Endothelial-to-Mesenchymal Transition as Underlying Mechanism for the Formation of Double-Chambered Right Ventricle.

Double-chambered right ventricle (DCRV) is a progressive division of the right ventricular outflow tract (RVOT) often associated with a subaortic ventricular defect (VSD). The septation is caused by a mixture of hypertrophied muscle bundles and fibrous tissue, whereof the latter is of unclear pathogenesis. Our group has previously reported that flow disturbances lead to formation of fibroelastic tissue through a process called endothelial-to-mesenchymal transition (EndMT) but it is unclear whether the same mechanism exists in the RV. Tissue from patients undergoing repair of DCRV was examined to identify the histomorphological substrate of this tissue. Demographic and pre-/post-operative echocardiographic data were collected from nine patients undergoing surgery for DCRV. RVOTO tissue samples were histologically analyzed for myocardial hypertrophy, fibrosis, elastin content, and active EndMT (immunohistochemical double-staining for endothelial and mesenchymal markers and transcription factors Slug/Snail) and compared to four healthy controls. Indication for surgery were symptoms and progressive RVOT gradients. A highly turbulent flow jet through the RVOTO and VSD was observed in all patients with a preoperative median RVOT peak gradient of 77 mmHg (IQR 55.0-91.5), improved to 6 mmHg (IQR 4.5-17) postoperatively. Histological analysis revealed muscle and thick infiltratively growing fibroelastic tissue. EndMT was confirmed as underlying patho-mechanism of this fibroelastic tissue but the degree of myocardial hypertrophy was not different compared to controls (P = 0.08). This study shows for the first time that an invasive fibroelastic remodeling processes of the endocardium into the underlying myocardium through activation of EndMT contributes to the septation of the RVOT.

Human endothelial colony-forming cells provide trophic support for pluripotent stem cell-derived cardiomyocytes via distinctively high expression of neuregulin-1.

The search for a source of endothelial cells (ECs) with translational therapeutic potential remains crucial in regenerative medicine. Human blood-derived endothelial colony-forming cells (ECFCs) represent a promising source of autologous ECs due to their robust capacity to form vascular networks in vivo and their easy accessibility from peripheral blood. However, whether ECFCs have distinct characteristics with translational value compared to other ECs remains unclear. Here, we show that vascular networks generated with human ECFCs exhibited robust paracrine support for human pluripotent stem cell-derived cardiomyocytes (iCMs), significantly improving protection against drug-induced cardiac injury and enhancing engraftment at ectopic (subcutaneous) and orthotopic (cardiac) sites. In contrast, iCM support was notably absent in grafts with vessels lined by mature-ECs. This differential trophic ability was due to a unique high constitutive expression of the cardioprotective growth factor neuregulin-1 (NRG1). ECFCs, but not mature-ECs, were capable of actively releasing NRG1, which, in turn, reduced apoptosis and increased the proliferation of iCMs via the PI3K/Akt signaling pathway. Transcriptional silencing of NRG1 abrogated these cardioprotective effects. Our study suggests that ECFCs are uniquely suited to support human iCMs, making these progenitor cells ideal for cardiovascular regenerative medicine.

Autogenous mitochondria transplantation for treatment of right heart failure.

