Sarco/endoplasmic Reticulum Calcium ATPase Research Articles

Myocardial Posttranscriptional Landscape in Peripartum Cardiomyopathy

BACKGROUND: Pregnancy imposes significant cardiovascular adaptations, including progressive increases in plasma volume and cardiac output. For most women, this physiological adaptation resolves at the end of pregnancy, but some women develop pathological dilatation and ultimately heart failure late in pregnancy or in the postpartum period, manifesting as peripartum cardiomyopathy (PPCM). Despite the mortality risk of this form of heart failure, the molecular mechanisms underlying PPCM have not been extensively examined in human hearts. METHODs: Protein and metabolite profiles from left ventricular tissue of end-stage PPCM patients (N=6–7) were compared with dilated cardiomyopathy (DCM; N=5–6) and nonfailing donors (N=7–18) using unbiased quantitative mass spectrometry. All samples were derived from the Sydney Heart Bank. Data are available via ProteomeXchange with identifier PXD055986. Differential protein expression and metabolite abundance and Kyoto Encyclopedia of Genes and Genomes pathway analyses were performed. RESULTS: Proteomic analysis identified 2 proteins, SBSPON (somatomedin B and thrombospondin type 1 domain-containing protein precursor) and TNS3 (tensin 3), that were uniquely downregulated in PPCM. SBSPON, an extracellular matrix protein, and TNS3, involved in actin remodeling and cell signaling, may contribute to impaired tissue remodeling and fibrosis in PPCM. Metabolomic analysis revealed elevated levels of homogentisate and deoxycholate and reduced levels of lactate and alanine in PPCM, indicating disrupted metabolic pathways and glucose utilization. Both PPCM and DCM shared pathways related to inflammation, immune responses, and signal transduction. However, thyroid hormone signaling was notably reduced in PPCM, affecting contractility and calcium handling through altered expression of PLN (phospholamban) and Sarcoendoplasmic Reticulum Calcium ATPase (SERCA). Enhanced endoplasmic reticulum stress and altered endocytosis pathways in PPCM suggested additional mechanisms of energy metabolism disruption. CONCLUSIONS: The present study reveals unique posttranslational molecular features of the PPCM myocardium, which mediates cellular and metabolic remodeling, and holds promise as potential targets for therapeutic intervention.

Quinoline- and pyrimidine-based allosteric modulators of the sarco/endoplasmic reticulum calcium ATPase.

Small-molecule allosteric activators of the enzyme sarco/endoplasmic reticulum calcium ATPase (SERCA) hold promise as novel experimental tools to manipulate intracellular calcium concentrations and as therapeutic agents to treat medical conditions associated with elevated cytosolic calcium levels. Here, we synthesized and characterized 20 analogs of the known allosteric SERCA activator CDN1163 and tested their ability to stimulate SERCA activity. The structures of the compounds varied in the alkyl group of the parent scaffold's ether moiety as well as in the composition of the nitrogenous aromatic ring system. The most active compounds exhibited potencies in the sub-micromolar range while increasing enzyme activity by more than 25%. The observed structure-activity relationships indicated that bulky alkyl groups in the ether moiety along with a quinoline ring methyl substituent were beneficial for activity. Replacement of the quinoline by a pyrimidine ring system reduced activity. To conceive a potential mechanism of action, we generated a molecular model of the transition state of SERCA when undergoing the rate-limiting step of its catalytic cycle. Subsequent blind docking with CDN1163 identified a high-affinity binding site close to the enzyme's ATP binding pocket, suggesting that the activators may accelerate SERCA's catalytic cycle by aiding in ATP binding and positioning.

NetSci: A Library for High Performance Biomolecular Simulation Network Analysis Computation.

We present the NetSci program-an open-source scientific software package designed for estimating mutual information (MI) between data sets using GPU acceleration and a k-nearest-neighbor algorithm. This approach significantly enhances calculation speed, achieving improvements of several orders of magnitude over traditional CPU-based methods, with data set size limits dictated only by available hardware. To validate NetSci, we accurately compute MI for an analytically verifiable two-dimensional Gaussian distribution and replicate the generalized correlation (GC) analysis previously conducted on the B1 domain of protein G. We also apply NetSci to molecular dynamics simulations of the Sarcoendoplasmic Reticulum Calcium-ATPase (SERCA) pump, exploring the allosteric mechanisms and pathways influenced by ATP and 2'-deoxy-ATP (dATP) binding. Our analysis reveals distinct allosteric effects induced by ATP compared to dATP, with predicted information pathways from the bound nucleotide to the calcium-binding domain differing based on the nucleotide involved. NetSci proves to be a valuable tool for estimating MI and GC in various data sets and is particularly effective for analyzing intraprotein communication and information transfer.

