Neuregulin-1 enhances cell-cycle activity, delays cardiac fibrosis, and improves cardiac performance in rat pups with right ventricular pressure load.

OBJECTIVES: Right ventricular (RV) failure is a leading cause of death in patients with congenital heart disease. RV failure is kept at bay during childhood. Limited proliferation of cardiomyocytes is present in the postnatal heart. We propose that cardiomyocyte proliferation improves RV adaptation to pressure load (PL). We studied adaptation in response to increased RV PL and the role of increased cardiomyocyte cell cycle activity (CCA) in rat pups growing into adulthood. METHODS: We induced RV PL at day of weaning in rats (3 weeks; 30-40 g) by pulmonary artery banding and followed rats into adulthood (300 g). We performed histological analyses and RNA sequencing analysis. To study the effects of increased cardiomyocyte cell cycle activity, we administered neuregulin-1 (NRG1), a growth factor involved in cardiac development. RESULTS: PL induced an increase in CCA, with subsequent decline of CCA (sham/PL at 4 weeks: 0.14%/0.83%; P = .04 and 8 weeks: 0.00%/0.00%; P = .484) and cardiac function (cardiac index: control/PL 4 weeks: 4.41/3.29; P = .468 and 8 weeks: 3.57/1.44; P = .024). RNA sequencing analysis revealed delayed maturation and increased CCA pathways. NRG1 stimulated CCA (PL vehicle/NRG1 at 2 weeks: 0.62%/2.28%; P = .003), improved cardiac function (cardiac index control vs vehicle/NRG1 at 2 weeks: 4.21 vs 3.07/4.17; P = .009/.705) and postponed fibrosis (control vs vehicle/NRG1 at 4 weeks: 1.66 vs 4.82%/2.97%; P = .009/.078) in RV PL rats during childhood. CONCLUSIONS: RV PL during growth induces a transient CCA increase. Further CCA stimulation improves cardiac function and delays fibrosis. This proof-of-concept study shows that stimulation of CCA can improve RV adaptation to PL in the postnatal developing heart and might provide a new approach to preserve RV function in patients with congenital heart disease.

Egr-1 identifies neointimal remodeling and relates to progression in human pulmonary arterial hypertension.

BACKGROUND: Pulmonary arterial hypertension (PAH) is hallmarked by the development of neointimal lesions. The transcription factor Egr-1 seems to play a critical role in neointimal formation in experimental PAH and was identified as a putative target for intervention. In this study we investigated whether Egr-1 is also associated with neointimal-type vascular remodeling in different forms of human PAH or pulmonary hypertension. METHODS: Using immunohistochemistry, we studied Egr-1 expression specifically in a wide morphologic spectrum of pulmonary arteries in the lung tissue of 72 patients with different forms and stages of PAH, specifically idiopathic PAH (n = 18), advanced-stage congenital heart disease‒associated PAH (PAH-CHD) (n = 21), early-stage PAH-CHD (n = 19) and non-neointimal hypoxic pulmonary hypertension (PH) (n = 4), and controls (n = 10). RESULTS: In PAH patients, pulmonary vascular expression of Egr-1 protein was abundant, whereas it was sporadic in non-neointimal (hypoxic) PH patients and controls. In PAH-CHD, protein expression was more pronounced in patients with advanced vascular lesions compared to those with less advanced lesions, such as medial hypertrophy. CONCLUSIONS: Pulmonary vascular Egr-1 expression is significantly increased in patients with PAH, appears specifically associated with neointimal-type vascular remodeling, and correlates with disease progression. These data translate the critical role of Egr-1 in the development of experimental PAH to human pulmonary vascular disease forms.

Right ventricular failure due to chronic pressure load: What have we learned in animal models since the NIH working group statement?

Right ventricular (RV) failure determines outcome in patients with pulmonary hypertension, congenital heart diseases and in left ventricular failure. In 2006, the Working Group on Cellular and Molecular Mechanisms of Right Heart Failure of the NIH advocated the development of preclinical models to study the pathophysiology and pathobiology of RV failure. In this review, we summarize the progress of research into the pathobiology of RV failure and potential therapeutic interventions. The picture emerging from this research is that RV adaptation to increased afterload is characterized by increased contractility, dilatation and hypertrophy. Clinical RV failure is associated with progressive diastolic deterioration and disturbed ventricular-arterial coupling in the presence of increased contractility. The pathobiology of the failing RV shows similarities with that of the LV and is marked by lack of adequate increase in capillary density leading to a hypoxic environment and oxidative stress and a metabolic switch from fatty acids to glucose utilization. However, RV failure also has characteristic features. So far, therapies aiming to specifically improve RV function have had limited success. The use of beta blockers and sildenafil may hold promise, but new therapies have to be developed. The use of recently developed animal models will aid in further understanding of the pathobiology of RV failure and development of new therapeutic strategies.

Clinical symptoms of right ventricular failure in experimental chronic pressure load are associated with progressive diastolic dysfunction.

