Carlin S. Long

Right Ventricular Function and Failure Report of a National Heart, Lung, and Blood Institute Working Group on Cellular and Molecular Mechanisms of Right Heart Failure

Knowledge about the role of the right ventricle in health and disease historically has lagged behind that of the left ventricle. Less muscular, restricted in its role to pumping blood through a single organ, and less frequently or obviously involved than the left ventricle in diseases of epidemic proportions such as myocardial ischemia, cardiomyopathy, or valvulopathy, the right ventricle has generally been considered a mere bystander, a victim of pathological processes affecting the cardiovascular system. Consequently, comparatively little attention has been devoted to how right ventricular dysfunction may be best detected and measured, what specific molecular and cellular mechanisms contribute to maintenance or failure of normal right ventricular function, how right ventricular dysfunction evolves structurally and functionally, or what interventions might best preserve right ventricular function. Nevertheless, even the proportionately limited information related to right ventricular function, its impairment in various disease states, and its impact on the outcome of those diseases suggests that the right ventricle is an important contributor and that further understanding of these issues is of pivotal importance. For this reason, the National Heart, Lung, and Blood Institute convened a working group charged with delineating in broad terms the current base of scientific and medical understanding about the right ventricle and identifying avenues of investigation likely to meaningfully advance knowledge in a clinically useful direction. The following summary represents the presentations and discussions of this working group. The right ventricle is affected by and contributes to a number of disease processes, including perhaps most notably pulmonary hypertension caused by a variety of lung or pulmonary vascular diseases (cor pulmonale). Other diseases affect the right ventricle in different ways, including global, left ventricular–, or right ventricular–specific cardiomyopathy; right ventricular ischemia or infarction; pulmonary or tricuspid valvular heart disease; and left-to-right shunts. The right ventricle pumps the same …

Chronic Pulmonary Artery Pressure Elevation Is Insufficient to Explain Right Heart Failure

Background— The most important determinant of longevity in pulmonary arterial hypertension is right ventricular (RV) function, but in contrast to experimental work elucidating the pathobiology of left ventricular failure, there is a paucity of data on the cellular and molecular mechanisms of RV failure. Methods and Results— A mechanical animal model of chronic progressive RV pressure overload (pulmonary artery banding, not associated with structural alterations of the lung circulation) was compared with an established model of angioproliferative pulmonary hypertension associated with fatal RV failure. Isolated RV pressure overload induced RV hypertrophy without failure, whereas in the context of angioproliferative pulmonary hypertension, RV failure developed that was associated with myocardial apoptosis, fibrosis, a decreased RV capillary density, and a decreased vascular endothelial growth factor mRNA and protein expression despite increased nuclear stabilization of hypoxia-induced factor-1α. Induction of myocardial nuclear factor E2-related factor 2 and heme-oxygenase 1 with a dietary supplement (Protandim) prevented fibrosis and capillary loss and preserved RV function despite continuing pressure overload. Conclusions— These data brought into question the commonly held concept that RV failure associated with pulmonary hypertension is due strictly to the increased RV afterload.

Expression and Regulation of Adhesion Molecules in Cardiac Cells by Cytokines Response to Acute Hypoxia

Adhesion molecules mediate inflammatory myocardial injury after ischemia/reperfusion. Cytokine release and hypoxia are features of acute ischemia that may influence expression of these molecules. Accordingly, we studied intercellular adhesion molecule (ICAM) and vascular cell adhesion molecule (VCAM) responses to cytokines and acute hypoxia in cultured myocardial cells. Northern blot analysis and immunoassay showed that the proinflammatory cytokines interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha stimulated concentration-dependent increases in ICAM and VCAM mRNA and protein. In both cardiac myocytes and fibroblasts, pretreatment with a specific inhibitor of nuclear transcription factor-kappaB (NF-kappaB) prevented cytokine induction of both molecules. We also found that inhibition of tyrosine kinase and p38/RK (stress-activated protein kinase) pathways prevented IL-1beta-induced ICAM and VCAM protein synthesis, whereas extracellular signal-regulated protein kinase (ERK1/ERK2) inhibition did not. Neither hypoxia (0% O2 for 6 hours) alone nor hypoxia/reoxygenation had any significant effect on ICAM and VCAM mRNA. However, hypoxia did enhance IL-1beta-induced ICAM mRNA expression in myocytes. As a possible mechanism of this synergistic action on CAM expression, hypoxia induced a time-dependent increase in the DNA binding activity of both NF-kappaB and activator protein-1 (AP-1), two transcription factors important for cell adhesion molecule expression. In contrast to the enhanced ICAM mRNA induced by IL-1beta during hypoxia, however, protein levels for this adhesion molecule were unchanged beyond IL-1beta-stimulated levels, suggesting posttranscriptional and/or posttranslational control mechanisms. We conclude that cytokines regulate ICAM and VCAM mRNA and protein in both cardiac myocytes and fibroblasts. Furthermore, adhesion molecule induction requires translocation of at least two transcription factors, NF-kappaB and AP-1.

