The atrial natriuretic factor promoter is a downstream target for Nkx-2.5 in the myocardium.

The recently described NK2 family of homeodomain proteins are key developmental regulators. In Drosophila melanogaster, two members of this family, bagpipe and tinman, are required for visceral and cardiac mesoderm formation, respectively. In vertebrates, tinman appears to represent a family of closely related NK2 genes, including Nkx-2.5, that are expressed at an early stage in precardiac cells. Consistent with a role for Nkx-2.5 in heart development, inactivation of the Nkx-2.5 gene in mice causes severe cardiac malformations and embryonic lethality. However, little is known about the molecular action of Nkx-2.5 and its targets in cardiac muscle. In this paper, we report the identification and characterization of a functional and highly conserved Nkx-2.5 response element, termed the NKE, in the proximal region of the cardiac atrial natriuretic factor (ANF) promoter. The NKE is composed of two near-consensus NK2 binding sites that are each able to bind purified Nkx-2.5. The NKE is sufficient to confer cardiac cell-specific activity to a minimal TATA-containing promoter and is required for Nkx-2.5 activation of the ANF promoter in heterologous cells. Interestingly, in primary cardiocyte cultures, the NKE contributes to ANF promoter activity in a chamber- and developmental stage-specific manner, suggesting that Nkx-2.5 and/or other related cardiac proteins may play a role in chamber specification. This work provides the identification of a direct target for NK2 homeoproteins in the heart and lays the foundation for further molecular analyses of the role of Nkx-2.5 and other NK2 proteins in cardiac development.     

Effects of cardiac hormones on arterial pressure and sodium excretion in NPRA knockout mice

These studies were designed to determine if the atria contains natriuretic substances that act through a non-natriuretic peptide type A (NPRA) receptor mechanism. C57BL/6 mice, either wild-type NPRA++ (WT) or NPRA-- knockout (KO), were anesthetized with pentobarbital. Catheters were placed in the trachea, carotid artery, jugular vein, and bladder. Urine was collected for six 30-min periods. Both groups received an iv injection of 100 ng of rat atrial natriuretic peptide (rANP) in 200 microl of saline after the first period (30 mins) and 200 microl of rat atrial extract after the fourth period (120 mins). ANP injection increased urine flow (UF) to 2.7 +/- 0.5 microl/min in the WT versus 1.9 +/- 0.2 in KO. Extract increased UF to 7.9 +/- 1.5 microl/min in WT versus 2.7 +/- 0.4 in KO (P < 0.01). ANP increased sodium excretion (ENa) to 0.47 +/- 0.10 micromoles/min in WT versus 0.27 +/- 0.04 in KO (P < 0.05). Extract increased ENa to 1.44 +/- 0.47 micromoles/min in WT versus 0.26 +/- 0.06 in KO (P < 0.05). Extract decreased mean arterial pressure (MAP) to 62 +/- 3 mm Hg in the WT versus 81 +/- 5 in KO (P < 0.01). ENa and MAP responses to extract in KO were not different from responses to 200 microl of saline. A constant 150-min infusion of rat atrial extract increased urine flow by 3-fold and ENa by 5-fold (both P < 0.05) in the WT mice but had no significant effect in the KO mice. Thus, acute renal and MAP responses to atrial extracts require the NPRA receptor.     

Chromogranin A: Immunocytochemical localization in secretory granules of frog atrial cardiomyocytes

Chromogranin A (CgA) belongs to the granin family of acidic proteins that are present in the secretory granules of many endocrine, neuroendocrine, and nerve cells. CgA has been shown to be stored in cardiomyocyte secretory granules of the rat heart atrium together with atrial natriuretic peptide (ANP). CgA-derived peptides (vasostatins) are known to produce a cardiosuppressive effect on isolated and working in vitro frog and rat hearts. Recently, CgA-derived vasostatin-containing peptides have been identified in rat hearts, whereas no data are available so far about the presence of CgA in the frog heart. In our work, we have studied the subcellular CgA localization in atrial myocytes of the adult frog R. temporaria heart by using an ultraimmunocytochemical method. Immunocytochemical staining of the frog atrial tissue for CgA and ANP showed the presence of the CgA-immunoreactive material in two types (A and B) of large specific atrial secretory granules, whereas no gold particles were revealed over the small granules (D) with a high electron density core. Similar results were obtained during the immunocytochemical staining by an antibody to ANP of the drog atrial cardiomyocytes. The data of the present work allow for the suggestion that CgA revealed in frog atrial cardiomyocytes, like CgA in rat cardiomyocytes, can be considered to be a precursor of intracardial vasostatins that, together with ANP, can play an important cardioprotector role under conditions of stress.