LncRNA sequencing in human cardiac myocytes (HCMs), human cardiac fibroblasts-adult ventricular (HCF-av) and human cardiac microvascular endothelial cells (HCMECs) from a single fetal donor
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE124402 | GEO | 2019/01/15
REPOSITORIES: GEO
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Project description:LncRNA sequencing in human cardiac myocytes (HCMs), human cardiac fibroblasts-adult ventricular (HCF-av) and human cardiac microvascular endothelial cells (HCMECs) from a single fetal donor
| PRJNA511951 | ENA
Project description:Ventricular septal defects (VSD) are the most common form of congenital heart disease, which is the leading non-infectious cause of death in children; nevertheless, the exact cause of VSD is not yet fully understood. Long non-coding RNAs (lncRNAs) have been shown to play key roles in various biological processes, such as imprinting control, circuitry controlling pluripotency and differentiation, immune responses and chromosome dynamics. Notably, a growing number of lncRNAs have been implicated in disease etiology, although an association with VSD has not been reported. In the present study, we conducted an integrated analysis of dysregulated lncRNAs, focusing specifically on the identification and characterization of lncRNAs potentially involving in initiation of VSD. Comparison of the transcriptome profiles of cardiac tissues from VSD-affected and normal hearts was performed using a second-generation lncRNA microarray, which covers the vast majority of expressed RefSeq transcripts (29,241 lncRNAs and 30,215 coding transcripts). In total, 880 lncRNAs were upregulated and 628 were downregulated in VSD. Furthermore, our established filtering pipeline indicated an association of two lncRNAs, ENST00000513542 and RP11-473L15.2, with VSD. This dysregulation of the lncRNA profile provides a novel insight into the etiology of VSD and furthermore, illustrates the intricate relationship between coding and ncRNA transcripts in cardiac development. These data may offer a background/reference resource for future functional studies of lncRNAs related to VSD.
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Project description:Applications of human induced pluripotent stem cell derived-cardiac myocytes (hiPSC-CMs) would be strengthened by the ability to generate specific cardiac myocyte (CM) lineages. However, purification of lineage-specific hiPSC-CMs is limited by the lack of cell marking techniques. Here, we have developed an iPSC-CM marking system using recombinant adenoviral reporter constructs with atrial- or ventricular-specific myosin light chain-2 (MLC-2) promoters. MLC-2a and MLC-2v selected hiPSC-CMs were purified by fluorescence-activated cell sorting and their biochemical and electrophysiological phenotypes analyzed. We demonstrate that the phenotype of both populations remained stable in culture and they expressed the expected sarcomeric proteins, gap junction proteins and chamber-specific transcription factors. Compared to MLC-2a cells, MLC-2v selected CMs had larger action potential amplitudes and durations. In addition, by immunofluorescence, we showed that MLC-2 isoform expression can be used to enrich hiPSC-CM consistent with early atrial and ventricular myocyte lineages. However, only the ventricular myosin light chain-2 promoter was able to purify a highly homogeneous population of iPSC-CMs. Using this approach, it is now possible to develop ventricular-specific disease models using iPSC-CMs while atrial-specific iPSC-CM cultures may require additional chamber-specific markers.
| S-EPMC4148042 | biostudies-literature
Project description:Compartmentation of cAMP signaling is a critical factor for maintaining the integrity of receptor-specific responses in cardiac myocytes. This phenomenon relies on various factors limiting cAMP diffusion. Our previous work in adult rat ventricular myocytes (ARVMs) indicates that PKA regulatory subunits anchored to the outer membrane of mitochondria play a key role in buffering the movement of cytosolic cAMP. PKA can be targeted to discrete subcellular locations through the interaction of both type I and type II regulatory subunits with A-kinase anchoring proteins (AKAPs). The purpose of this study is to identify which AKAPs and PKA regulatory subunit isoforms are associated with mitochondria in ARVMs. Quantitative PCR data demonstrate that mRNA for dual specific AKAP1 and 2 (D-AKAP1 & D-AKAP2), acyl-CoA-binding domain-containing 3 (ACBD3), optic atrophy 1 (OPA1) are most abundant, while Rab32, WAVE-1, and sphingosine kinase type 1 interacting protein (SPHKAP) were barely detectable. Biochemical and immunocytochemical analysis suggests that D-AKAP1, D-AKAP2, and ACBD3 are the predominant mitochondrial AKAPs exposed to the cytosolic compartment in these cells. Furthermore, we show that both type I and type II regulatory subunits of PKA are associated with mitochondria. Taken together, these data suggest that D-AKAP1, D-AKAP2, and ACBD3 may be responsible for tethering both type I and type II PKA regulatory subunits to the outer mitochondrial membrane in ARVMs. In addition to regulating PKA-dependent mitochondrial function, these AKAPs may play an important role by buffering the movement of cAMP necessary for compartmentation.
