We have used phosphate affinity SDS-PAGE to separate the phosphorylated species of cardiac troponin I (cTnI). To test the method we phosphorylated pure cTnI with protein kinase A catalytic subunit and observed up to six bands corresponding to 0, 1P, 2P, 3P, 4P and 5P phospho-species. We examined the phospho-species of cTnI in human heart myofibrillar extracts by phosphate affinity SDS-PAGE and Western blotting with a non-specific troponin I (TnI) antibody. In donor heart samples the bis-phosphorylated species of cTnI predominated and no more highly phosphorylated species were not detectable (0P was 10.3±1.9%, 1P, 17.5±3.5%, 2P, 72.2±4.7%, 11 samples). Total phosphorylation was 1.62±0.06 molsPi/mol TnI. In myofibrils from end-stage failing hearts, the unphosphorylated cTnI species predominated (0P was 78.5±1.8%, 1P, 17.5±1.9%, 2P, 4.0±0.7%, total phosphorylation 0.26±0.02 molsPi/mol TnI, five samples). Muscle from patients with hypertrophic obstructive cardiomyopathy was also largely unphosphorylated (0P was 76.6±3.1%, 1P, 17.5±2.7%, 2P, 5.9±0.8%, total phosphorylation 0.29±0.04 molsPi/mol TnI, 19 samples). Using a range of phospho-specific antibodies we demonstrated that 3/4 of the bis-phosphorylated band of donor heart cTnI is phosphorylated at Ser22 and Ser23 in approximately equal amounts and that phosphorylation of Ser43 and Thr142 was not detected.

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Background-Familial dilated cardiomyopathy can be caused by mutations in the proteins of the muscle thin filament. In vitro, these mutations decrease Ca 2ϩ sensitivity and cross-bridge turnover rate, but the mutations have not been investigated in human tissue. We studied the Ca 2ϩ -regulatory properties of myocytes and troponin extracted from the explanted heart of a patient with inherited dilated cardiomyopathy due to the cTnC G159D mutation. Methods and Results-Mass spectroscopy showed that the mutant cTnC was expressed approximately equimolar with wild-type cTnC. Contraction was compared in skinned ventricular myocytes from the cTnC G159D patient and nonfailing donor heart. Maximal Ca 2ϩ -activated force was similar in cTnC G159D and donor myocytes, but the Ca 2ϩ sensitivity of cTnC G159D myocytes was higher (EC 50 G159D/donorϭ0.60). Thin filaments reconstituted with skeletal muscle actin and human cardiac tropomyosin and troponin were studied by in vitro motility assay. Thin filaments containing the mutation had a higher Ca 2ϩ sensitivity (EC 50 G159D/donorϭ0.55Ϯ0.13), whereas the maximally activated sliding speed was unaltered. In addition, the cTnC G159D mutation blunted the change in Ca 2ϩ sensitivity when TnI was dephosphorylated. With wild-type troponin, Ca 2ϩ sensitivity was increased (EC 50 P/unPϭ4.7Ϯ1.9) but not with cTnC G159D troponin (EC 50 P/unPϭ1.2Ϯ0.1). Conclusions-We propose that uncoupling of the relationship between phosphorylation and Ca 2ϩ sensitivity could be the cause of the dilated cardiomyopathy phenotype. The differences between these data and previous in vitro results show that native phosphorylation of troponin I and troponin T and other posttranslational modifications of sarcomeric proteins strongly influence the functional effects of a mutation. (Circ Heart Fail. 2009;2:456-464.) Key Words: cardiomyopathy Ⅲ contractility Ⅲ heart failure D ilated cardiomyopathy (DCM) is a common cause of sudden death and heart failure, and it is estimated that 20% to 30% of cases of DCM are caused by mutations in specific proteins. 1 Many cases of inherited "pure" DCM that are not associated with other symptoms, such as conduction disease, are caused by mutations in contractile proteins, including actin, myosin, tropomyosin, and all 3 subunits of troponin. 1,2 These cardiomyopathy-causing mutations present a unique opportunity to link genotype with phenotype. Because mutations at different sites in several different contractile proteins can produce a single phenotype, it has been proposed that mutations causing the same phenotype alter the contractile mechanism in the same way. In support of this hypothesis, a number of DCM mutations have been shown to cause decreased Ca 2ϩ sensitivity linked to reduced troponin C Ca 2ϩ -binding affinity and decreased cross-bridge turnover rate in vitro. [3][4][5][6][7][8][9] However, recent work, studying both recombinant proteins and intact myofibrils, has provided results that contradict the simple hypothesis and this raises doubts about the physiological relev...