Introduction

Dilated cardiomyopathy (DCM) is characterized by left ventricle dilatation, ventricular dysfunction, arrhythmias and heart failure. Up to 50% of DCM cases could be attributed to the genetic basis, called familial DCM. Lamin A/C (LMNA) mutations share about 6% of familial DCM. DCM arising from LMNA mutations tend to have an early onset, aggressive phenotypes and further comprise multiple system disorders such as heart, adipose tissue, nervous system and accelerated ageing syndrome. The first line of treatment is mostly symptomatic and based on standard heart failure therapies. A lack of a proper animal model further hinders the understanding of molecular mechanisms. We therefore aimed to recapitulate a patient specific LMNA-knock-in (KI) mutation causing severe DCM to analyse the molecular pathways, both in-vitro and in-vivo, that are characteristic of a LMNA-KI DCM patient.

 

Methods and results

A NYHA class IV DCM patient with a familial LMNA missense mutation (NM_170707.3: c481 G>A: p. Glu161Lys) presented in our hospital. The patient conditions deteriorated rapidly despite maximum heart failure and cardiac resynchronization therapy. With laboratory evidence of increased mTOR activity, off-label rapamycin, an mTOR inhibitor, was administered at a serum concentration of 8ng/ml as an individualized treatment approach with fair success. The conditions improved from class IV to class II, NT-BNP serum concentration dropped from 8120ng/l to 2210ng/l, and left-ventricular ejection fraction increased from 27.8% to 44.5%. To better understand mTOR signaling pathway in DCM associated with laminopathies, we exploited the CRISPR-Cas9 system to introduce the patient specific mutation at the corresponding site of the mouse genome. 

 

We designed single guide RNAs and ligated them into two different plasmids pSpCas9(BB)-2A-GFP (PX458) and pSpCas9 (BB)-2A-Puro V2.0 (PX459) and performed gene editing in R1/E mouse embryonic stem (mES) cells (ATCC). The generation of positive knock-in cells were confirmed by Sanger sequencing. Furthermore, wild-type and mutated mES cells were differentiated into cardiomyocytes. Our results show normal cardiomyocyte morphometry, however, differential expressions of mesoderm marker (Gata4), cardiomyocyte marker (cTNT) and markers specific to atrium and ventricle (Mlc2a and Mlc2v) from cardiomyocyte differentiated from LMNA-KI mES cells. Furthermore, mRNA as well as protein expressions of mTOR adaptor protein Rptor, subunits of mTOR - Mlst8, mTOR non-core component - PRAS40, and downstream mTOR effector - 4Ebp1 were upregulated. Angiotensin II stimulation (200nM) of differentiated cardiomyocytes from LMNA-KI mES cells resulted in augmented mTOR signaling pathway (increased phosphorylation of mTOR, p70S6K and 4E-BP1) which were effectively abbreviated by mTOR-inhibitor rapamycin (200nM). 

 

Conclusion

We have successfully generated a mouse LMNA-KI cell line using the CRISPR-Cas9 technology which simulates the exact patient-KI mutation for LMNA related DCM. Our in-vitro studies suggest involvement of mTOR signaling pathway in DCM associated with laminopathies. We further plan to generate and characterize LMNA-KI mouse line as a novel model to recapitulate the complex LMNA related DCM in human, further investigate the mTOR signaling in preclinical model and add to the current advancement on research and treatment of clinical DCM. mTOR inhibition might be a valuable strategy for specific DCM patients with laminopathy.