Poly (ADP-ribose) polymerase pathway inhibitor (Olaparib) upregulates SERCA2a expression and attenuates doxorubicin-induced cardiomyopathy in mice.

The cardiotoxicity induced by doxorubicin is dose-dependent. The present study tested the potential cardioprotective effect of Poly ADP Ribose Polymerase (PARP) pathway inhibitor "olaparib" in a mouse model of doxorubicin-induced cardiomyopathy (DOX-CM). Seventy-two male BALB/c mice were randomized into six equal groups; control, DOX-CM, dexrazoxane-treated, and three olaparib-treated groups (5, 10, and 50 mg/kg/day). Cardiomyopathy was assessed by heart weight/Tibial length (HW/TL) ratio, cardiac fibrosis, oxidative stress, and electron microscope. Myocardial expression of SERCA2a mRNA and cleaved PARP-1 protein were also assessed. Similar to dexrazoxane, olaparib (10 mg/kg/day) significantly ameliorated oxidative stress, and preserved cardiac structure. It also suppressed myocardial PARP-1 protein expression and boosted SERCA2a mRNA expression. Olaparib (5 or 50 mg/kg/day) failed to show comparable effects. The current study detected the cardioprotective effect of olaparib at a dosage of 10 mg/kg/day. Also, the present study discovered a new cardioprotective mechanism of dexrazoxane by targeting PARP-1 in the heart.

Therapeutic targeting of organelles for inhibition of Zika virus replication in neurons.

Zika virus (ZIKV) is an arbovirus belonging to the family Flaviviridae. Since 2015, ZIKV infection has emerged as a leading cause of virus-induced placental insufficiency, microcephaly and other neuronal complications. Currently, no therapeutics have been approved to treat ZIKV infection. In this study, we examined how targeted inhibition of cellular organelles or trafficking processes affected ZIKV infection and replication in neural progenitor cells. We found that blocking endocytosis, Golgi function or structural filaments like actin or microtubules had moderate effects on virus replication. However, inducing endoplasmic reticulum (ER) stress by treatment with Thapsigargin substantially inhibited virus production, suggesting the ER might be a candidate cellular target. Further analysis showed that sarcoplasmic/endoplasmic reticulum Ca2+-ATPases (SERCA) was important for ZIKV inhibition. Collectively, these studies indicate that targeting the SERCA-dependent ER stress pathway may be useful to develop antivirals to inhibit ZIKV replication.

A multiscale approach for bridging the gap between potency, efficacy, and safety of small molecules directed at membrane proteins.

Membrane proteins constitute a substantial fraction of the human proteome, thus representing a vast source of therapeutic drug targets. Indeed, newly devised technologies now allow targeting "undruggable" regions of membrane proteins to modulate protein function in the cell. Despite the advances in technology, the rapid translation of basic science discoveries into potential drug candidates targeting transmembrane protein domains remains challenging. We address this issue by harmonizing single molecule-based and ensemble-based atomistic simulations of ligand-membrane interactions with patient-derived induced pluripotent stem cell (iPSC)-based experiments to gain insights into drug delivery, cellular efficacy, and safety of molecules directed at membrane proteins. In this study, we interrogated the pharmacological activation of the cardiac Ca2+ pump (Sarcoplasmic reticulum Ca2+-ATPase, SERCA2a) in human iPSC-derived cardiac cells as a proof-of-concept model. The combined computational-experimental approach serves as a platform to explain the differences in the cell-based activity of candidates with similar functional profiles, thus streamlining the identification of drug-like candidates that directly target SERCA2a activation in human cardiac cells. Systematic cell-based studies further showed that a direct SERCA2a activator does not induce cardiotoxic pro-arrhythmogenic events in human cardiac cells, demonstrating that pharmacological stimulation of SERCA2a activity is a safe therapeutic approach targeting the heart. Overall, this novel multiscale platform encompasses organ-specific drug potency, efficacy, and safety, and opens new avenues to accelerate the bench-to-patient research aimed at designing effective therapies directed at membrane protein domains.

Cellular Mechanisms Underlying the Low Cardiotoxicity of Istaroxime.

