Dominic J. Autelitano

Direct Actions of Urotensin II on the Heart. Implications for Cardiac Fibrosis and Hypertrophy

Abstract— Urotensin II (UII) is a somatostatin-like peptide recently identified as a potent vasoconstrictor. In this study, we examined whether UII promotes cardiac remodeling through nonhemodynamic effects on the myocardium. In a rat model of heart failure after myocardial infarction (MI), increased UII peptide and UII receptor protein expression was observed in both infarct and noninfarct regions of the left ventricle compared with sham. Moreover, post-MI remodeling was associated with a significant 75% increase in UII receptor gene expression in the heart (P <0.05 versus sham controls), with this increase noted in both regions of the left ventricle. In vitro, UII (10−7 mol/L) stimulation of neonatal cardiac fibroblasts increased the level of mRNA transcripts for procollagens &agr;1(I), &agr;1(III), and fibronectin by 139±15% (P <0.01), 59±5% (P <0.05), and 141±14% (P <0.01), respectively, with a concomitant 23±2% increase in collagen peptide synthesis as determined by 3H-proline incorporation (P <0.01). UII had no effect on cellular hypertrophy, as determined by changes in total protein content in isolated neonatal cardiomyocytes. However, expression of recombinant rat UII receptor in neonatal cardiomyocytes resulted in significant UII-dependent activation of hypertrophic signaling as demonstrated by increased total protein content (unstimulated, 122.4±4.0 &mgr;g/well; rat UII, 147.6±7.0 &mgr;g/well; P <0.01) and activation of the hypertrophic phenotype through G&agr;q- and Ras-dependent pathways. These results indicate that, in addition to potent hemodynamic effects, UII may be implicated in myocardial fibrogenesis through increased collagen synthesis by cardiac fibroblasts and may also be an important determinant of pathological cardiac hypertrophy in conditions characterized by UII receptor upregulation.

β2-Adrenergic Receptor Overexpression Exacerbates Development of Heart Failure After Aortic Stenosis

Background—β-Adrenergic signaling is downregulated in the failing heart, and the significance of such change remains unclear. Methods and Results—To address the role of β-adrenergic dysfunction in heart failure (HF), aortic stenosis (AS) was induced in wild-type (WT) and transgenic (TG) mice with cardiac targeted overexpression of β2-adrenergic receptors (ARs), and animals were studied 9 weeks later. The extents of increase in systolic arterial pressure (P<0.01 versus controls), left ventricular (LV) hypertrophy (TG, 94±6 to 175±7 mg; WT, 110±6 to 168±10 mg; both P<0.01), and expression of ANP mRNA were similar between TG and WT mice with AS. TG mice had higher incidences of premature death and critical illness due to heart failure (75% versus 23%), pleural effusion (81% versus 45%), and left atrial thrombosis (81% versus 36%, all P<0.05). A more extensive focal fibrosis was found in the hypertrophied LV of TG mice (P<0.05). These findings indicate a more severe LV dysfunction in TG mice. In sham-operated...

Reduced Reperfusion–Induced Ins(1,4,5)P3 Generation and Arrhythmias in Hearts Expressing Constitutively Active α1B-Adrenergic Receptors

Reperfusion of globally ischemic rat hearts causes the generation of inositol(1,4,5)trisphosphate [Ins(1,4,5)P3] and the initiation of arrhythmias. These responses are mediated by alpha1-adrenergic receptors (ARs), but the subtype of receptor involved has not been identified. Under normoxic conditions, hearts from transgenic animals expressing constitutively active alpha1B-ARs in heart (alpha1B-constitutively active mutant [CAM]) showed higher [3H] inositol phosphate responses to norepinephrine (2.3-fold) than hearts from nontransgenic animals (alpha1B-WT) (1.6-fold). alpha1B-WT hearts responded to 2 minutes of reperfusion after 20 minutes of global ischemia by generation of Ins(1,4,5)P3 (5301+/-1310 to 11 413+/-1597 CPM/g tissue; mean+/-SEM; n=6; P<0.01 in [3H] labeling studies and 3.8+/-0.2 to 6.3+/-0.6 nmol/g by mass analysis, n=6; P<0.05). In contrast to findings in normoxia, hearts from alpha1B-CAM animals showed no Ins(1,4,5)P3 response in early reperfusion. In parallel studies, alpha1B-WT hearts developed ventricular tachycardia and ventricular premature beats (VPB) during 5 minutes of reperfusion after 20 minutes of ischemia. The incidence of these arrhythmias was reduced in the alpha1B-CAM hearts (95% to 62% for VPB and 47% to 12% for ventricular tachycardia; both P<0.05). The resistance of the alpha1B-CAM hearts was not due to alpha1B-AR-mediated preconditioning, as the Ins(1,4,5)P3 response to thrombin receptor activation during reperfusion was not different between the 2 groups. To investigate the possibility of reduced alpha1A-receptor activity in the alpha1B-CAM hearts, expression of the mRNA for alpha1A- and alpha1B-receptors was measured. alpha1B-WT hearts contained mRNA for both receptor subtypes, but the levels of alpha1B-receptor mRNA were 5-fold higher than alpha1A-receptor mRNA. alpha1B-CAM hearts contained very high levels of alpha1B-receptor mRNA (26-fold increase), but the expression of mRNA for the alpha1A-receptors (0.141+/-0.035 amol/ microg RNA; mean+/-SEM; n=6) was reduced by 50% relative to alpha1B-WT controls (0.276+/-0.046 amol/ microg RNA; n=6; P<0.01). The reduction in arrhythmogenic and Ins(1,4,5)P3 responses in alpha1B-CAM hearts provides evidence that these response are not mediated by alpha1B-receptors.

