Effects Of Angiotensin â…¡ On The Expression Of Adiponectin In Rat Myocardium And The Mechanism Research

Activation of the renin-angiotensin-aldosterone system (RAAS) and increased formation of cytokines have both been shown to play a critical role in left ventricular remodeling and chronic heart failure (CHF) progression. Adiponectin is one of the cytokines participating in the pathogenesis of CHF. A better understanding of the relationship between RAAS and adiponectin is of great importance in CHF treatment.Angiotensinâ…¡(Angâ…¡) is the major effector of RAAS. Elevations in Angâ…¡have been implicated in myocyte hypertrophy, interstitial fibrosis, and apoptosis, with its effects exerted through two receptors: angiotensin type-1(AT₁R) and angiotensin type-2 (AT₂R). Both of these receptor subtypes are G-protein-coupled receptors (GPCR), which differ in tissue distribution and cellular signaling pathways. It is recognized that the AT₁R mediates most of the deleterious effects of Angâ…¡, including vasoconstriction, endothelial damage, and cell growth. The AT₂R is now recognized as the counter-regulator of AT₁R function, exerting mostly beneficial actions such as vasodilatation, anti-proliferation, and tissue regeneration. Under pathological conditions such as cardiac hypertrophy, infarction, or a failing heart, the relative distribution ratio of AT₂R is increased. These AT₂-mediated effects play an important role in the Angâ…¡'s control of the cardiovascular system. In hypertrophic cardiomyocytes, which express both AT₁R and AT₂R, genes regulated by AT₂R are mainly related to four signalling pathways: Phospholipase C (PLC), Nitric Oxide/Cyclic Guanosine Monophosphate -Dependent Protein Kinase (NO/cGMP), Rho, and Janus kinases signal transducers and activators of transcription (JAK/STAT₎. From these pathways, it is clear that AT₂R stimulates nitric oxide (NO) production, resulting in an increased formation of the second messenger Cyclic Guanosine Monophosphate-Dependent Protein Kinase (cGMP). This messenger, in turn, mediates many of NO's actions, including vasodilatation and natriuresis, by activating cGMP-dependent protein kinase.Adiponectin is a circulating cytokine secreted predominantly by adipose tissue. Recently, it was shown that adiponectin is synthesized by cultured cardiomyocytes, acting in a paracrine/autocrine manner. It is known that adiponectin production is upregulated in patients with heart failure, with high plasma adiponectin levels an indicator of prognosis. Recently, increased levels of adiponectin seen in peripheral circulation were determined to be released from the failing heart. Pericardial fat tissue present in proportion to ventricular mass has been known to produce adiponectin; however, whether adiponectin produced in heart-failure cardiomyocytes contributes to peripheral circulating levels has not been determined. In adipocytes, it is reported that adiponectin protein expression was significantly induced by Angâ…¡. Activation of the AT₂R is required for Angâ…¡-induced adiponectin expression, AT₁R is not involved in Angâ…¡-mediated adiponectin induction. Cardiac natriuretic peptides such as atrial natriuretic peptide (ANP) and N-terminal brain natriuretic peptide (NT-proBNP) have been shown to be powerful risk factors for chronic heart failure, with plasma levels positively correlating with plasma adiponectin concentrations in patients with chronic heart failure. ANP and BNP have been reported to increase adiponectin mRNA in cultured adipocytes via a cGMP?PKG (protein kinase G) -dependent pathway. Upregulation of cGMP by hemin also has enhanced plasma adiponectin levels in spontaneously hypertensive rats (SHRs).Given these experimental and clinical findings, we hypothesized that Angâ…¡could increase adiponectin production in neonatal rat cardiomyocytes (NRVMs) through AT₂R?NO?cGMP?PKG signaling.Experimental models of CHF can be induced by several methods. ISOproterenol(ISO) induced HF model is used spreadly with its simple operation, low cost and representative pathophysiologic changes. Subcutaneous administration of beta adrenoceptor agonist ISO produces patchy myocardial necrosis in a dose-related way with intact coronary vasculature. The