Pitx2 Differentially Regulates the Distinct Phases of Myogenic Program and Delineates Satellite Cell Lineages During Muscle Development

The knowledge of the molecular mechanisms that regulate embryonic myogenesis from early myogenic progenitors to myoblasts, as well as the emergence of adult satellite stem cells (SCs) during development, are key concepts to understanding the genesis and regenerative abilities of the skeletal muscle. Several previous pieces of evidence have revealed that the transcription factor Pitx2 might be a player within the molecular pathways controlling somite-derived muscle progenitors’ fate and SC behavior. However, the role exerted by Pitx2 in the progression from myogenic progenitors to myoblasts including SC precursors remains unsolved. Here, we show that Pitx2 inactivation in uncommitted early myogenic precursors diminished cell proliferation and migration leading to muscle hypotrophy and a low number of SCs with decreased myogenic differentiation potential. However, the loss of Pitx2 in committed myogenic precursors gave rise to normal muscles with standard amounts of SCs exhibiting high levels of Pax7 expression. This SC population includes few MYF5+ SC-primed but increased amount of less proliferative miR-106b+cells, and display myogenic differentiation defects failing to undergo proper muscle regeneration. Overall our results demonstrate that Pitx2 is required in uncommitted myogenic progenitors but it is dispensable in committed precursors for proper myogenesis and reveal a role for this transcription factor in the generation of diverse SC subpopulations.BFU2015-67131 (Spanish Ministery of Economy and Competitiveness)PID2019- 107492GB-100 (Spanish Ministry of Science and Innovation

miRNAs and Muscle Stem Cells

Skeletal muscle represents between 30 and 38% of the human body mass. Both the maintenance and repair of adult muscle tissue are directed by satellite cells (SCs). SCs are located beneath the basal lamina of the skeletal muscle myofiber. They are quiescent for most of their life but, in response to physiological stimuli or muscle trauma, they activate, proliferate, and enter the myogenic program via generating myogenic progenitors (myoblasts) that fuse to existing myofibers or de novo myofibers. MicroRNAs (miRNAs or miRs) play a critical role in regulating muscle regeneration and stem cell behavior. In this chapter, we review the pivotal role in the regulation of SC quiescence, activation, and differentiation in the context of muscular dystrophies

Novel PITX2 Homeodomain-Contained Mutations from ATRIAL Fibrillation Patients Deteriorate Calcium Homeostasis

Atrial fibrillation (AF) is the most common cardiac arrhythmia in the human population, with an estimated incidence of 1¿2% in young adults but increasing to more than 10% in 80+ years patients. Pituitary Homeobox 2, Paired Like Homeodomain 2 (PITX2c) loss-of-function in mice revealed that this homeodomain (HD)-containing transcription factor plays a pivotal role in atrial electrophysiology and calcium homeostasis and point to PITX2 as a candidate gene for AF. To address this issue, we recruited 31 AF patients for genetic analyses of both the known risk alleles and PITX2c open reading frame (ORF) re-sequencing. We found two-point mutations in the homedomain of PITX2 and three other variants in the 5¿untranslated region. A 65 years old male patient without 4q25 risk variants but with recurrent AF displayed two distinct HD-mutations, NM_000325.5:c.309G>C (Gln103His) and NM_000325.5:c.370G>A (Glu124Lys), which both resulted in a change within a highly conserved amino acid position. To address the functional impact of the PITX2 HD mutations, we generated plasmid constructs with mutated version of each nucleotide variant (MD4 and MD5, respectively) as well as a dominant negative control construct in which the PITX2 HD was lacking (DN). Functional analyses demonstrated PITX2c MD4 and PITX2c MD5 decreased Nppa-luciferase transactivation by 50% and 40%, respectively, similar to the PITX2c DN (50%), while Shox2 promoter repression was also impaired. Co-transactivation with other cardiac-enriched co-factors, such as Gata4 and Nkx2.5, was similarly impaired, further supporting the pivotal role of these mutations for correct PITX2c function. Furthermore, when expressed in HL1 cardiomyocyte cultures, the PITX2 mutants impaired endogenous expression of calcium regulatory proteins and induced alterations in sarcoplasmic reticulum (SR) calcium accumulation. This favored alternating and irregular calcium transient amplitudes, causing deterioration of the beat-to-beat stability upon elevation of the stimulation frequency. Overall this data demonstrate that these novel PITX2c HD-mutations might be causative of atrial fibrillation in the carrier.This work was supported by grants from The Spanish Ministry of Science Innovation and Universities [SAF2017-88019-C3-1-R] to L.H.-M. V.J.-S. was employed by CIBERCV [RD12/0042/0002] grant. Work was also supported by a PhD scholarship [FPU18/01250] to S.C., and partially funded by grants from Generalitat de Catalunya [SGR2017-1769] and Fundació Marato TV3 [20152030] to L.H.-M., a translational CNIC grant [2009/08] to D.F., R.C. and L.H.-M. and a grant-in-aid from the Junta de Andalucia Regional Council to D.F. and A.A. [CTS-446]

