Personalized Medicine Approach in a DCM Patient with LMNA Mutation Reveals Dysregulation of mTOR Signaling

Background: Mutations in the Lamin A/C (LMNA) gene are responsible for about 6% of all familial dilated cardiomyopathy (DCM) cases which tend to present at a young age and follow a fulminant course. Methods: We report a 47-year-old DCM patient with severely impaired left ventricular ejection fraction and NYHA functional class IV despite optimal heart failure treatment. Whole-exome sequencing revealed an LMNA E161K missense mutation as the pathogenetic cause for DCM in this patient. We generated a patient-specific LMNA-knock in (LMNA-KI) in vitro model using mES cells. Results: Beta adrenergic stimulation of cardiomyocytes derived from LMNA-KI mES cells resulted in augmented mTOR signaling and increased dysregulation of action potentials, which could be effectively prevented by the mTOR-inhibitor rapamycin. A cardiac biopsy confirmed strong activation of the mTOR-signaling pathway in the patient. An off-label treatment with oral rapamycin was initiated and resulted in an improvement in left ventricular ejection fraction (27.8% to 44.5%), NT-BNP (8120 ng/L to 2210 ng/L) and NYHA functional class. Conclusion: We have successfully generated the first in vitro model to recapitulate a patient-specific LMNA E161K mutation which leads to a severe form of DCM. The model may serve as a template for individualized and specific treatment of heart failure.


Highlights
Elevated mTOR signaling was evident in a DCM patient with an LMNA E161K mutation as well as in cardiomyocytes derived from mouse embryonic stem (mES) cells with the patient-specific LMNA mutation. LMNA E161K mutation resulted in impaired electrophysiological properties and disorganisation of nuclear lamin.
The mTOR-inhibitor rapamycin lead to a rapid reversal of the phenotype in vitro and also improved the clinical status of the patient with DCM and an LMNA E161K mutation.

Introduction
Dilated cardiomyopathy (DCM) is characterized by left ventricular dilatation and impaired contractility, ventricular and atrial arrhythmias, and a shortened life expectancy due to sudden death or progressive heart failure [1]. Around 20-30% of cases of DCM are thought to be attributable to familial forms. The molecular mechanisms and their genetic origins driving the DCM pathology are complex, due to the possible involvement of more than 40 genes encoding proteins of diverse function and localization such as cytoskeleton, sarcomere, nuclear membrane proteins, and transmembrane phosphoproteins [2,3]. One well-recognized genetic abnormality is that of lamin (LMNA), a series of proteins that provide structural support to the nucleus and assist in chromatin organization, gene regulation, DNA replication, and RNA splicing [4][5][6]. When compared with other known genetically-driven cardiomyopathies, those associated with LMNA mutations are associated with a poor clinical outcome [7]. Although a comprehensive analysis of familial history and a genetic assessment are important in assessing the prognosis of the proband and other family members [8] despite considerable enthusiasm for what might be possible, currently this information has no bearing on treatment [9].
Genetic defects result in a wide variety of alterations of proteins which are thought to either lead directly to, or contribute to susceptibility to, pathological cardiac remodeling. However, there is a significant knowledge gap in the nature of the molecular abnormalities and how these translate to the physiological or pathological remodeling process. As a result, there is as yet, little practical utility of genetic approaches in the treatment of DCM [10]. Specifically, in vivo research on LMNA mutations is further hindered by the lack of an optimal animal model for LMNA cardiomyopathies and also that the clinical phenotypes resulting from LMNA mutations, also known as laminopathies, are highly variable due to the involvement of other organ systems and accelerated aging syndrome (progeria) [7]. A hierarchical cluster analysis of 91 mutations in the LMNA gene revealed that the organ involvement, type, and severity depend upon the region of mutation in the gene [11].
Here, we report a case of a 47-year-old male patient with severe heart failure due to reduced ejection fraction (HFrEF). He had marked left ventricular (LV) dilatation and systolic dysfunction, New York Heart Association functional class IV symptoms, and persistently raised natriuretic peptide despite optimally tolerated medical therapy.
Whole-exome sequencing revealed a missense mutation (LMNA E161K) as the molecular cause for DCM in the family. This particular mutation is associated with a fulminant course [12] fitting with the clinical situation. This mutation has been associated with abnormal nuclear membrane assembly, genomic clustering of mutated genes, significant changes in nuclear localization and redirected internal areas of the nucleus [13].
Given the severe phenotype associated with this mutation, we aimed to create a patient-specific knock in (KI)-in vitro model to serve as a unique model with which to explore pathological mechanisms and to test individualized treatment approaches.

