Ma et al. Lipids in Health and Disease (2020) 19:189 https://doi.org/10.1186/s12944-020-01368-7 RESEARCH Open Access Atherogenic L5 LDL induces cardiomyocyte apoptosis and inhibits KATP channels through CaMKII activation Yanzhuo Ma1,2, Nancy Cheng2, Junping Sun2, Jonathan Xuhai Lu3,4, Shahrzad Abbasi5, Geru Wu2, An-Sheng Lee6,7, Tatsuya Sawamura8,9, Jie Cheng2, Chu-Huang Chen3,8* and Yutao Xi1,10* Abstract Background: Cardiac Ca2+/calmodulin-dependent protein kinase II (CaMKII) activation plays a critical role in cardiomyocyte (CM) apoptosis and arrhythmia. Functional ATP-sensitive potassium (KATP) channels are essential for cardiac protection during ischemia. In cultured CMs, L5 low-density lipoprotein (LDL) induces apoptosis and QTc prolongation. L5 is a highly electronegative and atherogenic aberrant form of LDL, and its levels are significantly higher in patients with cardiovascular-related diseases. Here, the role of L5 in cardiac injury was studied by evaluating the effects of L5 on CaMKII activity and KATP channel physiology in CMs. Methods: Cultured neonatal rat CMs (NRCMs) were treated with a moderate concentration (ie, 7.5 μg/mL) of L5 or L1 (the least electronegative LDL subfraction). NRCMs were examined for apoptosis and viability, CaMKII activity, phox and the expression of phosphorylated CaMKIIδ and NOX2/gp91 . The function of KATP and action potentials (APs) was analyzed by using the patch-clamp technique. Results: In NRCMs, L5 but not L1 significantly induced cell apoptosis and reduced cell viability. Furthermore, L5 decreased Kir6.2 expression by more than 50%. Patch-clamp analysis showed that L5 reduced the KATP current (IKATP) density induced by pinacidil, a KATP opener. The partial recovery of the inward potassium current during pinacidil washout was susceptible to subsequent inhibition by the IKATP blocker glibenclamide. Suppression of IKATP by L5 significantly prolonged the AP duration. L5 also significantly increased the activity of CaMKII, the phosphorylation of CaMKIIδ, and the expression of NOX2/gp91phox. L5-induced apoptosis was prevented by the addition of the CaMKII inhibitor KN93 and the reactive oxygen species scavenger Mn (III)TBAP. Conclusions: L5 but not L1 induces CM damage through the activation of the CaMKII pathway and increases arrhythmogenicity in CMs by modulating the AP duration. These results help to explain the harmful effects of L5 in cardiovascular-related disease. Keywords: Action potential, ATP-sensitive potassium, Ca2+/calmodulin-dependent protein kinase II, Cardiomyocytes, Electronegative low-density lipoprotein * Correspondence: [email protected]; [email protected] 3Vascular and Medicinal Research, Texas Heart Institute, 6770 Bertner Avenue, Houston, TX 77030, USA 1Department of Cardiology, Bethune International Peace Hospital, 398 Zhongshan Xilu, Shijiazhuang 050082, Hebei, China Full list of author information is available at the end of the article © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Ma et al. Lipids in Health and Disease (2020) 19:189 Page 2 of 10 Background multistep sodium chloride gradient, as previously de- Low-density lipoprotein (LDL) can be resolved into five scribed [17]. charge-defined subfractions, L1 to L5. L1 is the least negatively charged and most abundant subfraction of Isolation of neonatal rat CMs (NRCMs) circulating LDL and represents the healthy and harmless NRCMs were isolated from neonatal Sprague Dawley LDL class [1, 2]. L5 is the most negatively charged sub- rats [18]. Experiments were conducted according to The fraction of circulating LDL and has been shown to in- Code of Ethics of the World Medical Association (Dec- duce cell apoptosis [3, 4]. Although L5 is nearly laration of Helsinki), and the animal procedures were undetectable in healthy individuals, its concentrations approved by the Institutional Animal Care and Use are significantly higher in patients with cardiovascular- Committee at the Texas Heart Institute (Houston, related diseases. Studies have indicated that L5 increases Texas, USA, protocol number #2011–18). Phenobarbital the incidence of thrombosis that contributes to ST- (10 mg/kg, administered intraperitoneally, Sigma-Aldrich elevation myocardial infarction (STEMI) [5, 6] and that Corp., St. Louis, MO, USA) was used to anesthetize the patients with STEMI have an increased risk of fatal neonatal rats, and the left ventricle was quickly excised arrhythmia [7]. In addition, L5 has been considered a and rinsed three times with ice-cold Dulbecco’s novel