The Ubiquitin-Proteasome Proteolytic Pathway in Heart vs Skeletal Muscle: Effects of Acute Diabetes Zhenqi Liu, Wendell R. Miers, Liping Wei, and Eugene J. Barrett Division of Endocrinology and Metabolism, Department of Medicine, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908 Received August 25, 2000 The ubiquitin-proteasome system is thought to play a major role in normal muscle protein turnover and to contribute to diabetes-induced protein wasting in skeletal muscle. However, its importance in cardiac muscle is not clear. We measured heart muscle mRNA for ubiquitin and for the C2 and C8 proteasomal sub- units, the amount of free ubiquitin and the protea- some chymotrypsin-like proteolytic activity in control and diabetic rats. Results were compared to those in skeletal muscle (rectus). Heart ubiquitin, C2 and C8 subunit mRNA and proteolytic activity were signifi- cantly greater than in skeletal muscle (P < 0.05). This suggests that the ubiquitin proteasomal pathway may also be important for normal heart muscle turnover. Diabetes increased ubiquitin mRNA by 50% in heart (P < 0.03) and by 100% in skeletal muscle (P < 0.005). It remained high after 3 days of insulin treatment in both tissues. C2 and C8 subunit mRNA did not change with diabetes or insulin treatment. Diabetes did not change the amount of free ubiquitin or the proteaso- mal (lactacystin-inhibitable) chymotrypsin-like pepti- dase activity in heart or skeletal muscle. In conclu- sions, gene expression for several components of the ubiquitin-proteasome proteolytic pathway is signifi- cantly higher in cardiac than in skeletal muscle, as is the proteasome chymotrypsin-like peptidase activity. Diabetes increases the expression of ubiquitin but not C2 or C8 subunit mRNA, nor does it significantly alter the amount of free ubiquitin or the proteasome chymotrypsin-like peptidase activity. The rate- limiting step of enhanced protein degradation in dia- betic rat heart and skeletal muscle may be located at ubiquitin conjugation and/or its binding to protea- some, not at the ubiquitin availability or the protea- some itself. © 2000 Academic Press Key Words: heart; protein; diabetes; ubiquitin; pro- teasome; muscle; messenger RNA. At least four different proteolytic processes are present in the skeletal muscles. These include a lyso- somal, a calcium-dependent (1), an ATP-dependent (ubiquitin-proteasome pathway), and an ATP- independent proteolytic process (2). Recent studies suggest that in rat skeletal muscle the ubiquitin- proteasome system is the major pathway of protein degradation (3). This is a multistep pathway that first involves a complex of ubiquitin-conjugating enzymes that couple ubiquitin to proteins targeted for degrada- tion (4). The ubiquitin-tagged proteins are subse- quently degraded by the 26S proteasome (5). The reg- ulation of this pathway is not well understood. Studies have shown that in skeletal muscle mRNAs coding for some components of this pathway increase in response to pathologic/physiologic manipulations, such as fast- ing and glucocorticoids (6, 7), tumor implantation (8), muscle denervation (9), burn (10), sepsis (11), acidosis (12), uremia (13) and diabetes (14, 15). Despite the major role of the ubiquitin-proteasome system in the turnover of skeletal muscle protein, and despite evidence that the activity of the entire pathway is increased in a variety of conditions associated with muscle wasting, the normal physiology of this proteo- lytic pathway has not been well studied in heart mus- cle. Furthermore, there is limited data on protein ex- pression or proteasome activity, even in skeletal muscle. As a result, the importance of this pathway to proteolysis in cardiac and skeletal muscle is still un- clear. In the present study, we compared the amount of mRNA coding for ubiquitin, C2 and C8 proteasome subunits, the amount of free ubiquitin and the chymotrypsin-like peptidase activity of the proteasome in heart muscle vs. skeletal muscle in rats. We then examined the effects of acute diabetes on this system. Our results indicate that in cardiac muscle compared to skeletal muscle there is greater expression of com- ponents of the ubiquitin-proteasome proteolytic path- way and a higher enzymatic activity. This suggests that as in skeletal muscle this pathway has an impor- tant role in protein degradation in heart. In addition, our findings suggest that the rate-limiting step of en- hanced protein degradation in diabetic rat heart and Biochemical and Biophysical Research Communications 276, 1255–1260 (2000) doi:10.1006/bbrc.2000.3609, available online at http://www.idealibrary.com on 1255 0006-291X/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.