Rhododendron dauricum L. is an ancient Chinese traditional herb. The pharmacological effects of R. dauricum extract have been shown in chronic tracheitis. The aim of this study was to investigate the cardiovascular effects of Rhododendron dauricum L. flavonoids (RF) on rats and its mechanisms. This study was performed in isolated vascular rings and a rat model of myocardial infarction and isolated myocytes. RF (0.5 – 4 mg/mL) induced a concentration-dependent relaxant effect on the phenylephrine (10-5 M) and KCl (60 mM) contracted aortic rings, with or without intact endothelium. This effect was attenuated by pretreated with L-NAME (10-5 M) and K+ channel inhibitor 4 - AP (1 mM) and TEA (1 mM). The Ca2+-induced contraction and PE-induced contraction were obviously attenuated after pretreated with RF (2 mg/mL) for 30 min in Krebs solution, without Ca2+, containing 10-4 mol EGTA. KCl (60 mM) significantly increased the intracellular free Ca2+ concentration ([Ca2+]i) and RF inhibited the changes induced by KCl in single cardiac myocytes. RF obviously prolonged the survival time of hypoxia mice pretreated with isoprenaline and reduced the myocardial infarction size in rat coronary artery ligation. These findings suggest that RF induces concentration-dependent vasodilation and myocardial preservation. Rhododendron dauricum L., referred to as “Man-shan-hong” in Chinese, belongs to the family of Ericaceae, the genera of Rhododendron L. Rhododendron dauricum L. is distributed in the northern part of China, and is a type of medicinal herb often used to cure chronic tracheitis. Chinese Pharmacopoeia (1977 edition) has listed R. dauricum as an official drug  on respiratory system have been investigated, in which a decrease in [Ca2+]i of bronchial smooth muscle seems to be involved. It is also well known that intracellular calcium-induced calcium release (CICR) is involved not only in bronchial and vascular smooth muscle, but also in myocardial cells, which could induce the smooth muscle and myocardial cell constriction. It has also been reported that [Ca2+]i overload causes cell damage, as a result of cell undergoing hypoxic and ischemia. Should RF be capable of inhibiting influx of Ca2+ and intracellular calcium release in order to reduce [Ca2+]i, it would be able to avoid damage by calcium overload. The mechanisms of in vivo and in vitro. Hence, the present study was designed to examine the effects of on the vascular tone and explore its cardiovascular protective effects, both Extraction of RF Rhododendron dauricum (300 g) was extracted twice at 80-100°C, using 3000 mL water for 2 h each time. The extracts were then combined and filtered through filter paper. The filtrate was concentrated at 50-60°C by rotary evaporator to 1800 mL. This crude extract was then dissolved in three volumes 95% ethanol, under constant stirring. The ethanol suspension was laid overnight and then concentrated. Polyamide column chromatography  was used to enrich the flavonoid components. Non-flavonoid components were first eluted with 1200 mL distilled water and then 1500 mL 95 % ethanol was used to elute the flavonoid components, until reaching a negative reaction of hydrochloric acid-magnesium. The elution speed was about 400 mL/h. The flavonoids suspension was concentrated and then vacuum dried. The flavonoids powder was about 6 g and included hyperoside, quercetin, azaleatin and isorhamnetin. Assay of flavonoids The experiments consisted of 3 parts.(І) Assay of hyperosideThe condition of chromatography as below:stationary phase: ODS volume ; mobile phase: methanol: 0.5% phosphoric acid (45: 55) aqueous solution (pH was adjusted with triethylamine to 3); speed was 0.8 mL/min; wave length of detection was 355 nm; and sample size was 10-20 μL. The content of hyperoside was 16.6%.(ІІ) Assay of azaleatin The condition of chromatography as below:stationary phase: ODS volume ; mobile phase: methanol: water (60: 40); speed was 1 mL/min; wave length of detection was 296 nm; and sample size was 10-20 μL. The content of azaleatin was 1.1%.(ІІІ) Assay of quercetin The condition of chromatography as below:stationary phase: ODS volume ; mobile phase: methanol: water: glacial acetic acid (50: 50: 2); speed was 1 mL/min; wave length of detection was 370 nm; and sample size was 10-20 μL. The content of quercetin was 3.8%.Preparation of thoracic arterial rings 4, 1.2 mmol/L KH2PO4, 1.5 mmol/L CaCl2, 25 mmol/L NaHCO3, and 10 mmol/L glucose. The thoracic aortas were cleaned of connective tissue, and cut into approximately 3-4 mm-wide strips. For endothelium-intact strips, extreme care was taken to avoid endothelium injury. For endothelium-denuded strips, the endothelium was removed by rubbing the vessel interior with wet filter paper. Each vascular strip was mounted in a 5-mL organ bath containing K-H buffer solution. One end of the aortic strip was attached to a metal hook and the other end was connected to an isometric force transducer  in a bath containing K-H buffer solution, maintained at 37°C and bubbled with 95% O2 and 5% CO2. Rings were equilibrated for 45 min at 1.5 g resting tension, and then challenged with PE (1.0×10-5 M) until a maximal contractile response was obtained. The integrity of the endothelium was assessed in all preparations by determining the ability of carbachol (1.0×10-5 M), to induce more than 80% relaxation of rings. The endothelium was considered to be removed when there was less than 10% relaxation response to carbachol. The isometric tension was recorded with MedLab BL-420E+ recording system .Male Wistar rats were purchased from Harbin Medical University animal center. The rats (each weighing 250 - 300 g) were sacrificed by decapitation. The thoracic aortas were rapidly and carefully dissected and placed into ice-cold and oxygen-saturated K-H buffer solution (pH=7.4) containing 118 mmol/l NaCl, 4.7 mmol/l KCl, 1.1 mmol/L MgSOEffects of RF on vascular tone The experimental set up were consisted of 4 groups, with each group including six test animal.(I) Endothelium-dependent or independent effects of RF on isolated aortic rings-5 M) or KCl (60 mM), RF  was cumulatively added into the organ bath at 4 min intervals. The rings with intact or denuded endothelium were always tested in parallel.When the tension was at resting state or reached a plateau induced by PE (1.0×10(II) Effect of L-NAME (10-5 M) pretreatment on RF induced vasorelaxation in rat aortic rings with endothelium L-NAME (10-5 M) 30 min before contraction with PE. Then RF  was cumulatively added into the organ bath at 4 min intervals to examine a role of L-NAME. Aortic rings were pretreated with NOS inhibitor (III) Effect of potassium channels blocking agents pretreatment on RF induced vasorelaxation in rat aortic rings without endothelium 2 (10 μM) 30 min before contraction with PE. Then RF  was cumulatively added into the organ bath at 4 min intervals to examine the role of potassium channel blocking agents.Aortic rings were pretreated respectively with potassium channels blocking agents, including 4-AP (1 mM), TEA (1 mM), Gli (10 μM) and BaCl(IV) Effect of RF and KCl pretreatment in K-H buffer solution without Ca2+ on Ca2+-induced contraction in isolated aortic rings 2+ 30 min before contraction with Ca2+  was cumulatively added into the organ bath at 4 min intervals to examine the role of RF on Ca2+- induced contraction.Aortic rings were pretreated with RF (2 mg/mL) and KCl (60 mM) in K-H buffer solution without Caith Ca2+ . Then Ca(V) Effect of RF pretreatment in K-H buffer solution without Ca2+ on PE-induced contraction in isolated aortic rings 2+ 30 min before contraction with PE. Then PE (1.0×10-5 M) was added into the organ bath at 4 min intervals to examine the role of RF on PE- induced contraction.Aortic rings were pretreated with RF (2 mg/mL) in K-H buffer solution without CaPreparation of rat cardiomyocytes et al.  for 5 min, and calcium-free Tyrode’s solution until it stopped beating. The heart was then enzymatically digested with the calcium-free Tyrode’s solution, containing collagenase type II and BSA. The ventricular tissue was minced after being softened and placed in the KB medium . Single cells were obtained by gentle pipetting. It was stored at 4°C for 1–2 h, and then gassed with 95% oxygen and 5% carbon dioxide and warmed to 37 ± 0.5°C. Only rod-shaped myocytes with clear cross-striations were studied.Myocytes were isolated according to the method established by Yang et al. . Briefly[Ca2+]i measurement 0) and after (Fi) the drug administration, were recorded. The change in [Ca2+] i was represented by the ratio of F i /F0.After the isolation of single ventricular myocytes, they were adhered to the cover-slips of the chamber. Cells were then rinsed once with the normal Tyrode’s solution and subsequently incubated with a working solution containing Fluo-3/AM (20 mM) and Pluronic F-127 (0.03%) at 37°C for 45 min. After loading, the cells were washed once with the standard Tyrode’s solution to remove the extracellular Fluo- 3/AM. Fluorescent changes of the Fluo-3/AM-loaded cells were detected by a laser scanning confocal microscope at 488 nm for excitation from an argon ion laser and 530 nm for emission and inverted microscope with 20 × objective. Drugs were added between scans 3 and 4, and the images stored on disks. The fluorescent intensities, both before  Effect of KCl on [Cai was measured after adding KCl (60 mM) to the normal Tyrode’s solution containing 1.0 mmol/ L Ca2+.F 2+]i elevation induced by KCl (60 mM) (ІІ) Effect of RF on [Cai was then measured after adding 60mN KCl. The preparation was pretreated with RF (1~2 mg/mL) for 10 min. F i elevation induced by KCl (60 mM) (ІІІ) Effect of verapamil on [Ca2+]i was then measured after adding 60 mM KCl.The preparation was pretreated with calcium channel inhibitor verapamil (10 μM) for 10 min. F Effect of RF on hypoxia mice Fourty male mice (average weight of 20±2 g) were divided into 4 groups: 180 mg/kg RFH group, 90 mg/kg RFL group, control group  and 10 mg/kg BN52021 group. Each group received intraperitoneal injection with isoprenaline (0.05 mg/10 g) 15 min after injecting drugs through vena caudalis and then each animal moved into a wide-mouthed bottle (180 mL) containing 10 g nitrica calx to examine the survival time, through the observation of asphyxia.Rat model of myocardial infarction Male Wistar rats weighing 230–250 g were randomly divided into five groups: control, ischemia (MI), ischemia-low RF , ischemia-high RF  and BN52021 (10 mg/kg), respectively. The rats were heparinized (300 U) and anesthetized with IP sodium pentobarbital (40 mg/kg), and were then ventilated using a small animal ventilator at a frequency of 70/min and a tidal volume of 3 mL. The chest was surgically opened and the body temperature was maintained at 37°C by placing the animal on a heating pad. The standard limb lead ECG, together with the arterial pressure, was continuously recorded on a recorder . A left thoracotomy was performed via the fourth rib intercostal space and a segment of saline-soaked 5-0 suture was looped around the left anterior descending (LAD) coronary artery, near its origin from the left coronary artery and then the chest was closed quickly.Measurements of infarction size The heart was removed from the animal after 60 min of infarction, and then the ventricular tissue was dissected and kept at −4°C overnight. The frozen ventricles were sliced into 2 mm thick sections, and then incubated in 1% triphenyltetrazolium chloride at 37°C in 0.2 M Tris buffer (pH=7.4) for 30 min. While normal myocardium was stained brick red, the infarcted areas remained unstained. The size of the infarcted area was estimated by its volume and weight, as a percentagee of the left ventricle . Statistical analysis Data were presented as mean ± SD. Statistical comparison was performed by the Student’s t - test and with an analysis of variance (ANOVA), with a value of p < 0.05 considered as significant. Effects of RF on isolated aortic rings induced by PE and KCl -5 M)-precontracted aortic rings in a dose-dependent manner (RF relaxed PE (10t manner . 