factor for predicting coronary vascular disease [8] phosphate-buffered saline (PBS; Invitrogen, Carlsbad, and stroke [9], and its concentrations are increased in CA, USA). The ventricles were minced by using micro- individuals with cardiovascular risk factors. In cultured dissection in a dry petri dish and then by enzymatic di- cardiomyocytes (CMs), L5 has been reported to induce gestion with type II collagenase (Worthington cell apoptosis via cytokines released from vascular endo- Biochemical Co, Lakewood, NJ, USA) and procaine thelial cells [10]. Recently, it has been demonstrated that (Sigma-Aldrich Corp). After digestion, the cells were L5 causes prolongation of the corrected QT interval harvested and suspended in Dulbecco’s Modified Eagle’s (QTc) by mediating the current of L-type calcium and medium (DMEM, Gibco, Carlsbad, CA, USA) with 10% transient activated potassium channels [11]. Functional fetal bovine serum (FBS; Invitrogen, Carlsbad, CA, ATP-sensitive potassium channels (KATP) are essential USA), penicillin and streptomycin (100 U/mL, Invitro- for ischemic preconditioning, post-ischemia cardiac pro- gen, Grand Island, NY, USA), and 2 mM glutamate tection, and regulation of atrial and ventricular rhythm (Invitrogen). Next, the cells were placed on 100-mm cul- during cardiac ischemia [12, 13]. However, the effect of ture dishes, and the culture dishes were placed in a L5 on KATP current (IKATP) has not been studied. 37 °C incubator with 5% CO2. After fibroblasts settled, Cardiac Ca2+/calmodulin-dependent protein kinase II the supernatants were collected and replated for the fol- (CaMKII) is an underlying mechanism of CM apoptosis lowing experiments. and arrhythmia [14, 15]. CaMKII oxidation serves as an intermediate of oxidative stress and correlates with in- Cell apoptosis assays creased apoptosis in cardiac cells [16]. In this study, the NRCMs were starved overnight by using serum-free cul- effects of L5 on the electrophysiologic properties of CMs ture medium and were subjected to incubation with L5 were examined, as well as whether L5 induces CM dam- or L1 for 24 h. Cell apoptosis was analyzed by using age through CaMKIIδ via the oxidation and phosphoryl- staining. Briefly, NRCMs (1.0 × 105 cells/mL) were ation of its regulatory domain. treated with different concentrations of L5 (0.075, 0.25, 0.75, 2.5, 7.5, 25, 75, or 250 μg/mL) overnight. Parafor- Methods maldehyde (5%) was used to fix the NRCMs. Then, the L5 preparation and ion-exchange purification cells were incubated with Triton X-100 (0.1%) for 5 min. Whole LDL was respectively collected from 10 pa- Next, the cells were stained with Hoechst (blue) for 15 tients with STEMI and metabolic syndrome, as de- min and were observed by using a fluorescence micro- scribed previously [1], with approval from the scope. For Annexin V-Alexa 488/ propidium iodide (PI) Institutional Review Board at the Texas Heart Insti- staining (Invitrogen), NRCMs (1.0 × 105 cells/mL) were tute (Houston, Texas, USA). Plasma samples were placed in a 12-well culture plate and were treated with equilibrated by performing dialysis in a column various concentrations of L5 (0.075, 0.25, 0.75, 2.5, 7.5, loaded with buffer A (20 mmol/L Tris HCl [pH 8.0], 25, 75, or 250 μg/mL) overnight. NRCMs were collected 0.5 mmol/L EDTA, and 1% penicillin-streptomycin). and rinsed with ice-cold PBS, followed by centrifugation Approximately 100 mg of LDL was injected onto a and resuspension with 100 μL annexin binding buffer. UnoQ12 anion-exchange column (Bio-Rad, Richmond, Next, 5 μL Annexin V mixed with 1 μL PI (100 μg/mL) CA) by using a GE AKTA fast-protein liquid chroma- was added to the binding buffer. The cells were placed tography system (GE Healthcare Life Sciences, Logan, in the darkroom at room temperature. Then, 400 μL Utah, USA). LDL subfractions were eluted by using a annexin binding buffer was added 15 min later, and cell Ma et al. Lipids in Health and Disease (2020) 19:189 Page 3 of 10 apoptosis was measured by using a BD Biosciences im- 10 HEPES, and 10 glucose (pH 7.35 adjusted with aging system. Cells staining positive for PI were ex- NaOH). Calcium current blocker, nifedipine (2 nM, cluded, and the ratio of Annexin V–positive cells to Sigma), Glibenclamide transient potassium current nuclei was quantified. blocker, and 4-aminopyridine (2 mmol/L, Sigma-Aldrich Apo-ONE Homogeneous Caspase-3/7 Assay (Pro- Corp) were added. A − 40 mV prepulse was used to de- mega, Madison, WI, USA) was used to examine cell activate sodium currents.