Ryanodine Receptor Expression Research Articles

Effect of endocrine-disrupting chemicals on the expression of a calcium ion channel receptor (ryanodine receptor) in the mud crab (Macrophthalmus japonicus)

Endocrine-disrupting chemicals (EDCs) are toxic pollutants generated by artificial activities. Moreover, their hormone-like structure induces disturbances, such as mimicking or blocking metabolic activity. Previous studies on EDCs have focused on the adverse effect of the endocrine system in vertebrates, with limited investigations conducted on ion channels in invertebrates. Thus, in this study, we investigated the potential adverse effects of exposure to bisphenol-A (BPA) and di-(2-ethylhexyl) phthalate (DEHP) at the molecular level on the ryanodine receptor (RyR), a calcium ion channel receptor in Macrophthalmus japonicus. In the phylogenetic analysis, the RyR amino acid sequences in M. japonicus clustered with those in the Crustacean and formed separated branches for RyR in insects and mammals. When exposed to 1 μg L−1 BPA, a significant increase in RyR mRNA expression was observed in the gills on day 1, although a similar level to the control group was observed from day 4 to day 7. However, the RyR expression due to DEHP exposure decreased on days 1 and 4, although it increased on day 7 following exposure to 10 μg L−1. The RyR expression pattern in the hepatopancreas increased for up to 4 days, depending on the BPA concentration. However, there was a tendency for the expression to decrease gradually after the statistical significance increased during the early stage of DEHP exposure (D1). Hence, the transcriptional alterations in the M. japonicus RyR gene observed in the study suggest that exposure toxicities to EDCs, such as BPA and DEHP, have the potential to disrupt calcium ion channel signaling in the gills and hepatopancreas of M. japonicus crabs.

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

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

Potential therapeutic targets for hypotension in duchenne muscular dystrophy

Duchenne Muscular Dystrophy (DMD) is marked by genetic mutations occurring in the DMD gene, which is widely expressed in the cardiovascular system. In addition to developing cardiomyopathy, patients with DMD have been reported to be susceptible to the development of symptomatic hypotension, although the mechanisms are unclear. Analysis of single-cell RNA sequencing data has identified potassium voltage-gated channel subfamily Q member 5 (KCNQ5) and possibly ryanodine receptor 2 (RyR2) as potential candidate hypotension genes whose expression is significantly upregulated in the vascular smooth muscle cells of DMD mutant mice. We hypothesize that heightened KCNQ5 and RyR2 expression contributes to decreased arterial blood pressure in patients with DMD. Exploring pharmacological approaches to inhibit the KCNQ5 and RyR2 channels holds promise in managing the systemic hypotension observed in individuals with DMD. This avenue of investigation presents new prospects for improving clinical outcomes for these patients.

5-Methoxytryptophan alleviates atrial structural remodeling in ibrutinib-associated atrial fibrillation

BackgroundIbrutinib is an effective and well-tolerated treatment for B-cell lymphomas but is associated with an increased risk of atrial fibrillation (AF) by altering the structure of the atrium. 5-Methoxytryptophan (5-MTP) inhibits inflammatory and fibrotic processes. This study aimed to determine the effects and mechanisms of 5-MTP on the underlying mechanisms of AF caused by ibrutinib. MethodsThe effect of 5-MTP on ibrutinib-related AF was investigated in male Sprague Dawley rats using echocardiographic, electrophysiological, immunofluorescent, Masson staining, and molecular analyses. RusultsThe ibrutinib+5-MTP group showed (1) a lower incidence and shorter duration of AF and accelerated atrial conduction; (2) a decreased left atrial mass and left atrial diameter; (3) decreased myocardial fibrosis in the left atrium; (4) lower atrial inflammation; (5) increased sarcoplasmic reticulum Ca2+-ATPase 2a protein expression, decreased phosphorylation of phospholamban at Thr17, and decreased sodium/calcium exchanger 1 protein expression and phosphorylation of ryanodine receptor 2 at S2814; and (6) decreased phosphorylation of CaMKII expression. 5-MTP treatment markedly activated the PI3K-Akt signaling. Inhibiting PI3K-Akt signaling significantly reversed the protective effect of 5-MTP on ibrutinib-related AF. ConclusionsThese findings suggest that 5-MTP administration decreases the vulnerability of ibrutinib-related AF mainly caused by ameliorated maladaptive left atrial remodeling and dysregulation of calcium handling proteins. Mechanistically, 5-MTP treatment markedly enhanced the activation of cardiac PI3K-Akt signaling.

