Cigarette smoke extract (CSE) was generated as previously described with slight modifications [22]. Briefly, one 2RF4 research cigarette (University of Kentucky, Lexington, KY) was continuously smoked by connecting the filtered end of the cigarette to a vacuum pump, pulling the particles into 5 ml of DMEM/F12 and the resulting medium was defined as 100% CSE. The total particulate matter content of 2RF4 cigarettes is 11.7 mg/cigarette, tar is 9.7 mg/cigarette, and nicotine is 0.85 mg/cigarette. Dilutions were made using DMEM/F12 + 10% FBS. Human type II–like pulmonary adenocarcinoma cells (A-549; passage 10-15) were maintained in DMEM/F12 supplemented with 10% FBS (Invitrogen) and antibiotics. Cells were split into 6-well dishes and grown to 80% confluence. H9C2 cardiomyocytes were maintained in DMEM +10% FBS. For differentiation into myotubes, cells were grown to confluency and the medium was replaced with DMEM +10% horse serum (Invitrogen, Grand Island, NY). Myotubes were used for experiments on day 3 of differentiation. A-549 cultures were exposed to media supplemented with 10% CSE or media alone for 4 h, after which the medium was transferred to differentiated H9C2 cardiomyocytes (termed “conditioned medium”) for 12 h. Where indicated, cells were treated with myriocin (10 μM, Sigma, M1177), a known and widely used inhibitor of ceramide biosynthesis. Muscle cells were harvested for RNA, protein, and lipid isolation following treatments. Cell survival during treatments was confirmed by trypan blue exclusion.

For isolation of lipids, cell pellets or tissue were suspended in 900 μl ice-cold chloroform/methanol (1:2) and incubated for 15 minutes on ice, then briefly vortexed. Separation of aqueous and organic phases required addition of 400 μl of ice-cold water and 300 μl of ice-cold chloroform. The organic phase was collected into a fresh vial, and lipids were dried in a vacuum centrifuge (Eppendorf Concentrator Plus). Lipids were characterized and quantified using a shotgun lipidomics technique on a Thermo Scientific LTQ Orbitrap XL mass spectrometer. Evaporated lipid samples were re-suspended in a 2:1 chloroform: methanol Folch solution (200 μL). The re-suspended lipids were then combined with a modified 2:1:1.25 chloroform: methanol: isopropanol Bligh and Dyer solution (800 μL) with 15 mM ammonium acetate acting as an ionizing adduct. A 1.74 μM phosphatidylethanolamine, 1 μM C-17 ceramide, 1 μM tripalmitin internal standard cocktail (1 μL) was spiked into each sample for mass calibration and characterization data alignment. Samples were analyzed using a 2.5-minute mass-window scanning method in positive-ion mode at a resolution of 100,000 (FWHM at 400 m/z) for all primary MS¹ scans. MS² fragmentation data was also collected (PQD at relative intensity of 35) and manually verified for each mass window to give additional confidence to the correct identification of abundant lipid species. Three technical replicate mass spectrometer runs were performed on each sample. Samples were injected at 8 μL/min using a direct-inject electrospray ionization (ESI) soft-ionization spray head from a Hamilton GASTIGHT glass syringe. A nitrogen sheath gas spray flow rate of 8 (arb. Units) was used for all runs. The spray voltage and capillary temperature were maintained at 5.0 KV and 275°C respectively. Each technical replicate was run in random order to reduce systematic bias. Data were analyzed using in-house developed peak summarization, recalibration, and lipid identification software using lipid database information from the LIPID Metabolites and Pathways Strategy (Lipid MAPS) Lipidomics Gateway database [23]. To ensure high-confidence identifications, an intensity threshold estimated to be 5% above instrumental static signal was implemented. Lipid identities were only assigned when significantly observable peaks were identified in at least two of the three technical replicate runs. Non-zero lipid quantities were averaged from the replicate runs. The lipid species identified across different ionization states or with adducts were totaled together. Quantification was completed by normalizing total ion counts to the relative abundance of the internal standard that was spiked into each sample.

For cells, H9C2 cardiomyocytes were detached in culture dishes with 0.05% trypsin-EDTA (Sigma) and growth medium was added to the culture. Contents were transferred to a tube and centrifuged for 10 min at 1000 × g at RT. After removal of supernatant, cells were lifted in MiR05 [0.5 mM EGTA, 3 mM MgCl₂, 60 mM K-lactobionate, 20 mM taurine, 10 mM KH₂PO₄, 20 mM HEPES, 110 mM sucrose, and g/l BSA (Sigma; A3803) adjusted to pH 7.1] plus 1 mg/ml digitonin and gently rocked at RT for 5 min before centrifugation at 1000 × g for 5 min. After discarding supernatant, cells were then suspended in 2.2 ml warm MiR05 and transferred to chambers in the O2K (Oroboros Instruments, Innsbruck, Austria). Following respiration protocol (outlined below), cells were removed from the chambers and used for further analysis, including protein quantification. For myocardial mitochondrial respiration, left ventricle was quickly removed from euthanized mice and immediately placed in ice-cold buffer X (60 mM K-MES, 35 mM KCl, 7.23 mM K₂EGTA, 2.77 mM CaK₂EGTA, 20 mM imidazole, 20 mM tuarine, 5.7 mM ATP, 15 mM PCr, 6.56 mM MgCl₂-6H₂O, pH 7.1) and trimmed of connective tissue. Small fiber bundles were prepared and gently separated along their longitudinal axis under a surgical scope (Olympus, ST) to 1-2 mg. Bundles were then transferred to a tube with chilled buffer X and 50 μg/ml saponin and rocked at 4°C for 30 min, then washed in buffer Z (105 mM K-MES, 30 mM KCl, 10 mM KH₂PO₄, 5 mM MgCl₂-6H₂O, 0.5 mg/ml BSA, pH 7.1) at 4°C for at least 15 min. Samples were then blotted dry and weighed.

