Correction: Preservation of myocardial contractility during acute hypoxia with OMX-CV, a novel oxygen delivery biotherapeutic.

[This corrects the article DOI: 10.1371/journal.pbio.2005924.].

Preservation of myocardial contractility during acute hypoxia with OMX-CV, a novel oxygen delivery biotherapeutic.

The heart exhibits the highest basal oxygen (O2) consumption per tissue mass of any organ in the body and is uniquely dependent on aerobic metabolism to sustain contractile function. During acute hypoxic states, the body responds with a compensatory increase in cardiac output that further increases myocardial O2 demand, predisposing the heart to ischemic stress and myocardial dysfunction. Here, we test the utility of a novel engineered protein derived from the heme-based nitric oxide (NO)/oxygen (H-NOX) family of bacterial proteins as an O2 delivery biotherapeutic (Omniox-cardiovascular [OMX-CV]) for the hypoxic myocardium. Because of their unique binding characteristics, H-NOX-based variants effectively deliver O2 to hypoxic tissues, but not those at physiologic O2 tension. Additionally, H-NOX-based variants exhibit tunable binding that is specific for O2 with subphysiologic reactivity towards NO, circumventing a significant toxicity exhibited by hemoglobin (Hb)-based O2 carriers (HBOCs). Juvenile lambs were sedated, mechanically ventilated, and instrumented to measure cardiovascular parameters. Biventricular admittance catheters were inserted to perform pressure-volume (PV) analyses. Systemic hypoxia was induced by ventilation with 10% O2. Following 15 minutes of hypoxia, the lambs were treated with OMX-CV (200 mg/kg IV) or vehicle. Acute hypoxia induced significant increases in heart rate (HR), pulmonary blood flow (PBF), and pulmonary vascular resistance (PVR) (p < 0.05). At 1 hour, vehicle-treated lambs exhibited severe hypoxia and a significant decrease in biventricular contractile function. However, in OMX-CV-treated animals, myocardial oxygenation was improved without negatively impacting systemic or PVR, and both right ventricle (RV) and left ventricle (LV) contractile function were maintained at pre-hypoxic baseline levels. These data suggest that OMX-CV is a promising and safe O2 delivery biotherapeutic for the preservation of myocardial contractility in the setting of acute hypoxia.

Analysis of the microRNA signature driving adaptive right ventricular hypertrophy in an ovine model of congenital heart disease.

The right ventricular (RV) response to pulmonary arterial hypertension (PAH) is heterogeneous. Most patients have maladaptive changes with RV dilation and RV failure, whereas some, especially patients with PAH secondary to congenital heart disease, have an adaptive response with hypertrophy and preserved systolic function. Mechanisms for RV adaptation to PAH are unknown, despite RV function being a primary determinant of mortality. In our congenital heart disease ovine model with fetally implanted aortopulmonary shunt (shunt lambs), we previously demonstrated an adaptive physiological RV response to increased afterload with hypertrophy. In the present study, we examined small noncoding microRNA (miRNA) expression in shunt RV and characterized downstream effects of a key miRNA. RV tissue was harvested from 4-wk-old shunt and control lambs ( n = 5), and miRNA, mRNA, and protein were quantitated. We found differential expression of 40 cardiovascular-specific miRNAs in shunt RV. Interestingly, this miRNA signature is distinct from models of RV failure, suggesting that miRNAs might contribute to adaptive RV hypertrophy. Among RV miRNAs, miR-199b was decreased in the RV with eventual downregulation of nuclear factor of activated T cells/calcineurin signaling. Furthermore, antifibrotic miR-29a was increased in the shunt RV with a reduction of the miR-29 targets collagen type A1 and type 3A1 and decreased fibrosis. Thus, we conclude that the miRNA signature specific to shunt lambs is distinct from RV failure and drives gene expression required for adaptive RV hypertrophy. We propose that the adaptive RV miRNA signature may serve as a prognostic and therapeutic tool in patients with PAH to attenuate or prevent progression of RV failure and premature death. NEW & NOTEWORTHY This study describes a novel microRNA signature of adaptive right ventricular hypertrophy, with particular attention to miR-199b and miR-29a.

