pPKCα mediated-HIF-1α activation related to the morphological modifications occurring in neonatal myocardial tissue in response to severe and mild hyperoxia

Abstract

In premature babies birth an high oxygen level exposure can occur and newborn hyperoxia exposure can be associated with free radical oxygen release with impairment of myocardial function, while in adult animal models short exposure to hyperoxia seems to protect heart against ischemic injury. Thus, the mechanisms and consequences which take place after hyperoxia exposure are different and related to animals age. The aim of our work has been to analyze the role played by HIF-1α in the occurrence of the morphological modifications upon hyperoxia exposure in neonatal rat heart. Hyperoxia exposure induces connective compartment increase which seems to allow enhanced blood vessels growth. An increased hypoxia inducible factor-1α (HIF-1α) translocation and vascular endothelial growth factor (VEGF) expression has been found upon 95% oxygen exposure to induce morphological modifications. Upstream pPKC-α expression increase in newborn rats exposed to 95% oxygen can suggest PKC involvement in HIF-1α activation. Since nitric oxide synthase (NOS) are involved in heart vascular regulation, endothelial NOS (e-NOS) and inducible NOS (i-NOS) expression has been investigated: a lower eNOS and an higher iNOS expression has been found in newborn rats exposed to 95% oxygen related to the evidence that hyperoxia provokes a systemic vasoconstriction and to the iNOS pro-apoptotic action, respectively. The occurrence of apoptotic events, evaluated by TUNEL and Bax expression analyses, seems more evident in sample exposed to severe hyperoxia. All in all such results suggest that in newborn rats hyperoxia can trigger oxygen free radical mediated membrane injury through a pPKCα mediated HIF-1α signalling system, even though specificity of such response could be obtained by in vivo administration to the rats of specific inhibitors of PKCα. This intracellular signalling can switch molecular events leading to blood vessels development in parallel to pro-apoptotic events due to an immature anti-oxidant defensive system in newborn rat hearts.

Figure 1

Hematoxylin-eosin staining of neonatal rat heart; A) ambient air; B) 60% hyperoxia; C) 95% hyperoxia; D) ambient air 6 weeks; E) 60% hyperoxia and ambient air 4 weeks. Scale bar 50 µm.

Figure 2

Morphometric analysis of neonatal rat heart performed on hematoxylin-eosin stained slides; a) ambient air; b) 60% hyperoxia; c) 95% hyperoxia; d) ambient air 6 weeks; e) 60% hyperoxia and ambient air 4 weeks. A) Fiber diameter measurements expressed as mean values (±SD) assessed by direct visual measurement of ten muscle fibers for each of three slides per each of 5 samples at 40× magnification; *ambient air 6 weeks vs ambient air P<0.05; *60% hyperoxia and ambient air 4 weeks vs ambient air P<0.05. B) Blood vessels, connective and muscle compartment measurements expressed as % area mean (±SD) assessed by direct visual counting of ten fields for each of three slides per each of 5 samples; *60% hyperoxia connective compartment % area vs ambient air connective compartment % area P<0.05; *95% hyperoxia connective compartment vs ambient air connective compartment % area P<0.05; ** 95% hyperoxia blood vessels % area vs ambient air blood vessels % area P<0.05. C) Western blotting analysis of collagen III and α-actin expression; each membrane has been probed with anti β-tubulin antibody to verify loading evenness. The most representative out of three separate experiments is shown. Data are the densitometric measurements of protein bands expressed as Integrated Optical Intensity (IOI) mean (±SD) of three separate experiments; **60% hyperoxia collagen III vs ambient air collagen III P<0.05; **95% hyperoxia collagen III vs ambient air collagen III P<0.05; **60% hyperoxia collagen III vs 95% hyperoxia collagen III; ***95% hyperoxia α-actin vs ambient air α-actin P<0.05.

