Katrin Schäfer

Differential Cardiac Remodeling in Preload Versus Afterload

Background— Hemodynamic load regulates myocardial function and gene expression. We tested the hypothesis that afterload and preload, despite similar average load, result in different phenotypes. Methods and Results— Afterload and preload were compared in mice with transverse aortic constriction (TAC) and aortocaval shunt (shunt). Compared with sham mice, 6 hours after surgery, systolic wall stress (afterload) was increased in TAC mice (+40%; P<0.05), diastolic wall stress (preload) was increased in shunt (+277%; P<0.05) and TAC mice (+74%; P<0.05), and mean total wall stress was similarly increased in TAC (69%) and shunt mice (67%) (P=NS, TAC versus shunt; each P<0.05 versus sham). At 1 week, left ventricular weight/tibia length was significantly increased by 22% in TAC and 29% in shunt mice (P=NS, TAC versus shunt). After 24 hours and 1 week, calcium/calmodulin-dependent protein kinase II signaling was increased in TAC. This resulted in altered calcium cycling, including increased L-type calcium current, calcium transients, fractional sarcoplasmic reticulum calcium release, and calcium spark frequency. In shunt mice, Akt phosphorylation was increased. TAC was associated with inflammation, fibrosis, and cardiomyocyte apoptosis. The latter was significantly reduced in calcium/calmodulin-dependent protein kinase IIΔ-knockout TAC mice. A total of 157 mRNAs and 13 microRNAs were differentially regulated in TAC versus shunt mice. After 8 weeks, fractional shortening was lower and mortality was higher in TAC versus shunt mice. Conclusions— Afterload results in maladaptive fibrotic hypertrophy with calcium/calmodulin-dependent protein kinase II–dependent altered calcium cycling and apoptosis. Preload is associated with Akt activation without fibrosis, little apoptosis, better function, and lower mortality. This indicates that different loads result in distinct phenotype differences that may require specific pharmacological interventions.

Elevated heart-type fatty acid-binding protein levels on admission predict an adverse outcome in normotensive patients with acute pulmonary embolism.

OBJECTIVES We assessed the predictive value of heart-type fatty acid-binding protein (H-FABP) in normotensive patients with acute pulmonary embolism (PE). BACKGROUND Risk stratification of initially normotensive patients with PE on the basis of right ventricular dysfunction or injury remains controversial. Previous studies investigating biomarkers or imaging modalities included unselected patients, some of whom presented with cardiogenic shock. METHODS We included 126 consecutive normotensive patients with confirmed PE. Complicated 30-day outcome was defined as death, resuscitation, intubation, or use of catecholamines. Long-term survival was assessed by follow-up clinical examination. RESULTS During the first 30 days, 9 (7%) patients suffered complications. These patients had higher baseline H-FABP values (median, 11.2 ng/ml [interquartile range: 8.0 to 36.8 ng/ml]) compared with patients with an uncomplicated course (3.4 ng/ml [2.1 to 4.9 ng/ml]; p < 0.001). H-FABP values were above the calculated (by receiver operating characteristic curve analysis) cutoff value of 6 ng/ml in 29 patients. Eight (28%) of them suffered complications versus 1 of 97 patients with low H-FABP (negative predictive value, 99%; p < 0.001). By logistic regression, elevated (> or =6 ng/ml) H-FABP was associated with a 36.6-fold increase in the death or complication risk. The combination of H-FABP with tachycardia was a particularly useful prognostic indicator. H-FABP also predicted long-term mortality over 499 (interquartile range: 204 to 1,166) days (hazard ratio: 3.6; 95% confidence interval: 1.6 to 8.2; p = 0.003). CONCLUSIONS The H-FABP might be a useful biomarker for risk stratification of normotensive patients with acute PE.

