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Acantholytic skin disorders, by definition, compromise intercellular adhesion between epidermal keratinocytes. The root cause of blistering in these diseases traces back to direct disruption of adhesive cell-cell junctions, exemplified by autoantibody-mediated attack on desmosomes in pemphigus. However, genetic acantholytic disorders originate from more indirect mechanisms. Darier disease and Hailey-Hailey disease arise from mutations in the endoplasmic reticulum calcium pump, SERCA2, and the Golgi calcium/manganese pump, SPCA1, respectively. Though the disease-causing mutations have been known for nearly 25 years, the mechanistic linkage between dysregulation of intracellular ion stores and weakening of cell-cell junctions at the plasma membrane remains puzzling. The molecular underpinnings of a related idiopathic disorder, Grover disease, are even less understood. Due to an incomplete understanding of acantholytic pathology at the molecular level, these disorders lack proven, targeted treatment options, leaving patients with the significant physical and psychological burdens of chronic skin blistering, infections, and pain. This article aims to review what is known at the molecular, cellular, and clinical levels regarding these under-studied disorders and to highlight knowledge gaps and promising ongoing research. Armed with this knowledge, our goal is to aid investigators in defining essential questions about disease pathogenesis and to accelerate progress toward novel therapeutic strategies., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Background and Aim: Our previous research indicates that sarcoplasmic/endoplasmic reticulum calcium ATPase 2 (SERCA2) dysfunction facilitates the phenotypic transformation of aortic smooth muscle cells (ASMCs) and intensifies aortic aneurysm through the regulation of calcium-dependent pathways and endoplasmic reticulum stress. Our hypothesis is that additional mechanisms are involved in aortic aneurysm and atherosclerosis induced by SERCA2 dysfunction from the perspective of ASMC phenotypic transformation., Methods & Results: In SERCA2 dysfunctional mice and their control littermates, ASMCs were isolated to analyze protein expression and cell functions, and angiotensin II was infused into these mice that were backcrossed into LDL receptor deficient background to induce aortic aneurysm and atherosclerosis. In ASMCs from SERCA2 dysfunctional mice, the cell cycle was accelerated, and proliferation and migration were enhanced, which could be reversed by SERCA agonist CDN1163 or calcium chelator BAPTA-AM. In ASMCs, SERCA2 dysfunction increased reactive oxygen species (ROS) production, activating extracellular signal-regulated kinases 1 and 2 (ERK1/2) and angiotensin II/angiotensin II type 1 receptor (AT1R) pathways. Both ERK1/2 and angiotensin II/AT1R activations are implicated in SERCA2 dysfunction-induced ASMC phenotypic transformation and ROS production. The redox modulator Tempol suppressed ERK1/2 and angiotensin II/AT1R pathways, inhibiting ASMC phenotypic transformation and alleviating angiotensin II-induced aortic aneurysm and atherosclerosis., Conclusion: SERCA2 dysfunction accelerates aortic aneurysm and atherosclerosis by inducing oxidative stress in ASMCs, with activations of ERK1/2 and angiotensin II/AT1R involved in ASMC phenotypic transformation. Inhibition of oxidative stress in ASMCs is beneficial in alleviating angiotensin II-induced aortic aneurysm and atherosclerosis caused by SERCA2 dysfunction., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2025. Published by Elsevier Ltd.)

The regulation of calcium signaling within cardiomyocytes is pivotal for maintaining cardiac function, with disruptions in sarcoplasmic reticulum (SR) calcium handling linked to various heart diseases. This review explores the emerging role of microRNAs (miRNAs) in modulating SR calcium dynamics, highlighting their influence on cardiomyocyte maturation, function, and disease progression. We present a comprehensive overview of the mechanisms by which specific miRNAs, such as miR-1, miR-24, and miR-22, regulate key components of calcium handling, including ryanodine receptors, SERCA, and NCX. Notably, we identify critical research gaps, particularly the inconsistent findings regarding miRNA expression in heart disease and the need for standardized experimental conditions. Furthermore, we emphasize the potential of miRNAs as therapeutic targets, given their ability to influence calcium handling pathways and cardiac remodeling. The review also discusses the challenges in translating miRNA research into clinical applications, including the need for safe and effective delivery methods. By synthesizing current knowledge and identifying areas for future investigation, this review aims to provide insights into the therapeutic potential of miRNAs in diagnosing and treating heart diseases, ultimately contributing to improved patient outcomes., Competing Interests: Declaration of competing interest There is no conflict of interest among authors., (Copyright © 2025. Published by Elsevier Inc.)

To determine how regulation of the sarco(endo)plasmic reticulum calcium ATPase (SERCA) affects the Ca 2+ content of the endoplasmic reticulum (ER), we developed a ratiometric ER-localized Ca 2+ indicator to rapidly quantify Ca 2+ stores and assess SERCA function in live cells. This assay enables screening of membrane micropeptides and small molecules that modulate SERCA and Na + /K + -ATPase activity and may facilitate development of therapies that target cellular Ca 2+ handling. Of the micropeptides tested, phospholamban (PLB) had the greatest degree of inhibition of SERCA, as measured by a decrease in ER Ca 2+ content compared to control. Sarcolipin (SLN), endoregulin (ELN), and another-regulin (ALN) also decreased ER Ca 2+ content, though less potently than PLB. We also investigated micropeptides that have been shown to have a positive effect on ER Ca 2+ uptake. Dwarf open reading frame (DWORF), a positive modulator of SERCA activity, and phospholemman (PLM), an inhibitor of the Na + /K + -ATPase, both increased ER Ca 2+ content compared to control. A superinhibitory variant of PLM, R70C, further increased ER Ca 2+ load compared to wild type PLM. Overall, our findings indicate that the inhibitory potency of micropeptides is governed by their relative binding affinities to SERCA. This allows for finely tuned modulation of Ca 2+ handling in different tissues based on differential expressions of micropeptide species. Understanding the contribution of each micropeptide to SERCA regulation may reveal novel strategies for therapeutic intervention in conditions where calcium dysregulation plays a role, such as heart disease, vascular disease, or neurodegenerative disorders., Competing Interests: Declaration of competing interest Seth L. Robia is a member of the scientific advisory board of Neurodon LLC. The authors declare no other competing interests., (Copyright © 2025. Published by Elsevier Ltd.)

Background: The EF-hand Ca 2+ sensor protein S100A1 has been identified as a molecular regulator and enhancer of cardiac performance. The ability of S100A1 to recognize and modulate the activity of targets such as SERCA2a (sarcoplasmic reticulum Ca 2+ ATPase) and RyR2 (ryanodine receptor 2) in cardiomyocytes has mostly been ascribed to its hydrophobic C-terminal α-helix (residues 75-94). We hypothesized that a synthetic peptide consisting of residues 75 through 94 of S100A1 and an N-terminal solubilization tag (S100A1ct) could mimic the performance-enhancing effects of S100A1 and may be suitable as a peptide therapeutic to improve the function of diseased hearts., Methods: We applied an integrative translational research pipeline ranging from in silico computational molecular modeling and in vitro biochemical molecular assays as well as isolated rodent and human cardiomyocyte performance assessments to in vivo safety and efficacy studies in small and large animal cardiac disease models., Results: We characterize S100A1ct as a cell-penetrating peptide with positive inotropic and antiarrhythmic properties in normal and failing myocardium in vitro and in vivo. This activity translates into improved contractile performance and survival in preclinical heart failure models with reduced ejection fraction after S100A1ct systemic administration. S100A1ct exerts a fast and sustained dose-dependent enhancement of cardiomyocyte Ca 2+ cycling and prevents β-adrenergic receptor-triggered Ca 2+ imbalances by targeting SERCA2a and RyR2 activity. In line with the S100A1ct-mediated enhancement of SERCA2a activity, modeling suggests an interaction of the peptide with the transmembrane segments of the sarcoplasmic Ca 2+ pump. Incorporation of a cardiomyocyte-targeting peptide tag into S100A1ct (cor-S100A1ct) further enhanced its biological and therapeutic potency in vitro and in vivo., Conclusions: S100A1ct is a promising lead for the development of novel peptide-based therapeutics against heart failure with reduced ejection fraction., Competing Interests: Drs Most, Busch, and Katus are cofounders and shareholders of AaviGen GmbH.

Background: Nucleoside diphosphate kinase B (NDPKB) deficiency in endothelial cells (ECs) promotes the activation of the hexosamine biosynthesis pathway (HBP), leading to vascular damage in the retina. The aim of this study was to investigate the consequences of NDPKB deficiency in the mouse heart., Methods: NDPKB deficient mice were used in the study. Echocardiography was employed to assess cardiac function in vivo. Characterization of contractility in hiPSC-derived cardiomyocytes (hiPSC-CMs) was measured with the IonOptix contractility system. Immunoblotting and immunofluorescence were carried out to analyze the expression and localization of proteins in cultured cells and left ventricles (LVs)., Results: NDPKB deficient mice displayed impaired glucose tolerance and increased heart weight compared to controls. Echocardiographic analysis revealed an increase in the diastolic diameter of the left ventricular posterior wall (LVPW), a decrease in the early diastolic mitral valve E and E' wave, and in the ratios of E/A and E'/A' in NDPKB deficient hearts, suggesting cardiac hypertrophy and diastolic dysfunction. In line with cardiac dysfunction, the phosphorylation of myocardial phospholamban (PLN) and the expression of sarcoplasmic/endoplasmic reticulum Ca 2+ -ATPase 2 (SERCA2) in the NDPKB deficient LVs were significantly reduced. Moreover, the accumulation of collagen, fibronectin as well as the upregulation of transforming growth factor β (TGF-β), were detected in NDPKB deficient LVs. In addition, activation of the HBP and its downstream O-GlcNAc cycle was observed in the LVs and cardiac ECs (CECs) isolated from the NDPKB -/- mice. Furthermore, a bipolar O-GlcNAc regulation was identified in CMs. O-GlcNAc was decreased in NDPKB-depleted CMs, while conditioned medium from NDPKB-depleted ECs significantly increased O-GlcNAc levels, along with contractile and relaxation dysfunction of the hiPSC-CMs, which was attenuated by inhibiting endothelial HBP activation., Conclusions: Deficiency in NDPKB leads to endothelial activation of the HBP and cardiac dysfunction. Our findings may highlight the crucial role of proper endothelial HBP in maintaining cardiovascular homeostasis., Competing Interests: Declarations. Ethics approval and consent to participate: Animal care and experiments were approved by Regierungspräsidium Karlsruhe (reference number: G-165/20) and Medical Faculty Mannheim, Heidelberg University (reference number I-22/09) and in accordance with the "2010/63/EU" statement of the European Parliament. The application of hiPSCs for this study has been approved by Ethics Committee of the Medical Faculty Mannheim, University of Heidelberg (approval number: 2018-565N-MA). Consent for publication: Not applicable. Competing interests: The authors declare that they have no competing interests., (© 2025. The Author(s).)

