Introduction: Receptor tyrosine kinases (RTK) are a potential group of targets for the treatment of ischemic heart disease. For example, activation of ErbB2 and ErbB4 improves regeneration of myocardium and decreases infarction scar size after ischemic injury in mice. RTK family consists of 55 members, most of which have not yet been characterized in ischemic heart. In order to discover potential ischemia treatment targets, we systematically studied RTK regulation in the heart.
Methods: Expression levels of 52 RTK genes from human heart samples representing healthy tissue (n = 62), acute myocardial infarction (AMI, n = 12), or ischemic cardiomyopathy (ICM, n = 63) were analyzed using IST Medisapiens Database. As an experimental model, ischemia-reperfusion (I/R) injury by cardiopulmonary bypass in the whole pig heart was used. Phosphorylation status of 49 RTKs was assessed with phospho-RTK array on 3 healthy and 4 I/R-injured pig heart samples. Expression and function of one RTK, ROR1, was studied in further detail in pig samples with Western blotting and RT-PCR, and in vitro using HL-1 cardiomyocytes. Cells were exposed to hypoxia and reoxygenation (H/R) simulating the I/R injury. ROR1 protein level changes were analyzed by Western blotting. Cell viability after ROR1 siRNA knockdown or Wnt-5a ligand activation was assessed by MTT assay.
Results: There were 12 RTKs that were regulated in both the expression analysis of ischemia-regulated RTKs in human and the analysis of phosphorylated RTKs in pig. ROR1 was most significantly upregulated in human ICM samples compared to healthy heart, and it was among the most active receptors in the pig heart. Formerly undetected ROR1 expression in adult heart was confirmed on protein and mRNA level in the pig. In HL-1 cells, ROR1 protein level was down- and subsequently upregulated by hypoxia followed by reoxygenation. ROR1 knockdown with siRNAs increased, and activation with Wnt-5a reduced the viability of the cells significantly, both in normoxia and H/R.
Conclusions: Several novel RTKs were found to be regulated in expression or activity in ischemic heart. ROR1 was one of the most significantly regulated hits. The in vitro findings suggest a role for ROR1 as a potential novel target for the treatment for ischemic heart injury.