Analysis of heroin effects on calcium channels in rat cardiomyocytes based on transcriptomics and metabolomics

2.1 Materials

The 3-day-old SPF-class Sprague-Dawley (SD) neonatal rats were provided by the Experimental Animal Center of Xinjiang Medical University (Experimental animal ethical approval of the Medical Ethics Committee of the First Affiliated Hospital of Xinjiang Medical University: K201907-06). Heroin was provided by the Anti-Drug Corps of Xinjiang Uygur Autonomous Region. Anti-cardiac troponin T(ab209813) was provided by Abcam company, USA, secondary antibody to mouse (goat polyclonal secondary antibody to mouse IgG H&L) was provided by Alexa Flour^® 488, and ab150113 was provided by Abcam company, USA.

2.2 Cell culture

One hundred and twenty SD neonatal rats (1–3 days) were euthanatized, and their skin was disinfected with 75% alcohol under aseptic conditions. After cutting approximately half of the heart, the remaining heart was cut using an ophthalmic scissor into 7–8 petals along the tip of the heart in the radial shape. Then, 0.025% trypsin was added for digestion, and 0.6 mg/mL of type II collagenase was added for vibrating digestion for 7 min. Then, centrifugation was performed at 1,000 rpm for 5 min. The cells were counted and inoculated in a 100 mm culture dish at a cell density of 5 × 10⁵ cells/mL. In this way, the proliferation of cardiac fibroblasts was inhibited. The cells were linked and beaten at the same frequency after being cultured for 5–6 days. Then, they could be used for subsequent experiments.

2.3 Immunofluorescence

The cultured primary cardiomyocytes were seeded or placed in a polylysine-pretreated laser confocal culture dish. The cells were fixed with 4% tissue cell fixative for 40 min at room temperature. Then, 0.3% Triton X-100 working solution was added to the culture dish for perforation, followed by incubation for 30 min. The anti-cardiac troponin T (1:500) was diluted with 0.3% Triton X-100 working solution. Bovine serum albumin was used for blockage for 1 h and incubated with the primary antibody overnight at 4℃. Then, it was rinsed with phosphate-buffered saline (PBS) three times for 5 min. The secondary antibody, which is the FITC-labeled goat anti-mouse IgG antibody (goat antibody to mouse IgG H&L [Alexa Flour^® 488], 1:500), was incubated for 1 h at 37℃ in the dark. DAPI was used to stain the nucleus for 10 min. Under the confocal microscope, the purity of cardiomyocytes was identified and observed by taking five different visual fields. The expression of α-actin was observed at the excitation wavelength of 488 nm, and DAPI staining was observed under the excitation of ultraviolet wavelength. The positive cells were expressed, and the proportion of positive cells was counted by the two lasers that were superimposed at the same time.

2.4 Cell morphology

A total of 10⁻⁴ mol/L heroin was used for the intervention with cardiomyocytes. The concentration of heroin was 0 mol/L (N group) and 10⁻⁴ mol/L (H group). After the PBS solution in equivalent volume was added to the normal group and acted for 24 h, the changes in morphology and spontaneous beat frequency and rhythm of cardiomyocytes were observed under an inverted fluorescence microscope at different concentrations of drug interference.

2.5 Sample intervention and collection

Cultured primary rat cardiomyocytes were divided into the normal control group (Con) and drug intervention group (Drug). The Drug group was given 10⁻⁴ mol/L heroin for 24 h according to the optimal drug action concentration determined by a previous experiment, and an equal amount of PBS was added to the Con group. After 24 h, the cells were collected in 1.5 mL Eppendorf tubes, repeating each group for subsequent transcriptomics and targeted metabolomics experiments based on the multiple reaction monitoring (MRM) method.

2.6 Transcriptomic detection and analysis

Transcriptomic detection and analysis involved the following steps.

