Atrial Natriuretic Peptide Gene Research Articles

Therapeutic inhibition of the miR-34 family attenuates pathological cardiac remodeling and improves heart function

MicroRNAs are dysregulated in a setting of heart disease and have emerged as promising therapeutic targets. MicroRNA-34 family members (miR-34a, -34b, and -34c) are up-regulated in the heart in response to stress. In this study, we assessed whether inhibition of the miR-34 family using an s.c.-delivered seed-targeting 8-mer locked nucleic acid (LNA)-modified antimiR (LNA-antimiR-34) can provide therapeutic benefit in mice with preexisting pathological cardiac remodeling and dysfunction due to myocardial infarction (MI) or pressure overload via transverse aortic constriction (TAC). An additional cohort of mice subjected to MI was given LNA-antimiR-34a (15-mer) to inhibit miR-34a alone as a comparison for LNA-antimiR-34. LNA-antimiR-34 (8-mer) efficiently silenced all three miR-34 family members in both cardiac stress models and attenuated cardiac remodeling and atrial enlargement. In contrast, inhibition of miR-34a alone with LNA-antimiR-34a (15-mer) provided no benefit in the MI model. In mice subjected to pressure overload, LNA-antimiR-34 improved systolic function and attenuated lung congestion, associated with reduced cardiac fibrosis, increased angiogenesis, increased Akt activity, decreased atrial natriuretic peptide gene expression, and maintenance of sarcoplasmic reticulum Ca(2+) ATPase gene expression. Improved outcome in LNA-antimiR-34-treated MI and TAC mice was accompanied by up-regulation of several direct miR-34 targets, including vascular endothelial growth factors, vinculin, protein O-fucosyltranferase 1, Notch1, and semaphorin 4B. Our results provide evidence that silencing of the entire miR-34 family can protect the heart against pathological cardiac remodeling and improve function. Furthermore, these data underscore the utility of seed-targeting 8-mer LNA-antimiRs in the development of new therapeutic approaches for pharmacologic inhibition of disease-implicated miRNA seed families.

Effects of Ozone and Particulate Matter on Cardiac Mechanics: Role of the Atrial Natriuretic Peptide Gene

A positive association between air pollution exposure and increased human risk of chronic heart disease progression is well established. In the current study, we test two hypotheses: (1) the cardiac compensatory changes in response to air pollution are dependent on its composition and (2) specific cardiac adaptations are regulated by atrial natriuretic peptide (ANP). We address these hypotheses by initially examining the exposure effects of ozone (O(3)) and/or particulate matter (PM) on cardiac function in C57Bl/6J (B6) mice. Subsequently, the results are compared with cardiac functional changes to the same exposures in Nppa (the precursor gene for ANP) knockout (KO) mice. Separate groups of mice underwent 3 consecutive days of the same exposure sequence for 3h each consisting of the following: (1) 6h of filtered air (FAFA), (2) O(3) then FA (O(3)FA), (3) FA then carbon black (FACB), or (4) O(3) then CB. Cardiac function was assessed using a conductance catheter to generate cardiac pressure-volume loops 8-10h following each exposure sequence. As compared with FAFA, each sequence led to a substantial drop (as much as 33%) in stroke volume and cardiac output. However, these losses of cardiac function occurred by different compensatory mechanisms dependent on the pollutant composition. For example, O(3)FA exposure led to reductions in both end-systolic and end-diastolic left ventricular (LV) volumes, whereas FACB exposure led an increase in end-diastolic LV volume. These same cardiac compensatory changes were largely abolished in Nppa KO mice following O(3)FA or FACB exposure. These results suggest that cardiac functional changes in response to air pollution exposure are strongly dependent on the pollutant constituents, especially related to O(3) and/or PM. Furthermore, ANP regulation appears to be crucial to these cardiac compensatory mechanisms induced by air pollution.