Cell Culture
NIH/3T3 cells (ATCC, CRL-1658 TM ) were used for single guide RNA (sgRNA) optimization. Mouse embryonic fibroblasts (MEFs) (ATCC, SCRC-1008 TM ) were used as feeders for mouse embryonic stem (mES) cells. R1/E mES cells (ATCC, SCRC-1036 TM ) were used as wild type (WT) control and for the generation of a patient-specific knock in the LMNA mutation cell line. All cells were handled and cultured according to the protocols provided by ATCC. MEFs were mitotically arrested by irradiation.
Electroporation protocol: NIH/3T3 or WT mES cells were mixed with CRISPR-Cas9 components (IDT-DNA, Leuven, Belgium) and electroporated via Amaxa TM 4D-Nucleofector TM (Lonza, Cologne, Germany) using the manufacturer's guide. The extended electroporation protocol is included in the Supplementary Methods.
Surveyor nuclease assay: sgRNAs were first validated in NIH/3T3 cells via heteroduplexes formed by CRISPR-Cas9 mediated gene editing, reaction buffer, and T7E1 endonuclease (NEB, Frankfurt, Germany). sgRNA with the highest efficacy was used for WT mES cell gene edit.
After electroporation, mES cells were seeded, single colonies were picked, expanded, and further assessed via PCR and restriction digestion. This was done by the Hind III restriction enzyme (NEB, Frankfurt, Germany). Positive clones were sent for Sanger sequencing (LGC genomics, Berlin, Germany).

Cardiomyocyte Differentiation of mES Cells
WT or LMNA-KI mES cells were differentiated into cardiomyocytes by the previously described hanging drop method [14,15]. Briefly, mES cells were grown in mES cell medium on MEF feeder cells. After two passages, cells were trypsinized, centrifuged, re-suspended in differentiation medium, plated for 30 min to get rid of feeders, plated on the lid of Petri dishes, inverted, and cultured for 3 days. After 3 days, the embryoid bodies were transferred to gelatin-coated 6-well-plates and cultured for the next 12 days. The contracting cells were visible from day 4 onwards which peaked on days 10-12.

Real-Time PCR, Western Blot and Immunohistochemistry
Evaluation of mRNA expression by real-time PCR and detection of protein expressions by Western blot or immunohistochemistry were performed by standard methods. Extended protocols are included in the Supplementary Methods S1.

Electrophysiological Recordings
Standard whole-cell patch-clamp technique was used under the current-clamp configuration, to record repetitive action potentials during spontaneous contractions of WT cardiomyocytes (WT.CMs) or cardiomyocytes derived from LMNA-KI mES cell (LMNA-KI.CMs) at day 12. Before the patch clamp experiments, the cardiomyocytes were grown on coverslips coated with 0.2% gelatin. The coverslips were then mounted on the stage of an inverted phase-contrast microscope (Olympus IX71, Olympus Corporation, Tokyo, Japan) and prepared for patch clamp. Cells were perfused with Iscove's 20% FBS medium using an 8-channel gravity-automated perfusion system, cFlow (Cell MicroControls, Norfolk, VA, USA). A random subsample of cells was separated and incubated for 24 h with isoprenaline (2 µM final concentration) or with isoprenaline plus rapamycin (500 nM final concentration) before the measurements. Micropipettes (4-6 MΩ) were prepared from borosilicate glass capillaries with a micropipette puller (DMZ-Universal Electrode Puller; Zeitz Instruments, Martinsried, Germany) and filled with an intracellular solution containing (in mM): 120 KCl, 2 MgCl 2 , 10 HEPES, 10 EGTA and 3 Mg-ATP (pH 7.2). Action potentials were directly recorded from spontaneously firing cardiomyocytes by current clamp without electrical stimulus, using an Axon-200B amplifier (Molecular Devices; San Jose, CA, USA) and an Axon Digidata 1440 A acquisition system controlled by Clampex 10.0 (pClamp 10.0, Molecular Devices, Sunnyvale, CA, USA). Signals were recorded with a 5 kHz sampling rate, and digital data were stored in a compatible PC for off-line analysis using Clampfit 10.0 (pClamp 10.0, Molecular Devices, Sunnyvale, CA, USA).

