Ca(2+) release from the sarcoplasmic reticulum activated by the low affinity Ca(2+) chelator TPEN in ventricular myocytes

The Ca(2+) content of the sarcoplasmic reticulum (SR) of cardiac myocytes is thought to play a role in the regulation and termination of SR Ca(2+) release through the ryanodine receptors (RyRs). Experimentally altering the amount of Ca(2+) within the SR with the membrane-permeant low affinity Ca(2+) chelator TPEN could improve our understanding of the mechanism(s) by which SR Ca(2+) content and SR Ca(2+) depletion can influence Ca(2+) release sensitivity and termination. We applied laser-scanning confocal microscopy to examine SR Ca(2+) release in freshly isolated ventricular myocytes loaded with fluo-3, while simultaneously recording membrane currents using the whole-cell patch-clamp technique. Following application of TPEN, local spontaneous Ca(2+) releases increased in frequency and developed into cell-wide Ca(2+) waves. SR Ca(2+) load after TPEN application was found to be reduced to about 60% of control. Isolated cardiac RyRs reconstituted into lipid bilayers exhibited a two-fold increase of their open probability. At the low concentration used (20-40muM), TPEN did not significantly inhibit the SR-Ca(2+)-ATPase in SR vesicles. These results indicate that TPEN, traditionally used as a low affinity Ca(2+) chelator in intracellular Ca(2+) stores, may also act directly on the RyRs inducing an increase in their open probability. This in turn results in an increased Ca(2+) leak from the SR leading to its Ca(2+) depletion. Lowering of SR Ca(2+) content may be a mechanism underlying the recently reported cardioprotective and antiarrhythmic features of TPEN.

"Eventless" InsP 3 -dependent SR-Ca 2+ Release Affecting Atrial Ca 2+ Sparks

Augmented inositol 1,4,5-trisphosphate receptor (InsP3R) function has been linked to a variety of cardiac pathologies, including cardiac arrhythmia. The contribution of inositol 1,4,5-trisphosphate-induced Ca2+ release (IP3ICR) in excitation-contraction coupling (ECC) under physiological conditions, as well as under cellular remodelling, remains controversial. Here we test the hypothesis that local IP3ICR directly affects ryanodine receptor (RyR) function and subsequent Ca2+-induced Ca2+ release in atrial myocytes. IP3ICR was evoked by UV-flash photolysis of caged InsP3 under whole-cell configuration of the voltage-clamp technique in atrial myocytes isolated from C57/BL6 mice. Photolytic release of InsP3 was accompanied by a significant increase in the Ca2+ release event frequency (4.14±0.72 vs. 6.20±0.76 events (100 μm)−1 s−1). These individual photolytically triggered Ca2+ release events were identified as Ca2+ sparks, which originated from RyR openings. This was verified by Ca2+ spark analysis and pharmacological separation between RyR and InsP3R-dependent sarcoplasmic reticulum (SR)-Ca2+ release (2-aminoethoxydiphenyl borate, xestospongin C, tetracaine). Significant SR-Ca2+ flux but eventless SR-Ca2+ release through InsP3R were characterized using SR-Ca2+ leak/SR-Ca2+ load measurements. These results strongly support the idea that IP3ICR can effectively modulate RyR openings and Ca2+ spark probability. We conclude that eventless and highly efficient InsP3-dependent SR-Ca2+ flux is the main mechanism of functional cross-talk between InsP3Rs and RyRs, which may be an important factor in the modulation of ECC sensitivity.

