Familial hypertrophic cardiomyopathy (FHC) is an autosomal dominant disease, which in about 30% of the patients is caused by missense mutations in one allele of the β-myosin heavy chain (β-MHC) gene (MYH7). To address potential molecular mechanisms underlying the family-specific prognosis, we determined the relative expression of mutant versus wild-type MYH7-mRNA. We found a hitherto unknown mutation-dependent unequal expression of mutant to wild-type MYH7-mRNA, which is paralleled by similar unequal expression of β-MHC at the protein level. Relative abundance of mutated versus wild-type MYH7-mRNA was determined by a specific restriction digest approach and by real-time PCR (RT-qPCR). Fourteen samples from M. soleus and myocardium of 12 genotyped and clinically well-characterized FHC patients were analyzed. The fraction of mutated MYH7-mRNA in five patients with mutation R723G averaged to 66 and 68% of total MYH7-mRNA in soleus and myocardium, respectively. For mutations I736T, R719W and V606M, fractions of mutated MYH7-mRNA in M. soleus were 39, 57 and 29%, respectively. For all mutations, unequal abundance was similar at the protein level. Importantly, fractions of mutated transcripts were comparable among siblings, in younger relatives and unrelated carriers of the same mutation. Hence, the extent of unequal expression of mutated versus wild-type transcript and protein is characteristic for each mutation, implying cis-acting regulatory mechanisms. Bioinformatics suggest mRNA stability or splicing effectors to be affected by certain mutations. Intriguingly, we observed a correlation between disease expression and fraction of mutated mRNA and protein. This strongly suggests that mutation-specific allelic imbalance represents a new pathogenic factor for FHC.Electronic supplementary materialThe online version of this article (doi:10.1007/s00395-011-0205-9) contains supplementary material, which is available to authorized users.
Hypertrophic Cardiomyopathy (HCM) has been related to many different mutations in more than 20 different, mostly sarcomeric proteins. While development of the HCM-phenotype is thought to be triggered by the different mutations, a common mechanism remains elusive. Studying missense-mutations in the ventricular beta-myosin heavy chain (β-MyHC, MYH7) we hypothesized that significant contractile heterogeneity exists among individual cardiomyocytes of HCM-patients that results from cell-to-cell variation in relative expression of mutated vs. wildtype β-MyHC. To test this hypothesis, we measured force-calcium-relationships of cardiomyocytes isolated from myocardium of heterozygous HCM-patients with either β-MyHC-mutation Arg723Gly or Arg200Val, and from healthy controls. From the myocardial samples of the HCM-patients we also obtained cryo-sections, and laser-microdissected single cardiomyocytes for quantification of mutated vs. wildtype MYH7-mRNA using a single cell RT-qPCR and restriction digest approach. We characterized gene transcription by visualizing active transcription sites by fluorescence in situ hybridization of intronic and exonic sequences of MYH7-pre-mRNA. For both mutations, cardiomyocytes showed large cell-to-cell variation in Ca++-sensitivity. Interestingly, some cardiomyocytes were essentially indistinguishable from controls what might indicate that they had no mutant β-MyHC while others had highly reduced Ca++-sensitivity suggesting substantial fractions of mutant β-MyHC. Single-cell MYH7-mRNA-quantification in cardiomyocytes of the same patients revealed high cell-to-cell variability of mutated vs. wildtype mRNA, ranging from essentially pure mutant to essentially pure wildtype MYH7-mRNA. We found 27% of nuclei without active transcription sites which is inconsistent with continuous gene transcription but suggests burst-like transcription of MYH7. Model simulations indicated that burst-like, stochastic on/off-switching of MYH7 transcription, which is independent for mutant and wildtype alleles, could generate the observed cell-to-cell variation in the fraction of mutant vs. wildtype MYH7-mRNA, a similar variation in β-MyHC-protein, and highly heterogeneous Ca++-sensitivity of individual cardiomyocytes. In the long run, such contractile imbalance in the myocardium may well induce progressive structural distortions like cellular and myofibrillar disarray and interstitial fibrosis, as they are typically observed in HCM.
The aim of our study was to analyze the differential expression of miRNAs in the brains of BSEinfected cynomolgus macaques as a model for Creutzfeldt-Jakob disease (CJD). MicroRNAs (miRNAs) are small noncoding RNAs regulating gene expression by mRNA targeting. Among other functions they contribute to neuronal development and survival. Recently, the lack of miRNA processing has been shown to promote neurodegeneration and deregulation of several miRNAs has been reported to be associated with Scrapie in mice. Therefore, we hypothesized that miRNAs are also regulated in response to human prion disease. We have applied miRNA-microarrays to identify deregulated miRNA candidates in brains of BSE-infected macaques. Shock-frozen brain sections of six BSE-infected and five non-infected macaques were used to validate regulated miRNA candidates by two independent qRT-PCR-based methods. Our study revealed significant upregulation of hsa-miR-342-3p and hsa-miR-494 in the brains of BSE-infected macaques compared to non-infected animals. In a pilot study we could show that hsa-miR-342-3p was also upregulated in brain samples of human type 1 and type 2 sporadic CJD. With respect to the reported regulation of this miRNA in Scrapie-infected mice, we propose that upregulation of hsa-miR-342-3p may be a general phenomenon in late stage prion disease and might be used as a novel marker for animal and human TSEs.
