Sarco/endoplasmic Reticulum Ca2+-ATPase Research Articles

Mechanisms underlying the vasodilatory effects of canagliflozin in the rabbit thoracic aorta: Involvement of the SERCA pump and Kv channels

AimsCanagliflozin is an anti-diabetic agent and sodium glucose co-transporter-2 inhibitor. Despite numerous clinical trials demonstrating its beneficial effects on blood pressure, the cellular mechanisms underlying the effects of canagliflozin on vascular reactivity have yet to be clarified. We investigated the vasodilatory effect of canagliflozin on aortic rings isolated from rabbits. Main methodsWe used rabbit thoracic aortic rings and its arterial tone was tested by using wire myography system. Key findingsCanagliflozin caused concentration-dependent vasodilation in aortic rings pre-constricted with phenylephrine or high K+. However, the degree of canagliflozin-induced vasodilation of the aortic rings pre-constricted with high K+ was less than that of rings pre-constricted with phenylephrine. Application of 4-aminopyridine, a voltage-dependent K+ (Kv) channel inhibitor, reduced canagliflozin-induced vasodilation. However, pre-incubation of an inwardly rectifying K+ channel inhibitor, a large-conductance Ca2+-activated K+ channel inhibitor, and an ATP-sensitive K+ inhibitor did not modulate the vasodilatory effects of canagliflozin. Indeed, canagliflozin increased Kv currents in aortic smooth muscle cells. Pre-treatment with thapsigargin or cyclopiazonic acid, a sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) pump inhibitors, reduced the vasodilatory effects of canagliflozin. Conversely, pre-treatment with a Ca2+ channel inhibitor, adenylyl cyclase/PKA inhibitors, and guanylyl cyclase/PKG inhibitors did not modulate the vasodilatory effects of canagliflozin. Endothelium removal, and pre-treatment with the nitric oxide synthase inhibitor L-NAME, and small- and intermediate-conductance Ca2+-activated K+ channel inhibitor apamin and TRAM-34, did not diminish the vasodilatory effects of canagliflozin. SignificanceOur results indicate that canagliflozin induces vasodilation, which is dependent on the robust SERCA activity and Kv channel activation.

Genome-wide mRNA profiling identifies X-box-binding protein 1 (XBP1) as an IRE1 and PUMA repressor

Accumulation of misfolded proteins in ER activates the unfolded protein response (UPR), a multifunctional signaling pathway that is important for cell survival. The UPR is regulated by three ER transmembrane sensors, one of which is inositol-requiring protein 1 (IRE1). IRE1 activates a transcription factor, X-box-binding protein 1 (XBP1), by removing a 26-base intron from XBP1 mRNA that generates spliced XBP1 mRNA (XBP1s). To search for XBP1 transcriptional targets, we utilized an XBP1s-inducible human cell line to limit XBP1 expression in a controlled manner. We also verified the identified XBP1-dependent genes with specific silencing of this transcription factor during pharmacological ER stress induction with both an N-linked glycosylation inhibitor (tunicamycin) and a non-competitive inhibitor of the sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) (thapsigargin). We then compared those results to the XBP1s-induced cell line without pharmacological ER stress induction. Using next‐generation sequencing followed by bioinformatic analysis of XBP1-binding motifs, we defined an XBP1 regulatory network and identified XBP1 as a repressor of PUMA (a proapoptotic gene) and IRE1 mRNA expression during the UPR. Our results indicate impairing IRE1 activity during ER stress conditions accelerates cell death in ER-stressed cells, whereas elevating XBP1 expression during ER stress using an inducible cell line correlated with a clear prosurvival effect and reduced PUMA protein expression. Although further studies will be required to test the underlying molecular mechanisms involved in the relationship between these genes with XBP1, these studies identify a novel repressive role of XBP1 during the UPR.

Chemical Modification of the Tryptophan Residue in a Recombinant Ca2+-ATPase N-domain for Studying Tryptophan-ANS FRET.

The sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) is a P-type ATPase that has been crystallized in various conformations. Detailed functional information may nonetheless be obtained from isolated recombinant domains. The engineered (Trp552Leu and Tyr587Trp) recombinant nucleotide-binding domain (N-domain) displays fluorescence quenching upon ligand binding. An extrinsic fluorophore, namely, 8-anilino-1-naphthalene sulfonate (ANS), binds to the nucleotide-binding site via electrostatic and hydrophobic interactions with Arg, His, Ala, Leu, and Phe residues. ANS binding is evidenced by the increase in fluorescence intensity when excited at a wavelength (λ) of 370 nm. However, when excited at λ of 295 nm, the increase in fluorescence intensity seems to be coupled to the quenching of the N-domain intrinsic fluorescence. Fluorescence spectra display a Föster resonance energy transfer (FRET)-like pattern, thereby suggesting the presence of a Trp-ANS FRET pair, which appears to be supported by the short distance (~20 Å) between Tyr587Trp and ANS. This study describes an analysis of the Trp-ANS FRET pair by Trp chemical modification (and fluorescence quenching) that is mediated by N-bromosuccinimide (NBS). In the chemically modified N-domain, ANS fluorescence increased when excited at a λ of 295 nm, similar to when excited at a λ of 370 nm. Hence, the NBS-mediated chemical modification of the Trp residue can be used to probe the absence of FRET between Trp and ANS. In the absence of Trp fluorescence, one should not observe an increase in ANS fluorescence. The chemical modification of Trp residues in proteins by NBS may be useful for examining FRET between Trp residues that are close to the bound ANS. This assay will likely also be useful when using other fluorophores.