Video Imaging
The cardiomyocytes differentiated from WT and LMNA-KI mutant mES cells were seeded in 6-well-plates. Videos of each well for the contracting cardiomyocytes were taken after 12 days. Each beating was recorded for 1 min. Videos were captured with a camera (The Imaging Source DFK 72AUC02) mounted onto a Leica DM IL LED microscope. The analysis of the video recordings took place in a blinded manner to remove any risk of bias in the assessment.

Patient Study
The patient gave his informed consent to participate in this study. The study was approved by the local ethics committee of the University of Tübingen (Approval code: 270/2011 B01). The investigation conformed to the principles outlined in the Declaration of Helsinki.

Statistical Analysis
All values are presented as mean ± SEM. Unpaired t-test or one-way ANOVA with Tukey's post hoc analysis was used. A p-value of <0.05 was considered to be significant. All data were analysed using GraphPad Prism 9.0 (GraphPad Software, Inc., San Diego, CA, USA).

Identification of a DCM Patient with an LMNA Mutation
A 47-year-old male patient was referred to our hospital for further evaluation with a long-standing diagnosis of DCM since 2011. Despite optimal guideline-directed heart failure therapy (ramipril 5 mg od, bisoprolol 5 mg od, spironolactone 25 mg od, and torasemide 20 mg od) and the implantation of a cardiac resynchronisation (CRT-D) device in 2015, the patient had severe symptoms, persistent left ventricular dilation (LV end diastolic diameter 58 mm) and reduced left ventricular ejection fraction of 27%. Immunoadsorption of beta-1 adrenergic autoantibodies also failed to improve the clinical status which was further exacerbated by co-morbidities, including myelodysplastic syndrome and recurrent pulmonary embolisms. In addition, coronary angiography excluded the presence of coronary artery disease.
Since the patient had a family history of DCM with his father and his daughter also affected, whole-exome sequencing was performed, which revealed a missense mutation in the Lamin A/C (LMNA) gene (NM_170707.3: c481 G > A: p. E161K) as the molecular cause for DCM in the family ( Figure 1). To further understand the mechanisms of the disease and to establish an individualized treatment approach, we decided to analyse this specific patient mutation in vitro using an mES cell line.  Figure 2d and e represent WT mES cells colonies and the hanging drop method respectively. The WT mES cells from now on would be referred to as WT and the patient-specific mutation will be referred to as LMNA-KI.   Figure 2d and e represent WT mES cells colonies and the hanging drop method respectively. The WT mES cells from now on would be referred to as WT and the patient-specific mutation will be referred to as LMNA-KI.

Generation of CRISPR-Cas9 Mediated Patient Specific Knock-in Mouse Embryonic Stem Cells and Cardiomyocyte Differentiation
Efficacies of different sgRNAs were validated in NIH/3T3 cells via the Surveyor nuclease assay (Figure 2a Figure 2d and e represent WT mES cells colonies and the hanging drop method respectively. The WT mES cells from now on would be referred to as WT and the patient-specific mutation will be referred to as LMNA-KI.    Table S1, Supplementary Result S1, and Supplementary Figure S1). WT.CMs are depicted as seen by brightfield microscopy (Figure 2f) and by fluorescence microscopy after cardiomyocyte specific actinin antibody staining (Figure 2g).

