Fractional Sarcoplasmic Reticulum Ca 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.

Abstract 12951: Electrical and Calcium-Dependent Mechanisms That Contribute to Increase the Risk for Atrial Fibrillation in Obesity

Obesity (OB) is an independent predictor for atrial fibrillation (AF), but mechanisms by which OB increases AF risk remain poorly characterized. We hypothesized that OB modulates AF risk through electrical remodeling and impaired Ca 2+ dynamics that can be reversed after weight loss (WL). Using transesophageal rapid pacing we characterized AF inducibility and burden in lean, OB, and OB mice that lost 20% of body weight after modified vertical sleeve gastrectomy. Local field fluorescence microscopy was used to simultaneously record action potentials (APs) (di-8-ANEPPS or glass microelectrodes) and Ca 2+ transients (CaT) (Rhod-2) in Langendorff heart preparations. We evaluated AP duration (APD) dependency of frequency from 4 to 14 Hz (1 Hz incremental steps). AP parameters analyzed included APD30, APD50, APD90 and spontaneous arrhythmogenic events. We assessed Ca 2+ dynamics by studying CaT and fractional sarcoplasmic reticulum Ca 2+ release in the absence and presence of ryanodine (Ry) and thapsigargin (Tg). Since CaT Ry+Tg represents sarcolemmal Ca 2+ influx, we calculated the gain of Ca 2+ induced Ca 2+ release (CICR gain ) as the ratio between CaT in the absence and presence of Ry+Tg. Experiments were done in the presence of blebbistatin at 37° C. Mean±SE, t-test. After burst pacing, both AF prevalence and burden were higher in OB mice vs lean (94 % vs 47 %, 227±19 vs 20±1.2 s, n=16 mice/group, p<0.0001). About 76 % of OB hearts showed afterdepolarizations and fibrillation vs no events in lean. APD50 and APD90 significantly decreased as stimulation frequency increased from 4 to 14 Hz in OB hearts vs lean (n=6 mice/group, p<0.05). Peak CaT from OB hearts were significantly decreased compared to peak CaT from lean (0.3±0.05 vs. 0.8±0.06, n=9, p<0.001). CICR gain in OB hearts was also significantly compromised compared to lean (5.2±1 vs 23.3±1.4, n=7 mice/group, p<0.0001). After WL, OB mice showed a decreased AF burden (21.7±8 s), increased APD, increased peak CaT (0.8±0.15) and increased CICR gain (20±2.3) back to lean levels (n=8, p<0.001). In conclusion, shortened APs, spontaneous afterdepolarizations and impaired CICR significantly contribute to the development of AF in OB. WL reversed the electrical phenotype and the impaired Ca 2+ dynamics and prevented AF.

The Role of T-Tubule Organization in Regulation of Intracellular Ca2+ Handling by Muscarinic Receptor Stimulation in Ventricular Myocytes

Muscarinic receptors expressed in cardiac myocytes play critical role in mediating regulation of heart function by the parasympathetic nervous system. How structural organization of cardiac myocytes affects muscarinic receptor mediated regulation of Ca2+ handling is not well defined. Using confocal Ca2+ imaging and patch-clamp techniques we studied the relationships between t-tubule density and cholinergic regulation of intracellular Ca2+ handling in normal murine ventricular myocytes and in myocytes in which the t-tubule system was disrupted by formamide treatment. Muscarinic receptor agonist CCh (10 μM) significantly inhibited amplitude of Ca2+ transients evoked by electrical field-stimulation in the presence of 100 nM isoproterenol (Iso), a beta-adrenergic agonist, in formamide-treated but not in control ventricular myocytes. Following Iso stimulation, formamide-treated cells exhibited reduced density of the inward Ca2+ current (ICa), decreased sarcoplasmic reticulum (SR) Ca2+ content, and enhanced fractional SR Ca2+ release when compared to control myocytes. CCh did not change SR Ca2+ content in Iso-stimulated formamide-treated myocytes but significantly reduced fractional release. This effect was associated with reduced Ca2+ release at coupled SR Ca2+ release sites and decreased Ca2+ sensitivity of the uncoupled release sites. CCh inhibited peak ICa recorded in a presence of Iso by ∼20 % in both control and detubulated ventricular myocytes. Reducing ICa amplitude by up to 40% by changing voltage step levels from 0 to −25 mV did not affect Ca2+ transients in control myocytes in the presence of Iso. In contrast, in formamide-treated myocytes similar decrease in ICa significantly diminished Ca2+ transients. We conclude that downregulation of the t-tubule system, as often observed in failing hearts, augments the inhibitory effects of muscarinic receptor agonists on Ca2+ transients by reducing functional reserve of excitation-contraction coupling.

