Electrophysiological deterioration and resurrection in the scarred heart. Pijnappels, D.A.

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Citation

Pijnappels, D. A. (2009, June 18). Electrophysiological deterioration and resurrection in the scarred heart. Retrieved from

https://hdl.handle.net/1887/13851

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III

Chapter

R esynchronization of separated rat cardio- myocyte fields with genetically modified human ventricular scar fibroblasts

Daniël A. Pijnappels,¹ John van Tuyn,^1,2 Antoine A.F. de Vries,² Robert W. Grauss,¹ Arnoud van der Laarse,¹ Dirk L. Ypey,¹ Douwe E. Atsma,¹ Martin J. Schalij¹

Departments of Cardiology,¹ and Molecular Cell Biology,² Leiden University Medical Center, Leiden, The Netherlands

Circulation. 2007;116:2018-28

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III

Chapter

R esynchronization of separated rat cardio- myocyte fields with genetically modified human ventricular scar fibroblasts

Daniël A. Pijnappels,¹ John van Tuyn,^1,2 Antoine A.F. de Vries,² Robert W. Grauss,¹ Arnoud van der Laarse,¹ Dirk L. Ypey,¹ Douwe E. Atsma,¹ Martin J. Schalij¹

Departments of Cardiology,¹ and Molecular Cell Biology,² Leiden University Medical Center, Leiden, The Netherlands

Circulation. 2007;116:2018-28

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Abstract

Background: Non-response to cardiac resynchronization therapy (CRT) is associated with the presence of slow or non-conducting scar tissue. Genetic modification of scar tissue, aimed at improving conduction, may be a novel approach to achieve effective resynchronization. Therefore, the feasibility of resynchronization using genetically modified human ventricular scar fibroblasts was studied in a co-culture model.

Methods and Results: An in vitro model was used to study the effects of forced ex- pression of the myocardin (MyoC) gene in human ventricular scar fibroblasts (hVSFs) on resynchronization of two rat cardiomyocyte (CMC) fields, separated by a strip of hVSFs. Furthermore, the effects of MyoC expression on the capacity of hVSFs to serve as pacing site were studied. MyoC-dependent gene activation in hVSFs was examined by gene and immunocytochemical analysis.

Forced MyoC expression in hVSFs decreased dyssynchrony, expressed as the activation delay between two CMC fields (control hVSFs: 27.6±0.2 ms, n=11, vs MyoC- hVSFs: 3.6±0.3 ms, n=11, at day 8, p<0.01). Also, MyoC-hVSFs could be electrically stimulated, resulting in simultaneous activation of the two adjacent CMC fields. For- ced MyoC expression in hVSFs led to the expression of various connexin and cardiac ion channel genes. Intracellular measurements of MyoC-hVSFs coupled to surrounding CMCs showed strongly improved action potential conduction.

Conclusions: Forced MyoC gene expression in hVSFs allowed electrical stimulation of these cells and conferred the ability to conduct an electrical impulse at high velocity, which resulted in resynchronization of 2 separated cardiomyocyte fields. Both phenomena appear mediated mainly by MyoC-dependent activation of genes that encode connexins, strongly enforcing intercellular electrical coupling.

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Introduction

M

yocardial infarction results in replacement of well-coupled electrically active cardiomyocytes (CMCs) by scar tissue, containing primarily unexcitable fibroblasts and an electrically insulating extracellular matrix.¹ Consequently, coordinated impulse propagation across scarred myocardium is impaired due to local conduction block and slow conduction.² This leads to inefficient and inhomogeneous electrical and mechanical activation of different parts of the myocardium, resulting in increased left ventricular (LV) dyssynchrony and diminished LV contractile performance.^3,4 Ul- timately this may result in clinical symptoms of heart failure.

Cardiac resynchronization therapy (CRT) is a successful treatment modality for patients with drug-refractory heart failure and significant LV dyssynchrony.^5-7 However, about ~30% of the patients does not respond. Recently, amongst others, ischemic etiology of heart failure was considered as a predictor of non-responsiveness to CRT.⁸ More specifically, the extent of both viable and scarred myocardium,⁹ as well as the location of scar tissue,¹⁰ were shown to be predictive for the response to CRT. Impor- tantly, scar burden appears to be involved in both the cause of ventricular dyssynchrony as well as the lack of response to CRT.

