Propofol Induces Cardioprotection Against Ischemia-Reperfusion Injury via Suppression of Transient Receptor Potential Vanilloid 4 Channel

Ca2+ entry via the transient receptor potential vanilloid 4 (TRPV4) channel contributes to Ca2+ overload and triggers many pathophysiological conditions, including myocardial ischemia/reperfusion (I/R) injury. Propofol, a widely used intravenous anesthetic, attenuates myocardial I/R injury. However, the mechanism of propofol remains to be examined. The present study aims to test the hypothesis that propofol attenuates myocardial I/R injury through the suppression of TRPV4. We used a murine ex vivo model of myocardial I/R and in vitro cultured myocytes subjected to hypoxia/reoxygenation (H/R). Propofol or TRPV4 antagonist, HC-067047, attenuates myocardial I/R injury in isolated hearts. In addition, propofol, HC-067047, or TRPV4-siRNA attenuates H/R-induced intracellular Ca2+ concentration ([Ca2+]i) increase and cell viability reduction. On the contrary, TRPV4 agonist GSK1016790A exacerbates both ex vivo and in vitro myocardial injury. Pretreatment with propofol reverses the myocardial injury and intracellular Ca2+ overload induced by GSK1016790A at least in vitro. However, neither the combination of propofol and HC-067047 nor applying propofol to cells transfected with TRPV4-siRNA creates additional protective effects. In addition, propofol dose-dependently inhibits TRPV4-mediated Ca2+ entry induced by GSK1016790A and 4α-PDD. Propofol attenuates myocardial I/R injury partially through the suppression of TRPV4 channel and the subsequent inhibition of intracellular Ca2+ overload.

An increased level of intracellular Ca 2+ is detrimental to cardiomyocytes and results in myocardial cell death during I/R injury (Garcia-Dorado et al., 2012). One of our recent studies has shown that Ca 2+ entry via the transient receptor potential vanilloid 4 (TRPV4) channel in cardiomyocytes plays a critical role in mediating Ca 2+ overload and ROS release during the process of myocardial I/R injury . Similar to the cardioprotective effects of propofol, TRPV4 blockage reduces infarct size and troponin T (TnT) and improves in vivo heart function . TRPV4 protein levels have also been found to increase in the brain I/R model (Jie et al., 2016), while TRPV4 selective antagonist HC-067047 (Everaerts et al., 2010) attenuates I/R-induced brain injury . In addition, excessive Ca 2+ influx through TRPV4 induced by TRPV4 agonists GSK1016790A and 4α-PDD leads to the apoptosis of retinal ganglion cells and neuronal death in the hippocampus (Ryskamp et al., 2011;Jie et al., 2016). Based on these findings, TRPV4 channel is a promising target in the treatment of I/R-induced myocardial injury (Jones et al., 2019;Wu et al., 2019). Moreover, propofol has been found to directly interact with TRP channels, including TRPC5 and TRPA1 (Bahnasi et al., 2008;Ton et al., 2017). However, there is no direct evidence showing that propofol inhibits TRPV4mediated Ca 2+ entry. Therefore, in this study, we hypothesize that propofol activates protective mechanisms through the suppression of TRPV4 channel and the subsequent inhibition of intracellular Ca 2+ overload in ex vivo isolated hearts under I/R and in vitro cell models under H/R.
We first investigated the dose-dependent protective effects of propofol against reperfusion-induced myocardial injury. We subsequently confirmed the involvement of TRPV4 channel in reperfusion-induced myocardial injury using pharmacological approaches. Furthermore, we observed the effects of propofol on TRPV4 agonist-induced myocardial I/R injury and examined the cardioprotective effects of propofol in TRPV4-siRNA transfected cells. Finally, we analyzed the effects of propofol on TRPV4-mediated Ca 2+ influx and intracellular Ca 2+ concentration ([Ca 2+ ] i ) in HEK 293 cells, H9C2 cells, and adult rat ventricle myocytes (ARVMs).

Animals
Male C57BL/6 mice were purchased from Vital River Laboratories, Beijing, China, and Sprague-Dawley rats were purchased from the Experimental Animal Center, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China. All animals were kept in the Experimental Animal Center of Tongji Medical College. This study was carried out in accordance with the recommendations of the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 8023, revised 1978). The protocol was approved by the the Animal Research Ethics Committee of Tongji Medical College, Huazhong University of Science and Technology. All experiments were conducted on age and body weight-matched animal groups.
In the experimental protocol, we divided the mice hearts into 14 treatment groups (Figure 1)

Myocardial Infarct Size
Myocardial infarction size was measured by 2,3,5triphenyltetrazolium chloride (Sigma Aldrich, St. Louis, MO, USA) staining. At the end of reperfusion, the left ventricle was cut into 1 mm-thick transverse slices from the apex to the base. The size of 2,3,5-triphenyltetrazolium chloride -negative areas (infarct area, white) was calculated using the planimetry function in Image-Pro Plus v 6.0 (Media Cybernetics, MD, USA). The myocardial infarct size is presented as the percentage of infarct area size in the total left ventricle.
HEK-293T cells were purchased from ATCC and maintained in the same conditions as H9C2 cells. HEK-293T cells were transiently transfected with hTRPV4/pCDNA 4.0 (generously provided by Dr. Ke-wei Wang, Peking University School of Pharmaceutical Sciences, Qingdao University School of Pharmacy, China) using the Lipofectamine 3000 method as described previously (Fu et al., 2013).

