Rows 1 to 8 in Table 1 show selected discoveries related to the ability of the heart atrial muscle cells of mammals to synthesize, store within specific atrial granules, and release cardiac natriuretic peptides (cNPs). The mechanism of action of cNPs is summarized in Box 1. These findings by the research team of Dr. de Bold were the first direct demonstration that the heart has an endocrine function (summarized in Table 1, rows 1 to 8). In Table 2, we summarize selected observations describing how the cNP system influences hypertrophic growth, fibrosis, and cardiac remodeling/ dysfunction. For a summary of the influences of cNPs on adipose tissue biology and metabolism, please see Table 3.
Molecules investigated; Research question; Authors Author's main results and conclusions ANP; What are the acute renal effects of the extract of rat atrial myocardium? (de Bold AJ et al., 1981) The atrial extract (ⅰ) decreases blood pressure and slightly increases hematocrits; (ⅱ) rapidly increases the concentration and urinary excretion of sodium and chloride (≥30-fold), the urine volume (~10-fold), the potassium excretion (two-fold); (ⅲ) contains a potent natriuretic and chloriuretic factor, which strongly inhibits the renal tubular NaCI reabsorption. ANP; Can extracts from other sources induce a natriuretic and diuretic response? (de Bold AJ and Salerno TA, 1983) Natriuresis and diuresis is induced by atrial extracts from all mammalian species, frog atrial and ventricular extracts, hen ventricle extracts (only diuresis), and not by hen atrial extracts or rat tissue extracts other than the atrial extract; (ⅱ) Natriuretic activity is restricted to heart. ANP; What are the molecules responsible for these activities? (de Bold AJ and Flynn TG, 1983) Cardionatrin I, which also has effect on vascular smooth muscle tone, has a molecular mass of 5.1 kDa by urea-SDS-PAGE and 49 amino acid residues one of which is cysteine. ANP; What is the common precursor of Cardionatrin I and other atrial peptides? (Flynn TG et al., 1985) (ⅰ) Cardionatrin IV, consisting of 126 amino acids, has a molecular mass of 19 kDa by urea-SDS-PAGE, and begins immediately after the signal peptide sequence of procardionatrin at residue 25. It does not contain residues 151 and 152, which are arginines; (ⅱ) Cardionatrin III begins at residue 73 and Cardionatrin I begins at residue 123; (ⅲ) Cardionatrins I, III, many cleavage fragments thereof and numerous versions of the carboxyl terminal portion of Cardionatrin I are products derived from a common precursor, Cardionatrin IV; (ⅳ) Cardionatrins I-IV peptides are derived from preprocardionatrin, a common precursor of 152 amino acids (in the rat); (ⅴ) The biologically active sequences of the atrial natriuretic factor is contained in the COOH terminal portion of the molecule. BNP; Identification in porcine brain of a novel natriuretic peptide (Sudoh T et al., 1988) BNP contains 26 amino acid residues, two Cys residues, seven amino acid substitutions and one addition of (Arg) compared to α-ANP; (ⅱ) BNP possesses diuretic-natriuretic (e.g., increase in urine output, Na+, K+, Cl– excretion) and hypotensive (decrease in mean blood pressure) responses similar to that of ANP; (ⅲ) There may be a dual mechanism involving both ANP and BNP to control physiological functions such as water intake and salt appetite. BNP; What is the intracellular localization of BNP in human cardiac myocytes? (Nakamura S et al., 1991) BNP is specifically localized in only some of the secretory granules of the human atrium and ventricle that contain ANP, as shown with different patients (with aortic regurgitation, mitral regurgitation or autoptic); (ⅱ) The atrium is the major production site of BNP; (ⅲ) Together, ANP and BNP allows the human heart to regulate blood pressure and body fluid. ANP and BNP; Natriuretic peptides circulate in blood (Clerico et al., 2011 and citations therein) (ⅰ) Posttranslational processing of proBNP is required for secretion and bioactivity- this process is impaired in patients with heart failure leading to biologically inactive BNP; (ⅱ) proBNP-derived fragments (e.g., the intact and glycosylated forms of precursor proBNP, NH2-terminal-truncated BNP form 3-32) circulate in human plasma in addition to bioactive BNP1-32; (ⅲ) In plasma of patients with heart failure, a significant portion of immunoreactive B-type related peptides is comprised of intact or glycosylated forms of proBNP this suggests that plasma proteases cleave the circulating proBNP to produce biologically active BNP; (ⅳ) In experimental models and in patients with