Key pointsr The R403Q mutation, located in the S1 domain of the β-myosin heavy chain, is associated with a severe phenotype of hypertrophic cardiomyopathy (HCM).r Increased cross-bridge relaxation kinetics caused by the R403Q mutation might underlie increased energetic cost of sarcomeric tension generation; however, direct evidence is absent.r We studied the relationship between cross-bridge kinetics and energetics in single cardiac myofibrils and multicellular cardiac muscle strips in human HCM tissue with and without the R403Q mutation.r In human HCM with the R403Q mutation, cross-bridge relaxation was faster and correlated well with a rise in energetic cost of tension generation.r Our data suggest that an increase in tension cost is one of the causes underlying cardiomyopathy development in patients with the R403Q mutation.Abstract The first mutation associated with hypertrophic cardiomyopathy (HCM) is the R403Q mutation in the gene encoding β-myosin heavy chain (β-MyHC). R403Q locates in the globular head of myosin (S1), responsible for interaction with actin, and thus motor function of myosin. Increased cross-bridge relaxation kinetics caused by the R403Q mutation might underlie increased energetic cost of tension generation; however, direct evidence is absent. Here we studied to what extent cross-bridge kinetics and energetics are related in single cardiac myofibrils and multicellular cardiac muscle strips of three HCM patients with the R403Q mutation and nine sarcomere mutation-negative HCM patients (HCM smn ). Expression of R403Q was on average 41 ± 4% of total MYH7 mRNA. Cross-bridge slow relaxation kinetics in single R403Q myofibrils was significantly higher (P < 0.0001) than in HCM smn myofibrils (0.47 ± 0.02 and 0.30 ± 0.02 s −1 , respectively). Moreover, compared to HCM smn , tension cost was significantly higher in the muscle strips of the three R403Q patients (2.93 ± 0.25 and 1.78 ± 0.10 μmol l -1 s −1 kN −1 m −2 , respectively) which showed a positive linear correlation with relaxation kinetics in the corresponding myofibril preparations. This correlation suggests that faster cross-bridge relaxation kinetics results in an increase in energetic cost of tension generation in human HCM with the R403Q mutation compared to HCM smn . Therefore, increased tension cost might contribute to HCM disease in patients carrying the R403Q mutation.
Familial Hypertrophic Cardiomyopathy (FHC) is frequently caused by mutations in the β-cardiac myosin heavy chain (β-MyHC). To identify changes in sarcomeric function triggered by such mutations, distinguishing mutation effects from other functional alterations of the myocardium is essential. We previously identified a direct effect of mutation R723G (MyHC723) on myosin function in slow Musculus soleus fibers. Here we investigate contractile features of left ventricular cardiomyocytes of FHC-patients with the same MyHC723-mutation and compare these to the soleus data. In mechanically isolated, triton-permeabilized MyHC723-cardiomyocytes, maximum force was significantly lower but calcium-sensitivity was unchanged compared to donor. Conversely, MyHC723-soleus fibers showed significantly higher maximum force and reduced calcium-sensitivity compared to controls. Protein phosphorylation, a potential myocardium specific modifying mechanism, might account for differences compared to soleus fibers. Analysis revealed reduced phosphorylation of troponin I and T, myosin-binding-protein C, and myosin-light-chain 2 in MyHC723-myocardium compared to donor. Saturation of protein-kinaseA phospho-sites led to comparable, i.e., reduced MyHC723-calcium-sensitivity in cardiomyocytes as in M. soleus fibers, while maximum force remained reduced. Myofibrillar disarray and lower density of myofibrils, however, largely account for reduced maximum force in MyHC723-cardiomyocytes. The changes seen when phosphorylation of sarcomeric proteins in myocardium of affected patients is matched to control tissue suggest that the R723G mutation causes reduced Ca(++)-sensitivity in both cardiomyocytes and M. soleus fibers. In MyHC723-myocardium, however, hypophosphorylation can compensate for the reduced calcium-sensitivity, while maximum force generation, lowered by myofibrillar deficiency and disarray, remains impaired, and may only be compensated by hypertrophy.
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