Caffeine-induced Ca2 Research Articles

Kinetics on Demand Is a Simple Mathematical Solution that Fits Recorded Caffeine-Induced Luminal SR Ca2+ Changes in Smooth Muscle Cells.

The process of Ca2+ release from sarcoplasmic reticulum (SR) comprises 4 phases in smooth muscle cells. Phase 1 is characterized by a large increase of the intracellular Ca2+ concentration ([Ca2+]i) with a minimal reduction of the free luminal SR [Ca2+] ([Ca2+]FSR). Importantly, active SR Ca2+ ATPases (SERCA pumps) are necessary for phase 1 to occur. This situation cannot be explained by the standard kinetics that involves a fixed amount of luminal Ca2+ binding sites. A new mathematical model was developed that assumes an increasing SR Ca2+ buffering capacity in response to an increase of the luminal SR [Ca2+] that is called Kinetics-on-Demand (KonD) model. This approach can explain both phase 1 and the refractory period associated with a recovered [Ca2+]FSR. Additionally, our data suggest that active SERCA pumps are a requisite for KonD to be functional; otherwise luminal SR Ca2+ binding proteins switch to standard kinetics. The importance of KonD Ca2+ binding properties is twofold: a more efficient Ca2+ release process and that [Ca2+]FSR and Ca2+-bound to SR proteins ([Ca2+]BSR) can be regulated separately allowing for Ca2+ release to occur (provided by Ca2+-bound to luminal Ca2+ binding proteins) without an initial reduction of the [Ca2+]FSR.

Development and Characterization of a Scaffold-Free 3D Spheroid Model of Induced Pluripotent Stem Cell-Derived Human Cardiomyocytes.

Cardiomyocytes (CMs) are terminally differentiated cells in the adult heart, and ischemia and cardiotoxic compounds can lead to cell death and irreversible decline of cardiac function. As testing platforms, isolated organs and primary cells from rodents have been the standard in research and toxicology, but there is a need for better models that more faithfully recapitulate native human biology. Hence, a new in vitro model comprising the advantages of 3D cell culture and the availability of induced pluripotent stem cells (iPSCs) of human origin was developed and characterized. Human CMs derived from iPSCs were studied in standard 2D culture and as cardiac microtissues (MTs) formed in hanging drops. Two-dimensional cultures were examined using immunofluorescence microscopy and western blotting, while the cardiac MTs were subjected to immunofluorescence, contractility, and pharmacological investigations. iPSC-derived CMs in 2D culture showed well-formed myofibrils, cell-cell contacts positive for connexin-43, and other typical cardiac proteins. The cells reacted to prohypertrophic growth factors with a substantial increase in myofibrils and sarcomeric proteins. In hanging drop cultures, iPSC-derived CMs formed spheroidal MTs within 4 days, showing a homogeneous tissue structure with well-developed myofibrils extending throughout the whole spheroid without a necrotic core. MTs showed spontaneous contractions for more than 4 weeks that were recorded by optical motion tracking, sensitive to temperature and responsive to electrical pacing. Contractile pharmacology was tested with several agents known to modulate cardiac rate and viability. Calcium transients underlay the contractile activity and were also responsive to electrical stimulation, caffeine-induced Ca(2+) release, and extracellular calcium levels. A three-dimensional culture using iPSC-derived human CMs provides an organoid human-based cellular platform that is free of necrosis and recapitulates vital cardiac functionality, thereby providing a new and promising relevant model for the evaluation and development of new therapies and detection of cardiotoxicity.

Channel Gating Dependence on Pore Lining Helix Glycine Residues in Skeletal Muscle Ryanodine Receptor

Type 1 ryanodine receptors (RyR1s) release Ca(2+) from the sarcoplasmic reticulum to initiate skeletal muscle contraction. The role of RyR1-G4934 and -G4941 in the pore-lining helix in channel gating and ion permeation was probed by replacing them with amino acid residues of increasing side chain volume. RyR1-G4934A, -G4941A, and -G4941V mutant channels exhibited a caffeine-induced Ca(2+) release response in HEK293 cells and bound the RyR-specific ligand [(3)H]ryanodine. In single channel recordings, significant differences in the number of channel events and mean open and close times were observed between WT and RyR1-G4934A and -G4941A. RyR1-G4934A had reduced K(+) conductance and ion selectivity compared with WT. Mutations further increasing the side chain volume at these positions (G4934V and G4941I) resulted in reduced caffeine-induced Ca(2+) release in HEK293 cells, low [(3)H]ryanodine binding levels, and channels that were not regulated by Ca(2+) and did not conduct Ca(2+) in single channel measurements. Computational predictions of the thermodynamic impact of mutations on protein stability indicated that although the G4934A mutation was tolerated, the G4934V mutation decreased protein stability by introducing clashes with neighboring amino acid residues. In similar fashion, the G4941A mutation did not introduce clashes, whereas the G4941I mutation resulted in intersubunit clashes among the mutated isoleucines. Co-expression of RyR1-WT with RyR1-G4934V or -G4941I partially restored the WT phenotype, which suggested lessening of amino acid clashes in heterotetrameric channel complexes. The results indicate that both glycines are important for RyR1 channel function by providing flexibility and minimizing amino acid clashes.

