SERCA Research Articles

Melatonin, Circadian Rhythms, and Type 2 Diabetes Mellitus: A Comprehensive Review of Epidemiology, Molecular Mechanisms, and Therapeutic Implications

This extensive review delves into the epidemiology, risk factors, historical background, and molecular mechanisms of Type 2 Diabetes Mellitus (T2DM). The term "diabetes mellitus" dates back to ancient times and the Greek physician Aertaeus is credited with coining the term. T2DM, which is defined by hyperglycemia, insulin resistance, and relative insulin deficiency, is a global public health concern that affects millions of people worldwide. A combination of genetic, environmental, and lifestyle factors play a role in the development of T2DM. The distinction between Type 1 and Type 2 diabetes was established in 1936, and Type 2 diabetes was identified as a component of the metabolic syndrome in 1988. T2DM is becoming more and more common; it is expected to affect 552 million people by 2030, which means that research and interventions are desperately needed. The risk factors for T2DM include age, genetics, obesity, sedentary lifestyle, and components of the metabolic syndrome. Prediabetes, which is a precursor to T2DM, must be detected early in order to reduce the risk of progression. This review explores the physiological and molecular mechanisms underlying T2DM, with a focus on the complex interactions between melatonin, circadian rhythms, and diabetes. Melatonin, this is primarily synthesized by the pineal gland, is essential for the regulation of circadian rhythms and many other physiological processes. Research has shown how melatonin affects insulin secretion and glucose metabolism, suggesting that it may have therapeutic uses in the treatment of diabetes. Circadian rhythm disturbances, particularly those brought on by shift work, are linked to a higher risk of type 2 diabetes, underscoring the importance of comprehending these intricate relationships. The review highlights the need for more research to fully understand the complex relationships between melatonin, circadian rhythms, and diabetes. Pharmacological interventions, such as the synthetic melatonin agonist agomelatine, offer promising avenues for therapeutic exploration. Thapsigargin, an antagonist of sarco-endoplasmic reticulum Ca2+-ATPase (SERCA), reveals the role of calcium signaling in diabetes research.

Thapsigargin: a promising natural product with diverse medicinal potential - a review of synthetic approaches and total syntheses.

Thapsigargin, a sesquiterpene lactone, naturally occurring in the roots and fruits of the Mediterranean shrub Thapsia garganica L, is known to the practitioners of traditional medicines since the medieval ages as a cure for rheumatic pain, lung diseases, and female infertility. This naturally occurring guaianolide has shown remarkable activity for Sarco endoplasmic reticulum Ca2+ ATPase inhibition, which eventually renders it fit as a potential candidate for anti-cancer drugs. Mipsagargin, a prodrug derived from thapsigargin, is under clinical trials for the treatment of glioblastoma. Recently, thapsigargin has shown promise as an antiviral against SARS-CoV-2. Limited natural availability and challenging synthesis have prompted research into new synthetic pathways. This review discusses significant synthetic approaches and total syntheses of thapsigargin reported to date.

Vascular dysfunction caused by loss of Brn-3b/POU4F2 transcription factor in aortic vascular smooth muscle cells is linked to deregulation of calcium signalling pathways

Phenotypic and functional changes in vascular smooth muscle cells (VSMCs) contribute significantly to cardiovascular diseases (CVD) but factors driving early adverse vascular changes are poorly understood. We report on novel and important roles for the Brn-3b/POU4F2 (Brn-3b) transcription factor (TF) in controlling VSMC integrity and function. Brn-3b protein is expressed in mouse aorta with localisation to VSMCs. Male Brn-3b knock-out (KO) aortas displayed extensive remodelling with increased extracellular matrix (ECM) deposition, elastin fibre disruption and small but consistent narrowing/coarctation in the descending aortas. RNA sequencing analysis showed that these effects were linked to deregulation of genes required for calcium (Ca2+) signalling, vascular contractility, sarco-endoplasmic reticulum (S/ER) stress responses and immune function in Brn-3b KO aortas and validation studies confirmed changes in Ca2+ signalling genes linked to increased intracellular Ca2+ and S/ER Ca2+ depletion [e.g. increased, Cacna1d Ca2+ channels; ryanodine receptor 2, (RyR2) and phospholamban (PLN) but reduced ATP2a1, encoding SERCA1 pump] and chaperone proteins, Hspb1, HspA8, DnaJa1 linked to increased S/ER stress, which also contributes to contractile dysfunction. Accordingly, vascular rings from Brn-3b KO aortas displayed attenuated contractility in response to KCl or phenylephrine (PE) while Brn-3b KO-derived VSMC displayed abnormal Ca2+ signalling following ATP stimulation. This data suggests that Brn-3b target genes are necessary to maintain vascular integrity /contractile function and deregulation upon loss of Brn-3b will contribute to contractile dysfunction linked to CVD.

