Reperfusion of Rat Heart After Brief Ischemia Induces Proteolysis of Calspectin (Nonerythroid Spectrin or Fodrin) by Calpain

Abstract

Rat myocardium expresses the 240- and 235-kD polypeptides antigenically related to alpha- and beta-subunits of brain calspectin (nonerythroid spectrin or fodrin), respectively. In the subcellular fractions of the myocardium, alpha-calspectin was found in the 600g, 10,000g, and 100,000g pellets, whereas beta-calspectin was localized to the 10,000g pellet. On the basis of the Na+,K(+)-ATPase activity and the contents of a gap junction protein, the sarcolemma was distributed to the 10,000g and 100,000g pellets, and the intercalated disks were enriched in the 10,000g pellet. Both alpha- and beta-calspectin were proteolyzed by calpain in vitro. The two subunits were also proteolyzed in vivo, when the rat hearts underwent 10 to 60 minutes of global ischemia followed by 30 minutes of reperfusion. The reperfusion following the ischemia induced the proteolysis of alpha-calspectin in the 10,000g and 100,000g pellets, producing the 150-kD fragment. A synthetic calpain inhibitor, calpain inhibitor-1, suppressed the degradation of calspectin in vivo, which indicates that calpain is responsible for the reperfusion-induced proteolysis of calspectin. The inhibitor also improved myocardial stunning. Immunohistochemical study revealed that the proteolysis of alpha-calspectin occurs at the intercalated disks and the sarcolemma after postischemic reperfusion, in accord with the biochemical data. These results suggest that degradation of calspectin partly accounts for the contractile failure of the myocardium after postischemic reperfusion by disrupting the membrane skeleton and the intercalated disks.

Overexpression of TRPC3 increases apoptosis but not necrosis in response to ischemia-reperfusion in adult mouse cardiomyocytes

“…5B). Taken together, these data support the concept that in cardiomyocytes overexpressing TRPC3, Ca 2ϩ entry via CCE contributes to increased apoptosis, at least in part by calpain activation; however, whereas there are considerable data demonstrating that ␣-fodrin cleavage is calpain specific (37,40,41), a direct measure of calpain activity would have further substantiated this conclusion. Nakayama et al (28) have shown that TRPC3 overexpression was also associated with increased calcineurin activity; consequently, it is possible that calcineurin inhibition may also attenuate the increased apoptosis seen in the TRPC3 group.…”

“…Elevated intracellular Ca 2ϩ levels activate numerous Ca 2ϩ -regulated enzymes, including protein kinases, protein phosphatases, phospholipases, NO synthase, Ca 2ϩ /calmodulindependent protein kinase II, and the cysteine protease calpain (42). There is increasing evidence that calpain plays a major role in postischemic injury (15,36), in part because of proteolysis of structural proteins (10,11) including the cytoskeletal protein ␣-fodrin (37,40,41). We found that in WT cardiomyocytes, I/R did not significantly increase cleavage of ␣-fodrin; in contrast, TRPC3 overexpression was associated with an approximately threefold increase in ␣-fodrin cleavage, consistent with increased calpain activity (Fig.…”

“…One pathway by which increased cytosolic Ca 2ϩ contributes to apoptosis is by activation of the calcium-sensitive protease calpain, leading to proteolysis of structural proteins (10,11). The cytoskeletal protein ␣-fodrin is a wellcharacterized substrate for calpain, and increased calpain activity has been shown to lead to the formation of 140/150 kDa cleavage fragment of ␣-fodrin (37,40,41). In Fig.…”

Activity profile of calpains I and II in chronically infarcted rat myocardium – influence of the calpain inhibitor CAL 9961

“…Whereas some studies showed that several calpain inhibitors such as E-64, CI-1 and NCO-700 inhibit the activity of the calpains in the ischaemicreperfused rat heart (Yoshida et al, 1995b;Toda et al, 1989;Cuzzocrea et al, 2000;Toyo-Oka et al, 1982), the present study has demonstrated for the ®rst time the inhibitory action of the calpain inhibitor, CAL 9961, on the activity of the calpains in an animal model of permanent coronary occlusion in rats. Earlier studies demonstrated a contribution of activated calpains I and II in the proteolytic digestion of microtubules (Billger et al, 1988;Sato et al, 1993), actinbinding protein (Wencel-Drake et al, 1991), fodrin (Yoshida et al, 1995a) and vinculin (Steenbergen et al, 1987a) in the ischaemic myocardium which was associated with subsequent myocyte death and loss of myocardial structural integrity and, eventually, cardiac dysfunction. Additionally, the synthetic calpain inhibitor, NCO-700, has been shown to reduce the size of the acute myocardial infarcted region in rabbits with coronary artery ligation, indicating that calpain is involved in myocardial degradation in the ischaemic heart (Toyo-Oka et al, 1982;.…”

“…transcription factors) (Saido et al, 1994). Thus, during cardiac ischaemia, the increase in myocardial [Ca 2+ ] i can activate the calpains causing damage to myocardial proteins (Yoshida et al, 1995a) leading to myocyte death and, consequently, to loss of myocardial structure and function (Reimer & Jennings, 1986). Indeed, evidence suggests the involvement of the calpains in myocardial ischaemia-reperfusion injury (Iizuka et al, 1993;Yoshida et al, 1995b), myocardial stunning (Arthur & Belcastro, 1997) and cardiac hypertrophy (Maki et al, 1990).…”