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1.  Combination of Whole Genome Sequencing, Linkage and Functional Studies Implicates a Missense Mutation in Titin as a Cause of Autosomal Dominant Cardiomyopathy with Features of Left Ventricular Non-Compaction 
High throughput next generation sequencing techniques have made whole genome sequencing accessible in clinical practice, however, the abundance of variation in the human genomes makes the identification of a disease-causing mutation on a background of benign rare variants challenging.
Methods and Results
Here we combine whole genome sequencing with linkage analysis in a three-generation family affected by cardiomyopathy with features of autosomal dominant left-ventricular non-compaction cardiomyopathy. A missense mutation in the giant protein titin is the only plausible disease-causing variant that segregates with disease amongst the eight surviving affected individuals, with interrogation of the entire genome excluding other potential causes. This A178D missense mutation, affecting a conserved residue in the second immunoglobulin-like domain of titin, was introduced in a bacterially expressed recombinant protein fragment and biophysically characterised in comparison to its wild-type counterpart. Multiple experiments, including size exclusion chromatography, small angle X-ray scattering and circular dichroism spectroscopy suggest partial unfolding and domain destabilisation in the presence of the mutation. Moreover, binding experiments in mammalian cells show that the mutation markedly impairs binding to the titin ligand telethonin.
Here we present genetic and functional evidence implicating the novel A178D missense mutation in titin as the cause of a highly penetrant familial cardiomyopathy with features of left-ventricular non-compaction. This expands the spectrum of titin’s roles in cardiomyopathies. It furthermore highlights that rare titin missense variants, currently often ignored or left un-interpreted, should be considered to be relevant for cardiomyopathies and can be identified by the approach presented here.
PMCID: PMC5068189  PMID: 27625337
genetics; basic science research; left ventricular noncompaction; cardiomyopathy; whole genome sequencing; titin; telethonin; missense mutation
2.  Combination of Whole Genome Sequencing, Linkage, and Functional Studies Implicates a Missense Mutation in Titin as a Cause of Autosomal Dominant Cardiomyopathy With Features of Left Ventricular Noncompaction 
Supplemental Digital Content is available in the text.
High throughput next-generation sequencing techniques have made whole genome sequencing accessible in clinical practice; however, the abundance of variation in the human genomes makes the identification of a disease-causing mutation on a background of benign rare variants challenging.
Methods and Results—
Here we combine whole genome sequencing with linkage analysis in a 3-generation family affected by cardiomyopathy with features of autosomal dominant left ventricular noncompaction cardiomyopathy. A missense mutation in the giant protein titin is the only plausible disease-causing variant that segregates with disease among the 7 surviving affected individuals, with interrogation of the entire genome excluding other potential causes. This A178D missense mutation, affecting a conserved residue in the second immunoglobulin-like domain of titin, was introduced in a bacterially expressed recombinant protein fragment and biophysically characterized in comparison to its wild-type counterpart. Multiple experiments, including size exclusion chromatography, small-angle x ray scattering, and circular dichroism spectroscopy suggest partial unfolding and domain destabilization in the presence of the mutation. Moreover, binding experiments in mammalian cells show that the mutation markedly impairs binding to the titin ligand telethonin.
Here we present genetic and functional evidence implicating the novel A178D missense mutation in titin as the cause of a highly penetrant familial cardiomyopathy with features of left ventricular noncompaction. This expands the spectrum of titin’s roles in cardiomyopathies. It furthermore highlights that rare titin missense variants, currently often ignored or left uninterpreted, should be considered to be relevant for cardiomyopathies and can be identified by the approach presented here.
PMCID: PMC5068189  PMID: 27625337
cardiomyopathy; left ventricular noncompaction; missense mutation; telethonin; titin; whole genome sequencing
3.  Cardiac cytoarchitecture — why the “hardware” is important for heart function!☆ 
Biochimica et Biophysica Acta  2016;1863(7Part B):1857-1863.
Cells that constitute fully differentiated tissues are characterised by an architecture that makes them perfectly suited for the job they have to do. This is especially obvious for cardiomyocytes, which have an extremely regular shape and display a paracrystalline arrangement of their cytoplasmic components. This article will focus on the two major cytoskeletal multiprotein complexes that are found in cardiomyocytes, the myofibrils, which are responsible for contraction and the intercalated disc, which mediates mechanical and electrochemical contact between individual cardiomyocytes.
Recent studies have revealed that these two sites are also crucial in sensing excessive mechanical strain. Signalling processes will be triggered that## lead to changes in gene expression and eventually lead to an altered cardiac cytoarchitecture in the diseased heart, which results in a compromised function. Thus, understanding these changes and the signals that lead to them is crucial to design treatment strategies that can attenuate these processes. This article is part of a Special Issue entitled: Cardiomyocyte Biology: Integration of Developmental and Environmental Cues in the Heart edited by Marcus Schaub and Hughes Abriel.
•General description of cardiac cytoarchitecture during development and disease with an emphasis on cytoskeletal proteins•Cardiac cytoarchitecture and signalling in cardiomyopathy
PMCID: PMC5104690  PMID: 26577135
Intercalated disc; Myofibril; M-band; Formin; Dilated cardiomyopathy
4.  MLP and CARP are linked to chronic PKCα signalling in dilated cardiomyopathy 
Nature Communications  2016;7:12120.
