The assembly of 80S ribosomes requires joining of the 40S and 60S subunits, which is triggered by the formation of an initiation complex on the 40S subunit. This event is rate-limiting for translation, and depends on external stimuli and the status of the cell. Here we show that 60S subunits are activated by release of eIF6 (also termed p27BBP). In the cytoplasm, eIF6 is bound to free 60S but not to 80S. Furthermore, eIF6 interacts in the cytoplasm with RACK1, a receptor for activated protein kinase C (PKC). RACK1 is a major component of translating ribosomes, which harbour significant amounts of PKC. Loading 60S subunits with eIF6 caused a dose-dependent translational block and impairment of 80S formation, which were reversed by expression of RACK1 and stimulation of PKC in vivo and in vitro. PKC stimulation led to eIF6 phosphorylation, and mutation of a serine residue in the carboxy terminus of eIF6 impaired RACK1/PKC-mediated translational rescue. We propose that eIF6 release regulates subunit joining, and that RACK1 provides a physical and functional link between PKC signalling and ribosome activation.

Rationale: In the present study we explored the mechanisms behind excitation-contraction (EC) coupling defects in cardiomyocytes from mice with type-2 diabetes (db/db 1 This has severe implications, because cardiovascular mortality is Ϸ2-to 4-fold higher in diabetic compared to nondiabetic patients 2 and accounts for Ϸ80% of the mortality in type 2 diabetes, 3 of which Ϸ50% die of sudden cardiac death. 4 Furthermore, diabetics are 2.5 times more likely to develop congestive heart failure compared to nondiabetics. 5 The db/db diabetic mouse model develops cardiomyopathy in a similar manner as type 2 diabetes in humans, 6 and presents with reduced whole-heart 7 and isolated cardiomyocyte 8 11,12 In contrast, exercise training in healthy mice increases the level of phosphorylated cytosolic CaMKII␦ and in so doing increases CaMKII␦ activity. Under these circumstances, increased phosphorylation of CaMKII␦ was associated with an increased cardiac performance. 13 Alongside increased SR Ca 2ϩ leak, reduced transverse (T)-tubule structure leading to less synchronous SR Ca 2ϩ release contributes further to the depressed EC coupling in models of cardiac dysfunction. 14 The mechanism for increased SR Ca 2ϩ leak, and whether T-tubule structure in diabetic cardiomyopathy is conserved, has currently not been studied.In the present study, we explored the mechanisms behind the impaired cardiomyocyte function and increased SR Ca 2ϩ leak in db/db cardiomyocytes, and then reexamined the same parameters in the db/db mice after an aerobic interval exercise training program. Because the activities of both CaMKII␦ and PKA are associated with both pathological and physiological remodeling, we also investigated the contributions of CaMKII␦ and PKA for the observed exercise training-induced changes. MethodsFor a detailed description, see the data supplement (available online at http://circres.ahajournals.org). Mouse Model of Diabetes and Exercise TrainingThe db/db mice model has been proven to be a suitable model to study the consequences of diabetes on the heart. Here we studied the male diabetic (BKS.Cg-m ϩ/ϩ Lepdb/Bom Tac; 20 exercised and 20 sedentary mice) and sedentary (nϭ23) and exercise trained (nϭ6) nondiabetic healthy heterozygote (BKS.Cg-m ϩ/ϩ Lepdb/ϩ lean); all age-matched (7 weeks at study start). To determine maximal oxygen uptake (VO 2max ), mice ran until exhaustion on a customized treadmill in a metabolic chamber, and high-intensity aerobic interval training was performed as uphill running, alternating between 4 minutes at 85% to 90% of VO 2max and 2 minutes at 50% of VO 2max for 80 minutes/day, 5 days/wk, for 13 weeks. 15 Cardiomyocyte Isolation and Ca 2؉ MeasurementsLeft ventricular myocytes were isolated as previously described. 15 Fura-2/AM-loaded cardiomyocytes were stimulated by bipolar electric pulses for Ca 2ϩ handling measurements including SR Ca 2ϩ leak. CaMKII inhibitor and PKA inhibitor were used to determine the influence of the 2 kinases. Contractility was recorded by video-based sarcomere spacing. ...