Molecular characterization and expression patterns of a ryanodine receptor in soybean looper, Chrysodeixis includens.

Diamide insecticides, such as chlorantraniliprole, have been widely used to control insect pests by targeting the insect ryanodine receptor (RyR). Due to the efficacious insecticidal activity of diamides, as well as an increasing number of resistance cases, the molecular structure of RyR has been studied in many economically important insects. However, no research has been conducted on diamide resistance and RyR in the soybean looper, Chrysodeixis includens, a significant crop pest. In this study, we found moderate resistance to chlorantraniliprole in a field population from Puerto Rico and sequenced the full-length cDNA of the C. includens RyR gene, which encodes a 5124 amino acid-long protein. Genomic analysis revealed that the CincRyR gene consists of 113 exons, one of the largest exon numbers reported for RyR. Alternative splicing sites were detected in the cytosolic region. The protein sequence showed high similarity to other noctuid RyRs. Conserved structural features included the selectivity filter motif critical for ryanodine binding and ion conduction, as well as various domains involved in ion transport. Two mutation sites associated with diamide resistance in other insects were screened but not found in the Puerto Rico field populations or in the susceptible lab strain. Gene expression analysis indicated high expression of RyR in the third instar larval stage, particularly in muscle-containing tissues. Furthermore, exposure to a sublethal dose of chlorantraniliprole reduced RyR expression levels after 96 h. This study provides a molecular basis for understanding RyR structure and sheds light on potential mechanisms of diamide resistance in C. includens.

Effects of Triiodothyronine Treatment in an Animal Model of Heart Failure with Preserved Ejection Fraction.

Background: Low levels of triiodothyronine (T3) are common in patients with heart failure (HF). Our aim was to evaluate the effects of supplementation with low and replacement doses of T3 in an animal model of HF with preserved ejection fraction (HFpEF). Methods: We evaluated four groups: ZSF1 Lean (n = 8, Lean-Ctrl), ZSF1 Obese (rat model of metabolic-induced HFpEF, n = 13, HFpEF), ZSF1 Obese treated with a replacement dose of T3 (n = 8, HFpEF-T3high), and ZSF1 Obese treated with a low-dose of T3 (n = 8, HFpEF-T3low). T3 was administered in drinking water from weeks 13 to 24. The animals underwent anthropometric and metabolic assessments, echocardiography, and peak effort testing with maximum O2 consumption (VO2max) determination at 22 weeks, and a terminal hemodynamic evaluation at 24 weeks. Afterwhile myocardial samples were collected for single cardiomyocyte evaluation and molecular studies. Results: HFpEF animals showed lower serum and myocardial thyroid hormone levels than Lean-Ctrl. Treatment with T3 did not normalize serum T3 levels, but increased myocardial T3 levels to normal levels in the HFpEF-T3high group. Body weight was significantly decreased in both the T3-treated groups, comparing with HFpEF. An improvement in glucose metabolism was observed only in HFpEF-T3high. Both the treated groups had improved diastolic and systolic function in vivo, as well as improved Ca2+ transients and sarcomere shortening and relaxation in vitro. Comparing with HFpEF animals, HFpEF-T3high had increased heart rate and a higher rate of premature ventricular contractions. Animals treated with T3 had higher myocardial expression of calcium transporter ryanodine receptor 2 (RYR2) and α-myosin heavy chain (MHC), with a lower expression of β-MHC. VO2max was not influenced by treatment with T3. Myocardial fibrosis was reduced in both the treated groups. Three animals died in the HFpEF-T3high group. Conclusions: Treatment with T3 was shown to improve metabolic profile, myocardial calcium handling, and cardiac function. While the low dose was well-tolerated and safe, the replacement dose was associated with increased heart rate, and increased risk of arrhythmias and sudden death. Modulation of thyroid hormones may be a potential therapeutic target in HFpEF; however, it is important to take into account the narrow therapeutic window of T3 in this condition.