Cytotoxic effect of carbohydrate derivatives of digitoxigenin involves modulation of plasma membrane Ca2+ -ATPase.

Cardiac glycosides, such as digoxin and digitoxin, are compounds that interact with Na+ /K+ -ATPase to induce anti-neoplastic effects; however, these cardiac glycosides have narrow therapeutic index. Thus, semi-synthetic analogs of digitoxin with modifications in the sugar moiety has been shown to be an interesting approach to obtain more selective and more effective analogs than the parent natural product. Therefore, the aim of this study was to assess the cytotoxic potential of novel digitoxigenin derivatives, digitoxigenin-α-L-rhamno-pyranoside (1) and digitoxigenin-α-L-amiceto-pyranoside (2), in cervical carcinoma cells (HeLa) and human diploid lung fibroblasts (Wi-26-VA4). In addition, we studied the anticancer mechanisms of action of these compounds by comparing its cytotoxic effects with the potential to modulate the activity of three P-type ATPases; Na+ /K+ -ATPase, sarco/endoplasmic reticulum Ca2+ -ATPase (SERCA), and plasma membrane Ca2+ -ATPase (PMCA). Briefly, the results showed that compounds 1 and 2 were more cytotoxic and selectivity for HeLa tumor cells than the nontumor cells Wi-26-VA4. While the anticancer cytotoxicity in HeLa cells involves the modulation of Na+ /K+ -ATPase, PMCA and SERCA, the modulation of these P-type ATPases was completely absent in Wi-26-VA4 cells, which suggest the importance of their role in the cytotoxic effect of compounds 1 and 2 in HeLa cells. Furthermore, the compound 2 inhibited directly erythrocyte ghosts PMCA and both compounds were more cytotoxic than digitoxin in HeLa cells. These results provide a better understanding of the mode of action of the synthetic cardiac glycosides and highlights 1 and 2 as potential anticancer agents.

A novel derivative of picolinic acid induces endoplasmic reticulum stress-mediated apoptosis in human non-small cell lung cancer cells: synthesis, docking study, and anticancer activity

Thirteen new derivatives of picolinic acid (4–7) were designed and synthesized from the starting parent molecule, picolinic acid. The new compounds were characterized by ATR-FTIR, 1HNMR, and CHNS analysis. A molecular docking study was performed to evaluate the binding affinity of the synthesized compounds toward EGFR kinase domain that indicated occupation of the critical site of EGFR kinase pocket and excellent positioning of the compounds in the pocket. The cytotoxic activity of the compounds against two human cancer cell lines (A549 and MCF-7), the non-tumorigenic MCF10A cell line, and white blood cells (WBC) was evaluated using the MTT assay. Compound 5 showed anticancer activity against A549 lung cancer cells (IC50 = 99.93 µM) but not against MCF-7 breast cancer cells or normal cells. Compound 5 mediated cytotoxicity in A549 lung cancer cells by inducing apoptotic cell death, as suggested by fragmented nuclei after DAPI staining, and agarose gel electrophoresis. Moreover, compound 5 triggered the activation of caspases 3, 4 and 9. However, compound 5 treatment did not affect the release of cytochrome c from the mitochondria to the cytosol, as compared to the vehicle-treated control cells. Nevertheless, compound 5-treated cells reported greater release of smac/DIABLO to the cytosol. In the same context, both compound 5 and thapsigargin (specific inhibitor of sarco/endoplasmic reticulum Ca2+-ATPase (SERCA)) enhanced eIF2 phosphorylation, reflecting the activation of the atypical ER stress pathway and the potential applicability of compound 5 in lung cancer treatment.

