We measured LV pressure (LVP) and [Ca2+] as a function of intra-LV stretch in guinea pig intact hearts before and after 15 min global stunning ± perfusion with streptomycin (STM), a stretch-activated channel blocker. LV wall [Ca2+] was measured by indo-1 fluorescence and LVP by a saline-filled latex balloon inflated in 50 μL steps to stretch the LV. We implemented a mathematical model to interpret cross-bridge dynamics and myofilament Ca2+ responsiveness from the instantaneous relationship between [Ca2+] and LVP ± stretching. We found that: (1) stretch enhanced LVP but not [Ca2+] before and after stunning in either control (CON) and STM groups, (2) after stunning [Ca2+] increased in both groups although higher in STM versus CON (56% vs. 39%), (3) STM-enhanced LVP after stunning compared to CON , and (4) stretch-induced effects on LVP were independent of [Ca2+] before or after stunning in both groups. Mathematical modeling suggested: (1) cooperativity in cross-bridge kinetics and myofilament Ca2+ handling is reduced after stunning in the unstretched heart, (2) stunning results in depressed myofilament Ca2+ sensitivity in the presence of attached cross-bridges regardless of stretch, and (3) the initial mechanism responsible for increased contractility during stretch may be enhanced formation of cross-bridges. Thus stretch-induced enhancement of contractility is not due to increased [Ca2+], whereas enhanced contractility after stunning in STM versus CON hearts results from improved Ca2+ handling and/or enhanced actinomyosin cross-bridge cycling.Stretching the cardiac left ventricle (LV) enhances contractility but its effect on myoplasmic [Ca These are (1) the upstream mechanism that is related to the gross concentration of [Ca2+], (2) the central mechanism that is related to the sensitivity of the regulatory protein troponin C (TnC) to the available [Ca2+], and lastly, (3) the downstream mechanism that is related to the interaction between contractile proteins actin and myosin, and actinomyosin cross-bridge cycling. Myocardial stretch improves contractile function acutely by the Frank-Starling mechanism and more slowly due to the slow force response , a known blocker of sarcolemmal stretch-activated cation channels , which can irreversibly damage the heart, short-term ischemia, that is, stunning, and reperfusion is reversible with no permanent tissue damage.We also implemented a mathematical model to interpret cross-bridge dynamics and myofilament CaGuide for the Care and Use of Laboratory Animals from the National Institutes of Health . Prior approval was obtained from the Medical College of Wisconsin Institutional Animal Care and Use (IACUC) Committee. We have described the preparation of guinea pig hearts for Ca2+ measurements previously  at an aortic root perfusion pressure of 55 mmHg and was then rapidly excised. The KR perfusate  was filtered (5 μm pore size) in-line and had the following calculated composition in mmol/L (nonionized): Na+ 138, K+ 4.5, Mg2+ 1.2, Ca2+ 2.5, Cl− 134, HCO3− 15, H2PO4− 1.2, glucose 11.5, pyruvate 2, mannitol 16, EDTA 0.05, probenecid 0.1, and insulin 5 (U/L). Perfusate and bath temperatures were maintained at 37.2 ± 0.1°C using a thermostatically controlled water circulator.The investigation conformed to the Left ventricular pressure (LVP) was measured isovolumetrically with a transducer connected to a thin and compliant saline-filled latex balloon inserted into the left ventricle (LV) through the mitral valve from a cut in the left atrium. Balloon volume was initially adjusted to a diastolic LVP of zero mmHg so that any subsequent increase in diastolic LVP reflected an increase in LV wall stiffness or diastolic contracture. Saline was added in 50 μL increments to a maximum of 200 μL to stretch the LV. At each volume level the heart was allowed to stabilize for 2 min. Saline was removed in the same way to return the LV volume to baseline values after each volume increase. Pairs of bipolar electrodes were placed in the right atrial appendage, right ventricular apex, and LV base to monitor spontaneous heart rate (HR). Coronary flow  was measured at a constant perfusion pressure (55 mmHg) by an ultrasonic flowmeter  placed directly into the aortic inflow line.2) was placed gently against the LV epicardial surface through a hole in the bath. A rubber O ring was placed over the fiberoptic tip to seal the hole and netting was applied around the heart for optimal contact with the fiber optic tip. This maneuver did not affect LVP. Background auto fluorescence was determined for each heart after initial perfusion and equilibration at 37°C.Experiments were carried out in a light-blocking Faraday cage. The heart was partially immobilized by hanging it from the aortic cannula, the pulmonary artery catheter, and the LV balloon catheter. The heart was immersed continuously in the bath at 37°C. The distal end of a trifurcated fiber silica fiberoptic cable  were recorded using a modified luminescence spectrophotometer . The LV region of the heart was excited with light from a xenon arc lamp and the light filtered through a 350 nm monochromator with a bandwidth of 16 nm. The beam was focused onto the in-going fibers of the optic bundle. The arc lamp shutter was opened only for 2.5 sec recording intervals to prevent photobleaching. Emission fluorescence was collected by fibers of the remaining two limbs of the cable and filtered by square interference filters  at 405 and 460 nm. Although both F405 and F460 decline over time, time control studies showed that the F405/F460 ratio remains stable indicating no change in effective measured myoplasmic [Ca2+] during the course of these studies. The F405/F460 ratio was converted to [Ca2+] after correcting to account for the nonmyoplasmic fraction as described previously  that arise from single or clusters of sarcoplasmic reticular Ca2+ release channels at the T-tubule-SR microdomain due to influx of Ca2+ via L-type Ca2+ channels  or with KR solution alone  for the remainder of the experimental protocol. Saline was added to, and removed, from the balloon in the LV in steps of 50 μL to a maximal added volume of 200 μL to stretch and release the LV muscle, respectively; at each volume level the heart was allowed to stabilize for 2 min. Hearts were then subjected to 15 min global ischemia followed by 30 min reperfusion ± STM and then restretched and relaxed again as described before. Finally, 100 μmol/L MnCl2 was infused for 10 min to quench the myoplasmic indo-1 Ca2+ transients so that phasic Ca2+ disappeared. This allowed for correction of any nonmyoplasmic contribution to the measured Ca2+ transients as described earlier .Initial background data were obtained after 30 min stabilization of the heart Fig.. After d2+] and LVP were obtained at selected intervals before and during the stretch protocol and stunning . Because we could not directly measure interactions between Ca2+ and troponin C, or kinetics of cross-bridge cycling, we used a previously developed mathematical model to help elucidate effects of changes in myofilament interaction that contribute to changing the dynamic relationship between LVP and Ca2+  myofilament (TnCA) and myosin (M) for cross-bridge formation and force development in the presence of CaBurkhoff , 2006a. Burkhoff , 2 consiMod), is proportional to the number of cross-bridges formed: [Ca·TnCA·M] + [TnCA·M]  and actinomyosin cross-bridges (αa). The β parameter represents the static value of K1 and Ka at 0 LVP; an increase in β indicates an increase in the resting value of the affinity of TnCA and Ca2+ (β1) and cross-bridge attachment (βa).The 2+ dissociates from TnCA before the M head detaches from the A molecule  with a 0.4 msec step size equal to the postinterpolation sampling interval and the following initial conditions  error between LVPα1 and αa must be greater than 0. Kd′ must be greater than Kd since cross-bridges dissociate more readily in the absence of attached Ca2+; and Kd′ accounts for the physiological difference between contraction and relaxation kinetics ; this concept incorporates the idea of a positive feedback mechanism to explain the delay in rise in LVP during contraction (Hill Lower and upper bounds for optimization of 1st order rate constants were set at 0 and 2000 sec (0.5 msec step size) respectively. Initial values for the parameters were obtained from Baran et al. . Severalion Hill .t-test. Differences among means were considered statistically significant at P < 0.05 (two-tailed). All experimental measurements and model rate constants were expressed as means ± SE.Functional indices and model rate constants, computed before and during the stretch protocol and stunning, were compared using one-way ANOVA followed by Dunnett’s comparison of means post hoc test . Poststunning values from STM and CON groups were compared to their respective prestunning values using Student’s paired 2+] from hearts in the CON and STM groups before and after stunning . Systematic stretch caused an increase in phasic LVP in CON and STM hearts both before and after stunning. Also, the increase in phasic LVP by stretch in the CON group was greater than that in the STM group before stunning. But no concurrent increase in phasic [Ca2+] was observed in either group by stretch before stunning. After stunning, phasic LVP in the CON hearts was decreased. However, there was no significant difference in phasic LVP before or after stunning in the STM treated hearts.Summarized data of phasic  LVP and [Caning Fig. showed n2+] loops for representative cardiac beats at increasing stretch before and after stunning in CON and STM hearts are shown , which characterize the changes in loop shape , but not in the interaction between TnCA and Ca2+ (α1); (2) a decrease in the resting (static) value of affinity for cross-bridge attachment (βa); (3) no change in the interaction between TnCA and Ca2+ (β1), and (4) no changes in the association rate constant of Ca2+ to A·M (K2), or in the dissociation rate constants of Ca2+ from TnCA (K3), of Ca2+ from A·M (K4), or of A·M with attached Ca2+ (Kd) or unattached Ca2+ (Kd′).Tableαa) and in the dynamic interaction between TnCA and Ca2+ (α1); (2) a large decrease in the resting (static) value of affinity for cross-bridge attachment (βa); (3) an increase in the resting interaction between TnCA and Ca2+ (β1), (4) decreases in the association rate constant of Ca2+ to A·M (K2) and in the dissociation rate constants of Ca2+ from TnCA (K3), (5) an increase in the dissociation rate constant of Ca2+ from A·M (K4), and (6) decreases in the dissociation rate constants of A·M with attached Ca2+ (Kd) and with unattached Ca2+ (Kd′).Stunning itself (BL 0 μL) suggested: (1) decreases in dynamic A·M cross-bridge formation (αa) and a marked decrease in the dynamic interaction between TnCA and Ca2+ (α1); (2) a further marked decrease in the resting (static) value of affinity for cross-bridge attachment (βa) but no change in the resting interaction between TnCA and Ca2+ (β1), (3) marked decreases in the association rate constant of Ca2+ to A·M (K2) and in the dissociation rate constants of Ca2+ from TnCA (K3), (4) an increase of Ca2+ from A·M (K4), and decreases in the dissociation rate constants of A·M with attached Ca2+ (Kd) and with unattached Ca2+ (Kd′).After stunning, stretching the myocardium suggested: (1) an added increase in dynamic actinomyosin cross-bridge formation (Stretch-activated (gated) ion channels (SAIC) are mechanotransducers that conduct ionic currents only when a stretch stimulus occurs in the cell membrane (Sukharev and Sachs 2+] and cardiac contractility (phasic LVP) before and after short ischemic stunning. We blocked these channels with STM and used a mathematical model of Ca2+ handling and actinomyosin cross-bridge cycling to aid in assessing the mechanism of stretch-induced changes in contractility and myoplasmic Ca2+ transients. Overall, our results demonstrate that: (1) Increasing balloon volume to stretch the LV is a valid model of enhanced contractility. (2) Stretch-induced enhanced contractility is not a result of increased myoplasmic [Ca2+]. (3) Improved return of contractile function after stunning when stretch activated channels are blocked may be mediated by improved Ca2+ handling and/or increased actinomyosin cross-bridge cycling (enhanced sensitivity). (4) Cardioprotection imparted by STM may also be related to attenuated Ca2+ loading on reperfusion after ischemic stunning. The cardioprotective effects of STM are also noted in the change in loop orientation .  