Male Sprague-Dawley rats weighing 200–250 g (Charles River, Burlington, NJ, USA) were used in this study. The rats were adapted for one week in conditions appropriate to their species and ensuring their welfare. The animals were housed in cages (two rats per cage) and kept at controlled temperature (22 $\pm$ 2 ${}^{\circ }$C), humidity (55 $\pm$ 5%) and light/dark (12/12 hours) cycle. An ad libitum access to a diet of standard laboratory chow and water was provided. All rat experiments were conducted according to the Guide to the Care and Use of Experimental Animals published by the Canadian Council on Animal Care and reviewed by the Animal Research Ethics Board, University of Saskatchewan, Canada (Resolution no. 20060054).

Before heart surgery, rats were desensitised with buprenorphine (0.05 mg/kg, i.p.), and anaesthetised with sodium pentobarbital (0.5 mL/kg i.p.). Then, the chest was opened and the heart was rapidly excised from the animal. The spontaneously beating hearts were rinsed by immersion in an ice-cold Krebs-Henseleit Buffer (118 mmol/L NaCl, 4.7 mmol/L KCl, 1.2 mmol/L KH${}_2$PO${}_4$, 1.2 mmol/L MgSO${}_4$, 3.0 mmol/L CaCl${}_2$, 25 mmol/L NaHCO${}_3$, 11 mmol/L glucose, and 0.5 mmol/L EDTA, pH 7.4), and cannulated in the Langendorff system (EMKA Technologies, Paris, France) by the aorta. This procedure took no more than 30 sec to reduce the time of uncontrolled ischemia. Then, hearts were perfused at constant pressure (60 mmHg) with a Krebs-Henseleit Buffer (pH 7.4, 37 ${}^{\circ }$C) and gassed continuously (5% CO${}_2$/95% O${}_2$). The following haemodynamic parameters were monitored using an EMKA recording system with IOX2 software (EMKA Technologies, Paris, France): coronary flow (CF), heart rate (HR), left ventricular developed pressure (LVDP). LVDP and HR were measured with the help of a water-filled latex balloon connected to a pressure transducer and inserted through an incision in the left atrium into the left ventricle via the mitral valve. The volume was adjusted at the beginning of the perfusion period to achieve an end-diastolic pressure of 8–10 mmHg. LVDP was calculated as the difference between peak systolic and diastolic pressures.

Exclusion criteria: hearts that present atrial or ventricular fibrillation during ex vivo procedure as well as hearts that showed CF $>$28 mL/min or 10 mL/min when cannulated in EMKA system were excluded from the study.

The experimental rats were randomly allocated to the following groups: the aerobic control group, I/R group and I/R with $\mathrm{\Delta }$${}^9$-THC 0.1–1.0 $\mu$M groups.

Hearts from the I/R group were subjected to 25 min of aerobic stabilisation, 25 min of global no-flow ischemia (by cessation of the buffer flow) and 30 min of reperfusion (by buffer flow restoration), according to previous protocols [19, 20, 21]. The hearts from the aerobic control were perfused aerobically for 80 min. THC (Sigma Aldrich cat.no. T-005, Sigma-Aldrich, St. Louis, MO, USA‎) was administered with the Krebs-Henseleit buffer into the hearts during the last 10 min of aerobic stabilisation and in the first 10 min of reperfusion (after global ischemia) [19, 22] (Fig. 1A). Methyl alcohol (MetOH) was used as a vehicle for THC. Maximal MetOH infused 0.001% (v/v). To determine cardiac mechanical function, the recovery of rate pressure product (RPP) was expressed as the product of HR and LVDP and evaluated at experiment’s 25 min (the end of aerobic perfusion) and 80 min (the end of reperfusion) marks [19, 22]. After the experimental protocol, isolated hearts were immediately immersed in liquid nitrogen and stored at –80 ${}^{\circ }$C for further investigations.

Fig. 1.

Experimental protocol schemes. Experimental protocol scheme for heart I/R injury (A) and isolated cardiomyocytes chemical I/R injury (B).

Human Cardiac Myocytes (HCM) were purchased from ScienCell Research Laboratories (Carlsbad, CA, USA). The cells were grown in flasks covered with poly-lysine (final concentration at 0.014 mg/mL) at 37 ${}^{\circ }$C in a water-saturated, 5% CO${}_2$ atmosphere in Dulbecco’s Modified Eagle’s Medium (Sigma-Aldrich, St. Louis, MO, USA) containing Cardiac Myocyte Growth Supplement (ScienCell Research Laboratories, Carlsbad, CA, USA), 5% fetal bovine serum, 100 U/mL penicillin, 100 $\mu$g/mL streptomycin (all from Sigma-Aldrich, St. Louis, MO, USA) to 90% confluence.

