1726 GUO R et al. Circulation Journal ORIGINAL ARTICLE Circ J 2019; 83: 1726 – 1736 doi: 10.1253/circj.CJ-18-1391 Ischemic Heart Disease Withaferin A Prevents Myocardial Ischemia/Reperfusion Injury by Upregulating AMP-Activated Protein Kinase- Dependent B-Cell Lymphoma2 Signaling Rui Guo, MD, PhD; Lu Gan, PhD; Wayne Bond Lau, MD; Zheyi Yan, MD, PhD; Dina Xie, MD; Erhe Gao, MD, PhD; Theodore A Christopher, MD; Bernard L. Lopez, MD; Xinliang Ma, MD, PhD; Yajing Wang, MD, PhD Background: Withaferin A (WFA), an anticancer constituent of the plant Withania somnifera, inhibits tumor growth in association with apoptosis induction. However, the potential role of WFA in the cardiovascular system is little-studied and controversial. Methods and Results: Two different doses of WFA were tested to determine their cardioprotective effects in myocardial ischemia/ reperfusion (MI/R) injury through evaluation of cardiofunction in wild-type and AMP-activated protein kinase domain negative (AMPK- DN) gentransgenic mice. Surprisingly, cardioprotective effects (improved cardiac function and reduced infarct size) were observed with low-dose WFA (1 mg/kg) delivery but not high-dose (5 mg/kg). Mechanistically, low-dose WFA attenuated myocardial apoptosis. It decreased MI/R-induced activation of caspase 9, the indicator of the intrinsic mitochondrial pathway, but not caspase 8. It also upregulated the level of AMP-activated protein kinase (AMPK) phosphorylation and increased the MI/R inhibited ratio of Bcl2/Bax. In AMPK-deficient mice, WFA did not ameliorate MI/R-induced cardiac dysfunction, attenuate infarct size, or restore the Bcl2/Bax (B-cell lymphoma2/Mcl-2-like protein 4) ratio. Conclusions: These results demonstrated for the first time that low-dose WFA is cardioprotective via upregulation of the anti- apoptotic mitochondrial pathway in an AMPK-dependent manner. Key Words: AMP-activated protein kinase; Apoptosis; Bcl-2/Bax; Cardiac function; Withaferin A ithaferin A (WFA), a steroidal lactone purified positive effects in oncology, the possible worrisome manifes- from Withania somnifera, is one of the most active tation of toxicity in the cardiovascular system is a concern. components found in the Indian winter cherry, Recent in vitro studies have revealed antiapoptotic effects of W 14 which is an important medicinal plant in the Ayurvedic and WFA on cardiomyocytes in the regulation of Akt activation. Uniai systems of medicine. Prospective studies have reported However, WFA exerts potent antitumor activity in vivo at that WFA has antitumor and immunomodulatory activities.1–4 doses that are different from those previously reported for Experimental studies have demonstrated that WFA inhibits antiangiogenic activity.15,16 Because this discrepancy remains nuclear factor κ-B (NF-κB)-regulated gene expression,5–7 and unexplained, there is a great need for clarification of WFA’s activates the MAPK family in multiple experimental models,8–10 influence in the cardiovascular system in vivo in order to displaying anti-inflammatory and antioxidant properties when explore a safer therapeutic strategy for cancer patients with tested on human cancer cells in vitro. Pharmacological levels cardiovascular complications by determining the therapeutic of WFA triggered anticancer effects specific to several breast effects and toxicity of WFA. cancer cell lines.11 Although these results suggest that WFA is Reducing inflammation and oxidative stress reduces ischemia associated with a positive treatment effect in promoting apoptosis reperfusion-induced cardiomyocyte apoptosis. Clarifying the in cancer cells, other researchers have reported that it is mechanism by which WFA acts upon the cardiovascular system responsible for an antiapoptotic effect in brain cells;12 these will contribute to our understanding of its biological function discrepant roles of WFA in vivo have been not addressed and provide insight into a new, potential therapeutic modality. completely and are even unclear in the cardiovascular system. Because necrosis and apoptosis appear to be ongoing during Myocardial infarction (MI) is a leading cause of death and ischemia, while apoptosis is boosted by the reperfusion a major health problem worldwide.13 Despite WFA possessing event,17 we explored an ischemia/reperfusion (I/R) model to Received January 8, 2019; revised manuscript received April 30, 2019; accepted May 21, 2019; J-STAGE Advance Publication released online June 20, 2019 Time for primary review: 21 days Department of Physiology, Shanxi Medical University, Shanxi (R.G., Z.Y., Y.W.), China; Department of Emergency Medicine, Thomas Jefferson University, Philadelphia, PA (R.G., L.G., W.B.L., Z.Y., D.X., T.A.C., B.L.L., X.M., Y.W.); and Center for Translational Medicine, Temple University, Philadelphia, PA (E.G.), USA Mailing address: Yajing Wang, MD, PhD, Department of Emergency Medicine, Thomas Jefferson University, Philadelphia, PA, USA. E-mail: [email protected] ISSN-1346-9843 All rights are reserved to the Japanese Circulation Society. For permissions, please e-mail: [email protected] Circulation