Tetrodotoxin attenuates isoproterenol-induced hypertrophy in H9c2 rat cardiac myocytes

Cardiac hypertrophy is often associated with an increased sympathetic drive, and both in vitro and in vivo studies have demonstrated the development of cardiomyocyte hypertrophy in response to either α- or β- adrenergic stimulation. The present study was carried out to determine whether the reversible sodium channel blocker tetrodotoxin (TTX) exerts a direct anti-hypertrophic effect on isoproterenol (ISO)-induced cell hypertrophy and find the underlying mechanism that regulate [Na(+)]( i ). The experiments were performed on cultured H9c2 cells exposed to ISO (10 μM) alone or combined with TTX (1 μM) for 48 h. Our results showed that ISO significantly increased cell surface area by 30 % and atrial natriuretic peptide gene expression by nearly twofold (p < 0.05 for both). These effects were associated with a significant reduction in the gene expression of Na(+)/K(+)-ATPase isoforms α2 and α3, whereas the α1 isoform was unaffected. Conversely, ISO increased Na(+)-H(+) exchanger 1 (NHE-1) gene expression by approximately 40 % and significantly increased [Na(+)]( i ) level by 50 % (p < 0.05 for both). ISO was also found to significantly increase aquaporin 4 gene expression by nearly ninefold (p < 0.05). All these effects were prevented when identical experiments were carried out in the presence of TTX, but the expression of NHE-1. The expression of sodium channel protein type 5 subunit alpha was unaffected by either ISO or TTX. When taken together, these studies show that TTX attenuates the hypertrophic effect of ISO and suggest a possible approach to limiting ISO-induced hypertrophy in clinical treatment.

Cardiac adaptive mechanisms of Tibetan antelope (Pantholops hodgsonii) at high altitudes

To identify cardiac mechanisms that contribute to adaptation to high altitudes in Tibetan antelope (Pantholops hodgsonii). 9 male Tibetan antelope and 10 male Tibetan sheep (Ovis aries). Tibetan antelope and Tibetan sheep inhabiting a region with an altitude of 4,300 m were captured, and several cardiac variables were measured. Expression of genes for atrial natriuretic peptide, brain natriuretic peptide, and calcium-calmodulin-dependent protein kinase II δ was measured via real-time PCR assay. Ratios of heart weight to body weight for Tibetan antelope were significantly greater than those of Tibetan sheep, but ratios of right-left ventricular weights were similar. Mean ± SD baseline heart rate (26.33 ± 6.15 beats/min) and systolic arterial blood pressure (97.75 ± 9.56 mm Hg) of antelope were significantly lower than those of sheep (34.20 ± 6.57 beats/min and 130.06 ± 17.79 mm Hg, respectively). The maximum rate of rise in ventricular pressure in antelope was similar to that in Tibetan sheep, but after exposure to air providing a fraction of inspired oxygen of 14.6% or 12.5% (ie, hypoxic conditions), the maximum rate of rise in ventricular pressure of the antelope increased significantly to 145.1% or 148.1%, respectively, whereas that of the sheep decreased to 68.4% or 70.5%, respectively. Gene expression of calcium-calmodulin-dependent protein kinase II δ and atrial natriuretic peptide, but not brain natriuretic peptide, in the left ventricle of the heart was significantly higher in antelope than in sheep. Hearts of the Tibetan antelope in this study were well adapted to high-altitude hypoxia as shown by higher heart weight ratios, cardiac contractility in hypoxic conditions, and expression of key genes regulating cardiac contractility and cardiac hypertrophy, compared with values for Tibetan sheep.

Influence of rs5065 Atrial Natriuretic Peptide Gene Variant on Coronary Artery Disease

