The heart glycoside, ouabain (30 nM), and a recently developed inhibitor of diacylglycerol lipases, DO34 (1 μM), cause similar hypertrophy responses, and both responses are accompanied by equivalent increases of basal Ca2+ and diacylglycerol. These results together suggest that basal Ca2+ and diacylglycerol form a positive feedback signaling loop that promotes execution of cardiac growth programs in these human myocytes. Given that basal Ca2+ in myocytes depends strongly on the Na+ gradient, we also tested whether nanomolar ouabain concentrations might stimulate Na+/K+ pumps, as described by others, and thereby prevent hypertrophy. However, stimulatory effects of nanomolar ouabain (1.5 nM) were not verified on Na+/K+ pump currents in stem cell–derived myocytes, nor did nanomolar ouabain block hypertrophy induced by endothelin-1. Thus, low-dose ouabain is not a “protective” intervention under the conditions of these experiments in this human myocyte model. To summarize, the major aim of this study has been to characterize the progression of hypertrophy in human embryonic stem cell–derived cardiac myocytes in dependence on diacylglycerol and Na+ gradient changes, developing a case that positive feedback coupling between these mechanisms plays an important role in the initiation of hypertrophy programs.Human embryonic stem cell–derived cardiomyocytes develop pronounced hypertrophy in response to angiotensin-2, endothelin-1, and a selected mix of three fatty acids. All three of these responses are accompanied by increases in both basal cytoplasmic Ca Details of this coupling and the major signaling pathways leading to cardiac myocyte growth are summarized subsequently.Pathological cardiac myocyte hypertrophy  is a com2+- and DAG-dependent mechanisms are definitively engaged. Local and global Ca2+ signals are implicated to promote growth programs via the NFAT transcription factors, whose phosphorylation state and presence in the cytoplasm are under control of Ca2+/calmodulin (CAM)-dependent protein kinases and the Ca2+-dependent phosphatase, calcineurin [In the early stages, both Cacineurin . Accumulcineurin , especiacineurin , 72, leacineurin . Notablycineurin .2+-dependent signals represents a potentially very powerful positive feedback loop in the initiation of hypertrophy responses. Most DAG that activates PKCs is generated by phospholipase C (PLC) activities under the control of G protein–coupled receptors (GPCRs) and growth factor receptors [2+ signals during which PLC activity is promoted by the direct binding of Ca2+ to C2 domains [2+ signals generated by IP3-dependent Ca2+ release at the nuclear envelope are suggested to become importantly involved [The molecular and cellular coupling of DAG- and Caeceptors . The acteceptors  becomes  domains  of PLCs  domains . Local Cinvolved , 57. Thainvolved , 55.2+ signaling, as part of its role in hypertrophy signaling in myocytes, is also unambiguously established. First, L-type CaV Ca2+ channel activity has been shown to be specifically enhanced by DAG generated within the cytoplasm, as opposed to exogenous DAG applied to the outside of cells [2+ and Na+ influx that is demonstrated to be essential for the initiation of hypertrophy programs [2+ signals generated directly by the influx of Ca2+ through TRPC channels, and it may involve the indirect enhancement of global myocyte Ca2+ signaling via cytoplasmic Na+ loading with concomitant Ca2+ loading via Na+/Ca2+ exchange [That DAG signaling promotes Caof cells . Second,programs , 51, 58.exchange .+ and heart glycoside signaling beyond Na+ gradient coupling to Ca2+ via Na+/Ca2+ exchange. It must suffice here to point out that a great deal of evidence in fact supports the idea that Na+/K+ pumps can act as signaling molecules with interactions to other proteins that are highly dependent on heart glycosides [+/Ca2+ exchanger [+ gradient. Interventions that inhibit constitutive Na influx, such as NHE1 Na+/H+ exchange knockdown [+ influx in response to stretch of cardiac myocytes is documented to increase and likely mediate the initiation of hypertrophy programs [2+ and Na+ influx in cardiac myocytes; the simple hypothesis clearly emerges that hormonal activators of hypertrophy programs, such as endothelin-1 (ET-1), angiotensin-2 (AT-2), and catecholamines, promote joint Ca2+ and DAG signaling that becomes linked in a positive feedback loop that supports the initiation of cardiac hypertrophy programs.It would exceed drastically the scope of this article to consider mechanisms of cytoplasmic Naycosides , 79–81. xchanger  and thernockdown , have opprograms . Given e2+ handling in hESC-derived myocyte cultures have been shown to be qualitatively comparable to adult myocytes [2+ and increased DAG. Finally, we have addressed the question of whether basal  myocyte Ca2+, as determined by the Na+ gradient, plays a role in the initiation of hypertrophy signaling in these cell cultures.The cardiac myocyte models commonly employed in hypertrophy studies, such as cultured adult and neonatal rodent myocytes, have distinct limitations. These include questions about their relevance to human myocyte function, the control of key signaling variables, e.g., spontaneous activity, and finally, their limited viability in culture which often limits chronic experiments. Accordingly, we have chosen to employ human embryonic stem cell (hESC)–derived cardiomyocytes to address the working hypothesis outlined above. hESC-derived myocytes are cost-effective , 46 and myocytes , 63, 70.myocytes . Neverthmyocytes . In brie+ gradient changes, developing a case that positive feedback coupling between these mechanisms plays an important role in the initiation of hypertrophy programs. To this end, we have demonstrated that cardiac glycosides, which with strong certainty increase the resting cytoplasmic free Ca2+ by decreasing the Na+ gradient of myocytes [2+ feedback mechanisms.To reiterate the goal of this study, our central aim has been to characterize the progression of hypertrophy in human embryonic stem cell–derived cardiac myocytes in dependence on DAG and Namyocytes , activatFemale embryonic stem cells (H9) cells are maintained in mTeSR1  on growth factor–reduced Matrigel-coated dishes. When H9 cells reach 85% confluency, cells are split 1:10 using EDTA solution (Versene) and maintained in mTeSR for 3 days until they reach ~ 85–90% confluency. Differentiation into human stem cell–derived cardiac myocytes is performed as described . In brieFor chronic treatments, cells were seeded on growth factor–reduced Matrigel-coated glass cover slips or growth factor–reduced Matrigel-coated cell culture dishes and hypertrophic stimuli were added for a duration of 5 days with daily medium exchange. For life cell imaging, cells were grown on growth factor–reduced Matrigel-coated 35-mm glass bottom dishes  for at least 1 week prior to experiments.Cells were used between 2 and 4 months post differentiation, and all experiments were performed on cells originating from multiple differentiations.2O at 2× concentration. Fatty acid–free bovine serum albumin  was prepared at 2× concentration and warmed to 37 °C. Under constant stirring, the warm free fatty acid (FFA) mixture was added dropwise to the BSA. Aliquots were stored in glass vials at − 80 °C. The medium containing the FFA triple mix was sterile filtered using a 0.2-μm sterile filter.Palmitoleic acid , palmitic acid, and myristic acid were dissolved in warm dHg for 15 min and subjected to SDS-PAGE and subsequent immunoblotting. α-Actinin, actin, and GAPDH anti-sera were used at 1:1000, and β-MHC antiserum was used at 1:5000.For protein isolation, cells were washed 3 times with ice-cold PBS and homogenized in ice-cold RIPA buffer ) and lysed for 1 h at 4 °C. Lysates were cleared at 20,000×Immunofluorescence studies were performed as described elsewhere . In brieWheat germ agglutinin  staining was performed according to the manufacturer’s instructions. In brief, cells were incubated for 10 min with 1 μg/mL WGA in HBSS at 37 °C. Subsequently, cells were fixed in 4% paraformaldehyde  in 1× PBS for 10 min and mounted on glass slides using VECTASHIELD containing DAPI. Images were obtained using a Zeiss Observer Z1 microscope equipped with an AxioCam MRm camera and a ×20 DIC objective (software: AxioVision (Release 4.8.2)). Per condition, a minimum of 10 pictures were taken. Analysis of cell area was carried out using ImageJ in a blinded fashion and with image regions selected in a random fashion. Pictures were thresholded, and area was measured and divided by the number of cells to obtain the average cell size.2, 2 CaCl2, and 15 glucose, pH 7.4. Fifty micromolar of lidocaine were added to stop cell contractions. Cells were mounted on a Nikon Eclipse TE2000-S microscope equipped with a Photometrics CoolSNAP ES2 camera and a ×10 DIC objective, and the baseline Ca2+ was measured for 10 min at RT. Subsequently, cells were treated and placed at 37 °C for 15 min. Cells were mounted on the microscope again, and the same cell cluster was imaged for another 30 min. For maximal Ca2+ increases, 25 μM ferutinin was added. After background subtraction, data were normalized to the maximal signal and expressed as percentage of maximal arbitrary fluorescence units (AFUs). A minimum of 3 experiments was performed per condition.Cells were loaded with 5 μM Fluo-4  at 37 °C for 25 min. Subsequently, cells were washed 3× and placed in a buffer containing the following (in mM): 130 NaCl, 5 KCl, 10 HEPES, 1.6 MgCl+/K+ pump currents were monitored as described previously [Myocytes were patch clamped, and Naeviously , 43.2, 2 CaCl2, and 15 glucose, pH 7.4. Cells were mounted on a Nikon Eclipse TE2000-S microscope equipped with a Photometrics CoolSNAP ES2 camera and a ×10 DIC objective, and the baseline DAG signal was measured for 10 min at RT. Subsequently, cells were treated and placed at 37 °C for 15 min. Cells were mounted on the microscope again, and the same cell cluster was imaged for another 30 min. For maximal DAG signals, 1 μM phorbol myristate acetate (PMA) was added. After background subtraction (baseline measurement), data were normalized to the maximal signal and expressed as percentage of maximal arbitrary fluorescence units (AFUs). A minimum of 3 experiments were performed per condition.Cells were transduced with a DAG sensing protein  and grown for 7 days before being employed in experiments. Cells were washed and stimulated with a buffer containing the following (in mM): 130 NaCl, 5 KCl, 10 HEPES, 1.6 MgClCell culture reagents and media were obtained from Gibco/Life Technologies  unless otherwise stated. All other reagents were obtained from Millipore Sigma  in the highest available quality unless otherwise stated.Anti-actin antibody, clone C4 (Millipore Sigma MAB150), α-actinin (sarcomeric) antibody (Millipore Sigma A7732), anti-myosin  (Millipore Sigma M8421), and GAPDH antibody (G-9) .t test or, in occasional cases of inappropriate variance differences, by the Mann–Whitney rank-sum test.Unless stated otherwise, error bars represent standard deviations. Significance was assessed by Student’s 2O), for 5 days. After fixation and immunofluorescent staining, analysis of the identically treated cultures revealed substantial increases in the expression of β-MHC in response to both agents   are of sIt is reasonably established that DAG signaling in cardiac myocytes is terminated by roughly equal activities of DAG lipases, cleaving DAG to monoacylglycerol and fatty acid, and DAG kinases that phosphorylate DAG to phosphatidate , 27. A r+/K+ pump activity [+/K+ pump activity [A number of reports suggest that ouabain is a cardioprotective agent when employed in low nanomolar concentrations that putatively stimulate Naactivity , 62. Theactivity , 21. The+/K+ pump activity in hESC myocytes. In these recordings, we defined the magnitudes of Na+/K+ pump currents in hESC-derived myocytes, removed from dishes, and their responses to different concentrations of ouabain. The recording conditions were identical to those employed in recent Na+/K+ pump studies of murine myocytes [+ and 120 mM extracellular Na+. As illustrated in Fig. +/K+ pump currents activated by 5 mM K+ by about 90% and the inhibitory effect did not reverse after several minutes of superfusion with ouabain-free solution. To determine whether pump activity could