👤 Lambert Naudin ten Cate

Mutations in the MYBPC3 gene, encoding cardiac myosin binding protein C (cMyBP-C) are frequent causes of hypertrophic cardiomyopathy (HCM). Previously, we have presented evidence for reduced cMyBP-C expression (haploinsufficiency), in patients with a truncation mutation in MYBPC3. In mice, lacking cMyBP-C cross-bridge kinetics was accelerated. In this study, we investigated whether cross-bridge kinetics was altered in myectomy samples from HCM patients harboring heterozygous MYBPC3 mutations (MYBPC3mut). Isometric force and the rate of force redevelopment (k tr) at different activating Ca(2+) concentrations were measured in mechanically isolated Triton-permeabilized cardiomyocytes from MYBPC3mut (n = 18) and donor (n = 7) tissue. Furthermore, the stretch activation response of cardiomyocytes was measured in tissue from eight MYBPC3mut patients and five donors to assess the rate of initial force relaxation (k 1) and the rate and magnitude of the transient increase in force (k 2 and P 3, respectively) after a rapid stretch. Maximal force development of the cardiomyocytes was reduced in MYBPC3mut (24.5 ± 2.3 kN/m(2)) compared to donor (34.9 ± 1.6 kN/m(2)). The rates of force redevelopment in MYBPC3mut and donor over a range of Ca(2+) concentrations were similar (k tr at maximal activation: 0.63 ± 0.03 and 0.75 ± 0.09 s(-1), respectively). Moreover, the stretch activation parameters did not differ significantly between MYBPC3mut and donor (k 1: 8.5±0.5 and 8.8 ± 0.4 s(-1); k 2: 0.77 ± 0.06 and 0.74 ± 0.09 s(-1); P 3: 0.08 ± 0.01 and 0.09 ± 0.01, respectively). Incubation with protein kinase A accelerated k 1 in MYBPC3mut and donor to a similar extent. Our experiments indicate that, at the cMyBP-C expression levels in this patient group (63 ± 6 % relative to donors), cross-bridge kinetics are preserved and that the depressed maximal force development is not explained by perturbation of cross-bridge kinetics. Show less

Hypertrophic cardiomyopathy (HCM) is predominantly caused by mutations in genes encoding sarcomeric proteins. One of the most frequent affected genes is MYBPC3, which encodes the thick filament protein cardiac myosin binding protein C. Despite the prevalence of HCM, disease pathology and clinical outcome of sarcomeric mutations are largely unknown. We hypothesized that microRNAs (miRNAs) could play a role in the disease process. To determine which miRNAs were changed in expression, miRNA arrays were performed on heart tissue from HCM patients with a MYBPC3 mutation (n=6) and compared with hearts of non-failing donors (n=6). 532 out of 664 analyzed miRNAs were expressed in at least one heart sample. 13 miRNAs were differentially expressed in HCM compared with donors (at p<0.01, fold change ≥ 2). The genomic context of these differentially expressed miRNAs revealed that miR-204 (fold change 2.4 in HCM vs. donor) was located in an intron of the TRPM3 gene, encoding an aspecific cation channel involved in calcium entry. RT-PCR analysis revealed a trend towards TRPM3 upregulation in HCM compared with donor myocardium (fold change 2.3, p=0.078). In silico identification of mRNA targets of differentially expressed miRNAs showed a large proportion of genes involved in cardiac hypertrophy and cardiac beta-adrenergic receptor signaling and we showed reduced phosphorylation of cardiac troponin I in the HCM myocardium when compared with donor. HCM patients with MYBPC3 mutations have a specific miRNA expression profile. Downstream mRNA targets reveal possible involvement in cardiac signaling pathways. Show less