Heart Failure in Rats Causes Changes in Skeletal Muscle Morphology and Gene Expression That Are Not Explained by Reduced Activity

In patients with congestive heart failure, skeletal muscle is characterized by a smaller proportion of slow-twitch oxidative fibers and reduced oxidative enzyme activity. However, whether these changes result from disuse or occur as a direct consequence of heart failure is unresolved. To address this issue, 18 rats with heart failure 8 weeks after left coronary artery ligation and 13 sham-operated control rats underwent quantification of locomotor activity by a photocell activation technique, measurements of hemodynamics and infarct size, histochemical and morphological analyses of the soleus and plantaris muscles, and Northern analyses of muscle contractile protein and oxidative enzyme mRNA expression. Although the rats with heart failure had elevated left ventricular end-diastolic pressures (24.1 +/- 2.6 mm Hg) and a mean infarct size of 35.1 +/- 4.1%, activity levels were similar to those found in the sham-operated rats (3849 +/- 304 versus 3526 +/- 130 counts per hour). With heart failure, there was a significant reduction of type I fibers in the soleus muscle and type IIa fibers in the plantaris muscle, with corresponding increases in intermediate staining of type IIab fibers in both muscles. This was associated with a 17% decrease in citrate synthase activity in both the soleus and plantaris muscles (26.2 +/- 1.6 versus 30.7 +/- 3.4 and 29.1 +/- 2.4 versus 35.7 +/- 3.4 mumol/L per minute per gram, respectively [P < .05]). In the soleus muscle, mRNA for both beta-myosin heavy chains and cytochrome C oxidase III (normalized to 18S RNA) was reduced (0.27 +/- 0.02 versus 0.65 +/- 0.02 and 0.23 +/- 0.04 versus 0.64 +/- 0.02 U), whereas the messages for IIx and IIb myosin heavy chains were increased. A similar decrease in messages for cytochrome oxidase and the primary myosin isoform was observed in the plantaris muscle. Both soleus beta-myosin heavy chain and cytochrome C oxidase expression show significant inverse relationships to left ventricular end-diastolic pressure and infarct size. In contrast, there was no relationship between either beta-myosin heavy chain or cytochrome C oxidase expression and locomotor activity. These results indicate that in rats heart failure produces changes in skeletal muscle gene expression at the pretranslational level that cannot be explained by inactivity.

-Adrenergic Receptor Subtype mRNAs Are Differentially Regulated by -Adrenergic and Other Hypertrophic Stimuli in Cardiac Myocytes in Culture and In Vivo REPRESSION OF α AND α BUT INDUCTION OF α

The three cloned α-adrenergic receptor (AR) subtypes, α, α, and α, can all couple to the same effector, phospholipase C, and the reason(s) for conservation of multiple subtypes remain uncertain. All three α-ARs are expressed natively in cultured neonatal rat cardiac myocytes, where chronic exposure to the agonist catecholamine norepinephrine (NE) induces hypertrophic growth and gene transcription. We show here, using RNase protection, that the α-AR subtype mRNAs respond in distinctly different ways during prolonged NE exposure (12-72 h). α and α mRNA levels were repressed by NE, whereas α mRNA was induced. Changes in mRNA levels were mediated by an α-AR, were not explained by altered mRNA stability, and were reflected in receptor proteins by [3H]prazosin binding. α-AR-stimulated phosphoinositide hydrolysis and myocyte growth were not desensitized. Three other hypertrophic agonists in culture, endothelin-1, PGF2α, and phorbol 12-myristate 13-acetate, also induced α mRNA and repressed α mRNA. In myocytes from hearts with pressure overload hypertrophy, α mRNA changes were identical to those produced by NE in culture. These results provide the first example of a difference in regulation among α-AR subtypes expressed natively in the same cell. Transcriptional induction of the α-AR could be a mechanism for sustained growth signaling through this receptor and is a common feature of a hypertrophic phenotype in cardiac myocytes.

