SERCA Research Articles

Dwarf Open Reading Frame (DWORF) Gene Therapy Ameliorated Duchenne Muscular Dystrophy Cardiomyopathy in Aged mdx Mice.

Background Cardiomyopathy is a leading health threat in Duchenne muscular dystrophy (DMD). Cytosolic calcium upregulation is implicated in DMD cardiomyopathy. Calcium is primarily removed from the cytosol by the sarcoendoplasmic reticulum calcium ATPase (SERCA). SERCA activity is reduced in DMD. Improving SERCA function may treat DMD cardiomyopathy. Dwarf open reading frame (DWORF) is a recently discovered positive regulator for SERCA, hence, a potential therapeutic target. Methods and Results To study DWORF's involvement in DMD cardiomyopathy, we quantified DWORF expression in the heart of wild-type mice and the mdx model of DMD. To test DWORF gene therapy, we engineered and characterized an adeno-associated virus serotype 9-DWORF vector. To determine if this vector can mitigate DMD cardiomyopathy, we delivered it to 6-week-old mdx mice (6×1012 vector genome particles/mouse) via the tail vein. Exercise capacity, heart histology, and cardiac function were examined at 18 months of age. We found DWORF expression was significantly reduced at the transcript and protein levels in mdx mice. Adeno-associated virus serotype 9-DWORF vector significantly enhanced SERCA activity. Systemic adeno-associated virus serotype 9-DWORF therapy reduced myocardial fibrosis and improved treadmill running, electrocardiography, and heart hemodynamics. Conclusions Our data suggest that DWORF deficiency contributes to SERCA dysfunction in mdx mice and that DWORF gene therapy holds promise to treat DMD cardiomyopathy.

Calsequestrin in Purkinje cells of mammalian cerebellum

Cerebellum is devoted to motor coordination and cognitive functions. Endoplasmic reticulum is the largest intracellular calcium store involved in all neuronal functions. Intralumenal calcium binding proteins play a pivotal role in calcium storage and contribute to both calcium release and uptake. Calsequestrin, a key calcium binding protein of sarco-endoplasmic reticulum in skeletal and cardiac muscles, was identified in chicken and fish cerebellum Purkinje cells, but its expression in mammals and human counterpart has not been studied in depth. Aim of the present paper was to investigate expression and localization of Calsequestrin in mammalian cerebellum. Calsequestrin was found to be expressed at low level in cerebellum, but specifically concentrated in Calbindin D28- and zebrin- immunopositive-Purkinje cells. Two additional fundamental calcium store markers, sarco-endoplasmic calcium pump isoform 2, SERCA2, and Inositol-trisphosphate receptor isoform 1, IP3R1, were found to be co-expressed in the region, with some localization peculiarities. In conclusion, a new marker was identified for Purkinje cells in adult mammals, including humans. Such a marker might help in staminal neuronal cells specification and in dissection of still unknown neurodegeneration and physio-pathological effects of dysregulated calcium homeostasis.

PS-B01-10: ULTRASTRUCTURAL CHANGES IN SYSTEMIC RESISTANCE ARTERIES OF HYPERTENSIVE AND NORMOTENSIVE WOMEN WITH UTERINE FIBROIDS

Background: Uterine fibroids, the most common benign neoplasm in women, are associated with hypertension, but the nature of this association is not known. To assess a possible shared pathology of smooth muscle, we studied the ultrastructure of systemic resistance arteries of women with fibroids. Methods: Resistance-sized arteries (200 to 400 micrometer) were harvested from omental samples obtained during fibroid surgery. Transmission electron microscopic images were taken at different magnifications and locations of the vessel wall to assess qualitative and morphological characteristics. Results: We obtained resistance arteries of 19 women (9 hypertensive). Vessels of 17 women (8 hypertensive, 4 treated, none controlled) were available for analysis (Table). The arteries of normotensive and hypertensive women showed myocyte-like cells in the intima forming focal neo-intima plaques. In the media, myocytes showed a reduction in contractile fibers and prominent, dilated sarco-endoplasmic reticulum, with elongated mitochondria, and abundant autophagic and extracellular vacuoles. More profound ultrastructural changes were seen in hypertensives. Untreated hypertensives had a significantly higher media-to-lumen ratio than normotensives. Conclusion: To our knowledge these are the first data on the ultrastructure of systemic resistance-sized arteries in women with fibroids, showing vascular remodeling in both normotensives and hypertensives, with a shift from a contractile phenotype to a myocyte with prominent organelles involved in biosynthesis (synthetic phenotype). These abnormalities in systemic resistance-sized arteries may indicate that uterine fibroids are a manifestation of underlying systemic pathology affecting smooth muscle, or that fibroids induce systemic effects on the arterial wall, either directly or through factors that increase blood pressure, which needs exploration in further studies.

