Toon A.B. van Veen

TGF-β1 induces efficient differentiation of human cardiomyocyte progenitor cells into functional cardiomyocytes in vitro

The adult mammalian heart has limited regenerative capacity and was generally considered to contain no dividing cells. Recently, however, a resident population of progenitor cells has been identified, which could represent a new source of cardiomyocytes. Here, we describe the efficient isolation and propagation of human cardiomyocyte progenitor cells (hCMPCs) from fetal heart and patient biopsies. Establishment of hCMPC cultures was remarkably reproducible, with over 70% of adult atrial biopsies resulting in robustly expanding cell populations. Following the addition of transforming growth factor beta, almost all cells differentiated into spontaneously beating myocytes with characteristic cross striations. hCMPC-derived cardiomyocytes showed gap-junctional communication and action potentials of maturing cardiomyocytes. These are the first cells isolated from human heart that proliferate and form functional cardiomyocytes without requiring coculture with neonatal myocytes. Their scalability and homogeneity are unique and provide an excellent basis for developing physiological, pharmacological, and toxicological assays on human heart cells in vitro.

Cardiac gap junction channels: modulation of expression and channel properties

In the heart, intercellular gap junction channels constructed from connexin molecules are crucial for conduction of the electric impulse. Cardiomyocytes can be interconnected by channels composed of three types of connexin proteins: Cx40, Cx43 or Cx45. In mammalian hearts, these three isoforms are regionally differently expressed and even between the species differences exist. Each of these channel-types possesses specific properties and are susceptible to modulation by various mechanisms. In this paper we compare the differences in properties of these channels as deduced from studies on transfected cells and isolated cardiomyocytes and discuss the factors involved in modulation of channel properties. Next, we evaluate the consequences of alterations in expression and modulation of channel properties for cardiac function. Therefore, we have compared reports on genetically engineered animals and discuss this information in relation to various pathophysiological disorders.

Mouse model of SCN5A-linked hereditary Lenegre's disease - Age-related conduction slowing and myocardial fibrosis

Background—We have previously linked hereditary progressive cardiac conduction defect (hereditary Lenègre’s disease) to a loss-of-function mutation in the gene encoding the main cardiac Na+ channel, SCN5A. In the present study, we investigated heterozygous Scn5a-knockout mice (Scn5a+/− mice) as a model for hereditary Lenègre’s disease. Methods and Results—In Scn5a+/− mice, surface ECG recordings showed age-related lengthening of the P-wave and PR- and QRS-interval duration, coinciding with previous observations in patients with Lenègre’s disease. Old but not young Scn5a+/− mice showed extensive fibrosis of their ventricular myocardium, a feature not seen in wild-type animals. In old Scn5a+/− mice, fibrosis was accompanied by heterogeneous expression of connexin 43 and upregulation of hypertrophic markers, including &bgr;-MHC and skeletal &agr;-actin. Global connexin 43 expression as assessed with Western blots was similar to wild-type mice. Decreased connexin 40 expression was seen in the atria. Using pangenomic microarrays and real-time PCR, we identified in Scn5a+/− mice an age-related upregulation of genes encoding Atf3 and Egr1 transcription factors. Echocardiography and hemodynamic investigations demonstrated conserved cardiac function with aging and lack of ventricular hypertrophy. Conclusions—We conclude that Scn5a+/− mice convincingly recapitulate the Lenègre’s disease phenotype, including progressive impairment with aging of atrial and ventricular conduction associated with myocardial rearrangements and fibrosis. Our work provides the first demonstration that a monogenic ion channel defect can progressively lead to myocardial structural anomalies.

Triplex protein quantification based on stable isotope labeling by peptide dimethylation applied to cell and tissue lysates

Stable isotope labeling is at present one of the most powerful methods in quantitative proteomics. Stable isotope labeling has been performed at both the protein as well as the peptide level using either metabolic or chemical labeling. Here, we present a straightforward and cost‐effective triplex quantification method that is based on stable isotope dimethyl labeling at the peptide level. Herein, all proteolytic peptides are chemically labeled at their α‐ and ϵ‐amino groups. We use three different isotopomers of formaldehyde to enable the parallel analysis of three different samples. These labels provide a minimum of 4 Da mass difference between peaks in the generated peptide triplets. The method was evaluated based on the quantitative analysis of a cell lysate, using a typical “shotgun” proteomics experiment. While peptide complexity was increased by introducing three labels, still more than 1300 proteins could be identified using 60 μg of starting material, whereby more than 600 proteins could be quantified using at least four peptides per protein. The triplex labeling was further utilized to distinguish specific from aspecific cAMP binding proteins in a chemical proteomics experiment using immobilized cAMP. Thereby, differences in abundance ratio of more than two orders of magnitude could be quantified.