Agonist specific L-type Ca(2+)-current stimulation in ventricular myocytes by a novel steroid-like compound

In cardiac muscle the amplitude of Ca(2+) transients can be increased by enhancing Ca(2+) influx. Among the processes leading to increased Ca(2+) influx, agonists of the L-type Ca(2+)-channel can play an important role. Known pharmacological Ca(2+)-channel agonists act on different binding sites on the channel protein, which may lead not only to enhanced peak currents, but also to distinct changes in other biophysical characteristics of the current. In this study, membrane currents were recorded with the patch-clamp technique in the whole-cell configuration in guinea pig isolated ventricular myocytes in combination with confocal fluorescence Ca(2+) imaging techniques and a variety of pharmacological tools. Testing a new positive inotropic steroid-like compound, we found that it increased the L-type Ca(2+)-current by 2.5-fold by shifting the voltage-dependence of activation by 20.2 mV towards negative potentials. The dose-response relationship revealed two vastly different affinities (EC(50(high-affinity))=4.5+/-1.7 nM, EC(50(low-affinity))=8.0+/-1.1 microM) exhibiting differential pharmacological interactions with three classes of Ca(2+)-current antagonists, suggesting more than one binding site on the channel protein. Therefore, we identified and characterized a novel positive inotropic compound (F90927) as a member of a new class of Ca(2+)-channel agonists exhibiting unique features, which set it apart from other presently known L-type Ca(2+)-channel agonists.

Intracellular Ca(2+) operates a switch between repair and lysis of streptolysin O-perforated cells

Pore-forming (poly)peptides originating from invading pathogens cause plasma membrane damage in target cells, with consequences as diverse as proliferation or cell death. However, the factors that define the outcome remain unknown. We show that in cells maintaining an intracellular Ca(2+) concentration [Ca(2+)](i) below a critical threshold of 10 microM, repair mechanisms seal off 'hot spots' of Ca(2+) entry and shed them in the form of microparticles, leading to [Ca(2+)](i) reduction and cell recovery. Cells that are capable of preventing an elevation of [Ca(2+)](i) above the critical concentration, yet are unable to complete plasma membrane repair, enter a prolonged phase of [Ca(2+)](i) oscillations, accompanied by a continuous shedding of microparticles. When [Ca(2+)](i) exceeds the critical concentration, an irreversible formation of ceramide platforms within the plasma membrane and their internalisation drives the dying cells beyond the 'point of no return'. These findings show that the extent of [Ca(2+)](i) elevation determines the fate of targeted cells and establishes how different Ca(2+)-dependent mechanisms facilitate either cell survival or death.

[(99m)Tc]Demomedin C, a radioligand based on human gastrin releasing peptide(18-27): synthesis and preclinical evaluation in gastrin releasing peptide receptor-expressing models

The synthesis and preclinical evaluation of [(99m)Tc]Demomedin C in GRPR-expressing models are reported. Demomedin C resulted by coupling a Boc-protected N(4)-chelator to neuromedin C (human GRP(18-27)), which, after (99m)Tc-labeling, afforded [(99m)Tc]Demomedin C. Demomedin C showed high affinity and selectivity for the GRPR during receptor autoradiography on human cancer samples (IC(50) in nM: GRPR, 1.4 ± 0.2; NMBR, 106 ± 18; and BB(3)R, >1000). It triggered GRPR internalization in HEK-GRPR cells and Ca(2+) release in PC-3 cells (EC(50) = 1.3 nM). [(99m)Tc]Demomedin C rapidly and specifically internalized at 37 °C in PC-3 cells and was stable in mouse plasma. [(99m)Tc]Demomedin C efficiently and specifically localized in human PC-3 implants in mice (9.84 ± 0.81%ID/g at 1 h pi; 6.36 ± 0.85%ID/g at 4 h pi, and 0.41 ± 0.07%ID/g at 4 h pi block). Thus, human GRP-based radioligands, such as [(99m)Tc]Demomedin C, can successfully target GRPR-expressing human tumors in vivo while displaying attractive biological features--e.g. higher GRPR-selectivity--vs their frog-homologues.