Cardiopr [Ca2+].  Improvedroup Fig., which w2+] and LVP. We reported that IR injury resulted in reduced Ca2+ affinity for TnCA and decreased cross-bridge kinetics. In this report we examined for changes in Ca2+-contraction coupling due to stretch in normal and stunned cardiac muscle. It is evident that stretch does not affect this relationship via the upstream mechanism since there is no change in [Ca2+] with stretch either before or after stunning, although stunning itself does can result in an increase in phasic [Ca2+]. Consequently, we focused on the changes in central (Ca2+ affinity for TnCA) and downstream (A·M cross-bridge interaction) using the model.In a prior report  in a regulatory unit composed of actin (A) monomers, TnC, TnI (inhibitory subunit) and Tm, was sufficient to explain cooperativity. The model analysis suggested that Tm–Tm coupling potentiates the activating effects of strongly bound cross-bridges and contributes to force–Ca2+ dynamics in intact cardiac muscle.There are several proposals for cooperative mechanisms of cross-bridge formation  Stunning (IR) results in greater myoplasmic [Ca2+] but reduced Ca2+ affinity in the unstretched myocardium. However, stretch itself does not result in significant changes in measured myoplasmic [Ca2+] before or after stunning. (2) Cooperativity in cross-bridge kinetics and myofilament Ca2+ handling is depressed after as compared to before stunning in the unstretched myocardium. This may be related to fewer functioning cross-bridges. (3) Stunning results in depressed myofilament Ca2+ sensitivity in the presence of attached cross-bridges regardless of stretch as interpreted from the model parameters in Table2+ sensitivity is depressed while cross-bridge association increases dramatically in the early stages of stretch. (4) Cross-bridge dissociation rates Kd and Kd′ are largely depressed after stunning which might be related to decreased ATP synthesis. The stretch-related decrease in cross-bridge dissociation during moderate stretch exhibited by stunned myocardium may be a compensatory mechanism by functioning myocytes to improve contractile function.Our interpretation of the stretch-receptor–myoplasmic Ca+ relatio2+-contraction coupling relied on Ca2+ transients obtained via an optic probe with a transmural measurement field and surface area of approximately 3 mm2, under which the detected signal diminished proportional to depth. The model parameters were optimized to best-fit isovolumic LVP, a more global measurement. One of the implicit assumptions of this model was that the local Ca2+ transient is a true representation of Ca2+ kinetics in the entire ventricular wall regardless of orientation of the individual myocytes, which are known to be markedly different from endocardium to epicardium  also inhibited myogenic tone and K+-induced isometric force largely by blocking L-type, dihydropyridine-sensitive Ca2+ channels (Miller and Langton This model of Ca


Secondary intracellular calcium (Ca2+ cycling suppresses secondary Cai rise and genesis of EADs.To test whether inhibition of sarcoplasmic reticulum (SR) Cai optical mapping were conducted in 10 rabbit hearts. Atrioventricular block (AVB) was created by radiofrequency ablation. After baseline studies, E4031, SR Ca2+ cycling inhibitors (ryanodine plus thapsigargin) and nifedipine were then administrated subsequently, and the protocols were repeated.Langendorff perfusion and dual voltage and Cai rise was enhanced, and 7 (70%) rabbits developed spontaneous or pacing-induced TdP. In the presence of ryanodine plus thapsigargin, TdP inducibility was significantly reduced . Although APD was significantly prolonged  by ryanodine plus thapsigargin, the secondary Cai rise was suppressed . Nifedipine inhibited TdP inducibility in all rabbit hearts.At baseline, there was no spontaneous or pacing-induced TdP. After E4031 administration, action potential duration (APD) was significantly prolonged and the amplitude of secondary Ca2+ cycyling reduces the inducibility of TdP. The mechanism might be suppression of secondary Cai rise and genesis of EADs.In this AVB and long QT rabbit model, inhibition of SR Ca Kr) [Kr is encoded by the hERG gene, which is the gene encoding mutation in long QT syndrome type 2 [Drug-induced acquired long QT syndrome and torsades de pointes (TdP) is one of the most serious adverse effects of medications. There is close relationship between risk of prolonged QT interval/TdP and inhibition of rapidly activating delayed rectifier potassium current (IKr) ,2. IKr ie type 2 . HoweverCa,L) reactivation is required in the initiation of EADs [i) rise in a rabbit heart failure model [Ca,L blockade abolished EAD development in heart failure animal models [2+cycling also plays a role in genesis of EADs and secondary Ca2+ rise is still unclear. In this study, we hypothesized that SR Ca2+ cycling inhibition suppressed secondary Cai rise and development of EADs. We used a long QT syndrome rabbit model with atrioventricular block (AVB) creation and E4031 containing low-K+-low-Mg2+ Tyrode’s solution perfused to test the hypothesis. This study followed the previous studies of long QT rabbit models [Kr inhibitor and has been used for creation of long QT animal model.Early afterdepolarizations (EADs) are secondary depolarization during repolarization of action potential and usually developed in the presence of prolonged action potential duration (APD). L-type calcium current  of Chang Gung Memorial Hospital (Permit Number: 2012121704) and conformed to the Guide for Use of Laboratory Animals. All surgery was performed under general anesthesia with ketamine, rompun and isoflurane, and all efforts were made to minimize suffering. Ten adult New Zealand white rabbits (2.5–3.5 kg) were used in this study.2, 24 mM NaHCO3, 1.8 mM NaH2PO4, 1.8 mM CaCl2, 5.5 mM glucose and 100 mg/L albumin, equilibrated with 95% O2 and 5% CO2 in de-ionized water and with a pH of 7.40. We then performed radiofrequency ablation to create AVB using a 7Fr. quadripolar large-tip ablation catheter  at a output energy of 10~20W generated by a radiofrequency generator . The target ventricular escape rate after AV node ablation was less than 60 beats per minute.We created AVB after harvesting rabbit hearts in this study using a modified atrioventricular node ablation method  and the i and RH-237 (Molecular Probes) for membrane potential (Vm). We used a laser light at a wavelength of 532 nm  to excite the fluorescence dyes. The emitted fluorescence was acquired and filtered (715 mm for Vm and 580 nm for Cai) with twocharge-coupled device cameras  at 4 ms/frame temporal resolution and 128 x 128 pixels with spatial resolution of 0.35 x 0.35 mm2 per pixel. To provoke development of TdP, the perfused solution was changed to a modified low-K+ (2.25 mM)/low Mg2+ (0.25 mM) Tyrode’s solution after fluorescence dye staining. Motion artifacts were suppressed by blebbistatin .The hearts were stained with Rhod-2-AM  for CaPseudo-electrocardiography (pECG) was recorded using three electrodes placed at the left atrium, the posterior wall of the left ventricle and the right ventricle. A bipolar catheter was inserted into the right ventricular apex for pacing at twice threshold. Because of significantly prolonged APD in this long-QT syndrome model, the optical mapping signals were acquired at cycle lengths of 1000, 800, 600, 500, 400, 350, 300 ms and then down to the shortest 1:1 captured cycle length with a 20 ms step. A S1/S2/S3 short-long-short pacing protocol  was used to induce TdP. Some TdP episodes were also induced by a short burst pacing following a long pause in this AVB model. After the baseline studies, E4031 (0.5 μM), ryanodine (1.0 μM) plus thapsigargin (1.0 μM), and nifedipine (2.0 μM) were administered subsequently, and the experimental protocols were repeated after each set of medications.80 was measured at the level of 80% repolarization of action potential. Secondary rise of Cai is defined as the spontaneous increase of the Cai at the downslope of the primary Cai released. The amplitude of secondary Cai rises is defined as the largest deviation from a line drawn between the onset and offset of the secondary Cai rise as previously described [APDescribed . TdP wasi rise and Cai oscillation (red trace) during the spontaneous TdP. After ryanodine plus thapsigargin administration, the inducibility of TdP was significantly suppressed  in the presence of E4031. 80 was longer in the presence of E4031 and was further prolonged after administration of ryanodine plus thapsigargin at both 1000 ms and 500 ms pacing cycle lengths (PCLs). Nifedipine shortened APD80. Because of significantly prolonged APD80, we were not able to pace the hearts at pacing cycles shorter than 400 ms after applying E4031 in most of the hearts. Statistical tests showed that E4031 significantly prolonged APD80 . Nifedipine significantly shortened the extremely prolonged APD80 . Although inhibition of SR Ca2+ cycling further prolonged APD80, the inducibility of TdP was suppressed as shown in i rise and EADs in failing hearts [i rise. Therefore, we analyzed the amplitude of secondary Cai rise and the relationship between secondary Cai rise and TdP inducibility. m-Cai traces and secondary Cai rise maps. Significant secondary Cai rise developed at the basal area of the left ventricle in the presence of E4031. The amplitude of secondary Cai rise was positively correlated with the APD with E4031. i rise at baseline, in the presence of E4031, further administration of ryanodine plus thapsigargin, and then nifedipine. At baseline, there was only minimal secondary Cai rise (1.0 ± 1.0%), which was significantly enhanced by E4031 . Ryanodine plus thapsigargin prolonged APD but significantly suppressed secondary Cai rise . Nifedipine partially restored the extremely prolonged APD and further suppressed secondary Cai rise (0.4 ± 0.4%). As shown in i rise site presented the longest APD (point 1), and vice versa, the amplitude of secondary Cai rise was less obvious at the shorter APD area (point 3). i rise in each heart. The filled bars indicate hearts with inducible TdP and the unfilled bars are hearts without TdP. Note the 3 non-inducible hearts had relatively lower amplitude, although the animal number was too small for statistical testing.Previously we reported that apamin induced secondary Cag hearts . Opticali rise and APD maps. The greater-secondary Cai rise area co-localized with the longer-APD