(1) RNA extraction: the basic principle of extraction is to prevent RNA degradation in the process of extraction, maximize the efficiency of RNA extraction, and ensure the extraction of high-quality RNA with good integrity and high purity from the target sample. (2) Sample testing: high-quality RNA is the basis for the success of the whole project. We inspected the quality of the samples to ensure the accuracy of sequencing data, and the test results could only meet the requirements of sequencing and database construction. Sample test results are shown in the quality inspection report. (3) Library construction: after the sample test was qualified, magnetic beads with oligo (dT) were used to enrich the mRNA of eukaryotes. The fragmentation buffer was subsequently added to randomly interrupt the mRNA. Using mRNA as a template with six-base random primers (random hexamers), the second strand of cDNA was synthesized by buffer and dNTPs and DNA polymerase I, and the double-stranded cDNA was subsequently purified using AMPure XP beads. Purified double-stranded cDNA was then subjected to end repair, a tail addition, and sequencing joint ligation, followed by fragment size selection with AMPure XP beads. Finally, polymerase chain reaction enrichment was performed to obtain the final cDNA library. (4) Library quality control: after the library construction was completed, the insert length (insert size) and the effective concentration of the library were tested to ensure the library quality. (5) Computer sequencing: after the library inspection was qualified, different libraries were pooled according to the target data amount and computer sequencing. (6) Bioinformatics analysis included GO analysis, KEGG analysis, protein interaction network analysis, and transcription factor analysis.

2.7 Analysis of targeted metabolomics tests based on MRM

Energy metabolites were analyzed in rat cardiomyocytes based on the MRM method, and all samples were prepared as QC samples using QC samples to evaluate the stability and reproducibility of the data. The relative standard deviation (RSD) results of QC samples are shown in Figure 7, where the RSD is less than 30% energy metabolism, and the data are stable and reliable in the samples. The main steps are as follows: (1) Metabolite extraction: samples were removed at −80℃, and 1 mL of methanol acetonitrile solution (2:2:1, v/v) was added. The samples were vortexed for 60 s, underwent low-temperature sonication for 30 min twice, put at −20℃ for 1 h for protein precipitation, and centrifuged at 14,000 RCF at 4℃ for 20 min. The supernatant was frozen to get dry samples at −80℃. (2) Chromatography-mass spectrometry analysis: samples were separated using an Agilent 1290 Infinity LC ultra-HPLC system. Mass spectrometry was performed using a 5500 QTRAP mass spectrometer (AB SCIEX) in negative ion mode. (3) Data processing: the spectral peak area and retention time were extracted using MultiQuant software.

2.8 Cell patch clamp

After the treatment with 10⁻⁴ mol/L heroin for 24 and 48 h, the action potential and calcium channel current were detected. After the whole-cell recording mode was formed, the voltage clamp mode was changed to the current clamp mode, and the action potential was recorded. The patch clamp system was controlled by patch clamp amplifiers EPC10 and pCLAMP8.0 (Axon Instrument) software. The digital-to-analog converter performed I _Ca-L to stimulate signal generation, feedback signal acquisition, and data analysis. The following solution formulation was used for detecting action potential: extracellular fluid: 140 mmol/L NaCl, 3.5 mmol/L KCl, 10 mmol/L HEPES, 10 mmol/L glucose, 1.25 mmol/L NaH₂PO₄, 1 mmol/L MgCl₂, 2 mmol/L CaCl₂, NaOH, and pH = 7.4. Intracellular fluid: 5 mmol/L NaCl, 140 mmol/L KCl, 0.1 mmol/L CaCl₂, 1 mmol/L MgCl₂, 10 mmol/L HEPES, 2 mmol/L Mg-ATP, KOH, and pH = 7.2. The following solution formulation was used for detecting calcium channels: extracellular fluid: 140 mmol/L TEA-Cl, 2 mmol/L MgCl₂, 10 mmol/L HEPES, 10 mmol/L glucose, CsOH, and pH = 7.4. Intracellular fluid: 120 mmol/L CsCl, 1 mmol/L MgCl₂, 10 mmol/L HEPES, 4 mmol/L Mg-ATP, CsOH, and pH = 7.2.

2.9 Statistical analysis

The statistical results concerning measurement data and enumeration data were tested by SPSS 23.0 software. Data were presented as the mean value ± standard error of the mean from at least three independent experiments performed in duplicate or triplicate. Statistical analysis was performed by one-way ANOVA with Bonferroni post hoc test, and the test level was set at α = 0.05.