MLP (muscle LIM protein)-deficient mice count among the first mouse models for dilated cardiomyopathy (DCM), yet the exact role of MLP in cardiac signalling processes is still enigmatic. Elevated PKCα signalling activity is known to be an important contributor to heart failure. Here we show that MLP directly inhibits the activity of PKCα. In end-stage DCM, PKCα is concentrated at the intercalated disc of cardiomyocytes, where it is sequestered by the adaptor protein CARP in a multiprotein complex together with PLCβ1. In mice deficient for both MLP and CARP the chronic PKCα signalling chain at the intercalated disc is broken and they remain healthy. Our results suggest that the main role of MLP in heart lies in the direct inhibition of PKCα and that chronic uninhibited PKCα activity at the intercalated disc in the absence of functional MLP leads to heart failure.
Altered function of the muscle LIM protein (MLP) causes dilated cardiomyopathy in mice and humans. Lange et al. explain the molecular role of MLP in the heart by showing that it affects the signalling complex at the intercalated discs of failing hearts that consists of PKCα, PLCβ1 and CARP by inhibiting PKCα auto-phosphorylation and function.
PMCID: PMC4931343  PMID: 27353086
5.  Sarcoplasmic reticulum is an intermediary of mitochondrial and myofibrillar growth at the intercalated disc 
In cardiomyocytes columns of intermyofibrillar mitochondria run up to the intercalated disc (ID); half are collinear with those in the neighbouring cell, suggesting coordinated addition of sarcomeres and mitochondria both within and between cells during cardiomyocyte growth. Recent evidence for an association between sarcoplasmic reticulum (SR) and mitochondria indicates that the SR may be an intermediary in this coordinated behaviour. For this reason we have investigated the arrangement of SR and t tubules with respect to mitochondria and myofibrils, particularly at the ID. In the body of the cardiomyocyte the mitochondrial columns are frequently intersected by transverse tubules. In addition, we find that a majority of axial tubules are sandwiched between mitochondria and myofibril. No tubules are found at the ID. SR coats mitochondrial columns and fibrils throughout their length and reaches towards the peaks of the ID membrane where it attaches in the form of junctional (j)SR. These peripheral ID couplings are often situated between mitochondria and ID membrane, suggesting an SR connection between the two. In dilated cardiomyopathy (DCM) the mitochondria are somewhat disordered and clumped. In a mouse model for DCM, the muscle LIM protein KO, we find that there is a lack of mitochondria near the ID, suggesting the uncoupling of the myofibril/mitochondria organisation during growth. SR still coats the fibrils and reaches the ID folds in a jSR coupling. Unlike in control tissue, however, loops and long fingers of ID membrane penetrate into the proximal sarcomere suggesting a possible intermediary state in cardiomyocyte growth.
PMCID: PMC5010836  PMID: 27329158
Dilated cardiomyopathy; Heart; Mitochondria; Intercalated disc; T tubules; Electron tomography
6.  α5β1 integrin recycling promotes Arp2/3-independent cancer cell invasion via the formin FHOD3 
The Journal of Cell Biology  2015;210(6):1013-1031.
Rab-coupling protein–mediated integrin trafficking promotes filopodia formation via RhoA-ROCK-FHOD3, generating non-lamellipodial actin spike protrusions that drive cancer cell migration in 3D extracellular matrix and in vivo.
Invasive migration in 3D extracellular matrix (ECM) is crucial to cancer metastasis, yet little is known of the molecular mechanisms that drive reorganization of the cytoskeleton as cancer cells disseminate in vivo. 2D Rac-driven lamellipodial migration is well understood, but how these features apply to 3D migration is not clear. We find that lamellipodia-like protrusions and retrograde actin flow are indeed observed in cells moving in 3D ECM. However, Rab-coupling protein (RCP)-driven endocytic recycling of α5β1 integrin enhances invasive migration of cancer cells into fibronectin-rich 3D ECM, driven by RhoA and filopodial spike-based protrusions, not lamellipodia. Furthermore, we show that actin spike protrusions are Arp2/3-independent. Dynamic actin spike assembly in cells invading in vitro and in vivo is regulated by Formin homology-2 domain containing 3 (FHOD3), which is activated by RhoA/ROCK, establishing a novel mechanism through which the RCP–α5β1 pathway reprograms the actin cytoskeleton to promote invasive migration and local invasion in vivo.
PMCID: PMC4576860  PMID: 26370503
7.  The Cardiac Stress Response Factor Ms1 Can Bind to DNA and Has a Function in the Nucleus 
PLoS ONE  2015;10(12):e0144614.
Ms1 (also known as STARS and ABRA) has been shown to act as an early stress response gene in processes as different as hypertrophy in skeletal and cardiac muscle and growth of collateral blood vessels. It is important for cardiac development in zebrafish and is upregulated in mouse models for cardiac hypertrophy as well as in human failing hearts. Ms1 possesses actin binding sites at its C-terminus and is usually found in the cell bound to actin filaments in the cytosol or in sarcomeres. We determined the NMR structure of the only folded domain of Ms1 comprising the second actin binding site called actin binding domain 2 (ABD2, residues 294–375), and found that it is similar to the winged helix-turn-helix fold adopted mainly by DNA binding domains of transcriptional factors. In vitro experiments show specific binding of this domain, in combination with a newly discovered AT-hook motif located N-terminally, to the sequence (A/C/G)AAA(C/A). NMR and fluorescence titration experiments confirm that this motif is indeed bound specifically by the recognition helix. In neonatal rat cardiomyocytes endogenous Ms1 is found in the nucleus in a spotted pattern, reminiscent of PML bodies. In adult rat cardiomyocytes Ms1 is exclusively found in the sarcomere. A nuclear localisation site in the N-terminus of the protein is required for nuclear localisation. This suggests that Ms1 has the potential to act directly in the nucleus through specific interaction with DNA in development and potentially as a response to stress in adult tissues.
