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ABSTRACT: AIMS: Cardiac hypertrophy and fibrosis are associated with potentially lethal arrhythmias. As these substrates often occur simultaneously in one patient, distinguishing between pro-arrhythmic mechanisms is difficult. This hampers understanding of underlying pro-arrhythmic mechanisms and optimal treatment. This study investigates and compares arrhythmogeneity and underlying pro-arrhythmic mechanisms of either cardiac hypertrophy or fibrosis in in vitro models. METHODS AND RESULTS: Fibrosis was mimicked by free myofibroblast (MFB) proliferation in neonatal rat ventricular monolayers. Cultures with inhibited MFB proliferation were used as control or exposed to phenylephrine to induce hypertrophy. At Day 9, cultures were studied with patch-clamp and optical-mapping techniques and assessed for protein expression. In hypertrophic (n = 111) and fibrotic cultures (n = 107), conduction and repolarization were slowed. Triggered activity was commonly found in these substrates and led to high incidences of spontaneous re-entrant arrhythmias [67.5% hypertrophic, 78.5% fibrotic vs. 2.9% in controls (n = 102)] or focal arrhythmias (39.1, 51.7 vs. 8.8%, respectively). Kv4.3 and Cx43 protein expression levels were decreased in hypertrophy but unaffected in fibrosis. Depolarization of cardiomyocytes (CMCs) was only found in fibrotic cultures (-48 ± 7 vs. -66 ± 7 mV in control, P < 0.001). L-type calcium-channel blockade prevented arrhythmias in hypertrophy, but caused conduction block in fibrosis. Targeting heterocellular coupling by low doses of gap-junction uncouplers prevented arrhythmias by accelerating repolarization only in fibrotic cultures. CONCLUSION: Cultured hypertrophic or fibrotic myocardial tissues generated similar focal and re-entrant arrhythmias. These models revealed electrical remodelling of CMCs as a pro-arrhythmic mechanism of hypertrophy and MFB-induced depolarization of CMCs as a pro-arrhythmic mechanism of fibrosis. These findings provide novel mechanistic insight into substrate-specific arrhythmicity.
Cardiovascular research 09/2012; · 5.80 Impact Factor
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ABSTRACT: Gap junctional coupling is important for functional integration of transplanted cells with host myocardium. However, the role of gap junctions in cardiomyogenic differentiation of transplanted cells has not been directly investigated. The objective of this work is to study the role of connexin43 (Cx43) in cardiomyogenic differentiation of human mesenchymal stem cells (hMSCs). Knockdown of Cx43 gene expression (Cx43↓) was established in naturally Cx43-rich fetal amniotic membrane (AM) hMSCs, while Cx43 was overexpressed (Cx43↑) in inherently Cx43-poor adult adipose tissue (AT) hMSCs. The hMSCs were exposed to cardiomyogenic stimuli by coincubation with neonatal rat ventricular cardiomyocytes (nrCMCs) for 10 days. Differentiation was assessed by immunostaining and whole-cell current clamping. To establish whether the effects of Cx43 knockdown could be rescued, Cx45 was overexpressed in Cx43↓ fetal AM hMSCs. Ten days after coincubation, not a single Cx43↓ fetal AM hMSC, control adult AT MSC, or Cx43↑ adult AT mesenchymal stem cell (MSC) expressed α-actinin, while control fetal AM hMSCs did (2.2% ± 0.4%, n = 5,000). Moreover, functional cardiomyogenic differentiation, based on action potential recordings, occurred only in control fetal AM hMSCs. Of interest, Cx45 overexpression in Cx43↓ fetal AM hMSCs restored their ability to undergo cardiomyogenesis (1.6% ± 0.4%, n = 2,500) in coculture with nrCMCs. Gap junctional coupling is required for differentiation of fetal AM hMSCs into functional CMCs after coincubation with nrCMCs. Heterocellular gap junctional coupling thus plays an important role in the transfer of cardiomyogenic signals from nrCMCs to fetal hMSCs but is not sufficient to induce cardiomyogenic differentiation in adult AT hMSCs.
Stem Cells 03/2012; 30(6):1236-45. · 7.78 Impact Factor
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ABSTRACT: Arrhythmogenesis in cardiac fibrosis remains incompletely understood. Therefore, this study aims to investigate how heterocellular coupling between cardiomyocytes (CMCs) and myofibroblasts (MFBs) affects arrhythmogeneity of fibrotic myocardial cultures. Potentially, this may lead to the identification of novel anti-arrhythmic strategies.