area. Panel B shows serial Vm maps of initiation of an EAD (the numbers indicate the frame). Frame 308 shows that repolarization developed from the apex (red arrow) toward the middle LV. Frame 334 shows that the basal area sustained at plateau phase  and the apical area developed repolarization . Frame 336 shows that the earliest activation sites of this EAD beat was at the border of the high secondary Cai rises region , propagating to the apical area . Panel C shows the corresponding Vm and Cai traces of this particular EAD in panel B. The bottom and right Vm and Cai traces were zoomed-in signals of the black box in the top subpanel, representing the Vm and Cai signals of the corresponding sites on the phase map. Note that the Cai trace (red lines) shows earlier Cai pre-fluorescence (black arrow head) at site b, which co-localized with the initiation of the EAD in the Vm map (80. The average calcium decay were significantly prolonged by E4031 (2+ cycling further delayed the calcium decay, the inducibility of TdP was not further enhanced.Whether calcium decay is important in the development of TdP is not clear. i rise area. i rise region. Because of significant spatially heterogeneous prolongation of APD, the short-coupled EAD beat led to unidirectional conduction block and formed reentry. This arrhythmia pattern is consistent with a previous study reported by Asano et al [In this long-QT syndrome model, the TdP ventricular tachyarrhythmias usually resulted from beat to beat changes in wave propagation patterns initiated by EADs from the border of the largest Cano et al .2+ cycling suppressed the inducibility of TdP. In this model, inhibition of SR Ca2+ cycling did not shorten APD. The mechanism could be the suppression of secondary Cai rise. We also demonstrated that EADs was initiated from border of the high secondary Cai rises region with earlier Cai pre-fluorescence. These results indicate that both SR Ca2+ cycling and ICa,L are important in development of secondary Cai rise and EADs. Therfore, secondary Cai rise might be a marker of torsadogenesis risk.The major findings of this study include that inhibition of SR Ca2+ dynamics is complicated. APD and Cai are bidirectionally coupled in cardiac tissue: a longer APD usually triggers a larger Ca2+ release and a larger Ca2+ release can either shortens or prolongs APD [2+ release enhances Na+-Ca2+ exchange current to prolong APD, but it also potentiates Ca2+ induced ICa,L inactivation and enhances Ca2+-sensitive K+ and Cl- currents to shorten APD. Ryanodine has been shown to slow down ICa,L inactivation through blockade of SR Ca2+ release [2+ release leads with slow inactivation of ICa,L leads to longer APD. Thapsigargin had been also shown to prolong APD through depletion of SR Ca2+, and patch clamp recording showed an increased total influx of Ca2+ with a longer duration in the presence of thapsigargin [2+ cycling blockade include prolongation of APD, slowing down ICa,L inactivation and SR Ca2+ reuptake.The interaction between APD and Caongs APD ,13. A la release . Enhance release . The redsigargin . Therefo2+ cycling includes ICa,L, Na+-Ca2+ exchange current and SR Ca2+ release and reuptake. ICa,L reactivation plays a central role in the development of EADs and TdP, and inhibition of ICa,L is one of the preventive and therapeutic approaches to TdP [Ca,L may reactivate and reversely repolarize under the situation of reduced outward currents and/or increased inward currents [+-Ca2+ exchange current suppresses genesis of EADs without interfering the reactivation of ICa,L in a H2O2 oxidative cell model [+-Ca2+ exchange current may also play a role in EAD genesis. In this study, our data showed that SR Ca2+ cycling inhibition suppressed EAD genesis even in the situation of longer APD. A possible mechanism is that the inhibition of SR Ca2+ cycling leads to slowing of Ca2+-dependent ICa,L inactivation, which reduces the possibility of available L-type Ca2+ channels for reactivation and the degree of secondary rise of Cai to attenuate EAD genesis. This study also confirms previous findings that any interference among the Ca2+ cycling affects the genesis of TdP [2+ release-reuptake and Na+-Ca2+ exchanger is required to generate EADs.The chain of Cas to TdP ,17,18. Es of TdP . On the i rise is complicated. Priori et al. first proposed that abnormal Ca2+ cycling can be the mechanism of EADs [2+ influx during late portions of action potential in failing human cardiomyocytes using a voltage-clamp model [Ca,L is the mechanism of EADs [Ca,L plays the most important role in genesis of EADs. However, whether or not ICa,L is the only factor in the EAD genesis was not very clear. Qu et al. investigated the mechanisms of EADs and ultra-long APD using a Luo and Rudy simulation model [Ca,L, speed of IK activation, slope of the steady-state inactivation curve of ICa,L and pedestal ICa,L [2+ release or reuptake/extrusion can affect window ICa,L, IK activation, inactivation of ICa,L through Ca2+-dependent ICa,L inactivation, modulating Vm by Na+-Ca2+ exchange current, regulating IKs and Ca2+ activated potassium and chloride currents. The mechanisms of secondary Cai rise involve the interaction of ICa,L, SR Ca2+ cycling and Na+-Ca2+ exchange current: SR Ca2+ release causes inactivates ICa,L; then either sustained high Vm or spontaneous Ca2+ release-induced depolarization through Na+-Ca2+ exchange current reactivates ICa,L [2+ cycling blockade reduces Ca2+ release, subsequently slows ICa,L inactivation to reduce secondary Cai rise.The formation of secondary Ca of EADs . Piacentmp model . Zeng an of EADs . It seemon model . In the al ICa,L . SR Ca2+es ICa,L . SR Ca2+i rise in this study and the previous studies [i rise is correlated with the APD, and the excessive prolongation of APD prevents immediate re-initiation of an action potential. The nearby myocardium with relatively shorter APD is available to initiate an EAD. Prolonged APD also leads to unidirectional block during the episodes of TdP. Therefore, the pattern of arrhythmias was focal trigger of EADs and reentry due to heterogeneously prolonged APD in this model.Most EADs initiated from the border zone rather than the region with largest amplitude of Ca studies . The mecKr blockade activity at the therapeutic level [2+ channel and SR Ca2+ cycling [2+ homeostasis lead to anti-arrhythmic property with relatively less torsadogenic effects. The mechanisms can be explained partially by the effects of SR Ca2+ blockade on suppressing inducibility of TdP. SR Ca2+ homeostasis can also be one of the targets to manage TdP clinically.Prolongation of QT interval leading to TdP is one of the major adverse effects of medications. It has been reported that the risk of drug-induced QT prolongation and TdP is associated with Iic level . The conic level . Some me cycling ,26. It iThere are still some limitations in this study. We mapped only the epicardium of hearts and were not able to recognize some arrhythmias when EADs initiated from mid-myocardium, sub-endocardium or outside of mapping field. Purkinje-ventricular escape rhythm after AV node ablation depended on the level of ablation, and the escape rate was different among the hearts. Because of cardiac memory, the rate of escape rhythm might affect APD and the inducibility of TdP.2+ cycling reduces the inducibility of TdP. The mechanism might be suppression of secondary Cai rise, although inhibition of SR Ca2+ cycling does not shorten APD. Nifedipine further inhibits the inducibility of TdP. These results indicate that both SR Ca2+ cycling and ICa,L are important in EAD genesis.In this AVB and long QT rabbit model, inhibition of SR Ca


Cardiac muscle adapts to hemodynamic stress by altering myocyte size and function, resulting in cardiac hypertrophy. Alteration in myocyte calcium homeostasis is known to be an initial signal in cardiac hypertrophy signaling. Transient receptor potential melastatin 4 protein (TRPM4) is a calcium-activated non-selective cation channel, which plays a role in regulating calcium influx and calcium-dependent cell functions in many cell types including cardiomyocytes. Selective deletion of TRPM4 from the heart muscle in mice resulted in an increased hypertrophic growth after chronic angiotensin (AngII) treatment, compared to WT mice. The enhanced hypertrophic response was also traceable by the increased expression of hypertrophy-related genes like Rcan1, ANP, and α-Actin. Intracellular calcium measurements on isolated ventricular myocytes showed significantly increased store-operated calcium entry upon AngII treatment in myocytes lacking the TRPM4 channel. Elevated intracellular calcium is a key factor in the development of pathological cardiac hypertrophy, leading to the activation of intracellular signaling pathways. In agreement with this, we observed significantly higher Rcan1 mRNA level, calcineurin enzyme activity and protein level in lysates from TRPM4-deficient mice heart compared to WT after AngII treatment. Collectively, these observations are consistent with a model in which TRPM4 is a regulator of calcium homeostasis in cardiomyocytes after AngII stimulation. TRPM4 contributes to the regulation of driving force for store-operated calcium entry and thereby the activation of the calcineurin–NFAT pathway and the development of pathological hypertrophy.The online version of this article (doi:10.1007/s00395-015-0501-x) contains supplementary material, which is available to authorized users. Elevated systolic and/or diastolic [Ca2+]i can activate intracellular cascades leading to hypertrophic growth. Obviously, cardiomyocytes exhibit dramatic fluctuations of [Ca2+]i during the cardiac cycle, making it difficult to explain how Ca2+-activated signaling proteins function. One possibility is the presence of specialized pools of Ca2+ which are spatially separated from the bulk Ca2+. However, the source of this subcellular Ca2+ still remains doubtful. Growing evidence points to voltage-gated Ca2+ channels, i.e., L-type, T-type i after Ca2+ re-addition. The average fluorescent signals from WT and Trpm4/−− myocytes during Ca2+ re-addition is shown in Fig. 2+]i during the Ca2+ re-addition, we found a significantly higher increase in the AUC in Trpm4/−− myocytes compared to WT . In these experiments, we never observed depolarization beyond −60 mV, allowing us to exclude a significant contribution of L-type Ca2+ channel activity to the [Ca2+]i increase during Ca2+-reapplication. Furthermore, application of the L-type Ca2+ channel blocker nifedipine (1 µmol/L) had no effect on the [Ca2+]i increase in Ca2+ re-addition experiments . Application of 30 mmol/L K+ at the end of the experiment depolarized the membrane potential and induced a Ca2+ transient, indicating that the myocytes are intact and able to maintain their resting membrane potential.Based on our SOCE experiments, we hypothesized that during AngII application, the release of Cast cells . This hyque Fig. c. In WT tes Fig. d  . To further investigate calcineurin activity, we performed calcineurin-specific Western blot analysis on the same hearts. In line with the increased phosphatase activity, we observed ~1.6 times more protein in WT AngII-treated and ~2.4 times more in Trpm4cKO AngII-treated animals, whereas there was no significant difference in calcineurin protein level between the genotypes after sham treatment  Fig. a. Howeveent Fig. b. These 2+ influx via store-operated Ca2+ channels. The increased Ca2+ influx most likely explains the enhanced