Effect of menthol on two types of Ca currents in cultured sensory neurons of vertebrates

The effect of menthol on voltage-dependent Ca currents was investigated in cultured dorsal root ganglion cells from chick and rat embryos. Bath application of menthol (0.1–1 mM) had different effects on the various Ca currents present in these neurons. Below −20 mV, the low threshold Ca currents were reduced in amplitude in a dose-dependent manner by menthol with little changes of their activation kinetics. In contrast to this, the time course of inactivation of the high-threshold Ca currents, activated above −20 mV from a holding potential of −80 mV, was drastically accelerated by external menthol. The action of menthol was unchanged with more positive holding potentials (−50 mV). Thus, a proposed third type of Ca current with transient activation and complete deactivation below −50 mV was either not present or not affected by menthol. Menthol exerted its action only when applied from the outside. Its effect was completely reversible within 15–20 min of wash-out. Our findings are consistent with the idea that menthol acts on two types of Ca channels coexisting on the membrane of cultured sensory neurons. Menthol blocks currents through the low voltage-activated Ca channel, and facilitates inactivation gating of the classical high voltage-activated Ca channel.

Exercise Training Reduces Neointimal Growth and Stabilizes Vascular Lesions Developing After Injury in Apolipoprotein E–Deficient Mice

Background—Population-based studies have shown that exercise reduces cardiovascular morbidity and mortality. However, it is unknown whether these effects are solely a result of risk factor modification or whether exercise directly affects the homeostasis of the vessel wall. Methods and Results—We subjected 19-week-old apolipoprotein E (apoE)–knockout mice (apoE−/−; n=25) to a 6-week training program on a motorized treadmill. The control group consisted of 17 sedentary mice. After 3 weeks in the program, training and sedentary mice underwent carotid artery injury with ferric chloride. Training was then resumed for another 3 weeks. Exercise did not change body weight or lipid levels in apoE−/− mice but resulted in upregulated expression of nitric oxide synthase in the endothelium. Physical training did not significantly affect the thrombotic response to injury. However, morphometric analysis of vessels harvested 3 weeks after injury showed that neointima formation was reduced in the exercising group. This resulted in a lower intima/media ratio (0.29±0.03 versus 0.41±0.03 in sedentary mice; P =0.008) and less luminal stenosis (21±2.7% versus 33±2.3%; P =0.003). Importantly, exercise reduced the number of Mac-3–positive, oxidized LDL-containing macrophages in the vessel wall while increasing the content in collagen fibers (14.1±0.9% versus 4.8±0.8%; P <0.001). Plasminogen activator inhibitor-1, tissue factor, and fibrinogen were all significantly reduced in the lesions of trained mice. Conclusions—In the apoE−/− mouse, exercise training reduces neointimal growth and stabilizes vascular lesions after injury. These effects appear to be at least partly independent of changes in lipid levels or the initial thrombotic response to injury.

Telethonin Deficiency Is Associated With Maladaptation to Biomechanical Stress in the Mammalian Heart

Rationale: Telethonin (also known as titin-cap or t-cap) is a 19-kDa Z-disk protein with a unique &bgr;-sheet structure, hypothesized to assemble in a palindromic way with the N-terminal portion of titin and to constitute a signalosome participating in the process of cardiomechanosensing. In addition, a variety of telethonin mutations are associated with the development of several different diseases; however, little is known about the underlying molecular mechanisms and telethonins in vivo function. Objective: Here we aim to investigate the role of telethonin in vivo and to identify molecular mechanisms underlying disease as a result of its mutation. Methods and Results: By using a variety of different genetically altered animal models and biophysical experiments we show that contrary to previous views, telethonin is not an indispensable component of the titin-anchoring system, nor is deletion of the gene or cardiac specific overexpression associated with a spontaneous cardiac phenotype. Rather, additional titin-anchorage sites, such as actin–titin cross-links via &agr;-actinin, are sufficient to maintain Z-disk stability despite the loss of telethonin. We demonstrate that a main novel function of telethonin is to modulate the turnover of the proapoptotic tumor suppressor p53 after biomechanical stress in the nuclear compartment, thus linking telethonin, a protein well known to be present at the Z-disk, directly to apoptosis (“mechanoptosis”). In addition, loss of telethonin mRNA and nuclear accumulation of this protein is associated with human heart failure, an effect that may contribute to enhanced rates of apoptosis found in these hearts. Conclusions: Telethonin knockout mice do not reveal defective heart development or heart function under basal conditions, but develop heart failure following biomechanical stress, owing at least in part to apoptosis of cardiomyocytes, an effect that may also play a role in human heart failure.

