C. Ottenheijm

Right Ventricular Diastolic Impairment in Patients With Pulmonary Arterial Hypertension

Background— The role of right ventricular (RV) diastolic stiffness in pulmonary arterial hypertension (PAH) is not well established. Therefore, we investigated the presence and possible underlying mechanisms of RV diastolic stiffness in PAH patients. Methods and Results— Single-beat RV pressure-volume analyses were performed in 21 PAH patients and 7 control subjects to study RV diastolic stiffness. Data are presented as mean±SEM. RV diastolic stiffness (&bgr;) was significantly increased in PAH patients (PAH, 0.050±0.005 versus control, 0.029±0.003; P<0.05) and was closely associated with disease severity. Subsequently, we searched for possible underlying mechanisms using RV tissue of PAH patients undergoing heart/lung transplantation and nonfailing donors. Histological analyses revealed increased cardiomyocyte cross-sectional areas (PAH, 453±31 &mgr;m2 versus control, 218±21 &mgr;m2; P<0.001), indicating RV hypertrophy. In addition, the amount of RV fibrosis was enhanced in PAH tissue (PAH, 9.6±0.7% versus control, 7.2±0.6%; P<0.01). To investigate the contribution of stiffening of the sarcomere (the contractile apparatus of RV cardiomyocytes) to RV diastolic stiffness, we isolated and membrane-permeabilized single RV cardiomyocytes. Passive tension at different sarcomere lengths was significantly higher in PAH patients compared with control subjects (>200%; Pinteraction<0.001), indicating stiffening of RV sarcomeres. An important regulator of sarcomeric stiffening is the sarcomeric protein titin. Therefore, we investigated titin isoform composition and phosphorylation. No alterations were observed in titin isoform composition (N2BA/N2B ratio: PAH, 0.78±0.07 versus control, 0.91±0.08), but titin phosphorylation in RV tissue of PAH patients was significantly reduced (PAH, 0.16±0.01 arbitrary units versus control, 0.20±0.01 arbitrary units; P<0.05). Conclusions— RV diastolic stiffness is significantly increased in PAH patients, with important contributions from increased collagen and intrinsic stiffening of the RV cardiomyocyte sarcomeres.

Myocardial Titin Hypophosphorylation Importantly Contributes to Heart Failure With Preserved Ejection Fraction in a Rat Metabolic Risk Model

Background—Obesity and diabetes mellitus are important metabolic risk factors and frequent comorbidities in heart failure with preserved ejection fraction. They contribute to myocardial diastolic dysfunction (DD) through collagen deposition or titin modification. The relative importance for myocardial DD of collagen deposition and titin modification was investigated in obese, diabetic ZSF1 rats after heart failure with preserved ejection fraction development at 20 weeks. Methods and Results—Four groups of rats (Wistar-Kyoto, n=11; lean ZSF1, n=11; obese ZSF1, n=11, and obese ZSF1 with high-fat diet, n=11) were followed up for 20 weeks with repeat metabolic, renal, and echocardiographic evaluations and hemodynamically assessed at euthanization. Myocardial collagen, collagen cross-linking, titin isoforms, and phosphorylation were also determined. Resting tension (Fpassive)–sarcomere length relations were obtained in small muscle strips before and after KCl–KI treatment, which unanchors titin and allows contributions of titin and extracellular matrix to Fpassive to be discerned. At 20 weeks, the lean ZSF1 group was hypertensive, whereas both obese ZSF1 groups were hypertensive and diabetic. Only the obese ZSF1 groups had developed heart failure with preserved ejection fraction, which was evident from increased lung weight, preserved left ventricular ejection fraction, and left ventricular DD. The underlying myocardial DD was obvious from high muscle strip stiffness, which was largely (±80%) attributable to titin hypophosphorylation. The latter occurred specifically at the S3991 site of the elastic N2Bus segment and at the S12884 site of the PEVK segment. Conclusions—Obese ZSF1 rats developed heart failure with preserved ejection fraction during a 20-week time span. Titin hypophosphorylation importantly contributed to the underlying myocardial DD.