BACKGROUND: Right ventricular hypertrophy and failure are major causes of cardiac morbidity and mortality. A key event in the progression to right ventricular hypertrophy and failure is cardiomyocyte apoptosis due to mitochondrial dysfunction. We sought to determine whether localized intramyocardial injection of autologous mitochondria from healthy muscle treats heart failure. METHODS: Mitochondria transplanted from different sources were initially tested in cultured hypertrophic cardiomyocytes. A right ventricular hypertrophy/right ventricular failure model created through banding of the pulmonary artery in immature piglets was used for treatment with autologous mitochondria (pulmonary artery banded mitochondria injected/treated n = 6) from calf muscle, versus vehicle (pulmonary artery banded vehicle injected/treated n = 6) injected into the right ventricular free-wall, and compared with sham-operated controls (sham, n = 6). Animals were followed for 8 weeks by echocardiography (free-wall thickness, contractility), and dp/dt max was measured concomitantly with cardiomyocyte hypertrophy, fibrosis, and apoptosis at study end point. RESULTS: Internalization of mitochondria and adenosine triphosphate levels did not depend on the source of mitochondria. At 4 weeks, banded animals showed right ventricular hypertrophy (sham: 0.28 ± 0.01 cm vs pulmonary artery banding: 0.4 ± 0.02 cm wall thickness; P = .001), which further increased in pulmonary artery banded mitochondria injected/treated but declined in pulmonary artery banded vehicle injected/treated (0.47 ± 0.02 cm vs 0.348 ± 0.03 cm; P = .01). Baseline contractility was not different but was significantly reduced in pulmonary artery banded vehicle injected/treated compared with pulmonary artery banded mitochondria injected/treated and so was dp/dtmax. There was a significant difference in apoptotic cardiomyocyte loss and fibrosis in sham versus hypertrophied hearts with most apoptosis in pulmonary artery banded vehicle injected/treated hearts (sham: 1 ± 0.4 vs calf muscle vs vehicle: 13 ± 1.7; P = .001 and vs pulmonary artery banded mitochondria injected/treated: 8 ± 1.9, P = .01; pulmonary artery banded vehicle injected/treated vs pulmonary artery banded mitochondria injected/treated, P = .05). CONCLUSIONS: Mitochondrial transplantation allows for prolonged physiologic adaptation of the pressure-loaded right ventricular and preservation of contractility by reducing apoptotic cardiomyocyte loss.

Flow disturbances and the development of endocardial fibroelastosis.

OBJECTIVES: Endothelial-to-mesenchymal transition (EndMT) has been identified as the underlying mechanism of endocardial fibroelastosis (EFE) formation. The purpose of this study was to determine whether hemodynamic alterations due to valvar defects promote EndMT and whether age-specific structural changes affect ventricular diastolic compliance despite extensive surgical resection of EFE tissue. MATERIAL AND METHODS: We analyzed EFE tissue from 24 patients with hypoplastic left heart syndrome (HLHS) who underwent left ventricular (LV) rehabilitation surgery at Boston Children's Hospital between December 2011 and March 2018. Six patients with flow disturbances across the aortic valve and/or mitral valve but no HLHS diagnosis and macroscopic appearance of "EFE-like tissue" in the LV were included for comparison. All samples were examined for amount of collagen/elastin production and degradation, and presence of active EndMT by histologic analysis. RESULTS: EFE tissue from patients with and without HLHS consisted predominantly of elastin and collagen fibers. There was no alteration in degradation activity for collagen or elastin as shown by in situ zymography. Active EndMT was found in all patients with and without HLHS with flow disturbances ("EFE-like"). In patients with HLHS, EFE infiltrated into the underlying myocardium with increasing age. CONCLUSIONS: Patients with and without HLHS with flow disturbances due to stenotic or incompetent valves develop EndMT-derived fibrotic tissue covering the LV. When EFE recurs, it is directly associated with flow disturbances and switches to an infiltrative growth pattern with increasing age, leading to increased diastolic stiffness of the LV.

Flow disturbances and progression of endocardial fibroelastosis - a case report.

Endocardial fibroelastosis (EFE) is described as thickening of the endocardium and is associated with hypoplastic left heart syndrome (HLHS). The stimulus for EFE and the mechanism for recurrence and/or progression need to be investigated. In this report, we describe the case of a 4-year-old HLHS patient who underwent several surgeries with EFE resections due to recurrence of EFE. EFE recurrence was associated with flow disturbances due to valvar defects. At her latest follow-up 7 months after the last surgery, competent valves and no EFE were identified on all imaging study.

Vascular Endothelial Growth Factor Prevents Endothelial-to-Mesenchymal Transition in Hypertrophy.

