A crucial point in this respect is an increased spontaneous release of Ca2+ out of the sarcoplasmic reticulum during diastole via ryanodine receptors type 2 (RyR2). The consequence is a compromised sarcoplasmic reticulum Ca2+ storage capacity, which impairs systolic contractility and possibly diastolic cardiac function due to Ca2+ overload. Additionally, leaky RyR2 are more and more regarded to potently induce proarrhythmic triggers. Elimination of spontaneously released Ca2+ via RyR2 in diastole can cause a transient sarcolemmal inward current and hence delayed after depolarisations as substrate for cardiac arrhythmias. In this article, the pathological role and consequences of the SR Ca2+-leak and its regulation are reviewed with a main focus on protein kinase A and Ca2+-calmodulin-dependent kinase II. We summarise clinical consequences of “leaky RyR2” as well as possible therapeutic strategies in order to correct RyR2 dysfunction and discuss the significance of the available data.It has been persuasively shown in the last two decades that the development of heart failure is closely linked to distinct alterations in Ca Heart failure is characterised by a progressive deterioration of cardiac function and represents a major public health burden. Despite a number of well-established therapies, the prevalence as well as the mortality of heart failure are still alarmingly high. Heart failure remains the most frequent reason for hospital admission of patients older than 65 years . In the Huge efforts have been made to further elucidate underlying pathomechanisms and identify new targets for innovative therapies. The aetiologies of heart failure are various, stretching from coronary artery disease and heart valve dysfunctions to toxic influences on the myocardium or infectious diseases. Arterial hypertension could be identified as the most frequent risk factor in this context . The comOn a histological and cellular level, heart failure could be linked to several alterations including fibrosis, inflammation and apoptosis. Furthermore, it has become more and more obvious that a fully functional excitation–contraction coupling process in the cardiomyocyte is fundamental for both, a normal systolic and diastolic function.2+ release out of the sarcoplasmic reticulum (SR) in systole followed by a quick Ca2+ elimination out of the cytosol to induce relaxation in diastole  that are located on the surface of the SR and trigger the release of an even bigger amount of Ca2+ out of the SR. This mechanism of Ca2+-induced Ca2+ release leads to a prominent increase in cytosolic Ca2+ that binds to troponin C (TnC) and thereby induces actin–myosin interaction. In diastole, Ca2+ has to be eliminated from the cytosol to allow relaxation of the myofilaments. This is mainly achieved by an active, energy-consuming Ca2+-reuptake into the SR via the sarcoplasmic reticulum Ca2+-ATPase type 2a (SERCA2a). A smaller amount of Ca2+ is extruded out of the cell via the Na+/Ca2+-exchanger (NCX), which is a facilitated diffusion in which the electrochemical potential gradients of Na+ and Ca2+ are the source of energy to drive the transport. NCX in its “forward” mode produces an electrical current because 3 Na+ are exchanged for 1 Ca2+. In human cardiomyocytes, the reuptake of Ca2+ into the SR makes up for approximately 70 % of the systolic Ca2+ depending on the heart rate [2+ uptake into mitochondria and elimination via Ca2+-ATPases in the plasma membrane [During phase 0 of an action potential fast Naart rate . Around membrane .2+ concentrations make up the molecular trigger for cardiac contraction and relaxation, it is a matter of course that this system is elaborately regulated and highly adaptable to physical demands. SERCA2a activity is influenced by phospholamban (PLB) that is one of the major mediators of the cardiac contractility response upon β-adrenergic stimulation [2+/calmodulin-dependent protein kinase IIδ (CaMKIIδ) is able to phosphorylate PLB at specific residues  and thereby abandon its inhibitory effect on SERCA2a. A second subcellular microdomain is the RyR2-complex that consists of several proteins and whose Ca2+ release capacity and diastolic closure can be regulated via phosphorylation by PKA and CaMKII [2+ handling proteins [As the cyclic alterations of cytosolic Camulation . Inhibitd CaMKII –19 at Seproteins .2+ cycling is profoundly disturbed in heart failure  in the dyadic cleft. This locally restricted Ca2+ release is accompanied by a local decrease of SR Ca2+ content in the corresponding areas, the so-called Ca2+ blinks [2+ content reaches a threshold of approximately 60 % of the diastolic SR Ca2+ concentration [2+ ions, once released, are partly removed from the cytosol via the NCX, which was found to be overexpressed in heart failure [2+ ion) in exchange for three (3 Na+ ions) leads to a depolarisation of the cell membrane and can trigger delayed after depolarisations [On the other hand, there is an increased diastolic Ca+ blinks , and wasntration . The sta failure , 24, 29.isations . Thus, aisations , 31–37.2+ buffer thus influencing RyR2 behaviour. Furthermore, RyR2 function is crucially linked to its phosphorylation status, which can be seen as the net effect of a steady competition between two major cellular kinases, PKA and CaMKII, and two protein phosphatases, PP1 and PP2a, all of which are closely associated with the receptor [2+ sensitivity and thus the diastolic open probability as well as the systolic Ca2+ release capacity [2+ release pore and thus adapting Ca2+ release kinetics to cellular demands.Ryanodine receptors are the largest known ion channels. In the heart, ryanodine receptor type 2 is the predominantly expressed isoform. It is a huge macromolecular protein exceeding a molecular weight of 2 MDa. Structural analysis have shown that RyRs are mushroom-shaped assemblies of four identical subunits each comprising approximately 5,000 amino acids . This coreceptor . This clreceptor . The relcapacity , 45 by i2+-release channel [The mechanisms of RyR2 regulation have been subject of intense research in the last years due to their pathologic relevance. It has become undoubtedly clear that phosphorylation of the receptor protein is a key event in this context. The RyR2 complex thereby was found to be one of the first proteins that undergo phosphorylation upon beta-adrenergic stimulation . As earl channel . Further channel . From th2+ sensitivity and compromised diastolic RyR2 closure [2+ leak [2+ transients and unaltered overall cellular function. Obvious protection against maladaptive remodelling after TAC was absent. Moreover, they did not find a significant impact of RyR-S2808A mutation on the diastolic SR Ca2+ leak and showed that RyR2-S2808A mutants could still be phosphorylated by PKA, which might be attributed to another phosphorylation site at S2830. The authors further concluded that genetic ablation of one single phosphorylation site might lead to conformational changes and shift kinase activity to other, physiologically not equally relevant sites that in turn make up for the missing phosphorylation site. Furthermore, different amounts of phosphatases included in the isolated RyR2 complexes could, at least partially, explain the contradictory results of different works in the field. Another group just recently exposed RyR-S2808A-mutated mice to myocardial infarction. They, however, could not find any differences compared with wild-type littermates, neither as to global cardiac function and calcium cycling parameters after myocardial infarction nor as to infarct size [It was described by Marks and colleagues that PKA phosphorylation of the RyR2 leads to a dissociation of FKBP12.6 from the channel and consecutively to an increased Ca closure . Some ye closure . In cont closure . On acco closure . Other g[2+ leak . Further[2+ leak . In thisrct size .2+ release [2+ load but did not exhibit any attenuation of cardiac functional decline after aortic banding. Another aspect that argues against an important, physiologically relevant FKBP12.6-mediated RyR2 regulation is the small amount of FKBP12.6 normally existing in rat and mouse myocytes [2+ leak and SR Ca2+ content [Several studies strongly support the hypothesis that phosphorylation of RyR2 at Ser2808 by PKA causes the RyR2-associated protein FKBP12.6 to dissociate from the receptor with the result of an increased propensity for diastolic SR Ca release , 51, 52. release  examined release . Another release . They gemyocytes . FKBP12. content , 57.2+ channels [2+ cycling cannot be overestimated. It has been shown that mice with a conditional knockout of the NCX could escape death by an adaptive reduction of Ca2+ influx through L-type Ca2+ channels instead of upregulation of Ca2+ efflux mechanisms [Whereas the activating effect of PKA on L-type Cachannels  and SERCchannels  is indischanisms . This, h2+ leak due to CaMKII phosphorylation of the cardiac RyR2 [There is an increasing body of knowledge concerning the crucial role of CaMKII for modulation of RyR2 function. Different sequence analysis revealed that there are up to four serine and two threonine residues at the RyR2 that can be phosphorylated by CaMKII. The first report was published by Witcher et al. , showingiac RyR2 .2+ cycling and dilated heart failure [2+ leak in these mice lead to a severe depletion of SR Ca2+ storage and reduced systolic Ca2+ transients [After the discovery of RyR2 phosphorylation by CaMKII, several other groups confirmed the pathologically relevant impact of CaMKII on RyR2 function in animal models as well as in human disease , 34, 62. failure , 34. An ansients , 34.2+ leak in isolated cardiomyocytes from a rabbit heart failure model could be reduced by CaMKII inhibition but not by PKA inhibition. Curran et al. [2+ overload and shortage of energy supply in heart failure leading to chronic CaMKII activation. These findings could recently be confirmed for human heart failure by our group [2+ leak in human cardiomyocytes isolated from failing hearts and improved SR Ca2+ storage. Furthermore, positive inotropic effects could be detected in twitching