Cardiomyocyte-targeting exosomes from sulforaphane-treated fibroblasts affords cardioprotection in infarcted rats

Abstract

Background: Exosomes (EXOs), tiny extracellular vesicles that facilitate cell-cell communication, are being explored as a heart failure treatment, although the features of the cell source restrict their efficacy. Fibroblasts the most prevalent non-myocyte heart cells, release poor cardioprotective EXOs. A noninvasive method for manufacturing fibroblast-derived exosomes (F-EXOs) that target cardiomyocytes and slow cardiac remodeling is expected. As a cardioprotective isothiocyanate, sulforaphane (SFN)-induced F-EXOs (SFN-F-EXOs) should recapitulate its anti-remodeling properties.

Methods: Exosomes from low-dose SFN (3 μM/7 days)-treated NIH/3T3 murine cells were examined for number, size, and protein composition. Fluorescence microscopy, RT-qPCR, and western blot assessed cell size, oxidative stress, AcH4 levels, hypertrophic gene expression, and caspase-3 activation in angiotensin II (AngII)-stressed HL-1 murine cardiomyocytes 12 h-treated with various EXOs. The uptake of fluorescently-labeled EXOs was also measured in cardiomyocytes. The cardiac function of infarcted male Wistar rats intramyocardially injected with different EXOs (1·10¹²) was examined by echocardiography. Left ventricular infarct size, hypertrophy, and capillary density were measured.

Results: Sustained treatment of NIH/3T3 with non-toxic SFN concentration significantly enhances the release of CD81 + EXOs rich in TSG101 (Tumor susceptibility gene 101) and Hsp70 (Heat Shock Protein 70), and containing maspin, an endogenous histone deacetylase 1 inhibitor. SFN-F-EXOs counteract angiotensin II (AngII)-induced hypertrophy and apoptosis in murine HL-1 cardiomyocytes enhancing SERCA2a (sarcoplasmic/endoplasmic reticulum Ca²⁺ ATPase 2a) levels more effectively than F-EXOs. In stressed cardiomyocytes, SFN-F-EXOs boost AcH4 levels by 30% (p < 0.05) and significantly reduce oxidative stress more than F-EXOs. Fluorescence microscopy showed that mouse cardiomyocytes take in SFN-F-EXOs ~ threefold more than F-EXOs. Compared to vehicle-injected infarcted hearts, SFN-F-EXOs reduce hypertrophy, scar size, and improve contractility.

Conclusions: Long-term low-dose SFN treatment of fibroblasts enhances the release of anti-remodeling cardiomyocyte-targeted F-EXOs, which effectively prevent the onset of HF. The proposed method opens a new avenue for large-scale production of cardioprotective exosomes for clinical application using allogeneic fibroblasts.

Keywords: Allogeneic fibroblast; Cardioprotection; Exosomes; Myocardial infarction; Remodeling; Sulforaphane (PubChem CID: 5350).