The high mortality of these LMNA knockout mice restricted the possibility of chronic whole animal study. In addition, differences in cardiac electrophysiological behavior between humans and rodents may hinder the feasibility of translating pathophysiological discoveries into clinical practice. The mechanisms by which different LMNA mutations cause AV block or DCM remain uncertain. An in-vitro platform of human cardiomyocytes derived from patients with different LMNA mutations would be extremely useful for understanding disease mechanisms under stress conditions such as electrical field stimulation and mechanical stretch, as well as a hypoxic environment, and hence developing patient-specific therapies.

The recent breakthrough of human iPSCs generated from adult somatic tissues [

37

,

38

] provides a unique opportunity to produce patient-specific cardiomyocytes for disease modeling and drug screening [

39

41

] (Table

1

and Fig.

2

). Since iPSCs are genetically identical to the host bearing cardiac defeats, the iPSC-derived cardiomyoctes provide an attractive experimental platform to recapitulate cellular phenotypes of familial heart diseases such as arrhythmias and cardiomyopathies. This will provide new insights into disease-modifying mechanisms and enable the specific design of personalized therapeutic strategies.

Fig. 2

Schematic summary of existing cardiac laminopathy human iPSC modeling and future studies to understand the disease mechanism, drug screening, and interventions. HGPS Hutchinson Gilford progeria syndrome, miR microRNA, MLK Mixed-lineage kinases. [57, 58]

In 2011, Liu et al. [42] began to use human iPSCs for HGPS modeling. HGPS is caused by a single point mutation in the lamin A (LMNA) gene, resulting in the generation of progerin, a truncated splicing mutant of lamin A. The level of progerin accumulates with ages and leads to various ageing-associated nuclear defects including disorganization of the nuclear lamina and loss of heterochromatin. The reversible suppression of progerin expression by reprogramming was resumed upon differentiation with ageing-associated phenotypic consequences. The HGPS-iPSCs derived from skin fibroblasts showed an absence of progerin and more importantly lacked the nuclear envelope and epigenetic alterations normally associated with premature ageing. Nevertheless, the appearance of premature senescence phenotypes in HGPS-iPSC-derived smooth muscle cells (SMCs) was associated with vascular ageing. Additionally, they identified a DNA-dependent protein kinase catalytic subunit (DNAPKcs, also known as PRKDC) as a downstream target of progerin. The absence of nuclear DNAPK holoenzyme correlated with premature as well as physiological ageing. Others have reported the use of a human iPSC platform to model the disease phenotypes of HGPS in mesenchymal lineages and SMCs [42, 43]. Ho et al. as well as Liu et al. generated progeria iPSCs from skin fibroblasts of a patient bearing a mutation in LMNA [42, 44]. They proved that the human iPSC-derived fibroblasts are able to recapitulate the disease phenotype with prominent nuclear blebbing, are capable of cell senescence, and are susceptible to external stimulation (e.g., electrical field stimulation as the donor cells). Liu et al. showed that premature vascular ageing was probably due to accumulation of progerin in SMCs. Later, Blondel et al. in 2014 further investigated the translational aspect using iPSCs to reveal functional differences between drugs currently investigated in patients with HGPS. They trialed a farnesyltransferase inhibitor in combination with a statin (zoledronate and pravastatin), and the macrolide antibiotic rapamycin. This study revealed that a systematic cytostatic effect was observed in the treatment group with the farnesyltransferase inhibitor alone [45]. The investigators provide new insights into drug efficacy in functional improvement of prelamin A farnesylation that generates cytotoxic progerin, nuclear architecture, improvement in cell proliferation, as well as energy metabolism; in other words, ATP synthesis. This finding further proved iPSCs to be powerful tools for standardized and comparative pharmacological studies.

