Essential role of Cdc42 in cardiomyocyte proliferation and cell-cell adhesion during heart development.

Cdc42 is a member of the Rho GTPase family and functions as a molecular switch in regulating cell migration, proliferation, differentiation and survival. However, the role of Cdc42 in heart development remains largely unknown. To determine the function of Cdc42 in heart formation, we have generated a Cdc42 cardiomyocyte knockout (CCKO) mouse line by crossing Cdc42 flox mice with myosin light chain (MLC) 2a-Cre mice. The inactivation of Cdc42 in embryonic cardiomyocytes induced lethality after embryonic day 12.5. Histological analysis of CCKO embryos showed cardiac developmental defects that included thin ventricular walls and ventricular septum defects. Microarray and real-time PCR data also revealed that the expression level of p21 was significantly increased and cyclin B1 was dramatically decreased, suggesting that Cdc42 is required for cardiomyocyte proliferation. Phosphorylated Histone H3 staining confirmed that the inactivation of Cdc42 inhibited cardiomyocytes proliferation. In addition, transmission electron microscope studies showed disorganized sarcomere structure and disruption of cell-cell contact among cardiomyocytes in CCKO hearts. Accordingly, we found that the distribution of N-cadherin/ß-Catenin in CCKO cardiomyocytes was impaired. Taken together, our data indicate that Cdc42 is essential for cardiomyocyte proliferation, sarcomere organization and cell-cell adhesion during heart development.

Nicotine Promotes Cholangiocarcinoma Growth in Xenograft Mice.

Nicotine, the main addictive substance in tobacco, is known to play a role in the development and/or progression of a number of malignant tumors. However, nicotine's involvement in the pathogenesis of cholangiocarcinoma is controversial. Therefore, we studied the effects of nicotine on the growth of cholangiocarcinoma cells in vitro and the progression of cholangiocarcinoma in a mouse xenograft model. The predominant subunit responsible for nicotine-mediated proliferation in normal and cancer cells, the α7 nicotinic acetylcholine receptor (α7-nAChR), was more highly expressed in human cholangiocarcinoma cell lines compared with normal human cholangiocytes. Nicotine also stimulated the proliferation of cholangiocarcinoma cell lines and promoted α7-nAChR-dependent activation of proliferation and phosphorylation of extracellular-regulated kinase in Mz-ChA-1 cells. In addition, nicotine and PNU282987 (α7-nAChR agonist) accelerated the growth of the cholangiocarcinoma tumors in our xenograft mouse model and increased fibrosis, proliferation of the tumor cells, and phosphorylation of extracellular-regulated kinase activation. Finally, α7-nAChR was expressed at significantly higher levels in human cholangiocarcinoma compared with normal human control liver samples. Taken together, results of this study suggest that nicotine acts through α7-nAChR and plays a novel role in the pathogenesis of cholangiocarcinoma. Furthermore, nicotine may act as a mitogen in cholestatic liver disease processes, thereby facilitating malignant transformation.

Loss of myocardial retinoic acid receptor α induces diastolic dysfunction by promoting intracellular oxidative stress and calcium mishandling in adult mice.

Retinoic acid receptor (RAR) has been implicated in pathological stimuli-induced cardiac remodeling. To determine whether the impairment of RARα signaling directly contributes to the development of heart dysfunction and the involved mechanisms, tamoxifen-induced myocardial specific RARα deletion (RARαKO) mice were utilized. Echocardiographic and cardiac catheterization studies showed significant diastolic dysfunction after 16wks of gene deletion. However, no significant differences were observed in left ventricular ejection fraction (LVEF), between RARαKO and wild type (WT) control mice. DHE staining showed increased intracellular reactive oxygen species (ROS) generation in the hearts of RARαKO mice. Significantly increased NOX2 (NADPH oxidase 2) and NOX4 levels and decreased SOD1 and SOD2 levels were observed in RARαKO mouse hearts, which were rescued by overexpression of RARα in cardiomyocytes. Decreased SERCA2a expression and phosphorylation of phospholamban (PLB), along with decreased phosphorylation of Akt and Ca2+/calmodulin-dependent protein kinase II δ (CaMKII δ) was observed in RARαKO mouse hearts. Ca2+ reuptake and cardiomyocyte relaxation were delayed by RARα deletion. Overexpression of RARα or inhibition of ROS generation or NOX activation prevented RARα deletion-induced decrease in SERCA2a expression/activation and delayed Ca2+ reuptake. Moreover, the gene and protein expression of RARα was significantly decreased in aged or metabolic stressed mouse hearts. RARα deletion accelerated the development of diastolic dysfunction in streptozotocin (STZ)-induced type 1 diabetic mice or in high fat diet fed mice. In conclusion, myocardial RARα deletion promoted diastolic dysfunction, with a relative preserved LVEF. Increased oxidative stress have an important role in the decreased expression/activation of SERCA2a and Ca2+ mishandling in RARαKO mice, which are major contributing factors in the development of diastolic dysfunction. These data suggest that impairment of cardiac RARα signaling may be a novel mechanism that is directly linked to pathological stimuli-induced diastolic dysfunction.