HCM were subjected to in vitro chemical I/R in accordance with the guidelines for experimental models of myocardial ischemia [23]. Briefly, the cells underwent 15 min oxygenation, 15 min in vitro chemical ischemia and 20 min reperfusion [19], in the presence and absence of 10 $\mu$M THC (Sigma Aldrich cat.no. T-005, Sigma-Aldrich, St. Louis, MO, USA) (Fig. 1B). The aerobic stabilisation and reperfusion were performed in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer (5.5 mmol/L HEPES, 63.7 mmol/L CaCl${}_2$, 5 mmol/L KCl, 2.1 mmol/L MgCl${}_2$, 5.5 mmol/L glucose, 10 mmol/L taurine) containing an additional 55 $\mu$mol/L CaCl${}_2$ and 0.75 mg/mL BSA. The chemical ischemia group was incubated in a HEPES buffer containing 4.4 mmol/L 2-deoxyglucose (to inhibit glycolysis) and 4.0 mmol/L sodium cyanide (an inhibitor of cellular respiration) [19]. The optimal duration of ischemia (15 min) was previously established experimentally (data not shown). In the I/R group, after 15 min of aerobic stabilisation in the HEPES buffer at RT, the buffer was removed by centrifugation (1 min at 1500 $\times$ g) and the cell pellet was resuspended in the ischemia buffer and incubated for 15 min at RT. Then, the cells were centrifuged for 1 min at 1500 $\times$ g, the buffer was removed and cells were incubated in aerobic conditions with the HEPES buffer for 20 min at RT (reperfusion). After reperfusion, the buffer was removed by centrifugation at 1500 $\times$ g for 5 min, and the pellet was homogenised. The cells from the aerobic control group were incubated aerobically for 50 min in HEPES buffer at RT; however, they were centrifuge like the I/R group, and a new portion of an oxygenated HEPES buffer was administered as well. In the I/R+THC group, $\mathrm{\Delta }$${}^9$-THC (0.1–10 $\mu$M final concentration depending on the experiment) was added into the HEPES buffer.

Rat hearts were rapidly excised from rats anaesthetised with sodium pentobarbital (40 mg/kg, i.p.) as described above. The spontaneously beating hearts were immersed in an ice-cold Myocyte Isolation Buffer (MIB) containing 120 nM NaCl, 5 mM KCl, 2 mM NaAc, 2 mM MgCl${}_2$, 1 mM Na${}_2$HPO${}_4$, 20 mM NaHCO${}_3$, 5 mM glucose, 9 mM taurine and 10 mM CaCl${}_2$ at pH 7.4 immediately after removal. Then, hearts were suspended on a blunt-end needle of the Langendorff system by the aorta, perfused at a constant flow of 10 mL/min with an MIB buffer containing 10 mM CaCl${}_2$, pH 7.4, at 37 ${}^{\circ }$C, and gassed continuously with 5% carbogen for 5 min in the Langendorff system’s water-jacketed chamber.

After 5 min of stabilisation, the buffer was replaced with an MIB containing 5 $\mu$M CaCl${}_2$ and the hearts were perfused for 5 more minutes. The low concentration of CaCl${}_2$ resulted in the loss of cell contractility. After mild swelling of the myocardium with a HEPES buffer (120 mM NaCl 140, 5 mM KCl, 2 mM MgCl${}_2$, 5 mM glucose, 9 mM taurine, 5 mM HEPES) containing 40 $\mu$M CaCl${}_2$, 25 mg of collagenase and 2 mg of protease at pH 7.4, the right ventricle was excised from the heart, rinsed with a HEPES buffer containing 100 $\mu$M CaCl${}_2$, 150 mg bovine serum albumin (BSA), and then minced into small pieces in the digestion solution (a HEPES buffer containing 100 $\mu$M CaCl${}_2$, 150 mg BSA, 15 mg collagenase and 1 mg protease). The minced tissue was repeatedly digested (6 times for 20 and 10 min in a water bath (37 ${}^{\circ }$C)), and the 3rd–6th fraction was used for further experiments.

