scholarly journals Impact of site-specific phosphorylation of protein kinase A sites Ser23 and Ser24 of cardiac troponin I in human cardiomyocytes

2013 ◽  
Vol 304 (2) ◽  
pp. H260-H268 ◽  
Author(s):  
Paul J. M. Wijnker ◽  
D. Brian Foster ◽  
Allison L. Tsao ◽  
Aisha H. Frazier ◽  
Cristobal G. dos Remedios ◽  
...  
Keyword(s):  
Cardiac Troponin ◽  
Cardiac Tissue ◽  
Troponin I ◽  
Human Myocardium ◽  
Site Directed Mutagenesis ◽  
Adrenergic Stimulation ◽  
Human Cardiomyocytes ◽  
Health And Disease ◽  
Human Cardiac Tissue

PKA-mediated phosphorylation of contractile proteins upon β-adrenergic stimulation plays an important role in the regulation of cardiac performance. Phosphorylation of the PKA sites (Ser23/Ser24) of cardiac troponin (cTn)I results in a decrease in myofilament Ca2+ sensitivity and an increase in the rate of relaxation. However, the relation between the level of phosphorylation of the sites and the functional effects in the human myocardium is unknown. Therefore, site-directed mutagenesis was used to study the effects of phosphorylation at Ser23 and Ser24 of cTnI on myofilament function in human cardiac tissue. Serines were replaced by aspartic acid (D) or alanine (A) to mimic phosphorylation and dephosphorylation, respectively. cTnI-DD mimics both sites phosphorylated, cTnI-AD mimics Ser23 unphosphorylated and Ser24 phosphorylated, cTnI-DA mimics Ser23 phosphorylated and Ser24 unphosphorylated, and cTnI-AA mimics both sites unphosphorylated. Force development was measured at various Ca2+ concentrations in permeabilized cardiomyocytes in which the endogenous troponin complex was exchanged with these recombinant human troponin complexes. In donor cardiomyocytes, myofilament Ca2+ sensitivity (pCa50) was significantly lower in cTnI-DD (pCa50: 5.39 ± 0.01) compared with cTnI-AA (pCa50: 5.50 ± 0.01), cTnI-AD (pCa50: 5.48 ± 0.01), and cTnI-DA (pCa50: 5.51 ± 0.01) at ∼70% cTn exchange. No effects were observed on the rate of tension redevelopment. In cardiomyocytes from idiopathic dilated cardiomyopathic tissue, a linear decline in pCa50 with cTnI-DD content was observed, saturating at ∼55% bisphosphorylation. Our data suggest that in the human myocardium, phosphorylation of both PKA sites on cTnI is required to reduce myofilament Ca2+ sensitivity, which is maximal at ∼55% bisphosphorylated cTnI. The implications for in vivo cardiac function in health and disease are detailed in the discussion in this article.

scholarly journals A Possible Mechanism behind Faster Clearance and Higher Peak Concentrations of Cardiac Troponin I Compared with Troponin T in Acute Myocardial Infarction

2020 ◽  
Vol 66 (2) ◽  
pp. 333-341 ◽  
Author(s):  
Karin Starnberg ◽  
Vincent Fridén ◽  
Aida Muslimovic ◽  
Sven-Erik Ricksten ◽  
Susanne Nyström ◽  
...  
Keyword(s):  
Myocardial Infarction ◽  
Heart Failure ◽  
Acute Myocardial Infarction ◽  
Cardiac Troponin ◽  
Cardiac Tissue ◽  
Troponin I ◽  
Troponin T ◽  
Cardiac Troponin I ◽  
Human Cardiac Tissue

Abstract Background Although cardiac troponin I (cTnI) and troponin T (cTnT) form a complex in the human myocardium and bind to thin filaments in the sarcomere, cTnI often reaches higher concentrations and returns to normal concentrations faster than cTnT in patients with acute myocardial infarction (MI). Methods We compared the overall clearance of cTnT and cTnI in rats and in patients with heart failure and examined the release of cTnT and cTnI from damaged human cardiac tissue in vitro. Results Ground rat heart tissue was injected into the quadriceps muscle in rats to simulate myocardial damage with a defined onset. cTnT and cTnI peaked at the same time after injection. cTnI returned to baseline concentrations after 54 h, compared with 168 h for cTnT. There was no difference in the rate of clearance of solubilized cTnT or cTnI after intravenous or intramuscular injection. Renal clearance of cTnT and cTnI was similar in 7 heart failure patients. cTnI was degraded and released faster and reached higher concentrations than cTnT when human cardiac tissue was incubated in 37°C plasma. Conclusion Once cTnI and cTnT are released to the circulation, there seems to be no difference in clearance. However, cTnI is degraded and released faster than cTnT from necrotic cardiac tissue. Faster degradation and release may be the main reason why cTnI reaches higher peak concentrations and returns to normal concentrations faster in patients with MI.