The nitrodibenzofuran chromophore: a new caging group for ultra-efficient photolysis in living cells

Photochemical uncaging of bio-active molecules was introduced in 1977, but since then, there has been no substantial improvement in the properties of generic caging chromophores. We have developed a new chromophore, nitrodibenzofuran (NDBF) for ultra-efficient uncaging of second messengers inside cells. Photolysis of a NDBF derivative of EGTA (caged calcium) is about 16-160 times more efficient than photolysis of the most widely used caged compounds (the quantum yield of photolysis is 0.7 and the extinction coefficient is 18,400 M(-1) cm(-1)). Ultraviolet (UV)-laser photolysis of NDBF-EGTA:Ca(2+) rapidly released Ca(2+) (rate of 20,000 s(-1)) and initiated contraction of skinned guinea pig cardiac muscle. NDBF-EGTA has a two-photon cross-section of approximately 0.6 GM and two-photon photolysis induced localized Ca(2+)-induced Ca(2+) release from the sarcoplasmic recticulum of intact cardiac myocytes. Thus, the NDBF chromophore has great promise as a generic and photochemically efficient protecting group for both one- and two-photon uncaging in living cells.

Tyrosine kinase modulation of protein kinase C activity regulates G protein-linked Ca2+ signaling in leukemic hematopoietic cells

We have used a recombinant mouse pre-B cell line (TonB210.1, expressing Bcr/Abl under the control of an inducible promoter) and several human leukemia cell lines to study the effect of high tyrosine kinase activity on G protein-coupled receptor (GPCR) agonist-stimulated cellular Ca(2+) release and store-operated Ca(2+) entry (SOCE). After induction of Bcr/Abl expression, GPCR-linked SOCE increased. The effect was reverted in the presence of the specific Abl inhibitor imatinib (1microM) and the Src inhibitor PP2 (10microM). In leukemic cell lines constitutively expressing high tyrosine kinase activity, Ca(2+) transients were reduced by imatinib and/or PP2. Ca(2+) transients were enhanced by specific inhibitors of PKC subtypes and this effect was amplified by tyrosine kinase inhibition in Bcr/Abl expressing TonB210.1 and K562 cells. Under all conditions Ca(2+) transients were essentially blocked by the PKC activator PMA. In Bcr/Abl expressing (but not in native) TonB210.1 cells, tyrosine kinase inhibitors enhanced PKCalpha catalytic activity and PKCalpha co-immunoprecipitated with Bcr/Abl. Unlike native TonB210.1 cells, Bcr/Abl expressing cells showed a high rate of cell death if Ca(2+) influx was reduced by complexing extracellular Ca(2+) with BAPTA. Our data suggest that tonic inhibition of PKC represents a mechanism by which high tyrosine kinase activity can enhance cellular Ca(2+) transients and thus exert profound effects on the proliferation, apoptosis and chemotaxis of leukemic cells.

Calcium waves driven by "sensitization" wave-fronts

OBJECTIVE: Cellular Ca(2+) waves are understood as reaction-diffusion systems sustained by Ca(2+)-induced Ca(2+) release (CICR) from Ca(2+) stores. Given the recently discovered sensitization of Ca(2+) release channels (ryanodine receptors; RyRs) of the sarcoplasmic reticulum (SR) by luminal SR Ca(2+), waves could also be driven by RyR sensitization, mediated by SR overloading via Ca(2+) pump (SERCA), acting in tandem with CICR. METHODS: Confocal imaging of the Ca(2+) indicator fluo-3 was combined with UV-flash photolysis of caged compounds and the whole-cell configuration of the patch clamp technique to carry out these experiments in isolated guinea pig ventricular cardiomyocytes. RESULTS: Upon sudden slowing of the SERCA in cardiomyocytes with a photoreleased inhibitor, waves indeed decelerated immediately. No secondary changes of Ca(2+) signaling or SR Ca(2+) content due to SERCA inhibition were observed in the short time-frame of these experiments. CONCLUSIONS: Our findings are consistent with Ca(2+) loading resulting in a zone of RyR 'sensitization' traveling within the SR, but inconsistent with CICR as the predominant mechanism driving the Ca(2+) waves. This alternative mode of RyR activation is essential to fully conceptualize cardiac arrhythmias triggered by spontaneous Ca(2+) release.

