We examined ultrastructural alterations induced by high-dose CY administration in cardiac tissue using electron microscopic analysis. We found many structural changes in cardiomyocyte nuclear membranes, sarcoplasmic reticulum, mitochondria, and muscle fibers after high-dose CY administration. Changes in gene expression associated with high-dose CY administration were detected using comprehensive gene expression microarray analysis. In particular, most gene groups related to myocardial contraction and Ca2+ signaling pathways were suppressed. Changes observed in these gene groups were consistent with those observed in electron microscopy analysis. Genes that were altered by CY administration included oxidative stress-related genes, endoplasmic reticulum stress-related genes, apoptosis-related genes, p53 expression-related genes, p38MAPK, GSK-3β, and Akt/PI3K signaling-related genes, among others. However, there have been no reports on the myocardial contraction or Ca2+ signaling pathway[19–24], which may represent a mechanism of CY myocardial damage that has not been previously identified.
The pumping action of the heart is performed by the contraction and relaxation of cardiomyocytes, which constitute the heart. These mechanisms are mainly controlled by the increase and decrease of intracellular Ca2+ concentration. The regulation of intracellular Ca2+ concentration is important for the heart to continuously and smoothly pump the blood throughout the body[25, 26]. The increase in intracellular Ca2+ concentration in the myocardium is triggered by a Ca2+ influx through L-type Ca2+ channels (DHPR) and Ca2+ release from the sarcoplasmic reticulum (RyR2) upon stimulation[27, 28]. The increased intracellular Ca2+ binds to troponin C (TnC), a subunit of the contractile regulatory protein troponin, and changes the steric configuration of tropomyosin, resulting in an interaction between actin and myosin. A contraction is induced by the interaction between actin and myosin. The decrease in intracellular Ca2+ concentration is caused by the uptake of sarco/endoplasmic reticulum Ca2+-ATPase 2A into the sarcoplasmic reticulum, extracellular efflux of the Na+/Ca2+ exchange system, or efflux by cell membrane Ca2+ pumps, which results in relaxation of the myocardium[29, 30]. In our experiments, gene expression levels of DHPR, RyR2, and TnC, which are important proteins involved in the regulation of Ca2+ concentration, were all decreased. Disruption of calcium regulatory mechanisms may cause elevated intracellular calcium concentrations, resulting in inflammation and apoptosis of cardiomyocytes, leading to myocardial contractile dysfunction. Based on the new mechanism identified in this study, CY myocardial injury may be prevented with a Ca2+ signaling sensitizer, such as levosimendan and pimobendan.
Concurrently, gene expression analysis after the administration of acrolein, which is considered to be the main driver of CY-related myocardial damage in in vitro research, revealed patterns that were different from those observed after the administration of CY. Acrolein is a small molecule that is highly reactive when exposed to unsaturated aldehyde and is quickly adsorbed by proteins and other substances[16, 17]. Therefore, it may be necessary to reexamine whether the acrolein dosage and method of administration in this study were appropriate. Furthermore, given the metabolism of CY, the myocardial injury model of CY may not be truly replicated unless acrolein is present in the myocardial cells. The present findings preclude any conclusions regarding the contribution of acrolein to CY cardiotoxicity.
N-acetylcysteine treatment significantly altered gene expression related to pathways such as chemical cardiogenesis-DNA adducts, complement and coagulation cascades, and retinol metabolism. NAC treatment significantly altered gene expression related to dilated cardiomyopathy, hypertrophic cardiomyopathy, and arrhythmogenic right ventricular cardiomyopathy. Electron microscopic findings were comparable to those of CY administration. Experimental methods used to evaluate NAC treatment require further research.