activation of the calcineurin–NFAT pathway in Trpm4cKO myocytes.In this study, we demonstrated the effect of the calcium-activated non-selective cation channel TRPM4 on the development of cardiac hypertrophy upon chronic AngII infusion. In our experiments, mice lacking the channel exclusively in the heart were more sensitive to hypertrophic stimulus than WT mice. They displayed a significantly increased heart mass and myocyte cross-sectional area after 2 weeks of chronic AngII treatment. Furthermore, they showed a stronger re-activation of the fetal gene program than WT-treated mice. The increased sensitivity to AngII treatment was also present in vitro in cultured neonatal cardiac myocytes. Finally, we provide evidence that the increased response to AngII treatment was due to the lack of a TRPM4-dependent membrane depolarization leading to increased Ca3 and DAG. IP3 subsequently stimulates the release of Ca2+ from intracellular IP3-sensitive Ca2+ stores. It has been shown that IP3-mediated Ca2+ release plays a central role in regulating cardiomyocyte hypertrophy induced by different stimuli, including AngII infusion i elevation. In our previous work, we performed a whole-genome mRNA transcript analysis in WT and Trpm4/−− heart tissue where we found now major up- or downregulation of any genes involved in AngII receptor signaling [2+]i elevation via SOCE channels [2+ re-addition experiments as described earlier [Trpm4/−− myocytes. We observed increased [Ca2+]i in Trpm4/−− myocytes after store depletion by AngII compared to WT cells, which is in line with previous observations in mast cells i leads to an inward current and the subsequent depolarization can limit the driving force for calcium ions via voltage and non-voltage-gated calcium channels. This has been shown in different cell types including mast cells and cardiac myocytes i after Ca2+ re-addition was no longer present, suggesting that the original difference was mediated by a TRPM4-dependent membrane depolarization.Why do myocytes , 37. Furle cells . Taken t2+ entry through SOCE channels can activate the calcineurin–NFAT pathway and cause pathological cardiac hypertrophy . Subsequently, the heart was removed from the setup, atria were discarded and ventricular muscle was minced in collagenase solution (see above). The suspension was filtered through a nylon filter, and cells were centrifuged at 500×g for 2 min. The supernatant was discarded and the myocytes were resuspended in the first solution, but supplemented with 1 mmol/L CaCl2. Calcium-tolerant, rod-shaped myocytes with clear striations were used for measurements on the day of the isolation.8- to 12-week-old mice were heparinized and killed by intraperitoneal injection of pentobarbital . The hearts were removed and perfused retrograde for 2 min through the aorta with a Langendorff apparatus at 37 °C with oxygen-saturated nominally calcium-free solution containing (in mmol/L): 117 NaCl, 4 KCl, 1 KH2+ dynamics, isolated ventricular myocytes were loaded with the Ca2+ indicator Fluo-4-AM (1 μmol/L) for 15 min at room temperature. Cells were seeded on glass coverslips in extracellular solution containing (in mmol/L): 118 NaCl, 4.7 KCl, 2.52 CaCl2, 1.64 MgSO4, 24.88 NaHCO3, 1.18 KH2PO4, 6 glucose, 2 Na pyruvate. This solution was continuously bubbled with carbogen . Electrical stimulation was achieved using field stimulation. Fluorescence was detected with a photo-multiplier tube and acquired by the Patchmaster software. Excitation light (480 nm) was provided by a Polychrome V monochromator .For imaging of intracellular Ca2+-free buffer to deplete SR stores. Myocytes were then perfused with high Ca2+ (2.5 mmol/L), in the continuous presence of thapsigargin and AngII, and store-operated entry was measured as an increase in Fluo-4 fluorescence. For the measurement of the resting membrane potential during SOCE experiment, perforated patch technique was used in current clamp mode. In these experiments the pipette solution contained (in mmol/L) 10 KCl, 10 NaCl, 70 K2SO4, 1 MgCl2 10 Hepes and 240 µg/ml amphotericin B, pH 7.3.For store-operated calcium entry measurement, myocytes were handled as above. Myocytes were treated with 1 μmol/L AngII and 5 μmol/L thapsigargin for 5–6 min in nominally Ca8- to 12-week-old mice were anesthetized with isoflurane. Mini-osmotic pumps  filled with 3 mg/kg/day Ang II  or with saline were implanted subcutaneously. Two weeks after the operation, animals were killed by cervical dislocation, hearts were removed, cleaned from fat tissue and big vessels, washed in PBS and weighed. Subsequently, the left tibia was dissected and the length of the bone was measured.2+ and Mg2+. This step was repeated four to five times until the hearts were completely digested. The cells were then cultured in DMEM supplemented with 20 % FBS for 2–3 h, allowing the attachment of non-myocytes. Later, the cells in the supernatant were used and cultured in DMEM supplemented with 20 % FBS. After 1 day, the medium was changed to DMEM without FBS and supplemented with 1 µM AngII for 48 h.Neonatal mice (1- to 2-day-old) were used for isolation. Hearts were removed aseptically; atria and big vessels were discarded. The ventricles were cut into small pieces and digested in 0.25 % trypsin for 15 min at 37 °C. Subsequently, supernatants were removed and mixed with an equal volume of HBSS with CaTrpm4cKO mice were lysed in calcineurin assay buffer (Enzo Life Science) and centrifuged at 2000×g. The supernatant was then snap frozen at −70 °C. The phosphatase activity assay (BML-AK816) was performed based on the manufacturer’s instruction (Enzo Life Science). Calcineurin phosphatase activity was measured spectrophotometrically by detecting free phosphate released from the calcineurin-specific RII phosphopeptide.Tissue samples from freshly isolated hearts of WT and Trpm4cKO mice were lysed in 1 ml ice-cold lysis buffer  using a Polytron homogenizer. Subsequently, 4 ml saccharose buffer  was added to the lysate. The obtained homogenates were centrifuged at 3000×g for 15 min to remove any remaining large cellular fragments. The supernatants were ultracentrifuged at 200,000×g for 30 min. Pellets containing total membrane fractions were solubilized in a cold saccharose buffer. Protein concentrations were determined by the bicinchoninic acid assay method, using bovine serum albumin (BSA) as a standard. Samples (80 μg) were subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently transferred to a polyvinylidene fluoride (PVDF) membrane . The respective proteins were detected with purified rabbit polyclonal antibody (Ak578) directed against the amino-terminal end of mouse TRPM4 (27), isoform-specific mouse monoclonal calcineurin-Aß  and monoclonal mouse anti-Na+/K+ ATPase (1: 5000 dilution)  antibodies. Immunoreactive complexes were visualized by chemiluminescence, using anti-rabbit IgG  or anti-mouse IgG  antibodies conjugated to horseradish peroxidase.Proteins from freshly isolated heart of wild-type and Four mice of each genotype were perfused with saline. Then the hearts were fixed with zinc-based fixative solution . Subsequently, hearts were processed and embedded in paraffin wax. Serial sections were cut to a thickness of 5–7 μm and documented using a Zeiss microscope (Axiovert 40). For Masson’s trichrome staining, a commercially available kit (Sigma-Aldrich) was used based on the supplier’s instructions. For the analysis of the obtained pictures, ImageJ  software was used.2O, were performed with the 7500 Fast Real-Time PCR System (Life Technologies). mPGK1 and mTBP, selected using the geNorm application, were used as endogenous controls. TaqMan assays used: CACNA1H-Mm00445382_m1, TRPM4-Mm00613173_m1, ACTA1-Mm00808218_g1, ANP-Mm01255748_g1, RCAN1-Mm01213407_m1, MYH7-Mm00600555_m1.After euthanasia by cervical dislocation, hearts were removed, washed in ice-cold PBS, immediately snap frozen in liquid nitrogen and kept at −80 °C until final processing. Total RNA was extracted using the RNeasy Mini Kit (Qiagen), following the manufacturer’s protocol. RNA concentration and quality were assessed using the Experion RNA StdSens Analysis Kit (Bio-Rad). For cDNA synthesis, Ready-To-Go You-Prime First-Strand Beads  were used based on the supplier’s protocol. Generated cDNAs were stored at −20 °C. qPCR reactions, composed of cDNA template, Universal TaqMan MasterMix , TaqMan assay  and Ht test and two-way repeated measurement ANOVA followed by a Tukey post hoc test, unless mentioned otherwise. When assumptions were not valid, non-parametric tests were performed . A p value <0.05 was considered to be statistically significant.All statistical analyses were performed using Prism (Graphpad) or Origin  software. Data are expressed as mean ± SEM, unless otherwise specified. Differences between groups were compared by two-sample Supplementary material 1 (PDF 1084 kb)


Such fluxes are necessary for events like motility and egress from host cells. We used genetically encoded Ca2+ indicators (GCaMPs) to develop a cell-based phenotypic screen for compounds that modulate Ca2+ signaling in the model apicomplexan Toxoplasma gondii. In doing so, we took advantage of the phosphodiesterase inhibitor zaprinast, which we show acts in part through cGMP-dependent protein kinase  to raise levels of cytosolic Ca2+. We define the pool of Ca2+ regulated by PKG to be a neutral store distinct from the endoplasmic reticulum. Screening a library of 823 ATP mimetics, we identify both inhibitors and enhancers of Ca2+ signaling. Two such compounds constitute novel PKG inhibitors and prevent zaprinast from increasing cytosolic Ca2+. The enhancers identified are capable of releasing intracellular Ca2+ stores independently of zaprinast or PKG. One of these enhancers blocks parasite egress and invasion and shows strong antiparasitic activity against T. gondii. The same compound inhibits invasion of the most lethal malaria parasite, Plasmodium falciparum. Inhibition of Ca2+-related phenotypes in these two apicomplexan parasites suggests that depletion of intracellular Ca2+ stores by the enhancer may be an effective antiparasitic strategy. These results establish a powerful new strategy for identifying compounds that modulate the essential parasite signaling pathways regulated by Ca2+, underscoring the importance of these pathways and the therapeutic potential of their inhibition.The life cycles of apicomplexan parasites progress in accordance with fluxes in cytosolic Ca Toxoplasma gondii and Plasmodium spp., the causative agents of toxoplasmosis and malaria, require changes in cytosolic Ca2+ concentrations to egress from host cells and move within the infected organism . However, few of the molecules involved in regulating Ca2+ homeostasis and signaling have been identified in parasites, and their interplay is only evident in live cells. This has created a need for new methods to study Ca2+ signaling pathways in apicomplexan parasites, with the hope of defining the essential components and identifying novel inhibitors.Apicomplexan parasites, such as organism –6. These2+ entry into the cytoplasm and the physiologically relevant sources of Ca2+ remain poorly defined in apicomplexan parasites. Ca2+ can be mobilized from the parasite's intracellular stores, or it can be drawn from the environment. Current evidence points toward intracellular stores being sufficient for parasites to move between cells , which in other systems causes ion leakage from lysosomal compartments . This has been hypothesized