Cardiac dysfunction subsequent to chronic ozone exposure in rats.

A number of advancements have been made toward identifying the risk factors associated with cardiovascular disease (CVD) and have resulted in a decline in mortality. However, many patients with cardiac disease show no established previous risk. Thus, it appears that other unknown factors contribute to the pathophysiology of CVD. Out of 350,000 sudden cardiac deaths each year in the United States, 60,000 deaths have been linked to air pollution, suggesting a detrimental role of environmental pollutants in the development of CVD. This study tested the hypothesis that chronic ozone (O(3)) exposure diminishes myocardial function in healthy population. Male Sprague-Dawley rats were exposed 8 h/day for 28 and 56 days to filtered air or 0.8 ppm O(3). In vivo cardiac function was assessed by measuring LVDP, +dP/dt, -dP/dt, and LVEDP 24 h after termination of the O(3) exposure. Compared to rats exposed to filtered air, LVDP, +dP/dt, and -dP/dt were significantly decreased, and LVEDP was significantly increased in O(3) exposed animals. This attenuation of cardiac function was associated with increased myocardial TNF-alpha levels and lipid peroxidation as well as decreased myocardial activities of superoxidase dismutase and interleukin-10 levels. These novel findings suggest myocardial dysfunction subsequent to chronic O(3) exposure in normal adult rats may be associated with a decrease in antioxidant reserve and with an increased production of inflammatory mediators.

Castration inhibits biliary proliferation induced by bile duct obstruction: novel role for the autocrine trophic effect of testosterone.

Increased cholangiocyte growth is critical for the maintenance of biliary mass during liver injury by bile duct ligation (BDL). Circulating levels of testosterone decline following castration and during cholestasis. Cholangiocytes secrete sex hormones sustaining cholangiocyte growth by autocrine mechanisms. We tested the hypothesis that testosterone is an autocrine trophic factor stimulating biliary growth. The expression of androgen receptor (AR) was determined in liver sections, male cholangiocytes, and cholangiocyte cultures [normal rat intrahepatic cholangiocyte cultures (NRICC)]. Normal or BDL (immediately after surgery) rats were treated with testosterone or antitestosterone antibody or underwent surgical castration (followed by administration of testosterone) for 1 wk. We evaluated testosterone serum levels; intrahepatic bile duct mass (IBDM) in liver sections of female and male rats following the administration of testosterone; and secretin-stimulated cAMP levels and bile secretion. We evaluated the expression of 17ß-hydroxysteroid dehydrogenase 3 (17ß-HSD3, the enzyme regulating testosterone synthesis) in cholangiocytes. We evaluated the effect of testosterone on the proliferation of NRICC in the absence/presence of flutamide (AR antagonist) and antitestosterone antibody and the expression of 17ß-HSD3. Proliferation of NRICC was evaluated following stable knock down of 17ß-HSD3. We found that cholangiocytes and NRICC expressed AR. Testosterone serum levels decreased in castrated rats (prevented by the administration of testosterone) and rats receiving antitestosterone antibody. Castration decreased IBDM and secretin-stimulated cAMP levels and ductal secretion of BDL rats. Testosterone increased 17ß-HSD3 expression and proliferation in NRICC that was blocked by flutamide and antitestosterone antibody. Knock down of 17ß-HSD3 blocks the proliferation of NRICC. Drug targeting of 17ß-HSD3 may be important for managing cholangiopathies.

Isolation of Adult Mouse Cardiomyocytes Using Langendorff Perfusion Apparatus.