0) and after (Fi) the drug administration, were recorded. The change in [Ca2+] i was represented by the ratio of F i /F0.Compared with endothelium - denuded rings, the maximal relaxant effect of RF on rings with endothelium was increased by about 20 ± 1.8% and RF also relaxed KCl (60 mM)-precontracted aortic rings in a dose-dependent manner. Compared with endothelium-denuded rings, the maximal relaxant effect of RF on rings with endothelium was increased by about 22 ± 3/AM. Fluorescent changes of the Fluo-3/AM-loaded cells were detected by a laser scanning confocal microscope at 488 nm for excitation from an argon ion laser and 530 nm for emission and inverted microscope with 20 × objective. Drugs were added between scans 3 and 4, and the images stored on disks. The fluorescent intensities, both before  Effect of KCl on [Cai was measured after adding KCl (60 mM) to the normal Tyrode’s solution containing 1.0 mmol/ L Ca2+.F 2+]i elevation induced by KCl (60 mM) (ІІ) Effect of RF on [Cai was then measured after adding 60mN KCl. The preparation was pretreated with RF (1~2 mg/mL) for 10 min. F 2+]i elevation induced by KCl (60 mM) (ІІІ) Effect of verapamil on i   .Effect of RF on survival time of hypoxia mice Compared with the control group, the mice survival time of 180mg/kg RFH group and the positive control group were prolonged by 4 min and 6 min (p < 0.05), respectively. This finding suggested that RF could obviously prolong the survival time in hypoxia mice .Effect of RF on cardial infarction size during ischemia + channels inhibitor BaCl2 did not play a role on the relaxant effect of RF, compared with the control group  for 30 min in Krebs solution without Ca2+ containing 10-4 mol EGTA, compared with the control group  evoked a F i /F0 increase of 2.94 ± 0.11 fold, [Ca2+]i   .Effect of RF on survival time of hypoxia mice Compared with the control group, the mice survival time of 180mg/kg RFH group and the positive control group were prolonged by 4 min and 6 min (p < 0.05), respectively. This finding suggested that RF could obviously prolong the survival time in hypoxia mice .Effect of RF on cardial infarction size during ischemia The results, as shown in Rhododendron dauricum is a type of medicinal herb often used to cure chronic tracheitis. The related literatures have demonstrated that the flavonoids present in the leaves of R. dauricum (such as Farrerol) could, relieve coughs and move phlegm. These flavonoids are thought to reduce the [Ca2+]i , which can induce relaxation of tracheal smooth muscle and decrease gland excretion. There are several novel fingings in this present study: (i) the RF exerted a vasorelaxant effect on the phenylephrine and KCl contracted aortic ring with or without intact endothelium. (ii) Several mechanisms were involved, such as inhibition of influx of extracellular Ca2+ and release of Ca2+ from sarcoplasmic reticulum, contributing to NO release from intact endothelium, activation of voltage dependent K+ channels (KV) and calcium dependent K+ channels (KCa). (iii) In the rat myocardial cells, incubation with RF could dose-dependently decrease [Ca2+]i elevation by KCl, as observed by laser scanning confocal microscopy. (iv) In the animal experiments, RF could significantly prolong the hypoxia endurance time in mice and protect cardiac myocytes in the coronary artery ligation experiment. Based on these findings, we concluded that flavonoids are the active components of Rhododendron dauricum which can exert vasodilatation and myocardial preservation. The potentials of RF on cardiovascular disorder are closely related to a decreased [Ca2+]i content in vascular smooth and cardiac muscle cells.L-NAME. Endothelium-dependent relaxation of RF seemed to be associated with NO signaling, via guanylate cyclase activation, since L-NAME could attenuate this response. The direct endothelium-dependent and independent vasorelaxation should be taken into account, based on this study. Secondly, RF dose-dependently relaxed endothelium-denuded rings contracted with PE and KCl. The cellular mechanism of contraction involved in the response to KCl and PE is different. KCl induces Ca2+ influx via voltage-dependent Ca2+ channels, which further activates Ca2+-induced Ca2+ release through ryanodine-receptor. PE increases intracellular Ca2+ concentration via two mechanisms: (i) it activates receptor-gated Ca2+ channels, and (ii) it mobilizes Ca2+ from intracellular stores via the inositol-1, 4, 5- trisphosphate (IP3) receptor or induces secondly myosin light chain phosphorylation via activating PKC   , and mor2+]i, and these facts prompted us to testify the RF influence on isolated myocytes. Through the use of laser scanning confocal microscope, we observed that RF could dose-dependently inhibit the increase in [Ca2+]i induced by KCl, and the inhibitory effect did not change with 10 μM verapamil at the concentration of 2 mg/mL. This result confirmed that RF functioned to inhibit the [Ca2+]i on both vascular smooth muscle cells and myocytes, which supported the additional study, proposing that RF could exert cardioprotector effects. It has been proven that reducing the [Ca2+]i content protects myocytes during damage by hypoxia and ischemia (In this study, isolated vascular rings experiments indicated that RF-induced vasodilative effects partially contributed to the inhibition of [Caischemia , 12. TheR. dauricum exceed that of the traditional pharmacodynamic limitations. Hence, RF seem to have the potential for development into a potent drug for use in the cardiovascular system.In conclusion, it could be said that explored the cadiovascular potency of RF in isolated myocytes, and vascular tissue in this study. Based on these studies, it could be said that so far the pharmacological activities of


In the current study, we investigated the Ca2+-handling proteins. Detailed laser confocal Ca2+ imaging demonstrated spontaneous whole-cell [Ca2+]i transients. These transients required Ca2+ influx via L-type Ca2+ channels, as demonstrated by their elimination in the absence of extracellular Ca2+ or by administration of the L-type Ca2+ channel blocker nifedipine. The presence of a functional ryanodine receptor (RyR)-mediated sarcoplasmic reticulum (SR) Ca2+ store, contributing to [Ca2+]i transients, was established by application of caffeine (triggering a rapid increase in cytosolic Ca2+) and ryanodine (decreasing [Ca2+]i). Similarly, the importance of Ca2+ reuptake into the SR via the SR Ca2+ ATPase (SERCA) pump was demonstrated by the inhibiting effect of its blocker (thapsigargin), which led to [Ca2+]i transients elimination. Finally, the presence of an IP3-releasable Ca2+ pool in hiPSC-CMs and its contribution to whole-cell [Ca2+]i transients was demonstrated by the inhibitory effects induced by the IP3-receptor blocker 2-Aminoethoxydiphenyl borate (2-APB) and the phosopholipase C inhibitor U73122.RT-PCR and immunocytochemistry experiments identified the expression of key Ca2+ store in hiPSC-CMs. Furthermore, it demonstrates the dependency of whole-cell [Ca2+]i transients in hiPSC-CMs on both sarcolemmal Ca2+ entry via L-type Ca2+ channels and intracellular store Ca2+ release.Our study establishes the presence of a functional, SERCA-sequestering, RyR-mediated SR Ca The breakthrough technology introduced by Takahashi and Yamanka in 2006 enables the derivation of pluripotent stem cells by reprogramming of somatic cells with a set of transcription factors 2+-handling properties in-vitro models of human cardiac tissue). The suitability of hiPSC-CMs for such tasks depends, in part, on their contractile characteristics which in turn greatly depend on the Ca2+-handling nature of these cells.Only a limited number of studies described the ability to direct hiPSC differentiation towards the desired cardiac-lineage 2+-handling displays a well-defined sequence of events. Ca2+ influx into the cells via depolarization-activated L-type Ca2+ channels serves as an initial trigger that is then amplified several folds by sarcoplasmic reticulum (SR) Ca2+-store release via Ca2+-sensitive ryanodine receptors (RyRs); a process known as Ca2+ induced Ca2+ release (CICR) 2+]i transients being derived solely from Ca2+ influx through the membrane Ca2+ channels 2+ stores In adult ventricular cardiomyocytes, Ca2+]i transients in hiPSC-CMs are dependent on both transsarcolemmal Ca2+ entry via L-type Ca2+ channels and on intracellular store Ca2+ release. To test this hypothesis, we initially carried out gene expression and immunostaining studies to show that key Ca2+-handling proteins are expressed in hiPSC-CMs. To test for their functionality we then performed detailed laser-confocal Ca2+ imaging coupled with targeted pharmacological interventions. Initial studies confirmed the importance of transsarcolemmal Ca2+ entry through the L-type Ca2+ channels for modulation of the whole-cell [Ca2+]i transients in these cells. We then demonstrated that hiPSC-CMs display functional and loaded RyR-regulated intracellular Ca2+ stores that contribute as well to the whole-cell [Ca2+]i transient. In addition, we investigated the functionality of SR Ca2+ ATPase (SERCA) pumps, which serve as an important SR Ca2+ sequestration pathway. We found the SERCA pumps to be functional and responsible for the refilling of hiPSC-CMs' SR Ca2+ store content. Finally, we also present evidence showing the expression and functionality of inositol-1,4,5-trisphosphate receptors (IP3Rs) in hiPSC-CMs and demonstrate the important contribution of this alternative pathway to Ca2+-handling in these cells.In the current study, we tested the hypothesis that whole-cell i transients) as previously described 2-1.8, MgCl2-1, HEPES-10, glucose-10 at pH 7.4 via 1 mM NaOH. The tyrode solution was superfused at a rate of 1 ml/minute and at a temperature of 37°C. Intracellular Ca2+-transients were recorded using a confocal imaging system  mounted on an upright BX51WI Olympus microscope equipped with a X60 water objective Cells were loaded with 5 µM fluo-4 fluorescent Ca2+, temporally-limited puffs of caffeine were applied. This specific technique To investigate caffeine mobilization of store CaData are presented as mean ± standard error of mean (SEM). Student's paired t-test was used to compare between means. When the effects of multiple concentrations of the pharmacological agents were studied, then one way ANOVA was used followed by Dunnet's post-hoc comparison to baseline values. p<0.05 was considered