Increased expression of UBF is a critical determinant for rRNA synthesis and hypertrophic growth of cardiac myocytes

Recent evidence suggests that increased translational efficiency of existing ribosomes alone is insufficient to account for the hypertrophic growth of cardiomyocytes and that synthesis of new functional ribosomes must occur. The rate-limiting step in ribosome accumulation is the transcription of the ribosomal 45S genes (rDNA) by RNA polymerase I. Our previous studies have demonstrated that increases in the expression of the rDNA transcription factor UBF correlated with hypertrophy of neonatal cardiomyocytes. These studies expand this observation to examine directly the hypothesis that increased UBF levels are an essential requirement for the initiation of cardiac hypertrophy. We demonstrate that the introduction of UBF antisense RNA into myocytes, using adenovirus approaches, efficiently inhibits UBF accumulation during induction of cardiomyocyte hypertrophy. Moreover, this approach results in a significant reduction in rDNA transcription, rRNA levels, and protein accumulation, which are all the hallmarks of cardiac growth. Furthermore, UBF antisense RNA expression did not alter re-expression of the fetal gene program, which confirmed that the effect was specific for transcription by RNA polymerase I. These findings demonstrate that an increase in rRNA synthesis is required for hypertrophy of cardiomyocytes and also implicate UBF as a major regulatory factor in this process. Approaches that target UBF activity may be of therapeutic use in the regression of pathophysiological cardiac hypertrophy.

β2-Adrenergic receptor overexpression driven by α-MHC promoter is downregulated in hypertrophied and failing myocardium

Objective: The α-myosin heavy chain (α-MHC) promoter is frequently used to direct cardiac specific transgene expression. We studied whether transgene expression controlled by this promoter was altered under conditions of cardiac hypertrophy and failure. Methods: Transgenic (TG) mice overexpressing human β2-adrenergic receptors (β2AR) and wild type (WT) controls were subjected to thoracic aortic constriction (TAC) or sham operation and studied at 1, 3 and 8 weeks after surgery. Results: Sham operated TG mice had higher heart rates and left ventricular (LV) contractility than WT (all P <0.01), demonstrating enhanced βAR activation. TAC at 1, 3 and 8 weeks produced progressive LV hypertrophy which was similar between WT and TG mice. Evidence of heart failure was more marked in TG mice with a greater increase in weights of the right ventricle and lungs and a higher prevalence of atrial thrombus ( P <0.05 in each case). In hypertrophied TG hearts, endogenous α-MHC mRNA transcripts in LV were maintained at 1 and 3 weeks, but were reduced by approximately 40% relative to the sham-operated group at 8 weeks after TAC. Transgene expression, measured as human β2AR mRNA, was reduced by 45% at 1 and 3 weeks and by 70% at 8 weeks after TAC. β2AR binding sites were reduced by 35, 47 and 65%, respectively, at 1, 3 and 8 weeks. Conclusion: Cardiac hypertrophy and failure cause downregulation of the endogenous α-MHC as well as cardiac specific overexpression of the transgene directed by an α-MHC promoter.

Adrenomedullin inhibits angiotensin AT1A receptor expression and function in cardiac fibroblasts

Adrenomedullin (AM) is a multifunctional peptide hormone with wide-ranging actions related to cardiovascular homeostasis. AM receptors are highly expressed in the heart and AM has antihypertrophic and antiproliferative effects on cardiac myocytes and fibroblasts, respectively. We have investigated the interaction between AM and angiotensin II (Ang II) signalling in neonatal cardiac fibroblast cultures to determine whether the antagonistic effects of AM are mediated via the modulation of Ang II receptors. Cardiac fibroblasts exclusively expressed the Ang II type 1 receptor (AT(1)R) and binding to this site was downregulated by 35% following an 18-h incubation with 100 nM AM. Levels of AT(1A)R mRNA were dose-dependently lowered by AM, with a maximal 40-50% inhibition by 6 h. The decreases in both AT(1)R binding and AT(1A)R mRNA levels were mimicked by 8-Br-cAMP or forskolin, suggesting that the effects of AM were mediated via an elevation of cAMP. In cardiac fibroblasts pretreated with AM, the Ang II induction of collagen biosynthesis was attenuated, although basal collagen synthesis was unaffected. These data suggest that AM mediates the heterologous downregulation of AT(1)R expression via a relatively rapid decrease in AT(1A)R mRNA pools. This interaction may represent a relevant pathophysiological mechanism for modulating Ang II responsiveness in the diseased heart.

Cardiac hypertrophy in vivo is associated with increased expression of the ribosomal gene transcription factor UBF

The ribosomal DNA transcription‐specific factor, UBF, is a key target for the regulation of ribosomal RNA synthesis and hypertrophic growth of isolated neonatal cardiomyocytes. In this study, we have examined whether UBF expression is also an important determinant of cardiac growth rates in vivo. We show that rDNA transcription, rRNA synthesis and UBF expression in left ventricular myocytes isolated from mice 1–6 weeks following transverse aortic constriction were significantly increased (2.5–3.5‐fold) compared to the levels in myocytes from the left ventricle of sham‐operated mice.