Atrial natriuretic factor (ANF) is a cardiac hormone that helps maintain cardiovascular homeostasis. ANF secretion is linked to the constitutive, regulated and constitutive-like pathways. Presence of a monensin-sensitive pool that may follow constitutive-like secretion has previously been identified in an isolated atrial perfusion study. The intracellular ANF storage pools linked to each secretory pathway have not been identified. In this study, ANF storage and secretion was characterized in HL-1 atrial cardiomyocytes through the use of pharmacological agents, density gradient and RP- HPLC analysis. Treatment of HL-1 cells with monensin followed by cell fractionation was unsuccessful in identifying the monensin-sensitive pool. RP-HPLC analysis identified presence of low molecular weight ANF in low density gradient fractions that were defined by the presence of organelle markers of Golgi, early endosome, clathrin and corin. Since the monensin-sensitive pool was thought to be of a constitutive-like nature, targeting this pathway with pharmacological inhibitors of clathrin coat vesicle (CCV) formation and endosomal trafficking failed to prevent stimuli-independent secretion. Based on an inability to prevent ANF secretion by targeting the constitutive-like pathway and the presence of low molecular weight ANF in low density gradient fractions, stimuli- independent ANF secretion seems to be through a constitutive pathway.

The phosphoinositide 3-kinases (PI3K/Akt) dependent signaling pathway plays an important role in cardiac function, specifically cardiac contractility. We have reported that sepsis decreases myocardial Akt activation, which correlates with cardiac dysfunction in sepsis. We also reported that preventing sepsis induced changes in myocardial Akt activation ameliorates cardiovascular dysfunction. In this study we investigated the role of PI3K/Akt on cardiomyocyte function by examining the role of PI3K/Akt-dependent signaling on [Ca 2+]i, Ca2+ transients and membrane Ca2+ current, ICa, in cultured murine HL-1 cardiomyocytes. LY294002 (120 μM), a specific PI3K inhibitor, dramatically decreased HL-1 [Ca 2+]i, Ca2+ transients and ICa. We also examined the effect of PI3K isoform specific inhibitors, i.e. α (PI3-kinase α inhibitor 2; 28 nM); ? (TGX-221; 100 nM) and γ (AS-252424; 100 nM), to determine the contribution of specific isoforms to HL-1 [Ca 2+]i regulation. Pharmacologic inhibition of each of the individual PI3K isoforms significantly decreased [Ca2+]i, and inhibited Ca 2+ transients. Triciribine (120 μM), which inhibits AKT downstream of the PI3K pathway, also inhibited [Ca2+]i, and Ca 2+ transients and ICa. We conclude that the PI3K/Akt pathway is required for normal maintenance of [Ca2+]i in HL-1 cardiomyocytes. Thus, myocardial PI3K/Akt-PKB signaling sustains [Ca 2+]i required for excitation-contraction coupling in cardiomyoctyes.