pathophysiological and morphological changes observed in ISOproterenol-treated rats have been found to be similar to those observed in human myocardial infarction (MI). Myocardial infarction results in asymmetric cardiac remodeling that is caused by fibrosis and hypertrophy of the remnant viable myocardium. These cardiac structural adaptations facilitate compensated hemodynamic performance, but ultimately result in a high incidence of heart failure with death.Adiponectin acts as an anti-inflammatory, antidiabetic and antiatherogenic cytokine that has been demonstrated to suppresse cardiac hypertrophy in response to pressure overload. Another adipokine, TNF-α, is expressed and secreted from cardiomyocytes. A present study has shown that adiponectin are suppressed by TNF-αand locally generated TNF-αis involved in cardiac remodeling by stimulating apoptosis, inflammatory and fibrogenic responses in pressure-overloaded heart, which, in turn, contribute to cardiac dysfunction.The angiontensinâ…¡type 1 receptor system plays a crucial role in the regulation of blood pressure and contributes to a variety of physiological and pathophysiological processes, including cardiac remodeling. Angiotensin receptor blockers are known to reduce the infarct size and improve left ventricular function by antagonising angiotensinâ…¡type 1 receptor. Telmisartan being a selective angiotensinâ…¡type 1 receptor blocker is widely used in the treatment of hypertension, congestive heart failure and diabetic nephropathy and has been demonstrated to have beneficial effect on post-infarct ventricular remodeling. Recently, telmisartan has been reported to have additional peroxISOme proliferator -activated receptor-gamma (PPAR-γ) partial agonist activity which regulates metabolic and inflammatory pathways and improves left ventricular functions. Telmisartan with these dual actions, has been shown to have anti-inflammatory properties in vitro and animal studies. It can reduce the expression of inflammatory cytokines including tumor necrosis factor-α(TNF-α) and its transcriptional factors. ARB treatment increases circulating adiponectin concentrations in human and adiponectin mRNA level in fat tissue in hypertensive rats. ARB treatment also induces cardiac adiponectin expression in mice with viral myocarditis. however, till date the effect of telmisartan on cardiac adiponectin in postinfarction myocardium has not been studied.The present study investigated the effects of Angâ…¡on the expression of adiponectin in cultured neonatal rat ventricular cardiomyte with western immunoblotting and real time-PCR methods. Meanwhile, to explore whether the underlying mechanism was through the AT₂R?NO?cGMP?PKG singaling pathwaym, we have tested this hypothesis by stimulating cardiomyocytes with Angâ…¡in the presence of inhibitors of this pathway. The present study was also designed to investigate the cardioprotective effects of telmisartan in rat model with ISOproterenol-induced postinfarction remodeling and to investigate its protective effects on ISOproterenol-induced rat cardiac myocyte injury focusing on its effect on cardiac adiponectin as well as on cardiac TNF-α. In summary, the present study investigated the effects of Angâ…¡and telmisartan on the expression of cardiac adiponectin from mRNA expression and protein synthesis, demonstrated their cell and molecular biological mechanisms and signal transduction pathway involved, and to provided new ideas and experimental evidences for CHF therapy. The present study included four sections as following:Section 1: Effects of Angâ…¡on the expression of adiponectin in cultured neonatal rat ventricular myocytes (NRVMs).Objective: To investigate the effects of Angâ…¡on the expression of adiponectin in NRVMs and to explore the role of the angiotensin receptor that involved.Methods: NRVMs were treated with various concentrations of Angâ…¡, and adiponectin expression was measured by quantitative real-time reverse transcription-polymerase chain reaction and western immunoblotting.Results:1 Angâ…¡induces an increase in adiponectin mRNA level To determine the effect of Angâ…¡on adiponectin mRNA levels, NRVMs were incubated with various concentrations of Angâ…¡for 24 hours. Expression