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Background Receptor tyrosine kinases (RTK) are potential targets for the treatment of ischemic heart disease. The human RTK family consists of 55 members, most of which have not yet been characterized for expression or activity in the ischemic heart. Methods RTK gene expression was analyzed from human heart samples representing healthy tissue, acute myocardial infarction or ischemic cardiomyopathy. As an experimental model, pig heart with ischemia-reperfusion injury, caused by cardiopulmonary bypass, was used, from which phosphorylation status of RTKs was assessed with a phospho-RTK array. Expression and function of one RTK, ROR1, was further validated in pig tissue samples, and in HL-1 cardiomyocytes and H9c2 cardiomyoblasts, exposed to hypoxia and reoxygenation. ROR1 protein level was analyzed by Western blotting. Cell viability after ROR1 siRNA knockdown or activation with Wnt-5a ligand was assessed by MTT assays. Results In addition to previously characterized RTKs, a group of novel active and regulated RTKs was detected in the ischemic heart. ROR1 was the most significantly upregulated RTK in human ischemic cardiomyopathy. However, ROR1 phosphorylation was suppressed in the pig model of ischemia-reperfusion and ROR1 phosphorylation and expression were down-regulated in HL-1 cardiomyocytes subjected to short-term hypoxia in vitro. ROR1 expression in the pig heart was confirmed on protein and mRNA level. Functionally, ROR1 activity was associated with reduced viability of HL-1 cardiomyocytes in both normoxia and during hypoxia-reoxygenation. Conclusions Several novel RTKs were found to be regulated in expression or activity in ischemic heart. ROR1 was one of the most significantly regulated RTKs. The in vitro findings suggest a role for ROR1 as a potential target for the treatment of ischemic heart injury. Electronic supplementary material The online version of this article (10.1186/s12872-018-0933-y) contains supplementary material, which is available to authorized users.

Supplemental material, DS_10.13690022155419845373 for Decorin Expression in Human Vulva Carcinoma: Oncosuppressive Effect of Decorin cDNA Transduction on Carcinoma Cells by Marie C. Nyman, Anne B. Jokilammi, Pia C. Boström, Samu H. Kurki, Annele O. Sainio, Seija E. Grenman, Katri J. Orte, Sakari H. Hietanen, Klaus Elenius and Hannu T. Järveläinen in Journal of Histochemistry & Cytochemistry

ROR2 in cardiomyocytes. A) A representative Western analysis of ROR2 protein level in HL-1 cardiomyocytes after treatment with hypoxia and reoxygenation. All cells were first allowed to adhere for 24 h after plating in normoxic conditions. This was followed by culturing the cells in a hypoxic work station at 1% O2 (hypoxia) and subsequently again in the regular cell incubator in normoxia (reoxygenation) for the indicated periods of time. As different time points were distributed over three days after plating, control samples cultured in normoxia for 24, 48 or 72 h were also analyzed. Time points (hypoxia+reoxygenation) 1 + 0, 1 + 3, 3 + 0 and 3 + 3 are comparable to the 24 h control (lane 10), time points 1 + 24, 3 + 24, 24 + 0 and 24 + 3 to the 48 h control (lane 11), and time points 24 + 24 to the 72 h control (lane 12). B) A box plot presentation of densitometric quantitation of ROR1 bands from three replicate Western blots similar to the one shown in panel A. ROR1 band intensities were first normalized to each sample’s actin level, and subsequently divided by the control sample value of the respective time point. C) Effect of ROR2 knockdown on cellular viability. HL-1 cells were transfected with two different siRNAs targeting ROR2 (ROR2 siRNA #1 and #2) or negative control siRNA. Twenty-four hours after transfection, cells were either transferred into a hypoxic work station (1% O2) or were maintained in normoxia as controls. After another 24 h, all cells were returned to normoxia for 24 h to allow for reoxygenation. Cell viability was analyzed using the MTT assay. A box plot presentation is shown indicating cell viability as normalized to negative control siRNA-treated cells cultured in normoxia. Three independent experiments each including six replicates were carried out. D) Western analysis of ROR2 protein expression after ROR2 siRNA treatments. E) Western analyses of ROR1 and ROR2 protein expression after ROR1 siRNA treatment. (PDF 3815 kb)

Analysis of ROR1 phosphorylation in HL-1 cardiomyocytes after hypoxia and reoxygenation. A) Western analysis of tyrosine phosphorylation after ROR1 immunoprecipitation. Cells were first allowed to adhere for 24 h after plating in normoxic conditions. This was followed by culturing the cells in a hypoxic work station at 1% O2 (hypoxia) and subsequently again in the regular cell incubator in normoxia (reoxygenation) for the indicated periods of time. As different time points were distributed over two days after plating, control samples cultured in normoxia for 24 or 48 h were also analyzed. Time point of one hour of hypoxia (lane 1) is comparable to the 24 h control (lane 2) and time point of one hour of hypoxia and 24 h of reoxygenation (lane 3) is comparable to the 48 h control (lane 4). B) Quantitation of ROR1 phosphorylation relative to total protein. (PDF 179 kb)

Heatmaps of RTKs demonstrating significant changes in mRNA expression in either ischemic cardiomyopathy or acute myocardial infarction when compared to healthy heart. RTKs with significant expression level differences (FDR-corrected P values

Myocardial damage in ischemia-reperfusion-injured pig hearts. A) Plasma troponin T levels from four ischemia-reperfusion-injured pigs were collected at baseline, and 6 and 24Â h after reperfusion. Medians are indicated with horizontal lines. B) Representative HE-stained images from a healthy and ischemia-reperfusion-injured pig heart (sample collected 31Â h after reperfusion). (PDF 44885 kb)

Phospho-RTK array of ischemia-reperfusion-injured pig hearts. A) Representative phospho-RTK array blots from control and ischemia-reperfusion-injured pig heart samples. B) Array overlay and a corresponding coordinate table indicating the location of RTKs in the array. Each of the 49 RTKs included in the array are represented by two adjacent dots. (PDF 229 kb)

Phospho-RTK array of ischemia-reperfusion-injured pig hearts. A) Representative phospho-RTK array blots from control and ischemia-reperfusion-injured pig heart samples. B) Array overlay and a corresponding coordinate table indicating the location of RTKs in the array. Each of the 49 RTKs included in the array are represented by two adjacent dots. (PDF 229 kb)