Synergistic FRET assays for drug discovery targeting RyR2 channels.

A key therapeutic target for heart failure and arrhythmia is the deleterious leak through sarcoplasmic reticulum (SR) ryanodine receptor 2 (RyR2) calcium release channels. We have previously developed methods to detect the pathologically leaky state of RyR2 in adult cardiomyocytes by monitoring RyR2 binding to either calmodulin (CaM) or a biosensor peptide (DPc10). Here, we test whether these complementary binding measurements are effective as high-throughput screening (HTS) assays to discover small molecules that target leaky RyR2. Using FRET, we developed and validated HTS procedures under conditions that mimic a pathological state, to screen the library of 1280 pharmaceutically active compounds (LOPAC) for modulators of RyR2 in cardiac SR membrane preparations. Complementary FRET assays with acceptor-labeled CaM and DPc10 were used for Hit prioritization based on the opposing binding properties of CaM vs. DPc10. This approach narrowed the Hit list to one compound, Ro 90-7501, which altered FRET to suggest increased RyR2-CaM binding and decreased DPc10 binding. Follow-up studies revealed that Ro 90-7501 does not detrimentally affect myocyte Ca2+ transients. Moreover, Ro 90-7501 partially inhibits overall Ca2+ leak, as assessed by Ca2+ sparks in permeabilized rat cardiomyocytes. Together, these results demonstrate (1) the effectiveness of our HTS approach where two complementary assays synergize for Hit ranking and (2) a drug discovery process that combines high-throughput, high-precision in vitro structural assays with in situ myocyte assays of the pathologic RyR2 leak. These provide a drug discovery platform compatible with large-scale HTS campaigns, to identify agents that inhibit RyR2 for therapeutic development.

Cardiomyocyte Na+ and Ca2+ mishandling drives vicious cycle involving CaMKII, ROS, and ryanodine receptors.

Cardiomyocyte Na+ and Ca2+ mishandling, upregulated Ca2+/calmodulin-dependent kinase II (CaMKII), and increased reactive oxygen species (ROS) are characteristics of various heart diseases, including heart failure (HF), long QT (LQT) syndrome, and catecholaminergic polymorphic ventricular tachycardia (CPVT). These changes may form a vicious cycle of positive feedback to promote cardiac dysfunction and arrhythmias. In HF rabbit cardiomyocytes investigated in this study, the inhibition of CaMKII, late Na+ current (INaL), and leaky ryanodine receptors (RyRs) all attenuated the prolongation and increased short-term variability (STV) of action potential duration (APD), but in age-matched controls these inhibitors had no or minimal effects. In control cardiomyocytes, we enhanced RyR leak (by low [caffeine] plus isoproterenol mimicking CPVT) which markedly increased STV and delayed afterdepolarizations (DADs). These proarrhythmic changes were significantly attenuated by both CaMKII inhibition and mitochondrial ROS scavenging, with a slight synergy with INaL inhibition. Inducing LQT by elevating INaL (by Anemone toxin II, ATX-II) caused markedly prolonged APD, increased STV, and early afterdepolarizations (EADs). Those proarrhythmic ATX-II effects were largely attenuated by mitochondrial ROS scavenging, and partially reduced by inhibition of CaMKII and pathological leaky RyRs using dantrolene. In human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) bearing LQT3 mutation SCN5A N406K, dantrolene significantly attenuated cell arrhythmias and APD prolongation. Targeting critical components of the Na+-Ca2+-CaMKII-ROS-INaL arrhythmogenic vicious cycle may exhibit important on-target and also trans-target effects (e.g., INaL and RyR inhibition can alter INaL-mediated LQT3 effects). Incorporating this vicious cycle into therapeutic strategies provides novel integrated insight for treating cardiac arrhythmias and diseases.

Complex electrophysiological remodeling in postinfarction ischemic heart failure.

