| Object:It is widely recognized that the opening and closure of mitochondrial permeability transition pore (mPTP) plays an important role in cardiac ischemia/reperfusion injury (IRI). The function of mitochondria is also controlled by the dynamic structures of mitochondrial integrity and morphology, where the mitochondrial fission protein dynamin-related protein1(Drp1) plays a critical role although the underlying mechanism remains unknown. Dynasore is a cell-permeable small molecule that non-competitively inhibits the GTPase activity of dynaminl, dynamin2and Drp1. Most studies of Dynaosre focus on its role in endocytosis, and its function in cardiovascular disease is largely unaddressed. In our previous study on ion channel trafficking, when Dynasore was used to block endocytosis, we found Dynasore protects cardiomyocyte morphology during extended culture in vitro, indicating a potential cardioprotective effects of Dynasore. A critical yet unaddressed issue is whether Dynasore, through modulation of mitochondrial integrity and morphology by blocking mitochondrial fission protein Drp1, can increase cardiocmyocyte survival when stressed. Moeover, the role of dynamin2need to be further claritied in myocardial IRI. Methods:Isolated adult mouse cardiomyocytes were replenished with fresh medium (serum supplemented or depleted) and subjected to2hours of drug treatment (1μM、3μM、10μM Dynasore or vehicle) followed by oxidative stress (30mM H2O2for35min). Cardiomyocyte survival and viability were analyzed by trypan blue exclusion (TBE) assay. For ATP supplement experiments, the cells were treated with3mM ATP for30min before exposure to H2O2. A luminescence assay (Promega, Madison, WI) was used to quantify stressed cardiomyocyte and unstressed Hela cell ATP content. In Langendorff perfused whole mouse heart global IRI study, left ventricular end diastolic pressure (LVEDP), left ventricular end systolic pressure (LVESP) and heart rate were monitored and recorded continuously using PowerLab system (ADInstruments). The hearts were then randomized to one of the following two treatment groups:1μM Dynasore group (n=8, added into the recirculating perfusate in stepwise fashion to reach a final concentration of1μM within120min of recirculation) or DMSO control group (n=8, added in a similar manner of Dynasore). At the end of the reperfusion, Propidium iodide (excitation,535nm; emission,617nm) was used to determine infarct size. Cardiac effluent was collected from apex at baseline, before ischemia and during reperfusion. The samples were snap frozen immediately in liquid nitrogen and stored at-80℃for later analysis cardiac specific troponin I (cTnl) concentration using a commercial cTnI ELISA kit. The function of Dynasore on mitochondrial morphology was then studied in Organelle LightsTM Mito-RFP BacMam1.0(Invitrogen) transduced HeLa cells and mito-tracker treated neonatal mouse cardiomyocytes using Confocal Microscopy. Mitochondrial△Ψm were determined by TMRM using Confocal Microscopy. Finally, protein expression of Drpl and dynaimin2were determined by western blotting.Results:Our study found that:(1) In langendorff perfused adult mouse hearts subjected to IRI,1μM Dynasore significantly reduces infarct size from39.6%of PI positive infarct area to8.1%(80%reduction, p＜0.001), and a70%decrease in cTnI release in cardiac efflux(p＜0.05).(2) In langendorff perfused adult mouse hearts subjected to IRI, Dynasore significantly prevents LVEDP elevation during I/R injury without affecting LVDP.(mmHg, the time set of reperfusion15min,30min and60min compared with control:4.6±3.7,3.3±4.3,0.0±4.8vs21.6±3.7,25.2±6.6,24.9±4.8, p<0.05).(3) Oxidative stress causes a significant reduction in both adult mouse cardiomyocyte survival and viability. However the presence of Dynasore in the culture medium results in significantly improved survival and a major improvement in viability. The beneficial effects of Dynasore on viability and survival are dose dependent and are also more prominent in serum free conditions.(4) Dynasore increases cellular ATP content in live cardiomyocytes subjected to oxidative stress; direct supplementation with exogenous ATP has similar cardioprotective effect as Dyansore on hearts subjected to oxidative stress. Low dose Dynasore (1μM～3μM) does’t increase cellular ATP content in unstressed HeLa cells. The cardioprotective dose of Dynasore is significantly lower than the dose used to effectively block endocytosis.(5) Oxidative stress (30μM H2O2) causes mitochondrial fission and fragmentation in cultured Hela cells; In neonatal mouse cardiomyocytes,1μM Dynasore pretreatment has no significant effect on oxidative stress induced mitochondrial fission and fragmentation; mitochondrial fission and fragmentation is mildly decreased in3μM Dynasore pretreated cells; whereas mitochondrial integrity is well maintained in10μM Dynasore pretreated cells similar to50μM Mdivi-1pretreatment.(6) In neonatal mouse cardiomyocytes, oxidative stress (30μM H2O2) causes mitochondrial morphology undergoing fission and fragmentation, resulting in membrane depolarization, and loss of TMRM fluorescence labeled mitochondrial areas. In Dynasore and Mdivi-1pretreated cells, reticular mitochondrial networks are maintained, and TMRM fluorescence area is more preserved in10μM Dynasore pretreatment group, as compared to other pretreatment groups.(7) In neonatal mouse cardiomyocytes, oxidative stress (30μM H2O2) significantly increases protein expression of Dynamin2, and Drp1to a less extent. The increased protein expression of Dynamin2and Drp1are almost totally blocked by10μM dynasore pretreatment (protein expression almost back to pre-oxidative stress baseline levels). The increased protein expression of Drp1and Dynamin2are only partially inhibited by50μM Mdivi-1pretreatment (Drpl and Dynamin2are reduced to a level still above pre-oxidative stress baseline level).Conclusion:(1) Our study identified that Dynasore decreases myocardial death and myocardial infarction area in ex vivo Langendorff perfused mouse hearts subjected to global IRI.(2) Our study provides the first evidence that Dynasore has a potent lusitropic effect during I/R injury. The mechanism is mitochondrial protection and preservation of oxidative phosphorylation.(3) The cardioprotective function of Dynasore is dependent on inhibiting Drp1, decreasing mitochondrial fragmentation and fission, protecting mitochondrial△Ψm, and decreasing the opening of mPTP.(4) Our study indicates that Dynamin2inhibition may also contributed to cardioprotective function of Dynasore, which needs to be further investigated and evaluated.(5) The current study also indicates that pharmaceutical therapy that preserves mitochondrial function may not just benefit myocardial survival, but improves diastolic function as well. |
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