Jaana Rysä

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.

Distinct Upregulation of Extracellular Matrix Genes in Transition From Hypertrophy to Hypertensive Heart Failure

Cardiac hypertrophy in response to pressure overload is initially beneficial but eventually leads to heart failure, a major cause of morbidity and mortality in the Western countries. Although abnormalities in left ventricular (LV) diastolic filling are early features associated with pressure overload-induced LV hypertrophy, the molecular mechanisms regulating transition to diastolic heart failure are poorly understood. We analyzed global changes in gene expression in 12-, 16-, and 20-month-old spontaneously hypertensive rats (SHR) and their age-matched controls, Wistar Kyoto rats, using DNA microarrays. In SHR, a progressive LV hypertrophy was associated with increased expression of hypertrophy-associated genes including contractile protein and natriuretic peptide genes. Echocardiography indicated that 16-month-old SHR had features of diastolic dysfunction leading to diastolic failure at age 20 months without significant changes in LV systolic function. Comparison analysis revealed that the extracellular matrix genes strikingly dominated the list of altered genes after transition to the heart failure, whereas there was no major shift in gene expression patterns involved in calcium homeostasis and neurohumoral activation, as well as myofilament contractile and cytoskeletal proteins. The microarray analysis also revealed differential gene expression of several novel factors, such as thrombospondin-4 and matrix Gla protein, as well as unknown expressed sequence tags. Our data show that transition from LV hypertrophy to diastolic hypertensive heart failure is almost exclusively associated with progressive remodeling of the extracellular matrix and provide new insights into the pathogenesis of hypertrophy by suggesting existence of novel regulators of LV remodeling.

Identification of Cell Cycle Regulatory and Inflammatory Genes As Predominant Targets of p38 Mitogen-Activated Protein Kinase in the Heart

Mitogen-activated protein kinases (MAPKs) regulate cardiomyocyte growth and apoptosis in response to extracellular stimulation, but the downstream effectors that mediate their pathophysiological effects remain poorly understood. We determined the targets and role of p38 MAPK in the heart in vivo by using local adenovirus-mediated gene transfer of constitutively active upstream kinase mitogen-activated protein kinase kinase 3b (MKK3bE) and wild-type p38α in rats. DNA microarray analysis of animals with cardiac-specific overexpression of p38 MAPK revealed that 264 genes were upregulated more than 2-fold including multiple genes controlling cell division, cell signaling, inflammation, adhesion, and transcription. A large number of previously unknown p38 target genes were found. Using gel mobility-shift assays we identified several cardiac transcription factors that were directly activated by p38 MAPK. Finally, we determined the functional significance of the altered cardiac gene-expression profile by histological analysis and echocardiographic measurements, which indicated that p38 MAPK overexpression–induced gene expression results in myocardial cell proliferation, inflammation, and fibrosis. In conclusion, we defined the novel target genes and transcription factors as well as the functional effects of p38 MAPK in the heart. Expression profiling of p38 MAPK overexpression identified cell cycle regulatory and inflammatory genes critical for pathological processes in the adult heart.

p38 Kinase rescues failing myocardium after myocardial infarction: evidence for angiogenic and anti-apoptotic mechanisms

As a leading cause of heart failure, postinfarction left ventricular remodeling represents an important target for therapeutic interventions. Mitogen‐activated protein kinases regulate critical cellular processes including stress response and survival, but their role in left ventricular remodeling is unknown. In the present study, rats were subjected to myocardial infarction by ligating the left anterior descending coronary artery. Western blot and kinase assay analysis revealed an inactivation of p38 kinase after myocardial infarction. Local adenovirus‐mediated cotransfection of wild‐type (WT) p38 kinase and constitutively active MKK3b reduced infarct size (26±3% vs. 47±4%, P<0.05 vs. LacZ‐treated control) associated with improved ejection fraction (66.9±5.5% vs. 44.4±4.0%, P<0.001), fractional shortening (30.2±2.1% vs. 19.7±2.2%, P<0.001), and decreased left ventricular diastolic diameter (8.5±0.4 mm vs. 9.5±0.2 mm, P<0.01). p38 kinase gene transfer increased capillary density (2423±107/mm2 vs. 1934±86/mm2, P<0.001) and resulted in microvessel enlargement in the ischemic border zone. Apoptosis (35±7 vs. 69±13 cells, P<0.01) and fibrosis (16±3% vs. 34±8%, P<0.05) were reduced, while the number of c‐kit positive cardiac stem‐like cells remained unchanged. These results indicate that reduced p38 signaling predisposes to adverse postinfarction remodeling. The rescue of failing myocardium with p38 kinase may be a potential new therapy for heart failure after myocardial infarction. —Tenhunen, O., Soini, Y., Ilves, M., Rysä, J., Tuukkanen, J., Serpi, R., Pennanen, H., Ruskoaho, H., Leskinen, H. p38 Kinase rescues failing myocardium after myocardial infarction: evidence for angiogenic and anti‐apoptotic mechanisms. FASEB J. 20, E1276‐E1286 (2006)