Heart failure (HF) following myocardial infarction (MI) is associated with high incidence of cardiac arrhythmias. Development of therapeutic strategy requires detailed understanding of electrophysiological remodeling. However, changes of ionic currents in ischemic HF remain incompletely understood, especially in translational large-animal models. Here, we systematically measure the major ionic currents in ventricular myocytes from the infarct border and remote zones in a porcine model of post-MI HF. We recorded eight ionic currents during the cell's action potential (AP) under physiologically relevant conditions using selfAP-clamp sequential dissection. Compared with healthy controls, HF-remote zone myocytes exhibited increased late Na+ current, Ca2+-activated K+ current, Ca2+-activated Cl- current, decreased rapid delayed rectifier K+ current, and altered Na+/Ca2+ exchange current profile. In HF-border zone myocytes, the above changes also occurred but with additional decrease of L-type Ca2+ current, decrease of inward rectifier K+ current, and Ca2+ release-dependent delayed after-depolarizations. Our data reveal that the changes in any individual current are relatively small, but the integrated impacts shift the balance between the inward and outward currents to shorten AP in the border zone but prolong AP in the remote zone. This differential remodeling in post-MI HF increases the inhomogeneity of AP repolarization, which may enhance the arrhythmogenic substrate. Our comprehensive findings provide a mechanistic framework for understanding why single-channel blockers may fail to suppress arrhythmias, and highlight the need to consider the rich tableau and integration of many ionic currents in designing therapeutic strategies for treating arrhythmias in HF.

Stress Signaling JNK2 Crosstalk With CaMKII Underlies Enhanced Atrial Arrhythmogenesis.

RATIONALE: Atrial fibrillation (AF) is the most common arrhythmia, and advanced age is an inevitable and predominant AF risk factor. However, the mechanisms that couple aging and AF propensity remain unclear, making targeted therapeutic interventions unattainable. OBJECTIVE: To explore the functional role of an important stress response JNK (c-Jun N-terminal kinase) in sarcoplasmic reticulum Ca2+ handling and consequently Ca2+-mediated atrial arrhythmias. METHODS AND RESULTS: We used a series of cutting-edge electrophysiological and molecular techniques, exploited the power of transgenic mouse models to detail the molecular mechanism, and verified its clinical applicability in parallel studies on donor human hearts. We discovered that significantly increased activity of the stress response kinase JNK2 (JNK isoform 2) in the aged atria is involved in arrhythmic remodeling. The JNK-driven atrial proarrhythmic mechanism is supported by a pathway linking JNK, CaMKII (Ca2+/calmodulin-dependent kinase II), and sarcoplasmic reticulum Ca2+ release RyR2 (ryanodine receptor) channels. JNK2 activates CaMKII, a critical proarrhythmic molecule in cardiac muscle. In turn, activated CaMKII upregulates diastolic sarcoplasmic reticulum Ca2+ leak mediated by RyR2 channels. This leads to aberrant intracellular Ca2+ waves and enhanced AF propensity. In contrast, this mechanism is absent in young atria. In JNK challenged animal models, this is eliminated by JNK2 ablation or CaMKII inhibition. CONCLUSIONS: We have identified JNK2-driven CaMKII activation as a novel mode of kinase crosstalk and a causal factor in atrial arrhythmic remodeling, making JNK2 a compelling new therapeutic target for AF prevention and treatment.

Calcium-Dependent Arrhythmogenic Foci Created by Weakly Coupled Myocytes in the Failing Heart.

RATIONALE: Intercellular uncoupling and Ca2+ (Ca) mishandling can initiate triggered ventricular arrhythmias. Spontaneous Ca release activates inward current which depolarizes membrane potential (Vm) and can trigger action potentials in isolated myocytes. However, cell-cell coupling in intact hearts limits local depolarization and may protect hearts from this arrhythmogenic mechanism. Traditional optical mapping lacks the spatial resolution to assess coupling of individual myocytes. OBJECTIVE: We investigate local intercellular coupling in Ca-induced depolarization in intact hearts, using confocal microscopy to measure local Vm and intracellular [Ca] simultaneously. METHODS AND RESULTS: We used isolated Langendorff-perfused hearts from control (CTL) and heart failure (HF) mice (HF induced by transaortic constriction). In CTL hearts, 1.4% of myocytes were poorly synchronized with neighboring cells and exhibited asynchronous (AS) Ca transients. These AS myocytes were much more frequent in HF (10.8% of myocytes, P<0.05 versus CTL). Local Ca waves depolarized Vm in HF but not CTL hearts, suggesting weaker gap junction coupling in HF-AS versus CTL-AS myocytes. Cell-cell coupling was assessed by calcein fluorescence recovery after photobleach during intracellular [Ca] recording. All regions in CTL hearts exhibited faster calcein diffusion than in HF, with HF-AS myocyte being slowest. In HF-AS, enhancing gap junction conductance (with rotigaptide) increased coupling and suppressed Vm depolarization during Ca waves. Conversely, in CTL hearts, gap junction inhibition (carbenoxolone) decreased coupling and allowed Ca wave-induced depolarizations. Synchronization of Ca wave initiation and triggered action potentials were observed in HF hearts and computational models. CONCLUSIONS: Well-coupled CTL myocytes are effectively voltage-clamped during Ca waves, protecting the heart from triggered arrhythmias. Spontaneous Ca waves are much more common in HF myocytes and these AS myocytes are also poorly coupled, enabling local Ca-induced inward current of sufficient source strength to overcome a weakened current sink to depolarize Vm and trigger action potentials.

