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Background Mechanical right ventricular (RV) support in pulmonary arterial hypertension patients has been feared to cause pulmonary hemorrhage and to be detrimental for the after-load–sensitive RV. Continuous low-flow pumps offer promise but remain insufficiently tested. Methods The pulmonary artery was banded in 20 sheep in this study. Eight weeks later, a Synergy micro-pump (HeartWare International, Framingham MA) was inserted in 10 animals, driving blood from the right atrium to the pulmonary artery. After magnetic resonance imaging, hemodynamics and RV pressure–volume loop data were recorded. Eight weeks later, RV function was assessed in the same way, followed by histologic analysis of the ventricular tissue. Results During the 8 weeks of support, RV volumes and central venous pressure decreased significantly, whereas RV contractility increased. Pulmonary artery pressure increased modestly, particularly its diastolic component. RV contribution to total right-sided cardiac output increased from 12 ± 12% to 41 ± 9% ( p ). After pump inactivation, and compared with 8 weeks earlier, RV volumes had significantly decreased, tricuspid valve regurgitation had almost disappeared, and RV contractility had significantly increased, resulting in significantly increased RV forward power (0.25 ± 0.05 vs 0.16 ± 0.06 W, p = 0.014). Fulton index and RV myocyte size were significantly smaller, and without changes in fibrosis, when compared with controls. Conclusions Prolonged continuous low-flow RV mechanical support significantly unloads the chronic pressure–overloaded RV and improves cardiac output. After 8 weeks, RV hemodynamic recovery and reverse remodeling begin to occur, without increased fibrosis.

BACKGROUND: Interstitial fibrosis is an important component of diastolic, and systolic, dysfunction in heart failure (HF) and depends on activation and differentiation of fibroblasts into myofibroblasts (MyoFb). Recent clinical evidence suggests that in late-stage HF, fibrosis is not reversible. OBJECTIVES: The study aims to examine the degree of differentiation of cardiac MyoFb in end-stage HF and the potential for their phenotypic reversibility. METHODS: Fibroblasts were isolated from the left ventricle of the explanted hearts of transplant recipients (ischemic and dilated cardiomyopathy), and from nonused donor hearts. Fibroblasts were maintained in culture without passaging for 4 or 8 days (treatment studies). Phenotyping included functional testing, immunostaining, and expression studies for markers of differentiation. These data were complemented with immunohistology and expression studies in tissue samples. RESULTS: Interstitial fibrosis with cross-linked collagen is prominent in HF hearts, with presence of activated MyoFbs. Tissue levels of transforming growth factor (TGF)-β1, lysyl oxidase, periostin, and osteopontin are elevated. Fibroblastic cells isolated from HF hearts are predominantly MyoFb, proliferative or nonproliferative, with mature α-smooth muscle actin stress fibers. HF MyoFb express high levels of profibrotic cytokines and the TGF-β1 pathway is activated. Inhibition of TGF-β1 receptor kinase in HF MyoFb promotes dedifferentiation of MyoFb with loss of α-smooth muscle actin and depolymerization of stress fibers, and reduces the expression of profibrotic genes and cytokines levels to non-HF levels. CONCLUSION: MyoFb in end-stage HF have a variable degree of differentiation and retain the capacity to return to a less activated state, validating the potential for developing antifibrotic therapy targeting MyoFb. ispartof: JOURNAL OF THE AMERICAN COLLEGE OF CARDIOLOGY vol:73 issue:18 pages:2267-2282 ispartof: location:United States status: published

