Skeletal muscle myofibrillar protein oxidation and exercise capacity in heart failure

Background Heart failure is characterized by limited exercise tolerance and by a skeletal muscle myopathy with atrophy and shift toward fast fibres. An inflammatory status with elevated pro-inflammatory cytokines and exaggerated free radicals production can worsen muscle damage. We have previously demonstrated in a model of heart failure, the monocrotaline treated rat, that oxidation of skeletal muscle actin, tropomyosin and myosin produces a reduction of contractile efficiency, which may further depress muscle function and exercise capacity. Aims To investigate the presence of oxidized myofibrillar proteins in skeletal muscle of CHF patients by means of the Oxyblot technique and to correlate it with exercise capacity. Methods We have analyzed skeletal muscle biopsies taken from six patients with class III–IV NYHA CHF and four control patients (peak VO₂ 12.8 ± 1.9 vs. 29.7 ± 1.7 ml/kg/min, p < 0.0001). Results and conclusions A correlation between degree of myofibrillar oxidation and exercise capacity measured as peak VO₂ was obtained. In the skeletal muscle of CHF patient there was a much higher level of myofibrillar protein oxidation as expressed by the Oxyblot/Red Ponceau (Oxy/RP) ratio as compared to controls (2.1 ± 0.3 vs. 1.02 ± 0.09, p < 0.0001). The VO₂/Oxy/RP was significantly lower in the CHF patients. Higher levels of muscle oxidation were found in patients with lower exercise capacity with an inverse correlation between Oxyblot and VO₂ values (r ² = 0.83). Returned for 1. Revision: 6 August 2007 1. Revision received: 2 October 2007 Returned for 2. Revision: 8 November 2007 2. Revision received: 9 November 2007     

LAMP-2 deficient mice show depressed cardiac contractile function without significant changes in calcium handling

Objective Mutations in the highly glycosylated lysosome associated membrane protein-2 (LAMP-2) cause, as recently shown, familial Danon disease with mental retardation, mild myopathy and fatal cardiomyopathy. Extent and basis of the contractile dysfunction is not completely understood. Methods In LAMP-2 deficient mice, we investigated cardiac function in vivo using Doppler-echocardiography and contractile function in vitro in isolated myocardial trabeculae. Results LAMP-2 deficient mice displayed reduced ejection fraction (EF) (58.9±3.4 vs. 80.7±5.1%, P<0.05) and reduced cardiac output (8.3±3.1 vs. 14.7±3.6 ml/min, P<0.05) as compared to wild-type controls. Isolated multicellular muscle preparations from LAMP-2 deficient mice confirmed depressed force development (3.2±0.6 vs. 8.4±0.9 mN/mm², P<0.01). All groups showed similar force-frequency behaviour when normalised to baseline force. Post-rest potentiation was significantly depressed at intervals >15 s in LAMP-2 deficient mice (P<0.05). Although attenuated in absolute force development, the normalised inotropic response to increased calcium and β-adrenoreceptor stimulation was unaltered. Electron microscopic analysis revealed autophagic vacuoles in LAMP-2 deficient cardiomyocytes. Protein analysis showed unaltered levels of SERCA2a, calsequestrin and phospholamban. Conclusions Cardiac contractile function in LAMP-2 deficient mice as a model for Danon disease is significantly attenuated. The occurrence of autophagic vacuoles in LAMP-2 deficient myocytes is likely to be causal for the depressed contractile function resulting in an attenuated cardiac pump reserve. Returned for 1. revision: 5 December 2003 1. Revision received: 18 November 2005 Returned for 2. revision: 12 December 2005 2. Revision received: 23 December 2005 Returned for 3. revision: 1 February 2006 3.Revision received: 8 February 2006 Drs. Stypmann and Janssen contributed equally.     

Impaired endothelial progenitor cell function predicts age-dependent carotid intimal thickening

Objectives  We investigated whether qualitative or quantitative alterations of the endothelial progenitor cell (EPC) pool predict age-related structural vessel wall changes. Background  We have previously shown that age-related endothelial dysfunction is accompanied by qualitative rather than quantitative changes of EPCs. Animal studies suggest that impaired EPC functions lead to accelerated arterial intimal thickening. Methods  Intima-media thickness (IMT) was measured in the common carotid artery in our previously published groups of younger (25 ± 1 years, n = 20) and older (61 ± 2 years, n = 20) healthy non-smoking volunteers without arterial hypertension, hypercholesterolemia, and diabetes mellitus. Endothelial progenitor cells (EPCs, KDR⁺/CD34⁺ and KDR⁺/CD133⁺) were counted in peripheral blood using flow cytometry. In ex vivo expanded EPCs, the function was determined as chemotaxis to VEGF, proliferation, and survival. Results  We observed thicker IMT in older as compared to younger subjects (0.68 ± 0.03 mm Vs. 0.48 ± 0.02 mm, P < 0.001). Importantly, there were significant inverse univariate correlations between IMT, EPC chemotaxis, and survival (r = −0.466 P < 0.05; r = −0.463, P < 0.01). No correlation was observed with numbers of circulating EPCs. Multivariate regression analysis revealed that age, mean arterial pressure and migration of EPCs were independent predictors of IMT (R ² = 0.58). Conclusion  Impaired EPC function may lead to accelerated vascular remodeling due to chronic impairment of endothelial maintenance. Returned for 1. Revision: 13 December 2007 1. Revision received: 16 June 2008 Returned for 2. Revision: 20 June 2008 2. Revision received: 17 July 2008     

