Enhanced Proliferation of Monolayer Cultures of Embryonic Stem (ES) Cell-Derived Cardiomyocytes Following Acute Loss of Retinoblastoma

Background: Cardiomyocyte (CM) cell cycle analysis has been impeded because of a reliance on primary neonatal cultures of poorly proliferating cells or chronic transgenic animal models with innate compensatory mechanisms. Methodology/Principal Findings: We describe an in vitro model consisting of monolayer cultures of highly proliferative embryonic stem (ES) cell-derived CM. Following induction with ascorbate and selection with puromycin, early CM cultures are.98 % pure, and at least 85 % of the cells actively proliferate. During the proliferative stage, cells express high levels of E2F3a, B-Myb and phosphorylated forms of retinoblastoma (Rb), but with continued cultivation, cells stop dividing and mature functionally. This developmental transition is characterized by a switch from slow skeletal to cardiac TnI, an increase in binucleation, cardiac calsequestrin and hypophosphorylated Rb, a decrease in E2F3, B-Myb and atrial natriuretic factor, and the establishment of a more negative resting membrane potential. Although previous publications suggested that Rb was not necessary for cell cycle control in heart, we find following acute knockdown of Rb that this factor actively regulates progression through the G1 checkpoint and that its loss promotes proliferation at the expense of CM maturation. Conclusions/Significance: We have established a unique model system for studying cardiac cell cycle progression, and show in contrast to previous reports that Rb actively regulates both cell cycle progression through the G1 checkpoint an

Low-dose ramipril treatment improves relaxation and calcium cycling after established cardiac hypertrophy

Rapid cooling contractures were used in this study to test whether low-dose ramipril improves sarcoplasmic reticulum (SR) Ca2+ uptake and Na+/Ca2+ exchanger function in isolated hypertrophied rat myocytes. Compensated cardiac hypertrophy was induced by abdominal aortic constriction for 5 wk followed by administration of ramipril (50 μg·kg-1·day-1) or vehicle for 4 wk. Myocyte cell length and cell width were significantly (P < 0.05) increased in both hypertrophied groups (±ramipril). Myocytes were loaded with indo 1, and relaxation was investigated after rapid cooling. Hypertrophied myocyte relaxation in Na+-free/Ca2+-free solution was 63% slower (P < 0.01) and the fall in intracellular Ca2+ was 60% slower (P < 0.05) than the relaxation of control cells. After ramipril treatment both relaxation and the decline in intracellular Ca2+ returned to control rates through improved SR Ca2+-ATPase function. Relaxation in caffeine showed no change after hypertrophy; however, after ramipril treatment the time to 50% relaxation in caffeine decreased by 30% (P < 0.05). The improvement in Ca2+ extrusion across the sarcolemmal membrane occurred independently of changes in Na+/Ca2+ exchanger mRNA and protein abundance. These data demonstrate that ramipril improves both SR-dependent and non-SR-dependent calcium cycling after established cardiac hypertrophy. However, the improvements in function are independent of transcriptional activation and likely to involve altered intracellular ion concentrations.link_to_subscribed_fulltex

Patterns of expression of sarcoplasmic reticulum Ca2+-ATPase and phospholamban mRNAs during rat heart development

This study reports the clonal analysis and sequence of rat phospholamban (PLB) cDNA clones and the temporal appearance and patterns of distribution of the mRNAs encoding sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA2) and PLB in the developing rat heart determined by in situ hybridization. Both proteins play a critical role in the contraction-relaxation cycle of the heart. SERCA2 mRNA is already abundantly present in the first stage studied, in the cardiogenic plate of the 9-day-old presomite embryo, before the occurrence of the first contractions. This very early expression makes it an excellent marker for the study of early heart development. Subsequently, SERCA2 mRNA becomes expressed in a craniocaudal gradient, being highest at the venous pole and decreasing in concentration toward the arterial pole of the heart. PLB mRNA can be detected in hearts from 12 days of development onward in a virtually opposite gradient. In essence, these patterns do not change during further development. PLB mRNA levels remain highest in the ventricle and outflow tract, whereas SERCA2 mRNA prevails in the inflow tract and atrium, although the difference between atrium and ventricle becomes less pronounced. These observations are compatible with a model in which the upstream part of the heart (inflow tract and atrium) would have a greater capacity to clear calcium and hence would have a longer duration of the diastole than the downstream compartments (atrioventricular canal, ventricle, and outflow tract), similar to the observed pattern of contraction of the embryonic heart. The sinoatrial and atrioventricular nodes do not reveal an expression pattern of SERCA2 and PLB mRNA that allows one to distinguish them from the surrounding atrial working myocardium. However, the ventricular part of the conduction system, comprising atrioventricular bundle and bundle branches, are almost devoid of SERCA2 mRNA.link_to_subscribed_fulltex