Presenilin 1 is a direct regulator of the cardiac sarco/endoplasmic reticulum calcium pump

The gamma secretase catalytic subunit presenilin 1 (PS1) is expressed in the endoplasmic reticulum (ER) of neurons, where it regulates Ca2+ signaling. PS1 is also expressed in heart, but its role in regulation of cardiac Ca2+ transport remains unknown. Since the type 2 sarco/endoplasmic reticulum Ca2+ ATPase (SERCA2a) plays a central role in cardiac Ca2+ homeostasis, we studied whether PS1 regulates the cardiac SERCA2a function. The experiments were conducted in an inducible human SERCA2a stable T-Rex-293 cell line transfected with fluorescently labeled PS1 and the ER Ca2+ sensor R-CEPIA1er. Confocal imaging showed that that PS1 is localized predominantly in the ER membrane. Fluorescent resonance energy transfer (FRET) experiments in HEK293 cells transfected with fluorescently labeled SERCA2a and PS1 revealed that the two proteins directly interact with a 1:1 stoichiometry. The functional significance of this interaction was investigated in a heterologous cellular environment using a novel approach to directly measure ER Ca2+ dynamics. Measurements of SERCA2a-mediated Ca2+ transport showed that PS1 enhanced Ca2+ uptake at low ER Ca2+ loads (<0.15 mM) and reduced uptake at high loads (>0.35 mM). The results of this study revealed that PS1 could act as an important regulator of the cardiac Ca2+ pump function with a complex stimulatory/inhibitory profile.

Abstract P385: Sarcolipin Reduction Prevents Mitochondrial Dysfunction In Dystrophic Myocardium

Rationale &amp; Hypothesis: Sarcolipin (SLN), an inhibitor of sarco/endoplasmic reticulum Ca 2+ ATPase (SERCA) is abnormally high and enriched in the mitochondrial associated membrane (MAM) fractions of dystrophic hearts. We, therefore, hypothesized that in the dystrophic cardiomyocytes, SLN upregulation causes abnormal elevation of intracellular Ca 2+ (Ca 2+ i ) including Ca 2+ mishandling in the MAM region, resulting in sustained elevation of mitochondrial Ca 2+ (Ca 2+ m ) , which in turn affects mitochondrial structure and function. Approach: We chose dystrophin mutant and utrophin deficient, mdx:utr -/- mice for our studies and generated SLN haploinsufficient mdx:utr -/- (mdx:utr -/- :sln +/- ) mice. Single-cell Ca 2+ transients and Ca 2+ m efflux were measured in isolated cardiomyocytes to determine Ca 2+ i handling and Ca 2+ m content, respectively. Mitochondrial membrane potential was assessed with membrane-permeant dyes in isolated cardiomyocytes followed by confocal imaging. Mitochondrial respiration was measured using Complex activity assays and Seahorse metabolic profiling in ventricular tissue lysates and isolated mitochondria respectively. Mitochondrial structure, number, and area were evaluated using electron micrographs. Results: Heterozygous deletion of the SLN gene normalized SLN expression and improved Ca 2+ i cycling and prevented Ca 2+ m overload in dystrophic cardiac myocytes. Furthermore, reducing SLN expression prevents loss of membrane potential and improves mitochondrial respiration in the dystrophic cardiac myocytes. Transmission electron microscopic analyses of the dystrophic heart revealed significant cristae loss in the mitochondria globally as well as in the MAM-associated mitochondria. On the other hand, the reduction in SLN expression prevents these changes. Conclusions: In conclusion, reduction in SLN expression preserves the SR-mitochondrial interface and restores the mitochondrial function by mitigating the Ca 2+ overload, membrane potential loss, and structural damage. These changes improve cardiac function and prevent the development of cardiomyopathy in mdx:utr -/- mice. Our findings suggest that SLN reduction could be a potential therapeutic strategy for the treatment of Duchenne muscular dystrophy and associated cardiomyopathy.

Cryo-EM analysis provides new mechanistic insight into ATP binding to Ca2+ -ATPase SERCA2b.

Sarco/endoplasmic reticulum Ca2+ -ATPase (SERCA) 2b is a ubiquitous SERCA family member that conducts Ca2+ uptake from the cytosol to the ER. Herein, we present a 3.3Å resolution cryo-electron microscopy (cryo-EM) structure of human SERCA2b in the E1·2Ca2+ state, revealing a new conformation for Ca2+ -bound SERCA2b with a much closer arrangement of cytosolic domains than in the previously reported crystal structure of Ca2+ -bound SERCA1a. Multiple conformations generated by 3D classification of cryo-EM maps reflect the intrinsically dynamic nature of the cytosolic domains in this state. Notably, ATP binding residues of SERCA2b in the E1·2Ca2+ state are located at similar positions to those in the E1·2Ca2+ -ATP state; hence, the cryo-EM structure likely represents a preformed state immediately prior to ATP binding. Consistently, a SERCA2b mutant with an interdomain disulfide bridge that locks the closed cytosolic domain arrangement displayed significant autophosphorylation activity in the presence of Ca2+ . We propose a novel mechanism of ATP binding to SERCA2b.