Inhibition of apoptotic responses after ischemic stress in isolated hearts and cardiomyocytes

Recent findings on the induction of anti-apoptotic gene expression in ischemic/reperfused hearts encouraged us to investigate whether ischemic/reperfused hearts may be protected against apoptosis induction. To analyze this hypothesis we performed studies on isolated perfused hearts of rat. For apoptosis induction, hearts were perfused with the NO donor (±)-S-nitroso-N-acetylpenicillamine (SNAP, 10 μM) for 30 minutes. Four hours thereafter apoptosis was detected by DNA laddering and TUNEL assay. Under normoperfusion SNAP induced 5.5 ± 1.4 TUNEL-positive myocytes per tissue section (vs. 1.8 ± 0.5 in controls). But when hearts were subjected to 20 minutes of no flow ischemia, which was sufficient for energy depletion of the hearts without inducing severe necrotic or apoptotic cell death, reperfusion in the presence of SNAP did not induce apoptosis. To analyze if this mode of protection is a property of the cardiomyocytes, we performed corresponding experiments on ventricular cardiomyocytes of rat. Again, under normoxic conditions SNAP (100 (μM) increased the number of TUNEL-positive cells to 12.6 ± 4.9 % (vs. 5.4 ± 0.7 % in controls). But when SNAP was added after 3 h of simulated ischemia, which was sufficient for energy depletion of the cells without inducing apoptotic cell death, the number of apoptotic cells did not increase. The ischemia-induced protection of hearts and cardiomyocytes goes along with an increased expression of several anti-apoptotic genes, mainly of the bcl-2 family. This indicates that ischemic conditions induce an anti-apoptotic gene program in cardiomyocytes, which may also be responsible for the observed anti-apoptotic actions in the intact ischemic/reperfused myocardium. Received: 20 March 2002, Returned for 1. revision: 8 April 2002, 1. Revision received: 30 April 2002, Returned for 2. revision: 21 May 2002, 2. Revision received: 29 May 2002, Returned for 3. revision: 29 May 2002, 3. Revision received: 6 June 2002, Accepted: 12 June 2002 Correspondence to: Dr. G. Taimor     

Electrophysiology of single cardiomyocytes isolated from rabbit pulmonary veins: implication in initiation of focal atrial fibrillation

Pulmonary veins (PVs) are important foci in initiation of paroxysmal atrial fibrillation. However, the mechanisms of the high arrhythmogenic activity of PVs are unclear. This study aimed to isolate single cardiomyocytes from PVs and evaluate their electrophysiological characteristics and arrhythmogenic potential. Cardiomyocytes of rabbit PVs were isolated by retrograde perfusion with digestive enzymes from aorta via left ventricle and left atrium. The action potentials and ionic currents were investigated in isolated single PV cardiomyocytes using the whole-cell clamp technique. Dissociation of PVs yielded single pacemaker cardiomyocytes (76 %) and non-pacemaker cardiomyocytes with a fast response action potential. Both the pacemaker and non-pacemaker cardiomyocytes had similar inward Ca²⁺ currents and transient outward K⁺ currents. However, the pacemaker cardiomyocytes had a smaller inward rectifier K⁺ current (1.50 ± 0.22 versus 4.21 ± 1.15 pA/pF, P < 0.005) and a larger delayed rectifier K⁺ current (0.60 ± 0.05 versus 0.24 ± 0.05 pA/pF, P < 0.005) than non-pacemaker cardiomyocytes. Acetylcholine induced hyperpolarization and inhibited the spontaneous action potential. Isoproterenol (10 nM) accelerated the spontaneous activity and induced early or delayed afterdepolarization, which could be suppressed by nifedipine. The PV cardiomyocytes with early afterdepolarization have a greater prolongation of action potential duration (ΔAPD, + 67 ± 17 versus −109 ± 20 ms, P < 0.0001) and a greater increase of inward Ca²⁺ current (0.90 ± 0.23 versus 0.38 ± 0.08 pA/pF, P < 0.05) after isoproterenol than those without early afterdepolarization. These findings suggest that PV cardiomyocytes have distinct action potentials and ionic current profiles, which may be responsible for the high arrhythmogenic activity of the PVs. Received: 17 April 2001, Returned for revision: 23 May 2001, Revision received: 11 July 2001, Accepted: 17 July 2001     

