Salvia miltiorrhiza Bge) widely used in treatment of cardiovascular disorders. We investigated the effect of danshen or tanshinone IIA on blood pressure and its possible mechanisms. An i.p. injection of danshen at 10 mg kg−1 significantly lowered systolic blood pressure (SBP) of spontaneously hypertensive rats (SHRs) but failed to modify the SBP in normotensive Wistar-Kyoto rats (WKY). Oral administration of tanshinone IIA also decreased SBP in SHR but not in WKY. Tanshinone IIA produced a concentration-dependent relaxation in isolated SHR aortic rings precontracted with phenylephrine (10 nmol l−1) or potassium chloride (KCl) (40 mmol l−1). The relaxing effect of tanshinone IIA on tonic contraction of phenylephrine in isolated aortic rings without endothelium remained produced. Glibenclamide at concentration sufficient to block adenosine triphosphatase (ATP)-sensitive potassium (K+) channel attenuated this tanshinone IIA-induced relaxation that was not influenced by other inhibitors. We further investigated the effect of tanshinone IIA on the changes of intracellular calcium concentration ([Ca2+]i) in cultured aortic smooth muscle (A7r5) cells using fura-2 as indicator. Tanshinone IIA decreased [Ca2+]i elicited by phenylephrine (10 nmol l−1) or KCl (40 mmol l−1) in a concentration-dependent manner; glibenclamide, but not other inhibitors for K+ channel, abated this effect. Our results suggest that tanshinone IIA acts as an active principle of danshen showing vasodilation through ATP-sensitive K+ channel to lower [Ca2+]i.Tanshinone IIA is one of the active principles in danshen ( Salvia miltiorrhiza Bge (Labiatae), is one of the popular herbs used in China and the neighboring countries. This herb is widely applied in traditional Chinese medicine for promotion of blood flow to overcome blood stasis and to resolve abscesses i) to produce vasodilatation i was measured.The A7r5 line of rat aortic smooth muscle cells obtained from the Food Industry Institute  were incubated in DMEM containing 10% . Using external calibration, we then calculated [Ca2+]i according to the equation [Ca2+]i = [(R − Rmin)/(Rmax − R)×(Sf2/Sb2)×Kd], whereR is the fluorescence intensity of the Ca2+-sensitive dye fura-2 at excitation wavelengths of 340 and 380 nm, Rmin is the minimum fluorescence ratio of about 0.768 and Rmax is the maximum fluorescence ratio of about 35.1. The coefficient Sf2 indicates the free dye measured at wavelength of 380 nm and Sb2 indicates Ca2+-bound dye at 380 nm. According to experimental data, Sf2/Sb2 for fura-2 is about 15.3. Kd is the effective dissociation constant of fura-2, which was about 135 nmol l−1.The [Ca2+]i in response to phenylephrine or KCl was evaluated by using normal physiologic salt solution containing Ca2+. Pretreatment of tanshinone IIA was carried out to identify its antagonism of Ca2+. We administered the K+ channel blockers, then added tanshinone IIA to determine this inhibition of [Ca2+]i by tanshinone IIA that involved the opening of K+ channels.The change of i in A7r5 cells from 214.7 ± 34.2 to 440.2 ± 29.3 nmol l−1 (2+]i induced by phenylephrine (10 nmol l−1) in a concentration-dependent manner; the maximal inhibitory activity of tanshinone IIA was observed at 10 μmol l−1 (μmol l−1) reversed the inhibitory effect of tanshinone IIA (10 μmol l−1) on [Ca2+]i induced by phenylephrine (10 nmol l−1) (In Canmol l−1 . Tanshinμmol l−1 . Howevermol l−1) .−1) increased [Ca2+]i in A7r5 cells to 428.6 ± 27.4 nmol l−1 in Ca2+-containing medium (2+]i induced by KCl (40 mmol l−1) in a concentration-dependent manner (0.1–10 μmol l−1) parallel to its effects against the action of phenylephrine (10 nmol l−1)  , though mol l−1) .μmol l−1) nor charybdotoxin (0.1 μmol l−1) modify the inhibition of tanshinone IIA (10 μmol l−1)-induced changes in [Ca2+]i in A7r5 cells; the rise of [Ca2+]i in A7r5 cells by phenylephrine (10 nmol l−1) or KCl (40 mmol l−1) was not changed significantly (μmol l−1) or 4-aminopyridine (1 mmol l−1) did not influence the inhibitory effect of tanshinone IIA on [Ca2+]i in phenylephrine (10 nmol l−1)- or KCl (40 mmol l−1)-treated A7r5 cells  in vascular disease , 14. It  effects . We obse+ channels play an important role in the regulation of membrane potential in vascular smooth muscle [+ channels in vascular smooth muscle cell to elicit hyperpolarization and thereby a decline in [Ca2+]i may result in vasodilatation i related to the regulation of vascular tone in vascular smooth muscle i due to tanshinone IIA in phenylephrine- or KCl-pretreated A7r5 cells. However, apamin (SKCa channel blocker), charybdotoxin (LKCa channel blocker), barium chloride (KIR channel blocker) and 4-aminopyridine (KV channel blocker) were unable to interfere the ability of tanshinone IIA to relax tonic contraction of aortic rings isolated from SHR [2+]i induced by phenylephrine or KCl. Thus, the effect of tanshinone IIA on vasodilatation is not expected to be related to SKCa, LKCa, KIR or KV channels; selective opening of ATP-sensitive K+ channels can thus be considered for the action of tanshinone IIA regarding the reduction of [Ca2+]i to produce vasodilatation. Thus, it could be speculated that tanshinone IIA poses the ability to open ATP-sensitive K+ channels, which in turn leads to diffusion of K+ ions out of the vascular smooth muscle cells, then causes membrane hyperpolarization to close voltage-gated Ca2+ channels, thus resulting in decreased [Ca2+]i, and ultimately leads to vasodilatation , is similar to tanshinone IIA acting as the ATP-sensitive K+ channel opener i to induce vasodilatation.In conclusion, the opening of ATP-sensitive K


Excitation-contraction coupling involves the faithful conversion of electrical stimuli to mechanical shortening in striated muscle cells, enabled by the ubiquitous second messenger, calcium. Crucial to this process are ryanodine receptors (RyRs), the sentinels of massive intracellular calcium stores contained within the sarcoplasmic reticulum. In response to sarcolemmal depolarization, RyRs release calcium into the cytosol, facilitating mobilization of the myofilaments and enabling cell contraction. In order for the cells to relax, calcium must be rapidly resequestered or extruded from the cytosol. The sustainability of this cycle is crucially dependent upon precise regulation of RyRs by numerous cytosolic metabolites and by proteins within the lumen of the sarcoplasmic reticulum and those directly associated with the receptors in a macromolecular complex. In addition to providing the majority of the calcium necessary for contraction of cardiac and skeletal muscle, RyRs act as molecular switchboards that integrate a multitude of cytosolic signals such as dynamic and steady calcium fluctuations, β-adrenergic stimulation (phosphorylation), nitrosylation and metabolic states, and transduce these signals to the channel pore to release appropriate amounts of calcium. Indeed, dysregulation of calcium release via RyRs is associated with life-threatening diseases in both skeletal and cardiac muscle. In this paper, we briefly review some of the most outstanding structural and functional attributes of RyRs and their mechanism of regulation. Further, we address pathogenic RyR dysfunction implicated in cardiovascular disease and skeletal myopathies. In cardiac and skeletal muscle, depolarization of the external membrane and its invaginations, the T-tubules, elicits swift and massive Ca2+ release from the sarcoplasmic reticulum (SR), which in turn causes 'flooding' of the contractile myofilaments with Ca2+ and induction of contraction. This exquisitely coordinated series of events, in which an electrical stimulus (depolarization) is converted into a mechanical contraction, is collectively termed 'excitation-contraction (EC) coupling', and it has as central players the voltage-dependent Ca2+ channels/dihydropyridine receptors (DHPRs) as the sarcolemmal voltage sensors, and the Ca2+ release channels/ryanodine receptors (RyRs) as the SR Ca2+ release conduits. The structural and functional communication between the voltage sensor and the RyR dictate the magnitude of Ca2+ release from the SR, and thus the force of contraction. In fact, genetic mutations in either of these two proteins, or alterations in the environment that promotes their functional coupling, are known to cause ventricular arrhythmias, hypercontractures and/or pathological remodeling of cellular structures. Excellent reviews on DHPRs have appeared recently i), whose elevation triggers a cascade of events that culminates in, for example, neurotransmitter release, hormone secretion, lymphocyte activation and egg fertilization. To gain the functional flexibility necessary to respond to different triggering signals, at least three isoforms of RyRs are expressed in mammals, each one encoded by a distinct gene , residing on separate chromosomes. RyR1 is expressed predominantly in fast- and slow-twitch skeletal muscle and at lower densities in cerebellar Purkinje cells, gastric smooth muscle and B lymphocytes, among others. RyR2 was originally purified from cardiac muscle (it is the major isoform expressed there), but is also robustly expressed in neurons, and in visceral and arterial smooth muscle. RyR3 is the least understood of the RyR isoforms and seems to play its most important role during development, although in mature cells RyR3 is found in diaphragm, epithelial cells, brain, and smooth muscle i) fluctuates. According to this model, as apoCaM binds Ca2+ ions, it loses affinity for the apoCaM site and translocates to the nearby Ca2+-CaM site. The CaM binding sites identified by these studies was corroborated by a Förster resonance energy transfer (FRET)-based approach, which identified a CaM target helix that spanned residues 3614 to 3643. However, [Ca2+]-dependent translocation of CaM between adjacent or overlapping sites could not be detected by FRET, indicating that any such movements must be subtle. The authors pointed out that such structural rearrangements could also be explained by changes in the interaction of CaM with the RyR target sequence or by rotation of CaM around its major axis  in the medium to maintain the channel in its open state. However, despite the fact that RyRs in situ normally encounter fast and transient changes in [Ca2+], most of the functional characterizations of single RyR have been obtained under the continuous presence of a critical [Ca2+], that is, under steady-state conditions. How similar is this characterization to that obtained under a transient application of [Ca2+]? Fabiato, in his classic studies delineating CICR [2+ release was strongly dependent on the velocity of the Ca2+ pulse ((d[Ca2+]/dt) applied to skinned cardiac fibers. In light of these data, it is surprising that few studies have addressed the importance of dynamic fluctuations of Ca2+ on RyR kinetics. Three such studies have revealed that a fast Ca2+ stimulus elicits only a transient increase in the Po of RyRs; however, if Ca2+ is applied rapidly but sustained for long periods, RyR Po will 'relax' to a new steady-state that follows a bell-shaped curve as a function of the magnitude of Ca2+ stimulus. Thus, Po-[Ca2+] curves of greater magnitude emerge from titration of RyR activity with fast, calibrated Ca2+ pulses than those obtained under similar but steady applications of Ca2+  that its physiological relevance is questionable i transient , and decreases activity at high [Ca2+]  . Neverthh [Ca2+] . In skelh [Ca2+] . The phyh [Ca2+] . In singh [Ca2+] . CaM binh [Ca2+] -105. Alth [Ca2+] ,107. A rh [Ca2+] .Oxidative modifications of thiol residues in free cysteines, such as S-nitrosylation, S-glutathionylation and disulfide oxidation, can modulate RyR1 and RyR2 -111. TheS-nitroso-N-acetyl penicillamine (SNAP), which targets several EC-coupling proteins, increases inotropy of cardiac myocytes at low concentrations, but decreases it at high concentrations ,114. It In this section, we describe the role of RyR dysfunction in the generation of genetic and acquired diseases afflicting skeletal and cardiac muscle.RYR1, the gene encoding the skeletal isoform of RyRs, are associated with malignant hyperthermia (MH), a pharmacogenetic disease