Mouse Ventricular Myocytes Research Articles

Increased β-Adrenergic Inotropy in Ventricular Myocardium From Trpm4 −/− Mice

The Trpm4 gene has recently been associated with several disorders, including cardiac conduction diseases and Brugada syndrome. Transient receptor potential member 4 (TRPM4) proteins constitute Ca2+ -activated, but Ca2+ -impermeable, nonselective cation channels and are expressed both in atrial and in ventricular cardiomyocytes. The physiological function of TRPM4 in the heart remains, however, incompletely understood. To establish the role of TRPM4 in cardiac muscle function. We used TRPM4 knockout mice and performed patch-clamp experiments, membrane potential measurements, microfluorometry, contractility measurements, and in vivo pressure-volume loop analysis. We demonstrate that TRPM4 proteins are functionally present in mouse ventricular myocytes and are activated on Ca2+ -induced Ca2+ release. In Trpm4(-/-) mice, cardiac muscle displays an increased β-adrenergic inotropic response both in vitro and in vivo. Measurements of action potential duration show a significantly decreased time for 50% and 90% repolarization in Trpm4(-/-) ventricular myocytes. We provide evidence that this change in action potential shape leads to an increased driving force for the L-type Ca2+ current during the action potential, which explains the altered contractility of the heart muscle. Our results show that functional TRPM4 proteins are novel determinants of the inotropic effect of β-adrenergic stimulation on the ventricular heart muscle.

Modulation of intracellular calcium waves and triggered activities by mitochondrial ca flux in mouse cardiomyocytes.

Recent studies have suggested that mitochondria may play important roles in the Ca2+ homeostasis of cardiac myocytes. However, it is still unclear if mitochondrial Ca2+ flux can regulate the generation of Ca2+ waves (CaWs) and triggered activities in cardiac myocytes. In the present study, intracellular/cytosolic Ca2+ (Cai 2+) was imaged in Fluo-4-AM loaded mouse ventricular myocytes. Spontaneous sarcoplasmic reticulum (SR) Ca2+ release and CaWs were induced in the presence of high (4 mM) external Ca2+ (Cao 2+). The protonophore carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP) reversibly raised basal Cai 2+ levels even after depletion of SR Ca2+ in the absence of Cao 2+ , suggesting Ca2+ release from mitochondria. FCCP at 0.01 - 0.1 µM partially depolarized the mitochondrial membrane potential (Δψ m) and increased the frequency and amplitude of CaWs in a dose-dependent manner. Simultaneous recording of cell membrane potentials showed the augmentation of delayed afterdepolarization amplitudes and frequencies, and induction of triggered action potentials. The effect of FCCP on CaWs was mimicked by antimycin A (an electron transport chain inhibitor disrupting Δψ m) or Ru360 (a mitochondrial Ca2+ uniporter inhibitor), but not by oligomycin (an ATP synthase inhibitor) or iodoacetic acid (a glycolytic inhibitor), excluding the contribution of intracellular ATP levels. The effects of FCCP on CaWs were counteracted by the mitochondrial permeability transition pore blocker cyclosporine A, or the mitochondrial Ca2+ uniporter activator kaempferol. Our results suggest that mitochondrial Ca2+ release and uptake exquisitely control the local Ca2+ level in the micro-domain near SR ryanodine receptors and play an important role in regulation of intracellular CaWs and arrhythmogenesis.

Nitroxyl (HNO)

Background— The nitroxyl (HNO) donor, Angeli’s salt, exerts positive inotropic, lusitropic, and vasodilator effects in vivo that are cAMP independent. Its clinical usefulness is limited by chemical instability and cogeneration of nitrite which itself has vascular effects. Here, we report on effects of a novel, stable, pure HNO donor (CXL-1020) in isolated myoctyes and intact hearts in experimental models and in patients with heart failure (HF). Methods and Results— CXL-1020 converts solely to HNO and inactive CXL-1051 with a t 1/2 of 2 minutes. In adult mouse ventricular myocytes, it dose dependently increased sarcomere shortening by 75% to 210% (50–500 μmol/L), with a ≈30% rise in the peak Ca 2+ transient only at higher doses. Neither inhibition of protein kinase A nor soluble guanylate cyclase altered this contractile response. Unlike isoproterenol, CXL-1020 was equally effective in myocytes from normal or failing hearts. In anesthetized dogs with coronary microembolization-induced HF, CXL-1020 reduced left ventricular end-diastolic pressure and myocardial oxygen consumption while increasing ejection fraction from 27% to 40% and maximal ventricular power index by 42% (both P <0.05). In conscious dogs with tachypacing-induced HF, CXL-1020 increased contractility assessed by end-systolic elastance and provided venoarterial dilation. Heart rate was minimally altered. In patients with systolic HF, CXL-1020 reduced both left and right heart filling pressures and systemic vascular resistance, while increasing cardiac and stroke volume index. Heart rate was unchanged, and arterial pressure declined modestly. Conclusions— These data show the functional efficacy of a novel pure HNO donor to enhance myocardial function and present first-in-man evidence for its potential usefulness in HF. Clinical Trial Registration— URL: http://www.clinicaltrials.gov . Unique identifiers: NCT01096043, NCT01092325.

