CRISPR-mediated activation of endogenous gene expression in the postnatal heart [ChIP-seq]
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE137146 | GEO | 2021/08/02
REPOSITORIES: GEO
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Project description:Endogenous activation of Myocyte enhancer factor 2 D (Mef2d) in the postnatal mouse heart by cardiomyocyte-specific CRISPRactivation (Myh6-dCas9VPR) The aim of this study was to compare endogenous gene activation of the pro-hypertrophic factor Myocyte enhancer factor 2 D (Mef2d) in the postnatal mammalian heart and experimental control groups including wild-type litter mates injected with saline, wild-type litter mates injected with AAV9 TRISPR Mef2d and transgenic litter mates injected with saline. Mice were injected with saline/AAV9 TRISPR Mef2d at P4 and sacrificed 8 weeks later. RNA was isolated by TRIzol/Chloroform and isopropanol precipitation. Ventricular tissue mRNA profiles were obtained using deep sequencing, in triplicates, using Illumina HiSeq4000. The sequence reads that passed quality filters were analyzed at the transcript isoform level with TopHat followed by DESeq2. qPCR validation was performed using SYBR Green assays. We present in this study a mouse model for cardiomyocyte-specific endogenous gene activation in the postnatal mammalian heart by CRISPR/dCas9VPR. As a proof of concept, we activated the pro-hypertrophic factor Myocyte enhancer factor 2 D (Mef2d) leading to functional decline, increased marker expression of heart failure as well as cardiomyocyte hypertrophy over a time course of 8 weeks. We furthermore observed no phenotypic consequence of constitutive dCas9VPR transgene expression in the postnatal heart. We therefore conclude, that the Myh6-dCas9VPR mouse model is a suitable tool for endogenous gene activation studies in the postnatal heart.
2021-08-02 | GSE132992 | GEO
Project description:Sustained cardiac stress promotes the transition from an adaptive response to heart failure. Understanding of mechanisms governing this transition will assist in identifying targets that prevent this progression. Our study revealed age-specific transcriptional functions mediated by KLF15 that are crucial for cardiac homeostasis. We report that postnatally, KLF15 continuously activates cardiac metabolism, but represses pathological, hypertrophic pathways associated with cardiomyocyte de-differentiation and endothelial cell (EC) remodeling in an age-dependent manner. Our integrative genomic and transcriptomic analyses identified novel target genes directly bound, and either activated or repressed by KLF15 in vivo in the adult heart. We identified a cooperative program inducing aberrant EC remodeling, caused by a reduction of KLF15 and a concomitant activation of Wnt signaling. Within this program, we further identified a so far uncharacterized cardiac gene - Shisa3, which is expressed in the developing heart and is upregulated in cardiac hypertrophy, ischemia and failure. Importantly, we demonstrate that the KLF15- and Wnt co-dependent, SHISA regulation occurs also in the human myocardium. Altogether, our results unraveled and characterized a previously unknown cardiac gene Shisa3, and attributed its significance to EC homeostasis of the adult heart, controlled by KLF15-Wnt dynamics. Purpose: The aim of this study was to compare transcriptome profiles (RNA-seq) of heart tissue with a WT or KO Klf15 locus at different murine ages - postnatal day10 (p10), 4-week-old and 20-week-old mice. Methods: Cardiac tissue total RNA profiles for different groups were obtained using deep sequencing, in triplicates, using Illumina HiSeq4000. The sequence reads that passed quality filters were analyzed at the transcript isoform level with TopHat, followed by DESeq2. qPCR validation was performed using TaqMan and SYBR Green assays. Conclusions: Our study represents the first detailed analysis of the processes triggered upon Klf15 loss in hearts of different murine postnatal ages, which was so far not investigated. We report that this Klf15 loss first triggers Wnt canonical pathway activation, followed by activation of the non-canonical Wnt components, culminating in heart failure.