New Small-Molecule SERCA Inhibitors Enhance Treatment Efficacy in Lenvatinib-Resistant Papillary Thyroid Cancer.

Papillary thyroid cancer (PTC) is one of the most treatable forms of cancer, with many cases being fully curable. However, resistance to anticancer drugs often leads to metastasis or recurrence, contributing to the failure of cancer therapy and, ultimately, patient mortality. The mechanisms underlying molecular differences in patients with metastatic or recurrent PTC, particularly those resistant to anticancer drugs through epigenetic reprogramming, remain poorly understood. Consequently, refractory PTC presents a critical challenge, and effective therapeutic strategies are urgently needed. Therefore, this study aimed to identify small-molecule inhibitors to enhance treatment efficacy in lenvatinib-resistant PTC. We observed an increase in sarco/endoplasmic reticulum calcium ATPase (SERCA) levels in patient-derived lenvatinib-resistant PTC cells compared with lenvatinib-sensitive ones, highlighting its potential as a therapeutic target. We subsequently identified two SERCA inhibitors [candidates 40 (isoflurane) and 42 (ethacrynic acid)] through in silico screening. These candidates demonstrated significant tumor shrinkage in a xenograft tumor model and reduced cell viability in patient-derived lenvatinib-resistant PTC cells when used in combination with lenvatinib. Our findings have potential clinical value for the development of new combination therapies to effectively target highly malignant, anticancer drug-resistant cancers.

SARS-CoV-2 envelope protein alters calcium signaling via SERCA interactions

The clinical management of severe COVID-19 cases is not yet well resolved. Therefore, it is important to identify and characterize cell signaling pathways involved in virus pathogenesis that can be targeted therapeutically. Envelope (E) protein is a structural protein of the virus, which is known to be highly expressed in the infected host cell and is a key virulence factor; however, its role is poorly characterized. The E protein is a single-pass transmembrane protein that can assemble into a pentamer forming a viroporin, perturbing Ca2+ homeostasis. Because it is structurally similar to regulins such as, for example, phospholamban, that regulate the sarco/endoplasmic reticulum calcium ATPases (SERCA), we investigated whether the SARS-CoV-2 E protein affects the SERCA system as an exoregulin. Using FRET experiments we demonstrate that E protein can form oligomers with regulins, and thus can alter the monomer/multimer regulin ratio and consequently influence their interactions with SERCAs. We also confirm that a direct interaction between E protein and SERCA2b results in a decrease in SERCA-mediated ER Ca2+ reload. Structural modeling of the complexes indicates an overlapping interaction site for E protein and endogenous regulins. Our results reveal novel links in the host-virus interaction network that play an important role in viral pathogenesis and may provide a new therapeutic target for managing severe inflammatory responses induced by SARS-CoV-2.

PKM2 regulates metabolic flux and oxidative stress in the murine heart.

Cardiac metabolism ensures a continuous ATP supply, primarily using fatty acids in a healthy state and favoring glucose in pathological conditions. Pyruvate kinase muscle (PKM) controls the final step of glycolysis, with PKM1 being the main isoform in the heart. PKM2, elevated in various heart diseases, has been suggested to play a protective role in cardiac stress, but its function in basal cardiac metabolism remains unclear. We examined hearts from global PKM2 knockout (PKM2-/-) mice and found reduced intracellular glucose. Isotopic tracing of U-13C glucose revealed a shift to biosynthetic pathways in PKM2-/- cardiomyocytes. Total ATP content was two-thirds lower in PKM2-/- hearts, and functional analysis indicated reduced mitochondrial oxygen consumption. Total reactive oxygen species (ROS) and mitochondrial superoxide were also increased in PKM2-/- cardiomyocytes. Intriguingly, PKM2-/- hearts had preserved ejection fraction compared to controls. Mechanistically, increased calcium/calmodulin-dependent kinase II activity and phospholamban phosphorylation may contribute to higher sarcoendoplasmic reticulum calcium ATPase 2 pump activity in PKM2-/- hearts. Loss of PKM2 led to altered glucose metabolism, diminished mitochondrial function, and increased ROS in cardiomyocytes. These data suggest that cardiac PKM2 acts as an important rheostat to maintain ATP levels while limiting oxidative stress. Although loss of PKM2 did not impair baseline contractility, its absence may make hearts more sensitive to environmental stress or injury.