BACKGROUND: Right ventricular failure (RVF) due to pressure load is a major cause of death in congenital heart diseases and pulmonary hypertension. The mechanisms of RVF are unknown. We used an experimental approach based upon clinical signs of RVF to delineate functional and biological processes associated with RVF. METHODS AND RESULTS: Wistar rats were subjected to a pulmonary artery banding (PAB n=12) or sham surgery (CON, n=7). After 52±5days, 5/12 PAB rats developed clinical symptoms of RVF (inactivity, ruffled fur, dyspnea, ascites) necessitating termination (PAB+CF). We compared these to PAB rats with RVF without clinical symptoms (PAB-). PAB resulted in reduced cardiac output, RV stroke volume, TAPSE, and increased end diastolic pressure (all p<0.05 vs. CON) in all rats, but PAB+CF rats were significantly more affected than PAB-, despite similar pressure load (p=ns). Pressure-volume analysis showed enhanced contractility (end systolic elastance) in PAB- and PAB+CF, but diastolic function (end diastolic elastance, end diastolic pressure) deteriorated especially in PAB+CF. In PAB+CF capillary density was lower than in PAB-. Gene-array analysis revealed downregulation of both fatty acid oxidation and carbohydrate metabolism in PAB+CF. CONCLUSION: Chronic PAB led to different degrees of RVF, with half of the rats developing severe clinical symptoms of RVF, associated with progressive deterioration of diastolic function, hypoxia-prone myocardium, increased response to oxidative stress and suppressed myocardial metabolism. This model represents clinical RVF and allows for unraveling of mechanisms involved in the progression from RV adaptation to RV failure and the effect of intervention on these mechanisms.

A critical role for Egr-1 during vascular remodelling in pulmonary arterial hypertension.

AIMS: Pulmonary arterial hypertension (PAH) is characterized by the development of unique neointimal lesions in the small pulmonary arteries, leading to increased right ventricular (RV) afterload and failure. Novel therapeutic strategies are needed that target these neointimal lesions. Recently, the transcription factor Egr-1 (early growth response protein 1) was demonstrated to be up-regulated early in experimental neointimal PAH. Its effect on disease development, however, is unknown. We aimed to uncover a novel role for Egr-1 as a molecular inductor for disease development in PAH. METHODS AND RESULTS: In experimental flow-associated PAH in rats, we investigated the effects of Egr-1 down-regulation on pulmonary vascular remodelling, including neointimal development, and disease progression. Intravenous administration of catalytic oligodeoxynucleotides (DNA enzymes, DNAzymes) resulted in down-regulation of pulmonary vascular Egr-1 expression. Compared with vehicle or scrambled DNAzymes, DNAzymes attenuated pulmonary vascular remodelling, including the development of occlusive neointimal lesions. Selective down-regulation of Egr-1 in vivo led to reduced expression of vascular PDGF-B, TGF-ß, IL-6, and p53, resulting in a reduction of vascular proliferation and increased apoptosis. DNAzyme treatment further attenuated pulmonary vascular resistance, RV systolic pressure, and RV hypertrophy. In contrast, in non-neointimal PH rodents, DNAzyme treatment had no effect on pulmonary vascular and RV remodelling. Finally, pharmacological inhibition of Egr-1 with pioglitazone, a peroxisome proliferator activated receptor-γ ligand, attenuated vascular remodelling including the development of neointimal lesions. CONCLUSIONS: These results indicate that Egr-1 governs pulmonary vascular remodelling and the development of characteristic vascular neointimal lesions in flow-associated PAH. Egr-1 is therefore a potential target for future PAH treatment.

Distinct loading conditions reveal various patterns of right ventricular adaptation.

Right ventricular (RV) failure due to chronically abnormal loading is a main determinant of outcome in pulmonary hypertension (PH) and congenital heart disease. However, distinct types of RV loading have been associated with different outcomes. To determine whether the adaptive RV response depends on loading type, we compared hemodynamics, exercise, and hypertrophy in models of pressure overload due to pulmonary artery banding (PAB), pressure overload due to PH, combined pressure and volume overload, and isolated volume load. Ninety-four rats were subjected to either PAB, monocrotaline-induced PH (PH), aortocaval shunt (shunt), or combined monocrotaline and aortocaval shunt (PH + shunt). We performed pressure-volume analysis and voluntary exercise measurements at 4 wk. We compared PAB to PH (part I) and PH + shunt to either isolated PH or shunt (part II). In part I, enhanced contractility (end-systolic elastance and preload recruitable stroke work) was present in PH and PAB, but strongest in PAB. Frank-Starling mechanism was active in both PAB and PH. In PAB this was accompanied by diastolic dysfunction (increased end-diastolic elastance, relaxation constant), clinical signs of RV failure, and reduced exercise. These distinct responses were not attributable to differences in hypertrophy. In part II, in PH + shunt the contractility response was blunted compared with PH, which caused pseudonormalization of parameters. Additional volume overload strongly enhanced hypertrophy in PH. We conclude that different types of loading result in distinct patterns of RV adaptation. This is of importance for the approach to patients with chronically increased RV load and for experimental studies in various types of RV failure.