Recently, we demonstrated that over-expression of the gene encoding the transcripti- on factor myocardin (MyoC) induces the synthesis of cardiac proteins in human non- muscle cell types,¹¹ including those of cardiac ion channels and connexins (Cxs).¹² As these proteins are essential in cardiac action potential transmission and absent or only present at low levels in native human ventricular scar fibroblasts (hVSFs), it is therefore hypothesized that genetic modification of scar tissue cells with a recombinant MyoC gene might be a novel approach to treat conduction abnormalities.

In this study the potential of MyoC-transduced hVSFs to resynchronize and stimulate cardiac tissue in a standardized in vitro model were investigated.

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Materials & Methods

Model of Experimental Resynchronization

All animal experiments were approved by the Institutional Animal Experiments Committee and comply with the Guide for the Care and Use of Laboratory Animals as stated by the US National Institutes of Health.

Isolation, Culturing and Preparation of Cardiomyocytes and Myocardial Scar Fibroblasts CMCs were dissociated from ventricles of 2-day old male neonatal Wistar rats and grown in culture medium supplemented with 5% horse serum (Invitrogen, Carlsbad, CA, USA), penicillin (100 U/mL) and streptomycin (100 µg/mL) (all from Invitrogen, Carlsbad, CA, USA), as previously described.¹³

The hVSFs were isolated from human myocardial scar tissue of 8 different patients who underwent surgical reconstruction of the left ventricle. Each sample was dis- sected into small pieces, covered by glass coverslips and cultured in porcine gelatin- coated culture dishes containing Dulbecco’s modified Eagle’s medium supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin, and 10% fetal bovine serum (In- vitrogen). Outgrowth of cells was first seen after 2 days of culture, and after another 3 days coverslips and tissue pieces were removed, leaving the hVSFs attached to the culture dish. Next, hVSFs were trypsinized, counted, and replated at 70% confluency.

Cultures were refreshed every three days, and passage numbers 4-8 were used for further experiments. Importantly, culturing of the hVSFs on poly-D-lysine plates as described by Smith et al. did not give rise to cardiospheres, making contamination of the hVSFs with cardiac progenitor cells unlikely.¹⁴Prior to experiments, hVSFs were transduced with vesicular stomatitis virus G protein-pseudotyped lentivirus vectors encoding either human MyoC and enhanced green fluorescent protein (eGFP) (MyoC-hVSFs) or β-galactosidase and eGFP (LacZ-hVSFs) as control. To this pur- pose, hVSFs were seeded at a density 2x10⁴ cells per cm² in 10cm² culture dishes, and infected in the presence of 20µg/ml diethylaminoethyl-dextran sulfate (Pharmacia, Uppsala, Sweden), at a multiplicity of infection of 8 HeLa cell-transducing units per cell. After 4h of incubation, the cells were washed three times, and cultured for 5-7 days prior to seeding in micro-electrode array culture dishes or analysis of ion channel and connexin gene expression. The nucleotide sequences of the shuttle plasmids used to generate the lentivirus vectors are deposited in GenBank under the accession numbers EF205034 and EF205035.

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Micro-Electrode Arrays

CMCs were cultured in micro-electrode array culture dishes (MEAs, Multichannel Systems, Reutlingen, Germany). Prior to plating, MEAs were glow-discharged and coated with collagen to improve adhesion of cells. Electrical activation maps were generated after 2 days of culture to confirm the presence of a synchronously beating monolayer. After 3 days of culture, the monolayer was divided into two fields of CMCs by an a-cellular channel of either (A) 250-300 µm or (B) 300-350 µm wide, crossing the whole diameter of the culture dish. The a-cellular channel was generated by two pre-programmed linear laser dissections using a PALM Microlaser System, including PALM RoboSoftware Version 4.0, (Microlaser Technologies GmbH, Bern- ried, Germany). Removal of the strip of monolayer between the two laser dissection lines, created an a-cellular channel electrically separating the two CMC fields. Cells were applied in a channel-crossing pattern after ensuring that no cells or cell debris was present in the channel and after confirming the presence of a conduction block between the two CMC fields. Application of cells was achieved using a pipette moun- ted in a micro-manipulator and a light microscope (20x magnification). To study electrical dyssynchrony (group A), the channel was filled with 3x10⁴ eGFP-labeled LacZ-hVSFs, 3x10⁴ eGFP-labeled MyoC-hVSFs, or 3x10⁴ CMCs. In an additional set of control experiments no cells were applied. To investigate electrical stimulation (group B), 5x10⁴ labeled cells were applied to the channel to reach confluency. After 24h, the culture medium was refreshed to remove non-attached cells. In group A, the effect of cell transplantation on resynchronization was assessed for 8 days. In group B, driven resynchronization by electrical stimulation of the transplanted cells was investigated over the same period of time.

