CaT Amplitude Research Articles

Calcium Handling Remodeling Underlies Impaired Sympathetic Stress Response in Ventricular Myocardium from Cacna1c Haploinsufficient Rats.

CACNA1C encodes the pore-forming α1C subunit of the L-type Ca2+ channel, Cav1.2. Mutations and polymorphisms of the gene are associated with neuropsychiatric and cardiac disease. Haploinsufficient Cacna1c+/- rats represent a recently developed model with a behavioral phenotype, but its cardiac phenotype is unknown. Here, we unraveled the cardiac phenotype of Cacna1c+/- rats with a main focus on cellular Ca2+ handling mechanisms. Under basal conditions, isolated ventricular Cacna1c+/- myocytes exhibited unaltered L-type Ca2+ current, Ca2+ transients (CaTs), sarcoplasmic reticulum (SR) Ca2+ load, fractional release, and sarcomere shortenings. However, immunoblotting of left ventricular (LV) tissue revealed reduced expression of Cav1.2, increased expression of SERCA2a and NCX, and augmented phosphorylation of RyR2 (at S2808) in Cacna1c+/- rats. The β-adrenergic agonist isoprenaline increased amplitude and accelerated decay of CaTs and sarcomere shortenings in both Cacna1c+/- and WT myocytes. However, the isoprenaline effect on CaT amplitude and fractional shortening (but not CaT decay) was impaired in Cacna1c+/- myocytes exhibiting both reduced potency and efficacy. Moreover, sarcolemmal Ca2+ influx and fractional SR Ca2+ release after treatment with isoprenaline were smaller in Cacna1c+/- than in WT myocytes. In Langendorff-perfused hearts, the isoprenaline-induced increase in RyR2 phosphorylation at S2808 and S2814 was attenuated in Cacna1c+/- compared to WT hearts. Despite unaltered CaTs and sarcomere shortenings, Cacna1c+/- myocytes display remodeling of Ca2+ handling proteins under basal conditions. Mimicking sympathetic stress with isoprenaline unmasks an impaired ability to stimulate Ca2+ influx, SR Ca2+ release, and CaTs caused, in part, by reduced phosphorylation reserve of RyR2 in Cacna1c+/- cardiomyocytes.

Calcium handling maturation and adaptation to increased substrate stiffness in human iPSC-derived cardiomyocytes: The impact of full-length dystrophin deficiency.

Cardiomyocytes differentiated from human induced Pluripotent Stem Cells (hiPSC- CMs) are a unique source for modelling inherited cardiomyopathies. In particular, the possibility of observing maturation processes in a simple culture dish opens novel perspectives in the study of early-disease defects caused by genetic mutations before the onset of clinical manifestations. For instance, calcium handling abnormalities are considered as a leading cause of cardiomyocyte dysfunction in several genetic-based dilated cardiomyopathies, including rare types such as Duchenne Muscular Dystrophy (DMD)-associated cardiomyopathy. To better define the maturation of calcium handling we simultaneously measured action potential and calcium transients (Ca-Ts) using fluorescent indicators at specific time points. We combined micropatterned substrates with long-term cultures to improve maturation of hiPSC-CMs (60, 75 or 90 days post-differentiation). Control-(hiPSC)-CMs displayed increased maturation over time (90 vs 60 days), with longer action potential duration (APD), increased Ca-T amplitude, faster Ca-T rise (time to peak) and Ca-T decay (RT50). The progressively increased contribution of the SR to Ca release (estimated by post-rest potentiation or Caffeine-induced Ca-Ts) appeared as the main determinant of the progressive rise of Ca-T amplitude during maturation. As an example of severe cardiomyopathy with early onset, we compared hiPSC-CMs generated from a DMD patient (DMD-ΔExon50) and a CRISPR-Cas9 genome edited cell line isogenic to the healthy control with deletion of a G base at position 263 of the DMD gene (c.263delG-CMs). In DMD-hiPSC-CMs, changes of Ca-Ts during maturation were less pronounced: indeed, DMD cells at 90 days showed reduced Ca-T amplitude and faster Ca-T rise and RT50, as compared with control hiPSC-CMs. Caffeine-Ca-T was reduced in amplitude and had a slower time course, suggesting lower SR calcium content and NCX function in DMD vs control cells. Nonetheless, the inotropic and lusitropic responses to forskolin were preserved. CRISPR-induced c.263delG-CM line recapitulated the same developmental calcium handling alterations observed in DMD-CMs. We then tested the effects of micropatterned substrates with higher stiffness. In control hiPSC-CMs, higher stiffness leads to higher amplitude of Ca-T with faster decay kinetics. In hiPSC-CMs lacking full-length dystrophin, however, stiffer substrates did not modify Ca-Ts but only led to higher SR Ca content. These findings highlighted the inability of dystrophin-deficient cardiomyocytes to adjust their calcium homeostasis in response to increases of extracellular matrix stiffness, which suggests a mechanism occurring during the physiological and pathological development (i.e. fibrosis).

