Mutations in the non-lysosomal cysteine protease calpain 3 cause limb-girdle muscular dystrophy type 2A (LGMD2A). Our previous studies of the calpain 3 knockout mouse (C3KO) suggested a role for calpain 3 in sarcomere formation and remodeling. Calpain 3 may mediate remodeling by cleavage and release of myofibrillar proteins, targeting them for ubiquitination and proteasomal degradation. Loss of proper protein turnover may be the basis for this muscle disease. To test this hypothesis in vivo, we used an experimental model of hindlimb unloading and reloading that has been shown to induce sarcomere remodeling. We showed that the rate of atrophy and especially the rate of growth are decreased in C3KO muscles under conditions promoting sarcomere remodeling. In wild-type mice, an elevated level of ubiquitinated proteins was observed during muscle reloading, which is presumably necessary to remove atrophy-specific and damaged proteins. This increase in ubiquitination correlated with an increase in calpain 3 expression. C3KO muscles did not show any increase in ubiquitination at the reloading stage, suggesting that calpain 3 is necessary for ubiquitination and that it acts upstream of the ubiquitination machinery. We found upregulation of heat shock proteins in C3KO muscles following challenge with a physiological condition that requires highly increased protein degradation. Furthermore, old C3KO mice show evidence of insoluble protein aggregate formation in skeletal muscles. These studies suggest that accumulation of aged and damaged proteins can lead to cellular toxicity and a cell stress response in C3KO muscles, and that these characteristics are pathological features of LGMD2A.

The effects of Troponin T (TnT) mutants R141W and ⌬K210, the only two currently known mutations in TnT that cause dilated cardiomyopathy(DCM) independent of familial hypertrophic cardiomyopathy (FHC), and TnT-K273E, a mutation that leads to a progression from FHC to DCM, were investigated. Studies on the Ca 2؉ sensitivity of force development in porcine cardiac fibers demonstrated that TnT-⌬K210 caused a significant decrease in Ca 2؉ sensitivity, whereas the TnT-R141W did not result in any change in Ca 2؉ sensitivity when compared with human cardiac wild-type TnT (HCWTnT). TnT-⌬K210 also caused a decrease in maximal force when compared with HCWTnT and TnT-R141W. In addition, the TnT-⌬K210 mutant decreased maximal ATPase activity in the presence of Ca 2؉ . However, the TnT-K273E mutation caused a significant increase in Ca 2؉ sensitivity but behaved similarly to HCWTnT in actomyosin activation assays. Inhibition of ATPase activity in reconstituted actin-activated myosin ATPase assays was similar for all three TnT mutants and HCWTnT. Additionally, circular dichroism studies suggest that the secondary structure of all three TnT mutants was similar to that of the HCWTnT. These results suggest that a rightward shift in Ca 2؉ sensitivity is not the only determinant for the phenotype of DCM.

In this study we investigated the physiological role of the cardiac troponin T (cTnT) isoforms in the presence of human slow skeletal troponin I (ssTnI). ssTnI is the main troponin I isoform in the fetal human heart. In reconstituted fibers containing the cTnT isoforms in the presence of ssTnI, cTnT1-containing fibers showed increased Ca 2؉ sensitivity of force development compared with cTnT3-and cTnT4-containing fibers. The maximal force in reconstituted skinned fibers was significantly greater for the cTnT1 (predominant fetal cTnT isoform) when compared with cTnT3 (adult TnT isoform) in the presence of ssTnI. Troponin (Tn) complexes containing ssTnI and reconstituted with cTnT isoforms all yielded different maximal actomyosin ATPase activities. Tn complexes containing cTnT1 and cTnT4 (both fetal isoforms) had a reduced ability to inhibit actomyosin ATPase activity when compared with cTnT3 (adult isoform) in the presence of ssTnI. The rate at which Ca 2؉ was released from site II of cTnC in the cTnI⅐cTnC complex (122/s) was 12.5-fold faster than for the ssTnI⅐cTnC complex (9.8/s). Addition of cTnT3 to the cTnI⅐cTnC complex resulted in a 3.6-fold decrease in the Ca 2؉ dissociation rate from site II of cTnC. Addition of cTnT3 to the ssTnI⅐cTnC complex resulted in a 1.9-fold increase in the Ca 2؉ dissociation rate from site II of cTnC. The rate at which Ca 2؉ dissociated from site II of cTnC in Tn complexes also depended on the cTnT isoform present. However, the TnI isoforms had greater effects on the Ca 2؉ dissociation rate of site II than the cTnT isoforms. These results suggest that the different N-terminal TnT isoforms would produce distinct functional properties in the presence of ssTnI when compared with cTnI and that each isoform would have a specific physiological role in cardiac muscle.