Background Istaroxime is an inhibitor of Na+/K+ ATPase with proven efficacy to increase cardiac contractility and to accelerate relaxation attributable to a relief in phospholamban-dependent inhibition of the sarcoplasmic reticulum Ca2+ ATPase. We have previously shown that pharmacologic Na+/K+ ATPase inhibition promotes calcium/calmodulin-dependent kinase II activation, which mediates both cardiomyocyte death and arrhythmias. Here, we aim to compare the cardiotoxic effects promoted by classic pharmacologic Na+/K+ ATPase inhibition versus istaroxime. Methods and Results Ventricular cardiomyocytes were treated with ouabain or istaroxime at previously tested equi-inotropic concentrations to compare their impact on cell viability, apoptosis, and calcium/calmodulin-dependent kinase II activation. In contrast to ouabain, istaroxime neither promoted calcium/calmodulin-dependent kinase II activation nor cardiomyocyte death. In addition, we explored the differential behavior promoted by ouabain and istaroxime on spontaneous diastolic Ca2+ release. In rat cardiomyocytes, istaroxime did not significantly increase Ca2+ spark and wave frequency but increased the proportion of aborted Ca2+ waves. Further insight was provided by studying cardiomyocytes from mice that do not express phospholamban. In this model, the lower Ca2+ wave incidence observed with istaroxime remains present, suggesting that istaroxime-dependent relief on phospholamban-dependent sarcoplasmic reticulum Ca2+ ATPase 2A inhibition is not the unique mechanism underlying the low arrhythmogenic profile of this drug. Conclusions Our results indicate that, different from ouabain, istaroxime can reach a significant inotropic effect without leading to calcium/calmodulin-dependent kinase II-dependent cardiomyocyte death. Additionally, we provide novel insights regarding the low arrhythmogenic impact of istaroxime on cardiac Ca2+ handling.

Bay 60-7550, a PDE2 inhibitor, exerts positive inotropic effect of rat heart by increasing PKA-mediated phosphorylation of phospholamban.

This study investigated the hemodynamic effect of Bay 60-7550, a phosphodiesterase type 2 (PDE2) inhibitor, in healthy rat hearts both in vivo and ex vivo and its underlying mechanisms. In vivo rat left ventricular pressure-volume loop, Langendorff isolated rat heart, Ca2+ transient of left ventricular myocyte and Western blot experiments were used in this study. The results demonstrated that Bay 60-7550 (1.5 mg/kg, i. p.) increased the in vivo rat heart contractility by enhancing stroke work, cardiac output, stroke volume, end-diastolic volume, heart rate, and ejection fraction. The simultaneous aortic pressure recording indicated that the systolic blood pressure was increased and diastolic blood pressure was decreased by Bay 60-7550. Also, the arterial elastance which is proportional to the peripheral vessel resistance was significantly decreased. Bay 60-7550 (0.001, 0.01, 0.1, 1 µmol/l) also enhanced the left ventricular development pressure in non-paced and paced modes with a decrease of heart rate in non-paced model. Bay 60-7550 (1 µmol/l) increased SERCA2a activity and SR Ca2+ content and reduced SR Ca2+ leak rate. Furthermore, Bay 60-7550 (0.1 µmol/l) increased the phosphorylation of phospholamban at 16-serine without significantly changing the phosphorylation levels of phospholamban at 17-threonine and RyR2. Bay 60-7550 increased the rat heart contractility and reduced peripheral arterial resistance may be mediated by increasing the phosphorylation of phospholamban and dilating peripheral vessels. PDE2 inhibitors which result in a positive inotropic effect and a decrease in peripheral resistance might serve as a target for developing agents for the treatment of heart failure in clinical settings.

Chronic but not acute pharmacological activation of SERCA induces behavioral and neurochemical effects in male and female mice.

Intracellular calcium (Ca2+) homeostasis is a vital process to nerve cell survival and function with an intricate regulatory network. It is well established that the endoplasmic reticulum (ER) is a major intraneuronal Ca2+ storage and that the sarco/endoplasmic reticulum (SR/ER) calcium (Ca2+)-ATPase (SERCA) pump is a key regulator of cytosolic Ca2+ levels. SERCA pumps play a critical role in brain pathophysiology, thus SERCA comprises an emerging pharmacological target for the treatment of brain diseases. Interestingly, preclinical studies in rodents suggest that chronic pharmacological activation of SERCA2 by the quinoline derivative CDN1163 comprises a potential pharmacotherapeutic target in Alzheimer's and Parkinson's diseases. As little is known about the behavioral and neurochemical consequences of CDN1163 administration, in the current study we investigated the potential effects of acute (i.e., at 1 h) and chronic (i.e., 17 days) CDN1163 administration (i.e., 10 mg/kg and 20 mg/kg; intraperitoneally) on locomotor activity and relevant affective behaviors, as well as on monoaminergic neurotransmission in naïve C57BL/6J mice of both sexes. Interestingly, chronic, but not acute, CDN1163 administration induced anxiogenic and depressive-like behavioral effects in mice, as assessed in the open field (OF) test and the forced swim test (FST), respectively. In addition, chronic CDN1163 administration induced sustained sex- and brain region-dependent noradrenergic and serotonergic neurochemical effects ex vivo. Taken together, present findings support the critical role of SERCA-dependent Ca2+ handling in regulating behavior and neurochemical activity, and further highlight the need to consider sex in the development of SERCA-targeting pharmacotherapies for the treatment of debilitating brain disorders.

Phospholamban and sarcolipin prevent thermal inactivation of sarco(endo)plasmic reticulum Ca2+-ATPases.