The aim of this study was to investigate the impact of rs5065 atrial natriuretic peptide (ANP) gene variant on coronary artery disease (CAD) and its outcomes and to gain potential mechanistic insights on the association with CAD. Either modified ANP plasma levels or peptide structural alterations have been involved in development of cardiovascular events. Three hundred ninety-three control subjects and 1,004 patients undergoing coronary angiography for suspected CAD (432 stable angina [SA], 572 acute coronary syndrome [ACS]) were genotyped for rs5065 ANP gene variant. Data in SA and ACS groups were replicated in an independent population of 482 stable angina patients (rSA) and of 675 ACS patients, respectively. Clinical follow-up was available for both SA and rSA patients. Plasma N-terminal-proANP, myeloperoxidase, lipoprotein-associated phospholipase A2, and oxidized low-density lipoprotein were assessed in a subgroup of rSA patients. rs5065 minor allele (MA) was an independent predictor of ACS (odds ratio: 1.90; 95% confidence interval: 1.40 to 2.58, p < 0.001). At follow-up, rs5065 MA was independently associated with a significantly higher rate of major adverse cardiovascular events in the SA group, p < 0.001. Data were replicated in the rSA group at follow-up (p = 0.008). Cox proportional hazard analysis tested by 4 models confirmed higher major adverse cardiovascular events risk in rs5065 MA carriers in both SA and rSA cohorts. Significantly higher myeloperoxidase levels were detected in rs5065 MA carriers (n = 597 [345 to 832 μg/l] vs. n = 488 [353 to 612 μg/l], p = 0.038). No association of rs5065 was observed with N-terminal-proANP levels. The MA of rs5065 ANP gene variant associates with increased susceptibility to ACS and has unfavorable prognostic value in CAD.

Association of 2238T&gt;C Polymorphism of the Atrial Natriuretic Peptide Gene With Coronary Artery Disease in Afro-Caribbeans With Type 2 Diabetes

The atrial natriuretic peptide (ANP) is known mainly for its effects on kidney function and blood pressure homeostasis. We investigated the association between two ANP polymorphisms and pre-existing coronary artery disease (CAD) in patients of African descent with type 2 diabetes (T2D). We conducted a cross-sectional and retrospective study of 218 volunteer Afro-Caribbean patients with T2D. Two polymorphisms (rs5064, 708C>T; and rs5065, 2238T>C) of ANP were genotyped using PCR-restriction fragment length polymorphism analysis. ANCOVA, χ2-test, and logistic regression were used for statistical analysis. Among these patients (92 men; 128 women), 67 (30.7%) had CAD, of whom 75% had had myocardial infarction. The frequency of rs5065-C carriers (TC/CC) was significantly lower in patients with CAD than in those without CAD (24 vs. 41%, P = 0.01). The frequency of hypertension did not differ significantly according to genotype. Univariate logistic regression revealed that male sex, age, dyslipidemia, hypertension, and rs5065-C carrier status were associated significantly with CAD. After adjustment for the variables of interest, the odds ratio (ORs) of CAD for rs5065-C carriers (TC/CC) was 0.50 (0.26-0.96; P = 0.038). No association was found between the rs5064 (708C>T) single-nucleotide polymorphisms (SNPs) and pre-existing CAD or cardiovascular risk factors. The ANP rs5065 (2238T>C) C allele seems to exert a protective effect against CAD in T2D patients of African descent. The relevance of ANP polymorphisms for CAD should be determined in different populations.

Diurnal Plasma Concentrations of Natriuretic Propeptides in Healthy Young Males

To the Editor: Natriuretic peptides are a family of structurally related hormones encoded by genes for atrial natriuretic peptide (ANP),1 B-type natriuretic peptide (BNP), and C-type natriuretic peptide (CNP). Although the bioactive peptides are related, their tissue and receptor specificities differ. Natriuretic peptide production can also be assessed by measuring the N-terminal fragments from their prohormones in plasma, because these fragments constitute stable markers in vitro and in vivo. The molecular heterogeneity of the propeptides has proved to be complex, and cardiac biosynthesis involves both endoproteolytic cleavage and posttranslational modification 1. The BNP precursor, for instance, is a glycosylated polypeptide and variably matures to the C-terminal, bioactive BNP hormone. The CNP products encoded by the NPPC 2 (natriuretic peptide C) gene have been difficult to measure accurately because their plasma concentrations are considerably lower than for the ANP and BNP peptides. Diurnal hormone production is a hallmark of many endocrine systems. In mouse models, expression of the genes encoding ANP and BNP has been shown to be regulated in a circadian manner by clock genes. We recently reported that BNP mRNA contents, but not ANP mRNA contents, are produced in a circadian manner in the ventricles of wild-type mice and that the production pattern reflects the local expression of the central clock genes Per1 [period homolog 1 …

Angiotensin-converting enzyme 2 regulates renal atrial natriuretic peptide through angiotensin-(1–7)

