NOTCH1 is critical for fibroblast-mediated induction of cardiomyocyte specialization into ventricular conduction system-like cells in vitro
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ABSTRACT:
SUBMITTER: Ribeiro da Silva A
PROVIDER: S-EPMC7527973 | biostudies-literature | 2020 Jan
REPOSITORIES: biostudies-literature
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Project description:RationaleIn virtually all models of heart failure, prognosis is determined by right ventricular (RV) function; thus, understanding the cellular mechanisms contributing to RV dysfunction is critical. Whole organ remodeling is associated with cell-specific changes, including cardiomyocyte dedifferentiation and activation of cardiac fibroblasts (Cfib) which in turn is linked to disorganization of cytoskeletal proteins and loss of sarcomeric structures. However, how these cellular changes contribute to RV function remains unknown. We've previously shown significant organ-level RV dysfunction in a large animal model of pulmonary hypertension (PH) which was not mirrored by reduced function of isolated cardiomyocytes. We hypothesized that factors produced by the endogenous Cfib contribute to global RV dysfunction by generating a heterogeneous cellular environment populated by dedifferentiated cells.ObjectiveTo determine the effect of Cfib conditioned media (CM) from the PH calf (PH-CM) on adult rat ventricular myocytes (ARVM) in culture.Methods and resultsBrief exposure (<2 days) to PH-CM results in rapid, marked dedifferentiation of ARVM to a neonatal-like phenotype exhibiting spontaneous contractile behavior. Dedifferentiated cells maintain viability for over 30 days with continued expression of cardiomyocyte proteins including TnI and α-actinin yet exhibit myofibroblast characteristics including expression of α-smooth muscle actin. Using a bioinformatics approach to identify factor(s) that contribute to dedifferentiation, we found activation of the PH Cfib results in a unique transcriptome correlating with factors both in the secretome and with activated pathways in the dedifferentiated myocyte. Further, we identified upregulation of periostin in the Cfib and CM, and demonstrate that periostin is sufficient to drive cardiomyocyte dedifferentiation.ConclusionsThese data suggest that paracrine factor(s) released by Cfib from the PH calf signal a phenotypic transformation in a population of cardiomyocytes that likely contributes to RV dysfunction. Therapies targeting this process, such as inhibition of periostin, have the potential to prevent RV dysfunction.
| S-EPMC6677314 | biostudies-literature
Project description:RationaleThe heartbeat is organized by the cardiac conduction system (CCS), a specialized network of cardiomyocytes. Patterning of the CCS into atrial node versus ventricular conduction system (VCS) components with distinct physiology is essential for the normal heartbeat. Distinct node versus VCS physiology has been recognized for more than a century, but the molecular basis of this regional patterning is not well understood.ObjectiveTo study the genetic and genomic mechanisms underlying node versus VCS distinction and investigate rhythm consequences of failed VCS patterning.Methods and resultsUsing mouse genetics, we found that the balance between T-box transcriptional activator, Tbx5, and T-box transcriptional repressor, Tbx3, determined the molecular and functional output of VCS myocytes. Adult VCS-specific removal of Tbx5 or overexpression of Tbx3 re-patterned the fast VCS into slow, nodal-like cells based on molecular and functional criteria. In these cases, gene expression profiling showed diminished expression of genes required for VCS-specific fast conduction but maintenance of expression of genes required for nodal slow conduction physiology. Action potentials of Tbx5-deficient VCS myocytes adopted nodal-specific characteristics, including increased action potential duration and cellular automaticity. Removal of Tbx5 in vivo precipitated inappropriate depolarizations in the atrioventricular (His)-bundle associated with lethal ventricular arrhythmias. TBX5 bound and directly activated cis-regulatory elements at fast conduction channel genes required for fast physiological characteristics of the VCS action potential, defining the identity of the adult VCS.ConclusionsThe CCS is patterned entirely as a slow, nodal ground state, with a T-box dependent, physiologically dominant, fast conduction network driven specifically in the VCS. Disruption of the fast VCS gene regulatory network allowed nodal physiology to emerge, providing a plausible molecular mechanism for some lethal ventricular arrhythmias.