Nanojunctions: Specificity of Ca2+ signaling requires nano-scale architecture of intracellular membrane contact sites

Spatio-temporal definition of Ca2+ signals involves the assembly of signaling complexes within the nano-architecture of contact sites between the sarco/endoplasmic reticulum (SR/ER) and the plasma membrane (PM). While the requirement of precise spatial assembly and positioning of the junctional signaling elements is well documented, the role of the nano-scale membrane architecture itself, as an ion-reflecting confinement of the signalling unit, remains as yet elusive. Utilizing the Na+/Ca2+ Exchanger-1 / SR/ER Ca2+ ATPase-2-mediated ER Ca2+ refilling process as a junctional signalling paradigm, we provide here the first evidence for an indispensable cellular function of the junctional membrane architecture. Our stochastic modeling approach demonstrates that junctional ER Ca2+ refilling operates exclusively at nano-scale membrane spacing, with a strong inverse relationship between junctional width and signaling efficiency. Our model predicts a breakdown of junctional Ca2+ signaling with loss of reflecting membrane confinement. In addition we consider interactions between Ca2+ and the phospholipid membrane surface, which may support interfacial Ca2+ transport and promote receptor targeting. Alterations in the molecular and nano-scale membrane organization at organelle-PM contacts are suggested as a new concept in pathophysiology.

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.

Should women have lower thresholds for hypertension? Severe ultrastructural abnormalities in systemic arterioles of normotensive and hypertensive women

Abstract Background Hypertension in women is increasingly recognized to originate in the pre-menopause, but the factors that may affect the greater blood pressure increments observed in premenopausal women compared to age-matched men are understudied. Uterine fibroids, occurring in 30-50% of all women, are a strong sex-specific factor associated with hypertension and early cardiovascular events, but the nature of this association is not known. To assess a possible shared pathology of smooth muscle, we studied the ultrastructure of systemic resistance arteries of women with fibroids. Methods Resistance-sized arteries (200 to 400 μm) were harvested from omental samples obtained during fibroid surgery. Transmission electron microscopic images were taken at different magnifications and locations of the vessel wall to assess qualitative and morphological characteristics. Mean blood pressure was calculated as DBP+1/3(SBP-DBP). Results Resistance arteries of 17 women (8 hypertensive, 4 treated), mean age 42 (SE 1) y, were available for analysis. None had a history of cardiovascular disease. Mean blood pressure was 91.4 mm Hg in normotensives and 119.4 in hypertensives. The endothelium showed mitochondrial and endoplasmic reticulum stress in normotensives (Figure, Panel A), to necrosis and sloughing in hypertensives (B). Myocyte-like cells were observed to migrate across the internal elastic lamina, forming small aggregates of subendothelial myocytes in normotensives (C), and larger subendothelial myo-intimal "cushions" in hypertensives (D). Medial myocytes of all patients showed abnormalities similar to fibroids, with dilated sarco-endoplasmic reticulum, elongated mitochondria and myofilament loss involving focal areas or entire cells (E, clinically considered normotensive). Untreated hypertensives had a significantly higher intima-media thickness-to-lumen ratio (%) than normotensives and treated hypertensives as expected, respectively 15.9 (1.8), 12.5 (1.6) and 13.4 (2.1). Conclusion To our knowledge the first ultrastructure data of systemic resistance-sized arteries in women with fibroids, show that young premenopausal women considered clinically normotensive already have severe microvascular abnormalities that may cause or reflect perturbations in microvascular function and predispose to early end organ damage. While there is abundant clinical evidence that the association between blood pressure and cardiovascular events is stronger in women, hypertension in women is understudied. In particular women-specific risk factors are ill-understood. Our observations are relevant to the current discussion regarding whether the clinical threshold for hypertension treatment is too high for women and whether earlier management of cardiovascular risk is needed. Further studies are warranted in a woman-specific research agenda on cardiovascular risk factors in younger premenopausal women.Figure

Evaluation of the Anticancer and Biological Activities of Istaroxime via Ex Vivo Analyses, Molecular Docking and Conceptual Density Functional Theory Computations.