Co-cultures of neonatal rat CMCs and MFBs in a 1:1 ratio were used as a model of cardiac fibrosis, with purified CMC cultures as control. Arrhythmogeneity was studied at day 9 of culture by voltage-sensitive dye mapping. Heterocellular coupling was reduced by transducing MFBs with lentiviral vectors encoding shRNA targeting connexin43 (Cx43) or luciferase (pLuc) as control. In fibrotic cultures, conduction velocity (CV) was lowered (11.2 ± 1.6 cm/s vs. 23.9 ± 2.1 cm/s; P < 0.0001), while action potential duration and ectopic activity were increased. Maximal diastolic membrane potential (MDP) of CMCs was less negative in fibrotic cultures. In fibrotic cultures, (n = 30) 30.0% showed spontaneous re-entrant tachyarrhythmias compared with 5% in controls (n = 60). Cx43 silencing in MFBs made the MDP in CMCs more negative, increased excitability and CV by 51% (P < 0.001), and reduced action potential duration and ectopic activity (P < 0.01), thereby reducing re-entry incidence by 40% compared with pLuc-silenced controls. Anti-arrhythmic effects of Cx43 down-regulation in MFBs was reversed by depolarization of CMCs through I(k1) inhibition or increasing extracellular [K(+)].
Arrhythmogeneity of fibrotic myocardial cultures is mediated by Cx43 expression in MFBs. Reduced expression of Cx43 causes a more negative MDP of CMCs. This preserves CMC excitability, limits prolongation of repolarization and thereby strongly reduces the incidence of spontaneous re-entrant tachyarrhythmias.
Cardiovascular research 12/2011; 93(3):434-44. · 5.80 Impact Factor
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ABSTRACT: Cardiac fibrosis is associated with increased incidence of cardiac arrhythmias, but the underlying proarrhythmic mechanisms remain incompletely understood and antiarrhythmic therapies are still suboptimal. This study tests the hypothesis that myofibroblast (MFB) proliferation leads to tachyarrhythmias by altering the excitability of cardiomyocytes (CMCs) and that inhibition of MFB proliferation would thus lower the incidence of such arrhythmias.
Endogenous MFBs in neonatal rat CMC cultures proliferated freely or under control of different dosages of antiproliferative agents (mitomycin-C and paclitaxel). At Days 4 and 9, arrhythmogeneity of these cultures was studied by optical and multi-electrode mapping. Cultures were also studied for protein expression and electrophysiological properties. MFB proliferation slowed conduction from 15.3 ± 3.5 cm/s (Day 4) to 8.8 ± 0.3 cm/s (Day 9) (n = 75, P < 0.01), whereas MFB numbers increased to 37.4 ± 1.7 and 62.0 ± 2%. At Day 9, 81.3% of these cultures showed sustained spontaneous reentrant arrhythmias. However, only 2.6% of mitomycin-C-treated cultures (n = 76, P < 0.0001) showed tachyarrhythmias, and ectopic activity was decreased. Arrhythmia incidence was drug-dose dependent and strongly related to MFB proliferation. Paclitaxel treatment yielded similar results. CMCs were functionally coupled to MFBs and more depolarized in cultures with ongoing MFB proliferation in which only L-type Ca(2+)-channel blockade terminated 100% of reentrant arrhythmias, in contrast to Na(+) blockade (36%, n = 12).
Proliferation of MFBs in myocardial cultures gives rise to spontaneous, sustained reentrant tachyarrhythmias. Antiproliferative treatment of such cultures prevents the occurrence of arrhythmias by limiting MFB-induced depolarization, conduction slowing, and ectopic activity. This study could provide a rationale for a new treatment option for cardiac arrhythmias.
Cardiovascular research 02/2011; 90(2):295-304. · 5.80 Impact Factor
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Arti A Ramkisoensing,
Daniël A Pijnappels,
Saïd F A Askar,
Robert Passier, Jim Swildens,
Marie José Goumans,
Cindy I Schutte,
Antoine A F de Vries,
Sicco Scherjon,
Christine L Mummery,
Martin J Schalij,
Douwe E Atsma
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ABSTRACT: Mesenchymal stem cells (MSCs) show unexplained differences in differentiation potential. In this study, differentiation of human (h) MSCs derived from embryonic, fetal and adult sources toward cardiomyocytes, endothelial and smooth muscle cells was investigated. Labeled hMSCs derived from embryonic stem cells (hESC-MSCs), fetal umbilical cord, bone marrow, amniotic membrane and adult bone marrow and adipose tissue were co-cultured with neonatal rat cardiomyocytes (nrCMCs) or cardiac fibroblasts (nrCFBs) for 10 days, and also cultured under angiogenic conditions. Cardiomyogenesis was assessed by human-specific immunocytological analysis, whole-cell current-clamp recordings, human-specific qRT-PCR and optical mapping. After co-culture with nrCMCs, significantly more hESC-MSCs than fetal hMSCs stained positive for α-actinin, whereas adult hMSCs stained negative. Furthermore, functional cardiomyogenic differentiation, based on action potential recordings, was shown to occur, but not in adult hMSCs. Of all sources, hESC-MSCs expressed most cardiac-specific genes. hESC-MSCs and fetal hMSCs contained significantly higher basal levels of connexin43 than adult hMSCs and co-culture with nrCMCs increased expression. After co-culture with nrCFBs, hESC-MSCs and fetal hMSCs did not express α-actinin and connexin43 expression was decreased. Conduction velocity (CV) in co-cultures of nrCMCs and hESC-MSCs was significantly higher than in co-cultures with fetal or adult hMSCs. In angiogenesis bioassays, only hESC-MSCs and fetal hMSCs were able to form capillary-like structures, which stained for smooth muscle and endothelial cell markers.Human embryonic and fetal MSCs differentiate toward three different cardiac lineages, in contrast to adult MSCs. Cardiomyogenesis is determined by stimuli from the cellular microenvironment, where connexin43 may play an important role.