BACKGROUND: In hypertrophy, progressive loss of function caused by impaired diastolic compliance correlates with advancing cardiac fibrosis. Endothelial cells contribute to this process through endothelial-to-mesenchymal transition (EndMT) resulting from inductive signals such as transforming growth factor (TGF-ß). Vascular endothelial growth factor (VEGF) has proven effective in preserving systolic function and delaying the onset of failure. In this study, we hypothesize that VEGF inhibits EndMT and prevents cardiac fibrosis, thereby preserving diastolic function. METHODS: The descending aorta was banded in newborn rabbits. At 4 and 6 weeks, hypertrophied animals were treated with intrapericardial VEGF protein and compared with controls (n = 7 per group). Weekly transthoracic echocardiography measured peak systolic stress. At 7 weeks, diastolic stiffness was determined through pressure-volume curves, fibrosis by Masson trichrome stain and hydroxyproline assay, EndMT by immunohistochemistry, and activation of TGF-ß and SMAD2/3 by quantitative real-time polymerase chain reaction. RESULTS: Peak systolic stress was preserved during the entire observation period, and diastolic compliance was maintained in treated animals (hypertrophied: 20 ± 1 vs treated: 11 ± 3 and controls: 12 ± 2; p < 0.05). Collagen was significantly higher in the hypertrophied group by Masson trichrome (hypertrophied: 3.1 ± 0.9 vs treated: 1.8 ± 0.6) and by hydroxyproline assay (hypertrophied: 2.8 ± 0.6 vs treated: 1.4 ± 0.4; p < 0.05). Fluorescent immunostaining showed active EndMT in the hypertrophied group but significantly less in treated hearts, which was directly associated with a significant increase in TGF-ß/SMAD-2 messenger RNA expression. CONCLUSIONS: EndMT contributes to cardiac fibrosis in hypertrophied hearts. VEGF treatment inhibits EndMT and prevents the deposition of collagen that leads to myocardial stiffness through TGF-ß/SMAD-dependent activation. This presents a therapeutic opportunity to prevent diastolic failure and preserve cardiac function in pressure-loaded hearts.

Myocardial rescue with autologous mitochondrial transplantation in a porcine model of ischemia/reperfusion.

OBJECTIVE: To demonstrate the clinical efficacy of autologous mitochondrial transplantation in preparation for translation to human application using an in vivo swine model. METHODS: A left mini-thoracotomy was performed on Yorkshire pigs. The pectoralis major was dissected, and skeletal muscle tissue was removed and used for the isolation of autologous mitochondria. The heart was subjected to regional ischemia (RI) by temporarily snaring the circumflex artery. After 24 minutes of RI, hearts received 8 × 0.1 mL injections of vehicle (vehicle-only group; n = 6) or vehicle containing mitochondria (mitochondria group; n = 6) into the area at risk (AAR), and the snare was released. The thoracotomy was closed, and the pigs were allowed to recover for 4 weeks. RESULTS: Levels of creatine kinase-MB isoenzyme and cardiac troponin I were significantly increased (P = .006) in the vehicle-only group compared with the mitochondria group. Immune, inflammatory, and cytokine activation markers showed no significant difference between groups. There was no significant between-group difference in the AAR (P = .48), but infarct size was significantly greater in the vehicle group (P = .004). Echocardiography showed no significant differences in global function. Histochemistry and transmission electron microscopy revealed damaged heart tissue in the vehicle group that was not apparent in the mitochondria group. T2-weighted magnetic resonance imaging and histology demonstrated that the injected mitochondria were present for 4 weeks. CONCLUSIONS: Autologous mitochondrial transplantation provides a novel technique to significantly enhance myocardial cell viability following ischemia and reperfusion in the clinically relevant swine model.

A light-reflecting balloon catheter for atraumatic tissue defect repair.

A congenital or iatrogenic tissue defect often requires closure by open surgery or metallic components that can erode tissue. Biodegradable, hydrophobic light-activated adhesives represent an attractive alternative to sutures, but lack a specifically designed minimally invasive delivery tool, which limits their clinical translation. We developed a multifunctional, catheter-based technology with no implantable rigid components that functions by unfolding an adhesive-loaded elastic patch and deploying a double-balloon design to stabilize and apply pressure to the patch against the tissue defect site. The device uses a fiber-optic system and reflective metallic coating to uniformly disperse ultraviolet light for adhesive activation. Using this device, we demonstrate closure on the distal side of a defect in porcine abdominal wall, stomach, and heart tissue ex vivo. The catheter was further evaluated as a potential tool for tissue closure in vivo in rat heart and abdomen and as a perventricular tool for closure of a challenging cardiac septal defect in a large animal (porcine) model. Patches attached to the heart and abdominal wall with the device showed similar inflammatory response as sutures, with 100% small animal survival, indicating safety. In the large animal model, a ventricular septal defect in a beating heart was reduced to <1.6 mm. This new therapeutic platform has utility in a range of clinical scenarios that warrant minimally invasive and atraumatic repair of hard-to-reach defects.

