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African Journal of Pig Farming ISSN 2375-0731 Vol. 7 (10), pp. 001-005, October, 2019. Available online at 
www.internationalscholarsjournals.org © International Scholars Journals 

 

Author(s) retain the copyright of this article. 

 

Full Length Research Paper 

 

Expression of protein-gene peptide (PGP) 9.5 in 
myocardial sleeves around the pulmonary artery 

and aorta in pigs 

 
Jingping Lu, Dongsheng Zhao, Gang Zhang, Jie Gen and Qijun Shan* 

 
Department of Cardiology, First Affiliated Hospital of Nanjing Medical University, 300 Guangzhou Road, 

Nanjing, 210029, Jiangsu Province, China. 
 

Accepted 13 July, 2019 
 
A subgroup of outflow tract (OT) ventricular tachycardias (VT) originates from the aortic sinuses or the main 
stem of the pulmonary artery. Myocardial sleeves onto the pulmonary artery (PA) and aorta (Ao) have been 
recognized as a frequent site for the origin of arrhythmia. The aim of this study was to analyze PGP9.5 
expression in pulmonary artery and aorta from 100 pigs hearts and relate it to the mechanism of arrhythmia. 
The pulmonary artery and aorta were obtained from 100 healthy pigs; longitudinal strips of tissue containing 
each cusp, aortic, pulmonary artery walls, left and right ventricular outflow tracts were excised and 
histologically analyzed. PGP9.5 was studied immunohistochemically in myocardial sleeves. Myocardial 
sleeves were found in 88 of 100 Aos (88%) and 20 of 100 PAs (20%). PGP9.5 positivity was observed in 89 of 90 
(99%) pigs, which had myocardial sleeves. PGP9.5 immunopositivity can be detected in most pig myocardial 
sleeves around the PAs and Aos; this finding may contribute to the substrate for OT VTs. 

 
Key words: Myocardial sleeves, ventricular tachycardia, aortic sinus, pulmonary artery, protein-gene peptide (PGP) 
9.5. 

 
INTRODUCTION 

 
Ventricular tachycardia arising from the right or left 
ventricular outflow tract is a recognized arrhythmia in 
individuals with structurally normal hearts. The left and 
right outflow tracts share a common embryonic origin, 
which explains the similarity of the tachycardias (Lerman 
et al., 1996, 1997; Stevenson, 2005; Iwai et al., 2006; 
Bunch and Day, 2006). It has recently been shown that a 
subgroup of outflow tract ventricular tachycardias (OT 
VTs) originated from the aortic sinuses or the main stem 
of the pulmonary artery (Shimoike et al., 1999; Ouyang et 
al., 2002; Sadanaga et al., 1999; Tada et al., 2004; 
Kanagaratnam et al., 2001).  

A lot of studies have shown atrial myocardium extends 
into the pulmonary and caval veins plays a critical role in 
the initiation of atrial fibrillation (AF) (Haissaguerre et al., 
1998; Tsai et al., 2000; Mansour et al., 2002). Ventricular  
 
 
 
*Corresponding author: E-mail: qjshan@njmu.edu.cn. Tel: +86-
25-868136407. 

 
 
 

 
myocardial extensions onto the pulmonary artery (PA) and 
aorta (Ao) beyond the ventriculo-arterial junction are 
relatively common. It seems that their mere presence does 
not predispose to OT VTs. There are probably intrinsic 
arrhythmia properties in tissues specific to these regions in 
those patients who develop OT VTs (Hasdemir et al., 2007). 
The precise mechanism of these ventricular myocardial 
sleeves and their role in the causation of arrhythmia need to 
be explored further.  

PGP9.5 is a new cytoplasmic neuron-specific protein 
structurally and immunologically distinct from neuron-
specific enolase. Interestingly, PGP9.5 immunoreactivity 
has been described in morphologically dynamic 
myocardial regions during heart ontogenesis that may 
subsequently play a role in triggering arrhythmia. 
Expression of PGP9.5 has also been hypothesized to 
correspond with abnormal ventricular automaticity. In 
addition, some authors used PGP9.5 as a marker of the 
conduction system (El et al., 2001; Crick et al., 1999a, 
1996). The aim of this study therefore was to analyze 
PGP9.5 expression in pulmonary artery and aorta from 



 
 
 

 

100 pig hearts and relate it to the mechanism of 
arrhythmia. 
 
