A Family With A20 Haploinsufficiency Presenting With Novel Clinical Manifestations and Challenges for Treatment.

BACKGROUND: Tumor necrosis factor α-induced protein 3 gene (TNFAIP3, also called A20) haploinsufficiency (HA20) leads to autoinflammation and autoimmunity. We have recently shown that a p.(Lys91*) mutation in A20 disrupts nuclear factor κB signaling, impairs protein-protein interactions of A20, and leads to inflammasome activation. METHODS: We now describe the clinical presentations and drug responses in a family with HA20 p.(Lys91*) mutation, consistent with our previously reported diverse immunological and functional findings. RESULTS: We report for the first time that inflammasome-mediated autoinflammatory lung reaction caused by HA20 can be treated with interleukin 1 antagonist anakinra. We also describe severe anemia related to HA20 successfully treated with mycophenolate. In addition, HA20 p.(Lys91*) was found to associate with autoimmune thyroid disease, juvenile idiopathic arthritis, psoriasis, liver disease, and immunodeficiency presenting with specific antibody deficiency and genital papillomatosis. CONCLUSIONS: We conclude that HA20 may lead to combination of inflammation, immunodeficiency, and autoimmunity. The condition may present with variable and unpredictable symptoms with atypical treatment responses.

Prediction of renal outcome in Henoch-Schönlein nephritis based on biopsy findings.

BACKGROUND: In Henoch-Schönlein nephritis (HSN), a risk factor for unfavorable outcome is prolonged proteinuria, but the value of renal biopsies in prognosis assessment is debatable. METHODS: We evaluated serial renal biopsies from 26 HSN patients. Follow-up biopsy occurred at median 2.1 years after diagnostic biopsy. Patients formed two groups at the follow-up biopsy: patients without proteinuria (group I; n = 11) and with proteinuria (group II; n = 15). Biopsies underwent evaluation according to three classifications: International Study of Kidney Disease in Children (ISKDC), Oxford (MEST-C), and semiquantitative classification (SQC) including an activity and chronicity score. Analysis also included expression of pro-fibrotic (alpha-smooth muscle actin and vimentin) and inflammatory (P-selectin glycoprotein ligand-1) molecules in the diagnostic biopsy specimens. Definition of unfavorable outcome was active renal disease or reduced renal function at last follow-up. RESULTS: Between the biopsies, SQC chronicity score increased in 22 (85%) patients, whereas activity score and ISKDC grade decreased in 21 (81%) and 17 (65%), respectively. Of the MEST-C parameters, endocapillary proliferation (from 83 to 13%; p < 0.001) and crescents (from 63 to 25%; p = 0.022) showed significant reduction, and segmental glomerulosclerosis (from 38 to 79%; p = 0.006) significant increment. These changes occurred similarly in groups I and II. Expression of the pro-fibrotic and inflammatory molecules showed no clinically significant differences between groups I and II. None in group I and five (33%) patients in group II had unfavorable outcome (p = 0.053). CONCLUSIONS: Our results suggest that follow-up biopsies provide limited additional information to clinical symptoms in HSN outcome prediction.

Effects of oxonic acid-induced hyperuricemia on mesenteric artery tone and cardiac load in experimental renal insufficiency.

BACKGROUND: Recent studies suggest a causal role for increased plasma uric acid in the progression of chronic renal insufficiency (CRI). However, uric acid also functions as an antioxidant with possible beneficial effects. METHODS: We investigated the influence of hyperuricemia on mesenteric arterial tone (main and second order branch) and morphology in experimental CRI. Forty-four Sprague-Dawley rats were 5/6 nephrectomized (NX) or Sham-operated and fed 2.0% oxonic acid or control diet for 9 weeks. RESULTS: Oxonic acid feeding elevated plasma uric acid levels 2.4 and 3.6-fold in the NX and Sham groups, respectively. Plasma creatinine and urea were elevated 2-fold and blood pressure increased by 10 mmHg in NX rats, while hyperuricemia did not significantly influence these variables. Right and left ventricular weight, and atrial and B-type natriuretic peptide mRNA content were increased in NX rats, but were not affected by hyperuricemia. In the mesenteric artery, hyperuricemia did not influence vasoconstrictor responses in vitro to norepinephrine or potassium chloride. The small arteries of NX rats featured hypertrophic remodeling independent of uric acid levels: wall to lumen ratio, wall thickness and cross-sectional area were increased without changes in lumen diameter. In the main branch, vasorelaxations to acetylcholine were impaired in NX rats, but were not affected by hyperuricemia. In contrast, relaxations to the large-conductance Ca(2+)-activated K(+)-channel (BKCa) opener NS-1619 were reduced by oxonic acid feeding, whereas responses to nitroprusside were not affected. CONCLUSIONS: Experimental hyperuricemia did not influence cardiac load or vascular remodeling, but impaired BKCa -mediated vasorelaxation in experimental CRI.