Oxidation of ryanodine receptor after ischemia-reperfusion increases propensity of Ca2+ waves during β-adrenergic receptor stimulation.

β-Adrenergic receptor (β-AR) activation produces the main positive inotropic response of the heart. During ischemia-reperfusion (I/R), however, β-AR activation can trigger life-threatening arrhythmias. Because I/R is frequently associated with oxidative stress, we investigated whether ryanodine receptor (RyR) oxidation contributes to proarrythmogenic Ca2+ waves during β-AR activation. Measurements of contractile and electrical activity from Langendorff-perfused rabbit hearts revealed that I/R produces tachyarrhythmias. Ventricular myocytes isolated from I/R hearts had an increased level of oxidized glutathione (i.e., oxidative stress) and a decreased level of free thiols in RyRs (i.e., RyR oxidation). Furthermore, myocytes from I/R hearts were characterized by increased sarcoplasmic reticulum (SR) Ca2+ leak and enhanced fractional SR Ca2+ release. In myocytes from nonischemic hearts, β-AR activation with isoproterenol (10 nM) produced only a positive inotropic effect, whereas in myocytes from ischemic hearts, isoproterenol at the same concentration triggered spontaneous Ca2+ waves. β-AR activation produced a similar effect on RyR phosphorylation in control and I/R myocytes. Treatment of myocytes from I/R hearts with the reducing agent mercaptopropionylglycine (100 μM) attenuated RyR oxidization and decreased Ca2+ wave frequency during β-AR activation. On the other hand, treatment of myocytes from nonischemic hearts with H2O2 (50 μM) increased SR Ca2+ leak and triggered Ca2+ waves during β-AR activation. Collectively, these results suggest that RyR oxidation after I/R plays a critical role in the transition from positive inotropic to arrhythmogenic effects during β-AR stimulation. Prevention of RyR oxidation can be a promising strategy to inhibit arrhythmias and preserve positive inotropic effect of β-AR activation during myocardial infarction. NEW & NOTEWORTHY Oxidative stress induced by ischemia plays a critical role in triggering arrhythmias during adrenergic stimulation. The combined increase in sarcoplasmic reticulum Ca2+ leak (because of ryanodine receptor oxidation) and sarcoplasmic reticulum Ca2+ load (because of adrenergic stimulation) can trigger proarrythmogenic Ca2+ waves. Restoring normal ryanodine receptor redox status can be a promising strategy to prevent arrhythmias and preserve positive inotropic effect of adrenergic stimulation during myocardial infarction.

Computational modeling of amylin-induced calcium dysregulation in rat ventricular cardiomyocytes

Hyperamylinemia is a condition that accompanies obesity and precedes type II diabetes, and it is characterized by above-normal blood levels of amylin, the pancreas-derived peptide. Human amylin oligomerizes easily and can deposit in the pancreas [1], brain [2], and heart [3], where they have been associated with calcium dysregulation. In the heart, accumulating evidence suggests that human amylin oligomers form moderately cation-selective [4,5] channels that embed in the cell sarcolemma (SL). The oligomers increase membrane conductance in a concentration-dependent manner [5], which is correlated with elevated cytosolic Ca2+. These findings motivate our core hypothesis that non-selective inward Ca2+ conduction afforded by human amylin oligomers increase cytosolic and sarcoplasmic reticulum (SR) Ca2+ load, which thereby magnifies intracellular Ca2+ transients. Questions remain however regarding the mechanism of amylin-induced Ca2+ dysregulation, including whether enhanced SL Ca2+ influx is sufficient to elevate cytosolic Ca2+ load [6], and if so, how might amplified Ca2+ transients perturb Ca2+-dependent cardiac pathways. To investigate these questions, we modified a computational model of cardiomyocytes Ca2+ signaling to reflect experimentally-measured changes in SL membrane permeation and decreased sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA) function stemming from acute and transgenic human amylin peptide exposure. With this model, we confirmed the hypothesis that increasing SL permeation alone was sufficient to enhance Ca2+ transient amplitudes. Our model indicated that amplified cytosolic transients are driven by increased Ca2+ loading of the SR and that greater fractional release may contribute to the Ca2+-dependent activation of calmodulin, which could prime the activation of myocyte remodeling pathways. Importantly, elevated Ca2+ in the SR and dyadic space collectively drive greater fractional SR Ca2+ release for human amylin expressing rats (HIP) and acute amylin-exposed rats (+Amylin) mice, which contributes to the inotropic rise in cytosolic Ca2+ transients. These findings suggest that increased membrane permeation induced by oligomeratization of amylin peptide in cell sarcolemma contributes to Ca2+ dysregulation in pre-diabetes.