Electrical Stimulation of Cell Cultures and Assessment of Dyssynchrony

Cultures were stimulated via an external pipette electrode (diameter: 60µm) produ- cing bipolar rectangular pulses (1.5x threshold, pulse width: 10 ms, 1-2 Hz) placed

~1mm above the cell layer. Cultures were stimulated for at least 30s at a fixed location in the center of the upper CMC field before recordings were started. Two-dimensio- nal color-coded activation maps and conduction velocities (CV) were derived as described previously.¹³ Electrical re-coupling of the two CMC fields was defined as the presence of a consistent correlation between local electrical activation times recorded at both fields for 30s, while stimulating the upper CMC field.

The correlation between electrical activation of the CMC fields (local electrical activation time) and mechanical contraction (local mechanical activation time) of the CMC fields was assessed by high-frame rate (60 frames/s) video recordings of the cultures in the MEAs using a microscope equipped with a digital camera (Orca-RE, Hamamatsu

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Photonics Deutschland GmbH, Herrsching am Ammersee, Germany), and Openlab software (Improvision, Lexington, MA, USA).

Electrical Stimulation of Transplanted Cells and Assessment of Electrical Capture The above mentioned pipette electrode was used to stimulate at the site of hVSFs.

The threshold was defined as the minimal current necessary to capture the beating CMC culture. Stimulation was performed at twice the threshold. The electrode was placed ~1mm above the strip of hVSFs under microscopic control using a micro-manipulator and a focused bundle of external light to delineate the cell strip. Cell strips were stimulated for at least 30s at a fixed location before recordings were started and color coded activation maps were constructed. Electrical capture was considered to be present if a consistent phase relation between electrical stimulation of the cell strip and subsequent electrical activation of both the CMC fields was observed for a period of 30 s.

Immunocytochemical and Genetic Analysis Immunocytochemistry

Co-cultures of 2x10⁶ CMCs and 5x10⁴ eGFP-labeled MyoC-hVSFs or LacZ-hVSFs were stained on day 1 or day 8 using Cx40, Cx43-, or Cx45-specific antibodies (Saint Louis, MO, USA) to look for gap junctions. The primary antibodies were detected by goat anti-rabbit IgG conjugated to Alexa Fluor568 (Invitrogen). All antibodies were used at a dilution of 1:200. Staining was quantified in 6 cultures (60 cells per culture at 40x magnification) at each time point (threshold value 50 on a 0-255 gray inten- sity scale). A fluorescence microscope equipped with a digital camera (Eclipse, Nikon Europe, Badhoevedorp, The Netherlands) and dedicated software (Image-Pro Plus, Version 4.1.0.0, Media Cybernetics, Silver Spring, MD, USA) were used for data analysis. All co-cultures of CMCs and eGFP-labeled hVSFs were stained using the same solutions and equal exposure times were used for all recordings.

Reverse Transcription-Polymerase Chain Reaction Analysis

Semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) analyses were performed as previously reported,¹² including primer pairs targeting transcripts encoding the α-subunit of the cardiac voltage-gated sodium channel (SCN5A or Nav1.5), the α1C subunit of the voltage-dependent L-type calcium channel (CACNA1C or Cav1.2), and the inwardly rectifying potassium channel J3 (KCNJ3 also known as Kir3.1). An overview of all primer pairs used for RT-PCR is given in Table 1. As in-

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ternal controls for the quantity and quality of the RNA specimens, RT-PCR amplifi- cations targeting transcripts of the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were performed in parallel. PCRs carried out on cDNA derived from total RNA samples of human atrium, ventricle, vascular smooth muscle and skeletal muscle served as positive controls: PCRs in which the cDNA was repla- ced by water were included as negative controls. For comparing relative mRNA levels only PCR samples from within the linear range of amplification were used.

Table 1. Primer sequences and PCR conditions used for RT-PCR analysis.