Thymic neuroendocrine tumors in patients with multiple endocrine neoplasia type 1.

OBJECTIVE: MEN1 is associated with an increased risk of developing tumors in different endocrine organs. Neuroendocrine tumors of the thymus (TNETs) are very rare but often have an aggressive nature. We evaluated patients with MEN1 and TNET in three university hospitals in Finland. DESIGN/METHODS: We evaluated patient records of 183 MEN1-patients from three university hospitals between the years 1985-2019 with TNETs. Thymus tumor specimens were classified according to the new WHO 2021 classification of TNET. We collected data on treatments and outcomes of these patients. RESULTS: There were six patients (3.3%) with MEN1 and TNET. Five of them had the same common gene mutation occurring in Finland. They originated from common ancestors encompassing two pairs of brothers from sequential generations. The mean age at presentation of TNET was 44.7 ± 11.9 years. TNET was classified as atypical carcinoid (AC) in five out of six patients. One patient had a largely necrotic main tumor with very few mitoses and another nodule with 25 mitoses per 2 mm2, qualifying for the 2021 WHO diagnosis of large cell neuroendocrine carcinoma (LCNEC). In our patients, the 5-year survival of the TNET patients was 62.5% and 10-year survival 31.3%. CONCLUSION: In this study, TNETs were observed in one large MEN1 founder pedigree, where an anticipation-like earlier disease onset was observed in the most recent generation. TNET in MEN1 patients is an aggressive disease. The prognosis can be better by systematic screening. We also show that LCNEC can be associated with TNET in MEN1 patients.

Key roles of endothelin-1 and p38 MAPK in the regulation of atrial stretch response.

Mechanisms regulating stretch response in the left ventricle are investigated in detail but not well understood in atrial myocardium. Hypertrophic growth of atrial myocardium contributes to the pathogenesis of atrial fibrillation. In this study, we sought to elucidate mechanisms of stretch-induced activation of key signaling pathways and hypertrophy-associated genes in rat atria. Stretching of isolated atria induced a rapid increase in phosphorylation of p38 MAPK and ERK and induced a p38 MAPK-dependent increase in DNA binding activity of transcription factors Elk-1 and GATA-4. Inhibition of the ERK pathway had no effect on the cardiac transcription factors studied. Stretch-induced increase in atrial contractile function was substantially enhanced by inhibition of p38 MAPK. p38 MAPK also regulated stretch-induced increase in c-fos, ß-myosin heavy chain, B-type natriuretic peptide mRNA levels, and atrial natriuretic peptide secretion in isolated atria. Various autocrine/paracrine factors are known to mediate the stretch response in the left ventricle. Stretching of isolated atria resulted in a robust increase in endothelin-1 (ET-1) mRNA levels, while apelin and adrenomedullin signaling cascades were downregulated. Administration of mixed ET(A/B) receptor antagonist bosentan attenuated the stretch-induced activation of GATA-4 in isolated atria, whereas ANG II receptor type-1 antagonist CV-11974 had no effect. Moreover, analysis of RNA from intact atrial and ventricular myocardium revealed significantly higher mRNA levels of ET(A) receptor and ET converting enzyme-1 in atrial compared with ventricular myocardium. In conclusion, our findings identify the local ET-1 system and p38 MAPK as key regulators of load-induced hypertrophic response in isolated rat atria.

M-CAT element mediates mechanical stretch-activated transcription of B-type natriuretic peptide via ERK activation.