A unified theory of calcium alternans in ventricular myocytes.

Intracellular calcium (Ca2+) alternans is a dynamical phenomenon in ventricular myocytes, which is linked to the genesis of lethal arrhythmias. Iterated map models of intracellular Ca2+ cycling dynamics in ventricular myocytes under periodic pacing have been developed to study the mechanisms of Ca2+ alternans. Two mechanisms of Ca2+ alternans have been demonstrated in these models: one relies mainly on fractional sarcoplasmic reticulum Ca2+ release and uptake, and the other on refractoriness and other properties of Ca2+ sparks. Each of the two mechanisms can partially explain the experimental observations, but both have their inconsistencies with the experimental results. Here we developed an iterated map model that is composed of two coupled iterated maps, which unifies the two mechanisms into a single cohesive mathematical framework. The unified theory can consistently explain the seemingly contradictory experimental observations and shows that the two mechanisms work synergistically to promote Ca2+ alternans. Predictions of the theory were examined in a physiologically-detailed spatial Ca2+ cycling model of ventricular myocytes.

L-Type Calcium and NCX Currents during Ischemia and Reperfusion in Intact Mouse Hearts

Cardiac ischemia is a pathological condition in which the blood supply to the myocardial tissue is interrupted producing an impairment of the mechanical and electrical function of the heart. Although there is reduction of Ca2+ transients during ischemia, the underlying mechanism of this Ca2+ mishandling is not fully understood. Currently, no reports exist in which Ca2+ currents and SR Ca2+ release are measured simultaneously in situ during global ischemia. Recently, we found that global ischemia induces large increases in diastolic Ca2+ leading to an increase in sarcoplasmic reticulum (SR) Ca2+ content. Contrary to what we expected there was a smaller fractional SR Ca2+ depletion during an action potential (AP). However, Ca2+ sparks measured in intact hearts during ischemia are kinetically undistinguishable from preischemic recordings. Our idea is that depressed Ca2+ transients during ischemia may result from a reduction of L-type Ca2+ currents and/or coupling between plasma membrane Ca2+ influx and SR Ca2+ release. Recently, we found using loose patch photolysis that Ca2+ dependent currents during an AP showed a fast early component driven by an L-type Ca2+influx and a slower late component mediated by a Na+-Ca2+ exchanger (NCX) forward current. To understand why Ca2+ transient decreases, we performed experiments to address if ischemia induces changes in Ca2+ dependent currents. Our results indicate that ischemia produced a decrease of the early Ca2+ current and the late NCX current. However, the reduction of the NCX current occurs before the L-type current attenuation. Although both Ca2+ mediated currents recover during reperfusion after ischemia, L-type Ca2+ currents recuperate faster. We conclude that the impairment of NCX during ischemia is an early event in both the contractility reduction and diastolic Ca2+ increase and that this impairment is maintained during postischemic diastolic dysfunction.

Abstract 9785: The Contribution of CaMKII and PKA to the Development of an Increased RyR-dependent SR Ca Leak in Human Cardiac Pathology

The mechanisms of a progressive ryanodine receptor type 2 (RyR2) dysregulation in the transition from human cardiac hypertrophy (Hy) to heart failure (HF) are still unclear. Inhibition of Ca 2+ /calmodulin-dependent kinase II (CaMKII) reduces the sarcoplasmic reticulum (SR) Ca 2+- leak and improve contractility in human HF. However, the relative contribution of CaMKII and protein kinase A are still not clear. Western blot experiments from tissue samples (n=12) of patients with afterload-induced Hy and preserved ejection fraction (EF>50%) revealed an increase in RyR2 expression by 82±22% ( P =0.05) compared to healthy controls (n=5). The phosphorylation status of RyR2 at S2809 and S2815 was not altered. In human end-stage HF (n=8), a pronounced hyperphosphorylation of RyR2 could be detected at the CaMKII-dependent site S2815 (by 311±72%, P <0.05) whereas the PKA-dependent site S2809 lacked differential regulation. The comparison of HF patients with and without β-blocker medication (n=7 vs. 4) did not reveal any differences as to RyR2 phosphorylation status. When isolated myocytes were paced at 1 Hz and scanned for diastolic Ca 2+ sparks (confocal microscopy, Fluo 3) a higher Ca 2+ -spark frequency (by 82±26% P <0.05) was detected in HF (n=93) compared to Hy (n=94). Interestingly, PKA inhibition with H89 (5 μM) only accomplished a decrease of SR Ca 2+ -leak in Hy (by 59±13%, P =0.05, n=85 vs. 115) but not in HF (n=85 vs. 74). Stimulated Ca 2+ transients (0.5 Hz, n=31 vs. 23), SR Ca 2+ content (caffeine, n=23 vs. 22) and fractional SR Ca 2+ release (n=21 vs. 15) were not changed by H89 treatment in HF (epifluorescence microscopy, Fura 2). CaMKII inhibition by AIP (1 μM), in contrast, could significantly reduce SR Ca 2+ leak in Hy (by 63±14%, P <0.05, n=93 vs. 103) as well as in HF. Thus, in human Hy with preserved EF both kinases seem to functionally regulate RyR2 gating. In end-stage human HF, however, the diastolic SR Ca 2+ leak seems to be induced by CaMKII rendering an inhibition of CaMKII a promising pharmacologic toehold.

