It has been reported that opioid receptor activation mimics ischemic preconditioning, which may protect the heart from the development of infarction. Toll-like receptor 4 (TLR-4) during infarction stimulates cytokine production leading to inflammation and injury of the heart tissue. Our aim was to study the effect of morphine in vitro on the viability and oxidative state of H9c2 cells (rat cardiomyoblasts) and the role of TLR-4 during oxidative stress. Our experiments showed that pretreatment with morphine before tert-butylhydroperoxide (t-BHP)-, 2,2'-bipyridyl (BP)- and lipopolysaccharide (LPS)-induced oxidative stess had protective effect on the viability of H9c2 cells and markedly reduced the production of reactive oxygen species (ROS). The protective effect of morphine was diminished after naloxone treatment, which confirms the role of opioid receptors in preconditioning. TLR-4 inhibition by TAK-242 pretreatment and silencing TLR-4 by RNA interference resulted in a partial increase in cell viability but significant attenuation of ROS production after t-BHP and BP treatment. The action of LPS was reduced in response to TLR-4 silencing. Interestingly, naloxone pretreatment and suppression of TLR-4 markedly alleviated oxidative stress and resulted in a significant improvement of cell viability. We...

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.

Práce se zabývá zarovnáváním excitabilních buněk na multielektrodových polích. Nejprve bylo analyzováno zarovnávání excitabilních buněk. Byly použity embryonální neurony z hippocampusu potkanů a HL-1 buňky, které jsou odvozeny z AT-1 linie nádorových myších atriálních kardiomyocytů. Zarovnávání bylo testováno na drážkovaných površích a na površích s materiály s různou buněčnou afinitou. Bylo prokázáno, že na drážkových površích se ve směru drážek zarovnávají neurony i HL-1 buňky, ale na površích s různou chemickou affinitou se zarovnávají pouze neurony. Dále byly vyrobeny vlastní multielektrodová pole, na těchto multielektrodových polích byly kultivovány HL-1 buňky a byl změřen a analyzován akčních potenciál HL-1 buněk. Cílem bylo prokázat, že je možné měřit akční potenciál na vyrobených multielektrodových polích. Pro zarovnání buněk na multielektrodovém poli bylo vyrobeno speciální multieletrodové pole s uniformním povrchem. Toto multielektrodové pole je nazýváno planární multielektrodové pole. Planární multielektrodové pole bylo vyrobeno speciálním vyrobním procesem. Vrstvy planárního multielektrodového pole byly deponovány na pomocný substrát v opačném pořadí. Pomocný substrátem pro depozici byla křemíková deska, na který byla nadeponována další pomocná vrstva zlata. Horní izolační vrstva planárního multielektrodové pole byla deponována jako první a nejspodnější vrstva substrátu byla nadeponována jako poslední. Planární multielektrodové pole i s pomocnou zlatou vrstvou bylo strhnuto s křemíku díky nízké adhezi zlata ke křemíku a planární multielektrodové pole se otočilo vzhůru nohama. Pomocná zlatá vrstva byla odstraněna mokrým leptadlem a tím bylo planární multielektrodové pole dokončeno. Na planárním multielektrodovém poli byly zarovnány HL-1 buňky do pruhů chemickou metodou pomocí kombinace otisku adhezní látky a následným potažení neotisklých ploch anti-adhezní látkou. Elektrofyziologické vlastnosti zarovnaných HL-1 buněk byly změřeny pomocí planárního multielektrodového pole. Tímto experimentem byla představena výrobní technologie pro výrobu planárních multielektrodových polí a toto planární multielektrodové pole bylo úspěšně testováno pro zarovnání HL-1 buněk na jeho povrchu kombinací otisku adhezní látky a potahování antiadhezivním činidlem.