The most important function of collagen and elastin is to induce several mechanical parameters which are known to play a dominant role in governing mechanical properties of the blood vessels. The aortic tissue of rabbit is one of the important sources of collagen and elastin. The effects of high fat diet (HFD) on the hydroxyproline (Hyp) fractions in serum and aortic tissues of rabbits and collagen content in the aortic tissues of rabbits have not been documented before. The present study was undertaken to investigate the changes in Hyp fractions in serum and aortic tissues of rabbits and collagen content in the aortic tissues of rabbits during the progression of atherosclerosis. The atherosclerotic model used in this study was the New Zealand white rabbit . Twenty five rabbits were individually caged, and divided into control group  and HFD group . The control group was fed (100 g/day) of normal (NOR) diet for a period of 15 weeks. The HFD group was fed normal diet supplemented with 1.0% cholesterol plus 1.0% olive oil (100 g/day) for the same period of time.We found that the TC, LDLC, and TG (mg/dl) were significantly (p < 0.001) increased in HFD rabbits compared with control rabbits with percentage normalized changes of 1198%, 1591%, and 710%, respectively. The peptide-bound Hyp in the serum was significantly (P < 0.05) increased in HFD rabbits compared with control rabbits with percentage normalized change of 517% while it significantly (P < 0.01) decreased in aortic tissues of HFD rabbits compared with control rabbits with percentage normalized change of 65%. The protein-bound Hyp in the serum was significantly (P < 0.01) increased in HFD rabbits compared with control rabbits with percentage normalized change of 100%; the protein-bound Hyp in the aortic tissues of control rabbits was 235.30 ± 55.14 (Mean ± SD) while it was not detectable (ND) in HFD rabbits. Total serum Hyp showed no significant (P < 0.05) change in HFD rabbits compared with control rabbits while it was significantly (P < 0.05) decreased in aortic tissues of HFD rabbits compared with control rabbits with percentage normalized change of 73%. The total collagen was significantly (p < 0.01) decreased in aortic tissues of HFD rabbits compared with control rabbits with percentage normalized change of 73% which was supported by histological study.These results suggest that percentage decrease in various Hyp fractions in aortic tissue of HFD rabbits are closely related to percentage decrease of collagen content in aortic tissues of HFD rabbits. These results also suggest that it may be possible to use the changes in various Hyp fractions in aortic tissues of rabbits as an important risk factor during the progression of atherosclerosis. Gregory, 1999 . High TCThe atherosclerotic model used in this study was the New Zealand white rabbit , obtained from the Laboratory Animal Center . Twenty five rabbits were individually caged, and were divided into control group and HFD group. The control group  was fed on 100 g/day of NOR diet  for a period of 15 weeks. Chemical composition of the laboratory NOR rabbit diet (Purina Certified Rabbit Chow # 5321). The HFD group (n = 15) was fed on NOR Purina Certified Rabbit Chow # 5321 supplemented with 1.0% cholesterol plus 1.0% olive oil (100 g/day) for the same period of time. The animals were sacrificed by intravenous injection of Hypnorm (0.3 ml/kg) into the auricular vein in accordance with the guidelines approved by King Saud University Local Animal Care and Use Committee. The thoracic aorta tissues of control and HFD rabbits were removed with great care so as to avoid any damage for aortic tissues, and were placed in 10% buffered neutral formalin. The thoracic aortic tissues were stored in a refrigerator at a temperature of 4°C for a period less than 48 hrs until the staining was performed. Other specimens of thoracic aortic tissues were preserved in liquid nitrogen to determine Hyp concentration.Blood samples of 2 ml were obtained from the rabbits via venepuncture of an antecubital vein. Blood was collected into two polypropylene tubes viz., one for serum and the other for plasma. The blood for plasma was collected in heparin. Serum was prepared by allowing the blood to clot at 37°C and centrifuge at 3000 rpm for ten minutes.According to the routine procedures, thoracic aortic tissue specimens were stained by Victoria blue staining to examine fatty streaks, fibrous plaques, and any degenerative changes in collagen and elastin of the aortic tissues.Serum TC, LDLC, TG concentrations (mg/dl) were analyzed and determined by a clinical laboratory centre according to the previously reported method ,12.-1) using a stainless steel Omni-Mixer homogenizer . The homogenate was used for determination of Hyp concentrations. Further details about sample collection have been previously reported by [Dissected aortic tissue specimens were homogenized in normal saline . Values were considered significant if P < 0.05. Each sample was run in duplicate. The Hyp concentration and collagen content were expressed as mean ± SD . The Hyp concentration and collagen content in various aortic tissues of control and HFD rabbits were compared using one-way ANOVA analysis followed by Turkey's test for multiple comparisons. Bartlett's test was used for homogeneity of variances. Spearman correlation analysis was used to examine the association between variables.Statistical analysis was performed by means of InStatFigure Table Table Figure Figure In the present study, the rabbits were fed a HFD diet containing 1% cholesterol plus 1% olive oil for a feeding period of 15 weeks. The accompanying changes in TC, LDLC, and TG concentration in serum of control and HFD rabbits were estimated. The various Hyp fractions in serum and aortic tissues of control and HFD rabbits and collagen content in aortic tissues of control and HFD rabbits were studied. Thoracic aortic tissue specimens were stained by Victoria blue to examine fibrous plaques and any degenerative changes in collagen and elastin of the aortic tissues of HFD rabbits. This study suggests that HFD has a general tendency to induce increase in TC, LDLC, and TG concentration which may be deposited in the atheromatous lesions, and may be incorporated into atherosclerotic plaques. Moreover, as another possible cause, it is suggested that HFD diet may accelerate atherogenesis through disturbing the mechanical fragility of atherosclerotic plaques making them vulnerable to rupture and thrombosis. It has been reported that when LDLC is oxidized by macrophages in lesions, it becomes toxic to the endothelium, and thereby could injure endothelial cells. Thus, the effects of HFD diet are not only confined to deposition of lipids in atheromatous lesions, but it may also produce primary endothelial cell injury . In the These results were further confirmed by biochemical analysis which showed that feeding rabbits with a HFD caused a significant decrease of Hyp fractions and collagen content in aortic tissues of HFD rabbits compared with control rabbits. In the present study there was no significant change in free Hyp in serum and aortic tissues of HFD rabbits compared with control rabbits. This shows that the processes which contribute to free Hyp pool viz., mature collagen, newly synthesized collagen dietary collagen etc. were not affected by HFD diet. There was a significant decrease in peptide-bound Hyp and protein-bound Hyp was not detected in aortic tissues of HFD rabbits compared with control rabbits.Type I and III collagen plays an important role in arterial physiology by preventing arterial expansion beyond physiologic limits. Smooth muscle cells in the atherosclerotic arteries synthesize new extracellular matrix components, including collagen ,18. In aThe aortic tissue is among the most abundant tissue sources of collagen XVIII  and alsoTwenty five rabbits were individually caged, and divided into control group and HFD group. The control group was fed of normal (NOR) diet for a period of 15 weeks. The HFD group was fed normal diet supplemented with 1.0% cholesterol plus 1.0% olive oil for the same period of time. The TC, LDLC, and TG were determined in the serum of control and HFD rabbits. The various Hyp fractions were determined in the serum of control and HFD rabbits, and the various Hyp fractions and collagen content were determined in aortic tissues of control and HFD rabbits.We found that the TC, LDLC, and TG (mg/dl) were significantly (p < 0.001) increased in HFD rabbits compared with control rabbits with percentage normalized changes of 1198%, 1591%, and 710%, respectively. The peptide-bound Hyp in the serum was significantly (P < 0.05) increased in HFD rabbits compared with control rabbits with percentage normalized change of 517% while it significantly (P < 0.01) decreased in aortic tissues of HFD rabbits compared with control rabbits with percentage normalized change of 65%. The protein-bound Hyp in the serum was significantly (P < 0.01) increased in HFD rabbits compared with control rabbits with percentage normalized change of 100%; the protein-bound Hyp in the aortic tissues of control rabbits was 235.30 ± 55.14 (Mean ± SD) while it was not detectable (ND) in HFD rabbits. Total serum Hyp showed no significant (P < 0.05) change in HFD rabbits compared with control rabbits while it was significantly (P < 0.05) decreased in aortic tissues of HFD rabbits compared with control rabbits with percentage normalized change of 73%. The total collagen was significantly (p < 0.01) decreased in aortic tissues of HFD rabbits compared with control rabbits with percentage normalized change of 73% which was supported by histological study. These results suggest that percentage decrease in various Hyp fractions in aortic tissue of HFD rabbits are closely related to percentage decrease of collagen content in aortic tissues of HFD rabbits. These results also suggest that it may be possible to use the changes in various Hyp fractions in aortic tissues of rabbits as an important risk factor during the progression of atherosclerosis.TC: total cholesterol; LDLC: low-density lipoprotein cholesterol; TG: triglycerides; Hyp: hydroxyproline; NOR: normal; HFD (CHO): high fat diet; ND: not detectible; ns: not significant.The authors declare that they have no competing interests.MAKA, NJS, ASA and MSAA have participated in all experiments, data interpretation and analysis and drafting the manuscript. The atherosclerotic model used in this study was obtained from the Laboratory Animal Center . The control and HFD was prepared by Research Diet Inc., New Jersey; USA. The authors have conceived the study and its design, supervised all technical activities, and written the final version of the manuscript. All authors have read and approved the final manuscript.