Role of SERCA3 in the Prognosis and Immune Function in Pan-Cancer.

The sarcoendoplasmic reticulum calcium adenosine triphosphatase (ATPase) 3 (SERCA3), a member of the SERCA protein family, is located at the endoplasmic reticulum. Its main function is to pump Ca2+ into the endoplasmic reticulum and is involved in maintaining intracellular calcium homeostasis and signal transduction, which are very important factors impacting cancer development and progression. However, the specific role of SERCA3 in cancer remains unclear. Our study, for the first time, comprehensively analyzed the SERCA3 expression profile in multiple cancers and its prognostic value in different cancers using bioinformatics. Furthermore, TCGA database was applied to evaluate the certain correlation of SERCA3 expression with immune modulator genes, immune checkpoints, immune cell infiltration, TMB, and MSI. The results revealed that in many cancers, SERCA3 expression was markedly decreased, which was related to poor prognosis. Additionally, we noticed that SERCA3 expression was correlated with TNM classification and WHO cancer stages in some cancer types. The Pearson correlation analysis showed that SERCA3 expression was closely associated with chemokines, chemokine receptors, MHC, immune activation genes, and immunosuppressive genes. In most cancer types, SERCA3 expression was also associated with immune checkpoints, including PDCD1 and CTLA-4. Further analysis suggested that SERCA3 was significantly correlated with CD8+ T cells, and regulatory T cells. Additionally, pan-cancer analysis confirmed that SERCA3 expression was related to TMB and MSI. In conclusion, these results offer a new insight into the functions and effects of SERCA3 in pan-cancer, and further provide some basis for considering SERCA3 as a potential cancer treatment target and biomarker.

An S-glutathiomimetic Provides Structural Insights into Stromal Interaction Molecule-1 Regulation

Stromal interaction molecule 1 (STIM1) is an endo/sarcoplasmic reticulum (ER/SR) calcium (Ca2+) sensing protein that regulates store-operated calcium entry (SOCE). In SOCE, STIM1 activates Orai1-composed Ca2+ channels in the plasma membrane (PM) after ER stored Ca2+ depletion. S-Glutathionylation of STIM1 at Cys56 evokes constitutive SOCE in DT40 cells; however, the structural and biophysical mechanisms underlying the regulation of STIM1 by this modification are poorly defined. By establishing a protocol for site-specific STIM1 S-glutathionylation using reduced glutathione and diamide, we have revealed that modification of STIM1 at either Cys49 or Cys56 induces thermodynamic destabilization and conformational changes that result in increased solvent-exposed hydrophobicity. Further, S-glutathionylation or point-mutation of Cys56 reduces Ca2+ binding affinity, as measured by intrinsic fluorescence and far-UV circular dichroism spectroscopies. Solution NMR showed S-glutathionylated-induced perturbations in STIM1 are localized to the α1 helix of the canonical EF-hand, the α3 and α4 helices of the non-canonical EF-hand and α6 and α8 helices of the SAM domain. Finally, we designed an S-glutathiomimetic mutation that strongly recapitulates the structural, biophysical and functional effects within the STIM1 luminal domain and we envision to be another tool for understanding the effects of protein S-glutathionylation in vitro, in cellulo and in vivo.

Influence of vitamin D on sarcopenia pathophysiology: A longitudinal study in humans and basic research in knockout mice.

