Maximal Ca2 Research Articles

Eccentric Resistance Training Ameliorates Muscle Weakness in a Mouse Model of Idiopathic Inflammatory Myopathies.

High-force eccentric contractions (ECCs) have traditionally been excluded from rehabilitation programs that include patients with idiopathic inflammatory myopathies (IIMs) due to unverified fear of causing muscle damage and inflammation. In an IIM animal model that used mice with experimental autoimmune myositis (EAM), we undertook this study to investigate whether ECC training can safely and effectively be used to counteract muscle weakness in IIM. EAM was induced in BALB/c mice by immunization with 3 injections of myosin emulsified in Freund's complete adjuvant. Controls (n = 12) and mice with EAM (n = 12) were exposed to either an acute bout of 100 ECCs or 4 weeks of ECC training (20 ECCs every other day). To induce ECCs, plantar flexor muscles were electrically stimulated while the ankle was forcibly dorsiflexed. Less cell damage, as assessed by Evans blue dye uptake, was observed in the muscles of mice with EAM, compared to controls, after an acute bout of 100 ECCs (P < 0.05). Maximum Ca2+ -activated force was decreased in skinned gastrocnemius muscle fibers from mice with EAM, and this was accompanied by increased expression of endoplasmic reticulum (ER) stress proteins, including Gsp78 and Gsp94 (P < 0.05). ECC training prevented the decrease in force and the increase in ER stress proteins and also enhanced the expression and myofibrillar binding of small heat-shock proteins (HSPs) (P < 0.05), which can stabilize myofibrillar structure and function. ECC training protected against the reduction in myofibrillar force-generating capacity in an IIM mouse model, and this occurred via inhibition of ER stress responses and small HSP-mediated myofibrillar stabilization.

Relative statistics of Metals in a Smoker’s Gallbladder Stones using X-Ray Fluorescence

In this work, the X-ray fluorescence (XRF) technique was used to investigate the concentration of elements in gallbladder stones for smokers and non-smokers. Many elements have been detected in the non-smoker gallstone samples. The most important elements include calcium (Ca), potassium (K), phosphorous (P), magnesium (Mg), sodium (Na), iron (Fe) and lead (Pb). However, significantly, concentrations of the most toxic elements have been found within the group of the smokers. The maximum concentrations of toxic elements such as lead (Pb), cadmium (Cd) and cobalt (Co) were found in people older than 60 years. Furthermore, the minimum concentrations of the trace elements calcium (Ca), phosphorous (P) and sodium (Na) were detected in this age group. The relative statistics of Ca concentration in smokers’ and non-smokers’ were studied, and these groups were divided according to age. It was concluded that the maximum Ca concentration for non-smokers was found in the 20- to 40-year age group when compared with the other groups.

Phosphate has dual roles in cross-bridge kinetics in rabbit psoas single myofibrils.

In this study, we aimed to study the role of inorganic phosphate (Pi) in the production of oscillatory work and cross-bridge (CB) kinetics of striated muscle. We applied small-amplitude sinusoidal length oscillations to rabbit psoas single myofibrils and muscle fibers, and the resulting force responses were analyzed during maximal Ca2+ activation (pCa 4.65) at 15°C. Three exponential processes, A, B, and C, were identified from the tension transients, which were studied as functions of Pi concentration ([Pi]). In myofibrils, we found that process C, corresponding to phase 2 of step analysis during isometric contraction, is almost a perfect single exponential function compared with skinned fibers, which exhibit distributed rate constants, as described previously. The [Pi] dependence of the apparent rate constants 2πb and 2πc, and that of isometric tension, was studied to characterize the force generation and Pi release steps in the CB cycle, as well as the inhibitory effect of Pi. In contrast to skinned fibers, Pi does not accumulate in the core of myofibrils, allowing sinusoidal analysis to be performed nearly at [Pi] = 0. Process B disappeared as [Pi] approached 0 mM in myofibrils, indicating the significance of the role of Pi rebinding to CBs in the production of oscillatory work (process B). Our results also suggest that Pi competitively inhibits ATP binding to CBs, with an inhibitory dissociation constant of ∼2.6 mM. Finally, we found that the sinusoidal waveform of tension is mostly distorted by second harmonics and that this distortion is closely correlated with production of oscillatory work, indicating that the mechanism of generating force is intrinsically nonlinear. A nonlinear force generation mechanism suggests that the length-dependent intrinsic rate constant is asymmetric upon stretch and release and that there may be a ratchet mechanism involved in the CB cycle.

CMyBP-C phosphorylation modulates the time-dependent slowing of unloaded shortening in murine skinned myocardium.