Whole-Cell Patch-Clamp Measurements

Co-cultures of 2x10⁶ CMCs and 5x10⁴ eGFP-labeled MyoC-hVSFs or LacZ-hVSFs were used for intracellular measurements at day 1 and day 8 of culture. After iden- tification of hVSFs using fluorescence microscopy, action potentials (APs) in CMCs and hVSFs surrounded by 3-5 beating CMCs, were recorded in the current-clamp configuration.¹⁵

Whole-cell recordings were performed at 25°C using a L/M-PC patch-clamp amplifier (3kHz filtering) (List-Medical, Darmstadt, Germany). The pipette solution contained (in mM) 10 Na₂ATP, 115 KCl, 1 MgCl₂, 5 EGTA, 10 HEPES/KOH (pH 7.4). Tip and seal resistance was 2.0-2.5 MΩ and >1 GΩ, respectively. The bath solution contained (in mM) 137 NaCl, 4 KCl, 1.8 CaCl₂, 1 MgCl₂, and 10 HEPES (pH 7.4). For data acqui- sition and analysis pClamp/Clampex8 software (Axon Instruments, Molecular De- vices, Sunnyvale, CA, USA) was used. AP characteristics in CMCs, LacZ-hVSFs and MyoC-hVSFs were evaluated at day 1 and 8. At the same time points, input resistance of whole-cells coupled to their surrounding cells (3-5 in number) was measured in voltage-clamp by dividing the applied voltage steps (10 mV, 200 ms) by the stationary membrane current change and subtracting the access resistance (4-7 MΩ). The input conductance (reverse of resistance) was considered as an approximate measure of intercellular coupling under our conditions.¹⁶ Functionality of ion channels was assessed in single cells using appropriate voltage-clamp protocols during the 8 days of culture.¹⁷

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Statistics

Statistical analysis was performed using SPSS11.0 for Windows (SPSS Inc., Chicago, IL, USA). Data were compared with one-way or two-factor mixed ANOVA test with Bonferroni correction for multiple comparisons, and expressed as mean±SD. Linear correlation analysis was performed by calculating Pearson’s correlation coefficient.

P-values <0.05 were considered statistically significant. Calculation of sample size and power was less appropriate as the expected means and SDs were difficult to be estimated. However, the pronounced effects of myocardin suggest that only a limited number of experiments already would have rendered significant results.

Results

Myocardin Gene Expression in hVSFs and Resynchronization

Electrical activation of the CMC fields (local electrical activation time) was directly related to mechanical contraction (local mechanical activation time) of the CMC fields during the entire course of the experiments (Figure 1).

During this period no mechanical activity was observed in the center of cell strips consisting of LacZ-hVSFs or MyoC-hVSFs, as examined by light microscopy.

After 3 days of culture, only CMC monolayers with a high degree of structural and functional homogeneity, as determined by light microscopy and electrophysiological

Figure 1. Comparison of local electrical activation time based on electrograms with local mechanical activation time based on video recordings, described as Pearson’s correlation coefficient (R). Left, assessment at day 1 showed a direct relation (R²=0.98) between local electrical and mechanical activation time.

Right, a persistent direct relation (R²=0.97) between both activation times was found at day 8.

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mapping, were included in the study (yield: ~60%). By laser-dissection, an a-cellular channel (group A: 230±10 µm, group B: 335±15 µm) was created in the MEAs, resulting in two asynchronously beating CMC fields. Asynchronous beating was associated with maximal dyssynchrony (∞) between the two CMC fields. Application of LacZ-hVSFs (n=12) in group A resulted in resynchronization of the two CMC fields within 24h. Resynchronization was associated with a relatively large conduction delay (latency) of 28±1 ms and a low CV of 2.1±0.4 cm/s across the LacZ-hVSFs between the two CMC fields (Figure 2A, E, and F).

Figure 2. Extracellular recordings at day 1 after application of (A) human ventricular scar fibroblasts (LacZ-hVSFs), (B) MyoC-hVSFs, (C) no cells, and (D) cardiomyocytes (CMCs). (E) Response to electrical stimulation of one CMC field on distal CMC field at day 1. (F) Conduction velocity (CV) across LacZ- hVSFs, MyoC-hVSFs, and CMCs. A conduction block was present if no cells were applied in the channel between the two adjacent CMC fields. LacZ-hVSFs are presented as hVSFs in this figure. One-way ANO- VA with Bonferroni correction for multiple comparisons: *p<0.05 vs. MyoC-hVSFs, **p<0.01 vs CMCs.

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Electrograms derived from the LacZ-hVSF-strip showed clear signs of decremental conduction. During follow-up till day 8 of culture, conduction delay and CV did not change significantly (n=11) (Figure 3A, E, F, and 4).