Swelling-activated Cl- current (ICl,swell) is an outwardly rectifying Cl- current that influences cardiac electric activities and acts as a potential effector of mechanoelectrical feedback that antagonizes the effects of stretch-activated cation channels. Persistent activation of ICl,swell has been observed in multiple models of cardiovascular diseases. Previously we showed that angiotensin II (AngII) signaling and reactive oxygen species (ROS) produced by NADPH oxidase (NOX) are involved in the activation of ICl, swell by both beta1-integrin stretch and osmotic swelling. Because endothelin-1 (ET-1) is a potential downstream mediator of AngII and ETA receptor blockade abrogates AngII-induced ROS generation, we studied how ET-1 signaling regulates ICl,swell and the relationship between AngII and ET-1 signaling. Under isosmotic conditions, ET-1 elicited an outwardly rectifying Cl- current that was fully blocked by the highly selective ICl,swell inhibitor DCPIB and by osmotic shrinkage. Selective ETA blockade (BQ123), but not ETB blockade (BQ788), fully suppressed the ET-1-induced current. ET-1-induced ICl,swell was abolished by blockade of EGFR kinase (AG1478) and PI-3K inhibitors (LY294002 and wortmannin), which also suppress beta1-integrin stretch- and swelling-induced ICl,swell. ET-1-induced ICl,swell was abrogated by ebselen, a membrane-permeant glutathione peroxidase mimetic that dismutates H2O2 to H2O, suggesting that ROS were required intermediates in ET-1-induced activation of ICl,swell. Both NOX and mitochondria are important sources of ROS in cardiomyocytes. Blocking NOX with apocynin or mitochondrial complex I with rotenone both completely suppressed ET-1-induced ROS generation and activation of ICl,swell, indicating that ROS from both NOX and mitochondria were required to activate ICl,swell, and complete block by inhibitors of either ROS source suggests mitochondrial and NOX must act in series rather than in parallel. ICl,swell elicited by antimycin A, which stimulates superoxide production by mitochondrial complex III, was insensitive to NOX inhibitor apocynin and the NOX fusion peptide inhibitor gp91ds-tat. Activation of ICl,swell induced by diazoxide, which stimulates mitochondrial ROS production by opening mitochondrial KATP channels, was not affected by gp91ds-tat. These data suggests that mitochondrial ROS is downstream from NOX in the regulation of ICl,swell. Mitochondrial ROS production that is enhanced by NOX ROS is likely to be responsible for the activation of ICl,swell by ET-1. In order to determine the role of ERK in the proposed signaling pathway that regulates ICl,swell, we examined the effect of ERK inhibitors (PD 98059 and U0216) on the activation of ICl,swell elicited by ET-1, EGF, and H2O2. ERK inhibitors partially blocked ET-1-induced ICl,swell but fully inhibited activation of ICl,swell in response to EGF. However, ERK inhibitors did not affect ICl,swell elicited by exogenous H2O2. We also established the the relationship of ET-1 to AngII and osmotic swelling in the regulation of ET-1 ICl,swell. ETA blockade abolished ICl,swell elicited by both AngII and osmotic swelling, whereas AT1 blockade did not effect ET-1-induced ICl,swell, suggesting that ET-1 signaling is downstream from AngII and osmotic swelling. HL-1 cell is a murine atrial cell line that retain phenotypic characteristics of adult cardiomyocytes. We showed that osmotic swelling and ET-1 turned on DCPIB-sensitive outwardly rectifying Cl- current in HL-1 cells with both physiological and symmetrical Cl- gradients. The swelling-induced current was suppressed by gp91ds-tat and rotenone but insensitive to apocynin. Blockade of ETA receptor (BQ123) and NOX (gp91ds-tat) completely inhibited ET-1-induced ICl,swell in HL-1 cells. These data indicate that ICl,swell is present in HL-1 cell and regulated by similar mechanisms as in native cells. Finally, we confirmed the production of ROS by ET-1 signaling by flow cytometry of HL-1 cells using the nominally H2O2-selective fluorescent probe C H2DCFDA-AM. Exposure to ET-1 increased ROS production, as did H2O2, a positive control. ET-1-induced ROS production was fully suppressed by both gp91ds-tat and rotenone. HL-1 cell ROS production also was stimulated by the mitochondrial complex III inhibitor antimycin A, and antimycin A-induced ROS production was blocked by rotenone but not by gp91ds-tat. These data suggest that ET-1 ETA receptor signaling elicits ICl,swell by sequentially stimulating ROS production by NOX and mitochondria. ETA receptor signaling is down stream from AngII in the osmotic swelling-induced activation of ICl,swell and is upstream from EGFR kinase and PI-3K. Endothelin signaling is likely to be an important means of activating ROS production and ICl,swell in a variety of cardiovascular diseases.