to increase IP3, which causes release of Ca2+ from the ER through binding to the IP3 receptor, although such a channel remains to be identified in apicomplexan parasites , which regulates egress, invasion, and motility in T. gondii and Plasmodium spp.  grown in DMEM supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 10 μg/ml gentamicin. PKG-T and PKG-M alleles were constructed as described previously  and cloned directionally, replacing the CAT gene in pSAG1/2-CAT (pSAG1-GCaMP5. Recombinant human parasites were co-transfected with pSAG1-GCaMP5 and pSAG1/2-CAT and selected with chloramphenicol (40 μm), and clones were isolated by limiting dilution. The GCaMP6f strains was similarly derived, as described previously  or extracellular Ca2+ buffer , supplemented with 1% FBS when noted. For the Cpd1 inhibition experiments, parasites were suspended in Ringer's solution . In all cases, 100 μl of suspended parasites were applied to each well of a Cell Carrier 96-well plate (PerkinElmer Life Sciences). For store activation experiments, parasites were incubated on ice for 5 min before the addition of Enh1 or Enh2 , zaprinast , or DMSO  suspended in the same buffers as the parasites to which they were added. To examine the effect of Cpd1 on zaprinast or Enh1 treatment of GCaMP6-expressing parasites, fluorescence was recorded for 100 s before adding Cpd1 to a concentration of 1.2 μm. Fluorescence was recorded for an additional 300 s before treatment with 10 μm Enh1 or 100 μm zaprinast. Parasites were incubated on ice for 5 min before the addition of zaprinast to slow down the response and facilitate observation of peak fluorescence. Fluorescence was read with an excitation wavelength of 485 nm and an emission wavelength of 528 nm every 10 s in a BioTek Cytation 3. The assay plate was shaken for 1 s before each read.GCaMP6f-expressing 7 parasites/ml, and 50 μl of parasites were applied to each well of a 96-well plate . Parasites were treated with compounds from the GSK libraries PKIS and PKIS2 at 13.3 μm or 1.33% DMSO as a vehicle control and then incubated at 37 °C with 5% CO2 for 10 min. Parasites were then treated with 100 μm zaprinast or 0.1% DMSO as a vehicle control and incubated at 37 °C with 5% CO2 for an additional 4 min before measuring fluorescence with excitation and emission wavelengths of 485 and 525 nm, respectively, on a SpectraMax M3 (Molecular Devices). Basal fluorescence, measured from untreated parasites, was subtracted from all values, and results were expressed as -fold change from parasites treated only with zaprinast. Z′ factors were determined from (i) parasites treated with zaprinast versus untreated parasites and (ii) parasites treated with a final concentration of 1 μm Cpd2 or a vehicle control followed by zaprinast, using the formula, Z′ = 1 − 3(σp + σn)/(|μp − μn|), where σp and σn indicate S.D. values of positive and negative controls, and μp and μn indicate the means of positive and negative controls, respectively.GCaMP5-expressing parasites were suspended in Ringer's solution at 4 × 10T. gondii PKG was expressed using a baculovirus system. Synthetic DNA coding for the protein was amplified and subcloned into the pFBOH-MHL vector, which confers an N-terminal His6 tag with a tobacco etch virus cleavage site. The resulting plasmid was transformed into DH10BacTMEscherichia coli competent cells to produce recombinant viral DNA. P3 viral stocks were used to infect Sf9 insect cells grown in HyQ SFX insect serum-free medium (Thermo Fisher Scientific). The culture was incubated at 27 °C and shaken at 100 rpm. After 60–72 h, the cells were harvested. The His-tagged T. gondii PKG samples were purified by affinity chromatography and size exclusion chromatography using an ÄKTAxpress system equipped with a SuperdexTM 200 10/300 column . In vitro kinase assays were performed using a PKG assay kit (CycLex) as per the manufacturer's instructions. Inhibitors were tested against 6.76 nm recombinant PKG, which was active within the linear range of the assay.Recombinant Structures of various protein kinases in complex with pyrazolopyridazine (PP) and oxindole derivatives were obtained from the RCSB Protein Data Bank  and alig4 parasites/well. The HFF monolayer was washed once with Ringer's solution ∼18 h later, and 50 μl of Ringer's solution was applied to each well. Drugs suspended in Ringer's solution were applied at 1.33 times the indicated concentrations. 1.33% DMSO in Ringer's solution was used as a vehicle control. Cells were incubated at 37 °C with 5% CO2 for 20 min, stimulated with 500 μm zaprinast or 0.5% DMSO (vehicle), and incubated again for an additional 5 min. The cells were centrifuged at 400 × g for 5 min, before collecting 50 μl of each supernatant. The lactate dehydrogenase levels in the supernatant samples were quantified using the CytoTox 96 cytotoxicity assay (Promega) as per the manufacturer's instructions.Confluent HFF monolayers in 96-well plates were infected with 5 × 10T. gondii parasites were suspended in growth medium supplemented with Enh1, zaprinast, or DMSO alone at the indicated concentrations. The amount of DMSO in all treatment groups was normalized to 0.1%. Parasites were incubated at 37 °C with 5% CO2 for 20 min before infecting HFF monolayers in 6-well plates using 3 ml carrying 100 parasites/well. Medium containing the highest concentration of each drug was also applied to HFF cells in the absence of parasites to assess the effects of the drugs on host cell viability. The parasites were allowed to plaque for 8 days before fixing with 70% ethanol and staining with 0.1% crystal violet.6 parasites/ml. The maximum concentration of DMSO was added as a vehicle control: 1% DMSO in comparisons with zaprinast and 0.013% in comparisons with Enh1. Parasites were preincubated with the compounds for 20 min at 37 °C with 5% CO2, and then 200 μl/well (105 parasites) was added to host cell monolayers in 96-well plates. Assay plates were incubated at 37 °C with 5% CO2 for 3 days and then fixed in 70% ethanol and stained with 0.1% crystal violet. Absorbance at 590 nm was read as a measure of host cell lysis.Drugs were suspended at twice the indicated concentrations in growth medium and mixed with an equal volume of parasites at an initial concentration of 105 parasites/ml were pretreated with varying concentrations of zaprinast (as indicated) for 20 min at 37 °C with 5% CO2. 105 parasites/well were then applied to confluent host cell monolayers, and plates were centrifuged at 290 × g for 5 min. Following 1 h at 37 °C with 5% CO2, plates were centrifuged again at 500 × g for 5 min. 50 μl of supernatant were collected from each well, and lactate dehydrogenase was quantified using the CytoTox 96 cytotoxicity assay (Promega).Parasites suspended in Ringer's solution at 5 × 104 GFP-expressing parasites/well. 18 h postinfection, the medium was exchanged for Ringer's solution, and the intracellular parasites were treated with 10 μm Enh1, 500 μm zaprinast, or 0.5% DMSO. Images were acquired every 10 s for 30 min. To measure the effect of Enh1 on zaprinast and A23187-induced egress, the procedure was repeated, adding either 12.5 μm Enh1 or 0.13% DMSO and imaging for 10 min, before stimulation with 500 μm zaprinast or 1 μm A23187  and imaging for an additional 10 min. In all cases, images were acquired with a ×4 objective on a Cytation3 reader (BioTek) using excitation and emission wavelengths of 485 and 528 nm, respectively. Intact vacuoles were defined as objects of at least 78 μm2 with a circularity of at least 0.5, as determined in Fiji after default thresholding (Host cell monolayers in Cell Carrier 96-well plates (PerkinElmer Life Sciences) were infected with 5 × 10sholding .p30-DsRed and used to infect host cell monolayers in 3-cm glass bottom dishes (MatTek). Approximately 20 h after infection, medium was exchanged for Ringer's solution, and parasites were imaged on a Nikon Eclipse Ti epifluorescence microscope, equipped with an enclosure heated to 37 °C. Images were acquired every 5 s for 10 min following the addition of zaprinast to a final concentration of 100 μm. To quantify changes in fluorescence, videos were analyzed in Fiji to measure the average fluorescence intensity in specific circular regions of interest 10 μm in diameter. To examine the effects of zaprinast and Enh1 on mammalian cells, 6 × 103 HeLa cells were seeded in each well of a Cell Carrier 96-well plate. Approximately 24 h later, HeLa cells were transfected with 100 ng of the pCMV-R-GECO  using an excitation wavelength of 531 and an emission wavelength of 593. Higher resolution videos were similarly acquired from HeLa cells seeded in 3-cm glass bottom dishes (MatTek) and transfected with 200 ng of pCMV-R-GECO per dish. Cells were treated with either 500 μm zaprinast or 10 μm Enh1 in Ringer's solution and imaged every 250 ms using a Nikon Eclipse Ti microscope. Videos were analyzed in Fiji using default thresholding for the red channel to determine the mean gray value in each slice for individual cells. Kymographs were constructed from videos of intracellular GCaMP6f-expressing parasites, following the same treatment as for the video microscopy egress assays, except half as many parasites were used to infect the monolayers, and a ×20 objective was used to acquire the images. Regions of interest were defined by outlining parasites in Fiji and measuring the mean fluorescence of each region over time.Parasites expressing GCaMP5 were transfected with V-R-GECO  using Fum NaCl, 5.4 mm KCl, 0.8 mm MgSO4, 5.5 mmd-glucose, and 50 mm HEPES, pH 7.4) and resuspended to a final density of 1 × l09 parasites/ml in loading buffer (buffer A plus 1.5% sucrose and 5 μm Fura-2/AM). The suspension was incubated for 26 min at 26 °C with mild agitation. Subsequently, the parasites were washed twice with buffer A to remove extracellular dye, resuspended to a final density of 1 × 109 parasites/ml in buffer A, and kept in ice. Parasites are viable for a few hours under these conditions. For fluorescence measurements, 2 × 107 parasites/ml were placed in a cuvette with 2.5 ml of Ringer's solution. Fluorescence measurements were done in a thermostatically controlled Hitachi F-7000 spectrofluorometer using the Fura-2 conditions for excitation (340 and 380 nm) and emission (510 nm). The Fura-2 fluorescence response to Ca2+ was calibrated from the ratio of 340/380-nm fluorescence values after subtraction of the background fluorescence of the cells at 340 and 380 nm, as described previously  and cultured as described previously , at 2% hematocrit, in RPMI 1640 supplemented with HEPES (25 mm), hypoxanthine (50 mg/liter), sodium bicarbonate (2.42 mm), and Albumax (4.31 mg/ml). Cultures were incubated at 37 °C in microaerophilic atmospheric conditions  within modular incubator chambers.Blood stage eviously . Parasitg, 15 min, low acceleration and deceleration) on 4 ml of a 60% Percoll cushion  by centrifugation  at the f egress , 49.Invasion assays were performed as described previously . BrieflyT. gondii and P. falciparum egress in a PKG-dependent manner . This led to permeabilization of the vacuole membrane, as indicated by diffusion of DsRed, and subsequent egress of the parasites from the host cell .Previous work has shown that zaprinast triggers t manner , 22. Becg egress , we hypor GCaMP5  in the we egress . This se protein . Human f protein , A and B2+ represented release of intracellular stores or Ca2+ entry, we loaded wild-type parasites with the ratiometric Ca2+ indicator Fura-2/AM