Poster E6 - Utilizing CRISPR-Cas9 to permit new strategies in studying the H251N mutation in hiPSC-derived cardiomyocytes

Saturday, October 26, 2024

10:00 AM - 11:00 AM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

Mahadev Josyabhatla

Undergraduate Student
University of Michigan
Weston, Florida, United States

Primary Investigator(s)

NF

Nathan French (he/him/his)

Master's Student
Department of Biomedical Engineering, University of Michigan, Ann Arbor, USA
Ann Arbor, Michigan, United States

Co-Author(s)

AV

Alison Vander Roest (she/her/hers)

Assistant Professor
Department of Biomedical Engineering, University of Michigan, Ann Arbor, USA, Michigan, United States
GA

Georgina Aluoch

PhD Student
University of Michigan, United States

Introduction: The heart is composed of multiple cell types, including contractile cardiomyocytes (CMs). Certain mutations within CMs can alter their functionality which can lead to chronic cardiac problems for the patient, including hypertrophic cardiomyopathy (HCM). The Vander Roest Lab hopes to study the effects of specific sarcomeric mutations on the contractility and long-term health of CMs.

One mutation studied by this lab is the H251N mutation in the β-myosin of cardiomyocytes, which is generally seen in pediatric patients. Typically, this mutation is associated with increased contractility, force production, and hypertrophy of cardiomyocytes.

To study the effects of increased force production in cells with the H251N mutation, we are utilizing a vinculin tension-sensing probe flanked with two fluorophores, which when under low tension, allows energy transfer to occur, allowing for a measurement of force dynamics in the H251N cell line.

However, the H251N cell line (C3 cell line), and its respective control (C4 cell line), both have GFP-tagged alpha-actinin structure, preventing the usage of any fluorescent probes emitting this wavelength of light. To solve this issue, we designed and utilized CRISPR guides to knock out a region of the GFP-expressing gene in the genome of these cardiomyocytes, thereby preventing the expression of GFP and creating a new cell line where the alpha-actinin structure does not emit GFP. This would allow us to use the aforementioned fluorescent probes, allowing us to measure the effect of the H251N mutation on force production.

Materials and

Methods: For this experiment, the CRISPR-Cas 9 system was utilized to knock out the GFP gene in the GFP-tagged alpha-actinin cell lines. Using the alpha-actinin GFP gene sequence provided by the Allen Institute, we designed 2 guide RNA sequences that would induce a double stranded break over a 50 base pair region in the GFP sequence. We then annealed these gRNA sequences into the Cas-9 expressing plasmid (px459-puro) and prepared the plasmid for transfection into induced pluripotent stem cells (hiPSCs). Lipofectamine 3000 was used to transfect hiPSCs with this plasmid at varying concentrations. After a 48 hour incubation, puromycin was added to the media to kill off cells that were not successfully transfected.

On day 5 after puromycin treatment, colonies of surviving cells were replated. A few colonies were chosen for sequencing in order to verify the success of the CRISPR knockout. The positive colonies were expanded and differentiated into cardiomyocytes using the standard protocol for cardiomyocyte differentiation. At the same time, C3/C4 control lines (without transfection and CRISPR edits) were also differentiated.

Once the cardiomyocytes started beating, videos of their contractions were taken in the phase channel. These videos were then analyzed using Myocyter, a software that measures the contraction velocity and duration of cardiomyocytes.This analysis was performed to determine whether the genetic edit played any role in affecting the contractility of the GFP-knockout cells. Specifically, four cell lines were analyzed: C3 with a GFP knockout, C4 with a GFP knockout, C3 (control), and C4 (control).

Results, Conclusions, and Discussions: Based on the sequencing results (Figure 1), all tested clones appeared to have a 50 base-pair region in the sequenced area that was deleted, indicating that the CRISPR guides were able to successfully knock out the GFP tag. After imaging the differentiated cardiomyocytes under the GFP channel, we were unable to see the alpha-actinin in the GFP channel of the GFP knockout cells, indicating the genetic edit was successful. For the control cell lines, the alpha-actinin structures were visible under the GFP channel, as seen in Figure 2.

As seen in Figure 3, the decrease in contraction lengths between the C3 and C4 cell lines of the control group remained statistically significant in the knockout group. Meanwhile, the marked decrease in contraction lengths between the C3 and C4 cell lines of the control group became statistically significant when looking at the knockout group, which is expected based on prior results.

Based on the results, the GFP tag on the alpha-actinin was successfully knocked out in the cardiomyocytes. Additionally, the characteristics associated with the H251N phenotype appeared to be preserved within the C3 cell line.

In addition to the utilization of fluorescent probes in the CFP-YFP channels to measure the force production of these cells, we will also test the sensitivity of these cells to calcium and utilize fluorescent probes for intracellular calcium. Moreover, these results can be generalized to the unedited C3/C4 cell lines due to the relative similarity in contractility between the edited cells and the unedited cell lines.

This same gene editing strategy can also be applied to different hiPSC lines with different β-myosin mutations. Our studies of these varying mutations would be able to give new insights into the heterogeneity of HCM seen in patients. Additionally these genetically edited cell lines could be utilized to test the effectiveness of Mavacamten and other developing therapeutics in treating HCM.

Acknowledgements (Optional): We would like to acknowledge the funding sources that supported this works, specifically the NIH NHLBI K99/R00 Pathway to Independence, NIH NIGMS RM1 Early Stage Investigator Development grants.
Additionally, we would like to thank the entire Vander Roest Cardiac Mechanobiology Laboratory and the Department of Biomedical Engineering for their support!