Sarcoplasmic Reticulum Calcium Load Research Articles

Pitx2c deficiency confers cellular electrophysiological hallmarks of atrial fibrillation to isolated atrial myocytes

AimsAtrial fibrillation (AF) has been associated with altered expression of the transcription factor Pitx2c and a high incidence of calcium release-induced afterdepolarizations. However, the relationship between Pitx2c expression and defective calcium homeostasis remains unclear and we here aimed to determine how Pitx2c expression affects calcium release from the sarcoplasmic reticulum (SR) and its impact on electrical activity in isolated atrial myocytes. MethodsTo address this issue, we applied confocal calcium imaging and patch-clamp techniques to atrial myocytes isolated from a mouse model with conditional atrial-specific deletion of Pitx2c. ResultsOur findings demonstrate that heterozygous deletion of Pitx2c doubles the calcium spark frequency, increases the frequency of sparks/site 1.5-fold, the calcium spark decay constant from 36 to 42 ms and the wave frequency from none to 3.2 min−1. Additionally, the cell capacitance increased by 30% and both the SR calcium load and the transient inward current (ITI) frequency were doubled. Furthermore, the fraction of cells with spontaneous action potentials increased from none to 44%. These effects of Pitx2c deficiency were comparable in right and left atrial myocytes, and homozygous deletion of Pitx2c did not induce any further effects on sparks, SR calcium load, ITI frequency or spontaneous action potentials. ConclusionOur findings demonstrate that heterozygous Pitx2c deletion induces defects in calcium homeostasis and electrical activity that mimic derangements observed in right atrial myocytes from patients with AF and suggest that Pitx2c deficiency confers cellular electrophysiological hallmarks of AF to isolated atrial myocytes.

Expression and Impact of Adenosine A3 Receptors on Calcium Homeostasis in Human Right Atrium.

Increased adenosine A2A receptor (A2AR) expression and activation underlies a higher incidence of spontaneous calcium release in atrial fibrillation (AF). Adenosine A3 receptors (A3R) could counteract excessive A2AR activation, but their functional role in the atrium remains elusive, and we therefore aimed to address the impact of A3Rs on intracellular calcium homeostasis. For this purpose, we analyzed right atrial samples or myocytes from 53 patients without AF, using quantitative PCR, patch-clamp technique, immunofluorescent labeling or confocal calcium imaging. A3R mRNA accounted for 9% and A2AR mRNA for 32%. At baseline, A3R inhibition increased the transient inward current (ITI) frequency from 0.28 to 0.81 events/min (p < 0.05). Simultaneous stimulation of A2ARs and A3Rs increased the calcium spark frequency seven-fold (p < 0.001) and the ITI frequency from 0.14 to 0.64 events/min (p < 0.05). Subsequent A3R inhibition caused a strong additional increase in the ITI frequency (to 2.04 events/min; p < 0.01) and increased phosphorylation at s2808 1.7-fold (p < 0.001). These pharmacological treatments had no significant effects on L-type calcium current density or sarcoplasmic reticulum calcium load. In conclusion, A3Rs are expressed and blunt spontaneous calcium release at baseline and upon A2AR-stimulation in human atrial myocytes, pointing to A3R activation as a means to attenuate physiological and pathological elevations of spontaneous calcium release events.

L-type Cav1.3 calcium channels are key actors in beta-adrenergic triggered automaticity in dormant mouse sino-atrial myocytes