to be statistically significant.2+-handling molecular components are present in the hiPSC-CMs, we first used semi-quantitative RT-PCR analysis. We assessed for the expression of the following Ca2+-handling proteins: RyR2, IP3R2, SERCA2a, Cav1.2 (encoding the α-1C subunit of the L-type Ca2+ channel), Calsequestrin, and Phospholamban. As can be seen in To evaluate whether cardiac-related Ca2+]i transients in hiPSC-CMs were recorded from spontaneously beating dispersed single cells or small monolayered clusters, under native cytosolic conditions. These transients were monitored in Fluo-4 (a fluorescent Ca2+ indicator) loaded cells, examined under a laser scanning confocal microscope utilizing the line-scan mode. Line scans were adjusted to avoid the cell nuclei and were localized at mid-cell z-section depth. Under control conditions, in the presence of 1.8 mM external bath Ca2+, spontaneous whole-cell [Ca2+]i transients were recorded as cell-wide rhythmic events in all cells tested (n = 96) (Spontaneous whole-cell [Ca(n = 96) .2+ influx is an important initial trigger for excitation-contraction coupling in adult cardiomyocytes 2+]i transients require external Ca2+. To this end, we recorded whole-cell [Ca2+]i transients in the presence (1.8 mM bath Ca2+) and absence of Ca2+ in the bath solution (n = 6). As can be appreciated in 2+ the whole-cell [Ca2+]i transients were completely abolished.Transmembranal Ca2+ channel serves as an important transmembranal Ca2+ influx pathway in hiPSC-CMs, as documented in adult cardiomyocytes 2+ channel blocker. Whole-cell [Ca2+]i transients were recorded before and after the application of 1 µM Nifedipine (2+ (2+]i transients (n = 9). Dose-response studies using lower concentrations of nifedipine  demonstrated that the cells were extremely sensitive to L-type channel blockade with a steep decrease in [Ca2+]i transients amplitude observed at a very low concentration  application resulted in complete elimination of whole-cell [Ca2+]i transients in all cases  as well as from an additional well-characterized hiPSC line (hfib2-5) derived using the traditional 4-factors method ll cases .2+ influx and specifically Ca2+ entry via L-type Ca2+ channels are important requirements for the generation of whole-cell [Ca2+]i transients in hiPSC-CMs.Taken together these data confirm that transmembranal CaWe next conducted immunocytostaining studies of hiPSC-CMs probing for both RyR2 and sarcomeric α-actinin in small monolayered clusters . As prev2+ stores that release Ca2+ via functional RyRs we tested for caffeine responsiveness. Caffeine mobilization of store Ca2+ and its effect on whole-cell [Ca2+]i transients were measured by applying a pressure-ejected caffeine puff  on to fluo-4 loaded hiPSC-CMs. As can be observed in 2+ from the intracellular stores, resulting in a high amplitude caffeine-induced Ca2+ transient (n = 26). This was followed by reversible quiescence of whole-cell [Ca2+]i transients  induced an instantaneous rapid caffeine-induced [Ca2+]i transient  trials were conducted. These control puffs did not trigger any apparent intracellular Ca2+ response (data not shown).Next, it was important to validate that the caffeine-induced i transients before  with escalating doses  Mature molecular components RyR2, IP3R2, SERCA2a, CaV1.2, calsequestrin, and phospholamban are expressed in these cells; (2) hiPSC-CMs display spontaneous whole-cell [Ca2+]i transients; (3) Ca2+ entry via L-type Ca2+ channels is required for triggering whole-cell [Ca2+]i transients; (4) Caffeine-responsive and ryanodine-sensitive Ca2+ stores are loaded and functional; (5) RyR-mediated SR Ca2+ release contributes to whole-cell [Ca2+]i transients; (6) SERCA pumps are functional and enable the refilling of SR Ca2+ content, required for the modulation of whole-cell [Ca2+]i transients; (7) An IP3-releasable Ca2+ pool is expressed, functional, and contributes to whole-cell [Ca2+]i transients; and (8) the results obtained are comparable in cardiomyocytes derived from different differentiation experiments of the same hiPSC line, from different hiPSC clones , and from different hiPSCs lines established using different techniques (using either three or four reprogramming factors). Taken together we conclude that whole-cell [Ca2+]i transients in hiPSC-CMs depend on both Ca2+ influx via L-type Ca2+ channels and intracellular Ca2+ store release, as previously documented in mouse In the current study, we focused on studying the Ca2+ channels. The resulting Ca2+ influx serves as a trigger for activating RyR-mediated SR Ca2+ release by a mechanism known as CICR 2+]i transients were shown to be derived solely from Ca2+ influx through membrane Ca2+ channels. In contrast, global whole-cell [Ca2+]i transients in early cardiac cells derived from mouse ESC 2+ release from intracellular Ca2+ stores without the triggering of membrane Ca2+ currents.In adult cardiomyocytes the development of an action potential triggers the opening of L-type Ca2+ store in hESC-CMs and postulated that essentially all of the [Ca2+]i transients in these cells were a consequence of transsarcolemmal Ca2+ influx via membranal Ca2+ channels 2+ store in at least a subset The mechanism underlying E-C coupling in hESC-CMs is somewhat controversial. While some reports suggested the absence of a functional SR Ca2+ influx and intracellular Ca2+ store release to whole-cell [Ca2+]i transients in hiPSC-CMs. The importance of the L-type Ca2+ current in generating whole-cell [Ca2+]i transients in these cells was manifested by the elimination of these transients in the absence of external Ca2+ or in the presence of nifedipine, a selective L-type Ca2+ channel antagonist. A similar requirement for external Ca2+ and the consequent transsarcolemmal Ca2+ influx was documented in adult cardiomyocytes Our results support the contribution of both the transsarcolemmal Ca2+ release from intracellular SR Ca2+ stores to whole-cell [Ca2+]i transients was demonstrated by pharmacological studies interfering either with SR Ca2+ release (caffeine and ryanodine) or reuptake (thapsigargin). Caffeine increases RyR2s opening, leading to a single large-amplitude caffeine-induced Ca2+ transient, considered to be a descriptive index of the level of SR Ca2+ load 2+, followed by a short and reversible succession of whole-cell [Ca2+]i transients. These results suggest that caffeine induced depletion of the SR Ca2+ store and point to a whole-cell [Ca2+]i transient dependency on SR Ca2+ content.The contribution of Ca2+ source for the caffeine induced [Ca2+]i increase is RyR-mediated SR Ca2+ release and is not dominated by Ca2+ influx via voltage-gated Ca2+ channels. This was demonstrated by the similar caffeine-induced rise in intracellular Ca2+ documented in the absence of extracellular bath Ca2+ further confirming the presence of a caffeine-responsive intracellular Ca2+ store. Similar results were also acquired in hESC-CMs The main Ca2+ release. Ryanodine, has been reported to reduce by approximately twofold the conductance of RyRs in the SR 2+ release observed as a significant decrease in the amplitude of whole-cell [Ca2+]i transients. A similar ryanodine induced effect was also reported in pacemaker cells isolated from rabbit sinoatrial node 2+]i transients firing rate. This phenomenon was also previously documented in rabbit sinoatrial node pacemaker cells where a similar slowing in firing rate was detected in the presence of ryanodine We also applied the RyR antagonist, ryanodine, to further study hiPSC-CMs RyR-mediated SR Ca2+ must be removed from the cytosol. In adult cardiomyocytes, one of the main Ca2+ removal pathways is the SR Ca2+ ATPase pump (SERCA) 2+, by sequestering Ca2+ back into the SR, and in this manner also regulate SR Ca2+ load 2+ sequestration from the cytosol back into the SR 2+]i transients through their ability to reload the SR Ca2+ stores in hiPSC-CMs we applied the SERCA inhibitor thapsigargin. Thapsigargin acted slowly to progressively decrease the amplitude of whole-cell [Ca2+]i transients, eventually leading to their complete inhibition. A similar effect was observed in spontaneously beating fluo-4 loaded isolated mouse ESC-CMs34. An antagonistic effect of thapsigargin on [Ca2+]i transients was also reported in human ESC-CMs For cellular relaxation to take place Ca2+]i transients, was further demonstrated by the miniscule effect of caffeine in hiPSC-CMs pretreated with thapsigargin, as a result of a pronounced diminution in SR Ca2+ content. Interestingly, under conditions of SERCA uptake inhibition (thapsigargin) a low SR Ca2+ content was retained yet [Ca2+]i transients were completely abolished. This can be explained by reports showing that decrease in SR Ca2+ content can disproportionately inhibit SR Ca2+ release 2+]i transients. In an immediate subsequent caffeine puff the caffeine-induced [Ca2+]i transient was completely omitted. The absent caffeine-induced signal at this stage is postulated to be a consequence of caffeine induced depletion of the SR Ca2+ store and the inability of the SR to accumulate Ca2+ as a result of the thapsigargin treatment.The key role of SERCA in reloading the SR, and thereby indirectly modulating hiPSC-CMs whole-cell [Ca2+ release presents a fundamental pathway for intracellular Ca2+ release in electrically non-excitable adult cells 2+ release channel IP3-mediated Ca2+ pool to the modulation of Ca2+-handling in hiPSC-CMs we first examined the expression and localization of the IP3R at the protein level. Immunostainings of these hiPSC-CMs stained positive for IP3R with a strong subcellular distribution of the immunosignal around the nucleus  2+]i transients amplitude, as was also reported in human ESC-CMs under these conditions 2+]i transients frequency was observed under the influence of 2-APB. Next we applied U73122, a phosopholipase C blocker (PLC). Blocking the activation of PLC inhibits a receptor-stimulated increase in the production of the second messenger IP3 required as a trigger for IP3R-mediated Ca2+ release 2+]i transients amplitude and frequency. A U73122 PLC inhibitory effect was also reported in mouse ESC-CMs 2+ pool is expressed and functional in hiPSC-CMs and that the resulting IP3R-mediated Ca2+ release contributes to the modulation of Ca2+-handling of these cells.Next, to evaluate for IP3-releasable Ca2+-handling properties of cardiomyocytes differentiated from hiPSCs and demonstrated that they share components that are present in adult cardiomyocytes, such as functional RyR-mediated SERCA-sequestering SR Ca2+ stores. Importantly, the results of this study showing similar properties in cardiomyocytes derived from different differentiation batches, from different hiPSCs clones, and from different hiPSCs lines may have important implications for their potential use for the aforementioned tasks. The hiPSC-CMs may serve as attractive cell-candidates for myocardial cell replacement therapy because of their inherent cardiac-specific properties and the potential for autologous therapy. Nevertheless, since functional compatibility between donor hiPSC-CMs and host myocardium is likely to contribute to an improved functional outcome of the cell engraftment as well as a reduction in potential pro-arrhythmic risk, detailed characterization of their