Na+-K+-ATPase from mice lacking the γ subunit exhibits decreased thermal stability. Phospholamban (PLN) and sarcolipin (SLN) are small homologous proteins that regulate sarco(endo)plasmic reticulum Ca2+-ATPases (SERCAs) with properties similar to the γ subunit, through physical interactions with SERCAs. Here, we tested the hypothesis that PLN and SLN may protect against thermal inactivation of SERCAs. HEK-293 cells were co-transfected with different combinations of cDNAs encoding SERCA2a, PLN, a PLN mutant (N34A) that cannot bind to SERCA2a, and SLN. One-half of the cells were heat stressed at 40°C for 1 h (HS), and one-half were maintained at 37°C (CTL) before harvesting the cells and isolating microsomes. Compared with CTL, maximal SERCA activity was reduced by 25-35% following HS in cells that expressed either SERCA2a alone or SERCA2a and mutant PLN (N34A) whereas no change in maximal SERCA2a activity was observed in cells that co-expressed SERCA2a and either PLN or SLN following HS. Increases in SERCA2a carbonyl group content and nitrotyrosine levels that were detected following HS in cells that expressed SERCA2a alone were prevented in cells co-expressing SERCA2a with PLN or SLN, whereas co-expression of SERCA2a with mutant PLN (N34A) only prevented carbonyl group formation. In other experiments using knock-out mice, we found that thermal inactivation of SERCA was increased in cardiac left ventricle samples from Pln-null mice and in diaphragm samples from Sln-null mice, compared with WT littermates. Our results show that both PLN and SLN form a protective interaction with SERCA pumps during HS, preventing nitrosylation and oxidation of SERCA and thus preserving its maximal activity.

Assessment of Na+/K+ ATPase Activity in Small Rodent and Human Skeletal Muscle Samples.

INTRODUCTION: In skeletal muscle, the Na/K ATPase (NKA) plays essential roles in processes linked to muscle contraction, fatigue, and energy metabolism; however, very little information exists regarding the regulation of NKA activity. The scarcity of information regarding NKA function in skeletal muscle likely stems from methodological constraints, as NKA contributes minimally to total cellular ATP utilization, and therefore contamination from other ATPases prevents the assessment of NKA activity in muscle homogenates. Here we introduce a method that improves accuracy and feasibility for the determination of NKA activity in small rodent muscle samples (5-10 mg) and in human skeletal muscle. METHODS: Skeletal muscle homogenates from mice (n = 6) and humans (n = 3) were used to measure NKA and sarcoplasmic reticulum Ca ATPase (SERCA) activities with the addition of specific ATPase inhibitors to minimize "background noise." RESULTS: We observed that myosin ATPase activity was the major interfering factor for estimation of NKA activity in skeletal muscle homogenates, as the addition of 25 µM of blebbistatin, a specific myosin ATPase inhibitor, considerably minimized "background noise" (threefold) and enabled the determination of NKA maximal activity with values three times higher than previously reported. The specificity of the assay was demonstrated after the addition of 2 mM ouabain, which completely inhibited NKA. On the other hand, the addition of blebbistatin did not affect the ability to measure SERCA function. The coefficient of variation for NKA and SERCA assays were 6.2% and 4.4%, respectively. CONCLUSION: The present study has improved the methodology to determine NKA activity. We further show the feasibility of measuring NKA and SERCA activities from a common muscle homogenate. This methodology is expected to aid in our long-term understanding of how NKA affects skeletal muscle metabolic homeostasis and contractile function in diverse situations.

High glucose induced calcium overload via impairment of SERCA/PLN pathway and mitochondrial dysfunction leads to oxidative stress in H9c2 cells and amelioration with ferulic acid.

Oxidative stress and associated complications are the major pathological concerns of diabetic cardiomyopathy (DC). We aim to elucidate the mechanisms by which high glucose (HG) induced alteration in calcium homeostasis and evaluation of the beneficial effect of two concentrations (10 and 25 µm) of ferulic acid (FA). HG was induced in H9c2 cardiomyoblast by treating with glucose (33 mm) for 48 h, and FA was co-treated. Intracellular calcium ([Ca2+ ]i) overload was found increased significantly with HG. For elucidation of mechanism, the SERCA pathway and mitochondrial integrity (transmembrane potential and permeability transition pore) were explored. Then, we assessed oxidative stress, and cell injury with brain natriuretic peptide (BNP), atrial natriuretic peptide (ANP), and lactate dehydrogenase (LDH) release. HG caused significant [Ca2+ ]i overload through downregulation of SERCA2/1, pPLN, and pPKA C-α; and upregulation of PLN and PKA C-α and alteration in the integrity of mitochondria with HG. The [Ca2+ ]i overload in turn caused oxidative stress via generation of reactive oxygen species, lipid peroxidation, and protein carbonylation. This resulted in cell injury which was evident with significant release of BNP, ANP, and LDH. FA co-treatment was effective to mitigate all pathological changes caused by HG. From the overall results, we conclude that [Ca2+ ]i overload via SERCA pathway and altered mitochondrial integrity is the main cause for oxidative stress during HG. Based on our result, we report that FA could be an attractive nutraceutical for DC.