Myocardial ischemia-reperfusion injury (MIRI) often leads to irreversible myocardium dysfunction, while existing therapies are palliatives that transiently alleviate the disease symptoms. Repairing sarcoplasmic reticulum Ca 2+ -ATPase (SERCA) could reverse MIRI, which, however, requires precise drug delivery to the sarcoplasmic reticulum (SR). To this end, we leverage cell-cell "NETwork" of neutrophils to deliver SERCA activator-loaded SR-localized nanoparticles (L-P-NPs) to the damaged myocardial cells, following a hierarchical targeting process: (i) chemotactic neutrophils deliver L-P-NPs to ischemia-reperfused heart, achieving tissue level targeting; (ii) neutrophils produce neutrophil extracellular traps (NETs) to transport L-P-NPs to injured myocardial cell, achieving cellular level targeting; (iii) L-P-NPs escort therapeutic payloads to the SR, achieving subcellular targeting. We showed that this platform profoundly restored SERCA activity, augmented cardiac function, and ameliorated adverse heart remodeling. Our study provides insight into the direct restoration of SR for the effective treatment of MIRI and other muscle diseases.

This study investigated time-dependent changes in intracellular Ca 2 ⁺ levels in T cells, regulatory mechanisms, and functional effects after acute exercise. Male C57BL/6 mice were assigned to control and exercise groups, with the latter sacrificed at different intervals post-exercise. Murine splenic lymphocytes were isolated, and cytosolic Ca 2 ⁺ levels were measured using Fluo-3/AM. T-cell proliferation was assessed by flow cytometry and CFSE labeling, apoptosis by Annexin V/PI staining, and cytokine levels by CBA. RNA sequencing results were validated by qRT-PCR. The findings revealed that exercise significantly altered intracellular calcium oscillations in CD3 + cells, leading to reduced mitogen-stimulated proliferation, increased IL-6, IL-5, and IL-13 production, and decreased IL-2 secretion. Additionally, there was an increase in the apoptotic fraction of CD3 + cells, with upregulated expression of Cav1.1, Cav3.2, Cav3.3, SERCA2B, PKCθ, Bcl-xL, and FADD, and downregulated Ryr3 (p < 0.05). Transcriptomic analysis identified 607 differentially expressed genes involved in calcium ion binding and related pathways, including calcium signaling and cytokine-cytokine receptor interactions. Thus, acute exercise induces specific calcium oscillation patterns in T cells, mediated by PKCθ, affecting proliferation, apoptosis, and cytokine production. These changes are attributed to increased calcium influx through Cav1.1, Cav3.2, and Cav3.3 channels, decreased calcium reuptake via SERCA2B, and reduced calcium release through Ryr3. This research provides novel insights into how exercise modulates immune cell function by altering calcium levels, potential implications for enhancing immune responses or reducing inflammation., Competing Interests: Declaration of competing interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2025 Elsevier Inc. All rights reserved.)

Liquid storage is the primary preservation method in the swine breeding industry because of its advantages over cryopreservation. Calcium (Ca 2+ ), a key regulator of cell physiology, plays a crucial role during liquid preservation. Sarcoplasmic/Endoplasmic Reticulum Ca 2+ ATPases (SERCA) belong to a family of P-type ATPases that regulate Ca 2+ homeostasis within cells and have been previously described to play a function in the sperm of various mammalian species. Herein, we hypothesized that SERCA2 is present in pig sperm and is involved in the resilience of this cell to liquid preservation at 17 °C. For this purpose, sperm were incubated with different concentrations of thapsigargin (Thg; 0, 5, 25, and 50 µM) and stored at 17 °C for ten days. The presence and localization of SERCA2 were evaluated using immunoblotting and immunofluorescence, respectively. On days 0, 4, and 10, sperm motility was assessed using a computer-assisted sperm analysis (CASA) system, and sperm viability, membrane lipid disorder, acrosome integrity, mitochondrial membrane potential (MMP), and intracellular levels of Ca 2+ , superoxides and total reactive oxygen species (ROS) were evaluated by flow cytometry. We localized SERCA2 in the acrosome and midpiece of pig sperm. Furthermore, inhibition of SERCA with Thg resulted in reduced sperm viability and membrane stability, and increased MMP, and Ca 2+ and ROS levels. In conclusion, the activity of SERCA prevents the accumulation of intracellular Ca 2+ in sperm, which is detrimental to sperm quality and function during liquid storage at 17 °C. We thus suggest that the function of SERCA is crucial for the preservation of pig semen., Competing Interests: Declarations. Ethics approval and consent to participate: Not applicable. Consent for publication: Not applicable. Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Tetralogy of Fallot (TOF) is a common congenital heart disease. In this study, we proposed that cAMP response element-binding protein (CREB)-binding protein (CBP) regulates the proliferation and apoptosis in TOF by interacting with the sarco/endoplasmic reticulum Ca 2+ -ATPase (SERCA). To confirm this, we collected right ventricle tissue samples from TOF patients during surgery to correct the deformity and from the donors. We performed IHC, IF, RT-qPCR, WB and ChIP experiments. The analysis of these experiments shows that the expression of CBP is higher in TOF patients than in healthy individuals. Further, the RT-qPCR results indicated that the CBP and SERCA mRNA in TOF patients were significantly higher than in the healthy donors. Similarly, WB results suggested that the expression of CBP and SERCA was predominantly elevated in TOF patients compared to healthy individuals. Further, the AC16 cell line with CBP knockdown reveals high expression of the Edu compared to normal cells, and the percentage of the cell cycle in the M phase was elevated in the CBPi group. In addition, the CCK-8 cell viability assay showed more proliferation in the CBPi group than in the control group at different time points. Moreover, the RT-qPCR results indicated a lower expression of SERCA after the knockdown of CBP and CREB. Finally, the ChIP assay shows that CREB binds to the promoter of SERCA, and the CBP enrichment decreased after the CREB knockdown. In conclusion, these results suggest that CBP interacts with SERCA to regulate cell proliferation and apoptosis during heart development and that up-regulation of CBP leads to TOF., (© 2025 The Author(s). Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

Heart failure (HF) is one of the leading causes of death from cardiovascular disease among adults worldwide. The role of sex hormone receptors in the pathogenesis of HF and their regulatory mechanisms remain unclear. This study focused on investigating the localization and expression of sex hormone receptors (ERα, ERβ, AR and PR) and calcium handling proteins (SERCA2a and Cav1.2) in the left ventricle (LV) tissues of patients with HF, and to investigate their interactions. The LV tissues of HF patients were collected, and the localization of sex hormone receptors and calcium handling proteins was detected by immunofluorescence and immunohistochemistry. Western blotting was performed to study the expression levels of sex hormone receptors and calcium handling proteins. The interactions between these proteins were identified by immunofluorescence co-location and immunoprecipitation respectively. Compared with the control group, the expression levels of sex hormone receptors and calcium handling proteins in HF patients were significantly decreased. There was co-localization and interaction between protein ERα and Cav1.2, protein AR and SERCA2a, respectively. In summary, sex hormone receptors may be involved in regulating the progression of HF by interacting with calcium handling proteins.

This study explored the vasodilatory mechanisms of the sodium-glucose cotransporter-2 inhibitor remogliflozin using femoral arteries of rabbits. Remogliflozin dilated femoral arterial rings pre-contracted with phenylephrine in a concentration-dependent manner. Pretreatment with the Ca 2+ -sensitive K + channel inhibitor (paxilline), the ATP-sensitive K + channel inhibitor (glibenclamide), or the inwardly rectifying K + channel inhibitor (Ba 2+ ) did not alter the vasodilatory effect. However, vasodilation was significantly reduced by pretreatment with the voltage-dependent K + (Kv) channel inhibitor (4-AP) and with the Kv1.5 subtype inhibitor (DPO-1) but not with Kv2.1 or Kv7 subtype inhibitor. Neither endothelium removal nor the inhibition of nitric oxide production altered the vasodilatory effect of remogliflozin. However, pretreatment with the sarcoplasmic/endoplasmic reticulum Ca 2+ -ATPase (SERCA) pump inhibitors thapsigargin and cyclopiazonic acid effectively reduced the remogliflozin effect, as did pretreatment with cGMP/PKG-related but not cAMP/PKA-related signaling pathway inhibitors. These results indicate that remogliflozin-mediated dilation of the femoral artery occurs via the activation of Kv channels, mainly the Kv1.5 subtype, SERCA pump, and cGMP/PKG-related signaling pathways., Competing Interests: Declaration of competing interest The authors declare that there are no conflicts of interest., (Copyright © 2024. Published by Elsevier Inc.)

Aims: Sarcoendoplasmic reticulum Ca 2+ -ATPase 2 (SERCA2), encoded by ATP2A2, is a key protein involved in intracellular Ca 2+ homeostasis. The SERCA2a isoform is predominantly expressed in cardiomyocytes and type I myofibres. Variants in this gene are related to Darier disease, an autosomal dominant dermatologic disorder, but have never been linked to myopathy. We describe four patients suffering from a novel myopathy caused by a homozygous missense variant in ATP2A2., Methods: We studied a family with four individuals suffering from an adult-onset skeletal myopathy. We evaluated the clinicopathological phenotype, muscle imaging, and genetic workup including whole genome sequencing and segregation analysis. SERCA2 expression in skeletal muscle was assessed. Functional studies to evaluate Ca 2+ handling in patient myotubes in response to electrical stimulation or caffeine exposure were performed., Results: Four sisters developed slowly progressive proximal weakness in adulthood. Biopsy findings showed small vacuoles restricted to type I myofibres. Ultrastructural analysis showed sarcotubular dilation and autophagic vacuoles. Genome sequencing revealed a homozygous variant in ATP2A2 (c.1117G > A, p.(Glu373Lys)) which segregated with the disease. Immunohistochemistry suggested that there was SERCA2 mislocalisation in patient myofibres. Western blotting did not show changes in the amount of protein. In vitro functional studies revealed delayed sarcoendoplasmic reticulum Ca 2+ reuptake in patient myotubes, consistent with an altered pumping capacity of SERCA2 after cell stimulation., Conclusions: We report a novel adult-onset vacuolar myopathy caused by a homozygous variant in ATP2A2. Biopsy findings and functional studies demonstrating an impaired function of SERCA2 and consequent Ca 2+ dysregulation in slow-twitch skeletal myofibres highly support the pathogenicity of the variant., (© 2025 British Neuropathological Society.)