2019-10-07 | GSE113027 | GEO
Project description:An important event in the pathogenesis of heart failure is the development of pathological cardiac hypertrophy. In cultured cardiac cardiomyocytes, the transcription factor Gata4 is required for agonist-induced cardiomyocyte hypertrophy. We hypothesized that in the intact organism Gata4 is an important regulator of postnatal heart function and of the hypertrophic response of the heart to pathological stress. To test this hypothesis, we studied mice heterozygous for deletion of the second exon of Gata4 (G4D). At baseline, G4D mice had mild systolic and diastolic dysfunction associated with reduced heart weight and decreased cardiomyocyte number. After transverse aortic constriction (TAC), G4D mice developed overt heart failure and eccentric cardiac hypertrophy, associated with significantly increased fibrosis and cardiomyocyte apoptosis. Inhibition of apoptosis by overexpression of the insulin-like growth factor 1 receptor prevented TAC-induced heart failure in G4D mice. Unlike WT-TAC controls, G4D-TAC cardiomyocytes hypertrophied by increasing in length more than width. Gene expression profiling revealed upregulation of genes associated with apoptosis and fibrosis, including members of the TGF? pathway. Our data demonstrate that Gata4 is essential for cardiac function in the postnatal heart. After pressure overload, Gata4 regulates the pattern of cardiomyocyte hypertrophy and protects the heart from load-induced failure. Experiment Overall Design: We reasoned that if Gata4 was a crucial regulator of pathways necessary for cardiac hypertrophy, then modest reductions of Gata4 activity should result in an observable cardiac phenotype. To test this hypothesis, we used gene targeted mice that express reduced levels of Gata4. We characterized these mice at baseline and after pressure Experiment Overall Design: overload.
2007-11-13 | E-GEOD-5500 | biostudies-arrayexpress
Project description:Postnatal heart maturation is the basis of normal cardiac function and provides critical insights into heart repair and regenerative medicine. While static snapshots of the maturing heart have provided much insight into its molecular signatures, few key events during postnatal cardiomyocyte maturation have been uncovered.Here we processed single cell RNASeq of Cardiomyocyte at different postnatal time points.
2022-05-29 | GSE156482 | GEO
Project description:Postnatal heart maturation is the basis of normal cardiac function and provides critical insights into heart repair and regenerative medicine. While static snapshots of the maturing heart have provided much insight into its molecular signatures, few key events during postnatal cardiomyocyte maturation have been uncovered.Here we processed bulkRNASeq and ChIPSeq of Cardiomyocyte at different postnatal time points.
2022-05-29 | GSE155658 | GEO
Project description:Background: Discovering determinants of cardiomyocyte maturity will be critical to understanding the maintenance of differentiated states and potentially reawakening endogenous regenerative programs in adult mammalian hearts as a therapeutic strategy. Recent evidence has suggested that forced dedifferentiation paired with oncogene expression is sufficient to drive cardiac regeneration. However, elucidation of endogenous developmental determinants of the switch between regenerative and mature cardiomyocyte cell states is necessary for optimal design of regenerative approaches for heart disease. Muscleblind-like 1 (MBNL1) regulates both fibroblast and erythroid differentiation and proliferation. Hence, we examined whether MBNL1 promotes and maintains mature cardiomyocyte states while antagonizing cardiomyocyte proliferation. Methods: MBNL1 gain- and loss-of-function mouse models were studied at several developmental timepoints and in surgical models of heart regeneration. Multi-omics approaches were combined with biochemical, histological, and in vitro genetic work to determine the mechanisms through which MBNL1 exerts its effects. Results: MBNL1 is co-expressed with a maturation-association genetic program in the heart and is regulated by the MEIS1/Calcineurin signaling axis. Targeted MBNL1 overexpression early in development prematurely transitioned cardiomyocytes to hypertrophic growth, hypoplasia, and dysfunction, while loss of MBNL1 function increased cardiomyocyte cell cycle entry and proliferation through altered cell cycle inhibitor transcript stability. Moreover, MBNL1-dependent stabilization of estrogen-related receptor signaling was essential for maintaining cardiomyocyte maturity in adult myocytes. In accordance with these data, modulating MBNL1 dose tuned the temporal window of neonatal cardiac regeneration, where increased MBNL1 expression arrested myocyte proliferation and regeneration, and MBNL1 deletion promoted regenerative states with prolonged myocyte proliferation. However, MBNL1 deletion was not sufficient to promote regeneration in the adult heart due to cell-cycle checkpoint activation. Conclusions: Here, MBNL1 was identified as an essential regulator of cardiomyocyte differentiated states, their developmental switch from hyperplastic to hypertrophic growth, and their regenerative potential through controlling an entire maturation program by stabilizing adult myocyte mRNAs during postnatal development and throughout adulthood. Additionally, MBNL1-dependent perturbations of a myocyte’s preferred growth mechanism at a given developmental state had detrimental consequences to cardiac function, and targeting loss of maturity and removing cell cycle inhibitors was not insufficient to promote adult regeneration.