Chemical ischemia was induced after 15 min of $\mathrm{\Delta }$${}^9$-THC treatment in a HEPES buffer containing 100 $\mu$M CaCl${}_2$, 150 mg BSA, by covering the cell pellets with a HEPES buffer containing 4 mM 2-deoxyglucose and 40 mM sodium cyanide (2.5 $\mu$M). The optimal ischemia duration of 3 min was established in previous studies [20]. After 3 min chemical ischemia, the buffer containing sodium cyanide was removed by centrifugation (1 min at 1500 $\times$ g) and the cells pellet was suspended in the fresh portion of the HEPES buffer containing 100 $\mu$M CaCl${}_2$, 150 mg BSA and 10 $\mu$M $\mathrm{\Delta }$${}^9$-THC. After a 20 min reperfusion, the cells were centrifuged at 1500 $\times$ g for 5 min and the cell pellet suspended in HEPES buffer (100 $\mu$M CaCl${}_2$, 150 mg BSA) was used for contractility measurement. The aerobic control group was kept exposed to atmospheric air for 38 min, and the chemical ischemia control cardiomyocytes underwent the same experimental protocol without $\mathrm{\Delta }$${}^9$-THC treatment.

Next, the contractility of cardiomyocytes was measured. A 100 $\mu$L aliquot of cell suspension was placed in the rapid change stimulation chamber of the IonOptix Contractility System (IonOptix, Milton, MA, USA). After 3 min of stabilisation, cardiomyocytes were perfused with an oxygenated HEPES buffer containing 2 mM CaCl${}_2$ (4 mL/min) at 37 ${}^{\circ }$C. Cells were continuously paced with 1 Hz and 5 V (IonOptix MyoPacer, Milton, MA, USA), and the contractility—expressed as a per cent of peak shortening in comparison to the length of the diastolic cell—was measured on an average of 5 cells per sample. At least 5 samples per one experimental condition were evaluated.

Three cycles of freezing (in liquid nitrogen) and thawing (at 37 ${}^{\circ }$C) in the homogenisation buffer (50 mmol/L Tris-HCl (pH 7.4) containing 3.1 mmol/L sucrose, 1 mmol/L DTT, 10 $\mu$g/mL leupeptin, 10 $\mu$g/mL soybean trypsin inhibitor, 2 $\mu$g/mL aprotinin and 0.1% Triton X-100) were used to disorganise the cell pellet. Then, myocytes were homogenised mechanically on ice (three times for 30 seconds) with a hand-held homogeniser. Supernatants for further analysis (stored at –80 ${}^{\circ }$C) were obtained by a 5 min centrifugation at 10,000 $\times$ g 4 ${}^{\circ }$C.

The Bradford method was used to determine protein concentration in cell homogenates and supernatants. BSA (heat shock fraction, $\ge$98%, Sigma-Aldrich) at a final concentration of 1.0–0.0062 $\mu$g/$\mu$L served as the protein standard to create the standard curve. A Bio-Rad Protein Assay Dye Reagent (BioRad, Hercules, CA, USA) and a Spark multimode microplate reader (Tecan Trading AG, Switzerland) were used to measure the total protein concentration.

Fluorescein diacetate (FDA) and DAPI were used as the vital inclusion and vital exclusion dyes, respectively [24]. Viable and metabolically active cells can incorporate the nonpolar, nonfluorescent FDA and rapidly hydrolyse it by acetyl esterase to fluorescein, a polar, fluorescent compound retained in the cell. Damaged cells release esterified fluorescein to liquid medium, rendering cell visualisation impossible. Nonviable cell nuclei were stained with DAPI [25, 26]. Briefly, myocytes were seeded in a 96-well plate at a density of 2 $\times$ 10${}^4$ cells/well for 24 h and then subjected to in vitro chemical I/R injury in the presence and absence of $\mathrm{\Delta }$${}^9$-THC, according to the above protocol. The cells were washed three times with PBS and stained with 5 $\mu$g/mL FDA (cat. no. F7378, Sigma-Aldrich, St. Louis, MO, USA) and 1:1000 DAPI (cat.no. D8417-5MG, Sigma-Aldrich, St. Louis, MO, USA) for 15 min in the dark. ZOE Fluorescent Cell Imager (BioRad, Hercules, CA, USA) was used to estimate the bright green fluorescence for FDA and blue fluorescence for DAPI. Cells were considered metabolically active when stained only with FDA (FDA-positive, DAPI-negative), whereas cells that excluded FDA and stained only with DAPI were considered necrotic (FDA-negative, DAPI-positive) [22]. Image J 1.52a software (NIH, Bethesda, Maryland) was used to analyse the area of fluorescence of each image. To determine cell metabolic activity, green fluorescence (live cells) was normalised to the total number of cells (green + blue fluorescence) in each experiment [24]. Data were collected from three independent image trials for each experiment and shown as AU. The metabolic activity was compared in cells that were exposed to aerobic conditions and cells subjected to I/R and I/R with the addition of $\mathrm{\Delta }$${}^9$-THC.