Cytokine expression increases in nonmyocytes from rats with postinfarction heart failure

Growing evidence suggests that cardiac nonmyocyte cells may play an important regulatory role in the response to myocardial overload and injury via altered expression of paracrine products, such as cytokines and growth factors, but information concerning the cell-specific changes in the expression of these substances in heart-failure models is limited. Therefore, cardiac nonmyocytes were isolated from rats 1 day and 1 and 6 wk after left coronary artery ligation with resulting hemodynamic evidence of heart failure and in sham-operated control animals. mRNAs for tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-1beta, IL-6, transforming growth factors (TGF)-beta1 and TGF-beta3, and type I and type III collagen were measured by Northern analyses. The temporal and quantitative relationships between the expression of these cytokines and collagen and myocyte hypertrophy were determined. mRNA expression of IL-1beta was increased by 1.3-fold at 1 day and 1 wk, and expression of TNF-alpha, IL-1beta, IL-6, TGF-beta1, and TGF-beta3 were increased by 1.4- to 2.1-fold at the 1-wk time point before returning toward baseline at 6 wk. There were significant correlations between the expression of these cytokines and the expression of types I and III collagen, which also peaked at 1 wk. Myocyte hypertrophy was seen first at 6 wk. These observations are consistent with a hypothesis that nonmyocyte cells play a regulatory role in the extracellular matrix changes during postinfarction remodeling and highlight the importance of examining cell-specific changes in gene expression and elucidating the role of cell-to-cell interactions within the myocardium.Growing evidence suggests that cardiac nonmyocyte cells may play an important regulatory role in the response to myocardial overload and injury via altered expression of paracrine products, such as cytokines and growth factors, but information concerning the cell-specific changes in the expression of these substances in heart-failure models is limited. Therefore, cardiac nonmyocytes were isolated from rats 1 day and 1 and 6 wk after left coronary artery ligation with resulting hemodynamic evidence of heart failure and in sham-operated control animals. mRNAs for tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, IL-6, transforming growth factors (TGF)-β1 and TGF-β3, and type I and type III collagen were measured by Northern analyses. The temporal and quantitative relationships between the expression of these cytokines and collagen and myocyte hypertrophy were determined. mRNA expression of IL-1β was increased by 1.3-fold at 1 day and 1 wk, and expression of TNF-α, IL-1β, IL-6, TGF-β1, and TGF-β3 were increased by 1.4- to 2.1-fold at the 1-wk time point before returning toward baseline at 6 wk. There were significant correlations between the expression of these cytokines and the expression of types I and III collagen, which also peaked at 1 wk. Myocyte hypertrophy was seen first at 6 wk. These observations are consistent with a hypothesis that nonmyocyte cells play a regulatory role in the extracellular matrix changes during postinfarction remodeling and highlight the importance of examining cell-specific changes in gene expression and elucidating the role of cell-to-cell interactions within the myocardium.

Regulatory T Cells Limit Vascular Endothelial Injury and Prevent Pulmonary Hypertension

Rationale: Pulmonary arterial hypertension (PAH) is an incurable disease associated with viral infections and connective tissue diseases. The relationship between inflammation and disease pathogenesis in these disorders remains poorly understood. Objective: To determine whether immune dysregulation due to absent T-cell populations directly contributes to the development of PAH. Methods and Results: Vascular endothelial growth factor receptor 2 (VEGFR2) blockade induced significant pulmonary endothelial apoptosis in T-cell-deficient rats but not in immune-reconstituted (IR) rats. T cell–lymphopenia in association with VEGFR2 blockade resulted in periarteriolar inflammation with macrophages, and B cells even prior to vascular remodeling and elevated pulmonary pressures. IR prevented early inflammation and attenuated PAH development. IR with either CD8 T cells alone or with CD4-depleted spleen cells was ineffective in preventing PAH, whereas CD4-depleting immunocompetent euthymic animals increased PAH susceptibility. IR with either CD4+CD25hi or CD4+CD25− T cell subsets prior to vascular injury attenuated the development of PAH. IR limited perivascular inflammation and endothelial apoptosis in rat lungs in association with increased FoxP3+, IL-10- and TGF-&bgr;-expressing CD4 cells, and upregulation of pulmonary bone morphogenetic protein receptor type 2 (BMPR2)–expressing cells, a receptor that activates endothelial cell survival pathways. Conclusions: PAH may arise when regulatory T-cell (Treg) activity fails to control endothelial injury. These studies suggest that regulatory T cells normally function to limit vascular injury and may protect against the development of PAH.

Prevalence of Desmin Mutations in Dilated Cardiomyopathy

Background— Desmin-related myofibrillar myopathy (DRM) is a cardiac and skeletal muscle disease caused by mutations in the desmin (DES) gene. Mutations in the central 2B domain of DES cause skeletal muscle disease that typically precedes cardiac involvement. However, the prevalence of DES mutations in dilated cardiomyopathy (DCM) without skeletal muscle disease is not known. Methods and Results— Denaturing high-performance liquid chromatography was used to screen DES for mutations in 116 DCM families from the Familial Dilated Cardiomyopathy Registry and in 309 subjects with DCM from the Beta-Blocker Evaluation of Survival Trial (BEST). DES mutations were transfected into SW13 and human smooth muscle cells and neonatal rat cardiac myocytes, and the effects on cytoskeletal desmin network architecture were analyzed with confocal microscopy. Five novel missense DES mutations, including the first localized to the highly conserved 1A domain, were detected in 6 subjects (1.4%). Transfection of DES mutations in the 2B domain severely disrupted the fine intracytoplasmic staining of desmin, causing clumping of the desmin protein. A tail domain mutation (Val459Ile) showed milder effects on desmin cytoplasmic network formation and appears to be a low-penetrant mutation restricted to black subjects. Conclusions— The prevalence of DES mutations in DCM is between 1% and 2%, and mutations in the 1A helical domain, as well as the 2B rod domain, are capable of causing a DCM phenotype. The lack of severe disruption of cytoskeletal desmin network formation seen with mutations in the 1A and tail domains suggests that dysfunction of seemingly intact desmin networks is sufficient to cause DCM.

