Spark Activity Research Articles

EC Coupling Latency in Rat Ventricular Myocytes

Rapid activation of sarcolemmal L-type Ca channels (LCCs), Ca influx and activation of Ca-induced Ca release (CICR) from the sarcoplasmic reticulum (SR) are crucial to cardiac excitation-contraction (EC) coupling. The uniformity and duration of a Ca transient elicited by an action potential (AP) depends on LCC activation kinetics and ability of the consequent Ca influx to trigger Ca sparks. In contrast to some earlier work, recent studies have suggested that EC coupling ‘fidelity' is extremely low and that as many as 20-30 LCC openings are required to trigger a Ca spark. This result would suggest that the latency to Ca spark activation is dominated by LCC activation, (although this has not been shown). To investigate this idea, step voltage-clamp protocols were used to activate Ca transients and Ca sparks. In one series of experiments, LCCs were first activated by a pre-pulse. The latency of SR Ca release decreased with increasing depolarizing step potential, in qualitative agreement with the expected voltage-dependence of LCC activation. In contrast, the latency of SR Ca release increased with increasing step potential after a strong pre-pulse which should have removed the latency for LCC activation. The difference between these results reflects the influence of LCC gating kinetics on EC coupling latency. The results for Ca spark and Ca transient latency suggest that only a few LCC openings are required to trigger a Ca spark and that during a Ca transient, LCC availability was ∼3-fold larger. To test this interpretation, a computer model was constructed to simulate stochastic LCC openings, Ca appearance inside the dyad and Ca-dependent gating of ryanodine receptors. The model shows that the recorded Ca spark latencies can be reproduced when the number of LCCs that initiate SR Ca release is 1-2 and 5-6 for Ca transients.

Investigating the Effects of a Cardiotoxic Drug on Calcium Homeostasis in the Heart

Contractile dysfunction and arrhythmogenic activity can arise if there are perturbations to calcium (Ca) homeostasis in the heart, such as in response to cardiotoxic substances. This investigation focuses on changes in cellular Ca homeostasis in response to the acute application of the cardiotoxic drug Clozapine (an atypical anti-psychotic).Rat ventricular myocytes were isolated and stimulated under voltage clamp control. Various parameters of Ca handling were measured both in control conditions and in the presence of 10 μM clozapine. Ca-sensitive indicators fluo3-AM or fura-2 were used to quantify cytosolic Ca. Sarcoplasmic reticulum (SR) Ca content was measured by applying 10 mM caffeine. Activity of Ca extrusion mechanisms (SERCA - sarco-endoplasmic reticulum ATP-ase, NCX - sodium-calcium exchanger) were calculated from Ca transient decay rates. The current-voltage (I-V) relationship of the L-type Ca channel was also measured.Acute application of clozapine significantly reduced mean Ca transient amplitude by 48% and L-type Ca current (ICaL) density by 50%. SR content was decreased by 18 % and SERCA activity (rate constant) was reduced by 34 %. NCX rate constant was reduced by 17%. The effect of clozapine on SERCA was also present in phospholamban knockout myocytes suggesting that the effect on SERCA is not dependent on phospholamban.It remains unclear if or how the observed effects on Ca handling contribute to the cardiotoxicity of clozapine observed in the clinical setting (arrhythmogenesis and myocarditis). We speculate that effects on ICaL may predispose to refractory heterogeneity and reduced SERCA activity may increase the chance for Ca wave propagation. Current work focuses on effects of clozapine on calcium spark activity and SR threshold for Ca waves.

