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Acute Slowing of Cardiac Conduction in Response to Myofibroblast Coupling

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Acute Slowing of Cardiac Conduction in Response to Myofibroblast Coupling Journal of Molecular and Cellular Cardiology 68 (2014) 29–37 Contents lists available at ScienceDirect Journal of Molecular and Cellular Cardiology journal homepage: www.elsevier.com/locate/yjmcc Original article Acute slowing of cardiac conduction in response to myofibroblast coupling to cardiomyocytes through N-cadherin Susan A. Thompson a, Adriana Blazeski a, Craig R. Copeland b,DanielM.Cohenc, Christopher S. Chen c, Daniel M. Reich b,LeslieTunga,⁎ a Department of Biomedical Engineering, The Johns Hopkins University, Baltimore, MD, USA b Department of Physics and Astronomy, The Johns Hopkins University, Baltimore, MD, USA c Department of Bioengineering, University of Pennsylvania, Philadelphia, PA, USA article info abstract Article history: The electrophysiological consequences of cardiomyocyte and myofibroblast interactions remain unclear, and the Received 14 July 2013 contribution of mechanical coupling between these two cell types is still poorly understood. In this study, we ex- Received in revised form 24 December 2013 amined the time course and mechanisms by which addition of myofibroblasts activated by transforming growth Accepted 31 December 2013 factor-beta (TGF-β)influence the conduction velocity (CV) of neonatal rat ventricular cell monolayers. We ob- Available online 9 January 2014 served that myofibroblasts affected CV within 30 min of contact and that these effects were temporally correlat- fi Keywords: ed with membrane deformation of cardiomyocytes by the myo broblasts. Expression of dominant negative RhoA fi fi fi Cardiomyocyte in the myo broblasts impaired both myo broblast contraction and myo broblast-induced slowing of cardiac Myofibroblast conduction, whereas overexpression of constitutive RhoA had little effect. To determine the importance of me- Electrophysiology chanical coupling between these cell types, we examined the expression of the two primary cadherins in the Mechanobiology heart (N- and OB-cadherin) at cell–cell contacts formed between myofibroblasts and cardiomyocytes. Although Cadherin OB-cadherin was frequently found at myofibroblast–myofibroblast contacts, very little expression was observed Optical mapping at myofibroblast–cardiomyocyte contacts. The myofibroblast-induced slowing of cardiac conduction was not prevented by silencing of OB-cadherin in the myofibroblasts, and could be reversed by inhibitors of mechanosensitive channels (gadolinium or streptomycin) and cellular contraction (blebbistatin). In contrast, N-cadherin expression was commonly observed at myofibroblast–cardiomyocyte contacts, and silenc- ing of N-cadherin in myofibroblasts prevented the myofibroblast-dependent slowing of cardiac conduction. We propose that myofibroblasts can impair the electrophysiological function of cardiac tissue through the applica- tion of contractile force to the cardiomyocyte membrane via N-cadherin junctions. © 2014 Elsevier Ltd. All rights reserved. 1. Introduction fibers are regulated by phosphorylation of myosin light-chain (MLC) by Ca2+-dependent myosin light chain kinase or by inhibition of MLC Cardiac fibroblasts are commonly known for their maintenance of phosphatase by Rho/Rho-kinase (ROCK) [5]. Unlike in smooth or cardiac the extracellular matrix (ECM), balancing synthesis and degradation muscle cells, Rho/ROCK appears to be the main pathway that regulates of matrix components in the healthy myocardium and becoming key myofibroblast contraction [5], and ROCK regulation (which is Ca- participants in the process of fibrotic remodeling post injury [1].More independent) is capable of producing large, sustained contraction [6]. recently, however, substantial evidence suggests that fibroblasts also In addition to exerting strong contractile forces to stabilize scar tis- play a more active role in the electrical activity of the heart through sue, myofibroblasts persist in and around the scar following MI and in- cell–cell contacts with cardiomyocytes [2,3]. After cardiac injury, such fluence neighboring cardiomyocytes through biochemical, mechanical as myocardial infarction (MI), fibroblasts become activated by mechan- and electrical interactions. We studied this interaction previously ical and biochemical signaling and undergo a phenotypic change to be- in vitro by examining heterocellular junctions between myofibroblasts come α-smooth muscle actin (SMA)-positive cells. These contractile and cardiomyocytes in co-culture, and observed that heterocellular me- cells, known as myofibroblasts, increase the deposition of ECM chanical adherens junctions were more prevalent than electrical gap and compact newly-formed scars in the injured myocardium [4].Con- junctions [7]. On a functional level, slowing