Intravenous infusion of mesenchymal stem cells enhances regional perfusion and improves ventricular function in a porcine model of myocardial infarction

Transplantation of stem cells may improve regional perfusion and post-infarct ventricular function, but the optimal dose and efficacy of cell delivery via the intravenous route has not been determined. This study tested the hypothesis that intravenous infusion of bone marrow-derived mesenchymal stem cells (MSCs) enhances regional perfusion and improves ventricular function after myocardial infarction. In a closed-chest pig model, the LAD coronary artery was occluded for 75 min by angioplasty balloon inflation followed by 12 weeks of reperfusion. After 15 min of reperfusion, pigs randomly received 1 of 4 treatments: (1) Vehicle (Control, n = 10); (2) 1 × 10⁶ MSCs/kg (1 mill, n = 7); (3) 3 × 10⁶ MSCs/kg (3 mill, n = 8) and (4) 10 × 10⁶ MSCs/kg (10 mill, n = 8). Angiogenesis was demonstrated by immunohistochemical staining, myocardial blood flow (steady state and vasodilator reserve) was measured using 15 μm neutron-activated microspheres, and cardiac function was determined by contrast left ventriculography (ejection fraction) and pressure–volume relationships. After 12 week of reperfusion, von Willebrand Factor-positive vessels and tissue vascular endothelial growth factor (VEGF) expression in the scar zone was significantly greater in all MSCs-treated animals relative to Control. Steady state myocardial blood flow in the scar tissue was comparable among groups. However, adenosine recruited vasodilator reserve in the scar zone induced by intracoronary adenosine was significantly higher in the MSC-treated animals compared to Control. Furthermore, preload-recruitable stroke work and systolic performance were significantly greater compared to Control. In conclusion, these data demonstrate that intravenous delivery of MSCs during early reperfusion augments vasculogenesis, enhances regional perfusion, and improves post-infarct ventricular function. The results suggest that intravenous infusion of MSCs is an effective modality for the treatment of ischemia/reperfusion induced myocardial injury. Returned for 1. Revision: 11 April 2008 1. Revision received: 30 May 2008 Returned for 2. Revision: 11 June 2008 2. Revision received: 7 July 2008 Returned for 3. Revision: 9 July 2008 3. Revision received: 14 July 2008     

Effects of pentoxifylline on the vascular response to injury after angioplasty in rabbit iliac arteries

Pentoxifylline (PTX) inhibits the effects of several cytokines and reduces injury-related collagen accumulation. The aim of the present study was to investigate the effect of PTX on the vascular response to injury. We treated rabbits with PTX (100 mg/kg/day) or placebo (saline) subcutaneously from 2 days before angioplasty of an iliac artery until euthanasia 7 or 28 days later. At 7 days after injury, PTX treatment was associated with a more differentiated (less proliferation, more smoothelin-positive) intimal smooth muscle cell phenotype. Furthermore, PTX reduced myofibroblast accumulation in adventitia. At 28 days after injury, PTX-treated rabbits had a 48.5% larger lumen area (P = 0.03) and a 28.1% larger area within the external elastic lamina (P = 0.04). There were no significant differences between PTX-treated rabbits and the placebo group with regard to neointima and media area. Angioplasty induced marked neoadventitial hyperplasia, which was reduced by 20.5% (P = 0.01) in the PTX-treated group. Finally, PTX reduced collagen density in all three arterial layers. We conclude that PTX treatment induces less proliferation within the vessel wall early after angioplasty and increases late lumen size after angioplasty by a positive effect on vascular remodeling. Martin Busk and Michael Maeng contributed equally to this paper. Returned for 1. Revision: 6 September 2007 1. Revision received: 4 October 2007 Returned for 2. Revision: 24 October 2007 2. Revision received: 31 October 2007     

Effect of atrial dilatation on electrophysiologic properties and inducibility of atrial fibrillation

Introduction: Atrial dilatation may play an important role in the occurrence of atrial fibrillation (AF) in clinical situations. However, the electrophysiologic characteristics of dilated atria are still unclear. Methods and results: In 18 isolated Langendorff-perfused canine hearts (14.6 ± 2.2 kg), we measured atrial effective refractory periods (ERPs) at four different sites, conduction velocity and percentage of slow conduction on the right atrium (using a high-density electrode plaque), and assessed the inducibility of AF at the baseline (0 cm H₂O) and high (15 cm H₂O) atrial pressure. The atrial ERPs did not change significantly, but the dispersion of ERP increased significantly (40 ± 18 vs 25 ± 9 vs ms, p = 0.01) during high atrial pressure. The percentage of slow conduction (< 25 cm/s) over the mapping area, and the inducibility of AF increased during high atrial pressure (23.7 ± 10.2 % vs 32.1 ± 12.5 %, p = 0.02). The AF inducibility significantly correlated with the ERP dispersion (R = 0.75, p < 0.001) and maximal percentage of slow conduction (R = 0.88, p < 0.001). Furthermore, ERPs were significantly shorter in the induced AF group than those without induced AF (68 ± 17 vs 84 ± 16 ms, P < 0.05). Conclusions: The increased inhomogenity in atrial electrophysiological properties during atrial dilatation contributed to the inducibility of AF. Received: 26 November 2001?Returned for 1. revision: 2 January 2002?1. Revision received: 11 February 2002?Returned for 2. revision: 25 March 2002?2. Revision received: 6 May 2002?Returned for 3. revision: 10 June 2002?3. Revision received: 21 August 2002?Accepted: 11 September 2002     

