Characterization of Skeletal Muscle Regeneration Revealed a Novel Growth Network Induced by Molecular Acupuncture-like Transfection.

The low efficiency of in vivo transfection of a few fibres revealed a novel tissue network that temporally amplified growth stimulation in the entire regenerating rat soleus muscle. This acupuncture-like effect was demonstrated when the fibres began to grow after complete fibre degradation, synchronous inflammation, myoblast and myotube formation. Neonatal sarcoplasmic/endoplasmic reticulum ATPase (SERCA1b) was first detected in this system. The neonatal, fast and slow SERCA isoforms displayed consequent changes with innervation and differentiation, recapitulating events in muscle development. In vivo transfection of myotubes with plasmids expressing dominant negative Ras or a calcineurin inhibitor peptide (Cain/cabin) proved that expression of the slow myosin heavy chain and the slow muscle type SERCA2a are differentially regulated. In vivo transfection of a few nuclei of myotubes with dnRas or SERCA1b shRNA stimulated fibre size growth in the whole regenerating muscle but only until the full size had been reached. Growth stimulation by Ras and SERCA1b antisense was abolished by co-transfection of Cain or with perimuscular injection of IL4 antibody. This revealed a novel signalling network resembling scale-free networks which, starting from transfected fibre myonuclei as "hubs", can amplify growth stimulation uniformly in the entire regenerating muscle.

Effects of MiR-214-3p Regulation of SERCA2a Expression on Contractility of Cardiomyocytes in Heart Failure Model.

This study aimed to explore the targeted regulation of microRNA-214-3p (miR-214-3p) on sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase 2a (SERCA2a) and its mechanism on heart failure (HF). In this study, a rat model of HF was established by injecting isoproterenol to detect the changes in heart function. Then the primary rat cardiomyocytes were extracted and cultured. The cells were divided into the normal group, HF model group, miR-214-3p mimic group, and inhibitor group according to treatment methods. The expression differences of SERCA2a in each group were detected. The binding sites of miR-214-3p and SERCA2a were predicted, wild-type or mutant SERCA2a was prepared and co-transfected into cardiomyocytes with mimic, and the targeting effect was detected by the dual-luciferase reporter gene. Finally, the systolic function of each group was detected by a single-cell systolic dynamic edge detection system. The results showed that cardiac output and left ventricular ejection fraction of HF rats were significantly lower than those of normal rats (P<0.05). The results of the cell test showed that messenger ribonucleic acid (mRNA) and protein expression levels of SERCA2a in the model group and the mimic group were significantly lower than those in the mimic group (P<0.05), but there were no differences between normal group and inhibitor group (P>0.05). Target prediction revealed that miR-214-3p had a complementary pairing of 6 bases with the SERCA2a 3'non-coding region. After co-transfection with miR-214-3p mimic and wild-type SERCA2a expression vector, the dual-luciferase activity was significantly decreased (P<0.05). The percentage of maximal contraction amplitude, peak contraction time, and 50% diastolic time of cells in the model group and mimic group decreased significantly. The mimic group was significantly smaller (P<0.05), but there were no differences between the normal group and the inhibitor group (P>0.05). These results indicated that SERCA2a expression was significantly reduced in HF cells, and miR-214-3p could inhibit SERCA2a expression by targeting the SERCA2a 3'UTR region. Inhibition of miR-214-3p could promote the expression of SERCA2a, which in turn promoted the contractile function of HF rat cardiomyocytes.

A functional genomics pipeline identifies pleiotropy and cross-tissue effects within obesity-associated GWAS loci.

Genome-wide association studies (GWAS) have identified many disease-associated variants, yet mechanisms underlying these associations remain unclear. To understand obesity-associated variants, we generate gene regulatory annotations in adipocytes and hypothalamic neurons across cellular differentiation stages. We then test variants in 97 obesity-associated loci using a massively parallel reporter assay and identify putatively causal variants that display cell type specific or cross-tissue enhancer-modulating properties. Integrating these variants with gene regulatory information suggests genes that underlie obesity GWAS associations. We also investigate a complex genomic interval on 16p11.2 where two independent loci exhibit megabase-range, cross-locus chromatin interactions. We demonstrate that variants within these two loci regulate a shared gene set. Together, our data support a model where GWAS loci contain variants that alter enhancer activity across tissues, potentially with temporally restricted effects, to impact the expression of multiple genes. This complex model has broad implications for ongoing efforts to understand GWAS.