Figure 3. Extracellular electrophysiological recordings at day 8 after application of (A) human ventricu- lar scar fibroblasts (LacZ-hVSFs), (B) MyoC-hVSFs, (C) no cells, and (D) cardiomyocytes (CMCs). (E) Electrical stimulation of one CMC field resulted in activation of the distal CMC field after application of LacZ-hVSFs, MyoC-hVSFs, or CMCs. Electrical stimulation of a-cellular channel cultures resulted in non-response, associated with asynchrony throughout follow-up. (F) Conduction velocity (CV) across LacZ-hVSFs, MyoC-hMSCs, and CMCs. Conduction block was present in the absence of cells in the channel. LacZ-hVSFs are presented as hVSFs in this figure. One-way ANOVA with Bonferroni correction for multiple comparisons: *p<0.05 vs MyoC-hVSFs, **p<0.01 vs CMCs.

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Application of MyoC-hVSFs (n=11) in group A also resulted in synchronized beating of the two CMC fields within 24h, but with a smaller conduction delay of 20±1.5 ms and a CV of 3±0.7 cm/s (Figure 2B, E, and F). In contrast to the experiment car- ried out with LacZ-hVSFs, conduction delay between the two CMC fields coupled by MyoC-hVSFs decreased progressively to 4±1 ms at day 8 (n=11), with a correspon- ding increase in CV to 18±1.2 cm/s (p<0.01 vs. LacZ-hVSFs) (Figure 3B, E, F, and 4).

Furthermore, the decremental nature of conduction had disappeared. The a-cellular controls (n=12) showed asynchrony between the two CMC fields during follow-up (Figure 2C, E, and 3C, E). Application of CMCs (n=12), resulted in electrical coupling of the two CMC fields within 1 day, associated with a conduction delay of 3±1ms and a CV of 20.5±1 cm/s (Figure 2D, E, and F). No significant changes in conduction delay or CV were observed during follow-up in the CMC group (Figure 3D, E, F, and 4).

CV across CMC fields was 21.1±1.7 cm/s (n=70), which is comparable to CV across CMCs applied in-between CMC fields.

Figure 4. Conduction velocity (CV) across CMCs, LacZ-hVSFs (presented as hVSFs), and MyoC-hVSFs, with the associated dyssynchrony between two CMC fields. No differences were found in CV or dyssynchrony in the cultures re- ceiving CMCs or LacZ-hVSFs during follow-up.

However, in cultures having MyoC-hVSFs both CV and dyssynchrony were changed significantly in the first 8 days (p<0.01). One-way ANOVA with Bonferroni correction for multiple comparisons: †p<0.01 vs CMCs day 8, One-way repeated-measures ANOVA (with timepoint as factor) and Bonferroni-corrected: ‡p<0.01.

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Myocardin Gene Expression in hVSFs and Electrical Stimulation

In group B, a 330±15µm wide a-cellular channel was present after laser-dissection.

The pipette electrode was located successfully above the center of the hVSF strip in 7 out of 10 of the experiments. The strip of cells was visually distinguishable throughout follow-up. One day after cell application to the channel, electrical stimulation at LacZ-hVSFs site did not result in electrical capture (n=15). Also during follow-up (n=11), no electrical capture was observed (Figure 5B1).

However, electrical stimulation at the site of MyoC-hVSFs was successful in 2/10 experiments at day 1 (n=15), resulting in electrical capture and subsequent activation of both CMC fields (Figure 5B2). Interestingly, electrical stimulation at the site of MyoC-hVSFs was successful in 8/10 experiments at day 8 (n=10, p<0.001) (Figure 5A). After application of CMCs, electrical stimulation at day 1 (n=16) resulted in electrical capture in 100% of the attempts (Figure 5B3). As expected, during 8 days of

Figure 5. Capacity of transplanted cells to serve as electrical access tissue. (A, B1) No electrical capture was found after electrical stimulation of LacZ-hVSFs (presented as hVSFs), neither at day 1 nor at day 8.

(A, B2) However, electrical stimulation of MyoC-hVSFs at day 1 resulted in electrical capture in 20% of the attempts, which increased to 80% at day 8. (A, B3) Application of CMCs and subsequent electrical stimulation was associated with 100% success rate both at day 1 and day 8. Two-factor mixed ANOVA with Bonferroni correction for multiple comparisons: *p<0.01 vs MyoC-hVSFs, **p<0.01 vs day 1.

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follow-up (n=15), the 100% success rate was maintained, indicating stable activation of the CMC fields (Figure 5A).

Immunocytochemical and Genetic Analysis of Myocardin Gene Expression in hVSFs Immunocytochemistry

After day 1 of culture, Cx43 staining was found in the cytoplasm of LacZ-hVSFs as well as in-between adjacent LacZ-hVSFs where it displayed a punctuated pattern re- flecting the presence of gap junctions (Figure 6B1).