Ultrastructural study of calcium shift in ischemic/reperfused rat heart under treatment with dimethylthiourea, diltiazem and amiloride

Among factors underlying reperfusion injury are oxygen free radicals and Ca²⁺ influx via gated calcium channel or via Na⁺/H⁺–Na⁺/Ca²⁺ exchange which lead to calcium overload. The aim of the study was to ultrastructurally visualize the distribution of Ca²⁺ and to compare binding of calcium by the sarcolemma and calcium accumulation in mitochondria under therapy with an ·OH scavenger, dimethylthiourea (DMTU), Na⁺/H⁺ exchange inhibitor, amiloride, and calcium channel blocker, diltiazem, given alone or in combination to ischemic/reperfused hearts. Isolated working hearts subjected to 30 min ischemia and 30 min reperfusion were perfused with drugs added to the perfusate 15 min before ischemia and administered for the rest of the perfusion period. The cytochemical phosphate pyroantimonate method for localization of Ca²⁺ was used, and calcium distribution was analyzed with a computer image analyzer. All drugs given alone improved sarcolemmal ability to bind calcium. The best results were obtained with amiloride. All of the combined therapies gave even better results, but calcium accumulation in mitochondria diminished only with diltiazem therapy given alone or in combination with DMTU. Since the presence of Ca²⁺ deposits on the sarcolemma is believed to represent its normal function, and calcium sequestration by mitochondria reflects an increase in cytosolic calcium load, the lack of correlation between sarcolemmal and mitochondrial Ca²⁺ distribution might suggest impaired mechanisms of lowering cytoplasmic calcium or the existence of some mechanism other than Na⁺/Ca²⁺ exchange, mediated by activated Na⁺/H⁺ exchange. Received: 3 March 1997, Returned for 1. revision: 21 September 1997, 1. Revision received: 31 October 1997, Returned for 2. revision: 29 November 1997, 2. Revision received: 9 February 1998, Returned for 3. revision: 16 February 1998, 3. Revision received: 2 March 1998, Accepted: 3 March 1998     

Modulation of L-type Ca2+ channel current density and inactivation by β-adrenergic stimulation during murine cardiac embryogenesis

Background  L-type Ca²⁺ current (I _CaL) is a key regulatory and functional element during early embryonic cardiomyogenesis. As the embryonic heart underlies hormonal modulation, e.g. catecholamines, we aimed at studying effects of β-adrenergic stimulation on I _CaL densities and inactivation kinetics during murine heart development. Methods   I _CaL was recorded applying the whole-cell patch-clamp technique in ventricular myocytes of early embryonic (EDS, E9.5–11.5) and late developmental, fetal (LDS, E16.5–18.5) stages as well as adult cardiomyocytes. To distinguish between Ca²⁺-(CDI) and voltage-dependent inactivation (VDI), Ca²⁺ was replaced with Ba²⁺ in the extracellular recording solution. The β-adrenergic signaling pathway was simulated by applying isoproterenol (Iso). Results  Basal current densities showed an increase of I _CaL during development (EDS: −9.61 ± 1.97 pA/pF, n = 9; LDS: −13.2 ± 4.26 pA/pF, n = 12; adult: −16.1 ± 4.63 pA/pF, n = 5). Iso (1 μM) enhanced I _CaL density with low effects at EDS (17.1 ± 3.58%, n = 8, P > 0.05) but strong effects at LDS (74.1 ± 3.77%, n = 8, P < 0.01) and in adults (90.6 ± 7.01%, n = 6, P < 0.001). The current availability was significantly higher at LDS as compared to EDS and elevated after application of Iso. In the presence of Ca²⁺, the fast phase of I _CaL inactivation (τ_f) was significantly enhanced by Iso at LDS. The slow phase of inactivation (τ_s) was unaltered at both developmental stages. However, VDI was significantly reduced under Iso in LDS and adult cardiomyocytes. Conclusion  These results imply that β-adrenergic modulation becomes of importance especially during fetal heart development. CDI and VDI of I _CaL are modulated by β-adrenergic stimulation in fetal but not in early embryonic mouse cardiomyocytes. Furthermore our data suggest important changes of the L-type Ca²⁺ channel protein, and/or maturation of the Ca²⁺-induced Ca²⁺ release (CICR) machinery. Returned for 1. Revision: 7 March 2008 1. Revision received: 8 March 2008 Returned for 2. Revision: 15 August 2008 2. Revision received: 20 September 2008     

