Sebastian Kötter
University of Düsseldorf
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Featured researches published by Sebastian Kötter.
Circulation Research | 2008
Martina Krüger; Sebastian Kötter; Anika Grützner; Patrick Lang; Christian Andresen; Margaret M. Redfield; Elke Butt; Cris dos Remedios; Wolfgang A. Linke
The sarcomeric titin springs influence myocardial distensibility and passive stiffness. Titin isoform composition and protein kinase (PK)A-dependent titin phosphorylation are variables contributing to diastolic heart function. However, diastolic tone, relaxation speed, and left ventricular extensibility are also altered by PKG activation. We used back-phosphorylation assays to determine whether PKG can phosphorylate titin and affect titin-based stiffness in skinned myofibers and isolated myofibrils. PKG in the presence of 8-pCPT-cGMP (cGMP) phosphorylated the 2 main cardiac titin isoforms, N2BA and N2B, in human and canine left ventricles. In human myofibers/myofibrils dephosphorylated before mechanical analysis, passive stiffness dropped 10% to 20% on application of cGMP-PKG. Autoradiography and anti-phosphoserine blotting of recombinant human I-band titin domains established that PKG phosphorylates the N2-B and N2-A domains of titin. Using site-directed mutagenesis, serine residue S469 near the COOH terminus of the cardiac N2-B–unique sequence (N2-Bus) was identified as a PKG and PKA phosphorylation site. To address the mechanism of the PKG effect on titin stiffness, single-molecule atomic force microscopy force–extension experiments were performed on engineered N2-Bus–containing constructs. The presence of cGMP-PKG increased the bending rigidity of the N2-Bus to a degree that explained the overall PKG-mediated decrease in cardiomyofibrillar stiffness. Thus, the mechanically relevant site of PKG-induced titin phosphorylation is most likely in the N2-Bus; phosphorylation of other titin sites could affect protein–protein interactions. The results suggest that reducing titin stiffness by PKG-dependent phosphorylation of the N2-Bus can benefit diastolic function. Failing human hearts revealed a deficit for basal titin phosphorylation compared to donor hearts, which may contribute to diastolic dysfunction in heart failure.
Biophysical Journal | 2009
Anika Grützner; Sergi Garcia-Manyes; Sebastian Kötter; Carmen L. Badilla; Julio M. Fernandez; Wolfgang A. Linke
The giant protein titin is responsible for the elasticity of nonactivated muscle sarcomeres. Titin-based passive stiffness in myocardium is modulated by titin-isoform switching and protein-kinase (PK)A- or PKG-dependent titin phosphorylation. Additional modulatory effects on titin stiffness may arise from disulfide bonding under oxidant stress, as many immunoglobulin-like (Ig-)domains in titins spring region have a potential for S-S formation. Using single-molecule atomic force microscopy (AFM) force-extension measurements on recombinant Ig-domain polyprotein constructs, we show that titin Ig-modules contain no stabilizing disulfide bridge, contrary to previous belief. However, we demonstrate that the human N2-B-unique sequence (N2-B(us)), a cardiac-specific, physiologically extensible titin segment comprising 572 amino-acid residues, contains up to three disulfide bridges under oxidizing conditions. AFM force spectroscopy on recombinant N2-B(us) molecules demonstrated a much shorter contour length in the absence of a reducing agent than in its presence, consistent with intramolecular S-S bonding. In stretch experiments on isolated human heart myofibrils, the reducing agent thioredoxin lowered titin-based stiffness to a degree that could be explained (using entropic elasticity theory) by altered extensibility solely of the N2-B(us). We conclude that increased oxidant stress can elevate titin-based stiffness of cardiomyocytes, which may contribute to the global myocardial stiffening frequently seen in the aging or failing heart.
