CIRCULATIONAHA/2006/627588/R2 Ieda, et al.
On-line supplementary information
Supplementary Methods
Cell culture and immunostaining
DRG explants were removed from embryonic day (E) 12.5 or 14.5 mice embryos. The caspase inhibitor Q-VD-OPh (R&D systems) was included in the medium at a concentration of 5x10-5 M1. After 24 hours incubation, the explants were cultured with either cardiomyocyte-conditioned media or media containing various concentrations of NGF (Upstate) for 2 days. For assessment of axonal growth measurement, ganglia were immunostained with anti-neurofilament-M antibody (Chemicon) and the 20 longest axons per ganglion were measured in 10 ganglia. For assessment of CGRP expression, dissociated cultures of DRG neurons were incubated in cardiomyocyte-conditioned media for 5 days2. The percentage of CGRP-immunopositive neurons per total number of neurons was determined. A minimum of 200 neurons per well was counted and 4 wells per experiment were quantified from three independent experiments. PC12 cells were examined under a phase-contrast microscope, and the percentage of differentiated cells was counted as described previously3. Sigma-Aldrich supplied anti-NGF blocking antibody (1:10000).
RNA extraction and quantitative RT-PCR
RNA and quantitative RT-PCR were performed as described previously3. The primers and probes for NGF and neurotrophin-3 (NT-3) were as described previously3. The primers and probe for GDNF was Mm00599849_m1 and that for c-Fos was Mm00487425_m1 (Applied Biosystems). The mRNA levels were normalized by comparison to GAPDH mRNA.
Preparation of cardiomyocyte-conditioned media
Primary culture for cardiomyocytes was as described previously3. Cardiomyocytes were incubated in media containing serum for 24 hours and then the media was replaced with fresh serum-free media. After 24 hours with or without transfection of plasmids, the conditioned media was collected.
ELISA
ELISA kit for CGRP was purchased from SPI-BIO, and substance P was purchased from Cayman Chemical Company.
Induction of diabetes
Six-week-old ICR, wild-type (WT), NGFTG mice or eight-week-old Wistar rats were used. DM was induced via an intraperitoneal injection of 200 mg/kg or 65 mg/kg streptozotocin (STZ, Sigma-Aldrich) in either mice or rats as described previously4,5. Control animals received a mock injection only. Seven days later, tail-vein blood glucose was measured by a glucose stick and, and animals with blood glucose >300 mg/dl were presumed to be diabetic.
Coronary artery occlusion
Animals were intubated and anesthetized with 0.5% isofluorane gas. After left thoracotomy, the left ventricle was exposed and the left coronary artery was ligated as described previously6. Sham underwent the same procedure but did not have the left coronary artery ligated. The Keio University Ethics Committee for Animal Experiments approved all experiments in this study.
Ischemia-reperfusion animal model
Mice were subjected to transient coronary artery ligation for 30 min and reperfusion for 24 h. Area at risk and infarct size were determined by Evans blue and 2,3,5-triphenyltetrazolium chloride (TTC) staining7. Apoptotic cardiomyocytes in the ischemic area were detected using an ApopTag kit (Chemicon).
Immunohistochemistry of hearts
Paraffin-embedded sections were incubated with antibody against CGRP (Chemicon) to detect sensory nerve fibers, and with antibody against tyrosine hydroxylase (TH, Chemicon) to detect sympathetic nerve fibers. Following hybridization with the secondary antibody, sections were incubated with diaminobenzidine3. Cryostat sections were stained with antibodies against CGRP and P2X3 (Neuromics). The sections were incubated with HRP-conjugated secondary antibodies and visualized using the TSA direct kit (Perkin Elmer). Nuclei were stained with TOTO-3 (Molecular Probes). The sections were analyzed by confocal microscopy using an LSM 510 META (Carl Zeiss). Nerve density was determined using NIH Image, as described previously3.
Histology in DRG and DH
Paraffin-embedded sections were stained with Hematoxilin-Eosin (H&E）, and the antibody against CGRP. The total number of neurons and nerve density were determined as described previously1,3.
Supplementary references
1. Lonze BE, Riccio A, Cohen S, Ginty DD. Apoptosis, axonal growth defects, and degeneration of peripheral neurons in mice lacking CREB. Neuron. 2002;34:371-85.
2. Patel TD, Jackman A, Rice FL, Kucera J, Snider WD. Development of sensory neurons in the absence of NGF/TrkA signaling in vivo. Neuron. 2000;25:345-57.
3. Ieda M, Fukuda K, Hisaka Y, Kimura K, Kawaguchi H, Fujita J, Shimoda K, Takeshita E, Okano H, Kurihara Y, Kurihara H, Ishida J, Fukamizu A, Federoff HJ, Ogawa S. Endothelin-1 regulates cardiac sympathetic innervation in the rodent heart by controlling nerve growth factor expression. J Clin Invest. 2004;113:876-84.
4. Kanki H, Fukuda K, Okushi K, Ibata I, Toyama Y, Shimizu H, Arakawa Y, Ogawa S. Comparison of nerve growth factor mRNA expression in cardiac and skeletal muscle in streptozotocin-induced diabetic mice. Life Sci. 1999;65:2305-13.
5. Sasaki K, Chancellor MB, Goins WF, Phelan MW, Glorioso JC, de Groat WC, Yoshimura N. Gene therapy using replication-defective herpes simplex virus vectors expressing nerve growth factor in a rat model of diabetic cystopathy. Diabetes. 2004;53:2723-30.
6. Kawada H, Fujita J, Kinjo K, Matsuzaki Y, Tsuma M, Miyatake H, Muguruma Y, Tsuboi K, Itabashi Y, Ikeda Y, Ogawa S, Okano H, Hotta T, Ando K, Fukuda K. Nonhematopoietic mesenchymal stem cells can be mobilized and differentiate into cardiomyocytes after myocardial infarction. Blood. 2004;104:3581-7.
7. Matsushita K, Iwanaga S, Oda T, Kimura K, Shimada M, Sano M, Umezawa A, Hata J, Ogawa S. Interleukin-6/soluble interleukin-6 receptor complex reduces infarct size via inhibiting myocardial apoptosis. Lab Invest. 2005;85:1210-23.
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