triggered by inhalational anesthetics or depolarizing muscle relaxants [2+ 'leak' or excessive Ca2+ mobilization from the SR due to hyperactivity of RyR1  seems to be the underlying mechanism for this syndrome [2+] in human muscle has yet to be categorically demonstrated ryanodine binding of MH-susceptible pigs is wider than that of normal pigs, suggesting greater sensitivity of RyR1 to Ca2+ activation and increased resistance to Ca2+ inactivation [2+] for inactivation. All these findings point to greater Ca2+ mobilization from RyR1-controlled pathways during resting and contracting periods, with the resultant overflowing of cytosolic compartments with Ca2+, and the ignition of multiple signaling mechanisms. Heat seems to stem from the rampant hydrolysis of ATP by the SR Ca2+ pump and other active Ca2+ transporters trying to reestablish Ca2+ homeostasis. However, the pathway to MH-associated heat production may be much more complex, as shown by the finding that MH mutations cause not only Ca2+ dysregulation, but also an increased release of cytokines . In sufficiently large quantities, these cytokines can be pyrogenic [Porcine stress syndrome (PSS), which also results from point mutations of n R615C) , is a sihuman MH , and contivation . At the yrogenic .RYR1 [RYR1 mutation, as in MH, causes excessive Ca2+ leak. In the periphery of the fiber, Ca2+-extrusion mechanisms would be sufficient to remove the excess Ca2+, but the central areas would remain vulnerable to local Ca2+ gradients. Central mitochondria would, therefore, 'swallow' greater amounts of Ca2+ than they could handle, leading to swelling and death [Central core disease (CCD) is another autosomal-dominant myopathy resulting from mutations in RYR1 ,128. AltRYR1 . Type 1 RYR1 . The caund death .2+ leak in the SR triggers the release of reactive oxygen and nitrogen species (ROS/RNS), causing RYR1 S-nitrosylation and glutathionylation, modifications that further enhance SR Ca2+ leak and RyR heat sensitivity. The authors correlated the resulting cascade of deterioration with the formation of cores that progress through a series of histopathologically distinct stages as the mice age. They proposed that oxidative damage, Ca2+-dependent proteolysis, and extreme stretching of the myofilaments due to prolonged contracture in the presence of unsequestered Ca2+ may all contribute to the progression of the disease. Importantly, these mice are prone to fulminant MH-like responses to heat challenge and halothane exposure [An alternative model for core formation has been proposed, based on studies using a knock-in mouse heterozygously expressing a leaky-channel mutation in the RyR1 N-terminal region ,132-135.exposure ,135.2+ transients [2+ release, without change in either SR Ca2+ content or RyR1 sensitivity to activation by voltage or caffeine [An entirely different mechanism for the muscle weakness associated with CCD was revealed in recent studies using a mouse knock-in model expressing a heterozygous mutation in the RyR1 ion pore . Muscle ansients . Adult mansients . These rcaffeine ; this pacaffeine . Unlike RYR1 mutations cause MH in some human patients and CCD in others. The mechanism(s) that leads to the different phenotypes remains a mystery. Very interesting, however, is the observation that patients with MH who lack clinical myopathies are more likely to have mutations at the N-terminus of RYR1, whereas those with clinical myopathies are likely to have mutations at the C-terminus [2+ leakage, as the studies discussed above have shown that both C-terminal and N-terminal mutations are capable of producing this type of RyR1 dysfunction.Surprisingly, the same mutation produced dramatically different phenotypes in different strains of mice, experimental approaches, and expression systems -138. Simterminus . AlthougRYR2, the gene encoding the cardiac isoform of RyRs, have been associated with CPVT [2+ release from the SR, especially during diastole, seems to be the underlying mechanism that gives rise to the VT [2+ leak may overload the Na+/Ca2+ exchanger, which generates an inward current as it extrudes the released Ca2+. The inward current, in turn, gradually depolarizes the cell to threshold, favoring delayed after depolarizations (DADs) [2+-dependent inactivation of ICa due to previous depletion of the SR leads to higher Ca2+ entry and reloading of the SR, which triggers another DAD in the next beat. Successive repetition of this altered Ca2+ cycle could result in paroxysmal tachycardia and arrhythmias even if only a few foci of ventricular cells are involved.CPVT is an autosomal-dominant inherited cardiac disease characterized by exercise- or stress-induced tachyarrhythmia episodes in the absence of apparent structural heart disease or prolonged QT interval ,140. Theith CPVT , which iith CPVT . CPVT uso the VT ,144. Enhs (DADs) . During 1-adrenergic receptor activation to cellular effects, dissociates FKBP12.6 [RYR2 occur in domains corresponding exactly to mutation-containing domains that give rise to MH [2+ load and release seem to be crucial, because mutations in CSQ2, the gene encoding cardiac calsequestrin, an intraluminal Ca2+ buffering protein that regulates RyR2 activity, also generate CPVT [2+ load for spontaneous Ca2+ release, and that CPVT-associated mutations decrease the sensitivity to luminal Ca2+  [2+ load increases as a result of enhanced Ca2+ entry and uptake, CPVT mutant channels reach their threshold and generate spontaneous Ca2+-release waves, creating a substrate favorable for Ca2+-dependent arrhythmias. Alternatively, CPVT-related mutations could increase the SR load, making it easier to reach the threshold upon adrenergic stimulation [Although this hypothetical scheme logically relates RyR2 dysfunction with VT, it is unclear exactly what mechanism induces a group of apparently normal RyR2s to behave aberrantly and to generate sudden tachycardia. Because infusion of catecholamines also triggers CPVT, it is likely that activation of the β-adrenergic system plays an important role. In this regard, it has been suggested that phosphorylation of RyR2 by PKA, the kinase linking βFKBP12.6 ,91,146),FKBP12.6 ,148. Thuse to MH ,141. Altse to MH -151. Whaate CPVT . Accordireshold) ,153. Thumulation .2+ mishandling in HF leads to altered gene transcription, resulting in maladaptive structural transformations  that eventually hamper basic cardiac function [2+ leak seems to be central in the pathogenesis of some forms of HF [et al. [1-adrenergic receptors. PKA, in turn, phosphorylates RyR2-Ser2808 and dissociates FKBP12.6, increasing the probability of channel opening by inducing the appearance of long-lasting subconducting states. In this scheme, RyR2 hyperphosphorylation  causes  to RyR2 , several to RyR2 ,158,159. [et al.  reported [et al. . More re [et al. ,159. Fin [et al. ,162. Cle2+ necessary for contraction of cardiac and skeletal muscle, RyRs act as molecular switchboards that integrate a multitude of cytosolic signals such as dynamic and steady Ca2+ fluctuations, β-adrenergic stimulation (phosphorylation), nitrosylation and metabolic states, and transduce these signals to the channel pore to release appropriate amounts of Ca2+. Furthermore, because Ca2+ release is crucially modulated by luminal factors such as SR Ca2+ content and protein-protein interactions, RyRs play an additional role as integrative switch-valves that offset cytosolic-luminal Ca2+ imbalances. Thus, there is an ample margin to interfere with the activity of RyRs. In both experimental and natural conditions, such interference results in overt contractile dysfunction and gross morphological changes. MH, CCD and CPVT are among the most studied clinical presentations of RyR dysfunction, and the elucidation of the precise molecular mechanisms affected by this dysfunction is advancing with great strides.In addition to providing the majority of the CaThe authors declare that they have no competing interests.EM, RL and HV contributed equally to the writing of this review. All authors read and approved the manuscript.


Acute myeloid leukemia (AML) cells home to the endosteal region of the bone marrow. They interact with bone marrow stromal components including extracellular matrix proteins, glycosaminoglycans, and stromal cells, by which they derive proliferative and growth inhibitory signals. Furthermore, adhesion to marrow stroma confers chemotherapy drug resistance and thereby promotes leukemia survival. A subpopulation of the leukemic blasts, known as leukemia stem cells, that are capable of propagating the leukemia, remain sheltered in the bone marrow microenvironment, exhibit resistance to chemotherapy, and serve as the origin of relapse after a variable period of remission. Detachment of these cells from the bone marrow in combination with chemotherapy may improve the outcome of therapy for AML. CXCR4 is a chemokine receptor for stromal derived factor-1 (SDF-1) also known as CXCL12. CD44 is a hyaluronic acid receptor that is an E selectin ligand expressed by hematopoietic stem cells known as HCELL when properly glycosylated [Adhesive properties of leukemia cells are likely responsible for the complication of leukostasis in AML as well as leukemic meningitis, leukemia cutis, extramedullary leukemia, and formation of chloromas. Three receptors, VLA- (very late antigen-) 4, CXCR4, and CD44, play a critical role in normal stem cell homing and also appear to be paramount to the homing of AML cells to, or retention within, the bone marrow. VLA4 is the osylated , 4. The osylated . In contosylated . This fiosylated . Similarosylated . Furtherosylated . Thus, aNot only are leukemia cells dependent on the bone marrow stroma for survival, but also they are capable of distorting normal bone marrow niches in a manner that affects normal hematopoietic progenitor cells . The endβ1 integrins inhibits mitoxantrone- and etoposide-induced apoptosis [ Adhesion of acute myeloid leukemia cells confers resistance to several chemotherapy agents, including cytarabine, one of the most active agents in AML. This ability is known as environment-mediated drug resistance (EMDR) . The thepoptosis ; similarpoptosis . Adhesiopoptosis . Agents poptosis , antibodpoptosis , 16, solpoptosis , or a pepoptosis  all overpoptosis . The CXCpoptosis . Anotherpoptosis . In an ipoptosis . These lSeveral potential mechanisms have been proposed for the ability of integrin-mediated signaling to protect from chemotherapy toxicity that involve activation of survival pathways or inhibition of apoptosis. The specific pathways include activation of the PI3 K/Akt/bcl-2 pathway , an inteP < 0.001) [ Expression of each of the two receptors, VLA-4 or CXCR4, has been associated with prognosis in AML; the former is correlated with better survival, and the latter portends worse survival. Several studies showed that high-level expression of CXCR4 was associated with poor prognosis in AML. As described earlier, CXCR-4 was demonstrated to have a pivotal role in the homing, migration, and development of human AML in the NODscid murine mutant . Althoug< 0.001) . Further< 0.001) , and low< 0.001)  or highe< 0.001) . Presenc< 0.001) .  In contrast to CXCR4 expression, high-level VLA-4 expression has the opposite effect on prognosis in AML. Higher functional expression of VLA-4 was shown to correlate with longer survival for newly diagnosed adult AML . Further2 daily for three days. There is also an ongoing multicenter phase I trial of an anti-CXCR-4 antibody in combination with mitoxantrone, etoposide, and cytarabine for relapsed/refractory AML. As these inhibitors enter the clinic, we will ascertain their ability to mobilize AML out of the protected marrow microenvironment and determine if this approach improves outcome of patients with new diagnosis or relapsed/refractory AML.There are several ongoing clinical trials utilizing this novel concept of combining agents that mobilize leukemia with chemotherapy . For exa In summary, there may be several critical mechanisms for adhesion of AML within the bone marrow, and discovery of novel mechanisms and novel inhibitors targeting disruption of adhesion may provide a significant advance in the treatment of AML.