Figure 6. Comparison of immunocytochemical analysis of Cx40, Cx43 and Cx45 between LacZ-hVSFs (pre- sented as hVSFs) and MyoC-hVSFs, both at day 1 and day 8 after culture and CMCs after 8 days of culture.

Two-factor mixed ANOVA with Bonferroni correction for multiple comparisons: *p<0.001 vs hVSFs at day 1,

**p<0.05 vs MyoC-hVSFs at day 1, and one-way ANOVA, Bonferroni-corrected: ***p<0.05 vs CMCs.

A6 B6 C6

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Staining for Cx40 and Cx45 was only weakly present in the cytoplasm, with some Cx40 (Figure 6, A1) and Cx45 staining (Figure 6, C1) in-between or perinuclear in LacZ-hVSFs, respectively. MyoC-hVSFs displayed similar Cx40, Cx43 and Cx45 stai- ning patterns as LacZ-hVSFs at day 1 of culture (Figure 6A3-C3). However, in MyoC- hVSFs the amount of Cx40 and Cx45 were much higher (3266±802% and 2733±681%, respectively) than in LacZ-hVSFs (p<0.001) (Figure 6A6 and C6).

Cx levels in LacZ-hVSFs did not significantly change over time (Figure 6, A2-C2). In contrast, amounts of Cx40 and Cx45 in MyoC-hVSFs at day 8 were significantly hi- gher than at day 1 (5233±492% and 4233±351%, respectively; p<0.05) (Figure 6A3-4, C3-4, A6 and C6).

Comparison of the cardiac Cx levels between neonatal rat CMCs and MyoC-hVSFs at day 8 revealed that Cx43 amounts were more than 15 times higher in CMCs (P<0.01), while amounts of Cx40 and Cx45 were significantly higher in the MyoC-hVSFs (>40 and 10 times, respectively; p<0.001) (Figure 6A5-C5 and A6-C6).

Figure 7. Semi-quantitative RT-PCR analysis of ion channel gene expression in untreated (U) hVSFs or in hVSFs expressing eGFP and either LacZ (L), or MyoC (M) at day 1 and day 8 after culture. MyoC gene transduction in hVSFs resulted in (increased) expression of SCN5A, CACNA1C, KCNJ3, KCNJ8, and KCNMB1. These genes were exclusively or more expressed at day 8. In contrast, MyoC gene expression resulted in down-regulation of KCNA4 expression. Both expression of KCNH2 and KCNJ2 were not detectable in MyoC-hVSFs during follow-up. GAPDH and MyoC gene expression were used as reference.

The dilution factor of the template DNA is indicated at the bottom of the figure.

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Reverse Transcription-Polymerase Chain Reaction Analysis

Differences in gene expression were studied between untreated hVSFs, LacZ-hVSFs, and MyoC-hVSFs, both at day 1 and day 8 of culture (Figure 7). There were no or only minor differences in the expression of the analyzed genes between hVSFs and LacZ-hVSFs. However, forced expression of MyoC in hVSFs upregulated the transcription of the genes SCN5A and CACNA1C, encoding voltage-gated fast sodium channel and voltage-gated L-type calcium channel proteins, respectively. In addition, both the expression levels of SCN5A and CACNA1C increased in MyoC-hVSFs from day 1 to day 8 of culture. Furthermore, MyoC transduction led to a downregu- lation of KCNA4 expression, which was especially noticeable at day 8. No induction of KCNH2 or KCNJ2 was found in MyoC-hVSFs during follow-up. However, forced MyoC expression resulted in the time-dependent expression of the genes KCNJ3 and KCNJ8, both encoding inward rectifier potassium channel proteins. In addition, at day 8 MyoC-hVSFs but not LacZ-hVSFs were expressing KCNMB1, which encodings a voltage- and calcium-sensitive potassium channel protein. Analyses of MyoC gene transduction in hVSFs showed the increased expression of ion channels important for excitation,^17,18 and a significant increase in the expression of Cxs, components essential for inter-cellular electrical conduction.

Intracellular Electrical Recordings

Intracellular patch-clamp recordings were performed in co-cultures of LacZ-hVSFs (n=9) or MyoC-hVSFs (n=9) and CMCs (n=8) at day 1 and day 8 after culture initiati- on. Both LacZ-hVSFs and MyoC-hVSFs showed conducted APs. However, at day 1 of culture, MyoC-hVSFs showed larger conducted APs than LacZ-hVSFs (Figure 8A).