"Dirty Dancing" of Calcium and Autophagy in Alzheimer's Disease.

Alzheimer's disease (AD) is the most common cause of dementia. There is a growing body of evidence that dysregulation in neuronal calcium (Ca2+) signaling plays a major role in the initiation of AD pathogenesis. In particular, it is well established that Ryanodine receptor (RyanR) expression levels are increased in AD neurons and Ca2+ release via RyanRs is augmented in AD neurons. Autophagy is important for removing unnecessary or dysfunctional components and long-lived protein aggregates, and autophagy impairment in AD neurons has been extensively reported. In this review we discuss recent results that suggest a causal link between intracellular Ca2+ signaling and lysosomal/autophagic dysregulation. These new results offer novel mechanistic insight into AD pathogenesis and may potentially lead to identification of novel therapeutic targets for treating AD and possibly other neurodegenerative disorders.

PO-01-244 BMAL1 REGULATES THE CIRCADIAN RHYTHM OF RYR2 AND CONTRIBUTES TO CIRCADIAN VENTRICULAR ARRHYTHMIA IN CHRONIC HEART FAILURE AFTER MYOCARDIAL INFARCTION

Ventricular arrhythmia (VAs) is the major cause of sudden cardiac death in chronic heart failure (CHF) after myocardial infarction (MI), and previous studies have reported that VAs exist circadian oscillation in CHF. Peripheral circadian clock can regulate expression and function of ion channels in cardiomyocytes, which is associated with circadian rhythm of VAs in CHF. However, the regulation of peripheral circadian clock on Ryanodine Receptor 2 (RYR2), a calcium channel on cardiac sarcoplasmic reticulum, remain to be fully understood. We aimed to explore the regulation of peripheral circadian clock on RYR2 and VA events in CHF. CHF was induced by ligation of coronary artery of mice, and RT-PCR were performed to investigate the circadian rhythm of RYR2 in CHF myocardial tissue. Amplitude of calcium transient of ISO-induced cardiomyocytes at different time was measured to study the circadian rhythm of RYR2 function. To confirm the circadian oscillation of VAs, VAs incidences at different time of CHF wild type mice and cardiomyocyte-specific Bmal1 knockout (CKO) mice were investigated by programmed electrical stimulation. Infections of Ad-CLOCK and/or Ad-BMAL1 were applied to overexpress CLOCK, BMAL1 and CLOCK-BMAL1 in cardiomyocytes, and expression of RYR2 was measured by Western blot. Chromatin immunoprecipitation assay (ChIP) and luciferase (LUC) were applied to determine whether CLOCK-BMAL1 transcriptionally regulate RYR2 expression. CLOCK, BMAL1, and RYR2 exhibited notable circadian rhythm in CHF mice (Figure A, P<0.05). Amplitude of calcium transient at CT36 was lower than CT24 (Figure B, P<0.05). Compared to CT36, VAs had higher incidence at CT24(Figure C, P<0.05), and incidences of VAs had no significant difference between CT24 and CT36 in CKO Mice or using Dantrolene, an antagonist of RYR2. Ad-CLOCK and Ad-BMAL1 co-infection resulted in overexpression of RYR2 (Figure D, P<0.05), and BMAL1 bound to the enhancer of RYR2 gene and upregulated RYR2 expression transcriptionally (Figure E). In conclusion, BMAL1, as peripheral circadian clock, could contribute to circadian oscillation of VAs in CHF by regulating the circadian rhythm of RYR2.