Na+/K+-ATPase 2-isoform preferentially modulates Ca2+ transients and sarcoplasmic reticulum Ca2+ release in cardiac myocytes

Na(+)/K(+)-ATPase (NKA) is essential in regulating [Na(+)](i), and thus cardiac myocyte Ca(2+) and contractility via Na(+)/Ca(2+) exchange. Different NKA-α subunit isoforms are present in the heart and may differ functionally, depending on specific membrane localization. In smooth muscle and astrocytes, NKA-α2 is located at the junctions with the endo(sarco)plasmic reticulum, where they could regulate local [Na(+)], and indirectly junctional cleft [Ca(2+)]. Whether this model holds for cardiac myocytes is unclear. The ouabain-resistant NKA-α1 cannot be selectively blocked to assess its effect. To overcome this, we used mice in which NKA-α1 is ouabain sensitive and NKA-α2 is ouabain resistant (SWAP mice). We measured the effect of ouabain at low concentration on [Na(+)](i), Ca(2+) transients, and the fractional sarcoplasmic reticulum (SR) Ca(2+) release in cardiac myocytes from wild-type (WT; NKA-α2 inhibition) and SWAP mice (selective NKA-α1 block). At baseline, Na(+) and Ca(2+) regulations are similar in WT and SWAP mice. For equal levels of total NKA inhibition (~25%), ouabain significantly increased Ca(2+) transients (from ΔF/F(0)= 1.5 ± 0.1 to 1.8 ± 0.1), and fractional SR Ca(2+) release (from 24 ± 3 to 29 ± 3%) in WT (NKA-α2 block) but not in SWAP myocytes (NKA-α1 block). This occurred despite a similar and modest increase in [Na(+)](i) (~2 mM) in both groups. The effect in WT mice was mediated specifically by NKA-α2 inhibition because at a similar concentration ouabain had no effect in transgenic mice where both NKA-α1 and NKA-α2 are ouabain resistant. NKA-α2 has a more prominent role (vs. NKA-α1) in modulating cardiac myocyte SR Ca(2+) release.

Cardiac G-Protein–Coupled Receptor Kinase 2 Ablation Induces a Novel Ca 2+ Handling Phenotype Resistant to Adverse Alterations and Remodeling After Myocardial Infarction

Background— G-protein–coupled receptor kinase 2 (GRK2) is a primary regulator of β-adrenergic signaling in the heart. G-protein–coupled receptor kinase 2 ablation impedes heart failure development, but elucidation of the cellular mechanisms has not been achieved, and such elucidation is the aim of this study. Methods and Results— Myocyte contractility, Ca 2+ handling and excitation-contraction coupling were studied in isolated cardiomyocytes from wild-type and GRK2 knockout (GRK2KO) mice without (sham) or with myocardial infarction (MI). In cardiac myocytes isolated from unstressed wild-type and GRK2KO hearts, myocyte contractions and Ca 2+ transients were similar, but GRK2KO myocytes had lower sarcoplasmic reticulum (SR) Ca 2+ content because of increased sodium-Ca 2+ exchanger activity and inhibited SR Ca 2+ ATPase by local protein kinase A–mediated activation of phosphodiesterase 4 resulting in hypophosphorylated phospholamban. This Ca 2+ handling phenotype is explained by a higher fractional SR Ca 2+ release induced by increased L-type Ca 2+ channel currents. After β-adrenergic stimulation, GRK2KO myocytes revealed significant increases in contractility and Ca 2+ transients, which were not mediated through cardiac L-type Ca 2+ channels but through an increased SR Ca 2+ . Interestingly, post-MI GRK2KO mice showed better cardiac function than post-MI control mice, which is explained by an improved Ca 2+ handling phenotype. The SR Ca 2+ content was better maintained in post-MI GRK2KO myocytes than in post-MI control myocytes because of better-maintained L-type Ca 2+ channel current density and no increase in sodium-Ca 2+ exchanger in GRK2KO myocytes. An L-type Ca 2+ channel blocker, verapamil, reversed some beneficial effects of GRK2KO. Conclusions— These data argue for novel differential regulation of L-type Ca 2+ channel currents and SR load by GRK2. G-protein–coupled receptor kinase 2 ablation represents a novel beneficial Ca 2+ handling phenotype resisting adverse remodeling after MI.