SERCA2a-phospholamban interaction monitored by an interposed circularly permutated green fluorescent protein.

The interaction of phospholamban (PLB) and the sarcoplasmic reticulum Ca2+-ATPase (SERCA2a) is a key regulator of cardiac contractility and a therapeutic target in heart failure (HF). PLB-mediated increases in SERCA2a activity improve cardiac function and HF. Clinically, this mechanism can only be exploited by a general activation of the proteinkinase A (PKA), which is associated with side effects and adverse clinical outcomes. A selective interference of the PLB-SERCA2a interaction is desirable but will require novel tools that allow for an integrated assessment of this interaction under both physiological and pathophysiological conditions. A circularly permutated green fluorescent protein (cpGFP) was interposed between SERCA2a and PLB to result into a single SERCA2a-cpGFP-PLB recombinant protein (SGP). Expression, phosphorylation, fluorescence, and function of SGP were evaluated. Expression of SGP-cDNA results in a functional recombinant protein at the predicted molecular weight. The PLB domain of SGP retains its ability to polymerize and can be phosphorylated by PKA activation. This increases the fluorescent yield of SGP by between 10% and 165% depending on cell line and conditions. In conclusion, a single recombinant fusion protein that combines SERCA2a, a circularly permutated green fluorescent protein, and PLB can be expressed in cells and can be phosphorylated at the PLB domain that markedly increases the fluorescence yield. SGP is a novel cellular SERCA2a-PLB interaction monitor.NEW & NOTEWORTHY This study describes the design and characterization of a novel biosensor that can visualize the interaction of SERCA2a and phospholamban (PLB). The biosensor combines SERCA2a, a circularly permutated green fluorescent protein, and PLB into one recombinant protein (SGP). Proteinkinase A activation results in phosphorylation of the PLB domain and is associated with a marked increase in the fluorescence yield to allow for real-time monitoring of the SERCA2a and PLB interaction in cells.

Ginsenoside-Rb1 Improved Diabetic Cardiomyopathy through Regulating Calcium Signaling by Alleviating Protein O-GlcNAcylation.

Ginsenoside-Rb1 (Rb1), a major active component of ginseng, has many benefits for cardiovascular disease and diabetes mellitus (DM), but the effect and mechanism on diabetic cardiomyopathy are not clear. In the present study, we found that Rb1-feeding significantly improved cardiac dysfunction and abnormal cardiomyocytes calcium signaling caused by diabetes. This improved calcium signaling was because Rb1 reduced Ca2+ leakage caused by overactivated ryanodine receptor 2 (RyR2) and increased Ca2+ uptake by sarcoplasmic reticulum Ca2+-ATPase 2a (SERCA 2a). Furthermore, we found that Rb1 not only enhanced energy metabolism like metformin and eliminated O-GlcNAcylation of calcium handling proteins to regulate calcium signaling but also directly inhibited RyR2 activity to regulate calcium signaling. The present study indicated that as a health supplement or drug, Rb1 was a relatively effective auxiliary therapeutic substance for diabetic cardiomyopathy.

Investigating the functional link between TMEM165 and SPCA1.

TMEM165 was highlighted in 2012 as the first member of the Uncharacterized Protein Family 0016 (UPF0016) related to human glycosylation diseases. Defects in TMEM165 are associated with strong Golgi glycosylation abnormalities. Our previous work has shown that TMEM165 rapidly degrades with supraphysiological manganese supplementation. In this paper, we establish a functional link between TMEM165 and SPCA1, the Golgi Ca2+/Mn2+ P-type ATPase pump. A nearly complete loss of TMEM165 was observed in SPCA1-deficient Hap1 cells. We demonstrate that TMEM165 was constitutively degraded in lysosomes in the absence of SPCA1. Complementation studies showed that TMEM165 abundance was directly dependent on SPCA1's function and more specifically its capacity to pump Mn2+ from the cytosol into the Golgi lumen. Among SPCA1 mutants that differentially impair Mn2+ and Ca2+ transport, only the Q747A mutant that favors Mn2+ pumping rescues the abundance and Golgi subcellular localization of TMEM165. Interestingly, the overexpression of SERCA2b also rescues the expression of TMEM165. Finally, this paper highlights that TMEM165 expression is linked to the function of SPCA1.