of adiponectin mRNA was significantly increased by Angâ…¡at concentrations from 10-6 to 10-8 M, reaching a maximum of 10-7 M (4.5±0.2-fold induction vs. Vehicle-treated cells; p < 0.001, n=4). NRVMs were then incubated with 10-7 M Angâ…¡for 2, 4, 8, 12, 24, 48 and 72 hours. As adiponectin mRNA levels increased over time, a significant increase was seen after two hours of treatment and a maximal increase was seen at 24 hours (p < 0.05, n=4).2 Angâ…¡acts through the AT₂R to increase adiponectin mRNA levels We used the selective AT₂R antagonist PD123319 to examine AT₂R contributions to Angâ…¡-induced increases in adiponectin mRNA levels. NRVMs were pretreated for 1 hour with media alone or media containing PD123319 (10-5 M). With PD123319 still in the media, NRVMs were exposed to 10-7 M Angâ…¡for another 24 hours. We found that PD123319 alone had no significant effect on basal adiponectin mRNA expression; however, PD123319 completely prevented Angâ…¡-induced increases in adiponectin mRNA levels (p < 0.05, n=4).3 AT₁R is not involved in Angâ…¡-induced adiponectin upregulation. The ARB telmisartan prominently enhanced Angâ…¡-induced adiponectin protein expression at 10-7mol/L. telmisartan at concentrations sufficient to completely block AT₁Rs(10-9 and 10-7 mol/L) had no effect on adiponectin protein expression, suggesting that the AT₁R is not involved in Angâ…¡-mediated adiponectin induction, whereas telmisartan stimulated adiponectin expression might be independent of the AT₁R.4 Alterations in adiponectin mRNA correlate with changes in adiponectin protein levels To confirm whether changes in adiponectin mRNA correlated with changes in adiponectin protein, NRVMs were stimulated with 10-7 M Angâ…¡for 48 hours in the presence of either PD123319 (10-5 M). Although an increase in adiponectin mRNA was seen at 24 hours, adiponectin protein expression was increased at 48 hours in response to Angâ…¡stimulation. We determined that Angâ…¡increases adiponectin protein 3.9-fold (p < 0.05, n=4), which is attenuated by PD123319 treatment (p < 0.05).Conclusions: Angâ…¡upregulates adiponectin in NRVMs via the AT_<sub>2R activation.Section 2: Effects of NO?cGMP signal pathway on Angiotensinâ…¡upregulation of cardiomyocyte adiponectin productionObjective: To investigate the effects of NO?cGMP signal pathway on angiotensinâ…¡upregulation of cardiomyocyte adiponectin production.Methods: We stimulate cardiomyocytes with Angâ…¡in the presence of inhibitors of NO?cGMP signal pathway. Adiponectin expression was measured by quantitative real-time reverse transcription-polymerase chain reaction and western immunoblotting.Results:1 Angâ…¡increases adiponectin mRNA levels through NO activation We inhibited NO production with the nitric oxide synthase (NOS) inhibitor, Nx-Nitro-L-arginine methyl ester hydrochloride (L-NAME). NRVMs were preincubated for one hour with L-NAME (10-3 M) and then coincubated with the same inhibitor and 10-7 M Angâ…¡for another 24 hours. Cell monolayers were harvested and real-time PCR was performed. Angâ…¡-incubated cells had a twofold increase (p < 0.001, n = 4) in adiponectin mRNA at 24 hours compared with the control. This increase was completely abolished by L-NAME (p < 0.001, n=4). L-NAME alone had no effect on basal adiponectin mRNA levels;2 SNP induces an increase in adiponectin mRNA level To further investigate the role that NO plays in the regulation of adiponectin, we stimulated cells with a NO donor, sodium nitroprusside (SNP). We found that SNP produced the same effect as Angâ…¡on adiponectin levels. Cells were incubated with 10-6 M, 10-5 M and 10-4 M SNP for 24 hours. Cell monolayers were harvested and adiponectin mRNA was measured using real-time PCR. SNP induced a concentration-dependent increase in adiponectin with a similar magnitude as Angâ…¡. At the 10-5 M and10-4 M concentrations, we observed a statistically significant difference in Angâ…¡treatment (p < 0.01, n = 4) over control.3 Angâ…¡increases adiponectin mRNA levels through cGMP activation We inhibited cGMP with the cGMP antagonist analogue, Rp-8-Br-cGMP-S. NRVMs were preincubated for one hour with Rp-8-Br-cGMP-S (10-5 M) and then coincubated with the same inhibitor and 10-7 M Angâ…¡for another 24 