be stimulated by low ouabain concentrations, we first examined effects of ouabain concentrations from 0.5 to 15 nM. We were unable to detect any effect of ouabain at concentrations less than 5 nM, and we observed only inhibitory effects at higher concentrations. This is illustrated in the series of experiments shown in Fig. Given this outcome, we next attempted to verify that low-dose ouabain could stimulate Namyocytes , 43 usin2+ and DAG support hypertrophic growth in a positive feedback manner, we next analyzed the acute effects of several hypertrophic stimuli on the steady-state cytoplasmic Ca2+ and DAG levels in hESC-derived cardiomyocytes. To measure intracellular Ca2+, cells were loaded with the non-ratiometric Ca2+ dye, Fluo-4 [2+ and DAG measurements were carried out at multiple time points over a period of 45 min after addition of hypertrophic stimuli or vehicle. After 45 min, the nonselective ionophore, ferutinin (25 μM) [2+ response of Fluo-4, while 1 μM PMA was employed to determine the maximal DAG (C1 domain) response. The calibration of free Ca2+ concentrations, given in Fig. Kd of Fluo-4 for Ca2+ of 340 nM [2+ from less than 40 nM to about 500 nM, whereas Ca2+ in vehicle -treated cells rose to just 50 nM. As shown in Fig. 2+ rose over 45 min to 90 and 145 nM, respectively. During the same time frame, control basal Ca2+ levels remained at about 20 nM. DAG levels in both treatments rose to 21% of maximal FRET responses, whereas control DAG levels rose to 13% of maximal FRET responses.To test the working hypothesis that Ca, Fluo-4 , and to , Fluo-4 . Ca2+ an (25 μM) , was appf 340 nM  and the +/K+ pumps in these cells . DAG levels rose to 15 and 21% for low and high ouabain concentrations, respectively, compared to 11% of maximal FRET for control cells. We mention in closing the “Results for treatment of cell cultures with a low ouabain concentration (3 nM) and a moderate ouabain concentration (30 nM) are shown in Fig. lls Fig. . The low2+ and DAG signaling plays an important role in the initiation of cardiac hypertrophy programs. Specifically, we have demonstrated that basal Ca2+ and DAG increase to similar extents in remarkably diverse treatments that promote hypertrophy. Interventions expected to increase primarily Ca2+ increase DAG substantially and vice versa. This outcome has strong implications for an understanding of cardiac excitation–contraction coupling as well as the initiation of cardiac hypertrophy programs.This study documents further the utility and impressive advantages of hESC-derived cardiomyocytes for long-term studies of cell signaling mechanisms in cardiac myocytes. Obviously, the ability to maintain spontaneously beating syncytial human myocytes for periods of months in culture, in the absence of fibroblasts, is highly promising for a wide range of studies of long-term myocyte signaling programs, such as those originally envisioned by the Alliance for Cell Signaling . Our res3 receptors was negated. That AT-2 and ET-1 can increase Ca2+ transients and promote activation of specific PKCs in cardiac myocytes, notably PKCε [2+ in myocytes is increased by these reagents. Our results show unambiguously that both AT-2 and ET-1 increase basal Ca2+, determined after fully blocking spontaneous action potential firing  and/or may be acting by increasing the resting Na+ concentration of myocytes with concomitant rises in cytoplasmic Ca2+ by Na+/Ca2+ exchange. Consistent with the idea that a rise in resting Na+ can promote hypertrophy responses in these cells, ouabain induces robust hypertrophy responses in these myocytes at concentrations that inhibit Na+/K+ pump activity (Fig. + homeostasis to the forefront as an important factor in the initiation of pathological cardiac hypertrophy. In fact, a major role of the Na+/H+ exchanger, NHE1, in contributing Na+ load that promotes the initiation of cardiac hypertrophy, as well as its progression to cardiac failure, has long been indicated [+/K+ pump isoforms expressed in these cells and/or whether the cellular conditions of our experiments and the differentiation state of myocytes might play critical roles.These results indicate that growth-inducing hormones can be acting by increasing Caity Fig. . These rndicated , 34, 48.3, in initiating hypertrophy responses in these myocytes, we employed a new inhibitor of DAG lipases that profoundly affects brain function [2+ and DAG signaling becomes enhanced. These results support the idea that the termination of DAG signaling can be importantly influenced by DAG lipase activity in cardiac myocytes, as well as by the parallel function of DAG kinases that is already established to control the initiation of hypertrophy programs [To evaluate the potential triggering role of DAG per se, versus IPfunction , 56. Theprograms . How a mprograms . We poinprograms .2+ occurring with DAG lipase inhibition are of similar magnitude to those that occur with ET-1, and that changes in DAG with DAG lipase inhibition are not notably greater than those occurring with hormones (Fig. 2+ signaling mechanisms is quite strong over substantial signaling ranges. One factor that may contribute to coordinated Ca2+ and DAG signaling is that these hESC myocyte cultures are well coupled electrically and therefore behave in a substantially more coordinated fashion than traditional myocyte cultures used in hypertrophy studies. Another key factor may be that DAG acts primarily to increase cytoplasmic Na+ influx via TRP channels, rather than enhancing Ca2+ influx per se. In contrast to a direct Ca2+ influx mechanism, the accumulation of cytoplasmic Na+ may be expected to inhibit Ca2+ extrusion via Na+/Ca2+ exchange and thereby enhance the actions of all mechanisms that tend to increase myocyte Ca2+ signals.It is especially notable that the increases in Canes Fig. . These o2+- and DAG-dependent mechanisms in the initiation of cardiac hypertrophy programs. Ouabain-dependent signaling mechanisms may well modify this coupling and play some role in the outcomes observed. Certainly, it will be important to delineate the role specifically of Ca-independent ouabain signaling mechanisms [+ homeostatic mechanisms are likely at play constitutively in the control of cardiac signaling and myocyte phenotype.In summary, the present study highlights the potential importance of self-amplifying signaling via Cachanisms , 78. In


Ank1 gene that participate in the organization of the sarcoplasmic reticulum (SR) of striated muscles. Accordingly, the volume of SR tubules localized around the myofibrils is strongly reduced in skeletal muscle fibers of 4- and 10-month-old sAnk1 knockout (KO) mice, while additional structural alterations only develop with aging. To verify whether the lack of sAnk1 also alters intracellular Ca2+ handling, cytosolic Ca2+ levels were analyzed in stimulated skeletal muscle fibers from 4- and 10-month-old sAnk1 KO mice. The SR Ca2+ content was reduced in sAnk1 KO mice regardless of age. The amplitude of the Ca2+ transients induced by depolarizing pulses was decreased in myofibers of sAnk1 KO with respect to wild type (WT) fibers, while their voltage dependence was not affected. Furthermore, analysis of spontaneous Ca2+ release events (sparks) on saponin-permeabilized muscle fibers indicated that the frequency of sparks was significantly lower in fibers from 4-month-old KO mice compared to WT. Furthermore, both the amplitude and spatial spread of sparks were significantly smaller in muscle fibers from both 4- and 10-month-old KO mice compared to WT. These data suggest that the absence of sAnk1 results in an impairment of SR Ca2+ release, likely as a consequence of a decreased Ca2+ store due to the reduction of the SR volume in sAnk1 KO muscle fibers.Small Ankyrins (sAnk1) are muscle-specific isoforms generated by the  Two distinct SR regions can be described: the longitudinal SR (l-SR) and the junctional SR (j-SR). The l-SR surrounds each myofibril throughout its entire length and represents the major site for Ca2+ uptake from the cytosol due to the presence of the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) pumps. The j-SR is formed by the so-called terminal cisternae that are connected to the l-SR tubules. In skeletal muscle, two terminal cisternae are positioned on opposite sides of a transverse (T)-tubule, an in-folding of the sarcolemma, to form the triad. The membrane of the terminal cisternae facing the T-tubule accommodates the ryanodine receptor (RyR1) Ca2+ release channels, and additional SR proteins, including triadin, junctin, and calsequestrin, which, together with RyR1 channels, participate in the mechanisms of excitation–contraction (e–c) coupling. This mechanism transduces depolarization-induced activation of the dihydropyridine receptor (DHPR), the voltage sensor on T-tubules, into a release of Ca2+ from the SR to allow muscle contraction i in unstimulated Fura-2 loaded FDB fibers from sAnk1 KO and WT mice. These measurements indicated that the resting Ca2+ levels were not significantly different in both 4- and 10-month-old FDB fibers from sAnk1 KO mice compared to WT mice  was significantly smaller in fibers from 4- and 10-month-old KO mice with respect to age-matched control mice  was calculated in fibers from WT and sAnk1 KO mice. This analysis revealed that the Vmax progressively reduced in old mice compared to young animals, in both WT and sAnk1 KO mice . In addition, in old sAnk1 KO mice the Vmax was significantly lower compared to WT . This suggests that the remarkable but not statistically significant reduction in Ca2+ transients observed with age may be partially explained by a lower SERCA pump activity. However, the difference in Ca2+ transients observed between WT and sAnk1 KO muscle fibers at 4 months of age cannot be ascribed to SERCA pumps, since no difference could be observed between the two genotypes at this age .To evaluate whether the reduced Ca2+ release events in saponin-permeabilized muscle fibers offers the possibility to study the functional properties of RyR1 in its native environment, independently from the control of the DHPR i) in different extracellular solutions were measured using a Fura-2 fluorescent calcium indicator as described previously i was assessed as the ratio of measured fluorescence intensities (F340/F380). Resting [Ca2+]i was calculated from averaging the ratio in normal Tyrode’s. The SR Ca2+ content was calculated as the difference between resting [Ca2+]i and the maximum of the ratio measured during the application of the releasing cocktail.Changes in intracellular Caeviously . Briefly2, 5 Na2-ATP, 125 K-glutamate, 10 HEPES, 1 EGTA, 0.13 CaCl2, 10 glucose, 10 Na-phosphocreatine, pH 7.2) containing 0.002% saponin for 2–3 min. Permeabilization of the surface membrane by the detergent was monitored by imaging how the fluorescence of fluo-3 increases in the muscle fiber. This solution was then replaced by a recording solution . Images were recorded with a 40× oil immersion objective (NA = 1.3) on a Zeiss LSM 5 LIVE laser scanning confocal microscope . The 488 nm line of an argon laser excited fluo-3 in the fibers, and the emitted fluorescent light was collected above 505 nm wavelength. Fifteen minutes following the application of the recording solution, eight consecutive series of hundred and twenty 512 × 512  images captured every 67 ms were collected in each tested fiber. CRE detection and analysis were performed using modified methods and algorithms previously described in i(t) in a single compartment including quantitatively specified processes of removal, such as the maximal rate of the SERCA pump (Vmax) tot).Cap (Vmax) . The expChemicals, unless otherwise stated, were purchased from Sigma  and were of analytical grade.p value of less than 0.05 was considered statistically significant.Pooled data were expressed as mean ± standard error of the mean (SEM). The differences between control and sAnk1 KO mice with different ages were assessed using two-way analysis of variance (ANOVA) and all pairwise multiple comparison procedures (Student-Newman-Keuls Method) with Prism . An f-test was used to test the significance and a


