P2: cGMP/cGKI in cardiac fibroblast-to-myofibroblast differentiation and fibrosis

Aims

To explore the molecular and cellular mechanisms by which cGMP/cGKI counteract, or even reverse, cardiac fibrosis. We will characterize the heterogeneity and function of the cGMP cascade in distinct fibroblast populations of the injured heart.

Questions and Methods

cGMP and Cardiac Fibrosis

To establish a clinically relevant model of heart failure with preserved ejection fraction (HFpEF) by application of nitrosative stress (tool: high-fat diet (HFD) feeding and nitric oxide synthase (NOS) inhibition using L-NAME).
To define and compare cardiac remodeling, heart function, and exercise capacity in nitrosative stress-exposed conditional mouse mutants lacking cGKI (and selected cGMP generators) in distinct cardiac fibroblast and myofibroblast populations (tools: rotararod test, non-invasive cardiac echocardiography)
To genetically trace and map the fate of cGKI proficient versus deficient cardiac fibroblast and myofibroblast populations in HFpEF (tools: HFD feeding plus L-NAME and two-color fluorescent Cre-reporter mouse strain).
Investigate relevant components of the cGMP pathway (and other pro- and anti-fibrotic factors) by transcriptome profiling (tool: scRNAseq) in selected cardiac fibroblast and myofibroblast populations obtained from failing hearts.
Examine the molecular events upstream and downstream of cGMP/cGKI signaling in fibroblast versus myofibroblast cultures using biochemical approaches and live cell imaging. The latter approach will be applied to monitor cGMP responses upon pharmacological (e.g., NO-GC activator, CNP and its derivatives or antagonists, ODQ) and genetic manipulation of selected cGMP generators (tools: cGMP-based biosensors i.e., cGi500 and red/green cGull).
The ultimate goal is to address whether and how pharmacological or genetic interventions in the cGMP pathway influence the burden of disease due to HFpEF.

London Internship

Hobbs Lab

In the Hobbs lab in London, the doctoral researchers will study if CNP and its derivatives, potentially trough cardiac fibroblasts and/or myofibroblast cGMP/cGKI, beneficially modulate the progression of HFpEF.

Following technical guidance in London, cell-type specific cardiac fibroblast and myofibroblast CNP mutants, which are available in the Hobbs lab, will be subjected to the nitrosative stress-induced HFpEF model. In a first step, the doctoral researchers will then investigate how (endogenously produced versus exogenously applied) CNP is related to cGMP/cGKI signaling and whether this influences the function of cardiac fibroblasts and myofibroblasts in vitro.

London Co-mentor

Prof. Adrian Hobbs, PhD

Link to London researcher lab

Doctoral Students

Vittoria Bonetto

Vittoria Bonetto earned her Bachelor's degree in Biological Sciences from the University of Ferrara (Italy), where she completed her internship in the laboratory of Prof. G. Aguiari. Her Bachelor's thesis focused on exploring the role of the P53 protein in renal carcinoma. She then pursued a Master's degree in Molecular Biology at the University of Padua (Italy). She did her internship in the laboratory of Prof. I. Szabó, located in the Biology department A. Vallisneri. There, she contributed in elucidating the molecular mechanisms regulated by Kv1.3 voltage-gated potassium channel in macrophages and regulatory T lymphocytes, in the contest of the tumor microenvironment (TME). Following this, she joined the laboratory of Prof. Dr. Robert Lukowski for her PhD, working on the cGMP signalling pathway in cardiac fibrosis.

Melanie Cruz Santos (graduated in January 2024)

Melanie Cruz Santos studied pharmacy at the University of Tübingen and obtained her approbation as a pharmacist in 2017. During her pharmaceutical studies and her practical year, she developed a growing interest in physiological signaling pathways and their significance under pathophysiological conditions, leading her to subsequently complete a Master's degree in Pharmaceutical Sciences and Technologies with a focus on cardiovascular research at the University of Tübingen. A combination of her specific interests encouraged her to start her doctoral studies in Robert Lukowski's laboratory, where she investigates the therapeutic potential of cell-specific NO/cGMP mechanisms in cardiac recovery from myocardial ischemia and sudden cardiac arrest.