Abstract Mo119: PI3Kgamma regulates CamKII mediated phosphorylation of phospholamban and SR calcium cycling

Although phosphoinositide 3-kinase (PI3Kγ) null mice (PI3Kγ -/- ) show increased ventricular rate/contraction, it is unknown whether PI3Kγ regulates calcium recycling machinery underlying this phenotype. Primary adult cardiomyocytes from PI3Kγ -/- mice show altered sarcoendoplasmic reticulum (SR) calcium cycling following caffeine. Unexpectedly, PI3Kγ -/- cardiomyocytes showed significant reduction in phosphorylation of phospholamban (PLN) at Thr17, a key regulator of SR calcium re-uptake without changes in phosphorylation at Ser16. Furthermore, loss in PLN phosphorylation in PI3Kγ -/- cardiomyocytes was associated with augmented interaction with SR calcium ATPase (SERCA). Surprisingly, cardiomyocyte-specific overexpression of kinase-dead PI3Kγ (PI3Kγ inact ) in global PI3Kγ -/- mice (PI3Kγ inact /PI3Kγ -/- ) normalized caffeine-induced calcium re-uptake, PLN phosphorylation at Thr17 and decreased PLN-SERCA interaction. These data suggested kinase-independent function of PI3Kγ in regulation of SR calcium load and PLN phosphorylation. Since phosphorylation of Thr17 of PLN is carried out by Ca 2+ /Calmodulin dependent protein kinase (CamKII), we probed for the role of PI3Kγ in regulation of CamKII in PLN phosphorylation. Mechanistically, PI3Kγ exhibits scaffolding function in recruitment of CamKII to PLN at SR to mediate PLN phosphorylation. Furthermore, we showed that PI3Kγ directly interacts with CamKII. The study unravels a yet to be recognized kinase-independent role of PI3Kγ in regulating PLN with implications in cardiac function.

The molecular determinants of calcium ATPase inhibition by curcuminoids

The natural product curcumin and some of its analogs are known inhibitors of the transmembrane enzyme sarco/endoplasmic reticulum calcium ATPase (SERCA). Despite their widespread use, the curcuminoids' binding site in SERCA and their relevant interactions with the enzyme remain elusive. This lack of knowledge has prevented the development of curcuminoids into valuable experimental tools or into agents of therapeutic value. We used the crystal structures of SERCA in its E1 conformation in conjunction with computational tools such as docking and surface screens to determine the most likely curcumin binding site, along with key enzyme/inhibitor interactions. Additionally, we determined the inhibitory potencies and binding affinities for a small set of curcumin analogs. The predicted curcumin binding site is a narrow cleft in the transmembrane section of SERCA, close to the transmembrane/cytosol interface. In addition to pronounced complementarity in shape and hydrophobicity profiles between curcumin and the binding pocket, several hydrogen bonds were observed that were spread over the entire curcumin scaffold, involving residues on several transmembrane helices. Docking-predicted interactions were compatible with experimental observations for inhibitory potencies and binding affinities. Based on these findings, we propose an inhibition mechanism that assumes that the presence of a curcuminoid in the binding site arrests the catalytic cycle of SERCA by preventing it from converting from the E1 to the E2 conformation. This blockage of conformational change is accomplished by a combination of steric hinderance and hydrogen-bond-based cross-linking of transmembrane helices that require flexibility throughout the catalytic cycle.

Impact of RAAS Receptors and Membrane-Bound Transporter System in the Left Ventricle during the Long-Term Control of Hypertension.

