Abstract-Familial hypertrophic cardiomyopathy (HCM) is caused by mutations in at least 8 contractile protein genes, most commonly ␤ myosin heavy chain, myosin binding protein C, and cardiac troponin T. Affected individuals are heterozygous for a particular mutation, and most evidence suggests that the mutant protein acts in a dominant-negative fashion. To investigate the functional properties of a truncated troponin T shown to cause HCM, both wild-type and mutant human cardiac troponin T were overexpressed in Escherichia coli, purified, and combined with human cardiac troponins I and C to reconstitute human cardiac troponin. Significant differences were found between the regulatory properties of wild-type and mutant troponin in vitro, as follows. (1) In actin-tropomyosin-activated myosin ATPase assays at pCa 9, wild-type troponin caused 80% inhibition of ATPase, whereas the mutant complex gave negligible inhibition. (2) Similarly, in the in vitro motility assay, mutant troponin failed to decrease both the proportion of actin-tropomyosin filaments motile and the velocity of motile filaments at pCa 9. (3) At pCa 5, the addition of mutant complex caused a greater increase (21.7%) in velocity of actin-tropomyosin filaments than wild-type troponin (12.3%). These data suggest that the truncated troponin T prevents switching off of the thin filament at low Ca 2ϩ . However, the study of thin filaments containing varying ratios of wild-type and mutant troponin T at low Ca 2ϩ indicated an opposite effect of mutant troponin, causing enhancement of the inhibitory effect of wild-type complex, when it is present in a low ratio (10% to 50%). These multiple effects need to be taken into account to explain the physiological consequences of this mutation in HCM. Further, these findings underscore the importance of studying mixed mutant:wild-type preparations to faithfully model this autosomal-dominant disease. (Circ Res. 2000;86:1146-1152.) Key Words: familial hypertrophic cardiomyopathy Ⅲ troponin T Ⅲ cardiac muscle Ⅲ in vitro motility

Cardiac troponin I (cTnI), the inhibitory subunit of cardiac troponin (cTn), is phosphorylated by the cAMPdependent protein kinase A at two adjacently located serine residues within the heart-specific N-terminal elongation. Four different phosphorylation states can be formed. To investigate each monophosphorylated form cTnI mutants, in which each of the two serine residues is replaced by an alanine, were generated. These mutants, as well as the wildtype cardiac troponin I (cTnI-WT) have been expressed in Escherichia coli, purified and characterized by isoelectric focusing, MS and CD-spectroscopy. Monophosphorylation induces conformational changes within cTnI that are different from those induced by bisphosphorylation. Functionality was assessed by measuring the calcium dependence of myosin S1 binding to thin filaments containing reconstituted native, wild-type and mutant cTn complexes. In all cases a functional holotroponin complex was obtained. Upon bisphosphorylation of cTnI-WT the pCa curve was shifted to the right to the same extent as that observed with bisphosphosphorylated native cTnI. However, the absolute values for the midpoints were higher when recombinant cTn subunits were used for reconstitution. Reconstitution itself changed the calcium affinity of cTnC: pCa 50 -values were higher than those obtained with the native cardiac holotroponin complex. Apparently only bisphosphorylation of cTnI influences the calcium sensitivity of the thin filament, thus monophosphorylation has a function different from that of bisphosphorylation; this function has not yet been identified.Keywords: calcium regulation; cardiac troponin I mutants; cardiac troponin, phosphorylation; reconstitution.Cardiac troponin (cTn), the major regulatory protein of thin filaments, consists of the three subunits, cTnT (tropomyosin binding subunit), cTnI (inhibitory subunit) and cTnC (calcium binding subunit). Both the cTnT and cTnI subunits contain multiple phosphorylation sites. In the heart-specific N-terminus of cTnI two serine residues (amino acids 22 and 23 in the human sequence [1]) are phosphorylated by the cAMP-dependent protein kinase A (PKA) upon b-adrenergic stimulation. Phosphorylation by PKA as well as dephosphorylation by protein phosphatase 2A (PP2A) occur sequentially [2±4]. The distal serine, Ser23, is phosphorylated about 12 times faster and dephosphorylated about two times faster than the proximal serine, Ser22. Thus up to four phosphorylation states can be generated depending on the activity ratio of protein kinase to protein phosphatase, namely a bisphosphorylated, two monophosphorylated states and a dephosphorylated state [3]. All of these cTnI forms are found in different ratios in the various heart compartments (left or right ventricles and left or right atria [5]). Furthermore monophosphorylation and then bisphosphorylation of cTnI gradually reduces the affinity of cTnI to the other cTn subunits [6]. These results suggest specific functions for each of these forms which, to date, have not been elucidated. Bisphos...

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