Journal Vol.83, August 2019 Withaferin A’s AMPK-Dependent Cardioprotective Effect 1727 determine the effects of WFA on the cardiovascular apoptosis AFC by active caspase-3, was measured using Spectra- in vivo and sought to investigate the potential underlying the Max-Plus microplate spectrophotometer (Molecular cellular and molecular mechanisms of phenomena observed in Devices, Sunnyvale, CA, USA) with a fixed wavelength an animal model. (excitation wavelength, 400 nm; emission wavelength, The aims of the current study were to: (1) determine 505 nm). Caspase-3 activity was expressed as nmol AFC/h/ whether WFA modulates myocardial apoptosis in mice sub- mg protein. jected to myocardial I/R (MI/R); (2) determine whether WFA exerts regulatory effects in an AMP-activated protein kinase Determination of Cardiac Function (AMPK)-dependent fashion; and (3) to dissect the molecular Transthoracic echocardiographic analysis was performed mechanisms underlying WFA’s cardioprotective role. with a 12-MHz probe in a blinded manner while the mice were anesthetized with isoflurane (2% maintenance). Methods M-mode echocardiography was carried out in the paraster- nal short-axis in mice 24 h after reperfusion to assess heart Animals rate, LV ejection fraction (LVEF) and fractional shortening. Adult male wild-type (WT) mice and adult male AMPK- Hemodynamic analysis was conducted in a blinded 19 22 DN mice [dominant negative α2-subunit (D157A) of manner as described previously. , Briefly, a 1.4-Fr micro- AMPK] and respective male littermate controls were used manometer-tipped catheter (Millar Inc., Houston, TX, in this study. Generation, breeding, phenotype character- USA) was inserted into the right carotid artery and istics, and genotyping of AMPK-DN mice (80% inhibition advanced into the LV. Steady-state LV maximum (+dP/ of cardiac AMPK activity) have been previously described dTmax) and minimum (−dP/dTmax) was recorded in closed- in detail.18 All experiments were performed in adherence chest mode via PowerLab DAQ system (Millar Inc.). with the National Institutes of Health Guidelines on the Use of Laboratory Animals and were approved by the Primary Neonatal Cardiomyocyte Culture and siRNA Delivery Thomas Jefferson University Committee on Animal Care Neonatal rat ventricular myocytes (NRVMs) were isolated and Shanxi Medical University Committee on Animal from 1–2-day-old Sprague-Dawley rats (Envigo RMS, Care. Indianapolis, IN, USA) by enzymatic digestion method as previously described.23 Fibroblasts were removed by pre- Myocardial I/R plating for 2 h. NRVMs were cultured overnight in F10 Low-dose (1 mg/kg) or high-dose (5 mg/kg) WFA was medium (Mediatech, Manassas, VA, USA) containing administered by abdominal injection 90 min prior to sur- 10% horse serum, 5% fetal bovine serum (FBS), and 1% gery. Mice were anesthetized with 2% isoflurane. The penicillin/streptomycin/amphotericin B solution (Invitro- MI/R surgical procedure was performed as described pre- gen) at 37°C in a humidified incubator with 5% CO2. The viously.19 Briefly, the left anterior descending coronary following day, medium was replaced with F-10 medium artery was bound and 30 min later, the myocardium was containing 1% FBS. reperfused for 3 h (all assays except cardiac function and Transient siRNA transfections were conducted using infarct size) or 24 h (cardiac function and infarct size). scramble siRNA or siRNA AMPKα2 (final concentration Sham-operated control mice (Sham MI/R) underwent the 50 nmol/L) and TransMessenger transfection reagent (Qia- same surgical procedure, except the suture placed under gen) per 35-mm dish of NRVMs. Experiments were con- the left coronary artery was not tied. All assays utilized ducted on cells 48 h after transfection. tissue from the I/R area and the area-at-risk (AAR), iden- tified by Evans blue negative staining. Simulated I/R (SI/R) Prepared NRVMs were placed in a 5% CO2 incubator at Determination of Myocardial Apoptosis and Myocardial 37°C. NRVMs were subjected to SI/R as modified in our Infarct Size published study.21 In brief, glucose-free culture medium Myocardial apoptosis was determined by terminal deoxy- was first gassed for 5 min with a hypoxic gas mixture (95% nucleotidyl transferase-mediated dUTP nick-end labeling N2–5% CO2). Normal culture medium was quickly replaced (TUNEL) staining and caspase-3 activity, as described in with the hypoxic-hypoglycemic medium containing either our previous study.19,20 Mice hearts were fixed and sec- vehicle or WFA (500 nmol/L), and NRVMs were placed in tioned along the long axis (6 μm in thickness). TUNEL a HERAcell VIOS hypoxia (1% O2–5% CO2–94% N2) staining of the sections was performed with the In-Situ Cell incubator (Thermo Scientific, Waltham, MA, USA). After Death Detection Kit (Roche). 4 h of hypoxic-hypoglycemic culture, the hypoxic-hypogly- Apoptotic cells with green fluorescence were counted in cemic medium was replaced with normal culture medium a blinded fashion using Image J to assess the apoptotic (containing the same concentration of vehicle or WFA). index (TUNEL-positive cardiomyocytes).