Leiomodin-3 dysfunction results in thin filament disorganization and nemaline myopathy

Nemaline myopathy (NM) is a genetic muscle disorder characterized by muscle dysfunction and electron-dense protein accumulations (nemaline bodies) in myofibers. Pathogenic mutations have been described in 9 genes to date, but the genetic basis remains unknown in many cases. Here, using an approach that combined whole-exome sequencing (WES) and Sanger sequencing, we identified homozygous or compound heterozygous variants in LMOD3 in 21 patients from 14 families with severe, usually lethal, NM. LMOD3 encodes leiomodin-3 (LMOD3), a 65-kDa protein expressed in skeletal and cardiac muscle. LMOD3 was expressed from early stages of muscle differentiation; localized to actin thin filaments, with enrichment near the pointed ends; and had strong actin filament-nucleating activity. Loss of LMOD3 in patient muscle resulted in shortening and disorganization of thin filaments. Knockdown of lmod3 in zebrafish replicated NM-associated functional and pathological phenotypes. Together, these findings indicate that mutations in the gene encoding LMOD3 underlie congenital myopathy and demonstrate that LMOD3 is essential for the organization of sarcomeric thin filaments in skeletal muscle.

Nebulin Alters Cross-bridge Cycling Kinetics and Increases Thin Filament Activation A NOVEL MECHANISM FOR INCREASING TENSION AND REDUCING TENSION COST

Nebulin is a giant filamentous F-actin-binding protein (∼800 kDa) that binds along the thin filament of the skeletal muscle sarcomere. Nebulin is one of the least well understood major muscle proteins. Although nebulin is usually viewed as a structural protein, here we investigated whether nebulin plays a role in muscle contraction by using skinned muscle fiber bundles from a nebulin knock-out (NEB KO) mouse model. We measured force-pCa (−log[Ca2+]) and force-ATPase relations, as well as the rate of tension re-development (ktr) in tibialis cranialis muscle fibers. To rule out any alterations in troponin (Tn) isoform expression and/or status of Tn phosphorylation, we studied fiber bundles that had been reconstituted with bacterially expressed fast skeletal muscle recombinant Tn. We also performed a detailed analysis of myosin heavy chain, myosin light chain, and myosin light chain 2 phosphorylation, which showed no significant differences between wild type and NEB KO. Our mechanical studies revealed that NEB KO fibers had increased tension cost (5.9 versus 4.4 pmol millinewtons−1 mm−1 s−1) and reductions in ktr (4.7 versus 7.3 s−1), calcium sensitivity (pCa50 5.74 versus 5.90), and cooperativity of activation (nH 3.64 versus 4.38). Our findings indicate the following: 1) in skeletal muscle nebulin increases thin filament activation, and 2) through altering cross-bridge cycling kinetics, nebulin increases force and efficiency of contraction. These novel properties of nebulin add a new level of understanding of skeletal muscle function and provide a mechanism for the severe muscle weakness in patients with nebulin-based nemaline myopathy.

Experimentally increasing titin compliance in a novel mouse model attenuates the Frank-Starling mechanism but has a beneficial effect on diastole.

Background— Experimentally upregulating compliant titins has been suggested as a therapeutic for lowering pathological diastolic stiffness levels. However, how increasing titin compliance impacts global cardiac function requires in-depth study. We investigate the effect of upregulating compliant titins in a novel mouse model with a genetically altered titin splicing factor; integrative approaches were used from intact cardiomyocyte mechanics to pressure-volume analysis and Doppler echocardiography. Methods and Results— Compliant titins were upregulated through deletion of the RNA Recognition Motif of the splicing factor RBM20 (Rbm20&Dgr;RRMmice). A genome-wide exon expression analysis and a candidate approach revealed that the phenotype is likely to be dominated by greatly increased lengths of titin’s spring elements. At both cardiomyocyte and left ventricular chamber levels, diastolic stiffness was reduced in heterozygous (+/−) Rbm20&Dgr;RRMmice with a further reduction in homozygous (−/−) mice at only the intact myocyte level. Fibrosis was present in only −/− Rbm20&Dgr;RRM hearts. The Frank-Starling Mechanism was reduced in a graded fashion in Rbm20&Dgr;RRM mice, at both the cardiomyocyte and left ventricular chamber levels. Exercise tests revealed an increase in exercise capacity in +/− mice. Conclusions— Titin is not only important in diastolic but also in systolic cardiac function. Upregulating compliant titins reduces diastolic chamber stiffness owing to the increased compliance of myocytes, but it depresses end-systolic elastance; under conditions of exercise, the beneficial effects on diastolic function dominate. Therapeutic manipulation of the RBM20-based splicing system might be able to minimize effects on fibrosis and systolic function while improving the diastolic function in patients with heart failure.