Genetically Encoded Biosensors Reveal PKA Hyperphosphorylation on the Myofilaments in Rabbit Heart Failure.

RATIONALE: In heart failure, myofilament proteins display abnormal phosphorylation, which contributes to contractile dysfunction. The mechanisms underlying the dysregulation of protein phosphorylation on myofilaments is not clear. OBJECTIVE: This study aims to understand the mechanisms underlying altered phosphorylation of myofilament proteins in heart failure. METHODS AND RESULTS: We generate a novel genetically encoded protein kinase A (PKA) biosensor anchored onto the myofilaments in rabbit cardiac myocytes to examine PKA activity at the myofilaments in responses to adrenergic stimulation. We show that PKA activity is shifted from the sarcolemma to the myofilaments in hypertrophic failing rabbit myocytes. In particular, the increased PKA activity on the myofilaments is because of an enhanced ß2 adrenergic receptor signal selectively directed to the myofilaments together with a reduced phosphodiesterase activity associated with the myofibrils. Mechanistically, the enhanced PKA activity on the myofilaments is associated with downregulation of caveolin-3 in the hypertrophic failing rabbit myocytes. Reintroduction of caveolin-3 in the failing myocytes is able to normalize the distribution of ß2 adrenergic receptor signal by preventing PKA signal access to the myofilaments and to restore contractile response to adrenergic stimulation. CONCLUSIONS: In hypertrophic rabbit myocytes, selectively enhanced ß2 adrenergic receptor signaling toward the myofilaments contributes to elevated PKA activity and PKA phosphorylation of myofilament proteins. Reintroduction of caveolin-3 is able to confine ß2 adrenergic receptor signaling and restore myocyte contractility in response to ß adrenergic stimulation.

Quantitative analysis of the Ca2+ -dependent regulation of delayed rectifier K+ current IKs in rabbit ventricular myocytes.

KEY POINTS: [Ca2+ ]i enhanced rabbit ventricular slowly activating delayed rectifier K+ current (IKs ) by negatively shifting the voltage dependence of activation and slowing deactivation, similar to perfusion of isoproterenol. Rabbit ventricular rapidly activating delayed rectifier K+ current (IKr ) amplitude and voltage dependence were unaffected by high [Ca2+ ]i . When measuring or simulating IKs during an action potential, IKs was not different during a physiological Ca2+ transient or when [Ca2+ ]i was buffered to 500 nm. ABSTRACT: The slowly activating delayed rectifier K+ current (IKs ) contributes to repolarization of the cardiac action potential (AP). Intracellular Ca2+ ([Ca2+ ]i ) and ß-adrenergic receptor (ß-AR) stimulation modulate IKs amplitude and kinetics, but details of these important IKs regulators and their interaction are limited. We assessed the [Ca2+ ]i dependence of IKs in steady-state conditions and with dynamically changing membrane potential and [Ca2+ ]i during an AP. IKs was recorded from freshly isolated rabbit ventricular myocytes using whole-cell patch clamp. With intracellular pipette solutions that controlled free [Ca2+ ]i , we found that raising [Ca2+ ]i from 100 to 600 nm produced similar increases in IKs as did ß-AR activation, and the effects appeared additive. Both ß-AR activation and high [Ca2+ ]i increased maximally activated tail IKs , negatively shifted the voltage dependence of activation, and slowed deactivation kinetics. These data informed changes in our well-established mathematical model of the rabbit myocyte. In both AP-clamp experiments and simulations, IKs recorded during a normal physiological Ca2+ transient was similar to IKs measured with [Ca2+ ]i clamped at 500-600 nm. Thus, our study provides novel quantitative data as to how physiological [Ca2+ ]i regulates IKs amplitude and kinetics during the normal rabbit AP. Our results suggest that micromolar [Ca2+ ]i , in the submembrane or junctional cleft space, is not required to maximize [Ca2+ ]i -dependent IKs activation during normal Ca2+ transients.

