Working groups of the Molecular- and Cellphysiology
Deciphering motor function/dysfunction using single-molecule biophysics
Address:
Dr. rer. nat. Mamta-Amrute-Nayak
Institute for Molecular and Cell Physiology
Carl-Neuberg Str. 1, 30625 Hannover, Lower Saxony, Germany
Building: I3, level 03, room 1330
Telefon (+49) 511 532 2094
Fax (+49) 511 532 2754
E-Mail Mamta Amrute-Nayak
Research focus / Forschungsschwerpunkte
Cytoskeletal motors are ATP-dependent force generating biological machines that perform diverse tasks such as, intracellular cargo transport, muscle contraction, cell division, and whole cell movement etc.
The non-prcessive Myosin-II motors drives contraction of skeletal and cardic muscles, while the processive actin-based molecular motor proteins such as myosin V is involved in intracellular transport. The principle aim of our research is to gain detailed understanding of the mechanisms by which different motors perform diverse roles.
With vital roles in nearly all aspects of cellular physiology, motor protein dysfunctions are intricately linked to several myopathies including heart disorder Familial hypertrophic cardiomyopathy (FHC) that affects 1 in 200 individuals worldwide. Clinical phenotypes display a high variability ranging from being asymptomatic, to rapidly progressive failing heart or sudden cardiac death in young individuals and competitive athletes.
We aim to gain comprehensive understanding of functional domains in the myosin head, as a consequence of point mutations and to indentify the primary functional alteration resulting into myocardial disorganization leading to the hypertrophy of left ventricle.
Our experimental approaches include single molecule biophysical methods such as total internal reflection fluorescence microscopy (TIRFM), zero mode waveguides and optical trapping to obtain precise kinetic and mechanical insights of the motor proteins.
Our lab is funded by following grants:
- German Research Foundation (DFG)
- Hochschulinterne Förderung (HilF, MHH)
- Fritz Thyssen Foundation
Contraction-relaxation function and mechano-chemical coupling of myofibrils
Investigations of human, human pluripotent stem cell-derived cardiomyocytes and animal contractile models in non-pathologic and pathologic conditions
Address:
Apl. Prof. Dr. Bogdan Iorga
Institute for Molecular and Cell Physiology
Carl-Neuberg Str. 1, 30625 Hannover, Lower Saxony, Germany
Building: I3, level 03, room 1270
Tel: +49 511 532 -2753 (office), -3654 (lab)
Fax: +49 511 532 -4296
Email: Bogdan Iorga
Forschungsschwerpunkt
Contraction-relaxation function and mechano-chemical coupling of myofibrils.
The main function of cardiomyocytes (CMs) to generate force and shorten their length occurs when the subcellular myofibrils contract due to multiple interactions between ATPase-driven myosin motors and actin filaments. Myofibrils consist in many sarcomeres arranged in series driving directly the contraction-relaxation events of CMs upon cyclical variation of the intracellular Ca2+ concentration. Therefore, isolated myofibrils represent a contractile model used to understand sarcomeric protein-related processes that determine contractile function of CMs in the absence of Ca2+ handling systems and of upstream signaling.
Myofibrils can be investigated using fast kinetic micromechanical and chemical techniques because they are thin and in rapid diffusional equilibrium with their surrounding environment. We have established a micromechanical setup that uses an atomic force cantilever as a nN-sensitive force sensor. This setup allows rapid changes of the solutions to which myofibrils are exposed, and the force kinetic parameters of myofibrillar activation and relaxation at different Ca2+ concentrations can be analyzed with high time resolution. Different established biochemical methods can assess the steady-state or transient ATPase activity of the sarcomeric myosin motor using mechanical unloaded, native myofibrils or using myofibrils prevented from shortening by chemical cross-linking few of their myosin heads to the actin filaments. These investigations can allow correlating the biochemical events to the mechanical events during cross-bridge cycling based on isoform composition of sarcomeric proteins.