Deficiency of ACE2 (angiotensin-converting enzyme 2), which degrades Ang (angiotensin) II, promotes the development of glomerular lesions. However, the mechanisms explaining why the reduction in ACE2 is associated with the development of glomerular lesions have still to be fully clarified. We hypothesized that ACE2 may regulate the renoprotective actions of ANP (atrial natriuretic peptide). The aim of the present study was to investigate the effect of ACE2 deficiency on the renal production of ANP. We evaluated molecular and structural abnormalities, as well as the expression of ANP in the kidneys of ACE2-deficient mice and C57BL/6 mice. We also exposed renal tubular cells to AngII and Ang-(1-7) in the presence and absence of inhibitors and agonists of RAS (renin-angiotensin system) signalling. ACE2 deficiency resulted in increased oxidative stress, as well as pro-inflammatory and profibrotic changes. This was associated with a down-regulation of the gene and protein expression on the renal production of ANP. Consistent with a role for the ACE2 pathway in modulating ANP, exposing cells to either Ang-(1-7) or ACE2 or the Mas receptor agonist up-regulated ANP gene expression. This work demonstrates that ACE2 regulates renal ANP via the generation of Ang-(1-7). This is a new mechanism whereby ACE2 counterbalances the renal effects of AngII and which explains why targeting ACE2 may be a promising strategy against kidney diseases, including diabetic nephropathy.

Patent Highlights

Patent Highlights

Cardiomyocyte-Specific Deletion of the Vitamin D Receptor Gene Results in Cardiac Hypertrophy

A variety of studies carried out using either human subjects or laboratory animals suggest that vitamin D and its analogues possess important beneficial activity in the cardiovascular system. Using Cre-Lox technology we have selectively deleted the vitamin D receptor (VDR) gene in the cardiac myocyte in an effort to better understand the role of vitamin D in regulating myocyte structure and function. Targeted deletion of the exon 4 coding sequence in the VDR gene resulted in an increase in myocyte size and left ventricular weight/body weight versus controls both at baseline and following a 7-day infusion of isoproterenol. There was no increase in interstitial fibrosis. These knockout mice demonstrated a reduction in end-diastolic and end-systolic volume by echocardiography, activation of the fetal gene program (ie, increased atrial natriuretic peptide and alpha skeletal actin gene expression), and increased expression of modulatory calcineurin inhibitory protein 1 (MCIP1), a direct downstream target of calcineurin/nuclear factor of activated T cell signaling. Treatment of neonatal cardiomyocytes with 1,25-dihydroxyvitamin D partially reduced isoproterenol-induced MCIP1 mRNA and protein levels and MCIP1 gene promoter activity. Collectively, these studies demonstrate that the vitamin D-VDR signaling system possesses direct, antihypertrophic activity in the heart. This appears to involve, at least in part, suppression of the prohypertrophic calcineurin/NFAT/MCIP1 pathway. These studies identify a potential mechanism to account for the reported beneficial effects of vitamin D in the cardiovascular system.

In Vitro and In Vivo Actions of a Novel ANP Genetic Variant: Unique Actions on Human Endothelial Cells

In Vitro and In Vivo Actions of a Novel ANP Genetic Variant: Unique Actions on Human Endothelial Cells

A Genetic Variant of the Atrial Natriuretic Peptide Gene Is Associated With Cardiometabolic Protection in the General Community

We sought to define the cardiometabolic phenotype associated with rs5068, a genetic variant of the atrial natriuretic peptide (ANP) gene. The ANP and B-type natriuretic peptide play an important role in cardiorenal homeostasis but also exert metabolic actions. We genotyped 1,608 randomly selected residents from Olmsted County, Minnesota. Subjects were well-characterized. Genotype frequencies were: AA 89.9%, AG 9.7%, and GG 0.4%; all subsequent analyses were AA versus AG+GG. The G allele was associated with increased plasma levels of N-terminal pro-atrial natriuretic peptide (p = 0.002), after adjustment for age and sex. The minor allele was also associated with lower body mass index (BMI) (p = 0.006), prevalence of obesity (p = 0.002), waist circumference (p = 0.021), lower levels of C-reactive protein (p = 0.027), and higher values of high-density lipoprotein cholesterol (p = 0.019). The AG+GG group had a lower systolic blood pressure (p = 0.011) and lower prevalence of myocardial infarction (p = 0.042). The minor allele was associated with a lower prevalence of metabolic syndrome (p = 0.025). The associations between the G allele and high-density lipoprotein cholesterol, C-reactive protein values, myocardial infarction, and metabolic syndrome were not significant, after adjusting for BMI; the associations with systolic blood pressure, BMI, obesity, and waist circumference remained significant even after adjusting for N-terminal pro-atrial natriuretic peptide. In a random sample of the general U.S. population, the minor allele of rs5068 is associated with a favorable cardiometabolic profile. These findings suggest that rs5068 or genetic loci in linkage disequilibrium might affect susceptibility for cardiometabolic diseases and support the possible protective role of natriuretic peptides by their favorable effects on metabolic function. Replication studies are needed to confirm our findings.