Sevoflurane Protects Ventricular Myocytes against Oxidative Stress-induced Cellular Ca2+ Overload and Hypercontracture

Oxidative stress is implicated in pathogenesis of cardiac reperfusion injury, characterized by cellular Ca overload and hypercontracture. Volatile anesthetics protect the heart against reperfusion injury primarily by attenuating Ca overload. This study investigated electrophysiological mechanisms underlying cardioprotective effects of sevoflurane against oxidative stress-induced cellular injury. The cytosolic Ca levels and cell morphology were assessed in mouse ventricular myocytes, using confocal fluo-3 fluorescence imaging, whereas membrane potentials and L-type Ca current (ICa,L) were recorded using whole-cell patch-clamp techniques. Phosphorylation of Ca/calmodulin-dependent protein kinase II was examined by Western blotting. Exposure to H2O2 (100 μM) for 15 min evoked cytosolic Ca elevation and hypercontracture in 56.8% of ventricular myocytes in 11 experiments, which was partly but significantly reduced by nifedipine, tetracaine, or SEA0400. Sevoflurane prevented H2O2-induced cellular Ca overload in a concentration-dependent way (IC50 = 1.35%). Isoflurane (2%) and desflurane (10%) also protected ventricular myocytes by a degree similar to sevoflurane (3%). Sevoflurane suppressed H2O2-induced electrophysiological disturbances, including early afterdepolarizations, voltage fluctuations in resting potential, and abnormal automaticities. H2O2 significantly enhanced ICa,L by activating Ca/calmodulin-dependent protein kinase II, and subsequent addition of sevoflurane, isoflurane, or desflurane similarly reduced ICa,L to below baseline levels. Phosphorylated Ca/calmodulin-dependent protein kinase II increased after 10-min incubation with H2O2, which was significantly prevented by concomitant administration of sevoflurane. Sevoflurane protected ventricular myocytes against H2O2-induced Ca overload and hypercontracture, presumably by affecting multiple Ca transport pathways, including ICa,L, Na/Ca exchanger and ryanodine receptor. These actions appear to mediate cardioprotection against reperfusion injury associated with oxidative stress.

Localization of sodium channel subtypes in mouse ventricular myocytes using quantitative immunocytochemistry

Voltage-gated sodium channels are responsible for the rising phase of the action potential in cardiac muscle. Previously, both TTX-sensitive neuronal sodium channels (NaV1.1, NaV1.2, NaV1.3, NaV1.4 and NaV1.6) and the TTX-resistant cardiac sodium channel (NaV1.5) have been detected in cardiac myocytes, but relative levels of protein expression of the isoforms were not determined. Using a quantitative approach, we analyzed z-series of confocal microscopy images from individual mouse myocytes stained with either anti-NaV1.1, anti-NaV1.2, anti-NaV1.3, anti-NaV1.4, anti-NaV1.5, or anti-NaV1.6 antibodies and calculated the relative intensity of staining for these sodium channel isoforms. Our results indicate that the TTX-sensitive channels represented approximately 23% of the total channels, whereas the TTX-resistant NaV1.5 channel represented 77% of the total channel staining in mouse ventricular myocytes. These ratios are consistent with previous electrophysiological studies in mouse ventricular myocytes. NaV1.5 was located at the cell surface, with high density at the intercalated disc, but was absent from the transverse (t)-tubular system, suggesting that these channels support surface conduction and inter-myocyte transmission. Low-level cell surface staining of NaV1.4 and NaV1.6 channels suggest a minor role in surface excitation and conduction. Conversely, NaV1.1 and NaV1.3 channels are localized to the t-tubules and are likely to support t-tubular transmission of the action potential to the myocyte interior. This quantitative immunocytochemical approach for assessing sodium channel density and localization provides a more precise view of the relative importance and possible roles of these individual sodium channel protein isoforms in mouse ventricular myocytes and may be applicable to other species and cardiac tissue types.