H/R Model
Before hypoxia, the culture medium was changed to a fetal bovine serum-free DMEM. Hypoxia was achieved by placing the cells in a controlled hypoxic plastic chamber (HiTech Photelectricity Biotechnology Co., LTD., Guangzhou, China) containing 95% N 2 and 5% CO 2 for 6 h. Subsequently, the medium was replaced by a normal medium and the cells were incubated in 95% air and 5% CO 2 for a 6 h reoxygenation.
[Ca 2+ ] i Measurement [Ca 2+ ] i was measured according to previously described procedures . Cells were loaded with 2 μM Fluo-4 AM for 30 min. Florescence was captured with the Enspire Multimode Plate Reader (PerkinElmer, Boston, MA, USA). Relative changes in Ca 2+ influx stimulated by GSK1016790A (300 nM) or 4α-PDD (3 μM) are presented as (F/F0) or fold changes (∆F/F0), respectively. F represents fluorescence intensity, F0 represents the average fluorescence intensity before GSK1016790A or 4α-PDD stimulation, and ΔF represents the mean fluorescence intensity at the steady state after drug stimulation minus F0. Ionomycin (1 μM, Sigma, St. Louis, MO, USA) was set as a positive control.

Statistical Analysis
All values are presented as mean ± SD. Data was analyzed by a twotailed t test (data on Ca 2+ influx in H9C2 and ARVMs) or a oneway ANOVA followed by a Bonferroni method analysis (data on ex vivo and in vitro of I/R and H/R). Statistical analysis was performed using SigmaStat3.5 (Systat Software Inc, San Jose, California, USA). The dose-response curve was fitted using the Hill equation in Origin 9 (OriginLab Corporation, Northampton, MA, USA). IC 50 represents the propofol dosage that produces 50% of the maximal inhibitory effect. Only when p < 0.05 was the difference considered statistically significant.

RESULTS
As shown in the Supplementary Tables S1 and S2, all groups showed identical measured values (HR, LVDP, ± dP/dt max, and RPP) after 15 min of equilibration as well as before global ischemia. Original tracings of the left ventricle pressure for each experiment are shown in Supplementary Figures S2-S5.

Effects of the Combination of Propofol and TRPV4 Agonists/Antagonists on Myocardial I/R Injury in Isolated Mice Hearts
Next, we investigated the effect of propofol on TRPV4 agonistinduced myocardial I/R injury in isolated mice hearts. As shown in Figure 4 and Supplementary Figure S4, compared with INTRA+ DMSO, treatment with TRPV4 agonist GSK1016790A induced the detrimental effects. These effects, however, can be reduced by applying a combination of GSK1016790A and propofol at 50 μM. POP + DMSO had better recovery rates of cardiac function (LVDP, +dp/dt max, and RRR) and less release of LDH as well as infarct size compared with POP + GSK. It appears that propofol (50 μM) cannot completely reverse the detrimental effects induced by GSK1016790A in isolated mice hearts. We further investigated the effect of the combination of propofol and TRPV4 antagonists on myocardial I/R injury in isolated mice hearts. To leave sufficient room for HC-067047 to October 2019 | Volume 10 | Article 1150 Frontiers in Pharmacology | www.frontiersin.org   play an additive protective effect, we reduced the concentration of propofol to 25 μM. As shown in Figures 5 and 5S, combining TRPV4 antagonist HC-067047 and propofol produced no additional protective effect compared to propofol or HC-067047 alone. These results suggest that propofol attenuates myocardial I/R injury at least partially via the suppression of TRPV4 channel.
We also tested whether propofol had similar effects on ARVMs. ARVMs were isolated from adult rat hearts and were subjected to H/R after 1 day of incubation.

