Calsequestrins new calcium store markers of adult Zebrafish cerebellum and optic tectum

Calcium stores in neurons are heterogeneous in compartmentalization and molecular composition. Danio rerio (zebrafish) is an animal model with a simply folded cerebellum similar in cellular organization to that of mammals. The aim of the study was to identify new endoplasmic reticulum (ER) calcium store markers in zebrafish adult brain with emphasis on cerebellum and optic tectum. By quantitative polymerase chain reaction, we found three RNA transcripts coding for the intra-ER calcium binding protein calsequestrin: casq1a, casq1b, and casq2. In brain homogenates, two isoforms were detected by mass spectrometry and western blotting. Fractionation experiments of whole brain revealed that Casq1a and Casq2 were enriched in a heavy fraction containing ER microsomes and synaptic membranes. By in situ hybridization, we found the heterogeneous expression of casq1a and casq2 mRNA to be compatible with the cellular localization of calsequestrins investigated by immunofluorescence. Casq1 was expressed in neurogenic differentiation 1 expressing the granule cells of the cerebellum and the periventricular zone of the optic tectum. Casq2 was concentrated in parvalbumin expressing Purkinje cells. At a subcellular level, Casq1 was restricted to granular cell bodies, and Casq2 was localized in cell bodies, dendrites, and axons. Data are discussed in relation to the differential cellular and subcellular distribution of other cerebellum calcium store markers and are evaluated with respect to the putative relevance of calsequestrins in the neuron-specific functional activity

The role of connexin40 in developing atrial conduction

AbstractConnexin40 (Cx40) is the main connexin expressed in the murine atria and ventricular conduction system. We assess here the developmental role of Cx40 in atrial conduction of the mouse. Cx40 deficiency significantly prolonged activation times in embryonic day 10.5, 12.5 and 14.5 atria during spontaneous activation; the severity decreased with increasing age. In a majority of Cx40 deficient mice the impulse originated from an ectopic focus in the right atrial appendage; in such a case the activation time was even longer due to prolonged activation. Cx40 has thus an important physiological role in the developing atria

Structure and regulation of the mouse cardiac troponin I gene

The gene coding for mouse cardiac troponin I (TnI) has been cloned and sequenced. The cardiac TnI gene contains 8 exons and has an exon-intron organization similar to the quail fast skeletal TnI gene except for the region of exons 1-3, which is highly divergent. Comparative analysis suggests that cardiac TnI exon 1 corresponds to fast TnI exons 1 and 2 and that cardiac exon 3, which codes for most of the cardiac-specific amino-terminal extension and has no counterpart in the fast gene, evolved by exon insertion/deletion. The amino acid sequence of cardiac TnI exon 4 shows limited homology (36% identity) with fast TnI exon 4 but is remarkably similar (79% identity) to the corresponding sequence of slow TnI, possibly reflecting an isoform-specific TnC-binding site. The cardiac TnI gene is one of the very few contractile protein genes expressed exclusively in cardiac muscle. To identify the regulatory sequences responsible for the cardiac-specific expression of this gene we transfected cultured cardiac and skeletal muscle cells with fragments up to 4.0 kilobases of the 5'-flanking region linked to a reporter gene. Deletion analysis reveals four major regions in the 5'-flanking sequence, a minimal promoter region, which directs expression at low level in cardiac and skeletal muscle cells, and two upstream cardiac-specific positive regions separated by a negative region

Pitx2 Expression Defines a Left Cardiac Lineage of Cells: Evidence for Atrial and Ventricular Molecular Isomerism in the iv/iv Mice

AbstractThe homeobox gene Pitx2 has been characterized as a mediator of left-right signaling in heart, gut, and lung morphogenesis. However, the relationship between the developmental role of Pitx2 and its expression pattern at the organ level has not been explored. In this study we focus on the role of Pitx2 in heart morphogenesis. Chicken Pitx2 transcripts are present in the left portion of the cardiac crescent and in the left side of the heart tube. Through looping Pitx2 is present in the left atrium, in the ventral portion of the ventricles and in the left-ventral part of the outflow tract. Mouse Pitx2 shows a similar developmental profile of expression. To test whether Pitx2 represents a lineage marker we have tagged the left portion of the chicken cardiac tube with fluorescent DiD. Labeled cells were found at HH16 in the left atrium and in the ventral region of the ventricles and the outflow tract. In the iv/iv mouse model of cardiac heterotaxia Pitx2 was abnormally expressed in the atrial and in the ventricular chambers. Furthermore, altered Pitx2 expression correlated with the occurrence of DORV. Our data reveal the existence of molecular isomerism not only in the atrial, but also in the ventricular compartment of the heart

Optogenetic determination of the myocardial requirements for extrasystoles by cell type-specific targeting of ChannelRhodopsin-2