Seipin regulates lipid homeostasis by ensuring calcium-dependent mitochondrial metabolism.

Seipin, the gene that causes Berardinelli-Seip congenital lipodystrophy type 2 (BSCL2), is important for adipocyte differentiation and lipid homeostasis. Previous studies in Drosophila revealed that Seipin promotes ER calcium homeostasis through the Ca2+-ATPase SERCA, but little is known about the events downstream of perturbed ER calcium homeostasis that lead to decreased lipid storage in Drosophila dSeipin mutants. Here, we show that glycolytic metabolites accumulate and the downstream mitochondrial TCA cycle is impaired in dSeipin mutants. The impaired TCA cycle further leads to a decreased level of citrate, a critical component of lipogenesis. Mechanistically, Seipin/SERCA-mediated ER calcium homeostasis is important for maintaining mitochondrial calcium homeostasis. Reduced mitochondrial calcium in dSeipin mutants affects the TCA cycle and mitochondrial function. The lipid storage defects in dSeipin mutant fat cells can be rescued by replenishing mitochondrial calcium or by restoring the level of citrate through genetic manipulations or supplementation with exogenous metabolites. Together, our results reveal that Seipin promotes adipose tissue lipid storage via calcium-dependent mitochondrial metabolism.

THE M-RNA, EXPRESSION OF SERCA2 AND NCX1 IN THE PROCESS OF PHARMACOLOGICAL CELL PROTECTION IN EXPERIMENTAL ACUTE PANCREATITIS INDUCED BY TAUROCHOLATE.

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.

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.

Early Right Ventricular Apical Pacing-Induced Gene Expression Alterations Are Associated with Deterioration of Left Ventricular Systolic Function.

The chronic high-dose right ventricular apical (RVA) pacing may have deleterious effects on left ventricular (LV) systolic function. We hypothesized that the expression changes of genes regulating cardiomyocyte energy metabolism and contractility were associated with deterioration of LV function in patients who underwent chronic RVA pacing. Sixty patients with complete atrioventricular block and preserved ejection fraction (EF) who underwent pacemaker implantation were randomly assigned to either RVA pacing (n = 30) group or right ventricular outflow tract (RVOT) pacing (n = 30) group. The mRNA levels of OPA1 and SERCA2a were significantly lower in the RVA pacing group at 1 month's follow-up (both p < 0.001). Early changes in the expression of selected genes OPA1 and SERCA2a were associated with deterioration in global longitudinal strain (GLS) that became apparent months later (p = 0.002 and p = 0.026, resp.) The altered expressions of genes that regulate cardiomyocyte energy metabolism and contractility measured in the peripheral blood at one month following pacemaker implantation were associated with subsequent deterioration in LV dyssynchrony and function in patients with preserved LVEF, who underwent RVA pacing.

Coenzyme Q10 Improves Lipid Metabolism and Ameliorates Obesity by Regulating CaMKII-Mediated PDE4 Inhibition.

Our recent studies revealed that supplementation with the reduced form of coenzyme Q10 (CoQ10H2) inhibits oxidative stress and slows the process of aging in senescence-accelerated mice. CoQ10H2 inhibits adipocyte differentiation and regulates lipid metabolism. In the present study, we show that dietary supplementation with CoQ10H2 significantly reduced white adipose tissue content and improved the function of brown adipose tissue by regulating expression of lipid metabolism-related factors in KKAy mice, a model of obesity and type 2 diabetes. In the liver, CoQ10H2 reduced cytoplasmic Ca2+ levels and consequently inhibited the phosphorylation of CaMKII. CoQ10H2 also regulated the activity of the transcription factor C-FOS and inhibited gene expression of PDE4, a cAMP-degrading enzyme, via the CaMKII-MEK1/2-ERK1/2 signaling pathway, thereby increasing intracellular cAMP. This increased cAMP activated AMPK, enhanced oxidative decomposition of lipids, and inhibited de novo synthesis of fatty acids, inhibiting the development and progression of obesity and type 2 diabetes. These results suggest that CoQ10H2 supplementation may be useful as a treatment for metabolic disorders associated with obesity.