Measuring local gradients of intramitochondrial [Ca(2+)] in cardiac myocytes during sarcoplasmic reticulum Ca(2+) release.

RATIONALE: Mitochondrial [Ca(2+)] ([Ca(2+)](mito)) regulates mitochondrial energy production, provides transient Ca(2+) buffering under stress, and can be involved in cell death. Mitochondria are near the sarcoplasmic reticulum (SR) in cardiac myocytes, and evidence for crosstalk exists. However, quantitative measurements of [Ca(2+)](mito) are limited, and spatial [Ca(2+)](mito) gradients have not been directly measured. OBJECTIVE: To directly measure local [Ca(2+)](mito) during normal SR Ca release in intact myocytes, and evaluate potential subsarcomeric spatial [Ca(2+)](mito) gradients. METHODS AND RESULTS: Using the mitochondrially targeted inverse pericam indicator Mitycam, calibrated in situ, we directly measured [Ca(2+)](mito) during SR Ca(2+) release in intact rabbit ventricular myocytes by confocal microscopy. During steady state pacing, Δ[Ca(2+)](mito) amplitude was 29±3 nmol/L, rising rapidly (similar to cytosolic free [Ca(2+)]) but declining much more slowly. Taking advantage of the structural periodicity of cardiac sarcomeres, we found that [Ca(2+)](mito) near SR Ca(2+) release sites (Z-line) versus mid-sarcomere (M-line) reached a high peak amplitude (37±4 versus 26±4 nmol/L, respectively P<0.05) which occurred earlier in time. This difference was attributed to ends of mitochondria being physically closer to SR Ca(2+) release sites, because the mitochondrial Ca(2+) uniporter was homogeneously distributed, and elevated [Ca(2+)] applied laterally did not produce longitudinal [Ca(2+)](mito) gradients. CONCLUSIONS: We developed methods to measure spatiotemporal [Ca(2+)](mito) gradients quantitatively during excitation-contraction coupling. The amplitude and kinetics of [Ca(2+)](mito) transients differ significantly from those in the cytosol and are respectively higher and faster near the Z-line versus M-line. This approach will help clarify SR-mitochondrial Ca(2+) signaling.

Depolarization of cardiac membrane potential synchronizes calcium sparks and waves in tissue.

The diastolic membrane potential (Vm) can be hyperpolarized or depolarized by various factors such as hyperkalemia or hypokalemia in the long term, or by delayed afterdepolarizations in the short term. In this study, we investigate how Vm affects Ca sparks and waves. We use a physiologically detailed mathematical model to investigate individual factors that affect Ca spark generation and wave propagation. We focus on the voltage range of -90 ∼ -70 mV, which is just below the Vm for sodium channel activation. We find that Vm depolarization promotes Ca wave propagation and hyperpolarization prevents it. This finding is directly validated in voltage clamp experiments with Ca waves using isolated rat ventricular myocytes. Ca transport by the sodium-calcium exchanger (NCX) is determined by Vm as well as Na and Ca concentrations. Depolarized Vm reduces NCX-mediated efflux, elevating [Ca]i, and thus promoting Ca wave propagation. Moreover, depolarized Vm promotes spontaneous Ca releases that can cause initiation of multiple Ca waves. This indicates that during delayed afterdepolarizations, Ca release units (CRUs) interact with not just the immediately adjacent CRUs via Ca diffusion, but also further CRUs via fast (∼0.1 ms) changes in Vm mediated by the voltage and Ca-sensitive NCX. This may contribute significantly to synchronization of Ca waves among multiple cells in tissue.

Cardiac Na+-Ca2+ exchanger: dynamics of Ca2+-dependent activation and deactivation in intact myocytes.