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Project description:Sodium channel ?-subunits in ventricular myocytes (VMs) segregate either to the intercalated disc or to lateral membranes, where they associate with region-specific molecules.To determine the functional properties of sodium channels as a function of their location in the cell.Local sodium currents were recorded from adult rodent VMs and Purkinje cells by using the cell-attached macropatch configuration. Electrodes were placed either in the cell midsection (M) or at the cell end (area originally occupied by the intercalated disc [ID]). Channels were identified as tetrodotoxin (TTX)-sensitive (TTX-S) or TTX-resistant (TTX-R) by application of 100 nM of TTX.Average peak current amplitude was larger in ID than in M and largest at the site of contact between attached cells. TTX-S channels were found only in the M region of VMs and not in Purkinje myocytes. TTX-R channels were found in both M and ID regions, but their biophysical properties differed depending on recording location. Sodium current in rat VMs was upregulated by tumor necrosis factor-alpha. The magnitude of current increase was largest in the M region, but this difference was abolished by application of 100 nM of TTX.Our data suggest that (a) a large fraction of TTX-R (likely Na(v)1.5) channels in the M region of VMs are inactivated at normal resting potential, leaving most of the burden of excitation to TTX-R channels in the ID region; (b) cell-cell adhesion increases functional channel density at the ID; and (c) TTX-S (likely non-Na(v)1.5) channels make a minimal contribution to sodium current under control conditions, but they represent a functional reserve that can be upregulated by exogenous factors.
| S-EPMC3208741 | biostudies-literature
Project description:Cardiovascular disease is often related to defects of subcellular components in cardiac myocytes, specifically in the dyadic cleft, which include changes in cleft geometry and channel placement. Modeling of these pathological changes requires both spatially resolved cleft as well as whole cell level descriptions. We use a multiscale model to create dyadic structure-function relationships to explore the impact of molecular changes on whole cell electrophysiology and calcium cycling. This multiscale model incorporates stochastic simulation of individual L-type calcium channels and ryanodine receptor channels, spatially detailed concentration dynamics in dyadic clefts, rabbit membrane potential dynamics, and a system of partial differential equations for myoplasmic and lumenal free Ca2+ and Ca2+-binding molecules in the bulk of the cell. We found action potential duration, systolic, and diastolic [Ca2+] to respond most sensitively to changes in L-type calcium channel current. The ryanodine receptor channel cluster structure inside dyadic clefts was found to affect all biomarkers investigated. The shape of clusters observed in experiments by Jayasinghe et al. and channel density within the cluster (characterized by mean occupancy) showed the strongest correlation to the effects on biomarkers.
| S-EPMC6990380 | biostudies-literature
Project description:Persistent over-activation of CaMKII (Calcium/Calmodulin-dependent protein Kinase II) in the heart is implicated in arrhythmias, heart failure, pathological remodeling, and other cardiovascular diseases. Several post-translational modifications (PTMs)-including autophosphorylation, oxidation, S-nitrosylation, and O-GlcNAcylation-have been shown to trap CaMKII in an autonomously active state. The molecular mechanisms by which these PTMs regulate calmodulin (CaM) binding to CaMKIIδ-the primary cardiac isoform-has not been well-studied particularly in its native myocyte environment. Typically, CaMKII activates upon Ca-CaM binding during locally elevated [Ca]free and deactivates upon Ca-CaM dissociation when [Ca]free returns to basal levels. To assess the effects of CaMKIIδ PTMs on CaM binding, we developed a novel FRET (Förster resonance energy transfer) approach to directly measure CaM binding to and dissociation from CaMKIIδ in live cardiac myocytes. We demonstrate that autophosphorylation of CaMKIIδ increases affinity for CaM in its native environment and that this increase is dependent on [Ca]free. This leads to a 3-fold slowing of CaM dissociation from CaMKIIδ (time constant slows from ~0.5 to 1.5 s) when [Ca]free is reduced with physiological kinetics. Moreover, oxidation further slows CaM dissociation from CaMKIIδ T287D (phosphomimetic) upon rapid [Ca]free chelation and increases FRET between CaM and CaMKIIδ T287A (phosphoresistant). The CaM dissociation kinetics-measured here in myocytes-are similar to the interval between heartbeats, and integrative memory would be expected as a function of heart rate. Furthermore, the PTM-induced slowing of dissociation between beats would greatly promote persistent CaMKIIδ activity in the heart. Together, these findings suggest a significant role of PTM-induced changes in CaMKIIδ affinity for CaM and memory under physiological and pathophysiological processes in the heart.
