Sorbin and SH3 domain-containing protein 2 is released from infarcted heart in the very early phase: proteomic analysis of cardiac tissues from patients

Abstract

Background: Few proteomic studies have examined human cardiac tissue following acute lethal infarction. Here, we applied a novel proteomic approach to formalin-fixed, paraffin-embedded human tissue and aimed to reveal the molecular changes in the very early phase of acute myocardial infarction.

Methods and results: Heart tissue samples were collected from 5 patients who died within 7 hours of myocardial infarction and from 5 age- and sex-matched control cases. Infarcted and control myocardia were histopathologically diagnosed and captured using laser microdissection. Proteins were extracted using an originally established method and analyzed using liquid chromatography-tandem mass spectrometry. The label-free quantification demonstrated that the levels of 21 proteins differed significantly between patients and controls. In addition to known biomarkers, the sarcoplasmic protein sorbin and SH3 domain-containing protein 2 (SORBS2) was greatly reduced in infarcted myocardia. Immunohistochemical analysis of cardiac tissues confirmed the decrease, and Western blot analysis showed a significant increase in serum sorbin and SH3 domain-containing protein 2 in acute myocardial infarction patients (n=10) compared with control cases (n=11).

Conclusions: Our advanced comprehensive analysis using patient tissues and serums indicated that sarcoplasmic sorbin and SH3 domain-containing protein 2 is released from damaged cardiac tissue into the bloodstream upon lethal acute myocardial infarction. The proteomic strategy presented here is based on precise microscopic findings and is quite useful for candidate biomarker discovery using human tissue samples stored in depositories.

Keywords: SORBS2; myocardial infarction; proteomics; tissue.

Figure 1.

Hematoxylin and eosin (H&E) and phosphotungstic acid–hematoxylin (PTAH) staining of cardiac tissues. Two sampling areas were microscopically selected from the left ventricular free wall of each infarcted heart: area I and area II. Area I contains distinctive contraction bands (left) and wavy fibers (right). Area II is surrounded by area I and shows minor irregularities such as fragmented myocardia. From each control, cardiac tissue was taken from the left ventricular free wall. Magnification ×400. Scale bar=50 μm.

Figure 2.

Flow diagram of proteomic analysis. AMI indicates acute myocardial infarction; FDR, false discovery rate; FFPE, formalin‐fixed, paraffin‐embedded; LC‐MS/MS, liquid chromatography–tandem mass spectrometry.

Figure 3.

Differential levels of protein in acute myocardial infarction (AMI) and control tissues. Protein expression was compared between area I and control (left) and area II and control (right). Each point represents an individual protein. Vertical line represents 1.5‐fold change, and horizontal line denotes P=0.025 (Mann–Whitney U test followed by Bonferroni's correction). The most prominent proteins (P<0.025 and negative fold‐change <−1.5 or 1.5 <positive fold‐change) are labeled with their UniProt entry name. 3HIDH indicates 3‐hydroxyisobutyrate dehydrogenase; A1BG, alpha‐1B‐glycoprotein; ATPD ATP synthase subunit delta; DLDH, dihydrolipoyl dehydrogenase; FABPH, fatty acid–binding protein, heart (H‐FABP); IGKC, Ig kappa chain C region; ILK, integrin‐linked protein kinase; LONM, Lon protease homologue; MYH13, myosin heavy‐chain 13; MYO1C, myosin 1; MYOM2, myomesin‐2; ODPB, pyruvate dehydrogenase E1 component subunit beta; PACN3, protein kinase C and casein kinase substrate in neurons protein 3; PGAM1, phosphoglycerate mutase 1; SAMP, serum amyloid P‐component; SRBS2, sorbin and SH3 domain‐containing protein 2 (SORBS2); TINAL, tubulointerstitial nephritis antigen‐like; TMOD1, tropomodulin‐1.

Figure 4.

Immunostaining of SORBS2 in human heart tissues. A, SORBS2 regularly localizes along the Z‐line in the myocardial cytoplasm and is densely expressed in the intercalated disk in control heart tissue. B, In AMI tissue, the alignment of SORBS2 in the Z‐bands is disordered at the contraction bands in area I. C, Some wavy fibers show low staining in the Z‐lines (arrowheads) in area I. D, Sarcomeric arrays of SORBS2 disappear from cardiac myocytes (open arrows) in area II. Some intercalated disks appear to have burst (arrows). Magnification ×400. Scale bar=50 μm. AMI indicates acute myocardial infarction; SORBS2, sorbin and SH3 domain‐containing protein 2.

Figure 5.

Serum analyses. A, Representative serum ELISA against H‐FABP. In the commercial ELISA kit, “C” denotes positive control and “T” denotes true positive. AMI cases showed positive results, and control cases showed negative results. B, Representative Western blots against SORBS2 and transferrin. For each lane, 40 μg of serum protein was applied C, Relative intensity level of SORBS2 normalized to that of transferrin. AMI cases (n=10) and controls (n=11) were compared. Each bar represents mean±SEM. *P<0.05 (Mann–Whitney U test). AMI indicates acute myocardial infarction; H‐FABP, fatty acid–binding protein, heart; SEM, standard error of the mean; SORBS2, sorbin and SH3 domain‐containing protein 2.