muscle preparations after CaMKII inhibition during increasing stimulation frequencies. These data provide compelling evidence for an important pathological role of CaMKII in heart failure.Furthermore, Ai et al.  could shn et al.  proposedn et al. , 65 enabur group . CaMKII 2+ leak in the development of arrhythmias. Isolated cardiomyocytes from mice overexpressing CaMKIIδc [2+ signalling refractoriness [2+ leak, which is paralleled by an increased incidence of diastolic spontaneous Ca2+ waves [2+ leak and an increased CaMKII activity to atrial fibrillation (AF), the most common sustained cardiac arrhythmia [2+ levels were found to be increased as the result of an increased diastolic Ca2+ leak. Both findings could be normalised by CaMKII inhibition [2+ leak could be reduced in AF cells by CaMKII inhibition whereas PKA inhibition did not yield relevant effects on the SR Ca2+ leak again stressing the role of CaMKII under pathophysiological conditions as compared to PKA.There is also increasing evidence for the role of the CaMKII-induced diastolic CaCaMKIIδc  showed aCaMKIIδc . These pCaMKIIδc . A recentoriness . It coul2+ waves . The aet2+ waves . Accordirhythmia , 69. It rhythmia , 69. Diahibition . These dhibition , which a2+ release. One of the first compounds that combined both features was JTV519. It was shown to improve diastolic and systolic function in isolated human failing myocardium but also had unspecific effects on other ion channels and a narrow therapeutic range with negative inotropic effects at higher concentrations [The use of CaMKII inhibitors in patients is significantly hindered by the ubiquitous expression of this enzyme in different organs. Currently available CaMKII inhibitors cannot be administered to patients due to unspecific effects and pharmacodynamic problems . Major hurdles as to organ-specific attribution still have to be overcome. Gene therapy-based approaches might, however, be feasible in the near future. Additionally, several compounds directly leading to a stabilisation of diastolic RyR2 closure have already been developed. The major challenge thereby is to identify compounds that exert a beneficial effect on the diastolic leak without compromising systolic RyR2 opening and Catrations –73. A ne2+ leak has emerged as a pivot in the development of cardiac arrhythmias as well as heart failure under various conditions and therefore represents a promising toehold for future therapies. Whereas the direct contribution of PKA to the development of an increased SR Ca2+ leak is still controversial, the crucial role of CaMKII in this context seems to be more established. There are convincing data from animal models as well as human tissue showing that an increased CaMKII activity in cardiac pathologies contributes to the manifestation of pump failure and arrhythmias and that CaMKII inhibition can yield beneficial effects. Thus, to our current knowledge, the diastolic SR Ca2+ leak can be addressed by two different approaches: direct stabilisation of the leaky RyR2 via suitable new compounds (RyR stabilisers) on the one hand and reduction of its hyperphosphorylation by CaMKII inhibition on the other hand. The clinical implications for both approaches would be various, stretching from arrhythmias to systolic pump failure as well as diastolic dysfunction.Taken together, the RyR2-dependent SR Ca


The QC promoter contains a putative binding site for the Ca2+ dependent transcription factors c-fos and c-jun. C-fos and c-jun are induced by the same Ca2+-related stimuli as QC and their upregulation precedes QC expression. We show that in the human brain QC is predominantly expressed by neurons. Interestingly, the Ca2+- dependent regulation of both c-fos and QC is not observed in non-neuronal cells. Our results indicate that perturbed Ca2+ homeostasis results in upregulation of QC selectively in neuronal cells via Ca2+- dependent transcription factors. This suggests that disruption of Ca2+ homeostasis may contribute to the formation of the neurotoxic pE Aβ peptides in Alzheimer’s disease.A major neuropathological hallmark of Alzheimer’s disease (AD) is the deposition of aggregated β amyloid (Aβ) peptide in the senile plaques. Aβ is a peptide of 38–43 amino acids and its accumulation and aggregation plays a key role early in the disease. A large fraction of β amyloid is N-terminally truncated rendering a glutamine that can subsequently be cyclized into pyroglutamate (pE). This makes the peptide more resistant to proteases, more prone to aggregation and increases its neurotoxicity. The enzyme glutaminyl cyclase (QC) catalyzes this conversion of glutamine to pE. In brains of AD patients, the expression of QC is increased in the earliest stages of pathology, which may be an important event in the pathogenesis. In this study we aimed to investigate the regulatory mechanism underlying the upregulation of QC expression in AD. Using differentiated SK-N-SH as a neuronal cell model, we found that neither the presence of Aβ peptides nor the unfolded protein response, two early events in AD, leads to increased QC levels. In contrast, we demonstrated increased QC mRNA levels and enzyme activity in response to another pathogenic factor in AD, perturbed intracellular Ca The pE residue stabilizes the peptide by protection against degradation by aminopeptidases The formation of Aβ peptides from the amyloid precursor protein (APP) is a crucial event in Alzheimer’s disease (AD). Proteolytic processing of APP results in different forms of the Aβ protein with different characteristics 3(pE)-42 peptides, plaque formation as well as memory impairments in vitro and in vivoQC is abundantly expressed in the cortex and hippocampus and its expression correlates with the appearance of pE Aβ 2+ homeostasis, redox balance, lipid synthesis and the synthesis and folding of membrane bound and secretory proteins. We showed that increased production of Aβ 2+ homeostasis that have been suggested to be involved early in AD pathology are highly age related 2+ homeostasis - affect QC expression.Accumulation of Aβ is an early pathogenic event in AD. In particular, oligomeric assemblies of Aβ interfere with cellular physiology. AD is a multifactorial disease, and additional dysfunction that is not directly related to Aβ may converge with the toxic effects of Aβ. For example, our lab has previously shown that the unfolded protein response (UPR) is activated early in AD Elaborating on the previously reported QC mRNA expression data in the neocortex of AD patients 1–42 for 24 h and subsequently the expression level of QC mRNA was measured by semi-quantitative real-time PCR analysis , to induce the UPR in the differentiated SK-N-SH . All tre2+-dependent gene regulation since TG, a specific and irreversible sarco-endoplasmic reticulum ATPase (SERCA) pump inhibitor, blocks the re-uptake of cytosolic Ca2+ into the ER and as a consequence depletes the ER Ca2+ and increases the cytosolic Ca2+ as has been shown by fura-2 Ca2+ imaging To further explore the TG induced gene expression of QC, differentiated SK-N-SH cells were treated with increasing concentrations of TG. As shown in To investigate if TG also increased QC activity, we used an enzymatic activity assay. The activity of the enzyme is determined by the ability of QC to convert a glutaminyl residue to a pyroglutamyl residue on a fluorogenic substrate. Differentiated SK-N-SH cells were treated with 1 µM TG for 16 h. The fluorescence, indicative of the activity of QC, was measured and the data show that treatment with TG induces the fluorescence which is in line with the mRNA expression data .2+ pool depletion and/or (ii) TG-initiated rise in cytosolic Ca2+. To investigate which of these two mechanism might be responsible for the TG initiated increase in QC mRNA levels, differentiated SK-N-SH were pretreated (1 h) with the membrane permeable Ca2+ chelator BAPTA-AM (5 µM) before treatment with TG (16 h) (2+ concentration even upon subsequent stimulation with TG 2+ but rather the depletion of the ER Ca2+ that is responsible for the TG induced QC gene expression.Conceivably two TG-dependent events could regulate QC mRNA levels (i) TG-mediated ER CaG (16 h) . Once BA2+ -dependent transcription factor.To further analyze the effect of TG on QC expression we determined the expression of QC at different time points . The earwww.cbrc.jp/research/db/TFSEARCH.html) to identify possible Ca2+ -dependent transcription factor binding sites in the proximal promoter region of the QC gene. The program identified a putative binding site for heterodimers of the transcription factors encoded by the immediate early genes (IEGs) c-fos and c-jun. Disturbed ER Ca2+ homeostasis in neurons leads to the rapid activation of c-fos and c-jun We used Tfsearch  and ryanodine receptors (RyRs), whereas the SERCA pumps refill the store after depletion 2+ concentration and ER Ca2+ depletion. Our data show that chelation of intracellular Ca2+ using BAPTA-AM potentiates the QC induction by TG, but does not have an effect by itself. Combined use of TG and BAPTA-AM results in faster and increased release of Ca2+ from the ER. This indicates that depletion of ER Ca2+ stores is the main trigger to increase the expression of QC. To further define the mechanism we analysed the effect of SERCA inhibition in combinations with pharmacological inhibition of the IP3Rs and RyRs. Unfortunately these experiments were not informative, probably by artefacts generated by the use of combinations of different pharmacological inhibitors. In addition, it has been shown before that the ER Ca2+ leak is not affected by inhibition of the IP3Rs or of the RyRs 2+ leak mediators The expression of QC, the enzyme that catalyzes the conversion of Aβ to the more toxic pE Aβ, is upregulated in AD cortex. Here we demonstrate that also in hippocampus/entorhinal cortex the upregulation of QC occurs at the earliest stages of AD pathology. In this study we investigated whether early pathogenic events that occur in AD brain could be directly involved in this regulation. We demonstrated that QC mRNA expression was not increased by