alcohol-exposed or aged mice (where JNK2 is activated). We found that JNK2, but not JNK1 (c-Jun N-terminal kinase isoform 1), increased SERCA2 uptake and consequently elevated SR Ca2+ content load. JNK2 also associates with and phosphorylates SERCA2 proteins. JNK2 causally enhances SERCA2-ATPase activity via increased maximal rate, without altering Ca2+ affinity. Unlike the CaMKII (Ca2+/calmodulin-dependent kinase II)-dependent JNK2 action in SR Ca2+ leak, JNK2-driven SERCA2 function was CaMKII independent (not prevented by CaMKII inhibition). With CaMKII blocked, the JNK2-driven SR Ca2+ loading alone did not significantly raise leak. However, with JNK2-CaMKII-driven SR Ca2+ leak present, the JNK2-enhanced SR Ca2+ uptake limited leak-induced reduction in SR Ca2+, normalizing Ca2+ transient amplitude, but at a higher arrhythmogenic SR Ca2+ leak. JNK2-specific inhibition completely normalized SR Ca2+ handling, attenuated arrhythmic Ca2+ activities, and alleviated atrial fibrillation susceptibility in aged and alcohol-exposed myocytes and intact hearts. CONCLUSIONS: We have identified a novel JNK2-induced activation of SERCA2. The dual action of JNK2 in CaMKII-dependent arrhythmic SR Ca2+ leak and a CaMKII-independent uptake exacerbates atrial arrhythmogenicity, while helping to maintain normal levels of Ca2+ transients and heart function. JNK2 modulation may be a novel therapeutic target for atrial fibrillation prevention and treatment.

Loss of p21-activated kinase 1 (Pak1) promotes atrial arrhythmic activity.

BACKGROUND: Atrial fibrillation (AF) is initiated through arrhythmic atrial excitation from outside the sinus node or remodeling of atrial tissue that allows reentry of excitation. Angiotensin II (AngII) has been implicated in the initiation and maintenance of AF through changes in Ca2+ handling and production of reactive oxygen species (ROS). OBJECTIVE: We aimed to determine the role of p21-activated kinase 1 (Pak1), a downstream target in the AngII signaling cascade, in atrial electrophysiology and arrhythmia. METHODS: Wild-type and Pak1-/- mice were used to determine atrial function in vivo on the organ and cellular level by quantification of electrophysiological and Ca2+ handling properties. RESULTS: We demonstrate that reduced Pak1 activity increases the inducibility of atrial arrhythmia in vivo and in vitro. On the cellular level, Pak1-/- atrial myocytes (AMs) exhibit increased basal and AngII (1 µM)-induced ROS production, sensitivity to the NADPH oxidase-2 (NOX2) inhibitors gp91ds-tat and apocynin (1 µM), and enhanced membrane translocation of Ras-related C3 substrate 1 (Rac1) that is part of the multimolecular NOX2 complex. Upon stimulation with AngII, Pak1-/- AMs exhibit an exaggerated increase in the intracellular Calcium concentration ([Ca2+]i) and arrhythmic events that were sensitive to sodium-calcium exchanger (NCX) inhibitors (KB-R7943 and SEA0400; 1 µM) and suppressed in AMs from NOX2-deficient (gp91phox-/-) mice. Pak1 stimulation (FTY720; 200 nM) in wild-type AMs and AMs from a canine model of ventricular tachypacing-induced AF prevented AngII-induced arrhythmic Ca2+ overload by attenuating NCX activity in a NOX2-dependent manner. CONCLUSION: The experimental results support that Pak1 stimulation can attenuate NCX-dependent Ca2+ overload and prevent triggered arrhythmic activity by suppressing NOX2-dependent ROS production.

p21-Activated kinase1 (Pak1) is a negative regulator of NADPH-oxidase 2 in ventricular myocytes.