Heart disease is one of the leading causes of death in the United States. Isolation and culture adult cardiomyocytes are important for studying cardiomyocyte contractility, heart hypertrophy, and cardiac failure. In contrast to neonatal cardiomyocyte isolation, adult mice cardiomyocytes isolation is challenging due to firm connections among cardiomyocytes through intercalated discs. The availability of newly generated genetically modified mouse lines requires to establish protocols to isolation and culture adult mouse cardiomyocyte for in vitro studies. In this manuscript, we described a straightforward method of isolating adult mouse cardiomyocytes using Langendorff perfusion apparatus. Briefly, the hearts were harvested from adult mice and the heart was mounted to Lagendorff apparatus. After perfusion with calcium depletion and collagenase digestion, the left ventricles were minced and filtered. Lastly, the separated cardiomyocytes were treated with CaCl2. The isolated cardiac myocytes can be utilized in a broad range of experiments including screening for drugs.

Preparation of Neonatal Rat Papillary Muscles for Contractile Studies.

Isolated cardiac tissue allows investigators to study mechanisms underlying normal and pathological conditions, which would otherwise be difficult or impossible to perform in vivo. In contrast to ventricular muscle strip preparations, papillary muscles can be prepared without severely damaging the muscle tissue. In this preparation, the isolated papillary muscle is fixed in an environmentally controlled organ bath chamber and electrically stimulated. The evoked twitch force is recorded using a pressure transducer, and parameters such as twitch force amplitude and twitch kinetics are analyzed. A variety of experimental protocols can be performed to investigate the calcium- and frequency-dependent contractility as well as dose-response curves of contractile agents, as well as simulation of pathologic conditions such as acute cardiac ischemia. Mouse papillary muscle preparations have long been the mainstay for studying interactions between intracellular calcium regulation and contractile responses under a number of simulated pathophysiological conditions. These studies are often used to complement in vitro studies performed using isolated neonatal rat cardiac myocytes. In this procedure, we describe how neonatal rat papillary muscles can also be prepared for use in contractile studies.

Secretin inhibits cholangiocarcinoma growth via dysregulation of the cAMP-dependent signaling mechanisms of secretin receptor.

Secretin plays a key role in the regulation of normal cholangiocyte physiology via secretin receptor (SCTR). SCTR expression is upregulated during extrahepatic cholestasis induced by bile duct ligation and closely associated with cholangiocyte proliferative responses. Although well studied in normal cholangiocytes, the role of secretin and the expression of SCTR in the regulation of cholangiocarcinoma proliferation are unknown. In vitro, secretin (10(-7) M) displayed differential effects on normal cholangiocyte [H-69 and human intrahepatic biliary epithelial cell line (HIBEpiC)] and cholangiocarcinoma (Mz-ChA-1, HuH-28, TFK-1, SG231, CCLP1 and HuCC-T1) cell lines as such secretin is mitogenic for normal cholangiocytes and antiproliferative for cholangiocarcinoma. As expected in normal cholangiocytes (HIBEpiC), secretin increased intracellular cyclic adenosine monophosphate (cAMP) levels. However, the effect of secretin on intracellular cAMP levels was suppressed in Mz-ChA-1 cells. Secretin-stimulated intracellular cAMP levels in Mz-ChA-1 were restored by pretreatment with pertussis toxin, suggesting that the receptor coupled to Galpha(i) rather than Galpha(s). SCTR expression was found to be downregulated in 4 of the 6 cholangiocarcinoma cell lines evaluated and in human cholangiocarcinoma biopsy samples. In vivo, secretin significantly inhibited the tumor size and more than doubled tumor latency, which was associated with a decrease in proliferating cell nuclear antigen and an increase in cleaved-caspase 3 expression levels. Our results demonstrate that secretin and/or the modulation of SCTR expression might have potential as a therapeutic tool in the treatment of cholangiocarcinoma.

Morphological and functional heterogeneity of the mouse intrahepatic biliary epithelium.