The influence of $\mathrm{\Delta }$${}^9$-THC on cardiomyocyte morphology in chemical I/R was assessed in 9-well plates. HCM (4.8 $\times$ 10${}^4$ cells/well) were cultured for 24 h and subjected to chemical I/R, I/R + 10 $\mu$M $\mathrm{\Delta }$${}^9$-THC or maintained in aerobic conditions, as described above and shown in Fig. 1B. The structural changes in cardiomyocytes were photographed using a Live Cell Imaging Micriscope-3D Cell Explorer (Nanolive, Tolochenaz, Switzerland). The cells were analysed for cytoplasm vacuolisation, the size and the number of mitochondria, as well as the degree of degradation of the nucleus and cytoplasm.

To determine the activity of LDH in coronary effluents, a Lactate Dehydrogenase Activity Assay Kit (cat.no. MAK066-1KT, Sigma-Aldrich, St. Louis, MO, USA) was used, according to the manufacturer’s instruction. Since LDH is a stable cytosolic enzyme, it is released upon membrane damage/increased permeability or cell lysis and commonly serves as a marker of cell damage [19, 27, 28]. LDH activity in cultured cells was normalised to total protein.

Gelatine zymography was performed based on the Heussen and Dowdle protocol with our modifications, as previously described [22, 29]. 40 $\mu$g of heart homogenates were placed in a sample loading buffer and applied to polyacrylamide gel co-polymerised with gelatine. After electrophoresis, gels were rinsed in 2.5% Triton X-100 to remove SDS. Gels were incubated in an incubation buffer at 37 ${}^{\circ }$C overnight, stained in a staining solution and destained in a destaining solution. Zymograms were scanned with a VersaDoc 5000 (BioRad, Hercules, CA, USA) and the band intensities were analysed using Quantity One software v. 4.6.6 (BioRad, Hercules, CA, USA). MMP-2 activity was expressed as activity per microgram of protein.

A Human CERK (Ceramide kinase) ELISA Kit (cat.no. EH1538, Fine Test, Wuhan, Hubei, China) was used to evaluate the potential mechanism of $\mathrm{\Delta }$${}^9$-THC cardioprotection in I/R cardiomyocytes. The assay was used to quantitatively assesses ceramide kinase (CERK) in cell homogenates with the ELISA method, as per the manufacturer’s instructions. The level of CERK in cell homogenates was normalised to total protein concentration and expressed in pg/$\mu$g protein.

An OxiSelect™ In Vitro ROS/RNS Assay Kit (Cell Biolabs, San Diego, CA, USA) was used to explore the influence of $\mathrm{\Delta }$${}^9$-THC on the level of total reactive oxygen and nitrogen species (ROS/RNS) in cardiomyocytes. The assay is designed to measure the total ROS and RNS, including hydrogen peroxide, nitric oxide, peroxyl radicals, and peroxynitrite anions, with the use of a proprietary fluorogenic probe—dichlorodihydrofluorescin DiOxyQ (DCFH-DiOxyQ). DCFH-DiOxyQ is primed with a dequenching reagent to the highly reactive DCFH form. In the presence of ROS and RNS, the DCFH is quickly oxidised to the highly fluorescent 2, 7-dichlorodihydrofluorescein (DCF). Fluorescence intensity is proportional to the total ROS/RNS level within the sample. The total ROS/RNS level was assessed in cell homogenates and supernatants.

