Luteolin (>99% purity) was purchased from Hangzhou FST Pharmaceutical Co., Ltd. (Hangzhou, China). UW solution was purchased from Bristol-Myers Squibb (New York, NY, USA). Dulbecco's modified Eagle's medium (DMEM) solution, pentobarbital sodium and heparin were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). The calcium florescent detection kits for mitochondria (cat. no. HL10153.1) and sarcoplasmic reticulum (SR; cat. no. GMS10157.1), and intracellular calcium concentration fluorescent detection kits (cat. no. JM325) were purchased from Shanghai Haling Biological Technology Co., Ltd. (Shanghai, China). Fluo-3 acetoxymethyl (AM) was purchased from Beyotime Institute of Biotechnology (Haimen, China). Collagenase type I was purchased from Invitrogen; Thermo Fisher Scientific, Inc. (Waltham, MA, USA). Low Ca²⁺ enzymatic solution (50 µmol/l) was prepared by dissolving 20 mg collagenase type 1, 2 mg protease E (Sigma-Aldrich; Merck KGaA), 10 mg bovine serum albumin (MP Biomedicals, LLC, Santa Ana, CA, USA) and 72 mmol/l calcium chloride into 30 ml calcium free Tyrode's solution, which was composed of 140 mM sodium chloride, 5 mM potassium chloride, 10 mM HEPES, 2 mM BAPTA, 10 mM glucose, and 10 mM Na pyruvate (pH 7.4±0.2).

A total of 40 6–8 week old healthy male SD rats were purchased from Xi'an Jiaotong University Animal Center (Xi'an, China), weighing 250–300 g. Rats were housed in a pathogen-free environment (temperature, 20–30°C; relative humidity, 45–60%; lighting cycle, 12 h/day) and had ad lbitum access to food and drinking water. All animal experiments were performed according to the Guide of Care and Use of Laboratory Animals (20) and approved by the Animal Care and Use Committee of the Hainan Medical College (Haikou, China).

Following sacrifice, rat cardiomyocytes were isolated following a previously described protocol (21). In brief, 30 mg/kg pentobarbital sodium was intraperitoneally injected into the anesthetized rats, followed by 3 mg/kg heparin to prevent blood clot. Heart tissues were quickly isolated following a previously published protocol (10) and were then placed in calcium free Tyrode's solution at 4°C. Isolated rat hearts were first perfused with oxygenated calcium free Tyrode's solution at 37°C until cardiac arrest using the Langendorff technique (22). A low Ca²⁺ enzymatic solution (50 µmol/l) was then used to further perfuse the rat hearts for 20–40 mins. When the hearts were softened, they were then again perfused with 180–200 µmol/l low Ca²⁺ enzymatic solution for 5 min. Following the 20 min perfusion process, ventricles were chopped and filtered with Krebs-Bicarbonate (KB) solution composed of 50 mM L-glutamine, 20 mM potassium chloride, 1 mM HEPES, 10 mM glucose, 80 mM potassium hydroxide, 30 mM dipotassium phosphate, 20 mM taurine, 3 mM magnesium sulfate and 0.5 mM EDTA (all Sigma-Aldrich; Merck KGaA) and incubated for 2 h at 37°C. A total of 5×10⁶ harvested cardiomyocytes were then washed with KB solution and preserved in 5 ml UW solution or DMEM. Luteolin was used to supplement UW solution at a low dose of 7.5 µmol/l, middle dose of 15 µmol/l and a high dose of 30 µmol/l. The groups used were as follows: i) A UW group, consisting of cardiomyocytes preserved in UW solution only; ii) a DMEM group, consisting of cardiomyocytes preserved in DMEM only; iii) a 7.5 µmol/l luteolin group, consisting of cardiomyocytes preserved in UW solution supplemented with 7.5 µmol/l luteolin; iv) a 15 µmol/l luteolin group, consisting of cardiomyocytes preserved in UW solution supplemented with 15 µmol/l luteolin; and v) a 30 µmol/l luteolin group, consisting of cardiomyocytes preserved in UW solution supplemented with 30 µmol/l luteolin.

To detect calcium florescence, 2 µM Fluro-3 AM was incubated with cardiomyocytes for 1 h at 37°C. Samples were washed twice with PBS and observed under a Multiphoton microscope (710 NLO; Carl Zeiss AG, Oberkochen, Germany) with an excitation wavelength of 490 nm and an emission wavelength of 525 nm to detect the level of Ca²⁺ in the cytosol. DAPI purchased from Wuhan Boster Biological Technology, Ltd. (Wuhan, China) was used for nuclear staining at room temperature for 5 mins. The same wavelengths were applied for the mitochondria and SR calcium detection kits, which were used according to the manufacturer's protocols. Calcium florescence intensity was analyzed semi-quantitatively by a trained examiner in a blinded manner, with four sections per animal.