The endocrine heart synthesises and secretes two polypeptide hormones: the natriuretic peptides (NP) atrial natriuretic factor (ANF) and B-type natriuretic peptide (BNP). The biological actions of these hormones serve both acutely and chronically to reduce systemic blood pressure and hemodynamic load to the heart, thus contributing to the maintenance of cardiorenal homeostasis. Considerable effort has been focused on the elucidation of the mechanistic underlying ANF and BNP gene expression and secretion but much remains to be determined regarding specific molecular events involved in the cardiocyte secretory function. These hormones are produced by the atrial muscle cells (cardiocytes), which display a dual secretory/muscle phenotype. In contrast, ventricular cardiocytes display mainly a muscle phenotype. Comparatively little information is available regarding the genetic background for this important phenotypic difference with particular reference to the endocrine function of the heart. We postulated that comparison of gene expression profiles between atrial and ventricular muscles would help identify transcripts that underlie the phenotypic differences associated with the endocrine function of the heart as well as identify signaling pathways involved in its regulation. The cardiac atrial and ventricular transcriptomes were analyzed using oligonucleotide microarrays under normal or chronically induced aortocaval shunt volume-overload conditions. Transcriptional differences were validated by RT-PCR and transcripts of interest were knocked-down by RNAi. Comparison of gene expression profiles in the rat heart revealed a total of 1415 differentially expressed genes between normal atrial and ventricular tissues. Functional classification and pathway analysis identified numerous transcripts involved in mechanosensing, vesicle trafficking, hormone secretion, and G protein signaling. Volume-overloaded animals exhibited a progressive increase in cardiac mass over the four-week time course, an increase in expression of known hypertrophic genes, as well as the differential expression of 700 genes within the atria. Volume-overload specifically downregulated the accessory protein for heterotrimeric G protein signaling RASD1 in the atria. In vitro, knockdown of RASD1 in the atrial-derived HL-1 cells, significantly increased ANF secretion, demonstrating a previously unknown negative modulator role for RASD1. The data developed in this investigation provides insight into the expression profiles of genes particularly centered on the secretory function of the heart under normal and chronic hemodynamic overload conditions. Genome-wide expression profile analysis identified RASD1 as being differentially expressed between cardiac tissues as well as being modulated by chronic volume overload. RASD1 emerges as a tonic inhibitor of ANF secretion. The novel function identified herein for RASD1 in the atria is of considerable interest given the fact that secretory impairment of the cardiac natriuretic hormones can negatively impact cardiovascular homeostasis.

The endocrine heart synthesises and secretes two polypeptide hormones: the natriuretic peptides (NP) atrial natriuretic factor (ANF) and B-type natriuretic peptide (BNP). The biological actions of these hormones serve both acutely and chronically to reduce systemic blood pressure and hemodynamic load to the heart, thus contributing to the maintenance of cardiorenal homeostasis. Considerable effort has been focused on the elucidation of the mechanistic underlying ANF and BNP gene expression and secretion but much remains to be determined regarding specific molecular events involved in the cardiocyte secretory function. These hormones are produced by the atrial muscle cells (cardiocytes), which display a dual secretory/muscle phenotype. In contrast, ventricular cardiocytes display mainly a muscle phenotype. Comparatively little information is available regarding the genetic background for this important phenotypic difference with particular reference to the endocrine function of the heart. We postulated that comparison of gene expression profiles between atrial and ventricular muscles would help identify transcripts that underlie the phenotypic differences associated with the endocrine function of the heart as well as identify signaling pathways involved in its regulation. The cardiac atrial and ventricular transcriptomes were analyzed using oligonucleotide microarrays under normal or chronically induced aortocaval shunt volume-overload conditions. Transcriptional differences were validated by RT-PCR and transcripts of interest were knocked-down by RNAi. Comparison of gene expression profiles in the rat heart revealed a total of 1415 differentially expressed genes between normal atrial and ventricular tissues. Functional classification and pathway analysis identified numerous transcripts involved in mechanosensing, vesicle trafficking, hormone secretion, and G protein signaling. Volume-overloaded animals exhibited a progressive increase in cardiac mass over the four-week time course, an increase in expression of known hypertrophic genes, as well as the differential expression of 700 genes within the atria. Volume-overload specifically downregulated the accessory protein for heterotrimeric G protein signaling RASD1 in the atria. In vitro, knockdown of RASD1 in the atrial-derived HL-1 cells, significantly increased ANF secretion, demonstrating a previously unknown negative modulator role for RASD1. The data developed in this investigation provides insight into the expression profiles of genes particularly centered on the secretory function of the heart under normal and chronic hemodynamic overload conditions. Genome-wide expression profile analysis identified RASD1 as being differentially expressed between cardiac tissues as well as being modulated by chronic volume overload. RASD1 emerges as a tonic inhibitor of ANF secretion. The novel function identified herein for RASD1 in the atria is of considerable interest given the fact that secretory impairment of the cardiac natriuretic hormones can negatively impact cardiovascular homeostasis.