Monocyte chemoattractant protein-1 enhances and interleukin-10 suppresses the production of inflammatory cytokines in adult rat cardiomyocytes

Objective Chemokines control the migration of leukocytes to inflamed tissue, and in particular monocyte chemoattractant protein (MCP)-1 has been implicated in the pathogenesis of several cardiovascular disorders such as chronic heart failure (CHF) and myocarditis. We hypothesised that MCP-1 may directly contribute to an inflammatory response in the cardiomyocytes, and in the present study we examined in adult rat cardiomyocytes: (i) the effect of tumour necrosis factor (TNF)a on MCP-1 production, (ii) the effect of MCP-1 on production of other inflammatory cytokines, and (iii) if the anti-inflammatory cytokine interleukin (IL)-10 could suppress any TNFα-induced MCP-1 production. Methods We used enzyme immunoassays, RNase protection assays and slot blot analysis to measure protein and mRNA levels of various cytokines in adult rat cardiomyocyte cultures. Results (i) We found a ∼6.4-fold increase of the MCP-1 level accompanied by an increase in MCP-1 mRNA accumulation in cardiomyocyte cultures after TNFα stimulation. (ii) In contrast, TNFα had no effect on IL-10 and only a modest effect on IL-1β and IL-6 levels in these cells. (iii) Importantly, MCP-1 stimulated inflammatory response in cardiomyocytes by enhancing IL-1β and IL-6 levels in these cells as found at both the protein and mRNA level. (iv) Co-stimulation with IL-10 resulted in a ∼55 % reduction in TNFα-stimulated MCP-1 levels in cardiomyocyte culture supernatants. Conclusion The present study demonstrates for the first time that MCP-1 can directly affect cardiomyocytes, and we introduce MCP-1 as a potential enhancer and IL-10 as a potential suppresser of inflammatory responses within the myocardium. Received: 10 October 2000 / Returned for 1. revision: 2 November 2000 / 1. Revision received: 5 December 2000 / Returned for 2. revision: 2 January 2001 / 2. Revision received: 5 January 2001 / Accepted: 8 January 2001     

Omega-3 polyunsaturated fatty acid-enriched diet differentially protects two subpopulations of myocardial mitochondria against Ca2+-induced injury

Omega-3 polyunsaturated fatty acids (PUFA) confer protection against myocardial injury after ischemia-reperfusion. There are two subfractions of mitochondria located in different regions of the cell: subsarcolemmal mitochondria (SSM) and interfibrillar mitochondria (IFM). The present study explored possible differences between Ca²⁺-induced mitochondrial swelling in rat SSM and IFM fractions under control conditions (control group [CG]) and after dietary supplementation with omega-3 PUFA (experimental group [EG]). Changes in mitochondrial matrix volumes were measured using the light-scattering technique. In the CG, the time courses of swelling were comparable in both mitochondrial fractions, with no difference in Ca²⁺-induced swelling between the two mitochondrial fractions. In the SSM fraction, no difference in the time course of swelling in Ca²⁺-free solution between CG and EG was detected. In the EG, both SSM and IFM fractions demonstrated a decreased sensitivity to Ca²⁺; IFM fractions, however, exhibited significantly less pronounced swelling following Ca²⁺ addition. The authors conclude that IFM and SSM fractions do not differ in their sensitivity to Ca²⁺-induced swelling. While dietary omega-3 PUFA protected both mitochondrial fractions against Ca²⁺-evoked swelling, the protective effect appeared to be more pronounced for the IFM fraction than for the SSM fraction.     

Aging-associated insulin resistance predisposes to hypertension and its reversal by exercise: the role of vascular vasorelaxation to insulin