oligomeric or fibrillar Aβ aggregates. Also induction of the UPR, using TM and 2DG was without effect. Only when the UPR was induced by the SERCA pump inhibitor TG the QC mRNA level was increased, indicating the involvement of CaQC promoter showed the presence of a potential binding site for the heterodimer of the transcription factors c-fos and c-jun, both IEGs. We found that c-fos and c-jun mRNA are induced by the same stimuli as QC mRNA. It was reported before that c-fos is induced in neurons by treatment with TG 2+ depletion of the ER, rather than increased cytosolic Ca2+ levels. The induction of the IEGs is much faster and precedes the upregulation of QC. This is compatible with a model where the upregulation of QC by ER Ca2+ depletion is mediated by c-fos and c-jun, although this requires further study. Interestingly, both c-fos and c-jun are upregulated in AD hippocampus Activation of immediate early gene expression is a key event in stress-induced neuronal cell injury in situ hybridization we demonstrated that QC is predominantly expressed in neurons in the human brain, both in temporal cortex as well as in the earlier affected area in AD, the hippocampus/entorhinal cortex. The regulation of QC expression by TG appears to be specific for neuronal cells, because it is not observed in non-neuronal cells. Also c-fos is less responsive in non-neuronal cells, which strengthens the hypothesis that IEG induction is required for Ca2+ -regulated QC expression. BiP is upregulated in response to TG treatment in these cells, indicating that the treatment has resulted in depletion of ER Ca2+ levels. It is possible that non-neuronal cells have more resources to deal with disturbances in Ca2+ homeostasis, or that neuronal cells are more dependent on proper Ca2+ homeostasis for their function. It has been shown before that a different isoform of QC, isoQC, is involved in monocyte infiltration via modification of cytokines, and therefore it has a major role in cell types involved in the immune response Using 2+ homeostasis has been found in both sporadic and in familial (F)AD 2+ signalling 2+ leak channel and that the FAD mutations disrupt this Ca2+ leak function 2+ levels and lower cytosolic Ca2+ homeostasis levels and leads to exaggerated release of Ca2+ from the ER, both in in vitro and in vivo2+ hypothesis of aging emerged two decades ago 2+ homeostasis -regulating systems in neurons 2+ release from the ER stores through both the RyR and the IP3 receptors 2+ homeostasis facilitates the production of pE modified Aβ and may thereby contribute to the amyloid pathology in AD.Perturbed Catrans-retinoic acid  at a final concentration of 10 µM for 5 days. Differentiated cells were treated with Aβ1–42 oligomers or fibrils, TG, TM, DG  and BAPTA-AM  at the indicated concentrations and time points.SK-N-SH , 293 and Hela cells were cultured in Dulbecco’s modified Eagle’s medium with GlutaMAX  supplemented with 10% fetal calf serum , 100 µg/ml streptomycin and 100 U/ml penicillin. SK-N-SH cells were differentiated in culture medium supplemented with all 1–42 was purchased from Anaspec . Aβ1–42 was dissolved in hexafluorisopropanol (HFIP) at a final concentration of 1 mg/ml, aliquoted and dried under vacuum. The resulting peptide film was stored at −80°C until further use. To obtain low molecular weight Aβ, the peptide film was dissolved in DMSO (5 mM), and sonicated in a bath sonicator for 10 minutes. Aβ was subsequently diluted in PBS to a final concentration of 50 µM. To enrich for oligomers, this mixture was incubated at 4°C for 24 h. High order aggregates were removed by centrifugation  at 4°C. To obtain fibrillar Aβ, the peptide film was dissolved in DMSO (5 mM). Then 10 mM HCl was added to bring the peptide to 100 µM concentration and incubated for 24h at 37°C. The final concentration of the oligomeric and the fibrillar Aβ 1–42 preparations was determined using Bradford protein assay and the β-sheet content was determined by a Thioflavin T assay.Synthetic AβPost-mortem brain material was obtained from the Netherlands Brain Bank . All donors or their next of kin provided written informed consent for brain autopsy and use of tissue and medical records for research purposes. The parameters of all tissue samples used in this study are listed in Total RNA was isolated from differentiated SK-N-SH cells, untreated or treated, and post-mortem brain tissue with the RNAeasy minikit . cDNA synthesis was performed using Superscript II reverse transcriptase  and oligo-dT primers. Real-time quantitative PCR (qPCR) was performed using the Light Cycler 480 system . Reaction volumes of 5 µl contained cDNA, 0.1 µM Universal Probe Library probe , 0.4 µM forward primer, 0.4 µM reverse primer and 2.5 µl 2x LightCycler 480 Probes Master . After denaturation for 10 min at 95°C, amplification was performed using 35 cycles of denaturation (95°C for 10 s), followed by annealing (58°C for 15 s), and elongation (72°C for 15 s). Results were analysed using the LightCycler 480 software  version 1.5. Expression levels were normalized using eEF2α. Primers and probes used in this study are listed in Cells were washed with PBS and lysed in PBS+1%Triton. Subsequently the supernatant was collected after centrifugation 12000×g at 4°C for 10 minutes. The protein concentration was determined by the Bradford Assay . 100 µl lysate (0.5 mg/ml) was mixed with 100 µl of 200 µM Q-AMC  in assay buffer  and incubated in dark tubes at room temperature for 20 minutes. The reaction was stopped at 100°C for 5 minutes and the incubations were cooled on ice for 3 minutes. Subsequently 200 µl of rhPGPEP-1  was added to each vial and the mixture was incubated at room temperature for 10 minutes. Fluorescence was measured using a FLUOstar Omega .In situ hybridization was essentially performed as described before


However, their pathogenic role on SR Ca2+ handling in cardiac myocytes is unknown. Therefore, we investigated whether an AGE cross-link breaker could prevent the alterations in SR Ca2+ cycling that lead to in vivo cardiac dysfunction during diabetes. Streptozotocin-induced diabetic rats were treated with alagebrium chloride (ALT-711) for 8 weeks and compared to age-matched placebo-treated diabetic rats and healthy rats. Cardiac function was assessed by echocardiographic examination. Ventricular myocytes were isolated to assess SR Ca2+ cycling by confocal imaging and quantitative Western blots. Diabetes resulted in in vivo cardiac dysfunction and ALT-711 therapy partially alleviated diastolic dysfunction by decreasing isovolumetric relaxation time and myocardial performance index (MPI) . In cardiac myocytes, diabetes-induced prolongation of cytosolic Ca2+ transient clearance by 43% and decreased SR Ca2+ load by 25% (P < 0.05); these parameters were partially improved after ALT-711 therapy. SERCA2a and RyR2 protein expression was significantly decreased in the myocardium of untreated diabetic rats , but preserved in the treated diabetic group compared to controls. Collectively, our results suggest that, in a model of type 1 diabetes, AGE accumulation primarily impairs SR Ca2+ reuptake in cardiac myocytes and that long-term treatment with an AGE cross-link breaker partially normalized SR Ca2+ handling and improved diabetic cardiomyopathy.Diabetic heart disease is a distinct clinical entity that can progress to heart failure and sudden death. However, the mechanisms responsible for the alterations in excitation-contraction coupling leading to cardiac dysfunction during diabetes are not well known. Hyperglycemia, the landmark of diabetes, leads to the formation of advanced glycation end products (AGEs) on long-lived proteins, including sarcoplasmic reticulum (SR) Ca Several groups (including ours) have reported decreased expression of these SR Ca2+ regulatory proteins in type 1 diabetic rats with cardiac dysfunction  functions as the main regulator of intracellular Caon Bers, . Ca2+ en2+ homeostasis. However, the functional significance of AGEs on SR Ca2+ regulatory proteins in cardiac myocytes and thus on excitation-contraction coupling has not been determined. Our hypothesis was that treatment with an antiglycation therapeutic agent, dimethyl-3-phenacylthiazolium chloride , which chemically breaks AGE cross-links, will normalize SR Ca2+ reuptake in cardiac myocytes and therefore improve diastolic function in type 1 diabetes.Chronic hyperglycemia, the hallmark of diabetes, accelerates the reaction between glucose and proteins and leads to the formation of advanced glycation end products (AGEs). These AGEs form irreversible cross-links throughout the lifetime of many large proteins (such as collagen and hemoglobin), covalently modifying their structure and function Cooper, . Therefon = 11/group): untreated age-matched control group (CON); untreated diabetic group (DX); and ALT-711  treated diabetic group (DX-ALT). Diabetes was induced at 10 weeks of age in DX and DX-ALT groups by a single injection of streptozotocin . The control group received similar volume of vehicle. One diabetic group received dimethyl-3-phenacylthiazolium chloride  for 8 weeks. This therapeutic dose has been previously shown to significantly reduce cardiac AGE level in STZ-induced diabetes , and placed on a heating table to maintain normothermia. Examinations were done using a high-resolution high-frequency digital imaging system with a 21 MHz linear-array transducer and simultaneous ECG recording , following standard techniques as previously described  compound, frozen in dry ice and stained with Masson Trichrome staining.2 and 5% CO2), which contained (in mM): NaCl (135), KCl (5.4), MgCl2 (1), NaH2PO4 (0.33), Hepes (10), glucose (10), and CaCl2 (1). This initial perfusion was followed by a perfusion with tyrode buffer without any CaCl2. Subsequently, collagenase  was added to the calcium free tyrode buffer and recirculated for the rest of the perfusion period. When the heart was soft, the ventricles were minced and the cells were subsequently washed in tyrode solution containing CaCl2 (1). Only rod-shaped cells with sharp margins and clear striations were included in