To isolate mitochondria from left ventricle myocardium, the whole heart was placed in isolation buffer (300 mM sucrose, 10 mM Na-HEPES, 0.2 mM EDTA, pH 7.2) and the left ventricle isolated. After finely mincing the sample, trypsin (Sigma T9935) was added for two minutes before adding trypsin inhibitor (Sigma T9003). The sample was then transferred to a conical tube and allowed to settle. After removal of supernatant, samples were resuspended in isolation buffer with BSA (Sigma A3803) and homogenized with Teflon pestle before centrifugation (600 × g for 10 min at 4°C). Supernatant was then transferred to a new tube and centrifuged (8000 × g for 15 min at 4°C), then washed by gently adding isolation buffer and rotating the tube. A portion of this suspension is used for protein quantification. The pellet was then resuspended with isolation buffer + BSA.

High-resolution O₂ consumption was determined at 37°C in permeabilized cells and fiber bundles using the Oroboros O2K Oxygraph with MiR05 respiration buffer. Before addition of sample into respiration chambers, a baseline respiration rate was determined. After addition of sample, the chambers were hyperoxygenated to ~350 nmol/ml. Following this, respiration was determined by all or parts of the following substrate-uncoupler-inhibitor-titration (SUIT) protocol [24]: electron flow through complex I was supported by glutamate + malate (10 and 2 mM, respectively) to determine basal oxygen consumption (GM _B ). Following stabilization, ADP (2.5 mM) was added to determine oxidative phosphorylation capacity (GM _P ). Succinate was added (GMS _P ) for complex I + II electron flow into the Q-junction. To determine full electron transport system (F) capacity over oxidative phosphorylation, the chemical uncoupler carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP) was added (0.05 μM, followed by 0.025 μM steps until maximal O₂ flux was reached). Complex II-supported ETS was then measured by inhibiting complex I with rotenone (Rot; 0.5 μM). Mitochondrial membrane integrity was tested in all experiments by adding cytochrome c (10 μM; GMc _P ). Lastly, residual oxygen consumption was measured by adding antimycin A (2.5 μM) to block complex III action, effectively stopping any electron flow, which provides a baseline rate of respiration. Where indicated, C2-ceramide (Sigma A7191; 20 μM) was added to respiration chambers.

To determine the effects of ceramide on cardiac mitochondrial respiration, we utilized two models. First, mitochondria were isolated from left ventricle myocardium. During the course of the respiration protocol in the isolated mitochondria, C2-ceramide was added to one oxygraph chamber. The addition of ceramide (+C2) elicited a rapid and significant reduction in mitochondrial respiration that was sustained through maximal respiration with FCCP (Figure 1A; see Methods for protocol details). A second model was the use of permeabilized intact left ventricle myocardium. One sample was incubated with C2-ceramide throughout the length of the protocol, which elicited a sustained reduction in mitochondrial respiration throughout the entire protocol (Figure 1B).

The deleterious effect of cigarette smoking on heart function is undeniable, but the mechanism communicating the pulmonary insult to the heart is not clear. As proof of concept that the lung is capable of producing and secreting ceramides with smoke exposure, we treated A549 alveolar type 2 cells with cigarette smoke extract (CSE). Relative to PBS treatment, ceramides increased over 60% in CSE-treated lung cells, though this effect was mitigated with myriocin co-treatment (Figure 2A). Further, CSE treatment elicited an almost three-fold increase in ceramide secretion into medium compared with PBS treatment (Figure 2B). We also observed an upward trend (P = 0.069) in circulating ceramides from whole blood following 5 d of sidestream smoke exposure in adult mice (Figure 2C).

To determine the effects of lung cell-secreted ceramides on heart cell function, we utilized a conditioned medium in vitro model. Briefly, following incubation with PBS- or CSE-containing medium, with or without myriocin co-treatment, the conditioned medium from lung cells was transferred to H9C2 cardiomyotubes for 12 h. Following the incubation with conditioned medium, we determined cardiomyotube ceramide accrual and mitochondrial respiration. Ceramides were significantly increased in cardiomyotubes treated with conditioned medium from CSE-treated lung cells (Figure 3A). However, when lung cells received treatment with myriocin in addition to CSE, the conditioned medium had no effect on heart cell ceramides (Figure 3A). Mitochondrial respiration in the heart cells followed a similar trend as ceramides. Namely, respiration in heart cells receiving CSE conditioned medium was negatively affected, but not when myriocin was included in the lung cell culture medium (Figure 3B).

To more accurately determine the role of ceramides in mediating smoke-induced decayed heart mitochondrial respiration, we exposed animals to CS for 1 wk while receiving injections of PBS (vehicle) or myriocin every other day. Left ventricle ceramides increased four fold with CS compared with room air-exposed mice (Figure 4A) with vehicle injections, though myriocin prevented this effect. Moreover, respiration was protected from CS in myriocin-injected animals (Figure 4B).

Given the evidence suggesting the role of diet in increasing heart complications [25] and ceramide accrual [26], we provided a high-fat, high-sugar (Western diet; WD) to animals in conjunction with smoke (or room air) exposure over the course of an 8-wk study. Similar to before, animals received vehicle (PBS) or myriocin injections every other day. In addition to observing a roughly 6-fold increase in heart ceramides with smoke exposure, we found an equally great increase in heart ceramides in animals receiving WD diet and smoke (Figure 5A). However, ceramide inhibition was only partially successful in the WD + CS group. In general, ceramide accrual was associated with reduced myocardial mitochondrial respiration (Figure 5B).