chronic heart failure, a resistance to ANP and BNP is observed; possible mechanisms of resistance to biological effects of ANP and BNP may operate at: a) the pre-receptor level (e.g., existence of inactive natriuretic peptides in plasma, increase in inactivation and degradation of active natriuretic peptides, decreased renal filtration), b) receptor level (e.g., downregulation of NPR-A and NPR-B in target tissues, altered ANP/BNP receptor binding or desensitization, or c) post-receptor level (e.g., altered intracellular signaling). CNP; Are cardiomyocytes able to produce CNP? (Del Ry S et al., 2011) (ⅰ) Both HUVEC and H9c2 muscle cells express CNP (150 and 200 bp); which can be confirmed in neonatal rat primary cardiomyocytes; (ⅱ) CNP can be immunodetected in both H9c2 cells (by radioimmunologic assay) and cardiomyocytes of pig hearts; (ⅲ) CNP is constitutively expressed in cardiomyocytes. ANP and BNP; Biological factors and pathophysiological mechanisms that stimulate the production /release of natriuretic peptides (Clerico et al., 2011 and citations therein) (ⅰ) The production/release of cNPs is stimulated by: a) Ang II, ET1, α-adrenergic agonists, cytokines such as IL-1, IL-6 and TNF-α, and lipopolysaccharide (all of which signal throughout NF-kB activated by MAPK), b) arginine vasopressin (through Ca2+ influx and PKC), c) GFs (signaling through MAPK cascade), d) prostaglandins (through PLC, IP3, PKC, and MLCK), e) chromogranin B (through NF-kB and IP3/ Ca2+ influx), f) thyroid hormones (through thyroid hormone regulatory element), g) corticosteroids (through glucocorticoid responsive element), and h) estrogens; (ⅱ) The production and release of BNP from ventricular cardiomyocytes is stimulated by inflammation, ventricular hypertrophy, and fibrosis; (ⅲ) Even in isolated and cultured ventricular cells, myocardial ischemia can induce the synthesis/secretion of BNP and its related peptides; (ⅳ) Both ANP and BNP transcription may be activated by the hypoxia-inducible factor-1a (which is induced under low oxygen conditions). Cardiac specific miR-208a and MED13; How does cardiac MED13 influence whole body metabolism? (Grueter CE et al., 2012) (ⅰ) Pharmacologic inhibition of the cardiac-specific miR-208a confers resistance to diet-induced obesity (e.g., smaller visceral WAT and subscapular BAT, normal glucose response, lower fasting insulin levels) with beneficial metabolic effects (e.g., reduced serum triglyceride and cholesterol levels); (ⅱ) miR-208a is a negative transcriptional regulator of MED13 in the heart. Among the functions of MED13 are: (a) to inhibit expression of metabolic genes regulated by NRs (e.g., Gpd2, Thrsp, Cidea, Elovl6, Eno1, PPARγ Tkt), (b) to control whole-body metabolic homeostasis (e.g., αMHC-Med13 TG mice, with increased cardiac expression of MED13, show enhanced metabolic rate, diminished serum triglyceride and cholesterol levels, resistance to diet-induced obesity including less fat mass versus WT littermates, reduced visceral WAT and subscapular BAT mass as well as less adipocyte size and less lipid accumulation, improved glucose (tolerance) response, lowered plasma lipid levels, and improved whole-body insulin sensitivity, and (c) regulate energy expenditure (e.g., increased oxygen consumption, carbon dioxide production) in mice; (ⅲ) MED13 deficiency in the heart increases susceptibility to metabolic syndrome and diet-induced obesity in mice, as shown with Med13 cardiac knockout mice versus Med13fl/fl littermates on HF diet. (ⅳ) Circulating factors may relay MED13 activity from the heart to other organs but these factors remain elusive. Abbreviations: miR-208a: microRNA-208a; MED13: mediator complex subunit 13; ANP: atrial natriuretic peptide; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis; BNP: brain natriuretic peptide; proBNP: ventricular circulating inactive precursor of BNP; CNP: C-type natriuretic peptide; HUVEC: human umbilical vein endothelial cell; NRs: nuclear receptors; TG: transgenic; Gpd2: glycerol-3-phosphate dehydrogenase 2; Thrsp: thyroid hormone responsive; Cidea: cell death-inducing DFFA-like effector A; Elovl6: ELOVL fatty acid elongase 6; Eno1: Enolase 1; PPARγ: peroxisome proliferator activated receptor gamma; Tkt: Transketolase; WT: wild-type; WAT: white adipose tissue; BAT: brown adipose tissue; HF: high fat; Ang II: angiotensin II; ET1: endothelin-1; NF-kB: nuclear factor kappa-light-chain-enhancer of activated B cells; MAPK: p38 mitogen-activated protein kinase; PKC: protein kinase C; GFs: growth factors; MLCK: myosin light chain kinase; PLC: phospholipase C.
