Atrial chamber-specific expression of sarcolipin is regulated during development and hypertrophic remodeling

Intracellular Ca2+ regulation is critical in the normal cardiac function and development of pathologic hearts. Phospholamban, an endogenous inhibitor of sarcoplasmic reticulum Ca2+ ATPase in the sarcoplasmic reticulum, plays an important role in Ca2+ cycling in heart. Recently, sarcolipin has been identified as having a similar function as phospholamban in skeletal muscle. Because phospholamban is differentially expressed in atrial and ventricular myocardia and its expression is often altered in diseased hearts, we investigated the cardiac chamber specificity of sarcolipin expression and its regulation during development and hypertrophic remodeling. Northern blot analysis revealed that the expression of mouse sarcolipin mRNA was most abundant in the atria and was undetectable in the ventricles, indicating an atrial chamber-specific expression pattern. Atrial chamber-specific expression of sarcolipin mRNA was increased during development. These findings were confirmed by in situ hybridization studies. In addition, sarcolipin expression was down-regulated in the atria of hypertrophic heart when induced by ventricular specific overexpression of the activated H-ras gene. In humans, sarcolipin mRNA was also expressed in the atria but not detected in the ventricles, although sarcolipin expression was most abundant in skeletal muscle. Taken together, sarcolipin is likely to be an atrial chamber-specific regulator of Ca2+ cycling in heart.     

Temporal patterns of tyrosine nitration in embryo heart development

Tyrosine nitration is a biomarker for the production of peroxynitrite and other reactive nitrogen species. Nitrotyrosine immunoreactivity is present in many pathological conditions including several cardiac diseases. Because the events observed during heart failure may recapitulate some aspects of development, we tested whether nitrotyrosine is present during normal development of the rat embryo heart and its potential relationship in cardiac remodeling through apoptosis. Nitric oxide production is highly dynamic during development, but whether peroxynitrite and nitrotyrosine are formed during normal embryonic development has received little attention. Rat embryo hearts exhibited strong nitrotyrosine immunoreactivity in endocardial and myocardial cells of the atria and ventricles from E12 to E18. After E18, nitrotyrosine staining faded and disappeared entirely by birth. Tyrosine nitration in the myocardial tissue coincided with elevated protein expression of nitric oxide synthases (eNOS and iNOS). The immunoreactivity for these NOS isoforms remained elevated even after nitrotyrosine had disappeared. Tyrosine nitration did not correlate with cell death or proliferation of cardiac cells. Analysis of tryptic peptides by MALDI-TOF showed that nitration occurs in actin, myosin, and the mitochondrial ATP synthase α chain. These results suggest that reactive nitrogen species are not restricted to pathological conditions but may play a role during normal embryonic development.     

Dynamic expression of N‐myc in mouse embryonic development using an enhanced green fluorescent protein reporter gene in the N‐myc locus

N‐myc belongs to the Myc oncogene family and plays an essential role in mammalian embryonic development. The expression of N‐myc is dynamically regulated during embryonic development; however, its expression pattern has not been well characterized due to the lack of a suitable animal model. In this paper, a genetically modified mouse model was generated in which the enhanced green fluorescent protein (EGFP) coding sequence was inserted into the N‐myc locus, so that endogenous N‐myc expression could be traced by the signal of EGFP. The EGFP signal in the transgenic mouse was confirmed to be consistent with the expression pattern of endogenous N‐myc by fluorescence microscopy and immunohistochemical staining. Furthermore, the spatial and temporal expression of EGFP was observed in the central and peripheral nervous system, heart, lung and kidney, given the known indispensable role of N‐myc in their formation. EGFP was also strongly detected in the liver, paranephros and the epithelium of the intestine. The EGFP signal can be used to trace N‐myc expression in this transgenic mouse model. N‐myc expression was observed in specific locations and cell lineages, and dynamically changed during embryonic development. The changing N‐myc expression pattern seen in mouse embryonic development and the animal model described in this paper provide important insights and a new tool to research N‐myc function.     

A series of Cre‐ERT2 drivers for manipulation of the skeletal muscle lineage

We report the generation of five mouse strains with the tamoxifen‐inducible Cre (Cre‐ER^T2; CE) gene cassette knocked into the endogenous loci of Pax3, Myod1, Myog, Myf6, and Myl1, collectively as a resource for the skeletal muscle research community. We characterized these CE strains using the Cre reporter mice, R26R^LacZ, during embryogenesis and show that they direct tightly controlled tamoxifen‐inducible reporter expression within the expected cell lineage determined by each myogenic gene. We also examined a few selected adult skeletal muscle groups for tamoxifen‐inducible reporter expression. None of these new CE alleles direct reporter expression in the cardiac muscle. All these alleles follow the same knock‐in strategy by replacing the first exon of each gene with the CE cassette, rendering them null alleles of the endogenous gene. Advantages and disadvantages of this design are discussed. Although we describe potential immediate use of these strains, their utility likely extends beyond foreseeable questions in skeletal muscle biology. genesis 52:759–770, 2014. © 2014 Wiley Periodicals, Inc.     