PMCID: PMC4682817  PMID: 26656831
8.  OBSCN Mutations Associated with Dilated Cardiomyopathy and Haploinsufficiency 
PLoS ONE  2015;10(9):e0138568.
Studies of the functional consequences of DCM-causing mutations have been limited to a few cases where patients with known mutations had heart transplants. To increase the number of potential tissue samples for direct investigation we performed whole exon sequencing of explanted heart muscle samples from 30 patients that had a diagnosis of familial dilated cardiomyopathy and screened for potentially disease-causing mutations in 58 HCM or DCM-related genes.
We identified 5 potentially disease-causing OBSCN mutations in 4 samples; one sample had two OBSCN mutations and one mutation was judged to be not disease-related. Also identified were 6 truncating mutations in TTN, 3 mutations in MYH7, 2 in DSP and one each in TNNC1, TNNI3, MYOM1, VCL, GLA, PLB, TCAP, PKP2 and LAMA4. The mean level of obscurin mRNA was significantly greater and more variable in healthy donor samples than the DCM samples but did not correlate with OBSCN mutations. A single obscurin protein band was observed in human heart myofibrils with apparent mass 960 ± 60 kDa. The three samples with OBSCN mutations had significantly lower levels of obscurin immunoreactive material than DCM samples without OBSCN mutations (45±7, 48±3, and 72±6% of control level).Obscurin levels in DCM controls, donor heart and myectomy samples were the same.
OBSCN mutations may result in the development of a DCM phenotype via haploinsufficiency. Mutations in the obscurin gene should be considered as a significant causal factor of DCM, alone or in concert with other mutations.
PMCID: PMC4583186  PMID: 26406308
9.  Involvement of unconventional myosin VI in myoblast function and myotube formation 
Histochemistry and Cell Biology  2015;144(1):21-38.
The important role of unconventional myosin VI (MVI) in skeletal and cardiac muscle has been recently postulated (Karolczak et al. in Histochem Cell Biol 139:873–885, 2013). Here, we addressed for the first time a role for this unique myosin motor in myogenic cells as well as during their differentiation into myotubes. During myoblast differentiation, the isoform expression pattern of MVI and its subcellular localization underwent changes. In undifferentiated myoblasts, MVI-stained puncti were seen throughout the cytoplasm and were in close proximity to actin filaments, Golgi apparatus, vinculin-, and talin-rich focal adhesion as well as endoplasmic reticulum. Colocalization of MVI with endoplasmic reticulum was enhanced during myotube formation, and differentiation-dependent association was also seen in sarcoplasmic reticulum of neonatal rat cardiomyocytes (NRCs). Moreover, we observed enrichment of MVI in myotube regions containing acetylcholine receptor-rich clusters, suggesting its involvement in the organization of the muscle postsynaptic machinery. Overexpression of the H246R MVI mutant (associated with hypertrophic cardiomyopathy) in myoblasts and NRCs caused the formation of abnormally large intracellular vesicles. MVI knockdown caused changes in myoblast morphology and inhibition of their migration. On the subcellular level, MVI-depleted myoblasts exhibited aberrations in the organization of actin cytoskeleton and adhesive structures as well as in integrity of Golgi apparatus and endoplasmic reticulum. Also, MVI depletion or overexpression of H246R mutant caused the formation of significantly wider or aberrant myotubes, respectively, indicative of involvement of MVI in myoblast differentiation. The presented results suggest an important role for MVI in myogenic cells and possibly in myoblast differentiation.
PMCID: PMC4469105  PMID: 25896210
Actin cytoskeleton; Adhesion; Cardiomyocytes; Cell migration; Differentiation; Endoplasmic reticulum; Golgi apparatus; Neuromuscular junction; Talin
10.  Adaptations of cytoarchitecture in human dilated cardiomyopathy 
Biophysical Reviews  2014;7:25-32.
Hypertrophic cardiomyopathy is characterised by a histological phenotype of myocyte disarray, but heart tissue samples from patients with dilated cardiomyopathy (DCM) often look comparatively similar to those from healthy individuals apart from conspicuous regions of fibrosis and necrosis. We have previously investigated subcellular alterations in the cytoarchitecture of mouse models of dilated cardiomyopathy and found that both the organisation and composition of the intercalated disc, i.e. the specialised type of cell–cell contact in the heart, is altered. There is also is a change in the composition of the M-band of the sarcomere due to an expression shift towards the more extensible embryonic heart (EH)-myomesin isoform. Analysis of human samples from the Sydney Human Heart Tissue Bank have revealed similar structural findings and also provided evidence for a dramatic change in overall cardiomyocyte size control, which has also been seen in the mouse. Together these changes in cytoarchitecture probably contribute to the decreased functional output that is seen in DCM.
PMCID: PMC4322184  PMID: 25685241
Cytoskeleton; Intercalated disc; Dilated cardiomyopathy; Formin; M-band
11.  FHOD1 is needed for directed Forces and Adhesion Maturation during Cell Spreading and Migration 
Developmental cell  2013;27(5):545-559.