The modified Yi qi decoction protects cardiac ischemia-reperfusion induced injury in rats.

BACKGROUND: To investigate the effects and involved mechanisms of the modified Yi Qi decoction (MYQ) in cardiac ischemia-reperfusion (IR) induced injury. METHODS: Male Sprague-Dawley rats were subjected to a 30-min coronary arterial occlusion followed by reperfusion, low or high dose decoction of MYQ was administrated orally for 1 week or 1 month. RESULTS: Both in 1 week and 1 month IR rat groups, cardiac function indexes were significantly impaired compared with sham group rats, accompanied with higher ratio of infarct size to risk size, decreased expressions of sodium calcium exchanger (NCX1) and sarcoplasmic reticulum Ca2+-ATPase (Serca2a), and different expressions of autophagic proteins, Beclin-1 and LC3. Treatment with MYQ (low or high dose) for 1 week showed no marked beneficial effects on cardiac function and cardiac injury (ratio of infarct size to risk size), although expressions of anti-apoptotic protein, Bcl-2, NCX1 and Serca2a were increased. Treatment with MYQ (low or high dose) for 1 month showed significantly improved effects on cardiac function and cardiac injury (ratio of infarct size to risk size), accompanied with increase of Bcl-2, NCX1 and Serca2a expressions, and decrease of Bax (a pro-apoptotic protein) and Beclin-1 expressions. CONCLUSIONS: The results show that MYQ have potential therapeutic effects on IR-induced cardiac injury, which may be through regulation of apoptotic proteins, cytosolic Ca2+ handling proteins and autophagic proteins signal pathways.

Randomized Clinical Trials of Gene Transfer for Heart Failure with Reduced Ejection Fraction.

Despite improvements in drug and device therapy for heart failure, hospitalization rates and mortality have changed little in the past decade. Randomized clinical trials using gene transfer to improve function of the failing heart are the focus of this review. Four randomized clinical trials of gene transfer in heart failure with reduced ejection fraction (HFrEF) have been published. Each enrolled patients with stable symptomatic HFrEF and used either intracoronary delivery of a virus vector or endocardial injection of a plasmid. The initial CUPID trial randomized 14 subjects to placebo and 25 subjects to escalating doses of adeno-associated virus type 1 encoding sarcoplasmic reticulum calcium ATPase (AAV1.SERCA2a). AAV1.SERCA2a was well tolerated, and the high-dose group met a 6 month composite endpoint. In the subsequent CUPID-2 study, 243 subjects received either placebo or the high dose of AAV1.SERCA2a. AAV1.SERCA2a administration, while safe, failed to meet the primary or any secondary endpoints. STOP-HF used plasmid endocardial injection of stromal cell-derived factor-1 to promote stem-cell recruitment. In a 93-subject trial of patients with ischemic etiology heart failure, the primary endpoint (symptoms and 6 min walk distance) failed, but subgroup analyses showed improvements in subjects with the lowest ejection fractions. A fourth trial randomized 14 subjects to placebo and 42 subjects to escalating doses of adenovirus-5 encoding adenylyl cyclase 6 (Ad5.hAC6). There were no safety concerns, and patients in the two highest dose groups (combined) showed improvements in left ventricular function (left ventricular ejection fraction and -dP/dt). The safety data from four randomized clinical trials of gene transfer in patients with symptomatic HFrEF suggest that this approach can be conducted with acceptable risk, despite invasive delivery techniques in a high-risk population. Additional trials are necessary before the approach can be endorsed for clinical practice.

Caryocar brasiliense oil improves cardiac function by increasing Serca2a/PLB ratio despite no significant changes in cardiovascular risk factors in rats.