Aims: The present study investigated the vasorelaxant mechanisms of an oral antidiabetic drug, anagliptin, using phenylephrine (Phe)-induced pre-contracted rabbit aortic rings., Methods: Arterial tone measurement was performed in rabbit thoracic aortic rings., Results: Anagliptin induced vasorelaxation in a dose-dependent manner. Pre-treatment with the classical voltagedependent K + (Kv) channel inhibitors 4-aminopyridine and tetraethylammonium significantly decreased the vasorelaxant effect of anagliptin, whereas pre-treatment with the inwardly rectifying K + (Kir) channel inhibitor Ba 2+ , the ATP-sensitive K + (K ATP ) channel inhibitor glibenclamide, and the large-conductance Ca 2+ -activated K + (BKCa) channel inhibitor paxilline did not attenuate the vasorelaxant effect. Furthermore, the vasorelaxant response of anagliptin was effectively inhibited by pre-treatment with the sarco/endoplasmic reticulum Ca 2+ -ATPase (SERCA) pump inhibitors thapsigargin and cyclopiazonic acid. Neither cAMP/protein kinase A (PKA)-related signaling pathway inhibitors (adenylyl cyclase inhibitor SQ 22536 and PKA inhibitor KT 5720) nor cGMP/protein kinase G (PKG)-related signaling pathway inhibitors (guanylyl cyclase inhibitor ODQ and PKG inhibitor KT 5823) reduced the vasorelaxant effect of anagliptin. Similarly, the anagliptin-induced vasorelaxation was independent of the endothelium., Conclusions: Based on these results, we suggest that anagliptin-induced vasorelaxation in rabbit aortic smooth muscle occurs by activating Kv channels and the SERCA pump, independent of other vascular K + channels, cAMP/PKA- or cGMP/PKG-related signaling pathways, and the endothelium., Competing Interests: Declarations. Conflict of interest: The authors declare that there are no conflicts of interest. Ethical standard: All animal care and experimental procedures followed the guidelines of the Committee for Animal Experiments at Kangwon National University (approval No. KW-220627–2). Informed consent: Not applicable., (© 2024. Springer-Verlag Italia S.r.l., part of Springer Nature.)

The sarco(endo)plasmic reticulum Ca 2+ ATPase (SERCA) is a membrane transporter that creates and maintains intracellular Ca 2+ stores. In the heart, SERCA is regulated by an inhibitory interaction with the monomeric form of the transmembrane micropeptide phospholamban (PLB). PLB also forms avid homo-pentamers, and the dynamic exchange of PLB between pentamers and SERCA is an important determinant of cardiac responsiveness to exercise. Here, we investigated two naturally occurring pathogenic variants of PLB: a cysteine substitution of Arg9 (R9C) and an in-frame deletion of Arg14 (R14del). Both variants are associated with dilated cardiomyopathy. We previously showed that the R9C mutation causes disulfide crosslinking and hyperstabilization of pentamers. While the pathogenic mechanism of R14del is unclear, we hypothesized this mutation may also alter pentamer stability. Immunoblots revealed a significantly increased pentamer: monomer ratio for R14del-PLB compared to WT-PLB. We quantified homo-oligomerization and SERCA-binding in live cells using fluorescence resonance energy transfer (FRET) microscopy. R14del-PLB showed an increased affinity for homo-oligomerization and decreased binding affinity for SERCA compared to WT. The data suggest that, like R9C, the R14del mutation stabilizes PLB in pentamers, decreasing its ability to regulate SERCA. The R14del mutation reduced the rate of PLB unbinding from pentamers after transient elevations of Ca 2+ , limiting the recovery of PLB-SERCA complexes. A computational model predicted that hyperstabilization of PLB pentamers by R14del impairs the ability of cardiac Ca 2+ handling to respond to changing heart rates between rest and exercise. We postulate that impaired responsiveness to physiological stress contributes to arrhythmogenesis in human carriers of the R14del mutation., Competing Interests: Conflict of interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: S.L.R. is a member of the scientific advisory board of Neurodon LLC. Neurodon did not have any role in this study., (Copyright © 2025 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Aldosterone-producing adenomas (APAs) are a common cause of primary aldosteronism that can lead to cardiovascular complications if left untreated. Machine learning-based bioinformatics approaches have emerged as powerful tools for identifying potential disease markers, gaining widespread recognition in biomedical research. We aimed to use machine learning to discover novel biomarkers of APAs to identify new pathophysiological mechanisms., Methods: We applied 2 machine learning algorithms to published RNA sequencing data to identify APA feature genes. Validation was performed using APA tissue samples, spatial transcriptomics, pig adrenal glands, and in vitro assays in a human adrenocortical cell line., Results: Machine learning identified ATP2A3 as a key feature gene in APA, and its upregulation in APAs compared with the adjacent cortex was confirmed by spatial transcriptomics. In human adrenocortical cells, angiotensin II treatment increased ATP2A3 gene expression 9.15-fold. Silencing ATP2A3 decreased basal CYP11B2 expression and aldosterone secretion by 3.51-fold and 1.46-fold, respectively, and by 1.77-fold and 1.94-fold under angiotensin II stimulation. Dietary sodium restriction in pigs significantly increased ATP2A3 mRNA and protein levels. Spatial transcriptomics showed that APA cells exhibited higher ATP2A3 gene expression compared with all other adrenal cell types. The suppressive effect of ATP2A3 silencing on CYP11B2 expression was further enhanced by Ca 2+ inhibitors., Conclusions: The ATP2A3 gene is highly expressed in APA and is a key regulator of CYP11B2 expression and aldosterone production., Competing Interests: None.

Capsaicin, a polyphenol, is known to regulate energy expenditure and thermogenesis in adipocytes and muscles. However, its role in modulating uncoupling proteins (UCPs) and adenosine triphosphate (ATP)-dependent thermogenesis in muscles remains unclear. This study investigated the mechanisms underlying the role of capsaicin in modulating the UCP- and ATP-dependent thermogenesis in C2C12 myoblasts, as well as the gastrocnemius (GM) and soleus muscles (SM) of mice. We employed molecular dynamics (MD), quantitative real-time polymerase chain reactions (qRT-PCR), immunoblots, staining methods, and assay kits to investigate the role of capsaicin on thermogenesis and its modulatory roles on the transient receptor potential cation channel subfamily V member 1 (TRPV1) and α-/β-adrenergic receptors (ARs) using in vitro and in vivo models. Our findings demonstrate that capsaicin treatment in high-fat diet-induced obese mice reduces weight gain and elevates the expression of UCP- and ATP-dependent thermogenic effectors through ATP-consuming calcium and creatine futile cycles. In vitro and in vivo models capsaicin treatment elevated the expression of sarcoendoplasmic/endoplasmic reticulum calcium ATPases (SERCA-1 and -2), ryanodine receptors (RYR-1 and -2), uncoupling proteins (UCP-2 and -3), creatine kinase B (CKB), and creatine kinase mitochondrial 2 (CKMT2), through activation of TRPV1, α1-, β2-, and β3-AR as well as the suppressed expression of α2-AR. Furthermore, our results also indicate that capsaicin promotes myotube development and enhances lipid metabolism in C2C12 cells. We found that capsaicin increased intracellular Ca 2+ levels and the expression of the voltage-dependent anion channel (VDAC) and mitochondrial calcium uniporter (MCU), suggesting that elevated mitochondrial Ca 2+ levels boost the expression of oxidative phosphorylation protein complexes via the activation of the ATP-futile cycle. Mechanistic studies in C2C12 cells revealed that TRPV1 is likely dispensable for capsaicin-induced thermogenesis, and TRPV1 and α1-AR may synergistically induce thermogenesis. Collectively, our findings have uncovered a novel mechanism of UCP- and ATP-dependent thermogenesis and its associated pathways in both cellular and animal models which is crucial for designing therapeutic strategies to address obesity and associated metabolic diseases., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2025 Elsevier B.V. All rights reserved.)

Exercise-induced muscle damage (EIMD) is characterized by a severe and prolonged decline in force-generating capacity. However, the precise cellular mechanisms underlying the observed long-lasting decline in force-generating capacity associated with EIMD are still unclear. We investigated in vivo force generation and ex vivo Ca 2+ -activated force generation, Ca 2+ sensitivity, and myofiber Ca 2+ handling systems (SR and t-tubules) in human biceps brachii before and 2, 48, and 96 h after eccentrically muscle-damaging contractions and in non-exercised control arm. The force-generating capacity declined by 50 ± 13% 3 h after exercise and was still not recovered after 96 h. The force-Ca relationship of skinned myofibers revealed an impaired maximal Ca 2+ -activated force in MHC I-fibers, but not MHC II-fibers 48 h after exercise. Further, Ca 2+ sensitivity was increased in MHC II-fibers, which was reversed after incubation with a strong reductant. There was a biphasic increase in SERCA sulfonylation, and a parallel reduction in the SR Ca 2+ uptake rate, with no effects on SR vesicle leak or SR vesicle Ca 2+ release rate. T-tubules showed a progressive increase in the density of longitudinal tubules by 96 h after exercise. In conclusion, MHC II-fiber Ca 2+ sensitivity was increased 48 h after exercise, attributed to changes in the REDOX status. 96 h after exercise SR vesicle Ca 2+ uptake was impaired, and an increased number of longitudinal tubules were observed. These alterations may contribute to the impaired force generation evident at the late stage of recovery., (© 2025 The Author(s). Physiological Reports published by Wiley Periodicals LLC on behalf of The Physiological Society and the American Physiological Society.)