The 4q25 variant rs13143308T links risk of atrial fibrillation to defective calcium homeostasis

Aims: Single nucleotide polymorphisms on chromosome 4q25 have been associated with risk of atrial fibrillation (AF) but the exiguous knowledge of the mechanistic links between these risk variants and underlying electrophysiological alterations hampers their clinical utility. Here, we here tested the hypothesis that 4q25 risk variants cause alterations in the intracellular calcium homeostasis that predispose to spontaneous electrical activity. Methods and results: Western blotting, confocal calcium imaging, and patch-clamp techniques were used to identify mechanisms linking the 4q25 risk variants rs2200733T and rs13143308T to defects in the calcium homeostasis in human atrial myocytes. Our findings revealed that the rs13143308T variant was more frequent in patients with AF and that myocytes from carriers of this variant had a significantly higher density of calcium sparks (14.1±4.5 vs. 3.1±1.3 events/min, p¿=¿0.02), frequency of transient inward (ITI) currents (1.33±0.24 vs. 0.26±0.09 events/min, p¿<¿0.001) and incidence of spontaneous membrane depolarizations (1.22±0.26 vs. 0.56±0.17 events/min, p¿=¿0.001) than myocytes from patients with the normal rs13143308G variant. These alterations were linked to higher sarcoplasmic reticulum calcium loading (10.2±1.4 vs. 7.3±0.5amol/pF, p¿=¿0.01), SERCA2 expression (1.37±0.13 fold, p¿=¿0.03) and RyR2 phosphorylation at s2808 (0.67±0.08 vs. 0.47±0.03, p¿=¿0.01) but not at s2814 (0.28±0.14 vs. 0.31±0.14, p¿=¿0.61) in patients carrying the rs13143308T risk variant. Furthermore, the presence of a risk variant or AF independently increased the ITI frequency and the increase in the ITI frequency observed in carriers of the risk variants was exacerbated in those with AF. By contrast, the presence of a risk variant did not affect the amplitude or properties of the L-type calcium current in patients with or without AF. Conclusions: We here identify the 4q25 variant rs13143308T as a genetic risk marker for AF, specifically associated with excessive calcium release and spontaneous electrical activity linked to increased SERCA2 expression and RyR2 phosphorylationThis work was supported by multi-centric grants from Centro Nacional de Investigaciones Cardiovasculares [CNIC-2009-08 to L.H.-M. and D.F.]; a grant from Fundacio´ Marato´ TV3 [2015-20-30 to L.H.-M.]; and grants from the Spanish Ministry of Economy and Competition [SAF2014-58286-C2-1-R to L.H.-M.] and [DPI2013-44584-R to R.B.]; and from the Spanish Ministry of Health and Consume, Instituto de Salud Carlos III, Red de Investigacio´n Cardiovascular [RD12/0042/0002] and CIBERCV to J.C., and from Fondo Europeo de Desarrollo Regional (FEDER)

Tobacco cessation among smokers under substance use treatment for alcohol and/or cannabis: study protocol and pilot study

Background: Approximately 80% of people with a substance use disorder (SUD) are smokers. Starting SUD treatment offers the opportunity to also quit smoking. The ACT-ATAC project aims to identify the predictors associated with smoking cessation among persons treated for alcohol and/or cannabis use disorder in Barcelona. This manuscript reports its methodology and the experience of carrying it out during the COVID-19 pandemic. Methods: Mixed methods project with three substudies. Substudy 1 (S1) comprises heterogeneous discussion groups among clinicians. S2 has two prospective cohorts composed of smokers under treatment for alcohol and/or cannabis use disorder and the clinicians in charge of these patients. Participating smokers will be followed for 12 months and interviewed about their substance use and the tobacco cessation services received using the Spanish version of the users' Knowledge, Attitudes, and Services (S-KAS) scale. The clinicians will be asked about their self-reported practices in smoking cessation using the Knowledge, Attitudes, and Practices (S-KAP) scale. S3 comprises heterogeneous discussion groups with smokers. Data will be triangulated using qualitative and quantitative analyses. To facilitate the recruitment process, the researchers have introduced several strategies (design clear protocols, set monthly online meetings, extend the project, provide gift cards, etc.). Discussion: The results of S1 were used to develop the questionnaires. S2 required some adjustments due to the COVID-19 pandemic, particularly the follow-up interviews being conducted by phone instead of face-to-face, and the recruitment rhythm was lower than expected. Recruitment will last until reaching at least 200-250 users. The fieldwork could not have been possible without the collaboration of the ACT-ATAC team and the introduction of several strategies