In myocardium, phosphorylation of cardiac myosin-binding protein-C (cMyBP-C) is thought to modulate the cooperative activation of the thin filament by binding to myosin and/or actin, thereby regulating the probability of cross-bridge binding to actin. At low levels of Ca2+ activation, unloaded shortening velocity (Vo) in permeabilized cardiac muscle is comprised of an initial high-velocity phase and a subsequent low-velocity phase. The velocities in these phases scale with the level of activation, culminating in a single high-velocity phase (Vmax) at saturating Ca2+. To test the idea that cMyBP-C phosphorylation contributes to the activation dependence of Vo, we measured Vo before and following treatment with protein kinase A (PKA) in skinned trabecula isolated from mice expressing either wild-type cMyBP-C (tWT), nonphosphorylatable cMyBP-C (t3SA), or phosphomimetic cMyBP-C (t3SD). During maximal Ca2+ activation, Vmax was monophasic and not significantly different between the three groups. Although biphasic shortening was observed in all three groups at half-maximal activation under control conditions, the high- and low-velocity phases were faster in the t3SD myocardium compared with values obtained in either tWT or t3SA myocardium. Treatment with PKA significantly accelerated both the high- and low-velocity phases in tWT myocardium but had no effect on Vo in either the t3SD or t3SA myocardium. These results can be explained in terms of a model in which the level of cMyBP-C phosphorylation modulates the extent and rate of cooperative spread of myosin binding to actin.

Regional Citrate Anticoagulation Protocol for Patients with Presumed Absent Citrate Metabolism.

Regional citrate anticoagulation (RCA) is not recommended in patients with shock or severe liver failure. We designed a protocol with personalized precalculated flow settings for patients with absent citrate metabolism that abrogates risk of citrate toxicity, and maintains neutral continuous KRT (CKRT) circuit calcium mass balance and normal systemic ionized calcium levels. A single-center prospective cohort study of patients in five adult intensive care units triaged to the CVVHDF-RCA "Shock" protocol. Of 31 patients included in the study, 30 (97%) had AKI, 16 (52%) had acute liver failure, and five (16%) had cirrhosis at the start of CKRT. The median lactate was 5 mmol/L (interquartile range [IQR], 3.2-10.7), AST 822 U/L (IQR, 122-2950), ALT 352 U/L (IQR, 41-2238), total bilirubin 2.7 mg/dl (IQR, 1.0-5.1), and INR two (IQR, 1.5-2.6). The median first hemofilter life censored for causes other than clotting exceeded 70 hours. The cumulative incidence of hypernatremia (Na >148 mM), metabolic alkalosis (HCO3- >30 mM), and hypophosphatemia (P<2 mg/dl) were one out of 26 (4%), zero out of 30 (0%), and one out of 30 (3%), respectively, and were not clinically significant. Mild hypocalcemia occurred in the first 4 hours in two out of 31 patients, and corrected by hour 6 with no additional Ca supplementation beyond the per-protocol administered Ca infusion. The maximum systemic total Ca (tCa; mM)/ionized Ca (iCa; mM) ratio never exceeded 2.5. The Shock protocol can be used without contraindications and is effective in maintaining circuit patency with a high, fixed ACDA infusion rate to blood flow ratio. Keeping single-pass citrate extraction on the dialyzer >0.75 minimizes the risk of citrate toxicity even in patients with absent citrate metabolism. Precalculated, personalized dosing of the initial Ca-infusion rate from a table on the basis of the patient's albumin level and the filter effluent flow rate maintains neutral CKRT circuit calcium mass balance and a normal systemic iCa level.

Selective electrosorption of Ca2+ by MXene cathodes coupled with NiAl-LMO anodes through ion intercalation

Capacitive deionization (CDI), or electrosorption, is a desalination technology that exhibits significant potential; however, its major technical requirement of selective ion separation poses a challenge for its further practical application. Herein, a titanium carbide (MXene)–layered electrosorption electrode with high selectivity for Ca2+ was fabricated. The prepared MXene electrode had many surface hydroxyl functional groups that serve as adsorption sites for Ca2+. Ca2+ was successfully inserted into the interlayers of the MXene cathode and formed a strong interaction with [Ti-O] bonds during the capacitive deionization process. When a Ni-Al layered metal oxide anion intercalation electrode was employed as the counter electrode, Ca2+ adsorption by the MXene electrode was significantly enhanced due to the valence compensation balance effect. The maximum Ca2+ electrosorption capacity of the MXene electrode reached 1011.82 mg per gram effective MXene material, which is 6.3 times higher than that of Na+ based on the Langmuir adsorption isotherm model. The MXene electrode exhibited prominent selectivity for Ca2+ ions in the presence of Na+ and Mg2+. The Ca2+/Mg2+ selectivity factor for electrosorption reached 2.63, and Ca2+/Na+ selectivity factor could achieve 9.84, respectively. After five electrosorption/desorption cycles, the Ca2+ removal rate only decreased from 46.96% to 45.34%, suggesting that the MXene electrode has excellent stability. Our study demonstrated a novel CDI electrode and technical approach for softening water.

