huMSCs-exo showed cardioprotective effects in a sepsis model
huMSCs-exo were isolated and purified through differential ultracentrifugation, after which they were assess by TEM, exosome markers and particle size (Fig 1. A-C). We employed CLP to investigate the cardioprotective effects of huMSCs-exo in septic mice. A 2 µg/g concentration of huMSCs-exo was intraperitoneally injected at 0 hr and 6 hr after CLP, followed by testing at 12 hr after CLP. We found that the serum markers of cardiomyocyte injury (HBDH, CK and cTnI) increased significantly after CLP, which indicates that sepsis could induce cardiomyocyte injury in the first 12 hr. In contrast, these markers decreased markedly in the huMSCs-exo treatment group (Fig 1. D), which indicates that treatment with huMSCs-exo could effectively mitigate cardiomyocyte injury induced by sepsis. Next, we used the ejection fraction (EF), measured by echocardiography, to reflect heart function during sepsis. The results showed that EF decreased notably at 12 hr after CLP and that huMSCs-exo significantly improved EF in contrast with the CLP group (Fig 1. E, F), which indicated that huMSCs-exo could prevent heart function disorder induced by sepsis. In addition, we found that after huMSCs-exo treatment, CLP mouse survival significantly increased compared with the untreated group (Fig 1. G). Hence, we hypothesized that the increase in the mouse survival rate in the treatment group was highly correlated with heart function, which was protected by huMSCs-exo during sepsis.
huMSCs-exo protected cardiomyocyte mitochondria from damage induced by sepsis
To investigate the extent of cardiomyocyte injury in early sepsis, we used TUNEL to check cardiomyocyte apoptosis. We found that even though serum markers of cardiomyocyte injury increased significantly, there was almost no apoptosis in cardiomyocytes at 12 hr after CLP, and some cardiomyocytes did not undergo apoptosis until 24 hr after CLP (Fig 2. A). This suggests that at 12 hr of sepsis, cardiomyocytes were in the early stage of apoptosis, and some factors increased cardiomyocyte membrane permeability and resulted in cardiac-specific protein release.
To reveal the reason for increased injury markers and to find changes in cardiomyocytes during the first 12 hr of sepsis, we observed cardiomyocyte ultrastructure by TEM. We observed a disorderly myocardial myofibril arrangement, and that the band area was blurred, broken or dissolved at 12 hr after CLP, although cardiomyocyte apoptosis did not occur at this time. Furthermore, we also observed that cardiomyocyte mitochondrial swelling, cristae disorganization and cristae number decreased, which indicated that cardiomyocyte mitochondria were seriously injured during the first 12 hr of sepsis (Fig 2. B). Mitochondrial dysfunction is one of the most important pathways that induces cell apoptosis; therefore, mitochondrial injury is an upstream event of cardiomyocyte apoptosis in sepsis. One of the main characteristics of mitochondrial dysfunction is reduced ATP production, which matched our results, and we found that ATP production was significantly reduced at 12 hr after CLP (Fig 2. C).
We wondered whether the cardioprotective effects of huMSCs-exo were exerted through mitochondria protection. Therefore, we checked the cardiomyocytes’ ultrastructure by TEM after treatment with huMSCs-exo and found that myocardial myofibrils were arranged in order and that no broken or dissolved myofibrils were observed; in addition, the mitochondria morphology was nearly as normal as the sham group, with only a few mitochondria showing a decrease in the number of cristae (Fig 2. B). This observation indicates that huMSCs-exo could effectively prevent cardiomyocyte mitochondrial injury in the first 12 hr of sepsis. Furthermore, the ATP production of cardiomyocytes in the treatment group at 12 hr after CLP was increased, which was close to the level of the sham group (Fig 2. C), which might be a benefit from undamaged mitochondria protected by huMSCs-exo. According to these results, we hypothesized that huMSCs-exo could protect cardiomyocytes from damage induced by sepsis by preventing mitochondrial injury in the early stage of sepsis to subsequently reduce cardiomyocyte apoptosis.