The Renin-Angiotensin-Aldosterone System (RAAS) has been implicated in systemic and neurogenic hypertension. The infusion of RAAS inhibitors blunted arterial pressure and efficacy of use-dependent synaptic transmission in sympathetic ganglia. The current investigation aims to elucidate the impact of RAAS-mediated receptors on left ventricular cardiomyocytes and the role of the sarcolemma-bound carrier system in the heart of the hypertensive transgene model. A significant increase in mRNA and the protein expression for angiotensin II (AngII) receptor subtype-1 (AT1R) was observed in (mREN2)27 transgenic compared to the normotensive rodents. Concurrently, there was an upregulation in AT1R and a downregulation in the MAS1 proto-oncogene protein receptor as well as the AngII subtype-2 receptor in hypertensive rodents. There were modifications in the expressions of sarcolemma Na+-K+-ATPase, Na+-Ca2+ exchanger, and Sarcoendoplasmic Reticulum Calcium ATPase in the transgenic hypertensive model. These observations suggest chronic RAAS activation led to a shift in receptor balance favoring augmented cardiac contractility and disruption in calcium handling through modifications of membrane-bound carrier proteins and blood pressure. The study provides insight into mechanisms underlying RAAS-mediated cardiac dysfunction and highlights the potential value of targeting the protective arm of AngII in hypertension.

Branched-chain amino acids and L-alanine supplementation ameliorate calcium dyshomeostasis in sarcopenia: New insights for nutritional interventions.

Introduction: During aging, sarcopenia and decline in physiological processes lead to partial loss of muscle strength, atrophy, and increased fatigability. Muscle changes may be related to a reduced intake of essential amino acids playing a role in proteostasis. We have recently shown that branched-chain amino acid (BCAA) supplements improve atrophy and weakness in models of muscle disuse and aging. Considering the key roles that the alteration of Ca2+-related homeostasis and store-operated calcium entry (SOCE) play in several muscle dysfunctions, this study has been aimed at gaining insight into the potential ability of BCAA-based dietary formulations in aged mice on various players of Ca2+ dyshomeostasis. Methods: Seventeen-month-old male C57BL/6J mice received a 12-week supplementation with BCAAs alone or boosted with two equivalents of L-alanine (2-Ala) or with dipeptide L-alanyl-L-alanine (Di-Ala) in drinking water. Outcomes were evaluated on ex vivo skeletal muscles indices vs. adult 3-month-old male C57BL/6J mice. Results: Ca2+ imaging confirmed a decrease in SOCE and an increase of resting Ca2+ concentration in aged vs. adult mice without alteration in the canonical components of SOCE. Aged muscles vs. adult muscles were characterized by a decrease in the expression of ryanodine receptor 1 (RyR1), the Sarco-Endoplasmic Reticulum Calcium ATPase (SERCA) pump, and sarcalumenin together with an alteration of the expression of mitsugumin 29 and mitsugumin 53, two recently recognized players in the SOCE mechanism. BCAAs, particularly the formulation BCAAs+2-Ala, were able to ameliorate all these alterations. Discussion: These results provide evidence that Ca2+ homeostasis dysfunction plays a role in the functional deficit observed in aged muscle and supports the interest of dietary BCAA supplementation in counteracting sarcopenia-related SOCE dysregulation.

Aged garlic extract preserves beta-cell functioning via modulation of nuclear factor kappa-B (NF-κB)/Toll-like receptor (TLR)-4 and sarco endoplasmic reticulum calcium ATPase (SERCA)/Ca2+ in diabetes mellitus

