Heikki Tokola

Apelin, the Novel Endogenous Ligand of the Orphan Receptor APJ, Regulates Cardiac Contractility

Abstract— The orphan receptor APJ and its recently identified endogenous ligand, apelin, exhibit high levels of mRNA expression in the heart. However, the functional importance of apelin in the cardiovascular system is not known. In isolated perfused rat hearts, infusion of apelin (0.01 to 10 nmol/L) induced a dose-dependent positive inotropic effect (EC50: 33.1±1.5 pmol/L). Moreover, preload-induced increase in dP/dtmax was significantly augmented (P <0.05) in the presence of apelin. Inhibition of phospholipase C (PLC) with U-73122 and suppression of protein kinase C (PKC) with staurosporine and GF-109203X markedly attenuated the apelin-induced inotropic effect (P <0.001). In addition, zoniporide, a selective inhibitor of Na+-H+ exchange (NHE) isoform-1, and KB-R7943, a potent inhibitor of the reverse mode Na+-Ca2+ exchange (NCX), significantly suppressed the response to apelin (P <0.001). Perforated patch-clamp recordings showed that apelin did not modulate L-type Ca2+ current or voltage-activated K+ currents in isolated adult rat ventricular myocytes. Apelin mRNA was markedly downregulated in cultured neonatal rat ventricular myocytes subjected to mechanical stretch and in vivo in two models of chronic ventricular pressure overload. The present study provides the first evidence for the physiological significance of apelin in the heart. Our results show that apelin is one of the most potent endogenous positive inotropic substances yet identified and that the inotropic response to apelin may involve activation of PLC, PKC, and sarcolemmal NHE and NCX.

GATA-4 is a nuclear mediator of mechanical stretch-activated hypertrophic program

In overloaded heart the cardiomyocytes adapt to increased mechanical and neurohumoral stress by activation of hypertrophic program, resulting in morphological changes of individual cells and specific changes in gene expression. Accumulating evidence suggests an important role for the zinc finger transcription factor GATA-4 in hypertrophic agonist-induced cardiac hypertrophy. However, its role in stretch-induced cardiomyocyte hypertrophy is not known. We employed an in vitro mechanical stretch model of cultured cardiomyocytes and used rat B-type natriuretic peptide promoter as stretch-sensitive reporter gene. Stretch transiently increased GATA-4 DNA binding activity and transcript levels, which was followed by increases in the expression of B-type natriuretic peptide as well as atrial natriuretic peptide and skeletal α-actin genes. The stretch inducibility mapped primarily to the proximal 520 bp of the B-type natriuretic peptide promoter. Mutational studies showed that the tandem GATA consensus sites of the proximal promoter in combination with an Nkx-2.5 binding element are critical for stretch-activated B-type natriuretic peptide transcription. Inhibition of GATA-4 protein production by adenovirus-mediated transfer of GATA-4 antisense cDNA blocked stretch-induced increases in B-type natriuretic peptide transcript levels and the sarcomere reorganization. The proportion of myocytes with assembled sarcomeres in control adenovirus-infected cultures increased from 14 to 59% in response to stretch, whereas the values for GATA-4 antisense-treated cells were 6 and 13%, respectively. These results show that activation of GATA-4, in cooperation with a factor binding on Nkx-2.5 binding element, is essential for mechanical stretch-induced cardiomyocyte hypertrophy.

Involvement of Transcriptional and Posttranscriptional Mechanisms in Cardiac Overload–Induced Increase of B-Type Natriuretic Peptide Gene Expression

The induction of atrial and ventricular B-type natriuretic peptide (BNP) gene expression is one of the earliest events occurring during hemodynamic overload. To examine the molecular mechanisms for increased BNP gene expression during cardiac overload, we studied the induction of the BNP gene expression compared with that of atrial natriuretic peptide (ANP) in a modified perfused rat heart preparation. An increase in right atrial pressure of 5 mm Hg resulted in a 1.4-fold (P < .05) and 2.2-fold (P < .01) increase in BNP mRNA levels after 1 and 2 hours, respectively, whereas ANP mRNA levels remained unchanged. Stretching for up to 2 hours also significantly increased right atrial immunoreactive BNP (ir-BNP) levels (from 15.8 +/- 2.2 to 20.1 +/- 1.2 ng/mg, P < .05). Actinomycin D (10 micrograms/mL), a transcriptional inhibitor, completely inhibited the stretch-induced increase in atrial BNP mRNA levels at 1 hour (P < .05) and 2 hours (P < .001), whereas a protein synthesis inhibitor, cycloheximide (90 micrograms/mL), had no effect on basal or direct mechanical stretch-induced increase in right atrial BNP mRNA levels. Furthermore, we examined the role of tyrosine kinase and protein kinase C activities in acute mechanical stretch-induced increase in BNP synthesis. Tyrosine kinase inhibitors lavendustin A (1 mumol/L) and tyrphostin A25 (3 mumol/L) and protein kinase C inhibitors staurosporine (30 nmol/L) and chelerythrine (1 mumol/L) prevented the stretch-induced increase in right atrial ir-BNP concentrations at 2 hours. In addition, chelerythrine inhibited the increase of right atrial BNP mRNA levels stimulated by cardiac overload. These resuls demonstrate that the early increase of BNP mRNA levels by mechanical stretch results from increased transcriptional activation and is independent of protein synthesis. Our results also suggest that protein kinase C and tyrosine kinases activities may be involved in coupling cardiac overload to alterations in atrial BNP synthesis.