Cardiac pacemaking relies on the spontaneous electrical activity of sino-atrial myocytes (SANCs) in the right atrium arising from a robust interplay of membrane ion channels activity (membrane clock) and intracellular calcium dynamics (calcium clock). Strikingly, most of isolated SANCs exhibit a dormant state (no apparent spontaneous activity) whereas only a small fraction shows regular firing. It has recently been shown that these non-firing SANCs are also present in intact SAN and can switch into a firing mode under beta-adrenergic stimulation. However, their functional relevance in vivo is unknown. To study the role of L-type Ca v 1.3 calcium channels in dormant SANCs using a knock-in mouse strain in which the sensitivity of Ca v 1.2 α1 subunits to dihydropyridines (DHP) was inactivated (Ca v 1.2 DHP−/− ). We performed voltage and current-clamp recordings and confocal live imaging on isolated SANCs under isoprenaline (ISO, 100 nM) in the absence or presence of the DHP blocker Nifedipine (Nife, 3 μM). Preliminary results seem to show less L-type calcium current I Ca,L in dormant SANCs (D) compared to rhythmic SANCs (R) (D: −6.3 ± 1.2, R: −15.6 pA/pF). Dormant SANCs also have less diastolic calcium (D: 96.9 ± 12, R: 218 ± 51.2 a.u, P = 0.14) but significant higher sarcoplasmic reticulum calcium load (D: 6.3 ± 0.6, R: 3.1 ± 0.3 a.u, P = 0.004). Interestingly, ISO perfusion could only switch half of dormant SANCs into a firing mode in both action potential (AP) and calcium transients (CT) recordings whereas the others remained dormant. Further Nife perfusion completely stopped this ISO-induced firing switching the cells back into a dormant state for the vast majority of them (75% in AP-recorded SANCs and 100% in CT-recorded SANCs). Taken together, these results indicate that Cav1.3 channels play a key role in sustaining the coupling of membrane and calcium clock in dormant SANCs under beta-adrenergic stimulation.

Controllability of voltage- and calcium-driven cardiac alternans in a map model.

Certain cardiac arrhythmias are preceded by electrical alternans, a state characterized by beat-to-beat alternation in cellular action potential duration. Cardiac alternans may arise from different mechanisms including instabilities in voltage or intracellular calcium cycling. Although a number of techniques have been proposed to suppress alternans, these methods have mainly been tested using models that do not support calcium-driven alternans. Therefore, it is important to understand how control methods may perform when alternans is driven by instabilities in calcium cycling. In this study, we applied controllability analysis to a discrete map of alternans dynamics in a cardiac cell. We compared two different controllability measures to determine to what extent different control strategies could suppress alternans and tested these predictions using three feedback controllers. We found a modal controllability measure, unlike the minimum singular value of the controllability matrix, consistently indicated the control strategies requiring the least control effort and yielding the smallest closed-loop eigenvalue. In addition, action potential duration was identified as the most effective variable through which control can be applied, regardless of alternans mechanism, although sarcoplasmic reticulum calcium load was also useful for the calcium-driven alternans cases.

Modulation of Calcium Transients in Cardiomyocytes by Transient Receptor Potential Canonical 6 Channels.

Transient receptor potential canonical 6 (TRPC6) channels are non-selective cation channels that are thought to underlie mechano-modulation of calcium signaling in cardiomyocytes. TRPC6 channels are involved in development of cardiac hypertrophy and related calcineurin-nuclear factor of activated T cells (NFAT) signaling. However, the exact location and roles of TRPC6 channels remain ill-defined in cardiomyocytes. We used an expression system based on neonatal rat ventricular myocytes (NRVMs) to investigate the location of TRPC6 channels and their role in calcium signaling. NRVMs isolated from 1- to 2-day-old animals were cultured and infected with an adenoviral vector to express enhanced-green fluorescent protein (eGFP) or TRPC6-eGFP. After 3 days, NRVMs were fixed, immunolabeled, and imaged with confocal and super-resolution microscopy to determine TRPC6 localization. Cytosolic calcium transients at 0.5 and 1 Hz pacing rates were recorded in NRVMs using indo-1, a ratio-metric calcium dye. Confocal and super-resolution microscopy suggested that TRPC6-eGFP localized to the sarcolemma. NRVMs infected with TRPC6-eGFP exhibited higher diastolic and systolic cytosolic calcium concentration as well as increased sarcoplasmic reticulum (SR) calcium load compared to eGFP infected cells. We applied a computer model comprising sarcolemmal TRPC6 current to explain our experimental findings. Altogether, our studies indicate that TRPC6 channels play a role in sarcolemmal and intracellular calcium signaling in cardiomyocytes. Our findings support the hypothesis that upregulation or activation of TRPC6 channels, e.g., in disease, leads to sustained elevation of the cytosolic calcium concentration, which is thought to activate calcineurin-NFAT signaling and cardiac hypertrophic remodeling. Also, our findings support the hypothesis that mechanosensitivity of TRPC6 channels modulates cytosolic calcium transients and SR calcium load.

