Claudia Ferrara

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.

HDAC6 contributes to pathological responses of heart and skeletal muscle to chronic angiotensin-II signaling

Little is known about the function of the cytoplasmic histone deacetylase HDAC6 in striated muscle. Here, we addressed the role of HDAC6 in cardiac and skeletal muscle remodeling induced by the peptide hormone angiotensin II (ANG II), which plays a central role in blood pressure control, heart failure, and associated skeletal muscle wasting. Comparable with wild-type (WT) mice, HDAC6 null mice developed cardiac hypertrophy and fibrosis in response to ANG II. However, whereas WT mice developed systolic dysfunction upon treatment with ANG II, cardiac function was maintained in HDAC6 null mice treated with ANG II for up to 8 wk. The cardioprotective effect of HDAC6 deletion was mimicked in WT mice treated with the small molecule HDAC6 inhibitor tubastatin A. HDAC6 null mice also exhibited improved left ventricular function in the setting of pressure overload mediated by transverse aortic constriction. HDAC6 inhibition appeared to preserve systolic function, in part, by enhancing cooperativity of myofibrillar force generation. Finally, we show that HDAC6 null mice are resistant to skeletal muscle wasting mediated by chronic ANG-II signaling. These findings define novel roles for HDAC6 in striated muscle and suggest potential for HDAC6-selective inhibitors for the treatment of cardiac dysfunction and muscle wasting in patients with heart failure.

Deleting exon 55 from the nebulin gene induces severe muscle weakness in a mouse model for nemaline myopathy

Nebulin--a giant sarcomeric protein--plays a pivotal role in skeletal muscle contractility by specifying thin filament length and function. Although mutations in the gene encoding nebulin (NEB) are a frequent cause of nemaline myopathy, the most common non-dystrophic congenital myopathy, the mechanisms by which mutations in NEB cause muscle weakness remain largely unknown. To better understand these mechanisms, we have generated a mouse model in which Neb exon 55 is deleted (Neb(ΔExon55)) to replicate a founder mutation seen frequently in patients with nemaline myopathy with Ashkenazi Jewish heritage. Neb(ΔExon55) mice are born close to Mendelian ratios, but show growth retardation after birth. Electron microscopy studies show nemaline bodies--a hallmark feature of nemaline myopathy--in muscle fibres from Neb(ΔExon55) mice. Western blotting studies with nebulin-specific antibodies reveal reduced nebulin levels in muscle from Neb(ΔExon55) mice, and immunofluorescence confocal microscopy studies with tropomodulin antibodies and phalloidin reveal that thin filament length is significantly reduced. In line with reduced thin filament length, the maximal force generating capacity of permeabilized muscle fibres and single myofibrils is reduced in Neb(ΔExon55) mice with a more pronounced reduction at longer sarcomere lengths. Finally, in Neb(ΔExon55) mice the regulation of contraction is impaired, as evidenced by marked changes in crossbridge cycling kinetics and by a reduction of the calcium sensitivity of force generation. A novel drug that facilitates calcium binding to the thin filament significantly augmented the calcium sensitivity of submaximal force to levels that exceed those observed in untreated control muscle. In conclusion, we have characterized the first nebulin-based nemaline myopathy model, which recapitulates important features of the phenotype observed in patients harbouring this particular mutation, and which has severe muscle weakness caused by thin filament dysfunction.

Faster cross‐bridge detachment and increased tension cost in human hypertrophic cardiomyopathy with the R403Q MYH7 mutation

The R403Q mutation, located in the S1 domain of the β‐myosin heavy chain, is associated with a severe phenotype of hypertrophic cardiomyopathy (HCM). Increased cross‐bridge relaxation kinetics caused by the R403Q mutation might underlie increased energetic cost of sarcomeric tension generation; however, direct evidence is absent. 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. In human HCM with the R403Q mutation, cross‐bridge relaxation was faster and correlated well with a rise in energetic cost of tension generation. Our data suggest that an increase in tension cost is one of the causes underlying cardiomyopathy development in patients with the R403Q mutation.

