The effect of substrate stiffness on cardiac performance

The stiffness of the human myocardium increases as part of the ageing process. This can lead to ventricular pathologies such as heart failure with preserved ejection fraction (HFpEF), especially in elderly in the presence of co-morbidities. The mechanisms underlying increased myocardial stiffness are not understood. Moreover, atrial remodelling may be influenced by myocardial stiffness, potentially contributing to atrial fibrillation (AF). Both conditions comprise significant burdens on health care systems, with an estimate of 300-450,000 HFpEF patients in the UK by 2050 [1] and around 15 % of individuals over 75 years being affected by AF [2].
In addition to alterations in myocyte relaxation and metabolic imbalance, increased deposition of extracellular matrix proteins, particularly collagens, are believed to drive increased cardiac stiffness. The objectives of this project are to model increased matrix stiffness in vitro and to obtain insights into its molecular consequences in a human cellular model system, to better understand the predisposition to diseases such as HFpEF and AF in the elderly. The student will study the effects of matrix stiffness onto ventricular and atrial human cardiomyocytes, exploring various aspects of myocyte biology, such as metabolism, contractility, electrophysiology and mechanosignalling.
As the model system, induced pluripotent stem cells (from a normal individual) will be differentiated into cardiomyocytes (iPSC-CM). The student will use bespoke protocols, allowing the generation of either atrial or ventricular human myocytes of various “ages” (i.e. time from induction of differentiation).
These iPSC-CM will be cultured on substrates of embryonic, adult physiological and adult pathological stiffnesses (1 kPa to 130 kPa; collaboration with Dr T. Iskratsch at Queen Mary University London). Nano-patterned surfaces will additionally allow to control shape ratios and cell size.
The effect of substrate stiffness onto cardiomyocyte performance will be assessed by contractility measurements and electrophysiological assays. Molecular analyses, including Western blotting, real time PCR and high resolution (STORM) microscopy will identify the response of proteins involved in mechano-signalling as well as of ion channels to variation in substrate stiffness. Key players to be investigated are signalling proteins such as focal adhesion kinase, titin, filamin C and ion channels contributing to sodium and calcium homeostasis. Proteomics will identify further proteins responding to matrix stiffness changes, and these novel proteins will be included in the analyses above.
Moreover, a targeted metabolomics approach will be employed to assess shifts in substrate utilisation (glucose versus fatty acids; collaboration with Prof R. Dunn), thereby providing insights into the changes of cardiac metabolism with increased matrix stiffness.
In a last step, the student will reverse observed molecular changes with appropriate pharmacological interventions, providing proof-of-principle confirmation and additionally opening avenues for the pharmacological treatment of human pathologies associated with increased matrix stiffness.
In summary, the project will provide a better understanding of the role of matrix stiffness in the mal-adaptation of ventricular and atrial cardiomyocytes associated with ageing. Through the project, the student with obtain comprehensive training in a wide range of methods, covering bioengineering, microscopy, cell biology and electrophysiology. Working in an interdisciplinary environment, alongside clinicians, basic scientists and bioengineers, the student will get a thorough understanding of the mechanisms and translational implications.

List the techniques that will be undertaken during the project

Differentiation of iPSC into iPSC-CM
Bioengineering of substrates a various stiffnesses
Generating and application nano-patterned surfaces
High resolution microscopy (STORM)
Metabolomics, proteomics
Electrophysiology, real time PCR, Western blotting

Host Environment

Lab and office space will be provided within the ICVS. The student will be supported by experienced staff in the heart failure and arrhythmias cluster, https://www.birmingham.ac.uk/research/activity/cardiovascular-sciences/research/heart-failure-arrhythmias/index.aspx
established in 2011 by Larrisa Fabritz and Paulus Kirchhof, supported among others by EU funding (CATCH ME Horizon 2020 633196) and BHF (FS/13/32/30324). Electrophysiological methods, real time PCR and Western blotting are performed on a daily basis in the group in their IBR labs and the group has worked with high resolution microscopy available in COMPARE. The student will benefit from existing collaborations of Katja Gehmlich (Prof W. Dunn, UoB and Dr T. Iskratsch, QMUL) as well as the technical expertise on iPSC-CM in her group,, currently consisting of currently 3 postdocs, two research assistants, who will be able to support aspects of the project. Katja Gehmlich is supported by funding from the BHF (Fellowship FS/12/40/29712) and the Wellcome Trust (Collaborative Award 201543/B/16/Z).
The student will have access and make use of the BHF funded Centre of Membrane Proteins and Receptors (COMPARE) Imaging Facility at UoB. Working alongside clinicians, the student will be exposed to the translation relevance of the work.

Dr Fabritz: https://www.birmingham.ac.uk/staff/profiles/cardiovascular-sciences/Fabritz-Larissa.aspx
Dr Gehmlich: http://www.rdm.ox.ac.uk/principal-investigators/researcher/katja-gehmlich