Cardiomyocyte mitochondrial Ca2+ (mCa2+) efflux was obstructed during sepsis and reversed by huMSCs-exo
One study reported that in the early stage of sepsis, Ca2+ overinflows into the cytoplasm from the extracellular space of cardiomyocytes, and mitochondrial uptake overloads Ca2+ in the matrix, which ultimately induces mitochondrial calcium overload. Cardiomyocyte injury induced by mitochondrial calcium overload is an important mechanism that causes heart dysfunction in sepsis. To investigate how mitochondrial calcium overload of cardiomyocytes occurs in sepsis, we assessed the capacity of Ca2+ uptake and efflux in cardiomyocyte mitochondria. Adult cardiomyocytes (ACMs) isolated from sham-, CLP- and huMSCs-exo-treated mice were transferred to an intracellular-like medium, after which 1 μM Fura2-AM was added to monitor extramitochondrial Ca2+ and thapsigargin was added to prevent SR and ER Ca2+ uptake; hence, the changes in the fluorescence intensity indicated Ca2+ influx or efflux into/out of the mitochondria. The results showed that after the Ca2+ bolus was added, the time for extra-mitochondrial Ca2+ to reach the low point occurred earlier in the CLP group than in the sham group (Fig 3. A, B 60 s-260 s), which indicated that mitochondrial Ca2+ uptake in the CLP group was slightly faster than that in the sham group. After MCU was inhibited by Ru360 at 260 s-360 s, Ca2+ uptake in the mitochondria was blocked, and the increase in extramitochondrial Ca2+ was representative of mCa2+ efflux. The results showed that during the same period of time, the peak of extramitochondrial Ca2+ reached in the CLP group was much lower than that in the sham group (Fig 3. A, B 260 s-360 s, D), which indicates that the mCa2+ efflux rate decreased significantly in the CLP group. In contrast, we found that although Ca2+ uptake by mitochondria in the huMSCs-exo treatment group was not different from that in the CLP group (Fig 3. C 60 s-260 s), the mCa2+ efflux rate increased to the level of the sham group (Fig 3. C 260 s-360 s, D), suggesting that huMSCs-exo could keep the mCa2+ efflux of cardiomyocyte mitochondria normal during sepsis. According to these results, we hypothesize that abnormal cardiomyocyte mCa2+ efflux is an important reason for cardiomyocyte mitochondrial calcium overload, which causes mitochondrial dysfunction and finally induces cardiomyocyte apoptosis; furthermore, huMSCs-exo may protect heart function during sepsis by avoiding mitochondrial calcium overload induced by abnormal mCa2+ efflux.
Mitochondria regulate mCa2+ distribution in two ways: cytoplasmic Ca2+ uptake is driven by ΔΨm and mediated mainly by MCU and MICU119, and mCa2+ efflux is mediated by mitochondrial NCLX20. We assessed the expression of MCU, MICU1 and NCLX 12 hr after CLP, and there was little difference between the sham and CLP groups (Fig 3. E, F). This result indicates that the expression of these mCa2+ distribution regulators in cardiomyocytes was not affected during sepsis; therefore, in our opinion, the reason for abnormal mCa2+ efflux at 12 hr after CLP may be that the activity of NCLX was depressed.
huMSCs-exo increased the expression of PINK1 in cardiomyocytes during sepsis
Studies have indicated that the activity of NCLX is regulated by several proteins and kinases, such as stomatin-like protein 2 (SLP-2) and protein kinase C (PKC)21,22. Research has shown that in a cell model of Parkinson’s disease related to mutations in PINK1, the failure of mitochondrial Ca2+ efflux in neurons deficient in PINK1 is linked to the impaired activity of NCLX, which results in mitochondrial Ca2+ overload15,23. Therefore, we wondered whether PINK1 also regulated the activity of NCLX in cardiomyocytes. We found that at 12 hr after CLP, the expression of PINK1 decreased significantly (Fig 4. A), which matched the result that mCa2+ efflux was abnormal, suggesting that in cardiomyocytes, the decrease in PINK1 expression may be associated with a reduction in NCLX activity.
In addition, we found that after huMSCs-exo treatment, in addition to mCa2+ efflux being unaffected, PINK1 expression also increased in septic mouse cardiomyocytes (Fig 4. A), further confirming the relationship between PINK1 expression and NCLX activity. To further explore the source of increased PINK1 in cardiomyocytes after huMSCs-exo treatment, we detected Pink1 mRNA in huMSCs and their exosomes and found that huMSCs-exo carried more Pink1 mRNA than in the huMSCs (Fig 4. B). We hypothesized that this result indicates that in addition to meeting the physiological needs of huMSCs, Pink1 tended to be contained in exosomes and secreted extracellularly, which were used to regulate Pink1 expression in recipient cells.