Objective: To investigate the effects and mechanisms of miRNA 221 on myocardial ischemia/reperfusion injury (MIRI) in mice through the regulation of phospholamban (PLB) expression., Methods: The MIRI mouse model was created and mice were divided into sham, MIRI, MIRI+ 221, and MIRI+ scr groups, with miRNA 221 overexpression induced in the myocardium of MIRI mice by targeted myocardial injection. Quantitative RT-PCR analysis was performed to observe the variation in miRNA 221, PLB, SERCA2, RYR2, NCX1, Cyt C and caspase 3 mRNA levels in myocardium, while Western blot assessed the levels of PLB, p-PLB (Ser16), p-PLB (Thr17), SERCA2, RYR2, NCX1, Cyt C and caspase 3 proteins. Changes in the structural integrity of the mouse heart were identified with HE and MASSON staining, while TUNEL staining was used to evaluate the TUNEL-positive cells of cardiomyocytes. Changes in myocardium calcium concentration were detected with reagent kits and the targeting interaction between miRNA 221 and PLB was evaluated using a luciferase reporter assay., Results: In the myocardium of MIRI mice, miRNA 221 level was significantly reduced, while the levels of PLB, p-PLB (Ser16), p-PLB (Thr17), and apoptosis-related genes caspase 3, and Cyt C were increased markedly, as well as calcium levels in myocardium. Following the overexpression of miRNA 221 in myocardium, there was a marked alleviation of myocardial injury and cardiomyocyte apoptosis and necrosis, significant enhancement of left ventricular systolic function, and marked decrease in the levels of PLB, p-PLB (Ser16), p-PLB (Thr17), caspase 3 and Cyt C, as well as a significant decrease in total calcium levels in myocardium., Conclusions: miRNA 221 can alleviate myocardial injury in mouse myocardial ischemia/reperfusion by suppressing the expression of PLB, thus reducing calcium overload in myocardium., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2025 Li et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Nimodipine is the current gold standard in the treatment of subarachnoid hemorrhage, as it is the only known calcium channel blocker that has been proven to improve neurological outcomes. In addition, nimodipine exhibits neuroprotective properties in vitro under various stress conditions. Furthermore, clinical studies have demonstrated a neuroprotective effect of nimodipine after vestibular schwannoma surgery. However, the molecular mode of action of nimodipine pre-treatment has not been well investigated. In the present study, using real-time cell death assays, we demonstrated that nimodipine not only reduces cell death induced by osmotic and oxidative stress but also protects cells directly at the time of stress induction in Schwann cells. Nimodipine counteracts stress-induced calcium overload and the overexpression of the Cav1.2 calcium channel. In addition, we found nimodipine-dependent upregulation of sarcoplasmic/endoplasmic reticulum calcium ATPase 3 (SERCA3) and LIM domain only 4 (LMO4) protein. Analysis of anti-apoptotic cell signaling showed an inhibition of the pro-apoptotic protein glycogen synthase kinase 3 beta (GSK3β). Nimodipine-treated Schwann cells exhibited higher levels of phosphorylated GSK3β at serine residue 9 during osmotic and oxidative stress. In conclusion, nimodipine prevents cell death by protecting cells from calcium overload by fine-tuning intracellular calcium signaling and gene expression.

Rippling Muscle Disease (RMD) is a rare skeletal myopathy characterized by abnormal muscular excitability manifesting with wave-like muscle contractions and percussion-induced muscle mounding. Hereditary RMD is associated with caveolin-3 or cavin-1 mutations. Recently, we identified cavin 4 autoantibodies as a biomarker of immune-mediated RMD (iRMD), though the underlying disease-mechanisms remain poorly understood. Transcriptomic studies were performed on muscle biopsies of 8 patients (5 males; 3 females; ages 26-to-80) with iRMD. Subsequent pathway analysis compared iRMD to human non-disease control and disease control (dermatomyositis) muscle samples. Transcriptomic studies demonstrated changes in key pathways of muscle contraction and development. All iRMD samples had significantly upregulated cavin-4 expression compared to controls, likely compensatory for autoantibody-mediated protein degradation. Proteins involved in muscle relaxation (including SERCA1, PMCA and PLN) were significantly increased in iRMD compared to controls. Comparison of iRMD to dermatomyositis transcriptomics demonstrated significant overlap in immune pathways, and the IL-6 signaling pathway was markedly increased in all iRMD patient muscle biopsies and increased in the majority of iRMD patients' serum. This study represents the first muscle transcriptomic analysis of iRMD patients and dissects underlying disease mechanisms. Increase of sarcolemmal and cellular calcium channels as well as PLN, an inhibitor of the SERCA pump for calcium into the sarcoplasm, likely alters the calcium dynamics in iRMD. These changes in crucial components of muscle relaxation may underlie rippling by altering calcium flux. Our findings provide crucial insights into the differential expression of genes regulating muscle relaxation and highlight potential disease pathomechanisms., Competing Interests: Declarations. Ethics approval and consent to participate: This study was approved by the Mayo Clinic Institutional Review Board (IRB#18-010637). Consent for publication: All authors have reviewed and approved the final version of the manuscript and consent to its publication in Acta Neuropathologica Communications. The study was approved by the Mayo Clinic Institutional Review Board (IRB#18-010637). Competing interests: MM has received personal compensation for serving on a Scientific Advisory or Data Safety Monitoring board for Argenx on an unrelated topic and for serving as an Associate Editor for Neurology Genetics, AAN. SP has received personal compensation for consulting roles with Genentech, Sage Therapeutics, Prime Therapeutics, UCB, Roche/Genentech, and Arialys Therapeutics, as well as for serving on a Scientific Advisory or Data Safety Monitoring board for UCB, Inc., Genentech, and F. Hoffman/LaRoche, for advisory work with Hoffman/LaRoche AG and Alexion, for consulting roles with Astellas, Alexion, MedImmune/Viela Bio, and Roche/Genentech. His institution has received research support from Grifols, NIH, Viela Bio/MedImmune/Horizon, Alexion Pharmaceuticals, F. Hoffman/LaRoche/Genentech, NovelMed, and AstraZeneca. SP holds intellectual property interests in healthcare-related discoveries and technologies. D.D. has consulted for UCB, Immunovant, Argenx, Arialys and Astellas pharmaceuticals. All compensation for consulting activities is paid directly to Mayo Clinic. He is a named inventor on filed patent that relates to KLHL11 as marker of autoimmunity and germ cell tumor. He has patents pending for LUZP4-IgG, cavin-4-IgG and SKOR2 IgG as markers of neurological autoimmunity. He has received funding from the DOD (CA210208 & PR220430), David J. Tomassoni ALS Research Grant Program and UCB., (© 2025. The Author(s).)

The electrophysiological mechanisms underlying melatonin's actions and the electrophysiological consequences of superimposed therapeutic hypothermia (TH) in preventing cardiac ischemia-reperfusion (IR) injury-induced arrhythmias remain largely unknown. This study aimed to unveil these issues using acute IR-injured hearts. Rabbits were divided into heart failure (HF), HF+melatonin, control, and control+melatonin groups. HF was induced by rapid right ventricular pacing. Melatonin was administered orally (10 mg/kg/day) for four weeks, and IR was created by 60-min coronary artery ligation and 30-min reperfusion. The hearts were then excised and Langendorff-perfused for optical mapping studies at normothermia, followed by TH. Melatonin significantly reduced ventricular fibrillation (VF) maintenance. In failing hearts, melatonin reduced the spatially discordant alternans (SDA) inducibility mainly by modulating intracellular Ca 2+ dynamics via upregulation of sarcoplasmic reticulum Ca 2+ -ATPase (SERCA2a) and calsequestrin 2 and attenuating the downregulation of phosphorylated phospholamban protein expression. In control hearts, melatonin improved conduction slowing and reduced dispersion of action potential duration (APD dispersion ) by upregulating phosphorylated connexin 43, attenuating the downregulation of SERCA2a and phosphorylated phospholamban and attenuating the upregulation of phosphorylated Ca 2+ /calmodulin-dependent protein kinase II. TH significantly retarded intracellular Ca 2+ decay slowed conduction, and increased APD dispersion , thereby facilitating SDA induction, which counteracted the beneficial effects of melatonin in reducing VF maintenance.

Obesity is a chronic disease that contributes to the development of insulin resistance, type 2 diabetes (T2D), and cardiovascular risk. Glucose-dependent insulinotropic polypeptide (GIP) receptor (GIPR) and glucagon-like peptide-1 (GLP-1) receptor (GLP-1R) co-agonism provide an improved therapeutic profile in individuals with T2D and obesity when compared with selective GLP-1R agonism. Although the metabolic benefits of GLP-1R agonism are established, whether GIPR activation impacts weight loss through peripheral mechanisms is yet to be fully defined. Here, we generated a mouse model of GIPR induction exclusively in the adipocyte. We show that GIPR induction in the fat cell protects mice from diet-induced obesity and triggers profound weight loss (∼35%) in an obese setting. Adipose GIPR further increases lipid oxidation, thermogenesis, and energy expenditure. Mechanistically, we demonstrate that GIPR induction activates SERCA-mediated futile calcium cycling in the adipocyte. GIPR activation further triggers a metabolic memory effect, which maintains weight loss after the transgene has been switched off, highlighting a unique aspect in adipocyte biology. Collectively, we present a mechanism of peripheral GIPR action in adipose tissue, which exerts beneficial metabolic effects on body weight and energy balance., Competing Interests: Declaration of interests R.J.S., V.P., K.A.C., C.J., and M.C. are current or past employees of Eli Lilly and Company. This does not alter the authors’ adherence to science journal policies on sharing data and materials., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Ca2+ tunneling requires both store-operated Ca2+ entry (SOCE) and Ca2+ release from the endoplasmic reticulum (ER). Tunneling expands the SOCE microdomain through Ca2+ uptake by SERCA into the ER lumen where it diffuses and is released via IP3 receptors. In this study, using high-resolution imaging, we outline the spatial remodeling of the tunneling machinery (IP3R1; SERCA; PMCA; and Ano1 as an effector) relative to STIM1 in response to store depletion. We show that these modulators redistribute to distinct subdomains laterally at the plasma membrane (PM) and axially within the cortical ER. To functionally define the role of Ca2+ tunneling, we engineered a Ca2+ tunneling attenuator (CaTAr) that blocks tunneling without affecting Ca2+ release or SOCE. CaTAr inhibits Cl- secretion in sweat gland cells and reduces sweating in vivo in mice, showing that Ca2+ tunneling is important physiologically. Collectively our findings argue that Ca2+ tunneling is a fundamental Ca2+ signaling modality., (© 2024 Courjaret et al.)