Ischemic conditions reduce the activity of the p21-activated kinase (Pak1) resulting in increased arrhythmic activity. Triggered arrhythmic activity during ischemia is based on changes in cellular ionic balance and the cells Ca(2+) handling properties. In the current study we used isolated mouse ventricular myocytes (VMs) deficient for the expression of Pak1 (Pak1(-/-)) to determine the mechanism by which Pak1 influences the generation of arrhythmic activity during simulated ischemia. The Ca(2+) transient amplitude and kinetics did not significantly change in wild type (WT) and Pak1(-/-) VMs during 15 min of simulated ischemia. However, Pak1(-/-) VMs exhibited an exaggerated increase in [Ca(2+)]i, which resulted in spontaneous Ca(2+) release events and waves. The Ca(2+) overload in Pak1(-/-) VMs could be suppressed with a reverse mode blocker (KB-R7943) of the sodium calcium exchanger (NCX), a cytoplasmic scavenger of reactive oxygen species (ROS; TEMPOL) or a RAC1 inhibitor (NSC23766). Measurements of the cytoplasmic ROS levels revealed that decreased Pak1 activity in Pak1(-/-) VMs or VMs treated with the Pak1 inhibitor (IPA3) enhanced cellular ROS production. The Pak1 dependent increase in ROS was attenuated in VMs deficient for NADPH oxidase 2 (NOX2; p47(phox-/-)) or in VMs where NOX2 was inhibited (gp91ds-tat). Voltage clamp recordings showed increased NCX activity in Pak1(-/-) VMs that depended on enhanced NOX2 induced ROS production. The exaggerated Ca(2+) overload in Pak1(-/-) VMs could be mimicked by low concentrations of ouabain. Overall our data show that Pak1 is a critical negative regulator of NOX2 dependent ROS production and that a latent ROS dependent stimulation of NCX activity can predispose VMs to Ca(2+) overload under conditions where no significant changes in excitation-contraction coupling are yet evident.

The C-terminus of the long AKAP13 isoform (AKAP-Lbc) is critical for development of compensatory cardiac hypertrophy.

The objective of this study was to determine the role of A-Kinase Anchoring Protein (AKAP)-Lbc in the development of heart failure, by investigating AKAP-Lbc-protein kinase D1 (PKD1) signaling in vivo in cardiac hypertrophy. Using a gene-trap mouse expressing a truncated version of AKAP-Lbc (due to disruption of the endogenous AKAP-Lbc gene), that abolishes PKD1 interaction with AKAP-Lbc (AKAP-Lbc-ΔPKD), we studied two mouse models of pathological hypertrophy: i) angiotensin (AT-II) and phenylephrine (PE) infusion and ii) transverse aortic constriction (TAC)-induced pressure overload. Our results indicate that AKAP-Lbc-ΔPKD mice exhibit an accelerated progression to cardiac dysfunction in response to AT-II/PE treatment and TAC. AKAP-Lbc-ΔPKD mice display attenuated compensatory cardiac hypertrophy, increased collagen deposition and apoptosis, compared to wild-type (WT) control littermates. Mechanistically, reduced levels of PKD1 activation are observed in AKAP-Lbc-ΔPKD mice compared to WT mice, resulting in diminished phosphorylation of histone deacetylase 5 (HDAC5) and decreased hypertrophic gene expression. This is consistent with a reduced compensatory hypertrophy phenotype leading to progression of heart failure in AKAP-Lbc-ΔPKD mice. Overall, our data demonstrates a critical in vivo role for AKAP-Lbc-PKD1 signaling in the development of compensatory hypertrophy to enhance cardiac performance in response to TAC-induced pressure overload and neurohumoral stimulation by AT-II/PE treatment.

Ischemia/Reperfusion injury protection by mesenchymal stem cell derived antioxidant capacity.

