Figure 3. Cardiomyocyte-EV Specific Expression of POPDC2 and CHRNE Proteins in Cardiac-Specific Cre-Driven EXOMAP1 Mouse Model.

a. Schematic detailing the generation of cardiac-specific EXOMAP1 transgenic mice. Exomap1 mice, which express HsCD81mNG (humanized CD81 fused with mNeonGreen) in a Cre recombinase-dependent manner, were crossed with alpha myosin heavy chain (αMHC)-Cre mice, ensuring HsCD81mNG expression on cardiomyocyte membranes and their secreted EVs. b. Cardiac-specific hsCD81 expression as observed in immunohistochemistry staining of heart (positive expression), and absent in kidney and liver. c. Overview of the ExoCapture-MSB method for EV immunocapture using biotin-streptavidin affinity and Streptavidin Magnetic Beads targeting cardiac-tissue specific EVs from αMHC-Cre Exomap1 mice. d. Confirmation of canonical EV markers CD81 and CD9 enrichment in cardiac-derived EVs via Western blot assay, with controls (PD: Pulled-Down, FT: Flow-Through). e. UMAP legend of cardiac single nuclear RNA-seq data from Tabula muris. f. and g. UMAPs and Violin plots demonstrating enrichment of cardiomyocyte-specific transcripts Lrrc10, Myo18b, Ttn, and Fbxo40 from Tabula muris. h. Bar plots showing significant differences in cardiomyocyte-specific transcripts between humanized CD81-positive EV pulldown and isotype control, with respective p-values using Mann Whitney t-test (Lrrc10, p = 0.0482; Myo18b, p=0.0105; Ttn p=0.0459; FBxo40- p=0.0103) n= 4 mice. i. Confirmation of POPDC2, CHRNE, and TNNI3 enrichment in cardiac-derived EVs via Western blot assay, validating the selective presence of these key cardiomyocyte markers, affirming their cardiac origin. N= 4 mice.