Region-specific human cardiac extracellular vesicles differentially regulate mitochondrial function

Aim: The human heart contains two major mitochondrial subtypes, subsarcolemmal (SSL), fueling surface ion channels, and interfibrillar (IF), fueling interior contractile machinery. This study investigated whether the distinct extracellular vesicles (EVs) that we determined to be associated with these regions could influence cardiac energetics. Methods: Following mitochondrial extraction, Subsarcolemmal Heart Extracellular Vesicles (SHEVs) and Interfibrillar Heart Extracellular Vesicles (IHEVs) were extracted from nonfailing human left ventricular lysates using precipitation-based methods. We characterized these EVs by size, proteomic profile, phospholipid content, and their ability to modulate mitochondrial function in primary neonatal rat ventricular myocytes (NRVMs). Results: SHEVs (mean size: 148.9 ± 25.9 nm) and IHEVs (102.9 ± 31.0 nm) exhibited distinct proteomic signatures. SHEV and IHEV contained mostly structural proteins, but also contained 8% and 4% mitochondrial and glycolytic proteins, respectively. Lipidomic analysis revealed higher sphingomyelin and lower cardiolipin in EVs compared to whole tissue. SHEV treatment significantly increased mitochondrial ATP production, basal, and maximal respiration in NRVMs (P < 0.05). Conversely, IHEVs decreased maximal respiration without altering ATP production. Neither EV type exhibited intrinsic mitochondrial activity. Conclusion: Regionally distinct EVs within the human heart have the capacity to modulate mitochondrial function. These findings suggest that cardiac EVs may act as localized regulators of cardiomyocyte bioenergetics.