Matrix adhesions provide critical signals for cell growth or differentiation. They form through a number of distinct steps that follow integrin binding to matrix ligands. In an early step, integrins form clusters that support actin polymerization by an unknown mechanism. This raises the question of how actin polymerization occurs at the integrin clusters. We report here that a major formin in mouse fibroblasts, FHOD1 is recruited to integrin clusters, resulting in actin assembly. Using cell-spreading assays on lipid bilayers, solid substrates and high-resolution force sensing pillar arrays, we find that knockdown of FHOD1 impairs spreading, coordinated application of adhesive force and adhesion maturation. Finally we show that targeting of FHOD1 to the integrin sites depends on the direct interaction with Src family kinases, and is upstream of the activation by Rho Kinase. Thus our findings provide insights into the mechanisms of cell migration with implications for development and disease.
PMCID: PMC3890431  PMID: 24331927
12.  Two distinct phosphorylation events govern the function of muscle FHOD3 
Posttranslational modifications such as phosphorylation are universally acknowledged regulators of protein function. Recently we characterised a striated muscle-specific isoform of the formin FHOD3 that displays distinct subcellular targeting and protein half-life compared to its non-muscle counterpart, which is dependent on phosphorylation by CK2 (formerly casein kinase 2). We now show that the two isoforms of FHOD3 are already expressed in the vertebrate embryonic heart. Analysis of CK2alpha knockout mice showed that phosphorylation by CK2 is required for proper targeting of muscle FHOD3 to the myofibrils also in embryonic cardiomyocytes in situ. The localisation of muscle FHOD3 in the sarcomere varies depending on the maturation state, being either broader or restricted to the Z-disc proper in adult heart. Following myofibril disassembly such as in dedifferentiating adult rat cardiomyocytes in culture, the expression of non-muscle FHOD3 is up-regulated, which is reversed once the myofibrils are reassembled. The shift in expression levels of different isoforms is accompanied by an increased co-localisation with p62, which is involved in autophagy, and affects the half-life of FHOD3.
Phosphorylation of three amino acids in the C-terminus of FHOD3 by ROCK1 is sufficient for activation, which results in increased actin filament synthesis in cardiomyocytes and also a broader localisation pattern of FHOD3 in the myofibrils. ROCK1 can directly phosphorylate FHOD3 and FHOD3 seems to be the downstream mediator of the exaggerated actin filament formation phenotype that is induced in cardiomyocytes upon the overexpression of constitutively active ROCK1. We conclude that the expression of the muscle FHOD3 isoform is characteristic for the healthy mature heart and that two distinct phosphorylation events are crucial to regulate its activity in thin filament assembly and maintenance.
PMCID: PMC3696992  PMID: 23052206
myofibril; formin; cardiac cytoarchitecture; heart development
13.  Four-and-a-half LIM domains proteins are novel regulators of the protein kinase D pathway in cardiac myocytes 
Biochemical Journal  2014;457(Pt 3):451-461.
PKD (protein kinase D) is a serine/threonine kinase implicated in multiple cardiac roles, including the phosphorylation of the class II HDAC5 (histone deacetylase isoform 5) and thereby de-repression of MEF2 (myocyte enhancer factor 2) transcription factor activity. In the present study we identify FHL1 (four-and-a-half LIM domains protein 1) and FHL2 as novel binding partners for PKD in cardiac myocytes. This was confirmed by pull-down assays using recombinant GST-fused proteins and heterologously or endogenously expressed PKD in adult rat ventricular myocytes or NRVMs (neonatal rat ventricular myocytes) respectively, and by co-immunoprecipitation of FHL1 and FHL2 with GFP–PKD1 fusion protein expressed in NRVMs. In vitro kinase assays showed that neither FHL1 nor FHL2 is a PKD1 substrate. Selective knockdown of FHL1 expression in NRVMs significantly inhibited PKD activation and HDAC5 phosphorylation in response to endothelin 1, but not to the α1-adrenoceptor agonist phenylephrine. In contrast, selective knockdown of FHL2 expression caused a significant reduction in PKD activation and HDAC5 phosphorylation in response to both stimuli. Interestingly, neither intervention affected MEF2 activation by endothelin 1 or phenylephrine. We conclude that FHL1 and FHL2 are novel cardiac PKD partners, which differentially facilitate PKD activation and HDAC5 phosphorylation by distinct neurohormonal stimuli, but are unlikely to regulate MEF2-driven transcriptional reprogramming.
Protein kinase D has multiple roles in cardiac myocytes, where its regulatory mechanisms remain incompletely defined. In the present study we identify four-and-a-half LIM domains proteins 1 and 2 as novel binding partners and regulators of protein kinase D in this cell type.
