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Institut für Physiologie und Pathophysiologie

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Stem Cell-Derived Cardiomyocytes for Myocardial Repair


The regenerative potential of the adult heart is very limited and insufficient to replace damaged muscle mass in the diseased heart. Recent advances in cardiac cell therapy and tissue engineering fuel new hope for the development of novel therapeutic approaches with the aim to trigger myocardial regeneration after injury. Stem cell-derived cardiomyocytes represent ideal candidates for cardiac cell-based therapeutic strategies, and current research focuses on the development of cardiac constructs for implantation. Despite the cardiogenic properties of the newly generated cardiomyocytes, these cells present a heterogeneous and immature phenotype, which is more comparable with cardiomyocytes of early developmental stages. However, successful employment of these new cardiomyocytes for myocardial repair demands that the physiological profile of stem cell-derived cardiomyocytes matches with the functional complexity of mature cardiomyocytes. This is currently not the case. We are interested in gaining better insight into the mechanisms that drive cardiac maturation and follow different approaches to enhance the cardiac phenotype of stem cell-derived cardiomyocytes.

Shaping the Heart – Structural and Functional Characterization of Stem Cell-Derived Cardiomyocytes (SC-CMs)

 

The adult mammalian cardiomyocyte is a highly specialized and terminally differentiated cell with distinct structural features, such as a regular transverse (t)-tubular network (Figure 1A) and strict myofilament organization. This precise microarchitecture is a prerequisite for the sophisticated functional specialization of heart cells and provides the basis for local calcium signaling domains and protein interaction clusters for efficient excitation-contraction (EC-) coupling. Lack of this microarchitecture in premature SC-CMs (Figure 1B) strongly limits the efficiency of cell contraction and force production. These deficits may be overcome by triggering cellular maturation through optimized in vitro culture conditions, aiming at better mirroring the in vivo situation. In this project, we test the hypothesis that a specific cell geometry influences the subcellular microarchitecture of SC-CMs with the aim to trigger morphological and functional maturation in these cells and to approach a similar phenotype as mature myocytes.

 

Figure 1: Comparison of murine adult ventricular cardiomyocytes (A) and induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs, B). Membrane-specific stainings reveal clear differences in the structural organization of both cardiomyocytes. The membrane-specific indicator di-8-anepps was used to mark the sarcolemma. The rectangular shape and regularly spaced transverse (t)-tubular membrane invaginations (see insert) are characteristic for adult cardiomyocytes, while IPSC-CMs do not present any specific cell geometry or regular t-tubular network.

 

In a multi-disciplinary approach combining the expertise in material science and bio-engineering with cardiac cell physiology, we analyze the effects of material contact and specific predefined geometries on cell shape and function at the level of single cells (Figure 2).

 

Figure 2: Different geometries induce structural remodeling in SC-CMs (α-actinin stainings).

 

This project is part of the research bridge “Synthetic Biology” established by HEiKA and a collaboration with the lab of Prof. Dr. Martin Bastmeyer at the Karlsruhe Institute of Technology.

Intercellular Communication in Stem Cell-Derived Cardiomyocytes

 

In the light of novel treatment options for ischemic myopathies and arrhythmogenic diseases, the functional properties of newly developed cardiomyocytes derived from pluripotent stem cells are investigated and compared with primary cardiomyocytes at the cellular level and in multicellular preparations. One critical shortcoming of these cells is their limited potential to connect with native cardiomyocytes in order to establish a functional syncytium (Figure 3). We have recently shown that single SC-CMs present cardiogenic characteristics, however, in multicellular preparations cell-to-cell coupling is strongly compromised. Reduced coupling results in a strong reduction in electrical signal transmission and conduction velocity, which may increase the risk for the development of arrhythmias.

 

In this project, we are interested in the mechanisms that control intercellular communication with the aim to enhance signal transmission in SC-CMs and to improve heterocellular coupling with native cardiomyocytes (Figure 4).

 

Figure 3: Cardiomyocytes form intercellular gap junctions preferentially at the intercalated disks (ICDs), which are located at the end-to-end connections between cells. ICDs in SC-CMs are shown in the elelctron photomicrograph.


 

 

 

 

 

Figure 4: Illustration of the experimental setup to study intercellular coupling.

New Molecular Targets for the Treatment of Cardiac Diseases and Cellular Remodeling

 

In this project we are interested in the molecular determinants that underlie cardiomyocyte remodeling in hypertrophy and arrhythmogenic diseases. Chronic stress affects intracellular signaling cascades that are under control of adrenergic receptors, resulting in maladaptive functional changes and significant alterations of the Ca2+ handling machinery of the cardiomyocyte. We investigate the impact of stress-induced transcriptional changes on cardiac function at the level of sarcolemmal Ca2+-handling proteins and Ca2+ channels of the sarcoplasmic reticulum. Novel pharmacological compounds are tested for their capability of reversing pathological remodeling at the cellular level (Figure 5).

 

Figure 5: Cardiac excitation-contraction coupling and Ca2+ current properties. Left: Ca2+-induced Ca2+ release in adult ventricular cardiomyocytes. L-type Ca2+ currents are elicited by depolarization to different membrane potentials. In parallel, cytosolic Ca2+ levels are monitored in the line-scan mode using a Ca2+-sensitive fluorescent indicator. Right: activation and inactivation curves of the L-type Ca2+ channel under control conditions (black) and in the presence of two compounds with different effects on the channel kinetics (red, blue).