Abstract 366: Engineered Human Cardiac Tissue from Skeletal Muscle Derived Cells and Induced Pluripotent Stem Cell Derived Cardiomyocytes

2012 ◽  
Vol 111 (suppl_1) ◽  
Author(s):  
Kimimasa Tobita ◽  
Jason S Tchao ◽  
Jong Kim ◽  
Bo Lin ◽  
Johnny Huard ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Stem Cell ◽  
Pluripotent Stem Cell ◽  
Cardiac Tissue ◽  
Troponin I ◽  
Troponin T ◽  
Induced Pluripotent Stem Cell ◽  
Human Cardiac Tissue ◽  
Induced Pluripotent ◽  
Immature Myocardium

We have previously shown that rat skeletal muscle derived stem cells differentiate into an immature cardiomyocyte (CM) phenotype within a 3-dimensional collagen gel engineered cardiac tissue (ECT). Here, we investigated whether human skeletal muscle derived progenitor cells (skMDCs) can differentiate into a CM phenotype within ECT similar to rat skeletal muscle stem cells and compared the human skMDC-ECT properties with ECT from human induced pluripotent stem cell (iPSc) derived CMs. SkMDCs differentiated into a cardiac muscle phenotype within ECT and exhibited spontaneous beating activity as early as culture day 4 and maintained their activity for more than 2 weeks. SkMDC-ECTs stained positive for cardiac specific troponin-T and troponin-I, and were co-localized with fast skeletal muscle myosin heavy chain (sk-fMHC) with a striated muscle pattern similar to fetal myocardium. The iPS-CM-ECTs maintained spontaneous beating activity for more than 2 weeks from ECT construction. iPS-CM stained positive for both cardiac troponin-T and troponin-I, and were also co-localized with sk-fMHC while the striated expression pattern of sk-fMHC was lost similar to post-natal immature myocardium. Connexin-43 protein was expressed in both engineered tissue types, and the expression pattern was similar to immature myocardium. The skMDC-ECT significantly upregulated expression of cardiac-specific genes compared to conventional 2D culture. SkMDC-ECT displayed cardiac muscle like intracellular calcium ion transients. The contractile force measurements demonstrated functional properties of fetal type myocardium in both ECTs. Our results suggest that engineered human cardiac tissue from skeletal muscle progenitor cells mimics developing fetal myocardium while the engineered cardiac tissue from inducible pluripotent stem cell-derived cardiomyocytes mimics post-natal immature myocardium.

scholarly journals Heart Failure–Related Hyperphosphorylation in the Cardiac Troponin I C Terminus Has Divergent Effects on Cardiac Function In Vivo

2017 ◽  
Vol 10 (9) ◽  
Author(s):  
Yuejin Li ◽  
Guangshuo Zhu ◽  
Nazareno Paolocci ◽  
Pingbo Zhang ◽  
Cyrus Takahashi ◽  
...  
Keyword(s):  
Heart Failure ◽  
Cardiac Function ◽  
Cardiac Troponin ◽  
Troponin I ◽  
Cardiac Troponin I ◽  
C Terminus

HUMAN iPSC ENGINEERED CARDIAC TISSUE PLATFORM FAITHFULLY MODELS IMPORTANT CARDIAC PHYSIOLOGY

2021 ◽  
Author(s):  
Willem J. de Lange ◽  
Emily T. Farrell ◽  
Caroline R. Kreitzer ◽  
Derek R. Jacobs ◽  
Di Lang ◽  
...  
Keyword(s):  
Cardiac Tissue ◽  
Human Myocardium ◽  
Protein Isoforms ◽  
Calcium Handling ◽  
Unintended Effects ◽  
Cardiac Physiology ◽  
Adrenergic Stimulation ◽  
Cardiac Model ◽  
Short Period ◽  
Tubular System

Cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CM) may provide an important bridge between animal models and intact human myocardium. Fulfilling this potential is hampered by their relative immaturity. hiPSC-CMs grown in monolayer culture lack a t-tubular system, have rudimentary intracellular calcium-handling systems, express predominantly embryonic sarcomeric protein isoforms, and preferentially use glucose as energy substrate. Culturing hiPSC-CM in a 3D environment and the addition of nutritional, pharmacologic and electromechanical stimuli have proven to be beneficial for maturation. We present an assessment of a model in which hiPSC-CMs and hiPSC-derived cardiac fibroblasts are co-cultured in a 3D fibrin matrix to form human engineered cardiac tissue constructs (hECT).The hECT respond to physiological stimuli, including stretch, frequency and β-adrenergic stimulation, develop a t-tubular system, and demonstrate calcium-handling and contractile kinetics that compare favorably with ventricular human myocardium. Transcript levels of genes involved in calcium-handling and contraction are increased. These markers of maturation become more robust over a short period of time in culture (6 weeks vs. 2 weeks in hECT). A comparison of the hECT molecular and performance variables with those of human cardiac tissue and other available engineered tissue platforms is provided to highlight strengths and weaknesses of these preparations. Important and noteworthy aspects of this human cardiac model system are its reliance on 'off-the-shelf' equipment, ability to provide detailed physiological performance data, and the ability to achieve a relatively mature cardiac physiology without additional nutritional, pharmacological and electromechanical stimuli that may elicit unintended effects on function.

Decrease Tyrosine Phosphorylation of Stat3 in Human Myocardium with End-Stage Congestive Heart Failure

2000 ◽  
Vol 6 (S2) ◽  
pp. 596-597
Author(s):  
C. Wei ◽  
J. S. McLaughlin
Keyword(s):  
Heart Failure ◽  
Congestive Heart Failure ◽  
Protein Expression ◽  
Cardiac Tissue ◽  
Normal Subjects ◽  
Human Myocardium ◽  
Stat3 Phosphorylation ◽  
Normal Human ◽  
Human Cardiac Tissue ◽  
End Stage

Recent study demonstrated that decrease signal transducer and activator of transcription-3 (STAT3) phosphorylation and increase apoptosis might be a critical point in the transition between compensatory cardiac hypertrophy and heart failure. To date, the protein expression of STAT3 in normal and failing human heart remains unclear. Therefore, the current study was designed to investigate the protein expression of STAT3 in human myocardium with end-stage congestive heart failure (CHF) and compared with that in normal human cardiac tissue.Human cardiac atrial tissue was obtained from normal subjects (n=5) and end-stage CHF patients (n=5) during cardiac transplantation. To detect the DNA fragmentation, in situ terminal deoxymucleotidyl transferase dUTP nick end labeling (TUNEL) was performed. An average of 1000 nuclei was analyzed for TUNEL study. STAT3 protein expression and phosphorylation of STAT3 were determined by immunohistochemical staining (IHCS) with total STAT3 and phospho-specific STAT3 antibodies.

scholarly journals Cardiac Troponins are Among Targets of Doxorubicin-Induced Cardiotoxicity in hiPCS-CMs

2019 ◽  
Vol 20 (11) ◽  
pp. 2638 ◽  
Author(s):  
Michaela Adamcova ◽  
Veronika Skarkova ◽  
Jitka Seifertova ◽  
Emil Rudolf
Keyword(s):  
Cardiac Troponin ◽  
Troponin I ◽  
Troponin T ◽  
Early Recognition ◽  
Morphological Changes ◽  
Cancer Therapeutics ◽  
Diagnostic Strategies ◽  
Human Cardiomyocytes ◽  
Induced Pluripotent ◽  
Cardiac Troponins