Involvement of large-conductance Ca2+-activated K+ channels in chloroquine-induced force alterations in pre-contracted airway smooth muscle.

The participation of large-conductance Ca2+ activated K+ channels (BKs) in chloroquine (chloro)-induced relaxation of precontracted airway smooth muscle (ASM) is currently undefined. In this study we found that iberiotoxin (IbTx, a selective inhibitor of BKs) and chloro both completely blocked spontaneous transient outward currents (STOCs) in single mouse tracheal smooth muscle cells, which suggests that chloro might block BKs. We further found that chloro inhibited Ca2+ sparks and caffeine-induced global Ca2+ increases. Moreover, chloro can directly block single BK currents completely from the intracellular side and partially from the extracellular side. All these data indicate that the chloro-induced inhibition of STOCs is due to the blockade of chloro on both BKs and ryanodine receptors (RyRs). We also found that low concentrations of chloro resulted in additional contractions in tracheal rings that were precontracted by acetylcholine (ACH). Increases in chloro concentration reversed the contractile actions to relaxations. In the presence of IbTx or paxilline (pax), BK blockers, chloro-induced contractions were inhibited, although the high concentrations of chloro-induced relaxations were not affected. Taken together, our results indicate that chloro blocks BKs and RyRs, resulting in abolishment of STOCs and occurrence of contraction, the latter will counteract the relaxations induced by high concentrations of chloro.

Chloroquine Inhibits Ca2+ Signaling in Murine CD4+ Thymocytes

Background/Aims: Bitter-tasting chloroquine can suppress T cell activation by inhibiting Ca²⁺ signaling. However, the mechanism of inhibition remains largely unclear. Methods: In this study, CD4⁺ T cells were isolated from the thymus, and the calcium content of CD4⁺ thymocytes was measured using fura-2 AM and a TILL imaging system. Pyrazole-3 (Pyr3), thapsigargin (TG), and caffeine were used to assess the effects of chloroquine on the intracellular Ca²⁺ content of CD4⁺ T cells. Results: In murine CD4⁺ thymocytes, chloroquine decreased the TG-triggered intracellular Ca²⁺ increase in a dose-dependent manner. In the absence of chloroquine under Ca²⁺-free conditions (0 mM Ca²⁺ and 0.5 mM EGTA), TG induced a transient Ca²⁺ increase. After restoration of the extracellular Ca²⁺ concentration to 2 mM, a dramatic Ca²⁺ increase occurred. This elevation was completely blocked by chloroquine and was markedly inhibited by Pyr3, a selective antagonist of transient receptor potential C3 (TRPC3) channel and stromal interaction molecule (STIM)/Orai channel. Furthermore, the TG-induced transient Ca²⁺ increase under Ca²⁺-free conditions was eliminated in the presence of chloroquine. Chloroquine also blocked the dialyzed inositol-1,4,5-trisphosphate (IP₃)-induced intracellular Ca²⁺ increase. However, chloroquine was not able to decrease the caffeine-induced Ca²⁺ increase. Conclusion: These data indicate that chloroquine inhibits the elevation of intracellular Ca²⁺ in thymic CD4⁺ T cells by inhibiting IP₃ receptor-mediated Ca²⁺ release from intracellular stores and TRPC3 channel-mediated and/or STIM/Orai channel-mediated Ca²⁺ influx.

Abstract 18739: Acute Effect of the Na+/ca2+ Exchanger (ncx) Inhibitor Sea0400 on Diastolic Function in vivo and in vitro in a Model of Heart Failure With Preserved Ejection Fraction