Methods

 

Cell culture

 

  • Heart isolations using Langendorff apparatus (Figure 6)
  • Cardiac cell culture
  • Tissue engineering

Functional studies

 

  • Electrophysiology: patch clamp technique (voltage clamp and current clamp), electrical field stimulation
  • Confocal live cell imaging in parallel with electrophysiological measurements (Figure 7)
  • Contraction studies using edge detection, combined with cytosolic Ca2+ measurements
  • Pharmacological tests

Structural studies

 

  • Immunocytochemistry and confocal imaging
  • Electron microscopy (TEM, Figure 3)

Figure 6: Cardiac cell isolation by enzymatic dissociation.


Figure 7: Simultaneous recordings of membrane currents using voltage clamp and Ca2+ transients by confocal imaging of fluo-3 in the linescan mode. Ca2+ transients are elicited by depolarization (left panels) or caffeine application (right panels).


Neue Publikationen

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Temporal relations between cortical network oscillations and breathing frequency during REM sleep. J Neurosci. 2021 Jun 16;41(24):5229-5242. doi: 10.1523/JNEUROSCI.3067-20.2021. Epub 2021 May 7.

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AAV-mediated expression of NFAT decoy oligonucleotides protects from cardiac hypertrophy and heart failure. Basic Res Cardiol. 2021 Jun 4;116(1):38. doi: 10.1007/s00395-021-00880-w.

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Assessable learning outcomes for the EU Education and Training Framework core and Function A specific modules: Report of an ETPLAS WORKING Group. Lab Anim. 2021 Jun;55(3):215-232. doi: 10.1177/0023677220968589. Epub 2020 Dec 7.

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Trigger-Specific Remodeling of KCa2 Potassium Channels in Models of Atrial Fibrillation. Pharmgenomics Pers Med. 2021 May 20;14:579-590. doi: 10.2147/PGPM.S290291. eCollection 2021.

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TLR2- and TLR3-activated microglia induce different levels of neuronal network dysfunction in a context-dependent manner. Brain Behav Immun. 2021 May 17:S0889-1591(21)00194-X. doi: 10.1016/j.bbi.2021.05.013. Online ahead of print.

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Mechanobiology of Atherosclerosis. In: Vascular Mechanobiology in Physiology and Disease. Cardiac and Vascular Biology, vol 8. (Hecker M, Duncker DJ, eds.) Springer, Cham 2021, pp. 319-332. Hardcover ISBN 978-3-030-63163-5; eBook ISBN: 978-3-030-63164-2

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The Biomechanics of Venous Remodeling. In: Vascular Mechanobiology in Physiology and Disease. Cardiac and Vascular Biology, vol 8. (Hecker M, Duncker DJ, eds.) Springer, Cham 2021, pp. 167-189. Hardcover ISBN 978-3-030-63163-5; eBook ISBN: 978-3-030-63164-2

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Vascular Mechanobiology in Physiology and Disease. Cardiac and Vascular Biology, vol 8. Springer, Cham 2021, 352 pp. Hardcover ISBN 978-3-030-63163-5; eBook ISBN: 978-3-030-63164-2

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Alginate hydrogel polymers enable efficient delivery of a vascular-targeted AAV vector into aortic tissue. Mol Ther Methods Clin Dev. 2021 Jun 11;21:83-93. doi: 10.1016/j.omtm.2021.02.017. eCollection 2021 Jun 11.

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Debunking a myth: plant consciousness. Protoplasma. 2021 May;258(3):459-476. doi: 10.1007/s00709-020-01579-w. Epub 2020 Nov 16.

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Microglia and lipids: how metabolism controls brain innate immunity. Semin Cell Dev Biol. 2021 Apr;112:137-144. doi: 10.1016/j.semcdb.2020.08.001. Epub 2020 Aug 15.

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Editorial: Calcium Homeostasis in Skeletal Muscle Function, Plasticity, and Disease. Front Physiol. 2021 Mar 26;12:671292. doi: 10.3389/fphys.2021.671292. eCollection 2021.

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Absence of neocytolysis in humans returning from a 3-week high-altitude sojourn. Acta Physiol (Oxf). 2021 Mar 17:e13647. doi: 10.1111/apha.13647. Online ahead of print.

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Cardiomyocyte depolarization triggers NOS-dependent NO transient after calcium release, reducing the subsequent calcium transient. Basic Res Cardiol. 2021 Mar 17;116(1):18. doi: 10.1007/s00395-021-00860-0.

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Induced Pluripotent Stem Cell-derived cardiomyocytes (iPSC-CMs); generation and enrichment protocols, immature and mature structure and function. In: Recent Advances in iPSC-Derived Cell Types, Volume 4, 1st Edition (Birbrair A, ed.) Academic Press 2021, pp. 191-226. Paperback ISBN 9780128222300; eBook ISBN 9780128224540

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Simulation of Air Travel-Related Irradiation Exposure of Cryopreserved Mouse Germplasm Samples. Biopreserv Biobank. 2021 Mar 1. doi: 10.1089/bio.2020.0046. Online ahead of print.

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Astrocytes mediate the effect of oxytocin in the central amygdala on neuronal activity and affective states in rodents. Nat Neurosci. 2021 Apr;24(4):529-541. doi: 10.1038/s41593-021-00800-0. Epub 2021 Feb 15.

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Integrated information theory does not make plant consciousness more convincing. Biochem Biophys Res Commun. 2021 Jan 21:S0006-291X(21)00057-7. doi: 10.1016/j.bbrc.2021.01.022. Online ahead of print.

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AAV-mediated AP-1 decoy oligonucleotide expression inhibits aortic elastolysis in a mouse model of marfan syndrome. Cardiovasc Res. 2021 Jan 20:cvab012. doi: 10.1093/cvr/cvab012. Online ahead of print.


Institut für
Physiologie und Pathophysiologie

Universität Heidelberg

Im Neuenheimer Feld 326

69120 Heidelberg

Telefon:+49 6221 54-4056
Telefax:+49 6221 54-6364
E-Mail:susanne.bechtel@
physiologie.uni-heidelberg.de