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

Abstract

Heroin can cause damage to many human organs, possibly leading to different types of arrhythmias and abnormal electrophysiological function of the heart muscle and the steady state of calcium-ion channels. We explored cardiomyocytes treated with heroin and the effect on calcium-ion channels. Transcriptomics and metabolomics were used to screen for differential genes and metabolite alterations after heroin administration to jointly analyze the effect of heroin on calcium channels in cardiomyocytes. Cardiomyocytes from primary neonatal rats were cultured in vitro and were treated with different concentrations of heroin to observe the changes in morphology and spontaneous beat frequency and rhythm by a patch clamp technique. Transcriptomic studies selected a total of 1,432 differentially expressed genes, 941 upregulated and 491 downregulated genes in rat cardiomyocytes from the control and drug intervention groups. Gene Ontology 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, myofibril, inflammatory response, muscle system process, cardiac muscle contraction, etc. Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis indicated that these genes were mainly concentrated in cardiac muscle contraction, osteoclast differentiation, adrenergic signaling in cardiomyocytes, dilated cardiomyopathy, hypertrophic cardiomyopathy, and other important pathways. Metabolomic testing further suggested that cardiomyocyte metabolism was severely affected after heroin intervention. After the treatment with heroin, the L-type calcium channel current I-V curve was up-shifted, the peak value was significantly lower than that of the control group, action potential duration 90 was significantly increased in the action potential, resting potential negative value was lowered, and action potential amplitude was significantly decreased in cardiomyocytes. In this study, heroin could cause morphological changes in primary cardiomyocytes of neonatal rats and electrophysiological function. Heroin can cause myocardial contraction and calcium channel abnormalities, damage the myocardium, and change the action potential and L-type calcium channel.

Keywords: L-calcium channel; action potential; arrhythmia; heroin; transcriptomics.

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.

Figure 2

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

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.

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.

Figure 9

Trend diagram of group metabolite expression levels.

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.

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.