Previous functional analysis in pituitary melanotrophs described Rab3a as a positive regulator of Ca2+-dependent exocytosis. However, the precise role of the Rab3a isoform on the kinetics and intracellular [Ca2+] sensitivity of regulated exocytosis, which may affect the availability of two major peptide hormones, α-melanocyte stimulating hormone (α-MSH) and β-endorphin in plasma, remain elusive. We employed Rab3a knock-out mice (Rab3a KO) to explore the secretory phenotype in melanotrophs from fresh pituitary tissue slices. High resolution capacitance measurements showed that Rab3a KO melanotrophs possessed impaired Ca2+-triggered secretory activity as compared to wild-type cells. The hampered secretion was associated with the absence of cAMP-guanine exchange factor II/ Epac2-dependent secretory component. This component has been attributed to high Ca2+-sensitive release-ready vesicles as determined by slow photo-release of caged Ca2+. Radioimmunoassay revealed that α-MSH, but not β-endorphin, was elevated in the plasma of Rab3a KO mice, indicating increased constitutive exocytosis of α-MSH. Increased constitutive secretion of α-MSH from incubated tissue slices was associated with reduced α-MSH cellular content in Rab3a-deficient pituitary cells. Viral re-expression of the Rab3a protein in vitro rescued the secretory phenotype of melanotrophs from Rab3a KO mice. In conclusion, we suggest that Rab3a deficiency promotes constitutive secretion and underlies selective impairment of Ca2+-dependent release of α-MSH. Rab3a is a small GTPase of the Rab3 subfamily that acts during late stages of Ca Peptide hormones are packaged in specialized membrane compartments - secretory vesicles, which undergo a series of preparatory steps, including peptide expression, processing, maturation, storage and eventually Ca2+-dependent exocytosis. Dysregulation in vesicle biogenesis, docking, priming, and fusion may result in deficient or excessive peptide hormone release and the subsequent manifestation of neuro-/endocrine disorders.Release of transmitters and peptide hormones from neurons and neuroendocrine cells into the extracellular space is triggered by cytosolic Caper se and does not seem to be of crucial importance for nervous system function, since Rab3a-deficient mice are viable and do not show any apparent behavioral abnormalities [in vitro impairs the secretory output in the pituitary intermediate lobe cells – melanotrophs [2+]i sensitivity of the Ca2+-dependent secretory activity, and thus, the availability of the secreted pituitary hormones in plasma.Rab3 proteins are small regulatory GTPases expressed at high levels in most cell types that are specialized for secretion . Rab3 ismalities . In contnotrophs . These cnotrophs . Rab3a snotrophs . HoweverIn neurons and neuroendocrine cells, such as mouse pituitary cells, Rab3a has been shown to form complexes with cAMP-guanine exchange factor II (GEFII)/Epac2 and Rim2 . Yet, fuin vivo and in vitro, resulting in elevated plasma levels of α-MSH and reduced α-MSH cellular content in Rab3a KO pituitary gland, respectively. Overexpression of WT-Rab3a protein rescued both α-MSH expression and secretory phenotype in vitro. Our data suggest that α-MSH and β-endorphin release is differentially controlled by Rab3a, implying that release of vesicles with different content is regulated by different mechanisms. Here we have shown that ablation of Rab3a in mouse melanotrophs from pituitary tissue slices impairs the depolarisation-induced secretory activity due to the absence of GEFII/Epac2-dependent secretory component. The constitutive release of α-MSH was increased ad libitum. All animal procedures were performed in strict accordance with all national and European recommendations pertaining to work with experimental animals, and all efforts were made to minimize suffering of animals. The protocol was approved by the State of Lower Saxony, Germany, and the Veterinary Administration of the Republic of Slovenia (permit number: 34401-61-2009/2). Adult (age 8-20 weeks) Rab3a knock-out  and their wild-type  littermates (control) were used for experiments . PCR ana2-anaesthetized (2-min exposure) mice were sacrificed by cervical dislocation followed by decapitation. Pituitary glands were dissected carefully from the skull and pituitary slices were prepared for acute experiments or organotypic slice culture as described elsewhere i) measurements were performed as previously described i in WT cells  was significantly reduced in Rab3a KO cells  in respect to WT melanotrophs . Inclusion of cAMP in the pipette solution specifically increased the threshold (PKA-dependent) component in WT and Rab3a KO cells  compared to WT melanotrophs . As shown in The absence of α-MSH immunoreactivity in POMC-rich Rab3a KO melanotrophs could result from impaired vesicle maturation or peptide conversion, but might also suggest the possibility that these two processes were intact and that the stability of mature vesicles containing α-MSH was reduced. First, the degradation of vesicles may be increased. Alternatively a formation of a standing pool of docked vesicles could be prevented. In the latter scenario, the secretory vesicles fuse immediately after maturation following the constitutive-like route.P<0.05, P<0.05, 2+-dependent release of α-MSH from Rab3a KO slices (2.18±0.05 nM) compared to WT slices (2.41±0.04 nM), but this did not reach statistical significance  and KCl-triggered hormonal release. Plasma concentrations of α-MSH and β-endorphin in WT and Rab3a KO mice, the respective release of both hormones from incubated pituitary tissue slices as well as the cellular content of were determined using radioimmunoassay. Alpha-MSH plasma levels were significantly increased in Rab3a KO mice as compared to WT  was similar in Rab3a KO cells with an overexpressed WT-Rab3a mutant as compared with WT cells  had significantly decreased cumulative Cm change as compared to Rab3a KO cells overexpressing WT-Rab3a. Average slope of the linear component in Rab3aQ81L- and Rab3aT36N-infected Rab3a KO cells  was comparable to non-infected Rab3a KO cells  and a dominant-negative Rab3a mutant restricted to the GDP-bound form (Rab3aT36N) were used as controls. Rab3a KO melanotrophs infected with a WT-Rab3a mutant  as well e groups .2+-independent constitutive pathway by depleting the readily releasable pool of vesicles i was required to trigger a Cm change in Rab3a null melanotrophs compared to WT controls. Given that under physiological conditions [Ca2+]i probably does not exceed 1-2 µM [m change triggered by the slow photo-release of caged Ca2+ is physiologically relevant. We also showed that the changes in the secretory activity in Rab3A KO mice were primarily attributed to the pituitary cell and did not reflect increased hypothalamic GABAergic input, which might result in reduced excitability of the melanotrophs  manner [The increased constitutive exocytosis and reduced cellular content of α-MSH, together with the decreased initial Cing rate . We hypoted mice . Based o) manner . 2+-dependent exocytosis. In Rab3a KO mice, however, the genetic deletion of Rab3a may potentially lead to an up-regulation or abnormal subcellular localization of other Rab3 isoforms (including Rab3b), which may contribute to the increased constitutive secretion in Rab3a KO melanotrophs. However, our preliminary results on melanotrophs lacking other Rab3 isoform or combinations of Rab3 isoforms exclude this possibility (data not shown). Four Rab3 isoforms (Rab3a-d) are expressed in the mouse pituitary gland . PreviouAplysia neurons [2+ -triggered exocytosis in Rab3a KO cells. Our results are in accordance with a study by Lin et al. [2+-dependent exocytosis, whereas the mutant favoring the GDP-bound state inhibits regulated secretion [2+ delivery by means of slow dialysis vs. repetitive depolarizing stimulation), which were shown responsible for the activity-dependent modulation of release [Rab3 proteins cycle between GTP-bound (active) and GDP-bound (inactive) state to act as molecular switches, specifically associated only with the resting secretory vesicles ,31,32. I neurons , whereas neurons ,34,35. Wn et al.  , suggesecretion . This di2+-dependent secretion in both WT and Rab3a KO melanotrophs. However, a more precise analysis revealed that only the PKA-mediated exocytotic component was increased. In contrast, cAMP did not increase the amplitude or the rate of the first ATP-independent cAMP-GEFII/Epac2-dependent exocytotic phase in Rab3a KO melanotrophs. This suggests that the standing pool associated with a low Ca2+ threshold for release is dependent on the interaction of Rab3a with Rim2 and GEFII. This is consistent with a previous study, which demonstrated that cAMP-GEFII/Epac2 promotes fusion of vesicles with the plasma membrane by forming a complex with Rim2 and Rab3, both proteins implicated in the late stages of exocytosis [Previously we demonstrated that the depolarisation-induced secretory response comprises of an ATP-independent (linear) component, mediated through a PKA-independent (cAMP-GEFII/Epac2-dependent) pathway, and a subsequent ATP -dependent component mediated via PKA . Based oocytosis . 2+-affinity pool of docked granules. The absence of a functional Rab3a protein underlies loss of mature secretory granules containing α-MSH through constitutive exocytosis, resulting in increased levels of α-MSH in the plasma and depleted cellular content. The initial phases of Ca2+-dependent secretion likely involve tripartite Rab3a/rabphilin/Rim complex that operates through an ATP-independent but cAMP-GEFII/Epac2-dependent mechanism. Overexpression of GTP- or GDP-dominant Rab3a mutants rescued the standing pool of α-MSH vesicles, but they failed to support Ca2+-dependent exocytosis, suggesting the association of Rab3a with other protein complexes during vesicle priming and fusion. In summary, our data suggest that Rab3a is important for trapping the secretory vesicles into the high Ca