Cardiac Na(+)-Ca(2+) exchange (NCX) activity is regulated by [Ca(2+)]i. The physiological role and dynamics of this process in intact cardiomyocytes are largely unknown. We examined NCX Ca(2+) activation in intact rabbit and mouse cardiomyocytes at 37°C. Sarcoplasmic reticulum (SR) function was blocked, and cells were bathed in 2 mm Ca(2+). We probed Ca(2+) activation without voltage clamp by applying Na(+)-free (0 Na(+)) solution for 5 s bouts, repeated each 10 s, which should evoke [Ca(2+)]i transients due to Ca(2+) influx via NCX. In rested rabbit myocytes, Ca(2+) influx was undetectable even after 0 Na(+) applications were repeated for 2-5 min or more, suggesting that NCX was inactive. After external electric field stimulation pulses were applied, to admit Ca(2+) via L-type Ca(2+) channels, 0 Na(+) bouts activated Ca(2+) influx efficaciously, indicating that NCX had become active. Calcium activation increased with more field pulses, reaching a maximum typically after 15-20 pulses (1 Hz). At rest, NCX deactivated with a time constant typically of 20-40 s. An increase in [Na(+)]i, either in rabbit cardiomyocytes as a result of inhibition of Na(+)-K(+) pumping, or in mouse cardiomyocytes where normal [Na(+)]i is higher vs. rabbit, sensitized NCX to self-activation by 0 Na(+) bouts. In experiments with the SR functional but initially empty, the activation time course was slowed. It is possible that the SR initially accumulated Ca(2+) that would otherwise cause activation. We modelled Ca(2+) activation as a fourth-order highly co-operative process ([Ca]i required for half-activation K0.5act = 375 nm), with dynamics severalfold slower than the cardiac cycle. We incorporated this NCX model into an established ventricular myocyte model, which allowed us to predict responses to twitch stimulation in physiological conditions with the SR intact. Model NCX fractional activation increased from 0.1 to 1.0 as the frequency was increased from 0.2 to 2 Hz. By adjusting Ca(2+) activation on a multibeat time scale, NCX might better maintain a stable long-term Ca(2+) balance while contributing to the ability of myocytes to produce Ca(2+) transients over a wide range of intensity.

Ca2+/calmodulin-dependent protein kinase II (CaMKII) regulates cardiac sodium channel NaV1.5 gating by multiple phosphorylation sites.

The cardiac Na(+) channel Na(V)1.5 current (I(Na)) is critical to cardiac excitability, and altered I(Na) gating has been implicated in genetic and acquired arrhythmias. Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) is up-regulated in heart failure and has been shown to cause I(Na) gating changes that mimic those induced by a point mutation in humans that is associated with combined long QT and Brugada syndromes. We sought to identify the site(s) on Na(V)1.5 that mediate(s) the CaMKII-induced alterations in I(Na) gating. We analyzed both CaMKII binding and CaMKII-dependent phosphorylation of the intracellularly accessible regions of Na(V)1.5 using a series of GST fusion constructs, immobilized peptide arrays, and soluble peptides. A stable interaction between δ(C)-CaMKII and the intracellular loop between domains 1 and 2 of Na(V)1.5 was observed. This region was also phosphorylated by δ(C)-CaMKII, specifically at the Ser-516 and Thr-594 sites. Wild-type (WT) and phosphomutant hNa(V)1.5 were co-expressed with GFP-δ(C)-CaMKII in HEK293 cells, and I(Na) was recorded. As observed in myocytes, CaMKII shifted WT I(Na) availability to a more negative membrane potential and enhanced accumulation of I(Na) into an intermediate inactivated state, but these effects were abolished by mutating either of these sites to non-phosphorylatable Ala residues. Mutation of these sites to phosphomimetic Glu residues negatively shifted I(Na) availability without the need for CaMKII. CaMKII-dependent phosphorylation of Na(V)1.5 at multiple sites (including Thr-594 and Ser-516) appears to be required to evoke loss-of-function changes in gating that could contribute to acquired Brugada syndrome-like effects in heart failure.

Targeted ablation of the histidine-rich Ca(2+)-binding protein (HRC) gene is associated with abnormal SR Ca(2+)-cycling and severe pathology under pressure-overload stress.