The anti-diabetic drug trelagliptin induces vasodilation via activation of Kv channels and SERCA pumps

AimsIn this study, we investigated the vasodilatory effects of trelagliptin (a dipeptidyl peptidase-4 inhibitor) and its related mechanisms using rabbit aortic rings. Main methodsArterial tone measurement was performed in rabbit thoracic aortic rings. Key findingsTrelagliptin induced vasodilation in a dose-dependent manner. Pretreatment with the ATP-sensitive K+ channel inhibitor glibenclamide, large-conductance Ca2+-activated K+ channel inhibitor paxilline, and inwardly rectifying K+ channel inhibitor Ba2+ did not affect the vasodilatory effect of trelagliptin. However, pretreatment with the voltage-dependent K+ (Kv) channel inhibitors 4-aminopyridine and tetraethylammonium significantly attenuated the vasodilatory effect of trelagliptin, suggesting that the vasodilatory effect of trelagliptin is associated with Kv channel activation. Although pretreatment with Kv1.5 and Kv2.1 subtype inhibitors did not affect the response to trelagliptin, pretreatment with a Kv7.X subtype inhibitor effectively reduced the vasodilatory effect of trelagliptin. Furthermore, sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) pump inhibitors also significantly attenuated the vasodilatory effect of trelagliptin. These effects, however, were not affected by pretreatment with Ca2+ channel inhibitors, adenylyl cyclase/PKA inhibitors, guanylyl cyclase/PKG inhibitors, or removal of the endothelium. SignificanceFrom these results, we concluded that the vasodilatory effect of trelagliptin was associated with the activation of Kv channels (primary the Kv7.X subtype) and SERCA pump regardless of other K+ channels, Ca2+ channels, cAMP/PKA-related or cGMP/PKG-related signaling pathways, and the endothelium. Therefore, caution is required when prescribing trelagliptin to the patients with hypotension and diabetes.

Modulated Calcium Homeostasis and Release Events Under Atrial Fibrillation and Its Risk Factors: A Meta-Analysis

Background: Atrial fibrillation (AF) is associated with calcium (Ca2+) handling remodeling and increased spontaneous calcium release events (SCaEs). Nevertheless, its exact mechanism remains unclear, resulting in suboptimal primary and secondary preventative strategies.Methods: We searched the PubMed database for studies that investigated the relationship between SCaEs and AF and/or its risk factors. Meta-analysis was used to examine the Ca2+ mechanisms involved in the primary and secondary AF preventative groups.Results: We included a total of 74 studies, out of the identified 446 publications from inception (1982) until March 31, 2020. Forty-five were primary and 29 were secondary prevention studies for AF. The main Ca2+ release events, calcium transient (standardized mean difference (SMD) = 0.49; I2 = 35%; confidence interval (CI) = 0.33–0.66; p < 0.0001), and spark amplitude (SMD = 0.48; I2 = 0%; CI = −0.98–1.93; p = 0.054) were enhanced in the primary diseased group, while calcium transient frequency was increased in the secondary group. Calcium spark frequency was elevated in both the primary diseased and secondary AF groups. One of the key cardiac currents, the L-type calcium current (ICaL) was significantly downregulated in primary diseased (SMD = −1.07; I2 = 88%; CI = −1.94 to −0.20; p < 0.0001) and secondary AF groups (SMD = −1.28; I2 = 91%; CI = −2.04 to −0.52; p < 0.0001). Furthermore, the sodium–calcium exchanger (INCX) and NCX1 protein expression were significantly enhanced in the primary diseased group, while only NCX1 protein expression was shown to increase in the secondary AF studies. The phosphorylation of the ryanodine receptor at S2808 (pRyR-S2808) was significantly elevated in both the primary and secondary groups. It was increased in the primary diseased and proarrhythmic subgroups (SMD = 0.95; I2 = 64%; CI = 0.12–1.79; p = 0.074) and secondary AF group (SMD = 0.66; I2 = 63%; CI = 0.01–1.31; p < 0.0001). Sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) expression was elevated in the primary diseased and proarrhythmic drug subgroups but substantially reduced in the secondary paroxysmal AF subgroup.Conclusions: Our study identified that ICaL is reduced in both the primary and secondary diseased groups. Furthermore, pRyR-S2808 and NCX1 protein expression are enhanced. The remodeling leads to elevated Ca2+ functional activities, such as increased frequencies or amplitude of Ca2+ spark and Ca2+ transient. The main difference identified between the primary and secondary diseased groups is SERCA expression, which is elevated in the primary diseased group and substantially reduced in the secondary paroxysmal AF subgroup. We believe our study will add new evidence to AF mechanisms and treatment targets.

Angle change of the A-domain in a single SERCA1a molecule detected by defocused orientation imaging

The sarcoendoplasmic reticulum Ca2+-ATPase (SERCA) transports Ca2+ ions across the membrane coupled with ATP hydrolysis. Crystal structures of ligand-stabilized molecules indicate that the movement of actuator (A) domain plays a crucial role in Ca2+ translocation. However, the actual structural movements during the transitions between intermediates remain uncertain, in particular, the structure of E2PCa2 has not been solved. Here, the angle of the A-domain was measured by defocused orientation imaging using isotropic total internal reflection fluorescence microscopy. A single SERCA1a molecule, labeled with fluorophore ReAsH on the A-domain in fixed orientation, was embedded in a nanodisc, and stabilized on Ni–NTA glass. Activation with ATP and Ca2+ caused angle changes of the fluorophore and therefore the A-domain, motions lost by inhibitor, thapsigargin. Our high-speed set-up captured the motion during EP isomerization, and suggests that the A-domain rapidly rotates back and forth from an E1PCa2 position to a position close to the E2P state. This is the first report of the detection in the movement of the A-domain as an angle change. Our method provides a powerful tool to investigate the conformational change of a membrane protein in real-time.