BACKGROUND: Caryocar brasiliense (pequi) oil is high in monounsaturated fat acids (MUFA), especially oleic, and in carotenoids, which have been associated with protection against cardiovascular disease. However, this food is poorly studied in this context, especially in the cardiac function. Therefore, we investigated the effects of a long-term intake of pequi oil in systemic cardiovascular risk factors and in the ex vivo cardiac function of rats. METHODS: Previously, we determined fatty acids and carotenoids in pequi oil. Next, male rats were divided in C - control group feed a standard diet, and PO - pequi oil group fed the same diet added pequi oil (+2.25 g.100 g-1). After 15 weeks, plasma lipids, glucose, insulin, blood pressure, heart rate, hepatic lipids were accessed and visceral fat pads were harvested. Hearts were used for the ex vivo cardiac function, histologic assays, SERCA2a and phospholanban (PLB) determinations. RESULTS: In agreement with scientific data, pequi oil had expressive amounts MUFA, especially oleic acid, and carotenoids. Hepatic triglycerides (TG) were reduced by pequi oil intake (p < 0.05). All others cardiovascular risk factors were not changed. The intrinsic heart rate was lower in PO group (p < 0.05). SERCA2a content was higher in this group (p < 0.05), without affecting PLB. Also, SERCA2a/PLB ratio increased in PO group (p < 0.05). CONCLUSION: Pequi oil intake improved cardiac function ex vivo, despite no significant changes in systemic cardiovascular risk factors. The higher lipid offer in pequi oil diet, its composition in oleic acid and carotenoids could be related to those effects.

High-Throughput Spectral and Lifetime-Based FRET Screening in Living Cells to Identify Small-Molecule Effectors of SERCA.

A robust high-throughput screening (HTS) strategy has been developed to discover small-molecule effectors targeting the sarco/endoplasmic reticulum calcium ATPase (SERCA), based on a fluorescence microplate reader that records both the nanosecond decay waveform (lifetime mode) and the complete emission spectrum (spectral mode), with high precision and speed. This spectral unmixing plate reader (SUPR) was used to screen libraries of small molecules with a fluorescence resonance energy transfer (FRET) biosensor expressed in living cells. Ligand binding was detected by FRET associated with structural rearrangements of green fluorescent protein (GFP, donor) and red fluorescent protein (RFP, acceptor) fused to the cardiac-specific SERCA2a isoform. The results demonstrate accurate quantitation of FRET along with high precision of hit identification. Fluorescence lifetime analysis resolved SERCA's distinct structural states, providing a method to classify small-molecule chemotypes on the basis of their structural effect on the target. The spectral analysis was also applied to flag interference by fluorescent compounds. FRET hits were further evaluated for functional effects on SERCA's ATPase activity via both a coupled-enzyme assay and a FRET-based calcium sensor. Concentration-response curves indicated excellent correlation between FRET and function. These complementary spectral and lifetime FRET detection methods offer an attractive combination of precision, speed, and resolution for HTS.

Effect of electroacupuncture on gene expression in calcium signaling pathway in hippocampal cells in mice with cerebral ischemia reperfusion.

OBJECTIVE: To observe the regulation of electroacupuncture on gene expression at calcium signaling pathways in mice with cerebral ischemia reperfusion. METHODS: Sixty male, inbred Kunming mice were randomly assigned to three groups: repeated cerebral ischemia reperfusion group (RG, n = 24), sham-operated group (SG, n = 12), and electroacupuncture group (EG, n = 24). Mice in RG and EG groups were modeled by repeated cerebral ischemia reperfusion surgery, and EG mice were treated with electroacupuncture for 30 min after recovery from anesthesia. Changes in gene expression profile of mice hippocampi were analyzed by global expression profile microarray. Genes that were up-regulated or down-regulated greater than 1.5 folds were considered to be biologically meaningful. Real-time quantitative polymerase chain reaction (q-PCR) method was used to verify the expression of selected genes based on the algorithm [2^ (ΔΔCt)]. RESULTS: Compared with SG mice, 242 genes showed different in expressions in RG mice: 107 down-regulated and 135 up-regulated. Compared with RG mice, 609 genes showed a difference of expression in EG mice: 315 down-regulated and 375 up-regulated. Gene ontology and Kyoto Encyclopedia of Genes and Genomes analyses indicated two pathways: calcium signaling and long-term potentiation in which 11 differentially expressed genes selected. Six of the 11 genes in the calcium signaling pathway were verified after real-time q-PCR testing. CONCLUSION: Electroacupuncture treatment of cerebral ischemia reperfusion appears to regulate Atp2a2, Cacna1e, Camk2a, Gnas, Grm1, Rapgef3 genes in the calcium signaling pathway.

