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Nitric oxide production is higher in rat cardiac microvessel endothelial cells than ventricular cardiomyocytes in baseline and hypoxic conditions: a comparative study

Hans Strijdom^*,1, Sean Jacobs^*, Suzél Hattingh^*, Carine Page^* and Amanda Lochner^*,

^* Department of Medical Physiology and Biochemistry, Faculty of Health Sciences, Stellenbosch University, Tygerberg, Republic of South Africa; and
MRC Cape Heart Group, Tygerberg, Republic of South Africa

¹ Correspondence: Dept. of Medical Physiology and Biochemistry, Faculty of Health Sciences, Stellenbosch University, P.O. Box 19063, Tygerberg 7505, Republic of South Africa. E-mail: jgstr{at}sun.ac.za

SPECIFIC AIMS

We measured and compared baseline intracellular production of nitric oxide (NO) and total endothelial nitric oxide synthase (eNOS) content in cardiac microvessel endothelial cells (CMEC) and ventricular cardiomyocytes. In addition, hypoxia as a putative activator of NOS in both cell types and the relative responses of these cell types to hypoxia were investigated in terms of NO production.

PRINCIPAL FINDINGS

1. Baseline NO production and eNOS expression in CMEC and cardiomyocytes
Intracellular NO production was measured by flow cytometric analysis of cells treated with the fluorescent cell-permeable NO-specific probe, diaminofluorescein-diacetate (DAF-2/DA) (Fig. 1 A,B). Probe-specificity was validated by observing a dose-dependent increase in intracellular fluorescence in cardiomyocytes treated with the NO donor, DEA/NO, and an inability of authentic ONOO^– to increase fluorescence. Baseline production of NO was measured in two independent cell models, isolated and cultured. Isolated cell model: CMEC were isolated from culture by prior trypsinization, while cardiomyocytes were used in their fresh isolated state. The suspended cells were subsequently incubated. Results show that 180 min normoxic incubation produced intracellular NO in sufficient amounts in both cell types to be detected by the probe. CMEC produced 26-fold more NO per cell than cardiomyocytes (Fig. 1B ). Baseline expression of eNOS was measured by Western blot analysis of total eNOS content in isolated cells (Fig. 1C ). The eNOS expression was 22-fold higher in CMEC, which corresponded with the NO production in this cell model, suggesting that baseline NO production in both cell types was eNOS-dependent. Cultured cell model: to test whether differences in baseline NO production was cell model-dependent, we measured NO production in CMEC and cardiomyocytes subjected to overnight normoxic culture in fibronectin-plated dishes. Results indicated that the CMEC produced 7-fold more NO than the cardiomyocytes. These findings suggest that the production of NO in greater amounts compared with cardiomyocytes was an intrinsic property of CMEC, but that the degree of baseline production was dependent on the cell model used.

View larger version (23K): [in this window] [in a new window]   Figure 1. Comparative cell-to-cell data in cardiomyocytes and CMEC. A) A flow cytometry histogram depicting baseline DAF-2/DA fluorescence in cardiomyocytes (lower fluorescence intensity) and CMEC (higher fluorescence intensity). B) Combined bar chart demonstrating actual baseline and hypoxia DAF-2/DA fluorescence intensity readings in myocytes and CMEC (isolated cell model, hypoxia induced by mineral oil; see Fig. 2A for protocols used) as measured in the same number of cells per cell type. Baseline fluorescence signals were 26-fold stronger in CMEC than in cardiomyocytes (64.3±3.8 vs. 2.49±0.1, P<0.05), and hypoxia readings 52-fold stronger (185.1±24.1 vs. 3.55±0.3, P<0.05). C) Western blot analysis of total eNOS expression in cardiomyocytes and CMEC incubated for 180 min under normoxic conditions (isolated cell models; see Fig. 2A for protocols). Equal amounts of total protein per cell type (50µg) were loaded and analyzed on the same blot to enable direct comparisons (n=2, samples obtained from different preparations). Upper panel: Western blots depicting total eNOS expression in control cardiomyocytes (lanes 1, 2) and control CMEC (lanes 3, 4). Lower panel: bar chart of total pixels in oxygenated control samples of each cell type as measured by densitometry. Total eNOS protein content was 22-fold higher in CMEC than cardiomyocytes, which corresponds to the 28-fold increase observed in DAF-2/DA fluorescence measurements.