Ca2+-handling characteristics is mandatory.The hiPSC technology has raised significant excitement with regards to its unique potential for regenerative medicine and for the study of various genetic disorders as well as for drug discovery and screening. In the current work we focused on the characterization of the Ca2+-handling in these cells may be important if one wishes to utilize the iPSC technology for establishment of personalized in-vitro models of cardiac tissue for the development and testing of pharmacological compounds targeting these functional properties. Finally, detailed understanding of the Ca2+-handling properties of hiPSC-CMs generated from healthy individuals may be used as a future reference when studying E-C coupling in hiPSC-CMs derived from patients with genetic disorders involving Ca2+-handling. Such patient/disease-specific models can be established, for example, from families suffering from catecholinergic polymorphic ventricular tachycardia (CPVT) Similarly, phenotypic characterization of Ca2+-handling components of hiPSC-CMs. Our results show that hiPSC-CMs display functional and loaded RyR-regulated intracellular Ca2+ stores. These stores can release Ca2+ via RyRs and can reload their content through SR Ca2+ uptake utilizing functional SERCA pumps. We present evidence showing the expression and functionality of inositol-1,4,5-trisphosphate receptors (IP3Rs). Furthermore, our findings demonstrate that the observed whole-cell [Ca2+]i transients in hiPSC-CMs depend on both sarcolemmal Ca2+ entry via L-type Ca2+ channels and on intracellular store Ca2+ release. Taken together hiPSC-CMs recapitulate functional key Ca2+ handling proteins that have been shown to be expressed and functional in mouse ESC-CMs In the present work we investigated basic CaFigure S12+]i transients' requirement for Ca2+ influx via L-type Ca2+ channels as observed in cardiomyocytes derived from different hiPSCs clones and lines.Whole-cell [Ca Whole-cell [Ca2+]i transients recorded from (A): hIH1 clone 1, (B): hIH1 clone 2 and (C): hfib2-5 before (left) and after (right) application of nifedipine (1 µM). Abbreviations: F/Fo, fluorescence (F) normalized to baseline fluorescence (Fo); sec, seconds.(PDF)Click here for additional data file.Figure S22+ stores as displayed in cardiomyocytes derived from different hiPSCs clones and lines.Caffeine and Ryanodine-sensitive Ca (A) A line-scan presenting the effect of 20 mM caffeine puff application (noted by the arrows) in hIH1 clone 1 (left), hIH1 clone 2 (middle) and hfib2-5 (right). (B) Line-scan tracings of whole-cell [Ca2+]i transients recorded from hIH1 clone 1 (top), hIH1 clone 2 (middle) and hfib2-5 (bottom) under baseline conditions (left) and in the presence of 10 µM Ryanodine (right). (C) Dose-response curve for ryanodine  displayed as the percent decrease in [Ca2+]i transients amplitude from baseline values . Abbreviations: F/Fo, fluorescence (F) normalized to baseline fluorescence (Fo); sec, seconds.(PDF)Click here for additional data file.Figure S3The effects of SERCA inhibition in cardiomyocytes derived from the hfib2-5 line. (A) Line-scan tracings of whole-cell [Ca2+]i transients in a representative hfib2-5 hiPSC-CM under baseline conditions (left) and after 30 minutes (right) of constant 10 µM thapsigargin exposure. (B) Caffeine-induced Ca2+ transients (denoted by arrows) under baseline conditions (upper), after 30 minutes (middle), and 32 minutes (lower) of thapsigargin exposure. Abbreviations: F/Fo, fluorescence (F) normalized to baseline fluorescence (Fo); sec, seconds.(PDF)Click here for additional data file.Figure S4The effect of IP3R inhibition in cardiomyocytes derived from the hfib2-5 line. Whole-cell [Ca2+]i transients in representative hfib2-5 hiPSC-CMs before (left) and after (right) 2-APB (2 µM) application (A) and before (left) and after (right) U73122 (2 µM) application (B). Abbreviations: F/Fo, fluorescence (F) normalized to baseline fluorescence (Fo); IP3R, inositol-1,4,5-trisphosphate receptor; sec, seconds.(PDF)Click here for additional data file.


LY294002 (1–20 μM), a specific PI3K inhibitor, dramatically decreased HL-1 [Ca2+]i, Ca2+ transients and ICa. We also examined the effect of PI3K isoform specific inhibitors, i.e. α ; β  and γ , to determine the contribution of specific isoforms to HL-1 [Ca2+]i regulation. Pharmacologic inhibition of each of the individual PI3K isoforms significantly decreased [Ca2+]i, and inhibited Ca2+ transients. Triciribine (1–20 μM), which inhibits AKT downstream of the PI3K pathway, also inhibited [Ca2+]i, and Ca2+ transients and ICa. We conclude that the PI3K/Akt pathway is required for normal maintenance of [Ca2+]i in HL-1 cardiomyocytes. Thus, myocardial PI3K/Akt-PKB signaling sustains [Ca2+]i required for excitation-contraction coupling in cardiomyoctyes.The phosphoinositide 3-kinases (PI3K/Akt) dependent signaling pathway plays an important role in cardiac function, specifically cardiac contractility. We have reported that sepsis decreases myocardial Akt activation, which correlates with cardiac dysfunction in sepsis. We also reported that preventing sepsis induced changes in myocardial Akt activation ameliorates cardiovascular dysfunction. In this study we investigated the role of PI3K/Akt on cardiomyocyte function by examining the role of PI3K/Akt-dependent signaling on [Ca The phosphoinositide 3-kinases (PI3K) are a conserved family of signal transduction enzymes that are involved in regulating cellular proliferation and survival ,2. The P2+ channels, ryanodine receptors and sarcoplasmic reticulum Ca2+-ATPase 2a  was determined by interpolation from a standard curve generated by Ca2+ calibration buffer kit #2  and Fura-2/K5-salt. After correction for the individual background fluorescence, the ratio of the fluorescence at both excitation wavelengths (F340/F380) was monitored simultaneously in 30–40 cells, identified by their fluorescence within a single view field. Images were collected every 3.3 s. Each slide was perfused with standard external salt solution for 6 min for control measurements, followed by 10 min with the experimental solution. At 16 min, the slide was washed with standard external salt solution for 5 min, and at 21 min data collection was stopped. Data was then exported to MS Excel and graphed using Origin 7.0  and Sigmaplot 11.0 . For statistical analyses, average [Ca2+]i from 25–40 cells within a microscopic field were obtained during the control period of 1–5 min from each of 5 separate HL-1 cell preparations. These averages were then compiled to obtain average control values (n = 5), and comparisons were made on data collected similarly from the same microscopic fields 15 minutes after experimental additions. Statistical differences between control and experimental values were established at p < 0.05 (Student’s paired T-test).Cells were loaded with Fura-2/AM  by incubating them for 30 min at room temperature (22–23 °C) with a standard external salt solution containing 2-μM Fura-2/AM. Cells were then washed with the external salt solution and incubated at 37 °C with 5% CO2 1, CaCl2 1.5, N-2-Hydroxyethylpiperazine-N’-2-ethanesulfonic acid (HEPES) 10, glucose 10 (pH adjusted to 7.41 with NaOH). Pipette solution contained (mM): potassium aspartate 120, Na2GTP 0.4, Na2ATP 5, MgCl2 1, EGTA 5, CaCl2 0.1, HEPES 10 (pH adjusted to 7.2 with KOH). For whole-cell voltage clamp measurements of membrane Ca2+ currents external NaCl was substituted with (mM) n-methy-D-gluamine chloride (NMDG+) 150, and CaCl2 was increased to 5 to maximize Ca2+ current. All solution constituents were obtained from Sigma/Aldrich, St. Louis, MO.Standard external salt solution contained (mM): NaCl 150, KCl 6, MgClLY 294002 was obtained from Alomone Labs, LTD, Jerusalem, Israel. The PI3 kinase isoform inhibitors: PI3-kinase α inhibitor 2, TGX-221 β inhibitor, AS-252424 γ isoform inhibitor; and the AKT inhibitor, Triciribine were obtained from Cayman Chemical, Ann Arbor, MI. The dosages selected for the various inhibitors were based on the literature and the manufacturer’s instructions. All inhibitors were dissolved in DMSO in stock solutions and then diluted to final concentration. The highest final concentration of DMSO by this approach was 0.24% DMSO.2+, i resulted from inhibition of membrane Ca2+ channels, we determined the effect of LY 294002 and triciribine on membrane Ca2+ currents in HL-1 cells. For these measurements the pipette-membrane giga-ohm seal and whole-cell access were obtained with cells perfused with standard external solution. Once achieved, the external solution was exchanged to one in which Na+ was substituted with NMDG+ and [Ca2+]o increased from 1.5 to 5 mM. Following 5-min equilibration, voltage clamp step protocols were performed to generate current–voltage plots .As an initial step to determine whether the effects by inhibitors of PI3Ks and of Akt on Ca2+ in HL-1 cells, which constitute a murine-derived, immortalized cell line with phenotypes like those of adult cardiomyocytes i, intracellular Ca2+ transients and membrane ICa in a murine, immortalized cardiomyocyte cell line, HL-1 cells. These data demonstrate that PI3K/Akt dependent signaling is required for normal Ca2+ metabolism in murine cardiomyocytes. This extends our knowledge of the role of PI3K/Akt signaling in cardiovascular homeostasis. We conclude that maintaining myocardial PI3K/Akt signaling is essential for cardiomyocyte function in the presence and absence of disease.In sum, we have found that inhibitors of the PI3K/Akt signaling cascade decrease total i, Intracellular calcium ion; ATP, Adenosine triphosphate; GTP, Guanosine triphosphate; HEPES, N-2-Hydroxyethylpiperazine-N’-2-ethanesulfonic acid; ICa, Membrane calcium current; FBS, Fetal bovine serum; EGTA, Ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid; NMDG, n-methy-D-gluamine; I/V, Current–voltage; L-type, Long-lasting; T-type, Transient; If, Funny current.The authors declare that they have no competing interests.2+ measurements and analyzed data; TS analyzed data and contributed to writing and editing of the manuscript; CL contributed to the experimental design and writing of the manuscript; DLW designed experiments and wrote and revised the manuscript; RW performed all electrophysiology and wrote and edited the manuscript. All authors read and approved the final manuscript.BMG wrote the first draft of the manuscript, performed all Ca