| S-EPMC8716136 | biostudies-literature
Project description:Cardiac tissue undergoes renewal with low rates. Although resident stem cell populations have been identified to support cardiomyocyte turnover, the source of the cardiac stem cells and their niche remain elusive. Using Cre/Lox-based cell lineage tracing strategies, we discovered that labeling of endothelial cells in the adult heart yields progeny that have cardiac stem cell characteristics and express Gata4 and Sca1. Endothelial-derived cardiac progenitor cells were localized in the arterial coronary walls with quiescent and proliferative cells in the media and adventitia layers, respectively. Within the myocardium, we identified labeled cardiomyocytes organized in clusters of single-cell origin. Pulse-chase experiments showed that generation of individual clusters was rapid but confined to specific regions of the heart, primarily in the right anterior and left posterior ventricular walls and the junctions between the two ventricles. Our data demonstrate that endothelial cells are an intrinsic component of the cardiac renewal process.
| S-EPMC4101892 | biostudies-literature
Project description:Sarcomeres are the basic contractile units of cardiac myocytes. Recent studies demonstrated remodeling of sarcomeric proteins in several diseases, including genetic defects and heart failure. Here we investigated remodeling of sarcomeric α-actinin in two models of heart failure, synchronous (SHF) and dyssynchronous heart failure (DHF), as well as a model of cardiac resynchronization therapy (CRT). We applied three-dimensional confocal microscopy and quantitative methods of image analysis to study isolated cells from our animal models. 3D Fourier analysis revealed a decrease of the spatial regularity of the α-actinin distribution in both SHF and DHF versus control cells. The spatial regularity of α-actinin in DHF cells was reduced when compared with SHF cells. The spatial regularity of α-actinin was partially restored after CRT. We found longitudinal depositions of α-actinin in SHF, DHF and CRT cells. These depositions spanned adjacent Z-disks and exhibited a lower density of α-actinin than in the Z-disk. Differences in the occurrence of depositions between the SHF, CRT and DHF models versus control were significant. Also, CRT cells exhibited a higher occurrence of depositions versus SHF, but not DHF cells. Other sarcomeric proteins did not accumulate in the depositions to the same extent as α-actinin. We did not find differences in the expression of α-actinin protein and its encoding gene in our animal models. In summary, our studies indicate that HF is associated with two different types of remodeling of α-actinin and only one of those was reversed after CRT. We suggest that these results can guide us to an understanding of remodeling of structures and function associated with sarcomeres.
| S-EPMC4077200 | biostudies-literature
Project description:The N(1325)S mutation in the cardiac sodium channel gene SCN5A causes the type-3 long-QT syndrome but the arrhythmogenic trigger associated with N(1325)S has not been characterized. In this study, we investigated the triggers for cardiac events in the expanded N(1325)S family. Among 11 symptomatic patients with document triggers, six died suddenly during sleep or while sitting (bradycardia-induced trigger), three died suddenly, and two developed syncope due to stress and excitement (non-bradycardia-induced). Patch-clamping studies revealed that the late sodium current (I(Na,L)) generated by mutation N(1325)S in ventricular myocytes from TG-NS/LQT3 mice was reduced with increased pacing, which explains bradycardia-induced mortalities in the family. The non-bradycardic triggers are related to the finding that APD became prolonged and unstable at increasing rates, often with alternating repolarization phases which was corrected with verapamil. This implies that Ca2+ influx and intracellular Ca2+ ([Ca2+]i) ions are involved and that [Ca2+]i inhomogeneity may be the underlying mechanisms behind non-bradycardia LQT3 arrhythmogenesis associated with mutation N(1325)S.
| S-EPMC1773013 | biostudies-literature