Rat and human biliary epithelium is morphologically and functionally heterogeneous. As no information exists on the heterogeneity of the murine intrahepatic biliary epithelium, and with increased usage of transgenic mouse models to study liver disease pathogenesis, we sought to evaluate the morphological, secretory, and proliferative phenotypes of small and large bile ducts and purified cholangiocytes in normal and cholestatic mouse models. For morphometry, normal and bile duct ligation (BDL) mouse livers (C57/BL6) were dissected into blocks of 2-4 microm(2), embedded in paraffin, sectioned, and stained with hematoxylin and eosin. Sizes of bile ducts and cholangiocytes were evaluated by using SigmaScan to measure the diameters of bile ducts and cholangiocytes. In small and large normal and BDL cholangiocytes, we evaluated the expression of cholangiocyte-specific markers, keratin-19 (KRT19), secretin receptor (SR), cystic fibrosis transmembrane conductance regulator (CFTR), and chloride bicarbonate anion exchanger 2 (Cl(-)/HCO(3)(-) AE2) by immunofluorescence and western blot; and intracellular cyclic adenosine 3',5'-monophosphate (cAMP) levels and chloride efflux in response to secretin (100 nM). To evaluate cholangiocyte proliferative responses after BDL, small and large cholangiocytes were isolated from BDL mice. The proliferation status was determined by analysis of the cell cycle by fluorescence-activated cell sorting, and bile duct mass was determined by the number of KRT19-positive bile ducts in liver sections. In situ morphometry established that the biliary epithelium of mice is morphologically heterogeneous, with smaller cholangiocytes lining smaller bile ducts and larger cholangiocytes lining larger ducts. Both small and large cholangiocytes express KRT19 and only large cholangiocytes from normal and BDL mice express SR, CFTR, and Cl(-)/HCO(3)(-) exchanger and respond to secretin with increased cAMP levels and chloride efflux. Following BDL, only large mouse cholangiocytes proliferate. We conclude that similar to rats, mouse intrahepatic biliary epithelium is morphologically and functionally heterogeneous. The mouse is therefore a suitable model for defining the heterogeneity of the biliary tree.

Stretch-induced regulation of angiotensinogen gene expression in cardiac myocytes and fibroblasts: opposing roles of JNK1/2 and p38alpha MAP kinases.

The cardiac renin-angiotensin system (RAS) has been implicated in mediating myocyte hypertrophy, remodeling, and fibroblast proliferation in the hemodynamically overloaded heart. However, the intracellular signaling mechanisms responsible for regulation of angiotensinogen (Ao), a substrate of the RAS system, are largely unknown. Here we report the identification of JNK1/2 as a negative, and p38alpha as a major positive regulator of Ao gene expression. Isolated neonatal rat ventricular myocytes (NRVM) and fibroblasts (NRFB) plated on deformable membranes coated with collagen IV, were exposed to 20% equiaxial static-stretch (0-24 h). Mechanical stretch initially depressed Ao gene expression (4 h), whereas after 8 h, Ao gene expression increased in a time-dependent manner. Blockade of JNK1/2 with SP600125 increased basal Ao gene expression in NRVM (10.52+/-1.98 fold, P<0.001) and NRFB (13.32+/-2.07 fold, P<0.001). Adenovirus-mediated expression of wild-type JNK1 significantly inhibited, whereas expression of dominant-negative JNK1 and JNK2 increased basal and stretch-mediated (24 h) Ao gene expression, showing both JNK1 and JNK2 to be negative regulators of Ao gene expression in NRVM and NRFB. Blockade of p38alpha/beta by SB202190 or p38alpha by SB203580 significantly inhibited stretch-induced (24 h) Ao gene expression, whereas expression of wild-type p38alpha increased stretch-induced Ao gene expression in both NRVM (8.41+/-1.50 fold, P<0.001) and NRFB (3.39+/-0.74 fold, P<0.001). Conversely, expression of dominant-negative p38alpha significantly inhibited stretch response. Moreover, expression of constitutively active MKK6b (E) significantly stimulated Ao gene expression in the absence of stretch, indicating that p38 activation alone is sufficient to induce Ao gene expression. Taken together p38alpha was demonstrated to be a positive regulator, whereas JNK1/2 was found to be a negative regulator of Ao gene expression. Prolonged stretch diminished JNK1/2 activation, which was accompanied by a reciprocal increase in p38 activation and Ao gene expression. This suggests that a balance in JNK1/2 and p38alpha activation determines the level of Ao gene expression in myocardial cells.

Lethal and edema toxins of anthrax induce distinct hemodynamic dysfunction.

Fatalities due to anthrax are associated with severe hypotension suggesting that the toxins generated from Bacillus anthracis, lethal toxin (LeTx) and edema toxin (EdTx), have cardiovascular effects. Here, we demonstrate the effects of these toxins and characterize their effects by echocardiography. LeTx leads to a significant reduction in ejection fraction, decreased velocity of propagation (diastolic dysfunction), decreased velocity of circumferential fiber shortening (decreased contractility), and increased LV systolic area (pathophysiology). EdTx leads to a significant reduction in left ventricular volumes and cardiac output (reduced stroke volume) but does not cause significant change in ejection fraction or contractility. These results indicate that LeTx reduces left ventricular systolic function and EdTx reduces preload but does not have direct myocardial effects. Together, these findings suggest that LeTx and EdTx exert distinct hemodynamic dysfunction associated with anthrax infection.