Impaired Impulse Propagation in Scn5a-Knockout Mice Combined Contribution of Excitability, Connexin Expression, and Tissue Architecture in Relation to Aging

Background—The SCN5A sodium channel is a major determinant for cardiac impulse propagation. We used epicardial mapping of the atria, ventricles, and septae to investigate conduction velocity (CV) in Scn5a heterozygous young and old mice. Methods and Results—Mice were divided into 4 groups: (1) young (3 to 4 months) wild-type littermates (WT); (2) young heterozygous Scn5a-knockout mice (HZ); (3) old (12 to 17 months) WT; and (4) old HZ. In young HZ hearts, CV in the right but not the left ventricle was reduced in agreement with a rightward rotation in the QRS axes; fibrosis was virtually absent in both ventricles, and the pattern of connexin43 (Cx43) expression was similar to that of WT mice. In old WT animals, the right ventricle transversal CV was slightly reduced and was associated with interstitial fibrosis. In old HZ hearts, right and left ventricle CVs were severely reduced both in the transversal and longitudinal direction; multiple areas of severe reactive fibrosis invaded the myocardium, accompanied by markedly altered Cx43 expression. The right and left bundle-branch CVs were comparable to those of WT animals. The atria showed only mild fibrosis, with heterogeneously disturbed Cx40 and Cx43 expression. Conclusions—A 50% reduction in Scn5a expression alone or age-related interstitial fibrosis only slightly affects conduction. In aged HZ mice, reduced Scn5a expression is accompanied by the presence of reactive fibrosis and disarrangement of gap junctions, which results in profound conduction impairment.

Impaired Conduction in the Bundle Branches of Mouse Hearts Lacking the Gap Junction Protein Connexin40

Background—Connexin (Cx)40 and Cx45 are the major protein subunits of gap junction channels in the conduction system of mammals. To determine the role of Cx40, we correlated cardiac activation with C onnexin distribution in normal and Cx40-deficient mice hearts. Methods and Results—Epicardial and septal activation was recorded in Langendorff-perfused adult mice hearts with a 247-point compound electrode (interelectrode distance, 0.3 mm). After electrophysiological measurements, hearts were prepared for immunohistochemistry and histology to determine C onnexin distribution and fibrosis. In both wild-type and Cx40-deficient animals, epicardial activation patterns were similar. The right and left ventricular septum was invariably activated from base to apex. Histology revealed a continuity of myocytes from the common bundle to the septal myocardium. Within this continuity, colocalization was found of Cx43 and Cx45 but not of Cx40 and Cx43. Both animals showed similar His-bundle activation. In Cx40-deficient mice, the proximal bundle branches expressed Cx45 only. The absence of Cx40 in the proximal bundles correlated with right bundle-branch block. Conduction in the left bundle branch was impaired as compared with wild-type animals. Conclusions—Our data show that (1) in mice, a continuity exists between the common bundle and the septum, and (2) Cx40 deficiency results in right bundle-branch block and impaired left bundle-branch conduction.

Cardiac connexins and impulse propagation

Gap junctions form the intercellular pathway for cell-to-cell transmission of the cardiac impulse from its site of origin, the sinoatrial node, along the atria, the atrioventricular conduction system to the ventricular myocardium. The component parts of gap junctions are proteins called connexins (Cx), of which three main isoforms are found in the conductive and working myocardial cells: Cx40, Cx43, and Cx45. These isoforms are regionally expressed in the heart, which suggests a specific role or function of a specific connexin in a certain part of the heart. Using genetically modified mice, the function of these connexins in the different parts of the heart have been assessed in the past years. This review will follow the cardiac impulse on its path through the heart and recapitulate the role of the different connexins in the different cardiac compartments.