The muscle-CAT (M-CAT) promoter element is found on promoters of most muscle-specific cardiac genes, but its role in cardiac pathology is poorly understood. Here we studied whether the M-CAT element is involved in hypertrophic process activated by mechanical stretch, and identified the intracellular pathways mediating the response. When an in vitro stretch model of cultured neonatal rat cardiomyocytes and luciferase reporter construct driven by rat B-type natriuretic peptide (BNP) promoter were used, mutation of M-CAT element inhibited not only the basal reporter activity (88%), but also the stretch-activated BNP transcription (58%, p < 0.001). Stretch-induced BNP promoter activation was associated with an increase in transcriptional enhancer factor-1 (TEF-1) binding activity after 24 h mechanical stretch (p < 0.05). Inhibition of mitogen-activated protein kinases ERK, JNK, or p38 attenuated stretch-induced BNP activation. Interestingly, as opposed to p38 and JNK, inhibition of ERK had no additional effect on transcriptional activity of BNP promoter harboring the M-CAT mutation, suggesting a pivotal role for ERK in regulating stretch-induced BNP transcription via M-CAT binding site. Finally, immunoprecipitation studies showed that mechanical stretch induced myocyte enhancer factor-2 (MEF-2) binding to TEF-1. These data suggest a central role for M-CAT element in regulation of mechanical stretch-induced hypertrophic response via ERK activation.

A novel p38 MAPK target dyxin is rapidly induced by mechanical load in the heart.

Dyxin is a novel LIM domain protein acting as a transcriptional cofactor with GATA transcription factors. Here, we characterized dyxin as a p38 mitogen-activated protein kinase (MAPK) regulated gene, since combined upstream MAPK kinase 3b and wild-type p38 alpha MAPK gene transfer increased left ventricular dyxin mRNA and protein levels in vivo. We also studied cardiac dyxin expression in experimental models of pressure overload and myocardial infarction (MI) in vivo. Angiotensin II infusion increased left ventricular dyxin mRNA levels (9.4-fold, p<0.001) rapidly at 6 h followed by induction of protein levels. Furthermore, simultaneous administration of p38 MAPK inhibitor SB203580 abolished angiotensin II-induced activation of dyxin gene expression. During the post-infarction remodeling process, increased dyxin mRNA levels (7.7-fold, p<0.01) were noted at day 1 followed by the increase in proteins levels at 2 weeks after MI (1.5-fold, p<0.05). Moreover, direct wall stretch by using isolated rat heart preparation as well as direct mechanical stretch of cardiomyocytes in vitro activated dyxin gene expression within 1 h. Our results indicate that dyxin expression is rapidly upregulated in response to mechanical load, this increase being at least partly mediated by p38 MAPK. These results suggest that dyxin may play an important role in regulating hypertrophic process.

Mechanical stretch induced transcriptomic profiles in cardiac myocytes.

Mechanical forces are able to activate hypertrophic growth of cardiomyocytes in the overloaded myocardium. However, the transcriptional profiles triggered by mechanical stretch in cardiac myocytes are not fully understood. Here, we performed the first genome-wide time series study of gene expression changes in stretched cultured neonatal rat ventricular myocytes (NRVM)s, resulting in 205, 579, 737, 621, and 1542 differentially expressed (>2-fold, P < 0.05) genes in response to 1, 4, 12, 24, and 48 hours of cyclic mechanical stretch. We used Ingenuity Pathway Analysis to predict functional pathways and upstream regulators of differentially expressed genes in order to identify regulatory networks that may lead to mechanical stretch induced hypertrophic growth of cardiomyocytes. We also performed micro (miRNA) expression profiling of stretched NRVMs, and identified that a total of 8 and 87 miRNAs were significantly (P < 0.05) altered by 1-12 and 24-48 hours of mechanical stretch, respectively. Finally, through integration of miRNA and mRNA data, we predicted the miRNAs that regulate mRNAs potentially leading to the hypertrophic growth induced by mechanical stretch. These analyses predicted nuclear factor-like 2 (Nrf2) and interferon regulatory transcription factors as well as the let-7 family of miRNAs as playing roles in the regulation of stretch-regulated genes in cardiomyocytes.

