Final Activity Report Summary - FUGEN-FHC (Functional Genomics of Familial Hypertrophic Cardiomyopathy)
Familial hypertrophic cardiomyopathy (FHC) is a myocardial disease with the major feature of asymmetric septal hypertrophy (=hypertrophy of the wall between the two ventricles). It is the major cause of sudden death in the young and particularly young athletes, and is associated with a significant risk of heart failure without effective treatments. FHC is one of the most common monogenic diseases, and transmitted in an autosomal-dominant trait (1 mutated allele is sufficient to cause the disease). It involves more than 450 different mutations in 13 genes encoding proteins involved in cardiac contraction. Out of them, cardiac myosin-binding protein C (cMyBP-C) is the most frequently mutated gene in Europe. How mutations cause FHC and heart failure is incompletely understood and constitutes our major objective. Particularly, we investigated a new concept in cardiology emerging from our previous data that impairment of the ubiquitin-proteasome system (UPS=main protein degradation system in cells) by mutant cMyBP-C plays an own pathogenic role in FHC.
We generated a new cMyBP-C knock-in mouse (mutation is introduced in the mouse genome) and revealed an unanticipated complexity of the expression of a single point mutation in the whole animal. The mutation gives rise to 3 different mutant proteins: a missense (exchange of an amino acid by another), a nonsense (shorter protein) and another mutant containing deletion/insertion (almost full-length protein). These unexpected findings need to be taken into account in further genetic studies. In addition, using specific inhibitors in vivo, we showed that the nonsense-mediated mRNA decay and the UPS act as two quality control systems to eliminate mutant cMyBP-Cs. Both mechanisms lowered the level of mutant proteins, which may prevent these proteins to act as poison peptides. In contrast to humans who express the disease at the heterozygous state, heterozygous mice did not exhibit any cardiac phenotype. We hypothesised that mice have lower stress in cages. Therefore, heterozygous and wild-type mice were stressed with catecholamines. Adrenergic stress induced myocardial hypertrophy in all groups but septal hypertrophy only in heterozygous cMyBP-C knock-in and knock-out mice (both have one functional allele), and proteasome impairment only in heterozygous knock-in mice (also carry a mutated allele producing a (low amount) or mutant protein). These data provide evidence that stress is required to reveal septal hypertrophy and proteasome impairment in a mouse model of FHC. We also showed that alterations of the UPS are part of a general cardiac hypertrophy program in mouse models of FHC as shown previously in human and experimental models of heart failure. We identified atrogin-1 as an enzyme mediating the degradation of truncated cMyBP-C by the UPS, and MuRF1 as a negative regulator of cMyBP-C expression, underlying a new role for MuRF1.
We identified new genetic variants in two different genes. One of them is located in the gene encoding calmodulin III, which plays an important role in regulating calcium levels and cardiac hypertrophy. We found that the calmodulin variant (=point mutation) is more frequent in patients with FHC than in the control population and reduces the activity of the calmodulin. The calmodulin variant could be therefore a modifier gene for FHC (=it modulates the disease expression) potentially by affecting the level of calmodulin in the cell and therefore the regulation of intracellular calcium and the development of hypertrophy.
Finally, we showed that cMyBP-C plays a very important in the relaxation of cardiac myocytes (=contracting cells) and therefore of the heart. Taken together, our findings provide better understanding of the role of cMyBP-C in the normal and diseased hearts.
We generated a new cMyBP-C knock-in mouse (mutation is introduced in the mouse genome) and revealed an unanticipated complexity of the expression of a single point mutation in the whole animal. The mutation gives rise to 3 different mutant proteins: a missense (exchange of an amino acid by another), a nonsense (shorter protein) and another mutant containing deletion/insertion (almost full-length protein). These unexpected findings need to be taken into account in further genetic studies. In addition, using specific inhibitors in vivo, we showed that the nonsense-mediated mRNA decay and the UPS act as two quality control systems to eliminate mutant cMyBP-Cs. Both mechanisms lowered the level of mutant proteins, which may prevent these proteins to act as poison peptides. In contrast to humans who express the disease at the heterozygous state, heterozygous mice did not exhibit any cardiac phenotype. We hypothesised that mice have lower stress in cages. Therefore, heterozygous and wild-type mice were stressed with catecholamines. Adrenergic stress induced myocardial hypertrophy in all groups but septal hypertrophy only in heterozygous cMyBP-C knock-in and knock-out mice (both have one functional allele), and proteasome impairment only in heterozygous knock-in mice (also carry a mutated allele producing a (low amount) or mutant protein). These data provide evidence that stress is required to reveal septal hypertrophy and proteasome impairment in a mouse model of FHC. We also showed that alterations of the UPS are part of a general cardiac hypertrophy program in mouse models of FHC as shown previously in human and experimental models of heart failure. We identified atrogin-1 as an enzyme mediating the degradation of truncated cMyBP-C by the UPS, and MuRF1 as a negative regulator of cMyBP-C expression, underlying a new role for MuRF1.
We identified new genetic variants in two different genes. One of them is located in the gene encoding calmodulin III, which plays an important role in regulating calcium levels and cardiac hypertrophy. We found that the calmodulin variant (=point mutation) is more frequent in patients with FHC than in the control population and reduces the activity of the calmodulin. The calmodulin variant could be therefore a modifier gene for FHC (=it modulates the disease expression) potentially by affecting the level of calmodulin in the cell and therefore the regulation of intracellular calcium and the development of hypertrophy.
Finally, we showed that cMyBP-C plays a very important in the relaxation of cardiac myocytes (=contracting cells) and therefore of the heart. Taken together, our findings provide better understanding of the role of cMyBP-C in the normal and diseased hearts.