SENP2-mediated SERCA2a deSUMOylation increases calcium overload in cardiomyocytes to aggravate myocardial ischemia/reperfusion injury / 中华医学杂志(英文版)

BACKGROUND@#Sarcoplasmic reticulum calcium ATPase 2a (SERCA2a) is a key protein that maintains myocardial Ca 2+ homeostasis. The present study aimed to investigate the mechanism underlying the SERCA2a-SUMOylation (small ubiquitin-like modifier) process after ischemia/reperfusion injury (I/RI) in vitro and in vivo .@*METHODS@#Calcium transient and systolic/diastolic function of cardiomyocytes isolated from Serca2a knockout (KO) and wild-type mice with I/RI were compared. SUMO-relevant protein expression and localization were detected by quantitative real-time PCR (RT-qPCR), Western blotting, and immunofluorescence in vitro and in vivo . Serca2a-SUMOylation, infarct size, and cardiac function of Senp1 or Senp2 overexpressed/suppressed adenovirus infected cardiomyocytes, were detected by immunoprecipitation, triphenyltetrazolium chloride (TTC)-Evans blue staining, and echocardiography respectively.@*RESULTS@#The results showed that the changes of Fura-2 fluorescence intensity and contraction amplitude of cardiomyocytes decreased in the I/RI groups and were further reduced in the Serca2a KO + I/RI groups. Senp1 and Senp2 messenger ribose nucleic acid (mRNA) and protein expression levels in vivo and in cardiomyocytes were highest at 6 h and declined at 12 h after I/RI. However, the highest levels in HL-1 cells were recorded at 12 h. Senp2 expression increased in the cytoplasm, unlike that of Senp1. Inhibition of Senp2 protein reversed the I/RI-induced Serca2a-SUMOylation decline, reduced the infarction area, and improved cardiac function, while inhibition of Senp1 protein could not restore the above indicators.@*CONCLUSION@#I/RI activated Senp1 and Senp2 protein expression, which promoted Serca2a-deSUMOylation, while inhibition of Senp2 expression reversed Serca2a-SUMOylation and improved cardiac function.

Cenário Disfuncional dos Principais Componentes Responsáveis pelo Equilíbrio do Trânsito de Cálcio Miocárdico na Insuficiência Cardíaca Induzida por Estenose Aórtica / The Dysfunctional Scenario of the Major Components Responsible for Myocardial Calcium Balance in Heart Failure Induced by Aortic Stenosis

Resumo Fundamento O remodelamento cardíaco patológico se caracteriza por disfunção diastólica e sistólica, levando à insuficiência cardíaca. Neste contexto, o cenário disfuncional do trânsito de cálcio miocárdico (Ca2+) tem sido pouco estudado. Um modelo experimental de estenose aórtica tem sido extensamente utilizado para aprimorar os conhecimentos sobre os principais mecanismos do remodelamento patológico cardíaco. Objetivo Entender o processo disfuncional dos principais componentes responsáveis pelo equilíbrio do cálcio miocárdico e sua influência sobre a função cardíaca na insuficiência cardíaca induzida pela estenose aórtica. Métodos Ratos Wistar de 21 dias de idade foram distribuídos em dois grupos: controle (placebo; n=28) e estenose aórtica (EaO; n=18). A função cardíaca foi analisada com o ecocardiograma, músculo papilar isolado e cardiomiócitos isolados. No ensaio do músculo papilar, SERCA2a e a atividade do canal de Ca2+ do tipo L foram avaliados. O ensaio de cardiomiócitos isolados avaliou o trânsito de cálcio. A expressão proteica da proteínas do trânsito de cálcio foi analisada com o western blot. Os resultados foram estatisticamente significativos quando p <0,05. Resultados Os músculos papilares e cardiomiócitos dos corações no grupo EaO demonstraram falhas mecânicas. Os ratos com EaO apresentaram menor tempo de pico do Ca2+, menor sensibilidade das miofibrilas do Ca2+, prejuízos nos processos de entrada e recaptura de cálcio pelo retículo sarcoplasmático, bem como disfunção no canal de cálcio do tipo L (CCTL). Além disso, os animais com EaO apresentaram maior expressão de SERCA2a, CCTL e trocador de Na+/Ca2+. Conclusão Insuficiência cardíaca sistólica e diastólica devido à estenose aórtica supravalvular acarretou comprometimento da entrada de Ca2+ celular e inibição da recaptura de cálcio pelo retículo sarcoplasmático devido à disfunção no CCTL e SERCA2a, assim como mudanças no trânsito de cálcio e na expressão das principais proteínas responsáveis pela homeostase de Ca2+ celular.

