No abstract

Myocardial stunning is characterized by decreased myofilament Ca sup 2+ responsiveness. To investigate the molecular basis of stunned myocardium, we performed PAGE and Western immunoblot analysis of the contractile proteins. Isolated rat hearts were retrogradely perfused at 37 degrees C for either 50 minutes (control group) or for 10 minutes, followed by 20-minute global ischemia and 20-minute reperfusion (stunned group), or for 20-minute ischemia without reflow. Another group consisted of hearts subjected to 20-minute ischemia in which stunning was mitigated by 10-minute reperfusion with low Ca 2 +/low pH solution. Myocardial tissue samples subjected to PAGE revealed no obvious differences among groups. Western immunoblots for actin, tropomyosin, troponin C, troponin T, myosin light chain-1, and myosin light chain-2 showed highly selective recognition of the appropriate full-length molecular weight bands in all groups. Troponin I (TnI) Western blots revealed an additional band ([nearly =]26 kD, compared with 32 kD for the full-length protein) in stunned myocardial samples only. In parallel experiments, skinned trabeculae were treated with calpain I for 20 minutes; Western blots showed a TnI degradation pattern similar to that observed in stunned myocardium. Such TnI degradation was prevented by calpastatin, a naturally occurring calpain inhibitor. The results show that (1) TnI is partially and selectively degraded in stunned myocardium; (2) this degradation could be prevented by low Ca sup 2+/low pH reperfusion. which also prevented the contractile dysfunction of stunning; and (3) calpain I could similarly degrade TnI, supporting the idea that Ca 2 +-dependent myofilament proteolysis underlies myocardial stunning.

The Ca2' sensitivity of myofilaments was compared before and after skinning in the same rat trabeculae at a diastolic sarcomere length of 2.2 to 2.3 ,um. Trabeculae from rat right ventricle were loaded with fura-2 salt by iontophoretic microinjection, and [Ca 2+i was determined from the epifluorescence at 510 nm when excited at 340 and 380 nm. Steady (Circ Res. 1994;74:408-415.) Key Words * intracellular calcium * intracellular magnesium * excitation-contraction coupling * cardiac muscle and after skinning in the same cardiac muscles. Such an approach has several advantages: (1) maximal Ca2'-activated force can be compared directly before and after skinning; (2) sarcomere length, which is an important factor affecting force generation,8 can be controlled and kept at the same length before and after skinning; and (3) possible variations from muscle to muscle can be minimized. This study demonstrates that there is indeed a disparity in the force-[Ca21] relations obtained before and after skinning. Decreasing [Mg2+] in the bathing solution to the lowest level that we measured with Mg-fura-2 in this preparation shifted the force- [Ca2+] relation to the left, but important differences were still detectable after skinning. A preliminary report has appeared.9 Materials and Methods PreparationRats (LBN-F1 brown rats, 200 to 250 g; Harlan, Indianapolis, Ind) were anesthetized with ether and the hearts rapidly excised via a midsternal thoracotomy. Trabeculae from the right ventricle were dissected according to the method described previously.10 The dimensions of the trabeculae were 1.89±0.37 mm long, 0.127±0.045 mm wide, and 0.078+0.021 mm thick (mean±SD, n=22). After dissection, the trabeculae were mounted between a force transducer and a micromanipulator in a perfusion bath. The trabeculae were superfused with Krebs-Henseleit (K-H) buffer equilibrated with 95% 02/5% CO2 gas mixture. The K-H buffer was composed of (in mmol/L): Na+ 142, K' 5, Mg 2+ 1.2, Cl-127.4, P04-2, HCO3-20, CaCl2 0.5, and pH 7.35 to 7.4. The perfusion rate was =10 mL/min, and the preparations were stimulated at 0.5 Hz. All the experiments were performed at room temperature (20°C to 22°C).by guest on May 7, 2018