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.

Apelin, the novel endogenous ligand of the orphan receptor APJ, regulates cardiac contractility.

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/dt(max) 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.

Mitogen-activated protein kinase p38 target regenerating islet-derived 3γ expression is upregulated in cardiac inflammatory response in the rat heart.

Regenerating islet-derived 3γ (Reg3γ) is a multifunctional protein, associated with various tissue injuries and inflammatory states. Since chronic inflammation is characteristics also for heart failure, the aim of this study was to characterize Reg3γ expression in cardiac inflammatory conditions. Reg3γ expression was studied in experimental rat models of myocardial infarction (MI) and pressure overload in vivo. For cell culture studies neonatal rat cardiac myocytes (NRCMs) were used. In addition, adenovirus-mediated gene transfer of upstream mitogen-activated protein kinase (MAPK) kinase 3b and p38α MAPK in vivo and in vitro was performed. Reg3γ mRNA (12.8-fold, P < 0.01) and protein (5.8-fold, P < 0.001) levels were upregulated during the postinfarction remodeling at day 1 after MI, and angiotensin II (Ang II) markedly increased Reg3γ mRNA levels from 6 h to 2 weeks. Immunohistochemistry revealed that the Ang II-induced expression of Reg3γ was localized into the cardiac fibroblasts and myofibroblasts of the proliferating connective tissue in the heart. Stretching and treatments with endothelin-1, lipopolysaccharide (LPS), and fibroblast growth factor-1 increased Reg3γ mRNA levels in NRCMs. SB203580, a selective p38 MAPK inhibitor, markedly attenuated LPS and mechanical stretch-induced upregulation of Reg3γ gene expression. Moreover, combined overexpression of MKK3bE and WT p38α increased Reg3γ gene expression in cultured cardiomyocytes in vitro and in the rat heart in vivo. Our study shows that cardiac stress activates Reg3γ expression and p38 MAPK is an upstream regulator of Reg3γ gene expression in heart. Altogether our data suggest Reg3γ is associated with cardiac inflammatory signaling.

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 alpha-actin and beta-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.

Decoy oligonucleotide characterization of GATA-4 transcription factor in hypertrophic agonist induced responses of cardiac myocytes.

GATA-4 transcription factor is required for normal cardiac development. However, it is unknown whether GATA-4 is an essential mediator of hypertrophic responses in the heart. Rat B-type natriuretic peptide (BNP) gene promoter contains a region of two adjacent GATA binding sites (between -68 and -97) with high affinity for GATA-4. In order to block GATA-4 dependent signaling in cultured neonatal rat ventricular myocytes we administered a synthetic 30-bp phosphorothioated double-stranded DNA complementary to the rat BNP promoter region (between -68 and -97) as a "decoy" cis-element to bind GATA-4. GATA decoy oligodeoxynucleotide treatment of cardiomyocytes blocked GATA-4 DNA binding activity in electrophoretic mobility shift analysis and decreased baseline expression of cardiac natriuretic peptides and GATA-dependent promoter activity. In contrast, blocked GATA-4 DNA binding did not prevent endothelin-1 or phenylephrine induced expression of cardiac natriuretic peptides. Mutation of GATA binding sites at -80 and -91 rat BNP promoter downregulated baseline but did not affect endothelin-1 or angiotensin II induced promoter activity. Additively, GATA decoy oligodeoxynucleotide treatment was insufficient to block endothelin-1 induced activation of protein synthesis or sarcomeric protein assembly. In conclusion, a targeted disruption of GATA-4 DNA binding activity is insufficient to prevent hypertrophic agonist induced responses of ventricular myocytes.

GATA transcription factors in the developing and adult heart.