Background: Heart failure with preserved ejection fraction (HFPEF) is increasingly common but underlying cellular mechanisms are not well understood. We investigated cardiomyocyte function and the role of SEA0400, in vivo and in vitro in a rat model of diastolic heart failure. Methods: Young male Wistar rats were subjected to subtotal nephrectomy (NXT) or sham operation (SOP). After 24 weeks in vivo (pressure-volume loops) and in vitro characterization (cardiomyocyte function (Ca2+ transients, sarcoplasmic reticulum (SR) diastolic Ca2+ leak (Ca2+ sparks) and SR Ca2+ content)) as well as NCX function (caffeine-induced Ca2+ transient, TAU) and protein expression) were determined without an with the NCX inhibitor SEA0400 (in vivo: 1 mg/kgKG for 30 minutes/ in vitro: 300nM for 5 minutes incubation). Results: NXT rats showed stable compensated renal impairment and signs and symptoms of HFPEF (hypertrophied left ventricle (LV), left- and upward shift of end diastolic pressure (EDP) volume relationship, increased lung weight/body weight ratio indicating pulmonary congestion and preserved LV systolic function (EF, dP/dt)). In LV cardiomyocytes from NXT Ca2+ transient amplitude was unchanged but time for early (50%) decay was significantly prolonged at 24 weeks and correlated with diastolic dysfunction (EDP) in vivo. TAU of the caffeine transient was significantly prolonged at 24 weeks indicating reduced NCX forward mode activity, while NCX protein expression was up-regulated, suggesting increased NCX reverse mode activity. Ca2+ spark frequency was increased and SR Ca2+ content was decreased in NXT (p<0.05). SEA0400 significantly accelerated Ca2+ transient decay and reduced Ca2+ spark frequency in NXT. Acute in vivo treatment with SEA0400 significantly enhanced active relaxation (isovolumetric relaxation constant) of the LV in NXT. Conclusion: This model of HFPEF is associated with prolonged cytosolic Ca2+ decay and increased diastolic Ca2+ leak in LV cardiomyocytes. Acute treatment with NCX inhibitor SEA0400 normalized cytosolic Ca2+ transients, improved net transsarcolemmal Ca2+ export and decreased SR Ca2+ leak in NXT, in line with a role for reverse mode NCX activity in HFPEF. In vivo active relaxation was improved after acute SEA0400 treatment.

Caffeine-induced Ca2+ oscillations in type I horizontal cell of carp retina: A mathematical model

Oscillations in intracellular free Ca2+ concentration ([Ca2+]i) have been observed in a variety of cell types. In the present study, we constructed a mathematical model to simulate the caffeine-induced [Ca2+]i oscillations based on experimental data obtained from isolated type I horizontal cell of carp retina. The results of model analysis confirm the notion that the caffeine-induced [Ca2+]i oscillations involve a number of cytoplasmic and endoplasmic Ca2+ processes that interact with each other. Using this model, we evaluated the importance of store-operated channel (SOC) in caffeine-induced [Ca2+]i oscillations. The model suggests that store-operated Ca2+ entry (SOCE) is elicited upon depletion of the endoplasmic reticulum (ER). When the SOC conductance is set to 0, caffeine-induced [Ca2+]i oscillations are abolished, which agrees with the experimental observation that [Ca2+]i oscillations were abolished when SOC was blocked pharmacologically, verifying that SOC is necessary for sustained [Ca2+]i oscillations.

Aβ42 oligomers selectively disrupt neuronal calcium release

Accumulation of amyloid-β (Aβ) peptides correlates with aging and progression of Alzheimer's disease (AD). Aβ peptides, which cause early synaptic dysfunctions, spine loss, and memory deficits, also disturb intracellular Ca2+ homeostasis. By cytosolic and endoplasmic reticulum Ca2+ measurements, we here define the short-term effects of synthetic Aβ42 on neuronal Ca2+ dynamics. When applied acutely at submicromolar concentration, as either oligomers or monomers, Aβ42 did not cause Ca2+ release or Ca2+ influx. Similarly, 1-hour treatment with Aβ42 modified neither the resting cytosolic Ca2+ level nor the long-lasting Ca2+ influx caused by KCl-induced depolarization. In contrast, Aβ42 oligomers, but not monomers, significantly altered Ca2+ release from stores with opposite effects on inositol 1,4,5-trisphosphate (IP3)- and caffeine-induced Ca2+ mobilization without alteration of the total store Ca2+ content. Ca2+ dysregulation by Aβ42 oligomers involves metabotropic glutamate receptor 5 and requires network activity and the intact exo-endocytotic machinery, being prevented by tetrodotoxin and tetanus toxin. These findings support the idea that Ca2+ store dysfunction is directly involved in Aβ42 neurotoxicity and represents a potential therapeutic target in AD-like dementia.

Caffeine-induced Ca2+ oscillations in type I horizontal cells of the carp retina and the contribution of the store-operated Ca2+ entry pathway.

The mechanisms of release, depletion, and refilling of endoplasmic reticulum (ER) Ca2+ were investigated in type I horizontal cells of the carp retina using a fluo-3-based Ca2+ imaging technique. Exogenous application of caffeine, a ryanodine receptor agonist, induced oscillatory intracellular free Ca2+ concentration ([Ca2+]i) responses in a duration- and concentration-dependent manner. In Ca2+-free Ringer’s solution, [Ca2+]i transients could also be induced by a brief caffeine application, whereas subsequent caffeine application induced no [Ca2+]i increase, which implied that extracellular Ca2+ was required for ER refilling, confirming the necessity of a Ca2+ influx pathway for ER refilling. Depletion of ER Ca2+ by thapsigargin triggered a Ca2+ influx which could be blocked by the store-operated channel inhibitor 2-APB, which proved the existence of the store-operated Ca2+ entry pathway. Taken together, these results suggested that after being depleted by caffeine, the ER was replenished by Ca2+ influx via store-operated channels. These results reveal the fine modulation of ER Ca2+ signaling, and the activation of the store-operated Ca2+ entry pathway guarantees the replenishment of the ER so that the cell can be ready for response to the subsequent stimulus.