Changes in Ca2+ signaling and associated Ca2+-dependent processes are thought to underlie the high adaptive capacity of muscle fibers. Here we investigated the consequences and the involved mechanisms caused by the ectopic expression of the Ca2+-binding protein parvalbumin (PV) in C2C12 myotubes in vitro, and conversely, the effects caused by its absence in in fast-twitch muscles of parvalbumin null-mutant (PV−/−) mice in vivo. The absence of PV in fast-twitch muscle tibialis anterior (TA) resulted in an increase in the peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) and of its positive regulator, the deacetylase sirtuin 1 (SIRT1). TA muscles from PV−/− mice also have an increased mitochondrial volume. Mild ionophore treatment of control (PV-devoid) C2C12 myotubes causing a moderate elevation in [Ca2+]c resulted in an increase in mitochondrial volume, together with elevated PGC-1α and SIRT1 expression levels, whilst it increased PV expression levels in myotubes stably transfected with PV. In PV-expressing myotubes the mitochondrial volume, PGC-1α and SIRT1 were significantly lower than in control C2C12 myotubes already at basal conditions and application of ionophore had no effect on either one. SIRT1 activation causes a down-regulation of PV in transfected myotubes, whilst SIRT1 inhibition has the opposite effect. We conclude that PV expression and mitochondrial volume in muscle cells are inversely regulated via a SIRT1/PGC-1α signaling axis.Skeletal muscles show a high plasticity to cope with various physiological demands. Different muscle types can be distinguished by the force, endurance, contraction/relaxation kinetics (fast-twitch vs. slow-twitch muscles), oxidative/glycolytic capacity, and also with respect to Ca Cells make use of the Ca2+ signaling toolkit components 2+ concentration [Ca2+]c in a highly coordinated way. One of these components present in the cytosol of fast-twitch muscles is parvalbumin (PV), a member of the large family of EF-hand Ca2+-binding proteins 2+ buffer or more precisely Ca2+ signal modulator 2+ signaling needs to be finely tuned and it is not surprising that disturbances in the Ca2+-signaling machinery (e.g. due to genetic mutations) affects the physiology of muscle cells and leads to various muscle disorders, e.g. muscular dystrophies 2+ signaling toolkit not only necessitate functioning in a concerted manner, but also have to be expressed dynamically in such a way as to fulfill the requirements of various demands posed by the physical activity of muscles. That is, besides the functional crosstalk between Ca2+ signaling components, there also exists a complex crosstalk at the level of transcription/translation and the signaling pathways involved in this process are slowly emerging Excitable cells, including muscle and nerve cells, need to cope with a vast range of activities, from low activity under basal or “resting” conditions to maximal activity occurring during brief, fast movements or complex cognitive processes, for example. This requires rapidly adapting systems, both for energy metabolism and for intracellular signaling and evidently the two need to be interconnected. Cavice versa is not only accompanied by a switch in the expression of fiber-type specific filaments, in particular myosin heavy chain (MHC) isoforms, but those transitions also include shifting energy substrate (oxidative) metabolism and adaptation in mitochondrial biogenesis. The Ca2+-dependent mechanisms and the involved signaling pathways for the different processes are not entirely clear, but are likely to also involve Ca2+-dependent kinases , AMP-activated protein kinase (AMPK)) and phosphatases including calcineurin (CaN) Besides the modulation of contractile properties of muscle, skeletal muscles demonstrate a high degree of plasticity in order to adapt to altered physiological demands resulting from increased exercise or caloric restriction, for example. A change in muscle fiber type from fast to slow or 2+-binding proteins. PV−/− fast-twitch muscles maintain their fast-twitch phenotype with respect to MHC isoforms, but the mitochondria content is significantly increased 2+ signaling-dependent mitochondrial biogenesis, while not affecting fiber-type specificity. Moreover, an inverse correlation between PV content and mitochondria volume is also seen in PV-expressing neurons including cerebellar Purkinje cells tibialis anterior (TA) muscles from wildtype (WT) and PV−/− mice serving as a loss-of-function model. An ionophore-mediated persistent elevation in [Ca2+]c in PV-expressing myotubes increased PV expression and negligibly affected mitochondria biogenesis, whilst the rise in [Ca2+]c upregulated PGC-1α and mitochondria volume in PV-negative control C2C12 myotubes. Both PGC-1α and its regulator SIRT1 were also increased in TA of PV−/− mice, along with the increased mitochondria volume in these muscle fibers described before Elimination of PV in null-mutant mice (PV−/−) leads to cell-specific modifications/homeostatic adaptations that are not restricted to the level of other CaAll experiments were performed in accordance with the guidelines of the European Committee Council Directive of November 24, 1986 (86/609/EEC) and the study was approved by the Veterinary Office of Fribourg, Switzerland.DMEM, penicillin, streptomycin, horse and fetal calf serum and G418 geneticin were purchased from GIBCO Invitrogen, Basel, Switzerland. Cell culture plasticware was from Milian AG, Geneva, Switzerland. Anti-parvalbumin and anti-calcineurin A antibodies were from Swant, Bellinzona, Switzerland, anti-SIRT1, anti–PGC-1α and ferutinin were from Santa Cruz Biotechnology Inc., Santa Cruz, CA, U.S.A. Sirtinol, resveratrol were from Sigma. Calcein AM, rhodamin 123, rhod-2 AM, Fura-2 AM, MitoTracker Green FM, MitoTracker Red CMXRos, Hoechst and 4-bromo A23187 calcium ionophore were purchased from Molecular Probes BV, Leiden, The Netherlands. Glass coverslips were from Assistent. JetPEI™ transfection reagent was from Brunschwig, Basel, Switzerland. All other chemicals were of highest available grade.2 and perfused transcardially by ice-cold saline solution (0.9%) or phosphate-buffered saline (PBS). Muscles were immediately dissected, snap frozen in liquid nitrogen and stored at −70°C until analysis. Tissue used for RNA isolation was dissected from mice perfused with sterile ice-cold PBS prepared with diethyl pyrocarbonate (DEPC)-treated water.PV-deficient mice (PV−/−) were generated on a C57Bl/6J x 129S background by homologous recombination 2 in growth medium (GM): Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% heat-inactivated fetal calf serum (FCS), 100 units/ml penicillin, 100 µg/ml streptomycin. Cells were passaged by trypsinization every 2–3 days when approximately 60–70% confluence was reached. Differentiation into myotubes was induced using differentiation medium (DM), i.e. DMEM supplemented with 2% horse serum (HS) for 6–10 days.C2C12 mouse myoblast cells were obtained from ATCC . C2C12 cells were routinely cultured at 37°C in a humidified atmosphere of 95% air and 5% CO2+-binding properties of rat and mouse PV are almost identical, also based on the fact that the two Ca2+-chelating loops are 100% identical in rat and mouse PV and the overall identity at the amino acid level is 95%. The EGFP-C1 expression plasmid was obtained from Clontech . Myoblasts were seeded into 12-well plates at a concentration of 104 cells/well. Stable transfection was performed by the use of the JetPEI™ transfection reagent and a total of 2 µg linearized plasmid DNA per well. The day after transfection, the medium was changed to fresh GM. After an additional 24 h, cells were incubated with fresh GM supplemented by geneticin  for 10–12 days. Isolation of individual transfected G418-positive clones was obtained by serial dilution.The transfection procedure was performed as described before 6 cells were harvested and washed in PBS. Thawed muscles and harvested C2C12 cells were homogenized in ice-cold buffer 1  and centrifuged at 3000×g for 5 min at 4°C. Pellets were then resuspended in ice-cold buffer 2 , incubated 30 min on ice with occasional mixing and centrifuged at 3000×g for 5 min at 4°C. Supernatants from muscles or C2C12 cells were transferred to Amicon Ultra 5000 NMWL, 4 ml Ultrafree Filter Unit or Ultra 0.5 30 K centrifugal devices (Millipore), respectively and centrifuged at 7000×g for 1 h or 30 min at 14,000×g at 4°C in order to concentrate the solutes. Total protein concentration in all extracts was determined using the Bradford or Dc assays .Isolated muscles or C2C12 cell samples were sonicated in homogenization buffer . After centrifugation at 13,000 rpm for 5 min on a bench top centrifuge, the supernatant was retained for analysis of cytosolic proteins. To generate fractions enriched in nuclear proteins, samples were prepared as previously described Differentiated C2C12 myotubes were fixed with 4% PFA and immunostained with an antiserum against PV, using the polyclonal antiserum PV25  and Cy3-labeled donkey anti-rabbit secondary antibody . For Western blot analyses, proteins (20 µg or 50 µg) were separated on either 7.5% or 12.5% polyacrylamide gels (depending on protein of interest) and transferred onto nitrocellulose membranes using a semidry transfer protocol. After blocking with 5% solution of non-fat milk in TBS buffer, membranes were incubated with the relevant primary antibodies. Two types of secondary antibodies were used: either Li-Cor® infrared dye-labeled antibodies or biotinylated antibodies  accompanied with additional incubation with avidin–biotin-conjugated peroxidase solution (Vector). When the secondary antibodies were Li-Cor® antibodies, all antibodies were diluted in the Li-Cor® blocking reagent and bands were detected using the Li-Cor™ Odyssey system. All image analyses were performed with the free software ImageJ (NIH). All membranes were stained with Ponceau S to confirm equal loading and to enable normalization for purposes of densitometry. The integral of all proteins was used in order to prevent a bias from any single reference or house-keeping protein.Total RNA was extracted from samples using the Trizol reagent according to the manufacturer’s protocol. 2 µg of total RNA was subsequently reverse-transcribed using the RevertAid™ H Minus First Strand cDNA Synthesis Kit, again following the manufacturer’s instructions. An aliquot from this reaction, equivalent to 200 ng of un-transcribed RNA, was amplified with GoTaq™ Flexi DNA polymerase using conditions recommended by the manufacturer. Reactions were performed on an MJ Research PCR machine. Details of oligonucleotides sequences are shown in 2+ ionophore Br-A23187 (1 µM). Cells were trypsinized for 5 min and DMEM medium was added to stop the reaction. Cells were centrifuged at 1,000 rpm for 5 min, resuspended and counted. In order to assess the cellular mitochondrial mass, cells were stained with MitoTracker Green FM (200 nM), a mitochondrial specific fluorescent dye at 37°C for 30 min. After incubation, cells were washed and resuspended in PBS at a concentration of 0.5×106 cells/ml. Cell counts were performed on a FACS CyFlow space  using FloMax® software. The forward scatter vs. side scatter area was used to analyze a homogeneous population of live cells and to determine FACS size gating. One part of C2C12 cells was processed without MitoTracker Green incubation and was used as a negative control. FACS quantitative and statistical analyses were performed with the FlowJo software .Control C2C12 cells and PV-clones were incubated in basal media or media containing the CaC2C12 cells were transferred onto glass coverslips (4.2 cm diameter) and allowed to attach, proliferate in growth medium and then in differentiation medium. Calcein-AM (green), DAPI (blue) and Mito Tracker Red (red) signals were acquired with a Leica TCS SP5/DMI6000 confocal microscope using a 20x water-immersion HCX PL APO objective . Z-stacks were composed of 30–50 adjacent sections, of optical slices of 400–800 nm step size. Images were obtained from control and PV-expressing C2C12 cells at day 5 in differentiation medium (n>30 