However, CR expression is also observed in non-neuronal cells, e.g., during embryonic development and in mesothelioma cells. Of the 6 EF-hand domains, 5 are functional; the first 4 domains form 2 pairs showing high cooperativity within a pair that results in non-linear modulation of intracellular Ca2+ signals by CR. EF-hand domain 5 has a low affinity and represents the identified interaction site with CR-binding partners present in mouse cerebellar granule cells. CR binding to other targets including the pore-forming α1 subunit of the Ca2+ channel CaV2.1, as well as to huntingtin indicates additional Ca2+ sensor functions besides the well-known Ca2+-buffering functions. The absence of CR in cerebellar granule cells of CR−/− mice results in increased excitability and altered firing of Purkinje cells and promotes cerebellar 160-Hz oscillations impairing motor coordination. The putative role of CR in neuroprotection is still highly discussed. Altogether, CR emerges as a multi-functional protein also associated with development, i.e., cell proliferation, differentiation, and cell death.The hexa-EF-hand Ca CALB2), calbindin D-28k  and secretagogin  represent the 3 members of the hexa-EF-hand protein family, also named the calbindin sub-family i, CR behaves like a typical slow-onset buffer (EGTA). However, if the same increase occurs at elevated [Ca2+]i, in the order of 1 μM, when the first site of a pair is in the Ca2+-bound form, cooperativity sets in and CR functions almost like the fast buffer BAPTA . Results in support of CR acting as a Ca2+ sensor are presented in Section III.Calretinin  selectively binds a nuclear protein present in cerebellar granule cells and contains an “AP2-like” element . This channel is regulated in a dual fashion by Ca2+ ions, showing both, Ca2+-dependent inactivation (CDI) and Ca2+-dependent facilitation (CDF); both mechanisms influence synaptic plasticity in the nervous system. In cells expressing CaV2.1 in vitro, co-expression of CR inhibits CDI and enhances CDF via a direct interaction with the α12.1 subunit. The channel subunit α12.1 co-immunoprecipitates with CR antibodies using either extracts from HEK293T cells transfected with CR and CaV2.1 or mouse cerebellar extracts. In mouse cerebellum, both CR and CaV2.1 are strongly expressed in granule cells and the absence of CR in CR−/− mice causes impairment in motor control . The diffusion of fluorescence-labeled CR molecules is much slower than the one of freely diffusible molecules (fluorescein dextrans) of comparable size  of CaV2.1 channels and CR in granule cells indicate that channel numbers are too low to account for the strong effect on CR's mobility implicating additional, yet unidentified CR-binding partners in these neurons. A binding partner interacting with CR was found to be huntingtin (Htt), identified by tandem affinity purification  don't affect Ca2+ signals and e.g., don't protect PC12 cells against Ca2+ overload induced by ionophore treatment or trophic factor deprivation  terminals onto Purkinje cells) result in considerable nanodomain Ca2+-buffering by CR. As a consequence, a minor increase in the amplitude of AP-evoked Ca2+ signals in CR−/− PF boutons results in a considerably higher release probability  in hippocampal granule cells  evokes Ca2+ signals that are differently modulated by endogenous or synthetic Ca2+ buffers  decrease the amplitude of Ca2+ signals and favor “globalization” of spatially uniform Ca2+ signals, in particular, at high [IP3]. Interestingly, puffs are observed in the presence of CR at low stimulation intensities, i.e., at low [IP3], an effect never occurring in the presence of CB or BAPTA. Thus, under conditions of small elevations in [Ca2+]i from resting Ca2+ levels, CR has properties of a slow Ca2+ buffer such as PV or EGTA.The particular Ca−/− mice  “causes an early loss in proliferative capacity of the subgranular zone that is maintained into adult age, when it has a further impact on the migration/survival of newborn granule cells” (Todkar et al., 2+-binding protein-specific functions that cannot be shared and/or substituted even by apparently very similar proteins such as CB and CR.The detailed analyses of temporal and spatial expression of CR in the brain is the major focus of this Frontiers series, has been summarized in several papers and reviews Arai et al. ; Jacobow−/− mice are summarized. The decreased LTP in the DG is thought to be the result of an increased excitatory drive from CR-depleted mossy cells onto hilar interneurons (Schurmans et al., 2+ homeostasis of Purkinje cells and the emergence of cerebellar 160-Hz oscillations (Cheron et al., −/− mice. I am also not aware that anybody has attempted to manipulate CR expression levels of cortical interneurons, e.g., by shRNA and to investigate the functional consequences of CR down-regulation.As was previously reported for CB Schmidt, , CR cert1 block and in mesothelioma-derived cells leads to apoptosis via strong activation of the intrinsic caspase 9-dependent pathway (Blum and Schwaller, In CR-expressing mesothelial cells, CR down-regulation causes a GThe author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Consequently, intracellular [Ca2+] levels need to be tightly controlled. In the light-sensitive outer segments (OS) of photoreceptors, Ca2+ regulates the activity of retinal guanylate cyclases thus playing a central role in phototransduction and light-adaptation by restoring light-induced decreases in cGMP. In the synaptic terminals, changes of intracellular Ca2+ trigger various aspects of neurotransmission. Photoreceptors employ tonically active ribbon synapses that encode light-induced, graded changes of membrane potential into modulation of continuous synaptic vesicle exocytosis. The active zones of ribbon synapses contain large electron-dense structures, synaptic ribbons, that are associated with large numbers of synaptic vesicles. Synaptic coding at ribbon synapses differs from synaptic coding at conventional (phasic) synapses. Recent studies revealed new insights how synaptic ribbons are involved in this process. This review focuses on the regulation of [Ca2+] in presynaptic photoreceptor terminals and on the function of a particular Ca2+-regulated protein, the neuronal calcium sensor protein GCAP2 (guanylate cyclase-activating protein-2) in the photoreceptor ribbon synapse. GCAP2, an EF-hand-containing protein plays multiple roles in the OS and in the photoreceptor synapse. In the OS, GCAP2 works as a Ca2+-sensor within a Ca2+-regulated feedback loop that adjusts cGMP levels. In the photoreceptor synapse, GCAP2 binds to RIBEYE, a component of synaptic ribbons, and mediates Ca2+-dependent plasticity at that site. Possible mechanisms are discussed.Changes in intracellular calcium ions [Ca These include synaptic vesicles exocytosis and the mediation of various forms of synaptic plasticity  protein GCAP2 (guanylate cyclase-activating protein-2). GCAP2, an EF-hand-containing, Ca2+-binding protein is strongly expressed in photoreceptors and plays multiple roles in the outer segments (OS) and in the photoreceptor synapse.Changes in presynaptic free cytosolic Caasticity . Ca2+ ioFigure 1A). (1) An outer process forms the outer segment (OS) that is responsible for phototransduction. During this process, light-sensitive photopigments in the OS transform light quanta into a change of the photoreceptor membrane potential. (2) An inner cell process emerges from the vitread portion of the photoreceptor soma. This inner process ends in a specialized presynaptic terminal that contains synaptic ribbons in their active zones . At these ribbon-type active zones, signals generated by the absorption of light quanta in the OS are continuously transmitted to the inner retina  and communicate the information to the inner retina for further processing. Rod photoreceptors operate at low light intensities and are capable of single-photon-detection. Cone photoreceptors operate at daylight conditions and mediate color vision . Both tyFigure 1; for review, see +- and Ca2+-ions can pass from extra- to intracellular space along their electrochemical gradient. The light-induced closure of the CNG-channels generates a hyperpolarization response. The membrane potential ranges from ≈-40 mV in the dark to ≈-70 mV in bright light. The phototransduction system has a high dynamic range that is adjusted – not exclusively, but to a considerable extent – by Ca2+-dependent processes  thus restoring cGMP baseline levels . At the plasma membrane of the active zone, L-type voltage-gated Ca2+-channels (VGCC) are clustered. Ca2+-influx through these channels triggers release at the photoreceptor synapse (see below). The active zone with the attached synaptic ribbon is opposed by the tips of multiple postsynaptic dendritic endings from horizontal and bipolar cells. These dendritic tips are located in a cavity formed by an invagination of the presynaptic photoreceptor terminal. The dendritic tips contain metabotropic glutamate receptors (in case of invaginating ON bipolar cells) or ionotropic glutamate receptors . Other protein–protein interactions of RIBEYE do not depend on NADH, e.g., RIBEYE–Munc119 interaction -domain and CtBP1/CtBP2 bind NAD(H) . In caseeraction  or have eraction . While Reraction . Bassooneraction , though eraction . Therefoeraction . Proteineraction . These peraction ,b, 2013 2+-triggered exocytosis . As mentioned above, exocytosis of synaptic vesicles preferentially occurs at the base of the synaptic ribbon (2+-sensitivity of the release machinery and/or an efficient coupling of the release machinery to the presynaptic L-type Ca2+-channels (see below). In contrast, release probability in conventional, phasic synapses is typically much lower than in ribbon synapses . Continuous exocytosis in photoreceptor ribbon synapses caused depletion of synaptic vesicles at the base of the synaptic ribbon at physiological membrane potentials . This deDespite the large physiological differences between ribbon synapses and conventional synapses, these synapses do not differ strongly in the composition of the exocytotic machinery  and little voltage-dependent inactivation , Veli3, β-dystroglycan, bassoon, and RIM2. As consequence, the architecture of the synaptic terminal was profoundly altered.2+-channels via RIM-BPs  of VGCC . This in2+ . From this end of the bar-shaped ribbon, spherical ribbons, the synaptic spheres, detach . Thus, this illumination-dependent growth and disassembly of the synaptic ribbon appears to employ similar intermediate structures as in development . These spherical intermediates are similar in appearance as the spherical synaptic ribbons of hair cells in the inner ear . Spherical ribbons were added to remaining ribbons during darkness  of GCAP2   , NADH dey NAD(H)  which wo2+ could thus lead to a severing and subsequent disassembly of the ribbon body into spherical structures, the synaptic spheres . The immunogold electron microscopic studies by Figure 4).The conformational change of GCAP2 induced by low Ca2+ signals generated by influx through VGCC are modulated by various intracellular mechanisms that can subsequently increase or decrease cytosolic Ca2+-concentration. The final Ca2+-signal in the presynaptic terminal is shaped by the temporal and spatial interactions of these mechanisms (Cachanisms .2+-source (2+-ATPase (SERCA)-2 protein (2+-induced Ca2+-release (CICR) from the ER can amplify Ca2+-signals and enhance signal transmission  and IP3 receptors (IP3R) have been localized to photoreceptor terminals  is a main intracellular Ca+-source  and loca protein . Ca2+-insmission . Severalsmission . Both clerminals . CICR caterminal . Activatsynapses .2+-stores are exhausted, store-operated calcium entry (SOCE) mechanisms can replenish them -channels in case of store-depletion. CRAC channels contain Orai-proteins as major components that allow Ca2+ influx through the plasma membrane and subsequent Ca2+ replenishment of the emptied ER store  extrude Ca2+ from the cytosol into the extracellular space under physiological conditions (2+ by pumping Ca2+ from the cytosol into the ER (2+-extruding systems are important to restore resting levels of Ca2+ after depolarization-induced increases (2+ (2+-uptake by mitochondria was found to be only functionally relevant if the Ca2+-load was particularly high (The PMCAs represent a family of transmembrane proteins  that extt manner . The PMCt manner . Proper t manner . The PMCignaling  and alsoignaling . Deletioignaling . The sub ribbons . Na+/Ca2nditions . NCX pronditions . PMCA apnditions . The SERo the ER . In photo the ER . The desncreases . Photorencreases . Mitochoases (2+ . In ribbrly high .2+-levels, synaptic signaling and the synaptic ribbons. Ca2+-flux through VGCC as well as the subsequent cross-talk to other Ca2+-regulating systems in the presynaptic terminal affect structural plasticity and developmental maturation of the synaptic ribbon in photoreceptor synapses. In the light, when Ca2+ is low in presynaptic photoreceptor terminals (2+ is disturbed and presynaptic Ca2+ increased, synaptic ribbons were longer than in control animals (2+. A recent publication (2+-mediated dynamics of synaptic ribbons – at least in development – is more complex. Also gain-of-function mutations of the Cav1.4 channel, which lead to an activation of the Cav1.4 channel already at more negative membrane potentials, displayed shorter than normal synaptic ribbons (As summarized above, there are multiple links between Cav-channels, presynaptic Caerminals , synaptierminals . Similar animals . On the lication  indicate ribbons . Future 2+ exerts its influence on synaptic ribbons? Does Ca2+-dependent phosphorylation of ribbon proteins play a role? Why are there so many links between presynaptic Ca2+-channels and synaptic ribbons? How can very few ribbon synapse-specific components build such a fundamentally different synapse? How are the synaptic ribbon components mechanistically linked to the proposed functions of synaptic ribbons? The application of innovative physiological tools and the analysis of mouse knockout models are needed to answer these questions.Biochemical and molecular analyses identified the protein composition of synaptic ribbons to a large extent. Surprisingly, most of the ribbon proteins are not only present in ribbon synapses but also in conventional synapses. RIBEYE is the only major building block unique to synaptic ribbons. Still, many questions concerning the structure, assembly, and function of synaptic ribbons remain to be elucidated. How is the assembly of synaptic ribbons regulated? How do immature ribbons develop into mature ribbons? How does CaThe author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


