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

Abstract

1. The calpains have been proposed to be activated following cardiac ischaemia and to contribute to myocyte damage after myocardial infarction (MI). In this study, the activity of calpains I and II in the infarcted and non-infarcted rat myocardium and the action of the selective calpain inhibitor, CAL 9961, has been investigated. 2. MI was induced by permanent ligation of the left coronary artery. One, 3, 7 and 14 days post MI, the enzymes calpain I and II were separated from homogenates of the interventricular septum (IS) and left ventricular free wall (LVFW) by chromatography on DEAE-Sepharose. The activity of the calpains was measured in sham-operated and MI animals chronically treated with placebo or CAL 9961 (15 mg kg(-1) d(-1) s.c.) in a synthetic substrate assay. Treatment was started 3 days before MI induction. 3. Calpain I activity reached highest values in IS 14 days post MI, whereas maximum activity of calpain II was measured in LVFW 3 days post MI. In experiments in vitro, CAL 9961 completely inhibited both calpains. In vivo, chronic treatment of MI animals with CAL 9961 partially prevented the increase in calpain I activity in IS and reduced calpain II activity in LVFW to sham levels. 4. Our findings demonstrate that calpains I and II are activated after MI, however, both enzymes differ in their regional and temporal activation within the infarcted myocardium. Chronic inhibition of these enzymes with CAL 9961 might limit the calpain-induced myocardial damage and preserve cardiac structural integrity post MI.

Figure 1

Representation of the experimental protocol (study design).

Figure 2

The elution profile of calpain specific activity (Fluorescence_Ca2+-Fluorescence_EDTA; pmol min⁻¹) following anion exchanger chromatography on DEAE-Sepharose of supernatants of the interventricular septum (IS) (a) and of the left ventricular free wall (LVFW) (b) of placebo-treated MI animals 1, 3, 7 and 14 days post MI. Two peaks of activity were observed occurring in fractions 8 – 12 (180 – 240 mM NaCl) and in fractions 13 – 16 (260 – 320 mM NaCl). Peaks in IS elutions increased in the time course following MI showing highest values 14 days post MI. Maximum peak values in LVFW elutions were measured 3 days post MI. n=6 – 8.

Figure 3

Representative experiments showing Western blots of the calpains I and II in elutions of the infarcted rat myocardium following anion exchanger chromatography on DEAE-Sepharose. For both calpain isoforms, the 80 kDa subunit was detected. Peak 1 was identified to contain calpain I, whereas peak 2 was identified as calpain II, respectively.

Figure 4

Total calpains activity (pmol AMC min⁻¹ mg⁻¹ protein) in supernatants of homogenates of the interventricular septum (IS) (a) and of the left ventricular free wall (LVFW) (b) of sham-operated and infarcted animals with placebo and CAL 9961 treatment (15 mg kg⁻¹ d⁻¹ s.c.), measured 1, 3, 7 and 14 days post MI; ^*P<0.05 compared with sham; #P<0.05 compared with placebo; Data represent mean±s.e.mean; n=6 – 8.

Figure 5

Effects of chronic CAL 9961 treatment (15 mg kg⁻¹ d⁻¹ s.c.) on calpains I and II activity (pmol AMC min⁻¹ mg⁻¹ protein) in fractions following anion exchanger chromatography on DEAE-Sepharose of the interventricular septum (IS) of infarcted rat hearts 14 days post MI (a) and of the left ventricular free wall (LVFW) of infarcted rat hearts 3 days post MI (b) compared to fractions of sham-operated and placebo-treated infarcted rat hearts. ^*P<0.05 compared to sham; #P<0.05 compared to placebo; Data represent mean±s.e.mean; n=6 – 8.