Cancer arises when homeostasis in a susceptible cell is disrupted as a result of the mutation, upregulation and/or downregulation of critical genes. This gene dysregulation bestows upon affected cells such properties as independence from growth control and the ability to migrate to distant sites and grow there, which gives rise to metastasis. PHLPP1 is a member of the pleckstrin homology (PH) domain leucine-rich repeat protein phosphatase (PHLPP) family, which maintains the balanced level of protein  dephosphorization necessary to control a broad range of physiological processes, including cell proliferation, motility, migration, and survival. Changes in the level PHLPP1 or its mutation correlates with many pathophysiological processes, including the development and progression of cancer [min mice, which further confirms that PHLPP1 loss-of-function enhances tumor progression to metastasis. Clinically, analysis of 202 consecutive patients with gastric cancer showed that PHLPP1 expression correlates inversely with lymph node metastasis and that PHLPP1-negative patients had significantly shorter overall and relapse−free survival than PHLPP1−positive patients (both p < 0.001) [In a comparative study of high- and low-grade prostate cancers, Hellwinkel et al. found that levels of PHLPP1 transcription are significantly decreased in aggressive high-grade tumors , which s< 0.001) . In aggr< 0.001) .Cutaneous malignant melanoma is a genetically complex and highly aggressive disease, notorious for both its propensity for metastasis and its poor response to currently available therapy. Increasing evidence indicates that PI3K/AKT pathway activation acts as a driving force for melanoma metastasis. Within that context, we analyzed expression of PHLPP family proteins in poorly and highly metastatic melanoma cell lines and in primary and metastatic melanomas. Our findings revealed that PHLPP1 expression is significantly downregulated or lost in melanoma, and that PHLPP1 deficiency correlates with metastatic potential. Moreover, mutation of PHLPP1 in cutaneous melanoma (SKCM-TCGA) patients correlated significantly with poorer survival . StandarGiven that PHLPP1 dephosphorylates serine 474 of AKT2 and serine 472 of AKT3, thereby controlling PI3K/AKT kinase signaling [


Here, we devise a methodology based on Markov chain models that allows us to obtain analytic results for the expected rate. First, we analyze the property of the secretory complex obtained by coupling a single granule with one CaV. Then, we extend our results to a more general case where the granule is coupled with n CaVs. We investigate how the exocytosis rate is affected by varying the location of granules and CaVs. Moreover, we assume that the single granule can form complexes with inactivating or non-inactivating CaVs. We find that increasing the number of CaVs coupled with the granule determines a much higher rise of the exocytosis rate that, in case of inactivating CaVs, is more pronounced when the granule is close to CaVs, while, surprisingly, in case of non-inactivating CaVs, the highest relative increase in rate is obtained when the granule is far from the CaVs. Finally, we exploit the devised model to investigate the relation between exocytosis and calcium influx. We find that the quantities are typically linearly related, as observed experimentally. For the case of inactivating CaVs, our simulations show a change of the linear relation due to near-complete inactivation of CaVs.Hormones and neurotransmitters are released from cells by calcium-regulated exocytosis, and local coupling between Ca Molecules, e.g., neurotransmitters and proteins, are released from the cell by exocytosis . In this2+ levels, fuse with the cell membrane and release the hormone molecules. The main mechanisms regulating hormone exocytosis are shared with exocytosis of synaptic vesicles underlying neurotransmitter release in neurons [2+-sensing proteins such as synaptotagmins, which trigger exocytosis upon Ca2+ binding. Therefore, the local Ca2+ concentration at the Ca2+ sensor of the exocytotic machinery is a key factor determining the probability rate of exocytosis of the secretory granule [In most endocrine cells, the hormones are contained in secretory granules that, in response to a series of cellular mechanisms culminating with an increase in the intracellular Ca neurons , 5. The  neurons . SNARE c granule .2+ dynamics and exocytosis for the glucagon-secreting pancreatic alpha-cells and showed how exocytosis is dependent on calcium dynamics, in particular, on calcium levels surrounding the Ca2+ channels (CaVs) [Recently, we have devised a detailed model of Cas (CaVs) , the so-s (CaVs) . Here, is (CaVs) . We shows (CaVs) , we will2+ channel by using the 3-state Markov chain of C corresponds to the closed state, O to the open state, and B to the inactivated (blocked) state of the calcium channel [S={C, O, B} and its transition rate or generator matrix MCaV is given byα and β represent the voltage-dependent Ca2+ channel opening rate and closing rate, respectively, and have the following forms:We model the Ca channel . Then, tδ, is Ca2+-dependent and has the following form:CaCaV is the Ca2+ concentration at the Ca2+ sensor for inactivation and is given using reaction-diffusion theory [2+ current with VCa the reverse potential, and rCa represents the distance of the sensor for Ca2+-dependent inactivation from the channel pore. Finally, γ is the constant reverse reactivation rate. The rate for channel inactivation, n theory , 12, 13 h represents the fraction of Ca2+ channels not inactivated).The deterministic description of the 3-state Markov chain model for the CaV channel is given by the following ODE system:2+ loading, we compute the total charge entering via the Ca2+ channel at a given step voltage with time window, ts, asFinally, in order to investigate the relationship between exocytosis and Ca2+ ions bound to the Ca2+ sensor on the granule, likely synaptotagmin [G0 with no bound Ca2+ ions, or in G1 with one, or in G2 with two, or in G3 with three bound ions. Once it is in G3, the granule can fuse with the membrane and release its hormone content, assuming the final state Y [S={G0, G1, G2, G3, Y}, and its transition rate or generator matrix MG is given by2+ binding rate, with CaG the Ca2+ concentration at the granule sensor given by Equation . Then, by renaming the state variables asg2 and g3 as follows:For the above ODE model of Equations –13), we, we13), Equation  equal toquations  and 12)g3, sincS={G0, G1, G23, Y} ), as1 ∈ ℝ3×1.Using phase-type distribution results for Markov chains , we obtaDG:CaV, is as follows:MCaV is defined by Equation (kcCa(Ac) by Equation , and koCa(Ao) by Equation ), as1 ∈ ℝ9×1.By coupling the CaV and exocytosis models, we obtain the 12-state Markov chain model of Equation  (EquatioEquation ) with CaEquation  (EquatioEquation ) with CaEquation . Then, tEquation , assumin2+ channel can be only in C or in O). In this case, MCaV ∈ ℝ2×2 and is defined by Equation =(k2/2)+(3k/2)+1 possible states describing the k CaVs. In particular, the CaVs model takes values in the state space S={Ck−i−jOiBj} with j ∈ {0,…, k} and i ∈ {0,…, k − j}, and its generating matrix, MkCaV, is given byMk1×1=−kγ.In the following, we assume the case where the granule is coupled with more than one CaV. In particular, by considering nS-state Markov chain model. The model takes values in the state space S={Ck−i−jOiBjGl,…, Ck−i−jOiBjG23, Ck−i−jOiBjY}, with j ∈ {0,…, k}, i ∈ {0,…, k − j} and l ∈ {0,1}, and its transition matrix, DG:kCaV, can be written asThen, by coupling the CaVs and exocytosis models, we obtain a 4k CaVs, λYk, can be estimated by using Equation 1×(3), which yields1 ∈ ℝnS×13.Then, the expected exocytosis rate for the single granule coupled with Equation , assuminMkCaV=M0 by Equations ×3(k+1)3MkCaV=MEquation , belongsk of CaVs, we define the relative rate, ρλk, ask=1,…, n. Moreover, in order to compare the rate at different distances from the granule to CaVs, we define the relative distance rate, ρλd, asλYd is the rate computed at a given distance rG and λYdmin, the rate computed at rG=10 nm.In order to compare the rate for a granule coupled with different number k Ca2+ channels by using phase-type distribution results for Markov chains [k CaVs. Moreover, we consider for both the cases (1 or k CaVs) that the granule forms complexes with inactivating or non-inactivating CaVs. This scenario reflects, e.g., what it is observed in pancreatic beta-cells where the two main high voltage-activated Ca2+ channels, the L- and P/Q-type Ca2+ channels, are examples of inactivating and non-inactivating CaVs, respectively [We analyze the behavior of the devised exocytosis model where the single granule is coupled with v chains  . This is clearer from ρλd defined by Equation  while the exocytosis rate drops more than threefold for rG=20 nm  and more than fivefold for rG=10 nm .Figures Equation , for a gEquation  , while it goes down fivefold when the granule is far from CaVs . It seems that when the granule is surrounded by more non-inactivating CaVs, it is not necessary that the granule is very close to the CaVs for triggering exocytosis.As done for the case with one CaV, we performed the same analysis with  Figures –3. Also 2+ loading, we consider a set of scenarios where the granule is coupled with different number of non-inactivating or inactivating CaVs, placed very close (10 nm) or far (100 nm) from the granule. V=0 mV, for different numbers of non-inactivating CaVs, while pY (pY=P(S(t)=Y)) vs. the integral of the Ca2+ current, QCa, defined by Equation (rG=10 nm). For the case of non-inactivating CaVs  but also to near-complete inactivation of CaVs, in particular after 50 ms (pY vs. QCa when the granule is placed far from CaVs (rG=100 nm). Due to the distance to CaVs, the calcium concentration at the granule increases only modestly; hence, a greater calcium influx QCa is needed to allow the granule to move through the Markov chain from N0 to Y and undergoes exocytosis. This causes an evident initial delay for the granule to be released, resulting in an initial convex relation between pY and QCa. After this initial phase, for the case of non-inactivating CaVs . For inactivating CaVs . For iner 50 ms . Figuresing CaVs , pY raiing CaVs , as for 2+ channels and use phase-type distribution results [In this paper, we devise a strategy that allows us to characterize the local interactions between granules and CaVs. The methodology is similar to our approach for modelling the local effect of CaVs on whole-cell BK currents . We deve results  for esti2+ sensor of the) granule from CaVs. In particular, from our analysis, we find that the distance between the granule and CaVs is a major factor in determining the exocytosis rate, as we recently demonstrated and quantified explicitly [We investigate how the release probability of a granule can be affected by varying the number of CaVs and the distance of the (Caplicitly . Furtherplicitly , the sim2+ influx and exocytosis. The results of the devised exocytosis model confirm that the granule secretion is generally linearly related to the integral of Ca2+current, as experimentally observed [pY and QCa due to near-complete inactivation of CaVs. This fact is due to the rather complex exocytosis model where the efficacy of Ca2+ influx in triggering exocytosis depends on the number of active CaVs, as clearly seen in the case of non-inactivating CaVs (Figures 2+ bindings before exocytosis. During inactivation, the effective number of CaVs declines, which has a similar effect as reducing the number of CaVs, and hence the slope of the relation between exocytosis and QCa decreases. This finding reinforces the notion that a concave relation between exocytosis and Ca2+ influx does not necessarily reflect pool depletion [We also study the relationship between Caobserved –29 and tobserved . Surpris Figures  and 4, bepletion  and prov