Spatio-Temporal Properties of IP3 Receptor-Mediated Ca Release in Cardiac Myocytes

In cardiac myocytes cytosolic Ca increase and subsequent cell contraction are mainly determined by ryanodine receptor (RyR) mediated Ca release. However atrial cells are also equipped with IP3 receptors (IP3Rs), a second type of Ca release channels. We investigated IP3R-mediated Ca release events and their subcellular spatio-temporal properties in single membrane-permeabilized rabbit atrial myocytes. Local Ca release events were detected by confocal microscopy (fluo-4, longitudinal line scan mode). Local IP3R-mediated Ca release events were evoked by exposure to inositol-1,4,5-triphosphate (IP3) in the presence of tetracaine to inhibit RyR-mediated Ca spark activity, and could be inhibited by the IP3R blocker 2-APB. We classified IP3R-mediated Ca release events as subsarcolemmal, perinuclear (events 5μm) or nuclear. Nuclear events were considered membrane associated when occurring at a distance <1μm from the nuclear envelope or membranes of the nucleoplasmic reticulum. With tetracaine the Ca spark frequency decreased to 0.7±0.3 (from 8.8±1.2 in control), whereas the frequency of events detected after subsequent addition of IP3 increased to 2.9±1.3 (events/100μm/s). Subsequent exposure to 2-APB reduced the frequency to 1.8±0.8. Compared to Ca sparks, local Ca release events in the presence of IP3 were lower in amplitude, prolonged in duration and had a slower rise time. The increase in frequency of local Ca release events was particularly pronounced in the perinucler regions compared to the cytosol or the subsarcolemmal space. Furthermore, IP3-mediated Ca release events could also be detected within the nucleus. These nuclear events occurred at a higher incidence at locations that were closely associated with membrane structures of the nuclear envelope or the nucleoplasmic reticulum. In conclusion, in atrial myocytes IP3R-mediated Ca release is spatially inhomogeneous and preferentially occurs in the nuclear and perinuclear regions.

Maternal high-altitude hypoxia and suppression of ryanodine receptor-mediated Ca2+ sparks in fetal sheep pulmonary arterial myocytes

Ca(2+) sparks are fundamental Ca(2+) signaling events arising from ryanodine receptor (RyR) activation, events that relate to contractile and dilatory events in the pulmonary vasculature. Recent studies demonstrate that long-term hypoxia (LTH) can affect pulmonary arterial reactivity in fetal, newborn, and adult animals. Because RyRs are important to pulmonary vascular reactivity and reactivity changes with ontogeny and LTH we tested the hypothesis that RyR-generated Ca(2+) signals are more active before birth and that LTH suppresses these responses. We examined these hypotheses by performing confocal imaging of myocytes in living arteries and by performing wire myography studies. Pulmonary arteries (PA) were isolated from fetal, newborn, or adult sheep that lived at low altitude or from those that were acclimatized to 3,801 m for > 100 days. Confocal imaging demonstrated preservation of the distance between the sarcoplasmic reticulum, nucleus, and plasma membrane in PA myocytes. Maturation increased global Ca(2+) waves and Ca(2+) spark activity, with sparks becoming larger, wider, and slower. LTH preferentially depressed Ca(2+) spark activity in immature pulmonary arterial myocytes, and these sparks were smaller, wider, and slower. LTH also suppressed caffeine-elicited contraction in fetal PA but augmented contraction in the newborn and adult. The influence of both ontogeny and LTH on RyR-dependent cell excitability shed new light on the therapeutic potential of these channels for the treatment of pulmonary vascular disease in newborns as well as adults.

P6 Hydrogen sulfide activates Ca2+ sparks to induce cerebral arteriole dilation

Hydrogen sulfide (H 2 S) is a gaseous vasodilator produced by endothelial cells. Mechanisms by which H 2 S induce vasodilation are unclear. We tested the hypothesis that H 2 S dilates piglet cerebral arterioles by modulating local and global intracellular Ca 2+ signals in smooth muscle cells. High-speed confocal imaging revealed that H 2 S increased Ca 2+ spark frequency ∼1.43-fold and decreased global intracellular Ca 2+ concentration ([Ca 2+ ] i ) by ∼37 nM in smooth muscle cells of intact cerebral arteries. In contrast, H 2 S did not alter Ca 2+ wave frequency. In voltage-clamped (−40 mV) cells, H 2 S increased the frequency of Ca 2+ spark-induced transient Ca 2+ -activated K + (K Ca ) currents ∼ 1.8-fold, but did not alter the amplitude of these events. H 2 S did not alter the activity of single K Ca channels recorded in the absence of Ca 2+ sparks in arteriole smooth muscle cells. H 2 S increased SR Ca 2+ load ([Ca 2+ ] SR ), measured as caffeine (10 mM)-induced [Ca 2+ ] i transients, ∼1.5-fold. H 2 S dilated pressurized cerebral arterioles. Iberiotoxin, a K Ca channel blocker, and ryanodine, a ryanodine receptor (RyR) channel inhibitor, reduced H 2 S-induced vasodilation by ∼38 and ∼37%, respectively. In summary, our data indicate that H 2 S elevates [Ca 2+ ] SR , leading to Ca 2+ spark activation in cerebral arteriole smooth muscle cells. The subsequent elevation in transient K Ca current frequency leads to a reduction in global [Ca 2+ ] i and vasodilation.