Aims Differentiation of cardiac fibroblasts (Fbs) into myofibroblasts (MyoFbs) is responsible for connective tissue build-up in myocardial remodelling. We examined MyoFb differentiation and reversibility. Methods and results Adult rat cardiac Fbs were cultured on a plastic substratum providing mechanical stress, with conditions to obtain different levels of Fb differentiation. Fb spontaneously differentiated to proliferating MyoFb (p-MyoFb) with stress fibre formation decorated with alpha-smooth muscle actin (α-SMA). Transforming growth factor-β1 (TGF-β1) promoted differentiation into α-SMA-positive MyoFb showing near the absence of proliferation, i.e. non-p-MyoFb. SD-208, a TGF-β-receptor-I (TGF-β-RI) kinase blocker, inhibited p-MyoFb differentiation as shown by stress fibre absence, low α-SMA expression, and high proliferation levels. Fb seeded in collagen matrices induced no contraction, whereas p-MyoFb and non-p-MyoFb induced 2.5- and four-fold contraction. Fb produced little collagen but high levels of interleukin-10. Non-p-MyoFb had high collagen production and high monocyte chemoattractant protein-1 and tissue inhibitor of metalloproteinases-1 levels. Transcriptome analysis indicated differential activation of gene networks related to differentiation of MyoFb (e.g. paxilin and PAK) and reduced proliferation of non-p-MyoFb (e.g. cyclins and cell cycle regulation). Dedifferentiation of p-MyoFb with stress fibre de-polymerization, but not of non-p-MyoFb, was induced by SD-208 despite maintained stress. Stress fibre de-polymerization could also be induced by mechanical strain release in p-MyoFb and non-p-MyoFb (2-day cultures in unrestrained 3-D collagen matrices). Only p-MyoFb showed true dedifferentiation after long-term 3-D cultures. Conclusions Fb, p-MyoFb, and non-p-MyoFb have a distinct gene expression, ultrastructural, and functional profile. Both reduction in mechanical strain and TGF-β-RI kinase inhibition can reverse p-MyoFb differentiation but not non-p-MyoFb.

Scarring and remodeling of the left ventricle (LV) after myocardial infarction (MI) results in ischemic cardiomyopathy with reduced contractile function. Regional differences related to persisting ischemia may exist. We investigated the hypothesis that mitochondrial function and structure is altered in the myocardium adjacent to MI with reduced perfusion (MIadjacent) and less so in the remote, nonischemic myocardium (MIremote). We used a pig model of chronic coronary stenosis and MI ( n = 13). Functional and perfusion MR imaging 6 wk after intervention showed reduced ejection fraction and increased global wall stress compared with sham-operated animals (Sham; n = 14). Regional strain in MIadjacent was reduced with reduced contractile reserve; in MIremote strain was also reduced but responsive to dobutamine and perfusion was normal compared with Sham. Capillary density was unchanged. Cardiac myocytes isolated from both regions had reduced basal and maximal oxygen consumption rate, as well as through complex I and II, but complex IV activity was unchanged. Reduced respiration was not associated with detectable reduction of mitochondrial density. There was no significant change in AMPK or glucose transporter expression levels, but glycogen content was significantly increased in both MIadjacent and MIremote. Glycogen accumulation was predominantly perinuclear; mitochondria in this area were smaller but only in MIadjacent where also subsarcolemmal mitochondria were smaller. In conclusion, after MI reduction of mitochondrial respiration and glycogen accumulation occur in all LV regions suggesting that reduced perfusion does not lead to additional specific changes and that increased hemodynamic load is the major driver for changes in mitochondrial function.

Objective: Fibroblasts have been shown to couple to neonatal cardiomyocytes in heterocellular cultures through functional gap junctions. Our objective was to provide evidence for an additional type of heterocellular communication between fibroblasts and adult cardiomyocytes in vitro and in vivo. Methods: The contact areas in heterocellular co-cultures were evaluated by specific labeling and the intercellular communication was studied using preloading of fibroblasts with tracer molecules. Heterocellular fibroblast-cardiomyocyte contacts present in the in vitro setting and in the border zone of a rabbit myocardial infarction in vivo were further examined by electron microscopy. Results: Addition of fibroblasts preloaded with the fluorescent low molecular weight tracer calcein-AM to cultured myocytes indicated early dye transfer via connexin 43 functional gap junctions. At a later time-period after co-culturing, dye transfer of fibroblasts preloaded with the high molecular weight tracer dextran 10,000 suggested partial cell fusion. The membrane continuity giving rise to this partial cell fusion was confirmed by electron microscopy, clearly showing areas of intercytoplasmic contacts between fibroblasts and phenotypically adapted (dedifferentiated) cardiomyocytes. Fluorescein-labeled annexin V affinity studies revealed transient exposure of phosphatidylserine at the contact sites, suggesting that phosphatidylserine mediates the fusion process. Close contacts between cardiac fibroblasts and dedifferentiated cardiomyocytes accompanied by disruption of the basal lamina were observed in the border zone of a rabbit myocardial infarction in vivo. Conclusion: Our results suggest that the partial cell fusion-type of heterocellular communication in our co-culture model and the contacts observed in vivo may lead to new insights in cardiovascular disease.