LMNA-KI Cardiomyocytes Are Associated with Increased Nuclear Blebbing and Disorganized Lamin Distribution
Lamins are fundamental proteins that support nuclear stability, shape, structure, and pore positioning [12,16]. To test whether LMNA-KI affects nuclear morphometry, the organization of the nuclear lamina and the proportion of cardiomyocytes with nuclear blebbing were assessed (Figure 4a-c). Cardiomyocytes with LMNA-KI mutation had a significantly higher proportion of disorganized lamin distribution (WT 8.16%, LMNA-KI 17.71%, p < 0.0001) (Figure 4a,b) as evidenced by measuring the intensity of lamin through the nucleus (Figure 4d-g). Similarly, the percentage of nuclear blebbing was also significantly higher in cardiomyocytes derived from LMNA-KI mutated cells (WT 2.01%, LMNA-KI 11.34%, p < 0.0001) (Figure 4a,c).

LMNA-KI Cardiomyocytes Show Increased Functional Dysregulation under Isoprenaline Stress That Can Be Prevented by mTOR Inhibition
To characterize the effects of an mTOR inhibitor on the functional activity of WT.CMs and LMNA-KI.CMs, we examined the electrophysiological properties of single cells (Figure 5). Spontaneous action potential (AP) activity was recorded from cardiomyocytes without treatment and after 24 h of incubation with either β-adrenoceptor agonist isoprenaline alone or isoprenaline plus rapamycin (Figure 5a). Isogenic WT.CMs displayed regular APs with a firing frequency of 39.7 ± 1.6 APs/min, and contractions with a frequency of 34.8 ± 2.5 min −1 . In comparison, LMNA-KI.CMs exhibited an increased frequency of APs (49.5 ± 2.4 APs/min) and of contractions (40 ± 2.9 min −1 ) (Figure 5b,c, Supplementary videos S1-S5). Additionally, LMNA-KI.CMs also showed delayed afterdepolarization-like arrhythmias that appeared with a frequency of 13.5 ± 2.2 min −1 (Figure 5d). These afterdepolarization-like arrhythmias are illustrated in a phase plot in Figure 5e. The variability of individual APs of WT.CMs and LMNA-KI.CMs are illustrated in Supplementary Figure S2. The electrophysiological characteristics of LMNA-KI.CMs are consistent with a previous report of other LMNA mutants [17]. After 24 h of incubation with isoprenaline, WT.CMs displayed an increase in the frequency of APs (51.5 ± 2.2 APs/min)  (panels a,d,f). Additionally, nuclear lamin organization (b) and nuclear blebs (c) were quantified by counting the cells per field of the 40× microscopic view. Figures are representative of fully (e) or partially (g) organized nuclear lamin. Scale bar: 10 µm. An unpaired t-test was used. Data are shown as mean ± SEM (n = 80). **** p < 0.0001, ** p < 0.01.

