Sabine J. van Dijk

Cardiac Myosin-Binding Protein C Mutations and Hypertrophic Cardiomyopathy: Haploinsufficiency, Deranged Phosphorylation, and Cardiomyocyte Dysfunction

Background— 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 (MYBPC3mut). Methods and Results— Comparisons were made between cardiac samples from MYBPC3 mutant carriers (c.2373dupG, n=7; c.2864_2865delCT, 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 MYBPC3mut. Protein expression of cMyBP-C was significantly reduced in MYBPC3mut by 33±5%. Cardiac MyBP-C phosphorylation in MYBPC3mut 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 &bgr;-adrenergic pathway. Force measurements in mechanically isolated Triton-permeabilized cardiomyocytes demonstrated a decrease in maximal force per cross-sectional area of the myocytes in MYBPC3mut (20.2±2.7 kN/m2) compared with donor (34.5±1.1 kN/m2). Moreover, Ca2+ sensitivity was higher in MYBPC3mut (pCa50=5.62±0.04) than in donor (pCa50=5.54±0.02), consistent with reduced cardiac troponin I phosphorylation. Treatment with exogenous protein kinase A, to mimic &bgr;-adrenergic stimulation, did not correct reduced maximal force but abolished the initial difference in Ca2+ sensitivity between MYBPC3mut (pCa50=5.46±0.03) and donor (pCa50=5.48±0.02). Conclusions— Frameshift MYBPC3 mutations cause haploinsufficiency, deranged phosphorylation of contractile proteins, and reduced maximal force-generating capacity of cardiomyocytes. The enhanced Ca2+ sensitivity in MYBPC3mut is due to hypophosphorylation of troponin I secondary to mutation-induced dysfunction.

Contractile Dysfunction Irrespective of the Mutant Protein in Human Hypertrophic Cardiomyopathy With Normal Systolic Function

Background— 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. Methods and Results— Comparisons were made between cardiac samples from patients carrying a MYBPC3 mutation (MYBPC3mut; n=17), mutation negative HCM patients without an identified sarcomere mutation (HCMmn; 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 MYBPC3mut by 33±5%, and similar in HCMmn compared with donor. cMyBP-C phosphorylation in MYBPC3mut was similar to donor, whereas it was significantly lower in HCMmn. 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 MYBPC3mut and HCMmn, compared with donor (26.4±2.9, 28.0±3.7, and 37.2±2.3 kN/m2, respectively), and higher myofilament Ca2+-sensitivity (EC50=2.5±0.2, 2.4±0.2, and 3.0±0.2 &mgr;mol/L, respectively). The sarcomere length-dependent increase in Ca2+-sensitivity was significantly smaller in both patient groups compared with donor (&Dgr;EC50: 0.46±0.04, 0.37±0.05, and 0.75±0.07 &mgr;mol/L, respectively). Protein kinase A treatment restored myofilament Ca2+-sensitivity and length-dependent activation in both patient groups to donor values. Conclusions— 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.

Myofilament dysfunction in cardiac disease from mice to men

In healthy human myocardium a tight balance exists between receptor-mediated kinases and phosphatases coordinating phosphorylation of regulatory proteins involved in cardiomyocyte contractility. During heart failure, when neurohumoral stimulation increases to compensate for reduced cardiac pump function, this balance is perturbed. The imbalance between kinases and phosphatases upon chronic neurohumoral stimulation is detrimental and initiates cardiac remodelling, and phosphorylation changes of regulatory proteins, which impair cardiomyocyte function. The main signalling pathway involved in enhanced cardiomyocyte contractility during increased cardiac load is the β-adrenergic signalling route, which becomes desensitized upon chronic stimulation. At the myofilament level, activation of protein kinase A (PKA), the down-stream kinase of the β-adrenergic receptors (β-AR), phosphorylates troponin I, myosin binding protein C and titin, which all exert differential effects on myofilament function. As a consequence of β-AR down-regulation and desensitization, phosphorylation of the PKA-target proteins within the cardiomyocyte may be decreased and alter myofilament function. Here we discuss involvement of altered PKA-mediated myofilament protein phosphorylation in different animal and human studies, and discuss the roles of troponin I, myosin binding protein C and titin in regulating myofilament dysfunction in cardiac disease. Data from the different animal and human studies emphasize the importance of careful biopsy procurement, and the need to investigate localization of kinases and phosphatases within the cardiomyocyte, in particular their co-localization with cardiac myofilaments upon receptor stimulation.