Moreover, we show that the heat of interaction between two components is related to the partial enthalpy of interaction at infinite dilution of the titrant component, as well as to its partial volume of interaction at infinite dilution.We have performed a detailed study of the thermodynamics of the titration process in an isothermal titration calorimeter with full cells. We show that the relationship between the enthalpy and the heat measured is better described in terms of the equation Δ H = W In this case, the contributions to the heat measured are the heat of dilution of the component 3 and the interaction with the different parts of the experimental setup. In the third experiment the solution of component 2 in the solvent is titrated with the solvent and the heat of dilution of component 2 is the contribution to the heat measured. In the fourth experiment the solvent is titrated with the solvent. The fourth experiment takes part in the protocol because its contribution appears also in experiments one, second and three. From a practical point of view, the heats of experiments 3 and 4 are negligible since they are usually insignificant . In thisBecause all processes in the above protocol are carried out at constant pressure, the measured heat is usually interpreted in terms of the following Equation :(3)Q = It is interesting to note that the origin of the protocol shown in In this paper, we address the above problem and the physical meaning of the heat obtained from the given protocol based on the typical performance of an isothermal titration with full cells which is described in We observed that the heat measured in an experiment where solvent is titrated with itself has its origin in the work required for to inject the volume of titrant. For this reason it can be named as “heat of injection”. In addition, we see that the heat involved per mol of titrant when the titration is infinitesimally small is related to its partial molar enthalpy of interaction at infinite dilution and its molar partial volume of interaction also at infinite dilution. That is, using the full-cell method, the heat measured by the calorimeter when the above protocol is employed is the partial molar enthalpy of interaction only when the variation in the molar partial volume of interaction can be neglected. This fact is true in binding events where protein unfolding is involved.2.The calorimeter used was an ITC 4200 from CSC equipped to work with nanowatt sensitivity. The volume cell is 1,300 μL. The working temperature was in all cases 30 °C. The water used was bidestilated and the toluene (reagent grade) was obtained from Fermont.3.3.1.From a thermodynamic point of view, the process of titration shown in Thus, for a process in which the pressure and the volume are maintained constant, the variation in the enthalpy is:Substituting inj, will be named “injection work”; and the heat measured by the calorimeter is then:With  process  takes thNote that in 3.2.In this section, we will determinate the concentrations in experiments where a solution of components 2 and 3 in a solvent (component 1) is titrated with a stock solution of 3 in the same solvent. This titration experiment can be described as the combination of two simpler experiments. The first experiment that we will address is one in which a solution of component 3 in a solvent is titrated with a more concentrated stock solution of component 3 in the same solvent. Because the concentration of component 3 will increase in each titration, this type of experiment will be named “concentration experiment”. The other experiment is one in which a solution of component 2 in a solvent is titrated only with the solvent. In this case, the concentration of component 2 will decrease with each titration; for this reason this experiment will be named “dilution experiment”. The more complex experiment, in which a solution of components 2 and 3 in a solvent is titrated with a more concentrated stock solution of 3 in the same solvent, can be considered to be the combination of two simultaneous experiments: a dilution experiment component 2 and a concentration experiment for component 3. This experiment will named the “concentration-dilution experiment”.2 = n2/V and c3 = n3/V, with n2 and n3 being the numbers of moles of components 2 and 3, respectively.The concentrations of 2 and 3 in component 1 are expressed as c3.2.1.(i)3; and in the syringe, is present as a stock solution with a concentration cs3. We will consider the infinitesimal process with respect to the titration volume in which the solution of the vessel with concentration c3 is titrated with a volume dv of stock solution. The different steps of this infinitesimal process are shown in Let us now consider the system in 3, in the volume V is:In the first state, the number of moles of component 3, ns3. The number of moles of component 3 contained in the volume dv is:This solution V being the final number of moles of component 3 in the vessel. Balancing the number of moles for the titration process, we have:3 moles of component 3, dns3 moles were introduced into the vessel and dn3 moles were removed. Substituting the In state 2 see , the int3 = c3(v), with the initial condition:3 = c3(v) can be written as:3.2.2.s3 = 0 because the syringe holds only component 1, we then obtain the equation:In this experiment, we will consider that a solution of component 2 in component 1 is located in the vessel and that this solution is titrated with an amount of component 1. Assuming that there are similar states in this process as those presented in 3.2.3.(i)2 and c(i)3 respectively. This experiment can be considered as the sum of two experiments: the dilution the component 2 and the concentration of component 3. In the first, the concentration of 2 after titration is given by F and tf3 as:We consider the case in which the vessel contains a solution of components 2 and 3 in component 1 which is titrated with a solution of component 3 in component 1. The initial concentrations of 2 and 3 are cF(i) = c2(i) + c3(i).Upon substituting (16) and (17) into 3.3.In this section, we will determinate the heats that are involved in the different titration experiments: the concentration experiment, the dilution experiment and the concentration-dilution experiment. The heat of stirring (homogenization) is the same in all cases . Then it cancels into the thermomechanical balance.3.3.1.3 in the interior of the vessel; before the titration, a volume dv of solution stock with concentration cs3 is present at the end of the syringe needle. The enthalpy of the state 1, H1, is:In State 1 of s3 and a volume V-dv of solution with concentration c3; outside the vessel, in the drainage capillary, we have a volume dv of concentration c3. The enthalpy of state 2, H2, is:In State 2 of 3 + dc3 and the drainage capillary has a volume dv of concentration c3. The enthalpy of the state 3, H3, is:In State 3 of c1–2, for the process 1–2 between states 1 and 2 is defined as:c2–3, for the process 2–3 between states 2 and 3 is:c, for the entire process of titration between states 1 and 3 is:The variation in enthalpy, dHApplying the First Principle of Thermodynamics  in the dc1–2 can be calculated by substituting the values of H1 and H2 dH13,V) = hv(c3)V (Considering that H(c hv(c3)V ” one hasc1–2:By substituting (30) into (29), we obtain the value of dHThe applying in that case the First Principle of Thermodynamics  for the c1–2, comes from the work applied in order to introduce a volume dv of stock solution into the interior of the vessel while an equal volume dv of solution with concentration c3 is pushed out from the vessel.That is, the heat involved in the process 1–2, dQApplying the First Principle of Thermodynamics  to the pIn process 2–3, only a homogenizing process occurs in the vessel; thus, the work of injection is zero and:c2–3 by introducing the values of H2 and H3 dH23,V) = hv(c3)V V ”:(36)H and (36) in (34):The heat involved in the process 2–3, dQc represents the heat measured by the isothermal titration calorimeter in the experiment of concentration. From c1–2 and dHc2–3 calculated with respectively Now, we can apply the First Principle of Thermodynamics  to the cCombining c1–2 is the work of injection in the process of concentration; because dQc1–2 = -dWc1–2  can take the following form:Note that according to (41), dW -dWc1–2 , dQc1–2 c, inserting the value of dQc2–3 =hv(c2)V V ”:(51)dHWith this result, according to the First Principle of Thermodynamics  for the For process 2–3, in which only a homogenizing process occurs, the work is zero and the First Principle of Thermodynamics  for thisd2–3 by substituting the values Hd2 and Hd3 =hv(c2)V:1 and ρ1 are the enthalpy and the density, respectively, of component 1 in the pure state. Now, the First Principle of Thermodynamics  and (58) equal and reorganizing yields:3.3.3.2 = n2/V and c3 = n3/V, respectively. We consider that the volume dv, before it is introduced into the vessel, has a concentration cs3. For convenience, we consider the 3-component system as fractionalized, being composed of component 1 and a fraction F containing components 2 and 3. The composition of the fraction F will be expressed as a function of the variables cF and xf3, as defined by 1 of State 1 is:In this experiment, a solution of component 2 in a solvent (component 1) is titrated with a stock solution of component 3 in the same solvent. For State 1 as in s3 is titrated, an equal volume dv of solution with the composition cF and xf3, is pushed out from the vessel. The enthalpy of this state is:In State 2, while a volume dv of stock solution with a concentration cF + dcF and xf3 + dxf3 and a volume dv in the drainage capillary with the composition cF and xf3. In this way, the enthalpy of State 3 is:After homogenization, we have a volume V with composition cApplying the First Principle of Thermodynamics  to this Considering the processes 1–2 and 2–3 as in the above experiments, we arrive at the following equations:3.4.Next, we will discuss the protocol for measuring the heat of interaction between two components in solution in the high dilution region see “”. We ass2, and the concentration of component 3 in the stock solution is cs3, with dv being the volume of titration. The solvent in the two solutions is the same. The heat measured in this experiment is named dQ(3) where the superindex (3) indicates that a 3-component system is considered. The second experiment is a concentration experiment, in which the solvent is titrated with a volume dv of a stock solution of component 3. As in the first experiment the titrated volume of the stock solution of concentration cs3 is dv. In this case, the heat measured is dQ(2)c where the superindex (2) indicates that a 2-component system is considered. The third experiment is a dilution experiment, in which a solution of component 2 is titrated with the solvent. Initially, the concentration of component 2 in the solvent is c2. The heat measured in this case is dQ(2)d. The fourth experiment is the tritration of the solvent with itself. In this experiment the heat measured is dQinj(1) where the superindex (1) indicates that a 1-component system is considered in this experiment. We will define the following amounts:(3)inj, dQ(2)cinj, dQ(2)dinj are the heats of titration in the three firsts experiments. We suppose that the heats of injection can be estimated by the titration of component 1 with itself (fourth experiment), dQ(1)inj:The first experiment