Circulation Research | 2010
Ralph Knöll; Sawa Kostin; Stefanie Klede; K. Savvatis; Lars Klinge; Ina Stehle; Sylvia Gunkel; Sebastian Kötter; Kamila Babicz; Melanie Sohns; Snjezana Miocic; Michael Didié; Gudrun Knöll; Wolfram-Hubertus Zimmermann; Paul Thelen; Heike Bickeböller; Lars S. Maier; Wolfgang Schaper; Jutta Schaper; Theresia Kraft; Carsten Tschöpe; Wolfgang A. Linke; Kenneth R. Chien
Rationale: We previously discovered the human 10T→C (Trp4Arg) missense mutation in exon 2 of the muscle LIM protein (MLP, CSRP3) gene. Objective: We sought to study the effects of this single-nucleotide polymorphism in the in vivo situation. Methods and Results: We now report the generation and detailed analysis of the corresponding MlpW4R/+ and MlpW4R/W4R knock-in animals, which develop an age- and gene dosage–dependent hypertrophic cardiomyopathy and heart failure phenotype, characterized by almost complete loss of contractile reserve under catecholamine induced stress. In addition, evidence for skeletal muscle pathology, which might have implications for human mutation carriers, was observed. Importantly, we found significantly reduced MLP mRNA and MLP protein expression levels in hearts of heterozygous and homozygous W4R-MLP knock-in animals. We also detected a weaker in vitro interaction of telethonin with W4R-MLP than with wild-type MLP. These alterations may contribute to an increased nuclear localization of W4R-MLP, which was observed by immunohistochemistry. Conclusions: Given the well-known high frequency of this mutation in Caucasians of up to 1%, our data suggest that W4R-MLP might contribute significantly to human cardiovascular disease.
Cardiovascular Research | 2013
Sebastian Kötter; Laurence Gout; Marion von Frieling-Salewsky; Anna Eliane Müller; Stefan Helling; Katrin Marcus; Cristobal G. dos Remedios; Wolfgang A. Linke; Martina Krüger
AIMS Titin-based myofilament stiffness is defined by the expression levels of the cardiac titin-isoforms, N2B and N2BA, and by phosphorylation of the elastic titin domains N2-B unique sequence (N2-Bus) and PEVK. Phosphorylation of the N2-Bus by cGMP-dependent protein kinase (PKG) or cAMP-dependent protein kinase (PKA) decreases titin stiffness, whereas phosphorylation of the PEVK-domain by PKC increases it. We aimed to identify specific sites within the N2-Bus phosphorylated by PKA and PKG and to determine whether differential changes in titin domain phosphorylation could affect passive stiffness in human failing hearts. METHODS AND RESULTS Using mass spectrometry, we identified seven partly conserved PKA/PKG-targeted phosphorylation motifs in human and rat N2-Bus. Polyclonal antibodies to pSer4185, pSer4010, and pSer4099 in the N2-Bus, and to pSer11878 in the PEVK-region were used to quantify titin-domain phosphorylation by western blot analyses of a set of human donor and failing hearts with similar titin-isoform composition. Passive tension determined in skinned human myocardial fibre preparations was significantly increased in failing compared with donor hearts, notably at shorter sarcomere lengths where titin contributes most to total passive tension. Phosphorylation of Ser4185, Ser4010, and Ser4099 in the N2-Bus was significantly reduced in failing hearts, whereas phosphorylation of Ser11878 in the PEVK-region was increased compared with donor hearts. CONCLUSION We conclude that hypo-phosphorylation of the N2-Bus and hyper-phosphorylation of the PEVK domain can act complementary to elevate passive tension in failing human hearts. Differential changes in titin-domain phosphorylation may be important to fine-tune passive myocardial stiffness and diastolic function of the heart.
Frontiers in Physiology | 2016
Martina Krüger; Sebastian Kötter
Titin is a giant scaffold protein with multiple functions in striated muscle physiology. Due to the elastic I-band domains and the filament-like integration in the half-sarcomere titin is an important factor for sarcomere assembly and serves as an adaptable molecular spring that determines myofilament distensibility. Protein-interactions e.g., with muscle ankyrin repeat proteins or muscle LIM-protein link titin to hypertrophic signaling and via p62 and Muscle Ring Finger proteins to mechanisms that control protein quality control. This review summarizes our current knowledge on titin as a central node for exercise-induced mechanosignaling and remodeling and further highlights the pathophysiological implications.
Biological Chemistry | 2014
Sebastian Kötter; Christian Andresen; Martina Krüger
Abstract The giant sarcomeric protein titin has multiple important functions in striated muscle cells. Due to its gigantic size, its central position in the sarcomere and its elastic I-band domains, titin is a scaffold protein that is important for sarcomere assembly, and serves as a molecular spring that defines myofilament distensibility. This review focuses on the emerging role of titin in mechanosensing and hypertrophic signaling, and further highlights recent evidence that links titin to sarcomeric protein turnover.