 
MATERIALS AND METHODS 
 
Anatomy 
 
A hundred adult Chinese mongrel pigs of either sex weighing about 
90 kg were used in this study. In all subjects, the heart was excised 
together with PA and Ao. The ascending Aos and PAs were 
transected at the pericardial reflections. The aortic and pulmonary 
roots were opened by longitudinal incisions through the left aortic 
cusp and the anterior pulmonary cusp. The original strips of tissue 
samples were cut into three equal-sized pieces, containing each 
cusp, aortic and PA walls 20 mm above the ventriculo-arterial 
junction (VAJ), and left and right ventricular myocardial 20 mm 
below the VAJ, these were then fixed with 10% formalin. 

 

Histology 
 
Specimens were routinely dehydrated and embedded in paraffin 
using an automatic tissue processor. Serial sections were cut at 4 
to 5 μm thickness and mounted on poly L-lysine prepared slides. 
Serial sections were stained with haematoxylin and eosin (H & E) 
stain. The presence of myocardial sleeves was evaluated in each 
section microscopically. 

 

Immunohistochemistry 
 
Immunohistochemistry was performed with antibodies against 
Protein gene peptide 9.5 (PGP 9.5, rabbit polyclonal, dilution 1:200, 
abcam). Deparaffnized sections were rehydrated and 
immunostained according to the following protocol: endogenous 
peroxidase activity was inhibited, 30 min incubation in 0.3% 
hydrogen peroxide and methanol followed by tap water wash, the 

slides were incubated in a retrieval solution for 15 min at 95
o
C, 

which was cooled in the solution naturally and then washed in PBS 
(Phosphate Buffered Saline) 3 times, 3 min each. Subsequently, the 
sections were blocked with blocking buffer (from Boster, SA1022) 
and incubated for 20 min at room temperature in a humidified 
chamber and finally with avidin biotin peroxidase complex 
(Vectastain ABC kit, CA, USA) for 1 h. Peroxidase activity was 
detected by incubation of the slides with diaminobenzidine 
tetrahydrochloride (DAB) and H2O2 for 5 min. After washing with 
distilled water, sections were counterstained with haemalum, 
dehydrated and mounted with DPX. For negative controls, the first 
antibody was replaced by PBS. Staining intensity was evaluated as 
negative (-), faintly positive (+), moderately positive (++) and 
strongly positive (+++). 
 
 
RESULTS 
 
Morphological characteristics 
 

Myocardial sleeves were found in 90 of 100 (90%) pigs 
studied. Ventricular myocardial extensions beyond the 
ventriculo-arterial junction were found in 108 of 200 
(54%) of the great arteries examined.18 pigs had both 
aortic and pulmonary myocardial sleeves. Myocardial 
sleeves were continuous in 70 (78%), discontinuous in 13 
(14%), and both in 7 (8%). Myocardial sleeves were 
oriented obliquely in 16 (18%) and longitudinally in 

  
  

 
 

 

20 (22%), and both in 54 (22%). Myocellular hypertrophy 
and fibrosis were present in 69 (77%) and fatty tissue 
between the myocardial sleeve was 63 (70%).  

Myocardial sleeves were found in 88 of 100 Aos 
examined (88%): 71 around the right coronary sinus, 15 
around the left coronary sinus, and 59 around the 
noncoronary sinus (Figure 1).  

Myocardial sleeves were found in 20 of 100 PAs 
examined (20%). Myocardial sleeves in the PAs were 
located in the anterior pulmonary sinus in 9, in the left 
pulmonary sinus in 12 and in the right pulmonary sinus in 
1 (Figure 2). 
 

 

Expression of protein-gene peptide (PGP) 9.5 

 

PGP9.5 positivity was observed in 89 of 90 (99%) pigs, 
which had myocardial sleeves (Figure 3). PGP9.5 
negative was found in 5 of 59 (8%) in noncoronary sinus, 
4 of 71 (6%) in right coronary sinus, 1 of 9 (1%) in 
anterior pulmonary sinus. 
 