Endocardial fibroelastosis is caused by aberrant endothelial to mesenchymal transition.

RATIONALE: Endocardial fibroelastosis (EFE) is a unique form of fibrosis, which forms a de novo subendocardial tissue layer encapsulating the myocardium and stunting its growth, and which is typically associated with congenital heart diseases of heterogeneous origin, such as hypoplastic left heart syndrome. Relevance of EFE was only recently highlighted through the establishment of staged biventricular repair surgery in infant patients with hypoplastic left heart syndrome, where surgical removal of EFE tissue has resulted in improvement in the restrictive physiology leading to the growth of the left ventricle in parallel with somatic growth. However, pathomechanisms underlying EFE formation are still scarce, and specific therapeutic targets are not yet known. OBJECTIVE: Here, we aimed to investigate the cellular origins of EFE tissue and to gain insights into the underlying molecular mechanisms to ultimately develop novel therapeutic strategies. METHODS AND RESULTS: By utilizing a novel EFE model of heterotopic transplantation of hearts from newborn reporter mice and by analyzing human EFE tissue, we demonstrate for the first time that fibrogenic cells within EFE tissue originate from endocardial endothelial cells via aberrant endothelial to mesenchymal transition. We further demonstrate that such aberrant endothelial to mesenchymal transition involving endocardial endothelial cells is caused by dysregulated transforming growth factor beta/bone morphogenetic proteins signaling and that this imbalance is at least in part caused by aberrant promoter methylation and subsequent transcriptional suppression of bone morphogenetic proteins 5 and 7. Finally, we provide evidence that supplementation of exogenous recombinant bone morphogenetic proteins 7 effectively ameliorates endothelial to mesenchymal transition and experimental EFE in rats. CONCLUSIONS: In summary, our data point to aberrant endothelial to mesenchymal transition as a common denominator of infant EFE development in heterogeneous, congenital heart diseases, and to bone morphogenetic proteins 7 as an effective treatment for EFE and its restriction of heart growth.

Pressure overload induces IL-18 and IL-18R expression, but markedly suppresses IL-18BP expression in a rabbit model. IL-18 potentiates TNF-α-induced cardiomyocyte death.

Recurrent or sustained inflammation plays a causal role in the development and progression of left ventricular hypertrophy (LVH) and its transition to failure. Interleukin (IL)-18 is a potent pro-hypertrophic inflammatory cytokine. We report that induction of pressure overload in the rabbit, by constriction of the descending thoracic aorta induces compensatory hypertrophy at 4weeks (mass/volume ratio: 1.7±0.11) and ventricular dilatation indicative of heart failure at 6weeks (mass/volume ratio: 0.7±0.04). In concordance with this, fractional shortening was preserved at 4weeks, but markedly attenuated at 6weeks. We cloned rabbit IL-18, IL-18Rα, IL-18Rß, and IL-18 binding protein (IL-18BP) cDNA, and show that pressure overload, while enhancing IL-18 and IL-18R expression in hypertrophied and failing hearts, markedly attenuated the level of expression of the endogenous IL-18 antagonist IL-18BP. Cyclical mechanical stretch (10% cyclic equibiaxial stretch, 1Hz) induced hypertrophy of primary rabbit cardiomyocytes in vitro and enhanced ANP, IL-18, and IL-18Rα expression. Further, treatment with rhIL-18 induced its own expression and that of IL-18Rα via AP-1 activation, and induced cardiomyocyte hypertrophy in part via PI3K/Akt/GATA4 signaling. In contrast, IL-18 potentiated TNF-α-induced cardiomyocyte death, and by itself induced cardiac endothelial cell death. These results demonstrate that pressure overload is associated with enhanced IL-18 and its receptor expression in hypertrophied and failingrabbit hearts. Since IL-18BP expression is markedly inhibited, our results indicate a positive amplification in IL-18 proinflammatory signaling during pressure overload, and suggest IL-18 as a potential therapeutic target in pathological hypertrophy and cardiac failure.