Modulation of intracellular Ca2+ concentration ([Ca2+]i) is a major part of Ca2+-handling processes in cardiac pathophysiology. TRP channels including TRPM4 regulate [Ca2+]i, Ca2+-handling and cardiac contractility. The channels modulate flux of divalent cations, such as Ca2+ during Ca2+-handling and cardiac contractility. Seminal works implicate TRPM4 and TRPC families in intracellular Ca2+ homeostasis. Defective Ca2+-homeostasis through TRP channels interaction with Ca2+-dependent regulatory proteins such as sodium calcium exchanger (NCX) results in abnormal Ca2+ handling, contractile dysfunction and in spontaneous ectopy. This review provides insight into TRP channels mediated pathological Ca2+-handling and spontaneous ectopy.Ion channel biology offers great opportunity in identifying and learning about cardiac pathophysiology mechanisms. The discovery of transient receptor potential (TRP) channels is an add-on to the opportunity. Interacting with numerous signaling pathways, being activated multimodally, and having prescribed signatures underlining acute hemodynamic control and cardiac remodeling, TRP channels regulate cardiac pathophysiology. Impaired Ca The ionic movement is important. During action potential propagation, cell membranes depolarize, and Ca2+ enters into the cells. L-type calcium channel passes the Ca2+ influx as inward Ca2+ current , which contributes to the shape of action potential plateau. The Ca2+ entry initiates Ca2+ release from the sarcoplasmic reticulum (SR) by activation of ryanodine receptors (RyRs), thereby elevating [Ca2+]i in the so-called Ca2+-activated Ca2+ release. The raise in the [Ca2+]i, activated by a variety of ways, is a known mechanism of cell signaling. It enables Ca2+ to bind to contractile machineries such as myofilament protein and troponin C, and initiate contraction. The raise in the [Ca2+]i contrarily declines in relaxation. Intracellular Ca2+ is pumped from the SR by sarcoplasmic reticulum calcium pump (SERCA), and sarcolemmal efflux through sodium calcium exchanger (NCX), with little contribution by the SERCA during relaxation  channels seminal works in pathologic Ca2+-handling and spontaneous ectopy.Tremendous efforts at understanding the process is ongoing, but it is hampered by the complicated nature of changes in [Ca2+]i, that is part of CA2+ homeostasis, and governs Ca2+-handling in excitation and contraction coupling is an important process of cardiac contractile performance. Cardiac contractility is the intrinsic ability of the myocardium to contract. Readily occurrence of contraction depends on incremental degrees of binding between myosin (thick) and actin (thin) filaments, when the [Ca2+]i is elevated. Factors that promote Ca2+-handling and contractility do so by inducing increases in the [Ca2+]i. The heart is unable to contract in too little Ca2+ concentration  and unable to relax in too much Ca2+ concentration (large Ca2+ transient amplitude), indicating negative and positive inotropy, respectively. TRP channels as cell membrane ion channel fall into a category and were first identified in impaired Drosophila visual adaption, where photoreceptors carrying TRP gene mutations showed a transient voltage response to continuous light  is a family of TRP ion channels, that consists of eight different subfamilies (TRPM1-TRPM8). TRPM4 subfamily while permeable to Namplitude . The actduration . More so2+ entry . Therefo2+, and may be important in Ca2+-handling. TRPC6 promoter gene had two conserved nuclear factor of activated T-cell transcriptional factor (NFAT) consensus sites  infusion) or pressure overload induction  is another family of TRP channels. It consists of seven subfamilies (TRPC1-TRPC7). TRPCs are also permeable to but activated by Caus sites  and NFATexposure . Transgenduction . TRPC ch2+/calcineurin/NFAT signaling loop might regulate Ca2+-handling in diseases, how the signaling loop couples to [Ca2+]i and L-type Ca2+ channel activity is completely unknown. It appears in smooth muscle that entry of cations through a receptor operated TRPC6 caused membrane depolarization and consequent functional activity of L-type Ca2+ channels, Ca2+ influx, and smooth muscle contraction. However, whether this role is associated with ryanodine and NFAT is not known. Given that TRPC6 can operate as receptor-activated cation channels, which increases [Ca2+]i by Ca2+ entry across plasma membrane and/or by release of Ca2+ from intracellular stores such as the endoplasmic reticulum, TRPC6 contributes to Ca2+-handling and [Ca2+]i. The contribution can also be deduced on store-operation molecular standpoint. This is since store-operated and receptor-operated channels can come from the same proteins of the same family of TRP channels.Although it is possible that TRPC/Ca2+-dependent inactivation kinetics of STIM1 current is known to be critically dependent on cytosolic Ca2+ levels, which also regulates native SOCE and ROCE currents, through changes in the [Ca2+]i, similarly to that of TRP channel currents. Whether TRP channels kinetics are associated to SOCE and ROCE, is not elucidated. Native store-operated and receptor-operated complexes stoichiometry, if distinct from hetero/homo multimer variants Orai or TRP is also not known. Regardless, STIM1 indirectly activates TRPC3/6, but not TRPC7  direct effects of TRP channels and receptor operated calcium entry mechanisms on [Ca2+]i and Ca2+-handling may be questioned. (2) It is more likely that TRP channels regulate the process through effects on micro-domains or through direct interaction with Ca2+-dependent regulatory proteins. (3) It is noted that STIM1 is a Ca2+ sensor that relays Ca2+ load of the endoplasmic reticulum to store operated channels and may navigate some of the TRP channels into micro-domains to govern their roles in cardiovascular diseases through calcium signaling effectors.Nonetheless, TRP channels store operated and receptor operated calcium entry (SOCE) and (ROCE) are less clear. Discovery has it that stromal interaction molecule 1 (STIM1) and Orai 1 are mediators of SOCE. Fast Caot TRPC7 , and TRPot TRPC7 , and in ot TRPC7 . There mot TRPC7 . In fact-domains . DissociPC1-SOCE . Express2+ transients through mechanotransduction. Mechanical stretch is a determinant of atrial function such as contractility and TRPC-6 is a stretch responsive cation channel that governs mechanotransduction. To understand the roles of stretched-atrial endocardium, associated with physiological conditions and arrhythmogenesis Nikolova-Krstevski et al., tested the consequences of stretch-induced endocardial TRPC-6 activation on myocardial function and hypothesized that endocardial TRPC-6 is required for mechanical stretch responses in the atrium  communication/signaling was investigated in cultured human-induced pluripotent stem cell-derived cardiomyocytes before and after the addition of media collected from non-stretched and stretched atrial endocardial cells from porcine. The investigation revealed alteration of Ca2+ transient in human-induced pluripotent stem cell-derived cardiomyocytes with non-stretched but not with stretched atrial endocardial cells 2 with small interfering RNA (siRNA) increases TRPC levels  cell line was employed. The keratinocytes from DD patients have also one normal copy of SERCA2. Single copy of the gene is known to be inadequate, to appropriately account for proper amount of SERCA2, leading to changes in Ca2+ signaling in the cells. To examine this SERCA2 reduction in function, HaCaT cells were treated with adenovirus encoding either SERCA2-siRNA or control-siRNA and Ca2+ imaging was performed on the cells upon stimulation with thapsigargin, to deplete endoplasmic reticulum Ca2+  in the myocytes subjected to SERCA2-siRNA, there was no differences in ICa,L under the voltage clamp protocol tested. However, the myocytes were shown to have the ability to initiate transient elevations in cytosolic Ca2+ upon membrane stimulation, in compensatory mechanisms. To understand this process, the presence or absence of Na+ to examine the role of NCX to cytosolic Ca2+ removal under voltage-clamp depolarizations, and Ba2+ influx measurement in fura-2-loaded myocytes treated with thapsigargin to block SERCA pump activity to examine the role of TRPC channels, in another experiment, were together, employed to access the impact of SERCA2-siRNA on NCX, TRPC channels and Ca2+ signaling. This investigation revealed that reduced SERCA2 expression was associated with an elevation in TRPC expression and activity, as well as increased NCX expression and activity, that together may boost and normalize for reduced sarcoplasmic reticulum Ca2+ signaling following SERCA2 depletion  and sarcomeric scaffolding, and energetic agents referred to as cardiac myotropes and mitotropes, respectively, have been proposed as pharmacological agents that improve myocardial performance  that underlies the DADs, and contractile dysfunction (EADs are abnormally secondary cell membrane depolarizations during repolarization phases of action potential. Prolongation of APD is the principle factor that causes EAD, because reduced repolarization enables  arrange . Under ptivation . PKA hypactility . RYR2 chactility . Furtherfunction .2+ signaling can modulate action potential wave form and cause EADs. TRPM can also modulate diastolic Ca2+ release from sarcoplasmic reticulum Ca2+ stores and cause DADs. 9-Phenanthrol, an inhibitor of TRPM4 channel abolished hypoxia and re-oxygenation-induced EADs in a mouse model (2+ influx by regulating membrane potential, and intracellular Ca2+ increased through ICa,L upon TRPM4 protein depletion (EADs and DADs appear to be mediated by TRPM4 protein. TRPM mediated Case model . TRPM4-ise model . TRPM4 aepletion .ti initiated by Ca2+ waves that underline DAD. Iti has been described in Purkinje fibers, atrial and ventricular cardiomyocytes, and in sinoatrial node cells (ti is controversial, but it appears to reflect 3 Ca2+- dependent components: NCX, Ca2+-activated chloride current and current mediated by CAN channels such as TRPM4 (ti is mediated by TRPM4. However, that Iti is mediated by TRPM4 may be questioned by the fact that the single channel conductance of the Iti-mediating channel was 120 Ps (CAN channel activity had been suggested to contribute to Ide cells . In sinode cells , which sde cells . The molas TRPM4 . Put togs 120 Ps , which is 120 Ps .2+ is novel in understanding arrhythmogenesis. The TRP channels mediate Ca2+ flux and voltage changes across membranes. Regulation of Ca2+-handling by the TRP channels indicate they can potentially boost Ca2+ cycling disorders. Plasma membrane sensory and metabotropic TRPM4 subgroup is a drug candidate for Brugada syndrome and familial heart blocker (2+-handling and contractility is crucial.The past few decades have witnessed tremendous evolution in characterization of the molecular and genetic mechanisms of acquired and inherited arrhythmias. The mechanisms are as numerous as the phenotypes with which the arrhythmias present. Identifying the sources that control Ca blocker . Better 2+-handling and spontaneous ectopy are lacking. This study reflects on TRPM and TRPC seminal works, based on their biophysical properties, to state mechanistically their potential implications in Ca2+ signaling dynamics. Intracellular Ca2+ homeostasis show tremendous changes during cardiac cycle. Intracellular Ca2+ homeostasis is regulated by spatial signaling processes within restricted regions, rather than by changes in global cytosolic [Ca2+]i. [Ca2+]i is mediated by Ca2+ release from intracellular organelles, Ca2+ entry across plasma membrane through receptor agonists, receptor-activated Ca2+ channels, voltage-gated Ca2+ channels, and by ligand-gated cation channels. It appears that while overexpression of the TRPCs increase [Ca2+]i, TRPM4 depletion increases [Ca2+]i, through Ca2+ influx. This can be explained by the biophysical properties of the channels. Mechanization of [Ca2+]i, is defective in heart failure, failing hearts, cardiomyopathy, arrhythmia and sudden cardiac deaths. Defective intracellular Ca2+ homeostasis is responsible for pathological Ca2+-handling and contractile dysfunction. Hearts in these conditions show abnormal contractility, characterized by decreased SR Ca2+ sequestration, diminished intracellular Ca2+ transients, and enhanced diastolic SR Ca2+ leak activity. Sources that regulate these processes are unknown completely, and may include TRP channels and TRP channels SOCE associated mechanisms. Highlighting a potential therapeutic opportunity of the channels, this work discussed TRP channels mediated pathologic Ca2+-handling in spontaneous ectopy, and stated further scopes for TRP channels functions in cardiac pathophysiology.Essentially, studies illustrating the roles of TRP channels in pathological CaThe author confirms being the sole contributor of this work and has approved it for publication.The 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.