Cancer is a disease that occurs as a result of abnormal or uncontrolled growth of cells due to DNA damage, among many other causes. Certain cancer treatments aim to increase the excess of DNA breaks to such an extent that they cannot escape from the general mechanism of cell checkpoints, leading to the apoptosis of mutant cells. In this study, one of the Sarco-endoplasmic reticulum Ca2+ATPase (SERCA2a) inhibitors, Istaroxime, was investigated. There has been very limited number of articles so far reporting Istaroxime's anticancer activity; thus, we aimed to evaluate the anticancer effects of Istaroxime by cell proliferation assay and revealed the cytotoxic activity of the compound. We further determined the interaction of Istaroxime with topoisomerase enzymes through enzyme activity tests and detailed molecular modeling analysis. Istaroxime exhibited an antiproliferative effect on A549, MCF7, and PC3 cell lines and inhibited Topoisomerase I, suggesting that Istaroxime can act as a Topoisomerase I inhibitor under in vitro conditions. Molecular docking analysis supported the experimental observations. A chemical reactivity analysis of the Istaroxime molecule was made in the light of Density Functional Theory computations. For this aim, important chemical reactivity descriptors such as hardness, electronegativity, and electrophilicity were computed and discussed as detailed.

SEC61B Regulates Calcium Flux and Platelet Hyperactivity in Diabetes Mellitus

Background/Aim Platelets in individuals with diabetes mellitus show increased activity at baseline and in response to stimuli, which contributes to the increased cardiovascular events in this population. Endoplasmic reticulum (ER) stress, induced by misfolded proteins, has recently been identified as a mechanism of platelet hyperactivity in diabetes. Newly synthesized proteins enter the ER via the Sec61 translocon for folding. The Sec61 translocon is a heterotrimeric channel comprising of the pore-forming α, and peripheral β and γ subunits. In addition to its roles in proteostasis, it also acts as a leak channel to allow calcium efflux from the ER into the cytosol. However, a function for the Sec61 complex has not been previously described in platelets. Study design/Methods Platelets were isolated from 42 patients with and 34 patients without type 2 diabetes mellitus undergoing coronary angiography for suspected or known coronary artery disease. Patients were matched for age, sex, and coronary artery disease burden. Platelet proteins were identified by untargeted LC-MS/MS and analyzed by MaxQuant. Platelet RNA was isolated from a separate cohort of 5 patients with and 12 matched patients without type 2 diabetes. RNA-seq libraries were prepared with TruSeq V2, Illumina, and analyzed with the Deseq2 analysis package. Platelets and megakaryocytes were isolated from 2 mouse models of diabetes (Apoe-/- mice with induction of diabetes by streptozotocin injection) and outbred mice fed a high fat diet. HEK293 cells were generated with knockout or overexpression of SEC61B using CRISPR/Cas9 or cDNA transfection. Proteins of the ER stress response, including eukaryotic translation initiation factor 2A (EIF2A), inositol-requiring enzyme 1 (IRE1), and activating transcription factor 6 (ATF6), were determined by immunoblotting or immunostaining. Protein synthesis was measured using the Click-iT TM AHA Alexa Fluor 488 assay. Calcium flux was measured using fura-2-AM and expressed as normalised fluorescent 340/380 nm ratio. Calcium efflux from the ER was measured in the presence of translation inhibitors puromycin and eeyarestatin I and the sarcoendoplasmic reticulum calcium-ATPase inhibitor thapsigargin. Results Proteomics analysis identified an enrichment of proteins involved in response to oxidative stress in platelets of patients with diabetes. From the over 2600 intracellular proteins identified consistently in >50% of human platelet samples, SEC61B was the only protein significantly and positively correlated with serum fructosamine, which is a marker of glycaemic control (Spearman's rho = 0.33, p = 0.029). SEC61B protein abundance was increased in platelets with high fructosamine (>290 µmol/L) compared with normal fructosamine ( panel A). There were no differences between SEC61 subunits in patients with or without diabetes at the transcriptomic level. Increased SEC61B was confirmed in platelets and megakaryocytes of hyperglycemic mice. Induction of ER stress in healthy human platelets by incubation with thapsigargin significantly increased the phosphorylation of IRE1 and the expression of SEC61B but not of ATF6 or phosphorylated EIF2A. In HEK293 cells with SEC61B overexpression or knockout, protein synthesis rate was decreased (58-72% reduction knockout vs control, p<0.0001; 22% reduction overexpression vs control, p = 0.0005). SEC61B overexpression or knockout resulted in increased ER calcium efflux after treatment with puromycin or eeyarestatin I (which maintain the Sec61 translocon in an open, calcium permeable configuration ( panel B). Puromycin induced ER-mediated calcium leak in healthy human platelets (22% increase in maximal calcium efflux after puromycin treatment, p = 0.039). Conclusion Our data identify Sec61 translocon as a novel functional ER calcium leak channel in platelets. Diabetes mellitus is associated with upregulated megakaryocyte and platelet SEC61B. Dysregulated SEC61B expression is associated with increased calcium efflux from the ER. We propose a mechanism whereby ER stress-induced upregulation of platelet SEC61B leads to increased cytoplasmic calcium, potentially contributing to the platelet hyperactivity seen in diabetes mellitus.