A blood-resistant surgical glue for minimally invasive repair of vessels and heart defects.

Currently, there are no clinically approved surgical glues that are nontoxic, bind strongly to tissue, and work well within wet and highly dynamic environments within the body. This is especially relevant to minimally invasive surgery that is increasingly performed to reduce postoperative complications, recovery times, and patient discomfort. We describe the engineering of a bioinspired elastic and biocompatible hydrophobic light-activated adhesive (HLAA) that achieves a strong level of adhesion to wet tissue and is not compromised by preexposure to blood. The HLAA provided an on-demand hemostatic seal, within seconds of light application, when applied to high-pressure large blood vessels and cardiac wall defects in pigs. HLAA-coated patches attached to the interventricular septum in a beating porcine heart and resisted supraphysiologic pressures by remaining attached for 24 hours, which is relevant to intracardiac interventions in humans. The HLAA could be used for many cardiovascular and surgical applications, with immediate application in repair of vascular defects and surgical hemostasis.

Endogenous angiogenesis inhibitors prevent adaptive capillary growth in left ventricular pressure overload hypertrophy.

BACKGROUND: In left ventricular (LV) pressure-overload hypertrophy, lack of adaptive capillary growth contributes to progression to failure. Remodeling of the hypertrophied myocardium requires proteolysis of the extracellular matrix (ECM) carried out by matrix metalloproteinases (MMPs). MMPs, specifically MMP-9, are known to cleave ECM components to generate angiogenesis inhibitors (angiostatin, endostatin, tumstatin). We hypothesize that MMP-9 releases antiangiogenic factors during compensated and decompensated hypertrophy, which results in lack of adaptive capillary growth. METHODS: Newborn rabbits underwent aortic banding. Myocardial tissue from age-matched and banded animals at compensated (4 weeks) and decompensated hypertrophy (7 weeks), as identified by serial echocardiography, was analyzed by immunoblotting for angiostatin, endostatin, and tumstatin. MMP-9 activity was determined by zymography. A cell-permeable, potent, selective MMP-9 inhibitor was administered intrapericardially to animals with hypertrophied hearts and tissue was analyzed. RESULTS: MMP-9 is activated in hypertrophied myocardium versus in control hearts (22 ± 2 versus 16 ± 1; p = 0.04), which results in significantly increased levels of angiostatin (115 ± 10 versus 86 ± 7; p = 0.02), endostatin (33 ± 1 versus 28 ± 1; p = 0.006), and tumstatin (35 ± 6 versus 17 ± 4; p = 0.04). Zymography confirms inhibition of MMP-9 (hypertrophy + MMP-9 inhibitor, 14 ± 0.6 versus hypertrophy + vehicle, 17 ± 1; p = 0.01) and angiostatin, endostatin, and tumstatin are down-regulated, accompanied by up-regulation of capillary density (hypertrophy + MMP-9 inhibitor, 2.99 ± 0.07 versus hypertrophy + vehicle, 2.7 ± 0.05; p = 0.002). CONCLUSIONS: Up-regulation of angiogenesis inhibitors prevents adaptive capillary growth in pressure-overload hypertrophied myocardium. Therapeutic interventions aimed at inhibition of angiogenesis inhibitors are useful in maintaining capillary density and thereby preventing heart failure.

Up-regulation of soluble vascular endothelial growth factor receptor-1 prevents angiogenesis in hypertrophied myocardium.