During the past decade, emerging evidence has accumulated of different nuclear transcription factors in regulation of cardiac development and growth as well as in cardiac hypertrophy and heart failure. GATA-4, -5 and -6 are zinc finger transcription factors that are expressed in the developing heart and GATA-4 and -6 continue expression in the adult cardiac myocytes. GATA-4 and -6 regulate expression of several cardiac-specific genes, and during murine embryonic development, GATA-4 is essential for proper cardiac morphogenesis. In support of this, mutations of gene for GATA-4 or for its cofactors have been associated with human congenital heart disease. Pressure overload of the heart in vivo as well as hypertrophic stimulation of cardiac myocytes in vitro provide adequate stimulus for activation of GATA-4. Activity of GATA-4 transcription factor is subject to regulation at the level of gene expression and through post-translational modifications of GATA-4 protein. A number of genes induced during cardiac hypertrophy possess functional GATA sites in their promoter region and cardiac-specific overexpression of GATA-4 or -6 leads to cardiac hypertrophy. In addition, a pattern of interactions between GATA-4 and its numerous cofactors have been identified, showing an increasing complexity in regulatory mechanisms. The present review discusses current evidence of the role and regulation of GATA transcription factors in the heart, with an emphasis in the GATA-4 and development of cardiac hypertrophy.

Bone morphogenetic protein-2--a potential autocrine/paracrine factor in mediating the stretch activated B-type and atrial natriuretic peptide expression in cardiac myocytes.

Hemodynamic overload exposes the heart to variety of neural, humoral and mechanical stresses. Even without the neurohumoral control of the entire organism cardiac myocytes have the ability to sense mechanical stretch and convert it into adaptive intracellular signals. This process is controlled by several growth factors. Here we show that mechanical stretch in vitro and hemodynamic overload in vivo activated the expression of bone morphogenetic protein-2 (BMP-2), while expression of BMP-4 was temporarily attenuated by stretch. BMP-2 and BMP-4 alone stimulated B-type and atrial natriuretic peptide (BNP and ANP) expression and protein synthesis, and activated transcription factor GATA-4 resembling the effects of mechanical stretch of cultured cardiac myocytes. Further, BMP antagonist Noggin was able to inhibit stretch and hypertrophic agonist induced BNP and ANP expression. Together these data provide evidence for BMP-2 as a new autocrine/paracrine factor that regulates cardiomyocyte mechanotransduction and adaptation to increased mechanical stretch.

Mechanisms regulating adrenomedullin gene expression in the left ventricle: role of mechanical load.

Adrenomedullin (AM) may function as an autocrine and/or paracrine factor in the heart, but the exact mechanisms regulating cardiac AM gene expression are unknown. The aim of the present study was to characterize the role of mechanical load in regulating gene expression of AM by using two hypertensive rat strains as experimental models. Acute pressure overload was produced by arginine(8)-vasopressin (AVP, 0.05 microg/kg/min, i.v.) infusion in conscious spontaneously hypertensive rats (SHR) and double transgenic rats (dTGR) harboring both the human renin and angiotensinogen genes and in their respective normotensive strains. A significant increase in left ventricular AM mRNA levels was seen in the left ventricles of all rat strains, the increase being augmented in hypertensive strains. Direct left ventricular wall stretch in isolated, perfused rat heart preparation also activated AM gene expression. However, stretching of cultured neonatal ventricular myocytes resulted in inhibition of AM gene expression, and stretch also blocked hypoxia-induced increase in AM gene expression. The present study shows that cardiac AM gene expression is upregulated in response to pressure overload and that this upregulation may be mediated via cell types other than cardiac myocytes.

Plasma B-type natriuretic peptide reflects left ventricular hypertrophy and diastolic function in hypertension.

BACKGROUND: Hypertension is associated with changes in concentrations of vasoactive peptides and procollagen propeptides, but their relationships with left ventricular hypertrophy and cardiac function are unclear. METHODS: We measured plasma levels of atrial natriuretic peptide (ANP), its amino terminal propeptide (NT-proANP), B-type natriuretic peptide (BNP), endothelin-1 (ET-1), and serum levels of the aminoterminal propeptide of type I procollagen (PINP) and the aminoterminal propeptide of type III procollagen (PIIINP) and echocardiographic parameters in 97 patients with hypertension in the Anglo-Scandinavian Cardiac Outcomes Trial. RESULTS: Median values (reference values) of the peptides were: ANP 11.2 (6.9-14.9) pmol/l, NT-proANP 351 (143-311) pmol/l, BNP 1.1 (0.4-7.2) pmol/l, ET-1 8.7 (1.2-5.0) pmol/l, PIIINP 2.8 (1.7-4.2) microg/l and PINP 29 (19-84) microg/l. Plasma BNP levels in patients with left ventricular hypertrophy (1.2 pmol/l) and patients with echocardiographic signs of diastolic dysfunction (1.5 pmol/l) were greater than those in patients without hypertrophy (0.7 pmol/l) and normal diastolic parameters (0.9 pmol/l) (p<0.05). BNP was the only biochemical parameter that independently predicted interventricular septal diastolic diameter (p<0.05), left ventricular mass index (p<0.01) and ratio of the velocity-time integrals of the E and A waves of the mitral inflow in a stepwise logistic regression analysis (p<0.05). CONCLUSIONS: The results show that BNP reflects the remodelling process in hypertension.