Subcellular myofibrils can be obtained from human (e.g., biopsies), human-derived and non-human cardiac and skeletal small muscle samples (e.g., mouse, rat, rabbit, zebrafish and hESC-/hiPSC-CMs).
In vitro-differentiated cardiomyocytes.
Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) hold great potential for the treatment of cardiovascular diseases by cell transplantation or engineered cardiac tissue, for assessing efficiency and toxicity of pharmacological compounds, or to be used as cellular disease models in vitro. hPSC-CMs exhibit a series of immature features compared to ventricular CMs of an adult human heart. Therefore, characterization of hPSC-CMs at different hierarchically interrelated levels (molecular, subcellular, cellular and multicellular levels) and understanding how extracellular environment and intracellular factors may affect subcellular myofibrils and cell maturation of hPSC-CMs, in pathologic and non-pathologic conditions, represents important research objectives to us.
Hypertrophic cardiomyopathy: Contractile imbalance as a central pathomechanism? Investigations on human myocardium and cellular models.
Address:
Prof. Dr. Theresia Kraft
Institute for Molecular and Cell Physiology
Carl-Neuberg Str. 1, 30625 Hannover, Lower Saxony, Germany
Building: I3, level 03, room 1010
Phone (+49) 511 532 6396
Fax (+49) 511 532 4296
E-Mail Prof. Dr. rer. nat. T. Kraft
Research focus
One of our central goals is to elucidate how mutations in sarcomeric proteins disrupt sarcomere function in cardiomyocytes and lead to hypertrophic cardiomyopathy (HCM).
A key observation of our research was that the extent of functional changes caused by HCM mutations in cardiomyocytes of affected heterozygous patients is very different among cardiomyocytes isolated from the same tissue. For example, some cardiomyocytes showed significantly reduced force development at physiological calcium concentrations, while others behaved like healthy cardiomyocytes at the same calcium concentration as if no mutated protein was expressed in these cells. Meanwhile, we have been able to show for β-cardiac myosin heavy chain (β-MyHC) and cardiac troponin I (cTnI) that in each of the patients studied, individual cardiomyocytes from the same tissue express very different proportions of mutated and wild-type mRNA of the respective gene. This most likely leads to correspondingly different proportions of mutated and wild-type protein and thus to the observed variability of biomechanical function from cell to cell. Our hypothesis is that the resulting functional variability between neighboring cardiomyocytes in the cellular network of the myocardium causes distortions of the cardiomyocytes, which over time lead to typical features of HCM such as cellular and myofibrillar disarray, hypertrophy and fibrosis (“contractile imbalance” hypothesis).
So far, we were able to show that the observed cell-to-cell variability of the fraction of mutated mRNA is most likely a consequence of a burst-like transcription mechanism of mutated and wild-type alleles which is random and independent for both alleles. Model calculations confirmed the hypothesis that independent, burst-like transcription of the two alleles can lead to different proportions of mutated and wild-type protein in the individual cardiomyocytes and thus to the observed functional variability. According to this concept, a mutation in a sarcomeric or even non-sarcomeric protein (e.g. kinases), which produces a change in the biomechanical function of the sarcomere or myofibril, can lead to a functional imbalance between individual cardiomyocytes via random, burst-like transcription, and thus contribute to the development of HCM.
Aims are to identify primary functional effects of HCM mutations also in other sarcomeric proteins at the molecular level and to characterize them from cell to cell by investigating myocardial tissue from HCM patients. Furthermore, we analyze changes in signaling pathways at the single cell level, which are induced due to contractile imbalance and which can lead to hypertrophy and fibrosis. Finally, it is of great importance to elucidate mechanisms of burst-like transcription of the affected genes for sarcomeric proteins in cardiomyocytes to be able to modulate the expression, aiming at new therapeutic approaches.