hours. Cell monolayers were harvested and real-time PCR was performed. Angâ…¡-incubated cells had a twofold increase (p < 0.001, n = 4) in adiponectin mRNA at 24 hours compared with the control. This increase was completely abolished by Rp-8-Br-cGMP-S (p < 0.001, n=4). Rp-8-Br-cGMP-S alone had no effect on basal adiponectin mRNA levels;4 8-Br-cGMP induces an increase in adiponectin mRNA level We used a cGMP agonist, 8-Br-cGMP, to mimic the effect of Angâ…¡on adiponectin. Cells were exposed to 10-6 M, 10-5 M and10-4 M 8-Br-cGMP for 6 hours. We observed a concentration-dependent increase in adiponectin with a similar magnitude as seen with Angâ…¡and a statistically significant increase in adiponectin mRNA of 2.2-fold (p < 0.01, n = 4) with 10-4 M cGMP. These results suggest that the NO?cGMP pathway involves Angâ…¡-stimulated upregulation of adiponectin mRNA.5 Alterations in adiponectin mRNA correlate with changes in adiponectin protein levels To confirm whether changes in adiponectin mRNA correlated with changes in adiponectin protein, NRVMs were stimulated with 10-7 M Angâ…¡for 48 hours in the presence of 10-7 M Rp-8-Br-cGMP-S. Although an increase in adiponectin mRNA was seen at 24 hours, adiponectin protein expression was increased at 48 hours in response to Angâ…¡stimulation. We determined that Angâ…¡increases adiponectin protein 3.9-fold (p < 0.05, n=4), which is attenuated by Rp-8-Br-cGMP-S treatment (p <0.05). Rp-8-Br-cGMP-S alone had no effect on basal adiponectin protein levels;Conclusions: These data suggest a mechanism whereby Angâ…¡upregulates adiponectin in NRVMs via the AT₂R?NO?cGMP?PKG signaling pathway.Section 3: Telmisartan protects against ISOproterenol-induced rat cardiac myocyte injury via regulation of cardiac expression of adiponectin.Objective: To investigate whether telmisartan protects against ISOproterenol-induced rat cardiac myocyte injury via regulation of cardiac expression of adiponectin.Methods: The viability, activation of lactate dehydrogenase (LDH), and apoptosis rate were chosen for measuring the degree of cardiac myocytes injury. The apoptosis rate of cardiomyocytes were determined by flow cytometry. Cell growth was assessed by MTT. Cardiac adiponectin expression was detected by Western immunoblotting.Results:1 Telm inhibited ISO-induced cell death in cardiomyocytes Cardiomyocytes were exposed to ISO (10μM) alone, ISO (10μM) with telm (5-20μM), or no drug for 48 h. The cell death inhibition ratio of telm treated cells relative to ISO only treated cells ranged from 40.2±4.5% (5μM telm) to 92.6±7.1% (20μM telm). Telm dose-dependently protected ISO-treated cardiomyocytes from cell death and had no cytotoxic effects on cells.2 Telm inhibited ISO-induced LDH release in cardiomyocytes ISO (10μM) nearly doubled LDH release in cardiomyocytes relative to cells not exposed to any drug (P < 0.01). Pretreatment with telm for 48 h dose-dependently reduced ISO-induced LDH release; at 20μM, telm-pretreated, ISO-exposed cells had LDH release levels that were similar to that of non-drug-exposed cells. Telm alone (5-20μM) did not affect LDH release.3 Telm inhibited ISO-induced apoptosis in cardiomyocytes ISO (10μM) nearly doubled apoptosis rate in cardiomyocytes relative to cells not exposed to any drug (P < 0.01). Pretreatment with telm for 48 h dose-dependently reduced ISO-induced apoptosis; at 20μM, telm-pretreated, ISO-exposed cells had apoptosis rate that were similar to that of non-drug-exposed cells. Telm alone (5-20μM) did not affect apoptosis rate.4 Telm treatment blocked ISO effects on adiponectin protein expression in cardiomyocytes via a mechanism involving PPAR-γactivation After incubation with 10μM ISO, adiponectin protein expression in cardiomyocytes was decreased relative to that in cells not exposed to any drug. However, in cells that had been pretreated with telm (20μmol/L), this decrease did not occur. Treating ISO-exposed cells to the selective PPAR-γantagonist GW9662 (30μmol/L) potently blocked the effects of telm pretreatment on adiponectin expression. GW9662 alone in the absence of telm did not change ISO-induced decrease of adiponectin expression.5 Antagonizing PPAR-γactivity with the selective antagonist GW9662 blocked telm's