Familial hypertrophic cardiomyopathy (HCM), frequently caused by sarcomeric gene mutations, is characterized by cellular dysfunction and asymmetric left-ventricular (LV) hypertrophy. We studied whether cellular dysfunction is due to an intrinsic sarcomere defect or cardiomyocyte remodelling. Cardiac samples from 43 sarcomere mutation-positive patients (HCMmut: mutations in thick (MYBPC3, MYH7) and thin (TPM1, TNNI3, TNNT2) myofilament genes) were compared with 14 sarcomere mutation-negative patients (HCMsmn), eight patients with secondary LV hypertrophy due to aortic stenosis (LVHao) and 13 donors. Force measurements in single membrane-permeabilized cardiomyocytes revealed significantly lower maximal force generating capacity (Fmax) in HCMmut (21 ± 1 kN/m²) and HCMsmn (26 ± 3 kN/m²) compared with donor (36 ± 2 kN/m²). Cardiomyocyte remodelling was more severe in HCMmut compared with HCMsmn based on significantly lower myofibril density (49 ± 2 vs. 63 ± 5%) and significantly higher cardiomyocyte area (915 ± 15 vs. 612 ± 11 μm²). Low Fmax in MYBPC3mut, TNNI3mut, HCMsmn, and LVHao was normalized to donor values after correction for myofibril density. However, Fmax was significantly lower in MYH7mut, TPM1mut, and TNNT2mut even after correction for myofibril density. In accordance, measurements in single myofibrils showed very low Fmax in MYH7mut, TPM1mut, and TNNT2mut compared with donor (respectively, 73 ± 3, 70 ± 7, 83 ± 6, and 113 ± 5 kN/m²). In addition, force was lower in MYH7mut cardiomyocytes compared with MYBPC3mut, HCMsmn, and donor at submaximal [Ca²⁺]. Low cardiomyocyte Fmax in HCM patients is largely explained by hypertrophy and reduced myofibril density. MYH7 mutations reduce force generating capacity of sarcomeres at maximal and submaximal [Ca²⁺]. These hypocontractile sarcomeres may represent the primary abnormality in patients with MYH7 mutations. Show less

High-myofilament Ca(2+) sensitivity has been proposed as a trigger of disease pathogenesis in familial hypertrophic cardiomyopathy (HCM) on the basis of in vitro and transgenic mice studies. However, myofilament Ca(2+) sensitivity depends on protein phosphorylation and muscle length, and at present, data in humans are scarce. To investigate whether high myofilament Ca(2+) sensitivity and perturbed length-dependent activation are characteristics for human HCM with mutations in thick and thin filament proteins. Cardiac samples from patients with HCM harboring mutations in genes encoding thick (MYH7, MYBPC3) and thin (TNNT2, TNNI3, TPM1) filament proteins were compared with sarcomere mutation-negative HCM and nonfailing donors. Cardiomyocyte force measurements showed higher myofilament Ca(2+) sensitivity in all HCM samples and low phosphorylation of protein kinase A (PKA) targets compared with donors. After exogenous PKA treatment, myofilament Ca(2+) sensitivity was similar (MYBPC3mut, TPM1mut, sarcomere mutation-negative HCM), higher (MYH7mut, TNNT2mut), or even significantly lower (TNNI3mut) compared with donors. Length-dependent activation was significantly smaller in all HCM than in donor samples. PKA treatment increased phosphorylation of PKA-targets in HCM myocardium and normalized length-dependent activation to donor values in sarcomere mutation-negative HCM and HCM with truncating MYBPC3 mutations but not in HCM with missense mutations. Replacement of mutant by wild-type troponin in TNNT2mut and TNNI3mut corrected length-dependent activation to donor values. High-myofilament Ca(2+) sensitivity is a common characteristic of human HCM and partly reflects hypophosphorylation of PKA targets compared with donors. Length-dependent sarcomere activation is perturbed by missense mutations, possibly via posttranslational modifications other than PKA hypophosphorylation or altered protein-protein interactions, and represents a common pathomechanism in HCM. Show less

Differentiation of oligodendrocyte progenitor cells (OPCs) into mature oligodendrocytes is regulated by the interplay between extrinsic signals and intrinsic epigenetic determinants. In this study, we analyze the effect that the extracellular ligands sonic hedgehog (Shh) and bone morphogenetic protein 4 (BMP4), have on histone acetylation and gene expression in cultured OPCs. Shh treatment favored the progression toward oligodendrocytes by decreasing histone acetylation and inducing peripheral chromatin condensation. BMP4 treatment, in contrast, inhibited the progression toward oligodendrocytes and favored astrogliogenesis by favoring global histone acetylation and retaining euchromatin. Pharmacological treatment or silencing of histone deacetylase 1 (Hdac1) or histone deacetylase 2 (Hdac2) in OPCs did not affect BMP4-dependent astrogliogenesis, while it prevented Shh-induced oligodendrocyte differentiation and favored the expression of astrocytic genes. Transcriptional profiling of treated OPCs, revealed that BMP4-inhibition of oligodendrocyte differentiation was accompanied by increased levels of Wnt (Tbx3) and Notch-target genes (Jag1, Hes1, Hes5, Hey1, and Hey2), decreased recruitment of Hdac and increased histone acetylation at these loci. Similar upregulation of Notch-target genes and increased histone acetylation were observed in the corpus callosum of mice infused with BMP4 during cuprizone-induced demyelination. We conclude that Shh and Bmp4 differentially regulate histone acetylation and chromatin structure in OPCs and that BMP4 acts as a potent inducer of gene expression, including Notch and Wnt target genes, thereby enhancing the crosstalk among signaling pathways that are known to inhibit myelination and repair. Show less