Human ApoE2 Endows Stronger Contractility in Rat Cardiomyocytes Enhancing Heart Function

(1) Background: Apolipoprotein E (ApoE) is a critical plasma apolipoprotein for lipid transport and nonlipid-related functions. Humans possess three isoforms of ApoE (2, 3, and 4). ApoE2, which exhibits beneficial effects on cardiac health, has not been adequately studied. (2) Methods: We investigated the cardiac phenotypes of the humanized ApoE knock-in (hApoE KI) rats and compared to wild-type (WT) and ApoE knock-out (ApoE KO) rats using echocardiography, ultrasound, blood pressure measurements, histology strategies, cell culture, Seahorse XF, cardiomyocyte contractility and intracellular Ca2+ tests, and Western blotting; (3) Results: hApoE2 rats exhibited enhanced heart contractile function without signs of detrimental remodeling. Isolated adult hApoE2 cardiomyocytes had faster and stronger sarcomere contractility because of more mitochondrial energy generation and stimulation-induced fast and elevated intracellular Ca2+ transient. The abundant energy is a result of elevated mitochondrial function via fatty acid β-oxidation. The fast and elevated Ca2+ transient is associated with decreased sarcoplasmic reticulum (SR) Ca2+ ATPase (SERCA2) and increased expression of cardiac ryanodine receptor 2 (RyR2) conducting a potent Ca2+ release from SR.; (4) Conclusions: Our studies validated the association of polymorphic ApoEs with cardiac health in the rat model, and revealed the possible mechanisms of the protective effect of ApoE2 against heart diseases.

High oxygen-modified packaging (HiOx-MAP) mediates HIF-1α regulation of tenderness changes during postmortem aging of yak meat

In the present study, we studied the effect of high oxygen-modified packaging (HiOx-MAP) on yak meat tenderness and the underlying mechanism. HiOx-MAP significantly increased the myofibril fragmentation index (MFI) of yak meat. In addition, western blotting showed that the expression of hypoxia-inducible factor (HIF-1α) and ryanodine receptors (RyR) in the HiOx-MAP group was reduced. HiOx-MAP increased the activity of sarcoplasmic reticulum calcium-ATPase (SERCA). The energy disperse spectroscopy (EDS) mapping showed gradually reduced calcium distribution in the treated endoplasmic reticulum. Furthermore, HiOx-MAP treatment increased the caspase-3 activity and the apoptosis rate. The activity of calmodulin protein (CaMKKβ) and AMP-activated protein kinase (AMPK) was down-regulated leading to apoptosis. These results indicated that HiOx-MAP promoted apoptosis during postmortem aging to improve the tenderization of meat.

Cholecalciferol affects cardiac proteins regulating malonyl-CoA availability and intracellular calcium level.

Cholecalciferol improves insulin signaling and glucose metabolism in the heart and reduces circulating non-esterified fatty acids. Cholecalciferol effects on the cardiac fatty acid (FA) metabolism and the consequences on calcium handling were examined. Blood lipid profile was determined. Western blot and qRT-PCR were used to examine protein and mRNA expression. Cholecalciferoltreated rats had increased acetyl CoA carboxylase 2 protein expression and decreased expression of malonyl CoA decarboxylase. In addition, the expression of uncoupling protein 3 was elevated. Also, the level of peroxisome proliferator-activated receptor-gamma coactivator in the nucleus of heart cells was increased along with the level of sarcoplasmic/endoplasmic reticulum Ca2+ATPase in the microsomal fraction. In parallel, the L-type calcium channel and ryanodine receptor expression was reduced. In the heart of healthy rats, cholecalciferol affects proteins regulating malonyl CoA availability and intracellular Ca2+ handling proteins.

Ryanodine receptor 2 promotes colorectal cancer metastasis by the ROS/BACH1 axis.