Responses of sweet pepper (Capsicum annum L.) cultivated in a closed hydroponic system to variable calcium concentrations in the nutrient solution.

The use of water containing calcium bicarbonate (Ca(HCO3 )2 ) at excessively high concentrations in closed hydroponic crops can cause calcium ion (Ca2+ ) accumulation in the recycled nutrient solution (NS) and concomitantly negatively affect yield and product quality. The aim of the study was to determine maximum Ca2+ concentrations that do not harm the crop and to simulate the pattern of Ca2+ accumulation when the Ca2+ concentration in the irrigation water, and concomitantly in the replenishment nutrient solution (RNS), is excessive. In the current study, irrigation water containing 1.5, 3.0, 4.5 and 6.0 mmol L-1 Ca2+ was used to prepare the RNS supplied to pepper cultivated in a closed hydroponic system. At 1.5 mmol L-1 Ca2+ , no Ca2+ accumulation was observed in the recirculating NS. However, at 3.0, 4.5 and 6.0 mmol L-1 in the irrigation water, the Ca2+ concentration in the recirculating NS, increased by the latter cropping stages to 17, 28 and 37 mmol L-1 , corresponding to 6.4, 9.0 and 10.8 dS m-1 . The accumulation of Ca2+ in the recirculating NS affected both tissue nutrient concentrations and uptake concentrations of Ca2+ , sulphate ion (SO4 2- ) and magnesium ion (Mg2+ ), but this was not the case for nitrogen (N) or potassium ion (K+ ). Growth, yield and plant water uptake were restricted at moderate (3.0 and 4.5 mmol L-1 ) and high (6.0 mmol L-1 ) external Ca2+ levels. In soilless pepper crops with zero discharge of fertigation effluents, the Ca2+ concentration in the irrigation water and the RNS should be lower than 3.0 mmol L-1 to avoid yield restrictions due to salinity. © 2021 Society of Chemical Industry.

Water softening using graphene oxide/biopolymer hybrid nanomaterials

There is a growing interest in developing more environmentally friendly softeners for hardness reduction of water supplied for domestic consumption. This work focuses on exploring biopolymer-based softeners, through the surface modification of graphene oxide (GO) with the anionic biopolymer κ-carrageenan (GO-Si(κ)CRG). The performance of the modified GO to decrease the hardness of natural waters containing high levels of Ca2+ was assessed. The sorption efficiency was dependent on the initial Ca2+ concentration and on the sorbent dose, with 8–34% removal for GO and 21–100% for GO-Si(κ)CRG. The surface modification considerably improved the adsorptive efficiency and under certain experimental conditions, it was possible to convert very hard water (300 mg L−1 CaCO3) to soft water. Importantly, the performance was not affected by the presence of other ions typically found in natural bottled waters. The kinetics was well described by pseudo-first-order and diffusion models. The multi-linear nature observed in Boyd’s and Webber’s plots suggested that both film diffusion and pore diffusion controlled the sorption rate. The maximum Ca2+ sorption capacity at monolayer coverage of GO-Si(κ)CRG was 47.6 ± 3.2 mg g−1. The electrostatic attraction between sulfonate groups and calcium cations is likely to be the main mechanism involved in the sorption process of Ca2+ by GO-Si(κ)CRG. Overall, the results indicate good prospects for the development of a new class of softeners based on the GO modification described.

In Vitro Assessment of Intracanal Calcium Hydroxide Removal Using Various Irrigation Systems: An SEM Study