39-OR: Pancreatic Beta Cell SERCA2 Deficiency Impacts ER-Golgi Vesicle Trafficking and Ceramide Metabolism

Alterations in endoplasmic reticulum (ER) calcium (Ca2+) levels arising from reduced activity of the Sarcoendoplasmic Reticulum Ca2+ ATPase (SERCA) pump are associated with impaired insulin secretion and reduce β-cell survival in both major forms of diabetes. However, at present, the mechanisms of how reduced ER Ca2+ impairs β-cell function remain incompletely understood. To test this, we analyzed mice with β-cell specific SERCA2 deletion (βS2KO mice) and S2KO INS1 cells. βS2KO mice exhibited age-dependent glucose intolerance, reduced glucose-stimulated insulin secretion, and increased proinsulin secretion without evidence of impaired insulin sensitivity. Immunoblot analysis of βS2KO islets and S2KO INS-1 cells revealed reduced active forms of the proinsulin processing enzymes, PC1/3, PC2, and CPE, while immunofluorescence analysis suggested impaired ER-Golgi trafficking of the convertase enzymes and proinsulin. Because the ER serves as the main site of cellular lipid synthesis and changes in the β-cell lipidome have been linked with diabetes pathophysiology, we hypothesized that ER Ca2+ loss may alter protein trafficking in the secretory pathway through changes in ER lipid content. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was performed on the ER fractions of control and S2KO INS1 cells and revealed increased levels of 16:0, 24:0, 24:1, and total ceramides in S2KO cells. SERCA2 restoration partially rescued convertase enzyme expression, proinsulin processing, and ceramide content. Consistent with these findings, RNA sequencing analysis of βS2KO islets revealed changes in pathways related to vesicle transport, protein processing, and lipid metabolism. We conclude that SERCA2 in the β-cell plays a central role in the maintenance of insulin biosynthesis as well as ceramide metabolism. Disclosure T. Kono: None. H. Iida: None. C. Lee: None. P. Krishnan: None. P. Arvan: None. I. Lindberg: None. C. Evans-molina: Advisory Panel; Self; Provention Bio, Inc., Consultant; Self; Dompe, Other Relationship; Self; Bristol-Myers Squibb Company, Nimbus Pharmaceuticals, Pfizer Inc. Funding National Institutes of Health (R01DK093954)

Sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) activity is required for V(D)J recombination.

A whole-genome CRISPR/Cas9 screen identified ATP2A2, the gene encoding the Sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) 2 protein, as being important for V(D)J recombination. SERCAs are ER transmembrane proteins that pump Ca2+ from the cytosol into the ER lumen to maintain the ER Ca2+ reservoir and regulate cytosolic Ca2+-dependent processes. In preB cells, loss of SERCA2 leads to reduced V(D)J recombination kinetics due to diminished RAG-mediated DNA cleavage. SERCA2 deficiency in B cells leads to increased expression of SERCA3, and combined loss of SERCA2 and SERCA3 results in decreased ER Ca2+ levels, increased cytosolic Ca2+ levels, reduction in RAG1 and RAG2 gene expression, and a profound block in V(D)J recombination. Mice with B cells deficient in SERCA2 and humans with Darier disease, caused by heterozygous ATP2A2 mutations, have reduced numbers of mature B cells. We conclude that SERCA proteins modulate intracellular Ca2+ levels to regulate RAG1 and RAG2 gene expression and V(D)J recombination and that defects in SERCA functions cause lymphopenia.

Low-Dose Empagliflozin Improves Systolic Heart Function after Myocardial Infarction in Rats: Regulation of MMP9, NHE1, and SERCA2a.