The major goal of this study was to elucidate how troponin T (TnT) dilated cardiomyopathy (DCM) mutations in fetal TnT and fetal troponin affect the functional properties of the fetal heart that lead to infantile cardiomyopathy. The DCM mutations R141W and ⌬K210 were created in the TnT1 isoform, the primary isoform of cardiac TnT in the embryonic heart. In addition to a different TnT isoform, a different troponin I (TnI) isoform, slow skeletal TnI (ssTnI), is the dominant isoform in the embryonic heart. In skinned fiber studies, TnT1-wild-type (WT)-treated fibers reconstituted with cardiac TnI⅐troponin C (TnC) or ssTnI⅐TnC significantly increased Ca 2؉ sensitivity of force development when compared with TnT3-WT-treated fibers at both pH 7.0 and pH 6.5. Porcine cardiac fibers treated with TnT1 that contained the DCM mutations (R141W and ⌬K210), when reconstituted with either cardiac TnI⅐TnC or ssTnI⅐TnC, significantly decreased Ca 2؉ sensitivity of force development compared with TnT1-WT at both pH values. The R141W mutation, which showed no significant change in the Ca 2؉ sensitivity of force development in the TnT3 isoform, caused a significant decrease in the TnT1 isoform. The ⌬K210 mutation caused a greater decrease in Ca 2؉ sensitivity and maximal isometric force development compared with the R141W mutation in both the fetal and adult TnT isoforms. When complexed with cardiac TnI⅐TnC or ssTnI⅐TnC, both TnT1 DCM mutations strongly decreased maximal actomyosin ATPase activity as compared with TnT1-WT. Our results suggest that a decrease in maximal actomyosin ATPase activity in conjunction with decreased Ca 2؉ sensitivity of force development may cause a severe DCM phenotype in infants with the mutations.

Troponin (Tn) is made up of three subunits, troponin T (TnT), troponin I (TnI), and troponin C (TnC). In cardiac muscle, TnI can exist as two isoforms, slow skeletal TnI (ssTnI) or cardiac TnI (cTnI), whereas TnT occurs as multiple isoforms. The predominant form of TnI in fetal cardiac muscle is ssTnI, which is derived from a different gene than cTnI. However, the predominant form of cardiac TnT (cTnT) in fetal muscle is cTnT1, which is derived from the same gene that produces the adult cTnT isoform (cTnT3). Fetal cardiac muscle is more sensitive to Ca(2+) than adult muscle and this may be due in part to the fetal cTnT1 and ssTnI isoforms. cTnT1 and/or ssTnI by themselves cause a significant increase in Ca(2+) sensitivity when compared to cTnT3 and/or cTnI. Mutations in the gene for cTnT can cause hypertrophic cardiomyopathy or dilated cardiomyopathy (DCM). Investigation of DCM mutations in the fetal cTnT1 isoform showed that the cTnT isoform is an important determinant of the effect of the mutation. The TnI isoform also affects the physiological function of the cardiac muscle. The presence of both the fetal TnT isoform, containing a DCM mutation, and ssTnI results in larger changes in Ca(2+) sensitivity than the same DCM mutant in the adult TnT isoform and in the presence of cTnI (when compared to their respective wild-type TnT controls). These recent results suggest that some mutations may have different severities in fetal and adult hearts.