Modern diagnostic strategies for early recognition of cancer therapeutics-related cardiac dysfunction involve cardiac troponins measurement. Still, the role of other markers of cardiotoxicity is still unclear. The present study was designed to investigate dynamics of response of human cardiomyocytes derived from induced pluripotent stem cells (hiPCS-CMs) to doxorubicin with the special emphasis on their morphological changes in relation to expression and organization of troponins. The hiPCS-CMs were treated with doxorubicin concentrations (1 and 0.3 µM) for 48 h and followed for next up to 6 days. Exposure of hiPCS-CMs to 1 µM doxorubicininduced suppression of both cardiac troponin T (cTnT) and cardiac troponin I (cTnI) gene expression. Conversely, lower 0.3 µM doxorubicin concentration produced no significant changes in the expression of aforementioned genes. However, the intracellular topography, arrangement, and abundance of cardiac troponin proteins markedly changed after both doxorubicin concentrations. In particular, at 48 h of treatment, both cTnT and cTnI bundles started to reorganize, with some of them forming compacted shapes extending outwards and protruding outside the cells. At later intervals (72 h and onwards), the whole troponin network collapsed and became highly disorganized following, to some degree, overall changes in the cellular shape. Moreover, membrane permeability of cardiomyocytes was increased, and intracellular mitochondrial network rearranged and hypofunctional. Together, our results demonstrate complex effects of clinically relevant doxorubicin concentrations on hiPCS-CM cells including changes in cTnT and cTnI, but also in other cellular compartments contributing to the overall cytotoxicity of this class of cytostatics.

scholarly journals Atomic resolution probe for allostery in the regulatory thin filament

2016 ◽  
Vol 113 (12) ◽  
pp. 3257-3262 ◽  
Author(s):  
Michael R. Williams ◽  
Sarah J. Lehman ◽  
Jil C. Tardiff ◽  
Steven D. Schwartz
Keyword(s):  
Binding Site ◽  
Human Disease ◽  
Calcium Binding ◽  
Cardiac Troponin ◽  
Thin Filament ◽  
Troponin I ◽  
Troponin T ◽  
Calcium Binding Site

Calcium binding and dissociation within the cardiac thin filament (CTF) is a fundamental regulator of normal contraction and relaxation. Although the disruption of this complex, allosterically mediated process has long been implicated in human disease, the precise atomic-level mechanisms remain opaque, greatly hampering the development of novel targeted therapies. To address this question, we used a fully atomistic CTF model to test both Ca2+ binding strength and the energy required to remove Ca2+ from the N-lobe binding site in WT and mutant troponin complexes that have been linked to genetic cardiomyopathies. This computational approach is combined with measurements of in vitro Ca2+ dissociation rates in fully reconstituted WT and cardiac troponin T R92L and R92W thin filaments. These human disease mutations represent known substitutions at the same residue, reside at a significant distance from the calcium binding site in cardiac troponin C, and do not affect either the binding pocket affinity or EF-hand structure of the binding domain. Both have been shown to have significantly different effects on cardiac function in vivo. We now show that these mutations independently alter the interaction between the Ca2+ ion and cardiac troponin I subunit. This interaction is a previously unidentified mechanism, in which mutations in one protein of a complex indirectly affect a third via structural and dynamic changes in a second to yield a pathogenic change in thin filament function that results in mutation-specific disease states. We can now provide atom-level insight that is potentially highly actionable in drug design.

scholarly journals Length-dependent activation is modulated by cardiac troponin I bisphosphorylation at Ser23 and Ser24 but not by Thr143 phosphorylation

2014 ◽  
Vol 306 (8) ◽  
pp. H1171-H1181 ◽  
Author(s):  
Paul J. M. Wijnker ◽  
Vasco Sequeira ◽  
D. Brian Foster ◽  
Yuejin Li ◽  
Cristobal G. dos Remedios ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Cardiac Troponin ◽  
Troponin I ◽  
Isometric Force ◽  
Cardiac Troponin I ◽  
Human Cardiomyocytes ◽  
Kinase C ◽  
Myofilament Activation ◽  
Ventricular Filling ◽  
Length Dependent Activation

