Cemil Özcelik

Conditional mutation of the ErbB2 (HER2) receptor in cardiomyocytes leads to dilated cardiomyopathy

The ErbB2 (Her2) proto-oncogene encodes a receptor tyrosine kinase, which is frequently amplified and overexpressed in human tumors. ErbB2 provides the target for a novel and effective antibody-based therapy (Trastuzumab/Herceptin) used for the treatment of mammary carcinomas. However, cardiomyopathies develop in a proportion of patients treated with Trastuzumab, and the incidence of such complications is increased by combination with standard chemotherapy. Gene ablation studies have previously demonstrated that the ErbB2 receptor, together with its coreceptor ErbB4 and the ligand Neuregulin-1, are essential for normal development of the heart ventricle. We use here Cre-loxP technology to mutate ErbB2 specifically in ventricular cardiomyocytes. Conditional mutant mice develop a severe dilated cardiomyopathy, with signs of cardiac dysfunction generally appearing by the second postnatal month. We infer that signaling from the ErbB2 receptor, which is enriched in T-tubules in cardiomyocytes, is crucial for adult heart function. Conditional ErbB2 mutant mice provide a model of dilated cardiomyopathy. In particular, they will allow a rigorous assessment of the role of ErbB2 in the heart and provide insight into the molecular mechanisms that underlie the adverse effects of anti-ErbB2 antibodies.

RBM20 , a gene for hereditary cardiomyopathy, regulates titin splicing

Alternative splicing has a major role in cardiac adaptive responses, as exemplified by the isoform switch of the sarcomeric protein titin, which adjusts ventricular filling. By positional cloning using a previously characterized rat strain with altered titin mRNA splicing, we identified a loss-of-function mutation in the gene encoding RNA binding motif protein 20 (Rbm20) as the underlying cause of pathological titin isoform expression. The phenotype of Rbm20-deficient rats resembled the pathology seen in individuals with dilated cardiomyopathy caused by RBM20 mutations. Deep sequencing of the human and rat cardiac transcriptome revealed an RBM20-dependent regulation of alternative splicing. In addition to titin (TTN), we identified a set of 30 genes with conserved splicing regulation between humans and rats. This network is enriched for genes that have previously been linked to cardiomyopathy, ion homeostasis and sarcomere biology. Our studies emphasize the key role of post-transcriptional regulation in cardiac function and provide mechanistic insights into the pathogenesis of human heart failure.

Mutations in the Human Muscle LIM Protein Gene in Families With Hypertrophic Cardiomyopathy

Background—Muscle LIM protein (MLP) is an essential nuclear regulator of myogenic differentiation. Additionally, it may act as an integrator of protein assembly of the actin-based cytoskeleton. MLP-knockout mice develop a marked cardiac hypertrophy reaction and dilated cardiomyopathy (DCM). MLP is therefore a candidate gene for heritable forms of hypertrophic cardiomyopathy (HCM) and DCM in humans. Methods and Results—We analyzed 1100 unrelated individuals (400 patients with DCM, 200 patients with HCM, and 500 controls) for mutations in the human CRP3 gene that encodes MLP. We found 3 different missense mutations in 3 unrelated patients with familial HCM but detected no mutation in the DCM group or the controls. All mutations predicted an amino acid exchange at highly conserved residues in the functionally important LIM1 domain, which is responsible for interaction with &agr;-actinin and with certain muscle-specific transcription factors. Protein-binding studies indicate that mutations in the CRP3 gene lead to a decreased binding activity of MLP to &agr;-actinin. All 3 index patients were characterized by typical asymmetrical septal hypertrophy. Family studies revealed cosegregation of clinically affected individuals with the respective mutations in MLP. Conclusion—Here, we present evidence that mutations in the CRP3/MLP gene can cause HCM.

SDF-1α as a therapeutic stem cell homing factor in myocardial infarction.

Myocardial infarction is associated with persistent muscle damage, scar formation and depressed cardiac performance. Recent studies have demonstrated the clinical significance of stem cell-based therapies after myocardial infarction with the aim to improve cardiac remodeling and function by inducing the reconstitution of functional myocardium and formation of new blood vessels. Stem cell homing signals play an important role in stem cell mobilization from the bone marrow to the ischemic cardiac environment and are therefore crucial for myocardial repair. To date, the most prominent stem cell homing factor is the chemokine SDF-1α/CXCL12. This protein was shown to be significantly upregulated in many experimental models of myocardial infarction and in patients suffering from ischemic cardiac diseases, suggesting the involvement in the pathophysiology of these disorders. A number of studies focused on manipulating SDF-1α and its receptor CXCR4 as central regulators of the stem cell mobilization process. Targeted expression of SDF-1α after myocardial infarction was shown to result in increased engraftment of bone marrow-derived stem cells into infarcted myocardium. This was accompanied by beneficial effects on cardiomyocyte survival, neovascularization and cardiac function. Thus, the SDF-1/CXCR4 axis seems to be a promising novel therapeutic approach to improve post-infarction therapy by attracting circulating stem cells to remain, survive and possibly differentiate in the infarct area. This review will summarize clinical trials of stem cell therapy in patients with myocardial infarction. We further discuss the basic findings about SDF-1α in stem cell recruitment and its therapeutic implications in experimental myocardial infarction.