cells). For these measurements, only cells with a single nucleus and not multi-nucleated cells were analyzed. For measurements of the whole cell and nuclear volumes, complete series of z-stacks were processed using Imaris software  with the “isosurface” routine using automatic fluorescent threshold from the green and blue signal, respectively. The mitochondrial volume was determined from the detecting “spots” procedure by applying an identical intensity threshold to the red channel.2+]m, cells were loaded with the AM ester of rhodamin 123 and rhod-2  at 37°C for 30 min in culture medium, followed by a subsequent 30 min period in dye-free medium for hydrolysis of the dye. The positively charged rhodamin 123 preferentially accumulates in the mitochondria due to the negative mitochondrial membrane potential of 150–180 mV and its fluorescence is quenched inside these organelles. Upon intramitochondrial Ca2+ increase, Δψm collapses, rhodamin 123 is transported back to the cytoplasm and thus the fluorescence (F/F0) increases. Loaded cells were excited at 488 nm and 543 nm at a laser power of 20 and 25%, respectively, scan speed was at 8000 Hz and the pinhole was set at 1 Airy unit using a 20x water-immersion HCX PL APO objective . Rhodamin 123 and rhod-2 fluorescence emissions were simultaneously captured by 2 PMTs at 509–555 nm and 587–677 nm emission ranges, respectively. To measure cytosolic [Ca2+]c dynamics, cells were loaded with Fura-2 AM (5 µM) in Krebs Ringer (KR) medium for 30 min at 37°C. Prior to experiments, cells were rinsed twice with Ca2+-free KR solution to remove non-hydrolyzed indicator. Imaging was acquired with a digital cooled CCD camera  on an inverted stage microscope (Nikon Diaphot) using a 40x oil-immersion objective. Fura-2 was excited alternatively at 340 and 380 nm using a dual monochromator , emission fluorescence was selected through a dichroic mirror (DM400) and a wide-band filter (520–560 nm). Ca2+ signals were analyzed using the Metafluor software . Regions of interest (ROIs) were defined within individual cells. After background subtraction, the ratio F340nm/F380nm was calculated to estimate intracellular [Ca2+].C2C12 cells were grown and differentiated on glass coverslips. For measurements, coverslips were mounted onto a 37°C thermostatted chamber, which was continuously perfused with Krebs Ringer medium; individual cells were stimulated with the aid of an 8-channels computer-controlled multifunction superfusion system . On-line measurements were recorded using a confocal Leica DMI6000 inverted fluorescent microscope equipped with UV and visible laser lines. To investigate changes in mitochondrial membrane potential and in i (see below). Ionophore treatment increased PV protein expression levels in PV-clones in a time-dependent manner; a 1.77-fold±0.17 (p<0.05) increase at 48 h; results are the average from at least 2 control and 2 PV-transfected clones and protein extracts were isolated from at least 2 independent experiments  of PV knockout (PV−/−) mice is increased 2+-binding properties and high expression in rodent fast-twitch muscles, PV participates in shaping intracellular Ca2+ signals by sequentially acting as a Ca2+ sink (buffering phase) and a Ca2+ source (release phase) during each contraction-relaxation cycle. That mitochondria also behave in a similar way in skeletal muscle is a more recent finding 2+-buffering/uptake that only mildly affects the rising phase of [Ca2+]c transients, it was proposed that mitochondria might represent a valuable substitute for PV in fast-twitch muscles of PV−/− mice. Exposure of C2C12 myotubes to increased [Ca2+]c could hypothetically lead to adaptive mechanisms to increase the intracellular Ca2+-buffering capacity; in control cells, by increasing the mitochondrial mass/volume (2+]c.Due to PV’s Cas/volume  & 3 and s/volume . Results2+]c (i.e. by strong depolarization) was investigated. In C2C12 myotubes, changes in the mitochondrial membrane potential (ΔΨm) and in the mitochondrial Ca2+ concentration [Ca2+]m were measured by loading cells with the fluorescent dyes rhodamin 123 and rhod-2, respectively. As a paradigm to transiently elevate [Ca2+]c, cells were depolarized by local delivery of KCl-enriched buffer (concentration: 100 mM for 15 s and 300 mM for 60 s). The KCl-enriched buffers were applied via a thin glass pipette (flow: 0.1 ml/min) placed in the vicinity of the cells that were constantly perfused with KR solution (1.5 ml/min), thus the actual KCl concentration sensed by the cells was lower than the nominal concentration of applied KCl solutions, we estimated a dilution of at least 2-fold. The decrease in ΔΨm is reflected in the increase of the rhodamin 123 fluorescence  and the half-recovery time (τ1/2) were determined  in control myotubes than in those expressing PV  to deplete ER/SR stores and the kinetics and amplitude of cytosolic [Ca2+]c transients were evidenced by Fura-2 loading of cells. The ratio F340nm/F380nm before stimulation was identical in WT and PV-clones  confirming that the presence of a Ca2+ buffer didn’t affect basal [Ca2+]c (2+]c induced by thapsigargin application was not significantly different (0.612±0.052 in WT vs. 0.606±0.037) in PV-clones. Small differences in the shape of [Ca2+]c transients were discernable . In line with this hypothesis, CaN mRNA levels and CaN activity are increased in PV−/− TA 2+-dependent manner via an AMPK-SIRT1-PGC-1α signaling pathway 2+]c  or by subtle Ca2+ signal changes in the TA of PV−/− mice It was hypothesized that the induction of mitochondria biogenesis, i.e. an increase in mitochondrial volume caused by altered Ca2+ signaling toolkit components putatively linked to mitochondria biogenesis including kinases and phosphatases. Besides the previously reported increases in mRNA levels of CaN and CaMKII in PV-/- TA  and PV-null mutant (PV−/−) mice. Results obtained in the two models show that PV expression levels and mitochondria volume in skeletal muscle is inversely regulated likely in a bidirectional manner involving the PGC-1α/SIRT1 signaling axis.Muscle plasticity is characterized by adaptive changes including changes in muscle fiber type in response to various external stimuli such as physical exercise. Despite uncertainties about the precise signaling mechanisms involved, excitation-transcription (E–T) coupling translates the nerve-evoked electrical activity to a switch in fiber type-specific gene expression 2+]c are linked to muscle fiber transition, both from slow-to-fast, and fast-to-slow 2+ signaling that occurs in proliferating cells and/or cells subjected to differentiation from myoblasts to myotubes is sufficient to cause the down-regulation of mitochondria. Treatment with ionophore for 48 h had no additional effect on mitochondrial volume, while in PV-devoid (WT) myotubes subjected to the same treatment, mitochondrial volume was significantly upregulated. Mitochondria show a large plasticity with respect to protein composition, morphology and size and are tailored to the metabolic and signaling needs of each cell type and tissue 2+ uptake and resistance to K+-induced decrease of ψm between the PV-expressing and PV-devoid C2C12 myotubes is solely the result of increased mitochondrial volume, or is also caused by a selective (up)-regulation of one of the >1000 mitochondrial proteins described in MitoCarta 2+ handling remains to be investigated. Thus, C2C12 myotubes with or without PV expression may be considered as a valuable model system  what causes the changes in the Ca2+ signaling toolkit to adapt to changing demands; (ii) does PV itself possibly act as a local Ca2+ sensor? We hypothesize that upon increased muscle activity and associated increased Ca2+ signaling activity in PV-expressing muscle fibers an increase in PV expression in order to increase the slow-buffering capacity might be the least demanding choice from an energetic point of view. In fibers not expressing PV (e.g. slow-twitch) an increase in slow-buffering capacity could be mediated by increasing the mitochondrial volume and its associated Ca2+ handling capacity. Interestingly, there seems to exist a subtle balance, i.e. an inverse regulation of the two (PV and mitochondria) in all systems analyzed so far.Transient changes in [Cain vivo and in vitro situation with respect to CaN levels may be manifold and include I) chronically higher Ca2+-signaling activity in TA vs. acute elevation of [Ca2+]c in C2C12 myotubes and II) C2C12 myotube cultures representing only a partial (incomplete) model for an intact muscle fiber. However, the incongruent results from the two models hint that the post-transcriptional regulation of PV expression, at least in C2C12 myotubes, does not likely involve a CaN-dependent pathway. On the other hand, the highly similar pattern of mitochondrial volume and PGC-1α expression levels in both, control and PV-expressing myotubes , CaN levels were decreased, while in the PV-expressing myotubes CaN levels were not affected by either ionophore treatment. The cause for the apparent discrepancy between the  compare , as well compare , hinted in vivo and in vitro, we found SIRT1 expression to be elevated in association with PV-deficiency in TA muscles from PV−/−mice (Sirtuin 1 (SIRT1) is the main deacetylase of PGC-1α that positively regulates genes involved in fatty acid utilization and mitochondrial respiration V−/−mice  as well V−/−mice . UnexpecV−/−mice . HoweverPvalb) transfer leads to an improvement of diastolic function both in vitro and in vivo models, in normal and diseased conditions 2+ content hints towards adaptation/compensation mechanisms induced by ectopic PV expression also in the heart. Most importantly, based on this study, the long-term effects of PV expression on heart mitochondria biogenesis/function should be investigated; specifically if the therapeutic effects of PV expression will be aimed towards human applications. Focusing on the energy demand of cardiac function, PGC-1α has recently been suggested as a novel therapeutic candidate for metabolic modulation in cardiovascular diseases 2+ signaling might be modified, i.e. PV overexpression might lead to a decrease in heart mitochondrial content or possibly to other alterations of Ca2+ signaling components, e.g. linked to the decrease in the SR load The SIRT1/PGC-1α-mediated inverse regulation of PV and mitochondria has to be kept in mind when reflecting on the studies of ectopic PV expression in the heart; PV gene (