Autophagy-related gene 5 (Atg5) is required for maintaining subcellular calcium homeostasis during acute cardiac stress

Abstract Background Autophagy exerts protective effects during cardiac stress inflicted by β-adrenergic stimulation and pressure overload. Contrarily, deletion of the cardiac-specific Atg5 gene contributes to left ventricular hypertrophy and contractile dysfunction, resulting in dilated cardiomyopathy. Heart failure is characterized by perturbations of excitation-contraction coupling (ECC) underlying alterations in intracellular calcium signaling. Purpose In this study, we aimed to understand whether loss of basal autophagy due to Atg5 deletion specifically in cardiomyocytes contributes directly to disturbances in subcellular calcium cycling. Methods Cardiac-specific Atg5−/− and Atg5+/+ mice were used to isolate ventricular cardiomyocytes, which were loaded with 8 μM Ca2+ indicator Fluo4/AM. Isolated cells were electrically stimulated and subjected to elevated workload by increasing pacing frequencies from 1 to 4 Hz. Cytosolic and nuclear calcium transients (CaTs) were recorded in line-scan mode using a laser scanning confocal microscope. Pharmacological inhibition of autophagic flux was performed by an intraperitoneal injection of 40 mg/kg leupeptin to Atg5+/+ mice. Results At baseline (1 Hz) stimulation the amplitude and kinetics of calcium transients were comparable between Atg5−/− and Atg5+/+ cells. However, after progressive increase of the pacing rate from 1 Hz to 4 Hz, Atg5−/− cardiomyocytes displayed reduced cytoplasmic and nuclear CaT amplitude, but increased nuclear time-averaged CaTs compared to controls. Such increase in nuclear calcium load stimulates Ca2+-dependent transcriptional activity, and likely involves CaMKII-mediated pro-hypertrophic gene program thus contributing to cardiac remodelling. In addition, we observed increased occurrence of arrhythmic events in Atg5−/− hearts at high pacing frequency, indicative of intracellular calcium overload and disturbed ECC. Acute pharmacological autophagy inhibition failed to elicit direct changes in time-averaged CaTs in the cytoplasm and nucleoplasm, suggesting that loss of Atg5-dependent autophagy seems to induce adverse cardiac remodelling that involves increased CaMKII activity due to the imbalances in nuclear Ca2+ levels. Conclusion Specific loss of ATG5 protein impairs calcium cycling during acute stress exposure by reducing CaT amplitudes, promoting arrhythmia, and increasing nuclear calcium load, which may induce hypertrophic gene expression. Further work is needed to identify key components mediating autophagy-related cardioprotection to develop strategies against cardiac remodeling and its progression to heart failure. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Austrian Science Fund (FWF)

Transient Receptor Potential Vanilloid 4 channel participates in mouse ventricular electrical activity

Transient Receptor Potential Vanilloid 4 (TRPV4) channel is a calcium permeable channel (P Ca /P Na ∼ 10). Its expression was reported in ventricular myocytes where it is involved in several cardiac pathological mechanisms. In this study, we investigated the implication of TRPV4 in ventricular electrical activity. Left ventricular myocytes were isolated from trpv4 +/+ and trpv4 −/− mice. TRPV4 membrane expression and its colocalization with Ca v 1.2 was confirmed using western-blots biotinylation, immunoprecipitation and immunostaining experiments. Then, electrocardiograms (ECGs) and patch-clamp recordings showed shortened QTc and action potential (AP) duration in trpv4 −/− compared to trpv4 +/+ mice. Thus, TRPV4 activator GSK1016790A produced a transient and dose-dependent increase in AP duration at 90% of repolarization (APD 90 ) in trpv4 +/+ , but not in trpv4 −/− myocytes or when combined with TRPV4 inhibitor GSK2193874 (100 nM). Hence, GSK1016790A increased CaT amplitude in trpv4 +/+ but not in trpv4 −/− myocytes, suggesting that TRPV4 carries an inward Ca 2+ current in myocytes. Conversely, TRPV4 inhibitor GSK2193874 (100 nM) alone reduced APD 90 in trpv4 +/+ but not in trpv4 −/− myocytes, suggesting that TRPV4 prolongs AP duration (APD) in basal condition. Finally, introducing TRPV4 parameters in a mathematical model predicted the development of an inward TRPV4 current during repolarization that increases AP duration and CaT amplitude, in accordance with what found experimentally. This study shows for the first time that TRPV4 modulates AP and QTc durations. It would be interesting to evaluate whether TRPV4 could be involved in long QT-mediated ventricular arrhythmias.