The histidine-rich Ca(2+)-binding protein (HRC) is located in the lumen of the sarcoplasmic reticulum (SR) and exhibits high-capacity Ca(2+)-binding properties. Overexpression of HRC in the heart resulted in impaired SR Ca(2+) uptake and depressed relaxation through its interaction with SERCA2a. However, the functional significance of HRC in overall regulation of calcium cycling and contractility is not currently well defined. To further elucidate the role of HRC in vivo under physiological and pathophysiological conditions, we generated and characterized HRC-knockout (KO) mice. The KO mice were morphologically and histologically normal compared to wild-type (WT) mice. At the cellular level, ablation of HRC resulted in significantly enhanced contractility, Ca(2+) transients, and maximal SR Ca(2+) uptake rates in the heart. However, after-contractions were developed in 50 % of HRC-KO cardiomyocytes, compared to 11 % in WT mice under stress conditions of high-frequency stimulation (5 Hz) and isoproterenol application. A parallel examination of the electrical activity revealed significant increases in the occurrence of Ca(2+) spontaneous SR Ca(2+) release and delayed afterdepolarizations with ISO in HRC-KO, compared to WT cells. The frequency of Ca(2+) sparks was also significantly higher in HRC-KO cells with ISO, consistent with the elevated SR Ca(2+) load in the KO cells. Furthermore, HRC-KO cardiomyocytes showed significantly deteriorated cell contractility and Ca(2+)-cycling caused possibly by depressed SERCA2a expression after transverse-aortic constriction (TAC). Also HRC-null mice exhibited severe cardiac hypertrophy, fibrosis, pulmonary edema and decreased survival after TAC. Our results indicate that ablation of HRC is associated with poorly regulated SR Ca(2+)-cycling, and severe pathology under pressure-overload stress, suggesting an essential role of HRC in maintaining the integrity of cardiac function.

CaMKIIδC slows [Ca]i decline in cardiac myocytes by promoting Ca sparks.

Acute activation of calcium/calmodulin-dependent protein kinase (CaMKII) in permeabilized phospholamban knockout (PLN-KO) mouse myocytes phosphorylates ryanodine receptors (RyRs) and activates spontaneous local sarcoplasmic reticulum (SR) Ca release events (Ca sparks) even at constant SR Ca load. To assess how CaMKII regulates SR Ca release in intact myocytes (independent of SR Ca content changes or PLN effects), we compared Ca sparks in PLN-KO versus mice, which also have transgenic cardiac overexpression of CaMKIIδC in the PLN-KO background (KO/TG). Compared with PLN-KO mice, these KO/TG cardiomyocytes exhibited 1), increased twitch Ca transient and fractional release (both by ∼35%), but unaltered SR Ca load; 2), increased resting Ca spark frequency (300%) despite a lower diastolic [Ca]i, which also slowed twitch [Ca]i decline (suggesting CaMKII-dependent RyR Ca sensitization); 3), elevated Ca spark amplitude and rate of Ca release (which might indicate that more RyR channels participate in a single spark); 4), prolonged Ca spark rise time (which implies that CaMKII either delays RyR closure or prolongs the time when openings can occur); 5), more frequent repetitive sparks at single release sites. Analysis of repetitive sparks from individual Ca release sites indicates that CaMKII enhanced RyR Ca sensitivity, but did not change the time course of SR Ca refilling. These results demonstrate that there are dramatic CaMKII-mediated effects on RyR Ca release that occur via regulation of both RyR activation and termination processes.

Theoretical study of L-type Ca(2+) current inactivation kinetics during action potential repolarization and early afterdepolarizations.

Sarcoplasmic reticulum (SR) Ca(2+) release mediates excitation­contraction coupling (ECC) in cardiac myocytes. It is triggered upon membrane depolarization by entry of Ca(2+) via L-type Ca(2+) channels (LTCCs), which undergo both voltage- and Ca(2+)-dependent inactivation (VDI and CDI, respectively). We developed improved models of L-type Ca(2+) current and SR Ca(2+) release within the framework of the Shannon-Bers rabbit ventricular action potential (AP) model. The formulation of SR Ca(2+) release was modified to reproduce high ECC gain at negative membrane voltages. An existing LTCC model was extended to reflect more faithfully contributions of CDI and VDI to total inactivation. Ba(2+) current inactivation included an ion-dependent component (albeit small compared with CDI), in addition to pure VDI. Under physiological conditions (during an AP) LTCC inactivates predominantly via CDI, which is controlled mostly by SR Ca(2+) release during the initial AP phase, but by Ca(2+) through LTCCs for the remaining part. Simulations of decreased CDI or K(+) channel block predicted the occurrence of early and delayed after depolarizations. Our model accurately describes ECC and allows dissection of the relative contributions of different Ca(2+) sources to total CDI, and the relative roles of CDI and VDI, during normal and abnormal repolarization.

Subcellular Propagation of Cardiomyocyte ß-Adrenergic Activation of Calcium Uptake Involves Internal ß-Receptors and AKAP7.