The GCAP1−/− retinas expressed normal levels of RetGC isozymes and other phototransduction proteins, with the exception of GCAP2, whose expression was elevated in a compensatory fashion. RetGC activity in GCAP1−/− retinas became more sensitive to Ca2+ and slightly increased. The bright flash response in electroretinogram (ERG) recordings recovered quickly in GCAP1−/−, as well as in RetGC1−/−GCAP1−/−, and RetGC2−/−GCAP1−/− hybrid rods, indicating that GCAP2 activates both RetGC isozymes in vivo. Individual GCAP1−/− rod responses varied in size and shape, likely reflecting variable endogenous GCAP2 levels between different cells, but single-photon response (SPR) amplitude and time-to-peak were typically increased, while recovery kinetics remained faster than in wild type. Recovery from bright flashes in GCAP1−/− was prominently biphasic, because rare, aberrant SPRs producing the slower tail component were magnified. These data provide strong physiological evidence that rod photoresponse recovery is shaped by the sequential recruitment of RetGC isozyme activation by GCAPs according to the different GCAP sensitivities for Ca2+ and specificities toward RetGC isozymes. GCAP1 is the ‘first-response’ sensor protein that stimulates RetGC1 early in the response and thus limits the SPR amplitude, followed by activation of GCAP2 that adds stimulation of both RetGC1 and RetGC2 to speed-up photoreceptor recovery.Regulation of cGMP synthesis by retinal membrane guanylyl cyclase isozymes (RetGC1 and RetGC2) in rod and cone photoreceptors by calcium-sensitive guanylyl cyclase activating proteins (GCAP1 and GCAP2) is one of the key molecular mechanisms affecting the response to light and is involved in congenital retinal diseases. The objective of this study was to identify the physiological sequence of events underlying RetGC activation  RetGC), under the control of Ca2+ sensing, guanylyl cyclase activating proteins (GCAPs) 2+ levels promote the binding of Ca2+ to GCAPs, which then inhibit cGMP production, but when intracellular free Ca2+ is lowered by illumination, Mg2+ replaces the Ca2+ bound to GCAPs 2+ bound, GCAPs stimulate RetGC to synthesize cGMP at a faster rate. Rods of all vertebrate species express two guanylyl cyclases, RetGC1 and RetGC2 2+ – lower in GCAP1 and higher in GCAP2 2+ sensitivity of RetGC regulation in the retina. The SPR amplitude does not change but the kinetics of the recovery slow in GCAP2−/− rods 2+ feedback to RetGC in rod physiology could not be decisively validated without knowing how a selective disruption of GUCA1A gene coding for GCAP1 affects RetGC regulation and rod photoresponses. The presence of two different isozymes of RetGC – RetGC1 and RetGC2– further complicates understanding of the cGMP synthesis regulation in living photoreceptors. Although our recent study in vivo preferentially targets the RetGC1 isozyme, the in vivo specificity of GCAP2 toward a particular RetGC isozyme remains unclear.Guanylyl cyclase activating proteins (GCAP) play an essential physiological role in photoreceptors by accelerating the recovery of rods and cones from excitation by light. Photon absorption by rhodopsin triggers hydrolysis of cGMP and closes cGMP-gated cation channels in the rod plasma membrane, resulting in membrane hyperpolarization . The shape of their photon response shows that GCAP1 is essential for activation of the cyclase early in the course of the response and that restraint of the response amplitude and acceleration of the recovery kinetics in the rods is indeed achieved through the sequential regulation of RetGC activity by the two GCAPs. Moreover, the recovery from a bright flash of hybrid GCAP1−/− rods lacking one of the RetGC isozyme reveals that GCAP2 can effectively provide feedback to either RetGC isozyme in vivo. These findings allow us, for the first time, to reconstruct the sequence of the events underlying activation of cGMP synthesis with regard to the role of each GCAP isoforms and each RetGC isozyme in shaping the phases of photoresponse recovery. This study explains how complex relationships between sensor proteins and their target enzymes in a multi-component biochemical pathway tune the physiological function of the rod photoreceptor cell.By studying biochemical and physiological changes caused by elimination of GCAP1, we here demonstrate that even in the absence of the less CaAll animal procedures were approved by IACUC protocols AAMD0204 from Salus University or 95-06-006 from the Massachusetts Eye and Ear Infirmary, in compliance with NIH guidelines. In the experiments described below, mice of both sexes were used indiscriminately.PGK:Neo:tts cassette originating from Mulligan’s laboratory 5′- AGGAAGGTACCGTCTGCAGT TACTTCTGGTTCCCATTGT-3′ and 5′- ACCGAACGCGTATTGTCTCAAACTCGA GGTCAGTAGTCA-3′ primers and ligated into the KpnI/MluI sites of the pPNT plasmid. The 1.2-kb short arm was amplified using 5′-AAAACGCGGCCGCATATAAGGACATTGG AAGAAGGGAGTGT-3′ and 5′-AAAAACCTGCAGGGAAAGAAAGCAAGAGGATC ATGAAATG-3′ primers and inserted into the SbfI/NotI sites of the plasmid harboring the long arm. The resultant construct was verified by restriction nuclease digestion and DNA sequencing was linearized by NdeI digestion, purified by Whatman Elutip minicolumn chromatography and concentrated by ethanol precipitation. The purified linearized construct was electroporated into B6/129SVE mouse hybrid embryonic stem cells (Ingenious Targeting Laboratory) and 288 clones were screened by PCR for homologous recombination of the short arm using 5′-TGGCTATGGATTCCAGAAGATTAAAACAGG-3′ (“f1” in 5′-AGTGAGACGTGCTACTTCCATTTGTCA-3′ (“r1” in PGK:Neo:tts cassette) primers. Seven positive clones identified in the primary screen were also verified for homologous recombination of the long arm by PCR using 5′-TGATATTGCTGAAGAGCTTGGCGGCGAAT-3′ primer  and 5′-AAAAACGCGTGAACACAAACAGGCAGAAGTGAGGAGA-3′ . Five knockout-positive clones were expanded and two of them were injected into mouse blastocysts (service was provided by Ingenious Targeting Laboratory). Clone 13G3 effectively passed the KO allele to the progeny and was used to establish a hemizygous, GCAP1+/− line. After repetitive breeding to C57B6 WT mice (Taconic), GCAP1+/− mice were crossed to produce the GCAP1−/− genotype and the progeny were screened by PCR amplification from genomic tail DNA for the presence of the KO allele versus WT exon I using 5′-TCGGGAATCTTGCTTCATGGACATT-3′ and 5′- AGTGAGACGTGCTACTTCCATT TGTCA-3′ or 5′-CCTTGTGCAGGGGACATTA GAAAATAAG and 5′-CATCTGTTCCACATACTGGCTGGCT-3′ primers, respectively.The targeting construct for GUCA1A gene knockout (KO) was assembled in a pPNT vector harboring a −/− KO line, produced by simultaneous disruption of the neighboring GUCA1A and GUCA1B pair of genes (−/− (GC-E null) mice produced by the disruption of a mouse GUCY2E gene −/− mice, produced by disruption of GUCY2F gene −/− and RetGC2−/−lines were crossed with GCAP1−/− mice to produce RetGC1−/− GCAP1−/− and RetGC2−/−GCAP1−/− genotypes, respectively.WT C57BL6 mice originated from Taconic. The double GCAPs1,2of genes , was a gAntibodies against full-size recombinant mouse GCAP1 and GCAP2 were raised in rabbits and purified on the corresponding immobilized GCAP affinity matrix 1 and anti-PDE6α antibodies were from AbCam, anti-β-actin – from GeneTex, and anti-rhodopsin – was from Chemicon/Millipore. Secondary goat anti-rabbit antibodies were conjugated with either horseradish peroxidase for immunoblotting (Pierce) or FITC .Protein expression in the retina was compared by SDS-gel electrophoresis and immunoblotting of retinal samples as described in GC activity was assayed using [α-32P]GTP as a substrate 32P]cGMP was analyzed using polyethyleneimine cellulose TLC, as described previously Ca2+/EGTA buffers containing calibrated concentrations of free Ca2+ and Mg2+ were prepared using the method of Tsien and Pozzan 2+ indicator dyes as previously described 3 photons µm−2 at the cornea as a Ganzfeld, followed by an unfiltered white saturating “probe” flash delivering 5×105 photons µm−2. The amplitude of the a-wave evoked by the probe flash was normalized for each inter-stimulus time interval by dividing by the amplitude of the response to the probe flash given prior to the conditioning test flash.Scotopic ERG a-wave recovery was compared in different genotypes using the paired-flash approach 2 in 0.1 M cacodylate buffer for ∼24 hr at 4°C, washed with buffer and stored at 4°C. Eyes were post-fixed with 2% OsO4 for 90 min, dehydrated with ethanol, transitioned to propylene oxide and embedded in Epon resin. Sections from central retina were imaged on a Philips CM-10 transmission electron microscope and analyzed using ImageJ 1.42q (NIH) and PixelStick 1.1 . Measurements of the repeat distance for disks were made from arrays of 29 to 72 consecutive disks in rods whose disks were well organized. Rod diameter was determined from cross sections of rods with disks bearing an incisure.Following lethal injection of Ketamine/Xylazine mice were perfused through the heart, first with phosphate-buffered saline and then with freshly prepared 2.5% paraformaldehyde/2.5% glutaraldehyde mixture in phosphate-buffered saline. Enucleated eyes were then post-fixed in 2.5% glutaraldehyde/2.5% paraformaldehyde in sodium cacodylate buffer, pH 7.4 (Electron Microscopy Sciences), on ice for 4 hours, washed with 10 mM Na-phosphate/130 mM NaCl, pH 7.4, overnight, and processed for paraffin embedding (AML Laboratories). Sections, ∼3 µm in thickness, were stained with hematoxylin and eosine (AML Laboratories) and imaged using an Olympus BX51/Magnafire system. For electron microscopy, enucleated eyes were immersed in 2.5% glutaraldehyde/2% formaldehyde fixative and 0.08 M CaClEyes from 4% paraformaldehyde-perfused animals were fixed on ice for 6 hours with 4% paraformaldehyde in phosphate-buffered saline, washed with 10 mM Na-phosphate/130 mM NaCl, pH 7.4, overnight, impregnated with 30% sucrose solution for 48 hours at 4°C, and then frozen in OCT media (Electron Microscopy Sciences). Cryosections were taken using a Hacker-Bright OTF600 cryomicrotome, probed with antibodies as described in 2/5% CO2 at 37°C in an experimental chamber. The perfusion solution containing (mM): 139 Na+, 3.6 K+, 2.4 Mg2+, 1.2 Ca2+, 123.3 Cl−, 20 HCO3−, 10 HEPES, 3 succinate, 0.5 L-glutamate, 0.02 EDTA and 10 glucose, was supplemented with 0.1 mg/ml BSA , 1% (v/v) MEM amino acids (Invitrogen), and 1% (v/v) BMEM vitamins (Sigma). A rod outer segment was pulled into a glass pipette and responses to flashes  were recorded with a patch clamp amplifier . Light from a shuttered, xenon source passed through a 500 nm interference filter and neutral density filters. The pipette was filled with HEPES buffered Locke’s solution lacking amino acids and vitamins, in which bicarbonate was replaced with Cl-. Photoresponses were low pass filtered at 30 Hz  and digitized at 400 Hz on a MacIntosh computer . Responses were also recorded with a SCSI based data acquisition system  for later re-digitization. No corrections were made for the delay of ca. 17 ms introduced by low pass filtering. Recorded data were digitally filtered by convolution with a Gaussian , which smoothed the waveform without introducing any delay.Retinas from mice that were dark-adapted overnight were stored in chilled, oxygenated Leibovitz’s L-15 medium. Finely chopped pieces of retina were perfused with an enriched Locke’s solution equilibrated with 95% OGUCA1A gene together with the 5′-upstream fragment of the putative promoter region with the PGK:Neo:tts cassette tts) completely eliminated GCAP1 expression in mouse retinas  nmol cGMP min−1 retina−1 in WT (n = 5) to 0.8±0.09 nmol cGMP min−1 retina−1 in the KO (n = 4) . The [Ca]1/2 became significantly reduced , from 81±2  nM to 46±2 nM (n = 4), respectively  Fig. 3A,−/− retinas measured in vitro . Hence, the remaining GCAP2 efficiently activates both cyclase isozymes via negative Ca2+ feedback in living GCAP1−/− rods.The high cyclase activity in GCAP1in vitro Fig. 3 s−/− . The overshoot of the baseline was missing in these slow cells. Unlike WT rods, the