This crosstalk manifests as the orthograde Ca2+-induced Ca2+-release (CICR) mechanism triggered by Ca2+ influx, but also as the retrograde Ca2+-dependent inactivation (CDI) of LTCC, which depends on both Ca2+ permeating through the LTCC itself and on SR Ca2+ release through the RyR. This latter effect has been suggested to rely on local rather than global Ca2+ signaling, which might parallel the nanodomain control of CDI carried out through calmodulin (CaM). Analyzing the CICR in catecholaminergic polymorphic ventricular tachycardia (CPVT) mice as a model of RyR-generated Ca2+ leak, we evidence here that increased occurrence of the discrete local SR Ca2+ releases through the RyRs (Ca2+ sparks) causea depolarizing shift in activation and a hyperpolarizing shift inisochronic inactivation of cardiac LTCC current resulting in the reduction of window current. Both increasing fast [Ca2+]i buffer capacity or depleting SR Ca2+ store blunted these changes, which could be reproduced in WT cells by RyRCa2+ leak induced with Ryanodol and CaM inhibition.Our results unveiled a new paradigm for CaM-dependent effect on LTCC gating and further the nanodomain Ca2+ control of LTCC, emphasizing the importance of spatio-temporal relationships between Ca2+ signals and CaM function.Ca While such [Ca2+]i elevations are initiated by LTCC, they are also influenced by Ca2+ transporting organelles such as the mitochondria and the SR. Notably, in response to these increases in [Ca2+]i, Ca2+ binds to and activates RyRs thereby amplifying the initial Ca2+ signal through the locally controlled Ca2+-induced Ca2+-release (CICR) process to support the excitation-contraction coupling (ECC) and thus heart function 2+overload. A major intrinsic negative feedback mechanism is the Ca2+-dependent inactivation (CDI) of the widely distributed voltage-gated Ca2+ channels 2+ channel during double-pulse protocols. Early studies of CDI were mainly focused on Ca2+ entry, but SR Ca2+ release also contributes significantly to the CDI 2+ stores or abolition of SR Ca2+ release causes a reduction of CDI 2+ loading results in CDI enhancement 2+ release from the RyRs 2+ released, which might reduce Ca2+ influx during action potential up to 50% 2+ release dominates CDI initially, then, as [Ca2+]i decreases due to SR Ca2+ reuptake, the SR dependent contribution declines with participation from Ca2+ entry via CaI dominating 2+ release from the RyRs results in discrete and localized rises of [Ca2+]i (Ca2+ sparks) triggered by ICa2+during CICR modulate in turn LTCC 2+ cross-signaling occurs in the microdomains of LTCC-RyRs Dynamic modulation of cellular Ca2+channel as the specific resident Ca2+ sensor 2+ signal for regulation of Ca2+ channels 2+ channels 2+]i as a result of Ca2+ influx from the LTCC, while the N-lobe can operate as a tunable detector of global or local [Ca2+]i arising from distant Ca2+ sources 2+ signals in the immediate vicinity of the channel pore 2+ influx as source for modulation of the channel, formulated upon measurements from nonexcitable cells that lack CICR nanodomains. A pertinent question is, therefore, whether and how discrete and local SR Ca2+release participates to the Ca2+ control of LTCC?Over the past decades there has been rapid progress toward understanding the molecular basis for CDI, cumulating with the identification of constitutively complexed calmodulin (CaM) with the CaR4496C catecholaminergic polymorphic ventricular tachycardia (CPVT) mutant mice to their wild type littermates (WT) by simultaneous recordings of CaI and the evoked [Ca2+]i transients and Ca2+ sparks. CPVT is a severe inherited cardiac disorder that manifests as malignant exercise-emotion-triggered arrhythmias leading to syncope and sudden death. Mutations in the cardiac RyR account for an autosomal-dominant form in approximately 50% of CPVT cases. In knock-in transgenic mouse model (CPVT mice), the R4496C mutation of the RyR increased the Ca2+ sensitivity of RyR, leading to diastolic Ca2+ leak and arrhythmogenic triggered activity 2+ leak is increased in this CPVT mouse model we investigated several key steps in the process of ECC that might provide insights into local [Ca2+]i control of LTCC.We have chosen to further explore this question by comparing RyR2+ influx, or CICR-gain, reflects not only the operation of the fundamental processes that underlie normal ECC, but also those involved in important pathological conditions of the heart, such as triggered arrhythmias produced by uncontrolled SR Ca2+ release CaI traces  and line scan images of the evoked [Ca2+]i transients (using the fluorescence Ca2+ indicator fluo-3) recorded in freshly isolated ventricular myocytes from wild type  and CPVT (bottom) mouse hearts using simultaneous patch-clamp current recording and high resolution confocal Ca2+ imaging techniques 2+]i transient (F/F0) over the corresponding Ca2+ influx through the LTCC, calculated as the CIa integral, in response to voltage steps  The ability of the SR to amplify the trigger Cage steps  [22]. At2+ load, might reflect an increased efficiency of crosstalk between LTCCs and RyRs. 2+]i transient and CaI in WT and CPVT myocytes from experiments such as those shown in CaI and [Ca2+]i transients displayed bell-shaped, graded function with the membrane potential. Whereas no difference on [Ca2+]i transient was observed between WT and CPVT cells at any potential , which might rapidly and reversibly alter the Ca2+ concentration in the vicinity of the LTCCs 2+]i transient, as evaluated by synchronous confocal images, in pA/pF: −0.88±0.09 vs −0.82±0.13, in 10 WT vs 12 CPVT cells, respectively, P>0.05).These results indicated that the RyRR4496C mutation lowers CaI kinetics could account for the observed alteration of CaI. Over the whole voltage range, neither activation , or frequency-dependent facilitation, was not modified in CPVT cells (data not shown).A change in the time- and/or voltage-dependence of tivation  nor inactivation  kineticsCaI as function of the voltage might underlie the reduced peak current density at low voltages. The activation-voltage relationships were constructed by converting the peak current values from each current-voltage relationship data set to the chord conductance using the equation: G = I/(V-revE) and then the ratio maxG/G were plotted against the membrane potential (revE and maxG as determined by the current-voltage fits CaI in CPVT cells were significantly more positive than for WT cells (d ∞), curves through the data points were fitted by the Boltzmann function d ∞ (V ) = 1/{1+exp[(0.5V-V )/k]}, where 0.5V is the potential at which the conductance is half maximally activated and k is the slope factor describing the steepness of the curve. Whereas k was unchanged, a significant rightward shift in 0.5 Vwas observed in CPVT cells compared to WT  is proportional to the fraction of available channels at that given time. As shown in 2+-dependent processes  f∞(V) = (1-A)/{1+exp[(V-0.5V)/k]}+A; where 0.5V is the potential of half-maximal inactivation, k is the slope factor, A is the amplitude of the non-inactivating CaI. A significant leftward shift is observed for 0.5V in CPVT compared to WT cells  and the relative peak conductance using the classical Hodgkin and Huxley formulation: maxG.d∞.f∞.(V-revE). A marked reduction in maxG , no more differences in activation or inactivation parameters . These results lead us to probe the plausible Ca2+ dependence of the cross-signaling between CaI and RyR in the reduced 2+-buffering capacity of the cytoplasm through the introduction of exogenous buffer inside the patch pipette. We used the fast Ca2+chelator 1,2-bis . When compared to control conditions, increasing the cytoplasmic Ca2+-buffering capacity with BAPTA in CPVT cells completely blunted the shifts in d∞ activation and f∞ inactivation variables eliminating the difference with WT cells  to block the ability of the cell to pump Ca2+ into the SR and to deplete the store by consecutive depolarizations to 0 mV. Consequently, SR Ca2+ release was blunted. In the same way as for buffering [Ca2+]i, abolishing SR Ca2+ release restored d∞ and f∞ variables  has a central role in determining LTCC Ca2+ sensitivity N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide] , a water-soluble, cell membrane–permeant competitive antagonist of Ca2+/CaM in situ by confocal microscopy as spontaneous Ca2+ sparks frequencies  mice has revealed that in addition to the orthograde signal of ECC, there is also a retrograde signal whereby RyR promotes the Ca2+]i can participate to CDI, there is evidence suggesting that local Ca2+ signaling between RyRs and LTCCs might mediate a Ca2+ functional crosstalk between LTCCs and RyRs 2+ caused by the alteration of normal Ca2+ extrusion via NCX could be involved. But we observed no difference in NCX activity between WT and CPVT cells. Such an alteration in Ca2+ driving force, as well as in surface charges, will be difficult to reconcile with the absence of modification in peak CaI density, apparent reversal potential and opposite shifts of activation and inactivation voltage ranges. Long term changes are known to complicate the mechanistic attribution of cause and effect in genetically modified animals. One might thus suggest that the observed enhancement of inactivation of LTCCs could reflect a survival adaptation to increased [Ca2+]i, rather than a mechanistic insight into the functional coupling between LTCCs and RyRs. However, acute intervention buffering Ca2+  are closely interrelated. We reasoned on the possible crosstalk between the CDI and VDI gating of the LTCCs, such that a change in the stability of the open state is predicted to affect both VDI and CDI gating 2+ leak induced enhancement of inactivation might induce the rightward shift in the activation. In fact, conformational changes caused by Ca2+ binding to the resident CaM, the primary initiatory event for CDI CaI availability curve to more negative potential with calmidozolium, another CaM antagonist 2+ leak-dependent reduced 2+ buffer, an effect frequently overlooked 2+ buffer capacity of CaM normally prevents Ca2+ to access to other regulator sites, since free CaM is locally enriched in the vicinity of