The effects of the selective sodium-glucose cotransporter 2 (SGLT2) inhibitor empagliflozin in low dose on cardiac function were investigated in normoglycemic rats. Cardiac parameters were measured by intracardiac catheterization 30 min after intravenous application of empagliflozin to healthy animals. Empagliflozin increased the ventricular systolic pressure, mean pressure, and the max dP/dt (p < 0.05). Similarly, treatment with empagliflozin (1 mg/kg, p.o.) for one week increased the cardiac output, stroke volume, and fractional shortening (p < 0.05). Myocardial infarction (MI) was induced by ligation of the left coronary artery. On day 7 post MI, empagliflozin (1 mg/kg, p.o.) improved the systolic heart function as shown by the global longitudinal strain (−21.0 ± 1.1% vs. −16.6 ± 0.7% in vehicle; p < 0.05). In peri-infarct tissues, empagliflozin decreased the protein expression of matrix metalloproteinase 9 (MMP9) and favorably regulated the cardiac transporters sarco/endoplasmic reticulum Ca2+-ATPase (SERCA2a) and sodium hydrogen exchanger 1 (NHE1). In H9c2 cardiac cells, empagliflozin decreased the MMP2,9 activity and prevented apoptosis. Empagliflozin did not alter the arterial stiffness, blood pressure, markers of fibrosis, and necroptosis. Altogether, short-term treatment with low-dose empagliflozin increased the cardiac contractility in normoglycemic rats and improved the systolic heart function in the early phase after MI. These effects are attributed to a down-regulation of MMP9 and NHE1, and an up-regulation of SERCA2a. This study is of clinical importance because it suggests that a low-dose treatment option with empagliflozin may improve cardiovascular outcomes post-MI. Down-regulation of MMPs could be relevant to many remodeling processes including cancer disease.

Role of Reduced Sarco-Endoplasmic Reticulum Ca2+-ATPase Function on Sarcoplasmic Reticulum Ca2+ Alternans in the Intact Rabbit Heart.

Sarcoplasmic reticulum (SR) Ca2+ cycling is tightly regulated by ryanodine receptor (RyR) Ca2+ release and sarco-endoplasmic reticulum Ca2+-ATPase (SERCA) Ca2+ uptake during each excitation–contraction coupling cycle. We previously showed that RyR refractoriness plays a key role in the onset of SR Ca2+ alternans in the intact rabbit heart, which contributes to arrhythmogenic action potential duration (APD) alternans. Recent studies have also implicated impaired SERCA function, a key feature of heart failure, in cardiac alternans and arrhythmias. However, the relationship between reduced SERCA function and SR Ca2+ alternans is not well understood. Simultaneous optical mapping of transmembrane potential (Vm) and SR Ca2+ was performed in isolated rabbit hearts (n = 10) using the voltage-sensitive dye RH237 and the low-affinity Ca2+ indicator Fluo-5N-AM. Alternans was induced by rapid ventricular pacing. SERCA was inhibited with cyclopiazonic acid (CPA; 1–10 μM). SERCA inhibition (1, 5, and 10 μM of CPA) resulted in dose-dependent slowing of SR Ca2+ reuptake, with the time constant (tau) increasing from 70.8 ± 3.5 ms at baseline to 85.5 ± 6.6, 129.9 ± 20.7, and 271.3 ± 37.6 ms, respectively (p < 0.05 vs. baseline for all doses). At fast pacing frequencies, CPA significantly increased the magnitude of SR Ca2+ and APD alternans, most strongly at 10 μM (pacing cycle length = 220 ms: SR Ca2+ alternans magnitude: 57.1 ± 4.7 vs. 13.4 ± 8.9 AU; APD alternans magnitude 3.8 ± 1.9 vs. 0.2 ± 0.19 AU; p < 0.05 10 μM of CPA vs. baseline for both). SERCA inhibition also promoted the emergence of spatially discordant alternans. Notably, at all CPA doses, alternation of SR Ca2+ release occurred prior to alternation of diastolic SR Ca2+ load as pacing frequency increased. Simultaneous optical mapping of SR Ca2+ and Vm in the intact rabbit heart revealed that SERCA inhibition exacerbates pacing-induced SR Ca2+ and APD alternans magnitude, particularly at fast pacing frequencies. Importantly, SR Ca2+ release alternans always occurred before the onset of SR Ca2+ load alternans. These findings suggest that even in settings of diminished SERCA function, relative refractoriness of RyR Ca2+ release governs the onset of intracellular Ca2+ alternans.

TRPC7 regulates the electrophysiological functions of embryonic stem cell-derived cardiomyocytes