Different paths, same destination: divergent action potential responses produce conserved cardiac fight-or-flight response in mouse and rabbit hearts.

Cardiac electrophysiology and Ca2+ handling change rapidly during the fight-or-flight response to meet physiological demands. Despite dramatic differences in cardiac electrophysiology, the cardiac fight-or-flight response is highly conserved across species. In this study, we performed physiological sympathetic nerve stimulation (SNS) while optically mapping cardiac action potentials and intracellular Ca2+ transients in innervated mouse and rabbit hearts. Despite similar heart rate and Ca2+ handling responses between mouse and rabbit hearts, we found notable species differences in spatio-temporal repolarization dynamics during SNS. Species-specific computational models revealed that these electrophysiological differences allowed for enhanced Ca2+ handling (i.e. enhanced inotropy) in each species, suggesting that electrophysiological responses are fine-tuned across species to produce optimal cardiac fight-or-flight responses. Sympathetic activation of the heart results in positive chronotropy and inotropy, which together rapidly increase cardiac output. The precise mechanisms that produce the electrophysiological and Ca2+ handling changes underlying chronotropic and inotropic responses have been studied in detail in isolated cardiac myocytes. However, few studies have examined the dynamic effects of physiological sympathetic nerve activation on cardiac action potentials (APs) and intracellular Ca2+ transients (CaTs) in the intact heart. Here, we performed bilateral sympathetic nerve stimulation (SNS) in fully innervated, Langendorff-perfused rabbit and mouse hearts. Dual optical mapping with voltage- and Ca2+ -sensitive dyes allowed for analysis of spatio-temporal AP and CaT dynamics. The rabbit heart responded to SNS with a monotonic increase in heart rate (HR), monotonic decreases in AP and CaT duration (APD, CaTD), and a monotonic increase in CaT amplitude. The mouse heart had similar HR and CaT responses; however, a pronounced biphasic APD response occurred, with initial prolongation (50.9±5.1ms at t=0s vs. 60.6±4.1ms at t=15s, P<0.05) followed by shortening (46.5±9.1ms at t=60s, P=NS vs. t=0). We determined the biphasic APD response in mouse was partly due to dynamic changes in HR during SNS and was exacerbated by β-adrenergic activation. Simulations with species-specific cardiac models revealed that transient APD prolongation in mouse allowed for greater and more rapid CaT responses, suggesting more rapid increases in contractility; conversely, the rabbit heart requires APD shortening to produce optimal inotropic responses. Thus, while the cardiac fight-or-flight response is highly conserved between species, the underlying mechanisms orchestrating these effects differ significantly.

Calcium Transient Assays for Compound Screening with Human iPSC-derived Cardiomyocytes: Evaluating New Tools.

Calcium (Ca2+) plays a central role in regulating many biological processes in the cell from muscle contraction to neurotransmitter release. The need for reliable fluorescent calcium indicator dyes is of vast importance for studying many aspects of cell biology as well as screening compounds using phenotypic high throughput assays. We have assessed two of the latest generation of calcium indicator dyes, FLIPR Calcium 6 and Cal-520 AM for studying calcium transients (CaTs) in induced pluripotent stem cell (iPSC) -derived human cardiomyocytes. FLIPR Calcium 6 and Cal-520 dyes both displayed robust CaTs with a high signal-to-noise ratio (SNR) and were non-toxic to the cells. The analysis showed that CaT amplitudes were stable between measurements, but CaT duration was more variable and tended to increase between reads. Two methods were compared for drug-screening hit-selection; difference in average (unstandardized) and standardized difference. The unstandardized difference was better for assessing CaT amplitude, whereas standardized difference was equal to or better for assessing CaT duration. In summary, FLIPR Calcium 6 and Cal-520 are suitable dyes for drug-screening using iPSC-derived human cardiomyocytes.