The aim of this research was to assess the effectiveness of three different irrigation systems in elimination of calcium hydroxide [Ca(OH)2]from root canals. This in vitro research included ninety recently extracted mandibular premolar teeth with a solitary root. The sample teeth were subjected to disinfection employing sodium hypochlorite at 5.25%. The root canal preparation was performed followed by placement of premixed Ca(OH)2 within every canal. The sample teeth were then randomly allocated into one of the three experimental groups (each comprising 30 samples) as follows: Group 1, NaviTip FX irrigation system; group 2, Vibringe sonic irrigation system; and group 3, EndoVac apical negative pressure irrigation system. Following the preparation, each of the sample teeth was examined beneath a scanning electron microscope (SEM) at a magnification of 1000×. An intragroup comparative analysis revealed that the highest Ca(OH)2 elimination was seen at middle third (0.82 ± 0.09, 0.30 ± 0.11) in NaviTip FX irrigation system and Vibringe sonic irrigation system, respectively. Higher Ca(OH)2 was eliminated at the apical third (0.26 ± 0.02) by the EndoVac irrigation system. At coronal third, maximum Ca(OH)2 removal was seen in EndoVac irrigation system (0.49 ± 0.03). A statistically significant difference was noted amid Vibringe sonic irrigation and EndoVac irrigation systems. The intergroup evaluation of Ca(OH)2 elimination at coronal, middle, and apical third showed a statistically significant difference between NaviTip FX irrigation and Vibringe sonic irrigation as well as between NaviTip FX irrigation and EndoVac irrigation at a p value of 0.001. The difference between EndoVac irrigation and Vibringe sonic irrigation was not statistically significant. Amid the limitations of this research, this research concludes that none among the irrigation methods employed could totally eliminate the Ca(OH)2 off the root canals. Nevertheless, EndoVac apical negative pressure irrigation has slightly superior potential in eliminating Ca(OH)2 from the root canals in comparison with Vibringe sonic irrigation as well as the NaviTip FX irrigation system. Even though the Ca(OH)2 is largely suggested as medicament, its elimination at the point of canal obturation is likewise significant and complex as any remaining portion may avoid sealer diffusion inside the dentinal tubules, consequently substantially escalating the apical leakage of endodontically treated teeth.

Effectiveness of phytase pre-treatment on growth performance, nutrient digestibility and mineral status of common carp (Cyprinus carpio) juveniles fed Moringa by-product based diet.

Several anti-nutritional substances are found in plant derivatives for example phytate, that make the nutrients and minerals unavailable to fish, hence leading to poor growth performance. Presence of the anti-nutrient factor such as phytate is a chelated compound and need enzyme for its breakdown and availability of nutrients to improve fish growth. This research work was performed to check the improvement of overall performance of Cyprinus carpio fingerlings by the help of phytase addition in Moringa oleifera by- products based diet. Combination of Moringa seed meal and Moringa leaf meal was utilized as test ingredient to formulate seven test feeds, containing graded levels of phytase (0, 500, 650, 800, 950, 1100 and 1250 FTU kg−1). In feeding trial of 70 days, fingerlings were given feed two times in a day at the rate of 4% of wet weight of their bodies and faeces were collected. According to current results, it was found that growth performance parameters i.e. weight gain; 25 g, specific growth rate; 1.67 and feed conversion ratio; 1.10 were improved to maximum at 950 FTU kg−1. Digestibility of nutrients (crude protein; 73%, crude fat; 71% and gross energy; 67%) and minerals absorption was also maximum (Ca; 70%, Zn; 66%, K; 74%, Mn; 66% and P; 71%) at 950 FTU kg−1. Lowest growth efficiency, nutrient digestibility and mineral absorption were observed in fingerlings fed at control diet (0 FTU kg−1). Results of the current study, proved that 950 FTU kg−1 is the most optimum level of phytase to formulate economical and ecofriendly feed for improved growth of C. carpio fingerlings as it decreases the discharge of minerals and nutrients in water bodies.

Exercise-induced alterations of myocardial sarcomere dynamics are associated with hypophosphorylation of cardiac troponin I.

Although the knowledge of sports cardiology advanced significantly in the recent years, the molecular mechanisms by which exercise training augments cardiac performance is poorly understood. Here we aimed at determining left ventricular (LV) myocardial sarcomeric protein modifications in a rat model of exercise training and detraining. Young male Wistar rats were divided into exercised (Ex) and control (Co) groups. Trained rats swam 200 min/day for 12 weeks. Detrained (DEx) and control (DCo) rats remained sedentary for 8 weeks after completion of the 12-week-long protocol. Ca2+-regulated active force production (Fa⁢c⁢t⁢i⁢v⁢e), its Ca2+-sensitivity (pCa50) and Ca2+-independent passive tension (Fp⁢a⁢s⁢s⁢i⁢v⁢e) were determined in isolated permeabilized cardiomyocytes and phosphorylation levels of sarcomeric proteins were assayed by biochemical methods. Means of maximal Ca2+-activated isometric force (Fm⁢a⁢x) and pCa50 values were higher (p < 0.05) in the Ex group (28.0 ± 1.4 kN/m2 and 5.91 ± 0.03, respectively, mean ± SEM) than those in the Co group (15.8 ± 0.8 kN/m2 and 5.81 ± 0.03, respectively). Fp⁢a⁢s⁢s⁢i⁢v⁢e did not differ between these two groups. The level of cardiac troponin I (cTnI) phosphorylation decreased upon exercise (from 1.00 ± 0.02 to 0.66 ± 0.06, p < 0.05; in relative units). Site specific phosphorylation assays revealed cTnI hypophosphorylations at the protein kinase A (PKA)-specific Ser-22/23 sites and at the protein kinase C (PKC)-specific Thr-143 site. Mechanical and biochemical parameters of the DEx and DCo groups did not differ from each other following the detraining period. Exercise-induced hypertrophy is associated with reversible increases in Ca2+-dependent force production and its Ca2+-sensitivity in LV cardiomyocytes, which can be associated with changes in cTnI phosphorylation.