Hydrogen sulfide activates Ca2+ sparks to induce cerebral arteriole dilatation

Hydrogen sulfide (H₂S) is a gaseous vasodilator produced by endothelial cells. Mechanisms by which H₂S induces vasodilatation are unclear. We tested the hypothesis that H₂S dilates cerebral arterioles by modulating local and global intracellular Ca²⁺ signals in smooth muscle cells. High-speed confocal imaging revealed that Na₂S, an H₂S donor, increased Ca²⁺ spark frequency ∼1.43-fold and decreased global intracellular Ca²⁺ concentration ([Ca²⁺]i) by ∼37 nM in smooth muscle cells of intact piglet cerebral arterioles. In contrast, H₂S did not alter Ca²⁺ wave frequency. In voltage-clamped (-40 mV) cells, H₂S increased the frequency of iberiotoxin-sensitive, Ca²⁺ spark-induced transient Ca²⁺-activated K⁺ (KCa) currents ∼1.83-fold, but did not alter the amplitude of these events. H₂S did not alter the activity of single KCa channels recorded in the absence of Ca²⁺ sparks in arteriole smooth muscle cells. H₂S increased SR Ca²⁺ load ([Ca²⁺]SR), measured as caffeine (10 and 20mM)-induced [Ca²⁺]i transients, ∼1.5-fold. H₂S hyperpolarized (by ∼18 mV) and dilated pressurized (40 mmHg) cerebral arterioles. Iberiotoxin, a KCa channel blocker, reduced H₂S-induced hyperpolarization by ∼51%. Iberiotoxin and ryanodine, a ryanodine receptor channel inhibitor, reduced H₂S-induced vasodilatation by ∼38 and ∼37%, respectively. In summary, our data indicate that H₂S elevates [Ca²⁺]SR, leading to Ca²⁺ spark activation in cerebral arteriole smooth muscle cells. The subsequent elevation in transient KCa current frequency leads to membrane hyperpolarization, a reduction in global [Ca²⁺]i and vasodilatation.

Hydrogen sulfide activates Ca2+ sparks to induce cerebral arteriole dilation

Hydrogen sulfide (H2S) is a gaseous vasodilator produced by endothelial cells. Mechanisms by which H2S induce vasodilation are unclear. We tested the hypothesis that H2S dilates piglet cerebral arterioles by modulating local and global intracellular Ca2+ signals in smooth muscle cells. High‐speed confocal imaging revealed that H2S increased Ca2+ spark frequency ~1.43 fold and decreased global intracellular Ca2+ concentration ([Ca2+]i) by ~37 nM in smooth muscle cells of intact cerebral arteries. In contrast, H2S did not alter Ca2+ wave frequency. In voltage‐clamped (–40 mV) cells, H2S increased the frequency of Ca2+ spark‐induced transient Ca2+‐activated K+ (KCa) currents ~1.8 fold, but did not alter the amplitude of these events. H2S did not alter the activity of single KCa channels recorded in the absence of Ca2+ sparks in arteriole smooth muscle cells. H2S increased SR Ca2+ load ([Ca2+]SR), measured as caffeine (10 mM)‐induced [Ca2+]i transients, ~1.5 fold. H2S dilated pressurized cerebral arterioles. Iberiotoxin, a KCa channel blocker, and ryanodine, a ryanodine receptor (RyR) channel inhibitor, reduced H2S‐induced vasodilation by ~38 and ~37%, respectively. In summary, our data indicate that H2S elevates [Ca2+]SR, leading to Ca2+ spark activation in cerebral arteriole smooth muscle cells. The subsequent elevation in transient KCa current frequency leads to a reduction in global [Ca2+]i and vasodilation.NIH/NHLBI