Table 1. Discovery of cardiac natriuretic peptides and the heart-specific miR-208a/MED13 axis
Question Selected observations The role of ANP in cardiac hypertrophy and remodeling (Wang et al., 2005) The mechanism by which ANP protects against cardiac hypertrophy induced by pressure overload involves the negative regulation of genes encoding MMP-2 in mice subjected to transverse aortic constriction [e.g., 2-fold increase in Nppa+/+ and 3-fold increase in Nppa-/-] and TIMP-3 as well as other protein factors related to extra cellular matrix deposition (e.g., collagen I/III, osteopontin, periostin, thrombospondin). The role of NPRA/cGMP signaling in regulation of MMPs and other factors (e.g., proinflammatory mediators) (Vellaichamy et al., 2005) (ⅰ) There is a link between Npr1 gene disruption in mice and the expression and activation of matrix metalloproteinases (e.g., MMP-2, MMP-9) and pro-inflammatory cytokines that play critical roles in cardiac hypertrophy, fibrosis, and extra cellular matrix remodeling; (ⅱ) Hearts from Npr1-/-homozygous mice at an early age versus age-matched WT (Npr1+/+) control mouse hearts show strongly activated genes (e.g., MMP-2 and MMP-9 by 3-5-fold, TNF-α by 8-fold) - these genes remain activated in adult mice; (ⅲ) In Npr1-/- mice treated with GM6001, a MMP inhibitor, the activities of MMP-9 and MMP-2 are decreased (between 3 and 5 fold), fibrosis is reduced (75%), ventricular dilatation is attenuated, and fractional shortening is improved. These observations implicate MMPs in myocardial fibrosis and cardiac hypertrophy. The effect of Npr1 gene copy numbers on the expression of cardiac hypertrophic and fibrotic markers, proinflammatory mediators, and MMPs (Subramanian et al., 2016) (ⅰ) Npr1 gene-disrupted heterozygous (Npr1-/-, 1-copy) mice versus WT (Npr1-/-, 2-copy) mice and gene duplicated (Npr1-/-, 3-copy) mice, show an augmented heart weight to body weight ratio, elevated blood pressure, downregulated expression of TIMP-1 (by 36%) and TIMP-2 (by 40%) mRNA transcripts, and increased hypertrophic markers, proto-oncogenes, NF-kB, and MMPs (MMP-2 [e.g., 2.59-fold], MMP-9 [e.g., 1.93-fold]) - this leads to fibrosis and hypertrophic remodeling. (ⅱ) Retinoic acid and butyrate block histone deacetylases and activate histone acetyltransferases to activate Npr1 gene transcription. In Npr1-/- (1-copy) mice treated with all trans retinoic acid or sodium butyrate the expression of NF-kB and MMP-2/MMP-9 is reduced, the expression of TIMP-1 and TIMP-2 is upregulated, fractional shortening is increased, systolic and diastolic parameters of the Npr1-/- hearts are decreased; (ⅲ) In age-matched 2- and 3-copy cardiac tissues, the expression of fibrotic markers is strongly reduced and the expression of TIMPs activated, after drug treatment (with retinoic acid, sodium butyrate or in combination). Abbreviations: Nppa: pro-ANP gene; TIMP: tissue inhibitor of metalloproteinase; Npr1: NPR-A gene.