R4496C RyR2 mutation impairs atrial and ventricular contractility

A ryanodine receptor 2 mutation associated with catecholaminergic polymorphic ventricular tachycardia renders cardiomyocytes incapable of mediating a positive inotropic response.

Impact of E163R cTnT Mutation on Cardiac Mechanics and Energetics in a Murine Model

Introduction: Many of cTnT mutations linked to cardiomyopathies fall the TNT1 domain/N terminal tail region of unresolved high definition structure. This region (∼94-170) of cTnT is critical to Tm binding and contraction regulation. Here, the impact of the E163R mutation in cTnT-TNT1 on contractile function and tension cost was investigated using intact and skinned preparations from WT and transgenic mouse hearts.Methods: Left and right ventricular trabeculae were dissected from non-transgenic wild type (WT) and heterozygous (Δ160E or E163R) mouse hearts and mounted isometrically to record twitch tension or, when skinned, Ca2+ activated force. Myofibrillar ATPase activity was measured by fluorimetric enzyme coupled assay (de Tombe and Stienen, 1995).Results: Myocardium of E163R mice shows: (i) no change of myosin isoform expression (ii) maintained peak isomentric twitch tension at all stimulation frequencies, (iii) prolonged time to peak and time to 50% relaxation, with preserved rate-adaptation of twitch duration, (iv) changes of the short-term interval force relationship and increased occurrence of spontaneous contractions. No significant differences were found in maximum Ca2+ activated tension of E163R and WT skinned trabeculae. However, Ca2+ sensitivity of tension was significantly increased in E163R skinned trabeculae when compared with WT. As to the economy of force maintenance, preliminary experiments suggest an increase of tension cost in trabeculae from E163R hearts. Resting ATPase activity also tended to be higher in E163R preparations. Kinetics of force development and relaxation will be assessed on single myofibrils, isolated from the same hearts.Conclusions: Both primary sarcomeric changes and secondary E-C coupling alterations contribute to mechanical impairment in E163R cTnT mutant myocardium. Supported by: EC Grant n. 241577 (BIG-Heart)

P.9.10 Deleting exon 55 from the nebulin gene induces severe muscle weakness in a mouse model for nemaline myopathy

Nebulin – a giant sarcomeric protein – plays a pivotal role in skeletal muscle contractility by specifying thin filament length and function. Although mutations in the gene encoding nebulin (NEB) are a frequent cause of nemaline myopathy (NM), the most common non-dystrophic congenital myopathy, the mechanisms by which mutations in NEB cause muscle weakness remain largely unknown. To better understand these mechanisms, we have generated a mouse model in which Neb exon 55 is deleted (Neb Δex55 ) to replicate a founder mutation seen frequently in NM patients with Ashkenazi Jewish heritage. Neb Δex55 mice are born close to Mendelian ratios, but show growth retardation after birth. Electron microscopy studies show nemaline bodies – a hallmark feature of NM – in muscle fibers from Neb Δex55 mice. Western blotting studies with nebulin-specific antibodies reveal reduced nebulin levels in muscle from Neb ΔExon55 mice, and immunofluorescence confocal microscopy studies with tropomodulin antibodies and phalloidin reveal that thin filament length is significantly reduced. In line with reduced thin filament length, the maximal force generating capacity of permeabilized muscle fibers and single myofibrils is reduced in Neb Δex55 mice with a more pronounced reduction at longer sarcomere lengths. Finally, in Neb Δex55 mice the regulation of contraction is impaired, as evidenced by marked changes in cross bridge cycling kinetics and by a reduction of the calcium sensitivity of force generation. A novel drug that facilitates calcium binding to the thin filament significantly augmented the calcium sensitivity of submaximal force to levels that exceed those observed in untreated control muscle. In conclusion, we have characterized the first nebulin-based NM model, which recapitulates important features of the phenotype observed in patients harboring this particular mutation, and which has severe muscle weakness caused by thin filament dysfunction.

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.