Vitamin D is an essential nutrient in musculoskeletal function; however, its relationship to sarcopenia remains ambiguous, and the mechanisms and targets of vitamin D activity have not been elucidated. This study aimed to clarify the role of vitamin D in mature skeletal muscle and its relationship with sarcopenia. This epidemiological study included 1653 community residents who participated in both the fifth and seventh waves of the National Institute for Longevity Sciences, Longitudinal Study of Aging and had complete background data. Participants were classified into two groups: vitamin D-deficient (serum 25-hydroxyvitamin D<20ng/mL) and non-deficient (serum 25-hydroxyvitamin D≥20ng/mL); they underwent propensity-score matching for background factors (age, sex, height, weight, comorbidities, smoker, alcohol intake, energy intake, vitamin D intake, steps, activity, season and sarcopenia). Changes in muscle strength and mass over the 4-year period were compared. For basic analysis, we generated Myf6CreERT2 Vitamin D Receptor (VDR)-floxed (VdrmcKO ) mice with mature muscle fibre-specific vitamin D receptor knockout, injected tamoxifen into 8-week-old mice and analysed various phenotypes at 16weeks of age. Grip strength reduction was significantly greater in the deficient group (-1.55±2.47kg) than in the non-deficient group (-1.13±2.47kg; P=0.019). Appendicular skeletal muscle mass reduction did not differ significantly between deficient (-0.05±0.79kg) and non-deficient (-0.01±0.74kg) groups (P=0.423). The incidence of new cases of sarcopenia was significantly higher in the deficient group (15 vs. 5 cases; P=0.039). Skeletal muscle phenotyping of VdrmcKO mice showed no significant differences in muscle weight, myofibre percentage or myofibre cross-sectional area; however, both forelimb and four-limb muscle strength were significantly lower in VdrmcKO mice (males: forelimb, P=0.048; four-limb, P=0.029; females: forelimb, P<0.001; four-limb, P<0.001). Expression profiling revealed a significant decrease in expression of sarcoendoplasmic reticulum Ca2+ -ATPase (SERCA) 1 (P=0.019) and SERCA2a (P=0.049) genes in the VdrmcKO mice. In contrast, expression of non-muscle SERCA2b and myoregulin genes showed no changes. Vitamin D deficiency affects muscle strength and may contribute to the onset of sarcopenia. Vitamin D-VDR signalling has minimal influence on the regulation of muscle mass in mature myofibres but has a significant influence on muscle strength.

The SGLT2i Dapagliflozin Reduces RV Mass Independent of Changes in RV Pressure Induced by Pulmonary Artery Banding.

Sodium glucose linked transporter 2 (SGLT2) inhibition not only reduces morbidity and mortality in patients with diagnosed heart failure but also prevents the development of heart failure hospitalization in those at risk. While studies to date have focused on the role of SGLT2 inhibition in left ventricular failure, whether this drug class is efficacious in the treatment and prevention of right heart failure has not been explored. We hypothesized that SGLT2 inhibition would reduce the structural, functional, and molecular responses to pressure overload of the right ventricle. Thirteen-week-old Fischer F344 rats underwent pulmonary artery banding (PAB) or sham surgery prior to being randomized to receive either the SGLT2 inhibitor: dapagliflozin (0.5mg/kg/day) or vehicle by oral gavage. After 6weeks of treatment, animals underwent transthoracic echocardiography and invasive hemodynamic studies. Animals were then terminated, and their hearts harvested for structural and molecular analyses. PAB induced features consistent with a compensatory response to increased right ventricular (RV) afterload with elevated mass, end systolic pressure, collagen content, and alteration in calcium handling protein expression (all p < 0.05 when compared to sham + vehicle). Dapagliflozin reduced RV mass, including both wet and dry weight as well as normalizing the protein expression of SERCA 2A, phospho-AMPK and LC3I/II ratio expression (all p < 0.05). Dapagliflozin reduces the structural, functional, and molecular manifestations of right ventricular pressure overload. Whether amelioration of these early changes in the RV may ultimately lead to a reduction in RV failure remains to be determined.

A hydroalcoholic extract of Senecio nutans SCh. Bip (Asteraceae); its effects on cardiac function and chemical characterization

Ethnopharmacology relevanceThe plant Senecio nutans SCh. Bip. is used by Andean communities to treat altitude sickness. Recent evidence suggests it may produce vasodilation and negative cardiac inotropy, though the cellular mechanisms have not been elucidated. PurposeTo determinate the mechanisms action of S. nutans on cardiovascular function in normotensive animals. MethodsThe effect of the extract on rat blood pressure was measured with a transducer in the carotid artery and intraventricular pressure by a Langendorff system. The effects on sheep ventricular intracellular calcium handling and contractility were evaluated using photometry. Ultra-high-performance liquid-chromatography with diode array detection coupled with heated electrospray-ionization quadrupole-orbitrap mass spectrometric detection (UHPLC-DAD-ESI-Q-OT-MSn) was used for extract chemical characterization. ResultsIn normotensive rats, S. nutans (10 mg/kg) reduced mean arterial pressure (MAP) by 40% (p < 0.05), causing a dose-dependent coronary artery dilation and decreased left ventricular pressure. In isolated cells, S. nutans extract (1 μg/ml) rapidly reduced the [Ca2+]i transient amplitude and sarcomere shorting by 40 and 49% (p < 0.001), respectively. The amplitude of the caffeine evoked [Ca2+]i transient was reduced by 24% (p < 0.001), indicating reduced sarcoplasmic reticulum (SR) Ca2+ content. Sodium-calcium exchanger (NCX) activity increased by 17% (p < 0.05), while sarcoendoplasmic reticulum Ca2+-ATPase (SERCA) activity was decreased by 21% (p < 0.05). LC-MS results showed the presence of vitamin C, malic acid, and several antioxidant phenolic acids reported for the first time. Dihydroeuparin and 4-hydroxy-3-(3-methylbut-2-enyl) acetophenone were abundant in the extract. ConclusionIn normotensive animals, S. nutans partially reduces MAP by decreasing heart rate and cardiac contractility. This negative inotropy is accounted for by decreased SERCA activity and increased NCX activity which reduces SR Ca2+ content. These results highlight the plant's potential as a source of novel cardio-active phytopharmaceuticals or nutraceuticals.