Increased Na⁺/Ca²⁺ exchanger expression/activity constitutes a point of inflection in the progression to heart failure of hypertensive rats.

Spontaneously hypertensive rat (SHR) constitutes a genetic model widely used to study the natural evolution of hypertensive heart disease. Ca2+-handling alterations are known to occur in SHR. However, the putative modifications of Ca2+-handling proteins during the progression to heart failure (HF) are not well established. Moreover, the role of apoptosis in SHR is controversial. We investigated intracellular Ca2+, Ca2+-handling proteins and apoptosis in SHR vs. control Wistar rats (W) from 3 to 15 months (mo). Changes associated with the transition to HF (i.e. lung edema and decrease in midwall fractional shortening), occurred at 15 mo in 38% of SHR (SHRF). In SHRF, twitch and caffeine-induced Ca2+ transients, significantly decreased relative to 6/9 mo and 15 mo without HF signs. This decrease occurred in association with a decrease in the time constant of caffeine-Ca2+ transient decay and an increase in Na+/Ca2+ exchanger (NCX) abundance (p<0.05) with no changes in SERCA2a expression/activity. An increased Ca2+-calmodulin-kinase II activity, associated with an enhancement of apoptosis (TUNEL and Bax/Bcl2) was observed in SHR relative to W from 3 to 15 mo. Conclusions: 1. Apoptosis is an early and persistent event that may contribute to hypertrophic remodeling but would not participate in the contractile impairment of SHRF. 2. The increase in NCX expression/activity, associated with an increase in Ca2+ efflux from the cell, constitutes a primary alteration of Ca2+-handling proteins in the evolution to HF. 3. No changes in SERCA2a expression/activity are observed when HF signs become evident.

Increased Ca2+‐activated Ca2+ influx and impaired Ca2+ buffering in coronary smooth muscle from metabolic syndrome Ossabaw swine (1076.2)

Coronary smooth muscle (CSM) phenotype is affected by intracellular Ca2+ regulation and we have previously correlated changes in CSM Ca2+ signaling from Ossabaw swine with varying stages of coronary artery disease (CAD). Ossabaw swine are a model of metabolic syndrome (MetS) and CAD. Three year old pigs were fed an excess‐calorie, atherogenic or control diet for 6 months. CAD progression, coronary artery function, and CSM Ca2+ signaling were assessed. MetS coronaries had advanced, lipid‐laden atherosclerotic lesions containing calcium deposits, while lean had no CAD. In isometric tension studies diseased coronary rings generated significantly less tension than healthy rings in response to depolarization with 20 mM K+. CSM were enzymatically isolated and loaded with the Ca2+ indicator, fura‐2 AM. Diseased CSM showed less K+‐induced Ca2+ influx compared to healthy cells. Diseased cells recovered less from the cytosolic Ca2+ increase back to baseline, suggesting impaired Ca2+ buffering. In physiologic salt solution (PSS), there was no difference in peak Ca2+ transient elicited by caffeine‐induced intracellular Ca2+ store release. The caffeine‐induced Ca2+ transient in Ca2+‐free PSS, which represents exclusively sarcoplasmic reticulum (SR) Ca2+ release, was decreased in diseased cells. Taken together, these data suggest that lesser SR Ca2+ release elicited greater Ca2+‐activated Ca2+ influx in diseased CSM. After caffeine challenge healthy CSM buffered Ca2+ significantly below basal Ca2+ compared to diseased CSM. Overall, the data suggest impaired CSM SR Ca2+ buffering, decreased SR Ca2+ store, and increased Ca2+‐activated Ca2+ influx in CAD.Grant Funding Source: Supported by NIH HL062552, HL092245, NIH/NCATS CTSI TL1 TR000162

Natural Alkaloid Murrayafoline-A Sensitizes Ca2+ Release Sites via Adenylate Cyclase and Protein Kinase C Pathway in Rat Ventricular Myocytes