Pressure Overload Increases GATA4 Binding Activity via Endothelin-1

Background —The signaling cascades responsible for the activation of transcription factors in the hypertrophic growth of cardiac myocytes during hemodynamic overload are largely unknown. Several of the genes upregulated in the hypertrophied heart, including B-type natriuretic peptide (BNP) gene, are controlled by the cardiac-restricted zinc finger transcription factor GATA4. Methods and Results —An in vivo model of intravenous administration of arginine8-vasopressin (AVP) for up to 4 hours in conscious normotensive rats was used to study the signaling mechanisms for GATA activation in response to pressure overload. Gel mobility shift assays were used to analyze the trans-acting factors that interact with the GATA motifs of the BNP promoter. AVP-induced increase in mean arterial pressure was followed by a significant increase in the BNP and c-fos mRNA levels in both the endocardial and epicardial layers of the left ventricle, whereas GATA4 and GATA6 mRNA levels remained unchanged. Pressure overload within 15 to 60 minutes produced an increase in left ventricular BNP GATA4 but not GATA5 and GATA6 binding activity, and at 30 minutes a 2.2-fold increase (P <0.001) in GATA4 binding was noted. The mixed endothelin-1 ETA/ETB receptor antagonist bosentan but not the angiotensin II type 1 receptor antagonist losartan completely inhibited the pressure overload–induced increase in left ventricular BNP GATA4 binding activity. Bosentan alone had no statistically significant effect on GATA4 binding activity of the left ventricle in conscious animals. Conclusions —ET-1 is a signaling molecule that rapidly upregulates GATA4 DNA binding activity in response to pressure overload in vivo.

Evidence for a Functional Role of Angiotensin II Type 2 Receptor in the Cardiac Hypertrophic Process In Vivo in the Rat Heart

Background—The precise function of angiotensin II type 2 receptor (AT2-R) in the mammalian heart in vivo is unknown. Here, we investigated the role of AT2-R in cardiac pressure overload. Methods and Results—Rats were infused with vehicle, angiotensin II (Ang II), PD123319 (an AT2-R antagonist), or the combination of Ang II and PD123319 via subcutaneously implanted osmotic minipumps for 12 or 72 hours. Ang II–induced increases in mean arterial pressure, left ventricular weight/body weight ratio, and elevation of skeletal &agr;-actin and &bgr;-myosin heavy chain mRNA levels were not altered by PD123319. In contrast, AT2-R blockade resulted in a marked increase in the gene expression of c-fos, endothelin-1, and insulin-like growth factor-1 in Ang II–induced hypertension. In parallel, Ang II–stimulated mRNA and protein expression of atrial natriuretic peptide were significantly augmented by AT2-R blockade. Moreover, PD123319 markedly increased the synthesis of B-type natriuretic peptide. Furthermore, the expression of vascular endothelial growth factor and fibroblast growth factor-1 was downregulated by Ang II only in the presence of AT2-R blockade. Conclusions—Our results provide evidence that AT2-R plays a functional role in the cardiac hypertrophic process in vivo by selectively regulating the expression of growth-promoting and growth-inhibiting factors.

Endothelin-1-specific activation of B-type natriuretic peptide gene via p38 mitogen-activated protein kinase and nuclear ETS factors

Terminally differentiated cardiac myocytes adapt to mechanical and neurohumoral stress via morphological changes of individual cells accompanied by reactivation of fetal pattern of gene expression. Endothelin-1, a powerful paracrine mediator of myocyte growth, induces similar changes in cultured cardiac myocytes as those seen in hypertrophied heart in vivo. By using rat B-type natriuretic peptide promoter, we identified a novel ETS binding sequence, on which nuclear protein binding is activated in endothelin-1-treated cultured cardiac myocytes. This sequence binds ETS-like gene-1 transcription factor and mediates endothelin-1-specific activation of transcription, but not responses to increased calcium signaling via l-type calcium channels, angiotensin II treatment, or mechanical stretch of myocytes. Interestingly, endothelin-1 activated signaling converges via p38 mitogen-activated protein kinase-dependent mechanism on ETS binding site, whereas this element inhibits extracellular signal-regulated kinase activated transcription. In conclusion, given the fundamental role of the interaction of mitogen-activated protein kinases and ETS factors in regulation of eukaryotic cell differentiation, growth, and oncogenesis, these results provide the unique evidence of a endothelin-1- and mitogen-activated protein kinase-regulated ETS factor pathway for cardiac myocytes.