P1593VEGFR1-PLC signaling is implicated in normal spontaneous firing of cardiac pacemaker cells

Abstract Spontaneous firing of sinoatrial node cells (SANC) is regulated by sarcoplasmic reticulum (SR) generated local subsarcolemmal calcium releases (LCRs). LCRs appear during diastolic depolarization (DD) and activate an inward sodium-calcium exchange current to accelerate the DD rate and thus spontaneous SANC firing. Vascular endothelial growth factor (VEGF) receptors VEGFR1 and VEGFR2 activate PLC, and suppression of VEGFR-PLC signaling decreases calcium transients and contractility in ventricular myocytes. We tested the idea that VEGFR-PLC signaling may contribute to normal spontaneous beating of SANC. We observed that expression of VEGFR1 (assessed by RT-qPCR) in rabbit sinoatrial node was comparable to that of β1-adrenergic receptors, but less than that in ventricle. The pan VEGFR1/2/3 inhibitor PTK787/ZK222584 (10 μmol/L) in a time-dependent manner: (1) suppressed spontaneous SANC beating rate by ∼50% (perforated patch-clamp technique); (2) markedly decreased the LCR size and number per each spontaneous cycle (confocal microscopy, calcium indicator Fluo-3) and prolonged the LCR period (the interval between action potential-induced calcium transient and occurrence of subsequent LCR). The PTK787/ZK222584-induced increase in the LCR period (from 348.2±30.3 to 619.9±103.4 msec; P&lt;0.05) predicted the concomitant increase in the spontaneous cycle length (from 405.6±31.1 to 702.1±105.1 msec; P&lt;0.05), suggesting that calcium cycling could be a major target of VEGFR-dependent regulation of SANC firing. All effects of PTK787/ZK222584 were reversible upon washout. To elucidate whether signaling of VEGFR1 or VEGFR2 regulated spontaneous SANC firing, we employed a selective VEGFR2 inhibitor ZM-323881 (5 μmol/L), which suppressed spontaneous beating rate in only 2 of 10 SANC. These results indicate that VEGFR1, but not VEGFR2, is likely a key receptor that modulates automaticity in majority of SANC. To clarify downstream targets from VEGFR1 we employed PLC inhibitor U-73122, which decreased the LCR size, number and prolonged the LCR period. The inactive analog U-73343 was without effect. Because LCRs are critically dependent upon the SR calcium load, supplied by L-type calcium current (ICa,L), we examined effects of U-73122 on ICa,L. U-73122, but not U-73343, markedly suppressed ICa,L amplitude by ∼50%, leading to a decrease in the calcium influx and, as a result, to decrease in the LCR parameters, prolongation of the LCR period and spontaneous SANC cycle length. Thus, basal VEGFR1 signaling activates PLC, which modulates intracellular SR calcium cycling and LCR characteristics in SANC. We conclude that VEGFR1-PLC is a novel mechanism involved in the regulation of normal automaticity of cardiac pacemaker cells. Acknowledgement/Funding Intramural Research Program, National Institute on Aging, NIH

Targeting RyR2 with a phosphorylation site-specific nanobody reverses dysfunction of failing cardiomyocytes in rats.