Isolated neonatal rat papillary muscles: a new model to translate neonatal rat myocyte signaling into contractile mechanics.

Isolated cardiac tissue allows investigators to study mechanisms underlying normal and pathological conditions, which would otherwise be difficult or impossible to perform in vivo. Cultured neonatal rat ventricular cardiac myocytes (NRVM) are widely used to study signaling and growth mechanisms in the heart, primarily due to the versatility, economy, and convenience of this in vitro model. However, the lack of a well-defined longitudinal cellular axis greatly hampers the ability to measure contractile function in these cells, and therefore to associate signaling with mechanical function. In these methods, we demonstrate that this limitation can be overcome by using papillary muscles isolated from neonatal rat hearts. In the methods we describe procedures for isolation of right ventricular papillary muscles from 3-day-old neonatal rats and effects of mechanical and humoral stimuli on contraction and relaxation properties of these tissues.

p38α MAPK inhibits stretch-induced JNK activation in cardiac myocytes through MKP-1.

Mechanical stretch is a major determinant that leads to heart failure, which is associated with a steady increase in myocardial angiotensinogen (Aogen) expression and formation of the biological peptide angiotensin II (Ang II). c-jun NH2-terminal kinase (JNK) and p38α have been found to have opposing roles on stretch-induced Aogen gene expression in neonatal rat ventricular myocytes (NRVM). JNK negatively regulated Aogen expression in NRVM following acute stretch, whereas with prolonged stretch, JNK phosphorylation was downregulated and p38α was found responsible for upregulation of Aogen expression. However, the mechanisms responsible for regulation of these kinases, especially the cross-talk between p38 and JNK1/2, remain to be determined. In this study, a combination of pharmacologic and molecular approaches (adenovirus-mediated gene transfer) were used to examine the mechanisms by which p38 regulates JNK phosphorylation in NRVM under stretch and non-stretch conditions. Pharmacologic inhibition of p38 significantly increased JNK phosphorylation in NRVM at 15 min, whereas overexpression of wild-type p38α significantly decreased JNK phosphorylation. While p38α overexpression prevented stretch-induced JNK phosphorylation, pharmacologic p38 inhibition abolished the JNK dephosphorylation during 15-60 min of stretch. Expression of constitutively-active MKK3 (MKK3CA), the upstream activator of p38, abolished JNK phosphorylation in both basal and stretched NRVM. Pharmacologic inhibition of MAP kinase phosphatase-1 (MKP-1) or protein phosphatase-1 (PP1) increased JNK phosphorylation in NRVM, suggesting the involvement of these phosphatases on reversing stretch-induced JNK activation. Inhibition of MKP-1, but not PP1, reduced JNK phosphorylation in NRVM overexpressing MKK3CA under basal conditions (no-stretch). Inhibition of MKP-1 also enhanced stretch-induced JNK phosphorylation in NRVM at 15 to 60 min. In summary, these results indicate that MKP-1 inhibits JNK phosphorylation in stretched NRVM through p38 dependent and independent mechanisms, whereas PP1 regulates JNK through a p38-independent mechanism.

Activation of protein kinase A by atrial natriuretic peptide in neonatal rat cardiac fibroblasts: role in regulation of the local renin-angiotensin system.

There is an inverse relationship between renin and atrial natriuretic peptide (ANP) levels in the plasma. Since both the ANP and renin-angiotensin system (RAS) are upregulated in development and cardiac hypertrophy, we tested whether ANP differentially regulates RAS in cardiac cells. Cardiac fibroblasts isolated from neonatal rats were treated with ANP(1-28), a biologically active fragment of ANP. Renin and angiotensinogen (Ao) mRNA levels were measured by quantitative multiplex RT-PCR and protein levels determined by Western blot analysis. ANP(1-28) increased renin and Ao mRNA levels (737+/-131% and 178+/-51.3%) with EC50 values of 4.12+/-0.3 and 8.67+/-0.22 nmol/L, respectively. At the protein level, secretion of renin and Ao was significantly enhanced resulting in approximately 4-fold increase in ANG II level in the medium. The effect of ANP(1-28) on renin and Ao mRNA expression were reproduced by 8-bromo-cyclic GMP. Inhibition of protein kinase G (PKG) with KT5823 blunted ANP(1-28)-induced upregulation of renin, but not Ao mRNA, while inhibition of protein kinase A (PKA) with KT5720 attenuated the upregulation of both renin and Ao mRNA. These findings suggest that unlike in plasma, ANP positively regulates the RAS in cardiac fibroblasts, which may have a significant role in development of the fetal heart.