Basal and acidic fibroblast growth factor-induced atrial natriuretic peptide gene expression and secretion is inhibited by staurosporine

We examined the mechanisms involved in the activation of atrial natriuretic peptide (ANP) gene expression and secretion in response to acidic fibroblast growth factor (aFGF) by studying the effects of staurosporine, a protein kinase C inhibitor, and 12-O-tetradecanoyl phorbol 13-acetate (TPA), an activator of protein kinase C, on basal and AFGF-induced ANP messenger RNA (mRNA) and immunoreactive ANP (IR-ANP) levels in cultured neonatal rat cardiac myocytes. Acidic FGF caused a dose- and time-dependent increase in IR-ANP and immunoreactive N-terminal fragment of proANP (IR-NT-proANP) release into the culture medium from ventricular but not from atrial myocytes. In ventricular cells, 50 ng/ml aFGF for 24 or 48 h resulted in a 70% or 181% increase, respectively, in the accumulation of IR-ANP into the culture medium. Acidic FGF also stimulated ANP gene expression significantly; after 48 h of incubation, the ANP mRNA levels of aFGF-treated ventricular myocytes were 205% (P < 0.001) higher than those of control cells. Staurosporine alone at concentration of 10 nM significantly decreased the basal IR-ANP and IR-NT-proANP secretion, and inhibited the aFGF-induced increase in ANP mRNA and IR-ANP levels in ventricular myocytes. TPA (100 nM) alone significantly stimulated ANP gene expression and secretion but these effects were not augmented by combining aFGF with TPA. High performance liquid chromatographical analysis showed that atrial and ventricular myocytes maintained in serum-free medium were capable of secreting processed, ANP99-126 sized material, and that aFGF did not alter the processing of ANP in ventricular cultures. These results demonstrate that aFGF is a potent stimulator of ANP gene expression and secretion in cultured neonatal rat ventricular but not in atrial cells. The observations that (a) staurosporine completely abolished the effects of aFGF on ANP gene expression and release and (b) ANP secretory and gene expression inducing effects of phorbol ester were not augmented by aFGF, suggest an important role of protein kinase C in mediating aFGF-induced ANP gene expression and secretion.

Tumour necrosis factor-like weak inducer of apoptosis (TWEAK) and its receptor Fn14 during cardiac remodelling in rats.

Aim:  Accumulating evidence supports the concept that proinflammatory cytokines play an essential role in the failing heart. We examined the concomitant tumour necrosis factor‐like weak inducer of apoptosis (TWEAK)/Fn14 expression in myocytes in vitro as well as in vivo in cardiac remodelling.

Intramyocardial BNP Gene Delivery Improves Cardiac Function Through Distinct Context-Dependent Mechanisms

Background—B-type natriuretic peptide (BNP) is an endogenous peptide produced under physiological and pathological conditions mainly by ventricular myocytes. It has natriuretic, diuretic, blood pressure–lowering, and antifibrotic actions that could mediate cardiorenal protection in cardiovascular diseases. In the present study, we used BNP gene transfer to examine functional and structural effects of BNP on left ventricular (LV) remodeling. Methods and Results—Human BNP was overexpressed by using adenovirus-mediated gene delivery in normal rat hearts and in hearts during the remodeling process after infarction and in an experimental model of angiotensin II–mediated hypertension. In healthy hearts, BNP gene delivery into the anterior wall of the LV decreased myocardial fibrosis (P<0.01, n=7 to 8) and increased capillary density (P<0.05, n=7 to 8) associated with a 7.3-fold increase in LV BNP peptide levels. Overexpression of BNP improved LV fractional shortening by 22% (P<0.05, n=6 to 7) and ejection fraction by 19% (P<0.05, n=6 to 7) after infarction. The favorable effect of BNP gene delivery on cardiac function after infarction was associated with normalization of cardiac sarcoplasmic reticulum Ca2+-ATPase expression and phospholamban Thr17-phosphorylation. BNP gene delivery also improved fractional shortening and ejection fraction in angiotensin II–mediated hypertension as well as decreased myocardial fibrosis and LV collagen III mRNA levels but had no effect on angiogenesis or Ca2+-ATPase expression and phospholamban phosphorylation. Conclusions—Local intramyocardial BNP gene delivery improves cardiac function and attenuates adverse postinfarction and angiotensin II–induced remodeling. These results also indicate that myocardial BNP has pleiotropic, context-dependent, favorable actions on cardiac function and suggest that BNP acts locally as a key mechanical load–activated regulator of angiogenesis and fibrosis.

Cardiac BNP gene activation by angiotensin II in vivo

The transcription factors involved in the activation of cardiac gene expression by angiotensin II (Ang II) in vivo are not well understood. Here we studied the contribution of transcriptional elements to the activation of the cardiac B-type natriuretic peptide (BNP) gene promoter by Ang II in conscious rats and in angiotensin II type 1 receptor (AT1R) transgenic mice. Rat BNP luciferase reporter gene constructs were injected into the left ventricular wall. The mean luciferase activity was 1.8-fold higher (P<0.05) in the ventricles of animals subjected to 2-week Ang II infusion as compared with vehicle infusion. Our results indicate that GATA binding sites at -90 and -81 in the rat BNP promoter are essential for the in vivo response to Ang II. The GATA factor binding to these sites is GATA-4. BNP mRNA levels and GATA-4 binding activity are also increased in the hypertrophied hearts of aged AT1R transgenic mice.