The levels of CaM and MCU in the cardiomyocytes were examined using a Rat CaM ELISA kit (cat. no. E-EL-M0611) from Elabscience Biotechnology Co., Ltd. (Houston, TX, USA) and Rat MCU ELISA kit (cat. no. ABIN1744117) from Blue Gene Biotech Co., Ltd. (Shanghai, China), respectively. PKA and Ca²⁺-Mg²⁺-ATPase activity were quantified by Rat protein kinase A (cat. no. ADI-EKS-390A) and Ca²⁺-Mg²⁺-ATPase ELISA kits (cat. no. A070-3) purchased from Enzo Life Sciences, Inc. (Farmingdale, NY, USA) and Nanjing Jiancheng Bioengineering Institute (Nanjing, China), respectively. Kits were used according to the manufacturers' protocols.

All data are presented as the mean ± standard error of the mean. Two-way analysis of variance followed by Bonferroni's correction post hoc test was used for different preservation solutions and times to determine statistical differences between groups. All statistical analyses were performed using GraphPad Prism 6.0 for Macintosh (GraphPad Software, Inc., La Jolla, CA, USA). P<0.05 was considered to indicate a statistically significant difference.

To determine the effect of luteolin on modulating calcium ion cycling, 7.5, 15 and 30 µmol/l luteolin-supplemented UW solution was used to culture cardiomyocytes for 6 h and two control groups consisted of cardiomyocytes preserved in UW solution only or DMEM only. Following the labeling of cytosol free Ca²⁺ with a Fluro-3 AM fluorescence probe, the confocal microscopy images were captured following 1, 3 and 6 h preservation (Fig. 1). The results demonstrated that universally accepted heart preservation solution, UW solution, had a similar capacity to restrain cytosol free calcium ion load compared with DMEM media (Figs. 1A and B, and 2). There was a significant reduction in cytosol Ca²⁺ in the three groups using different doses of luteolin to supplement UW solution throughout the 6 h preservation period compared with the UW solution control group (all P<0.01; Figs. 1C-E and 2). The Ca²⁺ concentration in the cytosol of cardiomyocytes following 1 h UW solution preservation was increased by 31% compared with those preserved with 7.5 µmol/l luteolin for 6 h (Fig. 2). The results therefore demonstrated that luteolin significantly decreases the cytosol Ca²⁺ concentration in cardiomyocytes during hypothermic preservation (all P<0.01, Fig. 2).

It is known that calcium cycling in cardiomyocytes primarily occurs through calcium channels on the sarcolemma, which allows Ca²⁺ influx, SR involved intracellular calcium regulation and the transportation pathway of Ca²⁺ in mitochondria (18). A reduced Ca²⁺ presence was observed in the cytosol in the treatment groups, therefore, the influence of UW solution supplemented with luteolin on mitochondrial Ca²⁺ concentration and SR Ca²⁺ concentration was investigated (Fig. 3). In the DMEM and UW solution control groups, calcium ions accumulated inside the mitochondria during the 6 h cold preservation period (Figs. 3A and B, and 4). The concentration of mitochondrial Ca²⁺ remained at a significantly lower level following 3 h preservation in cardiomyocytes treated with different luteolin concentrations compared with the UW solution control group (all P<0.01; Figs. 3A and C-E, and 4). When preserved for 6 h in UW solution supplemented with 7.5 µmol/l luteolin, cardiomyocytes exhibited Ca²⁺ accumulation but remained slightly lower than that in the UW solution group, although this difference was not observed to be significant. The 15 and 30 µmol/l luteolin groups continued to effectively prevent Ca²⁺ overload in the mitochondria, as the Ca²⁺ accumulation was significantly decreased compared with that in the UW solution control group (both P<0.01; Figs. 3A, D and E, and 4). When the concentration of luteolin was increased from 7.5 to 15 µmol/l, there was a significant decrease in mitochondria calcium (P<0.01), most markedly following 6 h preservation (Fig. 4). However, the opposite effect was observed when the concentration of luteolin was increased from 15 to 30 µmol/l following 3 and 6 h preservation, suggesting a side effect of using high concentration luteolin for long-term preservation. (Fig. 4).

The Ca²⁺ concentration changes in the SR of cardiomyocytes during the 6 h cold preservation using the same solutions was then investigated (Fig. 5). Similar to the Ca²⁺ accumulation in the mitochondria, the Ca²⁺ concentration inside the SR accumulated over time when cardiomyocytes were preserved in UW, and this accumulation was significantly exacerbated in the DMEM group at all three time points (P<0.01; Figs. 5A and B, and 6). The Ca²⁺ accumulation inside the SR remained at a significantly decreased level in cardiomyocytes preserved in UW solution with 15 or 30 µmol/l luteolin throughout the 6 h preservation period compared with the UW solution control group (P<0.01; Figs. 5A and D, and 6). Even though cardiomyocytes preserved in UW solution with 7.5 µmol/l luteolin started to build up intracellular calcium slightly in the SR after 6 h, there was no significant difference between all three time points (Fig. 6). In addition, compared with the UW solution control group, Ca²⁺ concentration was significantly decreased in the 7.5 µmol/l luteolin group at all three time points (all P<0.01; Figs. 5A and C, and 6). Together, these results indicate that adding luteolin to UW solution to preserve heart myocytes may inhibit intracellular calcium overload in the SR.