Abstract 194: Phosphodiesterase 3 Controls Basal cGMP Levels And Regulates cGMP/cAMP Cross-talk In Adult Mouse Ventricular Cardiomyocytes

PURPOSE: cGMP is an important second messenger which is involved in the regulation of cardiac contractility and pathological hypertrophy. In cardiomyocytes, signaling by cGMP is organized in microdomains and is considered cardioprotective. Especially in adult cardiac myocytes, measurements of cGMP have been challenging and little is known about the spatio-temporal dynamics of cGMP. Here we developed a transgenic mouse model to visualize cGMP dynamis in adult cardiac myocytes. Methods: We generated transgenic mice with cardiomyocyte-specific expression of a highly sensitive fluorescence resonance energy transfer (FRET)-based cGMP biosensor red cGES-DE5 and performed FRET measurements in freshly isolated adult mouse ventricular myocytes. To analyze cGMP/cAMP crosstalk, FRET experiments were performed in cardiomyocytes isolated from mice transgenically expressing the cAMP sensor Epac1-camps. Results: Basal cytosolic cGMP levels were very low (~10 nM), but could be markedly increased by stimulation with natriuretic peptides (CNP>>ANP). In contrast, direct stimulation of the soluble guanylyl cyclase (sGC) with NO-donors such as SNAP showed no effect. However, constitutive activity of this cyclase contributes to basal cGMP production, since a clear decrease of basal cGMP levels was observed after stimulation with the sGC inhibitor ODQ. This basal cGMP production is regulated by phosphodiesterase (PDE) activity. Unexpectedly, PDE3 is most important in controlling basal cGMP levels, whereas PDE2 and PDE5 are much less active. We could also show that cGMP pools produced by GC-B after CNP stimulation are mainly regulated by PDE3, so that the receptor and this PDE form one functional unit important for the regulation of cGMP/cAMP cross-talk. Conclusion: In summary, we performed the first FRET-based measurements of cGMP in adult cardiomyocytes and we could highlight the key role of PDE3 in the regulation of basal cGMP levels and cGMP/cAMP cross-talk.

Abstract 045: Highly-reactive Lipid Aldehydes Are Downstream Mediators Of Oxidant-induced Spontaneous Ca Waves

Background: Reactive oxygen species (ROS) can activate ryanodine receptor (RyR2) Ca release channel and generate arrhythmogenic Ca waves (SCWs) in myocytes. ROS also cause lipid peroxidation in cell membranes that produce highly-reactive g-ketoaldehydes (IsoK), which can react immediately with proteins (RyR2) and accumulate in ischemic human heart. We previously found that specific IsoK scavengers reduce infarct size and prevent ventricular arrhythmia in an MI mouse model, suggesting a biological role of IsoK in oxidant heart injury. We hypothesize that IsoK mediate in part the ROS-induced SCW in myocytes. Methods: We compared the effect of H 2 O 2 , synthetic IsoK, the specific IsoK scavenger (SA, salicylamine), its inactive homolog (4-SA, 4-OH-salicylamine), and the ROS scavenger (MPG, 2-mercaptopropionyl glycine) in field-stimulated mouse ventricular myocytes loaded with Fura-2AM. Results: Thirty min exposure to H 2 O 2 (10μM) or IsoK (1μM) both generated SCWs (H 2 O 2 37%, IsoK 37%, p<0.05 vs.vehicle, VEH). MPG, (100μM, 60 min) prevented H 2 O 2 -induced SCWs, but had no effect on IsoK-induced SCWs ( Fig.A ). Pre-treatment with SA (8 μM, 60 min) significantly reduced H 2 O 2 -induced SCWs, whereas 4-SA had no effect on H 2 O 2 -induced SCWs (%SCWs, H 2 O 2 ; 53%, SA+H 2 O 2 ; 27%*, 4-SA+H 2 O 2 ; 56%. *p<0.05 vs. VEH, Fig. B ). In the absence of H 2 O 2 , MPG, SA, or 4-SA had no significant effect on Ca handling in myocytes. Conclusion: IsoK directly activate SCWs independent of ROS production in myocytes. Specific IsoK scavengers inhibit 50% of ROS-induced SCWs. Hence, our data suggested that IsoK are biologically-relevant downstream mediators of oxidant-induced RyR2 dysfunction.

