Dataset Information

Subcellular Proteomic Analyses Reveal REEP5 Knockdown in the Mouse Heart Disrupts Mitochondrial Networks

ABSTRACT: This study investigates the role of REEP5 in maintaining organelle integrity and redox homeostasis in cardiac tissue (specifically cardiomyocytes). Using AAV9-mediated shRNA knockdown in postnatal mice, we acheived a cardiac-specific depletion of REEP5. Hearts were harvested 4 weeks post-injection and subjected to subcellular fractionation to isolate microsomal (SR/ER-enriched), mitochondrial, and cytosolic cellular compartmetns. Each fraction was analyzed using data-independent acquisition (DIA) LC-MS/MS on a Q Exactive Plus instrument and the resulting dataset caputres an organelle-specific proteomic profile following depletion of REEP5. Loss of REEP5 leads to changes in the proteomic profiles of targets involved in regulating SR/ER membrane dynamics, ER-mitochondrial communications, mitochondrial morphology and oxidative stress. This work highlights REEP5 as an important regulator of ER-mitochondrial communication and cardiac homeostasis.

INSTRUMENT(S):

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Heart

SUBMITTER: Michelle Di Paola

LAB HEAD: Anthony Orlando

PROVIDER: PXD065280 | Pride | 2026-03-30

REPOSITORIES: Pride

ACCESS DATA

Dataset's files

Source:

Items per page:

1 - 5 of 52

Publications

Subcellular proteomic analyses reveal REEP5 knockdown in the mouse heart disrupts mitochondrial networks.

Di Paola Michelle M Reitz Cristine J CJ Kuzmanov Uros U Jia Kateleen K Gramolini Anthony O AO

Molecular & cellular proteomics : MCP 20260209

Receptor Expression-Enhancing Protein 5 (REEP5) is a cardiac-enriched, membrane-shaping protein localized to the sarco(endo)plasmic reticulum (SR/ER), where it supports membrane network architecture and cardiomyocyte function. While REEP5 has been implicated in calcium handling and contractility, its role in regulating inter-organelle communication and mitochondrial homeostasis remains less well-understood. In this study, we used recombinant adeno-associated virus serotype 9(rAAV9)-mediated shRN ...[more]

PMID: 41672147

Similar Datasets

Project description:Cardiac sarcoplasmic reticulum (SR) plays a central role in cellular Ca homeostasis, as does endoplasmic reticulum (ER) in the nonmuscle cell. The importance of SR Ca-handling function has led to detailed understanding of Ca-release and re-uptake protein complexes, but relatively little information regarding other known ER functions. We isolated cardiac membranes based upon their ability to efficiently accumulate Ca by the SR,ER-ATPase (SERCA-positive membranes), a process known to produce two characteristic SR membrane fractions: those enriched in known junctional SR markers, and those enriched in proteins originating from classic ER subdomains. Using standard proteomic techniques, we determined the SR proteins in three membrane preparations: 1) the highest-density SERCA-positive microsomes (HighSR); 2) the slightly less dense SERCA-positive microsomes that are constrained by a ryanodine-dependent leak (MedSR); and the original crude cardiac microsomes. Only a third of all crude microsomal proteins in heart were enriched in SERCA-positive membranes at least two fold. This protocol completely excluded known contractile, mitochondrial, sarcolemmal, and other major microsomal proteins, producing a far cleaner preparation of SR. Each SERCA-positive protein was distributed between HighSR and MedSR membrane vesicles in accordance with relative densities of SERCA2a versus ryanodine receptor. The distributions reinforced past findings on the distributions for several of the major known proteins. When combined with values for relative abundance, and enrichment over crude membranes, our protocol conveniently and effectively exposed multiple domains of the cardiac ER/SR. Most of the highly enriched and abundant cardiac SR/ER proteins represent proteins that have received little research attention. The data generate a useful quantitative analysis of intracellular membrane structure and function within cardiomyocytes, and may provide a reliable way of gauging broader changes in cardiac SR.