BackgroundBiological pacemakers consisting of pluripotent stem cell-derived cardiomyocytes are potentially useful for treating bradycardia. However, tachyarrhythmia caused by derived cardiomyocytes themselves is one of main barriers hampering their clinical translation. An in-depth understanding of the mechanisms underlying the spontaneous action potential (a.k.a. automaticity) might provide potential approaches to solve this problem. The aim of this project is to study the role of canonical transient receptor potential isoform 7 (TRPC7) channels in regulating the automaticity of embryonic stem cell-derived cardiomyocytes (ESC-CMs).Methods and resultsBy Western blotting, the expression of TRPC7 was found to be increased during the differentiation of mouse ESC-CMs (mESC-CMs). Adenovirus-mediated TRPC7 knockdown decreased while overexpression increased the frequency of Ca2+ transients (CaTs), local Ca2+ releases (LCRs), and action potentials (APs) as detected by confocal microscopy and whole-cell patch-clamping. TRPC7 was found to be positively associated with the activity of ryanodine receptor 2 (RyR2), sarco/endoplasmic reticulum Ca2+-ATPase (SERCA), and sodium-calcium exchanger (NCX) but not hyperpolarization-activated, cyclic nucleotide-gated channel (HCN), and inositol trisphosphate receptor (IP3R). Knockdown or overexpression of TRPC7 did not alter the expression of HCN4, Cav1.3, Cav3.1, Cav3.2, IP3R1, RyR2, and SERCA but positively regulated the phosphorylation of RyR2 at S2814 and phospholamban (PLN) at T17. Moreover, the positive regulation of APs by TRPC7 was Ca2+-dependent, as overexpression of N-terminus of TRPC7 (dominant negative of TRPC7) which diminished the Ca2+ permeability of TRPC7 decreased the AP frequency.ConclusionsTRPC7 regulates the automaticity of mESC-CMs through two mechanisms. On the one hand, TRPC7 positively regulates the intracellular Ca2+ clock through the regulation of activities of both RyR2 and SERCA; on the other hand, TRPC7 also positively regulates the membrane clock via its influence on NCX activity. Altogether, our study reveals that TRPC7 is a potential drug target to manipulate the action potential firing rate of pluripotent stem cell-derived cardiomyocyte-based biological pacemakers to prevent tachyarrhythmia, a condition that might be encountered after transplantation.

Heterozygous SOD2 deletion impairs SERCA function in soleus and heart muscles from female mice

Background Sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA) pumps actively transport intracellular Ca2+ back into the sarcoplasmic reticulum and are the primary facilitators of muscle relaxation, restoring intracellular [Ca2+] to resting levels. Like many proteins, SERCA pumps are subject to oxidation which may affect their functional capacity. Superoxide dismutase 2 (SOD2) is a critical antioxidant enzyme localised in the mitochondrial matrix which converts superoxide radicals into less reactive H2O2. The present study sought to explore the structural and functional consequences of halved SOD2 expression, particularly to SERCA isoforms, in skeletal and cardiac muscle. Specifically, we examined whether heterozygous SOD2 deletion would result in changes in levels of SERCA tyrosine nitration and S-glutathionylation. Tyrosine nitration is known to impair SERCA function, particularly SERCA2a (slow isoform) but not SERCA1a (fast isoform). Conversely, glutathionylation of SERCA may act as an adaptive modality to preserve function during moderate oxidative stress. Methods We obtained soleus, extensor digitorum longus (EDL), and left ventricle (LV) muscles from wild-type (WT) and SOD2 heterozygote (SOD2+/-) C57BL/6J female mice (n=8 per genotype, aged 6-7 months). Ca2+-dependent SERCA activity assays were performed along with immunoprecipitation experiments to examine SERCA2a and SERCA1a tyrosine nitration and S-glutathionylation. Results Heterozygous SOD2 deletion did not alter SERCA1a or SERCA2a expression in the soleus, EDL or LV compared with WT. However, relative to WT, SOD2+/- soleus muscles showed significant impairments in SERCA function, with a reduction in SERCA's affinity for Ca2+. This corresponded well with Western blot data showing significantly elevated SERCA2a tyrosine nitration and significantly reduced S-glutathionylation in the soleus. In contrast, we observed no change in SERCA function, SERCA1a tyrosine nitration, or S-glutathionylation in the EDL. Furthermore, there were no differences in SERCA1a tyrosine nitration in the soleus, which is consistent with the notion that the SERCA1a isoform is less susceptible to nitrosative modification. Together, these results suggest that the slow SERCA isoform is more susceptible to heterozygous SOD2 deletion, and this is further supported by a significant reduction in SERCA's affinity for Ca2+ in the LV where SERCA2a is the predominant isoform. Conclusions This is the first study showing the physiological effects of halved SOD2 expression on muscle SERCA function. Our results reinforce the idea that SERCA2a is more sensitive to nitrosative modification.

Sarcoplasmic Reticulum Ca 2+ Handling in Gastrocnemius Muscles from 10‐week‐old C57 and D2‐ mdx Mice