GW24-e0050 Stretch current-induced abnormal impulses in CaMKII & knockout mouse ventricular myocytes

ObjectivesCaMKII activation is pro-arrhythmic in heart failure where myocardium is stretched. However, the arrhythmogenic role of CaMKII in stretched ventricle has not been well understood. We tested abnormal impulse inducibility...

PDE3 is the major cGMP-PDE in adult mouse ventricular cardiomyocytes

Materials and methods We generated a transgenic mouse model with cardiomyocyte-specific expression of the highly sensitive fluorescence resonance energy transfer (FRET)-based cGMP biosensor red cGES-DE5. This sensor mouse allows us to performed FRET measurements of cGMP in freshly isolated adult mouse ventricular myocytes, to visulaize realtime dynamics of cGMP. To analyze cAMP and cGMP interactions (cGMP/cAMP signaling crosstalk), FRET experiments were performed in cardiomyocytes isolated from mice, transgenically expressing the FRET-based cAMP sensor Epac1-camps.

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.

Nanoscale Distribution of Ryanodine Receptors and Caveolin-3 in Mouse Ventricular Myocytes: Dilation of T-Tubules near Junctions

We conducted super-resolution light microscopy (LM) imaging of the distribution of ryanodine receptors (RyRs) and caveolin-3 (CAV3) in mouse ventricular myocytes. Quantitative analysis of data at the surface sarcolemma showed that 4.8% of RyR labeling colocalized with CAV3 whereas 3.5% of CAV3 was in areas with RyR labeling. These values increased to 9.2 and 9.0%, respectively, in the interior of myocytes where CAV3 was widely expressed in the t-system but reduced in regions associated with junctional couplings. Electron microscopic (EM) tomography independently showed only few couplings with caveolae and little evidence for caveolar shapes on the t-system. Unexpectedly, both super-resolution LM and three-dimensional EM data (including serial block-face scanning EM) revealed significant increases in local t-system diameters in many regions associated with junctions. We suggest that this regional specialization helps reduce ionic accumulation and depletion in t-system lumen during excitation-contraction coupling to ensure effective local Ca2+ release. Our data demonstrate that super-resolution LM and volume EM techniques complementarily enhance information on subcellular structure at the nanoscale.

Resolution of hyposmotic stress in isolated mouse ventricular myocytes causes sealing of t‐tubules

It has recently been shown that various stress-inducing manipulations in isolated ventricular myocytes may lead to significant remodelling of t-tubules. Osmotic stress is one of the most common complications in various experimental and clinical settings. This study was therefore designed to determine the effects of a physiologically relevant type of osmotic stress, hyposmotic challenge, to the integrity of the t-tubular system in mouse ventricular myocytes using the following two approaches: (1) electrophysiological measurements of membrane capacitance and inward rectifier (IK1) tail currents originating from K(+) accumulation in t-tubules; and (2) confocal microscopy of fluorescent dextrans trapped in sealed t-tubules. Importantly, we found that removal of '0.6 Na' (60% NaCl) hyposmotic solution, but not its application to myocytes, led to a ∼27% reduction in membrane capacitance, a ∼2.5-fold reduction in the amplitude of the IK1 tail current and a ∼2-fold reduction in the so-called IK1 'inactivation' (due to depletion of t-tubular K(+)) at negative membrane potentials; all these data were consistent with significant detubulation. Confocal imaging experiments also demonstrated that extracellularly applied dextrans become trapped in sealed t-tubules only upon removal of hyposmotic solutions, i.e. during the shrinking phase, but not during the initial swelling period. In light of these data, relevant previous studies, including those on excitation-contraction coupling phenomena during hyposmotic stress, may need to be reinterpreted, and the experimental design of future experiments should take into account the novel findings.