Extrasystoles lead to several consequences, ranging from uneventful palpitations to lethal ventricular arrhythmias, in the presence of pathologies, such as myocardial ischemia. The role of working versus conducting cardiomyocytes, as well as the tissue requirements (minimal cell number) for the generation of extrasystoles, and the properties leading ectopies to become arrhythmia triggers (topology), in the normal and diseased heart, have not been determined directly in vivo. Here, we used optogenetics in transgenic mice expressing ChannelRhodopsin-2 selectively in either cardiomyocytes or the conduction system to achieve cell type-specific, noninvasive control of heart activity with high spatial and temporal resolution. By combining measurement of optogenetic tissue activation in vivo and epicardial voltage mapping in Langendorff-perfused hearts, we demonstrated that focal ectopies require, in the normal mouse heart, the simultaneous depolarization of at least 1,300–1,800 working cardiomyocytes or 90–160 Purkinje fibers. The optogenetic assay identified specific areas in the heart that were highly susceptible to forming extrasystolic foci, and such properties were correlated to the local organization of the Purkinje fiber network, which was imaged in three dimensions using optical projection tomography. Interestingly, during the acute phase of myocardial ischemia, focal ectopies arising from this location, and including both Purkinje fibers and the surrounding working cardiomyocytes, have the highest propensity to trigger sustained arrhythmias. In conclusion, we used cell-specific optogenetics to determine with high spatial resolution and cell type specificity the requirements for the generation of extrasystoles and the factors causing ectopies to be arrhythmia triggers during myocardial ischemia

Chamber Formation and Morphogenesis in the Developing Mammalian Heart

AbstractIn this study we challenge the generally accepted view that cardiac chambers form from an array of segmental primordia arranged along the anteroposterior axis of the linear and looping heart tube. We traced the spatial pattern of expression of genes encoding atrial natriuretic factor, sarcoplasmic reticulum calcium ATPase, Chisel, Irx5, Irx4, myosin light chain 2v, and β-myosin heavy chain and related these to morphogenesis. Based on the patterns we propose a two-step model for chamber formation in the embryonic heart. First, a linear heart forms, which is composed of “primary” myocardium that nonetheless shows polarity in phenotype and gene expression along its anteroposterior and dorsoventral axes. Second, specialized ventricular chamber myocardium is specified at the ventral surface of the linear heart tube, while distinct left and right atrial myocardium forms more caudally on laterodorsal surfaces. The process of looping aligns these primordial chambers such that they face the outer curvature. Myocardium of the inner curvature, as well as that of inflow tract, atrioventricular canal, and outflow tract, retains the molecular signature originally found in linear heart tube myocardium. Evidence for distinct transcriptional programs which govern compartmentalization in the forming heart is seen in the patterns of expression of Hand1 for the dorsoventral axis, Irx4 and Tbx5 for the anteroposterior axis, and Irx5 for the distinction between primary and chamber myocardium

Pitx2 confers left morphological, molecular, and functional identity to the sinus venosus myocardium

AIMS: The sinus venous myocardium, comprising the sinoatrial node (SAN) and sinus horns (SH), is a region subject to congenital malformations and cardiac arrhythmias. It differentiates from symmetric bilateral mesenchymal precursors, but morphological, molecular, and functional left/right differences are progressively established through development. The role of the laterality gene Pitx2 in this process is unknown. We aimed to elucidate the molecular events driving left/right patterning in the sinus venosus (SV) myocardium by using a myocardial Pitx2 knockout mouse. METHODS AND RESULTS: We generated a myocardial specific Pitx2 knockout model (cTP mice). cTP embryos present several features of Pitx2 null, including right atrial isomerism with bilateral SANs and symmetric atrial entrance of the systemic veins. By in situ hybridization and optical mapping analysis, we compared throughout development the molecular and functional properties of the SV myocardium in wt and mutant embryos. We observed that Pitx2 prevents the expansion of the left-SAN primordium at the onset of its differentiation into myocardium; Pitx2 promotes expansion of the left SH through development; Pitx2 dose-dependently represses the autorhythmic properties of the left SV myocardium at mid-gestation (E14.5); Pitx2 modulates late foetal gene expression at the left SH-derived superior caval vein. CONCLUSION: Pitx2 drives left/right patterning of the SV myocardium through multiple developmental steps. Overall, Pitx2 plays a crucial functional role by negatively modulating a nodal-type programme in the left SV myocardium

Current Perspectives in Cardiac Laterality

The heart is the first organ to break symmetry in the developing embryo and onset of dextral looping is the first indication of this event. Looping is a complex process that progresses concomitantly to cardiac chamber differentiation and ultimately leads to the alignment of the cardiac regions in their final topology. Generation of cardiac asymmetry is crucial to ensuring proper form and consequent functionality of the heart, and therefore it is a highly regulated process. It has long been known that molecular left/right signals originate far before morphological asymmetry and therefore can direct it. The use of several animal models has led to the characterization of a complex regulatory network, which invariably converges on the Tgf-β signaling molecule Nodal and its downstream target, the homeobox transcription factor Pitx2. Here, we review current data on the cellular and molecular bases of cardiac looping and laterality, and discuss the contribution of Nodal and Pitx2 to these processes. A special emphasis will be given to the morphogenetic role of Pitx2 and to its modulation of transcriptional and functional properties, which have also linked laterality to atrial fibrillation

Homeobox transcription factor Pitx2: The rise of an asymmetry gene in cardiogenesis and arrhythmogenesis

The homeobox transcription factor Pitx2 displays a highly specific expression pattern during embryogenesis. Gain and loss of function experiments have unraveled its pivotal role in left-right signaling. Conditional deletion in mice has demonstrated a complex and intricate role for Pitx2 in distinct aspects of cardiac development and more recently a link to atrial fibrillation has been proposed based on genome-wide association studies. In this review we will revise the role of Pitx2 in the developing heart, starting from the early events of left-right determination followed by its role in cardiac morphogenesis and ending with its role in cardiac arrhythmogenesi