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Pro-regenerative fingerprints identified in a sub-population of adult mouse cardiomyocytes by integrative single-cell proteomics_FACS

ABSTRACT: We present a new experimental and computational workflow to analyze single-cell proteomics data applied to single-cardiomyocytes. By using an optimized isolation procedure and by taking advantage of the integrative capabilities of iSanXoT application, we eliminate batch effects, minimize cell size-related biases, obtain quantitative subcellular compartment information and detect protein alterations within subcellular compartments. This approach enhances data quantification accuracy and facilitates biological interpretation. We show that the overexpression of the Myc transcription factor switches the metabolism of adult cardiomyocytes and establishes a subpopulation of pro-regenerative cardiomyocytes. The reported advances pave the way for systematic analysis of single-cell proteomes, including those of sensitive cells, like cardiomyocytes.

INSTRUMENT(S):

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Heart, Cardiac Muscle Cell

SUBMITTER: Consuelo Marín-Vicente

LAB HEAD: Jesús Vázquez

PROVIDER: PXD064501 | Pride | 2026-04-06

REPOSITORIES: Pride

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			Action	DRS
	20221019_Ec_01_SC_CNIC-KI_TMT84.mzML	Mzml
	20221019_Ec_01_SC_CNIC-KI_TMT84.mzid	Mzid
	20221019_Ec_01_SC_CNIC-KI_TMT84.raw	Raw
	20221019_Ec_01_SC_CNIC-KI_TMT85.mzML	Mzml
	20221019_Ec_01_SC_CNIC-KI_TMT85.mzid	Mzid

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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.

Project description:Central questions like cardiomyocyte subtype emergence during cardiogenesis or availability of cardiomyocyte subtypes for cell replacement therapy require selective identification and purification of atrial and ventricular cardiomyocytes. However, characterization and implementation of pure cardiomyocyte subtypes is still challenging due to technical limitations. Our aim was to identify surface markers enabling the selective detection and purification of atrial and ventricular cardiomyocytes from mouse hearts. In a surface marker screen we found differential expression of CD49f in atrial and ventricular embryonic cardiomyocytes (E13.5). By flow cytometry we could correlate a high CD49f expression with MLC-2a on the single cell level; a low CD49f expression corresponded to MLC-2v. Based on the persisting differential CD49f expression we developed purification protocols for cardiomyocytes subtypes from the developing mouse heart. Flow sorting of E15.5 hearts into ErbB-2+/CD49flow and ErbB-2+/CD49fhigh cells led to a selective depletion (CD49flow) or enrichment of MLC-2a+ cells (CD49fhigh). We found a corresponding CD49f-dependent distribution of MLC-2a when pre-enriched neonatal cardiomyocytes (P2) were flow-sorted into CD49flow and CD49fhigh. Atrial and ventricular identity was confirmed by expression profiling and patch clamp analysis of sorted embryonic hearts, which unequivocally demonstrated that the sorted cells were viable and functional. For the first time, we introduce a non-genetic, antibody-based approach to specifically isolate atrial and ventricular cardiomyocytes from mouse hearts of various developmental stages. This newly gained capability of obtaining highly pure, viable cells will facilitate in-depths characterization of the individual cellular subsets and will aid translational research and therapeutic applications. The dataset comprises four different cardiomyocytes subtypes from the developing mouse heart. Embryonic (E15.5) hearts were dissociated and flow-sorted into ErbB-2+/CD49flow and ErbB-2+/CD49fhigh cardiomyocytes. Neonatal (P2) hearts were dissociated, contaminating non-myocytes were removed by MACS depletion, and the purified cardiomyocytes were flow-sorted into CD49flow and CD49fhigh cardiomyocytes. Four biological replicates were available for each sample groups. Microarray analysis was conducted on the Agilent Whole Mouse Genome Oligo Microarray 8x60K platform.

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Pro-regenerative fingerprints identified in a sub-population of adult mouse cardiomyocytes by integrative single-cell proteomics_FACS

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