Abstract Background Maladaptive cardiac remodelling is characterized by diastolic and systolic dysfunction, culminating in heart failure. In this context, the dysfunctional scenario of cardiac calcium (Ca2+) handling has been poorly studied. An experimental model of aortic stenosis has been extensively used to improve knowledge about the key mechanisms of cardiac pathologic remodelling. Objective To understand the dysfunctional process of the major components responsible for Ca2+ balance and its influence on cardiac function in heart failure induced by aortic stenosis. Methods Male 21-day-old Wistar rats were distributed into two groups: control (sham; n= 28) and aortic stenosis (AoS; n= 18). Cardiac function was analysed by echocardiogram, isolated papillary muscle, and isolated cardiomyocytes. In the papillary muscle assay, SERCA2a and L-type Ca2+ channel activity was evaluated. The isolated cardiomyocyte assay evaluated Ca2+ handling. Ca2+ handling protein expression was analysed by western blot. Statistical significance was set at p <0.05. Results Papillary muscles and cardiomyocytes from AoS hearts displayed mechanical malfunction. AoS rats presented a slower time to the Ca2+ peak, reduced Ca2+ myofilament sensitivity, impaired sarcoplasmic reticulum Ca2+ influx and reuptake ability, and SERCA2a and L-type calcium channel (LTCC) dysfunction. Moreover, AoS animals presented increased expression of SERCA2a, LTCCs, and the Na+/Ca2+ exchanger. Conclusion Systolic and diastolic heart failure due to supravalvular aortic stenosis was paralleled by impairment of cellular Ca2+ influx and inhibition of sarcoplasmic reticulum Ca2+ reuptake due to LTCC and SERCA2a dysfunction, as well as changes in Ca2+ handling and expression of the major proteins responsible for cellular Ca2+ homeostasis.

Calcium homeostasis behavior and cardiac function on left ventricular remodeling by pressure overload

Sarcoplasmic reticulum Ca2+-ATPase (SERCA2a) and sarcolemmal Na+/Ca2+ exchanger (NCX1) structures are involved in heart cell Ca2+ homeostasis. Previous studies have shown discrepancies in their function and expression in heart failure. The goal of this study was to evaluate heart function and hypertrophied muscle Ca2+-handling protein behavior under pressure overload. Twenty male Wistar rats were divided into two groups: Aortic stenosis (AoS), induced by a clip placed at the beginning of the aorta, and Control (Sham). After 18 weeks, heart function and structure were evaluated by echocardiogram. Myocardial function was analyzed by isolated papillary muscle (IPM) at basal condition and Ca2+ protein functions were evaluated after post-pause contraction and blockage with cyclopiazonic acid in IPM. Ca2+-handling protein expression was studied by western blot (WB). Echocardiogram showed that AoS caused concentric hypertrophy with enhanced ejection fraction and diastolic dysfunction inferred by dilated left atrium and increased relative wall thickness. IPM study showed developed tension was the same in both groups. AoS showed increased stiffness revealed by enhanced resting tension, and changes in Ca2+ homeostasis shown by calcium elevation and SERCA2a blockage maneuvers. WB revealed decreased NCX1, SERCA2a, and phosphorylated phospholambam (PLB) on serine-16 in AoS. AoS had left ventricular hypertrophy and diastolic dysfunction compared to Sham; this could be related to our findings regarding calcium homeostasis behavior: deficit in NCX1, SERCA2a, and phosphorylated PLB on serine-16.