PMCID: PMC3927927  PMID: 24219103
cardiac myocyte; four-and-a-half LIM (FHL); histone deacetylase; neurohormonal stimulation; protein kinase; signal transduction; ARVM, adult rat ventricular myocyte; BPKDi, bipyridyl PKD inhibitor; CaMK, Ca2+/calmodulin-dependent protein kinase; caPKD, constitutively active catalytic domain of PKD; cMyBP-C, cardiac myosin-binding protein C; CRM1, chromosome region maintenance 1; cTnI, inhibitory subunit of cardiac troponin; ERK, extracellular-signal-regulated kinase; ET1, endothelin 1; FHL, four-and-a-half LIM domains; HDAC, histone deacetylase; IVK, in vitro kinase; MEF2, myocyte enhancer factor 2; MOI, multiplicity of infection; MuRF, muscle RING finger; NRVM, neonatal rat ventricular myocyte; PE, phenylephrine; pfu, plaque-forming unit; PKC, protein kinase C; PKD, protein kinase D; TAC, transverse aortic constriction
14.  Cardiomyocyte growth and sarcomerogenesis at the intercalated disc 
Cardiomyocytes grow during heart maturation or disease-related cardiac remodeling. We present evidence that the intercalated disc (ID) is integral to both longitudinal and lateral growth: increases in width are accommodated by lateral extension of the plicate tread regions and increases in length by sarcomere insertion within the ID. At the margin between myofibril and the folded membrane of the ID lies a transitional junction through which the thin filaments from the last sarcomere run to the ID membrane and it has been suggested that this junction acts as a proto Z-disc for sarcomere addition. In support of this hypothesis, we have investigated the ultrastructure of the ID in mouse hearts from control and dilated cardiomyopathy (DCM) models, the MLP-null and a cardiac-specific β-catenin mutant, cΔex3, as well as in human left ventricle from normal and DCM samples. We find that the ID amplitude can vary tenfold from 0.2 μm up to a maximum of ~2 μm allowing gradual expansion during heart growth. At the greatest amplitude, equivalent to a sarcomere length, A-bands and thick filaments are found within the ID membrane loops together with a Z-disc, which develops at the transitional junction position. Here, also, the tops of the membrane folds, which are rich in αII spectrin, become enlarged and associated with junctional sarcoplasmic reticulum. Systematically larger ID amplitudes are found in DCM samples. Other morphological differences between mouse DCM and normal hearts suggest that sarcomere inclusion is compromised in the diseased hearts.
PMCID: PMC3889684  PMID: 23708682
Heart structure; Dilated cardiomyopathy; Adherens junction; Electron microscopy; Transitional junction
15.  Myosin VI in skeletal muscle: its localization in the sarcoplasmic reticulum, neuromuscular junction and muscle nuclei 
Histochemistry and Cell Biology  2012;139(6):873-885.
Myosin VI (MVI) is a unique unconventional motor moving backwards on actin filaments. In non-muscle cells, it is involved in cell migration, endocytosis and intracellular trafficking, actin cytoskeleton dynamics, and possibly in gene transcription. An important role for MVI in striated muscle functioning was suggested in a report showing that a point mutation (H236R) within the MVI gene was associated with cardiomyopathy (Mohiddin et al., J Med Genet 41:309–314, 2004). Here, we have addressed MVI function in striated muscle by examining its expression and distribution in rat hindlimb skeletal muscle. We found that MVI was present predominantly at the muscle fiber periphery, and it was also localized within muscle nuclei. Analysis of both the hindlimb and cardiac muscle longitudinal sections revealed ~3 μm striation pattern, corresponding to the sarcoplasmic reticulum. Moreover, MVI was detected in the sarcoplasmic reticulum fractions isolated from skeletal and cardiac muscle. The protein also localized to the postsynaptic region of the neuromuscular junction. In denervated muscle, the defined MVI distribution pattern was abolished and accompanied by significant increase in its amount in the muscle fibers. In addition, we have identified several novel potential MVI-binding partners, which seem to aid our observations that in striated muscle MVI could be involved in postsynaptic trafficking as well as in maintenance of and/or transport within the sarcoplasmic reticulum and non-sarcomeric cytoskeleton.
Electronic supplementary material
The online version of this article (doi:10.1007/s00418-012-1070-9) contains supplementary material, which is available to authorized users.
PMCID: PMC3656228  PMID: 23275125
Cytoskeleton; Muscle fiber; Myosin VI; Neuromuscular junction; Nucleus; Sarcoplasmic reticulum
16.  Knockdown of embryonic myosin heavy chain reveals an essential role in the morphology and function of the developing heart 
Development (Cambridge, England)  2011;138(18):3955-3966.
The expression and function of embryonic myosin heavy chain (eMYH) has not been investigated within the early developing heart. This is despite the knowledge that other structural proteins, such as alpha and beta myosin heavy chains and cardiac alpha actin, play crucial roles in atrial septal development and cardiac function. Most cases of atrial septal defects and cardiomyopathy are not associated with a known causative gene, suggesting that further analysis into candidate genes is required. Expression studies localised eMYH in the developing chick heart. eMYH knockdown was achieved using morpholinos in a temporal manner and functional studies were carried out using electrical and calcium signalling methodologies. Knockdown in the early embryo led to abnormal atrial septal development and heart enlargement. Intriguingly, action potentials of the eMYH knockdown hearts were abnormal in comparison with the alpha and beta myosin heavy chain knockdowns and controls. Although myofibrillogenesis appeared normal, in knockdown hearts the tissue integrity was affected owing to apparent focal points of myocyte loss and an increase in cell death. An expression profile of human skeletal myosin heavy chain genes suggests that human myosin heavy chain 3 is the functional homologue of the chick eMYH gene. These data provide compelling evidence that eMYH plays a crucial role in important processes in the early developing heart and, hence, is a candidate causative gene for atrial septal defects and cardiomyopathy.