is the titration of a solution of component 2 with a stock solution of component 3. Initially, the concentration of component 2 in the vessel is c3;1,2, measured from the protocol with component 3 as the titrant is defined as:The heat, dq3;1,2” means that a solution of components 1 and 2 is titrated with a stock solution of component 3. By substituting the values of dQ(3) ((2)c ((2)d [The notation “dqof dQ(3) , dQ(2)c 3) ((2)c  and dQ(2)c ((2)d , we arriCombining v as:2 and c3 can be written as functions of cF and xf3 as c2 = (1-xf3) cF and c2 = xf3 cF.For convenience, we define fWe are interested in the following amount:Substituting (72) and (73) into (74) yields:F (amount of fraction F) and xf3 (composition of F). In previous works [F close to zero. The region of concentrations for which this approximation holds is a high dilution region. Function fv in v, hv(c3), hv(c2) and h1ρ1. From v is a “volumetric enthalpy” since hv = H/V, H being the total enthalpy of the system and V the total volume of the system. consequently, hv is expressed in “units of enthalpy per unit of volume”. Furthermore, hv = h/v, where h is the molar enthalpy and v the molar volume, we can thus consider dilute solutions in fv by using the first order Taylor’s expansions of molar volumes and molar enthalpies. The details of our calculations are presented in “v, hv(c2) and hv(c3) for their dilute solutions [Now, we assume that the solutions in the cell are diluted solutions. In general, a molar property depends on xus works –6 we havolutions  in (73),As indicated in “The second is the contribution from the interactions between components of the fraction:Using (77) and (78), v shows the contribution of the interaction enthalpy and the interaction volume of the fraction F. The differential can be expressed as:Therefore, if the solutions are sufficiently diluted, the function fv/dv [v (F = cF(v) and tf3 = tf3(v) given in (20) and (21), we obtain:By combining the equation for dfv/dv , fv  takes the form:s3=cs3 dv, then:Since dnBy combining 4.i of the titrant volume would be less than the final temperature Tf. According to equation:inj is the heat obtained from the injection, m the mass of the titrant volume, cp the specific heat capacity and ΔT = Tf - Ti we would expect a heat positive. The heat shown on inj + Q [inj = -Winj, the heat measured must to be negative. The heat shown in As it has been stated previously, a measured heat is obtained experimentally when a liquid is titrated with itself.  inj + Q  is that inj can be several as for example the friction between liquids (relative viscosities) and the friction between the liquid and the narrow bore tube of the needle. Recently [3 min−1, ρ is the fluid density in g cm3, C is the fluid heat capacity in J g−1 K−1, and d is the tube diameter in cm. For water flowing through a 0.4 mm diameter tube 30 cm long at 1 cm3 min−1, ΔT = 0.002 K. As it is stated by inj. This fact was shown experimentally in the Figure 2.7 of ref. [Contributions to QRecently  the follTherefore we can state that a characteristic of isothermal titration calorimetry is the necessity of very small volume according to two considerations: first, with a large volume, the temperature of the experiment is not kept constant, second, the validity of Δ3;1,2). The second contribution is – ρ1h1ΔvΔ3;1,2. This term represents the enthalpy of a volume of solvent ΔvΔ3;1,2 as a consequence of the protocol employed. In addition to this, it is possible to demonstrate that when the interactions between two components are maximum, the heat dq3;1,2/dns3 obtained is zero. In a previous work [oF;1 as function of a variable of composition is linear for a range of compositions of F, then the interactions between the components of the fraction are maximum in that range. The composition variable employed was the mass fraction of component 3 in the fraction (tf3). f3, the behavior is non-linear. Considering that the value tcf3 in units of molar fractions is xcf3, at this composition the partial property of F takes the value:f2= 1-xf3. Above the value xcf3, joF;1 can be written as:f2= 1-Xf3. When we write jΔ2;1,3 and jΔ3;1,2, we assume [oF;1 = xf2 jΔ2;1,3 + xf3 jΔ3;1,2 indicates that, F is composed of components 2 and 3, which are interacting in a medium (component 1). On the other hand, jo2;1 and jo3;1 indicate that component 2 is alone in component 1 and that component 3 is alone in component 1. Therefore, if we write joF;1 = xf2 jo2;1 + xf3 jo3;1 we assume that fraction F is composed of components 2 and 3, which are not interacting.In ous work , we demoe assume –6 that coF;1(xcf3)) and an amount of component 3  and these components are not interacting. In other words [This is the case for er words ,6, in a er words –6.” ThisoF;1 in the region of saturation , dq3;1,2/dns3 in f3 and multiply by xf2, and we can also derivate dq2;1,3/dns2 with respect to xf3 and multiply by xf3. By adding the results and using Next we can derivate dqThis is an equation of the Gibbs-Duhem type that relates the heats of interaction obtained when components 2 and 3 are the titrant components.inj + Q, which involves a term of work. In addition, we have found that the heat obtained from the usual protocol employed in the determination of the heat of interaction dq3;1,2/dns3 between two components (Δ3;1,2 is not zero. As example of this, ΔF;1 can be neglected in the process of binding deciltrimethylammonium bromide to lysozyme [ΔF;1 ≈ 0 in From equation ΔH = Q it is commonly assumed the heat measured by an ITC can be related to the variation of enthalpy; many papers and books in biochemistry and biophysics have reported results on this link. In this work, we have demonstrated that the equation ΔH = Q does not hold for isothermal titration calorimetry and that the true equation is ΔH = Wmponents  involveslysozyme  (see Fig3;1,2/dns3 (Considering that 1,2/dns3  yields f1h1ΔvΔ3;1,2|≪ |ΔhΔ3;1,2| holds.Another possibility is that for processes of biophysical interest, the approximation |ρ5.inj + Q. The heat of interaction between two components is usually determined from a protocol composed of a number of simple titration experiments. Using the equation ΔH = Winj + Q and the thermodynamic tools developed in our previous works for multicomponent systems at infinite dilution, we show that in an infinitesimal titration, the heat of interaction per mole of titrant component is related to the partial enthalpy of interaction at infinite dilution and to the partial volume of interaction of the titrant component also at infinite dilution. This information can be essential in order to link theoretical models to experimental measurements. Another interesting conclusion is that for this type of calorimeters the variation in enthalpy equals the variation in internal energy.In this work we have studied in detail the thermodynamics of the titration process in isothermal titration calorimeters with full cells. We have shown that the equation ΔH = Q does not hold for this type of calorimeters because it cannot explain the heat obtained when a liquid is titrated with itself. In its place, we propose the equation ΔH = W


However, in contrast to the well-known effects of a high cytoplasmic Ca2+ concentration ([Ca2+]c) in the prefusion phase, the occurrence and significance of Ca2+ signals in the postfusion phase have not been described before.Ca2+- and actin-dependent process. Measurements of fusion pore formation by darkfield scattered light intensity decrease or FM 1-43 fluorescence intensity increase were combined with analysis of [Ca2+]c by ratiometric Fura-2 or Fluo-4 fluorescence measurements. We found that the majority of single lamellar body fusion events were followed by a transient (t1/2 of decay = 3.2 s) rise of localized [Ca2+]c originating at the site of lamellar body fusion. [Ca2+]c increase followed with a delay of ∼0.2–0.5 s (method-dependent) and in the majority of cases this signal propagated throughout the cell (at ∼10 µm/s). Removal of Ca2+ from, or addition of Ni2+ to the extracellular solution, strongly inhibited these [Ca2+]c transients, whereas Ca2+ store depletion with thapsigargin had no effect. Actin-GFP fluorescence around fused LBs increased several seconds after the rise of [Ca2+]c. Both effects were reduced by the non-specific Ca2+ channel blocker SKF96365.We studied isolated rat alveolar type II cells using previously developed imaging techniques. These cells release pulmonary surfactant, a complex of lipids and proteins, from secretory vesicles (lamellar bodies) in an exceptionally slow, CaFusion-activated Ca2+entry (FACE) is a new mechanism that leads to [Ca2+]c transients at the site of vesicle fusion. Substantial evidence from this and previous studies indicates that fusion-activated Ca2+ entry enhances localized surfactant release from type II cells, but it may also play a role for compensatory endocytosis and other cellular functions. This enabled a mapping of individual fusion events and [Ca2+]c on a spatial and temporal basis. Furthermore, we relied on the power of previously developed methods that allow precise and reliable determination of fusion pore formation. One of them, darkfield microscopy of scattered light intensity decrease (SLID), allows to separate the prefusion phase from hemifusion and postfusion, combined with the analysis of [Ca2+]c during all steps 2+]c. The possible modes of activation  and implications of fusion-activated Ca2+ entry (FACE) will be discussed below.To test the hypothesis of “hidden CaWe isolated alveolar type II cells from male Sprague-Dawley rats (150–200 g) as described 2, and 95% humidity and used for experiments 1–2 days after isolation. 4 Fluo-4 experiments on the darkfield microscope were done on cells which were frozen in liquid nitrogen immediately after cell isolation and thawed 2 days before the experiment.Rats were anesthetized (ketamin 10% and xylazil 2%), and injected with heparin (400 IU/kg). Lungs were perfused, resected, washed and incubated twice with elastase and trypsin at 37°C for 15 min. Lung tissue was immersed in DNase containing solution and sliced into ∼1 mm bits. Enzyme reaction was stopped by incubation with FCS . The tissue was filtered 3 times through gauze and nylon meshes  and the final filtrate centrifuged for 8 min at 130×g. After suspending in DMEM medium, the cells were put on IgG coated plastic dishes and incubated at 37°C for 15 min. Non-adherent cells were centrifuged for 8 min at 130×g, suspended in DMEM with 10% FCS and 1% Penicillin/Streptomycin  and seeded on glass cover slips. Cells were cultured at 37°C, 5% CO2, 2 CaCl2, 5 glucose, 10 Hepes; pH 7.4). Calcium free bath solution contained EGTA (2 mM) and no CaCl2. Lamellar body fusion was stimulated with ATP (100 µM) and phorbol 12-myristate 13-acetate  in darkfield/Fluo-4 and with ATP (10 µM) in FM 1-43/Fura-2 experiments, respectively. Secretagogues were added to the experimental bath solution directly, and immediately before the start of image acquisition. For the measurement of [Ca2+]c, the cells were incubated in DMEM with 5 µM Fluo-4 acetoxymethylester (AM) for 20 min or with 4 µM Fura-2 AM for 15 min. FM 1-43 was used at 2 µM in combination with Fura-2. In control experiments, cells were preincubated with CellTracker Green CMFDA (5-chloromethylfluorescein diacetate) at 800 nM for 10 minutes. To deplete Ca2+ stores, the cells were incubated with 150 nM thapsigargin for 20 min. In the experiments with the Ca2+ channel blocker SKF96365, 50 µM of this