Frontiers in Physiology | 2014
Anna Eliane Müller; Matthias Kreiner; Sebastian Kötter; Philipp Lassak; Wilhelm Bloch; Frank Suhr; Martina Krüger
Titin-based myofilament stiffness is largely modulated by phosphorylation of its elastic I-band regions N2-Bus (decreases passive stiffness, PT) and PEVK (increases PT). Here, we tested the hypothesis that acute exercise changes titin phosphorylation and modifies myofilament stiffness. Adult rats were exercised on a treadmill for 15 min, untrained animals served as controls. Titin phosphorylation was determined by Western blot analysis using phosphospecific antibodies to Ser4099 and Ser4010 in the N2-Bus region (PKG and PKA-dependent. respectively), and to Ser11878 and Ser 12022 in the PEVK region (PKCα and CaMKIIδ-dependent, respectively). Passive tension was determined by step-wise stretching of isolated skinned cardiomyocytes to sarcomere length (SL) ranging from 1.9 to 2.4 μm and showed a significantly increased PT from exercised samples, compared to controls. In cardiac samples titin N2-Bus phosphorylation was significantly decreased by 40% at Ser4099, however, no significant changes were observed at Ser4010. PEVK phosphorylation at Ser11878 was significantly increased, which is probably mediated by the observed exercise-induced increase in PKCα activity. Interestingly, relative phosphorylation of Ser12022 was substantially decreased in the exercised samples. Surprisingly, in skeletal samples from acutely exercised animals we detected a significant decrease in PEVK phosphorylation at Ser11878 and an increase in Ser12022 phosphorylation; however, PKCα activity remained unchanged. In summary, our data show that a single exercise bout of 15 min affects titin domain phosphorylation and titin-based myocyte stiffness with obviously divergent effects in cardiac and skeletal muscle tissues. The observed changes in titin stiffness could play an important role in adapting the passive and active properties of the myocardium and the skeletal muscle to increased physical activity.
PLOS ONE | 2013
Katharina Bottermann; Michael Reinartz; Marian Barsoum; Sebastian Kötter; Axel Gödecke
AKT2 is one of the three isoforms of the protein kinase AKT being involved in the modulation of cellular metabolism. Since protein-protein interactions are one possibility to convey specificity in signal transduction, we performed AKT2-protein interaction analysis to elucidate their relevance for AKT2-dependent cellular functions. We identified heat shock protein 90 kDa (HSP90), Cdc37, heat shock protein 70 kDa (HSP70), 78 kDa glucose regulated protein (GRP78), tubulin, GAPDH, α-enolase and elongation factor 2 (EF2) as AKT2-interacting proteins by a combination of tandem affinity purification and mass spectrometry in HEK293T cells. Quantitative MS-analysis using stable isotope labeling by amino acids in cell culture (SILAC) revealed that only HSP90 and Cdc37 interact stably with AKT2, whereas the other proteins interact with low affinity with AKT2. The interactions of AKT2 with α-enolase and EF2 were further analyzed in order to uncover the functional relevance of these newly discovered binding partners. Despite the interaction of AKT2 and α-enolase, which was additionally validated by proximity ligation assay (PLA), no significant impact of AKT on α-enolase activity was detected in activity measurements. AKT stimulation via insulin and/or inhibition with the ATP-competitive inhibitor CCT128930 did not alter enzymatic activity of α-enolase. Interestingly, the direct interaction of AKT2 and EF2 was found to be dynamically regulated in embryonic rat cardiomyocytes. Treatment with the PI3-kinase inhibitor LY294002 before stimulation with several hormones stabilized the complex, whereas stimulation alone led to complex dissociation which was analyzed in situ with PLA. Taken together, these findings point to new aspects of AKT2-mediated signal transduction in protein synthesis and glucose metabolism.