the channels 2+ leak in CPVT cells outdo this CaM effect, allowing Ca2+ feedback to other Ca2+ sensor domains involved in CDI Our results indicate that RyR Cas of ICa , similarvoltages . This em2+ regulatory mechanism acting as a powerful switch that determines LTCC gating by interfering with CaM modulation. This effect might serve as a compensatory response to counteract excessive SR Ca2+ release and SR store overload 2+ load decrease observed at high stimulation frequencies 2+ leakage on trigger activities for fatal acquired or genetic cardiac arrhythmias and sudden death. Elevation of diastolic [Ca2+]i through increased Ca2+ spark frequency is regarded as an arrhythmogenic mechanism: Ca2+ sparks are believed to participate as crucial events in the initiation and propagation of Ca2+ waves in cardiomyocytes and the elimination of cytosolic Ca2+ via the NCX generates a depolarizing current, which can give rise to delayed after depolarizations (DADs) 2+ release, triggered activity and promote reentrant arrhythmias such as Torsades de pointes 2+ leak would promote DADs Taken together, our study identifies a new CaR4497C mice (CPVT) and their WT littermates (F3 to F5 generation), as previously described Comité Régional d' Ethique sur l'expérimentation animale of Languedoc-Roussillon on the Use and Care of Animals. All persons who participated in the experiments had the training and authorization to do so .Experiments were performed on male and female heterozygous RyR2R4496C knock-in mice was previously described The generation of RyR-1) and anaesthetized with Na+ pentobarbital (50 mg/kg) administered intraperitoneally. The heart was excised rapidly via a thoracotomy and the pericardium was removed. The heart was placed in ice-cold (0 °C) oxygenated Tyrode solution containing (mmol/L): NaCl 130, NaH2PO4 0.4, NaHCO3 5.8, MgCl2 0.5, KCl 5.4, glucose 22, Hepes 25 and insulin 10−3 (titrated to pH 7.4 with NaOH). The aorta was cannulated above the aortic valve and was perfused by gravity (70 cm column height) with warm (37 °C), preoxygenated Tyrode solution supplemented with 0.1 mmol/L EGTA for 2 min. Enzyme solution containing 1 g/L collagenase Type II (Worthington) in Tyrode solution supplemented with 0.1 mmol/L CaCl2 was then perfused until the aortic valve was digested (attested by the increased outflow of perfusate). The heart was transferred to a Petri dish containing enzyme solution supplemented with 2 g/L bovine serum albumin (BSA) and gently shaken for 2–3 min at 37 °C to disperse individual myocytes. The resulting cell suspension was filtered through a 250 µm nylon mesh and centrifuged for 3 min at 20 g. The cell pellet was suspended in Tyrode solution supplemented with 0.5 mmol/L CaCl2 and 2 g/L BSA and was centrifuged again at the same speed. Finally, the cell pellet was suspended in storage solution comprising Tyrode solution supplemented with 1 mmol/L CaCl2 and 2 g/L BSA.Isolated ventricular myocytes from knock-in mouse-model carrier of the RyR R4496C mutation (CPVT) and their gender-matched littermates wild-type (WT) were prepared using an enzymatic perfusion method. Animals were treated with heparin  in line-scan mode (1.5 msec/line). CaI and fluorescence signals were simultaneously digitized  and acquired at sampling rate of 100 µsec using pClamp 8.1. During experiments, cells were superfused with an external solution containing (in mmol/L) 140 NaCl, 0.5 MgCl2, 5 CsCl, 1.8 CaCl2, 5.5 glucose, 5 Hepes (pH 7.4), while the patch pipette was filled with a solution containing (in mmol/L) 130 CsCl, 1 MgCl2, 1 NaH2PO4, 3.6 Na2 phosphocreatine, 5 MgATP, 10 HEPES and 0.05 Fluo-3 pentapotassium salt (Molecular Probes); pH 7.2. CaI and SR Ca2+ releases were elicited by 100 msec voltage steps in 10 mV increments from −50 to +60 mV, every 10 seconds. Prior to it, to allow steady-state SR load a voltage protocol including 4 voltage steps (150 msec) from −80 to 0 mV was apply and voltage-gated Na+ channels were inactivated by a 500-msec ramp from −80 to −42 mV Whole-cell currents were monitored with an Axopatch 200A patch-clamp amplifier. Capacitance compensation was optimized and series resistance was compensated by 40–80%. CaCaI was measured for every depolarizing step as the time from the onset of the voltage step to the peak of current.The activation kinetic of CaI was determined by analysis of the decay phase of current traces in response to voltage steps. Best fits were obtained with an equation including a sum of two exponentials plus a constant expressed as Afastexp(-t/fastτ)+Aslowexp(-t/slowτ)+A0, where τ and A are the time constant and the initial amplitude of the two components subscripted fast and slow, respectively, and A0 is the amplitude of the time-independent component.The time course of inactivation of CaI was maximum) and 100 msec in duration.Isochronic inactivation were performed with a double pulse protocol, in which a conditioning prepulse of variable amplitude  and 250 msec in duration  was followed by a test pulse to 0 mV .Fluo-3 fluorescence was excited with the 488-nm line of an argon ion laser. Emitted fluorescence was measured at wavelengths over 515 nm. Image acquisition was made in the line-scan mode. A single myocyte was scanned repetitively along a line parallel to the longitudinal cell axis. Image processing and analysis were performed using IDL software (Research Systems), as previously described 2+ load estimation, myocytes were previously stimulated (4 voltage 150 msec steps from −80 to 0 mV) for 1 min, then 10 mmol/L caffeine was added, the fluorescence image was recorded by confocal microscopy and the associated NCX current was recorded by patch clampand integrated to estimate the amount of Ca2+ released by the SR For SR Ca2+ sparks .Spontaneous Ca+ sparks  were imaBAPTA and W7 were purchased from Calbiochem; CALP2 and Ryanodine were from Tocris Bioscience; and other chemical products were from Sigma. Ryanodol was generated as previously described −4, Benzamidine 10−2, Leupeptin, 12 10∼3; PMSF 0.1, 0.5% sodium desoxicholate, 0.1% SDS, pH 7.2) using a Potter-Elvehjem and spun at 2,000 g for 10 min. Membrane fractions were isolated by ultracentrifugation at 40 000 g for 30 minutes at 4°C V1.2 policlonal antibody , in TBS buffer  with 1% w/v non-fat dried milk powder. Membranes were blocked overnight with 5% w/v non-fat dried milk powder in TBS buffer before primary antibody addition. Membranes were incubated 1 h with corresponding secondary peroxidase-conjugate goat antiserum . Signals were developed by chemiluminiscence . The relative amount of protein on the blots was determined by densitometry using KodakID Software . Actin signals were detected in the same blots with anti-actin serum  and used as loading controls.Mouse heart homogenates were prepared with homogenization buffer . Differences with values of P<0.05 were considered significant.Data are presented as means ± SEM and compared using t test or an unequal variance t statistic (Welch test), when appropriated, with the PAST program (


The selected modifications are associated with a broad range of cardiac diseases: three subtypes of familial cardiomyopathies  and ischemia-reperfusion injury. Consistent with previous studies, the majority of the protein modifications had no effect on the Ca2+ binding properties of the isolated troponin complex. However, when incorporated into the thin filament, dilated cardiomyopathy mutations desensitized (up to 3.3-fold), while hypertrophic and restrictive cardiomyopathy mutations, and ischemia-induced truncation of troponin I, sensitized the thin filament to Ca2+ (up to 6.3-fold). Kinetically, the dilated cardiomyopathy mutations increased the rate of Ca2+ dissociation from the thin filament (up to 2.5-fold), while the hypertrophic and restrictive cardiomyopathy mutations, and the ischemia-induced truncation of troponin I decreased the rate (up to 2-fold). The protein modifications also increased (up to 5.4-fold) or decreased (up to 2.5-fold) the apparent rate of Ca2+ association to the thin filament. Thus, the disease-related protein modifications alter Ca2+ binding by influencing both the association and dissociation rates of thin filament Ca2+ exchange. These alterations in Ca2+ exchange kinetics influenced the response of the thin filament to artificial Ca2+ transients generated in a stopped-flow apparatus. Troponin C may act as a hub, sensing physiological and pathological stimuli to modulate the Ca2+-binding properties of the thin filament and influence the contractile performance of the heart.The contractile response of the heart can be altered by disease-related protein modifications to numerous contractile proteins. By utilizing an IAANS labeled fluorescent troponin C,  Numerous cardiac diseases adversely alter the heart’s ability to properly maintain its performance 2+ sensor in cardiac muscle responsible for translating the intracellular Ca2+ signal into mechanical force 2+ sensitivity of TnC can be modulated by multiple factors, including its interactions with other myofilament proteins, post-translational modifications of the myofilament, as well as cardiac disease-related protein modifications 2+ signal. Instead, it may act as a central hub that integrates information from the myofilament  and adjusts its Ca2+ binding properties to regulate cardiac muscle mechanics Troponin C (TnC) is the myofilament Ca2+ sensitivity of TnC, much less is known regarding their effects on TnC’s Ca2+ exchange kinetics 2+ exchange with TnC may be even more significant to how the heart performs since the heart is dynamic and does not function in a static steady-state. Furthermore, it is the kinetics of Ca2+ exchange with TnC, including the rate of Ca2+ association to and dissociation from TnC that determine its steady state Ca2+ sensitivity. The rate of Ca2+ association is 3 to 4 orders of magnitude faster than the rate of Ca2+ dissociation, and considered diffusion controlled  and the thin filament using a fluorescently labeled TnC. The protein modifications include five dilated cardiomyopathy (DCM) mutations , two hypertrophic cardiomyopathy (HCM) mutations (TnI S166F and TnT R92Q), two restrictive cardiomyopathy (RCM) mutations (TnI D190H and TnI R192H), and an ischemia-induced truncation of TnI (residues 1-192). These protein modifications represent a broad spectrum of diseases that change the steady-state Ca2+ sensitivity of the actomyosin ATPase activity and/or force generation 2+ binding to TnC by influencing both its Ca2+ association and dissociation rates on the thin filament . These results suggest that TnC acts as a central hub on the thin filament by sensing pathological stimuli to alter cardiac contractile properties.In this work, disease-related protein modifications of troponin I (TnI) and troponin T (TnT) were selected to systematically study their effects on CaPhenyl-Sepharose CL-4B, Tween-20, and EGTA were purchased from Sigma Chemical Co. . IAANS and phalloidin were purchased from Invitrogen . Affi-Gel 15 affinity media was purchased from Bio-Rad .The pET3a plasmid encoding human cardiac TnC was a generous gift from Dr. Lawrence Smillie . The pET3a plasmids encoding human cardiac TnI and TnT were graciously provided by Dr. James Potter . TnC, TnI and TnT mutants were constructed from their respective pET3a plasmids using the primer-based QuikChange Site-Directed Mutagenesis Kit  as previously described E. coli BL21(DE3)pLysS cells , while the TnI and TnT plasmids were transformed into Rosetta™(DE3)pLysS cells . The proteins were expressed and purified as previously described The plasmid encoding human cardiac TnC was transformed into Rabbit skeletal actin and bovine ventricular tropomyosin (Tm) were purified from acetone powders as previously described C35S,T53C,C84S (herein denoted as TnCT53C) was labeled with the environmentally sensitive thiol-reactive fluorescent probe IAANS as previously described TnCThe reconstituted Tn complexes and regulated thin filaments were prepared as previously described 2 were added to 2 ml of each labeled Tn complex (0.15 µM) in a titration buffer (200 mM MOPS (to prevent pH changes upon addition of Ca2+), 150 mM KCl, 2 mM EGTA, 1 mM DTT, 3 mM MgCl2, 0.02% Tween-20, pH 7.0) with constant stirring. Reconstituted thin filaments were titrated in an identical buffer composition (excluding Tween-20). The [Ca2+]free was calculated using the computer program EGCA02 developed by Robertson and Potter 2+ sensitivities were reported as a dissociation constant Kd, representing a mean of three to four separate titrations ± S.E.M. The data were fit with a logistic sigmoid function , as previously described All steady-state fluorescence measurements were performed using a Perkin-Elmer LS55 spectrofluorimeter at 15°C. IAANS fluorescence was excited at 330 nm and monitored at 450 nm as microliter amounts of CaCl2+ exchange rates were characterized using an Applied Photophysics model SX.20 stopped-flow instrument with a dead time of 1.4 ms at 15°C. IAANS fluorescence was excited at 330 nm. The IAANS emission was monitored through either a 420-470 nm band-pass interference filter for 2, 0.02% Tween-20 (excluded for thin filament kinetic measurements), at pH 7.0. 