Adolfo J. de Bold

Investigations into the mixed muscle–secretory phenotype of cardiomyocytes from the atrial appendages of the heart led to the discovery that these cells produce, in a regulated manner, two polypeptide hormones — the natriuretic peptides — referred to as atrial natriuretic factor or atrial natriuretic peptide (ANP) and brain or B-type natriuretic peptide (BNP), thereby demonstrating an endocrine function for the heart. Studies on the gene encoding ANP (NPPA) initiated the field of modern research into gene regulation in the cardiovascular system. Additionally, ANP and BNP were found to be the natural ligands for cell membrane-bound guanylyl cyclase receptors that mediate the effects of natriuretic peptides through the generation of intracellular cGMP, which interacts with specific enzymes and ion channels. Natriuretic peptides have many physiological actions and participate in numerous pathophysiological processes. Important clinical entities associated with natriuretic peptide research include heart failure, obesity and systemic hypertension. Plasma levels of natriuretic peptides have proven to be powerful diagnostic and prognostic biomarkers of heart disease. Development of pharmacological agents that are based on natriuretic peptides is an area of active research, with vast potential benefits for the treatment of cardiovascular disease.

Techniques developed over the years in our laboratory for the study of tissue expression, storage, and secretion of the cardiac hormones ANF, BNP, and CNP are described below. They have proven highly reliable in our hands when the steps outlined are followed as described. Given the generic nature of the procedures, these should be applicable to other polypeptides.

Since their discovery in 1981, the cardiac natriuretic peptides (cNP) atrial natriuretic peptide (also referred to as atrial natriuretic factor) and brain natriuretic peptide have been well characterised in terms of their renal and cardiovascular actions. In addition, it has been shown that cNP plasma levels are strong predictors of cardiovascular events and mortality in populations with no apparent heart disease as well as in patients with established cardiac pathology. cNP secretion from the heart is increased by humoral and mechanical stimuli. The clinical significance of cNP plasma levels has been shown to differ in obese and non-obese subjects. Recent lines of evidence suggest important metabolic effects of the cNP system, which has been shown to activate lipolysis, enhance lipid oxidation and mitochondrial respiration. Clinically, these properties lead to browning of white adipose tissue and to increased muscular oxidative capacity. In human association studies in patients without heart disease higher cNP concentrations were observed in lean, insulin-sensitive subjects. Highly elevated cNP levels are generally observed in patients with systolic heart failure or high blood pressure, while obese and type-2 diabetics display reduced cNP levels. Together, these observations suggest that the cNP system plays a role in the pathophysiology of metabolic vascular disease. Understanding this role should help define novel principles in the treatment of cardiometabolic disease. Abstract Since their discovery in 1981, the cardiac natriuretic peptides (cNP) atrial natriuretic peptide (also referred to as atrial natriuretic factor) and brain natriuretic peptide have been well characterised in terms of their renal and cardiovascular actions. In addition, it has been shown that cNP plasma levels are strong predictors of cardiovascular events and mortality in populations with no apparent heart disease as well as in patients with established cardiac pathology. cNP secretion from the heart is increased by humoral and mechanical stimuli. The clinical significance of cNP plasma levels has been shown to differ in obese and non-obese subjects. Recent lines of evidence suggest important metabolic effects of the cNP system, which has been shown to activate lipolysis, enhance lipid oxidation and mitochondrial respiration. Clinically, these properties lead to browning of white adipose tissue and to increased muscular oxidative capacity. In human association studies in patients without heart disease higher cNP concentrations were observed in lean, insulin-sensitive subjects. Highly elevated cNP levels are generally observed in patients with systolic heart failure or high blood pressure, while obese and type-2 diabetics display reduced cNP levels. Together, these observations suggest that the cNP system plays a role in the pathophysiology of metabolic vascular disease. Understanding this role should help define novel principles in the treatment of cardiometabolic disease.

Expression of the G protein subunit Goα has been shown to be prominent in the atria of the rat heart and to be significantly associated with atrial natriuretic factor (ANF)-containing atrial-specific secretory granules by immunocytochemistry. In addition, differential expression profile analysis using oligonucleotide arrays has shown that the Goα isoform 1 (Goα1) is 2.3-fold more abundant in the atria than it is in the ventricles. In the present report, we show protein–protein interaction between Goα and ANF by yeast two-hybrid and by immunoprecipitation. A cardiac conditional Goα knockout model developed for the present study showed a 90% decrease in Goα expression and decreased atrial expression and ANF and brain natriuretic peptides (BNP) content. Expression of chromogranin A, a specific atrial granule core constituent, was not affected. Morphometric assessment of atrial tissue showed a very significant decrease in atrial-specific granule density as well as granule core electron density. Atrial electrical activity was not affected. The results obtained are compatible with the suggestion that Goα plays a role in ANF sorting during intracellular vectorial transport and with the presence of a mechanism that preserves the molar relationship between cellular ANF and BNP stores in the face of the decreased production of these hormones.

The concept of the heart as an endocrine organ arises from the observation that the atrial cardiomyocytes in the mammalian heart display a phenotype that is partly that of endocrine cells. Investigations carried out between 1971 and 1983 characterised, by virtue of its natriuretic properties, a polypeptide referred to atrial natriuretic factor (ANF). Another polypeptide isolated from brain in 1988, brain natriuretic peptide (BNP), was subsequently characterised as a second hormone produced by the mammalian heart atria. These peptides were associated with the maintenance of extracellular fluid volume and blood pressure. Later work demonstrated a plethora of other properties for ANF and BNP, now designated cardiac natriuretic peptides (cNPs). In addition to the cNPs, other polypeptide hormones are expressed in the heart that likely act upon the myocardium in a paracrine or autocrine fashion. These include the C-type natriuretic peptide, adrenomedullin, proadrenomedullin N-terminal peptide and endothelin-1. Expression and secretion of ANF and BNP are increased in various cardiovascular pathologies and their levels in blood are used in the diagnosis and prognosis of cardiovascular disease. In addition, therapeutic uses for these peptides or related substances have been found. In all, the discovery of the endocrine heart provided a shift from the classical functional paradigm of the heart that regarded this organ solely as a blood pump to one that regards this organ as self-regulating its workload humorally and that also influences the function of several other organs that control cardiovascular function.

The cardiac hormone atrial natriuretic factor (ANF or ANP) plays significant, well-established roles in a large number of physiological and pathophysiological processes, including water and electrolyte balance, blood pressure regulation, and cardiovascular growth. Understanding the regulation of its production and secretion by atrial cardiomyocytes is incomplete. We have previously established a significant role of G_i/oprotein signaling in modulating ANF secretion as promoted by stretch of the atrial myocardium. In the present study, we investigated the role of G_qprotein signaling and its relationship to G_i/oprotein signaling using pharmacological manipulation of proximal effectors of G_αqin an ex vivo model of spontaneously beating rat atria. Phospholipase C (PLC) and protein kinase C (PKC) inhibitors dramatically increased basal secretion of ANF. Furthermore, although atrial wall stretch is a potent stimulus for secretion, stretch unexpectedly reduced ANF secretion to basal levels under PLC and PKC inhibitory conditions. Inhibition of the inositol triphosphate receptor did not appear to affect basal secretion but dose-dependently blocked stretch-secretion coupling. The results obtained demonstrate that the PLC and PKC signaling cascades play important albeit unexpected roles in the regulation of basal and stimulated ANF secretion and suggest interplay between the G_qand G_i/oprotein signaling pathways.