PMCID: PMC3160091  PMID: 21862559
Atrial septal development; Cardiomyopathy; Myosin; Chick
17.  Formin follows function: a muscle-specific isoform of FHOD3 is regulated by CK2 phosphorylation and promotes myofibril maintenance 
The Journal of Cell Biology  2010;191(6):1159-1172.
Phosphorylation of the muscle-specific formin splice variant FHOD3 by CK2 regulates its stability, myofibril targeting, and myofibril integrity.
Members of the formin family are important for actin filament nucleation and elongation. We have identified a novel striated muscle–specific splice variant of the formin FHOD3 that introduces a casein kinase 2 (CK2) phosphorylation site. The specific targeting of muscle FHOD3 to the myofibrils in cardiomyocytes is abolished in phosphomutants or by the inhibition of CK2. Phosphorylation of muscle FHOD3 also prevents its interaction with p62/sequestosome 1 and its recruitment to autophagosomes. Furthermore, we show that muscle FHOD3 efficiently promotes the polymerization of actin filaments in cardiomyocytes and that the down-regulation of its expression severely affects myofibril integrity. In murine and human cardiomyopathy, we observe reduced FHOD3 expression with a concomitant isoform switch and change of subcellular targeting. Collectively, our data suggest that a muscle-specific isoform of FHOD3 is required for the maintenance of the contractile structures in heart muscle and that its function is regulated by posttranslational modification.
PMCID: PMC3002041  PMID: 21149568
18.  A novel desmocollin-2 mutation reveals insights into the molecular link between desmosomes and gap junctions 
Heart Rhythm  2011;8(5):711-718.
Cellular adhesion mediated by cardiac desmosomes is a prerequisite for proper electric propagation mediated by gap junctions in the myocardium. However, the molecular principles underlying this interdependence are not fully understood.
The purpose of this study was to determine potential causes of right ventricular conduction abnormalities in a patient with borderline diagnosis of arrhythmogenic right ventricular cardiomyopathy.
To assess molecular changes, the patient's myocardial tissue was analyzed for altered desmosomal and gap junction (connexin43) protein levels and localization. In vitro functional studies were performed to characterize the consequences of the desmosomal mutations.
Loss of plakoglobin signal was evident at the cell junctions despite expression of the protein at control levels. Although the distribution of connexin43 was not altered, total protein levels were reduced and changes in phosphorylation were observed. The truncation mutant in desmocollin-2a is deficient in binding plakoglobin. Moreover, the ability of desmocollin-2a to directly interact with connexin43 was abolished by the mutation. No pathogenic potential of the desmoglein-2 missense change was identified.
The observed abnormalities in gap junction protein expression and phosphorylation, which precede an overt cardiac phenotype, likely are responsible for slow myocardial conduction in this patient. At the molecular level, altered binding properties of the desmocollin-2a mutant may contribute to the changes in connexin43. In particular, the newly identified interaction between the desmocollin-2a isoform and connexin43 provides novel insights into the molecular link between desmosomes and gap junctions.
PMCID: PMC3085091  PMID: 21220045
Cardiomyopathy; Conduction; Connexin43; Desmocollin-2; Desmoglein-2; Desmosome; Functional studies; Gap junction; Mutation; Plakoglobin; ARVC, arrhythmogenic right ventricular cardiomyopathy; Cx43, connexin43; DAPI, 4′,6-diamidino-2-phenylindole; DSC2, desmocollin-2; DSG2, desmoglein-2; DSP, desmoplakin; GFP, green fluorescent protein; GST, glutathione-S-transferase; ICS, intracellular cadherin segment; PG, plakoglobin; PKP2, plakophilin-2; RV, right ventricle; YFP, yellow fluorescent protein
19.  Formin-g muscle cytoarchitecture 
Bioarchitecture  2011;1(2):66-68.
Striated muscle cells display an extremely regular assembly of their actin cytoskeleton that contributes to the contractile elements, the myofibrils. How this assembly is initiated and how these structures are maintained is still unclear. We have recently shown that striated muscle expresses a specific isoform of the formin protein family member FHOD3, which is characterised by the presence of a CK2 phosphorylation site at the C-terminal end of the formin homology domain 2 (FH2). Phosphorylated muscle FHOD3 displays a different subcellular localisation, namely to the myofibrils, and also has increased stability compared to un-phosphorylated or non muscle FHOD3. In addition, we could show that muscle FHOD3 is involved in myofibril maintenance in cultured cardiomyocytes and that its presence dramatically enhances the reconstitution of cardiac actin filaments after depolymerisation. Since FHOD3 expression levels and in particular that of the muscle isoform are also decreased in different types of cardiomyopathy, we postulate a crucial role for this protein in the maintenance of a fully functional cardiac cytoarchitecture.
PMCID: PMC3158626  PMID: 21866265
heart; development; actin filament; formin; sarcomere
20.  EH-myomesin splice isoform is a novel marker for dilated cardiomyopathy 
Basic Research in Cardiology  2010;106(2):233-247.