Frank-Starling's law reflects the ability of the heart to adjust the force of its contraction to changes in ventricular filling, a property based on length-dependent myofilament activation (LDA). The threonine at amino acid 143 of cardiac troponin I (cTnI) is prerequisite for the length-dependent increase in Ca2+sensitivity. Thr143 is a known target of protein kinase C (PKC) whose activity is increased in cardiac disease. Thr143 phosphorylation may modulate length-dependent myofilament activation in failing hearts. Therefore, we investigated if pseudo-phosphorylation at Thr143 modulates length dependence of force using troponin exchange experiments in human cardiomyocytes. In addition, we studied effects of protein kinase A (PKA)-mediated cTnI phosphorylation at Ser23/24, which has been reported to modulate LDA. Isometric force was measured at various Ca2+concentrations in membrane-permeabilized cardiomyocytes exchanged with recombinant wild-type (WT) troponin or troponin mutated at the PKC site Thr143 or Ser23/24 into aspartic acid (D) or alanine (A) to mimic phosphorylation and dephosphorylation, respectively. In troponin-exchanged donor cardiomyocytes experiments were repeated after incubation with exogenous PKA. Pseudo-phosphorylation of Thr143 increased myofilament Ca2+sensitivity compared with WT without affecting LDA in failing and donor cardiomyocytes. Subsequent PKA treatment enhanced the length-dependent shift in Ca2+sensitivity after WT and 143D exchange. Exchange with Ser23/24 variants demonstrated that pseudo-phosphorylation of both Ser23 and Ser24 is needed to enhance the length-dependent increase in Ca2+sensitivity. cTnI pseudo-phosphorylation did not alter length-dependent changes in maximal force. Thus phosphorylation at Thr143 enhances myofilament Ca2+sensitivity without affecting LDA, while Ser23/24 bisphosphorylation is needed to enhance the length-dependent increase in myofilament Ca2+sensitivity.

scholarly journals Pim-1 Kinase Phosphorylates Cardiac Troponin I and Regulates Cardiac Myofilament Function

2018 ◽  
Vol 45 (6) ◽  
pp. 2174-2186 ◽  
Author(s):  
Ni Zhu ◽  
Bing Yi ◽  
Zhifu Guo ◽  
Guanxin Zhang ◽  
Shengdong Huang ◽  
...  
Keyword(s):  
Growth Factor ◽  
Cardiac Troponin ◽  
Troponin I ◽  
Cardiac Troponin I ◽  
Site Directed Mutagenesis ◽  
Functional Assay ◽  
Protective Effects ◽  
Diabetic Mice ◽  
Myofilament Function ◽  
Cardiac Myofilament

Background/Aims: Pim-1 is a serine/threonine kinase that is highly expressed in the heart, and exerts potent cardiac protective effects through enhancing survival, proliferation, and regeneration of cardiomyocytes. Its myocardial specific substrates, however, remain unknown. In the present study, we aim to investigate whether Pim-1 modulates myofilament activity through phosphorylation of cardiac troponin I (cTnI), a key component in regulating myofilament function in the heart. Methods: Coimmunoprecipitation and immunofluorescent assays were employed to investigate the interaction of Pim-1 with cTnI in cardiomyocytes. Biochemical, site directed mutagenesis, and mass spectrometric analyses were utilized to identify the phosphorylation sites of Pim1 in cTnI. Myofilament functional assay using skinned cardiac fiber was used to assess the effect of Pim1-mediated phosphorylation on cardiac myofilament activity. Lastly, the functional significance of Pim1-mediated cTnI in heart disease was determined in diabetic mice. Results: We found that Pim-1 specifically interacts with cTnI in cardiomyocytes and this interaction leads to Pim1-mediated cTnI phosphorylation, predominantly at Ser23/24 and Ser150. Furthermore, our functional assay demonstrated that Pim-1 induces a robust phosphorylation of cTnI within the troponin complex, thus leading to a decreased Ca2+ sensitivity. Insulin-like growth factor 1 (IGF-1), a peptide growth factor that has been shown to stimulate myocardial contractility, markedly induces cTnI phosphorylation at Ser23/24 and Ser150 through increasing Pim-1 expression in cardiomyocytes. In a high-fat diabetic mice model, the expression of Pim1 in the heart is significantly decreased, which is accompanied by a decreased phosphorylation of cTnI at Ser23/24 and Ser150, further implicating the pathological significance of the Pim1/cTnI axis in the development of diabetic cardiomyopathy. Conclusion: Our results demonstrate that Pim-1 is a novel kinase that phosphorylates cTnI primarily at Ser23/24 and Ser150 in cardiomyocytes, which in turn may modulate myofilament function under a variety of physiological and pathophysiological conditions.

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