Atrial Fibrillation Activates AMP-Dependent Protein Kinase and its Regulation of Cellular Calcium Handling: Potential Role in Metabolic Adaptation and Prevention of Progression

BackgroundAtrial fibrillation (AF) is associated with metabolic stress, which activates adenosine monophosphate-regulated protein kinase (AMPK). ObjectivesThis study sought to examine AMPK response to AF and associated metabolic stress, along with consequences for atrial cardiomyocyte Ca2+ handling. MethodsCalcium ion (Ca2+) transients (CaTs) and cell shortening (CS) were measured in dog and human atrial cardiomyocytes. AMPK phosphorylation and AMPK association with Ca2+-handling proteins were evaluated by immunoblotting and immunoprecipitation. ResultsCaT amplitude and CS decreased at 4-min glycolysis inhibition (GI) but returned to baseline at 8 min, suggesting cellular adaptation to metabolic stress, potentially due to AMPK activation. GI increased AMPK-activating phosphorylation, and an AMPK inhibitor, compound C (CompC), abolished the adaptation of CaT and CS to GI. The AMPK activator 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) increased CaT amplitude and CS, restoring CompC-induced CaT and CS decreases. CompC decreased L-type calcium channel current (ICa,L), along with ICa,L-triggered CaT amplitude and sarcoplasmic reticulum (SR) Ca2+ content under voltage clamp conditions in dog cells and suppressed CaT and ICa,L in human cardiomyocytes. Small interfering ribonucleic acid-based AMPK knockdown decreased CaT amplitude in neonatal rat cardiomyocytes. L-type Ca2+ channel α subunits coimmunoprecipitated with AMPKα. Atrial AMPK-activating phosphorylation was enhanced by 1 week of electrically maintained AF in dogs; fractional AMPK phosphorylation was increased in paroxysmal AF and reduced in longstanding persistent AF patients. ConclusionsAMPK is activated by metabolic stress and AF, and helps maintain the intactness of atrial ICa,L, Ca2+ handling, and cell contractility. AMPK contributes to the atrial compensatory response to AF-related metabolic stress; AF-related metabolic responses may be an interesting new therapeutic target.

Variations in local calcium signaling in adjacent cardiac myocytes of the intact mouse heart detected with two-dimensional confocal microscopy.

Dyssynchronous local Ca release within individual cardiac myocytes has been linked to cellular contractile dysfunction. Differences in Ca kinetics in adjacent cells may also provide a substrate for inefficient contraction and arrhythmias. In a new approach we quantify variation in local Ca transients between adjacent myocytes in the whole heart. Langendorff-perfused mouse hearts were loaded with Fluo-8 AM to detect Ca and Di-4-ANEPPS to visualize cell membranes. A spinning disc confocal microscope with a fast camera allowed us to record Ca signals within an area of 465 μm by 315 μm with an acquisition speed of 55 fps. Images from multiple transients recorded at steady state were registered to their time point in the cardiac cycle to restore averaged local Ca transients with a higher temporal resolution. Local Ca transients within and between adjacent myocytes were compared with regard to amplitude, time to peak and decay at steady state stimulation (250 ms cycle length). Image registration from multiple sequential Ca transients allowed reconstruction of high temporal resolution (2.4 ± 1.3 ms) local CaT in 2D image sets (N = 4 hearts, n = 8 regions). During steady state stimulation, spatial Ca gradients were homogeneous within cells in both directions and independent of distance between measured points. Variation in CaT amplitudes was similar across the short and the long side of neighboring cells. Variations in TAU and TTP were similar in both directions. Isoproterenol enhanced the CaT but not the overall pattern of spatial heterogeneities. Here we detected and analyzed local Ca signals in intact mouse hearts with high temporal and spatial resolution, taking into account 2D arrangement of the cells. We observed significant differences in the variation of CaT amplitude along the long and short axis of cardiac myocytes. Variations of Ca signals between neighboring cells may contribute to the substrate of cardiac remodeling.

Na⁺ ions as spatial intracellular messengers for co-ordinating Ca²⁺ signals during pH heterogeneity in cardiomyocytes.