BackgroundPeripheral insulin resistance and compromised insulin secretion from pancreatic β-cells are significant factors and pathogenic hallmarks of diabetes mellitus (DM). NF-κβ/TLR-4 and SERCA/Ca2+ pathways have been identified as potential pathways regulating insulin synthesis by preserving pancreatic β-cell functioning. The current study aimed to evaluate the therapeutic effect of aged garlic extract (AGE) against DM in a streptozotocin (STZ)-induced rat model with particular emphasis on pancreatic β-cell functioning.MethodsAGE was characterized by gas chromatography-mass spectrometry (GC-MS), Fourier-transform infrared spectroscopy (FTIR), and scanning electron microscopy (SEM) to evaluate its physio-chemical characteristics followed by in-vitro anti-diabetic and antioxidant potential. This was followed by the induction of DM in laboratory animals for investigating the therapeutic action of AGE by evaluating the role of NF-κβ/TLR-4 and the SERCA/Ca2+ pathway. The parameters assessed in the present experimental setup encompassed antioxidant parameters, metabolic indicators, insulin concentration, intracellular calcium levels, apoptotic markers (CCK-8 and Caspase Glo-8), and protein expression (P-62 and APACHE-II).ResultsAGE characterization by SEM, GC-MS, and X-ray diffraction (XRD) revealed the presence of phenylalanine, alliin, S-allylmercaptocysteine (SAMC), tryptophan, 1-methyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid as major bioactive constituents of AGE. Metabolic studies, including intraperitoneal glucose tolerance test (IPGTT), revealed significantly lower blood glucose levels in the AGE group compared to the disease control group. In contrast, the intraperitoneal insulin tolerance test (ITT) exhibited no significant difference in insulin sensitivity between the AGE supplementation group and the DM control group. Interestingly, AGE was found to have no significant effect on fasting glucose and serum insulin levels. In contrast, AGE supplementation was found to cause significant hypoglycaemia in postprandial blood glucose and insulin levels. Importantly, AGE causes restoration of intracellular Ca2+ levels by modulation of SERCA/Ca2 functioning and inhibition NF-κB/TLR-4 pathway. AGE was found to interact with and inhibit the DR-5/ caspase-8/3 apoptotic complex. Furthermore, microscopic studies revealed degeneration and apoptotic changes in pancreatic β-cells of the DM control group, while supplementation of AGE resulted in inhibition of apoptotic pathway and regeneration of pancreatic β-cells.ConclusionThe current study suggests that AGE enhance glucose homeostasis by exerting their effects on pancreatic β-cells, without ameliorating peripheral sensitivity. Moreover, AGEs promote an increase in β-cell mass by mitigating the apoptosis of pancreatic β-cells. These findings suggest that AGE could aid in developing a viable alternative therapy for diabetes mellitus (DM).

Myocardial SERCA2 Protects Against Cardiac Damage and Dysfunction Caused by Inhaled Bromine.

Myocardial sarcoendoplasmic reticulum calcium ATPase 2 (SERCA2) activity is critical for heart function. We have demonstrated that inhaled halogen (chlorine or bromine) gases inactivate SERCA2, impair calcium homeostasis, increase proteolysis, and damage the myocardium ultimately leading to cardiac dysfunction. To further elucidate the mechanistic role of SERCA2 in halogen-induced myocardial damage, we used bromine-exposed cardiac-specific SERCA2 knockout (KO) mice [tamoxifen-administered SERCA2 (flox/flox) Tg (αMHC-MerCreMer) mice] and compared them to the oil-administered controls. We performed echocardiography and hemodynamic analysis to investigate cardiac function 24 hours after bromine (600 ppm for 30 minutes) exposure and measured cardiac injury markers in plasma and proteolytic activity in cardiac tissue and performed electron microscopy of the left ventricle (LV). Cardiac-specific SERCA2 knockout mice demonstrated enhanced toxicity to bromine. Bromine exposure increased ultrastructural damage, perturbed LV shape geometry, and demonstrated acutely increased phosphorylation of phospholamban in the KO mice. Bromine-exposed KO mice revealed significantly enhanced mean arterial pressure and sphericity index and decreased LV end diastolic diameter and LV end systolic pressure when compared with the bromine-exposed control FF mice. Strain analysis showed loss of synchronicity, evidenced by an irregular endocardial shape in systole and irregular vector orientation of contractile motion across different segments of the LV in KO mice, both at baseline and after bromine exposure. These studies underscore the critical role of myocardial SERCA2 in preserving cardiac ultrastructure and function during toxic halogen gas exposures. SIGNIFICANCE STATEMENT: Due to their increased industrial production and transportation, halogens such as chlorine and bromine pose an enhanced risk of exposure to the public. Our studies have demonstrated that inhalation of these halogens leads to the inactivation of cardiopulmonary SERCA2 and results in calcium overload. Using cardiac-specific SERCA2 KO mice, these studies further validated the role of SERCA2 in bromine-induced myocardial injury. These studies highlight the increased susceptibility of individuals with pathological loss of cardiac SERCA2 to the effects of bromine.

