Einfluss von PACAP (Pituitary Adenylate Cyclase-Activating Polypeptide) bzw. des PACAP Rezeptors PAC1 auf Morphologie und Expression Inflammations-/ Apoptose-/ Atherosklerose-relevanter Proteine/Zytokine in Mm. soleus/gastrocnemius bei ApoE defizienten Mäusen unter Standardfütterung
Das Hypophysen-Adenylat-Cyclase-aktivierende-Polypeptid (engl. „Pituitary Adenylate Cyclase-Activating Polypeptide“; PACAP) ist ein 38 AS großes Neuropeptid, das über die Bindung an verschiedene Rezeptoren vor allem dem PAC1-Rezeptor wirkt und seine Zielfunktionen über die Aktivierung einer Adenylat...
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Format: | Doctoral Thesis |
Language: | German |
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Philipps-Universität Marburg
2024
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The Pituitary Adenylate Cyclase-activating Polypeptide (PACAP) is a 38 amino acid neuropeptide that acts by binding to various receptors, most notably the PAC1 receptor, and performs its target functions via activation of an adenylate cyclase (AC). In the context of atherosclerosis, PACAP was shown to significantly decrease lipid peroxide formation and morphological changes induced by hyperlipidemia (ApoE-/-) in smooth muscle cells. Atherosclerosis plays an important pathophysiological role in several important diseases. In skeletal muscles, it can lead to increased oxidative stress, muscle fiber degeneration, fibrosis, as well as decreased capillarization of muscle fibers. This paper analyses the influence of PACAP or PAC1 deficiency on the morphology and expression of inflammation/apoptosis/atherosclerosis-related proteins/cytokines in Mm. soleus/gastrocnemius in hypercholesterolemic ApoE-/- mice under regular feed. For this purpose, the following genotypes were generated and studied: 1. PACAP+/+ / PAC1+/+ / ApoE-/--mice (short as "ApoE-/-"; control); 2. PACAP-/- / ApoE-/--mice; 3. PAC1-/- / ApoE-/--mice. All mice received regular feed for 30 weeks. Blood samples were taken from the animals for the measurement of triglyceride and cholesterol concentrations. The other parameters mentioned above were mainly examined by immunohistomorphometry. The body-weights of the PACAP-/PAC1-deficient mice were similar to those of the ApoE-/--mice. Plasma cholesterol- and triglyceride levels of PACAP-/PAC1-deficient mice were similar to those of ApoE-/- mice. In the gastrocnemius muscle, we found a significant increase in fiber density of 28.5% (p=0.042) with a concomitant decrease in fiber cross-sectional area of 22.9% (p=0.043) in PAC1-/- / ApoE-/- mice compared to ApoE-/-. In the soleus muscle of PAC1-/- / ApoE-/--mice fiber density was 19% (p=0.045) higher than in ApoE-/- mice. The fiber cross-sectional areas of all fibertypes were significantly decreased 15% and 17,2% in PACAP-/- / ApoE-/- as well as PAC1-/- / ApoE-/-- mice compared to ApoE-/- mice. The fiber cross-sectional areas of each fiber type were significantly decreased except for those of type I fibers. In addition, the soleus muscle of PACAP-/- / ApoE-/--mice showed a shift in fiber composition with 5% decrease in type I fibers and 5.9% increase in IIxa fibers compared 134 to ApoE-/--mice. The density of MuRF-1+ cells was significantly higher by 30.7% (p=0.018) in soleus muscle of PAC1-/- / ApoE-/--mice than ApoE-/--mice. Neuromuscular junctions, which are important for innervation, were significantly reduced by 33.3% in soleus muscle of PACAP-/- / ApoE-/- as well as PAC1-/- / ApoE-/-- mice. Our data show that in PAC1-/- / ApoE-/--mice, the density of COX-2+ cells in the soleus muscle was decreased by 18.3% (p=0.030) compared to ApoE-/- mice. The number of COX-2+ cells per fiber was also decreased by 33.2% in the soleus muscle respectively by 38% in the gastrocnemius muscle compared to ApoE-/- mice. IL-1β+ cells were significantly reduced in density and number/fiber in both muscle types and in PACAP- and PAC1-deficient mice compared with ApoE-/--mice. Our data show that in PACAP-/- / ApoE-/--mice, the density of IL-1ß+ cells was lower in soleus and gastrocnemius muscle by 24.6% (p=0.047) respectively by 19.6% (p=0.050) compared to ApoE-/-. The number of IL-1ß+ cells per fiber were also significantly reduced in both muscle types compared to ApoE-/- mice. We found a 30.4% (p=0.045) higher Capillary density in soleus muscles of PACAP-/- / ApoE-/--mice compared to ApoE-/--mice. Furthermore, we found significantly higher concentration of proteinogenic amino acids methionine, leucine, tyrosine and phenylalanine relative to total protein (nmol/mg protein) in the gastrocnemius muscle of PACAP-/- / ApoE-/-- and PAC1-/- / ApoE-/--mice. No significant changes in intracellular gluthation redox status were observed under PACAP or PAC1 deficiency compared to ApoE-/- mice. Our results show for the first time that PACAP-/PAC1-deficiency leads to a decrease in fiber cross-sectional area with a concomitant increase in fiber density, suggesting possible atrophy with accompanying hyperplasia. We observed markedly increased MuRF-1 expression in the soleus muscle in PAC1-/- / ApoE-/--mice, this could be a cause of the atrophic processes. Moreover, the decrease in innervation induced by PACAP or PAC1 deficiency could be responsible for the decrease in fiber cross-sectional area/increase in fiber density. Decreased blood flow as another possible cause of atrophy in soleus muscle could not be demonstrated in our studies, because PACAP/PAC1 deficiency rather showed an increase in capillary 135 density. Surprisingly, no concomitant increased pro-inflammatory environment was observed in skeletal muscle compared to cardiac muscle (Gohlke, 2017). The atrophic processes were accompanied by a reduced density of inflammatory parameters (COX-2; Il-1β). The increased concentrations of proteinogenic amino acids in the gastrocnemius muscle indicate increased proteolysis processes, which could be related to the reduced fiber cross-sectional area in PAC1-/- / ApoE-/--mice. In this case, the time of investigation in soleus muscle might have been too early, so that no change was observed yet. In summary, our assumption at the beginning of this work that PACAP-/PAC1 deficiency in skeletal muscle results in pathological changes in terms of atrophy, capillarization and inflammation is confirmed. From our data, we suggest that in skeletal muscle PACAP seems to have rather protective properties in terms of atrophy, innervation, and proteolysis within cells; here, it would be exciting to perform further studies with PACAP and PAC1 agonists. It is possible that these may be suitable for the treatment and regeneration of nervous muscle atrophies. The hyperplasia observed under PACAP deficiency suggests that PACAP and PAC1 agonists may also play a not insignificant role in neoplasia research.