Circulation Research | 2016
Sebastian Kötter; Malgorzata Kazmierowska; Christian Andresen; Katharina Bottermann; Maria Grandoch; Simone Gorressen; André Heinen; Jens M. Moll; Jürgen Scheller; Axel Gödecke; Jens W. Fischer; Joachim P. Schmitt; Martina Krüger
RATIONALE Myocardial infarction (MI) increases the wall stress in the viable myocardium and initiates early adaptive remodeling in the left ventricle to maintain cardiac output. Later remodeling processes include fibrotic reorganization that eventually leads to cardiac failure. Understanding the mechanisms that support cardiac function in the early phase post MI and identifying the processes that initiate transition to maladaptive remodeling are of major clinical interest. OBJECTIVE To characterize MI-induced changes in titin-based cardiac myocyte stiffness and to elucidate the role of titin in ventricular remodeling of remote myocardium in the early phase after MI. METHODS AND RESULTS Titin properties were analyzed in Langendorff-perfused mouse hearts after 20-minute ischemia/60-minute reperfusion (I/R), and mouse hearts that underwent ligature of the left anterior descending coronary artery for 3 or 10 days. Cardiac myocyte passive tension was significantly increased 1 hour after ischemia/reperfusion and 3 and 10 days after left anterior descending coronary artery ligature. The increased passive tension was caused by hypophosphorylation of the titin N2-B unique sequence and hyperphosphorylation of the PEVK (titin domain rich in proline, glutamate, valine, and lysine) region of titin. Blocking of interleukine-6 before left anterior descending coronary artery ligature restored titin-based myocyte tension after MI, suggesting that MI-induced titin stiffening is mediated by elevated levels of the cytokine interleukine-6. We further demonstrate that the early remodeling processes 3 days after MI involve accelerated titin turnover by the ubiquitin-proteasome system. CONCLUSIONS We conclude that titin-based cardiac myocyte stiffening acutely after MI is partly mediated by interleukine-6 and is an important mechanism of remote myocardium to adapt to the increased mechanical demands after myocardial injury.
Circulation Research | 2018
Anna-Eliane Hopf; Christian Andresen; Sebastian Kötter; Malgorzata Isić; Kamila Ulrich; Senem Sahin; Sabine Bongardt; Wilhelm Roell; Felicitas Drove; Nina Scheerer; Leni Vandekerckhove; Gilles W. De Keulenaer; Nazha Hamdani; Wolfgang A. Linke; Martina Krüger
Rationale: Increased titin-dependent cardiomyocyte tension is a hallmark of heart failure with preserved ejection fraction associated with type-2 diabetes mellitus. However, the insulin-related signaling pathways that modify titin-based cardiomyocyte tension, thereby contributing to modulation of diastolic function, are largely unknown. Objective: We aimed to determine how impaired insulin signaling affects titin expression and phosphorylation and thus increases passive cardiomyocyte tension, and whether metformin or neuregulin-1 (NRG-1) can correct disturbed titin modifications and increased titin-based stiffness. Methods and Results: We used cardiac biopsies from human diabetic (n=23) and nondiabetic patients (n=19), cultured rat cardiomyocytes, left ventricular tissue from apolipoprotein E–deficient mice with streptozotocin-induced diabetes mellitus (n=12–22), and ZSF1 (obese diabetic Zucker fatty/spontaneously hypertensive heart failure F1 hybrid) rats (n=5–6) and analyzed insulin-dependent signaling pathways that modulate titin phosphorylation. Titin-based passive tension was measured using permeabilized cardiomyocytes. In human diabetic hearts, we detected titin hypophosphorylation at S4099 and hyperphosphorylation at S11878, suggesting altered activity of protein kinases; cardiomyocyte passive tension was significantly increased. When applied to cultured cardiomyocytes, insulin and metformin increased titin phosphorylation at S4010, S4099, and S11878 via enhanced ERK1/2 (extracellular signal regulated kinase 1/2) and PKC&agr; (protein kinase C&agr;) activity; NRG-1 application enhanced ERK1/2 activity but reduced PKC&agr; activity. In apolipoprotein E–deficient mice, chronic treatment of streptozotocin-induced diabetes mellitus with NRG-1 corrected titin phosphorylation via increased PKG (protein kinase G) and ERK1/2 activity and reduced PKC&agr; activity, which reversed the diabetes mellitus–associated changes in titin-based passive tension. Acute application of NRG-1 to obese ZSF1 rats with type-2 diabetes mellitus reduced end-diastolic pressure. Conclusions: Mechanistically, we found that impaired cGMP–PKG signaling and elevated PKC&agr; activity are key modulators of titin-based cardiomyocyte stiffening in diabetic hearts. We conclude that by restoring normal kinase activities of PKG, ERK1/2, and PKC&agr;, and by reducing cardiomyocyte passive tension, chronic NRG-1 application is a promising approach to modulate titin properties in heart failure with preserved ejection fraction associated with type-2 diabetes mellitus.