Effects of propofol pretreatment on myocardial cell apoptosis and SERCA2 expression in rats with hepatic ischemia/reperfusion / Efeitos do pré-tratamento com propofol sobre a apoptose de células miocárdicas e expressão de SERCA2 em ratos com isquemia/reperfusão hepática

Abstract Introduction: Hepatic ischemia-reperfusion injury is a common pathophysiological process in liver surgery. Whether Propofol can reduce myocardial ischemia-reperfusion injury induced by hepatic ischemia-reperfusion injury in rats, together with related mechanisms, still needs further studies. Objective: To investigate if propofol would protect the myocardial cells from apoptosis with hepatic ischemia-reperfusion injury. Methods: Male Sprague-Dawley rats (n = 18) were randomly allocated into three groups: Sham Group (Group S, n = 6), Hepatic Ischemia-reperfusion Injury Group (Group IR, n = 6) and Propofol Group (Group P, n = 6). Group S was only subjected to laparotomy. Group IR was attained by ischemia for 30 min and reperfusion for 4 h. Group P was subjected identical insult as in Group IR with the administration of propofol started 10 min before ischemia with 120 mg.kg−1, following by continuous infusion at 20 mg.kg−1.h−1. Cell apoptosis was examined by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay. Endoplasmic reticulum Ca2+-ATPase2 (SERCA2) and cysteine-containing aspartic acid cleaved-caspase3 (cleaved-caspase3) were assayed by western blot and Altimeter polymerase chain reaction. Results: Apoptosis rate was increased, with mRNA and protein of SERCA2 down-regulated and cleaved-caspase3 up-regulated in Group IR compared with Group S (p < 0.01). Apoptosis rate was decreased, with mRNA and protein of SERCA2 up-regulated and cleaved-caspase3 down-regulated in Group P compared with Group IR (p < 0.01). Conclusions: Propofol can reduce hepatic ischemia-reperfusion injury-induced myocardial cell apoptosis, meanwhile, can up-regulate mRNA and protein of SERCA2 in rats.

Resumo Introdução: A lesão hepática por isquemia-reperfusão é um processo fisiopatológico comum em cirurgias hepáticas. Mais estudos ainda são necessários para avaliar se o propofol pode reduzir a lesão de isquemia-reperfusão miocárdica induzida pela lesão de isquemia-reperfusão hepática em ratos, juntamente com os mecanismos que estão relacionados. Objetivo: Investigar se propofol protege as células do miocárdio da apoptose com a lesão hepática por isquemia-reperfusão. Métodos: Ratos machos da raça Sprague-Dawley (n = 18) foram alocados aleatoriamente em três grupos: Grupo Sham (Grupo S, n = 6), Grupo Lesão Hepática por Isquemia-reperfusão (Grupo IR, n = 6) e Grupo Propofol (Grupo P, n = 6). O Grupo S foi submetido apenas à laparotomia. O grupo IR foi submetido à isquemia por 30 min e reperfusão por 4 h. O grupo P foi submetido à mesma isquemia do grupo IR, com a administração de 120 mg.kg-1 de propofol iniciada 10min antes da isquemia, seguida de infusão contínua a 20 mg.kg-1.h-1. A apoptose celular foi examinada por meio do ensaio de marcação de terminações dUTP pela deoxinucleotidil transferase. Retículo endoplasmático Ca2+-ATPase2 (SERCA2) e caspase-3 do ácido aspártico contendo cisteína (caspase-3 clivada) foram avaliados com o ensaio western blot e reação em cadeia da polimerase. Resultados: A taxa de apoptose foi maior com mRNA e proteína de SERCA2 regulados para baixo e caspase-3 clivada suprarregulada no Grupo IR, em comparação com o Grupo S (p < 0,01). A taxa de apoptose foi menor com mRNA e proteína de SERCA2 suprarregulada e caspase-3 clivada sub-regulada no Grupo P, em comparação com o Grupo IR (p < 0,01). Conclusões: O propofol pode reduzir a apoptose de células miocárdicas induzida por lesão hepática por isquemia-reperfusão. Entretanto, pode suprarregular o mRNA e a proteína de SERCA2 em ratos.

[Effects of propofol pretreatment on myocardial cell apoptosis and SERCA2 expression in rats with hepatic ischemia/reperfusion]. / Efeitos do pré­tratamento com propofol sobre a apoptose de células miocárdicas e expressão de SERCA2 em ratos com isquemia/reperfusão hepática.