Cardiac cell-cell junctions in health and disease: Electrical versus mechanical coupling

Intercalated discs are the membrane sites where individual cardiomyocytes are connected to each other. Adherens-, desmosomal-, and gap junctions are situated in the intercalated disc and ensure mechanical coupling between cells and enable propagation of electrical impulses throughout the heart. A number of cardiac disorders, for example arrhythmogenic right ventricular dysplasia/cardiomyopathy, have been described in which an impaired mechanical coupling leads to electrical dysfunction, with occurrence of fatal arrhythmias. In this article the interaction between electrical and mechanical coupling is explored by reviewing studies performed in patients, animals, and in vitro. In these studies the effect of changes in protein composition of a mechanical junction on the electrical junction, and vice versa were investigated. It is shown that impaired electrical coupling does not change mechanical coupling. However, impaired mechanical coupling largely affects electrical coupling.

Clinical Presentation, Long-Term Follow-Up, and Outcomes of 1001 Arrhythmogenic Right Ventricular Dysplasia/Cardiomyopathy Patients and Family Members

Background—Arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVD/C) is a progressive cardiomyopathy. We aimed to define long-term outcome in a transatlantic cohort of 1001 individuals. Methods and Results—Clinical and genetic characteristics and follow-up data of ARVD/C index-patients (n=439, fulfilling of 2010 criteria in all) and family members (n=562) were assessed. Mutations were identified in 276 index-patients (63%). Index-patients presented predominantly with sustained ventricular arrhythmias (268; 61%). During a median follow-up of 7 years, 301 of the 416 index-patients presenting alive (72%) experienced sustained ventricular arrhythmias. Sudden cardiac death during follow-up occurred more frequently among index-patients without an implantable cardioverter-defibrillator (10/63, 16% versus 2/335, 0.6%). Overall, cardiac mortality and the need for cardiac transplantation were low (6% and 4%, respectively). Clinical characteristics and outcomes were similar in index-patients with and without mutations, as well as in those with familial and nonfamilial ARVD/C. ARVD/C was diagnosed in 207 family members (37%). Symptoms at first evaluation correlated with disease expression. Family members with mutations were more likely to meet Task Force Criteria for ARVD/C (40% versus 18%), experience sustained ventricular arrhythmias (11% versus 1%), and die from a cardiac cause (2% versus 0%) than family members without mutations. Conclusions—Long-term outcome was favorable in diagnosed and treated ARVD/C index-patients and family members. Outcome in index-patients was modulated by implantable cardioverter-defibrillator implantation, but not by mutation status and familial background of disease. One third of family members developed ARVD/C. Outcome in family members was determined by symptoms at first evaluation and mutations.

Impact of genotype on clinical course in arrhythmogenic right ventricular dysplasia/cardiomyopathy-associated mutation carriers

AIMS We sought to determine the influence of genotype on clinical course and arrhythmic outcome among arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVD/C)-associated mutation carriers. METHODS AND RESULTS Pathogenic mutations in desmosomal and non-desmosomal genes were identified in 577 patients (241 families) from USA and Dutch ARVD/C cohorts. Patients with sudden cardiac death (SCD)/ventricular fibrillation (VF) at presentation (n = 36) were younger (median 23 vs. 36 years; P < 0.001) than those presenting with sustained monomorphic ventricular tachycardia (VT). Among 541 subjects presenting alive, over a mean follow-up of 6 ± 7 years, 12 (2%) patients died, 162 (30%) had sustained VT/VF, 78 (14%) manifested left ventricular dysfunction (EF < 55%), 28 (5%) experienced heart failure (HF), and 10 (2%) required cardiac transplantation. Patients (n = 22; 4%) with >1 mutation had significantly earlier occurrence of sustained VT/VF (mean age 28 ± 12 years), lower VT-/VF-free survival (P = 0.037), more frequent left ventricular dysfunction (29%), HF (19%) and cardiac transplantation (9%) when compared with those with only one mutation. Desmoplakin mutation carriers experienced more than four-fold occurrence of left ventricular dysfunction (40%) and HF (13%) than PKP2 carriers. Missense mutation carriers had similar death-/transplant-free survival and VT/VF penetrance (P = 0.137) when compared with those with truncating or splice site mutations. Men are more likely to be probands (P < 0.001), symptomatic (P < 0.001) and have earlier and more severe arrhythmic expression. CONCLUSIONS Presentation with SCD/VF occurs at a significantly younger age when compared with sustained monomorphic VT. The genotype of ARVD/C mutation carriers impacts clinical course and disease expression. Male sex negatively modifies phenotypic expression.