Introduction Our goal was to show that blunting of myocardial flow reserve is mainly involved in adaptive chronic myocardial hibernation without apparent cardiomyocyte degeneration. Methods and results Sheep chronically instrumented with critical multivessel stenosis and/or percutaneous transluminal coronary angioplasty (PTCA)-induced revascularization were allowed to run and feed in the open for 2 and 5 months, respectively. Regional myocardial blood flow (MBF) with colored microspheres, regional and global left ventricular function and dimensions (2D echocardiography), and myocardial structure were studied. In sheep with a critical stenosis, a progressive increase in left ventricular end-diastolic and end-systolic cavity area and a decrease in fractional area change were found. Fraction of wall thickness decreased in all left ventricular wall segments. MBF was slightly but not significantly decreased at rest at 2 months. Morphological quantification revealed a rather small but significant increase in diffusely distributed connective tissue, cardiomyocyte hypertrophy, and presence of viable myocardium of which almost 30 % of the myocytes showed depletion of sarcomeres and accumulation of glycogen. The extent of myolysis in the transmural layer correlated with the degree of left ventricular dilation. Structural degeneration of cardiomyocytes was not observed. Balloon dilatation (PTCA) of one of the coronary artery stenoses at 10 weeks revealed recovery of fraction of wall thickness and near normalization of global subcellular structure at 20 weeks. Conclusion These data indicate that chronic reduction of coronary reserve by itself can induce ischemic cardiomyopathy characterized by left ventricular dilatation, depressed regional and global function, adaptive chronic myocardial hibernation, reactive fibrosis and cardiomyocyte hypertrophy in the absence of obvious degenerative phenomena. Summary Reduction of myocardial flow reserve due to chronic coronary artery stenosis in sheep induces adaptive myocardial hibernation without involvement of degenerative phenomena.

Oxidative stress can modulate nitric oxide (NO) signalling pathways. Both pathways have been shown to be involved in the pathophysiology of atrial fibrillation (AF), but data are conflicting. We aimed to characterize the NO-pathway and its relation to oxidative stress in persistent AF in a sheep model.Persistent AF was induced by rapid atrial pacing for a mean of 136.5 ± 21.7 days. Non-stimulated sheep served as controls. Nicotine adenine dinucleotide phosphate (NADPH) oxidase-stimulated superoxide production was significantly increased in the AF group (+51.3 ± 23.2%, P0.01). Although there were no changes in mRNA expression of the different NADPH oxidase subunits, the increased activity was associated with markedly increased protein expression of the NADPH oxidase activator, Rac1 (+26 ± 4.6%, P0.05). No differences were seen in superoxide dismutase activity, but glutathione peroxidase activity was lower in the AF group. There was a marked accumulation of 3-nitrotyrosine, a biomarker for peroxynitrite, in atrial tissue of AF animals, as demonstrated by immunohistochemical staining and dot blot analysis (+15.6 ± 1.8%, P0.05). Expression of atrial NOS3 mRNA was 24.9 ± 4.4% lower in the AF group vs. control (P0.05), while NOS1 and 2 were unchanged. Immunoblot analysis revealed no changes in protein expression. Nitrite/nitrate levels were significantly lower during AF (-24.8 ± 5.8%, P0.05).In a sheep model of persistent AF, NOS3 transcript levels are attenuated and circulating NOx levels decreased. Persistent AF is associated with increased oxidative stress, probably resulting from increased NADPH oxidase activity, without major changes in anti-oxidant capacity of the atrial tissue.

Background Microvascular obstruction after reperfusion therapy of acute myocardial infarction is reported as an adverse promoter of left ventricular remodeling and is an important target to prevent deterioration into heart failure. In this study, we illustrate the early onset of a magnetic resonance imaged microvascular obstruction in a porcine model of acute myocardial infarction with the exact histological correlate. Methods Occlusion of the left anterior descending coronary artery followed by 3-h reperfusion was performed in 10 pigs. Microvascular obstruction was assessed by contrast-enhanced magnetic resonance imaging (MRI). After sacrifice, serial sectioned slices of the hearts matching the MRI were stained with Triphenyl tetrazolium chloride (TTC). Biopsies were fixed, embedded in paraffin, and stained for hematoxylin–eosin. Results Microvascular obstruction was defined with MRI as a hypoenhanced no-reflow area within the hyperenhanced infarct region. Erythrocyte plugging was consistently observed in the no-reflow area and was completely absent in the adjacent hyperenhanced infarct region. Conclusion This model of acute ischemia–reperfusion contributes to the histological comprehension of contrast-enhanced MRI during the early stages of myocardial infarction.