LMNA-KI Cardiomyocytes Show Increased Functional Dysregulation under Isoprenaline Stress That Can Be Prevented by mTOR Inhibition
To characterize the effects of an mTOR inhibitor on the functional activity of WT.CMs and LMNA-KI.CMs, we examined the electrophysiological properties of single cells ( Figure 5). Spontaneous action potential (AP) activity was recorded from cardiomyocytes without treatment and after 24 h of incubation with either β-adrenoceptor agonist isoprenaline alone or isoprenaline plus rapamycin (Figure 5a). Isogenic WT.CMs displayed regular APs with a firing frequency of 39.7 ± 1.6 APs/min, and contractions with a frequency of 34.8 ± 2.5 min −1 . In comparison, LMNA-KI.CMs exhibited an increased frequency of APs (49.5 ± 2.4 APs/min) and of contractions (40 ± 2.9 min −1 ) (Figure 5b,c, Supplementary Videos S1-S5). Additionally, LMNA-KI.CMs also showed delayed afterdepolarizationlike arrhythmias that appeared with a frequency of 13.5 ± 2.2 min −1 (Figure 5d). These afterdepolarization-like arrhythmias are illustrated in a phase plot in Figure 5e. The variability of individual APs of WT.CMs and LMNA-KI.CMs are illustrated in Supplementary Figure S2. The electrophysiological characteristics of LMNA-KI.CMs are consistent with a previous report of other LMNA mutants [17]. After 24 h of incubation with isoprenaline, WT.CMs displayed an increase in the frequency of APs (51.5 ± 2.2 APs/min) and of contractions (48.1 ± 4.4 min −1 , p < 0.05) and delayed afterdepolarizations appeared at a frequency of 7.5 ± 2.4 min −1 (Figure 5b-d). Stimulation of LMNA-KI.CMs with isoprenaline even further increased all the analysed parameters compared with WT.CMs (67 ± 4.9 APs/min; 60.9 ± 3.2 contractions/min, p < 0.05; 30.4 ± 7.8 afterdepolarizations/min, p < 0.01) (Figure 5b-d). Importantly, the higher frequency of APs and contractions of LMNA-KI.CMs treated with isoprenaline were completely prevented by rapamycin co-treatment (44.5 ± 6.0 APs/min, p < 0.01; 40.4 ± 0.9 contractions/min, p < 0.0001), such that the values were comparable to WT.CMs without isoprenaline stimulation (p < 0.9) (Figure 5b,c). Strikingly, after rapamycin treatment, we also observed almost no afterdepolarization-like arrhythmias (1.0 ± 0.5 min −1 ; Figure 5a,d). These results suggest that the mTOR signaling pathway could be a target to modify the pro-arrhythmic activity of LMNA-KI.CMs.

Relating the In-Vitro Data to Human Tissue
On the evidence of mTOR activation in our in vitro model, we obtained cardiac biopsies from the patient. Masson's trichrome staining revealed interstitial fibrosis (Figure 6a). Immunohistochemical stainings using an anti-phospho-mTOR antibody showed clear mTOR activation (Figure 6b), and macrophage infiltration as detected using an anti-HLA-DR antibody (Figure 6c) in the cardiac samples. Figure 6d-

Gradual Recovery of the Patient with Lamin-Based DCM by Off-Label mTOR-Inhibition
Since all conventional treatment regimens were exhausted (the patient was not suitable for heart transplantation or left ventricular assist device implantation due to his preexisting comorbidities) and the patient remained in severe clinical heart failure, an interdisciplinary team consisting of cardiologists, cardiac surgeons, geneticists, and anesthesiologists discussed the case in a "personalized medicine grand-round". It was decided that based on the findings, that mTOR inhibition with rapamycin could be attempted in this case.
Following informed, written consent, oral rapamycin at the serum concentration of 8

Gradual Recovery of the Patient with Lamin-Based DCM by Off-Label mTOR-Inhibition
Since all conventional treatment regimens were exhausted (the patient was not suitable for heart transplantation or left ventricular assist device implantation due to his pre-existing comorbidities) and the patient remained in severe clinical heart failure, an interdisciplinary team consisting of cardiologists, cardiac surgeons, geneticists, and anesthesiologists discussed the case in a "personalized medicine grand-round". It was decided that based on the findings, that mTOR inhibition with rapamycin could be attempted in this case.
Following informed, written consent, oral rapamycin at the serum concentration of 8 ng/mL was administered for a total of 6 months. After 6 months, the patient's symptoms had improved to NYHA class II and he was able to return to work. This was accompanied by a drop in the serum NT-pro-BNP concentration (from 8120 ng/L to 2210 ng/L), and an improvement in LVEF from 28% to 45% (Figure 6g,h). The patient was discharged from the hospital and was able to resume his work. Relevant side effects of rapamycin were not observed.