Long-term inactivity of skeletal muscle results in muscular disuse atrophy; however, hibernating animals do not experience muscular disuse atrophy during the hibernation period. The molecular mechanism underlining the anti-atrophy effect in these animals is unclear. O-linked N acetyl-β-D-glucosaminylation (O-GlcNAcylation) and its effect on cell signaling pathways are important mechanisms underlying muscular disuse atrophy; thus, in this study, we investigated O-GlcNAcylation changes during hibernation in Spermophilus dauricus to explore the role of O-GlcNAcylation in the muscle disuse atrophy resistance of hibernating animals. The results showed that during hibernation, the muscle fiber cross-sectional area and ratio of muscle fiber did not change, and the morphological structure of the muscle remained intact, with normal contractile function. The level of O-GlcNAcylation decreased during hibernation, but quickly returned to normal in the periodic arousal stage. The O-GlcNAcylation level of sarcoplasmic/endoplasmic reticulum calcium ATPase 1 (SERCA1) decreased, whereas its activity increased. The decrease in O-GlcNAcylation of SERCA could result in the decreased binding of phospholamban to SERCA1, thus decreasing its inhibition to SERCA1 activity. This in turn can inhibit muscle cell calcium overload, maintain muscle cell calcium homeostasis, and stabilize the calpain proteolytic pathway, ultimately inhibiting skeletal muscle atrophy. Our results demonstrate that periodic arousal along with returning O-GlcNAcylation level to normal are important mechanisms in preventing disuse atrophy of skeletal muscle during hibernation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Diabetes is a major risk factor for cardiovascular disease. However, the exact mechanism by which diabetes contributes to vascular damage is not fully understood. The aim of this study was to investigate the role of SUMO-1 mediated SERCA2a SUMOylation in the development of atherosclerotic vascular injury associated with diabetes mellitus. ApoE -/- mice were treated with streptozotocin (STZ) injection combined with high-fat feeding to simulate diabetic atherosclerosis and vascular injury. Human aortic vascular smooth muscle cells (HAVSMCs) were treated with high glucose (HG, 33.3 mM) and palmitic acid (PA, 200 µM) for 24 h to mimic a model of diabetes-induced vascular injury in vitro. Aortic vascular function, phenotypic conversion, migration, proliferation, intracellular Ca 2+ concentration, the levels of small ubiquitin-like modifier type 1 (SUMO1), SERCA2a and SUMOylated SERCA2a were detected. Diabetes-induced atherosclerotic mice presented obvious atherosclerotic plaques and vascular injury, companied by significantly lower levels of SUMO1 and SERCA2a in aorta. HG and PA treatment in HAVSMCs reduced the expressions of SUMO1, SERCA2a and SUMOylated SERCA2a, facilitated the HAVSMCs phenotypic transformation, proliferation and migration, attenuated the Ca 2+ transport, and increased the resting intracellular Ca 2+ concentration. We also confirmed that SUMO1 directly bound to SERCA2a in HAVSMCs. Overexpression of SUMO1 restored the function and phenotypic contractile ability of HAVSMCs by upregulating SERCA2a SUMOylation, thereby alleviating HG and PA-induced vascular injury. These observations suggest an essential role of SUMO1 to protect diabetes-induced atherosclerosis and aortic vascular injury by the regulation of SERCA2a-SUMOylation and calcium homeostasis., Competing Interests: Declarations. Competing interests: The authors declare no competing interests. Ethical approval: All experimental procedures and methods were approved by the Hebei University Animal Care and Use Committee (Approval No. IACUC-2021XG032). Consent to publish: We have obtained consent to publish this paper from all the participants of this research., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

SERCA2, a P-type ATPase located on the endoplasmic reticulum of cells, plays an important role in maintaining calcium balance within cells by transporting calcium from the cytoplasm to the endoplasmic reticulum against its concentration gradient. A multitude of studies have demonstrated that the expression of SERCA2 is abnormal in a wide variety of tumor cells. Consequently, research exploring compounds that target SERCA2 may offer a promising avenue for the development of novel anti-tumor drugs. This review has summarized the anti-tumor compounds targeting SERCA2, including thapsigargin, dihydroartemisinin, curcumin, galangin, etc. These compounds interact with SERCA2 on the endoplasmic reticulum membrane, disrupting intracellular calcium ion homeostasis, leading to tumor cell apoptosis, autophagy and cell cycle arrest, ultimately producing anti-tumor effects. Additionally, several potential research directions for compounds targeting SERCA2 as clinical anti-cancer drugs have been proposed in the review. In summary, SERCA2 is a promising anti-tumor target for drug discovery and development., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

While acute exercise affects sarcoplasmic reticulum (SR) function, the impact of resistance training remains unclear. The purpose of the present study was to investigate SR Ca 2+ handling plasticity in response to moderate- and high-volume strength training in elite rowers. Twenty elite male (n = 12) and female (n = 8) rowers performed three weekly strength training sessions for 8 weeks and were randomly allocated to either perform 3 sets (3-SET) or progressive increase from 5 to 10 sets (10-SET) of 10 repetitions during the training period. Skeletal muscle biopsies were collected before and after the intervention period and analyzed for SR vesicle Ca 2+ handling, SR related proteins, and myosin heavy chain (MHC) composition. Muscle strength was determined by isometric midthigh pull (IMTP). Training increased both the overall SR Ca 2+ release (19%) and uptake rates (34%), with no differences between groups. SR protein analysis revealed a high variability but suggests an increase of RYR1 and SERCA1, while SERCA2 decreased, corroborating changes in SR function, with no differences between groups. Regardless of training volume, a 9% higher relative MHCIIa proportion and a 7% decrease in the MHCI isoform was observed. There was an overall 8% increase of IMTP. Males exhibited higher SR Ca 2+ uptake and release rates compared to females, likely explained by a higher proportion of MHCII. These findings suggest that 8 weeks of moderate- or high-volume strength training enhances SR vesicle Ca 2+ uptake and release rates in elite male and female rowers, accompanied by a shift toward a larger proportion of MHCIIa fiber type., (© 2025 The Author(s). Scandinavian Journal of Medicine & Science In Sports published by John Wiley & Sons Ltd.)

Eccentric contractions (ECC) are accompanied by the accumulation of intracellular calcium ions ([Ca 2+ ] i ) and induce skeletal muscle damage. Suppressed muscle damage in repeated bouts of ECC is well characterized; however, whether it is mediated by altered Ca 2+ profiles remains unknown. We tested the hypothesis that repeated ECC suppresses Ca 2+ accumulation via adaptations in Ca 2+ regulation. Male Wistar rats were divided into two groups: ECC single bout (ECC-SB) and repeated bout (ECC-RB). Tibialis anterior (TA) muscles were subjected to ECC (40 times, 5 sets) once (ECC-SB) or twice 14 days apart (ECC-RB). Under anesthesia, the TA muscle was loaded with Ca 2+ indicator Fura 2-AM, and the 340/380 nm ratio was evaluated as [Ca 2+ ] i . Ca 2+ handling proteins were measured by Western blots. ECC induced [Ca 2+ ] i increase in both groups, but ECC-RB evinced a markedly suppressed [Ca 2+ ] i (Time: P < 0.01, Group: P = 0.0357). Five hours post-ECC, in contrast to the localized [Ca 2+ ] i accumulation in ECC-SB, ECC-RB exhibited lower and more uniform [Ca 2+ ] i ( P < 0.01). In ECC-RB, mitochondria Ca 2+ uniporter complex (MCU) components MCU and MICU2 were significantly increased pre-second ECC bout ( P < 0.01), and both SERCA1 and MICU1 were better preserved after contractions ( P < 0.01). Fourteen days after novel ECC, skeletal muscle mitochondrial Ca 2+ regulating proteins were elevated. Following subsequent ECC, [Ca 2+ ] i accumulation and muscle damage were suppressed and SERCA1 and MICU1 preserved. These findings suggest that tolerance to a subsequent ECC bout is driven, at least in part, by enhanced mitochondrial and sarcoplasmic reticulum Ca 2+ regulation. NEW & NOTEWORTHY We demonstrated a reduced [Ca 2+ ] i profile with suppressed muscle damage after a repeated bout of ECC in vivo: the ECC-induced immediate [Ca 2+ ] i increase was suppressed and the persistence of increased [Ca 2+ ] i with localized accumulation was diminished after repeated ECC. This effect occurred consonant with the upregulation of the mitochondrial Ca 2+ uniporter complex and better preservation of SERCA1 and MICU1. These findings suggest that the mechanistic bases for repeated bout protection involve adaptation of Ca 2+ regulation.

Background and Purpose: Sarcoplasmic reticulum Ca 2+ -ATPase (SERCA2a) is impaired in heart failure. Phosphodiesterases (PDEs) are implicated in the modulation of local cAMP signals and protein kinase A (PKA) activity essential for cardiac function. We characterise PDE isoforms that underlie decreased activities of SERCA2a and reduced cardiac contractile function in diabetic cardiomyopathy., Experimental Approach: Wild type mice were fed with either normal chow or a high-fat diet (HFD). Cardiomyocytes were isolated for excitation-contraction coupling (ECC), fluorescence resonant energy transfer PKA biosensor and proximity ligation assays., Key Results: The upregulated PDE4D3 and PDE4D9 isoforms in HFD cardiomyocytes specifically bound to SERCA2a but not ryanodine receptor 2 (RyR2) on the sarcoplasmic reticulum (SR). The increased association of PDE4D isoforms with SERCA2a in HFD cardiomyocytes led to reduced local PKA activities and phosphorylation of phospholamban (PLB) but minimally effected the PKA activities and phosphorylation of RyR2. These changes correlate with slower calcium decay tau in the SR and attenuation of ECC in HFD cardiomyocytes. Selective inhibition of PDE4D3 or PDE4D9 restored PKA activities and phosphorylation of PLB at the SERCA2a complex, recovered calcium decay tau, and increased ECC in HFD cardiomyocytes. Therapies with PDE4 inhibitor roflumilast, PDE4D inhibitor BPN14770 or genetical deletion of PDE4D restored PKA phosphorylation of PLB and cardiac contractile function., Conclusion and Implications: The current study identifies upregulation of specific PDE4D isoforms that selectively inhibit SERCA2a function in HFD-induced cardiomyopathy, indicating that this remodelling can be targeted to restore cardiac contractility in diabetic cardiomyopathy., (© 2024 The Author(s). British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