An OxiSelect™ Total Antioxidant Capacity (TAC) Assay Kit (cat.no. STA-360, Cell Biolabs, San Diego, CA,USA) was used to examine the influence of $\mathrm{\Delta }$${}^9$-THC on resistance to oxidative stress during I/R. Measurement of the total non-enzymatic antioxidant capacity (TAC) indicates the ability of cells to counteract induced oxidative stress. The TAC Assay is based on the reduction of copper (II) to copper (I) by the antioxidants present in the sample. After reduction, the copper (I) ion further reacts with the coupling chromogenic reagent which produces a color with a maximum absorbance of 490 nm. The absorbance values were proportional to the total reducing capacity of the cardiomyocytes. TAC levels were measured in cell homogenates and normalised to total protein concentration in each sample.

IL-6 concentration in cardiomyocytes homogenates was determined using Human IL-6 DuoSet ELISA Kit (cat.no. DY206-05, R&D Systems, a bio-techne brand, Minneapolis, Minnesota, USA), according to the manufacturer’s instructions. The minimum detectable concentration of IL-6 was 9.4 pg/mL. IL-6 concentration in cell homogenates was expressed as pg per $\mu$g of total protein.

GraphPad Prism 8.0 software (GraphPad Software, San Diego, CA, USA) was used for statistical analysis of the results. To assess normality of variance changes, the Shapiro-Wilk normality test or the Kolmogorov-Smirnov test was used. The Student t-test or the Mann–Whitney U test was used for data comparison of two groups. For multiple groups, ANOVA or nonparametric Kruskal-Wallis test with appropriate post hoc tests (Tukey-Kramer Multiple Comparisons test/Dunn test) were used. The correlation was assessed using the Spearman test. The results were expressed as mean $\pm$ SEM and a value of p 0.05 was regarded as statistically significant. Different sizes of animal groups (3–6) used in experiments depended on the exclusion criteria (presented above) and the Power Analysis test (alpha = 0.05, power = 0.80, $\pm$10%) as well as the principles of the 3Rs (replacement, reduction, refinement). The difference in N numbers between experiments also followed from the fact that some part of HCM died during cell culturing, and as such, the N number for selected tests had to be reduced. Some results had to be removed from the analysis as well, as they were significant outliers according to the Grubbs test.

To study the effect of $\mathrm{\Delta }$${}^9$-THC in hearts ex vivo, we used well established model of global ischemia with use of isolated rat hearts by inducing global, no-flow ischemia for 25 min, followed by 30 min reperfusion. The $\mathrm{\Delta }$${}^9$-THC was administered 10 min before ischemia and during the first 10 min of reperfusion (Fig. 1A).

Data showed that $\mathrm{\Delta }$${}^9$-THC 0.1–1 $\mu$M protected the heart, as evidenced by the improved recovery of cardiac function (p 0.0001), HR (p 0.0001) and LVDP (p = 0.001) analysis (Fig. 2A–C). We also noticed increased coronary flow in comparison to I/R hearts when 1 $\mu$M of $\mathrm{\Delta }$${}^9$-THC was used (Fig. 2D).

Fig. 2.

Protection of mechanical function of isolated rat hearts subjected to ischemia/reperfusion (I/R) injury and perfused with $\mathrm{\Delta }$${}^{\mathrm{9}}$-tetrahydrocannabinol ($\mathrm{\Delta }$${}^{\mathrm{9}}$-THC). (A) Recovery of heart rate pressure product (RPP). Recovery is expressed as a % of the pre-I/R mechanical function. (B) Left ventricular developed pressure (LVDP). (C) Heart rate. (D) Coronary flow. Results were expressed as mean $\pm$ SEM and p 0.05 was considered as statistically significant. * vs. Aerobic; # vs. 25/30 I/R; $ vs. 25/30 I/R = 0.1 $\mu$M THC. Aerobic, N = 6; Aerobic + 1 $\mu$M THC, N = 3; 25/30 I/R, N = 6; 25/30 I/R + 0.1 $\mu$M THC, N = 3; 25/30 I/R + 1 $\mu$M THC, N = 6. Methyl alcohol (MetOH) was used as a vehicle for THC. Maximal MetOH infused 0.001% (v/v).

As shown in Fig. 2, we constructed a stable model in which $\mathrm{\Delta }$${}^9$-THC did not affect hearts maintained in aerobic conditions. Hence, to study the mechanism of $\mathrm{\Delta }$${}^9$-THC action in cardiomyocytes, we constructed the model of chemical I/R, as shown in the scheme in Fig. 1B.