Table 2. Mechanisms linking the cardiac natriuretic system and heart issues and involving MMPs/cytokines
Question Selected observations The role of the cardiac natriuretic peptide system in adipose tissue biology and metabolism (Sarzani et al., 2008 and citations therein) (ⅰ) NPR-C is the second largest expressed receptor in adipose tissue; (ⅱ) NPR-C expression in adipose tissue is strongly and selectively downregulated by fasting; (ⅲ) Higher clearance and/or diminished activity of cNPs in the subcutaneous adipose tissue of obese hypertensive patients; (ⅳ) Decrease effectiveness of ANP in obese premenopausal women; (ⅴ) Increased effects (cGMP, natriuresis, diuresis, blood pressure) of ANP on obese hypertensive patients after a very-low-calorie diet; (ⅵ) In obese hypertensive subjects, a variant of the promoter of the NPR-C gene is linked to: a) higher blood pressure, b) lower ANP levels, c) augmented abdominal circumference, and d) the risk of developing abdominal obesity in male; (ⅶ) ANP inhibits the proliferation of human visceral differentiated pre-adipocytes and mature adipocytes; (ⅷ) Natriuretic peptides are strong lipolytic hormones, a property that is enhanced by diet and weight loss; (ⅸ) Natriuretic peptides regulate the supply of non-esterified fatty acids to the working muscles (both cardiac and skeletal); (ⅹ) An improved metabolism in the heart and muscles would result from the net effect of natriuretic peptides on adipose and muscle tissues; (ⅹⅰ) The inhibitory effect induced by ANP-induced lipolysis is counteracted by ANP-stimulated secretion of adiponectin; (ⅹⅱ) The secretion of cytokines such as IL-6 and TNF-α and several chemokines as well as adipokines (leptin, retinol-binding protein-4) involved in inflammation and insulin resistance is inhibited by ANP; (ⅹⅲ) The biological activity (local and systemic) of natriuretic peptides is increased by downregulation of NPR-C due to fasting and calorie restriction; (ⅹⅳ) Natriuretic peptides could behave as anti-obesity peptides in the presence of uncoupling activity involving non-esterified fatty acids and mitochondrial burning for ATP synthesis; (ⅹⅴ) Ang II may hamper the positive effects of natriuretic peptides; (ⅹⅵ) In obese subjects with and without hypertension or heart failure, circulating levels of natriuretic peptides and NH2-terminal-proANP and NH2-terminal-proBNP are reduced; (ⅹⅶ) Reduced NH2 terminal-proBNP levels correlate with hepatic steatosis and higher hematocrit values in elderly patients. Pathophysiological links between obesity (visceral fat distribution) and cardiac endocrine function (low plasma BNP levels) (Clerico et al., 2012 and citations therein) (ⅰ) There is an inverse correlation between BMI (e.g., ≥30 kg/m2) and plasma BNP and NH2-terminal-proBNP values, both in healthy subjects and in patients with heart failure; (ⅱ) The increased expression of NPR-C in the adipose tissue may lead to the peripheral clearance of BNP; nevertheless, this increase in NPR-C should have no influence in modulating the NH2- terminal-proBNP levels in obese subjects. (ⅲ) Some alterations of peripheral degradation of ANP and BNP in individuals having the corin I555 (P568) allele; (ⅳ) The production and secretion of BNP may be regulated by the gonadal function including the estrogens/androgens circulating ratio; (ⅴ) The activity of cardiac endocrine function is depressed by the presence of one or more circulating or tissue bioactive substances (e.g., leptin); (ⅵ) An efficacious (standard) pharmacological treatment of obese individuals or patients with hypertension and/or type-2 diabetes mellitus tends to decrease significantly the production/secretion of BNP and NT-proBNP; (ⅶ) The inverse correlation between BMI and BNP values in patients with heart failure (e.g., with hemodynamic impairment, activation of counteracting neurohormone, increased circulating levels of cytokines) may be partially explained by the effect of the malnutrition-inflammation complex syndrome. Effects of natriuretic peptide system in obesity, metabolic syndrome