Myocardial expression analysis of osteopontin splice variants in patients affected by end-stage dilated or ischemic cardiomyopathy

Background: Osteopontin (OPN) is a phosphoglycoprotein of cardiac extracellular matrix; its precursor mRNA could be subjected to alternative splicing, by thrombin, leading to full-length OPN-a (i.e., consist of all exons), OPN-b (lacks exon 5) and OPN-c (lacks exon 4). The OPN splice variants are differentally ex- pressed and may have functional heterogeneity in tumor specific manner but recently are beginning to be studied in other diseases. To date, no data are reported about OPN expression in different types of failing heart. Purpose: The aim of our study was to investigate the myocardial profile of OPN- a, thrombin, as well as of the two isoforms OPN-b and OPN-c in the left ventricle of patients affected by end-stage idiopathic or ischemic dilated cardiomyopathy. Methods: The full-length OPN-a and its isoforms (OPN-b, OPN-c) transcriptomic profile were evaluated in myocardium of dilated/ischemic cardiomyopathy (DCM n=8; LVEF%=17.5±3; ICM n=8; LVEF%=19.5±5.2) pts and in auricle of valvular pts (VLP n=5; LVEF%≥50), by Real-time PCR analysis. The protein levels of OPN-a were assessed by enzyme immunometric assay. Histological analysis was also performed and serial slices were stained by Masson’s trichrome to assess myocardial architecture and fibrosis. Results: OPN-a and thrombin mRNA levels resulted significantly higher in DCM compared to ICM patients (DCM:31.3±7.4, ICM:2.7±1.1, p=0.0002; DCM:19.1±4.9, ICM:5.4±2.2, p=0.007-respectively). Both biomarkers increased in patient with LVEF<50% (DCM+ICM) with respect to VLP with LVEF>50% but splitting in DCM and ICM, a significant increase in OPN (p=0.0004) and thrombin (p=0.001) expression was observed only in DCM. A correlation between OPN-a and thrombin was observed in patients with LVEF<50% (r=0.6; p=0.003). The mRNA pattern was confirmed by OPN-a cardiac protein concentration (VLP:1.127±0.26; DCM:1.29±0.22; ICM:1.00±0.077 ng/ml). The OPN splicevariants expression were detectable only in ICM (OPN-b: 0.357±0.273; OPN-c: 0.091±0.033) and not in DCM patients. Hystolgical analysis showed a larger myocardial amount of type I collagen, fibronectin and fibroblasts detectable in ICM rather than DCM hearts. A significant correlation was observed between collagen type I, evaluated by immunohistochemistry analysis, and both OPN-a mRNA expression (r=0.87, p=0.002) and OPN protein concentrations (r=0.77, p=0.016). Conclusion: OPN-a and thrombin mRNA resulted dependent on the origin of heart failure while OPN-b and OPN-c highlighted a different expression for DCM and ICM patients suggesting their correlation with different clinical-pathological outcomes.