Clinical and metabolic consequences of a historic pathogenic lamin A/C founder variant

Abstract

A novel LMNA p.(Glu105Leu) variant was identified in five families with dilated cardiomyopathy (DCM), revealed as a local founder variant originating approximately 650 years ago. Genetic testing and clinical analysis of 795 DCM patients demonstrated that probands with this variant typically present with severe DCM in their sixties, characterized by high prevalence of late gadolinium enhancement, arrhythmias, and conduction disorders. Time-to-event analysis suggested a later onset of clinical symptoms compared to other LMNA variants, with a trend towards longer event-free survival. Microscopic imaging of patient fibroblasts, induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs), and heart tissue confirmed structural nuclear LMNA-associated abnormalities. Patient iPSC-CMs exhibited distinct sarcomeric disorganization, increased glucose uptake and glycogen content, reduced mitochondrial function and biogenesis, and delayed contractile function. These findings support the pathogenicity of the variant and demonstrate its profound impact on structural and metabolic functions in cardiomyocytes.

Fig. 1

Pedigree of the families carrying the LMNA p.(Glu105Leu) variant. Squares represent males, circles represent females. Black symbol indicates an individual with a clinical diagnosis of dilated cardiomyopathy (DCM), a grey symbol indicates an individual with left ventricular dilatation, a crossed symbol indicates a deceased individual. ‘+’ indicates carriers of the variant.

Fig. 2

Age-dependent disease penetrance of LMNA variants. Kaplan-Meier curve showing the age-dependent penetrance of clinical symptoms in patients with the LMNA p.(Glu105Leu) variant (red) compared with a group of individuals with other P/LP LMNA variants (black). The time reflects the biological age of the individuals.

Fig. 3

Event-free survival analysis of LMNA variant carriers. Kaplan-Meier curve showing the event-free survival (heart failure hospitalization, cardiac death, or left-ventricular assist device implantation) in patients with the LMNA p.(Glu105Leu) variant (red) compared with a group of individuals with other P/LP LMNA variants (black). The time reflects the biological age of the individuals.

Fig. 4

Nuclear envelope imaging of LMNA p.(Glu105Leu) and wild-type fibroblasts, iPSC-CMs and heart tissues. Immunohistological staining for lamin A/C (green) and Hoechst (blue) was used to assess the nuclear morphology in patient and wild-type fibroblasts obtained from unrelated controls, undifferentiated iPSCs, wild-type and patient iPSC-CMs, and heart tissue. Two independent iPSC lines (clone 1 and clone 2) were generated, along with one healthy unrelated control. The overlay images show the combined image of lamin A/C and Hoechst staining, allowing for the visualization of both the nuclear envelope and the nucleus (A-C, E-H) LMNA p.(Glu105Leu) fibroblasts, iPSC-CMs and heart tissue display a higher number of abnormal nuclei, as compared to the wild-type. In both LMNA p.(Glu105Leu) and wild-type undifferentiated iPSCs, no expression of Lamin A/C was observed, as expected. (D, I) Heart tissue from LMNA p.(Glu105Leu) patient shows similar abnormalities as those observed in the iPSC-CM model. (J) The percentage of nuclear abnormalities (including nuclear blebbing, donut structure and honeycomb structure), was significantly higher in LMNA p.(Glu105Leu) fibroblasts and iPSC-CMs compared to the wildtype. Scale bars: 20 μm (A-C; F-H); 10 μm (D and I). Data are represented as mean ± standard error of mean (SEM). Statistical significance was assessed using ANOVA and unpaired t-test. #p < 0.0001.

Fig. 5

Transmission electron microscopy (TEM) imaging of wild-type (WT) and LMNA p.(Glu105Leu) iPSC-CMs. Wild-type iPSC-CMs show an (A) oval nucleus, (B) organized sarcomeric structures, (C) mitochondria aligned with the sarcomeres and (D) normal shaped mitochondria. In contrast, LMNA p.(Glu105Leu) iPSC-CMs show a (E) dysmorphic nucleus (F) disorganized sarcomeres surrounded with glycogen, (G) mitochondria clumped between sarcomeres and (H) mitochondria surrounded by glycogen. Annotation within figures: N = nucleus, mt = mitochondria, G = glycogen. Scale bars are indicated in each panel.

Fig. 6

LMNA p.(Glu105Leu) iPSC-CMs have an altered bioenergetic profile. Two independent iPSC lines (clone 1 and clone 2) were generated, along with one healthy unrelated control, and subsequently differentiated into cardiomyocytes for these experiments. (A) Normalized oxygen consumption (OCR), the rates of (B) ATP synthesis, (C) basal respiration, (D) maximal respiration, and (E) spare capacity, were significantly lower in LMNA p.(Glu105Leu) iPSC-CMs, indicating an impaired metabolic activity in the patient iPSC-CMs. (F) Flow-cytometry analysis of mitochondrial content using Mitotracker CMXRed shows the distribution of mitochondrial mass in wildtype and LMNA p.(Glu105Leu) iPSC-CMs. (G) Glucose uptake assay results comparing iPSC-CM WT with iPSC-CM LMNA p.(Glu105Leu) clones 1 and 2 LMNA p.(Glu105Leu) iPSC-CMs show a 2.48 0.46 fold increase in glucose uptake. Data are normalized to the total protein content, and the fold-change is relative to the condition ‘wildtype low palmitate’. (H) Decreased expression of mtND1 and mtND2, and (I) PGC-1α (assessed by qPCR), were substantially reduced in LMNA p.(Glu105Leu) iPSC-CMs, as compared to the wild-type. (J) An increase in ROS production was measured in LMNA p.(Glu105Leu) iPSC-CMs, as compared to the wild-type. Data were represented as mean ± SEM and plotted on Graphpad Prism (n = 3). Statistical significance was assessed using one-way ANOVA and unpaired t-test. *p < 0.05, **p < 0.01, ***p < 0.001.

Fig. 7

Analysis of contractile activity of both LMNA p.(Glu105Leu) iPSC-CMs clone 1 and clone 2 and wild-type iPSC-CMs. (A) Representative average contraction profiles. (B) Motion-based contractility analysis of iPSC-CMs reveals prolonged calcium transient duration, (C) prolonged relaxation time and (D) increased action potential duration (APD) in LMNA p.(Glu105Leu), as compared to the wild-type iPSC-CMs. (E–G) A particle image velocimetry (PIV)-determined map showing the spatiotemporal contraction of the iPSC-CMs. Data were represented as mean ± SEM and plotted on Graphpad Prism (n = 3). Statistical significance was assessed using one-way ANOVA and unpaired t-test. ***p < 0.001.