The M-band is the prominent cytoskeletal structure that cross-links the myosin and titin filaments in the middle of the sarcomere. To investigate M-band alterations in heart disease, we analyzed the expression of its main components, proteins of the myomesin family, in mouse and human cardiomyopathy. Cardiac function was assessed by echocardiography and compared to the expression pattern of myomesins evaluated with RT-PCR, Western blot, and immunofluorescent analysis. Disease progression in transgenic mouse models for dilated cardiomyopathy (DCM) was accompanied by specific M-band alterations. The dominant splice isoform in the embryonic heart, EH-myomesin, was strongly up-regulated in the failing heart and correlated with a decrease in cardiac function (R = −0.86). In addition, we have analyzed the expressions of myomesins in human myocardial biopsies (N = 40) obtained from DCM patients, DCM patients supported by a left ventricular assist device (LVAD), hypertrophic cardiomyopathy (HCM) patients and controls. Quantitative RT-PCR revealed that the EH-myomesin isoform was up-regulated 41-fold (P < 0.001) in the DCM patients compared to control patients. In DCM hearts supported by a LVAD and HCM hearts, the EH-myomesin expression was comparable to controls. Immunofluorescent analyses indicate that EH-myomesin was enhanced in a cell-specific manner, leading to a higher heterogeneity of the myocytes’ cytoskeleton through the myocardial wall. We suggest that the up-regulation of EH-myomesin denotes an adaptive remodeling of the sarcomere cytoskeleton in the dilated heart and might serve as a marker for DCM in mouse and human myocardium.
Electronic supplementary material
The online version of this article (doi:10.1007/s00395-010-0131-2) contains supplementary material, which is available to authorized users.
PMCID: PMC3032906  PMID: 21069531
Dilated cardiomyopathy; Heart failure; Sarcomere cytoskeleton; M-band; Myomesin
21.  Mechanistic insights into arrhythmogenic right ventricular cardiomyopathy caused by desmocollin-2 mutations 
Cardiovascular Research  2010;90(1):77-87.
Recent immunohistochemical studies observed the loss of plakoglobin (PG) from the intercalated disc (ID) as a hallmark of arrhythmogenic right ventricular cardiomyopathy (ARVC), suggesting a final common pathway for this disease. However, the underlying molecular processes are poorly understood.
Methods and results
We have identified novel mutations in the desmosomal cadherin desmocollin 2 (DSC2 R203C, L229X, T275M, and G371fsX378). The two missense mutations (DSC2 R203C and T275M) have been functionally characterized, together with a previously reported frameshift variant (DSC2 A897fsX900), to examine their pathogenic potential towards PG's functions at the ID. The three mutant proteins were transiently expressed in various cellular systems and assayed for expression, processing, localization, and binding to other desmosomal components in comparison to wild-type DSC2a protein. The two missense mutations showed defects in proteolytic cleavage, a process which is required for the functional activation of mature cadherins. In both cases, this is thought to cause a reduction of functional DSC2 at the desmosomes in cardiac cells. In contrast, the frameshift variant was incorporated into cardiac desmosomes; however, it showed reduced binding to PG.
Despite different modes of action, for all three variants, the reduced ability to provide a ligand for PG at the desmosomes was observed. This is in agreement with the reduced intensity of PG at these structures observed in ARVC patients.
PMCID: PMC3058729  PMID: 21062920
Arrhythmogenic right ventricular cardiomyopathy; Desmocollin-2; Desmosome; Functional studies; Mutation
22.  Normal passive viscoelasticity but abnormal myofibrillar force generation in human hypertrophic cardiomyopathy 
Hypertrophic cardiomyopathy (HCM) is characterized by left ventricular hypertrophy, increased ventricular stiffness and impaired diastolic filling. We investigated to what extent myocardial functional defects can be explained by alterations in the passive and active properties of human cardiac myofibrils. Skinned ventricular myocytes were prepared from patients with obstructive HCM (two patients with MYBPC3 mutations, one with a MYH7 mutation, and three with no mutation in either gene) and from four donors. Passive stiffness, viscous properties, and titin isoform expression were similar in HCM myocytes and donor myocytes. Maximal Ca2+-activated force was much lower in HCM myocytes (14 ± 1 kN/m2) than in donor myocytes (23 ± 3 kN/m2; P < 0.01), though cross-bridge kinetics (ktr) during maximal Ca2+ activation were 10% faster in HCM myocytes. Myofibrillar Ca2+ sensitivity in HCM myocytes (pCa50 = 6.40 ± 0.05) was higher than for donor myocytes (pCa50 = 6.09 ± 0.02; P < 0.001) and was associated with reduced phosphorylation of troponin-I (ser-23/24) and MyBP-C (ser-282) in HCM myocytes. These characteristics were common to all six HCM patients and may therefore represent a secondary consequence of the known and unknown underlying genetic variants. Some HCM patients did however exhibit an altered relationship between force and cross-bridge kinetics at submaximal Ca2+ concentrations, which may reflect the primary mutation. We conclude that the passive viscoelastic properties of the myocytes are unlikely to account for the increased stiffness of the HCM ventricle. However, the low maximum Ca2+-activated force and high Ca2+ sensitivity of the myofilaments are likely to contribute substantially to any systolic and diastolic dysfunction, respectively, in hearts of HCM patients.
Research Highlights
► The passive stiffness of skinned HCM cardiac myocytes was similar to that of normal (donor) myocytes. ► Maximum Ca-activated force production was reduced by 40% in HCM vs donor myocytes. ► This loss of force could contribute to systolic dysfunction in HCM hearts. ► Myofibrillar Ca sensitivity was higher in HCM than in donor myocytes. ► The enhanced Ca sensitivity could compensate for the smaller maximum force but would tend to cause diastolic dysfunction. ► These characteristics were common to all HCM patients studied, suggesting the changes were secondary consequence of the underlying genetic variants.