Mesenchymal stem cell (MSC) transplantation after ischemia/reperfusion (I/R) injury reduces infarct size and improves cardiac function. We used mouse ventricular myocytes (VMs) in an in vitro model of I/R to determine the mechanism by which MSCs prevent reperfusion injury by paracrine signaling. Exposure of mouse VMs to an ischemic challenge depolarized their mitochondrial membrane potential (Ψmito), increased their diastolic Ca(2+), and significantly attenuated cell shortening. Reperfusion of VMs with Ctrl tyrode or MSC-conditioned tyrode (ConT) resulted in a transient increase of the Ca(2+) transient amplitudes in all cells. ConT-reperfused cells exhibited a decreased number early after depolarization (EADs) (ConT: 6.3% vs. Ctrl: 28.4%) and prolonged survival (ConT: 58% vs. Ctrl: 33%). Ψmito rapidly recovered in Ctrl as well as ConT-treated VMs on reperfusion; however, in Ctrl solution, an exaggerated hyperpolarization of Ψmito was determined that preceded the collapse of Ψmito. The ability of ConT to attenuate the hyperpolarization of Ψmito was suppressed on inhibition of the PI3K/Akt signaling pathway or IK,ATP. However, protection of Ψmito was best mimicked by the reactive oxygen species (ROS) scavenger mitoTEMPO. Analysis of ConT revealed a significant antioxidant capacity that was linked to the presence of extracellular superoxide dismutase (SOD3) in ConT. In conclusion, MSC ConT protects VMs from simulated I/R injury by its SOD3-mediated antioxidant capacity and by delaying the recovery of Ψmito through Akt-mediated opening of IK,ATP. These changes attenuate reperfusion-induced ROS production and prevent the opening of the permeability transition pore and arrhythmic Ca(2+) release.

Functional integrity of the T-tubular system in cardiomyocytes depends on p21-activated kinase 1.

p21-activated kinase (Pak1), a serine-threonine protein kinase, regulates cytoskeletal dynamics and cell motility. Recent experiments further demonstrate that loss of Pak1 results in exaggerated hypertrophic growth in response to pathophysiological stimuli. Calcium (Ca) signaling plays an important role in the regulation of transcription factors involved in hypertrophic remodeling. Here we aimed to determine the role of Pak1 in cardiac excitation-contraction coupling (ECC). Ca transients were recorded in isolated, ventricular myocytes (VMs) from WT and Pak1(-/-) mice. Pak1(-/-) Ca transients had a decreased amplitude, prolonged rise time and delayed recovery time. Di-8-ANNEPS staining revealed a decreased T-tubular density in Pak1(-/-) VMs that coincided with decreased cell capacitance and increased dis-synchrony of Ca induced Ca release (CICR) at individual release units. These changes were not observed in atrial myocytes of Pak1(-/-) mice where the T-tubular system is only sparsely developed. Experiments in cultured rabbit VMs supported a role of Pak1 in the maintenance of the T-tubular structure. T-tubular density in rabbit VMs significantly decreased within 24h of culture. This was accompanied by a decrease of the Ca transient amplitude and a prolongation of its rise time. However, overexpression of constitutively active Pak1 in VMs attenuated the structural remodeling as well as changes in ECC. The results provide significant support for a prominent role of Pak1 activity not only in the functional regulation of ECC but for the structural maintenance of the T-tubular system whose remodeling is an integral feature of hypertrophic remodeling.

While systolic cardiomyocyte function is preserved, diastolic myocyte function and recovery from acidosis are impaired in CaMKIIδ-KO mice.

OBJECTIVE: CaMKII contributes to impaired contractility in heart failure by inducing SR Ca(2+)-leak. CaMKII-inhibition in the heart was suggested to be a novel therapeutic principle. Different CaMKII isoforms exist. Specifically targeting CaMKIIδ, the dominant isoform in the heart, could be of therapeutic potential without impairing other CaMKII isoforms. RATIONALE: We investigated whether cardiomyocyte function is affected by isoform-specific knockout (KO) of CaMKIIδ under basal conditions and upon stress, i.e. upon ß-adrenergic stimulation and during acidosis. RESULTS: Systolic cardiac function was largely preserved in the KO in vivo (echocardiography) corresponding to unchanged Ca(2+)-transient amplitudes and isolated myocyte contractility in vitro. CaMKII activity was dramatically reduced while phosphatase-1 inhibitor-1 was significantly increased. Surprisingly, while diastolic Ca(2+)-elimination was slower in KO most likely due to decreased phospholamban Thr-17 phosphorylation, frequency-dependent acceleration of relaxation was still present. Despite decreased SR Ca(2+)-reuptake at lower frequencies, SR Ca(2+)-content was not diminished, which might be due to reduced diastolic SR Ca(2+)-loss in the KO as a consequence of lower RyR Ser-2815 phosphorylation. Challenging KO myocytes with isoproterenol showed intact inotropic and lusitropic responses. During acidosis, SR Ca(2+)-reuptake and SR Ca(2+)-loading were significantly impaired in KO, resulting in an inability to maintain systolic Ca(2+)-transients during acidosis and impaired recovery. CONCLUSIONS: Inhibition of CaMKIIδ appears to be safe under basal physiologic conditions. Specific conditions exist (e.g. during acidosis) under which CaMKII-inhibition might not be helpful or even detrimental. These conditions will have to be more clearly defined before CaMKII inhibition is used therapeutically.