Chronic PKA phosphorylation of ryanodine receptor 2 (RyR2) has been shown to increase diastolic sarcoplasmic reticulum (SR) Ca2+ leakage and lead to cardiac dysfunction. We hypothesize that intracellular gene delivery of an RyR2-targeting phosphorylation site-specific nanobody could preserve the contractility of the failing myocardium. In the present study, we acquired RyR2-specific nanobodies from a phage display library that were variable domains of Camelidae heavy chain-only antibodies. One of the nanobodies, AR185, inhibited RyR2 phosphorylation in vitro and was chosen for further investigation. We investigated the potential of adeno-associated virus (AAV)9-mediated cardiac expression of AR185 to combat postischemic heart failure (HF). AAV gene delivery elevated the intracellular expression of the AR185 protein in a rat model of ischemic HF, and this treatment normalized the systolic and diastolic dysfunction of the failing myocardium in vivo by reversing myocardial Ca2+ handling. Furthermore, AR185 gene transfer to failing cardiomyocytes reduced the frequency of SR calcium leaks, thereby restoring the attenuated intracellular calcium transients and SR calcium load. Moreover, AR185 gene transfer inhibited the PKA-mediated phosphorylation of RyR2 in failing cardiomyocytes. Our results provide preclinical experimental evidence that the cardiac expression of RyR2 nanobodies with AAV9 vectors is a promising therapeutic strategy for HF.-Li, T., Shen, Y., Lin, F., Fu, W., Liu, S., Wang, C., Liang, J., Fan, X., Ye, X., Tang, Y., Ding, M., Yang, Y., Lei, C., Hu, S. Targeting RyR2 with a phosphorylation site-specific nanobody reverses dysfunction of failing cardiomyocytes in rats.

Dantrolene sodium increases calcium binding by human recombinant cardiac calsequestrin and calcium loading by sheep cardiac sarcoplasmic reticulum.

Dantrolene interacts with ryanodine receptors in skeletal and cardiac muscle affecting sarcoplasmic reticulum calcium release. Since dantrolene is lipophilic it could also affect intra-sarcoplasmic reticulum calcium handling proteins such as calsequestrin. This study investigated whether dantrolene (1-30µmol/L) alters the polymerization state and the calcium binding capacity of recombinant cardiac calsequestrin and cardiac sarcoplasmic reticulum calcium handling. Human recombinant cardiac calsequestrin was used to make simultaneous measurements of turbidity (to indicate calsequestrin polymerization) and calcium binding to calsequestrin in the presence and absence of dantrolene. Caffeine-induced Ca2+ transients were used to investigate the effects of dantrolene on sarcoplasmic reticulum calcium loading and release in saponin-permeabilized cardiomyocytes laid down in monolayers in 96-well array plates. Dantrolene (1-30µmol/L) increased the polymerization state of calsequestrin and its calcium binding capacity. In the presence of dantrolene, calsequestrin-dependent turbidity increased 2.5-11.5-fold at 1.0mmol/L calcium added to unbuffered Ca2+ solutions and 3-10-fold when calcium was raised from 0.06 to 30µmol/L. The dantrolene-dependent increase in turbidity at 30µmol/L dantrolene was associated with a 3-fold increase in the number of calcium ions bound per calsequestrin molecule at 30 and 100µmol/L calcium. The caffeine-induced releasable calcium loaded by the sarcoplasmic reticulum at 0.63µmol/L free calcium in permeabilized cardiomyocytes was also increased in the presence of 30µmol/L dantrolene. Dantrolene alters the polymerization state and the calcium binding properties of cardiac calsequestrin and increases sarcoplasmic reticulum calcium loading in permeabilized cardiomyocytes.

The 4q25 variant rs13143308T links risk of atrial fibrillation to defective calcium homoeostasis.