Aging is an independent risk factor for hypertension, and hypertension and insulin resistance commonly coexist in the elderly. This study was designed to examine the effects of aging-related insulin resistance on blood pressure (BP) and its underlying mechanisms, with specific focus on the role of exercise in reversing hypertensive response. Adult (6-month-old) and aging (24-month-old) male Sprague-Dawley rats were subjected to a 10 weeks free-of-loading swim training (60 min/day, 5 days/week). Arterial vasorelaxation, cardiac contraction, eNOS activation, and iNOS and gp91^phox expression were determined. Under aging-related insulin resistance conditions, insulin infusion significantly elevated BP (P < 0.05). Aging caused significant endothelial dysfunction (P < 0.05 − 0.01), which was responsible for decreased arterial vasorelaxation to insulin. Aging attenuated myocardial contractile response to insulin, decreased eNOS expression and its phosphorylation by insulin, and increased iNOS and gp91^phox expression in aging arteries (P < 0.01). Exercise improved insulin sensitivity, potentiated insulin’s positive inotropic effects, facilitated arterial vasorelaxation to insulin, increased arterial eNOS activation in adult and aging rats, and thus attenuated insulin resistance-related hypertensive response to insulin. Moreover, exercise markedly reversed increased iNOS and gp91^phox expression in aging arteries. Inhibition of eNOS with Cavtratin or L-NAME significantly blocked exercise-facilitated arterial vasorelaxation to insulin and exercise-lowered BP response to insulin. In conclusion, these results demonstrate that endothelial dysfunction in response to insulin, but not insulin’s positive inotropic effects, plays an important role in the development of aging-related hypertension. The reversal of hypertensive response to insulin by exercise is most likely associated with improved insulin sensitivity in an eNOS-dependent manner and reduced oxidative and nitrative stresses. Electronic supplementary material:  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Returned for 1. Revision: 18 February 2008 1. Revision received: 11 August 2008 Returned for 2. Revision: 10 September 2008 2. Revision received: 15 September 2008     

Ischemia-reperfusion injury and cardioprotection: investigating PTEN,the phosphatase that negatively regulates PI3K,using a congenital model of PTEN haploinsufficiency

Activation of the PI3K/Akt pathway protects the heart from ischemia-reperfusion injury (IRI). The phosphatase PTEN is the main negative regulator of this pathway. We hypothesized that reduced PTEN levels could protect against IRI. Isolated perfused mouse hearts from PTEN^+/− and their littermates PTEN^+/+ (WT), were subjected to 35 min global ischemia and 30 min reperfusion, with and without 2, 4 or 6 cycles ischemic preconditioning (IPC). The end point was infarct size, expressed as a percentage of the myocardium at risk (I/R%). PTEN and Akt levels were determined using Western blot analysis. Unexpectedly, there were no significant differences in infarction between PTEN^+/− and WT (42.1 ± 5.0% Vs. 45.6 ± 3.3%). However, the preconditioning threshold was significantly reduced in the PTEN^+/− Vs. WT, with 4 cycles of IPC being sufficient to reduce I/R%, compared to 6 cycles in the WT (4 cycles IPC: 29.8. ± 3.69% in PTEN^+/− Vs. 45.5. ± 5.08% in WT, P < 0.01). In addition, the ratio between the phospho/total Akt (Ser473 and Thr308) was slightly but significantly increased in the PTEN^+/− indicating an upregulation of PI3K/Akt pathway. Interestingly, the levels of the other phosphatases that may negatively regulate the PI3K/Akt pathway (PP2A, SHIP2 and PHLPP) were not significantly different between littermates and PTEN^+/−. In conclusion, PTEN haploinsufficiency alone does not induce cardioprotection in this model; however, it reduces the threshold of protection induced by IPC. Returned for 1. Revision: 29 November 2007 1. Revision received: 5 May 2008 Returned for 2. Revision: 2 June 2008 2. Revision received: 5 June 2008     

Lentiviral vectors for delivery of genes into neonatal and adult ventricular cardiac myocytes in vitro and in vivo

Vectors based on lentiviruses such as human immunodeficiency virus (HIV) type-1 have many advantages for gene therapy, including the ability to infect non-dividing cells, long-term transgene expression and the absence of induction of an inflammatory/immune response. This study was initiated to determine whether lentiviruses would efficiently transfer genes to both neonatal and adult cardiac cells in culture and, by direct injection, to the heart in vivo. A three-plasmid expression system, including a packaging defective helper construct, a plasmid coding for a heterologous (VSV-G) envelope protein and a vector construct harboring reporter genes –E-GFP (enhanced green fluorescent protein) and puro (puromycin-resistance protein) was used to generate pseudotyped HIV-1 particles by transient transfection of human embryonic kidney 293T cells. We demonstrated efficient gene transfer into neonatal and adult cardiac myocytes in vitro and identified conditions in which virtually 100 % of cultured neonatal and 70 % of adult cardiac myocytes express the reporter gene. Transduction of adult cardiac myocytes with high titre lentiviral vectors did not affect the cell number, morphology or viability compared to untransduced cells. We delivered HIV-1-based vectors to the intact heart by direct injection. Hearts transduced with pseudotyped HIV-1 vectors showed levels of transgene expression comparable to that achieved by adenovirus vectors. This study demonstrates for the first time that lentivirus-based vectors can successfully transduce adult cardiomyocytes both in vitro and in vivo, and opens up the prospect of lentivirus-based vectors becoming an important gene delivery system in the cardiovascular field. Received: 1 October 2001, Returned for 1. revision: 18 October 2001, 1. Revision received: 19 November 2001, Returned for 2. revision: 6 December 2001, 2. Revision received: 13 February 2002, Accepted: 6 March 2002     

Immunocytochemical evidence for inducible nitric oxide synthase and cyclooxygenase-2 expression with nitrotyrosine formation in human hibernating myocardium

