Myocardial Expression Analysis of Osteopontin and Its Splice Variants in Patients Affected by End-Stage Idiopathic or Ischemic Dilated Cardiomyopathy

Abstract

Osteopontin (OPN) is a phosphoglycoprotein of cardiac extracellular matrix and it is still poorly defined whether its expression changes in failing heart of different origin. The full-length OPN-a and its isoforms (OPN-b, OPN-c) transcriptomic profile were evaluated in myocardium of patients with dilated or ischemic cardiomyopathy (DCM n = 8; LVEF% = 17.5±3; ICM n = 8; LVEF% = 19.5±5.2) and in auricle of valvular patients (VLP n = 5; LVEF%≥50), by Real-time PCR analysis. 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). Although both genes' mRNA levels increased in patients with LVEF<50% (DCM+ICM) with respect to VLP with LVEF>50%, a significant increase in OPN (p = 0.0004) and thrombin (p = 0.001) expression was observed only in DCM. In addition, a correlation between OPN-a and thrombin was found 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 splice variants expression were detectable only in ICM (OPN-b: 0.357±0.273; OPN-c: 0.091±0.033) and not in DCM patients. 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). Concluding, 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-pathophysiological setting.

Fig 1. Transcriptional profile of OPN-a and thrombin in the heart of failing patients.

a) OPN-a and b) thrombin mRNA expression measured by Real Time PCR in DCM and ICM patients. The three most stably expressed genes (RPL13a, eEF1a, RPS4X) was used for normalization of mRNA expression results.

Fig 2. Transcriptional profile of OPN-a and thrombin in all group of patients studied.

a) OPN-a and b) thrombin mRNA expression measured by Real Time PCR in VLP patients with LVEF>50% and HF patients with LVEF<50%; c) OPN-a and d) thrombin mRNA expression measured by Real Time PCR in VLP patients with LVEF>50% and HF patients splitting in DCM and ICM. The three most stably expressed genes (RPL13a, eEF1a, RPS4X) was used for normalization of mRNA expression results.

Fig 3. Transcriptional profile of OPN- b and -c in all group of patients studied.

a) OPN-b and b) OPN-c mRNA expression measured by Real Time PCR in in VLP patients with LVEF>50% and HF patients splitting in DCM and ICM. The three most stably expressed genes (RPL13a, eEF1a, RPS4X) was used for normalization of mRNA expression results.

Fig 4. Methodological evaluation of OPN assay.

a) recovery test, evaluated adding known amounts of OPN standard (0–32 ng/ml, 1:10 dilution) to a plasma pool; b) dilution test, carried out using serial dilution of plasma pool.

Fig 5. Comparison of OPN-a at protein and at mRNA level.

Individual data plot of mRNA expression and protein concentration for VLP, DCM and ICM groups (Black rhombus:mRNA espression, grey square:protein concentration, ng/ml).

Fig 6. Histology.

Histological representation (Masson’s trichrome) of left ventricular mesocardial and sub-endocardial layers in DCM (left) and ICM (right).

Fig 7. Hystology.

Histological representation (Masson’s trichrome) of auricle in VLP.