 

DISCUSSION 
 

Ventricular tachycardia has diverse mechanisms and 
sites of origin. Most VT originate from ventricular 
myocardium, however, a subgroup of OT VTs originates 
from the aortic sinuses or the main stem of the PA 
(Sadanaga et al., 1999; Sekiguchi et al., 2005; Hachiya et 
al., 2002). VT originating above the level of the semilunar 
valves has also been described in a small number of 
case studies. Our study has shown that ventricular 
myocardial extensions extend into the aorta and 
pulmonary artery beyond the semilunar valves. 
Ventricular myocardium extending into the great vessels 
above the semilunar valves may be a trigger for the 
arrhythmia, similar to that observed from the superior 
vena cava and pulmonary veins in patients with atrial 
fibrillation (Haissaguerre et al., 1998; Tsai et al., 2000; 
Mansour et al., 2002). Nevertheless, in some cases, 
these potentials may result from bystander tissue to 
getting passively activated from the ventricle. The precise 
mechanism of these potentials and their role in the 
causation of arrhythmia need to be explored further.  

PGP9.5 was expressed by cardiac nerves and 
conducting system components, and successfully used to 
quantify the innervation of the conducting system of the 
adult calf, pig, guinea pig and human heart. It serves as 
useful markers for the cardiac conducting system (Crick 
et al., 1996, 1999a, 1999b). The sinus node, 
atrioventricular node, atrioventricular bundle and both left 
and right bundle branches all possessed a significantly 
higher density of PGP9.5-immunoreactive nerves relative 
to their surrounding myocardial tissues (El et al., 2001). 
PGP9.5 immunopositivity has been hypothesized to be 
associated with abnormal automaticity in human 
embryos, suggesting that this parameter could be used to



  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
Figure 1. Long axis view of the pulmonary root, stained with HE stain, myocardial 
sleeves is shown by the arrow. 

 
 

 

identify foci with a potential for abnormal automaticity in 
ventricular myocardium. These findings indicate that a 
nonreentrant mechanism of VT, like automaticity or 
triggered activity, would be an explanation for the 
mechanism responsible for the VT in this study.  

However, OT VTs that are based on a reentrant 
mechanism have been reported. The other hypothesis 
may reflect the fact that myocardial sleeves 
corresponding with PGP9.5 intensity may be a bystander 
tissue getting passively activated from the ventricle. Rich 
PGP9.5 positive innervation of all the components of the 
conduction tissues displayed a homogeneous distribution. 
In this respect, the presence of regions that possessed a 
high density of PGP 9.5-immunoreactive nerve trunks 
and fibres in the myocardial sleeves 

 
 
 

 

extending into the aorta and pulmonary artery beyond the 
semilunar valves, may provide some evidence for the 
existence of 'preferential conduction pathways' through 
the atrioventricular nodal region. The true relationship of 
these 'nerve pathways' to the preferential conduction 
routes into the node is, however, highly controversial and 
merits further investigation. Ventricular myocardial 
extensions extending into the aorta and pulmonary artery 
in pigs are relatively common. Although PGP9.5 
immunopositivity can be detected in some myocardial 
sleeves, this finding does not appear to reflect the 
prevalence of arrhythmogenic foci in OT VTs. The precise 
mechanism of these myocardial sleeves and their role in 
the causation of arrhythmia need to be explored further. 



   
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
Figure 2. Long axis view of the aortic root, stained with HE stain, myocardial sleeves is 
shown by the arrow.  

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
Figure 3. Myocardial sleeves with strong PGP9.5 immunopositivity in the compatible 
population of cells (black arrow). 



 
 
 

 

Conclusions 

 

Myocardial sleeves in pig arteries consist of myocytes 
different from normal ventricular myocytes, with abundant 
expression of PGP9.5, which serves as useful markers 
for the cardiac conducting system. The myocardial 
sleeves extending into the aorta and pulmonary artery 
may contribute to the substrate for OT VTs. 
 

 
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