Rhodiola Inhibits Atrial Arrhythmogenesis in a Heart Failure Model.

INTRODUCTION: Rhodiola, a popular plant in Tibet, has been proven to decrease arrhythmia. The aim of this study was to elucidate the molecular mechanism and electrophysiological properties of rhodiola in the suppression of atrial fibrillation. METHODS: This study consisted of 3 groups as follows: Group 1: normal control rabbits (n = 5); Group 2: rabbits with heart failure (HF) created by coronary ligation and who received 2 weeks of water orally as a placebo (n = 5); and Group 3: rabbits with HF who received 2 weeks of a rhodiola 270 mg/kg/day treatment orally (n = 5). The monophasic action potential, histology, and real-time polymerase chain reaction (RT-PCR) analysis of ionic channels and PI3K/AKT/eNOS were examined. RESULTS: Compared with the HF group, attenuated atrial fibrosis (35.4 ± 17.4% vs. 16.9 ± 8.4%, P = 0.05) and improved left ventricular (LV) ejection fraction (51.6 ± 3.4% vs. 68.0 ± 0.5%, P = 0.001) were observed in the rhodiola group. The rhodiola group had a shorter ERP (85.3 ± 6.8 vs. 94.3 ± 1.2, P = 0.002), APD90 (89.3 ± 1.5 vs. 112.7 ± 0.7, P < 0.001) in the left atrium (LA), and decreased AF inducibility (0.90 ± 0.04 vs. 0.42 ± 0.04, P < 0.001) compared with the HF group. The mRNA expressions of Kv1.4, Kv1.5, Kv4.3, KvLQT1, Cav1.2, and SERCA2a in the HF LA were up-regulated after rhodiola treatment. The rhodiola-treated HF LA demonstrated higher mRNA expression of PI3K-AKT compared with the HF group. CONCLUSIONS: Rhodiola reversed LA electrical remodeling, attenuated atrial fibrosis and suppressed AF in rabbits with HF. The beneficial electrophysiological effect of rhodiola may be related to upregulation of Kv1.4, Kv1.5, Kv4.3, KvLQT1, Cav1.2, SERCA2a, and activation of PI3K/AKT signaling.

Gene therapy for heart failure.

Heart failure is a major public health problem throughout the world and it is likely that its prevalence will continue to grow over the next several decades. Despite advances in the treatment of heart failure, morbidity and mortality remain unacceptably high. Gene transfer therapy provides a novel strategy for targeting abnormalities in cardiac cells that adversely affect cardiac function. New vectors for gene delivery, mainly adeno-associated viruses (AAVs) that are preferentially taken up by cardiomyocytes, can result in sustained transgene expression. The cardiac isoform of sarco(endo)plasmic reticulum Ca(2+)ATPase (SERCA2a) plays a major role in regulating calcium levels in cardiomyocytes. Abnormal calcium handling by the failing heart caused by a reduction in SERCA2a activity adversely affects both systolic and diastolic function. The Calcium Upregulation by Percutaneous Administration of Gene Therapy in Cardiac Disease (CUPID) study was a Phase 2a double-blind, randomized, placebo-controlled, dose-finding study that was performed in patients with advanced heart failure due to systolic dysfunction. Eligible patients received AAV/SERCA2a or placebo by direct antegrade infusion into the coronary circulation. At the end of 12 months, patients receiving high-dose therapy (i.e. 1×10(13) DNase Resistant Particles) had evidence of favorable changes in several clinically relevant domains compared to patients treated with placebo. There were no safety concerns at any dose of AAV/SERCA2a. Patients treated with AAV/SERCA2a exhibited a striking reduction in cardiovascular events that persisted through 36 months of follow-up compared to patients who received placebo. Transgene expression was detected in the myocardium of patients receiving AAV/SERCA2a gene therapy as long as 31 months after delivery. A second Phase 2b study, CUPID 2, designed to confirm this favorable effect on heart failure events, is currently underway with the results expected to be presented later in 2015. Additional studies using other vectors and targets are in planning or underway making gene transfer therapy one of the most exciting new approaches under development for treating heart failure.