Evolutionary implication of the absence of atrial natriuretic peptide (ANP) in euryhaline Oryzias fishes

The genus Oryzias contains nearly 20 species, including the Japanese medaka (Oryzias latipes). Because each species exhibits different adaptability to environmental salinity, Oryzias fishes offer unique opportunities for comparative studies. To understand the mechanisms of osmotic adaptation, we are studying the functional evolution of the natriuretic peptide (NP) family—a group of small peptide hormones involved in body fluid regulation—by using Oryzias fishes. Analysis of the Japanese medaka genome revealed that 7 NP subtypes, namely, Atrial NP (ANP), B-type NP (BNP), Ventricular NP (VNP), and 4 C-type NPs (CNP-1 through CNP-4) were generated from a CNP-4-like ancestral gene discovered in the cyclostomes before the ray-finned fish/lobe-finned fish divergence. This evolutionary history has been confirmed by the discovery of hidden NP genes in tetrapods. Through analyses of phylogenetic distribution of NP subtypes, we also found that specific losses of subtypes have occurred in each vertebrate lineage. For example, ANP is absent in the Japanese and Indian medaka and the flying fish, suggesting that loss of the ANP gene occurred after the divergence of Beloniformes from Cyprinodontiformes. This fact also supports the inclusion of Oryzias into Beloniformes as suggested by phylogenetic analysis using whole mitochondrial genome sequences. How Oryzias fishes have retained their euryhalinity with a reduced number of NPs is an interesting question. CNP-3, which is functionally flexible, may be a substitute for the lost cardiac NPs.

Current biochemistry, molecular biology, and clinical relevance of natriuretic peptides

The mammalian natriuretic peptide family consists of atrial (ANP), brain [B-type; BNP] and C-type natriuretic peptide (CNP) and three receptors, natriuretic receptors-A (NPR-A), -B (NPR-B) and -C (NPR-C). Both ANP and BNP are abundantly expressed in the heart and are secreted mainly from the atria and ventricles, respectively. By contrast, CNP is mainly expressed in the central nervous system, bone and vasculature. Plasma concentrations of both ANP and BNP are elevated in patients with cardiovascular disease, though the magnitude of the increase in BNP is usually greater than the increase in ANP. This makes BNP is a clinically useful diagnostic marker for several pathophysiological conditions, including heart failure, ventricular remodeling and pulmonary hypertension, among others. Recent studies have shown that in addition to BNP-32, proBNP-108 also circulates in human plasma and that levels of both forms are increased in heart failure. Furthermore, proBNP-108 is O-glycosylated and circulates at higher levels in patients with severe heart failure. In this review we discuss recent progress in our understanding of the biochemistry, molecular biology and clinical relevance of the natriuretic peptide system.