Background Duchenne Muscular Dystrophy (DMD) is an X-linked muscle wasting disease caused by an absence of dystrophin. Secondary mechanisms including elevated myoplasmic Ca2+ as a result of dysfunctional sarcoendoplasmic reticulum Ca2+-ATPase (SERCA) pumps and leaky ryanodine receptors (RyR) can perpetuate disease pathology. Although the widely used preclinical C57BL/10 (C57) mdx mouse model displays moderate muscle damage and weakness, this model falls short in its ability to recapitulate the more severe human phenotype. Conversely, the DBA/2J (D2) mdx mouse model displays severe and early-onset muscle weakness, damage and histopathology; and therefore, more closely mimics human DMD. To date, there has been no characterization of muscle Ca2+ handling in the D2-mdx mouse. Thus, we examined sarcoplasmic reticulum (SR) Ca2+ handling in both D2-mdx and C57-mdx mice. Methods Eight-week-old male C57-WT, C57-mdx, D2-WT and D2-mdx mice were purchased from Jackson Laboratories. All mice underwent a hangwire test and were temporarily housed (48 hr) in a Promethion Metabolic Cage System to measure energy expenditure and cage activity. At 10 weeks of age, mice were euthanized, and gastrocnemius muscles were collected to measure Ca2+ uptake and leak via an Indo-1 fluorimetric assay. Serum creatine kinase (CK) activity was measured using a commercially available kit. Results Consistent with previous literature, serum CK was elevated in both C57-mdx and D2-mdx mice compared with WT, but to a greater extent in the former (8.9-fold vs 3.3 fold, p = 0.04). While both C57-mdx and D2-mdx mice had lowered hangwire time, these impairments were more severe in the D2-mdx mouse (-30% in C57-mdx vs -50% in D2-mdx, p = 0.09). Similarly, while both C57-mdx (-15%) and D2-mdx (-33%) mice had lowered total cage activity, the D2-mdx mice were most affected (p = 0.03). Interestingly, metabolic cage analyses further revealed that D2-mdx (1.3-fold increase) but not C57-mdx mice, had significantly elevated daily energy expenditure compared to their respective WT groups. When examining SR Ca2+ uptake, we found that the D2-mdx mice had significantly elevated starting Ca2+ levels compared with D2-WT mice (5500 vs 2300 nM). When activated by ATP, there was significantly less removal of Ca2+ in the D2-mdx muscles compared with WT (2.6-fold greater area-under-the-curve). However, none of these effects were observed in the C57-mdx mice. Furthermore, when SERCA was inhibited with cyclopiazonic acid, the rate of SR Ca2+ leak (-50%) and the amount of SR Ca2+ released back into the cytosol (-48%) were significantly lowered in the D2-mdx but not in the C57-mdx mice. This is likely reflective of impaired SR Ca2+ uptake and filling. Conclusions Unlike the C57-mdx, the D2-mdx mice displayed early onset muscle weakness with lowered cage ambulation and hangwire time when compared with their respective WT groups. These findings were associated with impaired SR Ca2+ handling in the D2-mdx mice but not the C57-mdx mice. Together, the extensive muscle damage and impaired SR Ca2+ handling in the D2-mdx mice likely enhances their daily energy expenditure.

Piezo1 promotes ER Ca 2+ transport to regulate the cellular responses of endothelial cells to shear stress

Mechanotransduction is an essential mechanism by which endothelial cells (ECs) sense and adapt to the environment. Piezo1, a mechanosensitive cation channel, is responsible for sensing and transducing mechanical stimuli into chemical signals in endothelial cells. However, the signaling mechanism downstream of Piezo1 remains poorly understood. Here, we identified Piezo1-mediated ER Ca2+ transport as an essential element of endothelial cell adaptation to shear stress. Using the ER targeted calcium sensor G-Cepia1er, we showed that both laminal fluid shear and pharmacological activation of Piezo1 induced Ca2+ER transport. Activation of Piezo1 led to rapid influx of Ca2+ in the ER followed by Ca2+ER release via cAMP-sensitized Inositol TrisphosphateReceptor 2 (IP3R2), a calcium channel located in the ER membrane. Inhibition of sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) blocked Piezo1-induced Ca2+entry in the ER but did not affect the rate of Ca2+ER release. In contrast, depletion of soluble Adenylyl Cyclase (sAC) markedly reduced the rate of Piezo1-mediated Ca2+ER release without affecting Ca2+entry. These data indicate that the ER Ca2+ transport induced by shear stress is finely regulated by Piezo1 downstream signaling. Furthermore, depletion of Piezo1, IP3R2 and sAC blocked alignment of ECs in the direction of shear flow. Our results provide evidence on how mechanical forces are transmitted to cellular organelles such as the ER and, for the first time, establish the role of Piezo1-sAC-IP3R2 mediated ER Ca2+ transport as an essential signaling element of cell adaptation to mechanical cues.

The Crystal Structure of the Ca2+-ATPase 1 from Listeria monocytogenes reveals a Pump Primed for Dephosphorylation

Many bacteria export intracellular calcium using active transporters homologous to the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA). Here we present three crystal structures of Ca2+-ATPase 1 from Listeria monocytogenes (LMCA1). Structures with BeF3- mimicking a phosphoenzyme state reveal a closed state, which is intermediate between the outward-open E2P and the proton-occluded E2-P* conformations known for SERCA. It suggests that LMCA1 in the E2P state is pre-organized for dephosphorylation upon Ca2+ release, consistent with the rapid dephosphorylation observed in single-molecule studies. An arginine side-chain occupies the position equivalent to calcium binding site I in SERCA, leaving a single Ca2+ binding site in LMCA1, corresponding to SERCA site II. Observing no putative transport pathways dedicated to protons, we infer a direct proton counter transport through the Ca2+ exchange pathways. The LMCA1 structures provide insight into the evolutionary divergence and conserved features of this important class of ion transporters.