However, not much is known about the crosstalk between NO and Ca2+/CaM during the senescence of cut flowers. In this study, cut Oriental × Trumpet hybrid lily “Manissa” were used to investigate the roles and relationship between NO and Ca2+/CaM during postharvest freshness. The results show that the effects of CaCl2 or NO donor SNAP on the vase life, maximum flower diameter and hours until full opening were dose-dependent, with an optimum concentration of 20 mM CaCl2 or 100 μM SNAP. However, Ca2+ chelators EGTA or BAPTA/AM, Ca2+ channel inhibitors LaCl3 or nifedipine and CaM antagonists W-7 or TFP inhibited the promotion of SNAP. SNAP applied alone significantly increased the endogenous Ca2+/CaM contents in cut lily flowers, while EGTA, BAPTA/AM, LaCl3, nifedipine, W-7, and TFP decreased the advancement of SNAP. In addition, the SNAP-induced Ca2+-ATPase activity was more than twice as much as the control, but EGTA, BAPTA/AM, LaCl3, nifedipine, W-7, and TFP also reversed the enhancement. Moreover, EGTA, BAPTA/AM, LaCl3, nifedipine, W-7, and TFP prevented the SNAP-induced upregulation of gene expression of CaM, CBL1, and CBL3, which is associated with calcium signaling pathway. Overall, these results suggest that Ca2+/CaM may function as downstream molecules in NO-regulated senescence of cut flowers.Both nitric oxide (NO) and calcium ion (Ca Lilium spp.), a bulbous plant with large trumpet-shaped and typically fragrant flowers  may function as an influential plant growth regulator . It is e2+) plays critical roles in plant response to biotic and abiotic stresses, including light, unfavorable temperature, salt and osmotic stress, phytohormones, oxidative stress, wind stimulation, wounding, and anoxia. Ca2+ also plays an important role in plant membrane stability, cell wall stabilization, and cell integrity , calcineurin B-like (CBL) proteins, and calcium-dependent protein kinase (CDPK) are three main families of Ca2+ sensors  of lilies were rehydrated for 2 h, then the flowering stems were recut to a uniform length of 45 cm under distilled water to avoid air embolism. The blades of cutters were surface sterilized by rinsing in 95% (v/v) ethanol prior to use. The flowers were selected for uniformity of size, color, and free from any defects. All experiments were performed in the laboratory at temperature of 20 ± 2°C, relative humidity of 60 ± 10%, and light intensity of 12 μmol m2). The doses of SNAP solution were 50, 100, 200, and 400 μM while the concentration grads of CaCl2 solution were 5, 10, 20, 40, and 60 mM. After three replications of each treatment, 100 μM SNAP or 20 mM CaCl2 treatment was used in the following study.Experiment 1: after selecting the flowers, every 5 flowers were randomly put in a 2500 mL glass vase containing 1000 mL distilled water (control 1), 40 mM potassium chloride  or various concentrations of 1000 mL of test solutions. Unless otherwise specified, the word “control” refers to control 1 in the full text. Glass vases were surface sterilized with ethanol which was used for cutters as described above. There were two kinds of test solutions which were respectively made by NO donor S-nitro-N-acetyl-penicillamine (SNAP) or calcium dichloride (CaCl2); (4) 120 μM  (EGTA) + SNAP; (5) 30 μM   + SNAP; (6) 500 μM lanthanum chloride  + SNAP; (7) 150 μM nifedipine  +SNAP; (8) 80 μM [N-(6-aminohexyl)-5-chloro-1-naphtalene sulfonamide] + SNAP; (9) 100 μM trifluoperazine  + SNAP; (10) 150 μM nifedipine. Each treatment was conducted in three replications. The concentrations of these chemicals were selected based on the result of a recent experiment conducted in our laboratory (Experiment 2: 10 treatments were applied: (1) Control (distilled water); (2) 100 μM S-nitro-N-acetyl-penicillamine (SNAP); (3) 20 mM calcium dichloride . The flower diameter was defined as the maximum width of each flower and measured by a vernier caliper. The hours until full flower opening (HFO) was determined by the number of hours from the time when the flower was placed into the vase solution until the flower diameter reached the maximum value. There was an observation every 6 h at the same time of a day.2+ level in the cut lily leaves were measured by atomic absorption spectral analysis of nitric–perchloric acid digests. Soluble protein content in the extracts for CaM assay was determined according to 2+-ATPase activity was performed using the modified one-step lead method of The Ca260/OD280 ratio. About 2 μL of DNA-free total RNA was used for synthesis cDNA with a PrimeScript RT Master Mix kit . Quantitative real-time PCR reactions were performed with a Mastercycler®ep realplex real-time PCR system  with SYBR® Premix Ex TaqTM . The cDNA was amplified using specific primers (Supplementary Table S1). The amplification conditions were as follows: 95°C pre-degeneration for 30 s and repeat once, followed by 40 cycles of 95°C for 15 s, and then annealing at 60°C for 30 s. The expression levels of corresponding genes were presented as values relative to the corresponding control samples under the indicated conditions, with normalization of data to the geometric average of internal lily actin. Each sample was set three biological replicates.Total RNA was abstracted from healthy lily leaves with TaKaRa MiniBEST plant RNA extraction kit  according to the manufacturer’s instructions. The concentration of total RNA was measured using an ultraviolet spectrophotometer, and the quality of RNA was determined based on its ODP < 0.05). All statistical analysis was carried out using the statistical package for social science for windows .All of data presented in the tables and figures were expressed at the mean value ± SE from three independent duplications (five flowering stems per replication). Data collected were subjected to analysis of variance (ANOVA), and statistical divergence among treatments was analyzed through Duncan’s multiple range test (Table 1). CaCl2 treatments at the concentration of 10, 20, and 40 mM significantly extended the vase life of cut lily flowers compared to both control 1 and control 2. However, when tested at 5 mM and 60 mM CaCl2, each of the concentration had no significant effects on vase life. The longest vase life obtained by 20 mM CaCl2 treatment, which was 21 and 19% longer than the control 1 and control 2, respectively. As shown in Table 1, exogenous CaCl2 at proper levels  increased the maximum flower diameter of cut lily significantly in comparison to the control. Treatment with 20 mM CaCl2 created the maximum value. Placing cut lily flowers in the 20 mM CaCl2 solution created the maximum HFO, while 5 and 60 mM did not influence HFO (Table 1).There was no difference in the vase life of cut lilies between treatments with distilled water (control 1) and KCl  while higher dosage of SNAP (400 μM) was phytotoxic, thereby resulting in a shortened vase life. Treatment with 100 μM SNAP produced the longest vase life, which was about 132.29% of the control. Compared to the control, all various concentrations of treatments with SNAP markedly increased the maximum diameter (Table 2). Both treatments with 100 and 200 μM SNAP significantly extended the maximum flower diameter, whereas 100 μM is the best. Treatment with 100 μM SNAP extended HFO, which increased by 44.98% compared with the control. Therefore, 20 mM CaCl2 and 100 μM SNAP might delay senescence by slowing down flower opening, and they were used in further study in this experiment.As 2+ and CaM is involved in the NO-enhanced postharvest freshness of cut lily flowers, effects of Ca2+ chelators (EGTA and BAPTA/AM), Ca2+ channel inhibitors (LaCl3 and nifedipine), and CaM antagonists (W-7 and TFP) co-treated with SNAP were studied . When treated with SNAP solution alone, the vase life was the longest, which was 26% longer than that of the control. Treatments with SNAP plus Ca2+ chelators or Ca2+ channel inhibitors cut down the advancement of vase life, flower diameter and HFO caused by SNAP to some extent, thus produced significant effects on anti-aging of cut lily flowers. However, when CaM antagonists W-7 or TFP was added to SNAP containing solution, it reversed the SNAP-induced positive impact on cut flowers. In addition, when cut lilies were treated with nifedipine alone, the vase life and HFO was significant suppressed. These results indicate that NO-delayed senescence might relative to Ca2+ and CaM, at least partly.To further analyze whether Ca2+ concerned with NO-induced effect during the senescence process, the Ca2+ content in cut lily flowers of treatments with SNAP or SNAP in combination with EGTA, BAPTA/AM, LaCl3, nifedipine, W-7, and TFP were tested. Figure 2 shows that when SNAP applied alone, an obviously increase in Ca2+ level was detected, which was 144.45% of the control. When BAPTA/AM, LaCl3 or TFP was added to the SNAP-treated cut flowers, the effects of NO were weakened. However, SNAP treatment with EGTA, nifedipine or W-7 resulted in a significant reduction in Ca2+content. Placing CaM antagonists W-7 into SNAP solution maximally reversed the promotion induced by SNAP, which were 56.86% lower than that of the treatment with SNAP alone.To investigate the possible roles of CaFigure 3, the effects of different vase solutions  on CaM levels were determined. SNAP treatment gave the maximum CaM content, which was almost twice that of the control. The SNAP-induced increase on CaM content was inhibited by BAPTA/AM, LaCl3, nifedipine, W-7, or TFP, thereby resulted in similar contents to the control. Moreover, the vase solution including both SNAP and Ca2+ chelator EGTA caused a sharp decrease in endogenous CaM content, which was obviously lower than that of the control. These results indicate that SNAP promoted the CaM content of cut lily flowers, while Ca2+ chelators, Ca2+ channel inhibitors or CaM antagonists inhibited the effects.As shown in 2+-ATPase activity twice more than the control when SNAP applied alone . Placing EGTA, LaCl3, or TFP into SNAP solution partly reversed the promotion of SNAP. SNAP treatment with BAPTA/AM, nifedipine, or W-7 also caused a decrease in Ca2+-ATPase activity. The minimum Ca2+-ATPase activity were obtained by the SNAP plus Ca2+ channel inhibitor nifedipine, suggesting that nifedipine maximally prevented the NO-induced positive effects in the senescence process of cut lilies.There was a significant increase in the CaCaM was significantly increased by SNAP, resulted in a maximum value which was almost double the control; while the co-treatment with SNAP and EGTA, BAPTA/AM, LaCL3, nifedipine,W-7, or TFP obviously reduced the positive effects of SNAP . There was no difference in CaM expression between the control and SNAP plus EGTA, BAPTA/AM, LaCL3, or TFP. However, SNAP treatment in combination with nifedipine or W-7 caused a significant decrease compared to the control. Placing SNAP into the vase solution also upregulated CBL1 expression significantly, whereas there was a great inhibition when SNAP applied together with EGTA, BAPTA/AM, LaCL3, nifedipine, W-7, or TFP . Especially when EGTA, BAPTA/AM, LaCL3, W-7, or TFP was applied to SNAP-treated cut lily flowers, the expression of CBL1 was lower than that of the control. Similarly, EGTA, BAPTA/AM, LaCL3, nifedipine, W-7, or TFP obviously reduced the SNAP-induced increase on CBL3 expression .The gene expression of 2+ at proper dose could help against cut lily flowers senescence, while high concentration of NO and Ca2+ were invalid. This conforms to our previous study, which demonstrated that the effects of NO on vase life and maximum flower diameter of cut roses were dose-dependent  to eliminate the influence of chloride ion and further identify the promoting effects of Ca2+ on postharvest life of cut lily flowers. The results suggests that Ca2+ enhanced vase life because KCl had no significant effects on postharvest freshness in cut lily flowers compared with the control, while CaCl2 significantly increased vase life, flower diameter and HFO. Our results also supported by the observation of authors who indicated that Ca2+ could significantly increase vase life of cut gerbera flowers by delaying stem bending  and CaM antagonists  applied alone might inhibit adventitious rooting of marigold, while NO or Ca2+ promoted this process  variations have been reported in response to a large set of stimuli  and cyclic adenosine diphosphoribose , at least partly. Recent studies from our laboratory have shown that intracellular Ca2+ amount was increased by NO in cucumber hypocotyls during the development of adventitious roots under osmotic stress . This result was further confirmed by the use of Ca2+ chelators, Ca2+ channel inhibitors, and CaM antagonists, which resulted in a significant decrease in CaM content. This result is in conformity with our previous study which shows that the NO-induced CaM level was inhabited by Ca2+ chelators BAPTA or CaM antagonist W-7  on cellular membranes, is critical for maintaining Ca2+ homeostasis . A previous study shows that CaM3 expression was induced by heat and CaCl2 and correlated positively with thermotolerance of lily , Ca2+ channel inhibitors  and CaM antagonists  inhibited the NO-induced CaM gene expression. Interestingly, neither membrane-impermeable Ca2+ chelators EGTA nor membrane-permeable Ca2+ chelators BAPTA/AM inhibited CBL3 expression as much as CBL1 expression. Probably because CBL1 and CBL3 have different sensitivity to these two Ca2+ chelators and this possibility should be further investigated. Similarly, the CBL1 and CBL3 gene expression was also upregulated by NO, and the NO-enhanced gene expression was suppressed by these chelators, antagonists, or inhibitors . Notably, CaM antagonists W-7 and TFP also largely inhibited CBL1 and CBL3 expression. These results further verified the study of 2+-binding proteins such as CaM and CBLs (2+ signaling-regulated gene expression of CaM, CBL1, and CBL3.To further investigate the relationships of NO and Canist W-7 . Therefod plants . Thus, deostasis . Plant Ctoxicity . In the  of lily . Our resand CBLs . Thus, t2+ could prolong vase life of cut flowers. NO might promote postharvest freshness by increasing Ca2+/CaM content, and regulating gene expression of Ca2+ signaling-regulated proteins. Our data also revealed that Ca2+/CaM might act downstream molecules of NO signaling pathway. However, the interaction between NO and Ca2+/CaM seems to be very complex. Therefore, more efforts are needed to demonstrate the detailed mechanism during senescence processes of cut flowers.Altogether, the results of our study have shown that exogenous application of NO and CaWL designed the experiment. JZ, HF, JH, and DH preformed the experiment. JZ, HF, JH, and BW performed the data analysis. JZ wrote the manuscript. WL revised the manuscript. All the authors read and approved the final manuscript.The 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.


Patients harbor mild to moderate thrombocytopenia and a risk of around 35% to develop AML or myelodysplasia. This disease (OMIM 601399) is due to germline genetic anomalies affecting the transcription factor RUNX1. Missense, nonsense, and frameshift mutations of RUNX1 have been reported as well as small and large deletions. At the time of AML diagnosis, the most frequent acquired genetic alteration implicates a second alteration of RUNX1 ,3 attesting to the importance of RUNX1 dosage in AML development.4In 2016, the WHO classification added a new category entitled “myeloid neoplasms with germ line predisposition” to acute myeloid leukemia (AML) classification, reflecting the increasing importance of these malignancies. Diagnosis of such patients is of clinical relevance, as they can benefit of specific clinical care including hematopoietic stem cell transplantation in first complete remission and genetic counseling. Genetic analysis is imperative to avoid the use of hematopoietic stem cell transplantation from an asymptomatic mutation carrier family donor. Thus, genetic testing for genes implicated in hereditary myeloid malignancies has to be done, whatever the age of the patient, in case of suggestive personal and/or family history, this is facilitated by the expanded use of next-generation sequencing. Among these hereditary malignancies, the familial platelet disorder with predisposition to acute myelogenous leukemia (FPD/AML) was first described in 1999.NPM1 and FLT3-ITD mutations. He was in complete remission after a 3 + 7 induction therapy  complicated with an invasive pulmonary aspergillosis, followed by 4 ambulatory postremission chemotherapy  and a cord blood transplant. Unfortunately he relapsed and died in April 2011. The family and clinical history of the patient suggested inherited myeloid neoplasms associated with platelet disorders.A 62-year-old male Fig. , I-1 wasThe biological samples were collected after informed consent, in accordance with the Declaration of Helsinki. DNA was extracted from cell pellets using the QIAamp Tissue Kit (Qiagen) according to the manufacturer's instructions.RUNX1 gene was performed as previously described by Sanger sequencing.3Direct sequencing of all coding exons of the ASXL1, BCOR, BCORL1, CALR, CBL, CSF3R, DNMT3A, ETV6, EZH2, FLT3, GATA2, IDH1, IDH2, JAK2, KIT, KRAS, MPL, NIPBL, NPM1, NRAS, PHF6, PTPN11, RAD21, RIT1, RUNX1, SETBP1, SF3B1, SMC1A, SMC3, SRSF2, STAG2, TET2, TP53, U2AF1, WT1, ZRSR2 genes. The library was prepared using the Ampliseq System according to the manufacturer's instructions and run on Ion Proton . Raw data were analyzed with both Torrent Browser (Thermo Fisher) and SeqNext . A high depth of coverage (>1500) was obtained for all genes. A second panel of genes implicated in inherited myeloid malignancies including ANKRD26, CEBPA, DDX41, ETV6, GATA2, RUNX1, SRP72, TERC, TERT, and TP53 was sequenced according to the same protocol.For high-throughput sequencing (HTS), a panel of 36 genes recurrently mutated in myeloid malignancies was sequenced on genomic DNA isolated from mononuclear white blood cells. These include RUNX1 deletions, SNP-array karyotyping  was performed according to manufacturers’ instructions. Data were analyzed with the Chromosome Analysis Suite version 3.1 . The intragenic RUNX1 deletion was then confirmed by multiplex ligation probe amplification (MLPA) .To detect RUNX1 by the Sanger method at AML diagnosis and discovered a RUNX1 mutation (NM_001001890 exon 2 c.416G>A:p.R139Q). The R139Q mutation has been already described in FPD/AML patients.5 It affects the Runt homology domain (RHD) of RUNX1, which binds to DNA and to the other member of the CBF complex, CBFβ. After genetic counseling, we sequenced RUNX1 in the 2 adult's relative (II-1 and II-2) presenting thrombocytopenia but unexpectedly, we were not able to find the R139Q mutation or any other RUNX1 mutations. A clinical and biological follow-up was decided for the relatives. In 2016, we used a 36 genes-HTS panel to study the AML diagnosis sample of the proband. It confirmed the RUNX1 R139Q mutation with a variant allele frequency (VAF) at 50%. Additional somatic mutations were described as BCOR p.K1137delinsTX (VAF 54%), DNMT3A p.W795C (VAF 48%), NRAS p.Q61K (VAF 47%), and SRSF2 p.P95R (VAF 47%). Another HTS panel, including among others ANKRD26 and ETV6 which are also implicated in inherited myeloid malignancies with preexisting platelet disorders,9 was used but we did not find any additional mutation. Based on our recent paper describing a second aberration of RUNX1 in AML transformation in FPD/AML patients,2 we hypothesized that the RUNX1 mutation found at the AML stage was an acquired event and that we may have missed the inherited RUNX1 alteration.Based on patient's personal and familial history of thrombocytopenia, we first sequenced RUNX1 deletions have been already described in FPD/AML,14 we performed SNP-array and we were able to identify a microdeletion of 51 kb in the RUNX1 gene. This result was confirmed by MLPA in the patient and in the 2 adult's relative confirming the diagnosis of FPD/AML  and is associated with AML development.15 This suggests that such truncated mRNA could predispose to leukemia progression in FPD/AML. Besides large RUNX1 deletions, small deletions have been rarely described in FPD/AML, they were identified by MLPA or by CGH-array, confirming once again the importance of this type of genetic test for FPD/AML diagnosis.12 Additionally, we sequenced the proband's sample at complete remission using the 36 genes-HTS panel and did not find the mutations identified at AML stage, excepted the DNMT3A mutation with a lower VAF at 4%, which could represent the persistence of clonal hematopoiesis. We were not able to find the RUNX1 mutation anymore, confirming the acquired status of this genetic event.As germline RUNX1 at AML stage, confirming the association of this event with AML development. Of note, capture-based HTS may provide information on copy number variations for all homogeneously covered regions. This notion should be kept in mind in lab’ strategies especially for the assessment of haplo-insufficient genes status. In FPD/AML, as both deletions and mutations of RUNX1 have been reported in patients, biological tests should include other techniques such as SNP-array and/or MLPA in a second phase to identify microdeletions when FPD/AML is strongly suspected. Similar strategy should be applied to diagnose other inherited hematological disorders implicating not only mutations but also gene deletions.This report illustrates that, in front of a clinical and familial history evocating strongly inherited thrombocytopenia with predisposition to AML, all the biological examinations have to be conducted. Indeed, it is crucial to diagnose inherited diseases such as FPD/AML, as the management of the patient could differ from a patient with de novo AML, especially if hematopoietic stem cell transplant is considered. Moreover, it implicates a regular follow-up for patients within the family at thrombocytopenia stage, as they are at risk of developing AML. In our case, Sanger sequencing and amplicon-based HTS were not sufficient to establish the diagnosis but allowed to identify an acquired second hit affecting