the study. All recordings were made within 5 h of isolation . Fluo-3 was excited by the 488- nm beam of an argon-ion laser, and the fluorescence was acquired at wavelengths > 515 nm in the line scan mode, at a rate of 2 or 6 ms per scan. The magnitude of fluorescent signals was quantified in terms of F/F0, where F0 is baseline fluorescence  for the in vitro measurements were performed, as appropriate. Data were reported as means ± SE. Statistical significance was defined as P < 0.05.A Two-Way ANOVA (treatment and time factors) for the P < 0.05, Figure P < 0.1) for the treated diabetic rats to have a higher body weight than the untreated diabetic rats at 4 and 8 weeks after the induction of diabetes  diabetic and control groups. EF, a surrogate of systolic function, was mildly decreased at 8 weeks after the induction of diabetes compared to baseline values Table . ALT-7112+ into the SR or to histological changes in myocardium rendering it less compliant, hearts were stained with Masson's trichrome to quantify fibrosis. Masson trichrome staining did not reveal significant fibrosis of the left ventricle in (treated and untreated) diabetic rats compared with the control group, suggesting primarily an impaired ventricular relaxation rather than increased myocardial stiffness in our diabetic model  decrease in Ca2+ transient amplitude in isolated cardiac myocytes of (untreated and treated) diabetic rats when compared with controls  diabetes. STZ contains a glucose molecule with a highly reactive nitrosourea side chain, which initiates a specific cytotoxic action on the pancreatic β-cell. A few weeks after STZ injection, rodents develop biochemical and functional myocardial abnormalities, which are the result of chronic hyperglycemia rather than a direct effect of the drug itself. Therefore, diabetic rodents display clinical signs  and cardiovascular complications similar to those in human diabetic patients. Since a close relationship between the STZ dose and the severity of diabetes has been demonstrated and since other parameters  all significantly influence the severity of the model, we previously established in our laboratory a protocol (using a low dose of STZ) to induce a mild form of diabetes and to mimic the early metabolic and cardiac events that occur in diabetic subjects , by use of an antiglycation therapeutic agent: dimethyl-3-phenacylthiazolium chloride , which chemically breaks AGE cross-links. This compound has been tested in several pre-clinical animal studies, and has been shown to significantly reduce cardiac AGE level in STZ-induced diabetic rats and prevent diabetes-induced structural changes in the myocardium  in body weight. Importantly, ALT-711 therapy partially improved diastolic function, as evident by the attenuation of the prolongation in isovolumic relaxation time and MPI observed in treated diabetic rats. In addition, because of the lack of significant fibrosis in the diabetic myocardium, our data suggested that the beneficial therapeutic effects of ALT-711 were primarily due to improved ventricular relaxation.AGEs are proteins that accumulate in the plasma of diabetic patients as a result of the persistent hyperglycemia and are closely linked with cardiovascular diseases. During diabetes, and to a lesser extent during aging, AGEs also accumulate at an accelerated rate in various cell types (in days to weeks) and produce multiple-organ dysfunction Cooper, . In the 2+ transient decay, reduced intra-SR Ca2+ stores and Ca2+ transient amplitude and decreased SERCA2a protein content, which are all consistent with decreased SR Ca2+ reuptake during the relaxation phase, as previously reported by our group (Lacombe et al., 2+ homeostasis and to the diabetic cardiomyopathic phenotype, a complex multifactorial disorder (Poornima et al., 2+ regulatory proteins. Following ALT-711 therapy, we observed a normalization of the Ca2+ transient decay, and a partial restoration of intra-SR Ca2+ stores and SERCA2a protein expression in diabetic cardiac myocytes. These data suggested that ALT-711 treatment decreased excess accumulation of AGEs on the SERCA pump by breaking the cross-links that form during diabetic cardiomyopathy, resulting in partial improvement of SERCA activity and SR Ca2+ reuptake. The enhanced SR Ca2+ reuptake during the relaxation phase of cardiac myocytes resulted in partial improvement of in vivo diastolic function in treated diabetic subjects.In isolated cardiac myocytes of diabetic animals, we observed prolonged Ca2+ transient amplitude in isolated diabetic myocytes. In correlation with these in vitro findings, we observed a moderate, but persistent, reduction of cardiac contractility in diabetic animals treated with ALT-711, as evident by the mild decrease in EF in both untreated and treated diabetic animals. One surprising finding was the persistent decrease in Ca2+ transient amplitude in the face of partially restored SR Ca2+ load and RyR2 protein expression in the diabetic myocardium after ALT-711 treatment. Since abnormal SR Ca2+ release during diastole has been reported in diabetic myocytes (Shao et al., 2+ homeostasis by stabilizing RyR mediated SR Ca2+ release during the relaxation phase of diabetic cardiac myocytes and that AGE accumulation may also impair RyR function leading to diastolic SR Ca2+ leak. Therefore, these data further support the concept that AGE accumulation may play a larger pathogenic role during diastolic dysfunction and diastolic heart failure than during systolic dysfunction (Hartog et al., In contrast, treatment with the AGE cross-link breaker for 8 weeks did not normalize Ca2+ handling during diabetic cardiomyopathy. Since diabetic cardiomyopathy is a multifactorial disorder, these data suggest that AGE accumulation contributes to the impairment in excitation-contraction coupling by altering the function of SR Ca2+ regulatory proteins, leading to a decreased ability for the diabetic myocardium to relax. Therefore, findings from this study provide novel mechanistic insights related to the pathogenic role of AGE accumulation on SR Ca2+ handling in cardiac myocytes. Finally, since there is currently a lack of specific therapy to improve LV relaxation, findings from this study could have direct practical implications for the development of therapeutic strategies for patients with diabetic cardiomyopathy.Treatment with an AGE cross-link breaker partially attenuated the alterations associated with cardiac function and 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.


This paper tests a hypothesis that calpains are involved in Ca2+ -dependent increase of the late sodium current (INaL) in failing heart. Chronic heart failure (HF) was induced in 2 dogs by multiple coronary artery embolization. Using a conventional patch-clamp technique, the whole-cell INaL was recorded in enzymatically isolated ventricular cardiomyocytes (VCMs) in which INaL was activated by the presence of a higher (1μM) intracellular [Ca2+] in the patch pipette. Cell suspensions were exposed to a cell- permeant calpain inhibitor MDL-28170 for 1–2 h before INaL recordings. The numerical excitation-contraction coupling (ECC) model was used to evaluate electrophysiological effects of calpain inhibition in silico. MDL caused acceleration of INaL decay evaluated by the two-exponential fit . MDL significantly reduced INaL density recorded at –30 mV . Our measurements of current-voltage relationships demonstrated that the INaL density was decreased by MDL in a wide range of potentials, including that for the action potential plateau. At the same time the membrane potential dependency of the steady-state activation and inactivation remained unchanged in the MDL-treated VCMs. Our ECC model predicted that calpain inhibition greatly improves myocyte function by reducing the action potential duration and intracellular diastolic Ca2+ accumulation in the pulse train.Calpain is an intracellular CaNaL changes in failing dog ventricular cardiomyocytes in the presence of high intracellular Ca2+. Specifically it decreases INaL density and accelerates INaL kinetics resulting in improvement of myocyte electrical response and Ca2+ handling as predicted by our in silico simulations.Calpain inhibition reverses I NaL) in electrophysiological remodeling and arrhythmias in chronic heart failure (HF) has been extensively studied during the last decade. It has been shown that INaL is augmented and its decay slowed in failing human and dog ventricular cardiomyocytes (VCMs)(see for review NaL into HF mechanisms has been demonstrated in experiments where “correction” of INaL in failing VCMs resulted in: 1) rescue of normal repolarization, 2) decrease beat-to-beat action potential (AP) duration variability, and 3) improvement of Ca2+ handling and contractility NaL has emerged as a novel target for cardioprotection to treat the failing heart NaL, 2) targeting intracellular signaling pathways  that are altered in HF and may have modulatory effect on INaL, 3) modulation of altered Na+ channel (NaCh) microenvironment, such as different expression of auxiliary β-subunits and sub-sarcolemmal cytoskeleton that, in turn, may be responsible for the augmented slowed INaL in HF, 4) combination of two latter mechanisms. The new drug, ranolazine (RAN) that was developed as an antianginal agent, has been demonstrated to specifically inhibit INaLNaL. Ca2+, calmodulin and CaMKII and this Ca2+ signaling pathway can significantly amplify INaL in HF affecting both contractile and electrical performance 1 and β2 NaCh subunits, could be a plausible mechanism to modulate INaL in HF with the aim to improve both contractility and rhythm The role of the late sodium current  numerical model NaL alterations: 1) reduce AP duration, and 2) prevent diastolic intracellular Ca2+ accumulation during the excitation pulse train in silico.Calpain is an intracellular Ca2+-tolerant VCMs varied from 40 to 70%.The study conforms to the Guidelines for Care and Use of Laboratory Animals published by the US National Institutes of Health and was approved by the Animal Care and Use Committee (IACUC protocols 0816 and 0777) of the Henry Ford Health System. Chronic heart failure that is similar by vast array of functional and pathophysiological parameters NaL was measured using a whole-cell patch-clamp technique NaL was assessed by 2 s-long membrane depolarizations to various potentials from a holding potential of −130 mV applied with a stimulation frequency of 0.2 Hz. The bath solution contained (in mM):140 NaCl, 5.0 CsCl, 1.8 CaCl2, 2.0 MgCl2, 5 glucose, 0.002 nifedipine, and 5 HEPES-CsOH buffer (Ph 7.4). The pipette solution contained (in mM): 5 NaCl, 133 CsCl, 0.9 CaCl2 (free [Ca2+] = 1 μM) MgATP, 20 Tetraethylammonium chloride, 1.0 EGTA, and 5.0 HEPES-CsOH buffer. The free [Ca2+] of 1 μM was set in the pipette (and hence inside the cell) to exaggerate abnormal effect of Ca2+ on INaL in HF INaL decay has been approximated by a double exponential fit to INaL starting at 40 ms after the onset of depolarization to –30 mV as previously suggested 1τ and 2τ are the time constants, 40I is INaL instant value 40 ms after membrane depolarization, 1k and 2k are the contributions of each exponents (1 + k2k  = 1), respectively. 