SR-targeted CaMKII inhibition improves SR Ca²+ handling, but accelerates cardiac remodeling in mice overexpressing CaMKIIδC.

Cardiac myocyte overexpression of CaMKIIδ(C) leads to cardiac hypertrophy and heart failure (HF) possibly caused by altered myocyte Ca(2+) handling. A central defect might be the marked CaMKII-induced increase in diastolic sarcoplasmic reticulum (SR) Ca(2+) leak which decreases SR Ca(2+) load and Ca(2+) transient amplitude. We hypothesized that inhibition of CaMKII near the SR membrane would decrease the leak, improve Ca(2+) handling and prevent the development of contractile dysfunction and HF. To test this hypothesis we crossbred CaMKIIδ(C) overexpressing mice (CaMK) with mice expressing the CaMKII-inhibitor AIP targeted to the SR via a modified phospholamban (PLB)-transmembrane-domain (SR-AIP). There was a selective decrease in the amount of activated CaMKII in the microsomal (SR/membrane) fraction prepared from these double-transgenic mice (CaMK/SR-AIP) mice. In ventricular cardiomyocytes from CaMK/SR-AIP mice, SR Ca(2+) leak, assessed both as diastolic Ca(2+) shift into SR upon tetracaine in intact myocytes or integrated Ca(2+) spark release in permeabilized myocytes, was significantly reduced. The reduced leak was accompanied by enhanced SR Ca(2+) load and twitch amplitude in double-transgenic mice (vs. CaMK), without changes in SERCA expression or NCX function. However, despite the improved myocyte Ca(2+) handling, cardiac hypertrophy and remodeling was accelerated in CaMK/SR-AIP and cardiac function worsened. We conclude that while inhibition of SR localized CaMKII in CaMK mice improves Ca(2+) handling, it does not necessarily rescue the HF phenotype. This implies that a non-SR CaMKIIδ(C) exerts SR-independent effects that contribute to hypertrophy and HF, and this CaMKII pathway may be exacerbated by the global enhancement of Ca transients.

The IP3 receptor regulates cardiac hypertrophy in response to select stimuli.

RATIONALE: Inositol 1,4,5-trisphosphate (IP(3)) is a second messenger that regulates intracellular Ca(2+) release through IP(3) receptors located in the sarco(endo)plasmic reticulum of cardiac myocytes. Many prohypertrophic G protein-coupled receptor (GPCR) signaling events lead to IP(3) liberation, although its importance in transducing the hypertrophic response has not been established in vivo. OBJECTIVE: Here, we generated conditional, heart-specific transgenic mice with both gain- and loss-of-function for IP(3) receptor signaling to examine its hypertrophic growth effects following pathological and physiological stimulation. METHODS AND RESULTS: Overexpression of the mouse type-2 IP(3) receptor (IP(3)R2) in the heart generated mild baseline cardiac hypertrophy at 3 months of age. Isolated myocytes from overexpressing lines showed increased Ca(2+) transients and arrhythmias in response to endothelin-1 stimulation. Although low levels of IP(3)R2 overexpression failed to augment/synergize cardiac hypertrophy following 2 weeks of pressure-overload stimulation, such levels did enhance hypertrophy following 2 weeks of isoproterenol infusion, in response to Galphaq overexpression, and/or in response to exercise stimulation. To inhibit IP(3) signaling in vivo, we generated transgenic mice expressing an IP(3) chelating protein (IP(3)-sponge). IP(3)-sponge transgenic mice abrogated cardiac hypertrophy in response to isoproterenol and angiotensin II infusion but not pressure-overload stimulation. Mechanistically, IP(3)R2-enhanced cardiac hypertrophy following isoproterenol infusion was significantly reduced in the calcineurin-Abeta-null background. CONCLUSION: These results indicate that IP(3)-mediated Ca(2+) release plays a central role in regulating cardiac hypertrophy downstream of GPCR signaling, in part, through a calcineurin-dependent mechanism.