Discussion
Our work has shown four important observations. We found that cardiomyocytes differentiated from LMNA-KI mES cells containing the patient-specific LMNA E161K mutation showed increased mTOR phosphorylation when stimulated with isoprenaline. We were also able to reveal a higher percentage of nuclear disorganization and nuclear blebbing in LMNA-KI cardiomyocytes. Finally, we showed that mTOR inhibition prevented functional dysregulation in vitro (Figure 7), and that, using this information, mTOR inhibition led to a remarkable improvement in clinical status in a patient with the LMNA E161K mutation and severe heart failure. As such our data uniquely describe the complete process from genetic analysis through to bedside application of the findings. improvement in LVEF from 28% to 45% (Figure 6g,h). The patient was discharged from the hospital and was able to resume his work. Relevant side effects of rapamycin were not observed.

Discussion
Our work has shown four important observations. We found that cardiomyocytes differentiated from LMNA-KI mES cells containing the patient-specific LMNA E161K mutation showed increased mTOR phosphorylation when stimulated with isoprenaline. We were also able to reveal a higher percentage of nuclear disorganization and nuclear blebbing in LMNA-KI cardiomyocytes. Finally, we showed that mTOR inhibition prevented functional dysregulation in vitro (Figure 7), and that, using this information, mTOR inhibition led to a remarkable improvement in clinical status in a patient with the LMNA E161K mutation and severe heart failure. As such our data uniquely describe the complete process from genetic analysis through to bedside application of the findings. This dysregulation causes protein translation imbalance, lamin disorganization, and nuclear blebbing and adds to the disease phenotype. mTOR-inhibitor rapamycin is effective in checking the cellular effects of LMNA mutation.

Dilated cardiomyopathy (DCM)
Although DCM is a clinical syndrome with a characteristic phenotype, it can be the result of multiple underlying pathophysiologies most of which are poorly understood. Around 6% of cases of the familial form of DCM are the consequence of mutations in the nuclear membrane protein Lamin A/C [11]. Patients with LMNA mutations frequently Figure 7. Schematic diagram of a putative signaling pathway in cardiomyocytes with LMNA mutation. Factors like age or stimulants affect the gene expression of LMNA mutant cells. Dysregulation of gene expression from mutant alleles causes higher activation of the mTOR signaling pathway. This dysregulation causes protein translation imbalance, lamin disorganization, and nuclear blebbing and adds to the disease phenotype. mTOR-inhibitor rapamycin is effective in checking the cellular effects of LMNA mutation.

Dilated Cardiomyopathy (DCM)
Although DCM is a clinical syndrome with a characteristic phenotype, it can be the result of multiple underlying pathophysiologies most of which are poorly understood. Around 6% of cases of the familial form of DCM are the consequence of mutations in the nuclear membrane protein Lamin A/C [11]. Patients with LMNA mutations frequently suffer a fulminant heart failure syndrome along with multiple organ dysfunction [7]. There are no approved personalized approaches to the management of DCM, rather standard therapy follows the 'four pillars' approach recommended by the European Society of Cardiology for HFrEF (ESC HF guidelines 2021).

Generating the Specific Mutation in Wild-Type Cells
We exploited the CRISPR-Cas9 gene editing technology to generate the patient LMNA-KI mutation in WT mES cells. CRISPR-Cas9 is increasingly gaining popularity to create experimental models to study pathogenicity and cellular changes arising from gene mutations [18]. WT and LMNA-KI mutated mES cells were further differentiated into cardiomyocytes using the previously described hanging drop method [14]. This protocol is a standard method of cardiomyocyte differentiation via embryoid body formation [19,20]. Cardiomyocytes derived from both WT and LMNA-KI mutated mES cells were examined for markers of several cell types ranging from early embryonic ectoderm to cardiomyocytes. Significant upregulation of Oct3/4 and Nanog, both markers for early embryonic ectoderm [21,22], in undifferentiated cells, show the degree and specificity of mES cells. On the other hand, markers for cardiomyocyte differentiation, Gata4, Tbx20, cTNT, α-Myh6, Mlc2a, and Mlc2V, were significantly upregulated in differentiated WT and LMNA-KI cells. Tbx20, which is expressed within E7.5, has been known to interact with several other proteins such as Nkx2-5, Gata4, and Gata5 and regulates the expression of various cardiac genes as well as coordinates cell proliferation and the process of cardiac differentiation [23]. Also, in mature cardiomyocytes, the expression of cTNT and α-Myh6 is crucial to maintaining the normal heart function [21,24]. Atrial and ventricular subtypes of cardiomyocytes can be distinguished by the expression of either Mlc2a (for atrial cardiomyocytes) or Mlc2V (for ventricular cardiomyocytes). However, these markers are not completely specific, and can be present in both lineages [25]. Upregulation of cardiomyocyte-specific gene markers, thus, shows successful cardiomyocyte differentiation from WT or LMNA-KI mutated mES cells.

