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Acute myocardial hypoxia increases BNP gene expression

J. P. GOETZE^*, A. GORE^*, C. H. MØLLER, D. A. STEINBRÜCHEL, J. F. REHFELD^* and L. B. NIELSEN^*,1

Departments of
^* Clinical Biochemistry and
Thoracic Surgery, Rigshospitalet, University of Copenhagen, Copenhagen, Denmark

¹Correspondence: Department of Clinical Biochemistry, KB 3011 Rigshospitalet, University of Copenhagen, 9 Blegdamsvej, Copenhagen 2100, Denmark. E-mail: larsbo{at}rh.dk

SPECIFIC AIMS

It is well established that congestive heart failure increases cardiac BNP expression due to myocardial stretching. Accordingly, increased plasma concentrations of BNP and its precursor, proBNP, are markers of left ventricular systolic dysfunction. However, patients with ischemic heart disease also display increased plasma BNP and proBNP concentrations, despite preserved cardiac function. In this study, we explored whether myocardial hypoxia per se may increase cardiac BNP expression. Cardiac BNP mRNA and peptide expression were examined in pigs with a reduced blood flow to a restricted area of the left ventricle.

PRINCIPAL FINDINGS

1. Acute myocardial hypoxia increases the BNP mRNA expression
Surgical reduction of the blood flow to an area of the anterior ventricular wall in pigs reduced the myocardial oxygen tension from 46 ± 4 to 13 ± 5 mmHg. The mRNA content of the hypoxia-inducible VEGF gene increased 1.8-fold in hypoxic compared with normoxic ventricular myocardium after 2.2 ± 0.2 h (n=10, P<0.001). This finding corroborated the hypoxic effect on the cardiac gene expression. The tissue content of BNP mRNA increased 3.5-fold (Fig. 1 A) in hypoxic compared with normoxic ventricular myocardium; the magnitude of the increase in BNP mRNA expression in hypoxic myocardium was positively associated with that of VEGF (r=0.66, P<0.05). In support of increased BNP gene transcription, the content of a premature BNP mRNA was increased 4.8-fold in hypoxic compared with normoxic myocardium (Fig. 1B ). To examine whether acute hypoxia also increases myocyte BNP gene transcription in the absence of mechanical stimulation, freshly harvested ventricular cells were incubated under oxygen-deprived conditions. The content of premature BNP mRNA increased 2.2-fold after 3 h of oxygen-deprivation (n=8, P=0.002).

View larger version (16K): [in this window] [in a new window]   Figure 1. Effect of acute myocardial hypoxia on BNP gene transcription. A) Mature BNP mRNA content expressed as percent of the mean values in the normoxic region (ischemic pigs, n=10) or inferior ventricular myocardium (control pigs, n=4). B) Content of BNP pre-mRNA expressed as percent of the mean values in the normoxic (ischemic pigs) or inferior ventricular myocardium (control pigs). Horizontal lines indicate mean values and each point represents data obtained from individual pigs. All results are normalized by a housekeeping gene in the same sample.

2. Lack of storage of BNP and proBNP peptide in hypoxic ventricular myocardium
To assess whether the increased BNP gene expression in hypoxic myocardium lead to accumulation of BNP or proBNP peptide in ventricular myocardium, we analyzed BNP and proBNP by radioimmunoassays and immunohistochemistry. However, neither BNP nor proBNP was detectable in hypoxic or normoxic ventricular myocardium (Fig. 2 A, B). In contrast, atrial myocytes contained detectable amounts of BNP and proBNP peptides with a markedly lower BNP than proBNP concentration (Fig. 2C) . Confocal laser scanning microscopy of immunostained atrial sections showed a granular-like appearance of proBNP in the myocytes extending along the contractile apparatus (Fig. 2D ). Gel chromatography of medium from freshly harvested atrial cells revealed 2 forms of proBNP-derived peptides (i.e., the intact precursor and a smaller N-terminal fragment) (Fig. 2E ).

View larger version (40K): [in this window] [in a new window]   Figure 2. Myocardial BNP and proBNP peptide contents. A, C) BNP and proBNP contents as determined with radioimmunoassays in ventricular and atrial myocardium, respectively. B) Lack of proBNP contents in both normoxic (upper picture) and hypoxic (lower picture) ventricular myocytes by confocal microscopy. The corresponding differential contrast (Normasky) image of the basic morphology of the section is shown to the right of each frame. D) proBNP immunostaining in atrial myocytes. E) Elution of proBNP-derived peptides as determined with an antiserum directed toward the N terminus of proBNP (two peaks) and BNP immunoreactivity (one peak) after gel filtration chromatography of culture medium from atrial cells.

