Henk Granzier

Series of Exon-Skipping Events in the Elastic Spring Region of Titin as the Structural Basis for Myofibrillar Elastic Diversity

Titins are megadalton-sized filamentous polypeptides of vertebrate striated muscle. The I-band region of titin underlies the myofibrillar passive tension response to stretch. Here, we show how titins with highly diverse I-band structures and elastic properties are expressed from a single gene. The differentially expressed tandem-Ig, PEVK, and N2B spring elements of titin are coded by 158 exons, which are contained within a 106-kb genomic segment and are all subject to tissue-specific skipping events. In ventricular heart muscle, exons 101 kb apart are joined, leading to the exclusion of 155 exons and the expression of a 2.97-MDa cardiac titin N2B isoform. The atria of mammalian hearts also express larger titins by the exclusion of 90 to 100 exons (cardiac N2BA titin with 3.3 MDa). In the soleus and psoas skeletal muscles, different exon-skipping pathways produce titin transcripts that code for 3.7- and 3.35-MDa titin isoforms, respectively. Mechanical and structural studies indicate that the exon-skipping pathways modulate the fractional extensions of the tandem Ig and PEVK segments, thereby influencing myofibrillar elasticity. Within the mammalian heart, expression of different levels of N2B and N2BA titins likely contributes to the elastic diversity of atrial and ventricular myofibrils.

PKC Phosphorylation of Titin's PEVK Element. A Novel and Conserved Pathway for Modulating Myocardial Stiffness

Rationale: Protein kinase C (PKC) regulates contractility of cardiac muscle cells by phosphorylating thin- and thick- filament–based proteins. Myocardial sarcomeres also contain a third myofilament, titin, and it is unknown whether titin can be phosphorylated by PKC and whether it affects passive tension. Objective: The purpose of this study was to examine the effect of PKC on titin phosphorylation and titin-based passive tension. Methods and Results: Phosphorylation assays with PKC&agr; revealed that titin is phosphorylated in skinned myocardial tissues; this effect is exacerbated by pretreating with protein phosphatase 1. In vitro phosphorylation of recombinant protein representing titin’s spring elements showed that PKC&agr; targets the proline – glutamate – valine – lysine (PEVK) spring element. Furthermore, mass spectrometry in combination with site-directed mutagenesis identified 2 highly conserved sites in the PEVK region that are phosphorylated by PKC&agr; (S11878 and S12022); when these 2 sites are mutated to alanine, phosphorylation is effectively abolished. Mechanical experiments with skinned left ventricular myocardium revealed that PKC&agr; significantly increases titin-based passive tension, an effect that is reversed by protein phosphatase 1. Single molecule force-extension curves show that PKC&agr; decreases the PEVK persistence length (from 1.20 nm to 0.55 nm), without altering the contour length, and using a serially-linked wormlike chain model we show that this increases titin-based passive force with a sarcomere length dependence that is similar to that measured in skinned myocardium after PKC&agr; phosphorylation. Conclusions: PKC phosphorylation of titin is a novel and conserved pathway that links myocardial signaling and myocardial stiffness.

Genetic Variation in Titin in Arrhythmogenic Right Ventricular Cardiomyopathy–Overlap Syndromes

Background— Arrhythmogenic right ventricular cardiomyopathy (ARVC) is an inherited genetic myocardial disease characterized by fibrofatty replacement of the myocardium and a predisposition to cardiac arrhythmias and sudden death. We evaluated the cardiomyopathy gene titin (TTN) as a candidate ARVC gene because of its proximity to an ARVC locus at position 2q32 and the connection of the titin protein to the transitional junction at intercalated disks. Methods and Results— All 312 titin exons known to be expressed in human cardiac titin and the complete 3′ untranslated region were sequenced in 38 ARVC families. Eight unique TTN variants were detected in 7 families, including a prominent Thr2896Ile mutation that showed complete segregation with the ARVC phenotype in 1 large family. The Thr2896IIe mutation maps within a highly conserved immunoglobulin-like fold (Ig10 domain) located in the spring region of titin. Native gel electrophoresis, nuclear magnetic resonance, intrinsic fluorescence, and proteolysis assays of wild-type and mutant Ig10 domains revealed that the Thr2896IIe exchange reduces the structural stability and increases the propensity for degradation of the Ig10 domain. The phenotype of TTN variant carriers was characterized by a history of sudden death (5 of 7 families), progressive myocardial dysfunction causing death or heart transplantation (8 of 14 cases), frequent conduction disease (11 of 14), and incomplete penetrance (86%). Conclusions— Our data provide evidence that titin mutations can cause ARVC, a finding that further expands the origin of the disease beyond desmosomal proteins. Structural impairment of the titin spring is a likely cause of ARVC and constitutes a novel mechanism underlying myocardial remodeling and sudden cardiac death.Background Arrhythmogenic right ventricular cardiomyopathy (ARVC) is an inherited genetic myocardial disease characterized by fibrofatty replacement of the myocardium and a predisposition to cardiac arrhythmias and sudden death. We evaluated the cardiomyopathy gene titin (TTN) as a candidate ARVC gene because of its proximity to an ARVC locus at position 2q32 and the connection of the titin protein to the transitional junction at intercalated disks.