A naturally found alkaloid murrayafoline-A (Mu-A, 1-methoxy-3-methylcarbazole) acutely increases ventricle cell contractility partly via enhancement of L-type Ca2+ current. In the present study,we explored the effects of Mu-A on elementary Ca2+ release events (“Ca2+ sparks”) in cardiac myocytes and possible molecular mechanisms underlying the effect of Mu-A. Rapid two-dimensional confocal Ca2+ imaging and image analysis were used to assess spontaneous Ca2+ sparks in single adult rat ventricular myocytes. The frequency of Ca2+ sparks was dose-dependently increased by Mu-A. The duration and the size of single sparks were also significantly increased by Mu-A. The sarcoplasmic reticulum (SR) Ca2+ loading, measured as caffeine-induced Ca2+ transients, was increased by Mu-A. Neither SR Ca2+ clamping nor removal of external Ca2+ and Na+ affects the Mu-A-mediated enhancement in Ca2+ sparks. In contrast, prolongation and enlargement of individual Ca2+ sparks in Mu-A were attenuated by matching SR Ca2+ content to the control level. Higher propensity of Ca2+ sparks in the presence of Mu-A was not suppressed by the protein kinase A inhibitor, KT5720, or by the calmodulin-dependent protein kinase II inhibitor, KN93, whereas it was strongly reduced by the inhibitors of adenylate cyclase (MDL-12,330A) and protein kinase C (GF109203X). Our data suggest that Mu-A sensitizes ryanodine receptor (RyR) Ca2+ release channels via specialized signaling pathway involving adenylate cyclase and protein kinase C, and that larger SR Ca2+ loading in Mu-A may also play a role in enhanced Ca2+ release through the RyRs.

Ligand-Dependent Conformational Changes in the Cardiac Ryanodine Receptor

Global conformational changes in the three-dimensional (3D) structure of the calcium release channel/ryanodine receptor (RyR) occur upon ligand activation. Of several ligands that activate RyR, Ca2+ is the primary activator. Although RyR Ca2+ activation is well characterized functionally, little is known about the conformational changes in RyR induced by Ca2+. Here we generated three fluorescence resonance energy transfer (FRET)-based conformational probes. Each of these probes was constructed by inserting a CFP into one domain and a YFP into a neighboring domain in the cardiac RyR (RyR2) to yield a CFP- and YFP-dual labeled RyR2 (CFP/YFP FRET pair). These CFP/YFP FRET pairs were located in the “clamp” region (RyR2S2367-CFP/Y2801-YFP), the calmodulin binding region (RyR2R3595-CFP/K4269-YFP), and the “bridge” region (RyR2S437-YFP/S2367-CFP), respectively. We monitored the conformational changes in these regions by recording the FRET signals, and the extent of Ca2+ release by measuring store Ca2+ depletion in HEK293 cells expressing each of the CFP/YFP FRET pairs upon activation by Ca2+, caffeine, and ATP. Surprisingly, we found that different ligands induced different conformational changes in different regions of RyR2. For instance, we detected conformational changes in the clamp region for caffeine and ATP, but not for Ca2+, although they all induced Ca2+ release. Considering Ca2+ as the primary activator of RyR2, we determined the impact of cytosolic Ca2+ sensing mutation E3987A on conformational changes. Interestingly, this single mutation abolishes caffeine-induced conformational changes, but not caffeine-induced Ca2+ release. These observations demonstrate that conformational changes in RyR2 are ligand-dependent, and that E3987, which is critical for cytosolic Ca2+ sensing, is essential for ligand-induced conformational changes (Supported by CFI, CIHR, HSFC, NIH, and LCIA).

Inhibition of the NA+/CA2+ Exchanger (Ncx) Improves Cardiomyocyte Contractile Dysfunction in a Rat Model with Compensated Renal Failure and Heart Failure with Preserved Ejection Fraction

Background: Heart failure with preserved ejection fraction (HFPEF) and renal impairment are often associated but the underlying pathomechanisms are largely unknown. We investigated tissue fibrosis and cardiomyocyte contractile function in a rat model with HFPEF and compensated renal failure.Methods: Seventy young male Wistar rats were subjected to subtotal nephrectomy (NXT) or sham operation (SOP). Blood/urine samples, echocardiography, pressure-volume loops were performed after 8 and 24 weeks. Left ventricular (LV) hypertrophy, fibrosis (picosirius-red-staining) and NCX protein expression (Western blot) were determined after sacrification. Cardiomyocyte function (Ca2+ transients, cell shortening) was quantified in isolated cardiomyocytes without and with the NCX inhibitor SEA0400 (300nM).Results: NXT rats showed stable renal impairment with preserved urine excretion and elevated arterial pressure (148±8 and 154±7 vs. 105±3 and 109±2 mmHg, p<0.01) at 8 and 24 weeks, respectively. LV in NXT were significantly hypertrophied at 8 and 24 weeks, but LV systolic function (EF, dP/dt) were preserved. LVEDP, LA size and lung weight were significantly increased indicating HFPEF with pulmonary congestion. Fibrosis was increased in LV from NXT. LV cardiomyocytes showed significantly prolonged time for early (50%) relaxation and decay of the Ca2+ transient. Time constant of the caffeine-induced Ca2+ transient (TAU) was significantly prolonged indicating reduced NCX forward mode activity, while NCX protein expression was upregulated. Acute treatment with SEA0400 (300nM) significantly accelerated early relaxation in cardiomyocytes from NXT (8 weeks).Conclusion: In this model of compensated renal failure and HFPEF cardiomyocyte relaxation was prolonged already at an early stage. Our results suggest increased NCX activity may contribute to contractile dysfunction, likely through reverse mode Ca2+ influx. NCX represents a potential target to treat cardiomyocyte dysfunction in HFPEF.