Sp1 and Sp3 transcription factors are required for trans-activation of the human SERCA2 promoter in cardiomyocytes

Objectives: The sarcoplasmic reticulum (SR) Ca2+ ATPase (SERCA) is essential to the removal of cytosolic calcium following cardiac contraction, and its abundance and activity are significantly altered during perinatal development and in failing myocardium. The objective of the current study was to identify cis regulatory elements and nuclear transcription factors responsible for transactivating SERCA2 gene expression in cardiomyocytes. Methods: Primary cultures of neonatal rat ventricular myocytes were transiently transfected with luciferase (LUX) reporter gene constructs containing deletions of the SERCA2 promoter or which harbored mutations in consensus Sp1 transcription factor binding sites. Cotransfection assays, electrophoretic mobility shift, and supershift assays were also performed to delineate the regulatory role of specific transcription factors. Results: We identified a putative AP-1-like element and a consensus Egr-1 binding site, but neither Egr-1 nor 12-O-tetradecanoylphorbol 13-acetate (TPA) significantly modified human SERCA2 promoter activity in vitro. Maximal activity of the SERCA2 promoter required the proximal 177 bp, and strong activation was observed with a 125-bp construct, within which an Sp1 site and a CAAT box were important. Mutation analysis also revealed the importance of two Sp1 sites between -125 and -200. Sp1 and Sp3 transcription factors were subsequently identified to bind to oligonucleotide probes corresponding to only the two most proximal Sp1 sites. Conclusions: These studies provide direct evidence that regulation of human SERCA2 gene expression in cardiomyocytes depends on transactivation events elicited by Sp1 and Sp3 transcription factors. © 2003 European Society of Cardiology. Published by Elsevier B.V. All rights reserved.link_to_subscribed_fulltex

A distant upstream region of the rat multipartite Na+-Ca2+ exchanger NCX1 gene promoter is sufficient to confer cardiac-specific expression

The Na+-Ca2+ exchanger (NCX) regulates intracellular calcium homeostasis. We report on an upstream region of the rat NCX1 multipartite promoter that is active in cardiac myocytes. Although inactive in most non-cardiac cell lines, its activity can be rescued by cotransfection with GATA-4 and -6, but not GATA-5 transcription factors. In transgenic mice and similar to endogenous NCX1 mRNA expression, the upstream promoter region directs uniform β-galactosidase expression in cardiac myocytes from ∼7.75 dpc. In adult mouse hearts, promoter activity is, however, significantly reduced and heterogeneous, except in the conduction system (sinoatrial and atrioventricular node, atrioventricular bundles). The upstream NCX1 promoter region thus directs appropriate spatial and temporal control of cardiac expression throughout development.link_to_subscribed_fulltex

Inter-brain synchrony and cooperation context in interactive decision making

People engaged in interactive decision making rely on prior decision behaviors by other persons to make new choices and they exhibit inter-brain synchrony between each other. The functional meanings of such inter-brain synchrony, however, remains obscure. In the present study, dyads (15 pairs, all female) played the Prisoner's Dilemma game while their brain activities were recorded simultaneously by electroencephalography (EEG)-based hyperscanning technique. We manipulated the context of the game with higher versus lower cooperation index (HCI vs. LCI) and to each participant, we depicted the interaction as involving either another human partner or a machine (H-H vs. H-M). The results showed a higher cooperation rate and larger theta/alpha-band inter-brain synchrony in condition H-H than in H-M. In the condition H-H, there were larger centrofrontal theta-band and centroparietal alpha-band inter-brain synchrony in tasks set for high cooperation (HCI vs. LCI). Enhanced inter-brain synchrony covaried with increased cooperative choices observed between LCI and HCI. Furthermore, a subjective measure of perceived cooperativeness mediated the relationship between game context and inter-brain synchrony. These findings provide evidence for a role of cooperation on inter-brain synchrony during interactive decision making, and suggest distinct underlying neural processes recruited by cooperation contexts to enable high-level social cognitive processing in decision making.SCOPUS: ar.jinfo:eu-repo/semantics/publishe