Intracoronary sarcoplasmic reticulum calcium-atpase gene therapy in advanced heart failure patients with reduced ejection fraction: a prospective cohort study

OBJECTIVE: Heart failure is a progressive and debilitating disease. Intracoronary sarcoplasmic reticulum calcium-ATPase gene therapy may improve the function of cardiac muscle cells. This study aimed to test the hypothesis that intracoronary sarcoplasmic reticulum calcium-ATPase gene therapy can improve outcomes and reduce the number of recurrent and terminal events in advanced heart failure patients with reduced ejection fraction. METHODS: A total of 768 heart failure patients with reduced ejection fraction and New York Heart Association classification II to IV were included in this prospective cohort study. Patients either underwent intracoronary sarcoplasmic reticulum calcium-ATPase gene therapy (CA group, n=384) or received oral placebo (PA group; n=384). Data regarding recurrent and terminal event(s), treatment-emergent adverse effects, and outcome measures were collected and analyzed. RESULTS: After a follow-up period of 18 months, intracoronary sarcoplasmic reticulum calcium-ATPase gene therapy reduced the number of hospital admissions (p=0.001), ambulatory treatments (p=0.0004), and deaths (p=0.024). Additionally, intracoronary sarcoplasmic reticulum calcium-ATPase gene therapy improved the left ventricular ejection fraction (p<0.0001) and Kansas City Cardiomyopathy Questionnaire score (p<0.0001). The number of recurrent and terminal events/patients were higher in the PA group than in the CA group after the follow-up period of 18 months (p=0.015). The effect of the intracoronary sarcoplasmic reticulum calcium-ATPase gene therapy was independent of the confounding variables. No new arrhythmias were reported in the CA group. CONCLUSIONS: Intracoronary sarcoplasmic reticulum calcium-ATPase gene therapy reduces the number of recurrent and terminal events and improves the clinical course of advanced heart failure patients with reduced ejection fraction.

Detection and analysis of an ATP2A2 mutation in a family with Darier-White disease / 中华医学遗传学杂志

OBJECTIVE@#To explore the molecular basis for a pedigree affected with Darier-White disease.@*METHODS@#Genomic DNA was isolated from 3 patients and 1 unaffected member from the pedigree, as well as 80 healthy controls. Targeted sequence capture and next-generation sequencing were used to screen mutations of skin disease-related genes. Candidate mutations were verified by Sanger sequencing, and co-segregation analysis was carried out to confirm the pathogenicity of mutation. Conservation analysis and protein structure and function were also predicted with Bioinformatic tools.@*RESULTS@#A heterozygous mutation c.2246G>T (p.G749V) was identified in exon 15 of ATP2A2 gene in all 3 patients from the pedigree, but not in the unaffected member or 80 healthy controls. The corresponding amino acid was highly conserved, and mutation of which can lead to structural and functional changes of the protein.@*CONCLUSION@#The c.2246G>T missense mutation of the ATP2A2 gene probably underlies the Darier-White disease in this pedigree by causing damages to the structure and function of sarcoplasmic/endoplasmic reticulum calcium ATPase 2 (SERCA2).

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.

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.

The m-rna, expression of serca2 and ncx1 in the process of pharmacological cell protection in experimental acute pancreatitis induced by taurocholate / Expressão do rna-m, da serca2 e do ncx1 no processo de proteção celular farmacológica na pancreatite aguda experimental induzida por taurocolato

ABSTRACT Background: Intracellular calcium overload is known to be a precipitating factor of pancreatic cell injury in acute pancreatitis (AP). Intracellular calcium homeostasis depends of Plasmatic Membrane Calcium ATPase (PMCA), Sarcoplasmic Endothelial Reticulum Calcium ATPase 2 (SERCA 2) and the Sodium Calcium Exchanger (NCX1). The antioxidant melatonin (Mel) and Trisulfate Disaccharide (TD) that accelerates NCX1 action could reduce the cell damage determined by the AP. Aim: To evaluate m-RNA expressions of SERCA2 and NCX1 in acute pancreatitis induced by sodium taurocholate in Wistar rats pre-treated with melatonin and/or TD. Methods: Wistar rats were divided in groups: 1) without AP; 2) AP without pre-treatment; 3) AP and Melatonin; 4) AP and TD; 5) AP and Melatonin associated to TD. Pancreatic tissue samples were collected for detection of SERCA2 and NCX1 m-R NA levels by polymerase chain reaction (PCR). Results: Increased m-RNA expression of SERCA2 in the melatonin treated group, without increase of m-RNA expression of the NCX1. The TD did not affect levels of SERCA2 and NCX1 m-RNA expressions. The combined melatonin and TD treatment reduced the m-RNA expression of SERCA2. Conclusions: The effect of melatonin is restricted to increased m-RNA expression of SERCA2. Although TD does not affect gene expression, its action in accelerating calcium exchanger function can explain the slightest expression of SERCA2 m-RNA when associated with Melatonin, perhaps by a joint action of drugs with different and but possibly complementary mechanisms.