substance was either applied to the experimental bath solution directly, or the cells were additionally preincubated with it for 15 min at 37°C. OxiFluor  was added to experimental bath (1∶200) in darkfield experiments to prevent the fluorescence-induced cell damage. Actin coating of lamellar bodies was measured in cells transfected with β-actin-GFP adenovirus construct, as described before Experiments were performed at room temperature in experimental bath solution  in 5 and 10 µm linear distances from the site of fusion for events being sufficiently separated in time to avoid overlap of spreading Ca2+ signals from multiple events. The onset of the Ca2+ rise at these locations was determined as described above for the local Ca2+ increase.Measurements of FM 1-43 fluorescence in combination with Fura-2 were performed as described An upright Richardson RTM 2.5 microscope  was used for combined darkfield and fluorescence imaging. For darkfield illumination, a red LP-interference filter (>620 nm) in the condenser was used together with the RTM 3.0 blue filter cube for fluorescence imaging . Darkfield and fluorescence light was detected with a 3CCD color video camera . All images were acquired with the 63× Leica water immersion objective (NA = 0.90) and Volocity software . The darkfield and the fluorescent images were acquired simultaneously and were digitally separated based on different colors We defined a region of interest encircling the fusing LB in which darkfield light intensity change was measured. Fluo-4 fluorescence was measured within a ring-like region of interest surrounding the fusing LB. The size of selected regions of interest depended on LB size, whereas the width of the ring was set to approximately 1 µm. The lowest value of darkfield and fluorescent measurements was subtracted and the resulting change in light intensity was expressed in arbitrary units. Excel 2003 and SigmaPlot 10.0 were used for statistics and graphs. Unless otherwise stated, data are presented as mean±standard error of the mean. The alveolar type II cell photographs were corrected for brightness and contrast with Adobe Photoshop CS2. The background subtraction was made in ImageJ by subtracting the first image in an image sequence from the others with the Image Calculator feature.2+ dynamics in single cells, we combined two independent fusion- and Ca2+-indicators:To investigate fusion-related CaFM 1-43 fluorescence with the Fura-2 ratio SLID with Fluo-4 fluorescence In the Fura-2 experiments, LB fusion was detected with FM 1-43 added to the bath, as outlined in detail previously The Fura-2/FM 1-43 experiments revealed the following:n = 49). This indicates that a fusion pore sufficiently wide to allow FM 1-43 passage preceded the rise of [Ca2+]c.Vesicle fusions were followed by elevations of the peri-vesicular Fura-2 ratio , its ons2+ signal originated at the site of fusion but mostly  propagated across the cell. In this case a [Ca2+]c rise was also detectable at a distance from the fusion site, however with a considerable delay  to 16.7% (n = 24) when Ca2+ entry was blocked in the presence of 1 mM Ni2+, and was completely abolished when Ca2+ was removed from the bath .FACE was very frequently detected in LB-plasma membrane fusions, but not in compound (LB-LB) exocytoses see ; n = 10.2+ changes at the time of LB fusion was a combination of Fluo-4 fluorescence and darkfield microscopy. The latter approach, recently developed in our lab, is based on the observation that the intensity of scattered light produced by LBs decreases upon fusion with the plasma membrane The second method for detecting localized Ca2+ concentration was measured in a circular 1 µm wide region of interest  and initiated after the start of the second, steep phase of the scattered light intensity decrease (SLID2). Previous work showed that the start of the SLID2 corresponded to the fusion pore opening n = 21) which was longer than the delay between the FM 1-43 fluorescence and the Fura-2 ratio increase. Possible reasons for this discrepancy could be:The peri-vesicular increase of the Cainterest . The stainterest . Localiz2+ signal.different methods to analyze the instance of fusion pore opening. Previous work revealed that the onset of FM 1-43 fluorescence may be delayed by up to 0.5 s with regard to SLID2 or cell membrane capacitance steps, respectively d = 345 nM) and Fura-2 (Kd = 224 nM) for Ca2+ ionsdifferent affinities of Fluo-4 . The maximum observed half-time of decrease was 26.6 s. The major reason for the fast Fluo-4 fluorescence decrease in most experiments is probably photobleaching, which was present already before the fusion event , Caire cell . The ons n = 15, . The cal2+ wave propagated throughout the cytoplasm starting from one location (the fused LB), was the measurement of the slope coefficients for the Fluo-4 fluorescence increase. If the [Ca2+]c increase is actually starting from a point source in the cell, we would expect these slope coefficients to be steepest close to the Ca2+ source and less steep with increasing distance. A global [Ca2+]c increase (as caused by a homogeneous release from stores) should result in similar slope coefficients throughout the cell. The slope coefficients, calculated from linear regressions, for the local [Ca2+]c increase were significantly higher  than those of the [Ca2+]c increase 5 µm , and 10 µm away from the fused LB . In measurements combined with darkfield we did not observe an increase in CTG fluorescence in the vicinity of an LB at the time of fusion (measured in concentric areas as in Fluo-4 experiments), nor a change in fluorescence 5 and 10 µm away , we stained the cells with CellTracker Green (CTG), a cytoplasmic dye, which is not Ca2+]c increase. Fura-2 fluorescence experiments in the absence of bath Ca2+ showed the absolute requirement of extracellular Ca2+ for the initiation of the localized [Ca2+]c increase and for the Ca2+ spreading  were still accompanied by a localized [Ca2+]c increase (in control conditions 87.5%), which excludes the major role of the endoplasmic reticulum in initiation and propagation of the Ca2+ increase  do not contribute to this phenomenon.The first question from these experiments is the subcellular source or even molecular identity of this localized c increase may assist in actin coat formation and contraction in the course of surfactant secretion. To test the possible connection between the post-fusion [Ca2+]c increase and the actin coating we tried to block the post-fusion [Ca2+]c increase with the unspecific Ca2+ channel blocker SKF96365. By direct application of SKF96365 to the cells, the incidence of the localized post-fusion Fluo-4 fluorescence increase dropped from 87.7% to 35.7%  to 29.2%  Ca2+]c elevations, is the question of its origin: does Ca2+ come from the extracellular space exclusively, or also from the fusing LB? However, the distinction between LB and extracellular space is - in this context - somewhat semantic, because during the postfusion phase, the lumen of the LB is by definition part of the extracellular space , it is reasonable to assume that the crucial origin of Ca2+ is the extracellular space, for which “fusion-activated Ca2+ entry” (FACE) is the appropriate term. Ca2+-induced Ca2+ release may be an amplification mechanism that applies particularly for those cells, in which the signal spreads to sites remote from the location of fusion.A similar problem refers to the notion of “Ca2+ release from LBs. If Ca2+ stored in LBs was the major source of FACE, it should be greatly blocked by this drug, which depletes this store. However, we found that pretreatment with bafilomycin A1 (20 nM for 1h at 37°C) could not inhibit FACE . This further strengthens the notion that the major part of Ca2+ comes from the extracellular space  and also after stimulation of protein kinase A . Evidence suggesting that an ion channel is involved arises from the time-dependent inhibition by SKF96365 and from inhibition by NiAlso speculative are possible modes of activation of FACE. Two mechanisms are most likely:mechanical (“membrane stress”)chemical (“membrane merger”)The strongest argument in favor of a mechanical mode of activation is LB swelling. Many secretory vesicles swell after fusion through osmotic uptake of water by the vesicle matrix The fusion pore is a significant barrier for free lipid flow from the plasma into the LB membrane, such that swelling-induced LB membrane stress occurs. Recent observations of lipid-anchored fluorophore diffusion measurements suggest that this may be the case LB swelling leads to ion channel activation by channel-cytoskeleton interactions.Since both conditions are possible, mechanical activation of FACE is a reasonable working hypothesis.2+ channel.Recent studies indicate that lipid merger between the plasma and vesicle membranes cause translocation of lipid mediators and activation of signaling cascades that finally induce actin assembly 2+]c elevation caused by FACE in most cases spread out through the entire cell . Inhibition of both, FACE and actin coat formation, by this drug strengthens the notion that a major physiological role of FACE could be the facilitation of LB contraction and surfactant release. This function may gain particular importance if the fusion pore does not fully expand and remains a physical barrier for release, as demonstrated earlier 2+]c with the Ca2+ ionophore ionomycin showed that fusion pore expansion is enhanced by an increased Ca2+ concentration 2+ and the identity of molecular effectors involved in this process remain an open question. In addition, FACE may not only contribute to actin assembly, but also to active force generation and content release.Awareness is growing that postfusion regulation of content release may be relevant to cell physiology in various types of secretion . Its molecular pathway(s) and mode(s) of activation remain to be elucidated. Owing to common principles of exocytosis , FACE may also play an important role in other secretory cell types, in which time and space-resolved resolution of exocytotic fusion and Ca2+ dynamics are more difficult to detect than in the pulmonary alveolar type II cells.In this study, we have identified a new CaFigure S1Fluo-4 fluorescence increase after vesicle fusion was only detected on the area of the exocytosing cell. Left: Regions of Fluo-4 measurements. Orange circle: within the cell, green circle: outside the cell (see main text and (0.90 MB TIF)Click here for additional data file.Video S1Subtracted Fluo-4 fluorescence video for the fusion event shown in (6.60 MB AVI)Click here for additional data file.


Fifteen patients with acute myelogenous leukaemia were studied to determine if their remission blood leucocytes could be stimulated into taking up [3H] thymidine after in vitro culture with their own cryo-preserved irradiated AML leukaemia cells. In 6/15 patients it was possible to show autologous recognition and equal recognition of their stored leukaemia cells, even when they had previously been maintained in in vitro proliferative cultures in liquid suspension and undergoing myeloid maturation for one week. After in vitro proliferative culture, 4 populations of leukaemia cells produced material in the supernatant media between 3 and 7 days capable of inducing [3H] thymidine uptake in autologous  and allogeneic  AML remission lymphocytes, but not in normal donor lymphocytes. The relevance of these observations to tumour-associated AML antigen is discussed.


