REPERFUSION OF RAT-HEART AFTER BRIEF ISCHEMIA INDUCES PROTEOLYSIS OF CALSPECTIN (NONERYTHROID SPECTRIN OR FODRIN) BY CALPAIN

Rat myocardium expresses the 240- and 235-kD polypeptides antigenicall y related to alpha- and beta-subunits of brain calspectin (nonerythroi d spectrin or fodrin), respectively. In the subcellular fractions of t he myocardium, alpha-calspectin was found in the 600g, 10 000g, and 10 0 000g pellets, whereas beta-calspectin was localized to the 10 000g p ellet. 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 ca lpain in vitro. The two subunits were also proteolyzed in vivo, when t he rat hearts underwent 10 to 60 minutes of global ischemia followed b y 30 minutes of reperfusion. The reperfusion following the ischemia in duced the proteolysis of alpha-calspectin in the 10 000g and 100 000g pellets, producing the 15O-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-indu ced proteolysis of calspectin. The inhibitor also improved myocardial stunning. Immunohistochemical study revealed that the proteolysis of a lpha-calspectin occurs at the intercalated disks and the sarcolemma af ter postischemic reperfusion, in accord with the biochemical data. The se results suggest that degradation of calspectin partly accounts for the contractile failure of the myocardium after postischemic reperfusi on by disrupting the membrane skeleton and the intercalated disks.