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miRNA Expression Profiles in Isolated Ventricular Cardiomyocytes: Insights into Doxorubicin-Induced Cardiotoxicity
Yohana Domínguez Romero1,2, Gladis Montoya Ortiz2, Susana Novoa Herrán2
1Doctorate in Biotechnology Program, Faculty of Sciences, Universidad Nacional de Colombia, Bogotá 111321, Colombia.
Abstract:
Doxorubicin (DOX), widely used as a chemotherapeutic agent for various cancers, is limited in its clinical utility by its cardiotoxic effects. Despite its widespread use, the precise mechanisms underlying DOX-induced cardiotoxicity at the cellular and molecular levels remain unclear, hindering the development of preventive and early detection strategies. To characterize the cytotoxic effects of DOX on isolated ventricular cardiomyocytes, focusing on the expression of specific microRNAs (miRNAs) and their molecular targets associated with endogenous cardioprotective mechanisms such as the ATP-sensitive potassium channel (KATP), Sirtuin 1 (SIRT1), FOXO1, and GSK3β. We isolated Guinea pig ventricular cardiomyocytes by retrograde perfusion and enzymatic dissociation. We assessed cell morphology, Reactive Oxygen Species (ROS) levels, intracellular calcium, and mitochondrial membrane potential using light microscopy and specific probes. We determined the miRNA expression profile using small RNAseq and validated it using stem-loop qRT-PCR. We quantified mRNA levels of some predicted and validated molecular targets using qRT-PCR and analyzed protein expression using Western blot. Exposure to 10 µM DOX resulted in cardiomyocyte shortening, increased ROS and intracellular calcium levels, mitochondrial membrane potential depolarization, and changes in specific miRNA expression. Additionally, we observed the differential expression of KATP subunits (ABCC9, KCNJ8, and KCNJ11), FOXO1, SIRT1, and GSK3β molecules associated with endogenous cardioprotective mechanisms. Supported by miRNA gene regulatory networks and functional enrichment analysis, these findings suggest that DOX-induced cardiotoxicity disrupts biological processes associated with cardioprotective mechanisms. Further research must clarify their specific molecular changes in DOX-induced cardiac dysfunction and investigate their diagnostic biomarkers and therapeutic potential.
Insights
Doxorubicin chemotherapy damages heart cells by disrupting protective microRNAs and pathways like KATP channels and SIRT1. Understanding these molecular changes is key for preventing cardiotoxicity.
Area of Science:
- Cardiology
- Molecular Biology
- Oncology
Background:
- Doxorubicin (DOX) is a vital chemotherapy drug, but its use is restricted by cardiotoxicity.
- The exact molecular mechanisms of DOX-induced cardiotoxicity are not fully understood, impeding prevention and early detection.
Purpose of the Study:
- To investigate Doxorubicin's cytotoxic effects on cardiomyocytes.
- To analyze the expression of microRNAs (miRNAs) and their targets involved in cardioprotective pathways (KATP, SIRT1, FOXO1, GSK3β).
Main Methods:
- Isolated Guinea pig ventricular cardiomyocytes were exposed to DOX.
- Assessed cell viability, reactive oxygen species (ROS), intracellular calcium, and mitochondrial potential.
- Analyzed miRNA expression via small RNAseq and qRT-PCR.
- Quantified mRNA and protein levels of key cardioprotective molecules.
Main Results:
- DOX exposure caused cardiomyocyte shortening, increased ROS and calcium, and mitochondrial dysfunction.
- Significant alterations in specific miRNA expression were observed.
- Differential expression of KATP channel subunits, FOXO1, SIRT1, and GSK3β was detected.
- miRNA regulatory networks indicated disruption of cardioprotective biological processes.
Conclusions:
- DOX-induced cardiotoxicity involves the disruption of endogenous cardioprotective mechanisms at the molecular and miRNA levels.
- Further research is needed to explore these molecular changes for potential diagnostic biomarkers and therapeutic targets in DOX-induced cardiac dysfunction.