NO-dependent CaMKII activation during -adrenergic stimulation of cardiac muscle

AIMS:During β-adrenergic receptor (β-AR) stimulation, phosphorylation of cardiomyocyte ryanodine receptors by protein kinases may contribute to an increased diastolic Ca(2+) spark frequency. Regardless of prompt activation of protein kinase A during β-AR stimulation, this appears to rely more on activation of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII), by a not yet identified signalling pathway. The goal of the present study was to identify and characterize the mechanisms which lead to CaMKII activation and elevated Ca(2+) spark frequencies during β-AR stimulation in single cardiomyocytes in diastolic conditions. METHODS AND RESULTS:Confocal imaging revealed that β-AR stimulation increases endogenous NO production in cardiomyocytes, resulting in NO-dependent activation of CaMKII and a subsequent increase in diastolic Ca(2+) spark frequency. These changes of spark frequency could be mimicked by exposure to the NO donor GSNO and were sensitive to the CaMKII inhibitors KN-93 and AIP. In vitro, CaMKII became nitrosated and its activity remained increased independent of Ca(2+) in the presence of GSNO, as assessed with biochemical assays. CONCLUSIONS:β-AR stimulation of cardiomyocytes may activate CaMKII by a novel direct pathway involving NO, without requiring Ca(2+) transients. This crosstalk between two established signalling pathways may contribute to arrhythmogenic diastolic Ca(2+) release and Ca(2+) waves during adrenergic stress, particularly in combination with cardiac diseases. In addition, NO-dependent activation of CaMKII is likely to have repercussions in many cellular signalling systems and cell types.

Posttranslational modifications of cardiac ryanodine receptors: Ca2+ signaling and EC-coupling

In cardiac muscle, a number of posttranslational protein modifications can alter the function of the Ca(2+) release channel of the sarcoplasmic reticulum (SR), also known as the ryanodine receptor (RyR). During every heartbeat RyRs are activated by the Ca(2+)-induced Ca(2+) release mechanism and contribute a large fraction of the Ca(2+) required for contraction. Some of the posttranslational modifications of the RyR are known to affect its gating and Ca(2+) sensitivity. Presently, research in a number of laboratories is focused on RyR phosphorylation, both by PKA and CaMKII, or on RyR modifications caused by reactive oxygen and nitrogen species (ROS/RNS). Both classes of posttranslational modifications are thought to play important roles in the physiological regulation of channel activity, but are also known to provoke abnormal alterations during various diseases. Only recently it was realized that several types of posttranslational modifications are tightly connected and form synergistic (or antagonistic) feed-back loops resulting in additive and potentially detrimental downstream effects. This review summarizes recent findings on such posttranslational modifications, attempts to bridge molecular with cellular findings, and opens a perspective for future work trying to understand the ramifications of crosstalk in these multiple signaling pathways. Clarifying these complex interactions will be important in the development of novel therapeutic approaches, since this may form the foundation for the implementation of multi-pronged treatment regimes in the future. This article is part of a Special Issue entitled: Cardiomyocyte Biology: Cardiac Pathways of Differentiation, Metabolism and Contraction.

Hierarchical accumulation of RyR post-translational modifications drives disease progression in dystrophic cardiomyopathy

AIMS:Duchenne muscular dystrophy (DMD) is a muscle disease with serious cardiac complications. Changes in Ca(2+) homeostasis and oxidative stress were recently associated with cardiac deterioration, but the cellular pathophysiological mechanisms remain elusive. We investigated whether the activity of ryanodine receptor (RyR) Ca(2+) release channels is affected, whether changes in function are cause or consequence and which post-translational modifications drive disease progression. METHODS AND RESULTS:Electrophysiological, imaging, and biochemical techniques were used to study RyRs in cardiomyocytes from mdx mice, an animal model of DMD. Young mdx mice show no changes in cardiac performance, but do so after ∼8 months. Nevertheless, myocytes from mdx pups exhibited exaggerated Ca(2+) responses to mechanical stress and 'hypersensitive' excitation-contraction coupling, hallmarks of increased RyR Ca(2+) sensitivity. Both were normalized by antioxidants, inhibitors of NAD(P)H oxidase and CaMKII, but not by NO synthases and PKA antagonists. Sarcoplasmic reticulum Ca(2+) load and leak were unchanged in young mdx mice. However, by the age of 4-5 months and in senescence, leak was increased and load was reduced, indicating disease progression. By this age, all pharmacological interventions listed above normalized Ca(2+) signals and corrected changes in ECC, Ca(2+) load, and leak. CONCLUSION:Our findings suggest that increased RyR Ca(2+) sensitivity precedes and presumably drives the progression of dystrophic cardiomyopathy, with oxidative stress initiating its development. RyR oxidation followed by phosphorylation, first by CaMKII and later by PKA, synergistically contributes to cardiac deterioration.