AimsSingle nucleotide polymorphisms on chromosome 4q25 have been associated with risk of atrial fibrillation (AF) but the exiguous knowledge of the mechanistic links between these risk variants and underlying electrophysiological alterations hampers their clinical utility. Here, we tested the hypothesis that 4q25 risk variants cause alterations in the intracellular calcium homoeostasis that predispose to spontaneous electrical activity.Methods and resultsWestern blotting, confocal calcium imaging, and patch-clamp techniques were used to identify mechanisms linking the 4q25 risk variants rs2200733T and rs13143308T to defects in the calcium homoeostasis in human atrial myocytes. Our findings revealed that the rs13143308T variant was more frequent in patients with AF and that myocytes from carriers of this variant had a significantly higher density of calcium sparks (14.1 ± 4.5 vs. 3.1 ± 1.3 events/min, P = 0.02), frequency of transient inward currents (ITI) (1.33 ± 0.24 vs. 0.26 ± 0.09 events/min, P < 0.001) and incidence of spontaneous membrane depolarizations (1.22 ± 0.26 vs. 0.56 ± 0.17 events/min, P = 0.001) than myocytes from patients with the normal rs13143308G variant. These alterations were linked to higher sarcoplasmic reticulum calcium loading (10.2 ± 1.4 vs. 7.3 ± 0.5 amol/pF, P = 0.01), SERCA2 expression (1.37 ± 0.13 fold, P = 0.03), and RyR2 phosphorylation at ser2808 (0.67 ± 0.08 vs. 0.47 ± 0.03, P = 0.01) but not at ser2814 (0.28 ± 0.14 vs. 0.31 ± 0.14, P = 0.61) in patients carrying the rs13143308T risk variant. Furthermore, the presence of a risk variant or AF independently increased the ITI frequency and the increase in the ITI frequency observed in carriers of the risk variants was exacerbated in those with AF. By contrast, the presence of a risk variant did not affect the amplitude or properties of the L-type calcium current in patients with or without AF.ConclusionsHere, we identify the 4q25 variant rs13143308T as a genetic risk marker for AF, specifically associated with excessive calcium release and spontaneous electrical activity linked to increased SERCA2 expression and RyR2 phosphorylation.

The antiapoptotic protein HAX-1 mediates half of phospholamban's inhibitory activity on calcium cycling and contractility in the heart

The antiapoptotic protein HAX-1 (HS-associated protein X-1) localizes to sarcoplasmic reticulum (SR) in the heart and interacts with the small membrane protein phospholamban (PLN), inhibiting the cardiac sarco/endoplasmic reticulum calcium ATPase 2a (SERCA2a) in the regulation of overall calcium handling and heart muscle contractility. However, because global HAX-1 deletion causes early lethality, how much endogenous HAX-1 contributes to PLN's inhibitory activity on calcium cycling is unknown. We therefore generated a cardiac-specific and inducible knock-out mouse model. HAX-1 ablation in the adult heart significantly increased contractile parameters and calcium kinetics, associated with increased SR calcium load. These changes occurred without any changes in the protein expression of SERCA2a, PLN, and ryanodine receptor or in the PLN phosphorylation status. The enhanced calcium cycling in the HAX-1-depleted heart was mediated through increases in the calcium affinity of SERCA2a and reduced PLN-SERCA2a binding. Comparison of the HAX-1 deletion-induced stimulatory effects with those elicited by PLN ablation indicated that HAX-1 mediates ∼50% of the PLN-associated inhibitory effects in the heart. Stimulation with the inotropic and lusitropic agent isoproterenol eliminated the differences among wild-type, HAX-1-deficient, and PLN-deficient hearts, and maximally stimulated contractile and calcium kinetic parameters were similar among these three groups. Furthermore, PLN overexpression in the HAX-1-null cardiomyocytes did not elicit any inhibitory effects, indicating that HAX-1 may limit PLN activity. These findings suggest that HAX-1 is a major mediator of PLN's inhibitory activity and a critical gatekeeper of SR calcium cycling and contractility in the heart.

Up-regulation of Intracellular Calcium Handling Underlies the Recovery of Endotoxemic Cardiomyopathy in Mice.

In surviving patients, sepsis-induced cardiomyopathy is spontaneously reversible. In the absence of any experimental data, it is generally thought that cardiac recovery in sepsis simply follows the remission of systemic inflammation. Here the authors aimed to identify the myocardial mechanisms underlying cardiac recovery in endotoxemic mice. Male C57BL/6 mice were challenged with lipopolysaccharide (7 μg/g, intraperitoneally) and followed for 12 days. The authors assessed survival, cardiac function by echocardiography, sarcomere shortening, and calcium transients (with fura-2-acetoxymethyl ester) in electrically paced cardiomyocytes (5 Hz, 37°C) and myocardial protein expression by immunoblotting. Left ventricular ejection fraction, cardiomyocyte sarcomere shortening, and calcium transients were depressed 12 h after lipopolysaccharide challenge, started to recover by 24 h (day 1), and were back to baseline at day 3. The recovery of calcium transients at day 3 was associated with the up-regulation of the sarcoplasmic reticulum calcium pump to 139 ± 19% (mean ± SD) of baseline and phospholamban down-regulation to 35 ± 20% of baseline. At day 6, calcium transients were increased to 123 ± 31% of baseline, associated with increased sarcoplasmic reticulum calcium load (to 126 ± 32% of baseline, as measured with caffeine) and inhibition of sodium/calcium exchange (to 48 ± 12% of baseline). In mice surviving lipopolysaccharide challenge, the natural recovery of cardiac contractility was associated with the up-regulation of cardiomyocyte calcium handling above baseline levels, indicating the presence of an active myocardial recovery process, which included sarcoplasmic reticulum calcium pump activation, the down-regulation of phospholamban, and sodium/calcium exchange inhibition.