Effects of mavacamten on Ca2+ sensitivity of contraction as sarcomere length varied in human myocardium.

Heart failure can reflect impaired contractile function at the myofilament level. In healthy hearts, myofilaments become more sensitive to Ca2+ as cells are stretched. This represents a fundamental property of the myocardium that contributes to the Frank-Starling response, although the molecular mechanisms underlying the effect remain unclear. Mavacamten, which binds to myosin, is under investigation as a potential therapy for heart disease. We investigated how mavacamten affects the sarcomere-length dependence of Ca2+ -sensitive isometric contraction to determine how mavacamten might modulate the Frank-Starling mechanism. Multicellular preparations from the left ventricular-free wall of hearts from organ donors were chemically permeabilized and Ca2+ activated in the presence or absence of 0.5-μM mavacamten at 1.9 or 2.3-μm sarcomere length (37°C). Isometric force and frequency-dependent viscoelastic myocardial stiffness measurements were made. At both sarcomere lengths, mavacamten reduced maximal force and Ca2+ sensitivity of contraction. In the presence and absence of mavacamten, Ca2+ sensitivity of force increased as sarcomere length increased. This suggests that the length-dependent activation response was maintained in human myocardium, even though mavacamten reduced Ca2+ sensitivity. There were subtle effects of mavacamten reducing force values under relaxed conditions (pCa 8.0), as well as slowing myosin cross-bridge recruitment and speeding cross-bridge detachment under maximally activated conditions (pCa 4.5). Mavacamten did not eliminate sarcomere length-dependent increases in the Ca2+ sensitivity of contraction in myocardial strips from organ donors at physiological temperature. Drugs that modulate myofilament function may be useful therapies for cardiomyopathies.

Purification of sarcoplasmic reticulum vesicles from horse gluteal muscle

We have analyzed protein expression and enzyme activity of the sarcoplasmic reticulum Ca2+-transporting ATPase (SERCA) in horse gluteal muscle. Horses exhibit a high incidence of recurrent exertional rhabdomyolysis, with myosolic Ca2+ proposed, but yet to be established, as the underlying cause. To better assess Ca2+ regulatory mechanisms, we developed an improved protocol for isolating sarcoplasmic reticulum (SR) vesicles from horse skeletal muscle, based on mechanical homogenization and optimized parameters for differential centrifugation. Immunoblotting identified the peak subcellular fraction containing the SERCA1 protein (fast-twitch isoform). Gel analysis using the Stains-all dye demonstrated that calsequestrin (CASQ) and phospholipids are highly enriched in the SERCA-containing subcellular fraction isolated from horse gluteus. Immunoblotting also demonstrated that these horse SR vesicles show low content of glycogen phosphorylase (GP), which is likely an abundant contaminating protein of traditional horse SR preps. The maximal Ca2+-activated ATPase activity (Vmax) of SERCA in horse SR vesicles isolated using this protocol is 5‒25-fold greater than previously-reported SERCA activity in SR preps from horse skeletal muscle. We propose that this new protocol for isolating SR vesicles will be useful for determining enzymatic parameters of horse SERCA with high fidelity, plus assessing regulatory effect of SERCA peptide subunit(s) expressed in horse muscle.

Harnessing Environmental Ca2+ for Extracellular Protein Thermostabilization.