Inhibition of FcepsilonRI-dependent mediator release and calcium flux from human mast cells by sialic acid-binding immunoglobulin-like lectin 8 engagement

BACKGROUND: Sialic acid-binding immunoglobulin-like lectins (Siglecs) are a family of glycan-binding inhibitory receptors, and among them, Siglec-8 is selectively expressed on human eosinophils, basophils, and mast cells. On eosinophils, Siglec-8 engagement induces apoptosis, but its function on mast cells is unknown. OBJECTIVE: We sought to study the effect of Siglec-8 engagement on human mast cell survival and mediator release responses. METHODS: Human mast cells were generated from CD34+ precursors. Apoptosis was studied by using flow cytometry. Mast cell mediator release or human lung airway smooth muscle contraction was initiated by FcepsilonRI cross-linking with or without preincubation with Siglec-8 or control antibodies, and release of mediators was analyzed along with Ca++ flux. RBL-2H3 cells transfected with normal and mutated forms of Siglec-8 were used to study how Siglec-8 engagement alters mediator release. RESULTS: Siglec-8 engagement failed to induce human mast cell apoptosis. However, preincubation with Siglec-8 mAbs significantly (P < .05) inhibited FcepsilonRI-dependent histamine and prostaglandin D(2) release, Ca++ flux, and anti-IgE-evoked contractions of human bronchial rings. In contrast, release of IL-8 was not inhibited. Siglec-8 ligation was also shown to inhibit beta-hexosaminidase release and Ca++ flux triggered through FcepsilonRI in RBL-2H3 cells transfected with full-length human Siglec-8 but not in cells transfected with Siglec-8 containing a tyrosine to phenylalanine point mutation in the membrane-proximal immunoreceptor tyrosine-based inhibitory motif domain. CONCLUSION: These data represent the first reported inhibitory effects of Siglec engagement on human mast cells.

Short-term response of the Ca cycle of a montane forest in Ecuador to low experimental CaCl 2 additions

The tropical montane forests of the E Andean cordillera in Ecuador receive episodic Sahara-dust inputs particularly increasing Ca deposition. We added CaCl2 to isolate the effect of Ca deposition by Sahara dust to tropical montane forest from the simultaneously occurring pH effect. We examined components of the Ca cycle at four control plots and four plots with added Ca (2 × 5 kg ha–1 Ca annually as CaCl2) in a random arrangement. Between August 2007 and December 2009 (four applications of Ca), we determined Ca concentrations and fluxes in litter leachate, mineral soil solution (0.15 and 0.30 m depths), throughfall, and fine litterfall and Al concentrations and speciation in soil solutions. After 1 y of Ca addition, we assessed fine-root biomass, leaf area, and tree growth. Only < 3% of the applied Ca leached below the acid organic layer (pH 3.5–4.8). The added CaCl2 did not change electrical conductivity in the root zone after 2 y. In the second year of fertilization, Ca retention in the canopy of the Ca treatment tended to decrease relative to the control. After 2 y, 21% of the applied Ca was recycled to soil with throughfall and litterfall. One year after the first Ca addition, fine-root biomass had decreased significantly. Decreasing fine-root biomass might be attributed to a direct or an indirect beneficial effect of Ca on the soil decomposer community. Because of almost complete association of Al with dissolved organic matter and high free Ca2+ : Al3+ activity ratios in solution of all plots, Al toxicity was unlikely. We conclude that the added Ca was retained in the system and had beneficial effects on some plants.