AIMS: Inadequate capillary growth in pressure-overload hypertrophy impairs myocardial perfusion and substrate delivery, contributing to progression to failure. Capillary growth is tightly regulated by angiogenesis growth factors like vascular endothelial growth factor (VEGF) and endogenous inhibitors such as the splice variant of VEGF receptor-1, sVEGFR-1. We hypothesized that inadequate expression of VEGF and up-regulation of VEGFR-1 and its soluble splice variant, sVEGFR-1, restrict capillary growth in pressure-overload hypertrophy. METHODS AND RESULTS: Neonatal New Zealand White rabbits underwent aortic banding. mRNA (qRT-PCR) and protein levels (immunoblotting) were determined in hypertrophied and control myocardium (7/group) for total VEGF, VEGFR-1, sVEGFR-1, VEGFR-2, and phospho-VEGFR-1 and -R-2. Free VEGF was determined by enzyme-linked immunoassay (ELISA) in hypertrophied myocardium, controls, and hypertrophied hearts following inhibition of sVEGFR-1 with placental growth factor (PlGF). VEGFR-1 and sVEGFR-1 mRNA (seven-fold up-regulation, P = 0.001) and protein levels were significantly up-regulated in hypertrophied hearts vs. controls (VEGFR-1: 44 ± 8 vs. 23 ± 1, P = 0.031; sVEGFR-1: 71 ± 13 vs. 31 ± 3, P = 0.016). There was no change in VEGF and VEGFR-2 mRNA or protein levels in hypertrophied compared with controls hearts. A significant decline in free, unbound VEGF was found in hypertrophied myocardium which was reversed following inhibition of sVEGFR-1 with PlGF, which was accompanied by phosphorylation of VEGFR-1 and VEGFR-2. CONCLUSION: Up-regulation of the soluble VEGFR-1 in pressure-loaded myocardium prevents capillary growth by trapping VEGF. Inhibition of sVEGFR-1 released sufficient VEGF to induce angiogenesis and preserved contractile function. These data suggest sVEGFR-1 as possible therapeutic targets to prevent heart failure.

Electron transport chain dysfunction in neonatal pressure-overload hypertrophy precedes cardiomyocyte apoptosis independent of oxidative stress.

OBJECTIVES: We have previously shown in a model of pressure-overload hypertrophy that there is increased cardiomyocyte apoptosis during the transition from peak hypertrophy to ventricular decompensation. Electron transport chain dysfunction is believed to play a role in this process through the production of excessive reactive oxygen species. In this study we sought to determine electron transport chain function in pressure-overload hypertrophy and the role of oxidative stress in myocyte apoptosis. METHODS AND RESULTS: Neonatal rabbits underwent thoracic aortic banding at 10 days of age. Compensated hypertrophy (4 weeks of age), decompensated hypertrophy (6 weeks of age), and age-matched controls (n = 4-8 per group) as identified by serial echocardiography were studied. Electron transport chain complex activities were determined by spectophotometry in isolated mitochondria. Complex I was significantly decreased (P = .005) at 4 weeks and further decreased at 6 weeks (P = .001). Complex II was significantly decreased at both time points (4 weeks, P = .003; 6 weeks, P = .009). However, hyddrogen peroxide production, measured in isolated mitochondria by fluorescence spectroscopy, was significantly decreased at 4 weeks of age in banded animals compared with controls (P = .038), and mitochondrial DNA oxidative damage (measurement of 8- hydroxydeoxyguanosine by enzyme-linked immunosorbent assay) was also significantly decreased at 4 weeks of age (P = .031). Mitochondrial activated apoptosis was determined by Bax/Bcl-2 ratios (immunoblotting). Bax/Bcl-2 levels were significantly increased in banded animals at 6 weeks. CONCLUSIONS: In pressure-overload hypertrophy, the transition from compensated left ventricular hypertrophy to failure and cardiomyocyte apoptosis is preceded by mitochondrial complex I and II dysfunction followed by an increase in Bax/Bcl-2 ratios. The mechanism of apoptosis initiation is independent of increased oxidative stress.

Myocyte apoptosis occurs early during the development of pressure-overload hypertrophy in infant myocardium.