Myocardial Stiffness in Patients With Heart Failure and a Preserved Ejection Fraction Contributions of Collagen and Titin

Background— The purpose of this study was to determine whether patients with heart failure and a preserved ejection fraction (HFpEF) have an increase in passive myocardial stiffness and the extent to which discovered changes depend on changes in extracellular matrix fibrillar collagen and cardiomyocyte titin. Methods and Results— Seventy patients undergoing coronary artery bypass grafting underwent an echocardiogram, plasma biomarker determination, and intraoperative left ventricular epicardial anterior wall biopsy. Patients were divided into 3 groups: referent control (n=17, no hypertension or diabetes mellitus), hypertension (HTN) without (–) HFpEF (n=31), and HTN with (+) HFpEF (n=22). One or more of the following studies were performed on the biopsies: passive stiffness measurements to determine total, collagen-dependent and titin-dependent stiffness (differential extraction assay), collagen assays (biochemistry or histology), or titin isoform and phosphorylation assays. In comparison with controls, patients with HTN(–)HFpEF had no change in left ventricular end-diastolic pressure, myocardial passive stiffness, collagen, or titin phosphorylation but had an increase in biomarkers of inflammation (C-reactive protein, soluble ST2, tissue inhibitor of metalloproteinase 1). In comparison with both control and HTN(–)HFpEF, patients with HTN(+)HFpEF had increased left ventricular end-diastolic pressure, left atrial volume, N-terminal propeptide of brain natriuretic peptide, total, collagen-dependent, and titin-dependent stiffness, insoluble collagen, increased titin phosphorylation on PEVK S11878(S26), reduced phosphorylation on N2B S4185(S469), and increased biomarkers of inflammation. Conclusions— Hypertension in the absence of HFpEF did not alter passive myocardial stiffness. Patients with HTN(+)HFpEF had a significant increase in passive myocardial stiffness; collagen-dependent and titin-dependent stiffness were increased. These data suggest that the development of HFpEF depends on changes in both collagen and titin homeostasis.

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.

Recessive truncating titin gene, TTN, mutations presenting as centronuclear myopathy.

Objective: To identify causative genes for centronuclear myopathies (CNM), a heterogeneous group of rare inherited muscle disorders that often present in infancy or early life with weakness and hypotonia, using next-generation sequencing of whole exomes and genomes. Methods: Whole-exome or -genome sequencing was performed in a cohort of 29 unrelated patients with clinicopathologic diagnoses of CNM or related myopathy depleted for cases with mutations of MTM1, DNM2, and BIN1. Immunofluorescence analyses on muscle biopsies, splicing assays, and gel electrophoresis of patient muscle proteins were performed to determine the molecular consequences of mutations of interest. Results: Autosomal recessive compound heterozygous truncating mutations of the titin gene, TTN, were identified in 5 individuals. Biochemical analyses demonstrated increased titin degradation and truncated titin proteins in patient muscles, establishing the impact of the mutations. Conclusions: Our study identifies truncating TTN mutations as a cause of congenital myopathy that is reported as CNM. Unlike the classic CNM genes that are all involved in excitation-contraction coupling at the triad, TTN encodes the giant sarcomeric protein titin, which forms a myofibrillar backbone for the components of the contractile machinery. This study expands the phenotypic spectrum associated with TTN mutations and indicates that TTN mutation analysis should be considered in cases of possible CNM without mutations in the classic CNM genes.

Thin filament length dysregulation contributes to muscle weakness in nemaline myopathy patients with nebulin deficiency

Nemaline myopathy (NM) is the most common non-dystrophic congenital myopathy. Clinically the most important feature of NM is muscle weakness; however, the mechanisms underlying this weakness are poorly understood. Here, we studied the muscular phenotype of NM patients with a well-defined nebulin mutation (NM-NEB), using a multidisciplinary approach to study thin filament length regulation and muscle contractile performance. SDS-PAGE and western blotting revealed greatly reduced nebulin levels in skeletal muscle of NM-NEB patients, with the most prominent reduction at nebulins N-terminal end. Muscle mechanical studies indicated approximately 60% reduced force generating capacity of NM-NEB muscle and a leftward-shift of the force-sarcomere length relation in NM-NEB muscle fibers. This indicates that the mechanism for the force reduction is likely to include shorter and non-uniform thin filament lengths in NM-NEB muscle compared with control muscle. Immunofluorescence confocal microscopy and electron microscopy studies indicated that average thin filament length is reduced from approximately 1.3 microm in control muscle to approximately 0.75 microm in NM-NEB muscle. Thus, the present study is the first to show a distinct genotype-functional phenotype correlation in patients with NM due to a nebulin mutation, and provides evidence for the notion that dysregulated thin filament length contributes to muscle weakness in NM patients with nebulin mutations. Furthermore, a striking similarity between the contractile and structural phenotypes of nebulin-deficient mouse muscle and human NM-NEB muscle was observed, indicating that the nebulin knockout model is well suited for elucidating the functional basis of muscle weakness in NM and for the development of treatment strategies.

