Transgenic mice carrying the human PLN gene
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE20172 | GEO | 2011/12/15
SECONDARY ACCESSION(S): PRJNA125637
REPOSITORIES: GEO
Similar Datasets
Project description:These mice have been genetically modified so that the mouse PLN gene has been replace with the human PLN gene. Phopspholamban (PLN), the reversible inhibitor of the sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA2a), is a key regulator of myocyte Ca2+-cycling with a significant role in heart failure. We previously showed that the single amino acid difference between human and mouse PLN results in increased inhibition of Ca2+-cycling, cardiac remodeling and attenuated stress responses, in transgenic mice expressing the human PLN in the null background (hPLN). This set of microarray experiments aimed at deciphering the molecular mechanisms implicated in this process. 6 wild type, 6 transgenic mice. All at 11 weeks of age. FVB/N background. Whole hearts isolated, snap frozen and used for total RNA extraction.
2011-12-15 | E-GEOD-20172 | biostudies-arrayexpress
Project description:Background: The phospholamban (PLN) p.Arg14del mutation belongs to the established causes of dilated cardiomyopathy, with the molecular disease mechanisms incompletely understood. We used human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes, CRISPR/Cas9 gene editing and patient heart samples to investigate the molecular pathomechanisms of PLN p.Arg14del cardiomyopathy. Methods and results: Patient dermal fibroblasts carrying the PLN p.Arg14del mutation were reprogrammed into hiPSC, isogenic controls were established by CRISPR/Cas9. Cells were differentiated into cardiomyocytes and characterized in 2D and 3D-engineered heart tissue (EHT) format. Mutant cardiomyocytes revealed significantly prolonged Ca2+ transient decay time, Ca2+-load dependent irregular beating pattern and lower peak force compared to isogenic controls. While this data support the reported super-inhibitory effect of PLN p.Arg14del on the sarcoplasmic/endoplasmic reticulum Ca2+ ATPase, an unchanged SR Ca2+ content argued against disturbed SR Ca2+ cycling as the sole cause of contractile dysfunction. Label-free proteomic analysis of p.Arg14del EHTs revealed less endoplasmic reticulum (ER), ribosomal and mitochondrial proteins. Electron microscopy showed dilation of the rough ER, large lipid droplets in close association with mitochondria and reduced mitochondrial number. Follow-up experiments confirmed impairment of the rough ER/mitochondria compartment and enhanced oxidative stress in PLN p.Arg14del. Relevance for human disease was demonstrated by immunohistochemistry of human heart samples. PLN p.Arg14del end-stage heart failure samples revealed perinuclear aggregates positive for ER marker proteins and oxidative stress in comparison to ischemic heart failure - and non-failing donor heart samples. Surprisingly, transduction of PLN p.Arg14del EHTs with the Ca2+ binding protein GCaMP6f reversed the contractile, molecular and morphological disease phenotype. Conclusion: This study identified impairment of the rough ER/mitochondria compartment without SR dysfunction as a novel disease mechanism underlying PLN p.Arg14del cardiomyopathy. The pathology was improved by Ca2+-scavenging, suggesting impaired local Ca2+ cycling as an important disease culprit.
2021-04-12 | PXD020175 | Pride
Project description:Arrhythmogenic cardiomyopathy (ACM) is characterized by life-threatening ventricular arrhythmias and sudden cardiac death and affects hundreds of thousands of patients worldwide. The deletion of Arginine 14 (p.R14del) in the phospholamban (PLN) gene has been implicated in the pathogenesis of ACM. PLN is a key regulator of sarcoplasmic reticulum (SR) Ca2+ cycling and cardiac contractility. Despite global gene and protein expression studies, the molecular mechanisms of PLN-R14del ACM pathogenesis remain unclear. Using a humanized PLN-R14del mouse model and human induced pluripotent stem cell derived cardiomyocytes (iPSC-CMs), we investigated the transcriptome-wide mRNA splicing changes associated with the R14del mutation. We identified >200 significant alternative splicing (AS) events and distinct AS profiles were observed in the right (RV) and left (LV) ventricles in PLN- R14del compared to WT mouse hearts. Enrichment analysis of the AS events showed that the most affected biological process was associated with “cardiac cell action potential”, specifically in the RV. We found that splicing of 2 key genes, Trpm4andCamk2d, which encode proteins regulating calcium homeostasis in the heart, were altered in PLN-R14del mouse hearts and human iPSC-CMs. Bioinformatical analysis pointed to the tissue-specific splicing factors Srrm4 and Nova1 as likely upstream regulators of the observed splicing changes in the PLN-R14del cardiomyocytes. Our findings suggest that aberrant splicing may affect Ca2+- homeostasis in the heart, contributing to the increased risk of arrythmogenesis in PLN- R14del ACM.
