(G-267) Investigating Desmin Insufficiency in Induced Pluripotent Stem Cell Model of MYH7 Variant Dilated Cardiomyopathy

Introduction:: Dilated cardiomyopathy (DCM) is a type of genetic cardiomyopathy associated with a weakened left ventricle that can result in heart failure [1]. Missense mutations in the myosin heavy chain 7 (MYH7) gene have been identified as the 3rd most common genetic cause of DCM [2]. In previous work, we have established an induced pluripotent stem cell derived cardiomyocyte (hiPSC-CM) model of the pathogenic MYH7 E848G mutation to study hypocontractile DCM [3]. Using RNAseq, we found a key component of the sarcomere that allows cardiomyocytes to withstand mechanical stress during contraction, desmin, was transcriptionally downregulated in cells expressing the MYH7 E848G mutation. Protein expression of desmin was also reduced in patient cardiac tissue and hiPSC-CMs with MYH7 E848G. Interestingly, murine desmin knockout models develop DCM [4]. Therefore, I hypothesize that restoring typical desmin levels in cardiomyopathic cells with the MYH7 E848G mutation will help recover contractile function.




Materials and Methods:: Pathogenic iPSC-CMs were generated from a patient with dilated cardiomyopathy caused by an E848G MYH7 mutation (MYH7E848G/WT), and wild-type iPSC-CMs were generated from a healthy relative (MYH7WT/WT). Electroporating MYH7E848G/WT iPSC-CMs with a pJet-MYH7-EGFP-PGK-PuroR plasmid with or without the E848G mutation created isogenic lines expressing an enhanced green fluorescent protein (MYH7E848G-EGFP/WT and MYH7WT-EGFP/WT).

At day 33 of culture, RNA was isolated from lactate purified hiPSC-CMs and submitted for bulk RNA sequencing. Differentially expressed genes were identified within patient lines (MYH7E848G/WT relative to MYH7WT/WT ) and isogenic lines (MYH7E848G-EGFP/WT relative to MYH7WT-EGFP/WT). Protein levels were assessed with Western blotting using lysates from day 40 iPSC-CMs and patient cardiac tissue samples.


Results, Conclusions, and Discussions:: Using human induced pluripotent stem cells (hiPSCs) derived from a patient expressing the E848G variant, we established isogenic hiPSC-derived cardiomyocytes (hiPSC-CMs) with and without MYH7 E848G. Using RNAseq, we identified a deficiency in RNA transcripts for desmin in MYH7 E848G hiPSC-CMs (Figure 1A). Desmin protein levels were reduced in human myocardium (Figure 1B), patient hiPSC-CMs (Figure 1C), and isogenic hiPSC-CMs (Figure 1D) expressing the E848G mutation. Correction of the E848G mutation at the genetic level resulted in the recovery of contractile function in hiPSC-CMs and the recovery of both desmin RNA and protein compared to isogenic control hiPSC-CMs, which confirmed the role of the E848G mutation in inducing systolic dysfunction. Given previous studies establishing that a deficiency of desmin is sufficient to cause DCM, these results justify the further study of desmin overexpression in hiPSC-CMs derived from a patient with DCM to recover contractile function.
Ongoing work is focused on (1) restoring desmin protein levels in hiPSC-CMs expressing the MYH7 E848G mutation, and (2) assessing the contractile effects of desmin upregulation on iPSC-CMs by measuring single cell contractile force. I have generated a plasmid that overexpresses a cleavable fluorescent mApple-T2A-desmin fusion protein. The plasmid will be transfected into hiPSC-CMs, followed by flow sorting to isolate mApple+ cells. After confirming increased desmin protein levels, I will determine the effect of desmin overexpression on cardiomyocyte contractility with a single cell assay called traction force microscopy (TFM). TFM is a powerful technique that allows for the quantification of traction stresses generated by cells adherent to a two-dimensional substrate [5]. I will culture single hiPSC-CMs on a polyacrylamide substrate of physiological stiffness (10kPa) containing fluorescent beads which will be used to visualize and quantify the gel deformation caused by the adherent cells. The substrate is micropatterned with matrigel using aspect ratios that will promote physiological organization of sarcomeres [6]. Images of the gel beads will be taken during contraction and relaxation, and a traction force field will be calculated by tracking the fluorescent bead displacement. Thus, I will determine if the contractile function of pathogenic hiPSC-CMs can be restored by transient overexpression of desmin.







Acknowledgements (Optional): :

References (Optional): : 1. de Frutos F, Ochoa J, Navarro-Peñalver M, et al. Natural History of MYH7-Related Dilated Cardiomyopathy. J Am Coll Cardiol. 2022 Oct, 80 (15) 1447–1461.

https://doi.org/10.1016/j.jacc.2022.07.023



2. Yang K, Breitbart A, De Lange W, et al. Novel Adult-Onset Systolic Cardiomyopathy Due to MYH7 E848G Mutation in Patient-Derived Induced Pluripotent Stem Cells. J Am Coll Cardiol Basic Trans Science. 2018 Dec, 3 (6) 728–740.https://doi.org/10.1016/j.jacbts.2018.08.008



3. Ribeiro, A. J., Ang, Y.-S., Fu, J.-D., Rivas, R. N., Mohamed, T. M., Higgs, G. C., Srivastava, D., & Pruitt, B. L. (2015). Contractility of single cardiomyocytes differentiated from pluripotent stem cells depends on physiological shape and substrate stiffness. Proceedings of the National Academy of Sciences, 112(41), 12705–12710. https://doi.org/10.1073/pnas.1508073112

4. Ruppert, T., et al. “Aav-Mediated Cardiac Gene Transfer of Wild-Type Desmin in Mouse Models for Recessive Desminopathies.” Gene Therapy, vol. 27, no. 10–11, 2020, pp. 516–524, https://doi.org/10.1038/s41434-020-0147-7.

5. Kraning-Rush, Casey M., et al. “Quantifying Traction Stresses in Adherent Cells.” Methods in Cell Biology, 2012, pp. 139–178, https://doi.org/10.1016/b978-0-12-388403-9.00006-0.


6.Ribeiro, Alexandre J., et al. “Contractility of Single Cardiomyocytes Differentiated from Pluripotent Stem Cells Depends on Physiological Shape and Substrate Stiffness.” Proceedings of the National Academy of Sciences, vol. 112, no. 41, 2015, pp. 12705–12710, https://doi.org/10.1073/pnas.1508073112.