Pre- and post-synaptic stimulation by nicotine and noradrenalin on the spontaneous beating of cardiomyocytes.

<p><b>A)</b> Effects of nicotine (1 µM). Left: representative recording of extracellular potentials before and 6 min after application of nicotine to a mono-culture of VMs treated for 5 days with GDNF 10 ng/ml ([VM+GDNF]), co-cultures of VMs/SNs treated with vehicle only ([VM/SN+Vehicle], and co-cultures treated 5 days with GDNF 10 ng/ml ([VM/SN+GDNF]). Right: percent changes of spontaneous beating rate from baseline. Values are means±SD (n = 4), *p<0.05 vs. VM+GDNF. <b>B)</b> Effects of noradrenalin (10 µM). Left: representative extracellular potentials of VMs before and 3 min after application of noradrenalin to a mono-culture of VMs/SNs treated for 5 days with GDNF 10 ng/ml ([VM+GDNF]), co-cultures of VMs/SNs treated with vehicle only ([VM/SN+Vehicle]), co-cultures treated 5 days with GDNF 10 ng/ml ([VM/SN+GDNF]). Right: percent changes of spontaneous beating rate from baseline. [VM+Vehicle] : mono-cultures of VMs treated with vehicle only, [VM+NGF]: mono-cultures of VMs treated 5 days with NGF 50 ng/ml, [VM+GDNF]: mono-cultures of VMs treated 5 days with GDNF 10 ng/ml, respectively. Values are means±SD (n = 4). *Significantly different from baseline in each group (p<0.05, n = 4); †p<0.05 vs the VM/SN group (n = 4); #p<0.05 vs the VM/SN+NGF group (n = 4).</p

Axon Guidance of Sympathetic Neurons to Cardiomyocytes by Glial Cell Line-Derived Neurotrophic Factor (GDNF)

<div><p>Molecular signaling of cardiac autonomic innervation is an unresolved issue. Here, we show that glial cell line-derived neurotrophic factor (GDNF) promotes cardiac sympathetic innervation in vitro and in vivo. <i>In vitro</i>, ventricular myocytes (VMs) and sympathetic neurons (SNs) isolated from neonatal rat ventricles and superior cervical ganglia were cultured at a close distance. Then, morphological and functional coupling between SNs and VMs was assessed in response to GDNF (10 ng/ml) or nerve growth factor (50 ng/ml). As a result, fractions of neurofilament-M-positive axons and synapsin-I-positive area over the surface of VMs were markedly increased with GDNF by 9-fold and 25-fold, respectively, compared to control without neurotrophic factors. Pre- and post-synaptic stimulation of β₁-adrenergic receptors (BAR) with nicotine and noradrenaline, respectively, resulted in an increase of the spontaneous beating rate of VMs co-cultured with SNs in the presence of GDNF. GDNF overexpressing VMs by adenovirus vector (AdGDNF-VMs) attracted more axons from SNs compared with mock-transfected VMs. <i>In vivo</i>, axon outgrowth toward the denervated myocardium in adult rat hearts after cryoinjury was also enhanced significantly by adenovirus-mediated GDNF overexpression. GDNF acts as a potent chemoattractant for sympathetic innervation of ventricular myocytes, and is a promising molecular target for regulation of cardiac function in diseased hearts.</p></div

Three-dimensional (3D) imaging of sympathetic nerves and coronary vessels in the post-myocardial infarction (MI) mouse heart.

<p>(<b>A</b>) Three-D image of the heart 2 weeks after inducing MI. Heart samples are immunostained with tyrosine hydroxylase (green) and α-smooth muscle actin (red). Sympathetic nerves are abruptly extinguished at the site of the ligation. Arrowheads indicate the left anterior descending coronary artery. The dashed line indicates the site of ligation. The right panel shows a higher magnified view of the boxed region in the left panel. Images are obtained with a light-sheet microscope (Lightsheet Z.1, Carl Zeiss). Scale bars represent 500 μm. (<b>B</b>) Three-D image of the ischemic border zone in the post-MI heart. The right panel shows a higher magnified view of the boxed region in the left panel. In the ischemic border zone proximally adjacent to the site of ligation, numerous fine nerve fibers are distributed in a disordered manner. Images are obtained with a confocal microscope (LSM 700, Carl Zeiss). Scale bars represent 200 μm (left panel) and 100 μm (right panel). (<b>C</b>) Analyses of sympathetic nerves in post-MI mice. The nerve volume and surface area are significantly increased in the ischemic border zone and decreased in the infarct area of post-MI hearts compared to intact mice hearts (n = 5, *p<0.05, by Steel-Dwass test). Error bars represent standard error of the mean. IBZ, ischemic border zone.</p

Three-dimensional (3D) distribution of sympathetic nerves and coronary vessels in the heart.

<p>(<b>A</b>) X-Y plane image of tyrosine hydroxylase (TH, green)-stained heart sample. Sufficient transparency is achieved, and the transmural distribution of sympathetic nerves is visualized. (<b>B</b>) 3D images of TH (green)-stained and α-smooth muscle actin (red)-stained heart samples. (<b>C</b>) A higher magnified view of the boxed region in <b>B</b>. Cardiac nerves distributed along the epicardial surface with nerve branches penetrating into the myocardium and wrapping around coronary vessels. Images are obtained using a light-sheet microscopy (Lightsheet Z.1, Carl Zeiss). Scale bars represent 500 μm.</p