(K-416) Using Engineered Heart Tissues to Study the Role of Melusin in Humans

Thursday, October 12, 2023

9:30 AM – 10:30 AM PDT

Location: Exhibit Hall - Row K - Poster # 416

Presenting Author(s)

AG

Anika Ghelani

Undergraduate Student
University of Washington
Lynnwood, Washington, United States

Co-Author(s)

RP

Ruby Padgett

Research Scientist
University of Washington, United States
AG

Alex Goldstein

Graduate Student
University of Washington, United States

Primary Investigator(s)

NS

Nathan J. Sniadecki (he/him/his)

Professor
University of Washington Seattle, United States

Introduction::

Heart disease is the leading cause of death globally, taking an estimated 17.9 million lives each year [1]. Not only is heart disease incredibly prevalent, but treatment can be exceedingly expensive. With approximately 22 million US residents having prevalent cardiovascular disease (CVD), this extrapolates to direct costs of more than $400 billion [2]. Melusin, a chaperone protein found in the heart, holds potential as a target for heart failure therapeutics. Previous work has shown that melusin induces a protective hypertrophic response when the heart is subjected to chronic mechanical stress. This protective response helps prevent progression into cardiac failure. A previous study done in wild-type (WT) and melusin knockout (MelKO) mice used transverse aortic constriction (TAC) to band the aorta and induce mechanical stress on the heart. The absence of melusin was associated with a hypertrophic response indicative of heart failure [3]. I plan to further investigate the biomechanical role of melusin in humans using human-engineered heart tissues (EHTs) created from human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) that lack melusin and their isogenic controls. EHTs serve as a 3D in vitro model of the human heart, ideal for studying the role of melusin in humans. I hypothesize that WT EHTs subjected to mechanical stress, i.e., high afterload, will outperform the MelKO EHTs.

Materials and Methods::

EHTs are produced by differentiating hiPSCS to hiPSC-CMs using a well-established cardiac differentiation protocol [Figure 1]. On Day 21 of the protocol, we can produce EHTs consisting of a mixture of hiPSC-CMs, stromal cells, fibrinogen, and thrombin. The EHTs are suspended between one flexible and one rigid silicone post. The EHT displaces the flexible post as it contracts, from which the displacement can be measured to calculate various auxotonic properties of the tissue, including the amount of contractile force generated. [Figure 2]. To induce mechanical stress on the tissues, I used a brace to restrict the movement of the flexible post. The brace can be slid up the posts to further decrease the mobility of the flexible post and increase afterload on the tissues [Figure 3]. I plan to use histology to determine if there are any morphological differences between each type of tissue due to the brace.

Results, Conclusions, and Discussions:: Thus far, I have successfully differentiated high-purity WT and MelKO hiPSC-CMs and cast EHTs with and without the braces. I verified cardiomyocyte purity using flow cytometry for cardiac troponin T (cTnT). I used this to optimize the differentiation protocol for WT and MelKO cell lines [Figure 4]. After casting the EHTs, I completed twitch force measurements two and three weeks post casting. On week two, the braces were slid up from being at a height of 3 to 6 mm. Overall, I found that the MelKO EHTs demonstrated a lower contractile force than the WT EHTs [Figure 5]. In addition, more MelKO EHTs remained intact with the braces, while a couple of the WT tissues snapped. I plan to cast and collect more EHT data with and without braces for both cell types in order to provide insight on the role of melusin in humans. In the future, I aim to explore the impact of hormonal stress on WT and MelKO tissues using angiotensin II, a hormone that increases blood pressure. Furthering our understanding of the mechanotransduction properties of the heart using EHTs is important in expanding our knowledge about the heart's various hypertrophies and pathologies. Ultimately, studying the pressure overload pathways involving melusin can lead to the development of future therapies for cardiovascular disease.

Acknowledgements (Optional): :

References (Optional): :

[1]World Health Organization. (n.d.). Cardiovascular diseases. World Health Organization. https://www.who.int/health-topics/cardiovascular-diseases#tab=tab_1

[2] G. A. Nichols, T. J. Bell, K. L. Pedula, and M. O'Keefe-Rosetti, “Medical care costs among patients with established cardiovascular disease,” American Journal of Managed Care, pp. 86–93, Mar. 2010.

[3]M. Brancaccio, L. Fratta, A. Notte, E. Hirsch, R. Poulet, S. Guazzone, M. De Acetis, C. Vecchione, G. Marino, F. Altruda, L. Silengo, G. Tarone, and G. Lembo, “Melusin, a muscle-specific integrin β1–interacting protein, is required to prevent cardiac failure in response to chronic pressure overload,” Nature Medicine, vol. 9, no. 1, pp. 68–75, 2002.