OBJECTIVE: Abnormal hemodynamic loading often accompanies congenital heart disease both before and after surgical repair. Adaptive and maladaptive myocardial responses to increased load are numerous. This study examined the hypothesis that myocyte loss occurs during compensatory hypertrophic growth in the developing infant myocardium subjected to progressive pressure overload. METHODS: Pressure-overload left ventricular hypertrophy was induced in 7- to 10-day-old rabbits by banding the thoracic aorta. Left ventricular function and mechanics were quantified by serial echocardiography and noninvasive left ventricular wall stress analysis. Left ventricular tissue sections were examined for fibrosis by using Masson's trichrome stain and for myocyte apoptosis by using a myocyte-specific DNA fragmentation assay and caspase-3 activation (specific fluorescent substrate). RESULTS: Significant myocyte apoptosis (198 +/- 37/10(6) myocytes, P < .01 vs control) and caspase-3 activation were present in early hypertrophy when left ventricular contractility was preserved and compensatory hypertrophy had normalized wall stress. By 6 weeks, multiple indices of left ventricular contractility were reduced, and left ventricular wall stress was increased. Myocyte apoptosis was accelerated (361 +/- 56/10(6) myocytes), caspase-3 activity further increased, and the estimated total number of left ventricular myocytes was significantly reduced by 18% +/- 4%. CONCLUSION: In experimental infant left ventricular hypertrophy, myocyte apoptosis is initiated in the face of normalized wall stress and preserved contractility. The ongoing rate of apoptosis causes a measurable decrease in myocyte number that is coincident with the onset of ventricular dysfunction. It thus appears that pressure overload, even at its earliest stages, is not well tolerated by the developing ventricle.

Inhibition of glycogen synthase kinase-3beta improves tolerance to ischemia in hypertrophied hearts.

BACKGROUND: Hypertrophied myocardium is more susceptible to ischemia/reperfusion injury, in part owing to impaired insulin-mediated glucose uptake. Glycogen synthase kinase-3beta (GSK-3beta) is a key regulatory enzyme in glucose metabolism that, when activated, phosphorylates/inactivates target enzymes of the insulin signaling pathway. Glycogen synthase kinase-3beta is regulated upstream by Akt-1. We sought to determine whether GSK-3beta is activated in ischemic hypertrophied myocardium owing to impaired Akt-1 function, and whether inhibition with lithium (Li) or indirubin-3'-monoxime,5-iodo- (IMI), a specific inhibitor, improves post-ischemic myocardial recovery by improving glucose metabolism. METHODS: Pressure-overload hypertrophy was achieved by aortic banding in neonatal rabbits. At 6 weeks, isolated hypertrophied hearts underwent 30 minutes of normothermic ischemia and reperfusion with or without a GSK-3beta inhibitor (0.1 mM Li; 1 microM IMI) as cardioplegic additives. Cardiac function was measured before and after ischemia. Expression, activity of Akt-1 and GSK-3beta, and lactate were determined at end-ischemia. RESULTS: Contractile function after ischemia was better preserved in hypertrophied hearts treated with GSK-3beta inhibitors. Activity of Akt-1 was significantly impaired in hypertrophied myocardium at end-ischemia. Glycogen synthase kinase-3beta enzymatic activity at end-ischemia was increased in hypertrophied hearts and was blocked by Li or IMI concomitant with significantly increased lactate production, indicating increased glycolysis. CONCLUSIONS: Regulatory inhibition of GSK-3beta by Akt-1 in hypertrophied hearts is impaired, leading to activation during ischemia. Inhibition of GSK-3beta by Li or IMI improves tolerance to ischemia/reperfusion injury in hypertrophied myocardium. The likely protective mechanism is an increase in insulin-mediated glucose uptake, resulting in greater substrate availability for glycolysis during ischemia and early reperfusion.

Age-dependent expression of VEGF isoforms and receptors in the rabbit anterior cruciate ligament.

Vascular endothelial growth factor (VEGF) gene gives rise to several distinct isoforms of VEGF. Those isoforms differ in biochemical and biological properties, and it has been reported that their expression patterns are tissue and age specific as well. We investigated the expression levels of VEGF isoforms (VEGF121, VEGF165, VEGF183, VEGF189) and its receptors (VEGFR-1, flt-1 and VEGFR-2, flk-1/KDR) in the anterior cruciate ligament (ACL) of 2- to 3-week-, 2-month-, and 18-month-old New Zealand White rabbits using Sybr green Real-Time RT-PCR. VEGF isoforms and both receptors were expressed in the ACL at all investigated ages. VEGF121 was found to be the most abundant isoform at the ages under investigation, followed by VEGF165, VEGF189 and VEGF183. All isoforms showed decreased expression levels with age, however the larger membrane bound isoforms, VEGF183 and VEGF189, showed the most striking age-associated decrease in expression level. VEGFR-1 expression levels increased with age, while the expression level of VEGFR-2 expression was highest at 2-3 weeks and was significantly lower at 2 and 18 months of age. Distinct age-associated differences in the expression level of VEGF isoforms as well as their receptors suggest differential physiological functions during development, maturation and ageing of the ACL.