AIMS: Exercise started early after myocardial infarction (MI) improves in vivo cardiac function and myofilament responsiveness to Ca(2+). We investigated whether this represents partial or complete reversal of cellular remodelling. METHODS AND RESULTS: Mice with MI following left coronary ligation were given free access to a running wheel (MI(EXE), N = 22) or housed sedentary (MI(SED), N = 18) for 8 weeks and compared with sedentary sham-operated animals (SHAM, N = 11). Myocytes were enzymatically isolated from the non-infarcted left ventricle. Myocytes in MI were significantly longer and even more so with exercise (165 +/- 3 microm in MI(EXE) vs. 148 +/- 3 microm in MI(SED) and 136 +/- 2 microm in SHAM; P < 0.05, mean +/- SEM); cell width was not different. Contraction was measured during electrical field stimulation at 1, 2, and 4 Hz. Unloaded cell shortening was significantly reduced in MI(SED) (at 1 Hz, L/L(0)=4.4 +/- 0.3% vs. 6.7 +/- 0.4% in SHAM; P < 0.05, also at 2 and 4 Hz). Exercise restored cell shortening to SHAM values (MI(EXE), L/L(0)=6.4 +/- 0.5%). Membrane currents and [Ca(2+)](i) were measured via whole-cell patch clamping, with Fluo-3 as Ca(2+) indicator, all at 30 degrees C. Ca(2+) transient amplitude, I(CaL) and sarcoplasmic reticulum Ca(2+) content were not different between the three groups. Diastolic Ca(2+) levels at 4 Hz were significantly elevated in MI(SED) only, with a trend to increased spontaneous Ca(2+) release events (sparks). Action potential duration was increased and transient outward K(+) currents significantly reduced after MI; this was unaffected by exercise. CONCLUSIONS: Early voluntary exercise training after MI restores cell contraction to normal values predominantly because of changes in the myofilament Ca(2+) response and has a beneficial effect on diastolic Ca(2+) handling. However, the beneficial effect is not a complete reversal of remodelling as hypertrophy and loss of repolarizing K(+) currents are not affected. ispartof: Cardiovascular Research vol:86 issue:1 pages:72-81 ispartof: location:England status: published

Rationale : Persistent atrial fibrillation (AF) has been associated with structural and electric remodeling and reduced contractile function. Objective : To unravel mechanisms underlying reduced sarcoplasmic reticulum (SR) Ca 2+ release in persistent AF. Methods : We studied cell shortening, membrane currents, and [Ca 2+ ] i in right atrial myocytes isolated from sheep with persistent AF (duration 129±39 days, N=16), compared to matched control animals (N=21). T-tubule density, ryanodine receptor (RyR) distribution, and local [Ca 2+ ] i transients were examined in confocal imaging. Results : Myocyte shortening and underlying [Ca 2+ ] i transients were profoundly reduced in AF (by 54.8% and 62%, P 2+ ] i . SR Ca 2+ content was not different. Calculated fractional SR Ca 2+ release was reduced in AF (by 20.6%, P 2+ current density was modestly decreased (by 23.9%, P P 2+ release in AF was significantly reduced with increased areas of delayed Ca 2+ release. Propagation between RyR was unaffected but Ca 2+ release at subsarcolemmal sites was reduced. Rate of Ca 2+ extrusion by Na + /Ca 2+ exchanger was increased. Conclusions : In persistent AF, reduced SR Ca 2+ release despite preserved SR Ca 2+ content is a major factor in contractile dysfunction. Fewer Ca 2+ channel–RyR couplings and reduced efficiency of the coupling at subsarcolemmal sites, possibly related to increased Na + /Ca 2+ exchanger, underlie the reduction in Ca 2+ release.