5-15 experimental traces were averaged to improve the quality of analysis.The time course of INaL recordings were also analyzed to assess the current density (pA/pF), i.e. INaL  =  (whole cell INaL)/Cm, where Cm is cell electric capacitance that was measured by a voltage ramp (16) in each cell. The INaL data points in the current-voltage relationships were measured as the averaged current density within 200–220 ms after depolarization onset  ranging from –130 mV to −40 mV followed by a testing pulse to −30 mV. INa amplitudes were normalized to that measured at Vp =  −130 mV and the data points were fitted to a Boltzmann function A(Vp):The steady-state inactivation (SSI) was evaluated by a double-pulse protocol with 2 s-duration pre-pulses  and to contribute to diastolic Ca2+ accumulation in HF ventricular myocytes NaL data measured under voltage clamp at 24°C and then apply Q10 factors to calculate model parameters for our full INaL description at 37°C. The details of the model parameters calculations have been described in our previous publications 2+] of 0.125 μM as an initial value in both network SR and junctional SR before application of stimulation pulse train (at 1 Hz or 1.5 Hz).We simulated effect of selective inhibition of calpain on AP shape and diastolic CaMultiple comparisons between treatment groups were made using one-way analysis of variance (ANOVA) followed by Bonferroni's post hoc test or by the non-pared Student's t-test if appropriate. Data are reported as mean±SEM. The significance of SSA or SSI changes were evaluated using F-test  for tabulated values predicted by the model  at a confidence level of 0.95. Differences for both experimental data and model predictions were considered statistically significant for P<0.05.Collagenas type II (291 U/mg) was from Worthington . All other chemicals and enzymes, including calpain inhibitor MDL was purchased from Sigma .NaL decay in VCMs exposed to MDL with that in control, in the absence of the drug, at the intracellular Ca2+ = 1 μM . As it is shown in NaL decay becomes faster than in control cell. Shown are raw traces along with the two-exponential fit (solid lines). Statistical data are given in NaLI became significantly faster in MDL-treated cells as it is obvious from the reduction of the time constants τ1 (upper panel) and τ2 (lower panel).First we compare INaL density in VCMs compared with control and vehicle-treated cells . The solid lines represent theoretical fit to the Eq.2 (see Methods) with the aim to assess SSA parameters (Shown in the graph). The MDL does not affect the voltage-dependence of the SSA as it is evident of the mid-potential position and the slope of the curve, which we found to be unchanged. At the same time MDL reduced the maximum conductance, Gmax, for INaL, which is expected because of the density reduction  as we described in our previous studies 37  =  τ24τ ⋅2.2(24–37)/10 and max_37  =  Imax_24I ⋅1.5(37–24)/10. Finally, max  =  Imax_BM + Imax_LSMI and NaL_maxG are the maximal total INaL peak current density and conductance, respectively. NaL changes affect AP shape and duration at a physiological temperature of 37°C at a pacing rate of 1 Hz. Note that simulated APs are shorter with lower plateau in VCMs treated by MDL. Lower panel of NaL dynamics (profile) during the AP in control and after MDL treatment. Our simulations show that the amplitude and duration of INaL become substantially smaller in MDL-treated cells vs. control (untreated) cells. 2+] dynamics in VCMs of failing heart. In response to the pulse train stimulation with the rate of 1.5 Hz, the diastolic Ca2+ level gradually increases in control conditions. The MDL-induced changes in INaL amplitude and decay kinetics almost completely eliminate this diastolic Ca2+ accumulation pattern. Lower panel of We tested if these experimentally measured Irameters  requiredNaL alterations in amplitude and decay kinetics associated with chronic HF can be rescued by calpain inhibition. Our in silico simulations also demonstrate that the calpain modulation of INaL is physiologically important in HF myocytes, specifically, calpain inhibition greatly improves the myocyte function by reducing the action potential duration, and intracellular diastolic Ca2+ accumulation in the pulse train.For the first time we demonstrate at the single cell level that I2+-regulated cellular processes such as signal trasduction, secretion, cell proliferation, differentiation and apoptosis 2+ handling is one of the important mechanisms for the pathological processes such as apoptosis and necrosis, reperfusion-induced heart stunning, ischemia and hypoxia, hyperthrophy and heart failure, and atrial fibrillation The calpain family is a group of cysteine proteases unique in their dependency on calcium to attain functionally active forms NaL in HF. There are some indications of the involvement of calpain into ion channel gating regulation, namely L-type Ca2+ channels 2+ handling in failing heart and may be considered as a novel approach to modulate INaL current its related arrhythmias, and improve contractility NaL.Our findings thus suggest a novel cellular and molecular mechanism to modulate NaCh that could be targeted to prevent pathophysiological consequences related to the augmented INaL modulation has been confirmed in our previous studies 2+ handling can be mediated by calpain 2+- induced fodrin cytoskeleton degradation will likely improve Ca2+ handling in HF.Fodrin-based cytoskeleton, an important element of the NaCh microenvironment in heart, is a dynamic structure that is altered under a variety of pathological conditions  that are involved in INaL formation NaL decay in the presence of calpain inhibition  deficiencies caused by chronic HF NaL reported in the present study is physiologically relevant. To address this question, we have carefully measured and analyzed specific characteristics of INaL in control and in the presence of MDL  by about a factor of two during most of the plateau. This substantial “scaling” contributes not only to in AP duration shortening but also in to that AP plateau becomes substantially lower. This insight does not directly follow from the voltage clamp data and even not from AP simulations, because of a complex interplay of many Na+- and Ca2+- dependent mechanisms in ventricular cells reproduced by the dynamic ECC model.It has been established that Ie of MDL  and thenNaL in HF contributes to the diastolic [Ca2+]i accumulation in VCM of failing hearts during the pulse train NaL by the MDL significantly reduces this accumulation [Ca2+]i as it is predicted in silico  AP duration and prevents diastolic Ca2+ accumulation. Therefore, this Ca2+-dependent signaling cascade may serve as a plausible target to regulate INaL and its related electrical and contractile deficiencies in failing heart.Based on our present results with the specific calpain inhibitor MDL in ventricular cardiomyocytes isolated from failing dog hearts, we conclude that Ca


In ASM, plasma membrane Ca2+ fluxes form a key component of [Ca2+]i regulation. There is now growing evidence that the bidirectional plasma membrane Na+/Ca2+ exchanger (NCX) contributes to ASM [Ca2+]i regulation. In the present study, we examined NCX expression and function in human ASM cells under normal conditions, and following exposure to TNFα or IL-13. Western blot analysis showed significant expression of the NCX1 isoform, with increased NCX1 levels by both cytokines, effects blunted by inhibitors of nuclear factor NF-κB or mitogen-activated protein kinase. Cytokine-mediated increase in NCX1 involved enhanced transcription followed by protein synthesis. NCX2 and NCX3 remained undetectable even in cytokine-stimulated ASM. In fura-2 loaded human ASM cells, NCX-mediated inward Ca2+ exchange as well as outward exchange (measured as rates of change in [Ca2+]i) was elicited by altering extracellular Na+ and Ca2+ levels. Contribution of NCX was verified by measuring [Na+]i using the fluorescent Na+ indicator SBFI. NCX-mediated inward exchange was verified by demonstrating prevention of rising [Ca2+]i or falling [Na+]i in the presence of the NCX inhibitor KBR7943. Inward exchange-mode NCX was increased by both TNFα and IL-13 to a greater extent than outward exchange. NCX siRNA transfection substantially blunted outward exchange and inward exchange modes. Finally, inhibition of NCX expression or function blunted peak [Ca2+]i and rate of fall of [Ca2+]i following histamine stimulation. These data suggest that NCX-mediated Ca2+ fluxes normally exist in human ASM , and contribute to enhanced [Ca2+]i regulation in airway inflammation.Enhanced airway contractility following inflammation by cytokines such as tumor necrosis factor alpha (TNFα) or interleukin-13 (IL-13) involves increased intracellular Ca Following [Ca2+]i elevation, mechanisms to reduce Ca2+ levels are activated. In this regard, SR Ca2+ reuptake and plasma membrane Ca2+ ATPase are major mechanisms. However, an additional mechanism that received relatively