and type 2 diabetes (Moro, 2016 and citations therein) (ⅰ) Natriuretic peptides promotes fat mobilization and utilization as well as regulate energy metabolism and expenditure; (ⅱ) In obesity, metabolic syndrome and type 2 diabetes there is a state of natriuretic peptide system deficiency (natriuretic handicap); (ⅲ) Contributing factors to the natriuretic handicap are the elevated levels of glucose, insulin, fatty acids, proinflammatory cytokines; (ⅳ) The natriuretic handicap occurs despite a higher left-ventricular mass and end-diastolic pressure in people with obesity compared to lean individuals; (ⅴ) Human and mouse adipose tissue express NPR-C; high fat feeding up-regulates NPR-C and reduces NPRA mRNA – as confirmed in studies with mice; (ⅵ) Obese diabetic db/db mice show: a) white fat, brown fat and skeletal muscles containing strongly up-regulated NPR-C protein levels, b) down-regulated NPRA protein levels, and c) a strong decrease of plasma BNP levels; (ⅶ) Mice with diet-induced obesity show reduced NPRA expression (mRNA and protein); (ⅷ) Insulin up-regulates NPR-C expression in adipocytes; (ⅸ) ANP infusion in healthy volunteers increases adiponectin levels; (ⅹ) Natriuretic peptides induce the expression/secretion of adiponectin in cultured human adipocytes; (ⅹⅰ) In healthy Japanese men, serum NH2 terminal-proBNP is inversely associated with metabolic risk and positively with adiponectin; (ⅹⅱ) Systemic insulin sensitivity is enhanced by adiponectin and reduced by IL-6 and TNF-α secretion from adipose tissue; (ⅹⅲ) Humans volunteers show a blood glucose-lowering effect of infused BNP to an oral glucose challenge; (ⅹⅳ) In humans, whole-body insulin sensitivity correlates (positively) with NPR-A expression in adipose tissue and skeletal muscle; (ⅹⅴ) In adipocytes of obese individuals with and without type 2 diabetes, NPR-A is down-regulated and NPR-C is up-regulated (mRNA and protein); (ⅹⅵ) Natriuretic peptides protect human skeletal muscle against palmitate-induced lipotoxicity and insulin resistance; (ⅹⅶ) A robust link between natriuretic peptide signaling and type 2 diabetes is demonstrated by studies with transgenic mice of the natriuretic peptide system. The link between endocrine function, adipose tissue, and sex steroid hormones (Clerico et al., 2011 and citations therein) (ⅰ) Adipokines (e.g., leptin) can differently regulate the production/secretion of ANP and BNP by cardiomyocytes throughout multiple metabolic pathways; these pathways are activated or inhibited depending on different pathophysiological conditions; (ⅱ) A cross talk exits between the cardiac endocrine function and adipose tissue (e.g., ANP and BNP regulate fat tissue function and growth as well as exerting potent lipolytic effects in human fat cells, ANP increases production of adiponectin); (ⅲ) ANP gene expression is increased in a dosedependent manner by female sex steroids; (ⅳ) Estradiol and progesterone are required for normal ANP gene expression by rat cardiomyocytes; (ⅴ) In postmenopausal women, a stimulatory action on the production/secretion of ANP and BNP is induced by hormone replacement therapy with female steroid hormones; (ⅵ) There is debate on the effects of androgens on the production/secretion of ANP and BNP (e.g., in atrial cultured myocytes of newborn rats, the synthesis and secretion of both ventricular and atrial ANP is stimulated by testosterone; whereas in castrated male rats, the concentration of ANP in plasma and ANP atrial stores is increased; testosterone replacement reduces ANP levels in plasma, but not in ANP atrial stores; blocking the androgen receptor and, to a lesser extent, suppressing androgen in men with prostate cancer, increases NH2-terminal-proBNP levels in plasma; other studies [e.g., the Dallas Heart Study] in humans suggest that estrogens may stimulate whereas androgens inhibit the production/secretion of ANP and BNP). Abbreviations: ATP: adenosine 5'-triphosphate; BMI: body mass index.
Table 3. Effects of the cardiac natriuretic peptide system in the cardio-adipose axis