RESUMO Racional: A lesão celular da pancreatite aguda (PA) envolve sobrecarga de cálcio, regulada pela atividade da Cálcio ATPase de membrana (PMCA), Cálcio ATPase do Retículo (SERCA2) e pelo Trocador Sódio Cálcio (NCX1). A melatonina (antioxidante) e o Dissacarídeo Trissulfatado (acelerador do NCX1) poderiam reduzir a lesão celular na PA. Objetivo: Avaliar a expressão do RNAm da SERCA2 e NCX1 em modelo animal de pancreatite aguda tratados com melatonina e/ou dissacarídeo trissulfatado (DT). Método: Ratos Wistar foram divididos em grupos: 1) sem pancreatite aguda; 2) com pancreatite aguda por taurocolato; 3) PA e Melatonina; 4) PA e DT; 5) PA e Melatonina com DT. Amostras de tecido foram colhidas para detecção dos níveis de RNAm da SERCA2 e NCX1 por PCR. Resultados: Houve aumento da expressão do RNAm da SERCA2 no grupo com PA tratados com Melatonina, porém sem aumento de expressão do NCX1. O DT não afetou os níveis de SERCA2 e NCX1. O tratamento conjunto com Melatonina e DT diminuiu a expressão da SERCA2. Conclusões: O efeito da Melatonina é restrito ao aumento da expressão da SERCA2. O DT não tem ação na expressão gênica, porém sua ação na aceleração do trocador na retirada do cálcio pode explicar a menor expressão da SERCA2 quando associado à Melatonina, pela ação conjunta de drogas com mecanismos diferentes e possivelmente complementares.

Importance of SERCA2a on early isolated diastolic dysfunction induced by supravalvular aortic stenosis in rats

Cardiac remodeling is defined as changes in shape and function of the heart in response to aggression (pressure overload). The sarcoplasmic reticulum calcium ATPase cardiac isoform 2a (SERCA2a) is a known factor that influences function. A wide spectrum of studies report a decrease in SERCA2a in heart failure, but none evaluate it's the role in early isolated diastolic dysfunction in supravalvular aortic stenosis (AoS). Our hypothesis was that SERCA2a participates in such dysfunction. Thirty-day-old male Wistar rats (60-80 g) were divided into AoS and Sham groups, which were submitted to surgery with or without aorta clipping, respectively. After 6 weeks, the animals were submitted to echocardiogram and functional analysis by isolated papillary muscle (IPM) in basal condition, hypoxia, and SERCA2a blockage with cyclopiazonic acid at calcium concentrations of 0.5, 1.5, and 2.5 mM. Western-blot analyses were used for SERCA2a and phospholamban detection. Data analysis was carried out with Student's t-test and ANOVA. AoS enhanced left atrium and E and A wave ratio, with preserved ejection fraction. Basal condition in IPM showed similar increases in developed tension (DT) and resting tension (RT) in AoS, and hypoxia was similar between groups. After cyclopiazonic acid blockage, final DT was equally decreased and RT was similar between groups, but the speed of relaxation was decreased in the AoS group. Western-blot was uniform in all evaluations. The hypothesis was confirmed, since functional parameters regarding SERCA2a were changed in the AoS group.

Skeletal Muscle Thermogenesis and Its Role in Whole Body Energy Metabolism

Obesity and diabetes has become a major epidemic across the globe. Controlling obesity has been a challenge since this would require either increased physical activity or reduced caloric intake; both are difficult to enforce. There has been renewed interest in exploiting pathways such as uncoupling protein 1 (UCP1)-mediated uncoupling in brown adipose tissue (BAT) and white adipose tissue to increase energy expenditure to control weight gain. However, relying on UCP1-based thermogenesis alone may not be sufficient to control obesity in humans. On the other hand, skeletal muscle is the largest organ and a major contributor to basal metabolic rate and increasing energy expenditure in muscle through nonshivering thermogenic mechanisms, which can substantially affect whole body metabolism and weight gain. In this review we will describe the role of Sarcolipin-mediated uncoupling of Sarcoplasmic Reticulum Calcium ATPase (SERCA) as a potential mechanism for increased energy expenditure both during cold and diet-induced thermogenesis.