DIAPH1-MFN2 interaction regulates mitochondria-SR/ER contact and modulates ischemic/hypoxic stress

Inter-organelle contact and communication between mitochondria and sarco/endoplasmic reticulum (SR/ER) maintain cellular homeostasis and are profoundly disturbed during tissue ischemia. We tested the hypothesis that the formin Diaphanous-1 (DIAPH1), which regulates actin dynamics, signal transduction and metabolic functions, contributes to these processes. We demonstrate that DIAPH1 interacts directly with Mitofusin-2 (MFN2) to shorten mitochondria-SR/ER distance, thereby enhancing mitochondria-ER contact in cells including cardiomyocytes, endothelial cells and macrophages. Solution structure studies affirm the interaction between the Diaphanous Inhibitory Domain and the cytosolic GTPase domain of MFN2. In male rodent and human cardiomyocytes, DIAPH1-MFN2 interaction regulates mitochondrial turnover, mitophagy, and oxidative stress. Introduction of synthetic linker construct, which shorten the mitochondria-SR/ER distance, mitigated the molecular and functional benefits of DIAPH1 silencing in ischemia. This work establishes fundamental roles for DIAPH1-MFN2 interaction in the regulation of mitochondria-SR/ER contact networks. We propose that targeting pathways that regulate DIAPH1-MFN2 interactions may facilitate recovery from tissue ischemia.

Remote ischemic postconditioning alleviates cerebral ischemic injury through SERCA2/endoplasmic reticulum stress-mediated apoptosis.

Remote ischemic postconditioning (RIPostC) alleviates brain ischemic injury through several pathways, including endoplasmic reticulum (ER) stress modulation. Sarco endoplasmic reticulum Ca2+ -ATPase(SERCA2) which plays vital role in calcium homeostasis regulation could modulate ER stress logically. This study aimed to investigate whether RIPostC exerts its neuroprotective effect by reducing ER stress mediated by SERCA2. Male SD rats underwent transient middle cerebral artery occlusion (tMCAO) for 2 h followed by reperfusion, with the RIPostC group undergoing 3 cycles of bilateral femoral artery clamping and reperfusion at the beginning of reperfusion. Stroke outcome was assessed based on infarct volume and neurological function evaluation. Protein levels of SERCA2 and other ER stress markers were measured using Western blotting, immunofluorescence, and immunohistochemistry techniques. Compared to the sham group, we observed that RIPostC can effectively reduce cerebral infarct volume after I/R (34.55%: 21.03%; p = .004) and improve neurological function deficit (9.67:12.5; p = .029). Additionally, RIPostC increased SERCA2 protein expression and decreased the protein level of glucose-regulated protein 78 (GRP78), phosphorylation of eukaryotic translation initiation factor 2α (p-eIF2α) and CCAAT/EBP homologous protein (CHOP). Furthermore, B-cell lymphoma-2 (Bcl-2) expression was increased, while Bcl-2-associated X protein (Bax) and cleaved-caspase-3 was decreased in response to application of RIPostC. Our results suggest that RIPostC improves the prognosis of tMCAO rats, possibly by inhibiting the ER stress mediated by SERCA2, facilitating apoptosis downregulation. The significance of this study is to provide a theoretical basis for further exploring the protective mechanism of ischemic stroke by RIPostC. RESEARCH HIGHLIGHTS: Our results suggest that RIPostC improves the prognosis of tMCAO rats, possibly by inhibiting the ER stress mediated by SERCA2, facilitating apoptosis downregulation, thus achieving a neuroprotective effect.