KLF2-mediated disruption of PPAR-γ signaling in lymphatic endothelial cells exposed to chronically increased pulmonary lymph flow.

Lymphatic abnormalities associated with congenital heart disease are well described, yet the underlying mechanisms remain poorly understood. Using a clinically relevant ovine model of congenital heart disease with increased pulmonary blood flow, we have previously demonstrated that lymphatic endothelial cells (LECs) exposed in vivo to chronically increased pulmonary lymph flow accumulate ROS and have decreased bioavailable nitric oxide (NO). Peroxisome proliferator-activated receptor-γ (PPAR-γ), which abrogates production of cellular ROS by NADPH oxidase, is inhibited by Krüppel-like factor 2 (KLF2), a flow-induced transcription factor. We hypothesized that chronically increased pulmonary lymph flow induces a KLF2-mediated decrease in PPAR-γ and an accumulation of cellular ROS, contributing to decreased bioavailable NO in LECs. To better understand the mechanisms that transduce the abnormal mechanical forces associated with chronically increased pulmonary lymph flow, LECs were isolated from the efferent vessel of the caudal mediastinal lymph node of control ( n = 5) and shunt ( n = 5) lambs. KLF2 mRNA and protein were significantly increased in shunt compared with control LECs, and PPAR-γ mRNA and protein were significantly decreased. In control LECs exposed to shear forces in vitro, we found similar alterations to KLF2 and PPAR-γ expression. In shunt LECs, NADPH oxidase subunit expression was increased, and bioavailable NO was significantly lower. Transfection of shunt LECs with KLF2 siRNA normalized PPAR-γ, ROS, and bioavailable NO. Conversely, pharmacological inhibition of PPAR-γ in control LECs increased ROS equivalent to levels in shunt LECs at baseline. Taken together, these data suggest that one mechanism by which NO-mediated lymphatic function is disrupted after chronic exposure to increased pulmonary lymph flow is through altered KLF2-dependent PPAR-γ signaling, resulting in increased NADPH oxidase activity, accumulation of ROS, and decreased bioavailable NO. NEW & NOTEWORTHY Lymphatic endothelial cells, when exposed in vivo to chronically elevated pulmonary lymph flow in a model of congenital heart disease with increased pulmonary blood flow, demonstrate Krüppel-like factor 2-dependent disrupted peroxisome proliferator-activated receptor-γ signaling that results in the accumulation of reactive oxygen species and decreased bioavailable nitric oxide.

Disrupted NOS signaling in lymphatic endothelial cells exposed to chronically increased pulmonary lymph flow.

Associated abnormalities of the lymphatic circulation are well described in congenital heart disease. However, their mechanisms remain poorly elucidated. Using a clinically relevant ovine model of a congenital cardiac defect with chronically increased pulmonary blood flow (shunt), we previously demonstrated that exposure to chronically elevated pulmonary lymph flow is associated with: 1) decreased bioavailable nitric oxide (NO) in pulmonary lymph; and 2) attenuated endothelium-dependent relaxation of thoracic duct rings, suggesting disrupted lymphatic endothelial NO signaling in shunt lambs. To further elucidate the mechanisms responsible for this altered NO signaling, primary lymphatic endothelial cells (LECs) were isolated from the efferent lymphatic of the caudal mediastinal node in 4-wk-old control and shunt lambs. We found that shunt LECs (n = 3) had decreased bioavailable NO and decreased endothelial nitric oxide synthase (eNOS) mRNA and protein expression compared with control LECs (n = 3). eNOS activity was also low in shunt LECs, but, interestingly, inducible nitric oxide synthase (iNOS) expression and activity were increased in shunt LECs, as were total cellular nitration, including eNOS-specific nitration, and accumulation of reactive oxygen species (ROS). Pharmacological inhibition of iNOS reduced ROS in shunt LECs to levels measured in control LECs. These data support the conclusion that NOS signaling is disrupted in the lymphatic endothelium of lambs exposed to chronically increased pulmonary blood and lymph flow and may contribute to decreased pulmonary lymphatic bioavailable NO.