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.

Effects of Growth Hormone on Cardiac Remodeling During Resistance Training in Rats / Efeitos do Hormônio do Crescimento na Remodelação Cardíaca Durante Treinamento Resistido em Ratos

Abstract Background: Although the beneficial effects of resistance training (RT) on the cardiovascular system are well established, few studies have investigated the effects of the chronic growth hormone (GH) administration on cardiac remodeling during an RT program. Objective: To evaluate the effects of GH on the morphological features of cardiac remodeling and Ca2+ transport gene expression in rats submitted to RT. Methods: Male Wistar rats were divided into 4 groups (n = 7 per group): control (CT), GH, RT and RT with GH (RTGH). The dose of GH was 0.2 IU/kg every other day for 30 days. The RT model used was the vertical jump in water (4 sets of 10 jumps, 3 bouts/wk) for 30 consecutive days. After the experimental period, the following variables were analyzed: final body weight (FBW), left ventricular weight (LVW), LVW/FBW ratio, cardiomyocyte cross-sectional area (CSA), collagen fraction, creatine kinase muscle-brain fraction (CK-MB) and gene expressions of SERCA2a, phospholamban (PLB) and ryanodine (RyR). Results: There was no significant (p > 0.05) difference among groups for FBW, LVW, LVW/FBW ratio, cardiomyocyte CSA, and SERCA2a, PLB and RyR gene expressions. The RT group showed a significant (p < 0.05) increase in collagen fraction compared to the other groups. Additionally, the trained groups (RT and RTGH) had greater CK-MB levels compared to the untrained groups (CT and GH). Conclusion: GH may attenuate the negative effects of RT on cardiac remodeling by counteracting the increased collagen synthesis, without affecting the gene expression that regulates cardiac Ca2+ transport.

Resumo Fundamento: Apesar de os efeitos benéficos do treinamento resistido (TR) sobre o sistema cardiovascular estarem bem estabelecidos, poucos estudos têm investigado os efeitos crônicos da administração de hormônio do crescimento (GH) sobre a remodelação cardíaca durante um programa de TR. Objetivo: avaliar os efeitos do GH sobre a remodelação cardíaca em suas características morfológicas e na expressão dos genes do trânsito de Ca2+ em ratos submetidos ao TR. Métodos: Ratos Wistar machos foram divididos em 4 grupos (n = 7 por grupo): controle (CT), GH, TR e TR com GH (TRGH). A dose de GH foi de 0,2 UI/kg, a cada dois dias, por 30 dias. O modelo de TR utilizado foi o salto vertical em água (4 séries de 10 saltos, 3 vezes/semana) durante 30 dias consecutivos. Após o período experimental, as seguintes variáveis foram analisadas: peso corporal final (PCF), peso do ventrículo esquerdo (PVE), razão PVE/PCF, área seccional de cardiomiócitos (ASC), fração de colágeno, creatina quinase fração músculo-cérebro (CK-MB) e expressão gênica de SERCA2a, fosfolambam (PLB) e rianodina (RyR). Resultados: Não houve diferença significativa (p > 0,05) entre os grupos para PCF, PVE, razão PVE/PCF, ASC, e expressão gênica de SERCA2a, PLB e RyR. O grupo TR mostrou um significativo aumento (p < 0,05) da fração de colágeno em comparação aos outros. Além disso, os grupos treinados (TR e TRGH) apresentaram maiores níveis de CK-MB em comparação aos não treinados (CT e GH). Conclusão: Esses resultados indicam que o GH pode atenuar os efeitos negativos do TR na remodelação cardíaca por contrabalançar o aumento da síntese de colágeno, sem afetar a expressão de genes que regulam o trânsito de Ca2+ cardíaco.