These topics are being investigated together with the research groups Montag, Weber and Iorga on myocardial tissue from HCM patients and on cellular models based on stem cell-derived cardiomyocytes with HCM mutations. Apart from this, we are working with research group Montag on a pig model for HCM with a point mutation in β-cardiac myosin. The aim is to be able to examine different aspects of the course of the disease longitudinally. Our cell culture models as well as the animal model enable us to further investigate the contractile imbalance hypothesis, and to derive new treatment options for early prevention of the development of HCM. Last but not least, by investigating the primary functional effects of HCM mutations, we also gain insights into the relevance of individual proteins or their subdomains for the function of the sarcomere.
Unequal allelic expression (from cell to cell) as pathomechanism for hypertrophic cardiomyopathy
Address:
Dr. Judith Montag
Institute for Molecular and Cell Physiology
Carl-Neuberg Str. 1, 30625 Hannover, Lower Saxony, Germany
Building: I3, level 03, room 1340
Telefon (+49) 511 532 2094
Fax (+49) 511 532 4296
E-Mail 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.
Chromatin and SUMO physiology group
Address:
PhD. Arnab Nayak
Institute for Molecular and Cell Physiology
Carl-Neuberg Str. 1, 30625 Hannover, Lower Saxony, Germany
Building: I3, level 03, room1340
Telefon (+49) 511 532 2094
Fax (+49) 511 532 4296
E-Mail Arnab Nayak
Research focus / Forschungsschwerpunkte
Mobility is an indispensable feature that determines survival and success in the animal world. Skeletal muscle that allows this mobility accounts for 40% of human body mass. Apart from movement, another feature of the muscle power is to be able to bear the load. The skeletal muscle displaying a characteristic striated pattern is an array of linearly arranged units called ‘sarcomeres’. The individual sarcomere hosts highly organized structures including the actin, and myosin filaments. The cyclic interaction between these two types of filaments is responsible for generation of force and movement at the molecular to organismic level.
Precise molecular arrangement of sarcomere is central to the muscle function. Importantly, disorganization of sarcomere and thereby defective muscle function are the typical hallmarks of myopathies including cancer cachexia, prevalent in nearly 80% of patients.
Our group aims to understand how SUMO (Small Ubiquitin-like Modifier)-mediated epigenetic program regulates sarcomere organization and how it is deregulated in muscle atrophy/cachexia.
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Single molecule motility
Address:
Dr. rer. nat Tim Scholz
Institute for Molecular and Cell Physiology
Carl-Neuberg Str. 1, 30625 Hannover, Lower Saxony, Germany
Building: I3, level 03, room 1350
Tel: +49 511 532 -2737 (office), -9318 (lab)
Fax: +49 511 532 -4296
E-Mail: Tim Scholz
read more: Einzelmolekülmotilität von Motorproteinen
Characterization of cardiomyocytes from heart tissue and from human stem cells
Address
Dr. rer. nat. Joachim Meißner
Institute for Molecular and Cell Physiology
Carl-Neuberg Str. 1, 30625 Hannover, Lower Saxony, Germany
Building J03, level 03, room1270
Tel: +49 511 532 -2753
Fax: +49 511 532 -4296
E-Mail: Joachim Meißner
Focus of research
Maturation of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) and investigation of involved signaling pathways
We aim to investigate approaches to promote the maturation of human embryonic and human induced pluripotent stem cell-derived cardiomyocytes (hES- and hiPSC-CMs, resp.) towards an adult ventricle-like phenotype as well as involved signaling pathways in cooperation with the group of Dr. Robert Zweigerdt, LEBAO, MHH. Remarkably, hPSC-CMs demonstrate a pronounced heterogeneity, e.g. with regard to the expression of isoforms of the motor protein myosin (MyHC), a and β. MyHCb is predominantly expressed in ventricles of the human heart and therefore is together with other factors a marker for the maturation of hPSC-CMs towards an adult-like phenotype. A mature state of hPSC-CMs is important for modeling cardiac diseases like hypertrophic cardiomyopathy (HCM) in cell culture and for clinical applications like cell therapies in regenerative medicine or for drug screening.