TAT‐dextran–mediated mitochondrial transfer enhances recovery from models of reperfusion injury in cultured cardiomyocytes

Abstract

Acute myocardial infarction is a leading cause of death among single organ diseases.Despite successful reperfusion therapy, ischaemia reperfusion injury (IRI) can induce oxidative stress (OS), cardiomyocyte apoptosis, autophagy and release of inflammatory cytokines, resulting in increased infarct size. In IRI, mitochondrial dysfunction is a key factor, which involves the production of reactive oxygen species, activation of inflammatory signalling cascades or innate immune responses, and apoptosis.Therefore, intercellular mitochondrial transfer could be considered as a promising treatment strategy for ischaemic heart disease. However, low transfer efficiency is a challenge in clinical settings. We previously reported uptake of isolated exogenous mitochondria into cultured cells through co-incubation, mediated by macropinocytosis. Here, we report the use of transactivator of transcription dextran complexes (TAT-dextran) to enhance cellular uptake of exogenous mitochondria and improve the protective effect of mitochondrial replenishment in neonatal rat cardiomyocytes (NRCMs) against OS. TAT-dextran-modified mitochondria (TAT-Mito) showed a significantly higher level of cellular uptake. Mitochondrial transfer into NRCMs resulted in anti-apoptotic capability and prevented the suppression of oxidative phosphorylation in mitochondria after OS. Furthermore, TAT-Mito significantly reduced the apoptotic rates of cardiomyocytes after OS, compared to simple mitochondrial transfer.These results indicate the potential of mitochondrial replenishment therapy in OSinduced myocardial IRI.

From Cell Entry to Engraftment of Exogenous Mitochondria

“…In recent years, various methods have been developed to artificially introduce isolated exogenous mitochondria into cells ( Table 2). These methods, using cell penetrating peptides (CPPs) [7], centrifugal force [8], magnetic force [9], and vapor bubbles [10], successfully and more efficiently internalized exogenous mitochondria into cells. Putative intracellular trajectory of exogeneous internalized mitochondria, which originate from either isolated or exosome-derived mitochondria.…”

“…Maeda et al designed the Trans-Activator of Transcription protein (TAT) conjugated with dextran to enable complex formation and isolated mitochondria with TAT-dextran, which allowed easier penetration of the plasma membrane through the reduction of the net negative charges, and the properties of CPP and TAT, independent of macropinocytosis, demonstrated that the complex can more efficiently transfer exogenous mitochondria into cells [7]. The improvement of the transfer efficiency was verified by using primary rat neonatal cardiomyocytes that were exposed to oxidative stress based on the survival and the reduced apoptosis signals following stress.…”

“…Macropinocytosis is regulated by the sensing of environmental cues [ 5 ], and macropinosomes that are pinched off the plasma membrane might be leaky or fragile enough for the cargo to be liberated into the cytosol, as it has been reported from independent researchers that exogenously isolated mitochondria complement the respiratory deficits in host cells [ 4 , 6 ]. Currently, mitochondrial transfer that is mediated by either exogeneous [ 7 , 8 , 9 , 10 ] or intercellular transfer [ 11 ] is ensured not only in vitro but also in vivo in pathophysiological situations, ranging from fertilization [ 12 ] to cancer [ 13 ] and neurodegenerative diseases [ 14 ]. In particular, exogeneous mitochondrial transplantation has been intensively investigated in animal models of various diseases, including neurological and cardiovascular diseases, and major related reports are summarized in Table 1 .…”

Output Regulation and Function Optimization of Mitochondria in Eukaryotes

“…However, low transfer efficiency is a challenge in clinical settings. In the neonatal rat cardiomyocytes (NRCMs) with IRI, the use of transactivator of transcription dextran complex (TAT-dextran) enhanced the uptake of mitochondria into rat cardiomyocytes and protected the cardiomyocytes ( Maeda et al, 2020 ). The enhanced transfer of mitochondria to NRCMs can significantly reduce the apoptosis of cardiomyocytes after oxidative stress and prevent the inhibition of OXPHOS in mitochondria.…”

Generation of Somatic Mitochondrial DNA-Replaced Cells for Mitochondrial Dysfunction Treatment

“…Horizontal transfer of genetic materials is a common characteristic of bacteria such as E. coli; however, these phenomena are also observed in mammalian cells (Pace et al, 2008). Since mammalian horizontal transfer of mitochondria through a tunneling nanotube was reported in an in vitro experiment (Rustom et al, 2004), mitochondrial transfer has been demonstrated with various mechanisms, such as nanotube (Islam et al, 2012), extracellular vesicles (Hayakawa et al, 2016), and macropinocytosis (Kitani et al, 2014), as well as by using an artificial device (Wu et al, 2016) and a compound (Maeda et al, 2020a). Although the efficiency of mitochondrial transfer did not dominate over the pre-existing mtDNA but provided an additive functionality in the host cells, mechanistic insights of mitochondrial transfer with respect to cell entry and endosomal escape have been deepened (Kami & Gojo, 2020).…”