The effect of luteolin supplementation on calcium signaling-related proteins during preservation was investigated. CaM concentration in rat cardiomyocytes during 6 h hypothermic preservation was determined first (Fig. 7). Myocytes preserved with DMEM in the control group expressed a significantly higher CaM compared with UW solution (P<0.01). All three groups of cardiomyocytes preserved in UW solution supplemented with different doses of luteolin had a significantly lower CaM concentration compared with the UW solution control group (all P<0.01; Fig. 7A). The higher the dose of luteolin used to treat cardiomyocytes, the lower the CaM concentration throughout the 6 h preservation period. The differences between luteolin treatment groups at each time point were significant (P<0.01) except for cardiomyocytes preserved with 15 and 30 µmol/l luteolin during the 6 h preservation period (Fig. 7A). The differences between the CaM concentration of cardiomyocytes preserved in the UW solution control group at 1 h and the cardiomyocytes preserved in UW solution supplemented with 30 µmol/l luteolin for 6 h, or cardiomyocytes preserved in UW solution supplemented with 15 µmol/l luteolin for 5 h were not significant. This suggests that the presence of luteolin during hypothermic preservation may repress CaM accumulation in cardiomyocytes and thus prolong preservation time.

Luteolin had an effect on limiting the accumulation of Ca²⁺ in the mitochondria, therefore, it was hypothesized that MCU, which is a vital transporter for mitochondria Ca²⁺ entry, may be decreased during luteolin preservation to suppress Ca²⁺ influx into the mitochondria. MCU concentration was determined by ELISA and the results indicated that luteolin supplementation at all doses reduced MCU levels compared with the UW solution control group (all P<0.01; Fig. 7B). Compared with DMEM, utilizing UW solution reduced MCU expression over the 6 h preservation period, whereas luteolin supplemented UW solution further significantly reduced the MCU content in a dose dependent manner, from 3–6 h, for luteolin treated cardiomyocytes (all P<0.01; Fig. 7B). The concentration of MCU was significantly lower following the 6 h preservation period with 30 µmol/l luteolin compared with 1 h preservation in the UW solution control group (P<0.01). However, no significant difference was observed between myocytes preserved with 15 µmol/l luteolin for 5 h and the UW solution group following 1 h preservation. The results imply that cold preservation of cardiomyocytes in UW solution supplemented with luteolin results in decreased MCU concentration, which serves a role in restricting mitochondrial Ca²⁺ influx.

The role of PKA in luteolin prolonged heart preservation was investigated by measuring PKA enzymatic activity, as PKA is known to phosphorylate calcium regulatory proteins, including phospholamban (PLB) and ryanodine receptor (RyR) 2 (23,24). Cardiomyocytes preserved in UW solution supplemented with different doses of luteolin exhibited a reduction in PKA activity compared with the UW solution control group (Fig. 7C); however, unlike MCU and CaM, cardiomyocytes treated with 7.5 µmol/l luteolin exhibited the biggest reduction compared with cardiomyocytes treated with 15 and 30 µmol/l luteolin (Fig. 7C). Nevertheless, even in cardiomyocytes treated with 30 µmol/l luteolin, which may have a toxic effect on PKA activity, the PKA activity was significantly decreased compared with the UW solution control group at 1–2 h (P<0.01; Fig. 7C). Compared with the UW solution control group, PKA was significantly decreased in the 7.5 and 15 µmol/l luteolin treatment groups at all time points (P<0.01). When cardiomyocytes were preserved in DMEM, PKA activity was significantly increased compared with the UW solution control group (P<0.01), while adding luteolin to UW solution further suppressed PKA activity.

Ca²⁺-Mg²⁺-ATPase activity was measured using ELISA. The results indicated that luteolin supplementation increased Ca²⁺-Mg²⁺-ATPase activity, with the most marked differences compared with the UW control group observed with 15 and 30 µmol/l luteolin treatment, and significant differences at 4–6 h (P<0.01; Fig. 7D). There was a significant increase in Ca²⁺-Mg²⁺-ATPase activity when comparing the response in cardiomyocytes treated with 7.5 and 15 µmol/l at 1, 4 and 5 h (P<0.01), however, there was no difference in Ca²⁺-Mg²⁺-ATPase activity when comparing cardiomyocytes preserved with 15 to 30 µmol/l luteolin at the different time points during the 6 h preservation period.