Cyclic Nucleotides Cause Divergent Ryanodine Receptor Modulation in Pulmonary Arterial Myocytes from Immature Chronic Hypoxic Sheep

Calcium sparks are due to ryanodine receptor (RyR) activation and are intimate to local and global calcium signals in pulmonary arterial (PA) myocytes. RyRs are heavily regulated and cyclic nucleotides, including cAMP and cGMP, can increase RyR activity and thereby augment Ca2+ sparks and global Ca2+ waves. One possibility is that enhanced RyR activity contributes to altered pulmonary arterial reactivity responses found in sheep born at high altitude. Yet, the influence of chronic hypoxia (CH) on cyclic nucleotide regulation of RyRs is unknown. In these studies, we tested the hypothesis that cAMP and cGMP increase Ca2+ sparks and waves in PA myocytes from hypoxic sheep. Spark and wave activity was determined by visual analysis of line‐scan confocal recordings of Fluo‐4 or Fluo‐8 loaded PA from term‐fetal, ~ 10 day old, or adult sheep that lived at 3,200 meters for &lt;100 days. Similar percentages of fetal, newborn, and adult myocytes had sparks and waves before cyclic nucleotide treatment, although newborns had more sparks per recording. cAMP and cGMP increased the number of adult cells with sparks and waves, yet, they respectively decreased the number of newborn and fetal myocytes with sparks. Spark activity was increased in adult myocytes by cGMP. The data suggest that cyclic nucleotides cause divergent regulation of RyRs from PA myocytes of immature CH sheep. NSF MRI 092355 (SMW), NIH P01HD031226, R01HD003807 (LDL)

Acidosis Dilates Brain Parenchymal Arterioles by Conversion of Calcium Waves to Sparks to Activate BK Channels

Acidosis is a powerful vasodilator signal in the brain circulation. However, the mechanisms by which this response occurs are not well understood, particularly in the cerebral microcirculation. One important mechanism to dilate cerebral (pial) arteries is by activation of large-conductance, calcium-sensitive potassium (BK(Ca)) channels by local Ca(2+) signals (Ca(2+) sparks) through ryanodine receptors (RyRs). However, the role of this pathway in the brain microcirculation is not known. The objectives of this study were to determine the mechanism by which acidosis dilates brain parenchymal arterioles (PAs) and to elucidate the roles of RyRs and BK(Ca) channels in this response. Internal diameter and vascular smooth muscle cell Ca(2+) signals were measured in isolated pressurized murine PAs, using imaging techniques. In physiological pH (7.4), vascular smooth muscle cells exhibited primarily RyR-dependent Ca(2+) waves. Reducing external pH from 7.4 to 7.0 in both normocapnic and hypercapnic conditions decreased Ca(2+) wave activity, and dramatically increased Ca(2+) spark activity. Acidic pH caused a dilation of PAs which was inhibited by about 60% by BK(Ca) channel or RyR blockers, in a nonadditive manner. Similarly, dilator responses to acidosis were reduced by nearly 60% in arterioles from BK(Ca) channel knockout mice. Dilations induced by acidic pH were unaltered by inhibitors of K(ATP) channels or nitric oxide synthase. These results support the novel concept that acidification, by converting Ca(2+) waves to sparks, leads to the activation of BK(Ca) channels to induce dilation of cerebral PAs.