Impaired contractile function and calcium handling in hearts of cardiac-specific calcineurin b1-deficient mice.

To define the necessity of calcineurin (Cn) signaling for cardiac maturation and function, the postnatal phenotype of mice with cardiac-specific targeted ablation of the Cn B1 regulatory subunit (Ppp3r1) gene (csCnb1(-/-) mice) was characterized. csCnb1(-/-) mice develop a lethal cardiomyopathy, characterized by impaired postnatal growth of the heart and combined systolic and diastolic relaxation abnormalities, despite a lack of structural derangements. Notably, the csCnb1(-/-) hearts did not exhibit diastolic dilatation, despite the severe functional phenotype. Myocytes isolated from the mutant mice exhibited reduced rates of contraction/relaxation and abnormalities in calcium transients, consistent with altered sarcoplasmic reticulum loading. Levels of sarco(endo) plasmic reticulum Ca-ATPase 2a (Atp2a2) and phospholamban were normal, but phospholamban phosphorylation was markedly reduced at Ser(16) and Thr(17). In addition, levels of the Na/Ca exchanger (Slc8a1) were modestly reduced. These results define a novel mouse model of cardiac-specific Cn deficiency and demonstrate novel links between Cn signaling, postnatal growth of the heart, pathological ventricular remodeling, and excitation-contraction coupling.

Akt regulates L-type Ca2+ channel activity by modulating Cavalpha1 protein stability.

The insulin IGF-1-PI3K-Akt signaling pathway has been suggested to improve cardiac inotropism and increase Ca(2+) handling through the effects of the protein kinase Akt. However, the underlying molecular mechanisms remain largely unknown. In this study, we provide evidence for an unanticipated regulatory function of Akt controlling L-type Ca(2+) channel (LTCC) protein density. The pore-forming channel subunit Ca(v)alpha1 contains highly conserved PEST sequences (signals for rapid protein degradation), and in-frame deletion of these PEST sequences results in increased Ca(v)alpha1 protein levels. Our findings show that Akt-dependent phosphorylation of Ca(v)beta2, the LTCC chaperone for Ca(v)alpha1, antagonizes Ca(v)alpha1 protein degradation by preventing Ca(v)alpha1 PEST sequence recognition, leading to increased LTCC density and the consequent modulation of Ca(2+) channel function. This novel mechanism by which Akt modulates LTCC stability could profoundly influence cardiac myocyte Ca(2+) entry, Ca(2+) handling, and contractility.

Arrhythmogenic effects of beta2-adrenergic stimulation in the failing heart are attributable to enhanced sarcoplasmic reticulum Ca load.

Ventricular tachycardia in heart failure (HF) can initiate by nonreentrant mechanisms such as delayed afterdepolarizations. In an arrhythmogenic rabbit model of HF, we have shown that isoproterenol induces ventricular tachycardia in vivo and aftercontractions and transient inward currents in HF myocytes. To determine whether beta(2)-adrenergic receptor (beta(2)-AR) stimulation contributes, we performed in vivo drug infusion, in vitro myocyte and biochemical studies. Intravenous zinterol (2.5 microg/kg) led to ventricular arrhythmias, including ventricular tachycardia up to 13 beats long in 4 of 6 HF rabbits (versus 0 of 5 controls, P<0.01), an effect blocked by beta(2)-AR antagonist ICI-118,551 (0.2 mg/kg). In field-stimulated myocytes (0.5 to 4 Hz, 37 degrees C), beta(2)-AR stimulation (1 micromol/L zinterol+300 nmol/L beta(1)-AR antagonist CGP-29712A) induced aftercontractions and Ca aftertransients in 88% of HF versus 0% of control myocytes (P<0.01). beta(2)-AR stimulation in HF (but not control) myocytes increased Ca transient amplitude (by 29%), sarcoplasmic reticulum (SR) Ca load (by 28%), the rate of [Ca](i) decline (by 28%; n=12, all P<0.05), and phospholamban phosphorylation at Ser16, but Ca current was unchanged. All of these effects in HF myocytes were blocked by ICI-118,551 (100 nmol/L). Although total beta-AR expression was reduced by 47% in HF rabbit left ventricle, beta(2)-AR number was unchanged, indicating more potent beta(2)-AR-dependent SR Ca uptake and arrhythmogenesis in HF. Human HF myocytes showed similar beta(2)-AR-induced aftercontractions, aftertransients, and enhanced Ca transient amplitude, SR Ca load and twitch [Ca](i) decline rate. Thus, beta(2)-AR stimulation is arrhythmogenic in HF, mediated by SR Ca overload-induced spontaneous SR Ca release and aftercontractions.