Lowered sarcoendoplasmic reticulum calcium uptake and diaphragmatic SERCA1 expression contribute to diaphragmatic contractile and relaxation dysfunction in septic rats / 南方医科大学学报

<p><b>OBJECTIVE</b>The explore the mechanism responsible for diaphragmatic contractile and relaxation dysfunction in a rat model of sepsis.</p><p><b>METHODS</b>Thirty-six adult male Sprague-Dawley rats were randomized equally into a sham-operated group and two model groups of sepsis induced by cecal ligation and puncture (CLP) for examination at 6 and 12 h following CLP (CLP-6 h and CLP-12 h groups). The parameters of diaphragm contractile and relaxation were measured, and the calcium uptake and release rates of the diaphragmatic sarcoendoplasmic reticulum (SR) and the protein expressions of SERCA1, SERCA2 and RyR in the diaphragmatic muscles were determined.</p><p><b>RESULTS</b>The half-relaxation time of the diaphragm was extended in both the CLP-6 h and CLP-12 h groups with significantly reduced maximum tension declinerate and the peek uptake rate of SERCA (P<0.01). Diaphragmatic maximum twitch force development rate, the maximal twitch, tetanus tensions and the peek release rate of SR decreased only at 12h after CLP (P<0.01). The expression levels of SERCA1 protein decreased significantly in the diaphragmatic muscles at 12h following CLP (P<0.01) while SERCA2 expression level and SERCA activity showed no significant changes.</p><p><b>CONCLUSION</b>In the acute stage of sepsis, both the contractile and relaxation functions of the diaphragm are impaired. Diaphragmatic relaxation dysfunction may result from reduced calcium uptake in the SR and a decreased level of SERCA1 in the diaphragmatic muscles.</p>

Shen-Fu Injection () alleviates post-resuscitation myocardial dysfunction by up-regulating expression of sarcoplasmic reticulum Ca(2+)-ATPase / 中国结合医学杂志

<p><b>OBJECTIVE</b>To compare the effect of Shen-Fu Injection (SFI) and epinephrine on the expression of sarcoplasmic reticulum Ca(2+) ATPase 2a (SERCA2a) in a pig model with post-resuscitation myocardial dysfunction.</p><p><b>METHODS</b>Ventricular fibrillation (VF) was electrically induced in Wu-zhi-shan miniature pigs. After 8 min of untreated VF and 2 min of cardiopulmonary resuscitation (CPR), all animals were randomly administered a bolus injection of saline placebo (SA group, n=10), SFI (0.8 mg/kg, SFI group, n=10) or epinephrine (20 μg/kg, EPI group, n=10). After 4 min of CPR, a 100-J shock was delivered. If the defibrillation attempt failed to attain restoration of spontaneous circulation (ROSC), manual chest compressions were rapidly resumed for a further 2 min followed by a second defibrillation attempt. Hemodynamic variables were recorded, and plasma concentrations of catecholamines were measured. Adenylate cyclase (AC), cyclic adenosine monophosphate (cAMP) and the expressions of β1-adrenoceptor (AR) and SERCA 2a were determined.</p><p><b>RESULTS</b>Cardiac output, left ventricular dp/dtmax and negative dp/dtmax were significantly higher in the SFI group than in the SA and EPI groups at 4 and 6 h after ROSC. The expression of β1-AR and SERCA2a at 24 h after ROSC were significantly higher in the SFI group than in the SA and EPI groups (P<0.05 or P<0.01).</p><p><b>CONCLUSIONS</b>The administration of epinephrine during CPR decreased the expression of SERCA2a and aggravated postresuscitation myocardial function (P<0.01). SFI attenuated post-resuscitation myocardial dysfunction, and the mechanism might be related to the up-regulation of SERCA2a expression.</p>