Mouse and computational models link Mlc2v dephosphorylation to altered myosin kinetics in early cardiac disease.

Actin-myosin interactions provide the driving force underlying each heartbeat. The current view is that actin-bound regulatory proteins play a dominant role in the activation of calcium-dependent cardiac muscle contraction. In contrast, the relevance and nature of regulation by myosin regulatory proteins (for example, myosin light chain-2 [MLC2]) in cardiac muscle remain poorly understood. By integrating gene-targeted mouse and computational models, we have identified an indispensable role for ventricular Mlc2 (Mlc2v) phosphorylation in regulating cardiac muscle contraction. Cardiac myosin cycling kinetics, which directly control actin-myosin interactions, were directly affected, but surprisingly, Mlc2v phosphorylation also fed back to cooperatively influence calcium-dependent activation of the thin filament. Loss of these mechanisms produced early defects in the rate of cardiac muscle twitch relaxation and ventricular torsion. Strikingly, these defects preceded the left ventricular dysfunction of heart disease and failure in a mouse model with nonphosphorylatable Mlc2v. Thus, there is a direct and early role for Mlc2 phosphorylation in regulating actin-myosin interactions in striated muscle contraction, and dephosphorylation of Mlc2 or loss of these mechanisms can play a critical role in heart failure.

Physiological functions of the giant elastic protein titin in mammalian striated muscle

The striated muscle sarcomere contains the third filament comprising the giant elastic protein titin, in addition to thick and thin filaments. Titin is the primary source of nonactomyosin-based passive force in both skeletal and cardiac muscles, within the physiological sarcomere length range. Titins force repositions the thick filaments in the center of the sarcomere after contraction or stretch and thus maintains sarcomere length and structural integrity. In the heart, titin determines myocardial wall stiffness, thereby regulating ventricular filling. Recent studies have revealed the mechanisms involved in the fine tuning of titin-based passive force via alternative splicing or posttranslational modification. It has also been discovered that titin performs roles that go beyond passive force generation, such as a regulation of the Frank-Starling mechanism of the heart. In this review, we discuss how titin regulates passive and active properties of striated muscle during normal muscle function and during disease.

Heart rate reduction by If-inhibition improves vascular stiffness and left ventricular systolic and diastolic function in a mouse model of heart failure with preserved ejection fraction

AIMS In diabetes mellitus, heart failure with preserved ejection fraction (HFPEF) is a significant comorbidity. No therapy is available that improves cardiovascular outcomes. The aim of this study was to characterize myocardial function and ventricular-arterial coupling in a mouse model of diabetes and to analyse the effect of selective heart rate (HR) reduction by If-inhibition in this HFPEF-model. METHODS AND RESULTS Control mice, diabetic mice (db/db), and db/db mice treated for 4 weeks with the If-inhibitor ivabradine (db/db-Iva) were compared. Aortic distensibility was measured by magnetic resonance imaging. Left ventricular (LV) pressure-volume analysis was performed in isolated working hearts, with biochemical and histological characterization of the cardiac and aortic phenotype. In db/db aortic stiffness and fibrosis were significantly enhanced compared with controls and were prevented by HR reduction in db/db-Iva. Left ventricular end-systolic elastance (Ees) was increased in db/db compared with controls (6.0 ± 1.3 vs. 3.4 ± 1.2 mmHg/µL, P < 0.01), whereas other contractility markers were reduced. Heart rate reduction in db/db-Iva lowered Ees (4.0 ± 1.1 mmHg/µL, P < 0.01), and improved the other contractility parameters. In db/db active relaxation was prolonged and end-diastolic capacitance was lower compared with controls (28 ± 3 vs. 48 ± 8 μL, P < 0.01). These parameters were ameliorated by HR reduction. Neither myocardial fibrosis nor hypertrophy were detected in db/db, whereas titin N2B expression was increased and phosphorylation of phospholamban was reduced both being prevented by HR reduction in db/db-Iva. CONCLUSION In db/db, a model of HFPEF, selective HR reduction by If-inhibition improved vascular stiffness, LV contractility, and diastolic function. Therefore, If-inhibition might be a therapeutic concept for HFPEF, if confirmed in humans.