Altered skeletal muscle sarco-endoplasmic reticulum Ca2+-ATPase calcium transport efficiency after a thermogenic stimulus.

Exposure to predator threat induces a rapid and robust increase in skeletal muscle thermogenesis in rats. The central nervous system relays threat information to skeletal muscle through activation of the sympathetic nervous system, but muscle mechanisms mediating this thermogenesis remain unidentified. Given the relevance of sarcolipin-mediated futile calcium cycling through the sarco-endoplasmic reticulum Ca2+-ATPase (SERCA) pump to mammalian muscle nonshivering thermogenesis, we hypothesized that this plays a role in contextually induced muscle thermogenesis as well. This was assessed by measuring enzymatic activity of SERCA and sarcoplasmic reticulum Ca2+ transport, where the apparent coupling ratio (Ca2+ uptake rate divided by ATPase activity rate at a standard Ca2+ concentration) was predicted to decrease in association with muscle thermogenesis. Sprague-Dawley rats exposed to predator (ferret) odor (PO) showed a rapid decrease in the apparent coupling ratio in the soleus muscle, indicating SERCA uncoupling compared with control-odor-exposed rats. A rat model of high aerobic fitness and elevated muscle thermogenesis also demonstrated soleus muscle SERCA uncoupling relative to their obesity-prone, low-fitness counterparts. Both the high- and low-aerobic fitness rats showed soleus SERCA uncoupling with exposure to PO. Finally, no increase in sarcolipin expression in soleus muscle was detected with PO exposure. This dataset implicates muscle uncoupling of SERCA Ca2+ transport and ATP hydrolysis, likely through altered SERCA or sarcolipin function outside of translational regulation, as one contributor to the muscle thermogenesis provoked by exposure to predator threat. These data support the involvement of SERCA uncoupling in both muscle thermogenic induction and enhanced aerobic capacity.

Abstract 034: Absence Of Tachyphylaxis And Vasorelaxant Effect Induced By Nono2p, A New Nitric Oxide Donor

Introduction: Decreased bioavailability of nitric oxide (NO) plays a mechanistic role in hypertension and myocardial infarction. NO donors are potent vasodilators, but often exhibit toxicity or vascular tolerance. The aim of this study was to investigate the vascular activity possible tachyphylaxis of NONO2P, a novel NO donor. Methods: Male Wistar rats were euthanized, and the superior mesenteric artery was isolated for recordings of isometric force, and in vivo experiments were performed to evaluate blood pressure in non-anesthetized normotensive rats. Results: Cumulative administration of the NONO2P (10 -13 to 3x10 -6 M) induced endothelium-independent relaxation (Emax:111.51 ± 2.31%; pD2: 8.51 ± 0.08, n=9) in arterial rings pre-contracted with phenylephrine (Phe,1μM). However, the relaxation was reduced in pre-contracted rings with Phe exposed to Tyrode's solution containing 20 mM of K + (E max : 102.83 ± 2.10%; pD2: 7.73 ± 0.04, n=6), suggesting the participation of K + channels in the relaxation. The presence of the specific soluble guanylyl cyclase (sGC) inhibitor, ODQ (10μM), abolished the vasorelaxant effect (E max : 15.38 ± 11.85%, n=7). Pre-incubation with cyclopiazonic acid (CPA) (10μM), inhibitor of sarcoendoplasmic reticulum calcium ATPase (SERCA), shifted the relaxation concentration-response to the right (E max : 106.17 ± 3.06%; pD2: 7.69 ± 0.04, n=6). Interestingly, repeated NONO2P administration did not induce tachyphylaxis, and NONO2P presented similar maximum efficacy to sodium nitroprusside (SNP) (E max : 114.24 ± 3.47%; pD2: 9.40 ± 0.04, n=6). Moreover, NONO2P lowered blood pressure in normotensive rats. Conclusion: The endothelium-independent vasorelaxant effect induced by NONO2P involves both sGC, SERCA and K + channel activation, and NONO2P does not appear to cause tachyphylaxis. NONO2P is able to promote vasorelaxation with the same magnitude as SNP. Finally, NONO2P reduces blood pressure, becoming a promising molecule as a novel therapeutic alternative for the treatment of cardiovascular diseases.