IP3R1 underlies diastolic ANO1 activation and pressure-dependent chronotropy in lymphatic collecting vessels.

Pressure-dependent chronotropy of murine lymphatic collecting vessels relies on the activation of the Ca2+-activated chloride channel encoded by Anoctamin 1 (Ano1) in lymphatic muscle cells. Genetic ablation or pharmacological inhibition of ANO1 results in a significant reduction in basal contraction frequency and essentially complete loss of pressure-dependent frequency modulation by decreasing the rate of the diastolic depolarization phase of the ionic pacemaker in lymphatic muscle cells (LMCs). Oscillating Ca2+ release from sarcoendoplasmic reticulum Ca2+ channels has been hypothesized to drive ANO1 activity during diastole, but the source of Ca2+ for ANO1 activation in smooth muscle remains unclear. Here, we investigated the role of the inositol triphosphate receptor 1 (Itpr1; Ip3r1) in this process using pressure myography, Ca2+ imaging, and membrane potential recordings in LMCs of ex vivo pressurized inguinal-axillary lymphatic vessels from control or Myh11CreERT2;Ip3r1fl/fl (Ip3r1ismKO) mice. Ip3r1ismKO vessels had significant reductions in contraction frequency and tone but an increased contraction amplitude. Membrane potential recordings from LMCs of Ip3r1ismKO vessels revealed a depressed diastolic depolarization rate and an elongation of the plateau phase of the action potential (AP). Ca2+ imaging of LMCs using the genetically encoded Ca2+ sensor GCaMP6f demonstrated an elongation of the Ca2+ flash associated with an AP-driven contraction. Critically, diastolic subcellular Ca2+ transients were absent in LMCs of Ip3r1ismKO mice, demonstrating the necessity of IP3R1 activity in controlling ANO1-mediated diastolic depolarization. These findings indicate a critical role for IP3R1 in lymphatic vessel pressure-dependent chronotropy and contractile regulation.

Chronic pharmacological activation of SERCA with CDN1163 affects spatial cognitive flexibility but not attention and impulsivity in mice.

Intracellular calcium (Ca2+) homeostasis is critical for many neural processes, including learning, memory and synaptic plasticity. The sarco-endoplasmic reticulum Ca2+ ATPase (SERCA) is among the key regulators that preserve Ca2+ homeostasis in neurons. SERCAs comprise a set of ubiquitously expressed Ca2+ pumps that primarily function to sequester cytosolic Ca2+ into endoplasmic reticular stores. As SERCA has been implicated in the neurobiology of several neuropsychiatric and neurodegenerative diseases, pharmacological harnessing of its function is critical in understanding SERCA's role in brain physiology and pathophysiology. In the current study, we employed the Morris water maze and 5-choice serial reaction time task (5-CSRTT) to investigate the effects of chronic pharmacological activation of SERCA, using the small allosteric SERCA activator CDN1163, on spatial learning and memory, and executive functioning in naive C57BL/6J mice. Our data show that chronic pharmacological SERCA activation with CDN1163 (20 mg/kg) selectively impairs spatial cognitive flexibility and reversal learning in the Morris water maze while leaving executive functions such as attention and impulsivity intact. Present findings contribute to the growing field of the role of SERCA function in the brain and behavior and expand current knowledge on the use of the small allosteric activator CDN1163 as an investigational tool to study the role of SERCA in regulating neurobehavioral processes and as a potential therapeutic candidate for debilitating brain disorders.