Lena Birkenfeld

Lena Birkenfeld studied Biology at the Justus Liebig University of Gießen, Germany. There she focused on cell biology, microbiology and immunology. She earned her bachelor’s degree in the department of zoology for cellular recognition and defense processes. For her master’s degree, she continued her studies at the University in Gießen, where she concentrated on biomedicine and completed a semester abroad at the University College Dublin, Ireland. She did her master’s thesis at the Rudolf Buchheim Institute for Pharmacology in Gießen, where she investigated the protein:protein interactions of the JNK signalling pathway. She went to Tübingen to join Robert Lukowski’s laboratory. There she investigates the role of cGMP mechanisms in cardiac recovery.

Katharina Paulus (associated PhD student)

Katharina Paulus studied pharmacy at the University of Tübingen and received her approbation as a pharmacist in 2021. On top of that, she recently completed a Master’s degree in Pharmaceutical Sciences & Technologies. During her master thesis in the lab of Robert Lukowski, she investigated the impact of discrete potassium channels on the cellular metabolism which increased her interest in the field of pharmacological research. After finishing the practical year for pharmacists, Katharina returned to Robert Lukowski’s lab to start her doctoral studies with focus on the impact of cGMP pathway modulation in cardiac disease conditions.

David Spähn (associated PhD student)

David Spähn studied pharmacy at the University of Tübingen. He obtained his approbation as a pharmacist in 2021. Because of his interests in the molecular basis of diseases and their pharmacological treatments, he joined Robert Lukowski's group for his master's thesis to work on the characterization and function of intracellular i.e., nuclear ion channels. These studies strengthened his motivation to understand the mechanisms whereby ion channels and ion channel modulating drugs affect cell dys-/function. Consequently, he started his PhD studies in the same lab with the aim to characterize the function of BK channels in the metabolically highly active brown adipose tissue. The aim of his studies is to understand how K+ channels (and cGMP) contribute to morbid body weight gain.

Key Publications

Manke M-C, Roslan A, Walker B, Münzer P, Kollotzek F, Peng B, Mencl S, Coman C, Szepanowski R D, Schulze H, Lieberman A P, Lang F, Gawaz M, Kleinschnitz C, Lukowski R, Ahrends R, Bobe R, Borst O. Niemann-Pick C1 protein regulates platelet membrane-associated Ca²⁺ signalling in thrombo-occlusive diseases in mice. Journal of Thrombosis and Haemostasis, Accepted for publication March 29, 2023

Lukowski R, Cruz Santos M, Kuret A, Ruth P. cGMP and mitochondrial K⁺channels-compartmentalized but closely connected in cardioprotection. Br J Pharmacol. 2022 Jun;179(11):2344-2360. doi: 10.1111/bph.15536.

Bork NI, Kuret A, Cruz Santos M, Molina CE, Reiter B, Reichenspurner H, Friebe A, Skryabin BV, Rozhdestvensky TS, Kuhn M, Lukowski R, Nikolaev VO. Rise of cGMP by partial phosphodiesterase-3A degradation enhances cardioprotection during hypoxia. Redox Biol. 2021 Nov 6;48:102179. doi: 10.1016/j.redox.2021.102179.

Matt L, Pham T, Skrabak D, Hoffmann F, Eckert P, Yin J, Gisevius M, Ehinger R, Bausch A, Ueffing M, Boldt K, Ruth P, Lukowski R. The Na⁺-activated K⁺ channel Slack contributes to synaptic development and plasticity. Cell. Mol. Life Sci. 78, 7569–7587 (2021). https://doi.org/10.1007/s00018-021-03953-0

Längst N, Adler J, Schweigert O, Kleusberg F, Cruz Santos M, Knauer A, Sausbier M, Zeller T, Ruth P, Lukowski R. Cyclic GMP-dependent regulation of vascular tone and blood pressure involves cysteine-rich LIM-only protein 4 (CRP4). Int J Mol Sci. 2021 Sep 14;22(18):9925. doi: 10.3390/ijms22189925.