| S-EPMC8328577 | biostudies-literature
Project description:Rationale: In virtually all models of heart failure, prognosis is determined by right ventricular (RV) function; thus, understanding the cellular mechanisms contributing to RV dysfunction is critical. Whole organ remodeling is associated with cell-specific changes, including cardiomyocyte dedifferentiation and activation of cardiac fibroblasts (Cfib) which in turn is linked to disorganization of cytoskeletal proteins and loss of sarcomeric structures. However, how these cellular changes contribute to RV function remains unknown. We’ve previously shown significant organ-level RV dysfunction in a large animal model of pulmonary hypertension (PH) which was not mirrored by reduced function of isolated cardiomyocytes. We hypothesized that factors produced by the endogenous Cfib contribute to global RV dysfunction by generating a heterogeneous cellular environment populated by dedifferentiated cells. Objective: To determine the effect of Cfib conditioned media (CM) from the PH calf (PH-CM) on adult rat ventricular myocytes (ARVM) in culture. Methods and Results: Brief exposure (<2 days) to PH-CM results in rapid, marked dedifferentiation of ARVM to a neonatal-like phenotype exhibiting spontaneous contractile behavior. Dedifferentiated cells maintain viability for over 30 days with continued expression of cardiomyocyte proteins including TnI and α-actinin yet exhibit myofibroblast characteristics including expression of α-smooth muscle actin. Using a bioinformatics approach to identify factor(s) that contribute to dedifferentiation, we found activation of the PH Cfib results in a unique transcriptome correlating with factors both in the secretome and with activated pathways in the dedifferentiated myocyte. Further, we identified upregulation of periostin in the Cfib and CM, and demonstrate that periostin is sufficient to drive cardiomyocyte dedifferentiation. Conclusions: These data suggest that paracrine factor(s) released by Cfib from the PH calf signal a phenotypic transformation in a population of cardiomyocytes that likely contributes to RV dysfunction. Therapies targeting this process, such as inhibition of periostin, have the potential to prevent RV dysfunction.
2019-07-07 | GSE133904 | GEO
Project description:Cardiac Purkinje cells (PCs) comprise the most distal portion of the ventricular conduction system (VCS) and are essential for synchronous activation of the ventricular myocardium. Contactin-2 (CNTN2), a member of the immunoglobulin superfamily cell adhesion molecules (IgSF-CAMs), was previously identified as a marker of the VCS. Through differential transcriptional profiling we discovered two additional highly enriched IgSF-CAMs in the VCS, NCAM-1 and ALCAM. Immunofluorescence staining showed dynamic expression patterns for each IgSF-CAM during embryonic and early post-natal stages, but ultimately all three proteins became highly enriched in mature PCs. Mice deficient in NCAM-1, but not CNTN2 or ALCAM, exhibited defects in Purkinje cell gene expression and VCS patterning, as well as cardiac conduction disease. Moreover, using ST8sia2 and ST8sia4 knockout mice, we show that inhibition of post-translational modification of NCAM-1 by polysialic acid (PSA) disrupts trafficking of sarcolemmal intercalated disc proteins to Purkinje cell junctional membranes and abnormal expansion of the extracellular space between apposing Purkinje cells. Taken together, our data provide novel insights into the complex developmental biology of the ventricular conduction system.
2021-05-20 | GSE172267 | GEO
Project description:The cardiac conduction system (CCS) is composed of specialized cardiomyocytes that initiate and maintain cardiac rhythm. Any perturbation to the normal sequence of electrical events within the heart can result in cardiac arrhythmias. To understand how cardiac rhythm is established at the molecular level, several genetically modified mouse lines expressing Cre recombinase within specific CCS compartments have been created. In general, Cre driver lines have been generated either by homologous recombination of Cre into an endogenous locus or Cre expression driven by a randomly inserted transgene. However, haploinsufficiency of the endogenous gene compromises the former approach, while position effects negatively impact the latter. To address these limitations, we generated a Cre driver line for the ventricular conduction system (VCS) that preserves endogenous gene expression by targeting the Contactin2 (Cntn2) 3' untranslated region (3'UTR). Here we show that Cntn23'UTR-IRES-Cre-EGFP/+ mice recombine floxed alleles within the VCS and that Cre expression faithfully recapitulates the spatial distribution of Cntn2 within the heart. We further demonstrate that Cre expression initiates after birth with preservation of native Cntn2 protein. Finally, we show that Cntn23'UTR-IRES-Cre-EGFP/+ mice maintain normal cardiac mechanical and electrical function. Taken together, our results establish a novel VCS-specific Cre driver line without the adverse consequences of haploinsufficiency or position effects. We expect that our new mouse line will add to the accumulating toolkit of CCS-specific mouse reagents and aid characterization of the cell-autonomous molecular circuitry that drives VCS maintenance and function.