Effects of Obesity and Weight Loss on the Functional Properties of Early Outgrowth Endothelial Progenitor Cells

OBJECTIVES The purpose of this study was to examine the impact of obesity and weight loss on the angiogenic and regenerative capacity of endothelial progenitor cells (EPCs). BACKGROUND EPCs participate in angiogenesis and tissue repair. Several cardiovascular risk factors are associated with EPC dysfunction. METHODS Early outgrowth EPCs were isolated from 49 obese (age 42 +/- 14 years; body mass index 42 +/- 7 kg/m(2)) normoglycemic participants in a professional weight reduction program and compared with those from 49 age-matched lean controls. EPC function was tested both in vitro and in vivo. RESULTS EPCs expanded from the obese possessed reduced adhesive, migratory, and angiogenic capacity, and mice treated with obese EPCs exhibited reduced EPC homing in ischemic hind limbs in vivo. EPCs from the obese subjects failed to respond to conditioned medium of lean controls or to potent angiogenic factors such as vascular endothelial growth factor. Although no differences existed between lean and obese EPCs regarding the surface expression of vascular endothelial growth factor or chemokine receptors, basal p38 mitogen-activated protein kinase (MAPK) phosphorylation was elevated in obese EPCs (3.7 +/- 2.1-fold increase; p = 0.006). These cells also showed reduced secretion of the angiogenic chemokines interleukin-8 (p = 0.047) and monocyte chemoattractant protein-1 (p = 0.012). By inhibiting p38 MAPK, we could restore chemokine levels to those of lean control EPCs and also improve the angiogenic properties of obese EPCs. Accordingly, 6-month follow-up of 26 obese persons who achieved significant weight reduction revealed normalization of p38 MAPK phosphorylation levels and improved EPC function. CONCLUSIONS Obesity is associated with a reversible functional impairment of EPCs. This involves reduced secretion of angiogenic chemokines and increased basal phosphorylation of signaling molecules, notably p38 MAPK.

Unfavourable consequences of chronic cardiac HIF-1α stabilization

AIMS The hypoxia-inducible factor-1 (HIF-1) is the master modulator of hypoxic gene expression. The effects of chronically stabilized cardiac HIF-1α and its role in the diseased heart are not precisely known. The aims of this study were as follows: (i) to elucidate consequences of HIF-1α stabilization in the heart; (ii) to analyse long-term effects of HIF-1α stabilization with ageing and the ability of the HIF-1α overexpressing hearts to respond to increased mechanical load; and (iii) to analyse HIF-1α protein levels in failing heart samples. METHODS AND RESULTS In a cardiac-specific HIF-1α transgenic mouse model, constitutive expression of HIF-1α leads to changes in capillary area and shifts the cardiac metabolism towards glycolysis with a net increase in glucose uptake. Furthermore, Ca(2+) handling is altered, with increased Ca(2)(+) transients and faster intracellular [Ca(2+)] decline. These changes are associated with decreased expression of sarcoplasmic/endoplasmic reticulum calcium ATPase 2a but elevated phosphorylation of phospholamban. HIF-1α transgenic mice subjected to transverse aortic constriction exhibited profound cardiac decompensation. Moreover, cardiomyopathy was also seen in ageing transgenic mice. In parallel, we found an increased stabilization of HIF-1α in heart samples of patients with end-stage heart failure. CONCLUSION Changes induced with transgenic cardiac HIF-1α possibly mediate beneficial effects in the short term; however, with increased mechanical load and ageing they become detrimental for cardiac function. Together with the finding of increased HIF-1α protein levels in samples from human patients with cardiomyopathy, these data indicate that chronic HIF-1α stabilization drives autonomous pathways that add to disease progression.

Endothelial p53 deletion improves angiogenesis and prevents cardiac fibrosis and heart failure induced by pressure overload in mice.