Since LDH is a cellular protein that can be easily released from cells due to cell injury or increased permeability of the membrane [30], cells subjected to chemical I/R showed lower cytoplasmic LDH activity—as more of it was released into the extracellular space (here supernatants), while a 10 $\mu$M $\mathrm{\Delta }$${}^9$-THC treatment reduced cell injury and increased LDH content in cell homogenates to the level observed in the aerobic control (less LDH was released into supernatants) (p = 0.019) (Fig. 3A). At this point, we determined that 0.1 and 1.0 $\mu$M of $\mathrm{\Delta }$${}^9$-THC did not bring improvement in cardiomyocytes injury and thus assumed that the working dose of $\mathrm{\Delta }$${}^9$-THC was 10 $\mu$M, and the latter dose was used in further experiments.

To demonstrate the effect of $\mathrm{\Delta }$${}^9$-THC on impaired contractility, we measured the contractility of cardiomyocytes. Data showed that I/R attenuates cardiomyocytes contractility, but the treatment of the cells with 10 $\mu$M $\mathrm{\Delta }$${}^9$-THC ameliorated cell contractility, p = 0.024 (Fig. 3B).

Fig. 3.

Cardiomyocyte contractility and damage after I/R. (A) Cell injury and (B) cardiomyocyte contractility during I/R. I/R, ischemia/reperfusion; N = 6–9 per group. Results were expressed as mean $\pm$ SEM and p 0.05 was considered as statistically significant. * Aero vs. I/R; # I/R vs. I/R+10 $\mu$M THC. LDH, lactate dehydrogenase; Aero, aerobic; I/R, ischemia/reperfusion.

To further confirm that 10 $\mu$M $\mathrm{\Delta }$${}^9$-THC protected cardiomyocytes from I/R induced injury and then from apoptosis/necrosis, cell morphological changes have been analysed. In aerobic conditions, cells showed a morphologically normal state as it was expressed by a large surface area, spread out over the plate surface, with no signs of swelling and almost imperceptible mitochondria (about 0.3 $\mu$m in diameter) (Fig. 4A–C). In the I/R control group, the cells changed their shape into spherical due to oedema. A strong vacuolisation of the cytoplasm and many enlarged and swollen mitochondria (4.2 $\mu$m–3.0 $\mu$m) were observed. The cytoplasm was often fragmented, and the cell nucleus had a blurred structure and destructive features (Fig. 4D–F). In the group subjected to I/R in the presence of $\mathrm{\Delta }$${}^9$-THC at a dose of 10 $\mu$M, I/R lesions were also present, as in the I/R control, but they were less severe. The cells did not change their shape in any significant way. The swelling was smaller (2.3 $\mu$m–1.0 $\mu$m) and occurred in fewer mitochondria. The cytoplasm did not show any fragmentation features (Fig. 4G–I).

Fig. 4.

Cell morphology of I/R injured cardiomyocytes. (A–C) Aerobic control. Morphologically normal cells, with a large surface area, spread out over the plate surface, did not show signs of swelling, almost imperceptible mitochondria and vacuoles (mean ø 0.3 $\mu$m in diameter). (D–F) I/R cells. Cell shape changed into spherical due to oedema. A strong vacuolisation of the cytoplasm (mean ø 3.6 $\mu$m); numerous enlarged and swollen mitochondria with amorphous densities. The cytoplasm was fragmented, and the cell nucleus has a blurred structure and destructive features. (G–I) No significant change of shape. Swelling occurred in fewer mitochondria, a lower number of vacuoles (mean ø 1.6 $\mu$m) was observed. The cytoplasm does not show any fragmentation features. Live Cell Imaging Micriscope-3D Cell Explorer (Nanolive, Tolochenaz, Switzerland). N, nucleus; M, mitochondria; V, vacuoles; F, filopodium; LD, lipid droplets.

Cell viability was evaluated in cardiomyocytes subjected to chemical I/R, I/R with $\mathrm{\Delta }$${}^9$-THC, as well as in the aerobic control group, by vital staining with DAPI. I/R condition affected cell viability (p = 0.024) but 10 $\mu$M $\mathrm{\Delta }$${}^9$-THC decreased the number of dead cells (p = 0.005) (Fig. 5). The above results indicated that $\mathrm{\Delta }$${}^9$-THC exerts protection in the I/R damage of cardiomyocytes.

Fig. 5.

Cell viability after I/R. $\mathrm{\Delta }$${}^{\mathrm{9}}$-THC affects cell viability: graphical presentation of the result (A); microscopic photos (B–D). Nuclei in vivo staining with DAPI were considered necrotic. I/R, ischemia/reperfusion; N = 6–9 per group, *p = 0.024; #p = 0.005.