There is no targeted therapy for KRAS proto-oncogene, GTPase (KRAS)-mutant metastatic colorectal cancer (mCRC) because the underlying mechanism remains obscure. Based on bioinformatic analysis, this study aims to elucidate a potential gene target for which an approved drug is available, and to reveal the function as well as the underlying mechanism of the candidate gene. Here, we identified that ryanodine receptor 2 (RyR2) expression was upregulated in KRAS-mutant mCRC, and that this promoted cancer cell metastasis. S107, an approved drug to inhibit calcium release from RyR2 in the clinic, inhibited cancer cell metastasis both in vitro and in vivo. High expression of RyR2 predicts poor survival in our patient cohort. CRC patients with serosa invasion and vascular tumor thrombus are characterized by high RyR2 expression. Analysis of expression profiles upon RyR2 knockdown and inhibition, revealed a set of metastasis-related molecules, and identified BTB domain and CNC homolog 1 (BACH1) as the main transcription factor regulated by RyR2. RyR2 regulates cellular reactive oxygen species (ROS) levels, which activates nuclear factor erythroid 2-related factor 2 (Nrf2; also known as NFE2L2) and HMOX1 expression, and thus BACH1 accumulation. Collectively, this study provides evidence that the RyR2/ROS/BACH1 axis may be a potential intervention target for CRC metastasis.

Effect of unconventional feeds on production cost, growth performance and expression of quantitative genes in growing pigs

This study examined the effects of unconventional feed on performance characteristics and candi-date gene expression in growing pigs of 3-5months of age. A Total of forty-five (45) growing pigs were randomly allotted into five treatments consisting of whole maize (T1), brewer’s dried grain (T2), cassava peel meal (T3), plantain peel meal (T4), and corn husk meal (T5), at 35% inclusion rate. Per-formance data were collected for 12weeks, while duodenum and liver samples were collected after the experiment for gene expression analysis. Data were analyzed in a completely randomized design. Diets significantly (p&lt; 0.05) affected growth performance with treatment 2 recording the highest average daily weight gain of 0.21±0.02 whilecorn husk meal (T5)had the least 0.11±0.01. Feed efficiency was highest for whole maize (T1) and brewer’s dried grain (T2) (0.10±0.02) and lowest for t5 (0.07±0.17). Cost-benefit analysis were significantly different (p&lt;0.05) with t4 recording the highest gross margin. Insulin-like growth factor1 and leptin gene were differentially expressed in tissues, while no significant differences (p&gt;0.05) existed for growth hormone gene and ryanodine receptor1 gene. The results showed commendable gross margin, however, cognizance should be taken in the quantity of brewer’s dried grain fed to pigs since it increased the expression of ryanodine receptor 1 gene that leads to por-cine malignant hypertermia and pale soft exudates pork.

Dissecting the Component Parts of the Excitation‐Contraction Coupling Machinery in Drosophila melanogaster

The Drosophila neuromuscular system is widely used to characterize synaptic development and function. However, little is known about how specific synaptic and muscular alterations effect neuromuscular transduction and muscle contractility that ultimately dictate behavioral output. Here a system developed to characterize excitation‐contraction coupling at Drosophila larval neuromuscular junctions (NMJs) was exploited to examining how specific neuronal, synaptic, and muscle manipulations disrupt muscle contractility and locomotion. Increases in motor neuron stimulation frequency and duration increased muscle force in a predictable fashion, but show considerable plasticity between 5–40 Hz and saturating above 50 Hz. Fictive contraction recordings from larvae with CNS intact revealed motor neuron burst frequencies of 20‐30 Hz, consistent with the endogenous machinery operating within the range of contractility of greatest plasticity. Experimental parameters such as saline composition (e.g. calcium and magnesium) temperature, larval size and orientation, were explored to examine their impacts on muscle force production. Subsequently, genetic and pharmacological approaches were taken to dissect several molecular pathways which contribute to muscle contraction and modulation of force. First, well‐established calcium regulation pathways were manipulated via application of ryanodine, caffeine, thapsigargin, and felodipine to demonstrate the role of the ryanodine receptors, SERCA pump, and postsynaptic calcium channels in modulating the magnitude and time‐course of muscle contractions. Presynaptic calcium channels and ryanodine receptor expression were genetically altered to further examine their role in muscle force production. Next using both null and knock‐down (Gal4/UAS system) approaches combined with tissue‐specific expression of drivers, screens for motor, synaptic, and muscle proteins contributing to excitation‐contraction coupling (ECC) were conducted. Altering the expression of the motor proteins kinesin, dynein or dynactin all resulted in a significant reduction in muscle force production. Critical synaptic proteins like synaptotagmin, complexin, cacophony, gbb (homolog of BMP), significantly impacted ECC in Drosophila larvae. Several critical muscle‐specific proteins alter the magnitude and time‐course of muscle contractions when altering their expression in a sub‐set of muscle fibers. Lastly a screen for modulators of muscle contractility led to identification and characterization of the molecular and cellular pathway by which the FMRFa peptide, TPAEDFMRFa, increases muscle performance. These findings indicate Drosophila NMJs provide a robust system to correlate synaptic dysfunction, regulation, and modulation, to alterations in excitation‐contraction coupling.