Signaling Pathways Responsible for Fetal Gene Induction in the Failing Human Heart Evidence for Altered Thyroid Hormone Receptor Gene Expression

Background —We have previously demonstrated that changes in myosin heavy chain (MHC) isoforms that occur in failing human hearts resemble the pattern produced in rodent myocardium in response to hypothyroidism. Because thyroid hormone status is usually within normal limits in these patients, we hypothesized that failing/hypertrophied human myocardium might have a defect in thyroid hormone signaling due to alterations in expression of thyroid hormone receptors (TRs). Methods and Results —To examine this hypothesis, we used RNase protection assay to measure mRNA levels of TRs in failing left ventricles that exhibited a fetal pattern of gene expression, ie, decreased expression of &agr;-MHC with increased &bgr;-MHC expression compared with left ventricles from age-matched controls. We detected expression of TR-&agr;1, -&agr;2, and -&bgr;1 isoforms in human left ventricles. In failing left ventricles, TR-&agr;1 was downregulated, whereas TR-&agr;2, a splice variant that does not bind thyroid hormone but inhibits responses to liganded TRs, was increased. Expression levels of TR-&bgr;1 did not differ significantly between the 2 groups. According to linear regression analysis, expression levels of TR-&agr;1 and -&agr;2 were positively and negatively correlated with those of &agr;-MHC, respectively. Conclusions —We conclude that decreases in TR-&agr;1 and increases in TR-&agr;2 may lead to local attenuation of thyroid hormone signaling in the failing human heart and that the resulting tissue-specific hypothyroidism is a candidate for the molecular mechanism that induces fetal gene expression in the failing human ventricle.

Alpha 1-adrenergic receptor stimulation of sarcomeric actin isogene transcription in hypertrophy of cultured rat heart muscle cells.

During pressure-load hypertrophy of the adult heart in vivo, there is up-regulation of the mRNA encoding skeletal alpha-actin, the sarcomeric actin iso-mRNA characteristic of mature skeletal muscle and the fetal/neonatal heart. We have shown previously that during alpha 1-adrenergic receptor-stimulated hypertrophy of cultured rat heart myocytes, the induction of skeletal alpha-actin mRNA is greater than that of the mRNA encoding cardiac alpha-actin, the sarcomeric actin iso-mRNA characteristic of the adult heart. To determine if this actin iso-mRNA switch during cardiac hypertrophy reflects changes in the transcriptional status of the myocyte nucleus, we quantified the rate of transcription of actin mRNAs and total RNA, using an in vitro run-on transcription assay with nuclei isolated from the cultured myocytes after stimulation with norepinephrine (NE). Transcription of skeletal alpha-actin was increased at 3 h after NE, reached a maximum 6.1-fold increase at 12 h, and returned to the control level at 24 h. The EC50 for NE was 200 nM, and pharmacologic studies indicated alpha 1-receptor specificity. Transcription of cardiac alpha-actin was also increased rapidly by NE (maximum 4.6-fold vs. control at 3 h). However, cardiac alpha-actin transcription had returned to the control level at 6 h, when NE-stimulated skeletal alpha-actin transcription was still increasing. Transcription of the cytoskeletal (beta) actin gene was not changed significantly by NE treatment. Total RNA transcription was not increased until 6 h after NE (1.5-fold vs. control) and remained elevated through 24 h. Inhibition of protein synthesis did not attenuate NE-stimulated actin gene transcription. Thus the alpha 1-adrenoceptor mediates a rapid, transient, and selective increase in transcription of the sarcomeric actin isogenes during cardiac myocyte hypertrophy. Skeletal alpha-actin, the fetal/neonatal isogene, is induced preferentially to cardiac alpha-actin, the adult isogene. The different kinetics of actin isogene and total RNA transcription and the independence of transcription from protein synthesis suggest that transcriptional induction via the alpha 1 receptor is complex and may involve preexisting regulatory factors. These results are the first to demonstrate that the alpha 1-adrenergic receptor is a molecular mediator of transcriptional changes underlying an isogene switch that is known to be associated with cardiac myocyte hypertrophy.