2024-03-07 | GSE246743 | GEO
Project description:Background: Discovering determinants of cardiomyocyte maturity will be critical to understanding the maintenance of differentiated states and potentially reawakening endogenous regenerative programs in adult mammalian hearts as a therapeutic strategy. Recent evidence has suggested that forced dedifferentiation paired with oncogene expression is sufficient to drive cardiac regeneration. However, elucidation of endogenous developmental determinants of the switch between regenerative and mature cardiomyocyte cell states is necessary for optimal design of regenerative approaches for heart disease. Muscleblind-like 1 (MBNL1) regulates both fibroblast and erythroid differentiation and proliferation. Hence, we examined whether MBNL1 promotes and maintains mature cardiomyocyte states while antagonizing cardiomyocyte proliferation. Methods: MBNL1 gain- and loss-of-function mouse models were studied at several developmental timepoints and in surgical models of heart regeneration. Multi-omics approaches were combined with biochemical, histological, and in vitro genetic work to determine the mechanisms through which MBNL1 exerts its effects. Results: MBNL1 is co-expressed with a maturation-association genetic program in the heart and is regulated by the MEIS1/Calcineurin signaling axis. Targeted MBNL1 overexpression early in development prematurely transitioned cardiomyocytes to hypertrophic growth, hypoplasia, and dysfunction, while loss of MBNL1 function increased cardiomyocyte cell cycle entry and proliferation through altered cell cycle inhibitor transcript stability. Moreover, MBNL1-dependent stabilization of estrogen-related receptor signaling was essential for maintaining cardiomyocyte maturity in adult myocytes. In accordance with these data, modulating MBNL1 dose tuned the temporal window of neonatal cardiac regeneration, where increased MBNL1 expression arrested myocyte proliferation and regeneration, and MBNL1 deletion promoted regenerative states with prolonged myocyte proliferation. However, MBNL1 deletion was not sufficient to promote regeneration in the adult heart due to cell-cycle checkpoint activation. Conclusions: Here, MBNL1 was identified as an essential regulator of cardiomyocyte differentiated states, their developmental switch from hyperplastic to hypertrophic growth, and their regenerative potential through controlling an entire maturation program by stabilizing adult myocyte mRNAs during postnatal development and throughout adulthood. Additionally, MBNL1-dependent perturbations of a myocyte’s preferred growth mechanism at a given developmental state had detrimental consequences to cardiac function, and targeting loss of maturity and removing cell cycle inhibitors was not insufficient to promote adult regeneration.
2024-03-07 | GSE246741 | GEO
Project description:Background: Discovering determinants of cardiomyocyte maturity will be critical to understanding the maintenance of differentiated states and potentially reawakening endogenous regenerative programs in adult mammalian hearts as a therapeutic strategy. Recent evidence has suggested that forced dedifferentiation paired with oncogene expression is sufficient to drive cardiac regeneration. However, elucidation of endogenous developmental determinants of the switch between regenerative and mature cardiomyocyte cell states is necessary for optimal design of regenerative approaches for heart disease. Muscleblind-like 1 (MBNL1) regulates both fibroblast and erythroid differentiation and proliferation. Hence, we examined whether MBNL1 promotes and maintains mature cardiomyocyte states while antagonizing cardiomyocyte proliferation. Methods: MBNL1 gain- and loss-of-function mouse models were studied at several developmental timepoints and in surgical models of heart regeneration. Multi-omics approaches were combined with biochemical, histological, and in vitro genetic work to determine the mechanisms through which MBNL1 exerts its effects. Results: MBNL1 is co-expressed with a maturation-association genetic program in the heart and is regulated by the MEIS1/Calcineurin signaling axis. Targeted MBNL1 overexpression early in development prematurely transitioned cardiomyocytes to hypertrophic growth, hypoplasia, and dysfunction, while loss of MBNL1 function increased cardiomyocyte cell cycle entry and proliferation through altered cell cycle inhibitor transcript stability. Moreover, MBNL1-dependent stabilization of estrogen-related receptor signaling was essential for maintaining cardiomyocyte maturity in adult myocytes. In accordance with these data, modulating MBNL1 dose tuned the temporal window of neonatal cardiac regeneration, where increased MBNL1 expression arrested myocyte proliferation and regeneration, and MBNL1 deletion promoted regenerative states with prolonged myocyte proliferation. However, MBNL1 deletion was not sufficient to promote regeneration in the adult heart due to cell-cycle checkpoint activation. Conclusions: Here, MBNL1 was identified as an essential regulator of cardiomyocyte differentiated states, their developmental switch from hyperplastic to hypertrophic growth, and their regenerative potential through controlling an entire maturation program by stabilizing adult myocyte mRNAs during postnatal development and throughout adulthood. Additionally, MBNL1-dependent perturbations of a myocyte’s preferred growth mechanism at a given developmental state had detrimental consequences to cardiac function, and targeting loss of maturity and removing cell cycle inhibitors was not insufficient to promote adult regeneration.