Microarray Analysis of the Global Alterations in the Gene Expression in the Placentas From Cigarette-smoking Mothers

The effects of maternal cigarette smoking on the transcriptome of human full‐term placentas were investigated by a microarray analysis. QPCR was performed for a selected set of metabolizing genes. Differentially expressed genes were selected by fold change (±1.5‐fold) and analysis of variance (P<0.05) between the control and smoker groups. The expression of 174 probe sets was affected significantly. Chronic cigarette smoking induced the expression of CYP1A1. A trend toward a decrease in the expression of several steroid hormone‐metabolizing enzymes, including CYP19A1, was detected. The expression of phase II enzymes was not altered, and no enriched categories were observed among the regulated genes, except for aryl hydrocarbon receptor (AhR)‐CYP1A1. The unaltered expression of phase II enzymes may result in an increase in the levels of active metabolites and elevated oxidative chemical stress in the placenta and the fetus. On the basis of our results, it seems that cigarette smoke acts as a hormone disrupter in the placenta.

ABCG2/BCRP decreases the transfer of a food-born chemical carcinogen, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in perfused term human placenta.

We have studied the role of ATP binding cassette (ABC) transporters in fetal exposure to carcinogens using 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) a known substrate for ABC transporters as a model compound. In perfusion of human term placenta, transfer of (14)C-PhIP (2 microM) through the placenta resulted in fetal-to-maternal concentration ratio (FM ratio) of 0.72+/-0.09 at 6 h. The specific ABCG2 inhibitor KO143 increased the transfer of (14)C-PhIP from maternal to fetal circulation (FM ratio 0.90+/-0.08 at 6 h, p<0.05) while the ABCC1/ABCC2 inhibitor probenecid had no effect (FM ratio at 6 h 0.75+/-0.10, p=0.84). There was a negative correlation between the expression of ABCG2 protein in perfused tissue and the FM ratio of (14)C-PhIP (R=-0.81, p<0.01) at the end of the perfusion. The expression of ABCC2 protein did not correlate with FM ratio of PhIP (R: -0.11, p=0.76). In addition, PhIP induced the expression of ABC transporters in BeWo cells at mRNA level. In conclusion, our data indicates that ABCG2 decreases placental transfer of (14)C-PhIP in perfused human placenta. Also, PhIP may modify ABC transporter expression in choriocarcinoma cells.

GATA-4 Is an Angiogenic Survival Factor of the Infarcted Heart

Background—Recent data suggest that GATA-4 is an antiapoptotic factor required for adaptive responses and a key regulator of hypertrophy and hypertrophy-associated genes in the heart. As a leading cause of chronic heart failure, reversal of postinfarction left ventricular remodeling represents an important target for therapeutic interventions. Here, we studied the role of GATA-4 as a mediator of postinfarction remodeling in rats. Methods and Results—Myocardial infarction, caused by ligating the left anterior descending coronary artery, significantly decreased the DNA binding activity of GATA-4 at day 1, whereas at 2 weeks the GATA-4 DNA binding was significantly upregulated. To determine the functional role of GATA-4, peri-infarct intramyocardial delivery of adenoviral vector expressing GATA-4 was done before left anterior descending coronary artery ligation. Hearts treated with GATA-4 gene transfer exhibited significantly increased ejection fraction and fractional shortening. Accordingly, infarct size was significantly reduced. To determine the cardioprotective mechanisms of GATA-4, myocardial angiogenesis, rate of apoptosis, c-kit+ cardiac stemlike cells, and genes regulated by GATA-4 were studied. The number of capillaries and stemlike cells was significantly increased, and decreased apoptosis was observed. Conclusion—These results indicate that the reversal of reduced GATA-4 activity prevents adverse postinfarction remodeling through myocardial angiogenesis, antiapoptosis, and stem cell recruitment. GATA-4–based gene transfer may represent a novel, efficient therapeutic approach for heart failure.