In this study, however, we show that metabolic inhibition results in a biphasic effect on LTCC current (ICaL) in human and rat cardiac myocytes: an initial increase of ICaL is observed in the early phase of metabolic inhibition which is followed by the more classical and strong inhibition. We studied the mechanism of the initial increase of ICaL in cardiac myocytes during β-adrenergic stimulation by isoprenaline, a non-selective agonist of β-adrenergic receptors. The whole-cell patch–clamp technique was used to record the ICaL in single cardiac myocytes. The initial increase of ICaL was induced by a wide range of metabolic inhibitors . In rat cardiomyocytes, the initial increase of ICaL was eliminated when the cells were pre-treated with thapsigargin leading to the depletion of Ca2+ from the sarcoplasmic reticulum (SR). Similar results were obtained when Ca2+ release from the SR was blocked with ryanodine. These data suggest that the increase of ICaL in the early phase of metabolic inhibition is due to a reduced calcium dependent inactivation (CDI) of LTCCs. This was further confirmed in human atrial myocytes where FCCP failed to induce the initial stimulation of ICaL when Ca2+ was replaced by Ba2+, eliminating CDI of LTCCs. We conclude that the initial increase in ICaL observed during the metabolic inhibition in human and rat cardiomyocytes is a consequence of an acute reduction of Ca2+ release from SR resulting in reduced CDI of LTCCs.Metabolic inhibition is a common condition observed during ischemic heart disease and heart failure. It is usually accompanied by a reduction in L-type Ca During normal cardiac excitation–contraction coupling, a relatively small amount of Ca2+ enters the cell via the L-type current (ICaL) and this triggers the release of a larger amount of Ca2+ from the sarcoplasmic reticulum (SR) via ryanodine receptors (RyRs) in a process called Ca2+-induced Ca2+ release [2+ concentration in the vicinity of closely associated Ca2+ channels and RyRs leads to calcium dependent inactivation (CDI) of LTCCs [CaL [2+ channels [2+, which directly activates the contraction of myofilaments. The mechanisms of Ca2+ regulation in diseased cardiac cells may be disordered. Malfunction of LTTCs and RyRs interaction may affect the contractility of myocardium and, as a consequence, the main function of the heart, to pump blood, may be altered.The L-type Ca release . The incCCs [CaL . Hence, channels . The cloCaL during metabolic inhibition, however the mechanisms involved are not clear [CaL during β-adrenergic stimulation in human atrial and ventricular myocytes and rat ventricular myocytes. Surprisingly, we found that metabolic inhibition causes an acute transient increase of ICaL amplitude followed by a rapid reduction. This is consistent with the most recent study demonstrating AP prolongation during metabolic inhibition in the rabbit heart [CaL without the initial increase [CaL during metabolic inhibition. Our results point to a modulation of the Ca2+-dependent inactivation of LTCCs by the RyRs mediated Ca2+ release.Metabolic inhibition is commonly observed during ischemic heart disease and heart failure and is associated with biphasic changes in action potential (AP) duration in heart cells and whole heart ,6. Numerot clear ,9,10,11.ot clear ,13. ThusCaL in human atrial myocytes. The cardiac myocytes for the first series of experiments were derived from five patients. Metabolic inhibition was induced by the application of mitochondrial uncoupler carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) to isoprenaline  stimulated human atrial cells and changes of ICaL were monitored. Surprisingly, in most of the tested human atrial cells, FCCP induced a biphasic effect on ICaL, i.e., a rapid initial stimulation of ICaL that was followed by a strong inhibition of ICaL.The first series of experiments were set to determine the effect of metabolic inhibition on ICaL induced by FCCP was recorded in six out of nine (~67%) tested human atrial myocytes. In these cells, the application of 30 or 100 nmol/L of FCCP resulted in a transient increase of ISO-stimulated ICaL by 11.4 ± 2.5%  and 9.0 ± 2.8% , respectively. In all human atrial myocytes tested, metabolic inhibition led to a final suppression of ICaL by 65.5 ± 5.6%  and 55.8 ± 9.8%  with 30 and 100 nmol/L FCCP, respectively  on ISO (1 μmol/L) stimulated cells. We found that FCCP induced a rapid initial stimulation of ICaL in three out of six ventricular myocytes (five patients). Further, 100 nmol/L of FCCP increased ISO-stimulated ICaL by 24.8 ± 5.3% . The reduction of ICaL induced by FCCP was registered in all tested cells, i.e., ICaL was reduced by 42.5 ± 3.5% . Another uncoupler, DNP, also evoked a transient increase of ISO-stimulated ICaL in five ventricular myocytes from nine (three patients). Further, 100 µmol/L of DNP increased ISO-stimulated ICaL by 5.0 ± 0.9% . The reduction of ICaL by 44.8 ± 4.4% induced by DNP was registered in all  human ventricular myocytes (CaL induced by uncouplers FCCP and DNP was recorded in eight out of 15 (~53%) tested human ventricular myocytes.The next series of experiments were set to determine the effect of metabolic inhibition on Imyocytes c,d. The CaL suppression during metabolic inhibition was previously demonstrated by numerous studies [CaL amplitude during metabolic inhibition in cardiac myocytes has not previously been reported. To determine if this phenomenon is specific to human cardiomyocytes, we reproduced the same type of experiments in rat ventricular myocytes. Application of FCCP (100 nmol/L) in ISO-stimulated cells induced a transient increase in ICaL by 20.2 ± 2.7%  that was followed by ICaL reduction by 42.4 ± 4.6%   e,f. High < 0.05) e also inCaL were due to metabolic inhibition, effects of other metabolic inhibitors were also tested. Rotenone (30 µmol/L), an inhibitor of the complex I of the mitochondrial respiratory chain, also caused an initial increase of ISO-stimulated ICaL by 11.4 ± 0.2%  and later reduced ICaL by 28.2 ± 7.6% . Antimycin A (10 µmol/L), an inhibitor of the complex III of the mitochondrial respiratory chain, initially increased ISO stimulated ICaL by 8.3 ± 2.3%  and decreased by 35.5 ± 1.9%  and by characterizing two kinetically distinct fast  and slow  current components [CaL revealed that during the transient increase of ICaL with FCCP (100 nmol/L) in ISO stimulated cells, the time constant of the fast component τ1 increased from 5.3 ± 0.3 ms to 7.9 ± 1.1 ms , while the time constant of the slow component τ2 decreased from 48.7 ± 2.9 ms to 42.6 ± 2.5 ms . The changes in the amplitude of fast and slow components of ICaL were relatively small. During the inhibitory phase when the effect of FCCP reached steady-state, τ1 and τ2 were 13.8 ± 2.1 ms  and 42.7 ± 3.6 ms (n = 12), respectively, meanwhile the amplitude of both components of ICaL were reduced. The ICaL curves are presented in 1, suggesting a diminished calcium dependent inactivation (CDI) of the channels.To get an insight on the possible mechanisms of Imponents . Double CaL in rat ventricular cells , respectively. As expected, ISO significantly shifted I–V curve to the left [p < 0.05) while FCCP had no significant effect on the ICaL I–V relationship. The fit of the steady–state inactivation curves for control, ISO and FCCP led to V1/2 values of −29.20 ± 1.15 mV, −32.05 ± 1.60 mV and −32.75 ± 1.42 mV, respectively, and k was 6.2 ± 0.1, 5.9 ± 0.3 and 5.7 ± 0.1 (n = 3), respectively. Again, ISO significantly shifted the inactivation of ICaL to the left by ≈3 mV (p < 0.05), however the effect of FCCP was negligible. The changes of curve steepness k under the influence of ISO or FCCP were insignificant both for I–V and for steady–state inactivation curves. We also measured the current–voltage relationship (I–V) and the steady–state inactivation of Iar cells . AverageV curves b and inaV curves c in contthe left  by > 7 mCaL. In our previous study, we found no such stimulation during metabolic inhibition in frog cardiomyocytes [CaL in mammalian cells may involve metabolic sensitivity of SR and RyRs in regulation of calcium dependent inactivation (CDI) of LTCCs. To test this hypothesis, additional experiments were performed in rat cardiomyocytes in which Ca2+ release from SR was suppressed prior to metabolic inhibition.The above results showed that in human and rat cardiac cells, metabolic inhibition induced initial transient increase of Imyocytes  where SRmyocytes  and, in 2+ ATPase (SERCA) and impairs Ca2+ uptake by the SR and thus leads to the depletion of Ca2+ in the SR [CaL in rat ventricular myocytes to 142.1 ± 8.3%, i.e., from 17.7 ± 0.5 pA/pF to 25.1 ± 0.9 pA/pF . In the presence of thapsigargin, the initial ICaL increase was completely eliminated and only an inhibitory effect of FCCP on ISO stimulated ICaL was observed  , respectively. In the presence of thapsigargin, the effect of FCCP on τ1 was abolished. The time constant τ1 in such conditions remained similar to thapsigargin treated cells and was 26.4 ± 0.5 ms, while τ2 was similar to ISO stimulated ICaL and was 69.1 ± 3.7 ms (n = 4). The representative ICaL traces and their fits are shown in CaL and the application of FCCP failed to induce the initial ICaL increase and, thus, only an inhibition of ISO stimulated ICaL was observed . Thapsigargin inhibits SR Can the SR . As expe < 0.05) a,b. Doubobserved .CaL increase during the early phase of metabolic inhibition. To further confirm this hypothesis, we measured the changes of ICaL facilitation in rat cardiac cells in the absence and presence of FCCP. Increased frequency (to 1 Hz) of depolarization after a 20 s pause was used in these series of experiments. The peak amplitude of ICaL and the ICaL area were compared at the time of the first and fourth depolarizing pulse. In control conditions, increased stimulation frequency enhanced ICaL amplitude by 8.3 ± 2.1% and the area by 46.9 ± 6.3%  during the fourth depolarizing pulse. In ISO stimulated rat cardiac cells, increasing stimulation frequency to 1 Hz also induced ICaL facilitation and increased ICaL peak amplitude and area by 22.2 ± 5.3% and 42.0 ± 8.6% , respectively , while the time constant of the slow component τ2 decreased from 62.1 ± 4.8 ms to 51.3 ± 2.6 ms . The ICaL curves are presented in CaL facilitation was no longer observed and ICaL amplitude and area were reduced by 23.6 ± 4.1% and 23.1 ± 3.3%  when compared with the first depolarizing pulse. After the application of FCCP in ISO-stimulated and thapsigargin treated rat cells, the ICaL frequency-dependent facilitation remained abolished and the amplitude and area of ICaL were reduced during the fourth depolarizing pulse by 22.7 ± 1.8% and 20.0 ± 1.4%, respectively . The observation that the application of FCCP abolishes the facilitation of ICaL in a similar manner as the depletion of Ca2+ from the SR, indicates that FCCP affects CDI of LTCCs.Depletion of Calitation c and, duCaL during metabolic inhibition, we performed experiments in human atrial myocytes in which CaCl2 in the external solution was replaced by the equimolar BaCl2. LTCCs are permeable to Ba2+, however Ba2+ ions do not induce Ca2+ release from the SR and, thus, abolish CDI of LTCCs. As shown in 2+ current (IBa) without the transient increase observed in ICaL. At 100 nmol/L and 1 μmol/L concentrations, FCCP suppressed the ISO-stimulated IBa by 83.3 ± 1.6%  and 84.5 ± 3.5% , respectively , demonstrating that the observed effect is not dependent on the method used to induce metabolic inhibition. In the control experiments without metabolic inhibition, we observed only run-down of the ICaL and no spontaneous increase in ICaL was detected  [CaL in some of the cells, which was followed by a pronounced current inhibition. The authors have attributed this effect to a leftward shift of the Ca2+ channel activation curve. In our experiments, the leftward shift in the ICaL I–V relationship was induced only by application of ISO which is consistent with β-adrenergic stimulation [CaL I–V curve during the application of FCCP   found a mulation . However of FCCP . TherefoCaL in amphibian heart cells without the initial