Estimation of the fractional sarcoplasmic reticulum Ca2+ release in intact cardiomyocytes using integrated Ca2+ fluxes

The sarcoplasmic reticulum (SR) is the main source of contraction-activating Ca2+ in the adult mammalian myocardium. The fraction of the SR Ca2+ content released at a twitch (fractional SR Ca2+ release, FR) is an important parameter for assessing the efficiency of excitation-contraction coupling under physiological and pathophysiological conditions, as well as for identification of modulators of this process. We here describe and propose an approach for FR quantitation based on the estimation of integrated Ca2+ fluxes mediated by different transporters that remove the ion from the cytosol. These fluxes may be calculated solely from the measurement of cytosolic free Ca2+ concentration ([Ca2+]i) during Ca2+ transients evoked under selective inhibition of the transporters, and from the cell Ca2+ buffering parameters available in the literature. The FR values obtained with this approach in intact rat ventricular myocytes (0.63 ± 0.04; n = 12) were comparable to those estimated in the same cell type with an already established method, based on electrophysiological measurements with the patch-clamp technique, in addition to [Ca2+]i measurement (0.69 ± 0.05; n = 6; p > 0.40). We conclude that the proposed method might be a suitable and technically simpler alternative to the electrophysiological method for FR estimation.

The chronic effects of neonatal alloxan-induced diabetes mellitus on ventricular myocyte shortening and cytosolic Ca2+

Diabetes mellitus is a serious global health problem, and cardiovascular complications are the major cause of morbidity and mortality in diabetic patients. The chronic effects of neonatal alloxan- (ALX) induced diabetes mellitus on ventricular myocyte contraction and intracellular Ca(2+) transport have been investigated. Ventricular myocyte shortening was measured with a video edge detection system and intracellular Ca(2+) was measured in fura-2 loaded cells by fluorescence photometry. Diabetes was induced in 5-day old male Wistar rats by a single intraperitoneal injection of ALX (200 mg/kg body weight). Experiments were performed 12 months after ALX treatment. Fasting blood glucose was elevated and blood glucose at 60, 120 and 180 min after a glucose challenge (2 g/kg body weight, intraperitoneal) was elevated in diabetic rats compared to age-matched controls. Amplitude of shortening was significantly (P < 0.05) reduced in electrically stimulated myocytes from diabetic hearts (5.70 ± 0.24%) compared to controls (6.48 ± 0.28%). Amplitude of electrically evoked Ca(2+) transients was also significantly (P < 0.05) reduced in myocytes from diabetic hearts (0.11 ± 0.01 fura-2 ratio units) compared to controls (0.15 ± 0.01 fura-2 ratio units). Fractional sarcoplasmic reticulum Ca(2+) release was not significantly (P > 0.05) altered in myocytes from diabetic heart (0.70 ± 0.03 fura-2 ratio units) compared to controls (0.72 ± 0.03 fura-2 ratio units). Amplitude of caffeine-stimulated Ca(2+) transients was significantly (P < 0.05) reduced in myocytes from diabetic hearts (0.43 ± 0.02 fura-2 ratio units) compared to controls (0.51 ± 0.03 fura-2 ratio units). Area under the caffeine-evoked Ca(2+) transient was significantly (P < 0.05) reduced in myocytes from diabetic heart (0.77 ± 0.06 Vsec) compared to controls (1.14 ± 0.12 Vsec). Intracellular Ca(2+) refilling rate during electrical stimulation following application of caffeine was significantly (P < 0.05) slower in myocytes from diabetic heart (0.013 ± 0.001 V/sec) compared to controls (0.031 ± 0.007 V/sec). Depressed shortening may be partly attributed to depressed sarcoplasmic reticulum Ca(2+) transport in myocytes from neonatal ALX-induced diabetic rat heart.