Optimal T cell activation is critical to orchestrate adaptive immune responses. Calcium is critical for T cell activation and integrates signaling pathways necessary to activate key transcription factors. In fact, patients with calcium channelopathies are immunodeficient. Here, we investigated the effects of different concentrations of intracellular calcium on activation of mouse T cells. High intracellular calcium amounts inhibited in vitro T cell proliferation as evidenced by a decreased cell cycling-to-hypodiploidy ratio in two models of activation: the combination of phorbol 12-myristate 13-acetate (PMA) and Ionomycin (an ionophore)/Thapsigargin (a SERCA inhibitor) or plate bound anti-CD3 and anti-CD28. High intracellular calcium amounts increased the production of reactive oxygen species (ROS) in T cells activated with PMA and Ionomycin and scavenging excess ROS using N-acetyl cysteine (NAC) rescued the decrease in cycling-to-hypodiploidy ratio. To test the universality of our observations, we studied the effects of tert-Butylhydroquinone (tBHQ), a SERCA inhibitor and Nrf2 activator. tBHQ alone did not increase intracellular calcium amounts but the intracellular calcium amounts increased when tBHQ was used in combination with PMA. Also, tBHQ inhibited T cell activation in a dose-dependent manner in both in vitro models of T cell activation. Importantly, intraperitoneal injection of tBHQ ameliorated Dextran Sodium Sulfate (DSS)-induced colitis in mice as evidenced by rescue of colon length shortening and lower disease activity index. Overall, this study identifies high calcium amounts as a potential target to lower T cell activation. The implications of these observations are discussed in the context of calcium modulating drugs that are used to treat various diseases., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Homeothermic endotherms defend their body temperature in cold environments using a number of behavioral and physiological mechanisms. Maintaining a stable body temperature primarily requires heat production through shivering or non-shivering thermogenesis (NST). Although the use of NST is well established in mammalian systems, the mechanisms and extent to which NST is used in birds are poorly understood. In mammals, one well-characterized mechanism of NST is through uncoupling of Ca2+ transport from ATP hydrolysis by sarco/endoplasmic reticulum ATPase (SERCA) in the skeletal muscle, which generates heat and may contribute to Ca2+ signaling for fatigue resistance and mitochondrial biogenesis. Two small proteins-sarcolipin (SLN) and phospholamban (PLN)-are known to regulate SERCA in mammals, but recent work shows inconsistent responses of SLN to cold acclimation in birds. In this study, we measured SERCA uncoupling in the pectoralis flight muscle of control (18°C) and cold-acclimated (-8°C) dark-eyed juncos (Junco hyemalis) that exhibited suppressed SLN transcription in the cold. We measured SERCA activity and Ca2+ uptake rates for the first time in cold-acclimated birds and found greater SERCA uncoupling in the muscle of juncos in the cold. However, SERCA uncoupling was not related to SLN or PLN transcription or measures of mitochondrial biogenesis. Nonetheless, SERCA uncoupling reduced an individual's risk of hypothermia in the cold. Therefore, while SERCA uncoupling in the cold could be indicative of NST, it does not appear to be mediated by known regulatory proteins in these birds. These results prompt interesting questions about the significance of SLN and PLN in birds and the role of SERCA uncoupling in response to environmental conditions., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Society for Integrative and Comparative Biology.)

Disruption of calcium (Ca 2+ ) homeostasis in neurons is a hallmark of neurodegenerative diseases. Here, we investigate the mechanisms leading to Ca 2+ dysregulation and ask whether altered Ca 2+ dynamics impinge on neuronal stress and circuit dysfunction. Using two-photon microscopy, we show that ocular hypertension, a major risk factor in glaucoma, and optic nerve crush injury disrupt the capacity of retinal neurons to clear cytosolic Ca 2+ leading to impaired light-evoked responses. Gene- and protein expression analysis reveal the loss of the sarco-endoplasmic reticulum (ER) Ca 2+ -ATPase2 pump (SERCA2/ATP2A2) in injured retinal neurons from mice and patients with primary open-angle glaucoma. Pharmacological activation or neuron-specific gene delivery of SERCA2 is sufficient to rescue single-cell Ca 2+ dynamics and promote robust survival of damaged neurons. Furthermore, SERCA2 gene supplementation reduces ER stress, reestablishes circuit balance, and restores visual behaviors. Our findings reveal that enhancing the Ca 2+ clearance capacity of vulnerable neurons alleviates organelle stress and promotes neurorecovery., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The sarco (endo)plasmic reticulum Ca 2+ -ATPase 2a (SERCA2a)-phospholamban (PLN) system within the sarcoplasmic reticulum is crucial for regulating intracellular Ca 2+ cycling in ventricular cardiomyocytes. Given that impaired Ca 2+ cycling is associated with heart failure, modulating SERCA2a activity represents a promising therapeutic strategy. Previously, we engineered an RNA aptamer (Apt30) that binds to PLN, thereby activating SERCA2a by alleviating PLN's inhibitory effect. However, Apt30 alone cannot reach intracellular PLN, necessitating the development of a mechanism for its specific internalization into cardiomyocytes. Using the systematic evolution of ligands by exponential enrichment (SELEX) method, we isolated RNA aptamers capable of internalizing into cardiomyocytes. These aptamers demonstrated sub-micromolar EC 50 values for cardiomyocyte internalization and exhibited significantly reduced activity against various non-myocardial cells, highlighting their specificity for cardiomyocytes. Moreover, some of these cardiomyocyte-internalizing aptamers could be linked to Apt30 as a single RNA strand without compromising their internalization efficacy. Supplementing the culture medium with these hybrid aptamers enhanced Ca 2+ transients and contractile function in rat cardiomyocytes. These findings provide critical insights for developing novel therapeutics directly acting on PLN in cardiomyocytes, potentially compensating for the disadvantages of conventional methods that involve viral vector-mediated intracellular transduction or alterations in endogenous protein expression., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The type 2 ryanodine receptor (RyR2) is the major Ca 2+ release channel required for Ca 2+ -induced Ca 2+ release (CICR) and cardiac excitation-contraction coupling. The cluster organization of RyR2 at the dyad is critical for efficient CICR. Despite its central role in cardiac Ca 2+ signaling, the mechanisms that control CICR are not fully understood. As a single RyR2 Ca 2+ flux dictates local CICR that underlies Ca 2+ sparks, RyR2 density in a cluster, and therefore the distance between RyR2s, should have a profound impact on local CICR. Here, we studied the effect of the RyR2 expression level ([RyR2]) on CICR activation, termination, and amplitude. The endoplasmic reticulum (ER)-targeted Ca 2+ sensor RCEPIA-1er was used to directly measure the ER [Ca 2+ ] (Ca 2+ ] ER ) in the T-Rex-293 the sarco/endoplasmic reticulum Ca 2+ -ATPase (SERCA2a) stable cell line expressing human RyR2. Cells coexpressing RyR2 and SERCA2a produced periodic [Ca 2+ ] ER depletions in the form of spontaneous Ca 2+ waves due to propagating CICR. For each studied cell, the [Ca 2+ ] ER at which Ca 2+ waves are activated and terminated was analyzed as a function of [RyR2]. CICR parameters, such as [Ca 2+ ] ER activation, termination, and amplitude, were inversely proportional to [RyR2] at low-intermediate levels. Increasing the sensitivity of RyR2 to cytosolic Ca 2+ lowered the [Ca 2+ ] ER at which CICR is activated and terminated. Decreasing the sensitivity of RyR2 to cytosolic Ca 2+ had the opposite effect on CICR. These results suggest that RyR2 density in the release cluster should have a significant impact on local CICR activation and termination. Since SR Ca 2+ load is evenly distributed throughout the SR network, clusters with higher RyR2 density would have a higher probability of initiating spontaneous CICR., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Aims: Intracellular calcium (Ca2+) overload is known to play a critical role in the development of cardiac dysfunction. Despite the remarkable improvement in managing the progression of heart disease, developing effective therapies for heart failure (HF) remains a challenge. A better understanding of molecular mechanisms that maintain proper Ca2+ levels and contractility in the injured heart could be of therapeutic value., Methods and Results: Here, we report that transcription factor zinc finger E-box-binding homeobox 2 (ZEB2) is induced by hypoxia-inducible factor 1-alpha (HIF1α) in hypoxic cardiomyocytes and regulates a network of genes involved in Ca2+ handling and contractility during ischaemic heart disease. Gain- and loss-of-function studies in genetic mouse models revealed that ZEB2 expression in cardiomyocytes is necessary and sufficient to protect the heart against ischaemia-induced diastolic dysfunction and structural remodelling. Moreover, RNA sequencing of ZEB2-overexpressing (Zeb2 cTg) hearts post-injury implicated ZEB2 in regulating numerous Ca2+-handling and contractility-related genes. Mechanistically, ZEB2 overexpression increased the phosphorylation of phospholamban at both serine-16 and threonine-17, implying enhanced activity of sarcoplasmic reticulum Ca2+-ATPase (SERCA2a), thereby augmenting SR Ca2+ uptake and contractility. Furthermore, we observed a decrease in the activity of Ca2+-dependent calcineurin/NFAT signalling in Zeb2 cTg hearts, which is the main driver of pathological cardiac remodelling. On a post-transcriptional level, we showed that ZEB2 expression can be regulated by the cardiomyocyte-specific microRNA-208a (miR-208a). Blocking the function of miR-208a with anti-miR-208a increased ZEB2 expression in the heart and effectively protected from the development of pathological cardiac hypertrophy., Conclusion: Together, we present ZEB2 as a central regulator of contractility and Ca2+-handling components in the mammalian heart. Further mechanistic understanding of the role of ZEB2 in regulating Ca2+ homeostasis in cardiomyocytes is an essential step towards the development of improved therapies for HF., Competing Interests: Conflict of interest: none declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of the European Society of Cardiology.)

Background: Melatonin is an antioxidant that also has anti-inflammatory effects. It has been reported to delay the progression of age-related macular degeneration (AMD), however, the mechanism has not been fully recognized., Purpose: The aim of the present study was to investigate the effects of melatonin on sodium iodate (SI)-induced retinal degeneration and elucidate the specific mechanisms, then, provide novel targets in AMD treatment., Methods: Retinal degeneration mouse model and in vitro retinal pigment epithelium (RPE) death model were established by SI treatment. Melatonin was administrated intraperitoneally at a concentration of 20, 40 or 80 mg/kg for in vivo study or treated at 48 h before SI treatment. To confirm the therapeutic effects of melatonin on mouse, the retinal structure and visual function were evaluated. The specific cell death rates were determined by CCK-8 assay, PI staining and protein level of RIPK3. The cytosolic or mitochondrial calcium levels were determined by Fluo-4AM or Rhod-2AM staining. Mitochondrial functions including mitochondrial dynamics, mitochondrial membrane potential, or mitochondrial permeability pore opening were evaluated. The proteins involved in endoplasmic reticulum (ER) stress were measured by western blot assay while the genes expression in calcium signaling pathway were measured by RT-qPCR., Results: We show that melatonin protects RPE cells from necroptosis and NLRP3 inflammasome activation induced by SI. Mechanistically, melatonin suppresses ER stress and intracellular calcium overload triggered by SI through restoring the function of SERCA2. Silencing of SERCA2 or blocking of melatonin receptors inhibit the protective effects of melatonin. Melatonin reduces mitochondrial Ca 2+ levels and restores mitochondrial membrane potential. Constant mitochondrial Ca 2+ overload directly promote cell necroptosis through mitochondrial fission. Inhibition of mitochondrial fission by Mdivi-1 prevent necroptosis induced by SI without altering the level of mitochondrial Ca 2+ ., Conclusions: The results confirmed that melatonin protects RPE cells from SI-induced injury by regulates MT2/SERCA2/Ca 2+ axis. This study highlighted the potential of melatonin in the treatment of AMD and elucidated the mechanism and signaling pathway that mediate the protective effects., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2024 Elsevier GmbH. All rights reserved.)