Natural Polyphenols as SERCA Activators: Role in the Endoplasmic Reticulum Stress-Related Diseases.

Sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) is a key protein responsible for transporting Ca2+ ions from the cytosol into the lumen of the sarco/endoplasmic reticulum (SR/ER), thus maintaining Ca2+ homeostasis within cells. Accumulating evidence suggests that impaired SERCA function is associated with disruption of intracellular Ca2+ homeostasis and induction of ER stress, leading to different chronic pathological conditions. Therefore, appropriate strategies to control Ca2+ homeostasis via modulation of either SERCA pump activity/expression or relevant signaling pathways may represent a useful approach to combat pathological states associated with ER stress. Natural dietary polyphenolic compounds, such as resveratrol, gingerol, ellagic acid, luteolin, or green tea polyphenols, with a number of health-promoting properties, have been described either to increase SERCA activity/expression directly or to affect Ca2+ signaling pathways. In this review, potential Ca2+-mediated effects of the most studied polyphenols on SERCA pumps or related Ca2+ signaling pathways are summarized, and relevant mechanisms of their action on Ca2+ regulation with respect to various ER stress-related states are depicted. All data were collected using scientific search tools (i.e., Science Direct, PubMed, Scopus, and Google Scholar).

Abstract P1096: Kinase-independent Function Of PI3Kgamma Modulates Calcium Re-uptake By Regulating CamKII Mediated Phospholamban Phosphorylation

Although phosphoinositide 3-kinase (PI3Kγ) null mice (PI3Kγ -/- ) show increased ventricular rate/contraction, it is unknown whether PI3Kγ regulates calcium recycling machinery underlying this phenotype. Primary adult cardiomyocytes from PI3Kγ -/- mice show reduced calcium reuptake by sarcoendoplasmic reticulum (SR) following caffeine. Unexpectedly, PI3Kγ -/- cardiomyocytes showed significant reduction in phosphorylation of phospholamban (PLN) at Thr17, a key regulator of SR calcium re-uptake. Furthermore, loss in PLN phosphorylation in PI3Kγ -/- cardiomyocytes was associated with augmented interaction with SR calcium ATPase (SERCA). Surprisingly, cardiomyocyte-specific overexpression of kinase-dead PI3Kγ (PI3Kγ inact ) in global PI3Kγ -/- mice (PI3Kγ inact /PI3Kγ -/- ) normalized caffeine-induced calcium re-uptake, PLN phosphorylation at Thr17 and decreased PLN-SERCA interaction. These data suggested kinase-independent function of PI3Kγ in regulation of SR calcium load and PLN phosphorylation. Since phosphorylation of Thr17 of PLN is carried out by Ca 2+ /Calmodulin dependent protein kinase (CamKII) we probed for the role of PI3Kγ in regulation of CamKII in PLN phosphorylation. Mechanistically, PI3Kγ exhibits scaffolding function in recruitment of CamKII to PLN at SR to mediate PLN phosphorylation. The study unravels a yet to be recognized kinase-independent role of PI3Kγ in regulating PLN with implications in cardiac function.

Abstract P1090: Polycystin-1 Regulates Calcium Handling And Contractility In Human IPSC-derived Ventricular Cardiomyocytes

Mutations in polycystin-1 (PC1) cause 85% of autosomal dominant polycystic kidney disease cases. This disease is characterized by progressive renal disease and multiple cardiovascular alterations. Evidence suggests that cardiovascular disease precedes the onset of renal failure. We recently demonstrated that PC1 ablation shortens cardiomyocyte action potential, decreases Ca 2+ transient amplitude, and reduces contractility in adult cardiomyocytes isolated from cardiomyocyte-specific PC1 knock-out mice compared to control mice. Alterations in Ca 2+ cycling in PC1-deficient cardiomyocytes were demonstrated to be due to action potential duration shortening and impaired sarcoendoplasmic reticulum ATPase activity. However, mouse and human cardiomyocytes have considerable differences in their action potential shapes and the ion channels involved. It remains unknown whether PC1 regulates these events in human cardiomyocytes. Here, we sought to investigate whether PC1 regulates excitation-contraction coupling in human ventricular cardiomyocytes. We differentiated commercially available human-induced pluripotent stem cell lines (hiPSC) into ventricular cardiomyocytes (hiPSC-CM) using published protocols (measurements were performed 60-90 days after differentiation). We developed a protocol to simultaneously measure action potentials (FluoVolt), intracellular Ca 2+ (Calbryte-590) and contractility using the IonOptix algorithm to detect changes in contrast as contractility measurements. PC1 ablation using siRNA decreased action potential duration and impaired Ca 2+ transients and contractility in hiPSC-CMs. Because hiPSC-CMs have several limitations due to their immature state, we employed a well-validated method to improve their maturation that utilizes a Matrigel mattress and hormone supplementation (dexamethasone; Dexa / thyroid hormone; T3; PMID: 28974554; developed by Dr. Bjorn Knollmann’s lab). Using the Matrigel mattress/Dexa/T3 technique, we increased hiPSC-CM maturation measured as follows: cardiomyocytes were oriented and contracted in the longitudinal axis, ryanodine receptor type 2 and alpha-actinin localized with a striated pattern, exhibited robust inward K + rectifier currents (I K1 ) and fired action potentials that resemble human adult cardiomyocytes. In this model, PC1 knockdown also decreased action potential duration and impaired Ca 2+ transients and decreased contractility measured as fractional shortening. Altogether, this evidence shows for the first time that PC1 regulates human cardiomyocyte physiology and suggests that cardiac alterations in patients with autosomal dominant polycystic kidney disease may emerge as a result of impaired cardiomyocyte contractile function.