Diabetic Cardiomyopathy: An Immunometabolic Perspective.

The heart possesses a remarkable inherent capability to adapt itself to a wide array of genetic and extrinsic factors to maintain contractile function. Failure to sustain its compensatory responses results in cardiac dysfunction, leading to cardiomyopathy. Diabetic cardiomyopathy (DCM) is characterized by left ventricular hypertrophy and reduced diastolic function, with or without concurrent systolic dysfunction in the absence of hypertension and coronary artery disease. Changes in substrate metabolism, oxidative stress, endoplasmic reticulum stress, formation of extracellular matrix proteins, and advanced glycation end products constitute the early stage in DCM. These early events are followed by steatosis (accumulation of lipid droplets) in cardiomyocytes, which is followed by apoptosis, changes in immune responses with a consequent increase in fibrosis, remodeling of cardiomyocytes, and the resultant decrease in cardiac function. The heart is an omnivore, metabolically flexible, and consumes the highest amount of ATP in the body. Altered myocardial substrate and energy metabolism initiate the development of DCM. Diabetic hearts shift away from the utilization of glucose, rely almost completely on fatty acids (FAs) as the energy source, and become metabolically inflexible. Oxidation of FAs is metabolically inefficient as it consumes more energy. In addition to metabolic inflexibility and energy inefficiency, the diabetic heart suffers from impaired calcium handling with consequent alteration of relaxation–contraction dynamics leading to diastolic and systolic dysfunction. Sarcoplasmic reticulum (SR) plays a key role in excitation–contraction coupling as Ca2+ is transported into the SR by the SERCA2a (sarcoplasmic/endoplasmic reticulum calcium-ATPase 2a) during cardiac relaxation. Diabetic cardiomyocytes display decreased SERCA2a activity and leaky Ca2+ release channel resulting in reduced SR calcium load. The diabetic heart also suffers from marked downregulation of novel cardioprotective microRNAs (miRNAs) discovered recently. Since immune responses and substrate energy metabolism are critically altered in diabetes, the present review will focus on immunometabolism and miRNAs.

Atrial Spceific Pitx2 Insufficiencyincreases the Frequency of Calcium Sparks, Waves, and After-Depolarizations in Mouse Atrial Myocytes