Ca2+ is the third-most prevalent metal ion in the environment. EF hands are common Ca2+-binding motifs found in both extracellular and intracellular proteins of eukaryotes and prokaryotes. Cytoplasmic EF hand proteins often mediate allosteric control of signal transduction pathway components in response to intracellular Ca2+ concentration fluctuations by coupling Ca2+ binding to changes in protein structure. We show that an extracellular structural Ca2+-binding site mediates protein thermostabilization by such conformational coupling as well. Binding Ca2+ to the EF hand of the extracellular (periplasmic) Escherichia coli glucose-galactose binding protein thermostabilizes this protein by ∼17 K relative to its Ca2+-free form. Using statistical thermodynamic analysis of a fluorescent conjugate of ecGGBP that reports simultaneously on ligand binding and multiple conformational states, we found that its Ca2+-mediated stabilization is determined by conformational coupling mechanisms in two independent conformational exchange reactions. Binding to folded and unfolded states determines the maximum Ca2+-mediated stability. A disorder → order transition accompanies the formation of the Ca2+ complex in the folded state and dictates the minimum Ca2+ concentration at which the Ca2+-bound state becomes dominant. Similar transitions also encode the structural changes necessary for Ca2+-mediated control elements in signal transduction pathways. Ca2+-mediated thermostabilization and allosteric control, therefore, share a fundamental conformational coupling mechanism, which may have implications for the evolution of EF hands.

Effects of varying concentrations of different salts on mineral composition of leaves and roots of three pomegranate (Punica granatum L.) cultivars

The present investigation assesses the behavior of three pomegranate cultivars (Punica granatum L.) (‘Wonderful’, ‘Ermioni’ and ‘Grenada’) under increased concentrations of three salts (NaCl, KCl and K2SO4) after 150 days of growth in vivo conditions. The aim of this research was to study the nutrient accumulation and distribution in leaves and roots and its effects on total chlorophyll (a + b), carotenoids, photosynthetic rate and stomatal conductance of leaves. In general, high salt supply of KCl and K2SO4 led to significant decline in Ca and Mg concentration in leaves in all cultivars. ‘Grenada’ found with the highest concentration of Mn in leaves under all salt treatments. Regarding the roots, ‘Wonderful’ had the highest Mg concentration at 25, 50 and 75 mM of KCl and K2SO4, while ‘Ermioni’ had the maximum Ca concentration in roots. Chlorophyll concentration was reduced by the effect of KCl and K2SO4 salts in all cultivars. Furthermore, K2SO4 salt significantly reduced carotenoid concentration in leaves in all cultivars, especially ‘Wonderful’ showed 63.36 % reduction. ‘Grenada’ had the lowest photosynthetic rate and K2SO4 salt exerted a significant effect in all cultivars. Regarding stomatal conductance, ‘Ermioni’ had the highest values with KCl and K2SO4 salt treatments, while ‘Grenada’ was the most affected cultivar by all salts and at all concentrations. Our findings show the considerable potential of exploiting saline soils to obtain pomegranate plants with higher level of salt tolerance. Furthermore, our results suggest that ‘Wonderful’ and ‘Ermioni’ pomegranate cultivars may be cultivated also under marginal saline conditions.

Glycolaldehyde-Derived High-Molecular-Weight Advanced Glycation End-Products Induce Cardiac Dysfunction through Structural and Functional Remodeling of Cardiomyocytes.

High-molecular-weight advanced glycation end-products (HMW-AGEs) are abundantly present in our Western diet. There is growing evidence reporting that HMW-AGEs contribute to the development of cardiovascular dysfunction in vivo, next to the well-known low-molecular-weight AGEs. The goal of our study is to assess the ultrastructure and function of cardiomyocytes after chronic exposure to HMW-AGEs. A better understanding of underlying mechanisms is essential to create new opportunities for further research on the specific role of HMW-AGEs in the development and progression of cardiovascular diseases. Adult male rats were randomly assigned to daily intraperitoneal injection for six weeks with either HMW-AGEs (20 mg/kg/day) or a control solution. Hemodynamic measurements were performed at sacrifice. Single cardiomyocytes from the left ventricle were obtained by enzymatic dissociation through retrograde perfusion of the aorta. Unloaded cell shortening, time to peak and time to 50% relaxation were measured during field stimulation and normalized to diastolic length. L-type Ca2+ current density (ICaL) and steady-state inactivation of ICaL were measured during whole-cell ruptured patch clamp. Myofilament functional properties were measured in membrane-permeabilized cardiomyocytes. Ultrastructural examination of cardiac tissue was performed using electron microscopy. Rats injected with HMW-AGEs displayed in vivo cardiac dysfunction, characterized by significant changes in left ventricular peak rate pressure rise and decline accompanied with an increased heart mass. Single cardiomyocytes isolated from the left ventricle revealed concentric hypertrophy, indicated by the increase in cellular width. Unloaded fractional cell shortening was significantly reduced in cells derived from the HMW-AGEs group and was associated with slower kinetics. Peak L-type Ca2+ current density was significantly decreased in the HMW-AGEs group. L-type Ca2+ channel availability was significantly shifted towards more negative potentials after HMW-AGEs injection. The impact of HMW-AGEs on myofilament function was measured in membrane-permeabilized cardiomyocytes showing a reduction in passive force, maximal Ca2+ activated force and rate of force development. Ultrastructural examination of cardiac tissue demonstrated adverse structural remodeling in HMW-AGEs group characterized by a disruption of the cyto-architecture, a decreased mitochondrial density and altered mitochondrial function. Our data indicate that HMW-AGEs induce structural and functional cellular remodeling via a different working mechanism as the well-known LMW-AGEs. Results of our research open the door for new strategies targeting HMW-AGEs to improve cardiac outcome.