The phenotypic variability of hypertrophic cardiomyopathy (HCM) in patients with identical pathogenic mutations suggests additional modifiers. In view of the regulatory role in cardiac function, blood pressure, and electrolyte homeostasis, polymorphisms in the renin-angiotensin-aldosterone system (RAAS) are candidates for modifying phenotypic expression. In order to investigate whether RAAS polymorphisms modulate HCM phenotype, we selected a large cohort of carriers of one of the three functionally equivalent truncating mutations in the MYBPC3 gene. Family-based association analysis was performed to analyze the effects of five candidate RAAS polymorphisms (ACE, rs4646994; AGTR1, rs5186; CMA, rs1800875; AGT, rs699; CYP11B2, rs1799998) in 368 subjects carrying one of the three mutations in the MYBPC3 gene. Interventricular septum (IVS) thickness and Wigle score were assessed by 2D-echocardiography. SNPs in the RAAS system were analyzed separately and combined as a pro-left ventricular hypertrophy (LVH) score for effects on the HCM phenotype. Analyzing the five polymorphisms separately for effects on IVS thickness and Wigle score detected two modest associations. Carriers of the CC genotype in the AGT gene had less pronounced IVS thickness compared with CT and TT genotype carriers. The DD polymorphism in the ACE gene was associated with a high Wigle score (P=0.01). No association was detected between the pro-LVH score and IVS thickness or Wigle score. In conclusion, in contrast to previous studies, in our large study population of HCM patients with functionally equivalent mutations in the MYBPC3 gene we did not find major effects of genetic variation within the genes of the RAAS system on phenotypic expression of HCM. Show less

Hypertrophic cardiomyopathy (HCM), typically characterized by asymmetrical left ventricular hypertrophy, frequently is caused by mutations in sarcomeric proteins. We studied if changes in sarcomeric properties in HCM depend on the underlying protein mutation. Comparisons were made between cardiac samples from patients carrying a MYBPC3 mutation (MYBPC3(mut); n=17), mutation negative HCM patients without an identified sarcomere mutation (HCM(mn); n=11), and nonfailing donors (n=12). All patients had normal systolic function, but impaired diastolic function. Protein expression of myosin binding protein C (cMyBP-C) was significantly lower in MYBPC3(mut) by 33±5%, and similar in HCM(mn) compared with donor. cMyBP-C phosphorylation in MYBPC3(mut) was similar to donor, whereas it was significantly lower in HCM(mn). Troponin I phosphorylation was lower in both patient groups compared with donor. Force measurements in single permeabilized cardiomyocytes demonstrated comparable sarcomeric dysfunction in both patient groups characterized by lower maximal force generating capacity in MYBPC3(mut) and HCM(mn,) compared with donor (26.4±2.9, 28.0±3.7, and 37.2±2.3 kN/m(2), respectively), and higher myofilament Ca(2+)-sensitivity (EC(50)=2.5±0.2, 2.4±0.2, and 3.0±0.2 μmol/L, respectively). The sarcomere length-dependent increase in Ca(2+)-sensitivity was significantly smaller in both patient groups compared with donor (ΔEC(50): 0.46±0.04, 0.37±0.05, and 0.75±0.07 μmol/L, respectively). Protein kinase A treatment restored myofilament Ca(2+)-sensitivity and length-dependent activation in both patient groups to donor values. Changes in sarcomere function reflect the clinical HCM phenotype rather than the specific MYBPC3 mutation. Hypocontractile sarcomeres are a common deficit in human HCM with normal systolic left ventricular function and may contribute to HCM disease progression. Show less