3.1 Purity identification for primarily cultured cardiomyocytes of neonatal rats

The passaged-7 primarily cultured cardiomyocytes were identified by laser confocal microscopy. The expression of α-actin, which is a classical biomarker specific to cardiomyocytes, was detected in the primary cardiomyocytes. After the primary antibody FITC-labeled goat anti-mouse IgG combined with the myocardial cell-specific protein cardiac troponin T in the cytoplasm, the grid-like or filamentous-green fluorescence (Figure 1b) was excited by a 488 nm laser. DAPI dying was used to locate the nucleus, and it formed circular or elliptical blue fluorescence (Figure 1a) excited by the ultraviolet laser. The overlap figures showed the intact morphological structure of primary cardiomyocytes with various forms, including triangles, fusiforms, and polygons. The actin protein was filamentously distributed and interwoven into a network in the cells (Figure 1c). Five randfluorescent fields were selected from each dish, and cells expressing blue and green fluorescence were identified as positive cells. The number of blue fluorescence was recorded as the total number of cells, and the proportion of positive cells was calculated. The results showed that the purity of the primary cultured rat cardiomyocytes was >90%, which could be used in subsequent experiments.

Figure 1

Identification results of primary cardiomyocytes. (a) Anti-cardiac troponin T cytoplasm stained green; (b) DAPI nuclei are blue; and (c) A + B synthesis.

3.2 Morphological changes in cardiomyocytes treated with different concentrations of heroin

When the cardiomyocytes of primary neonatal rats were continuously cultured for 3 days, spontaneous pulsation occurred, and the pulsation frequency was slower. Then, the cells were cultured for 7 days, and most cells recovered to spontaneous pulsation, while some of the cardiomyocytes were connected into pieces and beaten with a regular rhythm. In the control group with PBS, the quantities of cultured cardiomyocytes were large, and their morphology was intact. Additionally, the cell morphology was diverse and triangular, long fusiform, and polygonal, with a clear structure, intact membrane, and bright membrane edge, and the nucleus was round or similar to round. The size was normal, the nuclear membrane was smooth, and the cells were connected to each other by pseudopods (Figure 2a). With the intervention of heroin, the number of cardiomyocytes sharply decreased, the cell membrane shrank, the pseudopods decreased, the nuclear structure was blurred, and the cell debris appeared in the cells (Figure 2b).

Figure 2

Morphological changes in myocardial cells after heroin intervention for 24 h. (a) Normal primary cardiomyocyte group; (b) Diacetylmorphine intervention group.

3.3 Transcriptomic screening for differential genes and signaling pathway changes before and after heroin intervention

A total of 1,432 differentially expressed genes were selected in the normal control group, 941 were upregulated and 491 were downregulated in the normal control and drug intervention groups, respectively. The direct embodiment of the gene expression level was the abundance of its transcripts, and the higher the transcript abundance, the higher the gene expression level. FPKM values for each gene in each sample were calculated using feature Counts software. FPKM represented the number of alignments to transcript per kilobase per million alignment fragments, eliminating the effect of gene length and sequencing quantity differences on the calculated gene expression. The calculated gene expression levels could be directly used to compare the gene expression differences between different samples. Table 1 shows the statistics of the number of different genes at different expression levels. A volcano plot can visually show the distribution of differential genes and their expression levels in the two groups (Figure 3). Differential gene clustering could be used to judge the changes in different genes between different groups. According to the similarity of the expression of genes in each sample, the genes were clustered to visually show the expression of genes in different samples to obtain information related to biological problems. As could be seen from the cluster map, most genes were significantly up or downregulated before and after the heroin intervention (Figure 4). We used p adj. of <0.05 and log 2 foldchange of >1 as the differential significance criterion. The top 30 significantly differentially expressed genes between the two groups were selected, and the analysis results are shown in Table 2. Gene Ontology (GO) functional enrichment showed that 1,432 differential genes selected by the two groups were mainly involved in the regulation of the multicellular organismal process, response to external stimulus, defense response, developmental process, myofibril, inflammatory response, circulatory system process, muscle system process, cardiac muscle contraction, cardiac muscle tissue development, etc. (Figure 5). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of 1,432 differential genes screened out the top 20 most significant differential signaling pathways. Cardiac muscle contraction, osteoclast differentiation, adrenergic signaling in cardiomyocytes, dilated cardiomyopathy (DCM), hypertrophic cardiomyopathy (HCM), and other important pathways were significantly upregulated. The NOD-like receptor signaling pathway, influenza A, TNF signaling pathway, Kaposi sarcoma-associated herpesvirus infection, herpes simplex virus 1 infection, and other important pathways were significantly downregulated (Figure 6; Table 3).

