Medizinische Hochschule Hannover : Group Montag

AG Montag

Unequal allelic expression (from cell to cell) as a pathomechanism for Hypertrophic Cardiomyopathy

Contact:

PD Dr. rer. nat. Judith Montag

Adress: MHH, Institut für Molekular- und Zellphysiologie
Carl-Neuberg-Straße 1, 30625 Hannover, Germany
Campus, Building J03, Block 01, Floor 03, Room 1340

Telephone    (+49) 511 532 2094
Fax            (+49) 511 532 4296
E-Mail      PD Dr. rer. nat. Judith Montag

Research focus

We examine the unequal expression of wildtype and mutant alleles in heterozygous patients with hypertrophic cardiomyopathy (HMC) as a potenial pathomechanism.

In previous work in our department, we could show that force generation differs severely between cardiomyocytes from the very same patient at the same calcium concentration. We hypothesized that this functional heterogeneity between neighboring cardiomyocytes may disrupt the myocardial network. This may lead to hallmarks of HCM such as cardiomyocyte and myofibrillar disarray, hypertrophy and fibrosis („contractile imbalance hypothesis”).

The special focus of our research group are the molecular mechanism underlying the contractile imbalance. We analyse the allele specific mRNA expression in single cardiomyocytes from cryosections of HCM patients and heart-healthy controls and in human induced pluripotent stem cells. We could show that individual cardiomyocytes express highly divergent fraction of mutant per wildtype mRNA. In addition, we visualize active transcription sites in individual nuclei to examine whether burst-like transcription, a stochastic and independent on and off switch of the alleles, may cause the allelic imbalance from cell to cell. To enable a longitudinal analysis of contractile imbalance and the underlying mechanisms and to test potential therapeutic approaches, we apply CRIPSR/Cas9 to to generate a genome edited pig model for HCM.

Current projects

Current projects

Unequal allelic expression in heterozygous patients as pathomechanism for Hypertrophic Cardiomyopathy
In previous work in our department, we could show that force generation differs severely between cardiomyocytes from the very same patient at the same calcium concentration. We hypothesized that this functional heterogeneity between neighboring cardiomyocytes may disrupt the myocardial network. This may lead to hallmarks of HCM such as cardiomyocyte and myofibrillar disarray, hypertrophy and fibrosis („contractile imbalance hypothesis”).

In our working group we provided evidence that individual cardiomyocytes express highly divergent fractions of mutant per wildtype mRNA. We have isolated single cardiomyocytes from cryosections of flash frozen tissue from HCM patients by laser microdissection and analyzed them using allele-specific single cell RT-PCR. We determined unequal allelic expression of both alleles from cell to cell for ß-cardiac myosin (MYH7) and cardiac troponin I (TNNI3). Some cells expressed essentially only wildtype mRNA, others almost only mutant mRNA and others different fractions of both alleles. Currently we extend our analysis for human induced pluripotent stem cells.

Burst-like transcription of HCM-genes

Our working group has shown that alleles of HCM-associated genes are transcribed burst-like. Using single molecule RNA fluorescence in situ hybridisation (smRNA-FISH) we determined nuclei with and without active transcription in the same tissue section in HCM-patients and in donor controls. This suggests that both alleles are switched on and off stochastically and independent from each other, indicating burst-like transcription.

Ongoing projects focus on the generation of cell culture systems for in vivo visualization of active transcription in order to monitor and modulate burst kinetics. In addition, we aim to differentiate mutant and wildtype active transcription sites.

In a ERA-CVD funded project (SCALE) we examine which influence allelic imbalance has on gene expression and whether a differential modification of histones plays a role in activation of each allele using hPSC-CMs with a mutation in MYH7. We will perform single cell RNA-Seq and RT-qPCR analysis.

Genome edited pig as large animal model for Hypertrophic Cardiomyopathy

For longitudinal analysis of disease development with a special focus on the progression of contractile imbalance and for testing of potential therapeutic approaches we aim to generate a large animal model for HCM. Pig’s cardiophysiology is similar to humans and they express mainly the b-MyHC isoform in the ventricles. Therefore we use designer nucleases (TALEN, CRISPR/Cas9) to introduce specific point mutations into the genome of porcine fibroblasts. Cells heterozygous for point mutations are subsequently used to clone genome edited pigs (in cooperation with the Friedrich-Löffler-Institute, Mariensee). We successfully generated pigs heterozygous for mutation R723G in MYH7. Neonatal animals showed mild symptoms of HCM but died within 24 h after birth.

Currently we use CRISPR/Cas9 to introduce different HCM-point mutations in porcine fibroblasts to generate genome edited pigs.

What causes allelic imbalance at tissue level?