Safety and efficacy of high-dose adeno-associated virus 9 encoding sarcoplasmic reticulum Ca(2+) adenosine triphosphatase delivered by molecular cardiac surgery with recirculating delivery in ovine ischemic cardiomyopathy.

OBJECTIVE: Therapeutic safety and efficacy are the basic prerequisites for clinical gene therapy. We investigated the effect of high-dose molecular cardiac surgery with recirculating delivery (MCARD)-mediated adeno-associated virus 9 (AAV9)/sarcoplasmic reticulum Ca(2+) adenosine triphosphatase (SERCA2a) gene delivery on clinical parameters, oxidative stress, humoral and cellular immune responses, and cardiac remodeling. METHODS: Ischemic cardiomyopathy was generated in a sheep model. The sheep were assigned to 1 of 2 groups: control (n = 10) and study (MCARD, n = 6). The control group underwent no intervention and the study group received 10(14) genome copies of AAV9/SERCA2a 4 weeks after infarction. RESULTS: Our ischemic model produced reliable infarcts leading to heart failure. The baseline ejection fraction in the MCARD group was 57.6% ± 1.6% versus 61.2% ± 1.9% in the control group (P > .05). At 12 weeks after infarction, the MCARD group had superior left ventricular function compared with the control group: stroke volume index, 46.6 ± 1.8 versus 35.8 ± 2.5 mL/m(2) (P < .05); ejection fraction, 46.2% ± 1.9% versus 38.7% ± 2.5% (P < .05); and left ventricular end-systolic and end-diastolic dimensions, 41.3 ± 1.7 versus 48.2 ± 1.4 mm and 51.2 ± 1.5 versus 57.6 ± 1.7 mm, respectively (P < .05). The markers of oxidative stress were significantly reduced in the infarct zone in the MCARD group. No positive T-cell-mediated immune response was seen in the MCARD group at any point. Myocyte hypertrophy was also significantly attenuated in the MCARD group compared with the control group. CONCLUSIONS: Cardiac overexpression of the SERCA2a gene by way of MCARD is a safe therapeutic intervention. It significantly improves left ventricular function, decreases markers of oxidative stress, abrogates myocyte hypertrophy, arrests remodeling, and does not induce a T-cell-mediated immune response.

Oestrogen upregulates the sarcoplasmic reticulum Ca(2+) ATPase pump in coronary arteries.

The presence of circulating plasma 17ß-oestradiol (E2) is beneficial in women against abnormal vascular tone development, such as coronary arterial vasospasms. Several vascular diseases have demonstrated that increased expression of the sarcoplasmic reticulum Ca(2+) -ATPase pump (SERCA2b) serves to limit the excessive accumulation of intracellular Ca(2+) . Therefore, the hypothesis of the present study was that E2 would increase SERCA2b expression in the coronary vasculature. Coronary arteries were dissected from hearts obtained from mature female pigs. Artery segments were cultured for 24 h in E2 (1 pmol/L or 1 nmol/L) and homogenized for western blot analysis. At 1 nmol/L, E2 induced an approximate 50% increase in immunoreactivity for SERCA2b. In addition, E2 increased the protein expression of the known SERCA regulatory proteins, protein kinase A (PKA) and protein kinase G (PKG). The E2-induced increase in SERCA2b was attenuated when the culture medium was supplemented with the oestrogen receptor (ER) α/ß antagonist ICI 182,780 and the PKG antagonist KT5823 (10 µmol/L, 24 h for both). The PKA antagonist (KT5720; 10 µmol/L, 24 h) had no effect on SERCA2b expression. Removal of the endothelium (using a wooden toothpick) from artery segments prior to culture decreased the E2-mediated increase in SERCA2b and PKG expression by 45% and 47%, respectively. Overall, the findings suggest that one of the potential cardiovascular benefits of E2 in women is upregulation of SERCA2b, via activation of the classic ERα and ERß pathway.