Background: Myocardial hibernation may result from repetitive episodes of transient ischaemia leading to prolonged dysfunction. Inducible nitric oxide synthase (iNOS) expression has been demonstrated in animals following brief, non-lethal ischaemia-reperfusion injury. We therefore, hypothesised that in human hibernating myocardium: 1) iNOS would be present; 2) the reaction of nitric oxide and superoxide would form the strong oxidant peroxynitrite; 3) that this process would be accompanied by the expression of cyclooxygenase-2 (Cox-2) which interacts with NOS and whose products could further affect myocardial function. Method and results: In sixteen patients with coronary artery disease (CAD), left ventricular biopsies were obtained from chronically dysfunctional segments subtended by a stenotic artery (> 75 %) and shown to be viable by ¹⁸F-fluorodeoxyglucose positron emission tomography. Comparison was made with myocardial biopsies (n = 8) from normally contracting myocardium in patients undergoing coronary surgery, from unused transplant donors and at post-mortem. Regional wall motion score improved in all patients 6 months post-revascularisation (from 2.7 ± 0.7 to 1.5 ± 0.5; p < 0.001), confirming hibernation. Immunocytochemistry localized reactivity to iNOS, Cox-2 and nitrotyrosine (a marker of peroxynitrite formation) to cardiomyocytes from hibernating segments. No difference in reactivity to endothelial NOS was seen between hibernating and control cardiomyocytes. Conclusion: Cox-2 and iNOS are co-expressed in hibernating myocardium with nitrotyrosine suggesting nitric oxide production and peroxynitrite formation. We propose that this is secondary to ischaemia-reperfusion and that the products of these enzymes may have consequences for myocardial contractile function and survival. Received: 11 February 2002, Returned for revision: 14 February 2002, Revision received: 4 March 2002, Accepted: 11 March 2002     

Recovery from sarafotoxin-b induced cardiopathological effects in mice following low energy laser irradiation

Objective: Low energy laser irradiation has been shown to accelerate various biological processes, including regeneration of injured tissues. In the present work we studied the effect of low energy laser irradiation on ischemic mice hearts, following administration of sarafotoxin-b, a powerful vasoconstrictor peptide of the endothelin/sarafotoxin family. Methods: Immediately after injection of the toxin and two days later, hearts were exposed to low energy laser irradiation. Eight days after the injection the mice were sacrificed and their hearts were processed for light and electron microscopy. Results:Sarafotoxin-b induced an approximate 2-fold increase in the relative cavity volume of the left ventricle. Low energy laser irradiation of the sarafotoxin-injected mice caused a significant decrease (62%) in the left ventricular dilatation. Electron microscopy of the sarafotoxin-injected mice hearts revealed that the extent of formation of large vacuoles in the cytoplasm of the cardiomyocytes as well as disorganization of the myofibrils were much higher than in the laser irradiated mice. Conclusions: Our study indicates that low energy laser irradiation enhanced recovery of the damaged cardiomyocytes from the ischemia induced by sarafotoxin-b. Received: 23 February 2000 Returned for revision: 29 March 2000 Revision received: 15 May 2000 Accepted: 24 May 2000     

Improved left ventricular function after transplantation of microspheres and fibroblasts in a rat model of myocardial infarction

As a novel and promising therapeutic strategy for heart failure, the application of different cell types is the subject of increasing research interest. In this study we investigated the effect of several cell types and microspheres (uniform polystyrene microspheres, 10 μm diameter) transplanted 4 weeks after induction of myocardial infarction in a rat model. Eight weeks after intramyocardial application of fibroblasts and microspheres, left ventricular function was significantly improved as demonstrated by isolated heart studies (Langendorff) and echocardiographic findings (LVDP fibroblasts 129 ± 32.9 mmHg, LVDP microspheres 119.2 ± 24.1 mmHg, fractional shortening (FS) microspheres 38.9 ± 4.6%, FS fibroblasts 36.84 ± 6.05%) in contrast to injection of macrophages or medium alone (LVDP medium 67 ± 22.6 mmHg, LVDP macrophages 75.9 ± 24.8 mmHg, FS macrophages 29.16 ± 8.7%, FS medium 27.2 ± 7.2%, P < 0.05). Signals of Bromodesoxy-Uridine (BrdU) labeled transplanted fibroblasts were detected in infarcted areas. Microspheres were recorded abundantly by autofluorescence. Significantly more apoptotic cells were observed in infarcted areas of macrophage (328.6 ± 37.4 cells/mm²) and medium (338.7 ± 16.5 cells/mm²; P < 0.05) treated hearts compared to microsphere (233.2 ± 16.8 cells/mm²) and fibroblast (232.2 ± 19.1 cells/mm²) injected hearts. Neovascularization, as reflected by the density of CD 31 positive vessels in the infracted area, did not differ between the four groups studied. The increased number of macrophages in infarcted areas after fibroblast and microsphere injection (fibroblasts 94.7 ± 7.1 cells/mm², microspheres 82.2 ± 3.0 cells/mm², macrophages 56.02 ± 9.93 cells/mm², medium 46.35 ± 9.03 cells/mm², P < 0.05) suggests that the underlying mechanism of augmented left ventricular function might be based on inflammatory processes. Returned for 1. Revision: 10 March 2008 1. Revision received: 11 September 2008 Returned for 2. Revision: 15 October 2008 2. Revision received: 28 October 2008 Dr. A. Schuh and Dr. E.A. Liehn equally contributed to this paper.     