Right ventricular nitric oxide signaling in an ovine model of congenital heart disease: a preserved fetal phenotype.

We recently reported superior right ventricle (RV) performance in response to acute afterload challenge in lambs with a model of congenital heart disease with chronic left-to-right cardiac shunts. Compared with control animals, shunt lambs demonstrated increased contractility because of an enhanced Anrep effect (the slow increase in contractility following myocyte stretch). This advantageous physiological response may reflect preservation of a fetal phenotype, since the RV of shunt lambs remains exposed to increased pressure postnatally. Nitric oxide (NO) production by NO synthase (NOS) is activated by myocyte stretch and is a necessary intermediary of the Anrep response. The purpose of this study was to test the hypothesis that NO signaling is increased in the RV of fetal lambs compared with controls and shunt lambs have persistence of this fetal pattern. An 8-mm graft was placed between the pulmonary artery and aorta in fetal lambs (shunt). NOS isoform expression, activity, and association with activating cofactors were determined in fetal tissue obtained during late-gestation and in 4-wk-old juvenile shunt and control lambs. We demonstrated increased RNA and protein expression of NOS isoforms and increased total NOS activity in the RV of both shunt and fetal lambs compared with control. We also found increased NOS activation and association with cofactors in shunt and fetal RV compared with control. These data demonstrate preserved fetal NOS phenotype and NO signaling in shunt RV, which may partially explain the mechanism underlying the adaptive response to increased afterload seen in the RV of shunt lambs.

Adaptive right ventricular performance in response to acutely increased afterload in a lamb model of congenital heart disease: evidence for enhanced Anrep effect.

Patients with pulmonary hypertension associated with congenital heart disease survive longer with preserved right ventricular (RV) function compared with those with primary pulmonary hypertension. The purpose of this study was to test the hypothesis that superior RV performance can be demonstrated, at baseline and when challenged with increased RV afterload, in lambs with chronic left-to-right cardiac shunts compared with control lambs. A shunt was placed between the pulmonary artery and the aorta in fetal lambs (shunt). RV pressure-volume loops were obtained 4 wk after delivery in shunt and control lambs, before and after increased afterload was applied using pulmonary artery banding (PAB). Baseline stroke volume (8.7 ± 1.8 vs. 15.8 ± 2.7 ml, P = 0.04) and cardiac index (73.0 ± 4.0 vs. 159.2 ± 25.1 ml·min(-1)·kg(-1), P = 0.02) were greater in shunts. After PAB, there was no difference in the change in cardiac index (relative to baseline) between groups; however, heart rate (HR) was greater in controls (168 ± 7.3 vs. 138 ± 6.6 beats/min, P = 0.01), and end-systolic elastance (Ees) was greater in shunts (2.63 vs. 1.31 × baseline, P = 0.02). Control lambs showed decreased mechanical efficiency (71% baseline) compared with shunts. With acute afterload challenge, both controls and shunts maintained cardiac output; however, this was via maladaptive responses in controls, while shunts maintained mechanical efficiency and increased contractility via a proposed enhanced Anrep effect-the second, slow inotropic response in the biphasic ventricular response to increased afterload, a novel finding in the RV. The mechanisms related to these physiological differences may have important therapeutic implications.

Differential responses of the right ventricle to abnormal loading conditions in vivo: possible pathophysiologic mechanisms.