and stimulated them with zaprinast in buffers containing either a basal Ca2+ concentration (100 nm free Ca2+) or one resembling the extracellular environment (1.8 mm). We found that cytosolic Ca2+ increased in response to zaprinast under both conditions (C), although the response was magnified in the presence of extracellular Ca2+. This indicates that zaprinast-mediated Ca2+ mobilization occurs through release of intracellular stores, which may enhance Ca2+ entry, as has been reported for other agonists capable of releasing intracellular stores . We also observed a slight decrease in the cytosolic Ca2+ level of the PKG-T strain after Cpd1 treatment, perhaps indicating that PKG regulates the basal Ca2+ level in addition to enhancing it before egress.Compound 1 (Cpd1), a specific inhibitor of apicomplexan PKG , blocks tekeeper . We thertekeeper . These s process D. We als2+, we assessed the involvement of various Ca2+ storage organelles. We loaded parasites with Fura-2/AM and suspended them in a buffer containing basal Ca2+ and then added the Ca2+ ionophore ionomycin. Binding of ionomycin to Ca2+ is pH-dependent and falls to negligible levels below pH 7 . Taken together, these results indicate that the zaprinast-mobilized store comprises a subset of the ionomycin-mobilized stores and as such is predicted to be neutral.To further characterize the source of zaprinast-mobilized Calow pH 7 . Ionomycan cells  and T. g. gondii . We obsecompound E. Taken 2+ stores, we treated parasites with GPN before or after the addition of zaprinast. GPN is specifically hydrolyzed in lysosomal compartments, leading to their leakage, as has been shown for the plantlike vacuole in T. gondii (F).To further rule out acidic Ca. gondii . As pred. gondii F.2+ store in many organisms . These results indicate either that zaprinast mobilizes a neutral store that is separate from the ER or that the ER is segmented into SERCA-dependent and SERCA-independent Ca2+ stores, as has been hypothesized  .Despite its central importance during infection, few compounds have been demonstrated to specifically interfere with the Ca2 (Cpd2) . These screening . This phignaling A.B and supplemental Table S1). The compounds were screened at a single 10 μm dose with two biological replicates. We identified 37 putative inhibitors and 14 putative enhancers, defined as compounds that resulted in fluorescence readings that were more than two S.D. values below or above the mean of all compounds, respectively. Compounds that interfered with accurate measurement of GCaMP5 fluorescence, including two putative enhancers, were identified and excluded from subsequent experiments.We obtained the compound libraries published kinase inhibitor sets PKIS1  and PKISFig. 22+ mobilization. To this end, we examined analogs of the most potent enhancer and inhibitor, which we here refer to as Enh1 (PKIS: GSK260205A) and Inh1 (PKIS: GW827099X) (C). Compounds with the same core structure as Enh1 generally produced a -fold change in fluorescence of >1 in our screen, indicating that parasite fluorescence was greater in the presence of such compounds than with zaprinast alone . A Kolmogorov-Smirnov test comparing the values obtained from all compounds with those obtained from the Enh1 analogs revealed a significant difference (p = 0.0042) . The combination of a phenyl group at the R1 position and a 3-aminopropyloxy group at the R3 position, as found in Enh1, appeared to produce a particularly effective enhancer; compounds having either group without the other produced only a mild effect. A similar analysis of Inh1 analogs revealed that these compounds tended to inhibit zaprinast-induced Ca2+ mobilization . The fluorophenyl group in the R1 position of Inh1 may be responsible for this compound's strong inhibitory effect (as discussed below). This 4-fluorophenyl is also found in Cpd1 and Cpd2. No other R1 side group produced a similarly strong response, although an analogue containing a trifluoromethyl group at the R1 position elicited the second largest response in the group. In summary, although Enh1 and Inh1 are unique within the set of compounds tested for their abilities to augment and repress cytosolic Ca2+, their core structures appear to be generally well suited to these purposes. Indeed, further exploration of these scaffolds could lead to the identification of improved modulators of parasite Ca2+ signaling.We wondered whether we could identify classes of molecules that inhibit or enhance Ca827099X) C. Compou2+ release (D), we hypothesized that the newly identified compounds could act in a similar manner. We tested the effects of Inh1 and Inh2 (compound GW407323A from the screen) on the in vitro activity of recombinant T. gondii PKG. Both compounds inhibited PKG activity with IC50 values of 580 nm for Inh1 and 670 nm for Inh2 (A), suggesting that PKG inhibition is a plausible explanation for their activity. Furthermore, neither inhibitor showed appreciable activity against human PKG or related AGC kinases . Several available kinase structures display a similar orientation of the PP scaffold in the ATP-binding pocket, with the C3 position engaging the hinge region and the C2 position oriented toward the gatekeeper residue. The structures of two such inhibitors complexed with human CDK2 demonstrate the proximity of the C2 position to the bulky phenylalanine-gatekeeper residue . Substitutions at this position decreased the activity of these inhibitors presumably due to steric clash with the gatekeeper  that extends the 4-fluorophenyl functional group into the hydrophobic pocket created by a threonine gatekeeper . Furthermore, the related human kinase ERK2, normally resistant to SB203580, can be rendered sensitive to this inhibitor by mutation of its gatekeeper glutamine to either an alanine or a threonine . Disubstituted and trisubstituted oxindoles are well studied protein kinase inhibitors. Several structures are available of kinases in complex with such inhibitors. In every instance, the oxindole is positioned such that the 2-oxygen and the nitrogen of the bicyclic scaffold engage the hinge residues . This configuration leaves the C3 and C5 positions pointing away from the binding pocket. Although the C6 substituent is inward pointing, it does not approach the direction of the gatekeeper. The binding configuration of oxindole derivatives is highly consistent across several different kinase structures, suggesting that Inh2  is likely to be gatekeeper-independent.Inh2 is an oxindole derivative, with substitutions at the C3 and C5 positions C. Disubsresidues C, left. D). As expected, PKG-T parasites did not egress after Cpd2 treatment, whereas the refractory PKG-M strain egressed similarly to vehicle-treated parasites. Both Inh1 and Inh2 reliably reduced egress relative to vehicle-treated parasites. The similar potencies of these compounds in vivo and in vitro are consistent with inhibition of PKG being the primary mechanism through which Inh1 and Inh2 block egress (D). As predicted from our structural analysis, Inh1 worked in a gatekeeper-dependent manner, reducing egress from cells infected with PKG-T parasites with an IC50 of 1.3 μm but leaving PKG-M parasites unaffected. In contrast, Inh2 reduced egress from cells infected with either strain, exhibiting an IC50 of 2.5 μm for PKG-T and 3.5 μm for PKG-M. These results were satisfying because we were able to recover inhibitors with distinct scaffolds and modes of inhibiting an enzyme known to be involved in the zaprinast response. Identification of two novel PKG inhibitors through a screen of only 823 compounds demonstrates the power of our approach to identify compounds with biological effects in pathways critical to parasite survival.To determine whether these inhibitors could interfere with other PKG-dependent processes, we next assessed their ability to block zaprinast-induced egress. We directly tested the structural predictions regarding the effect of the gatekeeper on Inh1, but not Inh2, by performing these experiments with the PKG-M and PKG-T strains. We measured zaprinast-induced egress by lactate dehydrogenase release from host cells infected with either strain D. As expk egress D. As pre2+ mobilization reveals novel PKG inhibitors, we turned our attention to Enh1 and another putative enhancer: Enh2, or GSK2188764A (C). During the course of this study, the genetically encoded Ca2+ indicator GCaMP6f became available. Because GCaMP6f has a broader dynamic range and faster kinetics than GCaMP5 .Having shown that a screen for modulators of zaprinast-induced Ca2188764A C. Duringn GCaMP5 , we usedaprinast A.2+ mobilized by the enhancers, we suspended parasites in buffers containing extracellular (1 mm) or basal (100 nm) levels of free Ca2+, treated these parasites with 10 μm Enh1 or Enh2, and monitored GCaMP6f fluorescence (A). Enh1 increased fluorescence under both conditions, indicating that it mobilizes intracellular stores. Surprisingly, we observed only weak mobilization of Ca2+ in response to Enh2 under both conditions. Because the original screen was performed in buffer containing 1% FBS, we tested the effect of this component. The addition of 1% FBS increased Enh2-mediated Ca2+ mobilization. We interpreted this to mean that Enh2 requires a cofactor found in FBS. Because the activity of Enh2 is relatively weak and depends on specific conditions, we focused subsequent experiments on Enh1.To determine the source of the Carescence A. Enh1 i2+. Significant overlap in the emission spectra of Enh1 and Fura-2 prevented us from using this ratiometric indicator. However, we were able to use GCaMP6f to make semiquantitative measurements. We recorded fluorescence from GCaMP6f-expressing parasites treated with Cpd1 or vehicle control, followed by stimulation with Enh1 or zaprinast. To record the peak fluorescence induced by zaprinast, we had to incubate parasites on ice before stimulation so as to slow down the response. In contrast to the zaprinast response, Cpd1-treated parasites responded to Enh1 in the same manner as vehicle-treated parasites, indicating that Enh1 does not act through PKG. Although Cpd1 accelerated the return to basal Ca2+ levels following zaprinast treatment, it only partially decreased the initial Ca2+ spike induced by zaprinast (B). This is reminiscent of results obtained from Fura-2-loaded parasites, where the increase in Ca2+ following zaprinast could never be fully suppressed by PKG inhibition (D).We wondered whether, like zaprinast, Enh1 functions through PKG to increase parasite Caaprinast B. This ihibition D.2+ fluxes in mammalian cells. We transfected HeLa cells with the plasmid CMV-R-GECO . Given that zaprinast is known to inhibit phosphodiesterases in mammalian cells (2+ (D), indicating that the effects of Enh1 are specific to Ca2+ signaling in the parasite.We wondered whether zaprinast or Enh1 could induce CaV-R-GECO , which et change C. Given an cells , we consells . These results for zaprinast agree with previously published work .Zaprinast and related phosphodiesterase inhibitors were recently shown to block roblasts . Alteriniability . We therle alone A. These hed work . In ordes 200 μm B.T. gondii in plaque assays (100 μm) and the amount needed in lytic assays (500 μm). Because a rise in cytosolic Ca2+ stimulates parasite motility . Due to the lower multiplicity of infection used in plaque assays compared with lytic assays, host cells in plaque assays would be unlikely to experience observable cell wounding. We therefore expect the