If allelic expression was dominated by stochastic (burst-like) transcription only we would expect an average ratio of 50:50 of mRNA from both alleles. However, we could show for several HCM-mutations that patients express fractions that deviate from this 50:50 ratio at mRNA and at protein level. Interestingly, the relative fractions of mutant mRNA and protein was almost always comparable between patients with the same mutation. E.g. all patients with mutation R723G express on average 68% mutant and 32% wildtype allele. Thus, additional mechanisms besides burst-like transcription must shift the allelic ratio towards one of the alleles. For R723G we could show that the mutation alters the mRNA secondary structure. This might increase the transcription rate of the mutant allele or stabilize the mRNA and decrease the degradation rate. These hypotheses are currently tested in cell culture models.

Allelic Imbalance as possible pathomechanism in PLN-cardiomyopathies

In a further project, we address the question whether allelic imbalance influences pathology of cardiomyopathies in patients with a deletion mutation (R14del) in the phospholamban gene PLN. Patients with this mutation show highly variable disease progression, ranging from asymptomatic mutation carriers to severe heart failure. We will examine whether these patients provide a differential allelic imbalance and whether increased expression of the mutated allele is associated with more severe functional alterations.

Influence of aipositas and aging on heterogeneity among cardiomyocytes

In collaboration with PD Dr. Julia Schipke (Funktionelle und angewandte Anatomie, MHH) we address the influence of aging and adipositas on cardiomyocytes in a mouse modell. A special focus lies on the heterogeneity among cardiomyocytes as potential mechanism underlying cardiovascular diseases upon aging and adipositas.

Methods

Laser microdissection
(Single cell-) Reverse Transcriptase PCR (RT-PCR)
single molecule RNA-fluorescence in situ hybridisation (smRNA-FISH)
Genome editing, knock-in of a point mutation (TALEN, CRISPR/Cas9)
cell culture (HeLa, AC16)
large animal model

Publications

2020

Stochastic allelic expression as trigger for contractile imbalance in hypertrophic cardiomyopathy. Montag J, Kraft T. Biophysical Reviews. 2020 July Epub ahead of print. Review
Advanced Single-Cell Mapping Reveals that in hESC Cardiomyocytes Contraction Kinetics and Action Potential Are Independent of Myosin Isoform. Weber N*, Kowalski K*, Holler T, Radocaj A, Fischer F, Thiemann S, de la Roche J, Schwanke K, Piep B, Peschel N, Krumm U, Lingk A, Wendland M, Greten S, Schmitto JD, Ismail I, Warnecke G, Zywietz U, Chichkov B, Meißner J, Haverich A, Martin U, Brenner B, Zweigerdt R, Kraft T. Stem Cell Reports. 2020 May 12;14(5):788-802 PubMed
Hypertrophic cardiomyopathy MYH7-mutation R723G alters mRNA secondary structure. Rose J, Kraft T, Brenner B, Montag J. Physiol Genomics. 2020 Jan 1;52(1):15-19

2019

Altered force generation and cell-to-cell contractile imbalance in hypertrophic cardiomyopathy; Kraft T, Montag J; Pflügers Arch: 471(5):719-733; 2019 May; doi: 10.1007/s00424-019-02260-9; Review

2018

Efficient Knock-in of a Point Mutation in Porcine Fibroblasts Using the CRISPR/Cas9-GMNN Fusion Gene; Gerlach M, Kraft T, Brenner B, Petersen B, Niemann H, Montag J.. Genes (Basel):13;9(6); 2018 Jun
Burst-like transcription of mutant and wildtype MYH7-alleles as possible origin of cell-to-cell contractile imbalance in hypertrophic cardiomyopathy; Montag J, Kowalski K, Makul M, Ernstberger P, Radocaj A, Beck J, Becker E, Tripathi S, Keyser B, Mühlfeld C, Wissel K, Pich A, van der Velden J, dos Remedios CG, Perrot A, Francino A, Navarro-López F, Brenner B, Kraft T; Front Physiol: 9:359. 2018 Apr
Successful knock-in of Hypertrophic Cardiomyopathy-mutation R723G into the MYH7 gene mimics HCM pathology in pigs; Montag J, Petersen B, Lucas-Hahn A, Becker E, Harries D, Niemeier H, Brenner B, Kraft T; Sci Rep: 8(1):4786; 2018 Mar

2017

Intrinsic MYH7 expression regulation contributes to tissue level allelic imbalance in hypertrophic cardiomyopathy; Montag J, Syring M, Rose J, Weber AL, Ernstberger P, Mayer AK, Becker E, Keyser B, Dos Remedios C, Perrot A, van der Velden J, Francino A, Navarro-Lopez F, Ho CY, Brenner B, Kraft T; J Muscle Res Cell Motil: 38 (3-4):291-302; 2017 Aug

2016

Kraft T, Montag J, Radocaj A, Brenner B. Hypertrophic Cardiomyopathy: Cell-to-Cell Imbalance in Gene Expression and Contraction Force as Trigger for Disease Phenotype Development; Kraft T, Montag J, Radocaj A, Brenner B; Circ Res: 119(9):992-995; 2016 Oct