Effects of reversible SERCA inhibition on catecholamine exocytosis and intracellular [Ca2+] signaling in chromaffin cells from normotensive Wistar Kyoto rats and spontaneously hypertensive rats

Intracellular Ca2+ ([Ca2+]i) signaling and catecholamine (CA) exocytosis from adrenal chromaffin cells (CCs) differ between mammalian species. These differences partly result from the different contributions of Ca2+-induced Ca2+-release (CICR) from internal stores, which boosts intracellular Ca2+ signals. Transient inhibition of the sarcoendoplasmic reticulum (SERCA) Ca2+ pump with cyclopiazonic acid (CPA) reduces CICR. Recently, Martínez-Ramírez et al. found that CPA had contrasting effects on catecholamine secretion and intracellular Ca2+ signals in mouse and bovine CCs, where it enhanced and inhibited exocytosis, respectively. After CPA withdrawal, exocytosis diminished in mouse CCs and increased in bovine CCs. These differences can be explained if mouse CCs have weak CICR and strong Ca2+ uptake, and the reverse is true for bovine CCs. Surprisingly, CPA slightly reduced the amplitude of Ca2+ signals in both mouse and bovine CCs. Here we examined the effects of CPA on stimulated CA exocytosis and Ca2+ signaling in rat CCs and investigated if it alters differently the responses of CCs from normotensive (WKY) or hypertensive (SHR) rats, which differ in the gain of CICR. Our results demonstrate that CPA application strongly inhibits voltage-gated exocytosis and Ca2+ transients in rat CCs, regardless of strain (SHR or WKY). Thus, despite the greater phylogenetic distance from the most recent common ancestors, suppression of endoplasmic reticulum (ER) Ca2+ uptake through CPA inhibits the CA secretion in rat CCs more similarly to bovine than mouse CCs, unveiling divergent evolutionary relationships in the mechanism of CA exocytosis of CCs between rodents. Agents that inhibit the SERCA pump, such as CPA, suppress catecholamine secretion equally well in WKY and SHR CCs and are not potential therapeutic agents for hypertension. Rat CCs display Ca2+ signals of varying widths. Some even show early and late Ca2+ components. Narrowing the Ca2+ transients by CPA and ryanodine suggests that the late component is mainly due to CICR. Simultaneous recordings of Ca2+ signaling and amperometry in CCs revealed the existence of a robust and predictable correlation between the kinetics of the whole-cell intracellular Ca2+ signal and the rate of exocytosis at the single-cell level.

The biophysical properties of TRIC-A and TRIC-B and their interactions with RyR2.

Trimeric intracellular cation channels (TRIC-A and TRIC-B) are thought to provide counter-ion currents to enable charge equilibration across the sarco/endoplasmic reticulum (SR) and nuclear membranes. However, there is also evidence that TRIC-A may interact directly with ryanodine receptor type 1 (RyR1) and 2 (RyR2) to alter RyR channel gating. It is therefore possible that the reverse is also true, where the presence of RyR channels is necessary for fully functional TRIC channels. We therefore coexpressed mouse TRIC-A or TRIC-B with mouse RyR2 in HEK293 cells to examine if after incorporating membrane vesicles from these cells into bilayers, the presence of TRIC affects RyR2 function, and to characterize the permeability and gating properties of the TRIC channels. Importantly, we used no purification techniques or detergents to minimize damage to TRIC and RyR2 proteins. We found that both TRIC-A and TRIC-B altered the gating behavior of RyR2 and its response to cytosolic Ca2+ but that TRIC-A exhibited a greater ability to stimulate the opening of RyR2. Fusing membrane vesicles containing TRIC-A or TRIC-B into bilayers caused the appearance of rapidly gating current fluctuations of multiple amplitudes. The reversal potentials of bilayers fused with high numbers of vesicles containing TRIC-A or TRIC-B revealed both Cl- and K+ fluxes, suggesting that TRIC channels are relatively non-selective ion channels. Our results indicate that the physiological roles of TRIC-A and TRIC-B may include direct, complementary regulation of RyR2 gating in addition to the provision of counter-ion currents of both cations and anions.

Targeting SERCA2 in organotypic epidermis reveals MEK inhibition as a therapeutic strategy for Darier disease.