The transcription factor Pitx2 has been proposed as a molecular link between single nucleotide polymorphisms on chromosome 4q25 and increased risk of atrial fibrillation in carriers of the risk variant. Since atrial fibrillation has been associated with calcium handling disturbances in isolated atrial myocytes, we here tested the hypothesis that Pitx2 insufficiency alters the calcium homeostasis in atrial myocytes.To test this hypothesis, we used right atrial myocytes from a transgenic mouse model with inducible atrial specific Pitx2 deletion. Spontaneous calcium release was detected with confocal calcium imaging and resulting ion currents or membrane depolarizations were measured with patch-clamp technique in myocytes from wild-type (Pitx2+/+) and heterozygous Pitx2+/- mice.Calcium imaging revealed that the frequency of calcium sparks (2.1±0.7 vs 0.2±0.1 events/cell/s, p<0.05) and waves (3.2±1.2 vs. 0 events/min, p<0.05) were significantly higher in Pitx2+/- mice. This was also true for the frequency of transient inward currants activated by calcium waves (2.8±0.5 vs 1.2±0.5 events/min, p<0.05). The higher frequency was of spontaneous calcium release was likely due to a higher caffeine releasable sarcoplasmic reticulum calcium load in the Pitx2+/- mice (23.8±5.8 vs 14.4±1.9 amol/pF, p<0.05). Moreover, only myocytes from Pitx2+/- had spontaneous action potentials at a resting potential of −80 mV (0.5±0.4/min). At −60 mV the frequency of spontaneous action potentials was 5.3±1.8/min for Pitx2+/- and 0.4±1.2/min for Pitx2+/+ mice. Importantly, spontaneous after-potentials were also recorded in myocytes subjected to field stimulation, but only in myocytes from Pitx2+/- mice. These results were not unique to right atrial myocytes as the calcium spark frequency was also 3.9 fold higher in left atrial myocytes from Pitx2+/- mice (p<0.001).Together, these results demonstrate that Pitx2 insufficiency promote both spontaneous calcium release and spontaneous action potentials. Both of these features are hallmarks of myocytes from patients with atrial fibrillation, suggesting that Pitx2-mediated modulation of intracellular calcium handling plays an important role in electrophysiological processes associated with atrial fibrillation.

Potential new mechanisms of pro-arrhythmia in arrhythmogenic cardiomyopathy: focus on calcium sensitive pathways.

Arrhythmogenic cardiomyopathy, or its most well-known subform arrhythmogenic right ventricular cardiomyopathy (ARVC), is a cardiac disease mainly characterised by a gradual replacement of the myocardial mass by fibrous and fatty tissue, leading to dilatation of the ventricular wall, arrhythmias and progression towards heart failure. ARVC is commonly regarded as a disease of the intercalated disk in which mutations in desmosomal proteins are an important causative factor. Interestingly, the Dutch founder mutation PLN R14Del has been identified to play an additional, and major, role in ARVC patients within the Netherlands. This is remarkable since the phospholamban (PLN) protein plays a leading role in regulation of the sarcoplasmic reticulum calcium load rather than in the establishment of intercellular integrity. In this review we outline the intracellular cardiac calcium dynamics and relate pathophysiological signalling, induced by disturbed calcium handling, with activation of calmodulin dependent kinase II (CaMKII) and calcineurin A (CnA). We postulate a thus far unrecognised role for Ca2+ sensitive signalling proteins in maladaptive remodelling of the macromolecular protein complex that forms the intercalated disk, during pro-arrhythmic remodelling of the heart.

Cardiac electrical defects in progeroid mice and Hutchinson–Gilford progeria syndrome patients with nuclear lamina alterations

Hutchinson-Gilford progeria syndrome (HGPS) is a rare genetic disease caused by defective prelamin A processing, leading to nuclear lamina alterations, severe cardiovascular pathology, and premature death. Prelamin A alterations also occur in physiological aging. It remains unknown how defective prelamin A processing affects the cardiac rhythm. We show age-dependent cardiac repolarization abnormalities in HGPS patients that are also present in the Zmpste24-/- mouse model of HGPS. Challenge of Zmpste24-/- mice with the β-adrenergic agonist isoproterenol did not trigger ventricular arrhythmia but caused bradycardia-related premature ventricular complexes and slow-rate polymorphic ventricular rhythms during recovery. Patch-clamping in Zmpste24-/- cardiomyocytes revealed prolonged calcium-transient duration and reduced sarcoplasmic reticulum calcium loading and release, consistent with the absence of isoproterenol-induced ventricular arrhythmia. Zmpste24-/- progeroid mice also developed severe fibrosis-unrelated bradycardia and PQ interval and QRS complex prolongation. These conduction defects were accompanied by overt mislocalization of the gap junction protein connexin43 (Cx43). Remarkably, Cx43 mislocalization was also evident in autopsied left ventricle tissue from HGPS patients, suggesting intercellular connectivity alterations at late stages of the disease. The similarities between HGPS patients and progeroid mice reported here strongly suggest that defective cardiac repolarization and cardiomyocyte connectivity are important abnormalities in the HGPS pathogenesis that increase the risk of arrhythmia and premature death.