ß-adrenergic receptor (ß-AR) signaling in cardiac myocytes is central to cardiac function, but spatiotemporal activation within myocytes is unresolved. In rabbit ventricular myocytes, ß-AR agonists or high extracellular [Ca] were applied locally at one end, to measure ß-AR signal propagation as Ca-transient (CaT) amplitude and sarcoplasmic reticulum (SR) Ca uptake. High local [Ca]o, increased CaT amplitude under the pipette faster than did ISO, but was also more spatially restricted. Local isoproterenol (ISO) or norepinephrine (NE) increased CaT amplitude and SR Ca uptake, that spread along the myocyte to the unexposed end. Thus, local [Ca]i decline kinetics reflect spatio-temporal progression of ß-AR end-effects in myocytes. To test whether intracellular ß-ARs contribute to this response, we used ß-AR-blockers that are membrane permeant (propranolol) or not (sotalol). Propranolol completely blocked NE-dependent CaT effects. However, blocking surface ß-ARs only (sotalol) suppressed only ∼50% of the NE-induced increase in CaT peak and rate of [Ca]i decline, but these changes spread more gradually than NE alone. We also tested whether A-kinase anchoring protein 7γ (AKAP7γ; that interacts with phospholamban) is mobile, such that it might contribute to intracellular spatial propagation of ß-AR signaling. We found AKAP7γ to be highly mobile using fluorescence recovery after photobleach of GFP tagged AKAP7γ, and that PKA activation accelerated AKAP7γ-GFP wash-out upon myocyte saponin-permeabilization, suggesting increased AKAP7γ mobility. We conclude that local ß-AR activation can activate SR Ca uptake at remote myocyte sites, and that intracellular ß-AR and AKAP7γ mobility may play a role in this spread of activation.

Beat-to-beat dynamic regulation of intracellular pH in cardiomyocytes.

The mammalian heart beats incessantly with rhythmic mechanical activities generating acids that need to be buffered to maintain a stable intracellular pH (pHi) for normal cardiac function. Even though spatial pHi non-uniformity in cardiomyocytes has been documented, it remains unknown how pHi is regulated to match the dynamic cardiac contractions. Here, we demonstrated beat-to-beat intracellular acidification, termed pHi transients, in synchrony with cardiomyocyte contractions. The pHi transients are regulated by pacing rate, Cl-/HCO3 - transporters, pHi buffering capacity, and ß-adrenergic signaling. Mitochondrial electron-transport chain inhibition attenuates the pHi transients, implicating mitochondrial activity in sculpting the pHi regulation. The pHi transients provide dynamic alterations of H+ transport required for ATP synthesis, and a decrease in pHi may serve as a negative feedback to cardiac contractions. Current findings dovetail with the prevailing three known dynamic systems, namely electrical, Ca2+, and mechanical systems, and may reveal broader features of pHi handling in excitable cells.

Targeting of protein phosphatases PP2A and PP2B to the C-terminus of the L-type calcium channel Ca v1.2.

The L-type Ca(2+) channel Ca(v)1.2 forms macromolecular signaling complexes that comprise the ß(2) adrenergic receptor, trimeric G(s) protein, adenylyl cyclase, and cAMP-dependent protein kinase (PKA) for efficient signaling in heart and brain. The protein phosphatases PP2A and PP2B are part of this complex. PP2A counteracts increase in Ca(v)1.2 channel activity by PKA and other protein kinases, whereas PP2B can either augment or decrease Ca(v)1.2 currents in cardiomyocytes depending on the precise experimental conditions. We found that PP2A binds to two regions in the C-terminus of the central, pore-forming α(1) subunit of Ca(v)1.2: one region spans residues 1795-1818 and the other residues 1965-1971. PP2B binds immediately downstream of residue 1971. Injection of a peptide that contained residues 1965-1971 and displaced PP2A but not PP2B from endogenous Ca(v)1.2 increased basal and isoproterenol-stimulated L-type Ca(2+) currents in acutely isolated cardiomyocytes. Together with our biochemical data, these physiological results indicate that anchoring of PP2A at this site of Ca(v)1.2 in the heart negatively regulates cardiac L-type currents, likely by counterbalancing basal and stimulated phosphorylation that is mediated by PKA and possibly other kinases.