Dimerization of SERCA2a Enhances Transport Rate and Improves Energetic Efficiency in Living Cells

The type 2a sarco/endoplasmic reticulum (ER) Ca2+-ATPase (SERCA2a) plays a key role in intracellular Ca2+ regulation in the heart. We have previously shown evidence of stable homodimers of SERCA2a in heterologous cells and cardiomyocytes. However, the functional significance of the pump dimerization remains unclear. Here, we analyzed how SERCA2a dimerization affects ER Ca2+ transport. Fluorescence resonance energy transfer experiments in HEK293 cells transfected with fluorescently labeled SERCA2a revealed increasing dimerization of Ca2+ pumps with increasing expression level. This concentration-dependent dimerization provided means of comparison of the functional characteristics of monomeric and dimeric pumps. SERCA-mediated Ca2+ uptake was measured with the ER-targeted Ca2+ sensor R-CEPIA1er in cells cotransfected with SERCA2a and ryanodine receptor. For each individual cell, the maximal ER Ca2+ uptake rate and the maximal Ca2+ load, together with the pump expression level, were analyzed. This analysis revealed that the ER Ca2+ uptake rate increased as a function of SERCA2a expression, with a particularly steep, nonlinear increase at high expression levels. Interestingly, the maximal ER Ca2+ load also increased with an increase in the pump expression level, suggesting improved catalytic efficiency of the dimeric species. Reciprocally, thapsigargin inhibition of a fraction of the population of SERCA2a reduced not only the maximal ER Ca2+ uptake rate but also the maximal Ca2+ load. These data suggest that SERCA2a dimerization regulates Ca2+ transport by improving both the SERCA2a turnover rate and catalytic efficacy. Analysis of ER Ca2+ uptake in cells cotransfected with human wild-type SERCA2a (SERCA2aWT) and SERCA2a mutants with different catalytic activity revealed that an intact catalytic cycle in both protomers is required for enhancing the efficacy of Ca2+ transport by a dimer. The data are consistent with the hypothesis of functional coupling of two SERCA2a protomers in a dimer that reduces the energy barrier of rate-limiting steps of the catalytic cycle of Ca2+ transport.

Abstract CT135: A phase 1b study of palbociclib + nab-paclitaxel in patients (pts) with metastatic adenocarcinoma of the pancreas (PDAC)