The Morphological Effects of LMNA-K1
Since lamins are important proteins to support the shape, size, and stability of the nucleus [12,17], we examined if the heterozygous allele impacts the organization of the nucleus. Interestingly, cardiomyocytes derived from LMNA-KI mES cells showed a higher proportion of disorganized lamin distribution as well as a higher percentage of nuclear blebbing. Morphological changes in abnormal forms such as nuclear blebbing, herniations, honeycomb/donut-like structures or any combinations of these categories in the nucleus due to mutations in the LMNA gene are well described. Smaller volumes of chromosome 13 and abnormal nuclear shapes have been described in LMNA E161K mutant fibroblast [13]. However, these abnormalities are dependent on the type of cells, and location of the mutation and are not observed in all laminopathic cells [26]. These dysmorphic nuclei are also reported in fibroblasts or lymphoblastoid cells obtained from an LMNA mutated patient [27] indicating their clinical significance.

The Electrophysiological Effects of LMNA-K1
Electrophysiologically, the LMNA-KI mutation generates an increased pro-arrhythmic activity in the cardiomyocytes derived from mES cells, characterized by an increased AP frequency, delayed afterdepolarizations, and an increase in the contraction frequency; this arrhythmic phenotype is similar to another LMNA mutation [17]. The pro-arrhythmic activity of LMNA-KI.CMs became severe after 24 h of treatment with the non-selective β-adrenoceptor agonist isoprenaline and with higher activation of the mTOR signaling pathway. Interestingly, we observed that with rapamycin treatment, we were able to completely recover the electrical activity of LMNA-KI.CMs to that of WT.CMs. Rapamycin, in this context, allowed us to explore the possibility to use a specific inhibitor of the mTOR signaling pathway as a pharmacological treatment for LMNA-related cardiomyopathy.
Cardiomyocytes generated from mES cells were further subjected to the non-selective β-adrenoceptor agonist isoprenaline. We have previously used isoprenaline as a factor to initiate DCM [1,28]. Cardiomyocytes derived from LMNA-KI mES cells showed increased phosphorylation of mTOR complex-1 (mTORC1) and 4EBP1 whereas, phosphorylation of mTOR complex-2 remained unaffected (Supplementary Figure S3). Thus, this study confirms an increased degree of the mTORC1 signaling pathway activation in the LMNA E161K mutation. We, therefore, opted for rapamycin to inhibit this signaling pathway. Our results showed a clear and strong inhibition of mTORC1, P70S6K, and 4EBP1 phosphorylation with rapamycin application in cardiomyocytes stimulated with isoprenaline. Preclinical studies in LMNA knock-out mice have shown the efficacy of rapamycin in preventing elevated mTORC1 signaling, thereby providing a potential remedy for the cure of DCM, skeletal muscle dystrophy, and lipodystrophy, and significantly increasing the survival time [29].