Characterization of the regulatory mechanisms of activating transcription factor 3 by hypertrophic stimuli in rat cardiomyocytes.

AIMS: Activating transcription factor 3 (ATF3) is a stress-activated immediate early gene suggested to have both detrimental and cardioprotective role in the heart. Here we studied the mechanisms of ATF3 activation by hypertrophic stimuli and ATF3 downstream targets in rat cardiomyocytes. METHODS AND RESULTS: When neonatal rat cardiomyocytes were exposed to endothelin-1 (ET-1, 100 nM) and mechanical stretching in vitro, maximal increase in ATF3 expression occurred at 1 hour. Inhibition of extracellular signal-regulated kinase (ERK) by PD98059 decreased ET-1- and stretch-induced increase of ATF3 protein but not ATF3 mRNA levels, whereas protein kinase A (PKA) inhibitor H89 attenuated both ATF3 mRNA transcription and protein expression in response to ET-1 and stretch. To characterize further the regulatory mechanisms upstream of ATF3, p38 mitogen-activated protein kinase (MAPK) signaling was investigated using a gain-of-function approach. Adenoviral overexpression of p38α, but not p38ß, increased ATF3 mRNA and protein levels as well as DNA binding activity. To investigate the role of ATF3 in hypertrophic process, we overexpressed ATF3 by adenovirus-mediated gene transfer. In vitro, ATF3 gene delivery attenuated the mRNA transcription of interleukin-6 (IL-6) and plasminogen activator inhibitor-1 (PAI-1), and enhanced nuclear factor-κB (NF-κB) and Nkx-2.5 DNA binding activities. Reduced PAI-1 expression was also detected in vivo in adult rat heart by direct intramyocardial adenovirus-mediated ATF3 gene delivery. CONCLUSIONS: These data demonstrate that ATF3 activation by ET-1 and mechanical stretch is partly mediated through ERK and cAMP-PKA pathways, whereas p38 MAPK pathway is involved in ATF3 activation exclusively through p38α isoform. ATF3 activation caused induction of modulators of the inflammatory response NF-κB and Nkx-2.5, as well as attenuation of pro-fibrotic and pro-inflammatory proteins IL-6 and PAI-1, suggesting cardioprotective role for ATF3 in the heart.

Distinct regulation of B-type natriuretic peptide transcription by p38 MAPK isoforms.

Persistent controversy underlies the functional roles of specific p38 MAPK isoforms in cardiac biology and regulation of hypertrophy-associated genes. Here we show that adenoviral gene transfer of p38ß but not p38α increased B-type natriuretic peptide (BNP) mRNA levels in vitro as well as atrial natriuretic peptide mRNA levels both in vitro and in vivo. Overexpression of p38α, in turn, augmented the expression fibrosis-related genes connective tissue growth factor, basic fibroblast growth factor and matrix metalloproteinase-9 both in vitro and in vivo. p38ß-induced BNP transcription was diminished by mutation of GATA-4 binding site, whereas overexpression of MKK6b, an upstream regulator of p38α and p38ß, activated BNP transcription through both GATA-4 and AP-1. Overexpression of MKK3, upstream regulator of p38α, induced BNP transcription independently from AP-1 and GATA-4. These data provide new evidence for diversity in downstream targets and functional roles of p38 pathway kinases in regulation of hypertrophy-associated cardiac genes.

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 Ca(2+)-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 Ca(2+)-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.