SERCA2a overexpression improves muscle function in a canine Duchenne muscular dystrophy model

Excessive cytosolic calcium accumulation contributes to muscle degeneration in Duchenne muscular dystrophy (DMD). Sarco/endoplasmic reticulum calcium ATPase (SERCA) is a sarcoplasmic reticulum (SR) calcium pump that actively transports calcium from the cytosol into the SR. We previously showed that adeno-associated virus (AAV)-mediated SERCA2a therapy reduced cytosolic calcium overload and improved muscle and heart function in the murine DMD model. Here we tested whether AAV SERCA2a therapy could ameliorate muscle disease in the canine DMD model. 7.83 x 1013 viral genome particles of the AAV vector were injected into the extensor carpi ulnaris (ECU) muscles of four juvenile-affected dogs. Contralateral ECU muscles received excipient. Three months later, we observed widespread transgene expression and significantly increased SERCA2a levels in the AAV-injected muscles. Treatment improved SR calcium uptake, significantly reduced calpain activity, significantly improved contractile kinetics, and significantly enhanced resistance to eccentric contraction-induced force loss. Nonetheless, muscle histology was not improved. To evaluate the safety of AAV SERCA2a therapy, we delivered the vector to the ECU muscle of adult normal dogs. We achieved strong transgene expression without altering muscle histology and function. Our results suggest that AAV SERCA2a therapy has the potential to improve muscle performance in a dystrophic large mammal.

The Ailing β-Cell in Diabetes: Insights From a Trip to the ER: The 2023 Outstanding Scientific Achievement Award Lecture.

The synthesis, processing, and secretion of insulin by the pancreatic β-cell is key for the maintenance of systemic metabolic homeostasis, and loss or dysfunction of β-cells underlies the development of both type 1 diabetes (T1D) and type 2 diabetes (T2D). Work in the Evans-Molina laboratory over the past 15 years has pioneered the idea that regulation of calcium dynamics is critical to β-cell biology and diabetes pathophysiology. In this article, I will share three vignettes from the laboratory that demonstrate our bench-to-bedside approach to determining mechanisms of β-cell stress that could improve therapeutic options and outcomes for individuals living with diabetes. The first of these vignettes will illustrate a role for the sarcoendoplasmic reticulum calcium ATPase (SERCA) pump in the regulation of endoplasmic reticulum (ER) calcium, protein trafficking, and proinsulin processing within the β-cell. The second vignette will highlight how alterations in β-cell calcium signaling intersect with T1D pathogenesis. The final vignette will demonstrate how activation of β-cell stress pathways may serve as an anchor to inform biomarker strategies in T1D. Lastly, I will share my vision for the future of diabetes care, where multiple biomarkers of β-cell stress may be combined with additional immune and metabolic biomarkers to better predict disease risk and improve therapies to prevent or delay T1D development.

A novel role for phospholamban in the thalamic reticular nucleus

The thalamic reticular nucleus (TRN) is a brain region that influences vital neurobehavioral processes, including executive functioning and the generation of sleep rhythms. TRN dysfunction underlies hyperactivity, attention deficits, and sleep disturbances observed across various neurodevelopmental disorders. A specialized sarco-endoplasmic reticulum calcium (Ca2+) ATPase 2 (SERCA2)-dependent Ca2+ signaling network operates in the dendrites of TRN neurons to regulate their bursting activity. Phospholamban (PLN) is a prominent regulator of SERCA2 with an established role in myocardial Ca2+-cycling. Our findings suggest that the role of PLN extends beyond the cardiovascular system to impact brain function. Specifically, we found PLN to be expressed in TRN neurons of the adult mouse brain, and utilized global constitutive and innovative conditional genetic knockout mouse models in concert with electroencephalography (EEG)-based somnography and the 5-choice serial reaction time task (5-CSRTT) to investigate the role of PLN in sleep and executive functioning, two complex behaviors that map onto thalamic reticular circuits. The results of the present study indicate that perturbed PLN function in the TRN results in aberrant TRN-dependent phenotypes in mice (i.e., hyperactivity, impulsivity and sleep deficits) and support a novel role for PLN as a critical regulator of SERCA2 in the TRN neurocircuitry.

Establishment and evaluation of targeted molecular screening model for the ryanodine receptor or sarco/endoplasmic reticulum calcium ATPase.