| S-EPMC5373586 | biostudies-literature
Project description:BackgroundMyocardial lipomatous metaplasia (LM) has been reported to be associated with post-infarct ventricular tachycardia (VT) circuitry.ObjectivesThis study examined the association of scar versus LM composition with impulse conduction velocity (CV) in putative VT corridors that traverse the infarct zone in post-infarct patients.MethodsThe cohort included 31 post-infarct patients from the prospective INFINITY (Intra-Myocardial Fat Deposition and Ventricular Tachycardia in Cardiomyopathy) study. Myocardial scar, border zone, and potential viable corridors were defined by late gadolinium enhancement cardiac magnetic resonance (LGE-CMR), and LM was defined by computed tomography. Images were registered to electroanatomic maps, and the CV at each electroanatomic map point was calculated as the mean CV between that point and 5 adjacent points along the activation wave front.ResultsRegions with LM exhibited lower CV than scar (median = 11.9 vs 13.5 cm/s; P < 0.001). Of 94 corridors computed from LGE-CMR and electrophysiologically confirmed to participate in VT circuitry, 93 traversed through or near LM. These critical corridors displayed slower CV (median 8.8 [IQR: 5.9-15.7] cm/s vs 39.2 [IQR: 28.1-58.5]) cm/s; P < 0.001) than 115 noncritical corridors distant from LM. Additionally, critical corridors demonstrated low-peripheral, high-center (mountain shaped, 23.3%) or mean low-level (46.7%) CV patterns compared with 115 noncritical corridors distant from LM that displayed high-peripheral, low-center (valley shaped, 19.1%) or mean high-level (60.9%) CV patterns.ConclusionsThe association of myocardial LM with VT circuitry is at least partially mediated by slowing nearby corridor CV thus facilitating an excitable gap that enables circuit re-entry.
| S-EPMC11168467 | biostudies-literature
Project description:The most distal portion of the ventricular conduction system (VCS) contains cardiac Purkinje cells (PCs), which are essential for synchronous activation of the ventricular myocardium. Contactin-2 (CNTN2), a member of the immunoglobulin superfamily of cell adhesion molecules (IgSF-CAMs), was previously identified as a marker of the VCS. Through differential transcriptional profiling, we discovered two additional highly enriched IgSF-CAMs in the VCS: NCAM-1 and ALCAM. Immunofluorescence staining showed dynamic expression patterns for each IgSF-CAM during embryonic and early postnatal stages, but ultimately all three proteins became highly enriched in mature PCs. Mice deficient in NCAM-1, but not CNTN2 or ALCAM, exhibited defects in PC gene expression and VCS patterning, as well as cardiac conduction disease. Moreover, using ST8sia2 and ST8sia4 knockout mice, we show that inhibition of post-translational modification of NCAM-1 by polysialic acid leads to disrupted trafficking of sarcolemmal intercalated disc proteins to junctional membranes and abnormal expansion of the extracellular space between apposing PCs. Taken together, our data provide insights into the complex developmental biology of the ventricular conduction system.