2022-11-22 | GSE217921 | GEO
Project description:Heart failure (HF) is a major cause of morbidity and mortality worldwide, but therapeutic intervention remains limited despite recent improvements. Aberrant Ca2+ handling is a key feature of HF pathophysiology. Restoring the Ca2+ regulating machinery is an attractive therapeutic strategy supported by genetic and pharmacological proof of concept studies. Here, we studied antisense oligonucleotides (ASOs) as a novel therapeutic modality, interfering with the PLN/SERCA2a interaction by targeting Pln mRNA for downregulation in the heart of murine HF models. Mice harboring the PLN R14del pathogenic variant recapitulate the human dilated cardiomyopathy (DCM) phenotype; subcutaneously administrated PLN-ASO prevented PLN protein aggregation, cardiac dysfunction, and led to a 3-fold increase in survival rate. In another genetic DCM mouse model, unrelated to PLN (as a disease driver) (Cspr3/Mlp-/-), PLN-ASO also reversed the HF phenotype. Finally, in rats with myocardial infarction, progression of left ventricular dilatation was prevented by PLN-ASO treatment, which also improved left ventricular contractility. Thus, our data establish PLN-ASO as a possible precision medicine for genetic cardiomyopathy as well as general HF.
2021-06-11 | GSE151156 | GEO
Project description:iPSC-derived cardiomyocytes (iPSC-CMs) have enormous potential for the study of human cardiac disorders. However, their physiological immaturity severely limits their utility as a model system and their adoption for drug discovery. In this study, we describe a maturation media (MM) designed to provide oxidative substrates adapted to the metabolic needs of hiPSC-CMs as compared to standard RPMI/B27 media. RNA sequencing was performed to gain insight into the underlying molecular basis for the changes. Compared to baseline (day 21) conditions, three weeks subsequent exposure to MM (day 41) caused a significant differential expression in genes. GO analysis of the MM-differentially upregulated genes were informative, mapping to mitochondrial metabolism. Additionally, multiple Ca2+ cycling/sarcoplasmic reticulum (SR), sarcomeric, and ion channel genes were differentially expressed. These gene expression changes indicated that supplementation with oxidative substrates and subsequent metabolic changes might affect cardiomyocyte electrophysiology, Ca2+ cycling and contractility.
2020-06-30 | GSE151279 | GEO
Project description:3’,5’-cyclic adenosine monophosphate (cAMP) is an ubiquitous second messenger which regulates multiple physiological functions by acting in distinct subcellular microdomains. Although several targeted cAMP biosensors have been developed and used in cell lines or neonatal cardiomyocytes, it is unclear whether such biosensors can be successfully used in vivo, especially in the context of disease. Here, we generated the first transgenic mouse model expressing a targeted cAMP sensor (Epac1-PLN) and analyzed microdomain-specific second messenger dynamics in the vicinity of the sarcoplasmic reticulum calcium ATPase (SERCA). We demonstrate the biocompatibility of this targeted sensor and its potential for real-time monitoring of compartmentalized cAMP signaling in adult cardiomyocytes isolated from healthy mouse heart and from in vivo cardiac disease model. Finally, we used gene arrays to verify that the mRNA expression levels of the main players involved in cAMP signaling such as beta-ARs, G-protein, adenylyl cyclases, PKAs, PDEs, and Epac were not significantly changed between wildtype and transgenic Epac1-PLN expressing cardiomyocytes. To compare cardiac specific mRNA expression between wildtype and Epac1-PLN transgenic mice, cardiomyocyte RNA from total of 5 individual wildtype and 5 Epac1-PLN transgenic mice was isolated and submitted for the Illumina gene array analysis to the Göttingen transcriptome analysis laboratory (TAL).
2015-03-02 | E-GEOD-65651 | biostudies-arrayexpress
Project description:AMP deaminase (AMPD) plays a crucial role in adenine nucleotide metabolism, and its upregulated activity contributes to diastolic dysfunction of the heart caused by type 2 diabetes mellitus (T2DM) via reduction of the adenine nucleotide pool. Here we examined the mechanism of AMPD upregulation by T2DM. The protein level of 90-kDa full-length AMPD3 was increased in whole myocardial lysates and in the sarcoplasmic reticulum (SR) fraction by 55% and 123%, respectively, in OLETF, a rat model of T2DM, compared to those in LETO, a non-diabetic control, though AMPD mRNA level was unchanged in OLETF. MicroRNA array analysis revealed downregulation (>50%) of 57 microRNAs in OLETF compared to those in LETO, among which miR-301b was predicted to interact with the 3’UTR of AMPD3 mRNA. The 90-kDa AMPD3 level was significantly increased by miR-301b inhibitor, but decreased by a miR-301b mimetic in H9c2 cells. A luciferase reporter assay indicated binding of miR-301b to the 3’UTR of AMPD3 mRNA. The results indicate that translational regulation by miR-301b mediates AMPD3 upregulation in the diabetic heart. Since ATP generated by SR-associated glycolytic enzymes contributes to Ca2+ uptake by SERCA2a, upregulated AMPD3 in the vicinity of the SR may be a therapeutic target for diabetic cardiomyopathy.