2. Hypoxia-induced NO production in CMEC and cardiomyocytes
The role of hypoxia as a putative NOS activator in CMEC and cardiomyocytes was investigated by subjecting cells to two different hypoxia protocols. Hypoxia induction by mineral oil layering: in this model, samples obtained from CMEC isolated by prior trypsinization or isolated cardiomyocytes were subjected to hypoxia by layering cell suspensions with mineral oil for 120 min. Cell viability tests showed that this model of hypoxia was effective in inducing cell injury (16 and 54% decrease in viable CMEC and cardiomyocytes, respectively), and also confirmed previous reports that endothelial cells are relatively resistant to hypoxic injury. Subsequent FACS analysis of DAF-2/DA fluorescence demonstrated that both cell types increased NO production over baseline (increase in myocytes: 56%, and in CMEC: 231%; Fig. 2 A, C), with CMEC generating 52-fold more intracellular NO per cell than cardiomyocytes (Fig. 1B ). Pretreatment of hypoxic cardiomyocytes with 50 µM L-NAME (non-specific NOS inhibitor) and 100 µM or 1 mM SMT (inducible NOS (iNOS) -specific inhibitor) significantly attenuated hypoxia-induced NO production, suggesting that NOS was activated by hypoxia, with a possible role for iNOS-activation (Fig. 2A ). In CMEC, L-NAME attenuated NO production, but SMT had no effect, which suggests that the increases observed in CMEC were not a result of iNOS-activation (Fig. 2C ). Hypoxia by PO₂ incubation: CMEC in culture and cardiomyocytes in suspension-culture were subjected to 18 h and 2 h hypoxic incubation, respectively. Cell viability decreased by 21% in CMEC, and 26% in cardiomyocytes, suggesting that cardiomyocytes required shorter exposure time to this model of hypoxia than CMEC for a similar degree of cell injury. FACS analysis of DAF-2/DA demonstrated that both cell types increased NO production over baseline (increase in cardiomyocytes: 9%, and CMEC: 41%; Fig. 2B, D ), although not to the same extent observed with the mineral oil technique. These results suggest that the degree of increased NO production was dependent on the hypoxia protocol used.

View larger version (26K): [in this window] [in a new window]   Figure 2. DAF-2/DA fluorescence in cardiomyocytes and CMEC. A) Subjecting cardiomyocytes to 120 min hypoxia induced with mineral oil resulted in enhanced mean DAF-2/DA fluorescence intensity. NOS inhibition with 50 µM L-NAME of 100 µM and 1 mM SMT during hypoxia reversed these effects, indicative of NOS activation by hypoxia in myocytes (*P<0.05 vs. hypoxia). B) Induction of a different model of hypoxia in cardiomyocytes also resulted in enhanced DAF-2/DA fluorescence, albeit not to the same extent than observed in mineral oil- treated cells. In this model of hypoxia, cardiomyocytes were suspended in substrate-free solution D for 120 min under a hypoxic atmosphere (1% O2 concentration; *P<0.05 vs. control). C) Subjecting CMEC isolated by prior trypsinization to 120 min hypoxia induced with mineral oil resulted in enhanced mean DAF-2/DA fluorescence intensity. NOS inhibition with 50 µM L-NAME, but not 100 µM or 1 mM SMT, during hypoxia reversed these effects, indicative of a degree of NOS-activation by hypoxia in CMEC (*P<0.05 vs. hypoxia). D) Induction of a different model of hypoxia in CMEC also resulted in enhanced DAF-2/DA fluorescence, albeit not to the same extent than observed in mineral oil-treated cells. In this model of hypoxia, cultured CMEC were incubated in serum-poor EGM for 18 h under a hypoxic atmosphere (PO2 hypoxia; *P<0.001 vs. control). Hyp, hypoxia; LN, L-NAME.