Preserved cardiac performance and adrenergic response in a rabbit model with decreased ryanodine receptor 2 expression

Ryanodine receptor 2 (RyR2) is an ion channel in the heart responsible for releasing into the cytosol most of the Ca2+ required for contraction. Proper regulation of RyR2 is critical, as highlighted by the association between channel dysfunction and cardiac arrhythmia. Lower RyR2 expression is also observed in some forms of heart disease; however, there is limited information on the impact of this change on excitation-contraction (e-c) coupling, Ca2+-dependent arrhythmias, and cardiac performance. We used a constitutive knock-out of RyR2 in rabbits (RyR2-KO) to assess the extent to which a stable decrease in RyR2 expression modulates Ca2+ handling in the heart.We found that homozygous knock-out of RyR2 in rabbits is embryonic lethal. Remarkably, heterozygotes (KO+/−) show ~50% loss of RyR2 protein without developing an overt phenotype at the intact animal and whole heart levels. Instead, we found that KO+/− myocytes show (1) remodeling of RyR2 clusters, favoring smaller groups in which channels are more densely arranged; (2) lower Ca2+ spark frequency and amplitude; (3) slower rate of Ca2+ release and mild but significant desynchronization of the Ca2+ transient; and (4) a significant decrease in the basal phosphorylation of S2031, likely due to increased association between RyR2 and PP2A.Our data show that RyR2 deficiency, although remarkable at the molecular and subcellular level, has only a modest impact on global Ca2+ release and is fully compensated at the whole-heart level. This highlights the redundancy of RyR2 protein expression and the plasticity of the e-c coupling apparatus.

Interleukin-6-Mediated-Ca2+ Handling Abnormalities Contributes to Atrial Fibrillation in Sterile Pericarditis Rats.

Pre-existing Ca2+ handling abnormalities constitute the arrhythmogenic substrate in patients developing postoperative atrial fibrillation (POAF), a common complication after cardiac surgery. Postoperative interleukin (IL)-6 levels are associated with atrial fibrosis in several animal models of POAF, contributing to atrial arrhythmias. Here, we hypothesize that IL-6-mediated-Ca2+ handling abnormalities contribute to atrial fibrillation (AF) in sterile pericarditis (SP) rats, an animal model of POAF. SP was induced in rats by dusting atria with sterile talcum powder. Anti-rat-IL-6 antibody (16.7 μg/kg) was administered intraperitoneally at 30 min after the recovery of anesthesia. In vivo electrophysiology, ex vivo optical mapping, western blots, and immunohistochemistry were performed to elucidate mechanisms of AF susceptibility. IL-6 neutralization ameliorated atrial inflammation and fibrosis, as well as AF susceptibility in vivo and the frequency of atrial ectopy and AF with a reentrant pattern in SP rats ex vivo. IL-6 neutralization reversed the prolongation and regional heterogeneity of Ca2+ transient duration, relieved alternans, reduced the incidence of discordant alternans, and prevented the reduction and regional heterogeneity of the recovery ratio of Ca2+ transient. In agreement, western blots showed that IL-6 neutralization reversed the reduction in the expression of ryanodine receptor 2 (RyR2) and phosphorylated phospholamban. Acute IL-6 administration to isolated rat hearts recapitulated partial Ca2+ handling phenotype in SP rats. In addition, intraperitoneal IL-6 administration to rats increased AF susceptibility, independent of fibrosis. Our results reveal that IL-6-mediated-Ca2+ handling abnormalities in SP rats, especially RyR2-dysfunction, independent of IL-6-induced-fibrosis, early contribute to the development of POAF by increasing propensity for arrhythmogenic alternans.