transient increase observed here. In frog cardiomyocytes, SR is scarce and lacks ryanodine channels [2+ for contraction in amphibian cardiomyocytes and Ca2+ release from the SR plays only a minor role, while in mammalian hearts, the SR serves as the main Ca2+ store and plays an important role in the regulation of the activity of LTCCs [2+ induced Ca2+ release (CICR) leading to diminished Ca2+ dependent inactivation (CDI) of LTCCs might underlie the transient increase in ICaL amplitude. It has previously been demonstrated that metabolic inhibition affects properties of intracellular Ca2+ release and decreases frequency of spontaneous Ca2+ waves [2+ load during metabolic inhibition are rather inconsistent as some studies have reported no change or a decrease in SR Ca2+ content [2+ load [2+ signaling, consisting of an initial inhibition followed by stimulation of SR Ca2+ release [2+ from SR as well as the reuptake of Ca2+ to the SR by SR Ca2+ ATP-ase, the activity of which may also be inhibited by deficiency of energy compounds. During metabolic inhibition, both Ca2+ release (RyRs) and reuptake (Ca2+ ATP-ase) mechanisms are inhibited to a different extent [CaL during ischemia may lead to early afterdepolarisation and long QT which can be followed by arrhythmias. In our study, double exponential fit to ICaL curves showed a significant increase of the fast component τ1 during the transient increase of ICaL in the presence of FCCP. It is known that the fast component of ICaL inactivation is dependent on the magnitude of Ca2+ release from SR [CaL and intracellular Ca2+ release during metabolic inhibition. In order to test the hypothesis that the changes of Ca2+ release from SR result in the potentiation of ICaL, we have performed a series of experiments where, prior to the application of metabolic inhibitors, SR Ca2+ release was suppressed. Suppression of SR Ca2+ release in cardiac cells reduces CICR and therefore leads to decreased CDI of LTCCs. Consequently, a significant augmentation of ICaL was observed in the presence of thapsigargin or ryanodine  was eliminated. We suggest that under these conditions, CDI of ICaL is abolished and, therefore, an increase in ICaL is not observed. For the same reason, there is a lack of ICaL facilitation in thapsigargin treated rat cardiac cells where the decay characteristics of fast and slow components of ICaL were significantly augmented and subsequent metabolic inhibition resulted only in a rapid decrease of ICaL without any initial ICaL increase. The finding that CDI plays a crucial role in the initial potentiation of ICaL during metabolic inhibition was also supported by the experiments where CDI was eliminated by equimolar substitution of extracellular Ca2+ for Ba2+ , where they describe the decay phases of ICaL at the stimulation frequency of 1 Hz.The double exponential fits and analysis of ISO stimulated Id to CDI . IncreasCaL amplitude was registered not in all human cardiac myocytes and this may point to possible impairment in cells derived from pathological cardiac specimens. However, we were not able to find a relation between the transient increase in ICaL (or its absence) and any pathology of the human heart. Moreover, we found that even myocytes derived from the same patient showed a different response to metabolic inhibition, i.e., in some cardiomyocytes, the transient increase of ICaL was registered, while in others it was not. We suggest that the observation that transient ICaL increase is induced just in some of human atrial and ventricular myocytes while all rat ventricular myocytes exhibit an increase in ICaL during metabolic inhibition, can be attributed to the pathophysiological state of the particular cell as well as to differences between species. The pathophysiological state of the cell and thus the different response to metabolic inhibition may be related with attenuation or downregulation of β-adrenergic signaling in human heart cells [2+ release from SR more homogeneous in rodents’ ventricular myocytes [2+ level in the micro-domain near SR RyRs and that they play an important role in the regulation of intracellular Ca2+ waves and arrhythmogenesis [CaL shows the abnormalities of Ca2+ cycling in heart cells during ischemia (metabolic inhibition) which may lead to the arrhythmogenic processes of the heart.The increase in Irt cells ,13. Rat rt cells  and to cmyocytes . There iogenesis . The ini2+-channel blockers, digitalis, β-AR antagonists, diuretics, ACE inhibitors, NO-donors and/or antiarrhythmic drugs) that was stopped 24 h before surgery. In addition, before and during surgery, all patients received sedatives, anesthesia and antibiotics. Information on patient group and specimens is provided in 2+ free Tyrode solution (in mmol/L): 136 NaCl, 5.4 KCl, 1.1 MgCl2, 10 HEPES, 20 taurine, 5 sodium pyruvate, 10 d-glucose, 0.3 NaH2PO4, pH 7.3 adjusted with NaOH at room temperature. Later, the tissue was cut into pieces of ~1 mm3 in Tyrode solution with 30 mmol/L 2,3-butanedionemonoxime added to the solution. Subsequently, a 30–40 min incubation with collagenase  and protease  in 10 mL Ca2+ free BSA (5 mg/mL) containing Tyrode solution was performed at 37 °C. The solution was then replaced by 10 mL of fresh solution containing collagenase (400 U/mL) and digestion was repeated for 20–30 min. Every five minutes, a small sample of solution was examined under a microscope to evaluate the stage of cell dissociation.This study was carried out in accordance with the recommendations of the principles outlined in the Declaration of Helsinki, Kaunas Regional Bioethics Committee (Lithuania) with written informed consent from all subjects. All subjects gave written informed consent in accordance with the Declaration of Helsinki. The protocol was approved by the Kaunas Regional Bioethics Committee (16 November 2015 No. BE-2-40). Specimens of human heart were obtained from 16 patients undergoing heart surgery for coronary artery diseases at the Hospital of Lithuanian University of Health Sciences . Most of the patients had received a pharmacological treatment (including Ca2+ free Ringer solution (in mmol/L): 117 NaCl, 5.8 KCl, 4.4 NaHCO3, 1.5 KH2PO4, 1.7 MgCl2, 11.7 d-glucose, 10 sodium phosphocreatine, 20 taurine, 21 HEPES, pH 7.1 adjusted with NaOH at room temperature. For heart myocyte, dissociation collagenase type A  was added to the Ca2+ free Ringer solution and the heart was perfused for 1 h. The ventricles were then separated from atria, cut to small pieces and agitated gently to dissociate the single cells.This study was carried out in accordance with the European Community guiding principles and recommendations of the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health and UK regulations on animal experimentation . The pro+ free control external solution containing (in mmol/L): 127 NaCl; 10 HEPES; 20 CsCl; 4 NaHCO3; 0.8 NaH2PO4; 1.8 MgCl2; 1.8 CaCl2; 5 d-glucose; 5 sodium pyruvate; pH 7.4 adjusted with NaOH. Patch pipettes were made from glass capillaries  and had resistances of 0.9–1.2 MΩ when filled with control internal solution. The myocytes were dialysed with control internal solution composed of (in mmol/L): 140 CsCl, 5 EGTA, 4 MgCl2, 0.062 CaCl2, 5 creatine phosphate disodium salt, 3.1 Na2ATP, 0.42 Na2GTP and 10 HEPES; pH to 7.3 was adjusted with CsOH. The whole-cell patch–clamp technique was used to record the ICaL in human and rat cardiac myocytes as described previously [CaL facilitation was generated after 20 s of rest applying four depolarizing pulses from −80 mV holding potential to 0 mV for 200 ms (frequency 1 Hz). Tetrodotoxin  and a 50 ms prepulse to −50 mV before every depolarizing pulse were used to eliminate sodium currents. Inward peak currents were measured as the difference between the maximal inward current amplitude and the current at the end of the test pulse [For electrophysiological experiments, a few drops of cells suspension were placed in a perfusion chamber mounted on an inverted microscope stage. After cells had settled to the bottom, the chamber was superfused with Keviously . Ionic cCaL is reached after four stimulations [CaL facilitation was assessed comparing the first ICaL trace with the fourth trace at 1 Hz. The peak of ICaL and the ICaL area, an integral of ICaL for 200 ms (pA x ms), were registered at the time of the first and fourth depolarizing pulse. The time integral of ICaL was registered in part of the experiments to elucidate the origin of ICaL increase during metabolic inhibition and to compare the initial effect of FCCP with frequency-dependent facilitation and reduced CDI.Equilibrium for steady-state frequency-dependent facilitation of Iulations , therefoCaL, a double pulse voltage-clamp protocol was applied every 4 s (see insert in To determine the current–voltage (I–V) relationship and inactivation of IAll experiments were performed at room temperature (18–24 °C) and the temperature did not change by more than 1°C during an experiment.p-trifluoromethoxyphenylhydrazone , 2,4-Dinitrophenol  in ethanol, rotenone (100 mmol/L) and antimycin A (100 mmol/L) in DMSO were used. The stock solution of the SR Ca2+ ATP-ase inhibitor thapsigargin (10 mmol/L) was prepared in ethanol. A stock solution of ryanodine (10 mmol/L) for blocking of RyR channels was prepared in distilled water. An aqueous stock solution of the isoprenaline  was freshly prepared every day prior to the experiments. When the stock solutions in DMSO or ethanol were used, the corresponding amount of solvent was also added to the external control solution.All drugs and chemicals were from Sigma-Aldrich  if not specified otherwise. All drugs tested in patch-clamp experiments were solubilized in experimental solutions just before application onto the cell studied, i.e., only fresh solutions were tested. To block oxidative phosphorylation stock solutions of carbonyl cyanide-CaL traces were best fitted by the sum of two sequential exponentials using the expression:CaL,1 and ICaL,2 are the amplitudes and τ1 and τ2 are the time constants of the fast and slow components of ICaL, respectively. The fitting of ICaL inactivation traces was performed using the modified protocol described by [Inactivation of Iribed by  in rat vCaL. The quality of the fits were evaluated by correlation coefficients (r) and in all of the experiments, the correlation coefficient was ≥0.985.The fitting time was set from ~1 ms after the peak of inward calcium current until the end of 400 ms pulse. The steady-state current was calculated at the end of 400 ms pulse and this helped to avoid residual leak current influence to the inactivation of I1) and I–V traces (B2) were fitted with Boltzmann equations:1/2 is 0.5 of the maximal value of steady-state activation and inactivation, Vrev is the reversal potential of Ca2+, g is the conductance of LTCC and k is the steepness parameter.Steady-state inactivation curves . The significance of differences was evaluated using one-way analysis of variance (ANOVA). The significance level was set at CaL which is due to a reduction in SR function and an alleviation of the Ca2+-dependent inactivation of the L-type Ca2+ channels. These mechanisms may contribute to the formation of arrhythmias in the ischemic heart and may be targets for new treatment methods of heart pathologies in the future.In conclusion, our data demonstrate that metabolic inhibition in mammalian cardiomyocytes causes an initial stimulation of I