amplitude of the GCAP1−/− response rose in proportion to the integration time (−/− rods showed higher amplitudes and faster recovery kinetics (−/− double KO rods recovered quite slowly and clustered around long integration times (−/− mice studied, for which three to nine rods were recorded per retina. In the remaining mouse, one fast rod and four medium rods were recorded. These changes were generally consistent with the altered Ca2+ sensitivity of the cyclase regulation, but the variability suggested that GCAP2 content varied considerably between GCAP1−/− outer segments. More subtle differences in GCAP2 content in WT rods may have contributed to the variability in their integration times (In addition, there were profound changes in the shape of the GCAP1−/− double KO mice, in which a portion of the chromosome coding for both GCAP isoforms was deleted, established that Ca2+ feedback to the cyclase is essential for the normal shape of the rod photoresponse −/− rods −/−. However, the two GCAP isoforms are not merely redundant Ca2+ sensors for RetGC regulation. GCAPs, due to their different Ca2+ sensitivities 2+ to suppress its ability to activate the cyclase, starts RetGC activation as Ca2+ concentrations begin to decline soon after photoexcitation, and therefore limits the amplitude of a single-photon response, while GCAP2 does not contribute to RetGC acceleration until Ca2+ concentrations fall to their minimal levels after the response peak so it serves to quicken the SPR recovery. Even though the two GCAPs have different specific effects on the SPR, both result in a shift of the rod’s operating range to higher intensities.Previous studies using GCAPs1,2in vitro studies of Ca2+ sensitivity of GCAP1 and GCAP2 −/− rods 1/2 of RetGC inhibition rose two-fold as a result of elimination of GCAP2, and in GCAP1−/− rods in our present study, where Ca2+ sensitivity shifted two-fold in the opposite direction, making RetGC more sensitive to inhibition by Ca2+ than normal (2+ sensitivity of the retinal cGMP synthesis in both KO models is consistent with the difference in Ca2+ sensitivity of mouse GCAP1 and GCAP2 observed in vitro−/− in a manner generally consistent with loss of the ‘first-response’ Ca2+ sensor (,,2+ concentration that occurred soon after CNG channel closure. While these findings, together with the previous observations in vivo (The relay model of sequential acceleration of RetGC activity accounts for the results of n normal Fig. 3. −/− rods remained fast, typically faster than in WT rods (, , 2+-sensitive GCAP2 isoform took over the entire regulation of RetGC in photoreceptors, because GCAP1−/− retinas expressed greater GCAP2 protein levels (−/− rod outer segments (−/− retinas increased (−/− retinas suggests that either transcriptional or translational regulation elevated GCAP2 synthesis in the absence of GCAP1, a phenomenon that deserves special study. Interestingly, there was no significant up-regulation of GCAP1 observed in GCAP2−/− retinas and flash recovery did slow down Deletion of GCAP1 might have been expected to slow flash response recovery, yet recovery kinetics in GCAP1T rods , , 6. Ain vitroin vivo preferentially activates RetGC1 −/− retinal biochemistry (in vitro but also maintains complexes with both of them in living photoreceptors. GCAP1, on the other hand, accelerates RetGC2 in vitro but fails to do so in vivoin vivoin vivo will be required to establish their role in shaping the photoresponse.For maximal activity to increase, GCAP2 must have taken over the regulation of both RetGC1 and RetGC2 isozymes. Both GCAP1 and GCAP2 are capable of activating mouse RetGC1 and RetGC2 isozymes in native mouse ROS membranes , −/− retinas (in vitroin vitro−/− retinas (in vivo.Even with the compensation by the increased levels of GCAP2 in rods , 2, the −/− rods within the same retina (, −/− mice. Interestingly, transgenic overexpression of GCAP2 in GCAPs1,2−/− rods driven by a rhodopsin promoter accelerates recovery after a strong flash and produces similar rod to rod variability in the dim flash response −/− retinas, either by a specific mechanism regulating the level of GCAPs expression or by our having inserted a construct containing PGK promoter in the vicinity of the chromosome region coding for GCAP2.Photon responses were highly variable in amplitude and duration between individual GCAP1retina , 6. This−/− rods (2+ than GCAP1 2+ are believed to equilibrate rapidly within mammalian outer segments in the transverse direction (cf. 2+ affects cGMP synthesis in homogenized ROS without a biochemically detectable lag phase 2+ for Ca2+ in EF-hands of GCAP2 bound to the cyclase in vivo occurs after a short delay. If GCAP2 levels approach 10 µM in the GCAP1−/− ROS as it was estimated for wild type 2+2+ and cessation of RetGC stimulation by GCAP2 in intact rods. According to the model in 2+ effect on GCAP2 at the end of the recovery makes both GCAPs become turned off nearly at the same time.The overshoot in response recovery, frequent in WT rods containing BAPTA, e.g. −/− rods Fig. 5. tion cf.  and that−/− rods, the aberrant responses appeared to be enlarged, underscoring the importance of GCAP1 in suppressing their size. It is not yet clear whether they also had a longer duration or occurred more frequently. The consequence of knocking out the “first response” Ca2+ sensor, GCAP1, even in the face of overexpression of GCAP2, was that the recovery after exposure to bright light was inordinately long.In mammalian rods, approximately one photoisomerization of rhodopsin out of several hundred gives rise to an aberrant response that rises to an amplitude that is 1.5−2× larger than normal and persists for an unpredictable period of time, lasting on average for about 3–4 seconds


P > 0.05). At 13 weeks old, the average live weights of rabbits were 1788.5 g, 1805.0 g, 1718.5 g, and 1801.3 g in respectively, F0, F5, F10 and F15 groups. No mortality of rabbits was recorded. Compared to F0, the feed conversion ratio and feeding cost decreased in the group of rabbits fed F15 diet. The carcass yield was similar between diets. An experiment was carried out to define the optimal rate of palm-press fibres in growing rabbits' diet. In total, 64 weaned rabbits (35 days old) of Beninese breed were divided in 16 groups of 4 rabbits  each. During six weeks, rabbits were fed with 4 complete diets containing 0% , 5% (F5), 10% (F10), and 15% (F15) of fibres from a palm oil industry. Results demonstrated that up to 15 of palm-press fibres can be included efficiently in growing rabbits' diet. The daily feed intake was not significantly affected by the diet ( In Benin, 64% of the population has consumed once rabbit meat at least, and 95% of them appreciated it positively . Thus, tUnfortunately, the availability of forages, main sources of fibre is low in peri-urban areas, and farmers have difficulties to provide rabbits with grass. The processing of complete diets with optimal level of fibres is therefore suitable to efficiently feed rabbits. Such feeding approach can be done by using different low cost and available feedstuffs. Palm-press fibres are available in many palm oil industries in Benin. They are extracted from the oil palm empty fruit bunches around the nut. Palm-press fibres contain about 86.2% of dry mater, 4% of crude proteins, 21% of fat, 0.31% of Calcium, 0.13% of Phosphate and 36.4% of crude fibre . Palm-prThe objective of this study was to contribute to the valorization of palm-press fibres in growing rabbits feeding by evaluating its optimal level in diets.A total of 64 rabbits weaned at 35 days old were divided in 16 groups of 4 rabbits each. At the beginning of the experiment, the average live body weight of these rabbits of local breed reared in Benin was 857.2 g. 3). Cages were installed in two rows of 8 cages each. They were at 70 cm from the ground and 180 cm from the roof. Each cage had a drinker and two feeders. Each group of 4 rabbits  was housed in a cage , 5% (F5), 10% (F10), and 15% (F15) of palm press fibres . Palm prP > 0.05) average live body weights of rabbits were recorded in F0 (858.1 g), F5 (836.3 g), F10 (880.0 g), and F15 (854.4 g) dietary treatments. The experiment took six weeks and finished when rabbits were thirteen weeks old.Each feed was delivered to four cages of four rabbits each. Per feed, two cages of rabbits were randomly selected in each row. At the beginning of the experiment, similar  were slaughtered per diet for carcass study. The full digestive tract, carcass, liver, and abdominal fat were weighted. Relative weights of liver and full digestive tract were calculated on carcass weight basis.P > 0.05). Thus, analyses were performed according to the model as follows:Yi is the observation for dependent variables; μ is the general mean; Fi is the fixed effect of the feed; εi is the residual error. Data were analyzed using general linear model (GLM) in SAS version 9.1.2 . The perP > 0.05). On average, daily feed intakes were, respectively, 78.4 ± 10.7 g (F0), 79.5 ± 10.2 g (F5), 79.7 ± 9.7 g (F10) and 79.94 ± 11.0 g (F15). In the sixth week of experiment (13 week old) the feed intake decreased in all dietary treatments. The decrease of feed intake was more important when the level of crude fibre in diet is low as it was the case in F0 and F5 diets (The daily feed intakes of rabbits fed with the four diets  were simF5 diets . P > 0.05). Thus, at the end of the experiment (77 days-old), the live body weights were not significantly different (P > 0.05) between rabbits fed F0 (1788.5 g), F5 (1805.0 g), F10 (1718.5 g) and F15 (1801.3 g).The growth of rabbits was regular . The daiP > 0.05). No mortality of rabbits was recorded during the study. Thus, included, up to 15% in balanced diet, palm-press fibres had no significant effect on the survivability of growing rabbits. Palm-press fibres-based diets were therefore efficient in growing rabbits feeding.The lowest feed conversion ratio and feeding cost were recorded in rabbits fed F15 diet . The ecoP > 0.05) between dietary treatments (P > 0.05). However, compared to the control diet (F0), the proportion of liver was significantly lower in rabbit fed F5 and F15 dietary treatments (P < 0.05). The carcass yields of rabbits were similar  were higher than the 7.48 to 10.91 g reported in Nigeria . CompareThe decrease of feed conversion ratio and feeding cost in F5 and F15 diets confirms the efficiency of these diets and the profitability of palm press fibres inclusion in growing rabbits' diets. Feed conversion ratios (3.78 to 4.64 g feed/g body weight gain) were lower than 5.81 to 8.11 feed/g weight gain, found in Nigeria .However, feeding costs (760 to 816 FCFA feed/kg body weight gain) were higher than about 596.4 to 685.1 FCFA feed/kg body weight gain . This ecThe carcass yields (60.8 to 62.9%) are higher than 57.1% ; but lowThis experiment suggests that growing rabbits can be fed exclusively with balanced palm press fibres-based diets. In such feeding strategy, up to 15% of palm press fibres can be included efficiently in the diets without any significant adverse effect on rabbits' survivability and their abdominal carcass fat. To reduce grass and forage need in peri-urban rabbitry, further investigations can be carried out on reproduction performance of rabbits.