Propofol Suppresses TRPV4 Channel Function in a Concentration-Dependent Manner in 293T Cells Transfected With hTRPV4
To test the hypothesis that propofol inhibits TRPV4 channel function, we measured the Ca 2+ influx induced by specific TRPV4 agonists GSK1016790A and 4α-PDD in HEK-293T cells transfected with hTRPV4. GSK1016790A and 4α-PDD both induced significant Ca 2+ influx, which is reduced by pretreatment with propofol (12.5, 25, 50, 100 µM, Figures 8A, B). The quantitative analysis of the relative changes (∆F/F0) in Ca 2+ influx is displayed in Figures 8 C, D. The concentration response data fitted by the Hill equation shows a propofol IC 50 of 39.1 ± 4.5 μΜ under GSK1016790A and 45.3 ± 6.9 μΜ under 4α-PDD (Figures 8E, F). Our results proved that propofol suppresses the function of TRPV4 channel in a concentration-dependent manner.

Propofol Suppresses TRPV4 Channel Function in Cardiomyocytes
Confirming our previous observation  H9C2 and ARVMs show obvious Ca 2+ influx after stimulation with 300 nM GSK1016790A (Figures 9A, D). Similar to our results from 293T cells transfected with hTRPV4, pretreatment with 50 μM propofol significantly reduced Ca 2+ influx induced by GSK1016790A, but had no effect on Ca 2+ influx induced by Ionomycin in H9C2 or ARVMs (Figures 9 B, E). The quantitative analysis of the relative changes (∆F/F0) in Ca 2+ influx in cardiomyocytes is presented in

