Using Next-generation RNA Sequencing to Examine Ischemic Changes Induced by Cold Blood Cardioplegia on the Human Left Ventricular Myocardium Transcriptome

Abstract

Background The exact mechanisms that underlie the pathological processes of myocardial ischemia in humans are unclear. Cardiopulmonary bypass with cardioplegic arrest allows us to examine the whole transcriptional profile of human left ventricular myocardium at baseline and after exposure to cold cardioplegia induced ischemia as a human ischemia model. Methods We obtained biopsies from 45 patients undergoing aortic valve replacement surgery at baseline and after an average of 79 min of cold cardioplegic arrest. Samples were RNA-sequenced and analyzed with the Partek Genomics Suite for differential expression. Ingenuity Pathway Analysis and Biobase Explain systems were used for functional and pathway analysis. Results Of the 4,098 genes with a mean expression value greater than 5, 90% were downregulated and 9.1% were upregulated. Of those, 1,241 were significantly differentially expressed. Gene ontology analysis revealed significant downregulation in immune inflammatory response and complement activation categories, and highly consistent was the downregulation of intelectin 1, proteoglycan, and SLPI. Upregulated genes of interest were FBJ murine osteosarcoma viral oncogene homolog (FOS) and the hemoglobin genes hemoglobin 1 (HBA1) and hemoglobin beta (HBB). In addition, analysis of transcription factor binding sites revealed interesting targets in factors regulating reactive oxygen species production, apoptosis, immunity, cytokine production, and inflammatory response. Conclusions We have shown that the human left ventricle exhibits significant changes in gene expression in response to cold cardioplegia induced ischemia during cardiopulmonary bypass which provides great insight into the pathophysiology of ventricular ischemia, and thus, may help guide efforts to reduce myocardial damage during surgery.

Transcriptome analysis in heart failure

“…Further methodological development in intact hearts (whether human or mouse) is still required to accurately define mRNAs affected in vivo by particular microRNAs, but promising techniques are available [54]. However, an interesting application of mRNA sequencing alone to better understand mechanisms of ischemia in human hearts is the measurement of gene expression changes in cold cardioplegia during cardiopulmonary bypass; insights obtained from coupled pathway analyses may be of assistance in minimizing surgery-induced myocardial damage [55]. Although patient numbers were small, similar mRNA sequencing was used to define and compare transcriptional profiles of heavily calcified vs. noncalcified diseased bicuspid aortic valves, finding divergent underlying mechanisms [56].…”

Allele-specific expression in the human heart and its application to postoperative atrial fibrillation and myocardial ischemia

“…This has thus far mostly been performed in animal models, exemplified by the assessment of changes in gene expression in mouse models of myocardial ischemia [3, 4]. In humans, we recently used this technique to measure the effects of ischemia on the gene expression of the left ventricle (LV) during cardiopulmonary bypass [5]. This indicated substantial changes in the expression of a wide variety of functional categories of genes between the baseline and post-ischemic samples in a short amount of time.…”

Next Generation of Cardiovascular Studies

“…, myocardial ischemia, acute kidney injury, acute lung injury), and subsequently examine adaptive responses that could be important in perioperative organ protection. As published in the current edition of Anesthesiology, Muehlschlegel et al took a different approach to identify transcriptional changes that could be targeted for perioperative organ protection 3 . Focusing on myocardial injury, they performed next-generation RNA sequencing to examine transcriptional changes that occur within the left ventricle of patients undergoing cardiac surgery.…”

“…display similar changes in gene expression and regulation as compared to humans. Finally, the authors were able to use the most important and relevant type of tissue – a freshly obtained biopsy of the left ventricle – to perform their studies 3 .…”

“…Similar approaches can now be taken in subsequent studies, based on the large array of data provided by Muehlschlegel et al . 3 . Indeed, changes in RNA content observed in the present study by Muehlschlegel et al can be further examined by taking those findings from bedside back to the bench.…”