Subcellular Heterogeneity of Ryanodine Receptor Properties in Ventricular Myocytes with Low T-Tubule Density

RationaleIn ventricular myocytes of large mammals, not all ryanodine receptor (RyR) clusters are associated with T-tubules (TTs); this fraction increases with cellular remodeling after myocardial infarction (MI).ObjectiveTo characterize RyR functional properties in relation to TT proximity, at baseline and after MI.MethodsMyocytes were isolated from left ventricle of healthy pigs (CTRL) or from the area adjacent to a myocardial infarction (MI). Ca2+ transients were measured under whole-cell voltage clamp during confocal linescan imaging (fluo-3) and segmented according to proximity of TTs (sites of early Ca2+ release, F>F50 within 20 ms) or their absence (delayed areas). Spontaneous Ca2+ release events during diastole, Ca2+ sparks, reflecting RyR activity and properties, were subsequently assigned to either category.ResultsIn CTRL, spark frequency was higher in proximity of TTs, but spark duration was significantly shorter. Block of Na+/Ca2+ exchanger (NCX) prolonged spark duration selectively near TTs, while block of Ca2+ influx via Ca2+ channels did not affect sparks properties. In MI, total spark mass was increased in line with higher SR Ca2+ content. Extremely long sparks (>47.6 ms) occurred more frequently. The fraction of near-TT sparks was reduced; frequency increased mainly in delayed sites. Increased duration was seen in near-TT sparks only; Ca2+ removal by NCX at the membrane was significantly lower in MI.ConclusionTT proximity modulates RyR cluster properties resulting in intracellular heterogeneity of diastolic spark activity. Remodeling in the area adjacent to MI differentially affects these RyR subpopulations. Reduction of the number of sparks near TTs and reduced local NCX removal limit cellular Ca2+ loss and raise SR Ca2+ content, but may promote Ca2+ waves.

Relationship between Ca2+sparklets and sarcoplasmic reticulum Ca2+load and release in rat cerebral arterial smooth muscle

Ca(+) sparklets are subcellular Ca(2+) signals produced by the opening of sarcolemmal L-type Ca(2+) channels. Ca(2+) sparklet activity varies within the sarcolemma of arterial myocytes. In this study, we examined the relationship between Ca(2+) sparklet activity and sarcoplasmic reticulum (SR) Ca(2+) accumulation and release in cerebral arterial myocytes. Our data indicate that the SR is a vast organelle with multiple regions near the sarcolemma of these cells. Ca(2+) sparklet sites were located at or <0.2 μm from SR-sarcolemmal junctions. We found that while Ca(2+) sparklets increase the rate of SR Ca(2+) refilling in arterial myocytes, their activity did not induce regional variations in SR Ca(2+) content or Ca(2+) spark activity. In arterial myocytes, L-type Ca(2+) channel activity was independent of SR Ca(2+) load. This ruled out a potential feedback mechanism whereby SR Ca(2+) load regulates the activity of these channels. Together, our data suggest a model in which Ca(2+) sparklets contribute Ca(2+) influx into a cytosolic Ca(2+) pool from which sarco(endo)plasmic reticulum Ca(2+)-ATPase pumps Ca(2+) into the SR, indirectly regulating SR function.

Urban School Children's Health-related Physical Fitness and Physical Activity Participation

PURPOSE: Although the interactive effects related to physical activity (PA) and health-related physical fitness have been well documented (Strong et al., 2005), such inquiries among underserved urban children remain largely unexplored. This study was designed to examine the relationship between urban elementary school children's health-related physical fitness and PA participation, as well as gender and grade differences of these study variables. METHODS: Participants were 196 third through fifth grade students (112 boys, 84 girls; Mage = 10.3 yrs) from an urban elementary school. The majority of participants (73%) were children of Latino immigrant families; 89% of them came from economically disadvantaged families. At the beginning of the school year, participants'physical fitness was measured through1-mile run (an indicator of cardiorespiratory fitness); body mass index (BMI), and percent of body fat (PBF) as measured by skinfold. They then responded to the self-reported SPARK activity checklist as a measure of their weekly PA levels, and the SPARK data was calculated into the MET scores (Barnara etc. 2000). RESULTS: Correlation analysis yielded that children's BMI and PBF was negatively related to 1-mile run score (p<.01). 1-mile run score was positively associated with weekly PA levels (p<.05). Regression analysis further indicated that children's 1-mile run score was the only significant predictor of their weekly PA participation (B=.16, p<.05). The 2-way MANOVA revealed significant main effects for gender (F =6.98, p<.01) and grade (F =6.40, p<.01). CONCLUSION: The findings suggested that urban children's 1-mile run score emerged as the positive predictor of their weekly PA participation. Additionally, boys had significantly lower PBF and performed better on 1-mile run than girls. Fourth graders had lower PBF yet performed worse on 1-mile run than fifth graders. Fifth graders were more physically active than the other graders.