Some family members are activated by increased intracellular Ca2+ concentration. Bestrophins feature a well conserved Asp-rich tract in their COOH terminus (Asp-rich domain), which is homologous to Ca2+-binding motifs in human thrombospondins and in human big-conductance Ca2+- and voltage-gated K+ channels (BKCa). Consequently, the Asp-rich domain is also a candidate for Ca2+ binding in bestrophins. Based on these considerations, we constructed homology models of human bestrophin-1 (Best1) Asp-rich domain using human thrombospondin-1 X-ray structure as a template. Molecular dynamics simulations were used to identify Asp and Glu residues binding Ca2+ and to predict the effects of their mutations to alanine. We then proceeded to test selected mutations in the Asp-rich domain of the highly homologous mouse bestrophin-2. The mutants expressed in HEK-293 cells were investigated by electrophysiological experiments using the whole-cell voltage-clamp technique. Based on our molecular modeling results, we predicted that Asp-rich domain has two defined binding sites and that D301A and D304A mutations may impact the binding of the metal ions. The experiments confirmed that these mutations do actually affect the function of the protein causing a large decrease in the Ca2+-activated Cl− current, fully consistent with our predictions. In addition, other studied mutations  did not decrease Ca2+-activated Cl− current in agreement with modeling results.Bestrophins are a recently discovered family of Cl In 1998, the gene VMD2, which encodes for the human bestrophin-1 (hBest1) protein, was found to be responsible for the inherited Best vitelliform macular dystrophy. This is an early-onset autosomal dominant maculopathy typically characterized by yellowish lesions in the central area of the retina − channels, likely to be composed by several subunits 2+2+-activated Cl− current in some epithelial cells 2+-activated Cl− channels All human and mouse members of the bestrophin family have been expressed in heterologous systems Although the link between the protein and the Best disease has not been clarified yet Clearly, knowledge of the structural determinants of the protein is crucial to understand its function both in health and in disease conditions. However, no 3-D structural information is available so far. Nevertheless, two topology models have been advanced 2+ sensor for bestrophins 2+-binding domains as the cytoplasmic Ca2+ bowl motif 2+- and voltage-gated K+ channels (BKCa) Ca). Site-directed mutagenesis experiments have confirmed the role of Ca2+ bowl in the binding of calcium ions Ca channels has been recently provided by us In an effort to characterize prominent structural and functional features of bestrophins, we noticed that the Asp-rich domain, which has been indicated as a possible Ca2+-binding domains, T3 repeats of thrombospondins, consists in tandems of Asp-rich motifs, which resemble EF hands in the spacing of acidic side chains 2+ ions The second type of Ca2+-binding domains, it is plausible to hypothesize that Ca2+ activation of bestrophins could involve, at least in part, Ca2+-binding to the Asp-rich domain. This hypothesis is fully consistent with the experimental evidence from mutations of amino acids constituting putative Ca2+ ligands  in the domain, which affect dramatically the measured current in the HEK-293 cells 2+-binding domains were not explored here. On the other hand, computer simulations of the entire channel, which would help to clarify how Ca2+ binding could affect channel gating, are not feasible due to the lack of structural information of the transmembrane region of the channel.Because of the significant sequence similarity between the Asp-rich domain and the aforementioned Ca2+ activation of bestrophins would be to mutate randomly its Asp/Glu residues and measure the functional properties of the channel. Here instead we followed a more rational approach, which uses computation to guide the experiments. We first constructed a structural model of hBest1 Asp-rich domain based on the TSP-1 T36 X-ray structure 2+-binding residues conserved across bestrophins. The models of WT and selected alanine mutants were then studied by molecular simulations. Mutations with the largest predicted effect on Ca2+ binding turned out to cause a dramatic decrease in the experimentally measured mBest2 Ca2+-activated current (more than 96% reduction with respect to the WT mBest2). The consistency between theory and experiments permits to have the first structural insights on the hBest1 protein.A straightforward approach to test if Asp-rich domain is involved in Ca2+ ions in the WT. Next, we predicted the effects of mutations to alanine of these residues on the structure and function of the Asp-rich domain. The effect is non-trivial, because residues, which bind Ca2+ ions in the WT, can be replaced by others when mutated to alanine.Our strategy to identify Asp and Glu amino acids important for the function of hBest1 was twofold. First, we attempted to identify residues that may bind the Ca6 repeat 2+-binding residues conserved across bestrophins family , four (M4), and three  Ca2+ ions  simulations, two ions  during the MD simulations for as long as 25 ns , running two independent MD simulations of the same length could result in slightly different events.In order to verify this, the experiments of the WT and mutant mBest2 were performed. The putative Caf hBest1 . To inve2+ coordination in the aforementioned mutations may affect the amplitude of bestrophin Ca2+-activated Cl− currents, which can be experimentally measured. This was done here for mBest2. WT and mutants currents were compared after their heterologous expression in the HEK-293 cell line . Therefore, the D312A mutation not only allowed a Ca2+-activated Cl− current, but it also modified channel gating increasing its current also in the absence of Ca2+.In contrast, D304A and D301A produced negligibly small currents in both Ca+ levels . Two oth+ levels , D312A sntration . In 22 µFinally, the double mutant D304A+E306A abolished the current, similarly to the single mutant D304A .2+-activated current, whereas D301A and D304A mutants produced a negligibly small Ca2+-activated current. These results were fully consistent with our theoretical predictions.Experiments showed that WT, E306A and D312A mutants allowed a substantial Ca2+ ions and the Asp-rich domain in bestrophins is an important step in the investigation of the physiological role of these proteins, because they are putatively involved in the Ca2+-activated Cl− current in some epithelial cells Understanding the interactions between the Ca2+ binding 2+-binding domains, as the Ca2+ bowl in BKCa2+ ligands in bestrophin Asp-rich domain affect dramatically the current We propose that the Asp-rich domain located at the C-terminal region of bestrophins can contribute to Ca2+ binding, we adopted a multifaceted strategy involving homology modelling, MD simulations, immunostaining and electrophysiological experiments of WT and mutated bestrophins.In order to identify negatively charged residues in the domain involved in Ca2+ binding. Simulations of D301A and D304A mutants point to the key role of these residues for binding. Measures of Ca2+-sensitive Cl− current of WT and mutant bestrophins expressed in HEK-293 cells show that D301A and D304A mutations do actually affect the functionality of the channel, dramatically reducing the Ca2+-activated current amplitude. These findings are fully consistent with our theoretical predictions.Theoretical data suggest that not all Asp and Glu residues have equal importance in Ca2+ binding, albeit smaller,  2+ bowl from human and mouse BKCa channels 2+ binding positions. Sequence similarity was determined considering amino acid pairs having positive values in BLOSUM62 6 motif as a template (PDB code: 1UX6) 2+ ions present in the template were kept. These are: (i) M5, with five Ca2+ ions coordinated. (ii) M4 is composed by four Ca2+ ions which are Ca1, Ca2, Ca4 and Ca5. (iii) M3′ and M3″ are including three Ca2+ ions, Ca1, Ca2, Ca4 or Ca1, Ca2, Ca5, respectively.The sequences of the human TSP-1 T3−  or Na+ (one in M3′ and M3″) counterions were added to ensure systems charge neutrality. The force fields for the protein, ions and water molecules were the Amber parm99 Models of the Asp-rich domain of hBest1 were inserted in a box of edges 49, 50 and 53 Å filled with ∼4,100 water molecules 2+ ions, with a force constant of value 25.0 kcal/mol Å−2. After the first 300 cycles of steepest descent minimization, conjugate gradient method was used until 50,000 cycles. Then steepest descent minimization of the system without restraints was performed for another 150 cycles, following by conjugate gradient minimization until 50,000 cycles. After that, constrained solute and Ca2+ ions underwent 60 ps of linear heating MD from 0 K till 300 K. Restraints on Asp-rich domain and Ca2+ ions corresponded to a force constant of 5.0 kcal/mol Å−2. A time step of 2 fs was used. Finally, the system underwent NPT MD simulations for 75 ps at 1 fs time step. The system was maintained at the reference pressure and temperature of 1 bar and 300 K, respectively, by coupling the system to a Berendsen thermostat and barostat For M5, energy minimization of water and counterions was performed with position restraints on the Asp-rich domain and CaFinally, unrestrained MD simulations were carried out for an overall length of 70 ns. A time step of 2 fs was used. All bond lengths were kept fixed applying the LINCS algorithm 2+ ions were built starting from the MD snapshot of M5 at 9 ns  by manually removing one or two ions (see above). In M4, M3′ and M3″ models Ca3 was always removed since it is immediately solvated during the simulation of M5  were built, based on the M5 model, using the program Molden The model of mBest2 Asp-rich domain underwent the same protocol of minimization and relaxation of hBest1, followed by a 60-ns MD run.All the systems underwent minimization using Amber 8.0 program 2 and Ca4 to Asp and Glu residues of the Asp-rich domain. They were defined as the number of contacts shorter than 4.3 Å among one Ca2+ ion and all carboxylate carbons of the studied domain:i runs over Ca2 and Ca4, j runs over all carboxylate carbons, ijR is the distance between the two atoms, R0 = 4.3 Å is a cutoff distance chosen in order to discriminate between bonded and non-bonded Ca2+ ions (this cutoff distance ensures that Ca2+ ion is considered bonded when it is at most 3 Å away from carboxylate oxygen atoms (see e.g.ijn is a function switching smoothly between 1 (bonded Ca2+ ion) and 0 (non-bonded Ca2+ ion). Similar CVs have been employed in a previous study of Ca2+-binding polymers 1 = σ2 = 0.2  and a height of 0.2 kcal/mol. A new Gaussian is added along the trajectory every 1 ps. Metadynamics is implemented in a modified version of the Gromacs-3.3.1 package A 60-ns long metadynamics simulation was performed by biasing two collective variables (CVs) with a history-dependent potential. These are the coordination numbers of Casee e.g., and nijThe clone of mBest2 in pCMV-Sport6 mammalian expression plasmid was obtained from the RZPD  collection (Deutsches Ressourcenzentrum für Genomforschung). Site-specific mutations of mBest2 were made using a PCR-based site-directed mutagenesis kit (Quiagen). Mutations were confirmed by DNA sequencing.mBest2 and its mutants were transfected into HEK-293 cells along with a vector that expressed EGFP  at a 1∶8 ratio using Fugene-6 transfection reagent (Roche). 2 days after transfection, cells were dissociated and replated. Transfected cells were identified by EGFP fluorescence and used for patch clamp experiments within 3 days after transfection.To test successful expression of proteins, as well as their spatial distribution in HEK-293 cells, we used co-staining of antibodies against mBest2 and labeled wheat germ agglutinin (WGA) as a marker of glycosylated surface-expressed proteins – a method previously used to show localization of proteins in cell membrane and/or close submembrane space Transfected cells (without the EGFP expressing vector) were fixed with 4% paraphormaldehyde for 20 min, washed, incubated with 5 µg/ml WGA conjugated to Alexa Fluor 488 (Invitrogen) for 20–30 min, incubated for 30 min in blocking solution containing 1% BSA (Sigma), 0.1% Triton X-100 (Sigma) and 1% FCS (Sigma), then for 2 hours with an antibody against mBest2 Currents were measured with an Axopatch 200B patch-clamp amplifier  in the whole-cell voltage-clamp mode. The amplifier was controlled via a Digidata 1440A .Patch pipettes were made using borosilicate capillaries  and pulled with a Narishige PP83 puller , using a double stage pull. The pipette resistance was 2–4 MΩ. Data were sampled at 20 kHz and low-pass filtered at 10 kHz. Acquisition and storage of data were performed with the PClamp 10 software . Data analysis and figures were made with the Clampfit 10 software  and Igor 6.0 software .− current blocker SITS. Current densities were calculated normalizing the current recorded at +60 mV to the capacitance of each cell. Data are shown as mean±standard error of the mean. N is the number of cells. Statistical significance was determined using un-paired t-tests. P values<0.05 were considered significant.The holding potential was 0 mV and a voltage protocol from −100 mV to +100 mV, with 20 mV steps of 200 ms duration was applied (starting 2 minutes after reaching the whole-cell configuration). Cell capacitance was estimated from a 10 mV step test protocol in the presence of the extracellular Cl2, 1 MgCl2, and 10 HEPES, 10 Glucose, pH = 7.4. The pipette solution contained (in mM): 130 CsCl, 2 MgCl2, 10 HEPES, 5 EGTA without added Ca2+ for the nominally 0 Ca2+ solution, or with 5 mM CaCl2 for the 22 µM Ca2+ free pipette solutions . pH was adjusted to 7.3 by adding HCl or CsOH. The osmolarity was adjusted with sucrose to 310 or 300 mOsm for the extracellular and intracellular solution, respectively.The standard extracellular solution contained (in mM): 140 NaCl, 5 KCl, 2 CaCl− currents, the extracellular blocker 4-acetamido-4′-isothiocyanato-stilben-2,2′-disulfonate (SITS) was directly dissolved in the extracellular solution at 2 mM. All chemicals were purchased from Sigma. All experiments were carried out at room temperature (20–22°C).To block ClText S1Test of the accuracy of the force field used in the MD simulations(0.11 MB PDF)Click here for additional data file.Table S12+ ions. Percentages were averaged over all MD simulations of all the studied models. Ca3 is not reported as it is never bound to the protein. “bb” indicates that the backbone carbonyl oxygen is involved in the binding.Percentage of time during which specific residue binds Ca(0.04 MB DOC)Click here for additional data file.Table S22+ protein coordination numbers in WT and the investigated mutants of the Asp-rich domain, as observed after 20 ns of MD simulations. First number refers to the number of peptide O-donors, whereas the second refers to the number of coordinated waters. Ca2+ ions were considered bound if they were coordinated with at least 5 protein O-donors and not more than 2 water molecules. Bound ions are highlighted (see text).MD simulations of the M3′ model and its alanine mutants. Ca(0.04 MB DOC)Click here for additional data file.Figure S11 through the metadynamics simulations of the Asp-rich domain of hBest1. As in standard MD, Ca1 is stably bound to the protein for most of the time. B. Number of peptide O-donors coordinating Ca2 (black) and Ca4 (green) plotted as a function of the metadynamics simulation time.Metadynamics simulation of M3′ model. A. Number of peptide O-donors (red) and water molecules (blue) coordinating Ca(1.19 MB TIF)Click here for additional data file.Figure S22+ ions are depicted. Ca2+ ions are represented as spheres  and numbered according to their binding positions as represented in the Most populated conformations of hBest1 Asp-rich domain as sampled by the metadynamics simulations. Asp/Glu residues coordinating Ca(2.44 MB TIF)Click here for additional data file.