Dopamine induces postischemic cardiomyocyte apoptosis in vivo: an effect ameliorated by propofol.

BACKGROUND: Dopamine is commonly used to improve postischemic myocardial contractile function. However, there is evidence that dopamine augments apoptosis after ischemia through increased intracellular calcium and opening of the mitochondrial permeability transition pore. Propofol (2,6-diisopropylphenol) is an anesthetic that has been shown to prevent mitochondrial permeability transition pore opening. We evaluated the effects of propofol given during reperfusion on dopamine-mediated apoptosis. METHODS: Hearts from 8-week-old inbred New Zealand White rabbit siblings were subjected to 2 hours of cold cardioplegic ischemia and 6 hours of reperfusion in a heterotopic transplant model. Controls consisted of the recipient rabbit's nonischemic heart. The ischemia-reperfusion (IR) group consisted of postischemic hearts reperfused with no drugs; the IR plus dopamine (IR+D) group received dopamine (20 microg x kg(-1) x min(-1)) continuously; the IR+D plus propofol (IR+D+P) group received dopamine (20 microg x kg(-1) x min(-1)) plus propofol (500 to 600 microg x kg(-1) x min(-1)); and the IR plus propofol (IR+P) group received propofol only (500 to 600 microg x kg(-1) x min(-1)) throughout reperfusion (n = 7 to 9 in each group). Myocardial function was measured using a left ventricular balloon; terminal nick-end labeling (TUNEL) staining, DNA electrophoresis, and immunoblotting for caspase-3 cleavage were performed at the end of reperfusion. RESULTS: Dopamine increased the number of TUNEL-positive nuclei significantly (14.0 +/- 2.0/1,000 for IR+D versus 6.7 +/- 2.0/1,000 for IR, p = 0.01). Propofol (IR+D+P) reduced the total number of apoptotic cells in hearts receiving dopamine (7.1 +/- 1.8/1,000, p = 0.01 versus IR+D) to the extent seen in IR alone. DNA laddering and caspase-3 cleavage were observed at greater frequency in the IR+D group compared with the IR and IR+D+P groups. Propofol had no effect on dopamine-mediated increased systolic function, but improved diastolic function after ischemia. CONCLUSIONS: Dopamine infusion has a positive inotropic effect on the postischemic heart at the expense of increased cardiomyocyte apoptosis. The addition of propofol prevents dopamine-induced apoptosis after ischemia while maintaining positive inotropy.

Cardiac conduction through engineered tissue.

In children, interruption of cardiac atrioventricular (AV) electrical conduction can result from congenital defects, surgical interventions, and maternal autoimmune diseases during pregnancy. Complete AV conduction block is typically treated by implanting an electronic pacemaker device, although long-term pacing therapy in pediatric patients has significant complications. As a first step toward developing a substitute treatment, we implanted engineered tissue constructs in rat hearts to create an alternative AV conduction pathway. We found that skeletal muscle-derived cells in the constructs exhibited sustained electrical coupling through persistent expression and function of gap junction proteins. Using fluorescence in situ hybridization and polymerase chain reaction analyses, myogenic cells in the constructs were shown to survive in the AV groove of implanted hearts for the duration of the animal's natural life. Perfusion of hearts with fluorescently labeled lec-tin demonstrated that implanted tissues became vascularized and immunostaining verified the presence of proteins important in electromechanical integration of myogenic cells with surrounding re-cipient rat cardiomyocytes. Finally, using optical mapping and electrophysiological analyses, we provide evidence of permanent AV conduction through the implant in one-third of recipient animals. Our experiments provide a proof-of-principle that engineered tissue constructs can function as an electrical conduit and, ultimately, may offer a substitute treatment to conventional pacing therapy.