Clinical Application
With laboratory evidence of increased mTOR activity and reversal of cellular functions by mTOR inhibitor, and supported by the hospital ethics board, we administered the mTOR inhibitor rapamycin, in a compassionate and off-label application to the LMNA-KI patient. This was followed by a steady improvement in clinical status and cardiac function on echocardiography.
We acknowledge that our work has limitations. First, although electrophysiological measurements revealed the typical character of cardiomyocytes, we cannot exclude that these cells have not or cannot become fully mature adults CMs under the current defined condition. Second, differentiation happened randomly with varying percentages of atrial or ventricular lineages. Third, an in vitro model cannot recapitulate the actual disease phenotype in an organism. Further, increased expression of microtubule-associated protein-1 light chain 3B (LC3B) (Supplementary Figure S4) and a study done by Choi et al. [30] suggest that modulation of defective autophagy might be an interesting target for pharmacological interventions in DCM arising from LMNA mutations. However, further research on this pathway was beyond the scope of this study.
The patient was under optimal medical treatment at the time of our study. Treatment guidelines for heart failure have changed and additional classes of agents have become the standard of care since our study. It is unclear, if mTOR inhibition would have had an additional benefit in the four-pillar model now proposed by the ESC.
Finally, the success of rapamycin treatment in one patient with a specific LMNA mutation may not be transferable to other LMNA mutations. Moreover, since treatment was terminated 6 months after initiation of rapamycin according to our ethics protocol, any conclusion on long-term effects cannot be made by our study. This requires further pre-clinical and clinical evaluation.

Conclusions
We successfully created and characterized a patient-specific LMNA-KI in vitro model that provided a comprehensive basis to understand cellular changes arising from an LMNA-KI mutation in a patient with severe and treatment-resistant familial DCM, and then allowed us to provide a well-tolerated, focused, and ultimately successful therapeutic approach. Our study describes how the future of personalized care for selected patients with HFrEF could look, with targeted therapies based upon genetic profiling and a comprehensive understanding of the molecular basis of their disease.

Clinical Competencies
This study describes a personalized medicine approach for a DCM patient with LMNArelated cardiomyopathy using mES cell-based disease modeling. This approach considers the genetic basis of the disease where clinical effects are often heterogeneous. The protocol may serve as a template for other patients with familial heart failure and stresses the need for consequent genetic screening in patients with unclear cardiomyopathy.

Translational Outlook
Increased mTOR signaling is evident in a DCM patient with an LMNA E161K mutation as well as in cardiomyocytes derived from mES cells with the patient-specific LMNA mutation. LMNA E161K mutation results in impaired electrophysiological properties and disorganisation of nuclear lamin in vitro. The mTOR-inhibitor rapamycin was able to rescue the phenotype in vitro and also improved the clinical conditions of a DCM patient with an LMNA E161K mutation. Further pre-clinical, as well as clinical studies, are required to establish a precision treatment.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/jpm12071149/s1, Table S1: List of primers used for expression analysis, Figure S1: Expression of markers for differentiated and undifferentiated WT and LMNA-KI mES cells, Figure S2: Action potential distribution plot of WT.CM and LMNA-KI.CM, Figure S3: Western blot images of protein expression in cardiomyocytes with and without isoprenaline (I) stimulation, Figure S4: Expression of autophagy markers in LMNA-KI cardiomyocytes, Supplementary Methods S1: Descriptive methodology used for electroporation, real-time PCR, Western blot, and immunohistochemistry, Supplementary Result S1: Validation of cardiomyocytes differentiated from WT and LMNA-KI mES cells, Video S1: The contractions of cardiomyocytes differentiated from wild type mouse embryonic stem cells, Video S2: The contractions of cardiomyocytes differentiated from wild type mouse embryonic stem cells and stimulated with isoprenaline (2 µM final concentration) for 24 h, Video S3: The contractions of cardiomyocytes differentiated from LMNA E161K mouse embryonic stem cells, Video S4: The contractions of cardiomyocytes differentiated from LMNA E161K mouse embryonic stem cells and stimulated with isoprenaline (2 µM final concentration) for 24 h, Video S5: The contractions of cardiomyocytes differentiated from LMNA E161K mouse embryonic stem cells and co-treated with isoprenaline (2 µM final concentration) and rapamycin (500 nM final concentration) for 24 h.