AimsContraction of the heart is regulated by electrically evoked Ca2+ transients (CaTs). H+ ions, the end products of metabolism, modulate CaTs through direct interactions with Ca2+-handling proteins and via Na+-mediated coupling between acid-extruding proteins (e.g. Na+/H+ exchange, NHE1) and Na+/Ca2+ exchange. Restricted H+ diffusivity in cytoplasm predisposes pH-sensitive Ca2+ signalling to becoming non-uniform, but the involvement of readily diffusible intracellular Na+ ions may provide a means for combatting this.Methods and resultsCaTs were imaged in fluo3-loaded rat ventricular myocytes paced at 2 Hz. Cytoplasmic [Na+] ([Na+]i) was imaged using SBFI. Intracellular acidification by acetate exposure raised diastolic and systolic [Ca2+] (also observed with acid-loading by ammonium prepulse or CO2 exposure). The systolic [Ca2+] response correlated with a rise in [Na+]i and sarcoplasmic reticulum Ca2+ load, and was blocked by the NHE1 inhibitor cariporide (CO2/HCO3−-free media). Exposure of one half of a myocyte to acetate using dual microperfusion (CO2/HCO3−-free media) raised diastolic [Ca2+] locally in the acidified region. Systolic [Ca2+] and CaT amplitude increased more uniformly along the length of the cell, but only when NHE1 was functional. Cytoplasmic Na+ diffusivity (DNa) was measured in quiescent cells, with strophanthidin present to inhibit the Na+/K+ pump. With regional acetate exposure to activate a local NHE-driven Na+-influx, DNa was found to be sufficiently fast (680 µm2/s) for transmitting the pH–systolic Ca2+ interaction over long distances.ConclusionsNa+ ions are rapidly diffusible messengers that expand the spatial scale of cytoplasmic pH–CaT interactions, helping to co-ordinate global Ca2+ signalling during conditions of intracellular pH non-uniformity.

Abstract 20662: Variations in Local Calcium Signaling in Adjacent Cardiomyocytes of the Intact Mouse Heart Detected With Two-Dimensional Confocal Microscopy

Coordinated Ca release in the heart ensures efficient myocardial contraction and electrical stability. Dyssynchronous Ca release within individual cardiomyocytes has been linked to cellular contractile dysfunction. Differences in Ca kinetics in adjacent cells may also provide a substrate for inefficient contraction and arrhythmias. We present an approach to quantify local Ca signals related to the geometry of adjacent cardiomyocytes within the intact heart using 2D confocal microscopy. We observe significant differences in Ca kinetics between adjacent cardiomyocytes. Methods: Ca transients (CaT, Fluo-8 AM) and cell membranes (Di-4 ANEPPS) were visualized in an epicardial area of 465 μm by 315 μm (12 to 20 cells) in Langendorff-perfused mouse hearts. Images (55/second) from a series of CaT at steady state stimulation (250 ms cycle length) were registered to their relative timepoint in the cardiac cycle to restore local CaT with a temporal resolution of 2 ms. Variation of local CaT within a cardiomyocyte (INTRA) and between adjacent cardiomyocytes (INTER) were compared with regard to amplitude, time to peak (TTP) and time constant of decay (TAU). Results: Temporal registration of images from successive CaT yielded local signals with good signal/noise ratio (N= 4 hearts, n= 8 regions). Along the long axis but not along the short axis of cardiomyocyte orientation the variation of CaT amplitude was significantly larger between that within cells (INTER 51.49 ±23.57% vs. INTRA &lt;0.01 ±0.001% of global Ca amplitude). Variation in TTP Ca was comparable in all directions, but TAU also tended to vary more along the long axis (INTER 68.50 ±27.03% vs. INTRA 37.74 ±13.17%). Isoproterenol (10 μM) significantly increased the differences in TAU between adjacent cardiomyocytes along the short axis but not along the long axis. Summary: We present an approach to detect and analyze local Ca signals in Langendorff-perfused intact mouse hearts with high temporal and spatial resolution, taking into account 2D orientation of the cells. We observed significant differences in the synchrony of Ca signals along the long and short axis of adjacent cardioymyocytes. Alterations in synchrony of Ca signals in neighboring cells may contribute to the substrate in cardiac remodeling.