In addition to increased cardiomyocyte viability, 10 $\mu$M $\mathrm{\Delta }$${}^9$-THC also enhanced the activity of intracellular acetyl esterase (p = 0.035) (Fig. 6A–D), which was able to rapidly hydrolyse fluorescein diacetate into polar fluorescein. This confirmed the cells’ ability to preserve their metabolic function. Increased metabolic activity of the cells positively correlated with cell contractility (Fig. 6E), providing evidence of a cardioprotective role of $\mathrm{\Delta }$${}^9$-THC.

Fig. 6.

The influence of $\mathrm{\Delta }$${}^{\mathrm{9}}$-THC on the metabolic activity of cardiomyocytes. Microscopic photos (A–C), graphical presentation of the result (D); Correlation of cardiomyocytes contractility and metabolic activity (E). I/R, ischemia/reperfusion; N = 6–9 per group, *p = 0.025, #p = 0.035.

To assess the potential mechanism of $\mathrm{\Delta }$${}^9$-THC cardioprotection, we analysed changes in MMP-2 activity using gelatine zymography. Data confirmed the decreased activity of MMP-2 (Fig. 7) in cells subjected to I/R (p = 0.019) compared to the Aero group. Moreover, the administration of 10 $\mu$M $\mathrm{\Delta }$${}^9$-THC into cells subjected to I/R (p = 0.040) restored MMP-2 activity to the level of the aerobic control. Decreased MMP-2 activity in cardiomyocytes was accompanied by an increased synthesis of IL-6, r = 0.998 p = 0.023.

Fig. 7.

MMP-2 activity in I/R cardiomiocytes. (A) MMP‑2 activity in HCM normalised to the protein content in each sample (N = 6–8 per group). (B) Representative zymogram of MMP‑2 activity in HCM. Data are presented as the mean $\pm$ SEM. HCM—human cardiomyocytes; Aero, aerobic control; I/R, chemical ischemia and reperfusion; AU, arbitrary units. 10 $\mu$M $\mathrm{\Delta }$${}^9$-THC was used. *p = 0.019; #p = 0.040.

The total level of CERK was lower in the I/R group, reflecting oxidative/nitrosative stress changes. The administration of 10 $\mu$M $\mathrm{\Delta }$${}^9$-THC effectively increased the production of CERK (Fig. 8A) to the level of the aerobic control. Since CERK induces cell survival [31], we showed a negative correlation between CERK and the number of dead cells, r = –0.53 p = 0.040 (Fig. 8B).

Fig. 8.

CERK activity in I/R cardiomyocytes. CERK, ceramide kinase; I/R, ischemia/reperfusion; N = 6–9 per group, *p = 0.028.

The total ROS/RNS level was significantly decreased in I/R cells (p = 0.007) and increased in I/R supernatants (p = 0.013) (Fig. 9A–B), confirming oxidative/nitrosative stress. The administration of 10 $\mu$M $\mathrm{\Delta }$${}^9$-THC significantly enhanced the total antioxidant capacity (TAC) in cardiomyocytes subjected to I/R (p = 0.010) (Fig. 9C).

Fig. 9.

Oxidative status in cardiomyocytes. (A) Total ROS/RNS level in cells expressed as nM of DCF normalised to $\mu$g protein, *p = 0.007. (B) total ROS/RNS level in cell supernatants expressed as nM of DCF, *p = 0.013. Supernatants were collected from the same number of cells. (C) Total antioxidant capacity of cells, *p = 0.010, #p = 0.010. TAC was expressed as $\mu$M of CRE and normalised to the total protein concentration. Aero, aerobic control group; I/R, ischemia/reperfusion; CRE, copper reducing equivalents; DCF, 2${}^{\prime }$, 7${}^{\prime }$-dichlorodihydrofluorescein; RNS, reactive nitrogen species; ROS, reactive oxygen species; TAC, total antioxidant capacity, N = 6–8 per group.

The IL-6 level in cells subjected to I/R as well as in those threated with $\mathrm{\Delta }$${}^9$-THC was increased, but this difference was not statistically significant (Fig. 10).

Fig. 10.

IL-6 concentration in cardiomyocytes. Aero—aerobic control group, I/R, ischemia/reperfusion; IL-6, interleukin 6. N = 5–8 per group.