Dox chelates iron ions and triggers the generation of free oxygen radicals, especially free hydroxyl radicals, resulting in lipid peroxidation of cardiomyocyte membrane and damage to the myocardial mitochondrial DNA. Mitochondria are essential organelles in cells and highly distributed in cardiomyocytes. Their functions include the generation of energy, regulation of cell survival (apoptosis), synthesizing ROS and controlling the concentration of intracellular calcium. Previous studies have shown that mitochondrial dysfunction after Dox employment plays a vital role in inducing cardiomyocyte apoptosis[20]. DOX accumulates in mitochondria through a variety of non-enzymatic mechanisms, leading to increased oxidative stress and mitochondrial dysfunction. Our study found a significant increase in the level of oxidative stress and the apoptosis of cardiomyocytes after Dox treatment, which is also consistent with findings from previous studies. Numerous studies have shown that mitochondrial dynamics play a unique role in mitochondrial-mediated cell death. Mitochondrial dynamics is defined as the adaptation of the mitochondria to changes in cells. The mitochondria continuously undergo fission and fusion. The imbalance in mitochondrial fission and fusion has been shown to promote sustained mitochondrial rupture, leading to the initiation of mitochondrial apoptosis. Several studies have described the interaction and colocalization of mitochondrial mitotic protein and pro-apoptotic protein (Bax) in mitochondrial mitotic foci, which suggests that mitochondrial dynamics are involved in mitochondrial-mediated cell death, which subsequently activates the pathway to cell death[21].
Mitochondrial fission begins with mitochondrial contraction caused by endoplasmic reticulum tubules. Endoplasmic reticulum tubules orient and determine the fission site, accompanied by mitochondrial fission[22]. Drp-1 is a dynamic protein GTPase protein located in the cytosol. After activation, it is transferred to the fission site on mitochondria for further mitochondrial separation[23]. Mfn2 and Opa1 are important mitochondrial fusion-related proteins[24, 25]. Previous studies have revealed that the conditional deletion of cardiac Mfn2 leads to mitochondrial dysfunction and resulting in myocardial hypertrophy and dysfunction[26, 27]. Since Mfn2 is responsible for mitochondrial fusion, the lowering of Mfn2 may impair the synergy of fission and fusion dynamics. In a previous study, Mfn2 knockout mice exhibited mitochondrial fragmentation, resulted in the reduction in the mitochondrial permeability transition pore(MPTP) and aggravated the mitochondrial dysfunction caused by ROS; another study showed that ischemia-reperfusion injury resulted in the low expression of the Mfn2, which resulted in mitochondrial and cardiac dysfunction[28]. There is further evidence that Mfn2-mediated restoration of mitochondrial fusion improved mitochondrial oxidative metabolism in Dox-treated cardiomyocytes, reduced cellular damage and decreased the level of mitochondria-derived oxidative stress[29]. Therefore, promoting Mfn2-mediated mitochondrial fusion may also be a potential strategy to mitigate Dox-induced cardiotoxicity.
Findings from our data indicate that the down-regulation of Mfn2 in the cardiomyocytes of mice treated with Dox promoted the fission of mitochondria. The average short-axis diameter of myocardial mitochondria in the Dox group was significantly shorter than that in the control group, and the mitochondria structure was abnormal, arranged disorderly, with a blurred or evenly broken mitochondria crest in the Dox group. However, there was a significant improvement after TSC-Exos intervention. In the treatment group receiving TSC-Exos therapy, the diameter of the mitochondria short axis was consistent with that of the control group; the myocardial mitochondria were still slightly damaged, with slightly clear mitochondrial cristae. It suggests that the exosomes secreted by the trophoblast stem cells have an inhibitory effect on myocardial mitochondrial damage induced by Dox.
Furthermore, this study showed mitochondrial fission could be improved by Dox through the downregulation of Mfn2 expression in cardiomyocytes, while the expression of Drp1 has no significant change compared with the control group, which is consistent with the previous findings reported[8]. However, this does not affect that Drp1 can be used as a potential therapeutic target. It could be attributed to the relative reduction in mitochondrial fusion, which makes the balance of mitochondrial dynamics to be more inclined towards fission. Therefore, some previous studies have also demonstrated the feasibility of reducing Dox-associated cardiotoxicity by inhibiting Drp1 expression, which could rebalance the scale between mitochondrial fission and fusion and ameliorate the Dox-induced decline in cardiac function[30]. Moreover, our study revealed that TSC-Exos could reduce the cardiotoxicity of Dox by affecting the mitochondrial dynamics. TSC-Exos enhances mitochondrial fusion by increasing the expression of Mfn2, reduces the increasing trend in mitochondrial fission in cardiomyocytes caused by Dox, rebalances the synergy of mitochondrial dynamics, and reduces the decline in the potential of the mitochondrial membrane and the production of ROS in cardiomyocytes caused by Dox. Previous studies have shown that TSC-Exos inhibits cardiomyocyte apoptosis by up-regulating the antiapoptotic protein Bcl-2 and reducing the expression of miR-200b in cardiomyocytes, our study validated the cardioprotective effect of TSC-Exos on this basis and further revealed that TSC-Exos could improve mitochondrial function by affecting the expression level of Mfn2. However, one of the limitations of this study is that it has not further explored the specific molecular mechanism. Perhaps further studies can be performed to explore particular pathway through what the exosomes secreted by the trophoblast stem cells alter the expression of Mfn2 and the changes in the mitochondrial dynamics.