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Pólos Miklós

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Kellermayer MIklós
Truncated titin is integrated into the human dilated cardiomyopathic sarcomere

Aug 29 - kedd

08:30 – 08:50

Molekuláris biofizika

E01

Truncated titin is integrated into the human dilated cardiomyopathic sarcomere

Dalma Kellermayer^1,2,3,$, Hedvig Tordai^2,$, Balázs Kiss², György Török², Dániel M. Péter², Alex Ali Sayour¹, Miklós Pólos¹, István Hartyánszky¹, Bálint Szilveszter¹, Siegfried Labeit⁴, Ambrus Gángó³, Gábor Bedics³, Csaba Bödör³, Tamás Radovits¹, Béla Merkely¹ and Miklós S.Z. Kellermayer²

¹Heart and Vascular Center, Semmelweis University, Budapest, Hungary

²Department of Biophysics and Radiation Biology, Semmelweis University, Budapest, Hungary

³1st Department of Pathology and Experimental Cancer Research, Semmelweis University, Budapest, Hungary

⁴DZHK Partnersite Mannheim-Heidelberg, Medical Faculty Mannheim, University of Heidelberg, Mannheim, Germany

Heteroyzgous truncating mutations in the TTN gene (TTNtv) encoding the giant protein titin are the most common genetic cause of dilated cardiomyopathy (DCM). However, the molecular mechanisms by which TTNtv mutations induce DCM are controversial. Here we investigated 118 clinically identified DCM human cardiac samples with next-generation sequencing, high-resolution gel electrophoresis, Western blot analysis and super-resolution microscopy in order to dissect the structural and functional consequences of TTNtv mutations. The occurrence of TTNtv was found to be 15% in the DCM cohort. Truncated titin proteins matching, by molecular weight, the gene-sequence predictions were detected in the majority of the TTNtv samples. The total amount of expressed titin, which includes the truncated fragments, was comparable in the TTNtv+ and TTNtv- samples, indicating that titin haploinsufficiency may not be the leading cause of the molecular pathogenesis. Proteomic analysis of washed cardiac myofibrils and STED super-resolution microscopy of myocardial sarcomeres labeled with sequence-specific anti-titin antibodies revealed that truncated titin is structurally integrated in the sarcomere. Sarcomere length-dependent anti-titin epitope position, shape and intensity analysis pointed at structural disarrangements in the I/A junction and the M-band of TTNtv+ sarcomeres, which may play a role, via faulty mechanosensor function, in the development of manifest DCM.

Acknowledgments

This research was funded by the ÚNKP-19-3-I New National Excellence Program of The Ministry for Innovation and Technology to D.K. and grants from the Hungarian National Research, Development and Innovation Office (K135360 to M.K., FK135462 to B.K., K135076 to B.M., Project no. NVKP_16-1–2016-0017 ’National Heart Program’, and the 2020-1.1.6-JÖVŐ-2021-00013 grant) and the Thematic Excellence Programme (2020-4.1.1.-TKP2020) of the Ministry for Innovation and Technology in Hungary, within the framework of the Therapeutic Development and Bioimaging thematic programs of Semmelweis University.

Sulea Cristina
Fibrillin-1 microfibrils in Marfan syndrome: nanoscale structural characterization using atomic force microscopy

Aug 29 - kedd

15:30 – 17:00

I. Poszterszekció

P27

Fibrillin-1 microfibrils in Marfan syndrome: nanoscale structural characterization using atomic force microscopy

Cristina M. Șulea^1,2,3, Zsolt Mártonfalvi¹, Csilla Csányi¹, Dóra Haluszka¹, Miklós Pólos^2,3, Kálmán Benke^2,3, Zoltán Szabolcs^2,3 and Miklós S. Z. Kellermayer¹

¹ Department of Biophysics and Radiation Biology, Semmelweis University, 1094 Budapest, Hungary

² Heart and Vascular Center, Semmelweis University, 1122 Budapest, Hungary

³ Hungarian Marfan Foundation, 1122 Budapest, Hungary

Fibrillin-1 microfibrils are essential elements of the extracellular matrix serving as a scaffold for the deposition of elastin and endowing connective tissues with tensile strength and elasticity. Mutations in the fibrillin-1 gene (FBN1) are linked to Marfan syndrome (MFS), a systemic connective tissue disorder that usually manifests in life-threatening aortic complications. The aortic involvement may be explained by a dysregulation in microfibrillar function and, conceivably, alterations in the microfibrils’ supramolecular structure.

The aim of the study was to perform a nanoscale structural characterization of fibrillin-1 microfibrils isolated from human aortic samples with different FBN1 gene mutations and to compare them with microfibrillar assemblies purified from non-MFS human aortic tissue.

Aortic wall samples were obtained from patients undergoing specific cardiovascular surgical interventions. Fibrillin-rich microfibrils were extracted by bacterial collagenase digestion and purified by size-exclusion chromatography. Atomic force microscopy was employed to visualize and study the microfibrillar assemblies.

Fibrillin-1 microfibrils displayed a characteristic “beads-on-a-string” appearance. The microfibrillar assemblies were investigated for bead geometry (height, length, and width), interbead region height, and periodicity. MFS fibrillin-1 microfibrils had a slightly higher mean bead height, but the bead length and width, as well as the interbead height, were significantly smaller in the MFS group. The mean periodicity varied around 50–52 nm among samples.

In conclusion, the data suggest an overall thinner and presumably more frail structure for the MFS fibrillin-1 microfibrils, which may play a role in the development of MFS-specific aortic symptomatology.

Acknowledgment

Funding sources: NRDI Office (ÚNKP-22-3-I-SE-49 to C.M.Ș.; K135360 to M.S.Z.K.; TKP2021-EGA-23), European Union (RRF-2.3.1-21-2022-00003 – National Cardiovascular Laboratory).