Nebulin, a major player in muscle health and disease

Nebulin is a giant 600‐ to 900‐kDa filamentous protein that is an integral component of the skeletal muscle thin filament. Its functions have remained largely nebulous because of its large size and the difficulty in extracting nebulin in a native state from muscle. Recent improvements in the field, especially the development of knockout mouse models deficient in nebulin (NEB‐KO mice), indicate now that nebulin performs a surprisingly wide range of functions. In addition to a major role in thin‐filament length specification, nebulin also functions in the regulation of muscle contraction, as indicated by the findings that muscle fibers deficient in nebulin have a higher tension cost, and develop less force due to reduced myofilament calcium sensitivity and altered crossbridge cycling kinetics. In addition, the function of nebulin extends to a role in calcium homeostasis. These novel functions indicate that nebulin might have evolved in vertebrate skeletal muscles to develop high levels of muscle force efficiently. Finally, the NEB‐KO mouse models also highlight the role of nebulin in the assembly and alignment of the Z disks. Notably, rapid progress in understanding the roles of nebulin in vivo provides clinically important insights into how nebulin deficiency in patients with nemaline myopathy contributes to debilitating muscle weakness.—Labeit, S., Ottenheijm, C. A. C., Granzier, H. Nebulin, a major player in muscle health and disease. FASEB J. 25, 822–829 (2011). www.fasebj.org

Contractile Dysfunction of Left Ventricular Cardiomyocytes in Patients With Pulmonary Arterial Hypertension

BACKGROUND After lung transplantation, increased left ventricular (LV) filling can lead to LV failure, increasing the risk of post-operative complications and mortality. LV dysfunction in pulmonary arterial hypertension (PAH) is characterized by a reduced LV ejection fraction and impaired diastolic function. OBJECTIVES The pathophysiology of LV dysfunction in PAH is incompletely understood. This study sought to assess the contribution of atrophy and contractility of cardiomyocytes to LV dysfunction in PAH patients. METHODS LV function was assessed by cardiac magnetic resonance imaging. In addition, LV biopsies were obtained in 9 PAH patients and 10 donors. The cross-sectional area (CSA) and force-generating capacity of isolated single cardiomyocytes was investigated. RESULTS Magnetic resonance imaging analysis revealed a significant reduction in LV ejection fraction in PAH patients, indicating a reduction in LV contractility. The CSA of LV cardiomyocytes of PAH patients was significantly reduced (~30%), indicating LV cardiomyocyte atrophy. The maximal force-generating capacity, normalized to cardiomyocyte CSA, was significantly reduced (~25%). Also, a reduction in the number of available myosin-based cross-bridges was found to cause the contractile weakness of cardiomyocytes. This finding was supported by protein analyses, which showed an ~30% reduction in the myosin/actin ratio in cardiomyocytes from PAH patients. Finally, the phosphorylation level of sarcomeric proteins was reduced in PAH patients, which was accompanied by increased calcium sensitivity of force generation. CONCLUSIONS The contractile function and the CSA of LV cardiomyocytes is substantially reduced in PAH patients. We propose that these changes contribute to the reduced in vivo contractility of the LV in PAH patients.

Muscle histopathology in nebulin-related nemaline myopathy: ultrastrastructural findings correlated to disease severity and genotype.

Nemaline myopathy (NM) is a rare congenital myopathy characterised by hypotonia, muscle weakness, and often skeletal muscle deformities with the presence of nemaline bodies (rods) in the muscle biopsy. The nebulin (NEB) gene is the most commonly mutated and is thought to account for approximately 50% of genetically diagnosed cases of NM. We undertook a detailed muscle morphological analysis of 14 NEB-mutated NM patients with different clinical forms to define muscle pathological patterns and correlate them with clinical course and genotype. Three groups were identified according to clinical severity. Group 1 (n = 5) comprises severe/lethal NM and biopsy in the first days of life. Group 2 (n = 4) includes intermediate NM and biopsy in infancy. Group 3 (n = 5) comprises typical/mild NM and biopsy in childhood or early adult life. Biopsies underwent histoenzymological, immunohistochemical and ultrastructural analysis. Fibre type distribution patterns, rod characteristics, distribution and localization were investigated. Contractile performance was studied in muscle fibre preparations isolated from seven muscle biopsies from each of the three groups. G1 showed significant myofibrillar dissociation and smallness with scattered globular rods in one third of fibres; there was no type 1 predominance. G2 presented milder sarcomeric dissociation, dispersed or clustered nemaline bodies, and type 1 predominance/uniformity. In contrast, G3 had well-delimited clusters of subsarcolemmal elongated rods and type 1 uniformity without sarcomeric alterations. In accordance with the clinical and morphological data, functional studies revealed markedly low forces in muscle bundles from G1 and a better contractile performance in muscle bundles from biopsies of patients from G2, and G3.In conclusion NEB-mutated NM patients present a wide spectrum of morphological features. It is difficult to establish firm genotype phenotype correlation. Interestingly, there was a correlation between clinical severity on the one hand and the degree of sarcomeric dissociation and contractility efficiency on the other. By contrast the percentage of fibres occupied by rods, as well as the quantity and the sub sarcolemmal position of rods, appears to inversely correlate with severity. Based on our observations, we propose myofibrillar dissociation and changes in contractility as an important cause of muscle weakness in NEB-mutated NM patients.