2024-03-07 | GSE246740 | GEO
Project description:Background: Discovering determinants of cardiomyocyte maturity will be critical to understanding the maintenance of differentiated states and potentially reawakening endogenous regenerative programs in adult mammalian hearts as a therapeutic strategy. Recent evidence has suggested that forced dedifferentiation paired with oncogene expression is sufficient to drive cardiac regeneration. However, elucidation of endogenous developmental determinants of the switch between regenerative and mature cardiomyocyte cell states is necessary for optimal design of regenerative approaches for heart disease. Muscleblind-like 1 (MBNL1) regulates both fibroblast and erythroid differentiation and proliferation. Hence, we examined whether MBNL1 promotes and maintains mature cardiomyocyte states while antagonizing cardiomyocyte proliferation. Methods: MBNL1 gain- and loss-of-function mouse models were studied at several developmental timepoints and in surgical models of heart regeneration. Multi-omics approaches were combined with biochemical, histological, and in vitro genetic work to determine the mechanisms through which MBNL1 exerts its effects. Results: MBNL1 is co-expressed with a maturation-association genetic program in the heart and is regulated by the MEIS1/Calcineurin signaling axis. Targeted MBNL1 overexpression early in development prematurely transitioned cardiomyocytes to hypertrophic growth, hypoplasia, and dysfunction, while loss of MBNL1 function increased cardiomyocyte cell cycle entry and proliferation through altered cell cycle inhibitor transcript stability. Moreover, MBNL1-dependent stabilization of estrogen-related receptor signaling was essential for maintaining cardiomyocyte maturity in adult myocytes. In accordance with these data, modulating MBNL1 dose tuned the temporal window of neonatal cardiac regeneration, where increased MBNL1 expression arrested myocyte proliferation and regeneration, and MBNL1 deletion promoted regenerative states with prolonged myocyte proliferation. However, MBNL1 deletion was not sufficient to promote regeneration in the adult heart due to cell-cycle checkpoint activation. Conclusions: Here, MBNL1 was identified as an essential regulator of cardiomyocyte differentiated states, their developmental switch from hyperplastic to hypertrophic growth, and their regenerative potential through controlling an entire maturation program by stabilizing adult myocyte mRNAs during postnatal development and throughout adulthood. Additionally, MBNL1-dependent perturbations of a myocyte’s preferred growth mechanism at a given developmental state had detrimental consequences to cardiac function, and targeting loss of maturity and removing cell cycle inhibitors was not insufficient to promote adult regeneration.
2024-03-07 | GSE246742 | GEO
Project description:Evidence implicating p38γ and p38δ (p38γ/p38δ) in inflammation are mainly based on experiments using p38γ/p38δ deficient (p38γ/δ-/-) mice, which show low levels of TPL2, the kinase upstream of MKK1-ERK1/2 in myeloid cells. This could obscure p38γ/p38δ roles, since TPL2 is essential for regulating inflammation. Here we generated a p38γD171A/D171A/p38δ-/- (p38γ/δKIKO) mouse, expressing kinase-inactive p38γ and lacking p38δ. This mouse exhibited normal TPL2 levels, making it an excellent tool to elucidate specific p38γ/p38δ functions. p38γ/δKIKO mice showed a reduced inflammatory response and less susceptibility to LPS-induced septic shock and Candida albicans infection than wild-type mice. Gene expression analyses in LPS-activated WT and p38γ/δKIKO macrophages revealed that p38γ/p38δ regulated numerous genes implicated in innate immune response. Additionally, phospho-proteomic analyses and in vitro kinase assays showed that the transcription factor myocyte enhancer factor-2D (MEF2D) was phosphorylated at Ser444 via p38γ/p38δ. Mutation of MEF2D Ser444 to the non-phosphorylatable residue Ala increased its transcriptional activity and the expression of iNOS and IL-1β mRNA. These results suggest that p38γ/p38δ govern innate immune responses by regulating MEF2D phosphorylation and transcriptional activity.
2023-08-10 | PXD042626 | Pride