GW27-e0368 Triggered Activity in Human Heart Is Determined by Sarcoplasmic Reticulum Calcium Load in Post Acidosis

GW27-e0368 Triggered Activity in Human Heart Is Determined by Sarcoplasmic Reticulum Calcium Load in Post Acidosis

Cardioprotective Effect of Ranolazine in the Process of Ischemia-reperfusion in Adult Rat Cardiomyocytes

Introduction and objectivesRanolazine is used as a complementary treatment for angina in symptomatic patients who are inadequately controlled with first-line antianginal therapies. Ranolazine inhibits sodium voltage-dependent channels, suggesting their possible involvement in the reperfusion process by preventing the sodium and calcium overload that occurs during ischemia. In this study, we characterized the effect of ranolazine on calcium homeostasis in isolated adult cardiac myocytes from rats subjected to a simulated ischemia and reperfusion protocol. MethodsThe effects of ranolazine on changes in intracellular calcium concentration were evaluated at different times using field electrostimulation. The study of intracellular calcium was performed using microfluorimetry with the fluorescent indicator, Fura-2, and by confocal microscopy with the indicator, Fluo-3. ResultsWe found that cardiomyocytes subjected to ischemia-reperfusion showed an increase in the diastolic calcium concentration and a decrease in the amplitude of intracellular calcium transients. The application of ranolazine during ischemia significantly improved intracellular calcium handling, preventing intracellular calcium overload, decreasing the diastolic calcium concentration, increasing the sarcoplasmic reticulum calcium load, and preserving the amplitude of the intracellular calcium transient, which was reflected by successful recovery in the process of excitation-contraction coupling during reperfusion. However, these effects of ranolazine did not occur when it was applied during reperfusion or when applied in both ischemia and reperfusion. ConclusionsRanolazine shows beneficial effects in cardiomyocytes exposed to ischemia/reperfusion but only when applied during ischemia. This effect is achieved through its improvement of calcium handling during ischemia.

Efecto cardioprotector de la ranolazina en el proceso de isquemia-reperfusión en cardiomiocitos de rata adultos

Introduction and objectivesRanolazine is used as a complementary treatment for angina in symptomatic patients who are inadequately controlled with first-line antianginal therapies. Ranolazine inhibits sodium voltage-dependent channels, suggesting their possible involvement in the reperfusion process by preventing the sodium and calcium overload that occurs during ischemia. In this study, we characterized the effect of ranolazine on calcium homeostasis in isolated adult cardiac myocytes from rats subjected to a simulated ischemia and reperfusion protocol. MethodsThe effects of ranolazine on changes in intracellular calcium concentration were evaluated at different times using field electrostimulation. The study of intracellular calcium was performed using microfluorimetry with the fluorescent indicator, Fura-2, and by confocal microscopy with the indicator, Fluo-3. ResultsWe found that cardiomyocytes subjected to ischemia-reperfusion showed an increase in the diastolic calcium concentration and a decrease in the amplitude of intracellular calcium transients. The application of ranolazine during ischemia significantly improved intracellular calcium handling, preventing intracellular calcium overload, decreasing the diastolic calcium concentration, increasing the sarcoplasmic reticulum calcium load, and preserving the amplitude of the intracellular calcium transient, which was reflected by successful recovery in the process of excitation-contraction coupling during reperfusion. However, these effects of ranolazine did not occur when it was applied during reperfusion or when applied in both ischemia and reperfusion. ConclusionsRanolazine shows beneficial effects in cardiomyocytes exposed to ischemia/reperfusion but only when applied during ischemia. This effect is achieved through its improvement of calcium handling during ischemia.Full English text available from:www.revespcardiol.org/en

Ibandronate and Ventricular Arrhythmia Risk

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

Abnormal calcium handling in the progeric mouse model Zmpste24-/- is linked to reduced sarcoplasmic reticulum calcium loading and depression of SERCA2 and calsequestrin-2 expression

Abnormal calcium handling in the progeric mouse model Zmpste24-/- is linked to reduced sarcoplasmic reticulum calcium loading and depression of SERCA2 and calsequestrin-2 expression