During 8 days of culture, the maximum diastolic potential (MDP) become more negative and the conducted APs of MyoC-hVSFs increased significantly in amplitude, and rate of rise, and became comparable to APs derived from CMCs (Figure 6B, D1-4).

This in contrast to MDPs and conducted APs of LacZ-hVSFs. Importantly, the estimated gap junctional conductance of the MyoC-hVSFs (n=9) increased significantly over time, from 18.1±5 nS to 80.3±18 nS, in contrast to LacZ-hVSFs (n=9) (Figure 8C), indicating a time-dependent increase in the degree of intercellular coupling after MyoC gene transfer in hVSFs. Surprisingly, inward currents of ion channels typically involved in excitation (SCN5A, CACNA1C) were not recorded in MyoC-hVSFs at day 1 (n=8) or day 8 (n=8) of culture, despite their increased gene expression (Figure 7).

These currents were easily measured in control CMCs (n=12).¹⁷

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Figure 8. Intracellular patch-clamp measurements in LacZ-hVSFs (presented as hVSFs), MyoC-hVSFs, and CMCs at day 1 and day 8 of culture. Gj=gap junctional conductance, MDP=maximum diastolic potential, APA=action potential amplitude, dV/dtmax=maximal upstroke velocity, APD90=action potential duration till 90% repolarization. Two-factor mixed ANOVA with Bonferroni correction for multiple comparisons: *p<0.05 vs MyoC-hVSFs, **p<0.05 vs Day 1.

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Discussion

The key findings of the present study are (1) application of MyoC gene–expressing hVSFs in an acellular channel between 2 cardiomyocyte fields resulted in resynchronization of 2 dyssynchronously beating cardiomyocyte fields, and (2) MyoC gene expression in hVSFs enabled simultaneous activation of the 2 adjacent cardiomyocyte fields by electrical stimulation of these cells, whereas it was not possible to stimulate via control hVSFs. Both phenomena appear to be mediated mainly by the MyoC- dependent activation of genes encoding connexins, strongly enforcing intercellular electrical coupling.

Role of Scar Tissue in Cardiac Dyssynchrony and Resynchronization

Scar tissue formation after myocardial infarction is associated with an accumulating population of electrically inert fibroblasts and extracellular matrix formation. Scar fibroblasts are not only unexcitable and poorly coupled, thereby creating areas of conduction block and zones of slow conduction, but they also modulate excitability and conduction properties of surrounding CMCs.¹⁹ Previous studies reported on the limited capacity of cardiac fibroblasts to conduct electrical current over extended distances.^20,21 As a result of modest expression levels of connexins in-between these cells and their non-excitability, conduction is not only limited in distance but also in velocity. Efficient mechanical activation of the myocardium depends on a coordinated and fast spread of electrical activation across well-coupled excitable CMCs. The- refore, the presence of poorly coupled, electrically inert scar fibroblasts contributes to increased dyssynchrony between ventricular segments.

Resynchronization after Transplantation of MyoC-Expressing hVSFs

We demonstrated that transplantation of MyoC-expressing hVSFs in an a-cellular channel between two CMC fields resulted in synchronization of the two fields, which was accompanied by an increase in CV, both at day 1 and 8 of co-culture as compared to control hVSFs.

Previously, Kizana et al. showed that forced expression in dermal fibroblasts of the gene encoding the myogenic transcription factor MyoD, resulted in excitability of some of these fibroblasts.²² However, as MyoD was used, the differentiated fibroblasts (now myotubes) did not express sufficient Cx43 to be electrically coupled to each other. Surprisingly, forced Cx43 expression in these myotubes even reduced the number of excitable cells (to less than 4%), possibly due to an increased electrotonic load.

In the present study we used human ventricular scar fibroblasts, and these cells already naturally express Cx43.

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Although the electrophysiological mechanisms responsible for resynchronization in our study remain to be determined in more detail, increased Cx expression in MyoC- hVSFs ensures improved intercellular coupling,^13,21 while a minor role might be attri- buted to ion channels. Although both mRNAs and proteins associated with a number of essential ion channels involved in excitation are present in hVSFs after myocardin gene transduction, their functionality could not be shown. However, ion channels are complex structures, and several requirements are needed to ensure functionality.²³ Nevertheless, the pore protein is present which is at least necessary for the expression of a functional ion channel.²⁴

Electrical Capture in hVSFs after Forced MyoC Expression

It was not possible to resynchronize the two CMC fields at any moment by electrical stimulation of control hVSFs. However, after forced expression of the MyoC gene in hVSFs, electrical capture of the CMC fields was observed in 20% of the experiments at day 1, and in 80% of the experiments at day 8. In contrast to control hVSFs, which play a rather depressing role in conduction,²⁵ hVSFs over-expressing MyoC allow ra- pid action potential transmission. We also showed that MyoC-hVSFs were able to conduct the electrical pacing pulse to adjacent CMC fields leading to their electrical and mechanical activation. Hence, relatively high expression levels of Cx seem to be mandatory in successful electrical capture.