Hydrogen Peroxide Induces Cell Cycle Arrest in Cardiomyoblast H9c2 Cells, Which Is Related to Hypertrophy

Cell cycle arrest is associated with differentiation, senescence and apoptosis. We investigated alterations in the cell cycle during the development of hypertrophy induced by hydrogen peroxide (H(2)O(2)) in the H9c2 clonal myoblastic cell line. H(2)O(2) induced hypertrophy in H9c2 cells that was indicated by an increase in atrial natriuretic peptide (ANP) gene expression, a marker of cardiomyocyte hypertrophy, and a larger cell size. On induction of hypertrophy by H(2)O(2) in H9c2 cells, cell proliferation was arrested, indicated by the number of cells remaining constant during a 72-h incubation period. The cell cycle was arrested at the G1 and G2/M phases with an increase in p21 expression, a negative cell cycle regulator. Cell cycle arrest and increase in p21 expression were significantly inhibited by 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra (acetoxymethyl) ester (BAPTA-AM), an intracellular calcium chelator. Although ANP gene expression was induced significantly, H(2)O(2) failed to induce hypertrophy in the presence of BAPTA-AM, and the cell cycle progressed. We concluded that H(2)O(2) induced cell cycle arrest in H9c2 cells, which was related to cellular hypertrophy.

RE1-silencing transcription factor (REST) and REST-interacting LIM domain protein (RILP) affect P19CL6 differentiation

During cardiac development, the heart produces the atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP). These peptides are found in high levels in cardiomyocytes and, like a number of other embryonic genes, are up-regulated in both failing and hypertrophied ventricles. At the transcriptional level, BNP and ANP genes are regulated through RE1 regulatory element, which binds RE1-silencing transcription factor (REST). REST/NRSF-interacting LIM domain protein (RILP) is required for the nuclear targeting and function of REST. In this study, the role of RILP and REST in cardiomyocyte development using a model system was studied by analyzing the expression of RILP and REST as well as several cardiac-specific genes during P19CL6 cell differentiation. Effects of RILP overexpression and transcriptional regulation of RILP in differentiating P19CL6 cells were also studied. RILP expression is transiently reduced during P19CL6 cell differentiation; however, REST expression remains unchanged. This transient reduction in RILP expression correlates with de-repression of sarcomeric myosin heavy chain, a marker for cardiomyocyte differentiation. Reporter gene analysis shows that RILP gene is down-regulated through 5'-regulatory elements before cardiac-specific gene expression. These results suggest that RILP expression and function control REST action more so than does REST expression and is an important regulatory role in cardiomyocyte differentiation.

Metoprolol Treatment Lowers Thrombospondin‐4 Expression in Rats with Myocardial Infarction and Left Ventricular Hypertrophy

Thrombospondins are matrix proteins linked to extracellular matrix remodelling but their precise role in the heart is not known. In this study, we characterised left ventricular thrombospondin-1 and -4 expression in rats treated with a beta-blocker metoprolol during the remodelling process in response to pressure overload and acute myocardial infarction. Left ventricular thrombospondin-1 and thrombospondin-4 mRNA levels increased 8.4-fold (p < 0.001) and 7.3-fold (p < 0.001) post-infarction, respectively. Metoprolol infusion by osmotic minipumps (1.5 mg/kg/hr) for 2 weeks after myocardial infarction decreased thrombospondin-1 and thrombospondin-4 mRNA levels (55% and 50%, respectively), improved left ventricular function, and attenuated left ventricular remodelling with reduction of left ventricular atrial natriuretic peptide and brain natriuretic peptide gene expression. Thrombospondin-1 and -4 mRNA levels correlated positively with echocardiographic parameters of left ventricular remodelling as well as with atrial natriuretic peptide and brain natriuretic peptide gene expression. Moreover, there was a negative correlation between left ventricular ejection fraction and thrombospondin-1 mRNA levels. In 12-month-old spontaneously hypertensive rats with left ventricular hypertrophy, metoprolol decreased left ventricular thrombospondin-4 levels and attenuated remodelling while thrombospondin-1, atrial natriuretic peptide and brain natriuretic peptide mRNA levels as well as left ventricular function remained unchanged. In metoprolol-treated spontaneously hypertensive rats, thrombospondin-4 gene expression correlated with parameters of left ventricular remodelling, while no correlations between thrombospondins and natriuretic peptides were observed. These results indicate that thrombospondin-1 expression is linked exclusively to left ventricular remodelling process post-infarction while thrombospondin-4 associates with myocardial remodelling both after myocardial infarction and in hypertensive heart disease suggesting that thrombospondins may have unique roles in extracellular matrix remodelling process.