A Mathematical Model of the Mouse Ventricular Myocyte Contraction

Mathematical models of cardiac function at the cellular level include three major components, such as electrical activity, Ca2+ dynamics, and cellular shortening. We developed a model for mouse ventricular myocyte contraction which is based on our previously published comprehensive models of action potential and Ca2+ handling mechanisms. The model was verified with extensive experimental data on mouse myocyte contraction at room temperature. In the model, we implemented variable sarcomere length and indirect modulation of the tropomyosin transition rates by Ca2+ and troponin. The resulting model described well steady-state force-calcium relationships, dependence of the contraction force on the sarcomere length, time course of the contraction force and myocyte shortening, frequency dependence of the contraction force and cellular contraction, and experimentally measured derivatives of the myocyte length variation. We emphasized the importance of the inclusion of variable sarcomere length into a model for ventricular myocyte contraction. Differences in contraction force and cell shortening for epicardial and endocardial ventricular myocytes were investigated. Model applicability for the experimental studies and model limitations were discussed.

A Compartmentalized Model of the Beta1-Adrenergic Signaling System in Mouse Ventricular Myocytes

The β1-adrenergic signaling system plays an important role in heart function. Activation of this system increases heart rhythm, heart contraction. It also increases blood flow throughout the body. However, in disease, stimulation of β1-adrenergic receptors can increase an occurrence of arrhythmias. Therefore, comprehensive experimental study and modeling of the β1-adrenergic signaling system in the heart is of significant importance. We developed an experimentally-based mathematical model of β1-adrenergic regulation of the action potential and Ca2+ dynamics in mouse ventricular myocytes. The model includes several modules that describe biochemical reactions, electrical activity, and protein phosphorylation during activation of β1-adrenergic receptors. The model cell consists of three major compartments: caveolae, extracaveolae, and cytosol. Concentrations of β1-adrenergic receptors, as well as other signaling molecules, vary across the different domains. In the model, β1-adrenergic receptors are stimulated by the catecholamine isoproterenol. This leads to the activation of the Gs-protein signaling pathway, which ultimately increases cyclic AMP concentration and activity of protein kinase A to different degrees in different domains. The catalytic subunit of protein kinase A phosphorylates ion channels and Ca2+ handling proteins, leading to an increase or decrease in their function. Dephosphorylation is performed by the protein phosphatases 1 and 2A. Our model reproduces time-dependent behavior of a number of biochemical reactions and voltage-clamp data on ionic currents in mouse ventricular myocytes. The model also predicts action potential prolongation as well as an increase in intracellular Ca2+ transients dependent upon stimulation of β1-adrenergic receptors. The accuracy of the model was tested against experimental findings.

Factors Influencing Ca2+ Spark Refractoriness in Mouse

In cardiac myocytes, Ca2+ sparks exhibit time-dependent refractoriness such that it is difficult to trigger a second spark soon after an initial spark has terminated. Recent studies in rat ventricular myocytes suggested that spark amplitude recovery is controlled by local sarcoplasmic reticulum (SR) refilling whereas refractoriness of Ca2+ spark triggering depends on both refilling and ryanodine receptor (RyR) sensitivity. Here we examined Ca2+ spark refractoriness in mouse ventricular myocytes by exposing fluo-3 loaded quiescent cells to 50 nM ryanodine, recording Ca2+ sparks with a confocal microscope, and analyzing the repeated sparks that were produced at a limited number of RyR clusters. The time constant of Ca2+ spark amplitude recovery was 70-80 ms in mouse versus 90-100 ms in rat, suggesting slightly faster SR refilling in mouse. Spark-to-spark delay histograms were similar in the two species. Depending on the conditions, incubation of mouse myocytes with Isoproterenol or the drug H89, an inhibitor of protein kinase A (PKA), led to faster or slower Ca2+ spark amplitude recovery and shorter or longer average spark-to-spark delays, respectively. Moreover, incubation with H89 caused a decrease in whole-cell Ca2+ transient amplitude, slower Ca2+ transient decay, and no apparent change in SR Ca2+ load, compared with control conditions. Together these results suggest that relatively high levels of endogenous PKA activity may act to shorten Ca2+ spark refractoriness in mouse ventricular myocytes.