Analysis of ATP2A2 gene mutations in a pedigree and a sporadic case with Darier disease / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To detect mutations of ATP2A2 gene in a pedigree and a sporadic case with Darier disease (DD) and explore the underlying molecular mechanism.</p><p><b>METHODS</b>Clinical data of the pedigree and the sporadic case were collected. Genomic DNA was extracted from blood samples of four members from the pedigree (including three patients and one healthy member), the sporadic case and 100 healthy controls. PCR was performed to amplify all coding exons of the ATP2A2 gene. And the products were directly sequenced to detect mutations.</p><p><b>RESULTS</b>A missense mutation c.1484C>T (p.S495L) in exon 12 was detected in all patients of the pedigree. For the sporadic case, a novel splicing mutation c.325-2A>G was detected at the junction between intron 4 and exon 5. The same mutations were not found in the 100 healthy controls.</p><p><b>CONCLUSION</b>Mutations of the ATP2A2 gene may lead to the occurrence of DD in both familial and sporadic cases with DD.</p>

Effects of hydrogen sulfide on contraction capacity of diaphragm from type 1 diabetic rats / 中南大学学报(医学版)

OBJECTIVE@#To investigate the effects of hydrogen sulfide (H2S) on contraction capacity of diaphragm in type 1 diabetic rats.
@*METHODS@#Thirty-two male SD rats were randomly divided into a normal group (NC), a diabetic group (DM), a NaHS treatment group (DM+NaHS) and a NaHS group (NaHS) (n=8). Intraperitoneal injection of streptozotocin was utilized to establish diabetic rat model. After the modeling, the rats in the DM+NaHS and the NaHS groups were intraperitoneally injected with 28 μmol/kg NaHS solution. 8 weeks later, the diaphragm contractility was assessed by isolated draphragm strips perfusion. The peak twitch tension (Pt), maximum tetanic tension (Po) and maximal rates of contraction/relaxation (±dT/dtmax) were determined. The alterations in diaphragm ultrastructure were observed under electron microscopy. The diaphragm weight/body weight (DW/BW) was measured. The activities of succinic dehydrogenase (SDH), lactate dehydrogenase (LDH) and sarcoplasmic reticulum Ca2+ ATPase (SERCA) were analyzed by spectrophotometric method. The mRNA levels of SERCA and prospholamban (PLB) in diaphragm were detected by RT-PCR.
@*RESULTS@#Compared with the NC group, there was no significant change in all measured index in the NaHS group (P>0.05), while Pt, Po and ±dT/dtmax were significantly decreased in the DM group (P<0.05). Transmission electron microscopy revealed obvious ultrastructural changes in the diaphragm. The DW/BW ratio and the activities of SDH, LDH and SERCA were decreased. The SERCA mRNA was decreased, while PLB mRNA was increased. Compared with the DM group, the diaphragm contractility and ultrastructure damage were improved in the DM+NaHS group. The DW/BW ratio and the activities of SDH, LDH and SERCA were increased. The SERCA mRNA was increased, while PLB mRNA was decreased (all P<0.05).
@*CONCLUSION@#H(2)S can enhance the contraction capacity of diaphragm in type 1 diabetic rats, which is involved in regulating the activities of biological enzymes and the gene expressions of calcium regulatory proteins.

Decreased expression of calcium-sensing receptor involved in the progression of diabetic cardiomyopathy / 中国应用生理学杂志

<p><b>OBJECTIVE</b>To observe the dynamic expression of calcium-sensing receptor(CaSR) in myocardium of diabetic rats.</p><p><b>METHODS</b>Thirty male Wistar rats were randomly divided into 3 groups including control, diabetic-4 week and diabetic-8 week groups(n = 10). The type 2 diabetes mellitus models were established by intraperitoneal injection of streptozotocin (STZ, 30 mg/kg) after high-fat and high-sugar diet for one month. The cardiac morphology was observed by electron microscope. Western blot analyzed the expression of CaSR, phospholamban (PLN), a calcium handling regulator, and Ca+-ATPase(SERCA) in cardiac tissues.</p><p><b>RESULTS</b>Compared with control group, the expressions of CaSR and SERCA were decreased, while the expression of PLN was significantly increased in a time-dependent manner in diabetic groups. Meanwhile diabetic rats displayed abnormal cardiac structure.</p><p><b>CONCLUSION</b>These results indicate that the CaSR expression of myocardium is reduced in the progression of DCM, and its potential mechanism may be related to the imnaired intracellular calcium homeostasis.</p>

Expression of sarco/endoplasmic reticulum Ca(2+)-ATPase from Bombyx mori by baculovirus expression system / 生物工程学报

