A novel phosphorylation site, Serine 199, in the C-terminus of cardiac troponin I regulates calcium sensitivity and susceptibility to calpain-induced proteolysis. (May 2015)

• Record Type: Journal Article
• Authors: Wijnker, Paul J.M.; Li, Yuejin; Zhang, Pingbo; Foster, D. Brian; Remedios, Cris dos; Van Eyk, Jennifer E.; Stienen, Ger J.M.; Murphy, Anne M.; van der Velden, Jolanda
• Is Part Of: Journal of molecular and cellular cardiology. Volume 82(2015:May)
• Page Start: 93
• Page End: 103
• Publication Date: 2015-05
• Subjects: A alanine -- cMyBP-C cardiac myosin-binding protein-C -- cTn cardiac troponin -- cTnC cardiac troponin C -- cTnI cardiac troponin I -- cTnT cardiac troponin T -- D aspartic acid -- Fmax maximal force -- Fpas passive force -- HFdys dyssynchronous pacing-induced heart failure -- IDCM idiopathic dilated cardiomyopathy -- ktr rate of tension redevelopment -- ktr-max maximal rate of tension redevelopment -- MLC2 myosin light chain 2 -- pCa50 −log10 of the calcium concentration at which 50% of maximal force is reached -- nHill steepness of the force–pCa relation -- PKA protein kinase A -- PKC protein kinase C -- Ser serine -- Tm tropomyosin -- 199A cTnI-Ser199 mutated into alanine (pseudo-dephosphorylated) -- 199D cTnI-Ser199 mutated into aspartic acid (pseudo-phosphorylated); Cardiomyocyte -- Heart failure -- Protein phosphorylation -- Proteolysis -- Cardiac troponin I -- Myofilament function; Cardiology -- Periodicals; Heart Diseases -- Periodicals; Molecular Biology -- Periodicals; Cardiologie -- Périodiques; Cardiology; Electronic journals; Periodicals; 616.12
• DOI: 10.1016/j.yjmcc.2015.03.006
• Languages: English
• ISSNs: 0022-2828

Abstract

Abstract: Phosphorylation of cardiac troponin I (cTnI) by protein kinase C (PKC) is implicated in cardiac dysfunction. Recently, Serine 199 (Ser199) was identified as a target for PKC phosphorylation and increased Ser199 phosphorylation occurs in end-stage failing compared with non-failing human myocardium. The functional consequences of cTnI-Ser199 phosphorylation in the heart are unknown. Therefore, we investigated the impact of phosphorylation of cTnI-Ser199 on myofilament function in human cardiac tissue and the susceptibility of cTnI to proteolysis. cTnI-Ser199 was replaced by aspartic acid (199D) or alanine (199A) to mimic phosphorylation and dephosphorylation, respectively, with recombinant wild-type (Wt) cTn as a negative control. Force development was measured at various [Ca 2 + ] and at sarcomere lengths of 1.8 and 2.2 μm in demembranated cardiomyocytes in which endogenous cTn complex was exchanged with the recombinant human cTn complexes. In idiopathic dilated cardiomyopathy samples, myofilament Ca 2 + -sensitivity (pCa50 ) at 2.2 μm was significantly higher in 199D (pCa50 = 5.79 ± 0.01) compared to 199A (pCa50 = 5.65 ± 0.01) and Wt (pCa50 = 5.66 ± 0.02) at ~ 63% cTn exchange. Myofilament Ca 2 + -sensitivity was significantly higher even with only 5.9 ± 2.5% 199D exchange compared to 199A, and saturated at 12.3 ± 2.6% 199D exchange. Ser199 pseudo-phosphorylation decreased cTnI binding to both actin and actin-tropomyosin. Moreover, altered susceptibility of cTnI to proteolysis by calpain I was found when Ser199 was pseudo-phosphorylated. Our data demonstrate that low levels of cTnI-Ser199 pseudo-phosphorylation (~ 6%) increase myofilament Ca 2 + -sensitivity in human cardiomyocytes, most likely by decreasing the binding affinity of cTnI for actin-tropomyosin. In addition, cTnI-Ser199 pseudo-phosphorylation or mutation regulates calpain I mediated proteolysis of cTnI. Highlights: cTnI-Ser199 pseudo-phosphorylation (199D) increases myofilament Ca 2 + -sensitivity. Low levels of 199D (~ 6%) already increase Ca 2 + -sensitivity in human cardiomyocytes. 199D decreases cTnI binding to actin-tropomyosin.