OBJECTIVE: The right ventricle (RV) demonstrates differential adaptations in response to pressure versus volume loading, a phenomenon that may be important in the management of children and adults with congenital heart disease (CHD). The purpose of this study is to elucidate possible transcriptional mechanisms of the RV response to pressure versus volume loading in vivo. METHODS: Fetal lambs had aortopulmonary shunting or pulmonary artery (PA) banding. Four weeks after spontaneous delivery, ovine hearts were evaluated for hemodynamic changes and changes in expression of sarcomeric gene proteins and transcriptional factors. Western blot densitometry and chromatin immunoprecipitation were applied using standard techniques. Transactivation assays were performed using transient transfections in Schneider's Drosophila line 2 cells in culture. RESULTS: After PA banding, the RV pressure increased from 36 ± 4 mm Hg (n = 4) to 96 ± 8 mm Hg (n = 4, P < .05). The RVs of shunted and banded animals showed significant increases in the expression levels and promoter binding of activators myocyte enhancer factor 2, GATA-4, Nkx2.5, transcriptional enhancer factor 1, and specificity protein (Sp) 1. The transcriptional repressor Sp3 was downregulated in shunted animals, but its expression was increased paradoxically in the RV of the PA band group. Immunoprecipitation of Sp3 showed posttranslational modification to the acetylated isoform. In transient transfections of Schneider's Drosophila line 2 cells, acetylation of Sp3 converted it from a transcriptional repressor to an activator. CONCLUSIONS: Posttranslational modifications of the transcriptional repressor Sp3, by acetylation, may be an important mechanism in the differential response of the RV to abnormal loading conditions. Sp3 may serve as a biomarker for RV failure for various heart defects in children and adults with CHD. These findings may have therapeutic implications in the management of right heart failure.

Growth of pseudomorphic structures through organic epitaxy.

The control of molecular orientation in thin solid film phases of organic semiconductors is a basic factor for the exploitation of their physical properties for optoelectronic devices. We compare structural and optical properties of thin films of the organic semiconductor α-quarterthiophene grown by molecular beam epitaxy on different organic substrates. We show how epitactic interactions, characteristic of the surface of organic crystals, can drive the orientation of the crystalline overlayer and the selection of specific polymorphs and new pseudomorphic phases. We identify a key role in this phenomenon played by the marked groove-like corrugations present in some organic crystal surfaces. Since different polymorphs possess rather different performance in terms of, e.g., charge carrier mobility, this strategy is demonstrated to allow for the growth of oriented phases with enhanced physical properties, while keeping the substrate at room temperature. These results provide useful guidelines for the design of technological substrates for organic epitaxy and they substantiate the adoption of an organic epitaxy approach for the fabrication of optoelectronic devices based on thin films of organic semiconductors.

Myocardial transcription factors are modulated during pathologic cardiac hypertrophy in vivo.

OBJECTIVES: In the current study we describe and characterize a novel ovine model of biventricular hypertrophy and heart failure and evaluate the role of selected cardiac transcription factors in the regulation of cardiac gene expression during pathologic hypertrophy in vivo. The cardiac troponin T promoter is used as a model gene. METHODS AND RESULTS: Transient transfections of ovine cardiomyocytes in culture show that Sp1, transcriptional enhancer factor-1, and myocyte enhancer factor-2 activate cardiac troponin T promoter constructs. Cotransfection of Sp3 inhibits cardiac troponin T promoter activity and represses Sp1-mediated activation of the cardiac troponin T promoter. By chromatin immunoprecipitation, transcriptional enhancer factor-1, myocyte enhancer factor-2, NKX2.5, GATA-4, and Sp factors bind the cardiac troponin T promoter in vivo. To assess the role of cardiac transcription during pathologic hypertrophy, in vivo, we created surgical aorta-pulmonary shunts in utero in fetal lambs. Two weeks after spontaneous delivery, shunted lambs showed failure to thrive, significant biventricular hypertrophy, and heart failure. Shunted hearts had significant increases in myosin and cardiac troponin T protein expression. There was a shift in expression to the high-molecular-weight fetal isoforms. Transcriptional enhancer factor-1, myocyte enhancer factor-2, GATA-4, NKX2.5, and Sp1 transcription factor levels were increased in all heart chambers of shunted animals. Sp3 expression was decreased in shunted ventricles. Immunoprecipitated Sp3 was associated with significant increases in histone acetyl transferase activity and decreases in histone-deacetylase activity. CONCLUSION: The shunted neonatal lamb is a valid, novel model of pathologic biventricular hypertrophy. During pathologic hypertrophy myocardial transactivators are upregulated while repressors are downregulated.

