Unknown,Transcriptomics,Genomics,Proteomics

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Gene expression profiling of wildtype vs. transgenic mice expressing the cardiac specific genetically encoded FRET biosensor Epac1-PLN

ABSTRACT: 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).

ORGANISM(S): Mus musculus

SUBMITTER: Gabriela Salinas-Riester

PROVIDER: E-GEOD-65651 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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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.

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Gene expression profiling of wildtype vs. transgenic mice expressing the cardiac specific genetically encoded FRET biosensor Epac1-PLN

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