In Vitro to in Vivo Translation

Genetic modification of cells requires efficient transfection to ensure optimal expression of the gene of interest. Several vectors are available, but adeno-associated virus (AAV) vectors seem be preferable with regard to cardiac gene therapy in vivo, as they demonstrated good tissue penetration and sustained transgene expression.²⁶

In this study we show the beneficial effects of myocardin gene overexpression in hVSFs on resynchronization. However, Badorff et al. showed that a similar overexpression in neonatal CMCs resulted in hypertrophy.²⁷ Therefore, not only the efficiency of infection is important in order to obtain an optimal effect, but also the site-directed introduction and cell type-specific expression of the gene is important to prevent a negative outcome. In addition, as the heart forms a functional electrical syncytium comprised of different cell types with different electrophysiological characteristics it is of importance to study the adaptation of genetically modified cells in relation to properties of the target tissue.²⁸

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Possible Clinical Implications

Slowing and even blocking of the spread of activation by scarred myocardium is not the only deleterious consequence of myocardial scar formation. In previous clinical studies we demonstrated the negative effects of scar tissue on the response to CRT.^9,10,29 A significant lower response rate was observed in patients with scars near the pacing electrode (14% vs. 81% in patients without detectable scar tissue).¹⁰ In the current study, we demonstrated the striking effects of forced MyoC gene expression in hVSFs on electrical conduction. Genetic modification of hVSFs with a recombinant MyoC gene resulted in both resynchronization of two adjacent beating CMC fields and endowed hVSFs with the capacity to serve as a pacing site to capture the cardiac impulse. Although conducted ex vivo, this study could provide a rationale for the treatment of scar-related myocardial dyssynchrony and contribute to increased efficiency of CRT in patients with scarred myocardium.

Study Limitations

In this study, myocardial scar tissue is represented by fibroblasts only. Although this model is sufficient to investigate the concept raised in this study, it does not fully mi- mic the composition of myocardial scar tissue in vivo. Excessive extracellular matrix and surviving CMCs might influence the effects of MyoC expression in the infarcted area. Furthermore, neonatal rat CMCs were used in this study. Although the use of human CMCs is preferable, the difficulties related to the availability of this cell type and their tendency to dedifferentiate during culture prevented the use of human CMCs.

Conclusions

Forced MyoC expression in hVSFs allowed resynchronization of two separated CMCs fields and establishment of interconnecting tissue for electrical pacing. Both phenomena primarily result from the MyoC-dependent activation of genes encoding connexins, thereby strongly enforcing inter-cellular electrical coupling.

Disclosures None.

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Clinical Perspectives

Myocardial scar tissue consists primarily of electrically inert fibroblasts. Consequent- ly, impulse propagation and subsequent mechanical activation is hampered, resulting in increased ventricular dyssynchrony. Cardiac resynchronization therapy (CRT) is a promising treatment for patients with drug-refractory heart failure and significant dyssynchrony, which often improves cardiac function and increases life expectancy.

However, ~30% of the patients subjected to CRT does not respond, which has been associated with the presence of ventricular scar tissue. Importantly, scar burden appears to be involved in both the cause of ventricular dyssynchrony as well as the lack of response to CRT. Modification of scar tissue, favoring electrical conduction, might therefore result in reduction of dyssynchrony and non-response. The present study provides a rationale for (i) the treatment of scar-related myocardial dyssynchrony and (ii) reduction of scar-related non-response to CRT. This was accomplished by genetic modification of human ventricular scar fibroblasts by myocardin gene transfer. Myo- cardin is a cardiac transcription factors known to induce or upregulate the expression of cardiac genes, including connexins which play a crucial role in electrical conduc- tion. This study shows that forced myocardin gene expression in scar fibroblasts al- lowed electrical stimulation of these cells and endowed them with the capability to conduct an electrical impulse at high velocity, resulting in resynchronization of two separated cardiomyocyte fields. Genetic modification of myocardial scar tissue might therefore be a promising therapy to resynchronize the infarcted myocardium, thereby improving cardiac function.

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