A role of the cryptic gene in the correct establishment of the left–right axis

During vertebrate embryogenesis, a left-right axis is established. The heart, associated vessels and inner organs adopt asymmetric spatial arrangements and morphologies. Secreted growth factors of the TGF-beta family, including nodal, lefty-1 and lefty-2, play crucial roles in establishing left-right asymmetries [1] [2] [3]. In zebrafish, nodal signalling requires the presence of one-eyed pinhead (oep), a member of the EGF-CFC family of membrane-associated proteins [4]. We have generated a mutant allele of cryptic, a mouse EGF-CFC gene [5]. Homozygous cryptic mutants developed to birth, but the majority died during the first week of life because of complex cardiac malformations such as malpositioning of the great arteries, and atrial-ventricular septal defects. Moreover, laterality defects, including right isomerism of the lungs, right or left positioning of the stomach and splenic hypoplasia were observed. Nodal gene expression in the node was initiated in cryptic mutant mice, but neither nodal, lefty-2 nor Pitx2 were expressed in the left lateral plate mesoderm. The laterality defects observed in cryptic(-/-) mice resemble those of mice lacking the type IIB activin receptor or the homeobox-containing factor Pitx2 [6] [7] [8] [9], and are reminiscent of the human asplenic syndrome [10]. Our results provide genetic evidence for a role of cryptic in the signalling cascade that determines left-right asymmetry.

Connective tissue growth factor overexpression in cardiomyocytes promotes cardiac hypertrophy and protection against pressure overload.

Connective tissue growth factor (CTGF) is a secreted protein that is strongly induced in human and experimental heart failure. CTGF is said to be profibrotic; however, the precise function of CTGF is unclear. We generated transgenic mice and rats with cardiomyocyte-specific CTGF overexpression (CTGF-TG). To investigate CTGF as a fibrosis inducer, we performed morphological and gene expression analyses of CTGF-TG mice and rat hearts under basal conditions and after stimulation with angiotensin II (Ang II) or isoproterenol, respectively. Surprisingly, cardiac tissues of both models did not show increased fibrosis or enhanced gene expression of fibrotic markers. In contrast to controls, Ang II treated CTGF-TG mice displayed preserved cardiac function. However, CTGF-TG mice developed age-dependent cardiac dysfunction at the age of 7 months. CTGF related heart failure was associated with Akt and JNK activation, but not with the induction of natriuretic peptides. Furthermore, cardiomyocytes from CTGF-TG mice showed unaffected cellular contractility and an increased Ca2+ reuptake from sarcoplasmatic reticulum. In an ischemia/reperfusion model CTGF-TG hearts did not differ from controls. Our data suggest that CTGF itself does not induce cardiac fibrosis. Moreover, it is involved in hypertrophy induction and cellular remodeling depending on the cardiac stress stimulus. Our new transgenic animals are valuable models for reconsideration of CTGFs profibrotic function in the heart.

ErbB2 pathways in heart and neural diseases.

The proto-oncogene ErbB2 (also known as c-neu or HER2 in humans) encodes a receptor tyrosine kinase that is frequently overexpressed in human tumors. It is the target of a novel and effective antibody-based therapy for malignant mammary tumors (trastuzumab/Herceptin). Biochemical and genetic experiments have shown that ErbB2 acts as a coreceptor for other members of the ErbB family of receptor tyrosine kinases. In particular, signals are transduced by ErbB2/ErbB4, ErbB2/ErbB3, and ErbB2/EGF receptor heteromers. ErbB2/4 and ErbB2/ErbB3 heteromers transmit neuregulin-1 signals in the developing and adult heart, and in the peripheral nervous system, respectively. Of particular medical relevance are recent findings that relied on tissue-specific mutation of ErbB2 in cardiomyocytes, which revealed an essential function of ErbB2 in normal heart physiology and demonstrated that loss of cardiac ErbB2 can cause dilated cardiomyopathy in adult mice. Thus, ErbB2 is important not only in development, but also for the correct functioning of the differentiated myocardium. The conditional ErbB2 mutant mice provide a model for the principal side effects--cardiomyopathy and heart failure--that can be observed in patients undergoing chemotherapy with Trastuzumab.

