Cardiomyocyte microvesicles contain DNA/RNA and convey biological messages to target cells

Abstract

Background: Shedding microvesicles are membrane released vesicles derived directly from the plasma membrane. Exosomes are released membrane vesicles of late endosomal origin that share structural and biochemical characteristics with prostasomes. Microvesicles/exosomes can mediate messages between cells and affect various cell-related processes in their target cells. We describe newly detected microvesicles/exosomes from cardiomyocytes and depict some of their biological functions.

Methodology/principal findings: Microvesicles/exosomes from media of cultured cardiomyocytes derived from adult mouse heart were isolated by differential centrifugation including preparative ultracentrifugation and identified by transmission electron microscopy and flow cytometry. They were surrounded by a bilayered membrane and flow cytometry revealed presence of both caveolin-3 and flotillin-1 while clathrin and annexin-2 were not detected. Microvesicle/exosome mRNA was identified and out of 1520 detected mRNA, 423 could be directly connected in a biological network. Furthermore, by a specific technique involving TDT polymerase, 343 different chromosomal DNA sequences were identified in the microvesicles/exosomes. Microvesicle/exosomal DNA transfer was possible into target fibroblasts, where exosomes stained for DNA were seen in the fibroblast cytosol and even in the nuclei. The gene expression was affected in fibroblasts transfected by microvesicles/exosomes and among 333 gene expression changes there were 175 upregulations and 158 downregulations compared with controls.

Conclusions/significance: Our study suggests that microvesicles/exosomes released from cardiomyocytes, where we propose that exosomes derived from cardiomyocytes could be denoted "cardiosomes", can be involved in a metabolic course of events in target cells by facilitating an array of metabolism-related processes including gene expression changes.

Figure 1. Transmission electron microscopy of purified microvesicles/exosomes.

A) Microvesicles/exosomes displaying an electron dense appearance, and B) electron lucent appearance. Bar represents 100 nm.

Figure 2. Detection of proteins on microvesicle/exosome surface with flow cytometry.

Microvesicles/exosomes prepared from Claycomb culture medium was incubated with antibodies conjugated with phycoerythrin (PE). A) Mouse anti-caveolin-3, was detected on approximately 30% of the microvesicles/exosomes. B) Mouse anti-flotillin-1, was detected on approximately 80% of the microvesicles/exosomes. C) Mouse anti-annexin-2, was not detected on the microvesicles/exosomes. D) Mouse anti-clathrin heavy chain, was not detected on the microvesicles/exosomes. The distribution of exosomes presenting caveolin-3 and flotillin-1 indicates that the sample contains more than one population of microvesicles/exosomes.

Figure 3. Flow cytometry of DNA-stained microvesicles/exosomes.

A) Enhanced fluorescence at the 530±15 nm channel of membrane permeable acridine orange-stained microvesicles/exosomes (below) in comparison with unstained microvesicles/exosomes (above). B) Weak or no fluorescence at 670 nm/LP channel of membrane impermeable propidium iodide-stained microvesicles/exosomes (below) not differing from unstained microvesicles/exosomes (above).

Figure 4. Confocal microscopy images of fibroblasts incubated with microvesicles/exosomes stained with acridine orange.

Confocal microscopy picture of DNA-stained microvesicles/exosomes after dialysis, ultracentrifugation and resuspension in DMEM. After incubation with fibroblasts for 3 h at 37°C the DNA-staining localizes in fibroblasts to and inside the nuclear membrane. Additional light microscopy was used to add a layer in images to visualize cell borders. Arrows in A) and B) indicate acridine orange staining inside nuclei. B) also visualizes red wave length which detects acridine orange staining for RNA. Yellow staining shows colocalization of DNA and RNA.