10 mM EGTA was utilized to remove 200 µM Ca2+ from the Tn complexes or thin filaments.Ca2+ association to the thin filament, a minimal concentration of EGTA was added to the thin filament mixture to remove contaminating Ca2+. The amount of EGTA added was determined by steady state titrations. For the control, TnI (1-192), TnT ΔK210, TnT R131W thin filaments, 5 µM EGTA was sufficient. For TnI R192H and TnI D190H thin filaments, 10 µM EGTA was required. The observed rates at various Ca2+ concentrations were plotted against the Ca2+ concentration and fit with a linear regression. The rate of Ca2+ association to the thin filament was obtained by calculating the slope of the fit 2+ association rate measurements was the same as that used for the Ca2+ dissociation rate measurements.In order to measure the rate of Ca2+ association rate to the thin filament, the IAANS labeled TnCs on the thin filament were exposed to artificial Ca2+ transients (ACTs) of increasing amplitude as previously described 2+ dissociation) in the presence of 500 µM EGTA (after mixing) against increasing [Ca2+]. This technique exposes the thin filament to rapid transient fluxes of Ca2+ . A relatively high concentration of EGTA (500 µM after mixing) was utilized to ensure that the thin filament is not pre-bound by Ca2+ and to provide a large enough Ca2+ sink to effectively remove the free [Ca2+] after mixing, effectively generating a Ca2+ transient. The amplitude of the Ca2+ transient can be adjusted by rapidly mixing the thin filament with different amounts of Ca2+. The ability of a Ca2+-binding protein  to respond to these rapid Ca2+ transients, or become transiently occupied by Ca2+, is a function of the Ca2+ association rate of the Ca2+-binding protein 2+, high [Ca2+] (1 mM after mixing) was used to exceed the [EGTA] and saturate the thin filament. Analysis of the response of the thin filament to the ACTs is described in the legend of In order to determine the potential effects of altering the CaStatistical significance was determined by ANOVA followed by a Dunnett’s post-hoc t-test using the statistical analysis software Minitab . Two means were considered to be significantly different when the P value was < 0.05. The data is shown as a mean value ± S.E.M.2+ binding properties of the Tn complex were examined since Tn is the simplest biochemical system to test the effects of TnI and TnT modifications on TnC. Our laboratory has developed a fluorescent TnC, 2+ binding and dissociation 2+ binding and exchange with isolated or thin filament bound Tn complexes.The data were divided into four subgroups according to the disease subtypes to facilitate data presentation. The effects of the disease-related protein modifications on the Ca2+ sensitivity of TnC within the various Tn complexes was measured by following the Ca2+ dependent decrease in IAANS fluorescence. Control 2+ induced half-maximal fluorescence decrease at 0.66±0.03 µM  hyper-sensitized Ca2+ binding to thin filament by ∼ 6.3-fold  between the changes in the thin filament Ca2+ sensitivities and the changes in the Ca2+ dissociation rates for the different protein modifications. These results suggest that the rate of Ca2+ association to the thin filament can also be altered by the protein modifications. To test this hypothesis, stopped-flow experiments were performed to measure the rate of Ca2+ association to the thin filaments containing TnI R192H, D190H, TnI (1-192), TnT ΔK210 and TnT R131W. Based on the calculated rate of Ca2+ association, TnT R131W was predicted to have little effect on the rate of Ca2+ association while the rest of the protein modifications were expected to significantly change the rate of Ca2+ association  increased the apparent rate of Ca2+ association to the thin filament by ∼ 4- to 5-fold  of increasing amplitude  until EGTA chelates the Ca2+ . Although these are not physiological Ca2+ transients, it is expected that for a fixed Ca2+ transient amplitude, a faster rate of Ca2+ association should lead to a higher level of transient occupancy, while a slower rate of Ca2+ association should lead to a lower level of transient occupancy. 2+] was successively increased from 12.5 µM to 25 µM and then to 50 µM (after mixing), the percentage of transient occupancy increased from 17±1%, to 30±2% and then to 47±3%, respectively  containing thin filament has an apparent faster rate of Ca2+ association, while the TnT ΔK210 containing thin filament has an apparent slower rate of Ca2+ association than the control thin filament.The Camplitude . Due to ectively . Comparetrations . Thus, c2+ binding properties of TnC in the Tn complex and on the thin filament. The selected ten protein modifications are associated with a broad range of cardiac diseases: three subtypes of familial cardiomyopathies , and ischemia-reperfusion injury. Most of the selected protein modifications have previously been shown to alter the Ca2+ sensitivity of skinned cardiac muscle force generation, and/or the actomyosin-ATPase activity 2+ sensitivity of force generation, some of these protein modifications also alter the kinetics and magnitude of contraction and/or relaxation 2+ sensitivity of TnC, but also the effects on the kinetics of Ca2+ exchange with TnC.In this study, we examined the effects of cardiac disease-related TnI and TnT modifications on the Ca2+ sensitivity of TnC 2+ binding properties of the Tn complex 2+ sensitivity of the thin filament, while most of the HCM and RCM mutations, as well as the ischemia-induced truncation of TnI, increased the Ca2+ sensitivity of the thin filament 2+ sensitivity observed for the same protein modifications in the current study, compared to previous studies Previous studies utilized IAANS labeled fluorescent TnCs (at either Cys 35 or Cys 84) to measure the effects of disease-related protein modifications on the steady-state Ca2+ sensitivity of TnC is determined by the kinetics of Ca2+ association and dissociation. It is generally assumed that alterations in the steady-state Ca2+ sensitivity of TnC operate exclusively through changes in the rate of Ca2+ dissociation, since Ca2+ association to TnC has been traditionally thought to be diffusion controlled  tend to alter the Ca2+ binding sensitivity by modulating both the association and dissociation rates; while the Ca2+ desensitizing mutations tend to predominantly affect the dissociation rates (with the exception of TnT ΔK210).The steady-state Ca2+ dissociation rate from TnC remains controversial 2+ dissociation rates prolonged or abbreviated relaxation 2+ dissociation from TnC could potentially contribute to the diastolic dysfunction typically observed with the various cardiomyopathies 2+ association to the thin filament could affect the amount of maximal force generation or myocyte shortening. For instance, despite similar intracellular Ca2+ transients, myocytes transfected with TnI R192H had a larger shortening amplitude than control myocytes, especially under high stimulating frequencies 2+ transient was higher than that of the control 2+ transient, the response of the thin filament to Ca2+ may also regulate the extent and duration of force development. Consistent with this idea, the current study demonstrates that both TnI (1-192) and TnT ΔK210 altered the response of the thin filament to non-physiological, artificial Ca2+ transients in a way consistent with their contractile effects.While the physiological significance of the Ca2+ binding properties of TnC on the thin filament 2+ sensitivity and kinetics of TnC 2+ binding properties of TnC, since it was the only protein modification that substantially altered the Ca2+ binding properties of the Tn complex. TnI S166F is located close to the switch region of TnI that binds to the hydrophobic pocket of the regulatory domain of TnC. Thus, TnI S166F might be influencing this critical TnC-TnI interaction to affect the Ca2+ binding properties of TnC.There are potentially multiple mechanisms that exist to alter the Ca2+ sensitivity of TnC 2+ only when the Tn complex is incorporated in the thin filament. Similarly, the TnT mutations may also change the ability of TnI to bind to actin-Tm. This hypothesis would be consistent with the TnT modifications only affecting the apparent Ca2+ sensitivity of the thin filament and not the Tn complex.The more distant C-terminal region of TnI (residues 188-210) is believed to contribute to the inhibition of actomyosin interactions during diastole by direct interactions with actin-Tm 2+ sensitivity of the thin filament by influencing both the association and dissociation rates of Ca2+ exchange. The protein modifications modified the Ca2+ exchange properties of TnC in a way consistent with their effect on cardiac muscle physiology. Therefore, TnC may act as a central hub that converges these pathological stimuli to affect cardiac contractile properties. The abnormal myofilament Ca2+ binding along with aberrant intra-cellular Ca2+ signaling could contribute to contractile dysfunctions leading to cardiac remodeling and disease phenotypes. Thus, pharmaceutically 2+ binding properties may represent a beneficial therapeutic strategy.In summary, the disease-related protein modifications studied in this work adversely altered the steady-state Ca