little attention is the bidirectional Na+/Ca2+ exchanger (NCX).In airway smooth muscle (ASM), regulation of intracellular Ca2+]i regulation of cardiac muscle is well-established + gradient to exchange 1 Ca2+ for 3 Na+ (electrogenic). Efflux mode NCX in cardiac muscle is widely accepted 2+ influx and contraction in vascular smooth muscle 2+]i homeostasis.A role for NCX in [Ca2+]i regulation in cow 2+]i regulation of human ASM has been barely examined. Recently, NCX-mediated Ca2+ influx was reported in human ASM, apparently linked to SR Ca2+ store depletion via the regulatory protein STIM1 2+ fluxes via NCX are present in human ASM, this would represent a potentially rapid mechanism for regulating SR Ca2+ content in opposing ways: 1) providing Ca2+ via influx mode, increasing [Ca2+]i and facilitating SR refilling; or 2) removing Ca2+ via efflux mode, decreasing [Ca2+]i, and SR refilling. Accordingly, a major goal of the present study was to establish the importance of influx vs. efflux modes of NCX in human ASM.In ASM, NCX has been reported to participate in [Ca2+]i regulation is a key component of the pathophysiology of airway diseases such as asthma 2+]i and contractility of ASM including in humans 2+]i levels as well as enhancing Ca2+ availability to refill the SR (and thus greater Ca2+ for agonist responses). In the present study, we used real-time imaging of [Ca2+]i and intracellular Na+ ([Na+]i) in human ASM cells to determine NCX fluxes under normal conditions and with exposure to TNFα and IL-13. Pharmacological inhibitors and siRNA were used to inhibit NCX expression or activity to determine the importance of this mechanism in [Ca2+]i regulation.It is well-recognized that altered i imaging of human ASM cells using fura-2 have been previously described 2+]i. However, due to the need for additionally altering the Na+ concentrations for examining NCX, the current study was conducted using Tyrodes' solution 2+ Tyrodes and baseline fluorescence established. A custom-built fluid level controller allowed cell perfusion with rapid exchange of perfusate (<300 ms). [Ca2+]i responses of at least 10 cells per chamber were obtained. Fura-2-loaded cells were alternately excited at 340 and 380 nm and emissions at 510 nm collected separately at 1 Hz . Results were expressed using the ratio of the 340 nm/380 nm wavelengths. Quantification of [Ca2+]i levels was performed from fura-2 levels using previously described calibration procedures The techniques for [Ca+]i in cardiac muscle 2+]i imaging. In a subset of preparations, calibration of [Na+]i was performed in vitro by exposing SBFI-loaded ASM cells to different extracellular Na+ . A mixture of two solutions with equal ionic strength in different proportions was used to vary extracellular Na+: one containing 145 mM Na+  and zero K+, and the other 145 mM K+  but zero Na+. Solutions were buffered with 10 mM HEPES and additionally contained glucose and EGTA. Ca2+ and Mg2+ levels were maintained as in normal Tyrodes' solution Other investigators have previously reported using the ratiometric cell-permeant dye SBFI/AM (Invitrogen) for real-time measurement of [Na2+/140 mM Na+) and baseline [Ca2+]i measured 2+ ([Ca2+]o) was then removed  to “Na+-load” cells via activation of outward Ca2+ exchange mode NCX (reflected by decreased [Ca2+]i). Inward Ca2+ influx via L-type channels was inhibited using 1 µM nifedipine. Reduced [Ca2+]i can result in Ca2+ release from the SR. Accordingly, SR Ca2+ release was simultaneously inhibited by exposing cells to 10 µM ryanodine (inhibiting Ca2+ release via ryanodine receptors) and 10 µM Xestospongin C . Thus, Na+ loading was achieved while preventing the activation of influx-mode NCX and SR Ca2+ dynamics. After 10 min, the perfusate was rapidly changed (<300 ms for complete replacement of bath solution) to 2 mM Ca2+/4 mM Na+ Tyrodes (zero-Na Tyrodes), activating inward exchange mode NCX. Nifedipine, ryanodine and Xest-C were maintained. To determine the mechanism underlying [Ca2+]i changes, cells were pre-exposed to the NCX inhibitor KBR7943 (10 µM) in zero-Ca Tyrodes for 5 min prior to activation of inward exchange mode NCX by zero-Na Tyrodes in the continued presence of KBR7943.Control, TNFα-exposed and IL-13-exposed ASM cells were loaded with fura-2 (as above). Cells were perfused with normal Tyrodes  along with 10 µM cyclopiazonic acid , which results in increased [Ca2+]i without activation of either NCX mode (due to continued SR Ca2+ leak in the absence of reuptake). Some decrease in the plateau of the [Ca2+]i response was expected due to eventual activation of the plasma membrane Ca2+ ATPase. When [Ca2+]i has more or less reached a plateau, the perfusate was rapidly changed (<300 ms for complete replacement of bath solution) to zero-Ca Tyrodes, activating outward exchange mode NCX.Following determination of baseline [Ca+]i was directly measured during activation of outward exchange mode NCX. Given the exchange of Na+ for Ca2+, [Na+]i was expected to fall when exposed to zero-Na, zero-Ca Tyrodes, but rapidly increase when exposed to zero-Ca Tyrodes. Specificity of NCX was verified in NCX1 siRNA transfected cells.In a second set of experiments using SBFI-loaded cells, [Na2+]i responses to 10 µM histamine were evaluated in fura-2 loaded ASM cells  perfused with normal Tyrodes' solution. Peak [Ca2+]i and the rate of fall of the response were measured. Following a thorough washout with Tyrodes' solution, cells were exposed to KBR7943, and [Ca2+]i responses to histamine reevaluated in the presence of KBR7943. In a second set of experiments, [Ca2+]i responses to histamine were compared in cells transfected with Lipofectamine alone, NCX1 siRNA or negative control siRNA.i/Na+ responses were analyzed using ANOVA with a Dunnett's post hoc test.Six bronchial samples were used to obtain ASM cells. All biochemical and molecular biology experiments  were repeated at least 3 times, and Western blot studies of human ASM cells showed significant expression of NCX1 protein , with noExposure to 20 ng/ml TNFα or 50 ng/ml IL-13 for 24 h significantly increased NCX1 protein expression in ASM .To determine potential mechanisms by which NCX1 protein expression is enhanced by cytokines, ASM cells were pre-exposed to SN50  or PD98059  for 1 h prior to TNFα or IL-13 exposure (in the continued presence of inhibitor). Subsequent Western analysis found that inhibition of either NF-κB or MEK1/2/ERK1/2 MAP kinases significantly blunted cytokine-induced increase in NCX1 ; p<0.05.To investigate whether cytokine-induced increase in NCX1 was due to newly synthesized protein, the effects of the protein synthesis inhibitor cycloheximide and the transcription inhibitor actinomycin D were assessed . Human A2+/140 mM Na+), baseline [Ca2+]i levels ranged between 80–140 nM (110 ± 10 nM). Following a slow increase in [Ca2+]i levels was observed with removal of Ca2+ by perfusion with zero-Ca Tyrodes  ranged from 8.3 to 49.3 nM s−1 in control cells. Repetition of the inward exchange protocol in control experiments resulted in a<10% decrease in inward exchange rate. Accordingly, time-related bias in the inward exchange protocol was ignored.In fura-2 loaded control ASM cells perfused with normal Tyrodes solution , and consistent with previous studies 2+ exchange following perfusion with zero-Na  Tyrodes in Na-loaded cells was significantly smaller in NCX siRNA transfected cells, but unaffected by TRPC3 siRNA  of Ca p<0.05; . Similar p<0.05; . Transfe p<0.05; .2+]i levels were comparable to those of controls. In these cytokine-exposed cells, following Na-loading using the protocol above, perfusion with zero-Na Tyrodes resulted in significantly faster inward Ca2+ exchange compared to controls , initial [Na+]i levels  was ∼10 mM. A slow increase in [Na+]i levels is observed with removal of Ca2+ using zero-Ca Tyrodes (with SR inhibition) . Under these conditions, estimated “peak” [Na+]i levels did not exceed 25 mM. Subsequent rapid perfusion with zero-Na Tyrodes resulted in a rapid decrease in [Na+]i levels  siRNA did not significantly influence changes in [Na+]i via NCX under control conditions, or the enhancing effect of cytokines , which eventually reached a plateau and then decreased slowly (likely reflecting continued efflux via plasma membrane Ca2+ ATPase). Rapidly reintroduction of Na+ (zero-Ca) Tyrodes resulted in a rapid decrease in [Ca2+]i levels  increased i response with an initial high peak (range 425–550 nM) which rapidly decayed to a lower plateau level . Peak [Ca2+]i was smaller and the rate of decay of the [Ca2+]i response were both significantly slower in the presence of KBR7943 , compared to Lipofectamine alone or negative siRNA , while the rate of fall of [Ca2+]i was significantly slower  providing Ca2+ via influx (i.e. inward Ca2+ exchange) mode, increasing [Ca2+]i and facilitating SR refilling; or 2) removing Ca2+ via efflux mode (i.e. outward Ca2+ exchange), decreasing [Ca2+]i, and SR refilling. Furthermore, we found that the pro-inflammatory cytokines TNFα and IL-13, both known to enhance ASM [Ca2+]i responses and airway contractility, increase NCX protein expression and enhance NCX-mediated Ca2+ fluxes. Thus, enhanced NCX expression and activity are potential mechanisms by which inflammation leads to increased airway contractility.In the present study, we demonstrate that human ASM cells express NCX protein, and display NCX-mediated inward Ca2+ exchange) being widely accepted 2+ influx can also occur in cardiac muscle, as demonstrated using inhibitors such as KBR7943 2+ regulatory mechanisms in smooth muscle. Accordingly, these data are considerably less on NCX in smooth muscle.The role of NCX has been best established in cardiac muscle 2+ fluxes have been most well-established in vascular smooth muscle: a) NCX-mediated Ca2+ influx contributes to myogenic vasoconstriction in rat cremaster muscle arterioles ++ levels 2+ influx facilitates SR Ca2+ refilling 2+]i homeostasis. A recent electrophysiology based study suggests that reverse (or influx) mode NCX is active in ASM 2+]i oscillations were largely unaffected by KBR7943 (in