INTRODUCTION: Hepatic ischemia-reperfusion injury is a common pathophysiological process in liver surgery. Whether Propofol can reduce myocardial ischemia-reperfusion injury induced by hepatic ischemia-reperfusion injury in rats, together with related mechanisms, still needs further studies. OBJECTIVE: To investigate if propofol would protect the myocardial cells from apoptosis with hepatic ischemia-reperfusion injury. METHODS: Male Sprague-Dawley rats (n=18) were randomly allocated into three groups: Sham Group (Group S, n=6), Hepatic Ischemia-reperfusion Injury Group (Group IR, n=6) and Propofol Group (Group P, n=6). Group S was only subjected to laparotomy. Group IR was attained by ischemia for 30min and reperfusion for 4h. Group P was subjected identical insult as in Group IR with the administration of propofol started 10min before ischemia with 120mg.kg-1, following by continuous infusion at 20mg.kg-1.h-1. Cell apoptosis was examined by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay. Endoplasmic reticulum Ca2+-ATPase2 (SERCA2) and cysteine-containing aspartic acid cleaved-caspase3 (cleaved-caspase3) were assayed by western blot and Altimeter polymerase chain reaction. RESULTS: Apoptosis rate was increased, with mRNA and protein of SERCA2 down-regulated and cleaved-caspase3 up-regulated in Group IR compared with Group S (p<0.01). Apoptosis rate was decreased, with mRNA and protein of SERCA2 up-regulated and cleaved-caspase3 down-regulated in Group P compared with Group IR (p<0.01). CONCLUSIONS: Propofol can reduce hepatic ischemia-reperfusion injury-induced myocardial cell apoptosis, meanwhile, can up-regulate mRNA and protein of SERCA2 in rats.

Melatonin-Induced Protective Effects on Cardiomyocytes Against Reperfusion Injury Partly Through Modulation of IP3R and SERCA2a Via Activation of ERK1.

BACKGROUND: Melatonin is a neuroendocrine hormone synthesized primarily by the pineal gland that is indicated to effectively prevent myocardial reperfusion injury. It is unclear whether melatonin protects cardiac function from reperfusion injury by modulating intracellular calcium homeostasis. OBJECTIVE: Demonstrate that melatonin protect against myocardial reperfusion injury through modulating IP3R and SERCA2a to maintain calcium homeostasis via activation of ERK1 in cardiomyocytes. METHODS: In vitro experiments were performed using H9C2 cells undergoing simulative hypoxia/reoxygenation (H/R) induction. Expression level of ERK1, IP3R and SERCA2a were assessed by Western Blots. Cardiomyocytes apoptosis was detected by TUNEL. Phalloidin-staining was used to assess alteration of actin filament organization of cardiomyocytes. Fura-2 /AM was used to measure intracellular Ca2+ concentration. Performing in vivo experiments, myocardial expression of IP3R and SERCA2a were detected by immunofluorescence staining using myocardial ischemia/ reperfusion (I/R) model in rats. RESULTS: In vitro results showed that melatonin induces ERK1 activation in cardiomyocytes against H/R which was inhibited by PD98059 (ERK1 inhibitor). The results showed melatonin inhibit apoptosis of cardiomyocytes and improve actin filament organization in cardiomyocytes against H/R, because both could be reversed by PD98059. Melatonin was showed to reduce calcium overload, further to inhibit IP3R expression and promote SERCA2a expression via ERK1 pathway in cardiomyocytes against H/R. Melatonin induced lower IP3R and higher SERCA2a expression in myocardium that were reversed by PD98059. CONCLUSION: melatonin-induced cardioprotection against reperfusion injury is at least partly through modulation of IP3R and SERCA2a to maintain intracellular calcium homeostasis via activation of ERK1.

The Anti-Cancer Multikinase Inhibitor Sorafenib Impairs Cardiac Contractility by Reducing Phospholamban Phosphorylation and Sarcoplasmic Calcium Transients.

Tyrosine-kinase inhibitors (TKIs) have revolutionized cancer therapy in recent years. Although more targeted than conventional chemotherapy, TKIs exhibit substantial cardiotoxicity, often manifesting as hypertension or heart failure. Here, we assessed myocyte intrinsic cardiotoxic effects of the TKI sorafenib and investigated underlying alterations of myocyte calcium homeostasis. We found that sorafenib reversibly decreased developed force in auxotonically contracting human myocardia (3 µM: -25 ± 4%, 10 µM: -29 ± 7%, 30 µM: -43 ± 12%, p < 0.01), reduced peak cytosolic calcium concentrations in isolated cardiomyocytes (10 µM: 52 ± 8.1% of baseline, p < 0.001), and slowed cytosolic calcium removal kinetics (RT50, RT10, Tau, p < 0.05). Beta-adrenergic stimulation induced augmentation of calcium transient (CaT) amplitude was attenuated in sorafenib-treated cells (2.7 ± 0.3-fold vs. 3.6 ± 0.2-fold in controls, p < 0.001). Sarcoplasmic reticulum (SR) calcium content was reduced to 67 ± 4% (p < 0.01), and SR calcium re-uptake slowed (p < 0.05). Sorafenib significantly reduced serine 16 phosphorylation of phospholamban (PLN, p < 0.05), while PLN threonine 17 and CaMKII (T286) phosphorylation were not altered. Our data demonstrate that sorafenib acutely impairs cardiac contractility by reducing S16 PLN phosphorylation, leading to reduced SR calcium content, CaT amplitude, and slowed cytosolic calcium removal. These results indicate myocyte intrinsic cardiotoxicity irrespective of effects on the vasculature and chronic cardiac remodeling.