Sarco/endoplasmic reticulum Ca(2+)-ATPase (Serca) is responsible for transporting Ca2+ into the endoplasmic reticulum and maintaining a suitable calcium environment in cells. The suitable calcium environment created by BmSerca is vital for the growth and development of silkworm. With a large molecular weight and 10 transmembrane domains, Serca is very difficult to express in Escherichia coli expression system. In order to obtain recombinant Serca with biological activity, pFastBac Dual vector was used to construct a binary baculovirus expression vector for expressing egfp and serca in cells. After transfection and infection, EGFP and Serca were expressed successfully in BmN-SWU1 cell line. Fluorescent observation revealed that the expression patterns of EGFP and Serca in infected cells were the same. Western blotting analysis showed that the recombinant proteins were about to express in cells 48 h post infection and highly expressed 96 h post infection. Ca(2+)-ATPase activities assays were used to evaluate the enzyme activities of recombinant Serca and found that the enzyme activities increased significantly after infection. The obtained data showed that this binary baculovirus expression system can be successfully used to express Serca with biological activity. The expression of Serca protein with this system is useful for further research on the function of Serca.

Alterations of the Ca²⁺ signaling pathway in pancreatic beta-cells isolated from db/db mice

Upon glucose elevation, pancreatic beta-cells secrete insulin in a Ca(2+)-dependent manner. In diabetic animal models, different aspects of the calcium signaling pathway in beta-cells are altered, but there is no consensus regarding their relative contributions to the development of beta-cell dysfunction. In this study, we compared the increase in cytosolic Ca(2+) ([Ca(2+)]i) via Ca(2+) influx, Ca(2+) mobilization from endoplasmic reticulum (ER) calcium stores, and the removal of Ca(2+) via multiple mechanisms in beta-cells from both diabetic db/db mice and non-diabetic C57BL/6J mice. We refined our previous quantitative model to describe the slow [Ca(2+)]i recovery after depolarization in beta-cells from db/db mice. According to the model, the activity levels of the two subtypes of the sarco-endoplasmic reticulum Ca(2+)-ATPase (SERCA) pump, SERCA2 and SERCA3, were severely down-regulated in diabetic cells to 65% and 0% of the levels in normal cells. This down-regulation may lead to a reduction in the Ca(2+) concentration in the ER, a compensatory up-regulation of the plasma membrane Na(+)/Ca(2+) exchanger (NCX) and a reduction in depolarization-evoked Ca(2+) influx. As a result, the patterns of glucose-stimulated calcium oscillations were significantly different in db/db diabetic beta-cells compared with normal cells. Overall, quantifying the changes in the calcium signaling pathway in db/db diabetic beta-cells will aid in the development of a disease model that could provide insight into the adaptive transformations of beta-cell function during diabetes development.

Rat vas deferens SERCA2 is modulated by Ca2+/calmodulin protein kinase II-mediated phosphorylation

Ca2+ pumps are important players in smooth muscle contraction. Nevertheless, little information is available about these pumps in the vas deferens. We have determined which subtype of sarco(endo)plasmic reticulum Ca2+-ATPase isoform (SERCA) is expressed in rat vas deferens (RVD) and its modulation by calmodulin (CaM)-dependent mechanisms. The thapsigargin-sensitive Ca2+-ATPase from a membrane fraction containing the highest SERCA levels in the RVD homogenate has the same molecular mass (∼115 kDa) as that of SERCA2 from the rat cerebellum. It has a very high affinity for Ca2+ (Ca0.5 = 780 nM) and a low sensitivity to vanadate (IC50 = 41 µM). These facts indicate that SERCA2 is present in the RVD. Immunoblotting for CaM and Ca2+/calmodulin-dependent protein kinase II (CaMKII) showed the expression of these two regulatory proteins. Ca2+ and CaM increased serine-phosphorylated residues of the 115-kDa protein, indicating the involvement of CaMKII in the regulatory phosphorylation of SERCA2. Phosphorylation is accompanied by an 8-fold increase of thapsigargin-sensitive Ca2+ accumulation in the lumen of vesicles derived from these membranes. These data establish that SERCA2 in the RVD is modulated by Ca2+ and CaM, possibly via CaMKII, in a process that results in stimulation of Ca2+ pumping activity.