DISCUSSION
Earlier studies have demonstrated that propofol protects the heart against I/R injury through inhibiting intracellular Ca 2+ overload (Kim et al., 2008). However, the specific mechanisms are not yet clear. Our previous research has shown that TRPV4 channel functional expression significantly increases in both the in vivo and in vitro model of myocardial I/R, resulting in intracellular Ca 2+ overload and myocardial injury Wu et al., 2017). This study is the first to prove that propofol attenuates myocardial I/R injury at least partially through the suppression of TRPV4 channel and the subsequent inhibition of intracellular Ca 2+ overload.
We first confirmed the previous finding that propofol protects isolated hearts from I/R injury in a concentration-dependent manner. However, our results showed that 50 μM propofol had the best protective effects on mice, while Shao et al. and Ko et al. found that 100 μM has the best results on rat (Ko et al., 1997;Shao et al., 2008). The reason might be that the concentration of 100 μM is too high for mice and that high dosage propofol suppresses heart contraction by inhibiting Ca 2+ influx as well as Ca 2+ -induced excitation-contraction coupling (Li et al., 1997;Han et al., 2016). Therefore, a concentration of 25 or 50 μM was chosen in the following experiment.
Using the Langendorff model, we found that 0.1 μM TRPV4 antagonist HC-067047 significantly promoted the recovery of LVDP, +dP/dt max, -dP/dt max, and RPP, and reduced LDH release as well as infarct size. On the contrary, 20 nM TRPV4 agonist GSK1016790A exacerbated myocardial I/R injury. These results demonstrated that TRPV4 channel activation contributed to myocardial I/R injury in ex vivo isolated mice hearts, which is consistent with the results of our previous in vivo and in vitro October 2019 | Volume 10 | Article 1150 Frontiers in Pharmacology | www.frontiersin.org  studies Wu et al., 2017). We further investigated the effects of the combination propofol and TRPV4 agonist/ antagonist on myocardial I/R injury. Our results show that the detrimental effects induced by GSK1016790A were reversed by pretreatment with propofol in vitro but not in ex vivo isolated hearts. TRPV4 activation during I/R is likely mediated by an endogenous mechanism that is less vigorous than GSK1067790A. Moreover, the exogenous GSK1067790A and the endogenous TRPV4 activators, e.g. EETs, may activate TRPV4 via different mechanisms. TRPV4 activation by I/R alone could be reduced by propofol. Furthermore, the combination of propofol and TRPV4 antagonist HC-067047 did not create additional protective effects ex vivo. Similar results were also found in H9C2 transfected with TRPV4-siRNA. This suggested that propofol reduced myocardial I/R injury at least partially via the suppression of TRPV4 channel.
Ca 2+ is vital for heart contraction. However, after myocardial I/R, intracellular Ca 2+ overload impairs left ventricular mechanical function (Steenbergen et al., 1993;Meissner and Morgan, 1995). A number of pathways have been found to mediate cardiomyocytes Ca 2+ overload, including  the Na + /Ca 2+ exchanger, Ca 2+ -ATPase, L-type Ca 2+ channel, sarcoplasmic reticulum Ca 2+ release channel, and TRPV4 channel (Takahashi et al., 1994;Garcia-Dorado et al., 2012;Wu et al., 2017;Jones et al., 2019). One of our previous studies shows that the activation of TRPV4 channel contributes to H/R-induced intracellular Ca 2+ overload in cardiomyocytes . In this study, we also measured the [Ca 2+ ] i in H9C2 cells and ARVMs subjected to H/R. Pretreatment with propofol inhibited H/R-induced [Ca 2+ ] i increase and also prevented the effect of TRPV4 agonist GSK1016790A on [Ca 2+ ] i . Furthermore, we found no obvious difference in [Ca 2+ ] i between H/R + POP, H/R + TRPV4 siRNA, and H/R + TRPV4 siRNA + POP groups. Therefore, we assume that propofol attenuates H/R-induced intracellular Ca 2+ overload through the suppression of TRPV4 channel. In addition, many previous studies have already demonstrated that the blockage of TRPV4 channel is a promising target for I/R therapy Jie et al., 2016;Dong et al., 2017;Jones et al., 2019;Wu et al., 2019).
In order to investigate whether propofol could inhibit TRPV4-mediared effects, we recorded the Ca 2+ signals in H9C2 cells, ARVMs, and HEK-293T cells transfected with hTRPV4. Our results showed that propofol suppressed the Ca 2+ influx induced by TRPV4 agonists GSK1016790A and 4α-PDD in a concentration-dependent manner. The IC 50 is 39.1 ± 4.5 μΜ and 45.3 ± 6.9 μΜ, respectively, both of which were close to the concentration levels of 35 μΜ in clinical applications (Mathur et al., 1999), indicating that propofol can suppress TRPV4 channel to induce cardioprotective effects under I/R. Propofol has also been reported to reduce Ca 2+ entry via TRPC5 (Bahnasi et al., 2008) with an estimated IC 50 value of 84.5 μΜ in transfected HEK293 cells. TRPC5 is present in the heart and is upregulated in the cardiac myocyte hypertrophy model and human heart failure conditions (Lau et al., 2016). Therefore, more research is required to determine whether propofol-induced cardiac protection acts through a TRPC5-dependent mechanism. Propofol, as a commonly used anesthetic in clinical practice, is recognized as a safe and effective drug. However, unwanted side effects with propofol Cells are pretreated with 50 μM propofol for 30 minutes. Ionomycin is set as a positive control. Values are presented as mean ± SD, n = 6 for all groups, a two-tailed t test, ***p < 0.001 vs. DMSO, NS. means no statistical significance use have been documented and include hypotension, postoperative pain, and propofol-related infusion syndrome, a rare but seriously life-threatening complication (Mirrakhimov et al., 2015). Activation of TRPA1 and TRPV1 has been demonstrated to contribute to propofol-induced vasodilation and pain (Nishimoto et al., 2015;Sinharoy et al., 2017). Interestingly, the concentrations of propofol that induced Ca 2+ influx via TRPA1 (EC 50 = 65.4 μM) and TRPV1 (90 μM) are similar to those that affect TRPV4. Moreover, TRPV4 is also abundant in smooth muscle and endothelial cells of blood vessels (Randhawa and Jaggi, 2015). TRPV4 activation involves in shear stress-induced vasodilation as well as the damage of the vascular barrier. Whether TRPV4 inhibition involved in propofolinduced vasoreactivity or propofol-related infusion syndrome requires further research.
Our study has a few limitations. First, ex vivo or in vitro findings might be different from in vivo results. Second, although we discovered that propofol suppresses TRPV4 channel, which has yielded new insights into how propofol induces cardioprotective effects, the binding sites of propofol and TRPV4 channel are still unclear. This requires further research that applies site-directed mutagenesis methods, gene delivery techniques, and advanced computational technologies. In addition, although common in other studies, the knockdown efficiency (50%) of TRPV4-siRNA in the present study is low (Adapala et al., 2013;Suresh et al., 2015).
In conclusion, we conducted a series of ex vivo and in vitro experiments and demonstrated that propofol directly inhibited Ca 2+ entry via TRPV4 channel, which is a newfound mechanism whereby propofol reduces intracellular Ca 2+ overload and subsequently attenuates myocardial I/R injury. Due to the widespread expression and numerous effects of TRPV4 channel, the findings of this research are also relevant to other organ systems, including the respiratory, digestive, and urinary systems (Randhawa and Jaggi, 2015). Recent evidence has demonstrated that TRPV4 channel activation is related to pulmonary edema, gastrointestinal disorders, and bladder dysfunction. Moreover, a recent review article has highlighted the importance of TRPV4 channel in the pathogenesis of various diseases (White et al., 2016). Therefore, we think the use of propofol during the perioperative period is potentially beneficial to the prevention of TRPV4 channel-related diseases. However, this is beyond the scope of this study and requires further research. In addition, future research will need to examine whether other general anesthetics, such as volatile anesthetics or etomidate, also suppress TRPV4 channel to produce cardioprotective effects.

DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher.

ETHICS STATEMENT
All procedures concerning animal use were performed in adherence to the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 8023, revised 1978) and approved by Tongji Medical College Committee on Animal Care.