contrast to inhibition of L-type Ca2+ channels) 2+ efflux appears to be important for [Ca2+]i homeostasis in guinea pig stomach 2+ influx is important for pacemaker activity 2+ influx and efflux in smooth muscle.Of the three major mammalian NCX isoforms, NCX1 has been shown to be expressed by a wide variety of tissues (including ASM) 2+]i in ASM involves SR Ca2+ release and reuptake as well as plasma membrane Ca2+ influx and efflux 2+ influx is known to occur through voltage-gated 2+ depletion (i.e. SOCE) 2+]i homeostasis involves mechanisms to reduce Ca2+ levels. Here, the role of SR Ca2+ reuptake is well-established, and some studies have established that ASM expresses functional plasma membrane Ca2+ ATPases It is now well-established that regulation of i response to histamine by both KBR7943 and NCX1 siRNA. Indeed, the significant NCX-mediated inward Ca2+ exchange that was noted in our study indicate a rapid mechanism for SR Ca2+ refilling, in addition to the recently established SOCE mechanism in ASM. As mentioned above, in vascular smooth muscle, it has been suggested that the proximity of NCX to perimembranous SR would allow for NCX-mediated influx to refill SR. Whether a similar situation occurs in ASM has not been directly demonstrated, but has been suggested by some investigators 2+ refilling following NCX activation. Indeed, in pilot studies, we have found that NCX1 is expressed within caveolar fractions of human ASM cells . Future studies will examine this important aspect of NCX function.Previous results regarding functional roles of NCX in ASM appear conflicting. NCX mediated Ca2+]i homeostasis in ASM is not clear. Even in vascular smooth muscle and other tissues where efflux mode NCX has been examined, interpretation may be limited by the fact that some were conducted in ASM tissues (i.e. not cells) where rapid changes in extracellular Ca2+ or Na+ required to verify NCX  cannot be satisfactorily achieved. Furthermore, pharmacological NCX inhibitors are not very specific 2+]i sensitivity of inward exchange vs. outward exchange mode NCX differs 2+]i as well as intracellular Na+ ([Na+]i) to determine NCX fluxes. Using pharmacological inhibitors as well as siRNA, we determined that NCX-mediated outward exchange does make a significant contribution to [Ca2+]i in human ASM, as evidenced by a) significant changes in outward Ca2+ exchange (or inward Na+ exchange) under conditions favoring NCX; b) significant effect of NCX1 siRNA; and c) the slowing of the decay of [Ca2+]i responses to histamine. These findings represent a novel mechanism for decreasing [Ca2+]i following agonist stimulation in addition to SR Ca2+ reuptake and plasma membrane Ca2+ ATPase. However, it must also be noted that the relative contribution of the outward vs. inward exchange modes of NCX to [Ca2+]i regulation in ASM may not be the same: inhibition of NCX (either by KBR7943 or siRNA) appeared to have a greater effect on the peak [Ca2+]i response to histamine  NCX siRNA suggest that upregulation of NCX expression and function may contribute to the altered ASM [Ca2+]i regulation that occurs in airway inflammation.Recent reviews have suggested that Na2+]i and [Na+]i changes in human ASM cells. Empirical calibrations of the ionic concentrations were performed to allow assessment of relative changes with inflammation or with drug exposures. However, it must be noted that the usual 3∶1 Na+: Ca2+ molar stoichiometry of NCX cannot be easily derived using fluorescent techniques such as these since only the overall [Na+]i (or [Ca2+]i) is being measured, rather than the dynamic peri-membranous ionic fluxes. Accordingly, calibrated signals provide values of nM Ca2+ (fura-2) but mM Na+ (SBFI). Therefore, we have used the terms “inward exchange” to represent NCX-mediated influx and “outward exchange” to represent NCX-mediated efflux, to highlight the fact that actual fluxes were not measured.In this study, we used fluorescent dyes such as fura-2 and SBFI to measure [Ca2+]i responses to agonist stimulation. These novel data point to a rapid mechanism for regulating [Ca2+]i in ASM. The pro-inflammatory cytokines TNFα and IL-13, both known to enhance ASM [Ca2+]i responses and airway contractility, increase NCX expression and enhance NCX-mediated Ca2+ fluxes. Thus, enhanced NCX expression and activity are potential mechanisms by which inflammation leads to increased airway contractility.In conclusion, in the present study, we demonstrate that human ASM cells express functional NCX1 protein with both inward exchange and outward exchange modes NCX contributing to [Ca


This high-throughput signaling consumes ATP.Ca2+ indirectly, regulate mitochondrial ATP production. While, clearly, Ca2+-calmodulin-CaMKII activity regulates ATP consumption, whether it has a role in the control of ATP production is unknown.We have previously demonstrated that basal AC-cAMP/PKA signaling directly, and Ca2+, flavoprotein fluorescence and spontaneous AP firing rate. We measured cAMP, ATP and O2 consumption in cell suspensions. Graded reductions in basal CaMKII activity by KN-93 (0.5–3 µmol/L) or AIP (2–10 µmol/L) markedly slow the kinetics of intracellular Ca2+ cycling, decrease the spontaneous AP firing rate, decrease cAMP, and reduce O2 consumption and flavoprotein fluorescence. In this context of graded reductions in ATP demand, however, ATP also becomes depleted, indicating reduced ATP production.We superfused single, isolated rabbit SANC at 37°C with physiological saline containing CaMKII inhibitors, (KN-93 or autocamtide-2 Related Inhibitory Peptide (AIP)), or a calmodulin inhibitor (W-7) and measured cytosolic CaCaMKII signaling, a crucial element of normal automaticity in rabbit SANC, is also involved in SANC bioenergetics. Experimental and computational data  protein phosphorylation  couple the function of proteins of both clocks to regulate SANC normal automaticity 2+ activated adenylyl cyclase produces a high basal level of cAMP compared to ventricular myocytes 2+ over the entire physiological Ca2+ range. Specifically, a reduction in intracellular Ca2+ by BAPTA reduced the cAMP level 2+ pumping by SR Ca2+-ATPase is regulated by phospholamban phosporylation of both Ser16 (PKA) and Thr17 (CaMKII) 2+ current amplitude and a reduction in Ca2+ influx 2+ and a decrease in the availability of Ca2+ for pumping into the SR. A reduction in cytosolic Ca2+ which leads to a reduction of Ca2+ activation of adenylate cyclase (AC), therefore, reduces cAMP activation of PKA, reduces phospholamban phosporylation and Ca2+ cycling kinetics. We have recently shown that this feed-forward basal Ca2+-cAMP/PKA signaling that drives spontaneous APs, not only regulates ATP consumption of SANC, but also regulates mitochondrial ATP production 2+ chelator, BAPTA, not only blocks Ca2+-dependent activation of CaMKII and suppresses AC/PKA signaling, but also reduces ATP in the context of a reduced ATP demand Both, cAMP-mediated, protein kinase A-dependent (PKA) protein phosphorylation and CaTo decrease CaMKII activity we chose two concentrations of CaMKII inhibitors that had been previously shown Concentrations of the calmodulin inhibitor, W-7, were chosen to match the reduction in AP firing rate achieved with inhibition of CaMKII activity. Graded concentrations of W-7 (20 to 50 µM) for 5 min, also produced graded reductions in the spontaneous AP firing rate . On aver2+ transients measured with indo-1 during partial inhibition of CaMKII or calmodulin . CaMKII or calmodulin inhibitors also markedly slow the kinetics of Ca2+ removal from the cytosol in single SANC . That the reduction in ATP effected by these agents is correlated with the decrease in O2 consumption strongly suggests that direct inhibition of Ca2+-cAMP/PKA-CaMKII reduces mitochondrial ATP production. Furthermore, application of AIP (10 µmol/L), to reduce CaMKII activity in single SANC decreases the flavoprotein fluorescence  or 3 µM KN-93 and glibenclamide (a K-ATP channel blocker) and compared it to the reduction in ATP in the presence of 3 µM KN-93 alone. In the presence of KN-93, ATP was reduced by 33±7% (n = 3), in the presence of KN-93 and pinacidil ATP was reduced by 27±3% (n = 3), and in the presence of KN-93 and glibenclamide ATP was reduced by 38±7% (n = 3). Therefore, during CaMKII inhibition, activation of K-ATP channels did not significantly prevent ATP depletion while blocking of K-ATP channels did not produce significant additive effects on ATP reduction.2+-cAMP/PKA signaling that has been documented recently 2+ pumping is reduced, myofilament contraction is reduced, Na+/K+ pump activity is reduced, and may lead to an increase in intracellular Na+ and a reduction in Na+-Ca2+ exchange current that is crucial to prompt spontaneous APs of SANC. However, in permeabilized SANC, in which the ATP level remains intact, both AIP (CaMKII blocker) and PKI (PKA inhibitor) markedly reduced the spontaneous local Ca2+ releases and SR Ca2+ content Basal CaMKII signaling in the absence of β-adrenergic stimulation is crucial for rabbit SANC to generate spontaneous rhythmic AP’s. The most important novel finding of our study is that when ATP demand in SANC is reduced by interfering with CaMKII or calmodulin activity, these cells become depleted of ATP, indicating concurrent reduction in both ATP generation and demand. Thus, CaMKII signaling, a crucial element of normal automaticity in rabbit SANC, is tightly linked to SANC bioenergetics, specifically to both ATP production and utilization. This result is similar to the reduction in ATP by interfering with basal Ca2+ influx into the cytosol triggered by each AP (reduced L-type Ca2+ channel phosphorylation), and 2. reduced Ca2+ release from the SR, related, in part at least, to reduced SR Ca2+ pumping 2+ loading and Ca2+ release, which leads to a reduction in Ca2+ activation of AC/cAMP-PKA signaling. Moreover a reduction in the ATP level, as noted, may directly reduce the activity of SR Ca2+ pump. Because CaMKII inhibition reduces cAMP, a direct activator of fI, a reduction in cAMP can decrease the fI activation, which could also be implicated in the