Pacific ciguatoxin-1B (P-CTX-1B) activates voltage-sensitive Na+ channels and promotes an increase in neurotransmitter release believed to underpin the symptoms associated with ciguatera. However, the mechanism through which slow Na+ influx promotes neurosecretion is not fully understood. In the present study, we used chromaffin cells as a model to reconstitute the sequence of events culminating in ciguatoxin-evoked neurosecretion. We show that P-CTX-1B induces a tetrodotoxin-sensitive rise in intracellular Na+, closely followed by an increase in cytosolic Ca2+ responsible for promoting SNARE-dependent catecholamine secretion. Our results reveal that brevenal and β-naphtoyl-brevetoxin prevent P-CTX-1B secretagogue activity without affecting nicotine or barium-induced catecholamine secretion. Brevenal is therefore a potent inhibitor of ciguatoxin-induced neurotoxic effect and a potential treatment for ciguatera.Ciguatoxins and brevetoxins are neurotoxic cyclic polyether compounds produced by dinoflagellates, which are responsible for ciguatera and neurotoxic shellfish poisoning (NSP) respectively. Recently, brevenal, a natural compound was found to specifically inhibit brevetoxin action and to have a beneficial effect in NSP. Considering that brevetoxin and ciguatoxin specifically activate voltage-sensitive Na Karenia brevis, has resulted in the discovery of natural and chemically modified compounds that may have the potential for the treatment of ciguatera.Ciguatera is the most widespread human syndrome of marine poisoning that occurs following consumption of fish from tropical and subtropical pacific regions contaminated with ciguatoxins that accumulate through the marine food chain Gambierdiscus toxicus and Karenia brevis, respectively, which bind competitively to the receptor-site 5 of voltage-sensitive Na+ channels + permeability and excitability of most excitable cells + channels activity by shifting their activation to more negative membrane potentials, and by inhibiting in part their inactivation + channel activation leads to asynchronous quantal spontaneous release. One of the possible hypotheses is that ciguatoxins promote Ca2+ entry in nerve terminals, which could in turn stimulate exocytosis. Importantly, the activation of Na+ channels by P-CTX-1B has been shown to lead to direct mobilisation of Ca2+ from internal stores in neuroblastoma cells and rat myotubes +/Ca2+ exchanger in the reversed mode in cholinergic synaptosomes, allowing Ca2+ influx against Na+ efflux Ciguatoxins and brevetoxins are lipid-soluble cyclic polyether compounds produced by the benthic dinoflagellates + influx in promoting Ca2+-dependent neuroexocytosis, by assessing the effect of P-CTX-1B on neuroendocrine cells. Chromaffin cells are a convenient model to study both Ca2+ and Na+ signalling together with catecholamine release In this study, we have clarified the role played by ciguatoxin-induced long lasting Na+ channels and membrane depolarization would be a marked increase in intracellular Na+. One way to verify this possibility was to measure cytosolic Na+ changes induced by P-CTX-1B. Intracellular Na+ was monitored in chromaffin cells pre-loaded with the cell-permeant Na+-indicator sodium green. Experiments were performed in a Ca2+-free medium containing EGTA (2 mM) to avoid activation of the Na+/Ca2+ exchanger + channel blocker tetrodotoxin  potently inhibited P-CTX-1B-evoked Na+ increase  resulted in a transient increase in intracellular fluorescence intensity  to chromaffin cells in the presence of 2 mM external Ca2+ promotes significant catecholamine secretion . The catecholamine secretion observed with P-CTX-1B was strongly inhibited by BoNT/A pre-treatment  by Botulinum neurotoxin type-A (BoNT/A). Cultured bovine chromaffin cells were treated reatment . This re2+ stores in P-CTX-1B-evoked Ca2+ rise , called receptor-site 5 Karenia brevis found to selectively displace brevetoxin from site 5 of the VSSC 50∼2 µM). This biphasic effect was accentuated by raising P-CTX-1B concentration to 50 nM  P-CTX-1B was extracted and highly purified from the Pacific moray-eel 0, where F0 is the initial cell fluorescence and F the monitored cell fluorescence.Chromaffin cells were loaded by incubation  with either fluo-3/AM or sodium green/AM in Krebs-Ringer solution. After washing the cells with a dye-free solution, the coverslips were placed on the stage of the microscope and imaged by confocal microscopy. A Sarastro 2000 or a Zeiss 510 META confocal laser scanning microscope  was used. The aperture setting of the confocal pinhole was 100 µm. Cells were either visualized using a 40× or a 60× water immersion objective . The 488 nm band of the Argon ion laser was used for excitation and the emitted light was collected through a 510 nm long-pass. All experiments were performed at room temperature and time series of cell confocal sections were acquired (using a time-lapse mode) with a frame interval of 5 s for fluo-3/AM and 5 min for sodium green/AM. Sections were collected using a standard scanning mode format of 512×512 pixels. The fluo-3/AM or sodium green/AM loaded cells were presented using a pseudocolor scale. The index of fluorescence variation was calculated and presented as F/F2PO4, 10 glucose, 20 HEPES (pH 7.4), and processed in quadruplicate. Aliquots (200 µl) of the medium were removed at the end of each experiment, and cells were lysed with Triton x-100 . Both set of samples were assayed fluorimetrically for catecholamine content. Amounts released were expressed as a percent of the total content of catecholamines present in the cells The catecholamine secretion was quantified as previously described t test. All experiments were performed at least 3 times. Values were expressed as mean±SEM and data were considered significant at *p<0.05 and **p<0.01.Data analysis was carried out using Student's


To investigate how the t-tubule microanatomy and the distribution of membrane Ca2+ flux affect cardiac excitation-contraction coupling we developed a 3-D continuum model of Ca2+ signaling, buffering and diffusion in rat ventricular myocytes. The transverse-axial t-tubule geometry was derived from light microscopy structural data. To solve the nonlinear reaction-diffusion system we extended SMOL software tool (http://mccammon.ucsd.edu/smol/). The analysis suggests that the quantitative understanding of the Ca2+ signaling requires more accurate knowledge of the t-tubule ultra-structure and Ca2+ flux distribution along the sarcolemma. The results reveal the important role for mobile and stationary Ca2+ buffers, including the Ca2+ indicator dye. In agreement with experiment, in the presence of fluorescence dye and inhibited sarcoplasmic reticulum, the lack of detectible differences in the depolarization-evoked Ca2+ transients was found when the Ca2+ flux was heterogeneously distributed along the sarcolemma. In the absence of fluorescence dye, strongly non-uniform Ca2+ signals are predicted. Even at modest elevation of Ca2+, reached during Ca2+ influx, large and steep Ca2+ gradients are found in the narrow sub-sarcolemmal space. The model predicts that the branched t-tubule structure and changes in the normal Ca2+ flux density along the cell membrane support initiation and propagation of Ca2+ waves in rat myocytes.The t-tubules of mammalian ventricular myocytes are invaginations of the cell membrane that occur at each Z-line. These invaginations branch within the cell to form a complex network that allows rapid propagation of the electrical signal, and hence synchronous rise of intracellular calcium (Ca 2+) is best known for its role in contraction activation. A remarkable amount of quantitative data on cardiac cell structure, ion-transporting protein distributions and intracellular Ca2+ dynamics has been accumulated. Various alterations in the protein distributions or cell ultra-structure are now recognized to be the primary mechanisms of cardiac dysfunction in a diverse range of common pathologies including cardiac arrhythmias and hypertrophy. Using a 3-D computational model, incorporating more realistic transverse-axial t-tubule geometry and considering geometric irregularities and inhomogeneities in the distribution of ion-transporting proteins, we analyze several important spatial and temporal features of Ca2+ signaling in rat ventricular myocytes. This study demonstrates that the computational models could serve as powerful tools for prediction and analyses of how the Ca2+ dynamics and cardiac excitation-contraction coupling are regulated under normal conditions or certain pathologies. The use of computational and mathematical approaches will help also to better understand aspects of cell functions that are not currently amenable to experimental investigation.In cardiac muscle cells, calcium (Ca We examined the Ca2+ signaling in rat ventricular myocytes that had been treated with ryanodine and thapsigargin to eliminate Ca2+ release and uptake by the SR. By using published electro-physiological data and laser-scanning confocal [Ca2+]i measurements, we were able to analyze several important spatial and temporal features of the Ca2+ signals in these cells. In this context, our goal was at least three-fold. The first aim was to develop a mathematical model that would be in qualitative or quantitative agreement with published experimental measurements on Ca2+ influx, and Ca2+ buffering and diffusion in rat ventricular cells with SR function inhibited 2+ flux distribution for the Ca2+ signals. The third task was to simulate the Ca2+ signals in the absence of fluorescent dye and to study the importance of the mobility of endogenous Ca2+ buffers  and altered extracellular Na+ ([Na+]e) for the Ca2+ signals. The analysis suggests that the quantitative understanding of the Ca2+ signaling requires more accurate knowledge of the t-tubule microanatomy and Ca2+ flux distribution along the sarcolemma. In agreement with experiment, with 100 µM Fluo-3, the lack of detectible differences in the depolarization-evoked Ca2+ transients was found when the Ca2+ flux was heterogeneously distributed along the sarcolemma. In the absence of Fluo-3, the predicted Ca2+ signals were strongly non-uniform. Even at modest elevation