The endocrine heart synthesises and secretes two polypeptide hormones: the natriuretic peptides (NP) atrial natriuretic factor (ANF) and B-type natriuretic peptide (BNP). The biological actions of these hormones serve both acutely and chronically to reduce systemic blood pressure and hemodynamic load to the heart, thus contributing to the maintenance of cardiorenal homeostasis. Considerable effort has been focused on the elucidation of the mechanistic underlying ANF and BNP gene expression and secretion but much remains to be determined regarding specific molecular events involved in the cardiocyte secretory function. These hormones are produced by the atrial muscle cells (cardiocytes), which display a dual secretory/muscle phenotype. In contrast, ventricular cardiocytes display mainly a muscle phenotype. Comparatively little information is available regarding the genetic background for this important phenotypic difference with particular reference to the endocrine function of the heart. We postulated that comparison of gene expression profiles between atrial and ventricular muscles would help identify transcripts that underlie the phenotypic differences associated with the endocrine function of the heart as well as identify signaling pathways involved in its regulation. The cardiac atrial and ventricular transcriptomes were analyzed using oligonucleotide microarrays under normal or chronically induced aortocaval shunt volume-overload conditions. Transcriptional differences were validated by RT-PCR and transcripts of interest were knocked-down by RNAi. Comparison of gene expression profiles in the rat heart revealed a total of 1415 differentially expressed genes between normal atrial and ventricular tissues. Functional classification and pathway analysis identified numerous transcripts involved in mechanosensing, vesicle trafficking, hormone secretion, and G protein signaling. Volume-overloaded animals exhibited a progressive increase in cardiac mass over the four-week time course, an increase in expression of known hypertrophic genes, as well as the differential expression of 700 genes within the atria. Volume-overload specifically downregulated the accessory protein for heterotrimeric G protein signaling RASD1 in the atria. In vitro, knockdown of RASD1 in the atrial-derived HL-1 cells, significantly increased ANF secretion, demonstrating a previously unknown negative modulator role for RASD1. The data developed in this investigation provides insight into the expression profiles of genes particularly centered on the secretory function of the heart under normal and chronic hemodynamic overload conditions. Genome-wide expression profile analysis identified RASD1 as being differentially expressed between cardiac tissues as well as being modulated by chronic volume overload. RASD1 emerges as a tonic inhibitor of ANF secretion. The novel function identified herein for RASD1 in the atria is of considerable interest given the fact that secretory impairment of the cardiac natriuretic hormones can negatively impact cardiovascular homeostasis.

The endocrine heart synthesises and secretes two polypeptide hormones: the natriuretic peptides (NP) atrial natriuretic factor (ANF) and B-type natriuretic peptide (BNP). The biological actions of these hormones serve both acutely and chronically to reduce systemic blood pressure and hemodynamic load to the heart, thus contributing to the maintenance of cardiorenal homeostasis. Considerable effort has been focused on the elucidation of the mechanistic underlying ANF and BNP gene expression and secretion but much remains to be determined regarding specific molecular events involved in the cardiocyte secretory function. These hormones are produced by the atrial muscle cells (cardiocytes), which display a dual secretory/muscle phenotype. In contrast, ventricular cardiocytes display mainly a muscle phenotype. Comparatively little information is available regarding the genetic background for this important phenotypic difference with particular reference to the endocrine function of the heart. We postulated that comparison of gene expression profiles between atrial and ventricular muscles would help identify transcripts that underlie the phenotypic differences associated with the endocrine function of the heart as well as identify signaling pathways involved in its regulation. The cardiac atrial and ventricular transcriptomes were analyzed using oligonucleotide microarrays under normal or chronically induced aortocaval shunt volume-overload conditions. Transcriptional differences were validated by RT-PCR and transcripts of interest were knocked-down by RNAi. Comparison of gene expression profiles in the rat heart revealed a total of 1415 differentially expressed genes between normal atrial and ventricular tissues. Functional classification and pathway analysis identified numerous transcripts involved in mechanosensing, vesicle trafficking, hormone secretion, and G protein signaling. Volume-overloaded animals exhibited a progressive increase in cardiac mass over the four-week time course, an increase in expression of known hypertrophic genes, as well as the differential expression of 700 genes within the atria. Volume-overload specifically downregulated the accessory protein for heterotrimeric G protein signaling RASD1 in the atria. In vitro, knockdown of RASD1 in the atrial-derived HL-1 cells, significantly increased ANF secretion, demonstrating a previously unknown negative modulator role for RASD1. The data developed in this investigation provides insight into the expression profiles of genes particularly centered on the secretory function of the heart under normal and chronic hemodynamic overload conditions. Genome-wide expression profile analysis identified RASD1 as being differentially expressed between cardiac tissues as well as being modulated by chronic volume overload. RASD1 emerges as a tonic inhibitor of ANF secretion. The novel function identified herein for RASD1 in the atria is of considerable interest given the fact that secretory impairment of the cardiac natriuretic hormones can negatively impact cardiovascular homeostasis.

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