A consequence of local control is that the whole-cell Ca2+ transient is a smooth continuous function of influx of Ca2+ through LCCs. While this so-called graded release property has been known for some time, its functional importance to the integrated behavior of the cardiac ventricular myocyte has not been fully appreciated. We previously formulated a biophysically based model, in which LCCs and RyRs interact via a coarse-grained representation of the dyadic space. The model captures key features of local control using a low-dimensional system of ordinary differential equations. Voltage-dependent gain and graded Ca2+ release are emergent properties of this model by virtue of the fact that model formulation is closely based on the sub-cellular basis of local control. In this current work, we have incorporated this graded release model into a prior model of guinea pig ventricular myocyte electrophysiology, metabolism, and isometric force production. The resulting integrative model predicts the experimentally observed causal relationship between action potential (AP) shape and timing of Ca2+ and force transients, a relationship that is not explained by models lacking the graded release property. Model results suggest that even relatively subtle changes in AP morphology that may result, for example, from remodeling of membrane transporter expression in disease or spatial variation in cell properties, may have major impact on the temporal waveform of Ca2+ transients, thus influencing tissue level electromechanical function.The local control theory of excitation-contraction (EC) coupling asserts that regulation of calcium (Ca However few models combine electrophysiology with contraction mechanics, mitochondrial energetics, or intracellular signaling due to the computational difficulty of combining disparate time and/or spatial scales. While the incorporation of more cellular component models increases the descriptive power of the combined model, the complexity of whole-cell model behavior and computational cost increase almost exponentially with the number of constitutive mechanisms represented.Since publication of the first computational model of the cardiac myocyte action potential (AP) in 1960 . LCCs are preferentially located in the t-tubules  is the portion of the SR most closely approximating the t-tubules. Ryanodine receptors (RyRs) are channels located in the JSR membrane in close opposition to LCCs in the dyad. During the initial phase of the AP, sarcolemmal membrane depolarization increases LCC open probability. The resulting flux of Ca2+ into the dyadic space (trigger flux) leads to Ca2+ binding to the RyRs. This increases RyR open probability, and when open, the resulting Ca2+ flux through RyRs (release flux) is directed into the dyadic space. There are thousands of dyads within the cardiac myocyte, and the net flux of Ca2+ from dyads into the cytosol triggers muscle contraction in a process known as excitation-contraction coupling (ECC). The ratio of release to trigger flux is typically large, and is referred to as ECC gain.The most fundamental property of cardiac myocytes is that they are electrically excitable cells (they generate APs). The rapid increase in membrane depolarization during the early phase of the AP increases the open probability of sarcolemmal L-Type Ca2+ release from JSR is a graded, smooth, continuous function of the amount of trigger Ca2+ entering the cell via LCCs. The majority of cardiac myocyte models lump all dyadic spaces together into a common pool known as the subspace, and net trigger flux through LCCs and net release flux through RyRs is directed into this common pool. In a landmark 1992 paper  results in all-or-none rather than graded release. More specifically, he showed that common pool models cannot reproduce both high gain and graded release. Despite this fact, early common pool models were able to reproduce a broad range of cardiac myocyte behaviors. However, Greenstein and Winslow (2+-dependent inactivation (CDI) of LCCs is stronger than voltage-dependent inactivation (VDI), into common pool models de-stabilized repolarization of the AP due to abnormal Ca2+ handling . Thus, common pool models not only fail to capture the graded release property, they cannot capture one of the most fundamental properties of normal cardiac myocytes at physiological pacing rates – stable APs.Graded release refers to the phenomenon, originally observed by Fabiato , whereby Winslow  showed t2+ release from their adjacent RyRs in the dyad. Under this assumption, graded release arises as the result of statistical recruitment of release clusters, a process known as local control of Ca2+ release. Guided by this insight, Greenstein and Winslow  can significantly affect timing of Ca2+ release. This model prediction is also validated by experimental data. These behaviors are specific to the graded release model, and cannot be revealed when using common pool models with all-or-none release. Finally, we show preliminary results indicating that the model predicts experimentally measured effects of mitochondrial Ca2+ uniporter block on amplitude of the cytosolic and mitochondrial Ca2+ transients, demonstrating the important role of beat-to-beat Ca2+ buffering by mitochondria.As a first step toward investigating these important questions, we present an extension to the ECME model incorporating the coupled LCC-RyR formulation of graded release and description of the local Ca2+ release unit (CaRU) is based on that presented previously for canine myocytes by Greenstein et al.  Ca2+ levels. In this minimal model, the single model RyR represents the estimated number of release channels per LCC measured in guinea pig  contribution to relaxation, resulting in Ca2+-cycling that is more similar to guinea pig than rat or mouse.Ryanodine receptor properties are those from our previous model  delayed outward rectifier currents. IKr and IKs current amplitudes were adjusted to match model APD to experimental data , in qualitative agreement when compared with experimental data exhibiting a tau of 36 s ) was also added using a model by Ferrero et al. (IK(ATP) was modified to take into account the range of experimental data  takes up 65.9% of the transported cytosolic Ca2+, NCX removes 28.9%, and the sarcolemmal (SL) Ca-pump removes 5.1%, close to estimates of 67% SERCa, 30% NCX, and 3% slow processes  channels, pumps, and exchangers are based on those of the 2006 ECME model . Equations and parameters for the model are also available in the online supplement.Model code was written in C++ using the SUNDIALS CVODE integration library and run using an IBM PC workstation with a 2.80-GHz processor and 2.5 GB of RAM. On such a workstation, this 75-state model takes approximately 15 s to compute 10 1 Hz APs with no output written to files. This is comparable to the performance of current models for which code is available . This quantity was calculated as unity minus the ratio of total systolic SR Ca2+ to total diastolic SR Ca2+. The 33% measurement given by the model agrees with 35% estimated in ferret , APD becomes constant from beat to beat. The time between the end of the AP and the onset of the next stimulus is the diastolic interval (DI). Immediately following an AP the cell will be in an inexcitable refractory state. As the DI increases beyond this refractory period toward the steady-state DI, an AP can be triggered, but its duration is less than that of the steady-state AP due to incomplete recovery from inactivation of 90 was measured. The results are compared to recordings under the same protocol from  strips of guinea pig ventricle at 37°C  varies as a function of pacing frequency  that initiates the AP and the export of Na+ by the Na+-K+ pump during diastole. In this model, NCX plays a large role in early repolarization of the plateau at slow frequencies. As [Na+]i continues to decrease with an increase in BCL, outward NCX current is reduced, allowing prolongation of the AP plateau phase.The model results presented in Figure 2+ concentration associated with increased SR load and gain. To compare the normalized force output of the model with those data the cross-sectional area used is 0.013 mm2, which is equivalent to a fiber with an elliptical profile of width 0.24 mm and thickness 0.069 mm, the minimum of the range of measurements for the Cortassa et al. . Both cytosolic Ca2+ and force transients show faster peaks at higher pacing frequencies, as has been shown experimentally , such that the time of decay to half peak amplitude is decreased with increasing frequency. Force transients have been shown to have a similar property  cycle, and through regulation of the F1-F0 ATPase by ADP. As pacing frequency increases, a higher ADP:ATP ratio results from the increased ATP consumption at rapid contraction rates. Increased mitochondrial ADP levels stimulate the F1-F0 ATPase to generate ATP by utilizing the proton-motive force as an energy source. Flux of electrons through the electron transport chain would in and of itself deplete the NADH pool. However, the amplitude of the cytosolic Ca2+ transient also increases with pacing frequency, and this Ca2+ signal is communicated to the mitochondria via Ca2+ uptake by the mitochondrial Ca2+ uniporter. Elevated mitochondrial Ca2+ levels stimulate the TCA cycle to increase production of NADH to sustain a higher rate of respiration. As in the original ECME model  and are followed by a recovery to rest levels at approximately 0.9 mN/mm2 during 0.25 Hz pacing. Mitochondrial Ca2+ levels  and does not require computationally expensive stochastic simulations of large numbers of individual ion channels  and consisting of one Hodgkin and Huxley .Many aspects of the canine and guinea pig local control models differ, including resting SR load, Cad Huxley  activatid Huxley D–F. Both Figures D–F. Addi2+ release, shape of the Ca2+ transient, and timing of force generation. AP clamp waveforms from guinea pig epicardial, midmyocardial, and endocardial cells as recorded by Sicouri et al. (2+ release and Ca2+ transient in each cell type (not shown). A 36% gradient of SERCa expression, measured with high resolution and accuracy by Anderson et al.  is much slower than that observed in experiments . In the coupled LCC-RyR formulation of Ca2+ release used in this model, JSR depletion cannot be tracked locally at individual release sites because the JSR and NSR are assumed to be in rapid equilibrium. Recent evidence suggests that the primary determinant of SR release termination and restitution of Ca2+ of sparks is the dynamics of local JSR refilling  descriptions to reduce the number of state variables could also be applied to the mitochondria. In a scenario where abnormal ATP supply does not need to be addressed, the Ca2+ depletion may be the major mechanism for release termination (Terentyev et al., The model presented here lacks a JSR compartment, which was combined with the network SR in developing the deterministic formulation of the CaRU model (Hinch et al., 2+ handling in pancreatic β-cells. Their Ca2+-regulation of the TCA cycle was the basis on which the energetics model used here was developed (Cortassa et al., 2+ handling between mitochondria and cytosol. However, this model is not able to capture the biphasic response of NADH levels to changes in pacing frequency. Another recent model (Hatano et al., 2+ and many metabolites. While this model supports the existence of subsarcolemmal Ca2+ gradients as predicted by NCX calculations (Weber et al., 2+ levels. The advantage of the model presented here is that its mechanistic description of ECC and Ca2+ cycling in the cell will provide a platform for future investigations in Ca2+-regulation of mitochondrial energy production.Other models that incorporate both ECC and mitochondrial energetics exist. Magnus and Keizer  develope2+ release and voltage-dependent gain. Additionally, the incorporation of mitochondrial energetics allows the model to qualitatively reproduce changes in NADH in response to changes in cardiac workload. Using this model we can improve our understanding of how changes in AP shape and Ca2+ transients affect energy supply and developed force in normal and failing myocytes.We have developed a mechanistically detailed description of ECC in the guinea pig cardiac myocyte combined with modules describing energetics and isometric force. This model successfully reproduces key ECC properties of graded SR CaThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.