protective effects against ISO-induced cell injury. Antagonism of PPAR-γactivity with the selective antagonist GW9662 (30μmol/L) potently blocked telm's protective effects against ISO-induced cell injury. GW9662 alone (in the absence of telm) had no observable effects on ISO-induced cell injury.Conclusion: Telm attenuated ISO-induced cell injury through a mechanism that may involve induction of expression of cardiac adiponectin.Section 4: Telmisartan attenuates ISOproterenol (ISO)-induced postinfarction remodeling in rats via regulation of cardiac expression of adiponectin.Objective: To investigate whether telm pre-treatment attenuates ISOproterenol(ISO)-induced postinfarction remodeling in rats via regulation of cardiac expression of adiponectin.Methods: The experimental animals were divided into the following treatment groups: 1) ISO, n = 10; 2) ISO + telm, n = 10; 3) telm, n = 10; and 4) control (vehicle), n = 10. Rats in the telm and ISO + telm groups were administered telm orally (10 mg/kg/d) by intragastric intubation for a period of 8 weeks. Postinfarction remodeling (PIR) was induced in male Wistar rats by two subcutaneous injections of 80 mg/kg homologous ISO or vehicle at 24 h intervals on 2 consecutive days. At week 8, Heart weight, body weight and left ventricular weight were measured. cardiac function was measured, including left ventricular systolic pressure (LVSP), left ventricular end-diastolic pressure (LVEDP) and maximum change velocity of left ventricular pressure in the ISOvolumic contraction or relaxation period (±dP/dtmax). Cardiac adiponectin and TNF-αexpression was measured by quantitative real-time polymerase chain reaction (qPCR), immunohistochemical findings and western immunoblotting.Results:1 Telm protected against ISO-induced effects on gravimetric parameters Rats treated with ISO alone showed significantly elevated heart mass normalized to body mass, while ISO-treated rats that had been pretreated with telm did not show these unfavorable changes. Telm pretreatment also reduced ISO-induced ventricular mass gain. Heart mass and ventricle/body mass ratio did not differ between rats treated with telm alone and control (no drug) rats.2 Telm protected against ISO-induced myocardial hypertrophy and interstitial fibrosis Compared to untreated PIR (ISOprotrenol alone) animals, PIR animals given telm showed significantly attenuated ISO-induced increases in mean myocyte diameter and collagen volume fraction. Treatment with telm alone had no effect on these values (P > 0.05 vs. control group).3 Telm protected against ISO-induced decrease in cardiac function Compared to untreated PIR (ISOprotrenol alone) animals, PIR animals given telm showed significantly attenuated ISO-induced increases in LVEDP and decrease in LVSP and±dp/dtmax. Treatment with telm alone had no effect on these values (P >0.05 vs. control group).4 Cardiac adiponectin and TNF-αmRNA expression by real-time RT-PCR Relative to intact hearts, TNF-αmRNA expression in hearts with PIR was increased by about three-fold (P < 0.01), while adiponectin mRNA expression in hearts with PIR was decreased by about one-half (P < 0.01). Oral administration of telm significantly attenuated these PIR-related changes in TNF-αand adiponectin mRNA expression.5 Cardiac adiponectin and TNF-αprotein expression by western blotting. Relative to intact hearts, TNF-αprotein expression in hearts with PIR was increased by about three-fold (P < 0.01), while adiponectin protein expression in hearts with PIR was decreased by about one-half (P < 0.01). Oral administration of telm significantly attenuated these PIR-related changes in TNF-αand adiponectin protein expression.6 Cardiac adiponectin and TNF-αprotein expression by immunohistochemical findings Relative to intact hearts, TNF-αprotein expression in hearts with PIR was increased by about three-fold (P < 0.01), while adiponectin protein expression in hearts with PIR was decreased by about one-half (P < 0.01). Oral administration of telm significantly attenuated these PIR-related changes in TNF-αand adiponectin protein expression.Conclusion: Telm attenuated ISO-induced cardiac remodeling through a mechanism that may involve induction of expression of cardiac adiponectin.