Vessel dilator and C-type natriuretic peptide enhance the proliferation of human osteoblasts

C-natriuretic peptide (CNP) has been shown to regulate proliferation of mouse and rat osteoblasts. Genetic deletion of CNP results in dwarfism. Overexposure of CNP has been associated with arachnodactyly of hands and feet with a very long hallux bilaterally in a 14-y-old girl. CNP effects on bone growth involve inhibition of MEK 1 and ERK 1/2 kinases mediated via the intracellular messenger cGMP. Vessel dilator is another natriuretic peptide synthesized by the atrial natriuretic peptide gene whose biologic half-life is 12 times longer than CNP. Vessel dilator's biologic effects on proliferating cells are mediated via inhibiting MEK 1/2 and ERK 1/2 kinases via cGMP. Vessel dilator has never been studied on osteoblasts. CNP at 10 (nanomolar) nM (p = 0.02) and vessel dilator at 10 nM, 1 nM, 100 (picomolar) pM, and 10 pM (p ≤ 0.01) in dose-response studies enhanced human osteoblasts' proliferation. This first study of human osteoblasts would suggest that vessel dilator with a much longer biologic half-life and with osteoblast-stimulatory effects at lower concentrations than CNP may have therapeutic potential in human achondroplasia, short stature, and osteoporosis. Vessel dilator stimulates osteoblast proliferation whereas most current therapies of osteoporosis target osteoclasts.

The effects of intra-cage aspen tube on cardiac morphology and gene expression

According to the European recommendations rodents should be provided with a nest box if there is insufficient nesting material to build a complete, covered nest. Rats are generally poor nest builders; hence an additional structure is needed. Optimally, housing refinement may be combined with better science; at least it should not detract from the scientific integrity. In order to evaluate these options, there is a need to assess the items used in individual research projects. Studies investigating molecular mechanisms of cardiac hypertrophy and heart failure are typically long-lasting studies; therefore, refinement of the housing of rats in these studies is important. The aim of this study was to evaluate in rats whether a wooden tube has any impact on cardiac morphology or on basal gene expression of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP); known markers of cardiac overload, hypertrophy and heart failure. The experimental protocol simulated cardiovascular studies, but without any surgical operations. A total of 42 male Hsd:SD rats were used in an eight-week experiment. After weaning, the experimental group was provided with a rectangular aspen tube and nesting material, and the control group with only nesting material. ANP and BNP gene expression were measured from the left ventricles with Northern blot analysis postmortem along with the absolute weights of the whole heart, left and right atria and left and right chambers. The weights of the whole heart and left chamber were also analysed in relation to body weight. No statistically significant differences were observed in any of these variables. The inter-individual variation was also unchanged by the cage item. In conclusion, the aspen tube does not disrupt research results or alter the number of animals needed and can therefore be recommended for enrichment purposes in cardiovascular studies.

Myogenic differentiation in atrium‐derived adult cardiac pluripotent cells and the transcriptional regulation of GATA4 and myogenin on ANP promoter

We established cardiac pluripotent stem-like cells from the left atrium (LA-PCs) of adult rat hearts. These cells could differentiate not only into beating myocytes but also into cells of other lineages, including adipocytes and endothelial cells in the methylcellulose-based medium containing interleukin-3 (IL-3), interleukin-6 (IL-6), and stem cell factor (SCF). In particular, IL-3 and SCF contributed to the differentiation into cardiac troponin I-positive cells. Notably, small population of LA-PCs coexpressed GATA4 and myogenin, which are markers specific to cardiomyocytes and skeletal myocytes, respectively, and could differentiate into both cardiac and skeletal myocytes. Therefore, we investigated the involvement of these two tissue-specific transcription factors in the cardiac transcriptional activity. Coexpression of GATA4 and myogenin synergistically activated GATA4-specific promoter of the atrial natriuretic peptide gene. This combinatorial function was shown to be dependant on the GATA site, but independent of the E-box. The results of chromatin immunoprecipitation and electrophoretic mobility shift assays suggested that myogenin bound to GATA4 on the GATA elements and the C-terminal Zn-finger domain of GATA4 and the N-terminal region of myogenin were required for this synergistic activation of transcription. Taken together, these two transcription factors could be involved in the myogenesis of LA-PCs.