Ca Release Unit Heterogeneity and Entrainment of Ca Waves in Cardiac Myocytes

Calcium (Ca) is a ubiquitous second messenger regulating many biological functions. The elementary events of local Ca signaling are Ca sparks, which occur randomly in time and space, and integrate to produce global signaling events including intracellular and intercellular Ca waves and whole-cell Ca oscillations. In a recent study using a computational model of Ca signaling in a cardiac myocyte and then experimentally in mouse ventricular myocytes, we demonstrated that criticality is the underlying theoretical mechanism that governs the transition from local Ca sparks to global Ca waves, analogous to a second-order phase transition in thermodynamics. Theoretically, criticality predicts that wave initiation sites should occur randomly and uniformly in space. While this has been demonstrated experimentally at relatively low Ca loads, recent experiments suggest that at higher Ca loads certain regions of the cell dominate the wave initiation process, acting as pacemakers to entrain whole cell oscillations. Here we show that the formation of these pacemaking sites is still governed by the theory of criticality, however, heterogeneities of the ryanodine receptor clusters result in different firing frequencies, with the fastest regions tending to entrain the whole cell. We demonstrate that there is a critical size and relative degree of heterogeneity that must be reached for entrainment to occur, with the novel finding that the degree of entrainment depends on the overall Ca load of the cell. Furthermore, we show that the stochastic nature of the ryanodine receptor channel is crucial to the wave generation process.

Electrophysiologic Effects of Azithromycin in Cardiomyocytes

The widely-used macrolide antibiotic azithromycin (AZ) increases risk of cardiovascular and sudden cardiac death. Case reports indicate that AZ can cause polymorphic ventricular tachycardia in the absence and presence of QT prolongation, implying a novel proarrhythmic syndrome. We investigated the electrophysiologic effects of AZ in vivo and in vitro using mice, cardiomyocytes, and heterologously-expressed human ion channels. After implanting an ECG telemeter, conscious adult mice received intraperitoneal injection of AZ (50 mg/kg, followed in 60 min by 100 mg/kg; n=7). With both doses of AZ, heart rate declined (from 685±24 to 489±20 and 481±21 bpm, for baseline, 50 and 100 mg/kg, respectively [mean±SEM]; P<0.001). In addition, AZ increased the PR interval (32.7±0.9 ms to 39.4±0.7 and 39.8±0.9 ms, respectively; P<0.001), QRS interval (10.2±0.4 ms to 12.5±0.4 and 13.3±0.5 ms, respectively; P<0.001), and QT interval (37.4±4 ms to 48.0±5 and 51.2±4 ms, respectively; P<0.01). In spontaneously-beating HL-1 cardiomyocytes, AZ (100 μM) significantly slowed beat rate (from 215±7 to 180±7 bpm; n=14; P<0.01), while increasing action potential rise time (23.0±3.0 to 36.2±3.8 ms; P<0.01) and duration (at 90% repolarization, 118.3±8 to 137.8±8.7ms; P<0.01). In HEK cells stably expressing SCN5A, AZ reduced Na+ currents (IC50 110±3 μM; n=14), while similar results were obtained using mouse ventricular myocytes (IC50 117±4 μM; n=6). In addition, AZ suppressed K+ currents recorded from HEK cells expressing hERG (IC50 219±21 μM; n=5) and CHO cells expressing KCNQ1 and KCNE1 (IC50 184±12 μM; n=6), as well as L-type Ca++ current in rabbit ventricular myocytes (IC50 67±4 μM; n=5). We conclude that azithromycin blocks multiple cardiac ion channels to prolong the PR, QRS, and QT interval in vivo, at concentrations achievable within the heart based on intracellular drug accumulation. These effects likely contribute to its novel proarrhythmic effect in humans.

Resolution of Hypo-Osmotic Stress in Isolated Mouse Ventricular Myocytes Leads to Detubulation

It has been recently shown that various stress-inducing manipulations in isolated ventricular cardiac myocytes may lead to significant remodeling of t-tubules. Osmotic stress is one of the most common complications in various experimental and clinical settings, and therefore, this study was designed to test a hypothesis that osmotic challenge may affect the integrity of t-tubules. T-tubular remodeling in mouse ventricular myocytes in response to various osmotic challenges was studied using two approaches: (1) electrophysiologically, by measuring membrane capacitance and IK1 tail currents originating from K+ accumulation in ttubules, and (2) using confocal microscopy of fluorescent dextrans trapped in vesiculated t-tubules. In particular, application and removal of 0.6 T (60% of NaCl) hypo-osmotic solution to myocytes led to ∼30% reduction in membrane capacitance, ∼3-fold reduction in the amplitude of IK1 tail current and ∼2-fold reduction in so-called IK1 ‘inactivation' (due to depletion of t-tubular K+) at negative membrane potentials – all being consistent with strong detubulation. Importantly, confocal imaging experiments showed that extracellularly applied dextrans become trapped inside the myocytes only upon removal of hypo-osmotic solutions (i.e. during shrinking phase) but not during initial swelling period. In light of these data some relevant previous studies, including those on EC coupling phenomena during hypo-osmotic stress, may need to be reinterpreted and the experimental design of future experiments should take into account the novel findings.