Endoplasmic reticulum/sarcoplasmic reticulum (ER/SR) is crucial for maintaining intracellular calcium homeostasis due to the calcium-signaling-related proteins on its membrane. While ryanodine receptors (RyR) on insect ER/SR membranes are well-known as targets for diamide insecticides, little is known about other calcium channels. Given the resistance of diamide insecticides, the establishment of molecular screening models targeting RyR or sarco/endoplasmic reticulum calcium ATPase (SERCA) is conducive to the discovery of new insecticidal molecules. The morphological features of Mythimna separata SR have closed vesicles with integrity and high density. The 282 proteins in the SR component contained RyR and SERCA. A measurement model for the release and uptake of calcium was successfully established by detecting calcium ions outside the SR membrane using a fluorescence spectrophotometer. In vitro testing systems using SR vesicles found that diamide insecticides could activate dose-dependently RyR, with EC50 values of 0.14 μM (Chlorantraniliprole), 0.21 μM (Flubendiamide), and 0.57 μM (Cyantraniliprole), respectively. However, dantrolene inhibited RyR-mediated calcium release with an IC50 value of 353.9 μM, suggesting that dantrolene can weakly antagonize RyR. Moreover, cyclopiazonic acid significantly reduced the enzyme activity and calcium uptake capacity of SERCA. On the contrary, CDN1163 markedly activated the enzyme activity and improved the calcium transport capacity of SERCA. SR vesicles can be used to study the function of unknown proteins on the SR membranes, as well as for high-throughput screening of highly active compounds targeting RyR or SERCA. © 2024 Society of Chemical Industry.

Pre-hibernation diet alters skeletal muscle relaxation kinetics, but not force development in torpid arctic ground squirrels.

During the hibernation season, Arctic ground squirrels (AGS) experience extreme temperature fluctuations (body temperature, Tb, as low as - 3°C), during which they are mostly physically inactive. Once Tb reaches ~ 15°C during interbout arousals, hibernators recruit skeletal muscle (SkM) for shivering thermogenesis to reach Tb of ~ 35°C. Polyunsaturated fatty acids (PUFA) in the diet are known to influence SkM function and metabolism. Recent studies in the cardiac muscle of hibernators have revealed that increased levels of ω-6 and the ω-6:ω-3 PUFA ratio correlate with sarco/endoplasmic reticulum calcium ATPase (SERCA) activity and hibernation status. We hypothesized that diet (increased ω-6:ω-3 PUFA ratio) and torpor status are important in the regulation of the SERCA pump and that this may improve SkM performance during hibernation. Ex vivo functional assays were used to characterize performance changes in SkM (diaphragm) from AGS fed the following diets. (1) Standard rodent chow with an ω-6:ω-3 ratio of 5:1, or (2) a balanced diet with an ω-6:ω-3 ratio of 1:1 that roughly mimics wild diet. We collected diaphragms at three different stages of hibernation (early torpor, late torpor, and arousal) and evaluated muscle function under hypothermic temperature stress at 4°C, 15°C, 25°C, and 37°C to determine functional resilience. Our data show that torpid animals fed standard rodent chow have faster SkM relaxation when compared to the balanced diet animals. Furthermore, we discovered that standard rodent chow AGS during torpor has higher SkM relaxation kinetics, but this effect of torpor is eliminated in balanced diet AGS. Interestingly, neither diet nor torpor influenced the rate of force development (rate of calcium release). This is the first study to show that increasing the dietary ω-6:ω-3 PUFA ratio improves skeletal muscle performance during decreased temperatures in a hibernating animal. This evidence supports the interpretation that diet can change some functional properties of the SkM, presumably through membrane lipid composition, ambient temperature, and torpor interaction, with an impact on SkM performance.