Background Cardiac dysfunction developing in response to chronic pressure overload is associated with apoptotic cell death and myocardial vessel rarefaction. We examined whether deletion of tumor suppressor p53 in endothelial cells may prevent the transition from cardiac hypertrophy to heart failure. Methods and Results Mice with endothelial‐specific deletion of p53 (End.p53‐KO) were generated by crossing p53fl/fl mice with mice expressing Cre recombinase under control of an inducible Tie2 promoter. Cardiac hypertrophy was induced by transverse aortic constriction. Serial echocardiography measurements revealed improved cardiac function in End.p53‐KO mice that also exhibited better survival. Cardiac hypertrophy was associated with increased p53 levels in End.p53‐WT controls, whereas banded hearts of End.p53‐KO mice exhibited lower numbers of apoptotic endothelial and non‐endothelial cells and altered mRNA levels of genes regulating cell cycle progression (p21), apoptosis (Puma), or proliferation (Pcna). A higher cardiac capillary density and improved myocardial perfusion was observed, and pharmacological inhibition or genetic deletion of p53 also promoted endothelial sprouting in vitro and new vessel formation following hindlimb ischemia in vivo. Hearts of End.p53‐KO mice exhibited markedly less fibrosis compared with End.p53‐WT controls, and lower mRNA levels of p53‐regulated genes involved in extracellular matrix production and turnover (eg, Bmp‐7, Ctgf, or Pai‐1), or of transcription factors involved in controlling mesenchymal differentiation were observed. Conclusions Our analyses reveal that accumulation of p53 in endothelial cells contributes to blood vessel rarefaction and fibrosis during chronic cardiac pressure overload and suggest that endothelial cells may be a therapeutic target for preserving cardiac function during hypertrophy.

Leptin promotes the mobilization of vascular progenitor cells and neovascularization by NOX2-mediated activation of MMP9

AIMS Bone marrow (BM) progenitors participate in new vessel formation and endothelial repair. The leptin receptor (ObR) is expressed on hematopoietic cells; however, the effects of leptin on BM progenitor cells and their angiogenic potential are unknown. METHODS AND RESULTS In the present study, we show that the short-term administration of leptin (over five consecutive days) into wild-type mice increased the number of circulating, BM-derived sca-1(+), flk-1(+) vascular progenitors, 95 ± 1.7% of which also expressed ObR. Ex vivo stimulation of BM cells with leptin enhanced the expression of NADPH oxidase isoform 2 (NOX2), and the leptin-induced increase in reactive oxygen species production, matrix metalloproteinase-9 (MMP9) expression and circulating soluble KitL levels was absent in mice lacking NOX2. Furthermore, intraperitoneal injections of leptin improved perfusion and increased the number of BM-derived, CD31-positive endothelial cells in ischaemic hindlimbs after femoral artery ligation. The effects of leptin on the mobilization of sca-1(+), flk-1(+) cells and neovascularization were abolished in mice transplanted with BM from ObR-deficient and in NOX2(-/-) mice. CONCLUSION Our findings suggest that the angiogenic effects of leptin involve sca-1(+), flk-1(+) vascular progenitor cells mobilized from the BM in response to ObR-mediated activation of NOX2, increased MMP9 expression, and sKitL release.

Melusin protects from cardiac rupture and improves functional remodelling after myocardial infarction

AIMS Melusin is a muscle-specific chaperone protein whose expression is required for a compensatory hypertrophy response to pressure overload. Here, we evaluated the consequences of melusin overexpression in the setting of myocardial infarction (MI) using a comprehensive multicentre approach. METHODS AND RESULTS Mice overexpressing melusin in the heart (TG) and wild-type controls (WT) were subjected to permanent LAD ligation and both the acute response (Day 3) and subsequent remodelling (2 weeks) were examined. Mortality in wild-type mice was significant between Days 3 and 7, primarily due to cardiac rupture, but melusins overexpression strongly reduced mortality (43.2% in wild-type vs. 27.3% in melusin-TG, P = 0.005). At Day 3 after MI, a time point preceding the mortality peak, TG hearts had increased heat shock protein 70 expression, increased ERK1/2 signalling, reduced cardiomyocyte hyper-contractility and inflammatory cell infiltrates, and increased matricellular protein expression in the infarcted area. At 2 weeks after MI, melusin overexpression conferred a favourable adaptive remodelling characterized by reduced left ventricle dilatation and better preserved contractility in the presence of a comparable degree of hypertrophy. Adaptive remodelling in melusin TG mice was characterized by reduced apoptosis and fibrosis as well as increased cardiomyocyte contractility. CONCLUSIONS Consistent with its function as a chaperone protein, melusin overexpression exerts a dual protective action following MI reducing an array of maladaptive processes. In the early phase after MI, reduced inflammation and myocyte remodelling protect against cardiac rupture. Chronically, reduced myocyte loss and matrix remodelling, with preserved myocyte contractility, confer adaptive LV remodelling.