Beyond the sarcomere: CSRP3 mutations cause hypertrophic cardiomyopathy

Hypertrophic cardiomyopathy (HCM) is a frequent genetic cardiac disease and the most common cause of sudden cardiac death in young individuals. Most of the currently known HCM disease genes encode sarcomeric proteins. Previous studies have shown an association between CSRP3 missense mutations and either dilated cardiomyopathy (DCM) or HCM, but all these studies were unable to provide comprehensive genetic evidence for a causative role of CSRP3 mutations. We used linkage analysis and identified a CSRP3 missense mutation in a large German family affected by HCM. We confirmed CSRP3 as an HCM disease gene. Furthermore, CSRP3 missense mutations segregating with HCM were identified in four other families. We used a newly designed monoclonal antibody to show that muscle LIM protein (MLP), the protein encoded by CSRP3, is mainly a cytosolic component of cardiomyocytes and not tightly anchored to sarcomeric structures. Our functional data from both in vitro and in vivo analyses suggest that at least one of MLPs mutated forms seems to be destabilized in the heart of HCM patients harbouring a CSRP3 missense mutation. We also present evidence for mild skeletal muscle disease in affected persons. Our results support the view that HCM is not exclusively a sarcomeric disease and also suggest that impaired mechano-sensory stress signalling might be involved in the pathogenesis of HCM.

A gain-of-function TBX20 mutation causes congenital atrial septal defects, patent foramen ovale and cardiac valve defects

Background Ostium secundum atrial septal defects (ASDII) account for approximately 10% of all congenital heart defects (CHD), and mutations in cardiac transcription factors, including TBX20, were identified as an underlying cause for ASDII. However, very little is known about disease penetrance in families and functional consequences of inherited TBX20 mutations. Methods The coding region of TBX20 was directly sequenced in 170 ASDII patients. Functional consequences of one novel mutation were investigated by surface plasmon resonance, CD spectropolarymetry, fluorescence spectrophotometry, luciferase assay and chromatin immunoprecipitation. Results We found a novel mutation in a highly conserved residue in the T-box DNA binding domain (I121M) segregating with CHD in a three generation kindred. Four mutation carriers revealed cardiac phenotypes in terms of cribriform ASDII, large patent foramen ovale or cardiac valve defects. Interestingly, tertiary hydrophobic interactions within the mutant TBX20 T-box were significantly altered leading to a more dynamic structure of the protein. Moreover, Tbx20-I121M resulted in a significantly enhanced transcriptional activity, which was further increased in the presence of co-transcription factors GATA4/5 and NKX2-5. Occupancy of DNA binding sites on target genes was also increased. Conclusions We suggest that TBX20-I121M adopts a more fluid tertiary structure leading to enhanced interactions with cofactors and more stable transcriptional complexes on target DNA sequences. Our data, combined with that of others, suggest that human ASDII may be related to loss-of-function as well as gain-of-function TBX20 mutations.

Mutations in GATA4, NKX2.5, CRELD1, and BMP4 Are Infrequently Found in Patients With Congenital Cardiac Septal Defects

Cardiac septal defects constitute the majority ofcongenitalheartdisease(CHD)inhumansandfamilialrecurrenceisreportedtoexceed5%[Burnetal.,1998].Previously, mutations in GATA4 and NKX2.5 havebeendescribedtobepathogenicforostiumsecundumatrial septal defects (ASDII) and ventricular septaldefects(VSD)[Schottetal.,1998;Gargetal.,2003].Incontrast, CRELD1 and BMP4 constitute functionalcandidatesforregulardevelopmentoftheendocardialcushionandmutationsinthesegenescause atrioven-tricular septal defects (AVSD) in animal models andhumans [Jiao et al., 2003; Robinson et al., 2003]. Wehypothesizedthatmutationsin GATA4(NM_002052),NKX2.5 (NM_004387), CRELD1 (NM_015513), andBMP4(NM_001202)canbeidentifiedinalargecohortof patients withcongenital septal defects with a focusonASDII.Weanalyzedthecodingregionofthesefourgenesin205patientswithcongenitalseptaldefectsbysinglestrandedconformationalpolymorphism(SSCP)and sequencing. The patient cohort was assembledout of 110 patients with isolated ASDII. Of these,four subjects (3.6%) mentioned a familial history andformal segregation analysis of pedigrees suggestedan autosomal dominant inheritance. However, familyrelatives were not studied systematically. To thishomogenous ASDII patient cohort we added agroupof95individualswithdifferentcongenitalseptaldefects (60 ASDII, 22 perimembranous VSD, and13 AVSD) and concomitant minor cardiac malforma-tions (Aortic coarctation ¼CoA, persistent ductusarteriosus¼PDA or partial anomalous venousreturn¼PAPVR). These patients were included as asubgroup in a candidate gene approach reportedpreviously [Ozcelik et al., 2006]. All patients wereattending the Department for Congenital Heart Dis-ease, German Heart Institute Berlin (GHIB). Patientswith syndromic appearance and/or limb malforma-tions were excluded from the genetic study andcontrolsubjectswerematchedforethnicity.Thestudyprotocol was approved by the Institutional ReviewBoard of the GHIB and Charite´.A heterozygous c.1750C>T mutation of GATA4,which predicts p.A411V, was identified in a cauca-sian patient with multiperforated ASDII and PAPVR.After exclusion in 600 control chromosomes weconsideredthevarianttobeanovelASDIIassociatedmutation representing the fifth GATA4 mutationidentified in a patient with ASDII. The carrier was a73-year-old female with ASDII and sustained atrial