Sp3 inhibits Sp1-mediated activation of the cardiac troponin T promoter and is downregulated during pathological cardiac hypertrophy in vivo.

Combinatorial interactions between cis elements and trans-acting factors are required for regulation of cardiac gene expression during normal cardiac development and pathological cardiac hypertrophy. Sp factors bind GC boxes and are implicated in recruitment and assembly of the basal transcriptional complex. In this study, we show that the cardiac troponin T (cTnT) promoter contains a GC box that is necessary for basal and cAMP-mediated activity of cTnT promoter constructs transfected in embryonic cardiomyocytes. Cardiac nuclear proteins bind the cTnT GC box in a sequence-specific fashion and consist of Sp1, Sp2, and Sp3 protein factors. By chromatin immunoprecipitation, Sp1 binds the cTnT promoter "in vivo." Cotransfected Sp1 trans-activates the cTnT promoter in cardiomyocytes in culture. Sp3 represses Sp1-mediated transcriptional activation of the cTnT gene in embryonic cardiomyocytes. Sp3 repression of Sp1-mediated cTnT promoter activation is dose dependent, inferring a mechanism of competitive binding/inhibition. To evaluate the role of Sp factors in cardiac gene expression in vivo, we have established a clinically relevant animal model of pathological cardiac hypertrophy where the fetal cardiac program is activated. In this animal model, cardiac hypertrophy results from increased left-right shunting, volume loading of the left ventricle, and pressure loading of the right ventricle. Sp1 expression is increased in all four hypertrophied cardiac chambers, whereas Sp3 expression is diminished. This observation is consistent with the in vitro activating function of Sp1 and inhibitory effects of Sp3 on activity of cTnT promoter constructs. Sp factor levels are modulated during the hypertrophic cardiac program in vivo.

Divergent transcriptional enhancer factor-1 regulates the cardiac troponin T promoter.

MCAT elements are essential for cardiac gene expression during development. Avian transcriptional enhancer factor-1 (TEF-1) proteins are muscle-enriched and contribute to MCAT binding activities. However, direct activation of MCAT-driven promoters by TEF-1-related proteins has not been uniformly achieved. Divergent TEF (DTEF)-1 is a unique member of the TEF-1 multigene family with abundant transcripts in the heart but not in skeletal muscle. Herein we show that DTEF-1 proteins are highly expressed in the heart. Protein expression is activated at very early stages of chick embryogenesis (Hamburger-Hamilton stage 4, 16-18 h), after which DTEF-1 becomes abundant in the sinus venosus and is expressed in the trabeculated ventricular myocardium and ventricular outflow tracts. By chromatin immunoprecipitation, DTEF-1 interacts with the cardiac troponin T (cTnT) promoter in vivo. DTEF-1 also interacts with MEF- 2 by coimmunoprecipitation and independently or cooperatively (with MEF-2) trans-activates the cTnT promoter. DTEF-1 isoforms do not activate the cTnT promoter in fibroblasts or skeletal muscle. DTEF-1 expression occurs very early in chick embryogenesis (16-18 h), preceding sarcomeric protein expression, and it activates cardiac promoters. As such, DTEF-1 may be an early marker of the myocardial phenotype. DTEF-1 trans-activates the cTnT promoter in a tissue-specific fashion independent of AT-rich, MEF-2, or GATA sites. The observed spatial pattern suggests decreasing levels of expression from the cardiac inlet to the ventricular outflow tracts, which may mark a cardiogenic or differentiation pathway that parallels the direction of flow through the developing chick heart.

The role of oxidative stress in the development of pulmonary arteriovenous malformations after cavopulmonary anastomosis.