Melatonin-Induced Protective Effects on Cardiomyocytes Against Reperfusion Injury Partly Through Modulation of IP3R and SERCA2a Via Activation of ERK1 / Efeitos Protetores Induzidos pela Melatonina nos Cardiomiócitos Contra Lesões de Reperfusão Parcialmente Através da Modulação Dde IP3R e SERCA por Meio da Ativação de ERK1

Resumo Background: Melatonin is a neuroendocrine hormone synthesized primarily by the pineal gland that is indicated to effectively prevent myocardial reperfusion injury. It is unclear whether melatonin protects cardiac function from reperfusion injury by modulating intracellular calcium homeostasis. Objective: Demonstrate that melatonin protect against myocardial reperfusion injury through modulating IP3R and SERCA2a to maintain calcium homeostasis via activation of ERK1 in cardiomyocytes. Methods: In vitro experiments were performed using H9C2 cells undergoing simulative hypoxia/reoxygenation (H/R) induction. Expression level of ERK1, IP3R and SERCA2a were assessed by Western Blots. Cardiomyocytes apoptosis was detected by TUNEL. Phalloidin-staining was used to assess alteration of actin filament organization of cardiomyocytes. Fura-2 /AM was used to measure intracellular Ca2+ concentration. Performing in vivo experiments, myocardial expression of IP3R and SERCA2a were detected by immunofluorescence staining using myocardial ischemia/ reperfusion (I/R) model in rats. Results: In vitro results showed that melatonin induces ERK1 activation in cardiomyocytes against H/R which was inhibited by PD98059 (ERK1 inhibitor). The results showed melatonin inhibit apoptosis of cardiomyocytes and improve actin filament organization in cardiomyocytes against H/R, because both could be reversed by PD98059. Melatonin was showed to reduce calcium overload, further to inhibit IP3R expression and promote SERCA2a expression via ERK1 pathway in cardiomyocytes against H/R. Melatonin induced lower IP3R and higher SERCA2a expression in myocardium that were reversed by PD98059. Conclusion: melatonin-induced cardioprotection against reperfusion injury is at least partly through modulation of IP3R and SERCA2a to maintain intracellular calcium homeostasis via activation of ERK1.

Resumo Fundamento: A melatonina é um hormônio neuroendócrino sintetizado principalmente pela glândula pineal que é indicado para prevenir efetivamente a lesão de reperfusão miocárdica. Não está claro se a melatonina protege a função cardíaca da lesão de reperfusão através da modulação da homeostase do cálcio intracelular. Objetivo: Demonstrar que a melatonina protege contra a lesão de reperfusão miocárdica através da modulação de IP3R e SERCA para manter a homeostase de cálcio por meio da ativação de ERK1 em cardiomiócitos. Métodos: Foram realizados experimentos in vitro usando células H9C2 submetidas a indução de hipoxia / reoxigenação simulada (H/R). O nível de expressão de ERK1, IP3R e SERCA foi avaliado por Western Blots. A apoptose de cardiomiócitos foi detectada por TUNEL. A coloração de faloidina foi utilizada para avaliar a alteração da organização de filamentos de actina dos cardiomiócitos. Fura-2 / AM foi utilizado para medir a concentração intracelular de Ca2+. Realizando experiências in vivo, a expressão miocárdica de IP3R e SERCA foi detectada por coloração com imunofluorescência usando modelo de isquemia miocárdica / reperfusão (I/R) em ratos. Resultados: resultados in vitro mostraram que a melatonina induz a ativação de ERK1 em cardiomiócitos contra H/R que foi inibida por PD98059 (inibidor de ERK1). Os resultados mostraram que a melatonina inibe a apoptose dos cardiomiócitos e melhora a organização do filamento de actina em cardiomiócitos contra H/R, pois ambas poderiam ser revertidas pela PD98059. A melatonina mostrou reduzir a sobrecarga de cálcio, além de inibir a expressão de IP3R e promover a expressão de SERCA através da via ERK1 em cardiomiócitos contra H/R. A melatonina induziu menor IP3R e maior expressão de SERCA no miocárdio que foram revertidas pela PD98059. Conclusão: a cardioproteção induzida pela melatonina contra lesão de reperfusão é pelo menos parcialmente através da modulação de IP3R e SERCA para manter a homeostase de cálcio intracelular via ativação de ERK1.

High-Salt Intake Augments the Activity of the RhoA/ROCK Pathway and Reduces Intracellular Calcium in Arteries From Rats.