Mutations in MYH7 reduce the force generating capacity of sarcomeres in human familial hypertrophic cardiomyopathy

AIMS 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. METHODS AND RESULTS 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⁺]. CONCLUSION 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.

The development of familial hypertrophic cardiomyopathy: from mutation to bedside

Eur J Clin Invest 2011; 41 (5): 568–578

Myocardial adaptations in the failing heart: cause or consequence?

Many changes in morphology, biochemical properties and myocyte function occur during development to heart failure. Most changes may be compensatory, yet unable to prevent cardiac dysfunction in the long run. This illustrates that it is important to carefully dissect the disease causing modifications from cardiac adaptation, in order to obtain a better understanding of the pathophysiological processes leading to heart failure.

The HCM-Associated Cardiac Troponin T Mutation K273N Accelerates Tension Generation and Relaxation in Human Cardiac Myofibrils

In spite of extensive work on the functional sequelae of Hypertrophic CardioMyopathy (HCM)-associated mutations in sarcomeric proteins, the mechanisms by which the mutant proteins cause the disease have not been definitely identified. Here we use the single myofibril technique (Tesi et al., Biophys. J., 2002, 83, 2142-2151) to compare the kinetics of contraction and relaxation of myofibrils isolated from frozen left ventricular samples of one homozygous HCM patient carrying the cardiac Troponin T (cTnT) mutation K273N (underwent heart transplantation) to those from “control” hearts. Preparations, mounted in a force recording apparatus (15 °C), were maximally Ca2+-activated (pCa 4.5) and fully relaxed (pCa 9) by rapid (<10 ms) solution switching. The rate constant of active tension generation following maximal Ca2+ activation (kACT) was markedly faster in the myofibrils from the K273N patient (1.7- 2 s−1) compared to controls (0.7-1 s−1). Replacement of the mutant protein by exchange with wild-type recombinant human cTn reduced kACT of HCM myofibrils close to control values (1 s−1). Force relaxation kinetics upon Ca2+ removal were also faster in K273N myofibrils than in controls, evidence that the apparent rate with which cross-bridges leave the force generating states is accelerated in the HCM preparations. The results indicate that the HCM-associated cTnT mutation K273N alters apparent cross-bridge kinetics. This can lead to increased energy cost of tension generation that may be central to the HCM disease process. Supported by the 7th Framework Program of the European Union, “BIG-HEART” grant agreement 241577, & Telethon-Italy GGP07133.

Myosin Binding Protein C Mutations and Hypertrophic Cardiomyopathy: Haploinsufficiency, Deranged Phosphorylation and Cardiomyocyte Dysfunction

Background Mutations in the MYBPC3 gene, encoding for cardiac myosin binding protein C (cMyBP-C), are a frequent cause of familial hypertrophic cardiomyopathy (FHCM). In the present study we investigated if protein composition and function of the sarcomere are altered in a homogenous FHCM patient group with truncating mutations in MYBPC3 (MYBPC3mut).Methods and Results Comparisons were made between cardiac samples from MYBPC3 mutant carriers (c.2373dupG, n=7; c.2864_2865delCT, n=4) and non-failing donors (n=8). Western Immunoblotting using antibodies directed against different parts of cMyBP-C did not reveal truncated cMyBP-C in MYBPC3mut. Protein expression of cMyBP-C was significantly reduced in MYBPC3mut by 33±5%. Cardiac MyBP-C phosphorylation in MYBPC3mut samples was similar to the values in donor samples, whereas the phosphorylation status of troponin I (cTnI) was reduced by 84±5%, indicating divergent phosphorylation of the two 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 MYBPC3mut (21.4±3.9 kN/m2) compared to donor (34.5±1.7 kN/m2). Moreover, Ca2+-sensititivy was higher in MYBPC3mut (pCa50=5.60±0.04) than in donor (pCa50=5.52±0.03), consistent with reduced cTnI phosphorylation. Treatment with exogenous protein kinase A, to mimic beta-adrenergic stimulation, did not correct reduced maximal force, but abolished the initial difference in Ca2+-sensititivy between MYBPC3mut (pCa50=5.46±0.03) and donor (pCa50=5.48±0.02).Conclusions Truncating MYBPC3 mutations cause haploinsufficiency, deranged phosphorylation of contractile proteins and reduced maximal force generating capacity of cardiomyocytes. The enhanced Ca2+-sensititivy in MYBPC3mut is due to hypophosphorylation of troponin I secondary to mutation-induced dysfunction.