3. Newly synthesized proBNP peptide is rapidly released by hypoxic ventricular myocytes
The absence of BNP and proBNP peptides in the ventricular myocardium may reflect rapid cellular release from the myocytes. To examine this possibility, freshly harvested ventricular cells were kept for 3 h in serum-free culture medium. Although the cells did not contain detectable amounts of BNP or proBNP peptides before or after incubation, proBNP was released into the culture medium (n=4, P<0.0001). Accordingly, the plasma proBNP concentration increased after 2 h of myocardial hypoxia in the experimental pigs (P=0.028).

CONCLUSIONS AND SIGNIFICANCE

The present study revealed that acute myocardial hypoxia results in increased cardiac BNP gene transcription. As a result of rapid release of proBNP peptides from the ventricular myocytes, this may confer a rise in the plasma proBNP concentrations.

It is well-established that cardiac secretion of BNP and proBNP is increased in congestive heart failure. The main stimulus for the increased BNP expression is ventricular dilation and concomitant myocyte stretching. The diagnostic specificity of increased plasma BNP and proBNP concentrations is nevertheless remarkably low in heart failure. This implies that other stimuli may be involved in regulating cardiac BNP gene expression. Plasma BNP and proBNP concentrations are also increased in patients with acute coronary syndromes and myocardial infarction. The increased plasma concentration precedes and even predicts later development of heart failure. We recently reported that ventricular BNP gene expression is associated with increased plasma BNP and proBNP concentrations in stable ischemic heart disease patients without ventricular dysfunction. The underlying mechanism, however, has not been resolved. We therefore examined the effect of acute myocardial hypoxia on cardiac BNP expression in a new porcine model with reduced blood supply to an area of the left ventricular myocardium. This approach allowed us to study the BNP gene and peptide expression in both normoxic and hypoxic ventricular myocardium in the same pig, thus minimizing possible biases from anesthesia or neurohormonal activation during surgery.

A principal finding of the study was a robust increase in the BNP mRNA contents in the left ventricular myocardium after only 2 h of hypoxia. In contrast, the BNP and proBNP peptide products were undetectable in normal and hypoxic ventricular biopsies. To seek evidence of a constitutive peptide release from the cardiac ventricle, we kept freshly harvested ventricular myocytes in a serum-free medium. While the ventricular cells were devoid of peptides, we found an accumulation of proBNP peptide in the medium. This strongly suggests that newly synthesized proBNP peptides are rapidly released from ventricular myocytes. In contrast, atrial myocytes stored proBNP in granulae-like organelles. Only proBNP (and not BNP) peptide was detectable in medium from cultured ventricular cells, indicating that proBNP may a more sensitive marker of rapid changes in ventricular BNP gene expression than BNP.

The present data suggest that the effect of acute myocardial hypoxia on BNP gene expression may be detectable by measuring proBNP concentrations in plasma. We found a significant rise of the plasma proBNP concentration in pigs with left ventricular hypoxia. Of note, though we cannot exclude that the rise in plasma proBNP partly reflects stretching of myocytes rather than hypoxia per se, we did not see an increase of the plasma proBNP concentration in control pigs. Although the rise in plasma proBNP concentrations was relatively small, it was achieved after only 2 h of hypoxia in a small area of the left ventricular wall. In patients with chronic myocardial hypoxia due to extensive coronary atherosclerosis, plasma BNP and proBNP concentrations are elevated to an extent that is similar to that in patients with severe systolic dysfunction. This may imply that prolonged oxygen-deprivation, despite preserved viability of the myocardium and despite lack of detectable cardiac systolic dysfunction, increases the plasma BNP and proBNP concentrations. BNP is a potent vasodilator in the coronary vasculature. The hypoxia-induced increase in BNP gene expression, therefore, makes teleological sense if BNP functions in a paracrine mechanism to increase the local blood supply to the hypoxic area. Thus, even modest increases of BNP gene expression may have important local functions.

The results provide an explanation for the increased plasma proBNP concentrations in patients with acute coronary syndromes. This could partially explain why the plasma proBNP concentration is prognostic of cardiac death, even in patients without cardiac dysfunction.

View larger version (34K): [in this window] [in a new window]   Figure 3. Hypothetical diagram of the established (heart failure with ventricular dysfunction) and proposed (myocardial hypoxia) regulatory pathways for increased cardiac BNP expression.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-1336fje;

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