plaque assays to provide a more accurate measure of zaprinast's antiparasitic activity.We noticed the discrepancy between the amount of zaprinast required to kill . gondii C. Due to2+ disregulation induced by this compound. We therefore characterized the Ca2+ response to Enh1 in more detail. We recorded videos of intracellular GCaMP6f-expressing parasites treated with Enh1 and compared the result with zaprinast treatment, which we have shown induces egress. We observed GCaMP6f activity in both cases, indicating that Enh1 can act on intracellular parasites. However, the profiles of zaprinast and Enh1-mediated Ca2+ mobilization were remarkably different. Whereas zaprinast induced a fast, strong Ca2+ peak , Enh1 elicited a slow, asynchronous effect in which some parasites appeared to experience multiple Ca2+ fluxes of similar magnitudes over the course of several minutes . The response to these compounds was similar in extracellular parasites (data not shown).We thought it likely that the plaquing defect caused by Enh1 was related to the Ca2+ agonists, we treated some of these parasites with zaprinast as well (B). The sporadic flashes of Ca2+ triggered by Enh1 were recapitulated, and all vehicle-treated parasites experienced an increase in Ca2+ immediately after the addition of zaprinast. However, Enh1-treated parasites varied in their responses to zaprinast, exhibiting asynchronous Ca2+ fluxes and inconsistent magnitudes in their Ca2+ increases. In rare cases, Ca2+ concentrations in Enh1-treated parasites even dropped upon zaprinast treatment. We quantified the changes in fluorescence of individual parasites over the 40 s following zaprinast addition and observed a more varied and overall diminished response to zaprinast in Enh1-treated parasites (C). Taken together, these results suggest that Enh1 may partially deplete the Ca2+ stores mobilized by zaprinast.We generated kymographs illustrating the responses of individual GCaMP6f parasites to Enh1. Wondering whether pretreatment with Enh1 would affect the ability of parasites to respond to other Ca as well B. The sparasites C. Taken C). To confirm these effects, we treated intracellular GFP-expressing parasites with Enh1 and quantified the number of intact vacuoles at various times after treatment, using fluorescence to monitor the infection using automated image analysis. This assay is capable of directly measuring egress for hundreds of vacuoles per sample. Surprisingly, despite its effects on extracellular parasites, Enh1 failed to stimulate egress beyond the spontaneous egress observed in the vehicle control. In contrast, zaprinast treatment resulted in ∼90% of vacuoles egressing within 30 min (A), as described previously . These results suggest that the antiparasitic activity of Enh1 is mediated by its inhibition of parasite egress.While examining Enh1-treated parasites, we noticed that this compound did not induce egress and in fact suppressed egress in response to zaprinast C. To conn 30 min A, as deseviously . We thenormation B. These 2+ ionophore A23187. Enh1 also produced a significant block on A23187-induced egress (C), demonstrating that its effects on parasite Ca2+ signaling extend to a variety of agonists.We wondered whether we could generalize the Enh1-associated egress defect beyond zaprinast-induced egress. We therefore tested the ability of Enh1 to block egress induced by the Cad egress C, demons2+-based signal transduction. Asexual, blood stage Plasmodium parasites undergo Ca2+-dependent egress from infected erythrocytes, followed by Ca2+-dependent invasion into new erythrocytes (m (D), the compound completely blocks invasion within the tested range (IC50 = 3.2 μm) (E). Wondering whether Enh1 could also inhibit invasion in T. gondii, we incubated tachyzoites with varying doses of Enh1 for 20 min and tested their ability to infect host cells. As in P. falciparum, Enh1 strongly inhibited T. gondii invasion (F). Furthermore, the IC50 of Enh1 in the invasion assay was 180 μm, similar to that in the egress assay, arguing for a common mechanism for the effect of Enh1 on both Ca2+-related phenotypes. In summary, Enh1 modulates Ca2+-dependent processes in diverse apicomplexan parasites.We tested whether Enh1 also affects other apicomplexan parasites that use Cahrocytes , 63. To hrocytes . We admihrocytes . Whereascytes  E. Wonderinvasion F. Furthe2+ signaling plays a central role in apicomplexan biology, yet few of the components that regulate Ca2+ uptake and release have been identified. In this study, we extend our understanding of these processes by demonstrating that PKG activity is needed for the robust Ca2+ response elicited by phosphodiesterase inhibitors. Furthermore, we determine the source of Ca2+ to be distinct from other previously described stores. We use this phenomenon as the basis of a phenotypic screen that allowed us to identify several novel inhibitors and enhancers of Ca2+ signaling. Two of the inhibitors could be shown to interfere with Ca2+ signaling by specifically targeting parasite PKG. In contrast, the enhancers could be shown to increase Ca2+ independently from zaprinast and in fact prevented parasite egress by apparently depleting intracellular Ca2+ stores. This compound displayed antiparasitic properties against both T. gondii and P. falciparum, establishing a new mechanism for interfering with apicomplexan parasitism.Ca2+ ionophores have long been known to stimulate egress in T. gondii  than when using GCaMP6 (B). This may result from the semiquantitative nature of GCaMP6 and the potentially non-linear relationship between fluorescence and Ca2+ concentration. Additionally, differences in the subcellular distribution of the two indicators may influence their responses to different sources of Ca2+. Ratiometric measurements with Fura-2 also revealed that inhibition of PKG by Cpd1 decreased basal Ca2+ concentrations in extracellular parasites, suggesting that PKG might also be necessary to maintain resting levels of Ca2+ in extracellular parasites. These changes in basal Ca2+ concentrations might not be evident with GCaMP6f due to its higher dK (375 nm) (m)   compared nm) (m) .2+ store that has been characterized in T. gondii. SERCA, the target of thapsigargin, localizes to the ER in intracellular parasites but redistributes so as to only partially colocalize with the ER in extracellular parasites . However, without a complete understanding of Enh1 function, we cannot rule out the possibility that inhibition of egress may be independent of its ability to modulate Ca2+ because (i) some parasites in which Enh1 did not elicit Ca2+ fluctuations still failed to egress in response to zaprinast, and (ii) A23187, which should equilibrate Ca2+ across membranes, failed to overcome Enh1 inhibition. Compounds similar to Enh1 have been shown to inhibit mammalian kinases belonging to the AGC family (T. gondii PKG, a member of the AGC kinase family, mediates Ca2+ release, it is an intriguing possibility that a related kinase might oppose its activity.In contrast to zaprinast and Careatment C. HoweveC family . Having P. falciparum to invade erythrocytes, although it did not affect egress. Despite the many parallels between egress of P. falciparum and T. gondii, substantial differences have also been uncovered. Plasmodium egress is a fast, highly synchronized process, dependent on a cascade of proteolytic activity (T. gondii but only invasion in P. falciparum (2+ stores of Plasmodium have not been investigated as thoroughly as those of T. gondii, but there is some evidence that it is not solely dependent on its intracellular Ca2+ stores and in fact utilizes Ca2+ found within the parasitophorous vacuole (P. falciparum and T. gondii to Enh1 are perhaps not surprising. That Enh1 blocks invasion of P. falciparum demonstrates this compound's ability to perturb Ca2+-dependent processes across multiple members of the Apicomplexa.Enh1 robustly inhibited the ability of activity . Geneticlciparum , mirrori vacuole . In lighToxoplasma Ca2+ signaling network shows evidence of conservation within apicomplexans while being dissimilar to human signaling pathways. By screening for modulation of this pathway, we have prioritized compounds that are likely to interfere selectively with infection without focusing on a single parasite protein. This screen can be performed with commonly available equipment and should be easily scalable to larger collections of compounds. Our success in identifying new modulators of Ca2+ signaling with effects that extend to multiple apicomplexans highlights the power of such an approach. In particular, Enh1 inhibits parasite viability with an EC50 in the nanomolar range and provides a good lead for the development of antiparasitic compounds. Further characterization of these compounds may identify novel components of apicomplexan Ca2+ signaling pathways and improve our ability to target these pathways specifically.Resistance to front line antimalarials is increasing , 72, andT. gondii strains with different PKG alleles. M. E. B., R. H., and W. J. Z. provided key reagents and advice. F. T. and N. J. W. synthesized Cpd2. M. T. D., S. N. J. M., and S. L. supervised the work in their respective laboratories and contributed to the analysis of experiments and writing of the manuscript.S. M. S., M. A. H. T., and A. S. P. designed and conducted the experiments and analyzed the data. S. M. S. wrote the majority of the manuscript, with specific sections contributed by M. A. H. T. and A. S. P. C. G. H. constructed the


Hypercholesterolemia alters intracellular Ca2+ signalling in vascular endothelial cells but our understanding of these changes, especially in the early stages of the disease process, is limited. We therefore determined whether carbachol‐mediated endothelial Ca2+ signals differ in plaque‐prone aortic arch compared to plaque‐resistant thoracic aorta, of wild‐type and ApoE−/− mice, and how this is affected by age and the presence of hypercholesterolemia. The extent of plaque development was determined using en‐face staining with Sudan IV. Tissues were obtained from wild‐type and ApoE−/− mice at 10 weeks (pre‐plaques) and 24 weeks (established plaques). We found that even before development of plaques, significantly increased Ca2+ responses were observed in arch endothelial cells. Even with aging and plaque formation, ApoE−/− thoracic responses were little changed, however a significantly enhanced Ca2+ response was observed in arch, both adjacent to and away from lesions. In wild‐type mice of any age, 1–2% of cells had oscillatory Ca2+ responses. In young ApoE−/− and plaque‐free regions of older ApoE−/−, this is unchanged. However a significant increase in oscillations (~13–15%) occurred in thoracic and arch cells adjacent to lesions in older mice. Our data suggest that Ca2+ signals in endothelial cells show specific changes both before and with plaque formation, that these changes are greatest in plaque‐prone aortic arch cells, and that these changes will contribute to the reported deterioration of endothelium in atherosclerosis.Apolipoprotein‐E knockout (ApoE We have investigated aortic endothelial cell calcium signalling changes in the Apolipoprotein E knockout mouse model of atherosclerosis. Our data show that calcium signals in endothelial cells undergo specific changes both before and with plaque formation, that these changes are greater in plaque‐prone aortic arch than in plaque‐resistant thoracic aorta, and that these changes will contribute to the reported