Combined influence of ontogeny and chronic hypoxia on ryanodine receptor function in sheep pulmonary arteries and myocytes

Ryanodine receptors (RyR) and associated Ca2+ spark events are important to hypoxic pulmonary vasoconstriction (HPV) and ET-1 dependent pulmonary arterial (PA) contractility. RyR function can change with development and chronic hypoxia (CH). Yet, the combined influence of CH and development on Ca2+ sparks is not known. We therefore tested the hypotheses that Ca2+ sparks are restricted before birth, that CH augments their activity, and that RyRs contribute to serotonin (5-HT)-mediated arterial stimulation. These hypotheses were tested on PA from near term fetal, 7–14 day old newborn and adult sheep that lived at low altitude or were maintained 100+ days at 3801 m (CH). Myography was performed on isolated PA and Ca2+ sparks were measured using fluo-4 loaded PA. Spark activity was greater in adults relative to fetuses and newborns. CH reduced spark activity in fetuses and newborns but not adults. Spark activity was reduced by 10 μM ryanodine (RY) in all groups except for CH fetuses. Selective RyR1/3 inhibition with 10 μM dantrolene suppressed sparks in normoxic fetuses and adults. Spark activity was not affected by 10 μM 5-HT in adult PA myocytes, and RY did not reduce 5-HT mediated PA contraction. Overall, RyR function increases following birth, CH differentially influences RyR function depending on developmental age, and 5-HT has little interaction with RyRs. NSF MRI 0923559 (SMW), NIH P01HD031226, R01HD003807 (LDL)

Role of ryanodine receptors in acidic pH‐induced dilation of brain parenchymal arterioles

Ryanodine receptors (RyRs) are Ca2+ permeable channels in the sarcoplasmic reticulum. RyRs regulate vascular smooth muscle cell (VSMC) membrane potential through their localized Ca2+‐release events, termed Ca2+ sparks, which activate hyperpolarizing BK currents leading to vasodilation. This mechanism is clearly demonstrated by the non‐additive constrictions induced by RyR or BK channel blockers in pressurized pial arteries. However these blockers have little effect on the diameter of pressurized parenchymal arterioles (PAs) from the brain, even though functional BK channels and RyRs are present. At normal pH (7.4), VSMCs in pressurized PAs exhibit largely Ca2+ waves but not Ca2+ sparks. Alkaline pH has been shown to shift Ca2+ sparks to Ca2+ waves in pial arteries. Therefore, we hypothesized that acidic pH might dilate PAs by reshaping the intracellular Ca2+ dynamic from Ca2+ waves to Ca2+ sparks, thereby activating BK channels. As predicted, reducing extracellular pH from 7.4 to 7.0 dramatically decreased Ca2+ wave activity, and increased spark activity. Acidic pH caused a dilation of up to 70% of the maximal diameter, and this dilation was inhibited by about 60% by BK or RyR blockers. These findings suggest that RyRs play a major role in the acidic pH‐induced dilation of PAs through Ca2+ spark‐mediated activation of BK channels. Supported by AHA 09POST2290090 (FD) and the NIH HL095488, HL44455 and HL58231.