14 Telomerase and myocardin co-expressing mesenchymal cells increase survival and induce cardiac and vascular markers in cardiac stromal cells undergoing simulated ischemia/reperfusion

Abstract Aims Cardiac stromal cells (CSCs) contain a pool of cells with supportive and paracrine functions. Various types of mesenchymal stromal cells (MSCs) can influence CSCs in the cardiac niche through their paracrine activity. Ischemia/reperfusion (I/R) leads to cell death and reduction of the paracrine activity of CSCs. The forced coexpression of telomerase reverse transcriptase (TERT) and myocardin (MYOCD), known to potentiate anti-apoptotic, pro-survival and pro-angiogenic activities of MSCs isolated from the adipose tissue (AT-MSCs), may increase CSC survival, favouring their paracrine activities. To investigate the hypothesis that CSCs feature improved resistance to simulated I/R (SI/R) and increased commitment toward the cardiovascular lineage when preconditioned with conditioned media (CM) or extracellular vesicles (EV) released from AT-MSCs overexpressing TERT and MYOCD (T/M AT-MSCs). Methods Murine CSCs were isolated with the cardiosphere (CSps) isolation technique. Results T/M AT-MSCs and their secretome improved spontaneous intracellular calcium changes and ryanodine receptor expression in aged CSps. The cytoprotective effect of AT-MSCs was tested in CSCs subjected to SI/R. SI/R induced cell death as compared to normoxia (28 ± 4 vs. 10 ± 3%, P = 0.02). Pre-treatment with CM (15 ± 2, P = 0.02) or with the EV-enriched fraction (10 ± 1%, P = 0.02) obtained from mock-transduced AT-MSCs in normoxia reduced cell death after SI/R. The effect was more pronounced with CM (7 ± 1%, P = 0.01) or the EV-enriched fraction (2 ± 1%, P = 0.01) obtained from T/M AT-MSCs subjected to SI/R. In parallel, we observed lower expression of the apoptosis marker cleaved caspase-3 and higher expression of cardiac and vascular markers eNOS, sarcomeric α-actinin and cardiac actin. Conclusions The T/M AT-MSCs secretome exerts a cytoprotective effect and promotes development of CSCs undergoing SI/R towards a cardiovascular phenotype.

Sulforaphane Effects on Cardiac Function and Calcium-Handling-Related Proteins in 2 Experimental Models of Heart Disease: Ischemia-Reperfusion and Infarction.

Sulforaphane (SFN) is a natural exogenous antioxidant from cruciferous vegetables already shown to improve cardiac function in cardiovascular diseases. The aim of this study was to analyze the effect of SFN treatment on the cardiac function in 2 experimental models of heart disease, ischemia/reperfusion (I/R) and myocardial infarction (MI), and whether an improvement of the cardiac function could be associated with a modulation of calcium-handling proteins. The study was divided into 2 main experiments: experiment 1, ex vivo with the I/R model and experiment 2, in vivo with the MI model. In the I/R model, rats were divided into control and SFN (0.5 mg/kg/d intraperitoneally for 3 days) groups, and the hearts were submitted to global ischemia (20 minutes) followed by reperfusion (20 minutes) in a Langendorff apparatus. SFN did not change left ventricle systolic and diastolic pressures but increased the contractility and relaxation indexes after 20 minutes of reperfusion. These functional changes were accompanied by a decreased protein expression of ryanodine receptor (RyR) and increased expression of p-phospholamban/phospholamban ratio, without alteration in the sarco/endoplasmic calcium ATPase expression. In the MI model, rats were randomly divided into Sham, MI (MI induced by left coronary artery ligation), Sham + SFN (5 mg/kg/d intraperitoneally for 25 days), and MI + SFN groups. Although SFN did not affect cardiac function, it led to a decreased RyR protein expression and reactive oxygen species levels in the left ventricular of the MI + SFN group. These data indicate that SFN modulates calcium-handling proteins and, thus, cardiac inotropism/lusitropism especially when administered previously to an ischemic event.