This study aimed to investigate the role of ITFG2, a protein highly expressed in cardiac tissues, in myocardial ischemic injury and its potential interactions with NEDD4-2. An in vivo myocardial infarction (MI) model was induced in mice via left anterior descending artery ligation, and ITFG2 expression was modulated using adeno-associated virus AAV9 vectors. Echocardiography was used to assess cardiac function, and primary mouse cardiomyocytes were cultured and subjected to hypoxia. ITFG2 expression was found to be significantly reduced following MI and in hypoxia-treated neonatal cardiomyocytes. Overexpression of ITFG2 improved cardiac contractility, reduced apoptosis, and stabilized calcium levels by inhibiting NEDD4-2-mediated ubiquitination of SERCA2a. Conversely, ITFG2 knockdown exacerbated calcium overload and cardiac dysfunction. Mechanistically, ITFG2 binds to NEDD4-2, decreasing its interaction with SERCA2a and preventing SERCA2a degradation. These findings suggest that ITFG2 acts as a critical inhibitor of NEDD4-2, preserving SERCA2a function and maintaining calcium homeostasis in cardiomyocytes under ischemic conditions. Therefore, ITFG2 may represent a potential therapeutic target for preventing myocardial ischemic injury and improving outcomes in MI patients., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Inc.)

Calcium (Ca 2+ ) dysregulation is a hallmark feature of cardiovascular disease. Intracellular Ca 2+ regulation is essential for proper heart function and is controlled by the sarco/endoplasmic reticulum Ca 2+ ATPase (SERCA2a). Another-regulin (ALN) is a newly discovered cardiomyocyte-expressed SERCA2a inhibitor, suggesting cardiomyocyte Ca 2+ -handling is more complex than previously appreciated. To study the role of ALN in cardiomyocytes, we generated ALN null mice (knockout, KO) and found that cardiomyocytes from these animals displayed enhanced Ca 2+ cycling and contractility compared to wildtype (WT) mice, indicating enhanced SERCA2a activity. In vitro and in vivo studies show that ALN is post-translationally modified via phosphorylation on Serine 19 (S19), suggesting this contributes to its ability to regulate SERCA2a. Immunoprecipitation and FRET analysis of ALN-WT, phospho-deficient ALN (S19A), or phosphomimetic ALN (S19D) revealed that S19 phosphorylation alters the SERCA2a-ALN interaction, leading to relief of its inhibitory effects. Adeno-associated virus mediated delivery of ALN-WT or phospho-mutant ALN-S19A/D in ALN KO mice showed that cardiomyocyte-specific expression of phospho-deficient ALN-S19A resulted in increased SERCA2a inhibition characterized by reduced rates of cytoplasmic Ca 2+ clearance compared to ALN-WT and ALN-S19D expressing cells, further supporting a role for this phosphorylation event in controlling SERCA2a-regulation by ALN. Levels of ALN phosphorylation were markedly increased in cardiomyocytes in response to Gα q agonists (angiotensin II, endothelin-1, phenylephrine) and Gα q -mediated phosphorylation of ALN translated to increased Ca 2+ cycling in cardiomyocytes from WT but not ALN KO mice. Collectively, these results indicate that ALN uniquely regulates Ca 2+ handling in cardiomyocytes via integration of neuroendocrine signaling with SERCA2a activity., Competing Interests: Declaration of competing interest None., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

An ellagitannin-derived metabolite, Urolithin A (UA), has emerged as a potential therapeutic agent for metabolic disorders due to its antioxidant, anti-inflammatory, and mitochondrial function-improving properties, but its efficacy in protecting against ER stress remains underexplored. The endoplasmic reticulum (ER) is a cellular organelle involved in protein folding, lipid synthesis, and calcium regulation. Perturbations in these functions can lead to ER stress, which contributes to the development and progression of metabolic disorders such as metabolic-associated fatty liver disease (MAFLD). In this study, we identified a novel target protein of UA and elucidated its mechanism for alleviating palmitic acid (PA)-induced ER stress. Cellular thermal shift assay (CETSA)-LC-MS/MS analysis revealed that UA binds directly to the sarcoplasmic/endoplasmic reticulum Ca 2+ -ATPase (SERCA), an important regulator of calcium homeostasis in mitochondria-associated ER membranes (MAMs). As an agonist of SERCA, UA attenuates abnormal calcium fluctuations and ER stress in PA-treated liver cells, thereby contributing to cell survival. The lack of UA activity in SERCA knockdown cells suggests that UA regulates cellular homeostasis through its interaction with SERCA. Collectively, our results demonstrate that UA protects against PA-induced ER stress and enhances cell survival by regulating calcium homeostasis in MAMs through SERCA. This study highlights the potential of UA as a therapeutic agent for metabolic disorders associated with ER stress.

The central aim of this study was to investigate whether male Wistar rats chronically fed a high-fat diet (HFD) over 106 days present high levels of interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α), and Na + and Ca 2+ transport alterations in the left ventricle, together with dyslipidemia and decreased glucose tolerance, and to investigate the influence of Ang-(3-4). The rats became moderately overweight with an expansion of visceral adiposity. Na + -transporting ATPases, sarco-endoplasmic reticulum Ca 2+ -ATPase (SERCA2a), and the abundance of Angiotensin II receptors were studied together with lipid and glycemic profiles from plasma and left-ventricle echocardiographic parameters fractional shortening (FS) and ejection fraction (EF). IL-6 and TNF-α increased (62% and 53%, respectively), but returned to normal levels with Angiotensin-(3-4) administration after 106 days. Significant lipidogram alterations accompanied a decrease in glucose tolerance. Angiotensin II receptors abundance did not change. (Na + + K + )ATPase and ouabain-resistant Na + -ATPase were downregulated and upregulated, respectively, but returned to normal values upon Angiotensin-(3-4) administration. SERCA2a lost its ability to respond to excess ATP. Echocardiography showed no changes in FS or EF. We conclude that being overweight causes an increase in Ang-(3-4)-sensitive IL-6 and TNF-α levels, and ion transport alterations in the left ventricle that could evolve into future heart dysfunction.

The neurodegenerative disorders, such as Alzheimer's disease (AD), Parkinson's disease (PD), Amyotrophic lateral sclerosis (ALS), Huntington's disease (HD) and Spinocerebellar ataxias (SCAs), present an enormous medical, social, financial and scientific problem. Despite intense research into the causes of these disorders, only marginal progress has been made in the clinic and no cures exist for any of them. Most of the scientific effort has been focused on identification of the major causes of these diseases and on developing ways to target them, such as targeting amyloid accumulation for AD or targeting expression of mutant Huntingtin for HD. Calcium (Ca 2+ ) signaling has long been proposed to play an important role in the pathogenesis of neurodegenerative disorders, but blockers of Ca 2+ channels and Ca 2+ signaling proteins have not been translated to clinic primarily due to side effects related to the important roles of target molecules for these compounds at the peripheral tissues. In this review article, we would like to discuss an idea that recently identified positive allosteric modulators (PAMs) of the sarco-endoplasmic reticulum calcium (SERCA) pump may provide a promising approach to develop therapeutic compounds for treatment of these disorders. This hypothesis is supported by the preclinical data obtained with animal models of AD and PD. The first critical test of this idea will be an imminent phase I study that will offer an opportunity to evaluate potential side effects of this class of compounds in humans., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: R.D. is an inventor of the patents US11,730,729, US11,821,626, US10,772,881, and WO2022/020261 “Quinolines that modulate SERCA and their use for treating disease” that describe all of the compounds disclosed herein and are assigned to Neurodon Corporation. Part of the cost of this research was paid for by Neurodon. R.D. is an employee of Neurodon. I.B. is a member of Neurodon SAB. R.D. and I.B. hold Neurodon stock or stock options, (Copyright © 2024 Elsevier Inc. All rights reserved.)

Aims: Hypertension is associated with an increased activity of matrix metalloproteinase (MMP)-2 in the vasculature, which, in turn, proteolyzes extra- and intracellular proteins that lead to vascular dysfunction. The activity of sarcoplasmic reticulum calcium ATPase (SERCA) is decreased in the aortas of hypertensive rats. Increased activity of MMP-2 proteolyzed SERCA in rat heart during ischemia and reperfusion injury, thus impairing cardiac function. Therefore, we examined whether increased activity of MMP-2 in early hypertension contributes to proteolyze SERCA in the aortas, thus leading to maladaptive vascular remodeling and dysfunction., Main Methods: Male Sprague-Dawley rats were submitted to two kidney-one clip (2K-1C) or Sham surgery and treated with doxycycline. Systolic blood pressure (SBP) was assessed by tail-cuff plethysmography. After 7 days, aortas were collected for zymography assays, Western blot to SERCA, ATPase activity assay, vascular reactivity, Ki-67 immunofluorescence and hematoxylin/eosin stain., Key Findings: SBP was increased in 2K-1C rats and doxycycline did not reduce it, but decreased MMP-2 activity and prevented SERCA proteolysis in aortas. Cross sectional area, media to lumen ratio and Ki-67 were all increased in the aortas of hypertensive rats and doxycycline decreased Ki-67. In 2K-1C rats, arterial relaxation to acetylcholine was impaired and doxycycline ameliorated it., Significance: doxycycline reduced MMP-2 activity in aortas of 2K-1C rats and prevented proteolysis of SERCA and its dysfunction, thus ameliorating hypertension-induced vascular dysfunction., Competing Interests: Declaration of competing interest We declare that there are no conflicts of interest associated with this publication., (Copyright © 2024. Published by Elsevier B.V.)