Impaired calcium signalling and neuropsychiatric disorders in Darier disease: An exploratory review.

Darier (Darier-White) disease (DD) is an autosomal dominant skin disorder caused by pathogenic mutations in the ATP2A2 gene which encodes a calcium ATPase in the sarco-endoplasmic reticulum (SERCA2). Defects in the SERCA2 protein lead to an impairment of cellular calcium homeostasis, which in turn, triggers cell death pathways. There is a high prevalence of neuropsychiatric disorders in patients affected by this condition, namely intellectual disability, bipolar disorder, schizophrenia, and suicidality. Though these associations have been well-documented over the years, little has been discussed or investigated regarding the pathophysiological mechanisms. The goal of this article is to review the literature related to the most commonly associated neuropsychiatric disorders found in patients with DD, highlight the pathophysiological mechanisms underlying each condition, and examine potential interventions that may be of interest for future development. A literature search was performed using PubMed to access and review relevant articles published in the last 40 years. Keywords searched included Darier disease neuropsychiatric, Darier disease pathophysiology, SERCA2 central nervous system, SERCA 2 skin, ATP2A2 central nervous system, ATP2A2 skin, sphingosine-1-phosphate signalling skin, sphingosine-1-phosphate signalling central nervous system, P2X7 receptor skin, and P2X7 receptor central nervous system. Our search resulted in 2692 articles, of which 61 articles were ultimately included in this review.

Primitive Phospholamban- and Sarcolipin-like Peptides Inhibit the Sarcoplasmic Reticulum Calcium Pump SERCA.

Intracellular calcium signaling is essential for all kingdoms of life. An important part of this process is the sarco-endoplasmic reticulum Ca2+-ATPase (SERCA), which maintains the low cytosolic calcium levels required for intracellular calcium homeostasis. In higher organisms, SERCA is regulated by a series of tissue-specific transmembrane subunits such as phospholamban in cardiac muscles and sarcolipin in skeletal muscles. These regulatory axes are so important for muscle contractility that SERCA, phospholamban, and sarcolipin are practically invariant across mammalian species. With the recent discovery of the arthropod sarcolambans, the family of calcium pump regulatory subunits appears to span more than 550 million years of evolutionary divergence from arthropods to humans. This evolutionary divergence is reflected in the peptide sequences, which vary enormously from one another and only vaguely resemble phospholamban and sarcolipin. The discovery of the sarcolambans allowed us to address two questions. How much sequence variation is tolerated in the regulation of mammalian SERCA activity by the transmembrane peptides? Do divergent peptide sequences mimic phospholamban or sarcolipin in their regulatory activities despite limited sequence similarity? We expressed and purified recombinant sarcolamban peptides from three different arthropods. The peptides were coreconstituted into proteoliposomes with mammalian SERCA1a and the effect of each peptide on the apparent calcium affinity and maximal activity of SERCA was measured. All three peptides were superinhibitors of SERCA, exhibiting either phospholamban-like or sarcolipin-like characteristics. Molecular modeling, protein-protein docking, and molecular dynamics simulations revealed novel features of the divergent peptides and their SERCA regulatory properties.