Activation of Calcium Sparks in Resting Cardiomyocytes by β-Adrenergic Stimulation May Involve CaMKII and nNOS

It has been reported that during β-adrenergic stimulation of cardiac myocytes, phosphorylation of Ca2+ release channels (ryanodine receptors, RyRs) by PKA and/or CaMKII may result in arrhythmogenic diastolic Ca2+ leak (as elementary Ca2+ release events, Ca2+ sparks) from intracellular Ca2+ stores (the sarcoplasmic reticulum, SR). Using confocal Ca2+ imaging, we have recently shown that β-adrenergic stimulation by 1 µM isoproterenol (ISO) increases the Ca2+ spark frequency several-fold in quiescent, whole-cell voltage-clamped guinea-pig myocytes, without altering SR Ca2+ content. As this occurs without variations of the diastolic intracellular Ca2+ concentration, this observation suggests a sensitization of the RyRs. Experiments with protein kinase inhibitors (KN-93 and H89) indicated an involvement of CaMKII in the change of spark frequency. Surprisingly, but in line with the kinase inhibitor experiments, increasing cAMP production and PKA activity by direct stimulation of adenylate cyclase with forskolin (1 µM) did not significantly elevate Ca2+ spark frequencies under the same experimental conditions. Further experiments revealed that the change in sensitivity of the RyRs upon β-adrenergic stimulation may be linked to nitric oxide (NO), as pre-incubation of the cells with the NOS inhibitor L-NAME (500 µM) prevented the increase of the Ca2+ spark frequency without dramatic changes of SR Ca2+ content. Using the nNOS specific inhibitor AAAN (100 µM) resulted in analogous observations, suggesting that the nNOS isoform, located in close proximity of the RyRs, may be involved in this signaling pathway. Taken together, the results suggest the presence of a non-classical pathway linking β-adrenergic stimulation of cardiac myocytes to enhanced activity of the RyRs. Preliminary pharmacological evidence indicates that the pathway includes both, CaMKII and nNOS as important components. Supported by SNF.

Ca2+spark-dependent and -independent sarcoplasmic reticulum Ca2+leak in normal and failing rabbit ventricular myocytes

Sarcoplasmic reticulum (SR) Ca²(+) leak is an important component of cardiac Ca²(+) signalling. Together with the SR Ca²(+)-ATPase (SERCA)-mediated Ca²(+) uptake, diastolic Ca²(+) leak determines SR Ca²(+) load and, therefore, the amplitude of Ca²(+) transients that initiate contraction. Spontaneous Ca²(+) sparks are thought to play a major role in SR Ca²(+) leak. In this study, we determined the quantitative contribution of sparks to SR Ca²(+) leak and tested the hypothesis that non-spark mediated Ca²(+) release also contributes to SR Ca²(+) leak. We simultaneously measured spark properties and intra-SR free Ca²(+) ([Ca²(+)](SR)) after complete inhibition of SERCA with thapsigargin in permeabilized rabbit ventricular myocytes. When [Ca²(+)](SR) declined to 279 ± 10 μm, spark activity ceased completely; however SR Ca²(+) leak continued, albeit at a slower rate. Analysis of sparks and [Ca²(+)](SR) revealed, that SR Ca²(+) leak increased as a function of [Ca²(+)](SR), with a particularly steep increase at higher [Ca²(+)](SR) ( >600 μm) where sparks become a major pathway of SR Ca²(+) leak. At low [Ca²(+)](SR) (< 300 μm), however, Ca²(+) leak occurred mostly as non-spark-mediated leak. Sensitization of ryanodine receptors (RyRs) with low doses of caffeine increased spark frequency and SR Ca²(+) leak. Complete inhibition of RyR abolished sparks and significantly decreased SR Ca²(+) leak, but did not prevent it entirely, suggesting the existence of RyR-independent Ca²(+) leak. Finally, we found that RyR-mediated Ca²(+) leak was enhanced in myocytes from failing rabbit hearts. These results show that RyRs are the main, but not sole contributor to SR Ca²(+) leak. RyR-mediated leak occurs in part as Ca²(+) sparks, but there is clearly RyR-mediated but Ca²(+) sparks independent leak.