deterioration of endothelium in atherosclerosis. The vascular endothelium plays a vital role in the maintenance of vascular health and homeostasis. In addition to regulating vascular tone and blood flow through the production of nitric oxide and other endothelial‐derived relaxing factors (EDRF), endothelial cells regulate cellular adhesion, thromboresistance, smooth muscle cell proliferation and inflammation. Endothelial dysfunction therefore is implicated in many pathological conditions such as atherosclerosis, hypertension, sepsis and inflammatory conditions  mice, a widely used model of atherosclerosis, spontaneously develop hypercholesterolemia and atherosclerotic plaques when fed a normal diet  and plasma membrane Ca2+ movements, shapes the Ca2+ signal amplitude and duration and also frequency, often referred to as Ca2+ oscillations  and old 24 week ApoE−/− mice (established plaques), and compare the data to that of WT controls.In this study we have used confocal microscopy to examine changes in intracellular Ca2+ responses in the straight thoracic aorta with those in the more plaque‐prone aortic arch in WT mice, to investigate if flow differences affect Ca2+ signalling in endothelial cells, as this appears to have been little investigated previously. We have also compared data obtained in WT and the ApoE−/− mice. Furthermore in older mice, where plaques were present, measurements were taken from endothelial cells both immediately adjacent to the plaques and at a distance, in order to determine whether the changes in Ca2+ response were dependent upon or altered by proximity to areas of plaque.Endothelial cells sense the shear stress generated by blood flow and their elicited signals evoke a cellular response  determine the time course of changes in Ca2+ signals in WT and ApoE−/− mice by studying different age groups and a range of agonist concentrations, (3) investigate if endothelial Ca2+ signals were affected by plaque formation, (4) test the hypothesis that Ca2+ signalling changes were greater in endothelial cells from aortic arch compared to thoracic aorta and (5) test the hypothesis that Ca2+ signals become more oscillatory with aging in ApoE−/− but not WT mice.The aims of this study were therefore to: (1) determine whether the endothelial CaApolipoprotein E knockout mice (homozygotes) were obtained from Charles River and a breeding colony established in‐house. Wild‐type C57BL/6J mice were obtained from Charles River as required. All were maintained on a normal chow diet. Young (8–10 weeks) and old (22–24 weeks) male mice were used in experiments.2) and humanely killed by cervical dislocation in accordance with Schedule 1 of the UK Animals (Scientific Procedures) Act of 1986.Mice were anaesthetized : 5 g Sudan IV, 500 mL acetone, 500 mL 70% EtOH) and gently agitated for 15 min and then destained in 80% EtOH for a further 5 min. Aortae were rinsed in PBS, dissected open along the outer edge of the arch and pinned out in a dissection dish containing PBS. Images were obtained using a dissecting microscope mounted with a Leica camera and plaque size assessed as a percentage of total surface area using ImageJ software. Plaques were not systematically investigated in the less plaque‐prone thoracic aorta, as they occur at too low a frequency.The presence or absence of plaques in the aorta of WT and ApoE47H2O 1.2, HEPES 10.9, Glucose 8, CaCl2 2 (adjusted to pH 7.4). The aorta was cleared of adhering tissue, divided into arch and thoracic sections and incubated with 23 μmol/L Fluo‐4 AM for 2 h at room temperature in the presence of 0.25% of the non‐ionic detergent Pluronic F‐127 and subsequently cut into strips (~1 × 6 mm). The tissue was then placed in physiological salt solution to allow de‐esterification of the dye. Strips were mounted, endothelium face down, under a small amount of isometric tension between two fixed aluminum foil clips at the bottom of the chamber, on the stage of an Olympus inverted microscope. The chamber was perfused with physiological salt solution at a constant flow rate (1 mL/min) and maintained at 30°C. Experiments were performed using an Ultraview LCI spinning (Nipkow) disc, wide‐field confocal microscope , equipped with an Orca ER cooled CCD camera  and a 20× objective . Once a stable baseline signal was established, the agonist carbachol was applied at 0.3, 1, 3 and 10 μmol/L to obtain a non‐cumulative dose‐response curve in both arch and thoracic aorta segments. The resulting Ca2+ signals were measured in terms of the amplitude of the initial peak response, the amplitude of the secondary plateau and the number of cells responding to CCh with an oscillatory Ca2+ response. Dose‐response curve data collected from plaque‐laden vessels was obtained from individual cells either immediately adjacent to plaques or in plaque‐free regions (no plaque present on entire strip), in order to compare whether this altered the Ca2+ signal observed. Similarly, Ca2+ oscillation data was collected from either totally plaque‐free strips or adjacent to plaques (either immediately adjacent or with plaque within the field of view). Data on Ca2+ oscillations from thoracic or arch regions were obtained and pooled with previously obtained data from WT  and ApoE−/−  animals, which had not previously been divided into region or proximity to plaque : NaCl 154, KCl 5.6, MgSOUnless otherwise specified, chemicals were obtained from Sigma (UK). Pluronic‐F127 and Fluo‐4 AM were obtained from Invitrogen.n = number of cells and N = number of mice. Statistical analysis and curve fitting were carried out using Graphpad Prism 5. The Student's t test and one‐way ANOVA were used for statistical comparisons. A value of P <0.05 was considered significant. In some cases, where Graphpad prism was unable to fit a curve to the mean data, a constraint was applied to the top of the curve , which allowed curve‐fitting to proceed and an EC50 estimate to be obtained. This value was chosen by examining all the examples where curves were successfully fitted to the experimental data and comparing the size of the response measured with 10 μmol/L CCh with the calculated maximum of the fitted curve, i.e. on average, the response to 10 μmol/L CCh was determined to be just 11% below the maximum response.Data are presented as mean ± SEM, where N = 10; 24 weeks, N = 5). In young ApoE−/− mice, no plaques were visible by eye, and Sudan IV staining revealed either no plaque or barely visible tiny areas (N = 10). In older ApoE−/− mice, all aortae exhibited clear atherosclerotic plaques . The percentage of the total surface area covered by plaque was calculated to be: 10 weeks, 0.08 ± 0.02% and 24 weeks, 2.8 ± 0.8%; Fig. No WT mice, young or old, exhibited atherosclerotic plaques in the aortic arch. This was confirmed by staining with Sudan IV  were investigated in endothelial cells from arch and thoracic sections of the aorta. 2+ in endothelial cells that are immediately adjacent to areas of plaque , as well as plaque‐free regions . As noted earlier, the components and hence shape of the endothelial Ca2+ signal elicited by carbachol depends on several mechanisms, which may be affected differently by age and dyslipidemia. We therefore went on to compare the Ca2+ responses in terms of their initial peak response, the magnitude of the plateau phase and the percentage of cells that respond to CCh with an oscillatory Ca2+ response, in the different age groups, between WT and ApoE−/− mice, and between endothelial cells in the arch and thoracic aorta.In all tissues CCh produced a concentration‐dependent rise in intracellular Calls Fig. , we note2+ signals were found between thoracic and arch regions of the aorta. In 10 week old WT mice, the amplitude of the Ca2+ response to CCh and the pEC50 was different in thoracic and arch sections of the aorta. In thoracic aorta, the amplitude of the response was greater  and the pEC50 more potent , however there was no difference in pEC50  and pEC50 was more potent  but the pEC50 was unaltered .In 24 week old ApoE−/− mice in order to investigate the effects of hypercholesterolemia.We have compared the CCh concentration‐response curve in WT and ApoE−/− mice plaques were absent and therefore all measurements were made from cells in plaque‐free areas. In thoracic aorta, there was no difference in the amplitude of the Ca2+ peak and plateau responses in WT and ApoE−/− cells, except at the highest CCh concentration of 10 μmol/L, where the ApoE−/− response was increased compared to WT  to those found in WT thoracic cells .Plaques were established in ApoEues Fig. Bi and thsee Fig. . In the 2+ response. In this study, we have examined whether the percentage of cells producing an oscillatory response varies depending on age and location . In WT mice of any age, ~1–2% of cells give an oscillatory Ca2+ response  is abundantly expressed in the vasculature, binds cholesterol and is necessary for caveolae formation. An important role for cav‐1 in atherosclerosis has been demonstrated using a cav‐1/ApoE−/− mice, we observed that hypercholesterolemia differentially affects Ca2+ signalling in endothelial cells from aortic arch and thoracic aorta. In thoracic aorta, the Ca2+ response was unaltered compared to WT, whereas in aortic arch sections, a widespread, significant increase in the Ca2+ response was apparent. This is despite the fact that these 10 week old ApoE−/− mice are not yet exhibiting plaques in the aorta. We have previously shown that this increased response is reversed by treatment with methyl‐β‐cyclodextrin and therefore directly related to the elevated levels of cholesterol present in these knockout mice  or a det et al. . Further−/− mice. Ca2+ responses were largely unaltered in thoracic aorta and significantly enhanced in aortic arch irrespective of the presence of plaque. This is in opposition to the findings of Crauwels et al. , transcription profiles of modifiers of SERCA function were found to be differentially regulated in plaque‐prone aortic smooth muscle compared to plaque‐resistant regions prior to plaque development. The combination of atherosusceptible gene changes and hypercholesterolemia leads to the significant Ca2+ signalling changes that we see in ApoE−/− aortic arch.We have demonstrated clear alterations in Ca2+ signalling in WT mice, that is modified in the hypercholesterolemic animals, (2) increasing age does not alter the Ca2+ response in WT mice, so the increased response to CCh in older ApoE−/− mice is attributable to hypercholesterolemia, (3) Ca2+ signalling changes are greater in endothelial cells from plaque‐prone aortic arch than plaque‐resistant thoracic aorta, (4) CCh concentration‐response curves were unaffected by plaque formation, but (5) Ca2+ signals become more oscillatory with age and in particular with plaque formation in ApoE−/− mice.We have observed that (1) there is a regional variation in Ca2+ signalling changes occur very early on in the development of atherosclerosis and are more pronounced in plaque‐prone areas than plaque‐resistant areas. Calcium signals become more oscillatory in nature as plaques develop, which may have implications for how well those calcium signals can stimulate generation of nitric oxide. By determining which specific aspects of Ca2+ signalling are first susceptible to atherosclerotic progression, we might gain insight into which points in the pathway may be therapeutically worthwhile targeting.In conclusion, CaNo conflicts of interest, financial or otherwise, are declared by the authors.