Vascular endothelial growth factor-B gene transfer prevents angiotensin II-induced diastolic dysfunction via proliferation and capillary dilatation in rats

AIMS heart growth and function are angiogenesis-dependent, but little is known concerning the effects of key regulators of angiogenesis on diastolic heart failure. Here, we tested the hypothesis that local vascular endothelial growth factor-B (VEGF-B) gene therapy prevents left ventricular diastolic dysfunction. METHODS AND RESULTS rats were subjected to pressure overload by infusing angiotensin II (33.3 microg/kg/h) for 2 weeks using osmotic minipumps. Intramyocardial delivery of adenoviral vector expressing VEGF-B(167A) improved the angiotensin II-induced diastolic dysfunction compared with LacZ control virus. Local VEGF-B gene transfer increased the mean capillary area in the left ventricle in control and angiotensin II-infused animals, whereas the density of capillaries was not affected. Interestingly, significant increases were noted in Ki67(+) proliferating cells, expression of interleukin1β, and c-kit(+) cells in response to VEGF-B gene transfer. The increase in cardiac c-kit(+) cells was not associated with an induction of stromal cell-derived factor 1α, suggesting no mobilization of cells from bone marrow. Also, the phosphatidylinositol 3-kinase/Akt pathway was activated. CONCLUSION VEGF-B gene transfer resulted in prevention of the angiotensin II-induced diastolic dysfunction associated with induction of the Akt pathway, increased proliferation and number of c-kit(+) cells, as well as an increase in the capillary area in the left ventricle. VEGF-B may offer novel therapeutic possibilities for the prevention of the transition from compensated to decompensated cardiac hypertrophy and thereby for the treatment of heart failure.

Thrombospondin-4 expression is rapidly upregulated by cardiac overload

The precise mechanisms regulating gene expression of thrombospondins (TSPs) in the heart remain incompletely understood. Here we characterized cardiac TSP-4 expression in response to pressure overload and myocardial infarction in vivo. Arginine(8)-vasopressin (AVP) infusion increased left ventricular (LV) TSP-4 mRNA levels within 30 min. Also angiotensin II infusion rapidly activated LV TSP-4 expression, TSP-4 mRNA levels being highest at 6h and protein at 72 h and 2 weeks. During remodeling process following myocardial infarction, LV TSP-4 mRNA levels increased at day one, as studied by quantitative RT-PCR. TSP-4 immunostaining was localized to endothelial cells in hypertrophied hearts of spontaneously hypertensive rats. AVP-infusion increased LV TSP-1 mRNA levels similarly to TSP-4 within 30 min showing that rapid induction of gene expression, well before the development of cardiac hypertrophy, is typical for the thrombospondin family. These results further suggest that TSP-4 may be an endothelial specific marker of cardiac overload.

Collagen XV Is Necessary for Modeling of the Extracellular Matrix and Its Deficiency Predisposes to Cardiomyopathy

Rationale: The extracellular matrix (ECM) is a major determinant of the structural integrity and functional properties of the myocardium in common pathological conditions, and changes in vasculature contribute to cardiac dysfunction. Collagen (Col) XV is preferentially expressed in the ECM of cardiac muscle and microvessels. Objective: We aimed to characterize the ECM, cardiovascular function and responses to elevated cardiovascular load in mice lacking Col XV (Col15a1−/−) to define its functional role in the vasculature and in age- and hypertension-associated myocardial remodeling. Methods and Results: Cardiac structure and vasculature were analyzed by light and electron microscopy. Cardiac function, intraarterial blood pressure, microhemodynamics, and gene expression profiles were studied using echocardiography, telemetry, intravital microscopy, and PCR, respectively. Experimental hypertension was induced with angiotensin II or with a nitric oxide synthesis inhibitor. Under basal conditions, lack of Col XV resulted in increased permeability and impaired microvascular hemodynamics, distinct early-onset and age-dependent defects in heart structure and function, a poorly organized fibrillar collagen matrix with marked interstitial deposition of nonfibrillar protein aggregates, increased tissue stiffness, and irregularly organized cardiomyocytes. In response to experimental hypertension, Col15a1 gene expression was increased in the left ventricle of wild-type mice, and mRNA expression of natriuretic peptides (ANP and BNP) and ECM modeling were abnormal in Col15a1−/− mice. Conclusions: Col XV is necessary for ECM organization in the heart, and for the structure and functions of microvessels. Col XV deficiency leads to a complex cardiac phenotype and predisposes the subject to pathological responses under cardiac stress.