Abstract 11237: Expression of Mitochondrial, Contractile, and Calcium Regulatory Proteins is Altered in Patients with Atrial Fibrillation

Introduction: Atrial fibrillation (AF) increases energy demand for contractile and electrical activity. Changes in left atrial (LA) protein expression of AF patients are poorly characterized. Hypothesis: Mitochondrial protein expression in patients with AF is altered in an attempt to meet increased energy demand. Methods: LA appendage tissue was obtained from 198 patients undergoing Maze surgery. At the time of surgery, 80 were in sinus rhythm (SR) (50 paroxysmal AF, 30 persistent AF) and 118 in AF (65 persistent AF, 53 permanent AF). Protein content was assessed by mass spectrometry and 2539 proteins were identified. Results: In AF compared to SR, 257 proteins were differentially expressed (q&lt;0.05); 44 of 62 mitochondrial proteins detected (MitoCarta 3.0) were increased. KEGG pathway analysis revealed Oxidative Phosphorylation was increased (p=1.01E-3) in AF, including 17 subunits of the electron transport chain (A). The Hypertrophic Cardiomyopathy pathway was decreased (p=1.01E-03). Expression of ryanodine receptor, and troponin and tropomyosin subunits decreased, but tropomyosin 4 and myosin heavy chain 9 and 10 increased (B), providing evidence of changes in myofibrillar and calcium regulatory proteins in AF. Among 39 putative AF risk genes detectable at the protein level, 8 were altered in AF (C). The Tricarboxylic Acid Cycle KEGG pathway was decreased (p=2.55E-3) in patients with permanent compared to persistent AF, with no significant changes in other cellular pathways or protein expression of putative AF risk genes. Conclusions: In one of the largest proteomic datasets in human LA to date, we find that expression of proteins in metabolic, myofibrillar, and calcium regulation pathways is altered in patients with AF. Additional metabolic changes were detected with progression to permanent AF. These data identify proteins that are altered in patients with AF providing insight into cellular pathways that may be targeted for AF prevention and therapy.

Arsenite impinges on endoplasmic reticulum-mitochondria crosstalk to elicit mitochondrial ROS formation and downstream toxicity.

Arsenite is an important carcinogen and toxic compound, causing various deleterious effects through multiple mechanisms. In this review, we focused on mitochondrial ROS (mitoROS) and discussed on the mechanisms mediating their formation. The metalloid promotes direct effects in mitochondria, resulting in superoxide formation only under conditions of increased mitochondrial Ca2+ concentration ([Ca2+]m). In this perspective, the time of exposure and concentration requirements for arsenite were largely conditioned by other effects of the metalloid in specific sites of the endoplasmic reticulum (ER). Arsenite induced a slow and limited mobilization of Ca2+ from IP3R via a saturable mechanism, failing to increase the [Ca2+]m. This effect was however associated with the triggering of an intraluminal crosstalk between the IP3R and the ryanodine receptor (RyR), causing a large and concentration dependent release of Ca2+ from RyR and a parallel increase in [Ca2+]m. Thus, the Ca2+-dependent mitoO2-. formation appears to be conditioned by the spatial/functional organization of the ER/mitochondria network and RyR expression. We also speculate on the possibility that the ER stress response might regulate the above effects on the intraluminal crosstalk between the IP3R and the RyR via oxidation of critical thiols mediated by the H2O2 locally released by oxidoreductin 1α.