Dual effect of polyaromatic hydrocarbons on sarco(endo)plasmic reticulum calcium ATPase (SERCA) activity of a teleost fish (Oncorhynchus mykiss)

Polycyclic aromatic hydrocarbons (PAHs) are embryo- and cardiotoxic to fish that might be associated with improper intracellular Ca2+ management. Since sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) is a major regulator of intracellular Ca2+, the SERCA activity and the contractile properties of rainbow trout (Oncorhynchus mykiss) ventricle were measured in the presence of 3- and 4-cyclic PAHs. In unfractionated ventricular homogenates, acute exposure of SERCA to 0.1–1.0 μM phenanthrene (Phe), retene (Ret), fluoranthene (Flu), or pyrene (Pyr) resulted in concentration-dependent increase in SERCA activity, except for the Flu exposure, with maximal effects of 49.7–83 % at 1 μM. However, PAH mixture did not affect the contractile parameters of trout ventricular strips. Similarly, all PAHs, except Ret, increased the myotomal SERCA activity, but with lower effect (27.8–40.8 % at 1 μM). To investigate the putative chronic effects of PAHs on SERCA, the atp2a2a gene encoding trout cardiac SERCA was expressed in human embryonic kidney (HEK) cells. Culture of HEK cells in the presence of 0.3–1.0 μM Phe, Ret, Flu, and Pyr for 4 days suppressed SERCA expression in a concentration-dependent manner, with maximal inhibition of 49 %, 65 %, 39 % (P < 0.05), and 18 % (P > 0.05), respectively at 1 μM. Current findings indicate divergent effects of submicromolar PAH concentrations on SERCA: stimulation of SERCA activity in acute exposure and inhibition of SERCA expression in chronic exposure. The depressed expression of SERCA is likely to contribute to the embryo- and cardiotoxicity of PAHs by depressing muscle function and altering gene expression.

Discovery of New Anti-Cancer Agents against Patient-Derived Sorafenib-Resistant Papillary Thyroid Cancer.

Thyroid cancer is the most well-known type of endocrine cancer that is easily treatable and can be completely cured in most cases. Nonetheless, anti-cancer drug-resistant metastasis or recurrence may occur and lead to the failure of cancer therapy, which eventually leads to the death of a patient with cancer. This study aimed to detect novel thyroid cancer target candidates based on validating and identifying one of many anti-cancer drug-resistant targets in patient-derived sorafenib-resistant papillary thyroid cancer (PTC). We focused on targeting the sarco/endoplasmic reticulum calcium ATPase (SERCA) in patient-derived sorafenib-resistant PTC cells compared with patient-derived sorafenib-sensitive PTC cells. We discovered novel SERCA inhibitors (candidates 33 and 36) by virtual screening. These candidates are novel SERCA inhibitors that lead to remarkable tumor shrinkage in a xenograft tumor model of sorafenib-resistant patient-derived PTC cells. These results are clinically valuable for the progression of novel combinatorial strategies that facultatively and efficiently target extremely malignant cancer cells, such as anti-cancer drug-resistant PTC cells.

Nongenetic Optical Modulation of Pluripotent Stem Cells Derived Cardiomyocytes Function in the Red Spectral Range.

Optical stimulation in the red/near infrared range recently gained increasing interest, as a not-invasive tool to control cardiac cell activity and repair in disease conditions. Translation of this approach to therapy is hampered by scarce efficacy and selectivity. The use of smart biocompatible materials, capable to act as local, NIR-sensitive interfaces with cardiac cells, may represent a valuable solution, capable to overcome these limitations. In this work, a far red-responsive conjugated polymer, namely poly[2,1,3-benzothiadiazole-4,7-diyl[4,4-bis(2-ethylhexyl)-4H-cyclopenta[2,1-b:3,4-b']dithiophene-2,6-diyl]] (PCPDTBT) is proposed for the realization of photoactive interfaces with cardiomyocytes derived from pluripotent stem cells (hPSC-CMs). Optical excitation of the polymer turns into effective ionic and electrical modulation of hPSC-CMs, in particular by fastening Ca2+ dynamics, inducing action potential shortening, accelerating the spontaneous beating frequency. The involvement in the phototransduction pathway of Sarco-Endoplasmic Reticulum Calcium ATPase (SERCA)and Na+ /Ca2+ exchanger (NCX) is proven by pharmacological assays and is correlated with physical/chemical processes occurring at the polymer surface upon photoexcitation. Very interestingly, an antiarrhythmogenic effect, unequivocally triggered by polymer photoexcitation, is also observed. Overall, red-light excitation of conjugated polymers may represent an unprecedented opportunity for fine control of hPSC-CMs functionality and can be considered as a perspective, noninvasive approach to treat arrhythmias.