Related Experiment Video
Updated: May 7, 2026
Identification of Post-translational Modifications of Plant Protein Complexes
Published on: February 22, 2014
PKCα-specific phosphorylation of the troponin complex in human myocardium: a functional and proteomics analysis
Viola Kooij1, Pingbo Zhang, Sander R Piersma
1Laboratory for Physiology, Institute for Cardiovascular Research, VU Medical Center, Amsterdam, The Netherlands ; Johns Hopkins Bayview Proteomics Center, Department of Medicine, School of Medicine, Johns Hopkins University, Baltimore, Maryland, United States of America.
Aims:
Protein kinase Cα (PKCα) is one of the predominant PKC isoforms that phosphorylate cardiac troponin. PKCα is implicated in heart failure and serves as a potential therapeutic target, however, the exact consequences for contractile function in human myocardium are unclear. This study aimed to investigate the effects of PKCα phosphorylation of cardiac troponin (cTn) on myofilament function in human failing cardiomyocytes and to resolve the potential targets involved.
Methods And Results:
Endogenous cTn from permeabilized cardiomyocytes from patients with end-stage idiopathic dilated cardiomyopathy was exchanged (∼69%) with PKCα-treated recombinant human cTn (cTn (DD+PKCα)). This complex has Ser23/24 on cTnI mutated into aspartic acids (D) to rule out in vitro cross-phosphorylation of the PKA sites by PKCα. Isometric force was measured at various [Ca(2+)] after exchange. The maximal force (Fmax) in the cTn (DD+PKCα) group (17.1±1.9 kN/m(2)) was significantly reduced compared to the cTn (DD) group (26.1±1.9 kN/m(2)). Exchange of endogenous cTn with cTn (DD+PKCα) increased Ca(2+)-sensitivity of force (pCa50 = 5.59±0.02) compared to cTn (DD) (pCa50 = 5.51±0.02). In contrast, subsequent PKCα treatment of the cells exchanged with cTn (DD+PKCα) reduced pCa50 to 5.45±0.02. Two PKCα-phosphorylated residues were identified with mass spectrometry: Ser198 on cTnI and Ser179 on cTnT, although phosphorylation of Ser198 is very low. Using mass spectrometry based-multiple reaction monitoring, the extent of phosphorylation of the cTnI sites was quantified before and after treatment with PKCα and showed the highest phosphorylation increase on Thr143.
Conclusion:
PKCα-mediated phosphorylation of the cTn complex decreases Fmax and increases myofilament Ca(2+)-sensitivity, while subsequent treatment with PKCα in situ decreased myofilament Ca(2+)-sensitivity. The known PKC sites as well as two sites which have not been previously linked to PKCα are phosphorylated in human cTn complex treated with PKCα with a high degree of specificity for Thr143.
Insights
Protein kinase Cα (PKCα) phosphorylation of cardiac troponin (cTn) in human heart failure reduces maximal force but increases Ca(2+) sensitivity. Subsequent PKCα treatment reverses this sensitivity change, highlighting specific phosphorylation targets.
Area of Science:
- Cardiovascular Physiology
- Molecular Cardiology
- Protein Phosphorylation
Background:
- Protein kinase Cα (PKCα) is a key enzyme implicated in heart failure pathogenesis.
- PKCα phosphorylates cardiac troponin (cTn), but its precise impact on human myocardial contractile function remains unclear.
- Understanding PKCα's role is crucial for developing targeted therapies for heart failure.
Purpose of the Study:
- To investigate the effects of PKCα-mediated cTn phosphorylation on myofilament function in human failing cardiomyocytes.
- To identify specific phosphorylation sites on cTn targeted by PKCα.
Main Methods:
- Human failing cardiomyocytes from patients with end-stage idiopathic dilated cardiomyopathy were utilized.
- Endogenous cTn was exchanged with recombinant human cTn engineered to prevent PKA site phosphorylation (cTn (DD)).
- Isometric force measurements at various calcium concentrations were performed before and after PKCα treatment; mass spectrometry identified phosphorylation sites.
Main Results:
- PKCα-treated cTn (cTn (DD+PKCα)) significantly reduced maximal force (Fmax) compared to control (cTn (DD)).
- Exchange with cTn (DD+PKCα) increased myofilament Ca(2+) sensitivity (pCa50).
- Subsequent in situ PKCα treatment decreased Ca(2+) sensitivity; Thr143 on cTnI and Ser179 on cTnT were identified as key phosphorylation sites.
Conclusions:
- PKCα phosphorylation of the cTn complex alters myofilament force and Ca(2+) sensitivity.
- Specific phosphorylation of cTnI at Thr143 by PKCα is a significant finding.
- These findings provide novel insights into the molecular mechanisms underlying cardiac dysfunction in heart failure.
More Related Videos
Related Concept Videos
02:54Protein Kinases and Phosphatases
Protein kinases
Many proteins in the cell are regulated by phosphorylation, the addition of a phosphate group. A family of enzymes called kinases...
01:02Phosphorylation
During phosphorylation, protein kinases transfer the terminal phosphate group of ATP to specific amino acid side chains of substrate proteins. Serine, threonine, and tyrosine are the most commonly...
01:20Blood Studies for Cardiovascular System I: Cardiac Biomarkers
The essential diagnostic tools for detecting myocardial necrosis and monitoring individuals suspected of having acute coronary syndrome (ACS) include:
Troponins
Troponins, particularly cardiac troponins I and T, are the most precise and sensitive markers of myocardial injury. They are detectable within 4-6 hours of myocardial injury and remain...
01:27Transducer Mechanism: Enzyme-Linked Receptors
Major types that are helpful drug targets include:
01:25cAMP-dependent Protein Kinase Pathways
01:16Calmodulin-dependent Signaling
The Ca2+-CaM complex does not have enzymatic activity by itself. Instead, the complex binds downstream target proteins, including membrane proteins or enzymes,...