of conduction in cardiac tractile forces produced by myofibroblasts expressing α-SMA stress monolayers induced by the supplementation of myofibroblasts can be fully restored to control levels by applying mechanosensitive channel (MSC) blockers or contraction blockers [7], but only partially restored ⁎ Corresponding author at: Department of Biomedical Engineering, The Johns Hopkins fi University, 720 Rutland Ave, Baltimore, MD 21205, USA. Tel.: +1 410 955 9603. by knockdown of connexin43 (Cx43) in the myo broblasts [7,8].Cor- E-mail address: [email protected] (L. Tung). roborating these findings is the observation that pharmacological 0022-2828/$ – see front matter © 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.yjmcc.2013.12.025 30 S.A. Thompson et al. / Journal of Molecular and Cellular Cardiology 68 (2014) 29–37 ablation of the protein responsible for exerting contractile forces, α- printing. Cardiac fibroblasts were separately pre-treated with 5 ng/ml SMA, abolishes the arrhythmogenic effect of myofibroblasts on cardio- TGF-β for at least 48 h to promote the cardiac myofibroblast phenotype myocyte conduction, even if heterocellular electrotonic coupling is and then added onto patterned NRVC monolayers of 1 million cells at sustained [9]. Taken together, these studies suggest a novel mechanism a concentration of 400,000 cells per monolayer. For time lapse experi- by which myofibroblasts may impair cardiomyocyte electrophysiologi- ments, monolayers were optically mapped with 10 μM voltage- cal function through the application of contractile force to the cardio- sensitive dye, di-4-ANEPPS, at designated time points following myocyte membrane and activation of MSCs [7]. myofibroblast supplementation (30 min, 1 h, 2 h, 3 h, 4 h, 8 h and Mechanical interactions between cardiomyocytes and myofibroblasts 24 h). Activation maps were obtained at 2 Hz pacing during constant are a possible contributing factor for the high risk of arrhythmia follow- superfusion (with bath volume exchange approximately every 2 min) ing MI, given the increased sensitivity of cardiomyocytes [10] and fibro- to determine myofibroblast impact on conduction velocity (CV) in the blasts [11] to mechanical perturbations post injury. The mechanical longitudinal (LCV) and transverse (TCV) directions. Dye transfer exper- interactions can be investigated more closely by examining the compo- iments, traction forces of myofibroblasts, and immunocytochemistry nents of the mechanical junctions that link these two cell types together. of adherens junction formation between myofibroblasts were also One class of proteins that constitute these intercellular adhesions is visualized on the same time scale. Live cell imaging was performed to the cadherins, which are transmembrane receptors that provide attach- characterize the interactions between myofibroblasts and cardio- ment points for adjoining cells. Cells exert strong contractile forces myocytes in real time. Fibroblasts were also transduced with OB- through cadherins [12,13], and cadherins allow for bi-directional trans- cadherin or N-cadherin shRNA lentiviral particles containing a puromy- mission of cytoskeletal tension between cells. There are two types of cin resistance gene. Two days later, cells stably expressing shRNA cadherins expressed in adherens junctions in the heart: OB-cadherin were selected with puromycin, and OB-cadherin or N-cadherin knock- and N-cadherin. OB-cadherin has been shown to be the dominant down was confirmed using Western blots. As a negative control, fibro- cadherin in myofibroblasts associated with wound healing [14,15] and blasts were transduced with shRNA lentiviral particles encoding a appears to be expressed at regions of focal contact between cardiac scrambled shRNA sequence. The transduced fibroblasts were treated myofibroblastsbothinvitroandinvivo[16]. N-cadherin is found in me- with 5 ng/ml TGF-β for 48 h prior to addition to initiate differentiation chanical junctions between adjoining cardiomyocytes [17] and between towards myo fibroblasts; 400,000 myofibroblasts were added onto adjoining myofibroblasts [14] and is upregulated in myofibroblasts dur- control NRVC monolayers for subsequent electrophysiological analysis. ing wound healing through transforming growth factor-β (TGF-β)over- Then, an excitation–contraction uncoupler (blebbistatin) or MSC expression [18]. Studies of postnatal cardiac development and of isolated blocker (gadolinium or streptomycin) was superfused over the mono- adult cardiomyocytes in culture have shown that adherens junctions layer to determine its impact on LCV and TCV. Fibroblasts were also precede gap junction formation in the intercalated disc [19,20],possibly transduced for 24 h with lentiviruses encoding activated RhoA (RhoA- because the adherens junctions tether the plus ends of microtubules that V14) or dominant negative RhoA (RhoA-N19) IRES-GFP
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