Msx1creERT2 knock‐In allele: A useful tool to target embryonic and adult cardiac valves

Summary : Heart valve development begins with the endothelial‐to‐mesenchymal transition (EMT) of endocardial cells. Although lineage studies have demonstrated contributions from cardiac neural crest and epicardium to semilunar and atrioventricular (AV) valve formation, respectively, most valve mesenchyme derives from the endocardial EMT. Specific Cre mouse lines for fate‐mapping analyses of valve endocardial cells are limited. Msx1 displayed expression in AV canal endocardium and cushion mesenchyme between E9.5 and E11.5, when EMT is underway. Additionally, previous studies have demonstrated that deletion of Msx1 and its paralog Msx2 results in hypoplastic AV cushions and impaired endocardial signaling. A knock‐in tamoxifen‐inducible Cre line was recently generated (Msx1^CreERT2) and characterized during embryonic development and after birth, and was shown to recapitulate the endogenous Msx1 expression pattern. Here, we further analyze this knock‐in allele and track the Msx1‐expressing cells and their descendants during cardiac development with a particular focus on their contribution to the valves and their precursors. Thus, Msx1^CreERT2 mice represent a useful model for lineage tracing and conditional gene manipulation of endocardial and mesenchymal cushion cells essential to understand mechanisms of valve development and remodeling. genesis 53:337–345, 2015. © 2015 Wiley Periodicals, Inc.     

Epidermal growth factor‐like domain 7 is a marker of the endothelial lineage and active angiogenesis

Epidermal growth factor‐like domain 7 (Egfl7) expression in the developing embryo is largely restricted to sites of mesodermal progenitors of angioblasts/hemangioblasts and the vascular endothelium. We hypothesize that Egfl7 marks the endothelial lineage during embryonic development, and can be used to define the emergence of endothelial progenitor cells, as well as to visualize newly‐forming vasculature in the embryo and during the processes of physiologic and pathologic angiogenesis in the adult. We have generated a transgenic mouse strain that expresses enhanced green fluorescent protein (eGFP) under the control of a minimal Egfl7 regulatory sequence (Egfl7:eGFP). Expression of the transgene recapitulated that of endogenous Egfl7 at sites of vasculogenesis and angiogenesis in the allantois, yolk sac, and in the embryo proper. The transgene was not expressed in the quiescent endothelium of most adult organs. However, the uterus and ovary, which undergo vascular growth and remodeling throughout the estrus cycle, expressed high levels of Egfl7:eGFP. Importantly, expression of the Egfl7:eGFP transgene was induced in adult neovasculature. We also found that increased Egfl7 expression contributed to pathologic revascularization in the mouse retina. To our knowledge, this is the first mouse model that enables monitoring of endothelial cells at sites of active vasculogenesis and angiogenesis. This model also facilitated the isolation and characterization of EGFL7⁺ endothelial cell populations by fluorescence activated cell sorting (FACS). Together, our results demonstrate that the Egfl7:eGFP reporter mouse is a valuable tool that can be used to elucidate the mechanisms by which blood vessels form during development and under pathologic circumstances. genesis 52:657–670, 2014. © 2014 Wiley Periodicals, Inc.     

Generation of a tamoxifen inducible Tnnt2MerCreMer knock‐in mouse model for cardiac studies

Tnnt2, encoding thin‐filament sarcomeric protein cardiac troponin T, plays critical roles in heart development and function in mammals. To develop an inducible genetic deletion strategy in myocardial cells, we generated a new Tnnt2:MerCreMer (Tnnt2^MerCreMer/+) knock‐in mouse. Rosa26 reporter lines were used to examine the specificity and efficiency of the inducible Cre recombinase. We found that Cre was specifically and robustly expressed in the cardiomyocytes at embryonic and adult stages following tamoxifen induction. The knock‐in allele on Tnnt2 locus does not impact cardiac function. These results suggest that this new Tnnt2^MerCreMer/+ mouse could be applied towards the temporal genetic deletion of genes of interests in cardiomyocytes with Cre‐LoxP technology. The Tnnt2^MerCreMer/+ mouse model also provides a useful tool to trace myocardial lineage during development and repair after cardiac injury. genesis 53:377–386, 2015. © 2015 Wiley Periodicals, Inc.     

Changes in short‐chain acyl‐coA dehydrogenase during rat cardiac development and stress

This study was designed to investigate the expression of short‐chain acyl‐CoA dehydrogenase (SCAD), a key enzyme of fatty acid β‐oxidation, during rat heart development and the difference of SCAD between pathological and physiological cardiac hypertrophy. The expression of SCAD was lowest in the foetal and neonatal heart, which had time‐dependent increase during normal heart development. In contrast, a significant decrease in SCAD expression was observed in different ages of spontaneously hypertensive rats (SHR). On the other hand, swim‐trained rats developed physiological cardiac hypertrophy, whereas SHR developed pathological cardiac hypertrophy. The two kinds of cardiac hypertrophy exhibited divergent SCAD changes in myocardial fatty acids utilization. In addition, the expression of SCAD was significantly decreased in pathological cardiomyocyte hypertrophy, however, increased in physiological cardiomyocyte hypertrophy. SCAD siRNA treatment triggered the pathological cardiomyocyte hypertrophy, which showed that the down‐regulation of SCAD expression may play an important role in pathological cardiac hypertrophy. The changes in peroxisome proliferator‐activated receptor α (PPARα) was accordant with that of SCAD. Moreover, the specific PPARα ligand fenofibrate treatment increased the expression of SCAD and inhibited pathological cardiac hypertrophy. Therefore, we speculate that the down‐regulated expression of SCAD in pathological cardiac hypertrophy may be responsible for ‘the recapitulation of foetal energy metabolism’. The deactivation of PPARα may result in the decrease in SCAD expression in pathological cardiac hypertrophy. Changes in SCAD are different in pathological and physiological cardiac hypertrophy, which may be used as the molecular markers of pathological and physiological cardiac hypertrophy.