Mutation of the ATP2A2 gene encoding sarco-endoplasmic reticulum calcium ATPase 2 (SERCA2) was linked to Darier disease more than 2 decades ago; however, there remain no targeted therapies for this disorder causing recurrent skin blistering and infections. Since Atp2a2-knockout mice do not phenocopy its pathology, we established a human tissue model of Darier disease to elucidate its pathogenesis and identify potential therapies. Leveraging CRISPR/Cas9, we generated human keratinocytes lacking SERCA2, which replicated features of Darier disease, including weakened intercellular adhesion and defective differentiation in organotypic epidermis. To identify pathogenic drivers downstream of SERCA2 depletion, we performed RNA sequencing and proteomics analysis. SERCA2-deficient keratinocytes lacked desmosomal and cytoskeletal proteins required for epidermal integrity and exhibited excess MAPK signaling, which modulates keratinocyte adhesion and differentiation. Immunostaining patient biopsies substantiated these findings, with lesions showing keratin deficiency, cadherin mislocalization, and ERK hyperphosphorylation. Dampening ERK activity with MEK inhibitors rescued adhesive protein expression and restored keratinocyte sheet integrity despite SERCA2 depletion or chemical inhibition. In sum, coupling multiomic analysis with human organotypic epidermis as a preclinical model, we found that SERCA2 haploinsufficiency disrupts critical adhesive components in keratinocytes via ERK signaling and identified MEK inhibition as a treatment strategy for Darier disease.

Seasonal cold induces divergent structural/biochemical adaptations in different skeletal muscles of Columba livia: evidence for nonshivering thermogenesis in adult birds.

Birds are endothermic homeotherms even though they lack the well-studied heat producing brown adipose tissue (BAT), found in several clades of eutherian mammals. Earlier studies in ducklings have demonstrated that skeletal muscle is the primary organ of nonshivering thermogenesis (NST) plausibly via futile calcium (Ca2+)-handling through ryanodine receptor (RyR) and sarco-endoplasmic reticulum Ca2+-ATPase (SERCA). However, recruitment of futile Ca2+-cycling in adult avian skeletal muscle has not been documented. Studies in mammals show remarkable mitochondrial remodeling concurrently with muscle NST during cold. Here, we wanted to define the mitochondrial and biochemical changes in the muscles in free-ranging adult birds and whether different skeletal muscle groups undergo similar seasonal changes. We analyzed four different muscles (pectoralis, biceps, triceps and iliotibialis) from local pigeon (Columba livia) collected during summer and winter seasons in two consecutive years. Remarkable increase in mitochondrial capacity was observed as evidenced from succinate dehydrogenase (SDH) and cytochrome c oxidase (COX) activity staining in all the muscles. Interestingly, fibers with low SDH activity exhibited greater cross-sectional area during winter in all muscles except iliotibialis and became peripherally arranged in individual fascicles of pectoralis, which might indicate increased shivering. Furthermore, gene expression analysis showed that SERCA, sarcolipin and RyR are up-regulated to different levels in the muscles analyzed indicating muscle NST via futile Ca2+-cycling is recruited to varying degrees in winter. Moreover, proteins of mitochondrial-SR-tethering and biogenesis also showed differential alterations across the muscles. These data suggest that tropical winter (∼15°C) is sufficient to induce distinct remodeling across muscles in adult bird.

Translocation of IGF-1R in endoplasmic reticulum enhances SERCA2 activity to trigger Ca2+ER perturbation in hepatocellular carcinoma.

The well-known insulin-like growth factor 1 (IGF1)/IGF-1 receptor (IGF-1R) signaling pathway is overexpressed in many tumors, and is thus an attractive target for cancer treatment. However, results have often been disappointing due to crosstalk with other signals. Here, we report that IGF-1R signaling stimulates the growth of hepatocellular carcinoma (HCC) cells through the translocation of IGF-1R into the ER to enhance sarco-endoplasmic reticulum calcium ATPase 2 (SERCA2) activity. In response to ligand binding, IGF-1Rβ is translocated into the ER by β-arrestin2 (β-arr2). Mass spectrometry analysis identified SERCA2 as a target of ER IGF-1Rβ. SERCA2 activity is heavily dependent on the increase in ER IGF-1Rβ levels. ER IGF-1Rβ phosphorylates SERCA2 on Tyr990 to enhance its activity. Mutation of SERCA2-Tyr990 disrupted the interaction of ER IGF-1Rβ with SERCA2, and therefore ER IGF-1Rβ failed to promote SERCA2 activity. The enhancement of SERCA2 activity triggered Ca2+ER perturbation, leading to an increase in autophagy. Thapsigargin blocked the interaction between SERCA2 and ER IGF-1Rβ and therefore SERCA2 activity, resulting in inhibition of HCC growth. In conclusion, the translocation of IGF-1R into the ER triggers Ca2+ER perturbation by enhancing SERCA2 activity through phosphorylating Tyr990 in HCC.