Suppressed RyR2 Function Represents a Common Cause of Idiopathic Ventricular Fibrillation and Sudden Cardiac Death

Cardiac Ryanodine Receptor (RyR2) is the major intracellular calcium release channel located in the sarcoplasmic reticulum. A large number of mutations in RyR2 have been linked to inherited cardiac arrhythmias (CPVT, catecholaminergic polymorphic ventricular tachycardia and IVF, idiopathic ventricular fibrillation) and sudden cardiac death. We previously demonstrated that in contrast to CPVT RyR2 mutations, the IVF RyR2 mutation A4860G suppresses RyR2 function. However, A4860G represents the only IVF RyR2 mutation that has been functionally characterized to date. To determine how common suppression-of-function RyR2 mutations are, we systematically characterized 27 RyR2 mutations that are associated with IVF or sudden cardiac death. Each of these mutations was generated by site-directed mutagenesis and expressed in HEK293 cells. Ca release assays revealed that 7 of these mutations suppress or abolish caffeine-induced Ca release. Single cell Ca imaging studies also indicated that these suppression- or loss-of-function RyR2 mutations reduce or abolish the propensity for store-overload induced Ca release (SOICR) in HEK293 cells. In addition, single channel analysis showed that these mutations significantly reduce the response of the channel to activation by cytosolic and luminal Ca. These observations demonstrate, for the first time, that suppressed or loss of RyR2 function may be a common mechanism underlying idiopathic ventricular fibrillation, which is opposite to the gain-of-function RyR2 mutations associated with CPVT. Thus, understanding the exact molecular defects of disease-causing RyR2 mutations will help to develop novel approaches to the diagnosis and personalized treatment of these lethal cardiac arrhythmias (Supported by CFI, CIHR, AIHS, and LCIA).

Abstract 18560: Defective Ca 2 + Homeostasis Contributes to Arrhythmia in Transgenic Rabbit Model of LQT2 Syndrome

Introduction: Loss-of function mutations in HERG potassium channels in Long QT2 syndrome are associated with sudden cardiac death triggered by sympathetic surge. In various models of cardiac disease changes in calcium (Ca) homeostasis, associated with upregulated plasmalemmal Na/Ca exchanger (NCX) and/or hyperactive ryanodine receptors (RyRs), have been linked to increased risk of arrhythmia. Methods and Results: The aims of this study were to investigate possible changes in Ca homeostasis in cardiomyocytes derived from from the hearts of LQT2 rabbits and to determine whether similar changes contribute to the triggering of early afterdepolarizations (EADs) characteristic of LQT2 myocytes under β-adrenergic stimulation. Parameters of Ca handling were measured in ventricular myocytes enzymatically isolated from LQT2 and littermate control hearts using confocal Ca imaging and whole cell patch clamp. Ca imaging revealed no changes in amplitude of Ca transients in intact myocytes and sarcoplasmic reticulum (SR) Ca content assessed by measuring amplitude of transients evoked by rapid application of caffeine. A two-fold increase in rate of decay of caffeine-induced Ca transients in LQT2 myocytes vs. controls indicated increased NCX activity. Experiments in saponin-permeabilized cells using a SR-entrapped low-affinity Ca indicator revealed ~2-fold increase in RyR-mediated SR Ca leak in LQT2 myocytes in comparison with controls. In contrast to controls, current-clamped LQT2 myocytes exposed to β-adrenergic agonist isoproterenol exhibited prolonged plateau of action potentials (AP) at -10 - 0 mV accompanied by aberrant Ca releases and phase 3 EADs. Importantly, complete exhaustion of SR [Ca] by incubation of LQT2 cells with SERCA inhibitor thapsigargin or 10 mM caffeine, shortened APD and completely abolished EADs. Conclusion: Functional changes in RyR and NCX underlie preserved systolic Ca transients in LQT2 myocytes under basal conditions and may contribute to stress-induced arrhythmia by maintaining the plateau of AP at voltages ideal for reactivation of plasmalemmal Ca channels and generation of EADs.