Modulation of ventricular transient outward K+ current by acidosis and its effects on excitation-contraction coupling

The contribution of transient outward current (Ito) to changes in ventricular action potential (AP) repolarization induced by acidosis is unresolved, as is the indirect effect of these changes on calcium handling. To address this issue we measured intracellular pH (pHi), Ito, L-type calcium current (ICa,L), and calcium transients (CaTs) in rabbit ventricular myocytes. Intracellular acidosis [pHi 6.75 with extracellular pH (pHo) 7.4] reduced Ito by ~50% in myocytes with both high (epicardial) and low (papillary muscle) Ito densities, with little effect on steady-state inactivation and activation. Of the two candidate α-subunits underlying Ito, human (h)Kv4.3 and hKv1.4, only hKv4.3 current was reduced by intracellular acidosis. Extracellular acidosis (pHo 6.5) shifted Ito inactivation toward less negative potentials but had negligible effect on peak current at +60 mV when initiated from -80 mV. The effects of low pHi-induced inhibition of Ito on AP repolarization were much greater in epicardial than papillary muscle myocytes and included slowing of phase 1, attenuation of the notch, and elevation of the plateau. Low pHi increased AP duration in both cell types, with the greatest lengthening occurring in epicardial myocytes. The changes in epicardial AP repolarization induced by intracellular acidosis reduced peak ICa,L, increased net calcium influx via ICa,L, and increased CaT amplitude. In summary, in contrast to low pHo, intracellular acidosis has a marked inhibitory effect on ventricular Ito, perhaps mediated by Kv4.3. By altering the trajectory of the AP repolarization, low pHi has a significant indirect effect on calcium handling, especially evident in epicardial cells.

Intracellular Na+ Spatially Controls Ca2+ Signaling during Acidosis in the Ventricular Myocyte

Myocardial intracellular pH (pHi) exerts major effects on cardiac function. Decreased pHi can depress Ca2+-transients (CaTs) via effects on SR CaATPase and ryanodine-receptors. The inhibition is typically offset by low pHi stimulation of sarcolemmal Na/H exchange (NHE). This raises [Na+]i sufficiently to increase SR Ca2+-loading via t-tubular Na/Ca exchange, thus increasing CaT-amplitude, which helps to protect contraction. We find that NHE activity (rat ventricular myocytes) also affects Ca2+-signalling spatially. A longitudinal pHi non-uniformity can be induced by microperfusion of the weak acid, acetate (80mM), over one end of the cell. pHi falls in the acetate-exposed region, resulting in a stable pHi-gradient of ∼0.6 units. With NHE inhibited (30μM cariporide), this procedure persistently reduced CaT amplitude, but only in the acidic region. With functional NHE (no cariporide), the same stably-imposed pHi-gradient increased CaT-amplitude uniformly throughout the myocyte. This occurred despite NHE being stimulated only at the acidic end of the cell. One explanation is that local Na+-influx during regional NHE-stimulation uniformly elevates [Na+]i, thus increasing SR Ca2+-loading in both acidic and non-acidic regions. This requires that cytoplasmic Na+ be readily diffusible, as NHE protein is mainly expressed at surface sarcolemma and intercalated-disks (not in transverse-tubules). We therefore imaged [Na+]i (AM-loaded SBFI) during localised NHE-stimulation (local acetate microperfusion, rest of cell exposed to Na-free solution; 10−6 M strophanthidin throughout to inhibit Na/K-transporters). This induced a local NHE-mediated [Na+]i-rise, which dissipated longitudinally, with an apparent diffusion-coefficient of 680μm2/s (∼50% of that in pure water), consistent with rapid Na+i-mobility. We therefore propose that high Na+i-mobility permits local NHE activity to unify CaT-amplitude spatially, even during pHi non-uniformity. NHE activity in the ventricular myocyte thus not only enhances Ca2+-signalling during acidosis, to protect contraction, but also spatially unifies Ca2+-signalling within the cell.