In 2012, our group subsequently generated another human iPSC platform from a patient bearing a premature termination codon in the LMNA gene, R225X. Although no clear nuclear phenotype was observed in iPSCs from the DCM patient with the LMNA mutation, several cellular phenotypes were observed in the human iPSC-derived cardiomyocytes, including nuclear morphology abnormality (blebbing), slow proliferation, improved cellular senescence, and increased incidence of apoptosis under electrical stimulation. Under field electrical stimulation to mimic the native cardiac environment, the percentage of LMNA‐mutated iPSC cardiomyocytes that exhibited nuclear senescence and cellular apoptosis markedly increased. shRNA knockdown of LMNA, resembling the halploinsufficiency situation of the R225X mutant, replicated those phenotypes of the mutated LMNA field electrical stress. We also demonstrated the central role of the MAPK–extracellular signal-regulated kinase-1 (MEK1) pathway in governing susceptibility to cardiac cell stress-response. Blockage of the extracellular signal-regulated kinase (ERK) pathway by MEK1 inhibitors attenuated the electrical stimulation-induced proapoptotic phenotypes of DCM iPSC cardiomyocytes [6]. ERK1/2 are activated directly by the upstream MEK1/2, which are dual-specificity protein kinases. Activated ERK1/2 kinases phosphorylate and activate a variety of substrates, which can be transcription factors, protein kinases and phosphatases, cytoskeletal and scaffold proteins, receptors and signaling molecules, and apoptosis-related proteins. Numerous MEK1/2 inhibitors have progressed into clinical trials since the identification of the first MEK inhibitor, PD098059 [46]. Most of these MEK1/2 inhibitors are ATP noncompetitive and bind to a unique allosteric site adjacent to the ATP site. Apart from pharmacological treatment of LMNA mutation-related disease, there were new breakthroughs in gene editing technologies for correction of laminopathy-associated LMNA mutations in patient-specific iPSCs. However, Liu et al. [42] discovered that the LMNA gene was transcriptionally inactive and would impede targeted gene editing. They further explored using helper-dependent adenoviral vectors (HDAdVs) as a robust and highly efficient vehicle for the delivery of gene editing tools. In comparison with the conventional piggybac method, the advantage of this system is the inclusion of a negative selection step by ganciclovir (GNAC) resistance to eliminate random insertion of clones that contain the HSVtk cassette. The resultant corrected HPGS iPSCs were essentially proved to be genetically identical to fibroblasts as well as epigenetically similar to the uncorrected clones. Such a new method would enhance the reliability of gene correction as a therapeutic tool to rescue the disease phenotype for cell therapies or to generate a patient-matched control for disease modeling and further the dissected disease causal target for drug discovery [47].

In fact, somatic reprogramming of the progeria patient-specific cell to a human iPSC is not an easy task with the considerable drawback of low efficiency of stem cell clone formation. The stress of premature aged cells was basically due to oxidative stress-related NF-kB activation, which blocks the generation of iPSCs and MSC differentiation. Soria-Valles et al. discovered that NF-kB repression occurred during reprogramming towards a pluripotent state. In contrast, the hyperactivation of NF-kB impaired the process though DOT1L, a histone H3 methyltransferase, which reinforced the senescence signals [48]. In the light of such observations, the authors demonstrated attenuating the NF-kB signal via direct or upstream DOT1L inhibition before somatic reprogramming, which also extended the lifespan and ameliorated the accelerated ageing phenotype in the animal model. Chronic treatment of NF-kB inhibition, an anti-inflammatory compound, may produce side effects. Besides, DOT1L inhibitors have recently been tested for the treatment of hematological malignancies, which suggests a better solution for age-associated diseases [49].

Apart from epigenetic profiling, the tissue-specific expression profile of miR may provide clues for laminopathy therapies. miR-9 was specifically expressed in neuronal cells derived from HGPS patients, which exerted a protective role of the miR specifically to preserve cognitive function [50]. The miR-9 acting 3′-untranslated region (UTR) of lamin A suppresses its expression level, thus reducing accumulation of prelamin A, which generates progerin. The direct role of miR-9 on lamin A gene expression was further confirmed by anti-miR-9 treatment (loss of function) or transfection with pre-miR-9 (gain of function) in the HGPS iPSC-MSC. Future studies on cardiac-specific laminopathy intervention could be focus on inhibiting miR-9 or other cardiac-specific miR targeting on the 3′-UTR of LMNA.