Figure 3

Volcano plot of the differential gene expression distribution. The abscissa indicates the fold change of gene expression in different experimental groups or in different samples; the ordinate indicates the statistically significant degree of gene expression change. Scatters in the plot represent individual genes, black dots indicate genes with no significant differences, red dots indicate upregulated genes with significant differences, and green dots indicate downregulated genes with significant differences.

Figure 4

Differential gene cluster map. Each column represents one sample, and each row represents one gene. Red indicates upregulation and blue downregulation. Above is the dendrogram of the sample clusters, two The closer the sample branches are, the closer the expression pattern of all different genes between these two samples is. The dendrogram of the gene clusters is shown on the left, two The closer the gene branches are, the closer their expression level is.

Figure 5

Bar graph of GO enrichment for differential genes.

Figure 6

KEGG enrichment bubble plot of the differentially expressed genes.

3.4 Metabolomic changes in cardiomyocytes after heroin intervention

The results of targeted metabolomics analysis based on the MRM methods revealed significant changes in multiple metabolites in SD rat cardiomyocytes following the heroin intervention, such as cis-aconitate, citrate, isocitrate, lactate, adenosine 5′-triphosphate (ATP), phosphoenolpyruvate (all the p-values were less than 0.05, and the difference was statistically significant). These metabolites are mainly related to mitochondrial oxidative phosphorylation. We found that the production of ATP was significantly reduced after diacetylmorphine intervention, suggesting that diacetylmorphine may affect the oxidative phosphorylation process of mitochondria, interfere with ATP production and then affect the myocardial contractile function (Figures 7–9).

Figure 7

The RSD distribution of the QC samples.

Figure 8

Results of the hierarchical clustering of the metabolites.

Figure 9

Trend diagram of group metabolite expression levels.

3.5 Action potential of myocardial cells

After 7 days of isolated culturing, the cardiomyocytes of the neonatal rats were treated with 10⁻⁴ mol/L heroin for 24 and 48 h, and the action potential was significantly changed compared with the N group. Compared with the normal control group, after the action of 10⁻⁴ mol/L heroin for 24 h, the APA and RP significantly decreased, and the APD was significantly prolonged, with the results being statistically significant (p < 0.05) (Figures 10 and 11). After 48 h of action, the negative value of the RP lowered, the APA significantly decreased, and the APD was significantly prolonged, with statistically significant results (p < 0.05) (Figures 10 and 11). The results showed that 10⁻⁴ mol/L heroin could change the AP of cardiomyocytes, decrease the RP and APA, increase the APD, and increase the APD with the extension of the action time.

Figure 10

Action potential of cardiomyocytes in different groups. Action potential of cardiomyocytes in control group, heroin intervened for 24 and 48 h.

Figure 11

The changes in action potential in control group and 10⁻⁴ mol/L heroin intervention 24 and 48 h group. (a) RP and APA values of each group; (b) Statistical chart of APD for each group.

3.6 L-type calcium channel current of cardiomyocytes

The activation potential of the normal control group was −40 mV, the maximum peak potential was −10 mV, and the inversion potential was +30 mV. The interference of 10⁻⁴ mol/L heroin for 24 h significantly up-shifted the current–voltage curve. The peak pA/pF decreased from −14.469 ± 3.611 to −4.966 ± 0.771 (p < 0.05). The current–voltage curve was significantly up-shifted by 48 h of interference, and the peak pA/pF decreased from −14.469 ± 3.611 to −3.757 ± 2.060 (p < 0.05). The membrane potential was plotted based on peak pA/pF, and the I–V curve of I _ca was plotted. After the interference of heroin, the Ca²⁺ current was significantly changed in the I–V curve compared with the control group, and I–V curve was significantly up-shifted, but the curve shape was unchanged (Table 4, Figure 12a–d), indicating that heroin only affected I _ca-L current but did not change the electrophysiological properties of the Ca²⁺ channel.

Figure 12

Changes in I _ca-L I−V curve and Ca²⁺ current intervened by heroin. (a) Changes in I _ca-L I−V curve in 10⁻⁴ mol/L heroin intervened group at different times. (b) Ca²⁺ current of cardiomyocytes after 10⁻⁴ mol/L heroin intervention 24 h. (c) Ca²⁺ current of cardiomyocytes after 10⁻⁴ mol/L heroin intervention 48 h. (d) Ca²⁺ current of cardiomyocytes after heroin intervention in different times.