reduction of spontaneous AP firing rate.Graded CaMKII inhibition by KN-93 or AIP causes graded reductions in cAMP in SANC suspensions . TherefoWe observed that in the presence of AIP the flavoprotein fluorescence is reduced by 52±9% from the control value . We measATP channels are activated with a decrease in intracellular ATP level. The activation of these KATP channels may play an important role during ischemia ATP channels under basal conditions is still controversial. There are no significant differences in the basal AP parameters between WT and Kir6.2 KO SANC mice Ca,L, If and SERCA pump. Depolarization of rabbit SANC membrane occurs not only when CaMKII activity is reduced (as documented in 2+ is buffered K2+ handling proteins and differences in the relative role of SERCA pump vs. Na+-Ca2+ exchange) in rabbit SANC or the genetic manipulation in mice account for the differences noted in AP firing rate. These factors might alter the requirement for basal CaMKII activity to maintain basal AP firing rates and compensatory mechanisms (e.g an increase in Ca2+ influx) in the knock-out mice.While the spontaneous AP firing rate reduction and ATP depletion demonstrated in the present study  are effe2+; since cytosolic Ca2+ is coupled to ATP hydrolysis via mitochondrial Ca2+ flux, a decrease in mitochondrial Ca2+ in response to CaMKII inhibition can decrease the activity of mitochondrial enzymes involved in ATP production. We have recently shown, however, that directly disabling mitochondrial Ca2+ influx or efflux in SANC does not affect the ATP level 2+ loading and release leads to a reduction in AC-cAMP/PKA-dependent phosphorylation . However, because both mitochondria and activated autophosphorylated CaMKII predominantly localize close to the SANC sarcolemmal membrane CaMKII activity can affect mitochondrial ATP demand coupling to ATP supply by impacting at least three inter-dependent signaling pathways : (i) a drylation  B–C of s2+ nor the ATP level Two different indirect mitochondrial mechanisms can also play a role in matching ATP supply to demand in the heart: (i) mitochondrial respiration and ATP synthesis in the heart are coupled to creatine and synthesis of phosphocreatine (PCr) by the mitochondrial creatine kinase (CK), a key component of the cellular system of creatine kinase CK. In the ventricular myocytes CK is localized to myofibrils at M-line and I-band of sarcomeres and subsarcolemmal space and serve as a control mechanism to match ATP supply to demand Ca+ currents 2 consumption measurements. But because KN-92 does not share the CaMKII-independent L-type Ca2+ channel antagonist actions of KN-93  It has been reported that KN-93 may affect I2+, and flavoprotein autofluorescence in single isolated rabbit SANC and ATP, cAMP and O2 consumption in SANC suspensions.In the presence and absence of specific CaMKII inhibitors, we measured the spontaneous AP firing rate from cell contraction or electrophysiology recordings, intracellular CaSingle, spontaneously beating, spindle-shaped SANC were isolated from New Zealand White rabbit hearts as previously described 2, 5 HEPES, 1.8 CaCl2, and 5.5 Glucose, pH 7.4 with NaOH. Spontaneous APs were measured via perforated patch-clamp technique with 35–50 µmol/L β-escin (Sigma) added to the pipette solution that contained (in mmol/L): 120 K-gluconate, 2.5 NaCl, 2.5 MgATP, 2.5 Na2ATP, 5 HEPES and 20 KCl, and titrated to pH 7.2 with KOH. APs were recorded using an Axopatch-200 B patch-clamp amplifier . To quantify the rate of spontaneous AP firing contraction, in cells in which APs were not measured directly, cells were imaged with an LSM-510 inverted confocal microscope using a 63x/1.4 N.A. oil immersion lens (Carl Zeiss), and 633 nm He-Ne laser excitation. The bath solution and the temperature were the same as in spontaneous AP recording experiments. Transmitted optics line-scan images (using 512x1 pixels at 21.5 pixel/µm and 0.8 ms/line) were recorded with a scan line oriented along the short axis of the cell to quantify the SANC contraction. The spontaneous AP firing rate was calculated as the time between the successive contraction periods. For AP and contraction recordings cells from at least 3 rabbits were used for the study of each drug.The cell spontaneous AP firing rate from contraction or electrophysiological recordings were measured in Tyrode solution at 35±0.5°C containing (in mmol/L): 140 NaCl, 5.4 KCl, 2 MgCl2+ was measured via calibrated Indo-1 fluorescence in Tyrode solution (described above) at 35±0.5°C as described before 2+]i was calculated according to the equation [Ca2+]i = βxKdx(R-Rmin)/(Rmax-R), using a Kd of 844 nM. The average Rmin , Rmax , and β  for the fluorescence system were determined by sequential exposure of SANC to a high potassium, zero- Ca2+ solution  containing metabolic inhibitors  (2), the same solution with 1 mM EGTA and 20 µM ionmycin (for Rmin). Measurements were taken when the fluorescence at both wavelengths reached stable values, and (3) high Ca2+ Tyrode’s solution (5 mM Ca2+ substitutes for EGTA) was used for determining Rmax. Average Rmax, Rmin and β were 1.9±0.16, 0.9±0.01, and 2.1±0.6, respectively (n = 10). All these measurements (step 1 to 3) were performed in Tyrode solution (described above) at 35±0.5°C. Note that this calibration method has two limitations: 1) a change in pH from the calibrated solution , 2) imprecision of the Kd of Indo-1. For Ca2+ recordings cells from at least 5 rabbits for each drug application were used.Intracellular CaThe endogenous autofluorescence of mitochondrial flavoproteins was imaged in Tyrode solution (see above) at 35±0.5°C by a LSM510 confocal microscope (see above) using a 40x/1.3 N.A. oil immersion lens, as previously described 2 consumption in aliquots of a given suspension containing an equal amount of protein and viable cells. Therefore, to compare results of different suspensions studied on different days, drug effects in aliquots from all suspensions were expressed as a % of their respective controls. Lack of significant contamination by atrial cells was verified by classifying morphology and counting cells under the microscope. Finally, to insure the validity of comparison between ATP, cAMP and spontaneous beating rate, the beating rates were measured in single SANC extracted from the same suspension in which ATP, cAMP and O2 were measured.Because the number of healthy and functional cells in cell suspensions varies from preparation to preparation (from 20 to 30%), comparability among responses to CaMKII or calmodulin inhibitors in different suspensions was insured by measuring ATP, cAMP and O2 consumption, were measured on single cells from the same cell suspension.Oxygen consumption was measured in cell suspensions of spontaneously beating SANC using Clark-type electrodes . SANC suspensions were centrifuged at 100 g for 5 min, the supernatant removed, and SANC were incubated in fresh Tyrode solution (as above) that was not oxygenated prior and during these experiments. The cell suspension was divided equally into 2 aliquots: the first aliquot for specific CaMKII or calmodulin inhibition protocols, and the second to serve as a control. Cell suspensions were stirred gently at 36°C, and measurements were acquired for 2 min under control conditions, and 3 min following application of CaMKII or calmodulin inhibitors. Measurements in the control were made for 5 min, and reported at the end of 5 min. Oxygen consumption in the presence of drug was normalized to the control level for each experiment . To guarantee that cells within the suspension were functioning normally and reacting to the drug application, spontaneous beating rates, prior and after specific pharmacological probes used to probe OThe cell suspension (incubated in fresh Tyrode solution (as above)) was divided equally into 6 aliquots: the first aliquot was treated with a CaMKII or calmodulin inhibitor, the second with 100 µmol/L DNP for 15 min, in order to uncouple SANC energetics and to decrease ATP level; the third and the fourth were used as controls, and the other two remaining aliquots were used to measure cAMP (see above). After a 5-min incubation period at 36°C  the reactions were stopped by adding to the cell suspensions, in a 1∶10 ratio, TRIS-lysis buffer that was heated in a boiling water bath. The TRIS-lysis buffer contained (in mmol/L) 100 TRIS and 4 EDTA, with pH adjusted to 7.75 with HCl. The extracts were heated in boiling water for 3-min and centrifuged at 12,755 g for 2-min at room temperature. Total protein concentration was determined by a BCA™ Protein Assay (Pierce), and the number of viable (spontaneously contracting) cells was determined from the original cell suspension before the reactions were stopped. The ATP concentration was determined by a bioluminescence assay kit, HS II (Roche), and normalized to the cell ATP level after 15-min incubation with 2,4-dinitrophenol, as previously described Two groups of cell suspensions (control and drug) were pretreated with 100 µmol/L IBMX prior to drug applications. The reaction was stopped by adding the cell suspensions, in 1∶1 ratio, to heated (in a boiling water bath) TRIS-lysis buffer (see above). The extracts were heated in boiling water bath for 5 min and centrifuged at 12,755 g for 2 min at room temperature. The supernatants were collected in fresh tubes and cAMP levels were measured by a LANCE cAMP detection kit (PerkinElmer) and expressed as percentage of the drug-free, control group.CaMKII inhibitor KN-93, CaMKII inactive analog KN-92 and a specific cell permeable PKA inhibitor peptide autocamtide-2 Related Inhibitory Peptide (AIP), Myristoylated were obtained from EMD Chemicals; 3-Isobutyl-1-methlxanthine (IBMX), N-(6-Aminohexyl)-5-chloro-1-naphthalensulfonamide hydrochloride (W-7), PKA inhibitor (H-89), NaCN and 2,4-dinitrophenol (DNP)) were obtained from Sigma.2+ measurements). ANOVA test was employed to compare paired samples with different drug concentration. P<0.05 was taken to indicate statistical significance.Data are presented as mean±SEM. T-test was employed to compare the drug effect in paired samples (Ca