2017-05-31 | GSE99410 | GEO
Project description:AMP deaminase (AMPD) plays a crucial role in adenine nucleotide metabolism, and its upregulated activity contributes to diastolic dysfunction of the heart caused by type 2 diabetes mellitus (T2DM) via reduction of the adenine nucleotide pool. Here we examined the mechanism of AMPD upregulation by T2DM. The protein level of 90-kDa full-length AMPD3 was increased in whole myocardial lysates and in the sarcoplasmic reticulum (SR) fraction by 55% and 123%, respectively, in OLETF, a rat model of T2DM, compared to those in LETO, a non-diabetic control, though AMPD mRNA level was unchanged in OLETF. MicroRNA array analysis revealed downregulation (>50%) of 57 microRNAs in OLETF compared to those in LETO, among which miR-301b was predicted to interact with the 3’UTR of AMPD3 mRNA. The 90-kDa AMPD3 level was significantly increased by miR-301b inhibitor, but decreased by a miR-301b mimetic in H9c2 cells. A luciferase reporter assay indicated binding of miR-301b to the 3’UTR of AMPD3 mRNA. The results indicate that translational regulation by miR-301b mediates AMPD3 upregulation in the diabetic heart. Since ATP generated by SR-associated glycolytic enzymes contributes to Ca2+ uptake by SERCA2a, upregulated AMPD3 in the vicinity of the SR may be a therapeutic target for diabetic cardiomyopathy.
2017-05-24 | GSE99203 | GEO
Project description:3’,5’-cyclic adenosine monophosphate (cAMP) is an ubiquitous second messenger which regulates multiple physiological functions by acting in distinct subcellular microdomains. Although several targeted cAMP biosensors have been developed and used in cell lines or neonatal cardiomyocytes, it is unclear whether such biosensors can be successfully used in vivo, especially in the context of disease. Here, we generated the first transgenic mouse model expressing a targeted cAMP sensor (Epac1-PLN) and analyzed microdomain-specific second messenger dynamics in the vicinity of the sarcoplasmic reticulum calcium ATPase (SERCA). We demonstrate the biocompatibility of this targeted sensor and its potential for real-time monitoring of compartmentalized cAMP signaling in adult cardiomyocytes isolated from healthy mouse heart and from in vivo cardiac disease model. Finally, we used gene arrays to verify that the mRNA expression levels of the main players involved in cAMP signaling such as beta-ARs, G-protein, adenylyl cyclases, PKAs, PDEs, and Epac were not significantly changed between wildtype and transgenic Epac1-PLN expressing cardiomyocytes.
2015-03-02 | GSE65651 | GEO
Project description:In differentiated skeletal muscle, intracellular Ca2+ concentrations rise dramatically upon membrane depolarization, constituting the link between excitation and contraction (EC). Transient rises in [Ca2+]i mainly emerge from Ca2+ released by the type 1 ryanodine receptor (RYR1) and, in non-adult muscle, by the inositol 1,4,5-triphosphate receptor (IP3R) of the sarcoplasmic reticulum (SR), the dominant Ca2+ store in skeletal muscle. While the IP3R-mediated, slow Ca2+ transients, have been implicated in cell signaling and development, RYR1’s non-contractile role(s) remain obscure. We used a homozygous mouse RyR1 knockout model (dyspedic) to investigate the effects of the absence of a functional RYR1 and, consequently, the lack of RyR1-mediated Ca2+ signaling during embryogenesis. While heterozygous mice of the model are undistinguishable from WT littermates, homozygous dyspedic mice die at birth from asphyxia, since their skeletal muscle does not support EC coupling. Furthermore, they display abnormal spine curvature, subcutaneous hematomas, small limbs, and enlarged neck. Skeletal muscles from front and hind limbs of dyspedic embryos (day E18.5) were subjected to microarray analyses, revealing 318 genes, significantly regulated by at least 50 % compared to control heterozygous littermates.
2016-02-01 | E-MTAB-3608 | biostudies-arrayexpress