Материал: nm1394

© 2006 Nature Publishing Group http://www.nature.com/naturemedicine

METHODS

Animal care and experimental protocol approval. We maintained experimental rats in accordance with the guiding principles of the American Physiological Society. The study was approved by local authorities (Regierung von Mittelfranken: 621.2531.31-2/00 and -17/01; BWG of the Freie und Hansestadt Hamburg: #54/04).

EHT construction. We constructed EHTs as previously described27. Briefly, we prepared EHT rings (reconstitution volume, 0.9 ml) by mixing isolated heart cells from neonatal rats (2.5 106 cells/EHT) with collagen type I from rat tails (0.8 mg/EHT), Engelbreth-Holm-Swarm tumor exudate (10% vol/vol; tebu) and concentrated serum-containing culture medium (2 DMEM, 20% horse serum, 4% chick embryo extract, 200 U/ml penicillin and 200 mg/ml streptomycin); we neutralized pH with 0.1 N NaOH. We transferred EHTs after 7 d onto custom-made stretch devices to facilitate static (110% of slack length), phasic (from 100 to 110% of slack length at 2 Hz) or auxotonic (culture on coils at 110% of slack length adjusted to deflect 1 mm at 1–1.5 mN) loading. We analyzed contractile parameters by isometric contraction experiments of single-loop EHTs as previously described27. We generated multiloop EHTs by stacking five single EHTs. To control for indirect paracrine or matrix-mediated effects, we used formaldehyde-fixed grafts and NCMGs.

Infarct model and EHT grafting. We generated myocardial infarctions in ventilated, isoflurane (2%)-anesthetized male Wistar rats (324 ± 2 g; n ¼ 343) by permanent LAD ligation (5-0, Prolene, Ethicon). We implanted EHTs and noncontractile grafts 14 d after infarct induction (inclusion criterion: FAS o40% by echocardiography; Supplementary Note and Supplementary Fig. 3 online) with six single-knot sutures (Fig. 1g; 5-0, Prolene, Ethicon). We placed the sutures in healthy myocardium adjacent to the visible infarct scars to arrange the center of the EHTs just above the latter. In the sham-operated group, sutures were placed as if EHTs were implanted. All rats received immunosuppressants as previously described (mg/kg body weight/d: azathioprine, 2; cyclosporin A, 5; methylprednisolone, 5)27.

Histology. We fixed hearts in neutral buffered 4% formaldehyde–1% methanol, pH 7.4, and subjected them to histological analysis as previously described27. Briefly, we stained paraffin-embedded sections (4 mm) with H&E. We stained cryosections (10 mm) with phalloidin–Alexa 488 (3.3 U/ml; Molecular Probes) to label F-actin, Bandeiraea simplicifolia lectin-TRITC (10 mg/ml; Sigma) to label endothelial cells and antibodies directed against ED2 (CD163 cell-surface glycoprotein, 1:100; Serotec) to label macrophages with appropriate secondary antibodies.

Epicardial mapping. We performed epicardial multielectrode mapping as previously described28 (Supplementary Methods online).

Ambulatory ECG telemetry. We inserted telemetry devices (PhysioTel CA-F40, DataSciences) into the abdominal cavity during sham operation (n ¼ 5) or EHT (n ¼ 5) operation to record electrocardiograms continuously for 2 (sham/EHT, n ¼ 2 per group) to 4 (n ¼ 3 per group) weeks. Data analysis was performed semiautomatically (ECG-auto, v.1.5.12.38, EMKA Technologies) using rat-specific waveform libraries (Supplementary Methods online).

Echocardiography. We performed echocardiography in volatile isoflurane (2%) anesthesia as described previously with a HP Sonos 7500 System (Philips) equipped with a 15 MHz linear array transducer27 or a Vevo660 ultrasound biomicroscopy system equipped with a 37.5 MHz scan head (RMV 710; VisualSonics). Interand intraobserver variation of reported data was o10%.

Magnetic resonance imaging. We performed MRI in rats under volatile isoflurane (2%) anesthesia with a Bruker 4.7T Biospect System using a fast gradient echo sequence with 21 ms repetition time (TR), 5 ms echo time (TE) and a flip angle of 301. Recordings were ECG triggered and breath triggered. We acquired a total of six to eight sequential movie frames with 256 128 pixels at a pixel resolution of 200 400 mm. We obtained a longitudinal view for orientation purposes. Based on the latter, 20–30 cross-sections (short axis) from apex to base were imaged (4D cine mode). Subsequently, we subjected data sets to off-line analysis with manual segmentation of the heart contours.

Left ventricular catheterization. Pressure-volume loops were recorded under isoflurane (2%) anesthesia with a Millar 2 Fr catheter (model SPR-838) connected to Aria/PowerLab data-acquisition hardware (Millar/PowerLab) by an investigator blinded to the experimental conditions. We performed volume calibration by equating catheter-recorded minimal and maximal conductances with minimal and maximal MRI volumes, respectively. We analyzed all data off-line with PVAN 3.2 software (Millar).

Statistical analysis. Data are presented as mean ± s.e.m. or box plots indicating the median, 25th and 75th percentiles, and the range. We determined statistical differences using paired and unpaired two-tailed Student t-tests (in vivo data), repeated-measures ANOVA (in vitro contraction experiment) or MannWhitney U-test (mapping and telemetry). A P value of o0.05 was considered statistically significant.

Note: Supplementary information is available on the Nature Medicine website.

ACKNOWLEDGMENTS

This work is part of the doctoral thesis of I.M. at the Universities of ErlangenNuremberg and Hamburg. We acknowledge the technical assistance of S. John and F. Bussmann (University of Erlangen; MRI data evaluation) and T. Mu¨ller (University of Erlangen; construction of stretch devices). The help from H. Ru¨tten and D. Gehling (Aventis, Frankfurt) in echocardiography and pressure-volume loop recordings is appreciated. This study was supported by the German Research Foundation (Deutsche Forschungsgemeinschaft; Es 88/8-2 to T.E. and FOR 604/1-1 to T.E. and H.E.), the German Ministry for Education and Research (Bundesministerium fu¨r Bildung und Forschung 01GN 0124 and BMBF 01GN 0520 to T.E. and W.-H.Z.), the Deutsche Stiftung fu¨r Herzforschung (W.-H.Z.) and the Minerva foundation (W.-H.Z.).

COMPETING INTERESTS STATEMENT

The authors declare that they have no competing financial interests.

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