| S-EPMC8217711 | biostudies-literature
Project description:RationaleZO-1 (Zonula occludens-1), a plasma membrane-associated scaffolding protein regulates signal transduction, transcription, and cellular communication. Global deletion of ZO-1 in the mouse is lethal by embryonic day 11.5. The function of ZO-1 in cardiac myocytes (CM) is largely unknown.ObjectiveTo determine the function of CM ZO-1 in the intact heart, given its binding to other CM proteins that have been shown instrumental in normal cardiac conduction and function.Methods and resultsWe generated ZO-1 CM-specific knockout (KO) mice using α-Myosin Heavy Chain-nuclear Cre (ZO-1cKO) and investigated physiological and electrophysiological function by echocardiography, surface ECG and conscious telemetry, intracardiac electrograms and pacing, and optical mapping studies. ZO-1cKO mice were viable, had normal Mendelian ratios, and had a normal lifespan. Ventricular morphometry and function were not significantly different between the ZO-1cKO versus control (CTL) mice, basally in young or aged mice, or even when hearts were subjected to hemodynamic loading. Atrial mass was increased in ZO-1cKO. Electrophysiological and optical mapping studies indicated high-grade atrioventricular (A-V) block in ZO-1cKO comparing to CTL hearts. While ZO-1-associated proteins such as vinculin, connexin 43, N-cadherin, and α-catenin showed no significant change with the loss of ZO-1, Connexin-45 and Coxsackie-adenovirus (CAR) proteins were reduced in atria of ZO-1cKO. Further, with loss of ZO-1, ZO-2 protein was increased significantly in ventricular CM in a presumed compensatory manner but was still not detected in the AV nodal myocytes. Importantly, the expression of the sodium channel protein NaV1.5 was altered in AV nodal cells of the ZO-1cKO versus CTL.ConclusionsZO-1 protein has a unique physiological role in cardiac nodal tissue. This is in alignment with its known interaction with CAR and Cx45, and a new function in regulating the expression of NaV1.5 in AV node. Uniquely, ZO-1 is dispensable for function of the working myocardium.
| S-EPMC7571547 | biostudies-literature
Project description:The cardiac conduction system (CCS) ensures regular contractile function, and injury to any of its components can cause cardiac dysrhythmia. Although all cardiomyocytes (CMs) originate from common progenitors, the CCS is composed of biologically distinct cell types with unique functional and developmental characteristics. In contrast to ventricular cardiomyocytes, which continue to proliferate after birth, most CCS cells terminally exit the cell cycle during fetal development. Although the CCS should thus provide a poor substrate for postnatal injury repair, its regenerative capacity remains untested. Here, we describe a genetic system for ablating CMs that reside within the atrioventricular conduction system (AVCS). Adult mouse AVCS ablation resulted in regenerative failure characterized by persistent atrioventricular conduction defects and contractile dysfunction. In contrast, AVCS injury in neonatal mice led to recovery in a subset of these mice, thus providing evidence for CCS plasticity. Furthermore, CM proliferation did not appear to completely account for the observed functional recovery, suggesting that mechanisms regulating recovery from dysrhythmia are likely to be distinct from cardiac regeneration associated with ventricular injury. Taken together, we anticipate that our results will motivate further mechanistic studies of CCS plasticity and enable the exploration of rhythm restoration as an alternative therapeutic strategy.
| S-EPMC8483757 | biostudies-literature
Project description:The ventricular conduction system (VCS) orchestrates the harmonious contraction in every heartbeat. Defects in the VCS are often associated with life-threatening arrhythmias and also promote adverse remodeling in heart disease. We have previously established that the Irx3 homeobox gene regulates rapid electrical propagation in the VCS by modulating the transcription of gap junction proteins Cx40 and Cx43. However, it is unknown whether other factors contribute to the conduction defects observed in Irx3 knockout (Irx3(-/-)) mice. In this study, we show that during the early postnatal period, Irx3(-/-) mice develop morphological defects in the VCS which are temporally dissociated from changes in gap junction expression. These morphological defects were accompanied with progressive changes in the cardiac electrocardiogram including right bundle branch block. Hypoplastic VCS was not associated with increased apoptosis of VCS cardiomyocytes but with a lack of recruitment and maturation of ventricular cardiomyocytes into the VCS. Computational analysis followed by functional verification revealed that Irx3 promotes VCS-enriched transcripts targeted by Nkx2.5 and/or Tbx5. Altogether, these results indicate that, in addition to ensuring the appropriate expression of gap junctional channels in the VCS, Irx3 is necessary for the postnatal maturation of the VCS, possibly via its interactions with Tbx5 and Nkx2.5.
| S-EPMC4726432 | biostudies-literature