Pitx2c modulates cardiac-specific transcription factors networks in differentiating cardiomyocytes from murine embryonic stem cells.

AIM: The knowledge of the molecular signals that control cell differentiation into cardiomyocytes is critical to apply cell-based therapies and repair an injured heart. The transcription factor Pitx2 has essential roles in the development of different organs including the heart. Although a direct role of Pitx2 in the developing myocardium has recently been reported, the molecular pathways driven by Pitx2 as well as its cardiac target genes remain largely unexplored. The aim of this study was to unravel the molecular mechanisms driven by Pitx2 during the process of cardiomyocyte differentiation in vitro in mouse embryonic stem cell-derived cardiomyocytes. METHODS AND RESULTS: Pitx2c was overexpressed in the R1-embryonic stem cell line. mRNA levels and protein distribution of several specific cardiac genes were analyzed by real-time PCR and immunohistochemistry experiments in R1-embryonic stem cell-derived beating areas at different stages of in vitro differentiation. Our results show that overexpression of Pitx2c in embryonic stem cell-derived cardiomyocytes is able to dynamically upregulate several cardiac-enriched transcription factors such as Isl1, Mef2c and Gata4. Additionally, Pitx2c induces the expression of chamber-specific cardiac genes such as Tbx5, Nppa and Cx40. These data were validated in an in vivo model of Pitx2 loss of function. CONCLUSION: Taken together, these results demonstrate that Pitx2 plays a major role reinforcing the transcriptional program of cardiac differentiation.

Designing and Validating a Basketball Learning and Performance Assessment Instrument (BALPAI).

INTRODUCTION: The assessment of learning in basketball in the PE class, and in training sessions of young players, requires valid, reliable, and trustworthy tools. The purpose of this research was to design and validate the Basketball Learning and Performance Assessment Instrument (BALPAI) that assesses simultaneously decision making, technical execution and efficacy. The play actions are codified using a categorical system, awarding a score for each category (1 = inadequate action; 2 = neutral action 3 = adequate action). An example of a summative procedure for assessing decision making in dribbling is: (1) Dribbling to a place where there is defensive pressure and there is a free teammate able to receive the pass; (2) Dribbling to a place where there is defensive pressure or a free teammate able to receive the pass; (3) Dribbling through a space where there is no defensive pressure and no free teammate able to receive the pass. METHODS: A pilot study was performed following this procedure. A group of 13 experts participated in the assessment of the 33 elements (66 items) included in the BALPAI. Aiken's V formula was used to analyze content validity, and internal consistency was calculated using Cronbach's α. Inter-observer reliability was determined among three observers who used the BALPAI to record the play actions in a 3 × 3 basketball match (N = 45 possessions) and was calculated with the Multirater κfree, obtaining an almost perfect agreement with values between 0.84 and 1. RESULTS: The BALPAI has very high internal consistency (0.969), Interobserver reliability was almost perfect (>0.84 in all items) and Aiken's V coefficient (>0.71 in all items) attained a high value. CONCLUSION: The BALPAI proved to be a valid tool, with high internal consistency and reliability that makes it possible to perform a complete assessment of basketball in PE classes.

A role for p38alpha mitogen-activated protein kinase in embryonic cardiac differentiation.

Cardiac differentiation involves cross-regulation of several transcription factors, such as Mef2C, regulated by p38alpha MAP kinase. We analysed the role of p38alpha in cardiac differentiation. Either the absence or inhibition of p38alpha impairs MEF2C nuclear localization in cardiomyocytes, colocalising with vimentin at the perinuclear region. As a consequence, expression of the Mef2C targets, ANF and myocardin, is drastically downregulated. In contrast, Mlc2v and crt are mainly unaltered, probably by the strong Mef2B upregulation, conpensating for the impaired Mef2C transactivity. In addition, p38alpha deficiency leads to a decrease in the phosphorylated Mlc2v fraction and alpha-actinin accumulation causing sarcomere disorganisation. We propose a critical role for p38alpha in early stages of cardiac differentiation by modulation of Mef2C localisation and sarcomeric assembly.

Pitx2c overexpression promotes cell proliferation and arrests differentiation in myoblasts.

Pitx2 is a paired-related homeobox gene that has been shown to play a central role during development. In the mouse, there are three isoforms, Pitx2a, b, and c, which differ only in their amino terminal regions. Pitx2 is expressed in myotomes, myoblasts, and myofibers and may be involved in muscle patterning. However, the mechanism by which Pitx2 acts in muscle cell lineages as well as the distinct functions of the individual isoforms have not been investigated. In this study, we used Sol8 myoblasts to investigate the function of Pitx2 in skeletal myogenesis. We found that Pitx2c is the main Pitx2 isoform present in Sol8 myoblasts. Overexpression of Pitx2c in Sol8 myoblasts inhibited myocyte differentiation and myotube formation. Furthermore, Sol8 cells overexpressing Pitx2c maintained high proliferative capacity and a significant up-regulation of the cell cycle genes cyclin D1, cyclin D2, and c-myc. Gene expression analysis for Pax3 and the s MyoD and myogenin showed that Pitx2c-overexpression caused Sol8 cells to remain as myoblasts, in an undifferentiated myogenic state. Furthermore, down-regulation of the muscle-specific genes sTnI and MyHC3 demonstrated that Sol8-overexpressing Pitx2c myoblasts failed to reach terminal differentiation. This study sheds light on previously unknown functions of the Pitx2c isoform in balancing proliferation vs. differentiation in a myogenic cell line.