👤 Nicolas Vignier

LMNA mutations in patients are responsible for a dilated cardiomyopathy. Molecular mechanisms underlying the origin and development of the pathology are unknown. Herein, using mouse pluripotent embryonic stem cells (ESCs) and a mouse model both harboring the p.H222P Lmna mutation, we found early defects in cardiac differentiation of mutated ESCs and dilatation of mutated embryonic hearts at E13.5, pointing to a developmental origin of the disease. Using mouse ESCs, we demonstrated that cardiac differentiation of LmnaH222P/+ was impaired at the mesodermal stage. Expression of Mesp1, a mesodermal cardiogenic gene involved in epithelial-to-mesenchymal transition of epiblast cells, as well as Snai1 and Twist expression, was decreased in LmnaH222P/+ cells compared with WT cells in the course of differentiation. In turn, cardiomyocyte differentiation was impaired. ChIP assay of H3K4me1 in differentiating cells revealed a specific decrease of this histone mark on regulatory regions of Mesp1 and Twist in LmnaH222P/+ cells. Downregulation or inhibition of LSD1 that specifically demethylated H3K4me1 rescued the epigenetic landscape of mesodermal LmnaH222P/+ cells and in turn contraction of cardiomyocytes. Inhibition of LSD1 in pregnant mice or neonatal mice prevented cardiomyopathy in E13.5 LmnaH222P/H222P offspring and adults, respectively. Thus, LSD1 appeared to be a therapeutic target to prevent or cure dilated cardiomyopathy associated with a laminopathy. Show less

This research investigated the impact of angiotensin AT1 receptor (Agtr1) blockade on left ventricular (LV) hypertrophy in a mouse model of human hypertrophic cardiomyopathy (HCM), which carries one functional allele of Mybpc3 gene coding cardiac myosin-binding protein C (cMyBP-C). Five-month-old heterozygous cMyBP-C knockout (Het-KO) and wild-type mice were treated with irbesartan (50 mg/kg/day) or vehicle for 8 weeks. Arterial blood pressure was measured by tail cuff plethysmography. LV dimension and function were accessed by echocardiography. Myocardial gene expression was evaluated using RT-qPCR. Compared with wild-type littermates, Het-KO mice had greater LV/body weight ratio (4.0 ± 0.1 vs. 3.3 ± 0.1 mg/g, P < 0.001), thicker interventricular septal wall (0.70 ± 0.02 vs. 0.65 ± 0.01 mm, P < 0.02), lower Mybpc3 mRNA level (-43%, P < 0.02), higher four-and-a-half LIM domains 1 (Fhl1, +110%, P < 0.01), and angiotensin-converting enzyme 1 (Ace1, +67%, P < 0.05), but unchanged Agtr1 mRNA levels in the septum. Treatment with irbesartan had no effect in wild-type mice but abolished septum-predominant LV hypertrophy and Fhl1 upregulation without changes in Ace1 but with an increased Agtr1 (+42%) in Het-KO mice. Thus, septum-predominant LV hypertrophy in Het-KO mice is combined with higher Fhl1 expression, which can be abolished by AT1 receptor blockade, indicating a role of the renin-angiotensin system and Fhl1 in cMyBP-C-related HCM. Show less

Hypertrophic cardiomyopathy (HCM) is frequently caused by mutations in MYBPC3 encoding cardiac myosin-binding protein C (cMyBP-C). The mechanisms leading from gene mutations to the HCM phenotype remain incompletely understood, partially because current mouse models of HCM do not faithfully reflect the human situation and early hypertrophy confounds the interpretation of functional alterations. The goal of this study was to evaluate whether myofilament Ca(2+) sensitization and diastolic dysfunction are associated or precede the development of left ventricular hypertrophy (LVH) in HCM. We evaluated the function of skinned and intact cardiac myocytes, as well as the intact heart in a recently developed Mybpc3-targeted knock-in mouse model carrying a point mutation frequently associated with HCM. Compared to wild-type, 10-week old homozygous knock-in mice exhibited i) higher myofilament Ca(2+) sensitivity in skinned ventricular trabeculae, ii) lower diastolic sarcomere length, and faster Ca(2+) transient decay in intact myocytes, and iii) LVH, reduced fractional shortening, lower E/A and E'/A', and higher E/E' ratios by echocardiography and Doppler analysis, suggesting systolic and diastolic dysfunction. In contrast, heterozygous knock-in mice, which mimic the human HCM situation, did not exhibit LVH or systolic dysfunction, but exhibited higher myofilament Ca(2+) sensitivity, faster Ca(2+) transient decay, and diastolic dysfunction. These data demonstrate that myofilament Ca(2+) sensitization and diastolic dysfunction are early phenotypic consequences of Mybpc3 mutations independent of LVH. The accelerated Ca(2+) transients point to compensatory mechanisms directed towards normalization of relaxation. We propose that HCM is a model for diastolic heart failure and this mouse model could be valuable in studying mechanisms and treatment modalities. Show less

Mutations in the MYBPC3 gene encoding cardiac myosin-binding protein (cMyBP)-C are frequent causes of hypertrophic cardiomyopathy, but the mechanisms leading from mutations to disease remain elusive. The goal of the present study was therefore to gain insights into the mechanisms controlling the expression of MYBPC3 mutations. We developed a cMyBP-C knock-in mouse carrying a point mutation. The level of total cMyBP-C mRNAs was 50% and 80% lower in heterozygotes and homozygotes, respectively. Surprisingly, the single G>A transition on the last nucleotide of exon 6 resulted in 3 different mutant mRNAs: missense (exchange of G for A), nonsense (exon skipping, frameshift, and premature stop codon) and deletion/insertion (as nonsense but with additional partial retention of downstream intron, restoring of the reading frame, and almost full-length protein). Inhibition of nonsense-mediated mRNA decay in cultured cardiac myocytes or in vivo with emetine or cycloheximide increased the level of nonsense mRNAs severalfold but not of the other mRNAs. By using sequential protein fractionation and a new antibody directed against novel amino acids produced by the frameshift, we showed that inhibition of the proteasome with epoxomicin via osmotic minipumps increased the level of (near) full-length mutants but not of truncated proteins. Homozygotes exhibited myocyte and left ventricular hypertrophy, reduced fractional shortening, and interstitial fibrosis; heterozygotes had no major phenotype. These data reveal (1) an unanticipated complexity of the expression of a single point mutation in the whole animal and (2) the involvement of both nonsense-mediated mRNA decay and the ubiquitin-proteasome system in lowering the level of mutant proteins. Show less