PMCID: PMC2954357  PMID: 20615414
Hypertrophic cardiomyopathy; Skinned cardiac myocytes; Viscoelasticity; Ca2+ sensitivity; Cross-bridge kinetics
23.  Novel missense mutations in exon 15 of desmoglein-2: Role of the intracellular cadherin segment in arrhythmogenic right ventricular cardiomyopathy? 
Heart Rhythm  2010;7(10):1446-1453.
The diagnosis of arrhythmogenic right ventricular cardiomyopathy can be challenging. Disease-causing mutations in desmosomal genes have been identified. A novel diagnostic feature, loss of immunoreactivity for plakoglobin from the intercalated disks, recently was proposed.
The purpose of this study was to identify two novel mutations in the intracellular cadherin segment of desmoglein-2 (G812S and C813R in exon 15). Co-segregation of the G812S mutation with disease expression was established in a large Caucasian family. Endomyocardial biopsies of two individuals showed reduced plakoglobin signal at the intercalated disk.
To understand the pathologic changes occurring in the diseased myocardium, functional studies on three mutations in exon 15 of desmoglein-2 (G812C, G812S, C813R) were performed.
Localization studies failed to detect any differences in targeting or stability of the mutant proteins, suggesting that they act via a dominant negative mechanism. Binding assays were performed to probe for altered binding affinities toward other desmosomal proteins, such as plakoglobin and plakophilin-2. Although no differences were observed for the mutated proteins in comparison to wild-type desmoglein-2, binding to plakophilin-2 depended on the expression system (i.e., bacterial vs mammalian protein expression). In addition, abnormal migration of the C813R mutant protein was observed in gel electrophoresis.
Loss of plakoglobin immunoreactivity from the intercalated disks appears to be the endpoint of complex pathologic changes, and our functional data suggest that yet unknown posttranslational modifications of desmoglein-2 might be involved.
PMCID: PMC2994644  PMID: 20708101
Arrhythmogenic right ventricular cardiomyopathy; Desmoglein-2; Desmosome; Genetics; Missense mutation; Plakophilin-2; ARVC, arrhythmogenic right ventricular cardiomyopathy; Cx43, connexin43; DSC2, desmocollin-2; DSG2, desmoglein-2; DSP, desmoplakin; GFP, green fluorescent protein; GST, glutathione-S-transferase; ICS, intracellular cadherin segment; PG, plakoglobin; PKP2, plakophilin-2; RV, right ventricle
24.  Proteomics Analysis of the Cardiac Myofilament Subproteome Reveals Dynamic Alterations in Phosphatase Subunit Distribution* 
Myofilament proteins are responsible for cardiac contraction. The myofilament subproteome, however, has not been comprehensively analyzed thus far. In the present study, cardiomyocytes were isolated from rodent hearts and stimulated with endothelin-1 and isoproterenol, potent inducers of myofilament protein phosphorylation. Subsequently, cardiomyocytes were “skinned,” and the myofilament subproteome was analyzed using a high mass accuracy ion trap tandem mass spectrometer (LTQ Orbitrap XL) equipped with electron transfer dissociation. As expected, a small number of myofilament proteins constituted the majority of the total protein mass with several known phosphorylation sites confirmed by electron transfer dissociation. More than 600 additional proteins were identified in the cardiac myofilament subproteome, including kinases and phosphatase subunits. The proteomic comparison of myofilaments from control and treated cardiomyocytes suggested that isoproterenol treatment altered the subcellular localization of protein phosphatase 2A regulatory subunit B56α. Immunoblot analysis of myocyte fractions confirmed that β-adrenergic stimulation by isoproterenol decreased the B56α content of the myofilament fraction in the absence of significant changes for the myosin phosphatase target subunit isoforms 1 and 2 (MYPT1 and MYPT2). Furthermore, immunolabeling and confocal microscopy revealed the spatial redistribution of these proteins with a loss of B56α from Z-disc and M-band regions but increased association of MYPT1/2 with A-band regions of the sarcomere following β-adrenergic stimulation. In summary, we present the first comprehensive proteomics data set of skinned cardiomyocytes and demonstrate the potential of proteomics to unravel dynamic changes in protein composition that may contribute to the neurohormonal regulation of myofilament contraction.
PMCID: PMC2849712  PMID: 20037178
25.  Prox1 maintains muscle structure and growth in the developing heart 
Development (Cambridge, England)  2008;136(3):495-505.
Impaired cardiac muscle growth and aberrant myocyte arrangement underlie congenital heart disease and cardiomyopathy. We show that cardiac-specific inactivation of the homeobox transcription factor Prox1 results in disruption of the expression and localisation of sarcomeric proteins, gross myofibril disarray and growth retarded hearts. Furthermore, we demonstrate that Prox1 is required for direct transcriptional regulation of structural proteins α-actinin, N-RAP and Zyxin which collectively function to maintain an actin-α-actinin interaction as the fundamental association of the sarcomere. Aspects of abnormal heart development and manifestation of a subset of muscular-based disease have previously been attributed to mutations in key structural proteins. Our study demonstrates an essential requirement for direct transcriptional regulation of sarcomere integrity, in the context of enabling fetal cardiomyocyte hypertrophy, maintenance of contractile function and progression towards inherited or acquired myopathic disease.
PMCID: PMC2655234  PMID: 19091769
Prox1; heart development; myocardium; sarcomere; hypertrophy; myopathy

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