Evidence of a novel intracrine mechanism in angiotensin II-induced cardiac hypertrophy.

Angiotensin II (Ang II) has a significant role in regulating cardiac homeostasis through humoral, autocrine and paracrine pathways, via binding to the plasma membrane AT1 receptor. Recent literature has provided evidence for intracrine growth effects of Ang II in some cell lines, which does not involve interaction with the plasma membrane receptor. We hypothesized that such intracrine mechanisms are operative in the heart and likely participate in the cardiac hypertrophy induced by Ang II. Adenoviral and plasmid vectors were constructed to express Ang II peptide intracellularly. Neonatal rat ventricular myocytes (NRVMs) infected with the adenoviral vector showed significant hypertrophic growth as determined by cell size, protein synthesis and enhanced cytoskeletal arrangement. Adult mice injected with the plasmid vector developed significant cardiac hypertrophy after 48 h, without an increase in blood pressure or plasma Ang II levels. This was accompanied by increased transcription of transforming growth factor-beta (TGF-beta) and insulin-like growth factor-1 (IGF-1) genes. Losartan did not block the growth effects, excluding the involvement of extracellular Ang II and the plasma membrane AT1 receptor. These data demonstrate a previously unknown growth mechanism of Ang II in the heart, which should be considered when designing therapeutic strategies to block Ang II actions.

Baseline echocardiographic values for adult male rats.

BACKGROUND: Because of safety, repeatability, and portability, clinical echocardiography is well established as a standard for cardiac anatomy, cardiac function, and hemodynamics. Similarly, application of echocardiography in commonly used rat experimental models would be worthwhile. The use of noninvasive ultrasound imaging in the rat is a potential replacement for more invasive terminal techniques. Although echocardiography has become commonly used in the rat, normal parameters for cardiac anatomy and function, and comparison with established human values, have not been reported. METHODS: A total of 44 Sprague-Dawley male rats had baseline echocardiography replicating a protocol for clinical echocardiography. RESULTS: Complete 2-dimensional echocardiography for cardiac anatomy and function was obtained in 44 rats. Hemodynamic parameters could be recorded in 85% of rats. The ejection fraction and fractional shortening values of the left ventricle were similar to those reported for healthy human beings. Pulsed Doppler velocities of atrial systole for mitral valve inflow, pulmonary vein reversal, and Doppler tissue of the lateral mitral valve annulus also had similar means as healthy human beings. The calculated left ventricular mass was at the same order of magnitude as a proportion of body weight of rat to man. All other observations in the clinical protocol were different from those reported in healthy human beings. CONCLUSION: The use of echocardiography for assessment of cardiac anatomy, function, and hemodynamics can be consistently applied to the rat and replicates much of the information used routinely in human echocardiography.

Mechanosensing and Regulation of Cardiac Function.

The role of mechanical force as an important regulator of structure and function of mammalian cells, tissues, and organs has recently been recognized. However, mechanical overload is a pathogenesis or comorbidity existing in a variety of heart diseases, such as hypertension, aortic regurgitation and myocardial infarction. Physical stimuli sensed by cells are transmitted through intracellular signal transduction pathways resulting in altered physiological responses or pathological conditions. Emerging evidence from experimental studies indicate that ß1-integrin and the angiotensin II type I (AT1) receptor play critical roles as mechanosensors in the regulation of heart contraction, growth and leading to heart failure. Integrin link the extracellular matrix and the intracellular cytoskeleton to initiate the mechanical signalling, whereas, the AT1 receptor could be activated by mechanical stress through an angiotensin-II-independent mechanism. Recent studies show that both Integrin and AT1 receptor and their downstream signalling factors including MAPKs, AKT, FAK, ILK and GTPase regulate heart function in cardiac myocytes. In this review we describe the role of mechanical sensors residing within the plasma membrane, mechanical sensor induced downstream signalling factors and its potential roles in cardiac contraction and growth.