BACKGROUND: We investigated the influence of salt overconsumption on the functionality of the RhoA/Rho-associated kinase (ROCK) pathway and calcium regulation in arteries. METHODS: The aorta and small mesenteric arteries from rats fed a chow containing 2%, 4%, or 8% NaCl were evaluated in organ baths for the activity of the RhoA/ROCK pathway and intracellular calcium mobilization. Components of these pathways and intracellular calcium levels were also assessed in samples from 4% NaCl group. RESULTS: In arteries from animals fed regular chow, the ROCK inhibitor Y-27632 reduced the responses to phenylephrine, even when the smallest concentrations (1 and 3 µM) were tested. However, only higher concentrations of Y-27632 (10 and 50 µM) reduced phenylephrine-induced contraction in vessels from high-salt groups. Immunoblotting revealed augmented phosphorylation of the myosin phosphatase targeting subunit 1 and increased amounts of RhoA in the membrane fraction of aorta homogenates from the 4% NaCl group. Under calcium-free solution, vessels from NaCl groups presented reduced contractile responses to phenylephrine and caffeine, compared with the regular chow group. Moreover, decreased intracellular calcium at rest and after stimulation with ATP were found in aortic smooth muscle cells from 4% NaCl-fed rats, which also showed diminished levels of SERCA2 and SERCA3, but not of IP3 and ryanodine receptors, or STIM1 and Orai1 proteins. CONCLUSIONS: Arteries from rats subjected to high-salt intake are unable to properly regulate intracellular calcium levels and present augmented activity of the calcium sensitization pathway RhoA/ROCK. These changes may precede the development of vascular diseases induced by high-salt intake.

Slow Phospholipid Exchange between a Detergent-Solubilized Membrane Protein and Lipid-Detergent Mixed Micelles: Brominated Phospholipids as Tools to Follow Its Kinetics.

Membrane proteins are largely dependent for their function on the phospholipids present in their immediate environment, and when they are solubilized by detergent for further study, residual phospholipids are critical, too. Here, brominated phosphatidylcholine, a phospholipid which behaves as an unsaturated phosphatidylcholine, was used to reveal the kinetics of phospholipid exchange or transfer from detergent mixed micelles to the environment of a detergent-solubilized membrane protein, the paradigmatic P-type ATPase SERCA1a, in which Trp residues can experience fluorescence quenching by bromine atoms present on phospholipid alkyl chains in their immediate environment. Using dodecylmaltoside as the detergent, exchange of (brominated) phospholipid was found to be much slower than exchange of detergent under the same conditions, and also much slower than membrane solubilization, the latter being evidenced by light scattering changes. The kinetics of this exchange was strongly dependent on temperature. It was also dependent on the total concentration of the mixed micelles, revealing the major role for such exchange of the collision of detergent micelles with the detergent-solubilized protein. Back-transfer of the brominated phospholipid from the solubilized protein to the detergent micelle was much faster if lipid-free DDM micelles instead of mixed micelles were added for triggering dissociation of brominated phosphatidylcholine from the solubilized protein, or in the additional presence of C12E8 detergent during exchange, also emphasizing the role of the chemical nature of the micelle/protein interface. This protocol using brominated lipids appears to be valuable for revealing the possibly slow kinetics of phospholipid transfer to or from detergent-solubilized membrane proteins. Independently, continuous recording of the activity of the protein can also be used in some cases to correlate changes in activity with the exchange of a specific phospholipid, as shown here by using the Drs2p/Cdc50p complex, a lipid flippase with specific binding sites for lipids.

Thyroxine increases Serca2 and Ryr2 gene expression in heart failure rats with euthyroid sick syndrome

ABSTRACT Objective The current study was aimed at analyzing sarcoplasmic reticulum Ca2+ ATPase (Serca2) and ryanodine receptor type 2 (Ryr2) gene expression in rats subjected to surgery that induced HF and were subsequently treated with T4 using physiological doses. Materials and methods HF was induced in 18 male Wistar rats by clipping the ascending thoracic aorta to generate aortic stenosis (HFS group), while the control group (9-sham) underwent thoracotomy. After 21 weeks, the HFS group was subdivided into two subgroups. One group (9 Wistar rats) with HF received 1.0 µg of T4/100 g of body weight for five consecutive days (HFS/T4); the other group (9 Wistar rats) received isotonic saline solution (HFS/S). The animals were sacrificed after this treatment and examined for signs of HF. Samples from the left ventricles of these animals were analyzed by RT-qPCR for the expression of Serca2 and Ryr2 genes. Results Rats with HF developed euthyroid sick syndrome (ESS) and treatment with T4 restored the T3 values to the Sham level and increased Serca2 and Ryr2 gene expression, thereby demonstrating a possible benefit of T4 treatment for heart function in ESS associated with HF. Conclusion The T4 treatment can potentially normalize the levels of T3 as well elevated Serca2 and Ryr2 gene expression in the myocardium in heart failure rats with euthyroid sick syndrome.

Thyroxine increases Serca2 and Ryr2 gene expression in heart failure rats with euthyroid sick syndrome.