Regulation of myocyte contraction via neuronal nitric oxide synthase: role of ryanodine receptor S‐nitrosylation

The sarcoplasmic reticulum (SR) Ca(2+) release channel (ryanodine receptor, RyR2) has been proposed to be an end target of neuronal nitric oxide synthase (NOS1) signalling. The purpose of this study is to investigate the mechanism of NOS1 modulation of RyR2 activity and the corresponding effect on myocyte function. Myocytes were isolated from NOS1 knockout (NOS1(/)) and wild-type mice. NOS1(/) myocytes displayed a decreased fractional SR Ca(2+) release, NOS1 knockout also led to reduced RyR2 S-nitrosylation levels. RyR2 channels from NOS1(/) hearts had decreased RyR2 open probability. Additionally, knockout of NOS1 led to a decrease in [(3)H]ryanodine binding, Ca(2+) spark frequency (CaSpF) and a rightward shift in the SR Ca(2+) leak/load relationship. Similar effects were observed with acute inhibition of NOS1. These data are indicative of decreased RyR2 activity in myocytes with NOS1 knockout or acute inhibition. Interestingly, the NO donor and nitrosylating agent SNAP reversed the depressed RyR2 open probability, the reduced CaSpF, and caused a leftward shift in the leak/load relationship in NOS1(/) myocytes. SNAP also normalized Ca(2+) transient and cell shortening amplitudes and SR fractional release in myocytes with NOS1 knockout or acute inhibition. Furthermore, SNAP was able to normalize the RyR2 S-nitrosylation levels. These data suggest that NOS1 signalling increases RyR2 activity via S-nitrosylation, which contributes to the NOS1-induced positive inotropic effect. Thus, RyR2 is an important end target of NOS1.

Spark-Induced Sparks As a Mechanism of Intracellular Calcium Alternans in Cardiac Myocytes

Intracellular calcium (Ca) alternans has been widely studied in cardiac myocytes and tissue, yet the underlying mechanism remains controversial. In this study, we used computational modeling and simulation to study how randomly occurring Ca sparks interact collectively to result in whole-cell Ca alternans. We developed a spatially distributed intracellular Ca cycling model in which Ca release units (CRUs) are locally coupled by Ca diffusion throughout the myoplasm and sarcoplasmic reticulum (SR) network. Ca sparks occur randomly in the CRU network when periodically paced with a clamped voltage waveform, but Ca alternans develops as the pacing speeds up. Combining computational simulation with theoretical analysis, we show that Ca alternans emerges as a collective behavior of Ca sparks, determined by 3 critical properties of the CRU network from which Ca sparks arise: "randomness" (of Ca spark activation), "refractoriness" (of a CRU after a Ca spark), and "recruitment" (Ca sparks inducing Ca sparks in adjacent CRUs). We also show that the steep nonlinear relationship between fractional SR Ca release and SR Ca load arises naturally as a collective behavior of Ca sparks, and Ca alternans can occur even when SR Ca is held constant. We present a general theory for the mechanisms of intracellular Ca alternans, which mechanistically links Ca sparks to whole-cell Ca alternans, and is applicable to Ca alternans in both physiological and pathophysiological conditions.

Na+-dependent SR Ca2+ overload induces arrhythmogenic events in mouse cardiomyocytes with a human CPVT mutation

Mutations in the cardiac ryanodine receptor Ca(2+) release channel, RyR2, underlie catecholaminergic polymorphic ventricular tachycardia (CPVT), an inherited life-threatening arrhythmia. CPVT is triggered by spontaneous RyR2-mediated sarcoplasmic reticulum (SR) Ca(2+) release in response to SR Ca(2+) overload during beta-adrenergic stimulation. However, whether elevated SR Ca(2+) content--in the absence of protein kinase A activation--affects RyR2 function and arrhythmogenesis in CPVT remains elusive. Isolated murine ventricular myocytes harbouring a human RyR2 mutation (RyR2(R4496C+/-)) associated with CPVT were investigated in the absence and presence of 1 micromol/L JTV-519 (RyR2 stabilizer) followed by 100 micromol/L ouabain intervention to increase cytosolic [Na(+)] and SR Ca(2+) load. Changes in membrane potential and intracellular [Ca(2+)] were monitored with whole-cell patch-clamping and confocal Ca(2+) imaging, respectively. At baseline, action potentials (APs), Ca(2+) transients, fractional SR Ca(2+) release, and SR Ca(2+) load were comparable in wild-type (WT) and RyR2(R4496C+/-) myocytes. Ouabain evoked significant increases in diastolic [Ca(2+)], peak systolic [Ca(2+)], fractional SR Ca(2+) release, and SR Ca(2+) content that were quantitatively similar in WT and RyR2(R4496C+/-) myocytes. Ouabain also induced arrhythmogenic events, i.e. spontaneous Ca(2+) waves, delayed afterdepolarizations and spontaneous APs, in both groups. However, the ouabain-induced increase in the frequency of arrhythmogenic events was dramatically larger in RyR2(R4496C+/-) when compared with WT myocytes. JTV-519 greatly reduced the frequency of ouabain-induced arrhythmogenic events. The elevation of SR Ca(2+) load--in the absence of beta-adrenergic stimulation--is sufficient to increase the propensity for triggered arrhythmias in RyR2(R4496C+/-) cardiomyocytes. Stabilization of RyR2 by JTV-519 effectively reduces these triggered arrhythmias.