Project description:MicroRNAs (miRNAs) are small non-coding RNAs that associate with Argonaute 2 protein to regulate gene expression at the post-transcriptional level in the cytoplasm. However, recent studies have reported that some miRNAs localize to and function in other cellular compartments. Mitochondria harbour their own genetic system that may be a potential site for miRNA-mediated post-transcriptional regulation. We aimed at investigating whether nuclear-encoded miRNAs can localize to and function in human mitochondria. To enable identification of mitochondrial-enriched miRNAs, we profiled the mitochondrial and cytosolic RNA fractions from the same HeLa cells by miRNA microarray analysis. Mitochondria were purified using a combination of cell fractionation and immunoisolation, and assessed for the lack of protein and RNA contaminants. We found 57 miRNAs differentially expressed in HeLa mitochondria and cytosol. Of these 57, a signature of 13 nuclear-encoded miRNAs was reproducibly enriched in mitochondrial RNA and validated by RT-PCR for hsa-miR-494, hsa-miR-1275 and hsa-miR-1974. This study provides the first comprehensive view of the localization of RNA interference components to the mitochondria. Our data outline the molecular bases for a novel layer of crosstalk between nucleus and mitochondria through a specific subset of human miRNAs that we termed ‘mitomiRs’. To assess whether nuclear-encoded miRNA are detectable in human mitochondria, we performed the following four steps approach. First, cultured HeLa cells were allowed to reach 80-100% confluence and subjected to fractionation in order to isolate the cytosolic fraction. From the same HeLa cells, mitochondria were isolated by immunomagnetic Anti-TOM22 MicroBeads from the Mitochondria Isolation Kit (Miltenyi Biotec). In total, six mitochondria preparations were perfromed, three of these were additionally treated with RNase A. Second, total RNA was extracted from the mitochondrial and cytosolic fractions. Third, mitochondrial and cytosolic RNA were respectively profiled by microRNA microarray analysis. Last, data were analyzed and normalized.Three independent assays were performed.

Project description:VEGF family members are important regulators of vascular functions. Promoting VEGFA signalling in aged mice has been shown to delay various aging phenotypes and extend the survival of aged mice. Although there is profound knowledge on functions of VEGFA, VEGFB has not been investigated in the context of cardiac aging. Our RNA data of aged mouse hearts revealed significant downregulation of Vegfb in the heart, specifically in endothelial cells and cardiomyocytes, while VEGFB expression was reduced in endothelial cells, fibroblasts and cardiomyocytes in aged human hearts. By contrast, VEGFB expression was exclusively reduced in cardiomyocytes of patients with cardiac hypertrophy. Hence, we investigated whether Vegfb gene therapy can revert age-dependent cardiac pathologies. We overexpressed Vegfb186, the soluble VEGFB isoform, via AAV9 vector transduction into 18-month-old C57Bl/6J male mice. AAV9-Vegfb treatment prevented progression age-related diastolic dysfunction and decreased cardiac fibrosis. We further found a rescue of aging-related left ventricular denervation in the hearts of AVV9-Vegfb treated old mice which was associated with an increase in heart rate variability. However, heart to body weight ratio and cardiomyocyte hypertrophy were increased in the AAV9-Vegfb treated mouse hearts, without alteration of cardiac systolic or diastolic function. Histological and transcriptomic analyses revealed that VEGFB186 induces compensatory cardiac hypertrophy which was accompanied by a rescued length-width-ratio, reduced fibrosis and the absence of cardiac inflammation. Cardiac single-nuclei RNA sequencing further suggested that AAV9-Vegfb treatment affects cardiac hypertrophy putatively via STAT3 which was validated in vitro. In conclusion, our data reveals that Vegfb overexpression partially reverses pathological alterations in the aging heart. Despite the overall improvement of the age-related cardiac phenotype, the AAV9-Vegfb-mediated induced cardiac hypertrophy which might reflect protective hypertrophy.

			Action	DRS
	MD_cyto_01.raw	Raw
	MD_cyto_02.raw	Raw
	MD_cyto_03.raw	Raw
	MD_cyto_04.raw	Raw
	MD_cyto_05.raw	Raw