CONCLUSIONS AND SIGNIFICANCE

Most studies comparing cardiac endothelial cells and cardiomyocytes with regards to their NO production, make use of eNOS-labeling and expression studies; however, studies that directly measure intracellular NO production are lacking. CMEC are important in understanding the actions of NO in the heart, as they are the endothelial cell type most exposed and in closest proximity to cardiomyocytes, which makes the existence of a CMEC-cardiomyocyte NO cross-talk mechanism very likely. However, despite extensive research, the relative importance of CMEC- and myocyte-derived NO remains unclear. Using a direct, intracellular NO detection method, this study showed that CMEC produce more NO per cell than ventricular cardiomyocytes under baseline conditions, using two independent cell models. In a cultured cell model, CMEC produced 7-fold more NO per cell than cardiomyocytes under baseline conditions. The relative production of NO in CMEC compared with myocytes (observed in the isolated cell models) was similar to the eNOS expression in these cell types, suggesting that baseline NO production was due to constitutive eNOS activity.

Studies have shown that NO exerts ambivalent actions on the heart in hypoxia and ischemia-reperfusion, ranging from harmful to protective. We have recently shown a protective role for NO in the ischemic preconditioned whole heart, however this could not be reproduced in an isolated cardiomyocyte model. These examples of contradictory effects may be explained partly by the differences in NO production by the various cell types in the heart, and needs further investigation. We studied the role of hypoxia as a putative activator of NOS in these two cell types. Results showed that hypoxia induced increased NO production in both CMEC and cardiomyocytes and that this was due to NOS activation. iNOS activation seemed to play a role in hypoxic myocytes, but not CMEC. The relative amount of NO produced by CMEC during hypoxia compared with myocytes increased significantly compared with the ratio observed under baseline conditions. It is possible that large amounts of CMEC-derived NO are released, which could result in spillover diffusion into the underlying cardiomyocytes (Fig. 3 ).

View larger version (37K): [in this window] [in a new window]   Figure 3. Schematic diagram summarizing the most important findings of this study and possible implications. A) The proximity of the two NO-producing cell types investigated in this study, CMEC (lining the myocardial capillaries) and the underlying ventricular cardiomyocytes, is shown here. Due to the short diffusion distance between these cell types (<1 µm), it is likely that a NO-cross-talk mechanism exists; however, the relative importance of CMEC- and cardiomyocyte-derived NO remains unknown. B) In the in vitro situation, results of this study show that CMEC produce 7- to 26-fold more NO per cell than cardiomyocytes, which corresponded with the relative eNOS expression observed. C) After exposure to hypoxia, NOS was activated in both cell types resulting in increased NO production over baseline. In the myocytes, this seemed to be partly iNOS driven, but a role for iNOS activation could not be shown in CMEC.

Using a relatively novel methodological approach, we have for the first time quantitated the in vitro cell-to-cell NO production in CMEC and ventricular cardiomyocytes. Baseline eNOS-expression data in CMEC and cardiomyocytes demonstrated a similar ratio than observed with NO production. Hypoxia activated NOS in both cell types, resulting in increased NO production. It is thought that CMEC comprise 33% of the total cell number in the ventricular wall, and the ratio of total cardiac endothelial cells (CMEC+endocardial endothelial cells) to cardiomyocytes is 3:1. CMEC are likely to produce significantly more NO than ventricular cardiomyocytes under baseline conditions (derived mainly from the constitutive activity of eNOS) and after exposure to hypoxia.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-4225fje;

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