CaMKII inhibition targeted to the sarcoplasmic reticulum inhibits frequency-dependent acceleration of relaxation and Ca2+ current facilitation.

Cardiac Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) in heart has been implicated in Ca(2+) current (I(Ca)) facilitation, enhanced sarcoplasmic reticulum (SR) Ca(2+) release and frequency-dependent acceleration of relaxation (FDAR) via enhanced SR Ca(2+) uptake. However, questions remain about how CaMKII may work in these three processes. Here we tested the role of CaMKII in these processes using transgenic mice (SR-AIP) that express four concatenated repeats of the CaMKII inhibitory peptide AIP selectively in the SR membrane. Wild type mice (WT) and mice expressing AIP exclusively in the nucleus (NLS-AIP) served as controls. Increasing stimulation frequency produced typical FDAR in WT and NLS-AIP, but FDAR was markedly inhibited in SR-AIP. Quantitative analysis of cytosolic Ca(2+) removal during [Ca(2+)](i) decline revealed that FDAR is due to an increased apparent V(max) of SERCA. CaMKII-dependent RyR phosphorylation at Ser2815 and SR Ca(2+) leak was both decreased in SR-AIP vs. WT. This decrease in SR Ca(2+) leak may partly balance the reduced SERCA activity leading to relatively unaltered SR-Ca(2+) load in SR-AIP vs. WT myocytes. Surprisingly, CaMKII regulation of the L-type Ca(2+) channel (I(Ca) facilitation and recovery from inactivation) was abolished by the SR-targeted CaMKII inhibition in SR-AIP mice. Inhibition of CaMKII effects on I(Ca) and RyR function by the SR-localized AIP places physical constraints on the localization of these proteins at the junctional microdomain. Thus SR-targeted CaMKII inhibition can directly inhibit the activation of SR Ca(2+) uptake, SR Ca(2+) release and I(Ca) by CaMKII, effects which have all been implicated in triggered arrhythmias.

Cardiac alternans do not rely on diastolic sarcoplasmic reticulum calcium content fluctuations.

Cardiac alternans are thought to be a precursor to life-threatening arrhythmias. Previous studies suggested that alterations in sarcoplasmic reticulum (SR) Ca2+ content are either causative or not associated with myocyte Ca2+ alternans. However, those studies used indirect measures of SR Ca2+. Here we used direct continuous measurement of intra-SR free [Ca2+] ([Ca2+]SR) (using Fluo5N) during frequency-dependent Ca2+ alternans in rabbit ventricular myocytes. We tested the hypothesis that alternating [Ca2+]SR is required for Ca2+ alternans. Amplitudes of [Ca2+]SR depletions alternated in phase with cytosolic Ca2+ transients and contractions. Some cells showed clear alternation in diastolic [Ca2+]SR during alternans, with higher [Ca2+]SR before the larger SR Ca2+ releases. However, the extent of SR Ca2+ release during the small beats was smaller than expected for the modest decrease in [Ca2+]SR. In other cells, clear Ca2+ alternans was observed without alternations in diastolic [Ca2+]SR. Additionally, alternating cells were observed, in which diastolic [Ca2+]SR fluctuations occurred interspersed by depletions in which the amplitude was unrelated to the preceding diastolic [Ca2+]SR. In all forms of alternans, the SR Ca2+ release rate was higher during large depletions than during small depletions. Although [Ca2+]SR exerts major influence on SR Ca2+ release, alternations in [Ca2+](SR) are not required for Ca2+ alternans to occur. Rather, it seems likely that some other factor, such as ryanodine receptor availability after a prior beat (eg, recovery from inactivation), is of greater importance in initiating frequency-induced Ca2+ alternans. However, once such a weak SR Ca2+ release occurs, it can result in increased [Ca2+]SR and further enhance SR Ca2+ release at the next beat. In this way, diastolic [Ca2+]SR alternans can enhance frequency-induced Ca2+ alternans, even if they initiate by other means.