2014

Familial hypertrophic cardiomyopathy: functional variance among individual cardiomyocytes as a trigger of FHC-phenotype development; Brenner B, Seebohm B, Tripathi S, Montag J, Kraft T; Front Physiol: 5:392; 2014 Oct
Faster cross-bridge detachment and increased tension cost in human hypertrophic cardiomyopathy with the R403Q MYH7 mutation. Witjas-Paalberends ER, Ferrara C, Scellini B, Piroddi N, Montag J, Tesi C, . Stienen GJM, Michels M, Ho CY, Kraft T, Poggesi C,van der Velden J; J Physiol: 592(Pt 15):3257-72; 2014 Aug
Gene expression profiling of brains from bovine spongiform encephalopathy (BSE)-infected cynomolgus macaques; Barbisin M, Vanni S, Schmädicke AC, Montag J, Motzkus D, Opitz L, Salinas-Riester G, Legname G; BMC Genomics:15:434, 2014 Jun

2013

Mapping the Small RNA Content of Simian Immunodeficiency Virions (SIV); Brameier M, Ibing W, Höfer K, Montag J, Stahl-Hennig C, Motzkus D; PLoS One: 8(9): e75063; 2013 Sept
Asynchronous onset of prion disease in bovine spongiform encephalopathy (BSE) infected macaques; Montag J, Schulz-Schäffer W, Schrod A, Hunsmann G, Motzkus D; Emerg Infect Dis: 19(7):1125-7;2013 Jul
Familial Hypertrophic Cardiomyopathy: functional effects of myosin mutation R723G in cardiomyocytes; Kraft T, Paalberends ER, Boontje N, Tripathi S, Brandis A, Montag J, Francino A, Navarro-Lopez F, Brenner B, Stienen GJM, van der Velden J; J Mol Cell Cardiol: 57:13-22; 2013 Jan

2012

A genome-wide survey for prion-regulated miRNAs associated with cholesterol homeostasis; Montag J, Brameier M, Schmädicke AC, Gilch S, Schätzl HM, Motzkus D; BMC Genomics: 13:486; 2012 Sep

2011

Unequal allelic expression of wild-type and mutated beta-myosin in familial hypertrophic cardiomyopathy; Tripathi S, Schultz I, Becker E, Montag J, Borchert B, Francino A, Navarro-Lopez F, Perrot A, Özcelik C, Osterziel KJ, McKenna WJ, Brenner B, Kraft T; Basic Res Cardiol; 106(6):1041-55; 2011 Nov
Bovine spongiform encephalopathy infection alters endogenous retrovirus expression in distinct brain regions of cynomolgus macaques (Macaca fascicularis), Greenwood AD, Vincendeau M, Schmädicke AC, Montag J, Seifarth W, Motzkus D. Mol Neurodegener: 6(1):44; 2011 Jun

2009

Upregulation of miRNA hsa-miR-342-3p in experimental and idiopathic prion disease; Montag J, Hitt R, Opitz L, Schulz-Schaeffer WJ, Hunsmann G, Motzkus D; Mol Neurodegener:4:36, 2009 Aug

Teaching

Practical courses for medical students, dentists and biologists
Seminars for medical students
laboratory projects for biomedical master students
Bachelor theses in Biology and Biochemistry
PhD theses in Biology and Medicine

Graduation theses

current:

PhD theses

Dr. rer. nat.

Valentin Burkart

Dr. med. (StrucMed-Program)

Felix Wilczak

completed:

PhD theses

Dr. rer. nat.

Kathrin Kowalski (2020)

Anne-Kathrin Mayer (2020)

Dr. med.

Anna-Lena Weber (2019)

Pia Ernstberger (2020)

Bachelor theses:

Maren Kestner (2012)

Stella Louise Reich (2015)

Anna Flögel (2016)

Max Gerlach (2017)

Ilario Kosoburd (2018)

Jirka Baustian (2018)

Dorina Lang (2019)

Group members

Group members:

Dr. rer. nat. Valentin Burkart

M.Sc. Alina Disch

Dr. rer. nat. Kathrin Kowalski

Philemon Libertus (StrukMed)

Benita Haß (StrukMed)

Sarah Menz

Karina Schlosser

FWJler:

Ilka Anja Kröber

Publication

2022

Transcriptional bursts and heterogeneity among cardiomyocytes in hypertrophic cardiomyopathy, Valentin Burkart, Kathrin Kowalski, David Aldag-Niebling, Julia Beck, Dirk Alexander Frick, Tim Holler, Ante Radocaj, Birgit Piep, Andre Zeug, Denise Hilfiker-Kleiner, Cristobal G. dos Remedios, Jolanda van der Velden, Judith Montag and Theresia Kraft, Front. Cardiovasc. Med., August 2022, Sec. Cardiovascular Biologics and Regenerative Medicin, doi: 10.3389/fcvm.2022.987889