of Ca2+, reached during Ca2+ influx, large and steep Ca2+ gradients may develop in the narrow sub-sarcolemmal space. The model also predicts that branched t-tubule topology and changes in the normal Ca2+ flux density along the cell membrane support Ca2+ waves initiated at the sarcolemma. Preliminary results of this work have been presented to the Biophysical Society in abstract form In the present study, we sought to develop a morphologically correct geometric model of the t-tubule and to use this model for computational studies of the intracellular Caet al.2+ dynamics. The gap between imaging and simulation involves two major steps: (1) extracting features (boundary or skeleton) from imaging data; (2) constructing geometric models (represented by meshes) from the detected features. In addition, image pre-processing is usually necessary for better feature extraction, when the original image is noisy or the contrast between features and background is low. With 3-D T-PM images, Yu and collaborators developed a set of image processing and analysis tools and using the mesh generator called GAMer Combining light microscopy (LM) and electron microscopy (EM) together with 3-D tomographic reconstruction, Hayashi et al.2+ signaling in rat ventricular myocytes, we considered a small compartment containing a single t-tubule and its surrounding half-sarcomeres for two reasons: (a) the entire t-tubular system in a ventricular myocyte forms a roughly periodic pattern corresponding to individual sarcomere; and (b) a small model contains much fewer number of mesh nodes that would render numerical simulation significantly faster and more feasible in ordinary computers. The surrounding half-sarcomeres were modeled as a rectangular-shaped box of 2 µm×2 µm in the plane of external sarcolemma and 5.96 µm in depth  and within the external membrane 36% (∼3.25 µm2), 2+ was estimated to be ∼35–37% of the total cytosolic volume (2+ (i.e. contains water); (3) mitochondria and nuclei are not rapidly accessible for Ca2+; (4) the SR lumen is not accessible to Ca2+ in the presence of ryanodine and thapsigargin The extreme intricacy of the t-tubule system in mice  has been observed in rat ventricular myocytes as well in depth . As Yu'sin depth  was assuin depth . These a2+-transporting protein complexes  along the cell membrane probably exist 2+ channels are concentrated in the t-tubules  Recent immunohistochemical studies have demonstrated that marked variations in the distribution of Ca2+ efflux pathway, the Na+/Ca2+ exchanger, are more controversial. All studies but one et al. data 2+ microdomains suggest that significant differences in the excitation-contraction coupling between the cell surface and cell interior may be exist. However how the localization of Ca2+- transporting protein complexes along the sarcolemma regulates the intracellular Ca2+ signaling still remains uncertain.Studies on the distribution of the main Ca2+ buffers  were considered  diffuse and react throughout the cytoplasm. The cell membrane and sarcomere box faces are subject to reflective boundary conditions. The nonlinear reaction-diffusion equations describing Ca2+ and buffers dynamics inside the model cell are:m] represents the concentration of mobile buffer Fluo-3, ATP or calmodulin; [Bs] is the concentration stationary buffer troponin C.In the current model, the effects of four exogenous and endogenous Cansidered . The end2+, CaATP, CaCal and CaFluo as well as the total buffer concentrations and buffer rate constants used in the model are shown in 2+ and all mobile buffers 2+ binds to Fluo-3, calmodulin, ATP, and TN without cooperativity; (3) the initial total concentrations of the mobile buffers are spatially uniform; (4) the diffusion coefficients of Fluo-3, ATP or calmodulin with bound Ca2+ are equal to the diffusion coefficients of free Fluo-3, ATP or calmodulin.The diffusion coefficients for Ca2+ flux  was converted to Ca2+ flux (Ji) by using the experimentally suggested surface to volume ratio  and whole-cell L-type CaIn finite element methods, a complex domain needs to be discretized into a number of small elements (such as triangles or tetrahedra). This process is usually referred to as mesh generation http://mccammon.ucsd.edu/smol/) with the time step of 4 ms. It takes around 20 minutes to run 400 ms snapshots with a single Intel Xeon X5355 processor. The SMOL program utilizes libraries from the finite element tool kit (FETK), which previously has been used in several molecular level studies The nonlinear reaction diffusion system was solved by a finite difference method in time and finite element method in space using our SMOL software tool  heterogeneously distributed along the cell surface; (2) six times higher and uniform in the t-tubule membrane; or (3) homogeneously distributed along the sacrolemma. In cases (1–2) the NCX flux density was assumed three times higher in the t-tubule and in case (3) NCX was evenly distributed along the sarcolemma. In this study, Ca2+ leak density was homogeneously distributed along the cell membrane with respect to all distribution choices of LCC and NCX. In case (1), the 3-D distribution of LCC current was computed by combining the cluster density and fluorescent intensity plots, see x is the distance from the external cell surface.In agreement with the reported experimental data j, j = 1–4) are shown in 2+ flux along the t-tubule membrane remained unchanged by redistributing the Ca2+ fluxes. To fit the whole-cell LCC current density to the reported data in rat myocytes with SR release inhibited The parameter values of the polynomial . The SCH is defined to be the difference of the maximal and minimal [Ca2+]i values, normalized by the maximal value at given reference point along the line-scan  in given moment tj,  by 1.6 folds, SCH(t70 ms) by 2.29 folds, SCH([Ca]i-peak) by 2.34 folds, SCH(t100ms) by 2.87 folds, and SCH(t200ms) by 8.45 folds. These findings demonstrate that the predicted [Ca2+]i distribution with LCC heterogeneous more closely resembles the Chang et al. experimental findings 2+ transients when the LCC, NCX and Ca2+ leak fluxes were uniformly distributed throughout the cell surface (see right panel) demonstrates that here the Ca2+ signal spreads from the external membrane to the cell center as a continuum wave but after LCC channel closing (t∼72 ms) this wave faltered.These results demonstrate that with LCC heterogeneous or LCC six times higher in the t-tubule: (1) predicted Ca100 ms,  . To deli2+]i see , we intr tj, see . High SC surface . In addi2+]i transient, Na+/Ca2+exchanger, and Ca2+ leak currents with LCC pathways heterogeneously distributed (as in 2+ entry mode) while when repolarization occurred the flow of Ca2+ through NCX was reversed again ; (2) upon returning to resting voltage of −50mV the exchanger's rate rapidly increased  Ca2+ diffused toward the cell center and when 2+]i (∼0.16 µM) was reached. Intracellular Ca2+ equilibrated faster when Ca2+ flux was more concentrated in the t-tubule membrane because [Ca2+]i gradient near the t-tubule mouth was lower there than [Ca2+]i gradient with Ca2+transporting complexes distributed homogeneously. Additional reasons for the observed rapid equilibrium of [Ca2+]i may be that [Na+]i was kept constant .This model is also able to predict how the Ca2+ concentration distributions and spatial Ca2+ profiles at Ca2+ peak (76 ms) are shown in 2+ concentration near the external membrane decreased while [Ca2+]i in the cell interior increased when Ca2+ transporters were uniformly distributed and after that heterogeneously redistributed. The jumps in 2+ flux ; (3) redistributing NCX flux uniformly via the sarcolemma was not able to alter significantly the predicted Ca2+ signals in data not shown).Additional interesting model findings are that: (1) large and steep i distributions (data not shown).We could not find experimental data suggesting 2+ release into the diadic cleft, a major effect of the stationary phospholipids Ca2+ binding sites has been suggested 2+ signals (arising from Ca2+ influx via Ca2+ current only), we included this buffer in our model. The results demonstrated that the phospholipids had only a limited effect on the calculated Ca2+ signals in the sub-sarcolemmal region  and that this effect was even smaller when 100 µM Fluo-3 was included (data not shown).During simulations of SR CaThis model is also able to predict the spatial [Ca2+]i signals that would occur in the absence of Fluo-3 . Since it has been suggested that the dye could not affect Ca2+ entry via L-type channels 2+ signals arising from the ionic influx via L-type Ca2+ channels during voltage-clamp stimulation with LCC and NCX pathways heterogeneously distributed . The removing of the mobile Ca2+ dye from the solution increased SCH(tIca-peak) by 1.635 folds, SCH(t70 ms) by 2.63 folds, SCH([Ca]i-peak) by 2.72 folds, SCH(t100ms) by 4.46 folds, and SCH(t200ms) by 8.65 folds  the reversal of current of NCX upon returning to resting voltage of −50mV increased additionally the current rate  when the line-scan was positioned 200 nm away from the t-tubule surface at angle 60° or at 50, 100, 200, 300 or 400 nm at different angles (data not shown).The model predicts here that the spread and buffer capacity of 100 µM Fluo-3 were able to mask completely the pronounced spatial non-uniformities in i-peak) by 1.49 folds, SCH(t100ms) by 1.92 folds, and SCH(t200 ms) by 3.54 folds  upon repolarization global 2+]i peak decreased but the time to peak remained unchanged (∼76 ms), (see dashed lines in 2+ buffers) Ca2+ could escape the tight control of membrane voltage in some sub-cellular regions giving rise of cytosolic Ca2+ wave initiated at the sarcolemma (see right panel - near the outer cell edge Ca2+ wave was initiated (t∼60 ms) but very soon after ∼12ms this wave faltered).The conjecture made in the present model, that the endogenous calmodulin and ATP are mobile Caolds see . The res2+ signals in the absence of fluorescent indicator. The inhibition of NCX forward mode was achieved by removing extracellular sodium (i.e. 0 mM [Na+]e). To adjust Ca2+ flux via Ca2+ leak to match Ca2+ influx via NCX, so that at rest no net movement across the cell membrane to