To investigate the outcome of cardiac evaluation and the risk stratification for sudden cardiac death (SCD) in asymptomatic hypertrophic cardiomyopathy (HCM) mutation carriers. Seventy-six HCM mutation carriers from 32 families identified by predictive DNA testing underwent cardiac evaluation including history, examination, electrocardiography, Doppler echocardiography, exercise testing, and 24 h Holter monitoring. The published diagnostic criteria for HCM in adult members of affected families were used to diagnose HCM. Thirty-three (43%) men and 43 (57%) women with a mean age of 42 years (range 16-79) were examined; in 31 (41%) HCM was diagnosed. Disease penetrance was age related and men were more often affected than women (P = 0.04). Myosin Binding Protein C (MYBPC3) mutation carriers were affected at higher age than Myosin Heavy Chain (MYH7) mutation carriers (P = 0.01). Risk factors for SCD were present in affected and unaffected carriers. Hypertrophic cardiomyopathy was diagnosed in 41% of carriers. Disease penetrance was age dependent, warranting repeated cardiologic evaluation. The MYBPC3 mutation carriers were affected at higher age than MYH7 mutation carriers. Risk factors for SCD were present in carriers with and without HCM. Follow-up studies are necessary to evaluate the effectiveness of risk stratification for SCD in this population. Show less

To test the hypothesis that carriers of Dutch founder mutations in cardiac myosin-binding protein C (MYBPC3), without left ventricular hypertrophy (LVH) or electrocardiographic abnormalities, have diastolic dysfunction on tissue Doppler imaging (TDI), which can be used for the screening of family members in the hypertrophic cardiomyopathy (HCM) population. TDI is a more sensitive technique for the assessment of left ventricular contraction and relaxation abnormalities than is conventional echocardiography. Echocardiographic studies including TDI were performed in genotyped hypertrophic cardiomyopathy patients (genotype-positive, G+/LVH+; n = 27), mutation carriers without LVH (G+/LVH-; n = 27), and healthy controls (n = 55). The identified mutations in MYBPC3 in the G+/LVH+ subjects were c.2864₂₈₆₅delCT (12 subjects), c.2373dupG (n = 8), and p. Arg943X (n = 7). In the G+/LVH- subjects, the following mutations were identified: c.2864₂₈₆₅delCT (n = 11), c.2373dupG (n = 8), and p. Arg943X (n = 8). Mean TDI-derived systolic and early and late diastolic mitral annular velocities were significantly lower in the G+/LVH+ subjects compared with the other groups. However, there was no difference between controls and G+/LVH- subjects. Mean TDI-derived late mitral annular diastolic velocities were significantly higher in the G+/LVH- subjects compared with controls and G+/LVH+ subjects. Using a cut-off value of mean +/- 2 SD, an abnormal late mitral annular diastolic velocity was found in 14 (51%) of G+/LVH- patients. There was no difference among the 3 different mutations. In contrast to earlier reports, mean mitral annular systolic velocity and early mitral annular diastolic velocity velocities were not reduced in G+/LVH- subjects, and TDI velocities were not sufficiently sensitive for determination of the affected status of an individual subject. Our findings, however, support the theory that diastolic dysfunction is a primary component of pre-clinical HCM. Show less

Mutations in the MYBPC3 gene, encoding cardiac myosin-binding protein C (cMyBP-C), are a frequent cause of familial hypertrophic cardiomyopathy. In the present study, we investigated whether protein composition and function of the sarcomere are altered in a homogeneous familial hypertrophic cardiomyopathy patient group with frameshift mutations in MYBPC3 (MYBPC3(mut)). Comparisons were made between cardiac samples from MYBPC3 mutant carriers (c.2373dupG, n=7; c.2864₂₈₆₅delCT, n=4) and nonfailing donors (n=13). Western blots with the use of antibodies directed against cMyBP-C did not reveal truncated cMyBP-C in MYBPC3(mut). Protein expression of cMyBP-C was significantly reduced in MYBPC3(mut) by 33+/-5%. Cardiac MyBP-C phosphorylation in MYBPC3(mut) samples was similar to the values in donor samples, whereas the phosphorylation status of cardiac troponin I was reduced by 84+/-5%, indicating divergent phosphorylation of the 2 main contractile target proteins of the beta-adrenergic pathway. Force measurements in mechanically isolated Triton-permeabilized cardiomyocytes demonstrated a decrease in maximal force per cross-sectional area of the myocytes in MYBPC3(mut) (20.2+/-2.7 kN/m(2)) compared with donor (34.5+/-1.1 kN/m(2)). Moreover, Ca(2+) sensitivity was higher in MYBPC3(mut) (pCa(50)=5.62+/-0.04) than in donor (pCa(50)=5.54+/-0.02), consistent with reduced cardiac troponin I phosphorylation. Treatment with exogenous protein kinase A, to mimic beta-adrenergic stimulation, did not correct reduced maximal force but abolished the initial difference in Ca(2+) sensitivity between MYBPC3(mut) (pCa(50)=5.46+/-0.03) and donor (pCa(50)=5.48+/-0.02). Frameshift MYBPC3 mutations cause haploinsufficiency, deranged phosphorylation of contractile proteins, and reduced maximal force-generating capacity of cardiomyocytes. The enhanced Ca(2+) sensitivity in MYBPC3(mut) is due to hypophosphorylation of troponin I secondary to mutation-induced dysfunction. Show less