More than Just a Simple Cardiac Envelope; Cellular Contributions of the Epicardium

The adult pumping heart is formed by distinct tissue layers. From inside to outside, the heart is composed by an internal endothelial layer, dubbed the endocardium, a thick myocardial component which supports the pumping capacity of the heart and exteriorly covered by a thin mesothelial layer named the epicardium. Cardiac insults such as coronary artery obstruction lead to ischemia and thus to an irreversible damage of the myocardial layer, provoking in many cases heart failure and death. Thus, searching for new pathways to regenerate the myocardium is an urgent biomedical need. Interestingly, the capacity of heart regeneration is present in other species, ranging from fishes to neonatal mammals. In this context, several lines of evidences demonstrated a key regulatory role for the epicardial layer. In this manuscript, we provide a state-of-the-art review on the developmental process leading to the formation of the epicardium, the distinct pathways controlling epicardial precursor cell specification and determination and current evidences on the regenerative potential of the epicardium to heal the injured heart

Non-Coding RNAs in the Cardiac Action Potential and Their Impact on Arrhythmogenic Cardiac Diseases

Cardiac arrhythmias are prevalent among humans across all age ranges, affecting millions of people worldwide. While cardiac arrhythmias vary widely in their clinical presentation, they possess shared complex electrophysiologic properties at cellular level that have not been fully studied. Over the last decade, our current understanding of the functional roles of non-coding RNAs have progressively increased. microRNAs represent the most studied type of small ncRNAs and it has been demonstrated that miRNAs play essential roles in multiple biological contexts, including normal development and diseases. In this review, we provide a comprehensive analysis of the functional contribution of non-coding RNAs, primarily microRNAs, to the normal configuration of the cardiac action potential, as well as their association to distinct types of arrhythmogenic cardiac diseases

Pitx2 in Embryonic and Adult Myogenesis

Skeletal muscle is a heterogeneous tissue that represents between 30 and 38% of the human body mass and has important functions in the organism, such as maintaining posture, locomotor impulse, or pulmonary ventilation. The genesis of skeletal muscle during embryonic development is a process controlled by an elaborate regulatory network combining the interplay of extrinsic and intrinsic regulatory mechanisms that transform myogenic precursor cells into functional muscle fibers through a finely tuned differentiation program. However, the capacity of generating muscle still remains once these fibers have matured. Adult myogenesis resembles many of the embryonic morphogenetic episodes and depends on the activation of satellite cells that have the potential to differentiate into new muscle fibers. Pitx2 is a member of the bicoid family of homeodomain transcription factors that play an important role in morphogenesis. In the last decade, Pitx2 has emerged as a key element involved in the fine-tuning mechanism that regulates skeletal-muscle development as well as the differentiation and cell fate of satellite cells in adult muscle. Here we present an integrative view of all aspects of embryonic and adult myogenesis in which Pitx2 is involved, from embryonic development to satellite-cell proliferation, fate specification, and differentiation. Those new Pitx2 functions on satellite-cell biology might open new perspectives to develop therapeutic strategies for muscular disorders

Genomic and Non-Genomic Regulatory Mechanisms of the Cardiac Sodium Channel in Cardiac Arrhythmias

Nav1.5 is the predominant cardiac sodium channel subtype, encoded by the SCN5A gene, which is involved in the initiation and conduction of action potentials throughout the heart. Along its biosynthesis process, Nav1.5 undergoes strict genomic and non-genomic regulatory and quality control steps that allow only newly synthesized channels to reach their final membrane destination and carry out their electrophysiological role. These regulatory pathways are ensured by distinct interacting proteins that accompany the nascent Nav1.5 protein along with different subcellular organelles. Defects on a large number of these pathways have a tremendous impact on Nav1.5 functionality and are thus intimately linked to cardiac arrhythmias. In the present review, we provide current state-of-the-art information on the molecular events that regulate SCN5A/Nav1.5 and the cardiac channelopathies associated with defects in these pathways

Cardiac conduction system anomalies and sudden cardiac death: insights from murine models

The cardiac conduction system (CCS) is a series of specialized tissues in the heart responsible for the initiation and co-ordination of the heartbeat. Alterations in the CCS, especially the His-Purkinje system, have been identified as an important player in the generation of lethal arrhythmias. Unstable arrhythmias secondary to channelopathies highly increase the risk of sudden cardiac death (SCD). Sudden cardiac death is a major contributor to mortality in industrialized nations, and most cases of SCD in the young are related to inherited ion channel diseases. In this review we examine how murine transgenic models have contributed to understanding that a broad variety of cardiac arrhythmias involve the cardiac specialized conduction system and may lead to sudden cardiac death