Platelet-derived microvesicles induce intracellular calcium mobilization in human platelets.

Platelet-derived microvesicles (PMVs) represent a significant proportion of microvesicles in circulation and have been linked to various pathophysiological complications. Recent research suggests that PMVs carry significant amounts of cargo that can affect cellular functions by influencing calcium oscillations in target cells. As calcium is involved in multiple cellular processes, including hemostasis and thrombosis, this study aimed to investigate the impact of PMVs on platelet calcium mobilization. The study found that PMVs increase platelet intracellular calcium levels via both intracellular storage and extracellular space in a dose-dependent manner. The study highlighted the critical role of the dense tubular system, acidic vacuoles, mitochondrial stores, and store-operated calcium entry (SOCE) in PMV-mediated calcium release in human platelets. Moreover, the study revealed that PMV-induced calcium rise in platelets does not occur via sarcoendoplasmic reticulum calcium ATPase, and extracellular calcium addition further increases the calcium level in platelets, demonstrating the involvement of SOCE. These findings provide insights into the platelet stimulation signaling mechanisms and contributes to our understanding of platelet and cell behavior when exposed to PMV-rich environments.

(2R,4aS,6aS,12bR,14aS,14bR)10-Hydroxy-N-(4-((6-methoxyquinolin-8-yl)amino)pentyl)-2,4a,6a,9,12b,14a-hexamethyl-11-oxo-1,2,3,4,4a,5,6,6a,11,12b,13,14,14a,14b-tetradecahydropicene-2-carboxamide

We herein report the synthesis of a derivative of the natural compound celastrol linked to the antimalarial drug primaquine through an amide obtained by the activation of the carboxylic acid with HOBt/EDC. The chemical structure of the new molecule was fully characterized by proton nuclear magnetic resonance (1H-NMR), carbon-13 nuclear magnetic resonance (13C-NMR), heteronuclear single quantum coherence (HSQC), correlation spectroscopy (1H-1H-COSY), distortionless enhancement by polarization transfer (DEPT), mass spectrometry, Fourier-transform infrared (FTIR), and ultraviolet (UV) spectroscopies. Computational studies were enrolled to predict the interaction of the synthesized compound with sarco-endoplasmic reticulum (SR) Ca2+ transport ATPase (SERCA), a target of relevance for developing new therapeutics against arthritis. The drug-likeness of the compound was also investigated by predicting its pharmacokinetic properties.

Echocardiographic evaluation of right heart failure which might be associated with DNA damage response in SU5416-hypoxia induced pulmonary hypertension rat model

Right heart failure is the leading cause of death in pulmonary hypertension (PH), and echocardiography is a commonly used tool for evaluating the risk hierarchy of PH. However, few studies have explored the dynamic changes in the structural and functional changes of the right heart during the process of PH. Previous studies have found that pulmonary circulation coupling right ventricular adaptation depends on the degree of pressure overload and other factors. In this study, we performed a time-dependent evaluation of right heart functional changes using transthoracic echocardiography in a SU5416 plus hypoxia (SuHx)-induced PH rat model. Rats were examined in 1-, 2-, 4-, and 6-week using right-heart catheterization, cardiac echocardiography, and harvested heart tissue. Our study found that echocardiographic measures of the right ventricle (RV) gradually worsened with the increase of right ventricular systolic pressure, and right heart hypofunction occurred at an earlier stage than pulmonary artery thickening during the development of PH. Furthermore, sarco-endoplasmic reticulum calcium ATPase 2 (SERCA2), a marker of myocardial damage, was highly expressed in week 2 of SuHx-induced PH and had higher levels of expression of γ-H2AX at all timepoints, as well as higher levels of DDR-related proteins p-ATM and p53/p-p53 and p21 in week 4 and week 6. Our study demonstrates that the structure and function of the RV begin to deteriorate with DNA damage and cellular senescence during the early stages of PH development.