PLoS ONE 01/2011; 6(9):e24164. · 4.09 Impact Factor
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ABSTRACT: Previously, our research group showed that integrin stimulation induces release of cardiac troponin I from viable neonatal rat ventricular cardiomyocytes (NRCMs), but would it also stimulate uptake of exogenous macromolecules? For this purpose, beating NRCMs were incubated without or with an RGD motif-containing peptide (GRGDS) to stimulate integrins in the presence of Texas Red-conjugated ovalbumin (OTR; 45 kDa) or dextran (DTR; 70 kDa). After incubation periods of 8, 16 and 24 hours endocytosis of red label was quantified by fluorescence microscopy. Uptake of OTR and DTR by NRCMs was intensified by GRGDS treatment (p for trend <0.001 and 0.019, respectively) and increased with duration of incubation (p<0.001 for both). The GRGDS-induced uptake of OTR by NRCMs correlated positively with OTR concentration (p<0.001). Experiments with pharmacological inhibitors of endocytosis indicated that in the absence of GRGDS, NRCMs take up OTR by the clathrin-mediated pathway of endocytosis while the GRGDS-dependent OTR uptake occurs by macropinocytosis. Cultures of NRCMs that were stretched cyclically showed ≍4-fold increased uptake of OTR compared to stationary NRCM cultures. Immunofluorescence microscopy revealed that the dysferlin-positive plasma membrane (PM) areas in beating GRGDS-treated NRCMs were ≍3-fold larger than in contracting NRCMs incubated with vehicle (p<0.001). However, in non-beating NRCMs exposure to GRGDS did not induce larger dysferlin-positive PM areas, nor did it stimulate uptake of OTR. After inhibition of dysferlin expression by short hairpin RNA-mediated RNA interference, OTR uptake by contracting NRCMs could no longer be stimulated via GRGDS treatment. We conclude that in NRCMs, stimulation of integrins by RGD motif-containing peptides or stretch cause uptake of labeled macromolecules. The latter process appears to depend on the contractile behavior of the NRCMs and on the PM repair protein dysferlin, probably because of its role in macropinocytosis.
Cellular Physiology and Biochemistry 01/2010; 26(6):999-1010. · 2.86 Impact Factor
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ABSTRACT: Duchenne muscular dystrophy (DMD) is caused by mutations in the X chromosome-linked DMD gene, which encodes the sarcolemma-stabilizing protein-dystrophin. Initial attempts at DMD therapy deployed muscle progenitor cells from healthy donors. The utilization of these cells is, however, hampered by their immunogenicity, while those from DMD patients are scarce and display limited ex vivo replication. Nonmuscle cells with myogenic capacity may offer valuable alternatives especially if, to allow autologous transplantation, they are amenable to genetic intervention. As a paradigm for therapeutic gene transfer by heterotypic cell fusion we are investigating whether human mesenchymal stem cells (hMSCs) can serve as donors of recombinant DMD genes for recipient human muscle cells. Here, we show that forced MyoD expression in hMSCs greatly increases their tendency to participate in human myotube formation turning them into improved DNA delivery vehicles. Efficient loading of hMSCs with recombinant DMD was achieved through a new tropism-modified high-capacity adenoviral (hcAd) vector directing striated muscle-specific synthesis of full-length dystrophin. This study introduces the principle of genetic complementation of gene-defective cells via directed cell fusion and provides an initial framework to test whether transient MyoD synthesis in autologous, gene-corrected hMSCs increases their potential for treating DMD and, possibly, other muscular dystrophies.
Molecular Therapy 05/2008; 16(4):741-8. · 6.87 Impact Factor