Figure 3

A) Immunohistochemical detection of HIF-1α expression in neonatal rat heart; a) ambient air; b) 60% hyperoxia; c) 95% hyperoxia; d) ambient air 6 weeks; e) 60% hyperoxia and ambient air 4 weeks; c(−) negative control; arrows indicate HIF-1α positive nuclei; scale bar 50 µm; inset shows HIF-1α nuclear staining. B) Graphic representation of HIF-1α positive nuclei % (±SD) densitometric analysis determined by direct visual counting of ten fields (mean values) for each of three slides per sample at 40× magnification;*95% hyperoxia vs ambient air P<0.05.

Figure 4

A) Immunohistochemical detection of VEGF expression in neonatal rat heart; a) ambient air; b) 60% hyperoxia; c) 95% hyperoxia; d) ambient air 6 weeks; e) 60% hyperoxia and ambient air 4 weeks; c(−) negative control; scale bar 50 µm. B) Graphic representation of VEGF % positive area (± SD) densitometric analysis determined by direct visual counting of ten fields (mean values) for each of three slides per sample at 40× magnification; *60% hyperoxia vs ambient air P<0.05; *95% hyperoxia vs ambient air P<0.05; *95% hyperoxia vs 60% hyperoxia P<0.05. C) Western blotting analysis of VEGF expression; each membrane has been probed with anti β-tubulin antibody to verify loading evenness. The most representative out of three separate experiments is shown. Data are the densitometric measurements of protein bands expressed as Integrated Optical Intensity (IOI) mean (±SD) of three separate experiments; **95% hyperoxia vs ambient air P<0.05; **95% hyperoxia vs 60% hyperoxia P<0.05.

Figure 5

Western blotting analysis of PKCα and p-PKCα expression in neonatal rat heart; a) ambient air; b) 60% hyperoxia; c) 95% hyperoxia; d) ambient air 6 weeks; e) 60% hyperoxia and ambient air 4 weeks. Each membrane has been probed with anti β-tubulin antibody to verify loading evenness. The most representative out of three separate experiments is shown. Data are the densitometric measurements of protein bands expressed as Integrated Optical Intensity (IOI) mean (± SD) of three separate experiments; *95% hyperoxia PKCα vs ambient air PKCα P<0.05; *60% hyperoxia PKCα vs ambient air PKCα P<0.05; **95% hyperoxia p-PKCα vs ambient air p-PKCα P<0.05.

Figure 6

Western blotting analysis of e-NOS and i-NOS expression in neonatal rat hearts; a) ambient air; b) 60% hyperoxia; c) 95% hyperoxia; d) ambient air 6 weeks; e) 60% hyperoxia and ambient air 4 weeks. Each membrane has been probed with anti β-tubulin antibody to verify loading evenness. The most representative out of three separate experiments is shown. Data are the densitometric measurements of protein bands expressed as Integrated Optical Intensity (IOI) mean (±SD) of three separate experiments; *95% hyperoxia e-NOS vs ambient air e-NOS P<0.05; **95% hyperoxia i-NOS vs ambient air i-NOS P<0.05.

Figure 7

A) TUNEL detection of apoptotic nuclei in neonatal rat hearts; a) ambient air; b) 60% hyperoxia; c) 95% hyperoxia; d) ambient air 6 weeks; e) 60% hyperoxia and ambient air 4 weeks; c(−) negative control. Arrows indicate TUNEL positive nuclei (brown), negative nuclei are blue; scale bar 50 µm; B) Graphic representation of TUNEL positive nuclei % (±SD) densitometric analysis determined by direct visual counting of ten fields (mean values) for each of three slides per sample; *95% hyperoxia vs ambient air P<0.05.

Figure 8

A) Immunohistochemical detection of Bax positive cells in different experimental points; magnification 40×; a) ambient air; b) 60% hyperoxia; c) 95% hyperoxia; d) ambient air 6 weeks; e) 60% hyperoxia and ambient air 4 weeks; c(−) negative control; scale bar 50 µm; B) Graphic representation of Bax positive cells % (±SD) determined by direct visual counting of ten fields (mean values) for each of three slides per sample; *95% hyperoxia vs ambient air P<0.05.