BACKGROUND: Cavopulmonary anastomosis is used for palliation of cyanotic heart disease. Clinically significant pulmonary arteriovenous malformations occur in up to 25% of patients after surgical intervention. Cavopulmonary anastomosis creates several modifications to pulmonary physiology that may contribute to the development of pulmonary arteriovenous malformations, including reduced pulmonary blood flow and the exclusion of inferior vena caval effluent. OBJECTIVE: By comparing the expression of angiogenic and stress-related proteins after cavopulmonary anastomosis and pulmonary artery banding, we sought to determine which genes were upregulated independent of reduced pulmonary blood flow. METHODS: Lambs aged 35 to 45 days were placed into 1 of 3 groups: cavopulmonary anastomosis (n = 6), pulmonary artery banding (n = 6), and sham control (n = 6) animals. In our model pulmonary arteriovenous malformations are detectable by means of bubble-contrast echocardiography 8 weeks after cavopulmonary anastomosis. Lung tissue was harvested for Western blotting at 2 and 5 weeks after surgery. RESULTS: Cavopulmonary anastomosis and pulmonary artery banding both increased angiogenic gene expression, but only cavopulmonary anastomosis induced the expression of endothelial stress-related genes. Vascular endothelial growth factor was upregulated 2.5-fold after both cavopulmonary anastomosis (P =.002) and pulmonary artery banding (P =.007). Only cavopulmonary anastomosis upregulated 2 stress-related genes, HO1 and GLUT1, 2.7-fold (P =.002) and 3.8-fold (P =.03), respectively. Hypoxia-inducible factor was upregulated 4-fold (P =.003) after cavopulmonary anastomosis. Pulmonary artery banding failed to induce the increased expression of any of these proteins. CONCLUSIONS: Reduced pulmonary blood flow induces a pulmonary angiogenic response but not an endothelial stress response. These results suggest that oxidative stress is more relevant to the formation of pulmonary arteriovenous malformations than angiogenic signaling alone because pulmonary artery banding does not result in pulmonary arteriovenous malformations. Oxidative stress of the pulmonary endothelium resulting from cavopulmonary anastomosis may predispose the affected vasculature to arteriovenous shunting.

Cavopulmonary anastomosis induces pulmonary expression of the angiotensin II receptor family.

BACKGROUND: Cavopulmonary anastomosis is used for palliation of cyanotic cardiac lesions. Postoperative development of pulmonary arteriovenous malformations can be significant in 10% to 25% of patients. To study the basis for formation of arteriovenous malformations, we developed an ovine model that reliably induces their development 8 weeks after cavopulmonary anastomosis. Previously, we found that cavopulmonary anastomosis inhibits the expression of pulmonary angiotensin-converting enzyme and suppresses angiotensin II production. OBJECTIVE: This study examines the role of the angiotensin II receptors, type 1 and type 2, in this setting of pulmonary vascular remodeling. METHODS: Lambs, aged 40 to 50 days, underwent cavopulmonary anastomosis. In age-matched control animals, a sham operation was performed. Messenger RNA and protein expression in lung specimens was measured at successive time points after cavopulmonary anastomosis or sham operations (n = 3 at each time point). RESULTS: Angiotensin type 1 mRNA was maximally upregulated 2-fold at 5 weeks after cavopulmonary anastomosis (P =.006). Expression of angiotensin type 1 protein was increased at least 2-fold at 2, 5, and 15 weeks after cavopulmonary anastomosis (P =.005). Cavopulmonary anastomosis also increased angiotensin type 2 mRNA and protein expression at least 2-fold at 2 and 5 weeks (P =.02) after surgical intervention. At 15 weeks, expression of angiotensin type 2 mRNA and protein was unchanged from that seen in control animals. Immunolocalization in pulmonary tissue sections 2 weeks after cavopulmonary anastomosis revealed markedly enhanced staining of angiotensin II receptor type 1 in vascular smooth muscle and angiotensin II receptor type 2 in the endothelium of pulmonary arteries. CONCLUSIONS: Rapid elevation in the expression of the type 1 and 2 angiotensin II receptors in the affected pulmonary vasculature after cavopulmonary anastomosis suggests their involvement in the pathologic vascular remodeling that occurs after cavopulmonary anastomosis.