Potential role of high molecular weight calmodulin-binding protein in cardiac injury

Abstract

Ca(2+) is a major determinant of many biochemical processes in various cell types and is a critical second messenger in cell signalling. High molecular weight calmodulin-binding protein (HMWCaMBP) was originally discovered and purified in the authors' laboratory. It was identified as a homologue of calpastatin - an inhibitor of Ca(2+)-activated cysteine proteases (calpains). Decreased expression of HMWCaMBP in ischemia suggests that it is proteolyzed by calpains during ischemia and reperfusion. In normal myocardial muscle, HMWCaMBP may protect its substrate from calpains, but during an early stage of ischemia/reperfusion with increased Ca(2+) influx, calpain activity exceeds HMWCaMBP activity, leading to proteolysis of HMWCaMBP and other protein substrates, resulting in cellular damage. The role of HMWCaMBP in ischemia/reperfusion is yet to be explored. The present review summarizes developments from the authors' laboratory in the area of HMWCaMBP.

Figure 1)

Identification and purification of high molecular weight calmodulin (CaM)-binding protein (HMWCaMBP) from cardiac muscle. Heart tissue was homogenized in buffer A (20 mM Tris-HCl, 1 mM magnesium acetate, 1 mM imidazole, pH 7.0, 10 mM 2-mercaptoethanol) and applied to a DEAE-Sepharose CL-6B column (Pharmacia Corp, Sweden); the eluate was applied to a CaM-Sepharose 4B column (15). Lanes A to C show the total CaM-binding protein fraction from the CaM-Sepharose 4B column eluted by 1.0 mM EGTA in buffer A in the presence of 0.2 M NaCl; 1.0 mM EGTA alone; and 1.0 mM EGTA containing 0.5 M NaCl, respectively. Lane D shows purified HMWCaMBP. Reproduced with the permission of Highwire Press from reference

Figure 2)

A Dose-dependent inhibition of calpain II (m-calpain) activity by high molecular weight calmodulin-binding protein (HMWCaMBP). HMWCaMBP inhibited the calpain II activity in a concentration-dependent manner in the presence of Ca²⁺. B Inhibition of human calpain I (μ-calpain) by both bovine cardiac HMWCaMBP and calpastatin. Purified calpain I at a concentration of 8 nM was assayed for caseinolytic activity in the presence of various concentrations of HMWCaMBP (•) and calpastatin (○). Both inhibited calpain I activity. Reproduced with the permission of the American Chemical Society from reference

Figure 3)

Western blot analysis of high molecular weight calmodulin-binding protein (HMWCaMBP) and calpastatin from bovine cardiac muscle. Purified HMWCaMBP and calpastatin from bovine cardiac muscle was subjected to 7.5% sodium dodecyl sulphate-polyacrylamide gel electrophoresis, transferred to a nitrocellulose membrane and probed with anti-HMWCaMBP antibody. Lane 1, molecular weight marker; lane 2, HMWCaMBP; lane 3, calpastatin. Reproduced with the permission of the American Chemical Society from reference

Figure 4)

Ultrastructural localization of high molecular weight calmodulin-binding protein (HMWCaMBP) in human cardiac myocytes using the immunogold labelling technique demonstrates cytoplasmic (arrow) and myofilament (circles) immunoreactivity with the anti-HMWCaMBP antibody. Reproduced with the permission of John Wiley & Sons Inc from reference

Figure 5)

Immunoblot of high molecular weight calmodulin-binding protein from normal human cardiac tissue and ischemic cardiac tissue supernatants. Reproduced with the permission of John Wiley & Sons Inc from reference

Figure 6)

Immunoperoxidase staining of high molecular weight calmodulin-binding protein (HMWCaMBP) in ischemic cardiac tissue demonstrated lack of staining, whereas surviving muscle showed positive staining with anti-HMWCaMBP antibody. Reproduced with the permission of John Wiley & Sons Inc from reference

Figure 7)

Expression of high molecular weight calmodulin-binding protein in rat cardiac tissues after ischemia (I) and reperfusion (R). A Control, no I or R; B 5 min I, 30 min R; C 15 min I, 30 min R; D 30 min I, 30 min R; E 15 min I, 0 min R; F 15 min I, 5 min R; G 15 min I, 15 min R; H Hearts were perfused with 100 μM calpain inhibitor in 0.1% dimethyl sulphoxide (DM) for 5 min before 15 min of I and again during 15 min of R; I Hearts were perfused with 0.1% DM alone for 5 min before 15 min of I and again during 15 min of R. Reproduced with the permission of Elsevier Ltd from reference

Figure 8)

Expression of calpains in rat cardiac tissues after ischemia (I) and reperfusion (R). Panel 1 μ-calpain; Panel 2 m-calpain. Details for groups A to I for each panel are provided in Figure 7. DM Dimethyl sulphoxide. Reproduced with the permission of Elsevier Ltd from reference