The role of disturbed blood flow in the development of pulmonary arterial hypertension: lessons from preclinical animal models.

Pulmonary arterial hypertension (PAH) is a progressive pulmonary vasoproliferative disorder characterized by the development of unique neointimal lesions, including concentric laminar intima fibrosis and plexiform lesions. Although the histomorphology of neointimal lesions is well described, the pathogenesis of PAH and neointimal development is largely unknown. After three decades of PAH pathobiology research the focus has shifted from vasoconstriction towards a mechanism of cancer-like angioproliferation. In this concept the role of disturbed blood flow is seen as an important trigger in the development of vascular remodeling. For instance, in PAH associated with congenital heart disease, increased pulmonary blood flow (i.e., systemic-to-pulmonary shunt) is an essential trigger for the occurrence of neointimal lesions and PAH development. Still, questions remain about the exact role of these blood flow characteristics in disease progression. PAH animal models are important for obtaining insight in new pathobiological processes and therapeutical targets. However, as for any preclinical model the pathophysiological mechanism and clinical course has to be comparable to the human disease that it mimics. This means that animal models mimicking human PAH ideally are characterized by: a hit recognized in human disease (e.g., altered pulmonary blood flow), specific vascular remodeling resembling human neointimal lesions, and disease progression that leads to right ventriclular dysfunction and death. A review that underlines the current knowledge of PAH due to disturbed flow is still lacking. In this review we will summarize the current knowledge obtained from PAH animal models associated with disturbed pulmonary blood flow and address questions for future treatment strategies for PAH.

Sildenafil enhances systolic adaptation, but does not prevent diastolic dysfunction, in the pressure-loaded right ventricle.

AIM: Right ventricular (RV) failure due to pressure or volume overload is a major risk factor for early mortality in congenital heart disease and pulmonary hypertension, but currently treatments are lacking. We aimed to demonstrate that the phosphodiesterase 5A inhibitor sildenafil can prevent adverse remodelling and improve function in chronic abnormal RV overload, independent from effects on the pulmonary vasculature. METHODS AND RESULTS: In rat models of either pressure or volume overload, we performed pressure-volume studies to measure haemodynamic effects and voluntary exercise testing as clinical outcome after 4 weeks of sildenafil (or vehicle) administration. In the pressure-loaded right ventricle, sildenafil enhanced contractility [end-systolic elastance (mmHg/mL) 247 ± 68 vs.155 ± 71, sildenafil vs. vehicle, P < 0.05], prevented RV dilatation [end-diastolic volume (µL) 733 ± 50 vs. 874 ± 39, P < 0.05], reduced wall stress [peak wall stress (mmHg) 323 ± 46 vs. 492 ± 62, P < 0.05], and partially preserved exercise tolerance [running distance (%) -33 ± 15 vs. -62 ± 12, P < 0.05]. Protein kinase A was not activated by sildenafil and thus did not mediate the observed effects. In contrast, protein kinase G-1 was activated by sildenafil, but hypertrophy was not inhibited. Importantly, sildenafil did not prevent diastolic dysfunction, whereas RV fibrosis appeared to be increased in sildenafil-treated rats. In the volume-loaded right ventricle, sildenafil treatment did not show any beneficial effects. CONCLUSION: We demonstrate sildenafil to have beneficial, afterload-independent effects on the pressure-loaded right ventricle, but not on the volume-loaded right ventricle. These results indicate that sildenafil may offer a specific treatment for the pressure-loaded right ventricle, although persistent diastolic dysfunction and RV fibrosis could be of concern.

Multiple voxel 1H MR spectroscopy of phosphorylase-b kinase deficient patients (GSD IXa) showing an accumulation of fat in the liver that resolves with aging.

BACKGROUND/AIMS: Phosphorylase-b deficient patients suffer from glycogen storage disease (GSD IXa) leading to liver enlargement which usually resolves during puberty and adolescence. This pathology has not yet been documented by (1)H MR spectroscopy (MRS) investigation. METHODS: MRS of eight GSD IXa patients was performed in this study to assess whether or not liver fat content is elevated in GSD IXa and decreases with aging. An improvement in our MRS method compared with previous liver fat MRS studies is that we measured a plane of liver voxels at once rather than a single MRS voxel, yielding a reliable determination of liver fat content. RESULTS: Fat contents of 3.4-10% were observed in young GSD IXa patients, as compared with 0.5-0.9% in controls, these dropped to control levels in patients past age 40 (r = -0.82; P < 0.01). CONCLUSIONS: Liver fat content is increased in glycogen storage disease (GSD IXa) and normalizes with ageing. Assessing liver fat levels in this population is a novel and interesting concept. This could potentially enhance the understanding of liver function in that 20% of the population who has increased liver fat.