Experimental and computational analyses of calcium dynamics in 22q11.2 deletion model astrocytes

Methods for deriving mechanistic information from intracellular calcium dynamics have largely been applied to neuronal data despite the knowledge of roles of glial cells in behavior, cognition, and psychiatric disorders. Using calcium imaging, computer vision, and Bayesian kinetic inference (BKI), we analyzed calcium dynamics in primary astrocytes derived from control or Df1/+ mice, a model of 22q11.2 deletion (DiGeorge syndrome). Inference of the highest-likelihood molecular kinetic characteristics of intracellular calcium dynamics identified changes in the activity of the sarcoendoplasmic reticulum calcium ATPase (SERCA). Application of a SERCA inhibitor to wild-type astrocytes reproduced the differences detected in Df1/+ astrocytes. Our work reveals the molecular changes driving the calcium kinetics in astrocytes from a 22q11.2 deletion model. BKI can be useful for mechanistically dissecting calcium dynamics in glial cells and formulating and testing hypotheses about underlying molecular mechanisms.

HuR Mediates Thermogenic Metabolism in Brown Adipose Tissue Through Control of Sarco‐Endoplasmic Calcium Cycling

Obesity is classified by the World Health Organization as one of eight principle causes of preventable chronic disease and is directly associated with decreased life expectancy due to increased risk of associated comorbidities. Brown adipose tissue (BAT) is a thermogenically active, endocrine organ regulating both glucose homeostasis and heat production, and the ability to induce BAT‐mediated energy expending represents a potential therapeutic avenue to reduce obesity.We recently showed that the RNA‐binding protein HuR (Human antigen R) plays a functional role in BAT‐mediated thermogenic metabolism. RNA sequencing analysis from mice with an adipocyte‐specific deletion of HuR (Adipo‐HuR‐/‐) revealed a BAT‐specific decrease in the expression of many genes responsible for sarco‐endoplasmic reticulum (SER) calcium cycling, including SERCA1a, RyR1, RyR2, and additional RyR complex proteins such as triadin, junctin, and CASR. Canonical thermogenesis in BAT occurs through uncoupling protein 1 (UCP1). However, several UCP1‐independent energy expending cycles, including intracellular calcium transport, have also been shown to regulate thermogenesis. SER calcium cycling has recently been established as a thermogenic mechanism in beige adipose tissue, but its functional contributions to thermogenesis in BAT have not yet been fully elucidated.To determine if the HuR‐dependent changes in SER calcium cycles manifest as a functional change in SER loading/cycling in BAT, we show that decreased cytosolic intracellular calcium levels in BAT isolated from Adipo‐HuR‐/‐mice. Additionally, we show that HuR is activated acutely downstream of beta‐adrenergic stimulation in BAT, and deletion of HuR blunts adrenergic‐induced increases in intracellular calcium. Furthermore, HuR directly binds RyR2 mRNA in brown adipocytes and regulates RyR2 expression through RNA stabilization. Taken together, this growing body of work suggests an important role for HuR in mediating thermogenesis through modulation of SER calcium cycling in brown adipose tissue.

Effects of Metabolic Syndrome and SIRT1 Mutation on Store‐Operated Ca2+ Entry in Peripheral Blood Mononuclear Cells in Ossabaw Miniature Swine

BackgroundMetabolic syndrome (MetS) impairs sarco‐endoplasmic reticulum Ca2+ ATPase (SERCA) activity that may decrease the ER Ca2+ store and increase store‐operated Ca2+ entry (SOCE) in a variety of cell types. Similar Ca2+ signaling via SOCE causes fibrosis, vascular disease, and may trigger immunogenic (type 1) diabetes by action on peripheral blood mononuclear cells (PBMCs). Sirtuin 1 (SIRT1) is a deacetylase that maintains healthy metabolism and generally attenuates vascular disease. SIRT1 impairment exacerbates MetS and vascular disease by hyper‐acetylation of SERCA, decreasing its activity and thereby potentially increasing SOCE.HypothesisThe SIRT1 mutation made in Ossabaw miniature swine would mimic the mutation found in humans and increase PBMC SOCE.MethodsUsing CRISPR/Cas9 methodology a point mutation (SIRT1L100P) was made in Ossabaw miniature swine to mimic the naturally occurring mutation in humans and decrease SIRT1 activity, thereby resulting in hyperacetylation of Ca2+transporters and impaired function. Four groups of pigs were used to analyze genotype and diet interactions: wild type lean, SIRT1 lean, wild type MetS, and SIRT1 MetS. Pigs were age 4 months at the start and fed normal chow (lean) or atherogenic diet (MetS) for 7 months. PBMCs were isolated and Ca2+ was measured with fura‐2. The ER Ca2+store was depleted by 10‐5M cyclopiazonic acid to inhibit SERCA in a Ca2+‐free extracellular solution to fully deplete the ER Ca2+store and then Ca2+ was returned to the solution to induce maximal SOCE. Cells were recovered in Ca2+‐free solution to partially re‐fill the ER Ca2+ store, then Ca2+ returned to the solution to observe submaximal SOCE.ResultsBoth MetS diet groups (wild type MetS, SIRT1 MetS) showed impaired glucose tolerance. Fasting glucose in SIRT1 mutants was not different from wild type pigs, thus SIRT1 does not cause immunogenic diabetes at this early stage. Robust coronary atherosclerosis was shown by angiography, intravascular ultrasound, and gross imaging in MetS groups, but there was no additive or synergistic effect of SIRT1 mutation. There was no effect of MetS or SIRT1 mutation on maximal SOCE. Two‐way ANOVA showed that MetS diet increased submaximal SOCE (p&lt;0.009), but there was no effect of SIRT1 genotype on submaximal SOCE (p=0.266). The percentage of PBMCs that responded to partial ER Ca2+ depletion was 45% in lean pigs compared to 81% in wild type and 63% in SIRT1 pigs fed MetS diet.ConclusionSIRT1L100P mutation is likely to require longer duration to manifest robust effects on PBMC SOCE that were found with MetS diet. PBMC activation and can be easily studied longitudinally in the SIRT1L100P mutant swine model due to the ease of blood sampling.