Multiple pterygium syndromes (MPS) comprise a group of multiple congenital anomaly disorders characterized by webbing (pterygia) of the neck, elbows, and/or knees and joint contractures (arthrogryposis). MPS are phenotypically and genetically heterogeneous but are traditionally divided into prenatally lethal and nonlethal (Escobar) types. Previously, we and others reported that recessive mutations in the embryonal acetylcholine receptor g subunit (CHRNG) can cause both lethal and nonlethal MPS, thus demonstrating that pterygia resulted from fetal akinesia. We hypothesized that mutations in acetylcholine receptor-related genes might also result in a MPS/fetal akinesia phenotype and so we analyzed 15 cases of lethal MPS/fetal akinesia without CHRNG mutations for mutations in the CHRNA1, CHRNB1, CHRND, and rapsyn (RAPSN) genes. No CHRNA1, CHRNB1, or CHRND mutations were detected, but a homozygous RAPSN frameshift mutation, c.1177-1178delAA, was identified in a family with three children affected with lethal fetal akinesia sequence. Previously, RAPSN mutations have been reported in congenital myasthenia. Functional studies were consistent with the hypothesis that whereas incomplete loss of rapsyn function may cause congenital myasthenia, more severe loss of function can result in a lethal fetal akinesia phenotype. Show less

Axon regeneration in the adult CNS is prevented by inhibitors in myelin. These inhibitors seem to modulate RhoA activity by binding to a receptor complex comprising a ligand-binding subunit (the Nogo-66 receptor NgR1) and a signal transducing subunit (the neurotrophin receptor p75). However, in reconstituted non-neuronal systems, NgR1 and p75 together are unable to activate RhoA, suggesting that additional components of the receptor may exist. Here we describe LINGO-1, a nervous system-specific transmembrane protein that binds NgR1 and p75 and that is an additional functional component of the NgR1/p75 signaling complex. In non-neuronal cells, coexpression of human NgR1, p75 and LINGO-1 conferred responsiveness to oligodendrocyte myelin glycoprotein, as measured by RhoA activation. A dominant-negative human LINGO-1 construct attenuated myelin inhibition in transfected primary neuronal cultures. This effect on neurons was mimicked using an exogenously added human LINGO-1-Fc fusion protein. Together these observations suggest that LINGO-1 has an important role in CNS biology. Show less

Hypertrophic cardiomyopathy (HCM) is caused by mutations in genes that encode sarcomeric proteins. In this study we investigated the involvement of the sarcomeric myosin binding protein C in the Dutch HCM population. We initially screened 22 Dutch index patients for mutations in the MYBPC3 gene, which revealed four different mutations in 14 patients. The 2373insG mutation was identified in 10 apparently unrelated patients. A subsequent screening for the 2373insG mutation in a group of another 237 unrelated HCM patients revealed 50 additional carriers of the same genetic defect. Genotyping with polymorphic repeat markers and intragenic SNPs of the 60 Dutch as well as two German and five North American 2373insG carriers indicated they all share the same haplotype. The 2373insG mutation accounts for almost one-fourth of all HCM cases in the Netherlands (60/259), which is predominantly present in the northwestern part of the country (22/66) and is a founder mutation probably originating from the Netherlands. Show less