K7del is a common TPM2 gene mutation associated with nemaline myopathy and raised myofibre calcium sensitivity

Mutations in the TPM2 gene, which encodes β-tropomyosin, are an established cause of several congenital skeletal myopathies and distal arthrogryposis. We have identified a TPM2 mutation, p.K7del, in five unrelated families with nemaline myopathy and a consistent distinctive clinical phenotype. Patients develop large joint contractures during childhood, followed by slowly progressive skeletal muscle weakness during adulthood. The TPM2 p.K7del mutation results in the loss of a highly conserved lysine residue near the N-terminus of β-tropomyosin, which is predicted to disrupt head-to-tail polymerization of tropomyosin. Recombinant K7del-β-tropomyosin incorporates poorly into sarcomeres in C2C12 myotubes and has a reduced affinity for actin. Two-dimensional gel electrophoresis of patient muscle and primary patient cultured myotubes showed that mutant protein is expressed but incorporates poorly into sarcomeres and likely accumulates in nemaline rods. In vitro studies using recombinant K7del-β-tropomyosin and force measurements from single dissected patient myofibres showed increased myofilament calcium sensitivity. Together these data indicate that p.K7del is a common recurrent TPM2 mutation associated with mild nemaline myopathy. The p.K7del mutation likely disrupts head-to-tail polymerization of tropomyosin, which impairs incorporation into sarcomeres and also affects the equilibrium of the troponin/tropomyosin-dependent calcium switch of muscle. Joint contractures may stem from chronic muscle hypercontraction due to increased myofibrillar calcium sensitivity while declining strength in adulthood likely arises from other mechanisms, such as myofibre decompensation and fatty infiltration. These results suggest that patients may benefit from therapies that reduce skeletal muscle calcium sensitivity, and we highlight late muscle decompensation as an important cause of morbidity.

Protein changes contributing to right ventricular cardiomyocyte diastolic dysfunction in pulmonary arterial hypertension.

Background Right ventricular (RV) diastolic function is impaired in patients with pulmonary arterial hypertension (PAH). Our previous study showed that elevated cardiomyocyte stiffness and myofilament Ca2+ sensitivity underlie diastolic dysfunction in PAH. This study investigates protein modifications contributing to cellular diastolic dysfunction in PAH. Methods and Results RV samples from PAH patients undergoing heart‐lung transplantation were compared to non‐failing donors (Don). Titin stiffness contribution to RV diastolic dysfunction was determined by Western‐blot analyses using antibodies to protein‐kinase‐A (PKA), Cα (PKCα) and Ca2+/calmoduling‐dependent‐kinase (CamKIIδ) titin and phospholamban (PLN) phosphorylation sites: N2B (Ser469), PEVK (Ser170 and Ser26), and PLN (Thr17), respectively. PKA and PKCα sites were significantly less phosphorylated in PAH compared with donors (P<0.0001). To test the functional relevance of PKA‐, PKCα‐, and CamKIIδ‐mediated titin phosphorylation, we measured the stiffness of single RV cardiomyocytes before and after kinase incubation. PKA significantly decreased PAH RV cardiomyocyte diastolic stiffness, PKCα further increased stiffness while CamKIIδ had no major effect. CamKIIδ activation was determined indirectly by measuring PLN Thr17phosphorylation level. No significant changes were found between the groups. Myofilament Ca2+ sensitivity is mediated by sarcomeric troponin I (cTnI) phosphorylation. We observed increased unphosphorylated cTnI in PAH compared with donors (P<0.05) and reduced PKA‐mediated cTnI phosphorylation (Ser22/23) (P<0.001). Finally, alterations in Ca2+‐handling proteins contribute to RV diastolic dysfunction due to insufficient diastolic Ca2+ clearance. PAH SERCA2a levels and PLN phosphorylation were significantly reduced compared with donors (P<0.05). Conclusions Increased titin stiffness, reduced cTnI phosphorylation, and altered levels of phosphorylation of Ca2+ handling proteins contribute to RV diastolic dysfunction in PAH.