We next used Pink1-specific siRNA to inhibit Pink1 expression in huMSCs to confirm whether huMSCs-exo increased PINK1 expression in recipient cardiomyocytes by transferring Pink1 mRNA from huMSCs. Pink1 expression was dramatically inhibited by Pink1 siRNA in both huMSCs and their exosomes (Fig 4. C), and PINK1 expression in ACMs isolated from mice treated with Pink1-inhibited huMSCs exosomes was still at a low level (Fig 4. D), suggesting that the increased PINK1 expression in recipient cardiomyocytes comes from Pink1 mRNA carried in huMSCs-exo.
huMSCs-exo with inhibited Pink1 could not reverse mCa2+ efflux obstruction and mitochondrial damage induced by sepsis
To further validate that PINK1 is related to NCLX-mediated mCa2+ efflux, we monitored the mCa2+ efflux rate of ACMs from mice treated with Pink1-inhibited huMSCs-exo. In our results, no significant difference was detected between the CLP and CLP treated with exopink1 siRNA groups (Fig 5. B, D, E). In contrast, the mCa2+ efflux rate in the CLP treated with exoNeg siRNA group was the same as that of the sham group (Fig 5. A, C, E). This suggests that the loss of Pink1 mRNA attenuated the ability of huMSCs-exo to recover mCa2+ efflux. Then, whether this loss also weakened the ability of huMSCs-exo to keep mitochondria from sepsis-induced damage remains unclear. We observed mitochondrial morphology with TEM and found that the mitochondrial cristae number still decreased and the arrangement was disordered in CLP mice with exopink1 siRNA treatment (Fig5. F). This indicates that mitochondrial protection of huMSCs-exo was greatly weakened after Pink1 mRNA inhibition. We confirmed from these results that PINK1 was important for the regulation of NCLX-mediated mCa2+ efflux, that cardiomyocyte mCa2+ efflux obstruction during sepsis was a crucial reason for mitochondrial damage, and that Pink1 mRNA contained in huMSCs-exo was a central factor that reversed mCa2+ efflux obstruction induced by sepsis to prevent mitochondrial damage.
PINK1 regulated NCLX-mediated mCa2+ efflux possibly by affecting PKA activity after transfer from huMSCs-exo to cardiomyocytes
The above results have confirmed the effect of PINK1 on NCLX-mediated mCa2+ efflux, but no evidence has shown that the interaction between PINK1 and NCLX is direct. In contrast, many studies argue against a direct interaction of PINK1 and NCLX. These studies have indicated that bioinformatic analysis failed to identify any PINK1 phosphorylation site on NCLX and that proteomic analysis of PINK1-interacting proteins found 14 candidates but not NCLX15,24. After transfer from huMSCs-exo to the cardiomyocytes, the pathway by which PINK1 regulates NCLX-mediated mCa2+ efflux is unknown. One study showed that PINK1-deficient cells exhibit PKA inhibition and NCLX-mediated mCa2+ efflux impairment, which could be fully rescued by activated PKA.
To determine whether PINK1 regulated NCLX-mediated mCa2+ efflux through PKA, we used forskolin (FSK) and H89 to activate and inhibit PKA, respectively. AC16 cells were treated with LPS and huMSCs-exo, after which we monitored the mCa2+ efflux rate. The results showed that PKA activation by FSK (Fig 6. A, B light green) could enhance the mCa2+ efflux rate compared with treatment with huMSCs-exo alone (Fig 6. A, B blue). In addition, consistent with a previous study15, we found that FSK greatly recovered the mCa2+ efflux rate decrease caused by the absence of PINK1 (Fig 6. C, D, gray and green) and that the ΔΨm also increased (Fig 6. E, G). However, coapplication of FSK and H89 completely abolished mCa2+ efflux activation (Fig 6. A, B light orange; C, D orange) and inhibited the increasing ΔΨm (Fig 6. E, F, G) both in the huMSCs-exo and huMSCs-exopink1 siRNA treatment groups. These results suggest that PKA is essential for PINK1-regulated NCLX-mediated mCa2+ efflux.