The inotropic effect of cardioactive glycosides in ventricular myocytes requires Na+-Ca2+ exchanger function.

Glycoside-induced cardiac inotropy has traditionally been attributed to direct Na(+)-K(+)-ATPase inhibition, causing increased intracellular [Na(+)] and consequent Ca(2+) gain via the Na(+)-Ca(2+) exchanger (NCX). However, recent studies suggested alternative mechanisms of glycoside-induced inotropy: (1) direct activation of sarcoplasmic reticulum Ca(2+) release channels (ryanodine receptors; RyRs); (2) increased Ca(2+) selectivity of Na(+) channels (slip-mode conductance); and (3) other signal transduction pathways. None of these proposed mechanisms requires NCX or an altered [Na(+)] gradient. Here we tested the ability of ouabain (OUA, 3 microm), digoxin (DIG, 20 microm) or acetylstrophanthidin (ACS, 4 microm) to alter Ca(2+) transients in completely Na(+)-free conditions in intact ferret and cat ventricular myocytes. We also tested whether OUA directly activates RyRs in permeabilized cat myocytes (measuring Ca(2+) sparks by confocal microscopy). In intact ferret myocytes (stimulated at 0.2 Hz), DIG and ACS enhanced Ca(2+) transients and cell shortening during twitches, as expected. However, prior depletion of [Na(+)](i) (in Na(+)-free, Ca(2+)-free solution) and in Na(+)-free solution (replaced by Li(+)) the inotropic effects of DIG and ACS were completely prevented. In voltage-clamped cat myocytes, OUA increased Ca(2+) transients by 48 +/- 4% but OUA had no effect in Na(+)-depleted cells (replaced by N-methyl-d-glucamine). In permeabilized cat myocytes, OUA did not change Ca(2+) spark frequency, amplitude or spatial spread (although spark duration was slightly prolonged). We conclude that the acute inotropic effects of DIG, ACS and OUA (and the effects on RyRs) depend on the presence of Na(+) and a functional NCX in ferret and cat myocytes (rather than alternate Na(+)-independent mechanisms).

Targeted inhibition of sarcoplasmic reticulum CaMKII activity results in alterations of Ca2+ homeostasis and cardiac contractility.

Transgenic (TG) mice expressing a Ca2+/calmodulin-dependent protein kinase II (CaMKII) inhibitory peptide targeted to the cardiac myocyte longitudinal sarcoplasmic reticulum (LSR) display reduced phospholamban phosphorylation at Thr17 and develop dilated myopathy when stressed by gestation and parturition (Ji Y, Li B, Reed TD, Lorenz JN, Kaetzel MA, and Dedman JR. J Biol Chem 278: 25063-25071, 2003). In the present study, these animals (TG) are evaluated for the effect of inhibition of sarcoplasmic reticulum (SR) CaMKII activity on the contractile characteristics and Ca2+ cycling of myocytes. Analysis of isolated work-performing hearts demonstrated moderate decreases in the maximal rates of contraction and relaxation (+/-dP/dt) in TG mice. The response of the TG hearts to increases in load is reduced. The TG hearts respond to isoproterenol (Iso) in a dose-dependent manner; the contractile properties were reduced in parallel to wild-type hearts. Assessment of isolated cardiomyocytes from TG mice revealed 40-47% decrease in the maximal rates of myocyte shortening and relengthening under both basal and Iso-stimulated conditions. Although twitch Ca2+ transient amplitudes were not significantly altered, the rate of twitch intracellular Ca2+ concentration decline was reduced by approximately 47% in TG myocytes, indicating decreased SR Ca2+ uptake function. Caffeine-induced Ca2+ transients indicated unaltered SR Ca2+ content and Na+/Ca2+ exchange function. Phosphorylation assays revealed an approximately 30% decrease in the phosphorylation of ryanodine receptor Ser2809. Iso stimulation increased the phosphorylation of both phospholamban Ser16 and the ryanodine receptor Ser2809 but not phospholamban Thr17 in TG mice. This study demonstrates that inhibition of SR CaMKII activity at the LSR results in alterations in cardiac contractility and Ca2+ handling in TG hearts.