Alteration of Ryanodine Receptor-Mediated Calcium Release in Heart Failure

The decrease in contractility in heart failure associates with impaired cellular Ca2+ homeostasis that is in part due to altered ryanodine receptor (RyR) function. We studied properties of sarcoplasmic reticulum (SR) Ca2+ release in normal and failing rabbit ventricular myocytes using simultaneous measurements of cytosolic ([Ca2+]i) and intra-SR free Ca2+ ([Ca2+]SR). At a given SR Ca2+ content, fractional SR Ca2+ release during action potential stimulation was higher in failing than nonfailing myocytes, suggesting increased sensitivity of RyRs in heart failure. In permeabilized myocytes, SR Ca2+ content and Ca2+ spark frequency were decreased in heart failure, while Ca2+ spark amplitude was similar between failing and nonfailing myocytes. To compare these two groups further, SR Ca2+ content was experimentally decreased in nonfailing myocytes to the level observed in failing myocytes using SERCA inhibition. When SR Ca2+ content was matched, both Ca2+ spark frequency and amplitude were markedly increased in failing myocytes, showing that RyRs are more sensitive to release activation. By monitoring [Ca2+]SR during Ca2+ sparks, we also observed that the [Ca2+]SR level for spark termination was significantly lower in myocytes from failing hearts. Because Ca2+ sparks are a major contributing factor to diastolic SR Ca2+ leak, we compared the properties of SR Ca2+ leak in normal and failing myocytes. In failing myocytes SR Ca2+ leak was significantly faster, particularly at high [Ca2+]SR where Ca2+ sparks are the predominant pathway for SR Ca2+ leak. These data show that during the progression of heart failure, modifications to RyRs alter both activation and termination of local SR Ca2+ release events. At a given SR Ca2+ content these effects may increase fractional SR Ca2+ release and preserve contractility during systole, however at the cost of increased diastolic SR Ca2+ leak and SR depletion.

Dynamic Changes of Calcium in Sarcoplasmic Reticulum of Rabbit Atrial Myocytes

With each contraction in atrial muscle, Ca2+ is released from the sarcoplasmic reticulum (SR) through ryanodine receptors and then reaccumulated by the activity of the SR Ca2+ ATPase (SERCA). Recent experiments by us and others have examined SR Ca2+ concentration ([Ca2+]SR) using low affinity fluorescent Ca2+ sensors (e.g. fluo-5N) in ventricular myocytes (Brochet et al. PNAS 2005 102(8) 3099-104) however, similar characterization in atrial cells is missing. Here we examine [Ca2+] SR dynamics in healthy adult rabbit atrial myocytes using fluo-5N. Fractional SR Ca2+release at room temperature was 30% (n=12), where caffeine was used to determine 100% release. Restoration of [Ca2+]SR by SERCA was consistent with the decline of [Ca2+]i with each beat (1 Hz). The interconnectivity of the cell-wide SR and nuclear envelope and endoplasmic reticulum was characterized using fluorescence recovery after photobleaching (FRAP). Additional diverse modulations of atrial cell [Ca2+]SR will be discussed.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Alteration of sarcoplasmic reticulum Ca2+ release termination by ryanodine receptor sensitization and in heart failure

Many physiological processes and pharmacological agents modulate the ryanodine receptor (RyR), the primary sarcoplasmic reticulum (SR) Ca(2+) release channel in the heart. However, how such modulations translate into functional effects during cardiac excitation-contraction coupling (ECC) is much less clear. Using a low dose (250 microM) of caffeine we sensitized the RyR and examined SR Ca(2+) release using dynamic measurements of cytosolic Ca(2+) ([Ca(2+)](i)) and free Ca(2+) within the SR ([Ca(2+)](SR)). In field stimulated (1 Hz) rabbit ventricular myocytes, application of 250 microM caffeine caused an initial 33% increase in SR Ca(2+) release, which was followed by a decrease in SR Ca(2+) load (28%) and steady-state SR Ca(2+) release (23%). To investigate the effects of caffeine on local SR Ca(2+) release, we measured [Ca(2+)](SR) from individual release junctions during ECC as well as during spontaneous Ca(2+) sparks. In intact myocytes during ECC, caffeine increased global fractional SR Ca(2+) release by decreasing the [Ca(2+)](SR) level at which local release terminated by 21%. Similarly, in permeabilized myocytes during spontaneous Ca(2+) sparks, caffeine decreased the [Ca(2+)](SR) level for release termination by 12%. Finally, we examined if Ca(2+) release termination was changed in myocytes from failing hearts, where remodelling processes lead to altered RyR function. In myocytes from failing rabbit hearts, the [Ca(2+)](SR) termination level for Ca(2+) sparks was 13% lower than that of non-failing myocytes. Collectively, these data suggest that altering the termination level for local Ca(2+) release may represent a novel mechanism to increase SR Ca(2+) release and contractility during ECC.