occur, we estimated Ca2+ leak constant (+]e zero (see 2+ entry mode while membrane leak pumped Ca2+ out of the cell (see dashed lines). see dashed lines) show the predicted local [Ca2+]i transients and the corresponding line-scan image. The line scan-image demonstrates that [Ca2+]i distribution was again non-uniform but rather different compared to that predicted with 140 mM [Na+]e  increased and that this increase was more pronounced near t-tubule mouth; (2) upon repolarization [Ca2+]i suddenly dropped because 2+ transient near the outer cell edge was slow; (4) [Ca2+]i in the featured spots, 3.09 µm and 5.45 µm, begun slowly to increase when t>70 ms because +]e zero versus 140 mM [Na+]e SCHs increased: SCH(tIca-peak) by 1.29 folds; SCH(t70 ms) by 1.4 folds; SCH(t[Ca]i-peak) by 1.43 folds; SCH(t100ms) by 1.65 folds; and SCH(t200ms) by 3.04 folds .In this study, we also examined the effects of NCX inhibition on the voltage-clamp induced Cazero see . Under t compare . The resolds see . Further2+ signaling in rat ventricular cells that incorporates the realistic transverse-axial t-tubule topology and considers geometric irregularities and inhomogeneities in the distribution of ion-transporting proteins. The t-tubule micro-architecture was extracted from the Hayashi et al. two-photon imaging data in mice et al. geometric model in mice 2+, was estimated to be ∼35–37% 2+ signaling in cells is largely governed by Ca2+ diffusion and binding to mobile and stationary Ca2+ buffers 2+ buffers  was considered.The current study attacks a difficult problem on how to incorporate the structural-based biological information, critical for the subcellular and cellular function, into sophisticated computational investigations. Pursuing this goal we developed a 3-D continuum model of Ca2+ influx, membrane protein distributions and Ca2+ diffusion in rat cells treated with ryanodine and thapsigargin to inhibit the SR Ca2+ metabolism et al. experimental data 2+]i profiles were within the range of experimental noise 2+ gradients and propagation of Ca2+ wave are predicted, not observed in Cheng et al. experiment, when the LCC and NCX were uniformly distributed along the sarcolemma.The model was validated against published experimental data on Ca2+]i gradients depend on the diffusion distances in the axial and cell surface directions. Thus, when the LCC were distributed uniformly the local Ca2+ peak in radial depth (5.96 µm) decreased from ∼1.5 fold while in the other cell directions (1 µm×1 µm) no significant changes were found. Redistributing the amount of Ca2+ pumped via the cell membrane  while keeping total Ca2+ flux unchanged, lowered Ca2+ gradients near the surface membrane and increased Ca2+ levels in the cell interior  is comparable to the measured of about 163 nM 2+]i profiles.The model studies with 100 µM Fluo-3 indicate also that the i gradients could occur throughout the whole cell volume et al. idealistic t-tubule model predicts high and steep sub-sarcolemmal [Ca2+]i gradients only in the transverse cell direction 2+]i distribution along the depth of the t-tubule when t<100 ms (see left panel) while this was not the case when the t-tubule geometry is assumed cylindrical  to simulate the Ca et al., . Third, 2+]i non-uniformities that would occur during the Ca2+ influx in the absence of dye . Here the simulations demonstrated that with zero Fluo-3 the local and global Ca2+ peaks increased while the time of Ca2+ rise remained unchanged. The predicted sub-sarcolemmal [Ca2+]i gradients were heterogeneous along the cell membrane and larger and steeper compared to those with 100 µM Fluo-3. The NCX and Ca2+ leak time-courses were also affected due to increased local free [Ca2+]i levels. It is interesting that under these conditions no differences in the local Ca2+ time-courses were found (as with 100 µM Fluo-3) when the line-scan was positioned at different angles and distances. To test further the model we also examined how the mobility of endogenous Ca2+ buffers  and altered extracellular Na+ ([Na+]e) would affect the Ca2+ signals in the absence of fluorescence dye when the Ca2+-transportes are heterogeneously distributed. The results showed that when ATP and calmodulin were immobilized Ca2+ diffuses slowly toward the center of the cell, resulting in higher Ca2+ concentrations near the outer cell edge. When the NCX forward mode was inhibited (assuming [Na+]e = 0 mM) the local [Ca2+]i peaks increased and this increase was more pronounced near the outer cell edge. New findings are that under these conditions near the outer cell edge Ca2+ wave was initiated while this was not the case when ATP and calmodulin were mobile and [Na+]e 140 mM  the use of 100 µM Fluo-3 during the experiment can sensitively alter the realistic Ca2+ distribution. A new the question, however, arises: Based on the above model findings what could be the underlying mechanism(s) for the predicted heterogeneous Ca2+ concentrations gradients in the absence of Fluo-3? A reasonable answer is that the Ca2+ movement and distribution inside the cell rely strongly not only on the specific cell micro-architecture and Ca2+ transporters distribution but also on the presence of endogenous mobile and stationary Ca2+ buffers  2+ buffer troponin C imposed stronger diffusion barrier for Ca2+ that resulted in larger and steeper sub-sarcolemmal Ca2+ gradients; (2) in the cell interior, owing on their sheer buffering capacity, Ca2+ buffers  tended to slow down additionally the propagation of Ca2+ so that ATP and calmodulin spreading alone was not able to contribute the spatially uniform Ca2+ profiles to be achieved; (3) immobilizing the endogenous Ca2+ buffers slowed down the Ca2+ movement from the cell periphery to the center leading to build-up of large sub-sarcolemmal Ca2+ gradients and subsequent initiation of Ca2+ wave. It is important to mention that the Lu et al. idealistic t-tubule model predicts completely different 3-D [Ca2+]i distributions with zero Fluo-3, SR Ca2+ metabolism inhibited and Ca2+ transporters heterogeneously distributed Taken together, these studies provide compelling evidence that (1) the exogenous Fluo-3 acts as a significant buffer and carrier for Ca2+-signaling in rat ventricular myocytes is regulated in the absence or presence of SR Ca2+ release and uptake 2+ signal  the relatively small size of the model compartment that contains only a single realistic t-tubule shape and spans by just a half-sarcomere inside the ventricular myocyte; and (2) the assumption that the model compartment is a repeating unit inside the cell. The structural studies, however, provide evidence that in rodent ventricular myocytes the realistic t-tubule network is quite complex, see , [6]. Th2+]i see . This as2+ channels appear to be concentrated as discrete clusters in the dyadic clefts (narrow spaces between LCC and RyR) distributed regularly along the t-tubule membrane at relatively small distances of ∼0.68 µm, et al. data in mice 2+ gradients and local membrane currents that will affect differently the spatial Ca2+ profiles as predicted with the current model. Further extending of our current t-tubule model toward more realistic geometric models containing dyadic cleft topology and L-type Ca 2+ channel clustering could help to better understand how the Ca2+ signaling is regulated in the heart.Important limitation of this study is also that we assume that the ion flux pathways are continuously distributed throughout the t-tubule membrane. Immunohistochemical studies, however, suggest that L-type Ca2+ mobile buffers  and one stationary Ca2+ buffer (troponin C) were considered only. The ventricular cells, however, contain other stationary Ca2+ buffers  or small and high mobile Ca2+ binding molecules  that were not included in the model (or may be other stationary and mobile buffers that have not been identified yet), Finally, in the present model the effects of two endogenous Ca2+ movement from the cell membrane to the cell interior relies also strongly on the presence of mobile and stationary Ca2+ buffers, including the Ca2+ indicator dye. The key findings are: (1) the model predicts lack of detectible differences in the fluorescence Ca2+ signals at the peripheral and deep myoplams when the membrane Ca2+ flux is heterogeneously distributed along the sarcolemma; (2) 100 µM mobile Fluo-3 is able to mask the pronounced spatial non-uniformities in the [Ca2+]i distribution that would occur when the SR Ca2+ metabolism is inhibited; (3) during the Ca2+ influx alone, large and steep Ca2+ gradients are predicted in the narrow sub-sarcolemmal space (∼40–50 nm in depth); (4) in rodents the branched t-tubule topology, the punctuate spatial distribution of Ca2+ flux along the sarcolemma and the endogenous Ca2+ buffers actually function to inhibit Ca2+ waves. Improved functional and structural computational models are needed to guide the experiments and to test further our understanding of how the t-tubule microanatomy and protein distributions regulate the normal cell function or cell cycle under certain pathologies. To our best knowledge, this study is the first attempt to use the finite element methods to investigate the intracellular Ca2+ responses in physiologically realistic transverse-axial t-tubule geometry.Simulations presented in this study demonstrate that the more accurate knowledge of transverse-axial t-tubule microanatomy and protein distributions along the sarcolemma is important to better understand the mechanisms regulating the excitation-contraction coupling in rat ventricular myocytes. The results demonstrate that CaVideo S1left panel); Calcium flux higher and uniform in the t-tubule (middle panel); Calcium flux uniformly distributed (right panel).100 µM Fluo-3: Calcium flux heterogeneously distributed ((5.45 MB AVI)Click here for additional data file.Video S2left panel); ATP and calmodulin immobile (right panel).Zero Fluo-3 and calcium flux heterogeneously distributed: ATP and calmodulin mobile ((4.05 MB AVI)Click here for additional data file.Video S3left panel); Zero extracellular sodium (right panel.)Zero Fluo-3 and calcium flux heterogeneously distributed: Extracellular sodium 140 mM ((4.07 MB AVI)Click here for additional data file.