Resveratrol Preserves Mitochondrial Function By Sirt3 Activation In A Right Ventricle Dysfunction Model

<h3>Introduction</h3> Pulmonary Arterial Hypertension (PAH) leads to right ventricular (RV) failure by increasing pulmonary vasculature pressure. Previously we showed that resveratrol (RES) has anti-hypertrophic properties and improved RV function in PAH. However, underlying mechanisms are not fully understood. Of note, sirtuins modulation has been identified as critical mediators that induce cardioprotection. <h3>Objective</h3> To elucidate the contribution of sirtuins in RV protection by RES in a monocrotaline-induced PAH rat model. <h3>Methodology</h3> The Internal Committee for Care and Handling of Laboratory Animals of the Tecnologico de Monterrey approved all animal procedures (protocols 2017-006, 2019-019). Myocyte function and mitochondrial membrane potential (ΔΨm) was characterized by confocal microscopy; mitochondrial respiration was assessed using high-resolution respirometry and calcium retention capacity (CRC) by fluorescence; Western Blot and immunoprecipitation evaluated post-translational modification. Data are presented as percentages, n>3. Statistical significance was set at <i>p</i>< 0.05. PAH is prone to mitochondrial permeability transition pore (mPTP) opening, thus decreasing the mitochondrial membrane potential. The compromised cellular energetics affect cardiomyocyte function by decreasing sarco-endoplasmic reticulum Ca2+-ATPase (SERCA) activity and delaying myofilament unbinding, disrupting cell relaxation. RES partially protects mitochondrial integrity by deacetylating cyclophilin-D, a critical component of the mPTP, increasing SIRT3 expression and activity and preventing mPTP opening. <h3>Results</h3> PAH is prone to mitochondrial permeability transition pore (mPTP) opening by decrease 81% CRC, thus reducing the mitochondrial membrane potential by 47%. The compromised cellular energetics showed by 26% decrease in mitochondrial oxidative phosphorylation affect cardiomyocyte function by decreasing shortening and less efficient with slower time to peak shortening (TTPS, 81%) and half relaxation (TTHR, 41%). RES partially protects mitochondrial integrity by decreasing 51% cyclophilin-D hiperacetylation, a critical component of the mPTP, and increasing 3.5 fold SIRT3 expression thus preventing mPTP opening. <h3>Conclusion</h3> Preventing CypD hyperacetylation through SIRT3 partially contributes to preserving mitochondrial function, which holds an energetic cellular capability to maintain myocyte function. These findings provide new insight into the mechanisms underlying RES protection.

Aberrations in T cell calcium signaling and mitochondrial function predict the presence of the gain-of-function mutation in the ryanodine receptor type 1

Gain-of-function RyR1-p.R163C mutation in ryanodine receptors type 1 (RyR1) deregulates Ca2+ signaling in skeletal muscle and causes malignant hyperthermia in humans under triggering conditions. T lymphocytes from heterozygous RyR1-p.R163C knock-in mutant mice (HET T cells) display measurable aberrations in resting cytosolic Ca2+ concentration ([Ca2+]i), Ca2+ release from the intracellular store, and store-operated Ca2+ entry (SOCE), compared with WT T cells. Pretreatment of HET T cells with ryanodine receptor inhibitors ryanodine or dantrolene reduced disparities between HET and WT T cells in the resting [Ca2+]i and the ability of thapsigargin, an inhibitor of the sarco-endoplasmic reticulum Ca2+ ATPase, to mobilize Ca2+ from the store.