Reduced Ca2+ Spark Activity after Subarachnoid Hemorrhage Disables BK Channel Control of Cerebral Artery Tone

Intracellular Ca(2+) release events ('Ca(2+) sparks') and transient activation of large-conductance Ca(2+)-activated potassium (BK) channels represent an important vasodilator pathway in the cerebral vasculature. Considering the frequent occurrence of cerebral artery constriction after subarachnoid hemorrhage (SAH), our objective was to determine whether Ca(2+) spark and BK channel activity were reduced in cerebral artery myocytes from SAH model rabbits. Using laser scanning confocal microscopy, we observed ∼50% reduction in Ca(2+) spark activity, reflecting a decrease in the number of functional Ca(2+) spark discharge sites. Patch-clamp electrophysiology showed a similar reduction in Ca(2+) spark-induced transient BK currents, without change in BK channel density or single-channel properties. Consistent with a reduction in active Ca(2+) spark sites, quantitative real-time PCR and western blotting revealed decreased expression of ryanodine receptor type 2 (RyR-2) and increased expression of the RyR-2-stabilizing protein, FKBP12.6, in the cerebral arteries from SAH animals. Furthermore, inhibitors of Ca(2+) sparks (ryanodine) or BK channels (paxilline) constricted arteries from control, but not from SAH animals. This study shows that SAH-induced decreased subcellular Ca(2+) signaling events disable BK channel activity, leading to cerebral artery constriction. This phenomenon may contribute to decreased cerebral blood flow and poor outcome after aneurysmal SAH.

Excitation–contraction coupling in human heart failure examined by action potential clamp in rat cardiac myocytes

The effect of the loss of the notch in the human action potential (AP) during heart failure was examined by voltage clamping rat ventricular myocytes with human APs and recording intracellular Ca 2+ with fluorescent dyes. Loss of the notch resulted in about a 50% reduction in the initial phase of the Ca 2+ transient due to reduced ability of the l-type Ca 2+ channel to trigger release. The failing human AP increased non-uniformity of cytosolic Ca 2+, with some cellular regions failing to elicit Ca 2+-induced Ca 2+ release from the sarcoplasmic reticulum. In addition, there was an increase in the occurrence of late Ca 2+ sparks. Monte-Carlo simulations of spark activation by l-type Ca 2+ current supported the idea that the decreased synchrony of Ca 2+ spark production associated with the loss of the notch could be explained by reduced Ca 2+ influx from open Ca 2+ channels. We conclude that the notch of the AP is critical for efficient and synchronous EC coupling and that the loss of the notch will reduce the SR Ca 2+ release in heart failure, without changes in (for example) SR Ca 2+-ATPase uptake.

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.

RyR function in sheep pulmonary arteries is differentially influenced by postnatal maturation and chronic hypoxia

Ryanodine receptors (RYR) and associated Ca2+ spark events are important to pulmonary arterial (PA) tone with potential differences in their role in fetuses as compared to adults. RyRs are implicated in hypoxic pulmonary vasoconstriction (HPV) and ET‐1 dependent contractility. Evidence also indicates HPV and PA tone are dysregulated by chronic hypoxia (CH) in fetuses and adults, and yet their combined influence on Ca2+ sparks and the relationship to PA tone is not known. We therefore tested the hypotheses that Ca2+ sparks are restricted before birth and that CH augments their function. These hypotheses were tested on PA from near term fetal sheep or adult ewes at low altitude or following 100+ days at 3801 m (CH). Ca2+ sparks were measured in fluo‐4 loaded PA strips while myography was performed on isolated PA rings. Ca2+ spark activity was far greater in adults relative to fetuses while CH accentuated spark activity in both groups. Selective RyR1 inhibition with 10 μM dantrolene (DAN) reduced spark activity in normoxic and CH fetuses and in normoxic but not CH adults, where 10 μM ryanodine (RY) restricted spark activity. Interestingly, DAN but not RY moderately reduced serotonin (5‐HT) mediated PA contraction in CH fetuses and adults. Overall, RyR function appears developmentally regulated and augmented by CH, and RyR function has modest influence on 5‐HT dependent contractility. NIH P01HD031226, R01HD003807 (LDL)