Investigating the influence of substrate stiffness and cardiovascular ageing: structural, functional and metabolic responses using induced Pluripotent stem cell-derived Cardiomyocytes

Patel, Leena (2025). Investigating the influence of substrate stiffness and cardiovascular ageing: structural, functional and metabolic responses using induced Pluripotent stem cell-derived Cardiomyocytes. University of Birmingham. Ph.D.

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Abstract

Background
Cardiovascular ageing is characterised by fibrosis and increased stiffening of the extracellular matrix (ECM) due to excessive deposition of ECM proteins. The structural remodelling of the ECM can largely impact cardiomyocyte (CMs) behaviour and function. Induced pluripotent stem cell derived cardiomyocytes (iPSC-CMs) have been widely used in cardiac research to model cardiovascular diseases. Current experiments utilise tissue culture plastics, which are far stiffer than the natural environment of CMs, thus are not representative of physiological conditions.

Modelling cardiac ageing by recapitulating the stiffness of the healthy and fi- brotic myocardium using biomaterials can provide insight into mechanisms and pathways altered by ageing, ultimately contributing to the development of novel therapeutic strategies. Furthermore, cardiac diseases that display accelerated ageing phenotypes, such as Alström Syndrome (AS), can also be studied to enhance our understanding of cardiac ageing disease mechanisms and pathways.

Aims: This work attempts to develop a representative model of the healthy and ageing myocardium, by mimicking stiffnesses using biomaterial hydrogels (Chapter 3). Characterise the effect of ECM stiffnesses on the structure of iPSC- CMs (Chapter 4). Characterise the effect of ECM stiffnesses on iPSC-CM metabolic function (Chapter 5). Assess whether AS is a model of accelerated ageing using Phenoage(Chapter 6). Investigate the cardiac role of ALMS1 in AS using ALMS1 knockout (KO) iPSC-CMs to assess changes in structure, senescence, metabolism and calcium (Ca2+) handling dynamics (Chapter 7).

Methods: Polydimethylsiloxane (PDMS) hydrogels of 20kPa (healthy) and 130kPa (fibrotic) stiffnesses were developed. Ventricular iPSC-CMs were differentiated and plated onto PDMS hydrogels of stiffnesses and plastic/glass. Transcriptional, structural and functional profiles of iPSC-CMs on stiffnesses were investigated using methods such as Quantitative polymerase chain reaction (qPCR), Western blotting and Ca2+ optical mapping. Metabolic function of iPSC-CMs wasassessed using several techniques, including isotope labelled mass spectrometry.
Phenoage, a measure of biological ageing, was compared to chronological age for a cohort of patients with AS. Retrospective analysis of cardiovascular changes over time in AS patients using echocardiography were also assessed. ALMS1 KO iPSC-CMs were assessed for molecular, functional and metabolic changes compared to wildtype iPSC-CMs.

Results: iPSC-CMs cultured on 20kPa PDMS mimicking the healthy myocardium exhibit greater structural alignment, expression of cardiac markers and altered Ca2+ handling dynamics, indicative of CM maturity. iPSC-CMs cultured on plastic substrates exhibit a preference to glycolytic metabolism, increased lactic acid production and signs of a disease profile compared to iPSC-CMs cultured on softer PDMS substrates.
Patients with AS displayed a higher Phenoage compared to their chronolog- ical age, indicating AS serves as a model of accelerated ageing. Furthermore, ALMS1 KO iPSC-CMs displayed altered Ca2+ handling dynamics, changes in cellular bioenergetics and increased senescence, providing insight into the molecular changes occurring in AS.

Conclusions: The work supports the idea that ageing and stiffness of the ECM affects structure, function and metabolism of iPSC-CMs. The pathological metabolic phenotype of iPSC-CMs cultured on traditional cell culture plastics indicates that plastic may not serve as an effective control condition for experiments. iPSC-CM culture on softer substrates provides data of greater physiological relevance and is representative of a healthy myocardium, thus highlighting the importance of substrate stiffness and the impact the environment can have on iPSC-CM behaviour. This research demonstrates softer physiological substrates serve as an effective model for healthier and mature iPSC-CM cultures and should be used in research involving iPSC-CMs to accurately reflect cardiac environments. Novel findings also display AS as a disease model of accelerated ageing. The research further highlights the benefits of physiological model development and the use of iPSC-CMs to model cardiovascular diseases, allowing investigation into molecular mechanisms of diseases.

Type of Work:

Thesis (Doctorates > Ph.D.)

Award Type:

Doctorates > Ph.D.

Supervisor(s):