Metabolic Maturation Media Improve Physiological Function of Human iPSC-Derived Cardiomyocytes.

Induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) have enormous potential for the study of human cardiac disorders. However, their physiological immaturity severely limits their utility as a model system and their adoption for drug discovery. Here, we describe maturation media designed to provide oxidative substrates adapted to the metabolic needs of human iPSC (hiPSC)-CMs. Compared with conventionally cultured hiPSC-CMs, metabolically matured hiPSC-CMs contract with greater force and show an increased reliance on cardiac sodium (Na+) channels and sarcoplasmic reticulum calcium (Ca2+) cycling. The media enhance the function, long-term survival, and sarcomere structures in engineered heart tissues. Use of the maturation media made it possible to reliably model two genetic cardiac diseases: long QT syndrome type 3 due to a mutation in the cardiac Na+ channel SCN5A and dilated cardiomyopathy due to a mutation in the RNA splicing factor RBM20. The maturation media should increase the fidelity of hiPSC-CMs as disease models.

Enhanced Depolarization Drive in Failing Rabbit Ventricular Myocytes: Calcium-Dependent and ß-Adrenergic Effects on Late Sodium, L-Type Calcium, and Sodium-Calcium Exchange Currents.

BACKGROUND: Heart failure (HF) is characterized by electrophysiological remodeling resulting in increased risk of cardiac arrhythmias. Previous reports suggest that elevated inward ionic currents in HF promote action potential (AP) prolongation, increased short-term variability of AP repolarization, and delayed afterdepolarizations. However, the underlying changes in late Na+ current (INaL), L-type Ca2+ current, and NCX (Na+/Ca2+ exchanger) current are often measured in nonphysiological conditions (square-pulse voltage clamp, slow pacing rates, exogenous Ca2+ buffers). METHODS: We measured the major inward currents and their Ca2+- and ß-adrenergic dependence under physiological AP clamp in rabbit ventricular myocytes in chronic pressure/volume overload-induced HF (versus age-matched control). RESULTS: AP duration and short-term variability of AP repolarization were increased in HF, and importantly, inhibition of INaL decreased both parameters to the control level. INaL was slightly increased in HF versus control even when intracellular Ca2+ was strongly buffered. But under physiological AP clamp with normal Ca2+ cycling, INaL was markedly upregulated in HF versus control (dependent largely on CaMKII [Ca2+/calmodulin-dependent protein kinase II] activity). ß-Adrenergic stimulation (often elevated in HF) further enhanced INaL. L-type Ca2+ current was decreased in HF when Ca2+ was buffered, but CaMKII-mediated Ca2+-dependent facilitation upregulated physiological L-type Ca2+ current to the control level. Furthermore, L-type Ca2+ current response to ß-adrenergic stimulation was significantly attenuated in HF. Inward NCX current was upregulated at phase 3 of AP in HF when assessed by combining experimental data and computational modeling. CONCLUSIONS: Our results suggest that CaMKII-dependent upregulation of INaL in HF significantly contributes to AP prolongation and increased short-term variability of AP repolarization, which may lead to increased arrhythmia propensity, and is further exacerbated by adrenergic stress.

Disease-associated mutations in Niemann-Pick type C1 alter ER calcium signaling and neuronal plasticity.

Niemann-Pick type C1 (NPC1) protein is essential for the transport of externally derived cholesterol from lysosomes to other organelles. Deficiency of NPC1 underlies the progressive NPC1 neurodegenerative disorder. Currently, there are no curative therapies for this fatal disease. Given the Ca2+ hypothesis of neurodegeneration, which posits that altered Ca2+ dynamics contribute to neuropathology, we tested if disease mutations in NPC1 alter Ca2+ signaling and neuronal plasticity. We determine that NPC1 inhibition or disease mutations potentiate store-operated Ca2+ entry (SOCE) due to a presenilin 1 (PSEN1)-dependent reduction in ER Ca2+ levels alongside elevated expression of the molecular SOCE components ORAI1 and STIM1. Associated with this dysfunctional Ca2+ signaling is destabilization of neuronal dendritic spines. Knockdown of PSEN1 or inhibition of the SREBP pathway restores Ca2+ homeostasis, corrects differential protein expression, reduces cholesterol accumulation, and rescues spine density. These findings highlight lysosomes as a crucial signaling platform responsible for tuning ER Ca2+ signaling, SOCE, and synaptic architecture in health and disease.