Danhong Injection and Trimetazidine Protect Cardiomyocytes and Enhance Calcium Handling after Myocardial Infarction

Abstract

Myocardial infarction (MI) is one of the leading causes of death worldwide. However, there is no effective treatment for MI. In this study, trimetazidine (TMZ) and Danhong injection (DHI), representing western medicine and traditional Chinese medicine for MI, were used as tools to identify vital processes in alleviating MI injury. Administration of DHI and TMZ obviously decreased myocardial infarct size, improved ultrasonic heart function, and reduced creatine kinase (CK), lactate dehydrogenase (LDH), and glutamic oxaloacetic transaminase (AST) levels after MI. RNA-seq results indicated calcium ion handling and negative regulation of apoptotic process were vital processes and DHI and TMZ obviously reduced the expression of CaMK II and inhibited cleaved caspase-3 and Bax. Furthermore, DHI and TMZ increased p-S16-PLB, p-S16T17-PLB, CACNA1C, p-RyR2, and p-PKA expression but did not affect SERCA2a expression. In addition to the enhancement of cardiac myocyte shortening amplitude, maximum shortening velocity, and calcium transients, DHI and TMZ increased sarcoplasmic reticulum calcium content and enhanced SERCA2a calcium uptake capability by upregulating the phosphorylation of PLB but did not affect calcium exclusion by NCX. In conclusion, DHI and TMZ protect against MI through inhibiting apoptosis by downregulating CaMKII pathway and enhancing cardiac myocyte contractile functions possibly through the PKA signaling pathway.

Figure 1

DHI and TMZ treatment reduced myocardial infarction and enhanced heart contractile functions. (a) TTC staining of heart tissues indicated reduction in myocardial infarction by DHI. (b) The statistical analysis of infarction rate in heart tissues as calculated by Image-J software (n = 6). (c) Echocardiography images under short-axis M-mode showed distinct heart functions after various treatments. (d) Left ventricular ejection fraction (LVEF) and (e) fractional shortening (FS) were calculated from echocardiography images (n = 6). Data are expressed as the mean ± SD, ^#P < 0.05 compared to sham, ^∗P < 0.05 compared to MI, and ^ΔP < 0.05 compared to D2.1 + MI.

Figure 2

Systematic investigation of the mechanism in DHI- and TMZ-mediated protection by by RNA-seq analysis: (a) alterations in the whole genome shown as a volcano plot. Red dots represent differentially expressed genes, and green dots represent nondifferentially expressed genes (n = 3). (b) Differentially expressed genes were clustered as a hierarchical heatmap.

Figure 3

The GO or KEGG pathways enriched by the differentially expressed genes (DEs) in DHI- and TMZ-mediated protection against MI. (a) The GO or KEGG pathways enriched by the DEs after MI surgery. (b) The GO or KEGG pathways enriched by the DEs in MI after DHI treatment. (c) The regulatory network of DEs involved in the terms of “cellular calcium ion homeostasis” and “negative regulation of apoptotic process” after DHI treatment (the DEs involved in “cellular calcium ion homeostasis” are labelled red, and the DEs involved in “negative regulation of apoptotic process” are labelled blue).

Figure 4

DHI and TMZ reduced myocardium damage, inhbited cell apoptosis, and decreased CaMKII. DHI and TMZ reduced myocardium damage, inhbited cell apoptosis, and decreased CaMKII. (a) Serum CK, AST, and LDH levels (n = 6). (b) TUNEL staining and immunofluorescence staining of Bax, Bcl-2; scale bar for TUNEL and Bax and Bcl-2 staining was 50 μm. (c) Representative western blotting for cleaved caspase-3 and its quantitation (n = 3). (d) Representative western blotting for CaMKII and its quantitation (n = 3). The data are shown as the mean ± SD, ^#P < 0.05 compared to sham, ^∗P < 0.05 compared to MI.

Figure 5

The effect of DHI and TMZ on cardiac myocyte sarcomere shortening and calcium transient at the infracted edge. (a) Sarcomere shortening amplitude; (b) maximum sarcomere shortening velocity; (c) maximum sarcomere relaxation velocity; (d) typical sarcomere shortening curve pacing at 1 Hz; (e) calcium transient amplitude (RU, 340/380 nm fluorescence ratio unit); (f) maximum calcium release velocity in calcium transients; (g) maximum calcium decline velocity in calcium transients; (h) typical calcium transient curve pacing at 1 Hz; (i) twitch-induced calcium decline time constant; (j) caffeine-induced calcium decline time constant; (k) sarcoplasmic reticulum calcium content (caffeine-induced); (l) typical caffeine-induced calcium release curves. The data are shown as the mean ± SD, n = 41–47 from 4 hearts for each group, ^#P < 0.05 compared to sham, ^∗P < 0.05 compared to MI.

Figure 6

DHI and TMZ improved Ca²⁺ handling-related protein levels in myocardial ischemia in rats. Western blotting results for (a) CACNA1C, SERCA2a, and p-RyR2 (n = 3) and (b) PLB, p-S16-PLB, and p-S16T17-PLB (n = 3) in ischemic heart tissues. Image-J was used to quantify the protein levels, and the data are shown as the mean ± SD, ^#P < 0.05 compared to sham, ^∗P < 0.05 compared to MI.