Abstract P482: Elucidating And Characterizing The Molecular Mechanistic Role Of Phospholamban L39 Stop In The Pathophysiology Of Cardiomyopathy Using Patient-derived Human Induced Pluripotent Stem Cells And Humanized Knock-in Mouse Model Systems

Abstract

Background: Heart failure (HF) is a complex clinical condition associated with substantial morbidity and mortality worldwide. The contractile dysfunction and arrhythmogenesis related to HF has been linked to the remodelling of calcium (Ca ++ ) handling. Phospholamban (PLN) has emerged as a key regulator of intracellular Ca ++ concentration. Of the PLN mutations, L39X is intriguing as it has not been fully characterized. This mutation is believed to be functionally equivalent to PLN null (KO) but contrary to PLN KO mice, L39X carriers develop a lethal cardiomyopathy (CMP). Our study aims at using induced pluripotent stem cell (iPSC)-derived cardiomyocytes (CMs) from homozygous L39X carriers to elucidate the role of L39X in human pathophysiology. Our plan also involves the characterization of humanized L39X knock-in mice (KM), which we hypothesize will develop a CMP from mis-localization of PLN and disruption of Ca ++ signalling. Methodology and Results: Mononuclear cells from Hom L39X carriers were obtained to generate 11 integration-free patient-specific iPSC clones. The iPSC-CMs were derived using established protocols. Compared to the WT iPSC-CMs, the Hom L39X derived-CMs PLN had an abnormal cytoplasmic distribution and formed intracellular aggregates, with the loss of perinuclear localization. There was also a 70% and 50% reduction of mRNA and protein expression of PLN respectively in L39X compared to WT iPSC-CMs. These findings indicated that L39X PLN is both under-expressed and mis-localized within the cell. To validate this observation in-vivo, we genetically modified FVB mice to harbour the human L39X. Following electroporation, positively transfected mouse embryonic stem cells were injected into host blastocysts to make humanized KM that were subsequently used to generate either a protamine-Cre (endogenous PLN driven expression) or a cardiac TNT mouse (i.e., CMP specific). Conclusion: Our data confirm an abnormal intracellular distribution of PLN, with the loss of perinuclear accumulation and mis-localization, suggestive of ineffective targeting to or retention of L39X. The mouse model will be critically important to validate the in-vitro observations and provides an ideal platform for future studies centred on the development of novel therapeutic strategies including virally delivered CRISPR/Cas9 for in-vivo gene editing and testing of biochemical signalling pathways.

The Role of Bone Morphogenetic Protein Receptor Type 2 (BMPR2) and the Prospects of Utilizing Induced Pluripotent Stem Cells (iPSCs) in Pulmonary Arterial Hypertension Disease Modeling

“…Additional studies using loss of function experiments with siRNA-mediated BIRC3 knock-down resulted in enhanced apoptosis in UMC-derived iPSC-ECs, demonstrating how BIRC3 is important for cell survival and as a protective genetic modifier in PAH [ 71 ]. These results are consistent with earlier reports demonstrating how decreased BMPR2 signaling in PAECs is related to elevated apoptosis in response to injury [ 108 ] as well as reduced adhesion and migration [ 109 ].…”

“…Following this remarkable discovery, iPSCs have been effectively differentiated into numerous cell types such as neurons [ 98 , 99 , 100 ], blood cells [ 101 , 102 ], adipocytes [ 103 ], fibroblasts [ 104 , 105 ], endothelial cells [ 101 , 106 ], smooth muscle cells [ 107 ], and cardiomyocytes [ 108 , 109 ]. They have also been originated from subjects with a variety of monogenic diseases [ 90 ] such as HPAH [ 99 ], for which iPSCs have been used to generate pulmonary vascular cells, thus providing a new avenue for disease modeling.…”

Phospholamban p.Leu39* Cardiomyopathy Compared with Other Sarcomeric Cardiomyopathies: Age-Matched Patient Cohorts and Literature Review

“…Different studies suggest that the p.Leu39* PLN mutant is expressed but mis-located within the cardiomyocytes [22,23]. The mis-location of PLN was associated to decreased SERCA2a expression and impaired Ca 2+ handling in human pathophysiology.…”