Transgenic CaMKIIdeltaC overexpression uniquely alters cardiac myocyte Ca2+ handling: reduced SR Ca2+ load and activated SR Ca2+ release.

Ca2+/calmodulin-dependent protein kinase II (CaMKII) delta is the predominant cardiac isoform, and the deltaC splice variant is cytoplasmic. We overexpressed CaMKIIdeltaC in mouse heart and observed dilated heart failure and altered myocyte Ca2+ regulation in 3-month-old CaMKIIdeltaC transgenic mice (TG) versus wild-type littermates (WT). Heart/body weight ratio and cardiomyocyte size were increased about 2-fold in TG versus WT. At 1 Hz, twitch shortening, [Ca2+]i transient amplitude, and diastolic [Ca2+]i were all reduced by approximately 50% in TG versus WT. This is explained by >50% reduction in SR Ca2+ content in TG versus WT. Peak Ca2+ current (ICa) was slightly increased, and action potential duration was prolonged in TG versus WT. Despite lower SR Ca2+ load and diastolic [Ca2+]i, fractional SR Ca2+ release was increased and resting spontaneous SR Ca2+ release events (Ca2+ sparks) were doubled in frequency in TG versus WT (with prolonged width and duration, but lower amplitude). Enhanced Ca2+ spark frequency was also seen in TG at 4 weeks (before heart failure onset). Acute CaMKII inhibition normalized Ca2+ spark frequency and ICa, consistent with direct CaMKII activation of ryanodine receptors (and ICa) in TG. The rate of [Ca2+]i decline during caffeine exposure was faster in TG, indicating enhanced Na+-Ca2+ exchange function (consistent with protein expression measurements). Enhanced diastolic SR Ca2+ leak (via sparks), reduced SR Ca2+-ATPase expression, and increased Na+-Ca2+ exchanger explain the reduced diastolic [Ca2+]i and SR Ca2+ content in TG. We conclude that CaMKIIdeltaC overexpression causes acute modulation of excitation-contraction coupling, which contributes to heart failure.

Frequency-dependent acceleration of relaxation in the heart depends on CaMKII, but not phospholamban.

Frequency-dependent acceleration of relaxation (FDAR) is an intrinsic physiological mechanism, which allows more rapid ventricular diastolic filling at higher heart rates. FDAR is also observed in isolated myocardial trabeculae and cardiac myocytes, but its mechanism is still poorly understood. We tested the hypothesis that FDAR results mainly from Ca/calmodulin-dependent protein kinase II (CaMKII) dependent stimulation of sarcoplasmic reticulum (SR) Ca transport, but does not require phospholamban. Experiments were performed at 23 or 35 degrees C in isolated ventricular muscle and single myocytes from wild-type (WT) and phospholamban knockout (PLB-KO) mice and rat ventricular myocytes. Isometric twitch force of muscles and unloaded shortening and Ca transients in myocytes were measured ([Ca](o)=1mM) in the absence and presence of CaMKII inhibitors (1 microM KN-93 or 20 microM autocamtide-2 related inhibitory peptide, AIP). Stimulation frequency was altered over a wide range (0.2-8Hz) and post-rest vs steady state twitches were also compared. In both WT and PLB-KO mouse muscles FDAR of twitch force was prominent, but was largely suppressed by KN-93. FDAR of twitch contractions was associated with FDAR of Ca transients in PLB-KO myocytes, and both were inhibited by KN-93. Similarly, a different CaMKII inhibitor (AIP) inhibited FDAR of contraction and Ca transients in rat ventricular myocytes. We conclude that FDAR results mainly from CaMKII-dependent stimulation of SR Ca transport, but does not require phospholamban.