Inefficient ventilation and reduced respiratory muscle capacity in congestive heart failure

The extent and time-course of changes in lung volumes, ventilatory efficiency at rest and during exercise, and respiratory muscle function and their influence on exercise limitation in congestive heart failure (CHF) are unclear. It is unknown whether respiratory muscle function may predict changes in exercise limitation or may be impaired in patients with poor outcome. 145 male patients (54±1 years) suffering from CHF (NYHA class I–III, mean 2.3±0.1), with a LVEF of 23±1 %, and a mean peak O₂ uptake (V_O2peak) 15.0±:0.5 mL×min⁻¹×kg⁻¹, were studied. They were grouped in Weber functional classes A to D according to their V_O2peak. Significant increases in ventilatory equivalents for O₂ and CO₂ (V_E/V_CO2peak) and in dead space ventilation at rest and during exercise were found with increasing exercise limitation. Moreover, there was a correlation of static and dynamic lung volumes (inspiratory vital capacity, IVC, r = 0.43, P < 0.01), as well as of maximal inspiratory pressure (MIP; r = 0.46, P < 0.01) with V_O2peak. Patients who died (n = 26) or were heart transplanted (n = 20) during a follow-up (mean 800 ± 10 days) had a reduced MIP (6.4 ± 0.4 kPa) as compared with survivors (n = 82; 9.3±0.7 kPa, P < 0.01). In a subgroup of 33 patients re-evaluated after six months, individual changes in IVC and V_E/V_CO2peak, but not in MIP, correlated to changes in V_O2peak (r = 0.69 and r = 0.72 respectively; P < 0.01). In CHF, exercise limitation is associated with reversible lung restriction and inefficient ventilation at rest and during exercise. Patientss with severe CHF have a significant reduction in MIP, a finding that is associated with poor outcome. Received: 12 July 1999, Returned for 1. revision: 26 August 1999, 1. Revision received: 23 November 1999, Returned for 2. Revision: 16 December 1999, 2. Revision received: 28 January 2000, Accepted: 8 February 2000     

Effects of different inotropic interventions on myocardial function in the developing rabbit heart

The development of the mammalian heart is characterized by substantial changes in myocardial performance. We studied the ontogeny of myocardial function with and without various inotropic interventions in the developing isolated, antegrade-perfused rabbit heart (2d, 8d, 14d, 28d, n = 96). Myocardial function was related to the protein expression of the sarcolemmal Na⁺-Ca²⁺ exchanger and to the sarcoplasmic Ca²⁺-ATPase. In neonatal hearts an age-dependent increase in maximal developed pressure velocity (dP/dt_max) by 45 % and peak negative pressure velocity (dP/dt_min) by 75 % within days 2 to 8 were observed. In response to inotropic intervention with isoproterenol, ouabain, calcium and the Na⁺-channel modulator BDF 9148, dP/dt_max and dP/dt_min increased in a concentration dependent manner. Significant differences between neonatal, juvenile and adult hearts could be demonstrated in a repeated measurement ANOVA model on the concentration-response curves for BDF 9148 (dP/dt_max and dP/dt_min), ouabain (dP/dt_min) and calcium (dP/dt_min), but not for isoproterenol. At the maximum isoproterenol concentration of 1 μmol/l, the increase in dP/dt_max and dP/dt_min was significantly higher in adult compared to neonatal hearts (t-test, p < 0.01). The significant decline of the Na⁺-Ca²⁺ exchanger protein expression from neonatal (1822 ± 171) to adult hearts (411 ± 96 S.E.M. [units per 20 μg protein], p < 0.01) was related to an increase in myocardial function (dP/dt_max r = 0.63, p < 0.01, dP/dt_min r = 0.62, p < 0.01). Contractility, relaxation and the observed positive inotropic effects were in general significantly lower in neonatal compared to adult hearts. In the individual heart an increase in contractility and relaxation was related to a decrease in Na⁺-Ca²⁺ exchanger expression. Received: 22 May 2000, Returned for 1. revision: 21 June 2000, 1. Revision received: 27 November 2000, Returned for 2. revision: 19 December 2000, 2. Revision received: 2 January 2001, Returned for 3. revision: 17 January 2001, 3. Revision received: 25 May 2001, Accepted: 11 June 2001