Dissecting the Calcium Transient Refractoriness in Mouse Ventricular Myocytes

The activation cascade of Ca2+ induced Ca2+ release (CICR) to produce a [Ca2+]i transient has been well described; on the other hand, mechanisms responsible for its termination and refractoriness are still a matter of debate. It is known that the ryanodine receptor (RyR) state and sarcoplasmic reticulum Ca2+ content ([Ca2+]SR) are major determinates in the termination and refractoriness of Ca2+ sparks at room temperature. The goal of this work was to evaluate [Ca2+]i transient refractoriness (CaTR) when RyR state and/or [Ca2+]SR was altered by pharmacological means at physiological temperature (36±1oC). CaTR was measured in mouse ventricular myocytes loaded with Fluo-5F and paced at 2Hz (field stimulation or depolarization through patch pipette) followed by an extra-stimuli with a decreased interval (ESI). This enabled us to measure a [Ca2+]i transient restitution curves. The [Ca2+]i transient peak recovered exponentially as ESI increased (τ ∼ 137ms). L-type Ca2+ channel current (ICa) recovery from inactivation was measured (τ ∼ 53ms, Vhold = −80mV) and found to be much faster than CaTR; suggesting a minimal role. Interestingly, decreasing RyR cytosolic Ca2+ sensitivity with tetracaine strongly shifted CaTR curve to the right. While increasing RyR Ca2+ sensitivity with caffeine only moderately shifted the CaTR curve to the left. These results were then compared to [Ca2+]SR measurements under similar conditions and times. The roles of RyR state and SR Ca2+ load in determining CaTR will be discussed. These findings are relevant to an investigation of the mechanisms of Ca2+ signaling instability and SR Ca2+ leak in arrhythmic conditions including those attributable to myocardial infarction, heart failure and inheritable diseases including catecholaminergic polymorphic ventricular tachycardia (CPVT).

Impact of SR Ca2+ Release on the Cardiac Action Potential Repolarization during Postnatal Development

In heart, the excitation-contraction coupling (ECC) mechanism changes from sarcolemmal Ca2+ diffusion in newborn, to Ca2+ induced Ca2+ release in adult. This implies different contribution of the sarcoplasmic reticulum (SR). Since SR Ca2+ release (SRCa) deeply influences sarcolemmal Ca2+ fluxes, we hypothesized that the differential contribution of the SRCa to ECC during postnatal development is critical in defining the properties of AP repolarization in epicardium. Consequently, the AP properties were evaluated during postnatal development in correlation with the SRCa contribution to ECC. To this end, the temperature dependence of the epicardical AP properties was determined with the local-field fluorescence detection method in intact mouse hearts, with/without SRCa, and at various ages after birth (7h, 1d, 2d, 7d, & AD). Our pharmacological (ryanodine+thapsigargin) and thermodynamic (temperature dependence from 21°-37°C) studies confirmed that the SRCa contribution becomes more prominent with development. More importantly, they showed that the impact of SRCa on the AP duration (measured at 25% & 75% of repolarization; APD25 & APD75) exhibits a biphasic effect. At day 2, SRCa significantly reduced the APD25 & APD75 by 38.7% & 31%, respectively; while in adult SRCa prolonged the APD75 by 49.7%; with day 7 being a transitional stage with a 44%-shortened APD25 and 25%-lengthened ADP75. Interestingly, the peak-and-dome AP morphology typical of mouse ventricular myocytes, became evident only in adult. A possible explanation for this dual effect on APD is that early after birth, SRCa promotes a Ca2+-dependent inactivation of the L-type ICa while in adult, SRCa activates the Na-Ca exchanger forward mode. Altogether, our results demonstrated that differences in SR Ca2+ release during development not only play a different role in ECC, but greatly impacts plasma membrane excitability as well. Supported by NIH R01-HL-084487 to AE.