Ultrastructural and Functional Remodeling of the Coupling Between Ca 2+ Influx and Sarcoplasmic Reticulum Ca 2+ Release in Right Atrial Myocytes From Experimental Persistent Atrial Fibrillation

Persistent atrial fibrillation (AF) has been associated with structural and electric remodeling and reduced contractile function. To unravel mechanisms underlying reduced sarcoplasmic reticulum (SR) Ca(2+) release in persistent AF. We studied cell shortening, membrane currents, and [Ca(2+)](i) in right atrial myocytes isolated from sheep with persistent AF (duration 129+/-39 days, N=16), compared to matched control animals (N=21). T-tubule density, ryanodine receptor (RyR) distribution, and local [Ca(2+)](i) transients were examined in confocal imaging. Myocyte shortening and underlying [Ca(2+)](i) transients were profoundly reduced in AF (by 54.8% and 62%, P<0.01). This reduced cell shortening could be corrected by increasing [Ca(2+)](i). SR Ca(2+) content was not different. Calculated fractional SR Ca(2+) release was reduced in AF (by 20.6%, P<0.05). Peak Ca(2+) current density was modestly decreased (by 23.9%, P<0.01). T-tubules were present in the control atrial myocytes at low density and strongly reduced in AF (by 45%, P<0.01), whereas the regular distribution of RyR was unchanged. Synchrony of SR Ca(2+) release in AF was significantly reduced with increased areas of delayed Ca(2+) release. Propagation between RyR was unaffected but Ca(2+) release at subsarcolemmal sites was reduced. Rate of Ca(2+) extrusion by Na(+)/Ca(2+) exchanger was increased. In persistent AF, reduced SR Ca(2+) release despite preserved SR Ca(2+) content is a major factor in contractile dysfunction. Fewer Ca(2+) channel-RyR couplings and reduced efficiency of the coupling at subsarcolemmal sites, possibly related to increased Na(+)/Ca(2+) exchanger, underlie the reduction in Ca(2+) release.

Negative Relationship Between Fractional SR Ca2+ Release and Stimulation Rate

Typically, contraction amplitude in rodent myocardium is inversely related to stimulation rate. We calculated Ca2+ fluxes across the sarcolemma and the sarcoplasmic reticulum (SR) membrane, and SR Ca2+ content to estimate the fraction of the SR Ca2+ load released at a twitch (FR) during steady-state (SS) stimulation at 0.2-2 Hz in intact rat cardiomyocytes. Cytosolic [Ca2+] ([Ca2+]i) was measured with indo-1. SR Ca2+ content was determined from the transient in response to 10 mM caffeine in Na+,Ca2+-free medium. From Ca2+ buffer parameters, and [Ca2+]i values and decline kinetics during transients in which SR Ca2+-ATPase and/or Na+-Ca2+ exchanger (NCX) were inhibited, SR- and NCX-mediated Ca2+ fluxes at each rate were estimated and integrated up to attainment of SS diastolic [Ca2+]i. Assuming equal inward and outward Ca2+ fluxes at SS, Ca2+ influx was considered as the NCX-mediated flux, whereas SR Ca2+ release and uptake fluxes were considered equivalent. FR was taken as the ratio of the integrated SR-mediated Ca2+ flux and SR Ca2+ content. Increasing rate did not affect significantly SR Ca2+ content (128±4 and 133±4 μM at 0.2 and 2 Hz, respectively), but decreased (p<0.01) both Ca2+ influx (16.9±1.4 vs. 6.7±0.3 μM at 0.2 and 2 Hz) and SR-dependent Ca2+ flux (93±4 vs. 77±3 μM at 0.2 and 2 Hz). Estimated FR showed a negative relationship with the stimulation rate (0.73±0.03 vs. 0.58±0.02, p<0.01), possibly due to decreased Ca2+ influx, although action potential duration was increased (APD90= 76±9 vs. 130±11 ms, p<0.01). Alternatively, incomplete time-dependent recovery from inactivation of SR Ca2+ channels might contribute to depress FR. These results support the proposal that changes in FR underlie the negative force-frequency relationship in rodents. (FAPESP, CNPq, FAEPEX).