Echocardiographic effects of eplerenone and aldosterone in hypertensive rats.

The effects of aldosterone receptor blockade on echocardiography in spontaneously hypertensive rats (SHR) are not fully characterized. In this study, multiple echocardiographic parameters were compared for 42 weeks between SHR versus Wistar-Kyoto rats (WKY) serving as normotensive controls. In addition, echocardiographic parameters were compared for 28 weeks between the SHR versus SHR treated with eplerenone 100 mg/kg/day or spironolactone 50 mg/kg/day. Compared to normotensive WKY rats, SHRs had significantly increased systolic blood pressure, increased cardiac mass, increased isovolumic relaxation time (IVRT), decreased E/A ratio, increased mitral closure opening time interval (MCO) and increased Tei index. Both eplerenone and spironolactone significantly decreased systolic blood pressure compared to the SHR controls. The spironolactone treatment group demonstrated significant increases in heart rate and cardiac output and a decrease in cardiac index compared to SHR controls. Any aldosterone blockade in SHR protected against the increased cardiac mass. Similar to clinical echocardiographic observations, hypertension in rats results in left ventricular hypertrophy (LVH) and diastolic dysfunction and aldosterone receptor blockade reduces LVH in SHR.

Caveolin and ß1-integrin coordinate angiotensinogen expression in cardiac myocytes.

BACKGROUND: The cardiac renin-angiotensin system (RAS) has been implicated in mediating myocyte hypertrophy and remodeling, although the biochemical mechanisms responsible for regulating the local RAS are poorly understood. Caveolin-1 (Cav-1)/Cav-3 double-knockout mice display cardiac hypertrophy, and in vitro disruption of lipid rafts/caveolae using methyl-ß-cyclodextrin (MßCD) abolishes cardiac protection. METHODS: In this study, neonatal rat ventricular myocytes (NRVM) were used to determine whether lipid rafts/caveolae may be involved in the regulation of angiotensinogen (Ao) gene expression, a substrate of the RAS system. RESULTS: Treatment with MßCD caused a time-dependent upregulation of Ao gene expression, which was associated with differential regulation of mitogen-activated protein (MAP) kinases ERK1/2, p38 and JNK phosphorylation. JNK was highly phosphorylated shortly after MßCD treatment (2-30 min), whereas marked activation of ERK1/2 and p38 occurred much later (2-4h). ß1D-Integrin was required for MßCD-induced activation of the MAP kinases. Pharmacologic inhibition of ERK1/2 and JNK enhanced MßCD-induced Ao gene expression, whereas p38 blockade inhibited this response. Adenovirus-mediated expression of wild-type p38α enhanced MßCD-induced Ao gene expression; conversely expression of dominant negative p38α blocked the stimulatory effects of MßCD. Expression of Cav-3 siRNA stimulated Ao gene expression, whereas overexpression of Cav-3 was inhibitory. Cav-1 and Cav-3 expression levels were found to be positively regulated by p38, but unaffected by ERK1/2 and JNK. CONCLUSION: Collectively, these studies indicate that lipid rafts/caveolae couple to Ao gene expression through a mechanism that involves ß1-integrin and the differential actions of MAP kinase family members.

Production of spontaneously beating neonatal rat heart tissue for calcium and contractile studies.

Neonatal rat ventricular myocytes (NRVM) and fibroblasts (FB) serve as in vitro models for studying fundamental mechanisms underlying cardiac pathologies, as well as identifying potential therapeutic targets. Typically, these cell types are separated using Percoll density gradient procedures. Cells located between the Percoll bands (interband cells [IBCs]), which contain less mature NRVM and a variety of non-myocytes, including coronary vascular smooth muscle cells and endothelial cells (ECs), are routinely discarded. However, we have demonstrated that IBCs readily attach to extracellular matrix-coated coverslips, plastic culture dishes, and deformable membranes to form a 2-dimensional cardiac tissue layer which quickly develops spontaneous contraction within 24 h, providing a robust coculture model for the study of cell-to-cell signaling and contractile studies. Below, we describe methods that provide good cell yield and viability of IBCs during isolation of NRVM and FB obtained from 0- to 3-day-old neonatal rat pups. Basic characterization of IBCs and methods for use in intracellular calcium and contractile experiments are also presented. This method maximizes the use of cells obtained from neonatal rat hearts.