Basic Science of Cardiac Resynchronization Therapy

Electromechanical dyssynchrony can markedly worsen heart failure (HF) morbidity and mortality, independent of traditional risk factors.1–4 Depending on the metric used, current estimates of the prevalence of dyssynchrony vary from 25–30% in patients with HF (based on QRS widening) up to 60%, based on tissue Doppler or MRI measures of dyssynchronous contraction of the left ventricle (LV).5,6 Cardiac resynchronization therapy (CRT) or biventricular pacing has emerged as a promising option to treat patients with HF and dyssynchronous contraction.7–9 The past few decades have seen the rise of pharmacotherapy, primarily through agents that antagonize the effect of excessive concentrations of circulating neurohormones, yet, HF-related morbidity and mortality remain high.3–6 Biventricular stimulation has been demonstrated to improve contractile performance in patients with mechanical dyssynchrony acutely and chronically while also prolonging long-term survival—something not yet achieved by drug therapy.10 Although the clinical and mechanical effectiveness of CRT are well described, 30% of patients do not benefit from CRT and clinical criteria to identify CRT nonresponders remain elusive8,11,12 Currently, the most widely used predictor of reverse remodeling is the presence of marked mechanical dyssynchrony before CRT, as indexed by the width of the QRS.13 Mechanical dyssynchrony seems important, yet imaging-based measures have not predicted response well14 and even improvement in dyssynchrony after initiation of CRT only weakly predicts chronic response.15 Limited understanding of the molecular mechanisms underlying reverse cardiac remodeling induced by CRT has hampered the selection of potential responders. In this review, we focus on the electrophysiological aspects and molecular networks underlying the benefits of CRT. We will review how CRT homogenizes regional differences in stress kinase signaling and electric remodeling and then review its global effect on myocyte function and its …

Ranolazine Antagonizes the Effects of Increased Late Sodium Current on Intracellular Calcium Cycling in Rat Isolated Intact Heart

Pathological conditions, including ischemia and heart failure, are associated with altered sodium channel function and increased late sodium current (I(Na,L)), leading to prolonged action potential duration, increased intracellular sodium and calcium concentrations, and arrhythmias. We used anemone toxin (ATX)-II to study the effects of increasing I(Na,L) on intracellular calcium cycling in rat isolated hearts. Cardiac contraction was abolished using paralytic agents. Ranolazine (RAN) was used to inhibit late I(Na). Hearts were loaded with fluo-4-acetoxymethyl ester, and myocyte intracellular calcium transients (CaTs) were measured using laser scanning confocal microscopy. ATX (1 nM) prolonged CaT duration at 50% recovery in hearts paced at a basal rate of 2 Hz and increased the sensitivity of the heart to the development of calcium alternans caused by fast pacing. ATX increased the time required for recovery of CaT amplitude following a previous beat, and ATX induced spontaneous calcium release waves during rapid pacing of the heart. ATX prolonged the duration of repolarization from the initiation of the activation to terminal repolarization in the pseudo-electrocardiogram. All actions of ATX were both reversed and prevented by subsequent or prior exposure, respectively, of hearts to RAN (10 microM). Most importantly, the increased vulnerability of the heart to the development of calcium alternans during rapid pacing was reversed or prevented by 10 microM RAN. These results suggest that enhancement of I(Na,L) alters calcium cycling. Reduction by RAN of I(Na,L)-induced dysregulation of calcium cycling could contribute to the antiarrhythmic actions of this agent in both reentrant and triggered arrhythmias.

Muscarinic cation current and suppression of Ca 2+ current in guinea pig ileal smooth muscle cells

Cationic current ( I cat) and inhibition of the voltage-dependent Ca 2+ current ( I Ca) evoked by muscarinic receptor activation with carbachol were studied using whole-cell patch clamp technique in smooth muscle cells isolated from longitudinal muscle of guinea pig small intestine. With low buffering of [Ca 2+] i (0.1 mM BAPTA [1,2-bis-(2-aminophenoxy)-ethane- N, N, N′, N′-tetraacetic acid] in pipette solution) I cat and I Ca inhibitory responses had a rapid onset to an initial peak followed by a sustained phase. The sustained phase of I Ca suppression was bigger than in the case when [Ca 2+] i was clamped to 100 nM, but decreased with repeated stimulation. Upon repeated stimulation with 50 μM carbachol in cells where [Ca 2+] i was clamped to 100 nM and when GTP was absent, I cat amplitude decreased strongly and more substantially compared to I Ca inhibition, but both responses declined only slightly when 1 mM GTP was present in the pipette solution. GDP- βS (1 or 5 mM) in pipette solution or pre-treatment of cells with pertussis toxin (6 μg/ml, for 4 h or longer) blocked I cat more than I Ca suppression by carbachol, whereas l-NAME ( N- Ω-nitro- l-arginine methyl ester hydrochloride) (100 μM in pipette solution) affected neither of them significantly. We conclude that the cationic current and the suppression of the voltage-dependent Ca 2+ current evoked by muscarinic receptor activation are mediated by pertussis toxin-sensitive G-protein(s) but the latter response was less sensitive to blockade by GDP- βS and to GTP deficiency in the cell.