OBJECTIVE: The current study was aimed at analyzing sarcoplasmic reticulum Ca2+ ATPase (Serca2) and ryanodine receptor type 2 (Ryr2) gene expression in rats subjected to surgery that induced HF and were subsequently treated with T4 using physiological doses. MATERIALS AND METHODS: HF was induced in 18 male Wistar rats by clipping the ascending thoracic aorta to generate aortic stenosis (HFS group), while the control group (9-sham) underwent thoracotomy. After 21 weeks, the HFS group was subdivided into two subgroups. One group (9 Wistar rats) with HF received 1.0 µg of T4/100 g of body weight for five consecutive days (HFS/T4); the other group (9 Wistar rats) received isotonic saline solution (HFS/S). The animals were sacrificed after this treatment and examined for signs of HF. Samples from the left ventricles of these animals were analyzed by RT-qPCR for the expression of Serca2 and Ryr2 genes. RESULTS: Rats with HF developed euthyroid sick syndrome (ESS) and treatment with T4 restored the T3 values to the Sham level and increased Serca2 and Ryr2 gene expression, thereby demonstrating a possible benefit of T4 treatment for heart function in ESS associated with HF. CONCLUSION: The T4 treatment can potentially normalize the levels of T3 as well elevated Serca2 and Ryr2 gene expression in the myocardium in heart failure rats with euthyroid sick syndrome.

Resveratrol protects against lipopolysaccharide-induced cardiac dysfunction by enhancing SERCA2a activity through promoting the phospholamban oligomerization.

The bacterial endotoxin lipopolysaccharide (LPS) is a main culprit responsible for cardiac dysfunction in sepsis. This study examined whether resveratrol could protect against LPS-induced cardiac dysfunction by improving the sarcoplasmic endoplasmic reticulum Ca2+-ATPase (SERCA2a) activity. Echocardiographic parameters, cardiomyocyte contractile and Ca2+ transient properties, markers for cardiac inflammation, cell death, and oxidative stress, SERCA2a activity, and the ratios of phospholamban (PLB) monomer to oligomer were measured. Cardiac function was decreased >50% after LPS challenge (6 mg/kg for 6 h), which was improved by resveratrol. There was neither difference in plasma tumor necrosis factor-α and troponin I levels nor in infiltration of CD45+ cells in cardiac tissue between resveratrol-treated and untreated groups. In cardiomyocytes, LPS significantly decreased contractile amplitude, elongated relengthening time, diminished Ca2+ transient, reduced SERCA2a activity, and increased superoxide generation. These pathological alterations were attenuated by resveratrol treatment. Immunoblot analysis showed that LPS-treated mice had increased levels of malondialdehyde (MDA), 4-hydroxynonenal (4-HNE), and the monomer form of PLB, along with decreases in the levels of SERCA2a, the oligomer form of PLB and nuclear factor erythroid 2-related factor (Nrf-2). Resveratrol treatment upregulated SERCA2a, the oligomer form of PLB, and Nrf-2 expression and function, and downregulated MDA, 4-HNE, and the monomer form of PLB. Our data suggest that the activity of SERCA2a in endotoxemia is inhibited, possibly due to increases in the monomer form of PLB. Resveratrol protects the heart from LPS-induced injuries at least in part through promoting the oligomerization of PLB that leads to enhanced SERCA2a activity.

Combination of angiotensin II and l-NG-nitroarginine methyl ester exacerbates mitochondrial dysfunction and oxidative stress to cause heart failure.

Mitochondrial dysfunction has been implicated as a cause of energy deprivation in heart failure (HF). Herein, we tested individual and combined effects of two pathogenic factors of nonischemic HF, inhibition of nitric oxide synthesis [with l-N(G)-nitroarginine methyl ester (l-NAME)] and hypertension [with angiotensin II (AngII)], on myocardial mitochondrial function, oxidative stress, and metabolic gene expression. l-NAME and AngII were administered individually and in combination to mice for 5 wk. Although all treatments increased blood pressure and reduced cardiac contractile function, the l-NAME + AngII group was associated with the most severe HF, as characterized by edema, hypertrophy, oxidative stress, increased expression of Nppa and Nppb, and decreased expression of Atp2a2 and Camk2b. l-NAME + AngII-treated mice exhibited robust deterioration of cardiac mitochondrial function, as observed by reduced respiratory control ratios in subsarcolemmal mitochondria and reduced state 3 levels in interfibrillar mitochondria for complex I but not for complex II substrates. Cardiac myofibrils showed reduced ADP-supported and oligomycin-inhibited oxygen consumption. Mitochondrial functional impairment was accompanied by reduced mitochondrial DNA content and activities of pyruvate dehydrogenase and complex I but increased H2O2 production and tissue protein carbonyls in hearts from AngII and l-NAME + AngII groups. Microarray analyses revealed the majority of the gene changes attributed to the l-NAME + AngII group. Pathway analyses indicated significant changes in metabolic pathways, such as oxidative phosphorylation, mitochondrial function, cardiac hypertrophy, and fatty acid metabolism in l-NAME + AngII hearts. We conclude that l-NAME + AngII is associated with impaired mitochondrial respiratory function and increased oxidative stress compared with either l-NAME or AngII alone, resulting in nonischemic HF.