Abstract Palbociclib (P), an oral cyclin-dependent kinase 4/6 (CDK4/6) inhibitor, has shown antitumor activity in preclinical PDAC models. In Avatar PDAC models, P+ nab-paclitaxel (A) was more effective than either agent alone and similar to P+A+gemcitabine. This study assessed safety, pharmacokinetics (PK), pharmacodynamics (PD), preliminary efficacy, and maximum tolerated dose (MTD) of P+A in pts with advanced PDAC. Pts with metastatic PDAC, Karnofsky performance status ≥70 and adequate organ function were eligible. In the dose escalation phase, P was administered orally once daily at doses ranging from 75-125 mg/d for 21 of every 28 days (3/1 schedule). A was administered intravenously at 100-125 mg on days 1, 8 and 15 every 28 days. Additional cohorts were P 75 mg (3/1 schedule or continuously) with A (100- and 125-mg dose, biweekly). The prespecified efficacy target was 12-month survival probability &amp;gt;65% at MTD. In total, 76 pts (median age 61 years [range 39-75], 45% female) received a median of 4 treatment cycles (range 1-21); 44 (57.9%) pts had liver disease and 61 (80.3%) had prior treatment for advanced disease. Four dose-limiting toxicities were observed: grade 3 mucositis (1), grade 4 neutropenia (2), and febrile neutropenia (1). Grade 3/4 adverse events were reported in 67 (88.2%) pts; events reported for ≥5% of pts were neutropenia (61.8%), leukopenia (26.3%), anemia (22.4%), asthenia (9.2%), lymphopenia (7.9%), and peripheral neuropathy (5.3%). PK demonstrated no significant drug-drug interactions. MTD was P 100 mg (3/1 schedule) and A 125 mg (days 1, 8 and 15 every 28 days). Objective response rate and disease control rate in the 23 pts treated at MTD were 13% and 65.2%, respectively. In these pts, median progression-free survival, median overall survival, and survival probability at 12 months were 5.3 months (95% CI: 3.5-9.7), 12.1 months (95% CI: 6.4-14.8), and 50% (95% CI: 29.9%-67.2%), respectively. Thirty of 57 analyzed pts (52.6%) had an on-treatment maximum Ca 19-9 reduction &amp;gt;50% from baseline. In paired skin punch biopsies (26 pts at any dose level, including 13 pts at MTD), pRb decreased during P dosing, increased above baseline during the week off P, and decreased again following P dosing, demonstrating PD modulation of Rb phosphorylation. CDKN2A, RAS, and TP53 mutations were analyzed in plasma DNA; clinical outcome of pts without alterations detected (n=58, 26, and 28, respectively) was superior to those with alterations detected (n=12, 44, and 42 pts, respectively). In conclusion, P+A was tolerated, with side effects as expected based on single agent profiles, and no significant PK interactions. MTD was P 100 mg (3/1 schedule) + A 125 mg (days 1, 8, and 15 every 28 days). PD observations were consistent with CDK4/6 inhibition. The combination regimen did not meet the prespecified efficacy threshold.Pfizer (NCT02501902) Citation Format: Manuel Hidalgo, Rocio Garcia Carbonero, Kian-Huat Lim, Wells Messersmith, Ignacio Garrido-Laguna, Erkut Borazanci, Andrew Lowy, Laura Medina Rodriguez, Daniel Laheru, Xin Huang, Meggan Tammaro, Jean-François Martini, Arthur Kudla, Kenneth Kern, Teresa Macarulla. A phase 1b study of palbociclib + nab-paclitaxel in patients (pts) with metastatic adenocarcinoma of the pancreas (PDAC) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr CT135.

Abstract MP105: Python Post-prandial Cardiac Adaptation is Supported by Enhanced Metabolism, Reduced Sarcomere Passive Tension, and Epigenetic Remodeling

Pythons are infrequent feeders that can ingest meals equal to their own body mass. The extreme metabolic response required to digest such large meals is associated with a dramatic increase in the mass of most organs, including the heart. Recently, we have been able to assess functional effects of feeding using isolated python cardiomyocytes and myofibrils, advancing our understanding of extreme cardiac adaptation in python ( Python regius ). Twenty-four hours after feeding, python cardiomyocytes showed prolonged Ca 2+ transients, increased maximal tension and Ca 2+ sensitivity of myofibrils as compared to fasted pythons. Post-prandial positive inotropy was accompanied by enhanced metabolic output via increased mitochondrial ATP production rate and by AMP-dependent kinase (AMPK) activation and phosphofructokinase-2 reduction, suggesting a key role for fatty acid, but not glucose, metabolism after feeding. In addition, 24h post-fed hearts had significantly reduced tissue stiffness and myofibril passive tension. Finally, chromatin condensation was reduced about 30% after feeding in python cardiomyocytes and confirmed by increased histone acetylation, indicating a predominant role for epigenetics in post-prandial adaptation. These results suggest that feeding promotes positive cardiac inotropy in python via a number of coordinated mechanisms to enhance energy production, increase myofibril and tissue compliance, and increase chromatin accessibility. As heart failure is commonly characterized by depressed contractility, compromised energetics, and increased tissue stiffness, assessing post-prandial adaptation in python hearts provides us with powerful insights that could inform the development of therapeutics for human heart diseases.

Research of Contact Response of an Elastic Sphere Impacting a Rigid Plate Based on the Mass-Spring-Damped Oscillator Model and Dimensionless Parameter \omega_\Omega

The contact theory, such as the Hertz theory and Hunt-Crossley model can just solve the contact response when the sphere goes to the maximum compression but can't solve the contact process. In traditional research the impact process is considered as a single degree of freedom dynamical spring-damping system, however, the calculated contact force is much greater than the actual one. Based on the shortage of the conducted studies, a new dimensionless parameter Omega is proposed in this paper, the mass-spring-damped oscillator model (MSDOM) and the new contact theory with the consideration of the sphere mass and Omega are established. The correctness of the theoretical model is verified by 3D simulation, and the optimal contact force model is obtained. Furthermore, influence rules of material properties and contact parameters on contact response were studied. The results show that the contact deformation, velocity, piecewise nonlinear contact force and acceleration based on MSDOM and Omega are effective to calculate the contact response. The contact stiffness, damping, material properties of sphere and elastic modulus of the plate have different influence on the contact response while the Poisson's ratio nu_2 has little effect on it.