Cellular and Molecular Mechanisms of Atrial Arrhythmogenesis in Patients With Paroxysmal Atrial Fibrillation

Electrical, structural, and Ca2+ -handling remodeling contribute to the perpetuation/progression of atrial fibrillation (AF). Recent evidence has suggested a role for spontaneous sarcoplasmic reticulum Ca2+ -release events in long-standing persistent AF, but the occurrence and mechanisms of sarcoplasmic reticulum Ca2+ -release events in paroxysmal AF (pAF) are unknown. Right-atrial appendages from control sinus rhythm patients or patients with pAF (last episode a median of 10-20 days preoperatively) were analyzed with simultaneous measurements of [Ca2+]i (fluo-3-acetoxymethyl ester) and membrane currents/action potentials (patch-clamp) in isolated atrial cardiomyocytes, and Western blot. Action potential duration, L-type Ca2+ current, and Na+ /Ca2+ -exchange current were unaltered in pAF, indicating the absence of AF-induced electrical remodeling. In contrast, there were increases in SR Ca2+ leak and incidence of delayed after-depolarizations in pAF. Ca2+ -transient amplitude and sarcoplasmic reticulum Ca2+ load (caffeine-induced Ca2+ -transient amplitude, integrated Na+/Ca2+ -exchange current) were larger in pAF. Ca2+ -transient decay was faster in pAF, but the decay of caffeine-induced Ca2+ transients was unaltered, suggesting increased SERCA2a function. In agreement, phosphorylation (inactivation) of the SERCA2a-inhibitor protein phospholamban was increased in pAF. Ryanodine receptor fractional phosphorylation was unaltered in pAF, whereas ryanodine receptor expression and single-channel open probability were increased. A novel computational model of the human atrial cardiomyocyte indicated that both ryanodine receptor dysregulation and enhanced SERCA2a activity promote increased sarcoplasmic reticulum Ca2+ leak and sarcoplasmic reticulum Ca2+ -release events, causing delayed after-depolarizations/triggered activity in pAF. Increased diastolic sarcoplasmic reticulum Ca2+ leak and related delayed after-depolarizations/triggered activity promote cellular arrhythmogenesis in pAF patients. Biochemical, functional, and modeling studies point to a combination of increased sarcoplasmic reticulum Ca2+ load related to phospholamban hyperphosphorylation and ryanodine receptor dysregulation as underlying mechanisms.

Endogenous and maximal sarcoplasmic reticulum calcium content and calsequestrin expression in type I and type II human skeletal muscle fibres

The relationship between sarcoplasmic reticulum (SR) Ca(2+) content and calsequestrin (CSQ) isoforms was investigated in human skeletal muscle. A fibre-lysing assay was used to quantify the endogenous Ca(2+) content and maximal Ca(2+) capacity of the SR in skinned segments of type I and type II fibres from vastus lateralis muscles of young healthy adults. Western blotting of individual fibres showed the great majority contained either all fast or all slow isoforms of myosin heavy chain (MHC), troponins C and I, tropomyosin and SERCA, and that the strontium sensitivity of the force response was closely indicative of the troponin C isoform present. The endogenous SR Ca(2+) content was slightly lower in type I compared to type II fibres (0.76 ± 0.03 and 0.85 ± 0.02 mmol Ca(2+) per litre of fibre, respectively), with virtually all of this Ca(2+) evidently being in the SR, as it could be rapidly released with a caffeine-low [Mg(2+)] solution (only 0.08 ± 0.01 and <0.07 mmol l(-1), respectively, remaining). The maximal Ca(2+) content that could be reached with SR Ca(2+) loading was 1.45 ± 0.04 and 1.79 ± 0.03 mmol l(-1) in type I and type II fibres, respectively (P < 0.05). In non-lysed skinned fibres, where the SR remained functional, repeated cycles of caffeine-induced Ca(2+) release and subsequent Ca(2+) reloading similarly indicated that (i) maximal SR Ca(2+) content was lower in type I fibres than in type II fibres (P < 0.05), and (ii) the endogenous Ca(2+) content represented a greater percentage of maximal content in type I fibres compared to type II fibres (∼59% and 41%, respectively, P < 0.05). Type II fibres were found on average to contain ∼3-fold more CSQ1 and ∼5-fold less CSQ2 than type I fibres (P < 0.001). The findings are consistent with the SR Ca(2+) content characteristics in human type II fibres being primarily determined by the CSQ1 abundance, and in type I fibres by the combined amounts of both CSQ1 and CSQ2.

Partial and Controlled Depletion of Sarcoplasmic Reticulum (SR) Calcium Prevents Spontaneous Calcium Release and Tachyarrhythmias in a Mouse Model of CPVT