The allosteric SERCA (Sarcoplasmic/Endoplasmic Reticulum Ca 2+ -ATPase) activator CDN1163 has been recently added to the group of pharmacological tools for probing SERCA function. We chose to investigate the effects of the compound on T lymphocyte Ca 2+ stores, using the well-described Jurkat T lymphocyte as a reliable cell system for Ca 2+ signaling pathways. Our study identified the lowest concentrations of the SERCA inhibitors thapsigargin (TG) and 2,5-di-( tert butyl)-1,4-benzohydroquinone (tBHQ) capable of releasing Ca 2+ , permitting the differentiation of the TG-sensitive SERCA 2b Ca 2+ store from the tBHQ-sensitive SERCA 3 Ca 2+ store. We proceeded to test the effects of CDN1163 on Ca 2+ stores, examining specific actions on the SERCA 2b and SERCA 3 Ca 2+ pools using our low-dose SERCA blocker regimen. In contrast to previous work, we find CDN1163 exerts complex time-sensitive and SERCA isoform-specific actions on Ca 2+ stores. Surprisingly, short-term exposure (0-30 min) to CDN1163 perturbs T cell Ca 2+ stores by suppressing Ca 2+ uptake with diminished Ca 2+ release from the SERCA 2b-controlled store. Concomitantly, we find evidence for a SERCA-activating effect of CDN1163 on the SERCA-3 regulated store, given the observation of increased Ca 2+ release inducible by low-dose tBHQ. Intriguingly, longer-term (>12 h) CDN1163 exposure reversed this pattern, with increased Ca 2+ release from SERCA 2b-regulated pools yet decreased Ca 2+ release responses from the tBHQ-sensitive SERCA 3 pool. Indeed, this remodeling of SERCA 2b Ca 2+ stores with longer-term CDN1163 exposure also translated into the compound's ability to protect Jurkat T lymphocytes from TG but not tBHQ-induced growth suppression.

Sarco/endoplasmic reticulum Ca 2+ -ATPase (SERCA) is an important regulatory protein responsible for maintaining calcium homeostasis within cells. Impairment of SERCA associated with activity/expression decrease has been implicated in multiple chronic conditions, including cardiovascular diseases, diabetes, cancer, neurodegenerative diseases, and skeletal muscle pathologies. Natural polyphenols have been recognized to interact with several target proteins involving SERCA. To date, only a limited number of polyphenolic compounds or their derivatives have been described either to increase SERCA activity/expression directly or to affect Ca 2+ signaling pathways. In this study, we tested polyphenols for their ability to activate SERCA1a in the absence or presence of methylglyoxal or palmitate and to impact insulin release in pancreatic beta cells. The protective effects of these compounds against methylglyoxal- or palmitate-induced injury were evaluated. Results indicate that 6-gingerol, resveratrol, and ellagic acid activate SERCA1a and protect against activity decrease induced by methylglyoxal and palmitate. Molecular docking analysis revealed the binding of these polyphenols to Glu439 in the SERCA1a P-domain, suggesting a critical role in the stimulation of enzyme activity. Ellagic acid was found to directly stimulate the activity of SERCA1a, marking the first instance of such an observation.

Electric pacing of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) has been increasingly used to simulate cardiac arrhythmias in vitro and to enhance cardiomyocyte maturity. However, the impact of electric pacing on cellular electrophysiology and Ca 2+ handling in differentiated hiPSC-CM is less characterized. Here we studied the effects of electric pacing for 24 h or 7 days at a physiological rate of 60 beats/min on cellular electrophysiology and Ca 2+ cycling in late-stage, differentiated hiPSC-CM (>90% troponin + , >60 days postdifferentiation). Electric culture pacing for 7 days did not influence cardiomyocyte cell size, apoptosis, or generation of reactive oxygen species in differentiated hiPSC-CM compared with 24-h pacing. However, epifluorescence measurements revealed that electric pacing for 7 days improved systolic Ca 2+ transient amplitude and Ca 2+ transient upstroke, which could be explained by elevated sarcoplasmic reticulum Ca 2+ load and SERCA activity. Diastolic Ca 2+ leak was not changed in line-scanning confocal microscopy, suggesting that the improvement in systolic Ca 2+ release was not associated with a higher open probability of ryanodine receptor (RyR)2 during diastole. Whereas bulk cytosolic Na + concentration and Na + /Ca 2+ exchanger (NCX) activity were not changed, patch-clamp studies revealed that chronic pacing caused a slight abbreviation of the action potential duration (APD) in hiPSC-CM. We found in whole cell voltage-clamp measurements that chronic pacing for 7 days led to a decrease in late Na + current, which might explain the changes in APD. In conclusion, our results show that chronic pacing improves systolic Ca 2+ handling and modulates the electrophysiology of late-stage, differentiated hiPSC-CM. This study might help to understand the effects of electric pacing and its numerous applications in stem cell research including arrhythmia simulation. NEW & NOTEWORTHY Electric pacing is increasingly used in research with human induced pluripotent stem cell cardiomyocytes (hiPSC-CM), for example to simulate arrhythmias but also to enhance maturity. Therefore, it is mandatory to understand the effects of pacing itself on cellular electrophysiology in late-stage, matured hiPSC-CM. This study provides an electrophysiological characterization of the effects of chronic electric pacing at a physiological rate on differentiated hiPSC-CM.

Anthracyclines' cardiotoxicity involves an accelerated generation of reactive oxygen species. This oxidative damage has been found to accelerate the expression of hexose-6P-dehydrogenase (H6PD), that channels glucose-6-phosphate (G6P) through the pentose phosphate pathway (PPP) confined within the endoplasmic/sarcoplasmic reticulum (SR). To verify the role of SR-PPP in the defense mechanisms activated by doxorubicin (DXR) in cardiomyocytes, we tested the effect of this drug in H6PD knockout mice (H6PD -/- ). Twenty-eight wildtype (WT) and 32 H6PD -/- mice were divided into four groups to be treated with intraperitoneal administration of saline (untreated) or DXR (8 mg/Kg once a week for 3 weeks). One week thereafter, survivors underwent imaging of 18 F-deoxyglucose (FDG) uptake and were sacrificed to evaluate the levels of H6PD, glucose-6P-dehydrogenase (G6PD), G6P transporter (G6PT), and malondialdehyde. The mRNA levels of SR Ca 2+ -ATPase 2 (Serca2) and ryanodine receptors 2 (RyR2) were evaluated and complemented with Hematoxylin/Eosin staining and transmission electron microscopy. During the treatment period, 1/14 DXR-WT and 12/18 DXR-H6PD -/- died. At microPET, DXR-H6PD -/- survivors displayed an increase in left ventricular size (p < 0.001) coupled with a decreased urinary output, suggesting a severe hemodynamic impairment. At ex vivo analysis, H6PD -/- condition was associated with an oxidative damage independent of treatment type. DXR increased H6PD expression only in WT mice, while G6PT abundance increased in both groups, mismatching a generalized decrease of G6PD levels. Switching-off SR-PPP impaired reticular accumulation of Ca 2+ decelerating Serca2 expression and upregulating RyR2 mRNA level. It thus altered mitochondrial ultrastructure eventually resulting in a cardiomyocyte loss. The recognized vulnerability of SR to the anthracycline oxidative damage is counterbalanced by an acceleration of G6P flux through a PPP confined within the reticular lumen. The interplay of SR-PPP with the intracellular Ca 2+ exchanges regulators in cardiomyocytes configure the reticular PPP as a potential new target for strategies aimed to decrease anthracycline toxicity., (© 2023. The Author(s).)

Previous studies have observed alterations in excitation-contraction (EC) coupling during end-stage heart failure that include action potential and calcium (Ca 2+ ) transient prolongation and a reduction of the Ca 2+ transient amplitude. Underlying these phenomena are the downregulation of potassium (K + ) currents, downregulation of the sarcoplasmic reticulum Ca 2+ ATPase (SERCA), increase Ca 2+ sensitivity of the ryanodine receptor, and the upregulation of the sodium-calcium (Na = -Ca 2+ ) exchanger. However, in human heart failure (HF), debate continues about the relative contributions of the changes in calcium handling vs. the changes in the membrane currents. To understand the consequences of the above changes, they are incorporated into a computational human ventricular myocyte HF model that can explore the contributions of the spontaneous Ca 2+ release from the sarcoplasmic reticulum (SR). The reduction of transient outward K + current (I to ) is the main membrane current contributor to the decrease in RyR2 open probability and L-type calcium channel (LCC) density which emphasizes its importance to phase 1 of the action potential (AP) shape and duration (APD). During current-clamp conditions, RyR2 hyperphosphorylation exhibits the least amount of Ca 2+ release from the SR into the cytosol and SR Ca 2+ fractional release during a dynamic slow-rapid-slow (0.5-2.5-0.5 Hz) pacing, but it displays the most abundant and more lasting Ca 2+ sparks two-fold longer than a normal cell. On the other hand, under voltage-clamp conditions, HF by decreased SERCA and upregulated I NCX show the least SR Ca 2+ uptake and EC coupling gain, as compared to HF by hyperphosphorylated RyR2s. Overall, this study demonstrates that the (a) combined effect of SERCA and NCX, and the (b) RyR2 dysfunction, along with the downregulation of the cardiomyocyte's potassium currents, could substantially contribute to Ca 2+ mishandling at the spark level that leads to heart failure.

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.

Heart failure with preserved ejection fraction (HFpEF), a complex disease that is increasingly prevalent due to population aging, pose significant challenges in its treatment. The present study utilized the HFpEF rat model and H9C2 cells as research subjects to thoroughly investigate the potential mechanisms of alarin in protecting cardiac function in HFpEF. The study shows that under HFpEF conditions, oxidative stress significantly increases, leading to myocardial structural damage and dysfunction of calcium ion channels, which ultimately impairs diastolic function. Alarin, through its interaction with NADPH oxidase 1 (NOX1), effectively alleviates oxidative stress and modulates the activities of type 2 ryanodine receptor (RyR2) and sarcoplasmic/endoplasmic reticulum calcium ATPase 2 (SERCA2), thereby facilitating the restoration of Ca2+ homeostasis and significantly improving cardiac function in the HFpEF model. This research not only uncovers the cardioprotective effects of alarin and its underlying molecular mechanisms but also provides new insights and potential therapeutic targets for HFpEF treatment strategies, suggesting a promising future for alarin and related therapies in the management of this debilitating condition.