Background SR Ca 2+ ([Ca 2+ ]SR) overload during β-adrenergic stimulation precipitates spontaneous Ca 2+ release (SCR) and tachyarrhythmias in cardiomyocytes harboring catecholaminergic polymorphic ventricular tachycardia (CPVT)-linked RyR2 mutations. We sought to determine whether imperacalcin (IpCa), a cell-permeable high-affinity agonist of RyR2 channels, prevents SCR and tachyarrhythmias in a mouse model of CPVT (RyR2-R4496C +/– ) by partially depleting [Ca 2+ ]SR during β-adrenergic stimulation. Methods Ca 2+ imaging was conducted by confocal microscopy in isolated ventricular myocytes from wild-type (WT) and RyR2-R4496C +/– mice. ECGs and left ventricular (LV) pressure were monitored in Langendorff-perfused beating WT and RyR2-R4496C +/– hearts stimulated with 300 nM isoproterenol (Iso). Cell penetration and topologic localization of IpCa was conducted in rat ventricular myocytes using Alexa546-IpCa. Results Alexa546-IpCa penetrated intact cardiomyocytes and localized in a striated pattern with ~2 μm interspacing, consistent with z-line labeling (n = 6 cells). Rapid perfusion of IpCa (1 µM) onto intact WT cardiomyocytes abruptly increased and then steadily decreased Ca 2+ transient amplitude, reflecting initial opening of RyR2 channels that gradually leads to partial depletion of [Ca 2+ ]SR (caffeine-induced Ca 2+ transient=7.6 ± 1.2 and 4.5 ± 0.82 F/Fo← before and after IpCa, respectively, n=15 cells, P = .001). The frequency of SCR waves in cardiomyocytes subjected to a stress protocol (5-second train of stimulations at 3 Hz under 300 nM Iso) was 3.6 ± 1.4 and 8.6 ± 5.3 waves/min in WT and RyR2-R4496C +/– cells, respectively, but decreased dramatically to 1.1 ± 0.9 and 1.6 ± 1.5 waves/min, respectively, after incubation with 1 µM IpCa (n = 10 cells each, P = 0.001). RyR2-R449C +/– hearts showed 56 ± 39 ectopic beats/min immediately ( Conclusions Partial depletion of [Ca 2+ ]SR with IpCa is a promising therapeutic alternative for Ca 2+ -triggered arrhythmias.

Cellular mechanisms of contractile dysfunction in a rat model with compensated renal failure and heart failure with preserved ejection fraction

Background: Renal impairment and heart failure with preserved ejection fraction (HFPEF) are often associated but the underlying mechanisms are largely unresolved. Therefore we investigated cardiomyocyte function in a rat model with compensated renal failure and diastolic dysfunction. Methods: Fifty-six young male Wistar rats were subjected to subtotal nephrectomy (NXT) or sham operation (SOP) and observed up to 24 weeks. Urine and blood samples for calculating the glomerular filtration rate (GFR) and echocardiography were performed during disease progression. Invasive hemodynamics (P/V-loops), morphometry, protein expression analysis and Ca2+ transients (confocal and ratiometric) as well as cell shortening (CS) were studied in isolated left ventricle (LV) cardiomyocytes in final experiments at 8 and 24 weeks. Results: Following NXT rats develop compensated renal failure (GFR 22±2 and 16±2 vs. 32±2 and 28±3 ml/min/m2 at 8 and 24 weeks, resp. p<0.01) with preserved urinary excretion and unchanged electrolytes. Arterial pressure is significantly increased in NXT at 8 and 24 weeks (148±8 and 154±7 vs. 105±3 and 109±2 mmHg, p<0.01) which is associated with pronounced LV hypertrophy in NXT (LV mass 708±21 and 866±34 vs. 637±12 and 660±7 mg, resp. p<0.05). Increased LV end-diastolic pressure (10.4±0.8 vs. 5.1±0.4 mmHg, p<0.01), left atrial enlargement (39±2 vs. 30±1 mg, p<0.01) and increased lung weight (1816±92 vs. 1414±39 mg, p<0.01) in NXT after 24 weeks reflect diastolic dysfunction with congestion indicating HFPEF (EF 50±5 vs. 56±4%). LV cardiomyocytes isolated from NXT compared to SOP showed no significant differences in cell shortening amplitude at 1 Hz (4.3±0.7 vs. 5.1±1.1%; n≥10) while time for early (50%) relaxation was significantly prolonged in NXT (CS RT50 42±3 vs. 34±1 ms; n≥10; p< 0.05) at 24 weeks. Cytosolic Ca2+ transients had similar amplitude (F/FO 4.2±0.1 vs. 4.1±0.1, n≥20). Sarcoplasmatic reticulum (SR) Ca2+ content was significantly reduced in NXT at 24 weeks (F/F0 5.3±0.2 vs. 6.0±0.2; n≥20; p<0.01) while it was unchanged after 8 weeks (F/F0 4.7±0.2 vs. 4.8±0.3; n≥10). Time constant of the caffeine-induced Ca2+ transient (TAU) was significantly prolonged in NXT after 24 weeks (1.77±0.07 vs. 1.22±0.06; n≥20; p< 0.01). NCX protein expression was unchanged in NXT, suggesting functional impairment of NCX1 activity. Conclusion: Subtotal nephrectomy in rats lead to compensated renal failure and an in vivo phenotype of HFPEF. Our in vitro results suggest early impairment of intracellular Ca2+ transients in HFPEF in NXT, leading to contractile dysfunction of the cardiac myocyte.