Albrecht Elsässer

Myocytes Die by Multiple Mechanisms in Failing Human Hearts

&NA; We tested the hypothesis that myocyte loss in failing human hearts occurs by different mechanisms: apoptosis, oncosis, and autophagic cell death. Explanted hearts from 19 patients with idiopathic dilated cardiomyopathy (EF≤20%) and 7 control hearts were analyzed. Myocyte apoptosis revealed by caspase‐3 activation and TUNEL staining occurred at a rate of 0.002±0.0005% (P<0.05 versus control) and oncosis assessed by complement 9 labeling at 0.06±0.001% (P<0.05). Cellular degeneration including appearance of ubiquitin containing autophagic vacuoles and nuclear disintegration was present at the ultrastructural level. Nuclear and cytosolic ubiquitin/protein accumulations occurred at 0.08±0.004% (P<0.05). The ubiquitin‐activating enzyme E1 and the ligase E3 were not different from control. In contrast, ubiquitin mRNA levels were 1.8‐fold (P<0.02) elevated, and the conjugating enzyme E2 was 2.3‐fold upregulated (P<0.005). The most important finding, however, is the 2.3‐fold downregulation of the deubiquitination enzyme isopeptidase‐T and the 1.5‐fold reduction of the ubiquitin‐fusion degradation system‐1, which in conjunction with unchanged proteasomal subunit levels and proteasomal activity results in massive storage of ubiquitin/protein complexes and in autophagic cell death. A 2‐fold decrease of cathepsin D might be an additional factor responsible for the accumulation of ubiquitin/protein conjugates. It is concluded that in human failing hearts apoptosis, oncosis, and autophagy act in parallel to varying degrees. A disturbed balance between a high rate of ubiquitination and inadequate degradation of ubiquitin/protein conjugates may contribute to autophagic cell death. Together, these different types of cell death play a significant role for myocyte disappearance and the development of contractile dysfunction in failing hearts. (Circ Res. 2003;92:715–724.)

Classically and alternatively activated macrophages contribute to tissue remodelling after myocardial infarction

An important goal in cardiology is to minimize myocardial necrosis and to support a discrete but resilient scar formation after myocardial infarction (MI). Macrophages are a type of cells that influence cardiac remodelling during MI. Therefore, the goal of the present study was to investigate their transcriptional profile and to identify the type of activation during scar tissue formation. Ligature of the left anterior descending coronary artery was performed in mice. Macrophages were isolated from infarcted tissue using magnetic cell sorting after 5 days. The total RNA of macrophages was subjected to microarray analysis and compared with RNA from MI and LV‐control. mRNA abundance of relevant targets was validated by quantitative real‐time PCR 2, 5 and 10 days after MI (qRT‐PCR). Immunohistochemistry was performed to localize activation type‐specific proteins. The genome scan revealed 68 targets predominantly expressed by macrophages after MI. Among these targets, an increased mRNA abundance of genes, involved in both the classically (tumour necrosis factor α, interleukin 6, interleukin 1β) and the alternatively (arginase 1 and 2, mannose receptor C type 1, chitinase 3‐like 3) activated phenotype of macrophages, was found 5 days after MI. This observation was confirmed by qRT‐PCR. Using immunohistochemistry, we confirmed that tumour necrosis factor α, representing the classical activation, is strongly transcribed early after ligature (2 days). It was decreased after 5 and 10 days. Five days after MI, we found a fundamental change towards alternative activation of macrophages with up‐regulation of arginase 1. Our results demonstrate that macrophages are differentially activated during different phases of scar tissue formation after MI. During the early inflammatory phase, macrophages are predominantly classically activated, whereas their phenotype changes during the important transition from inflammation to scar tissue formation into an alternatively activated type.

Tako-tsubo cardiomyopathy (apical ballooning)

In recent years, a new cardiac syndrome with transient left ventricular dysfunction has been described in Japanese patients. This new entity has been referred to as “tako-tsubo cardiomyopathy” or “apical ballooning”, named for the particular shape of the end-systolic left ventricle in ventriculography.1 To date, tako-tsubo cardiomyopathy has also been reported to occur in the western population. The following clinical characteristics of this phenomenon must be met: (1) transient akinesis or dyskinesis of left ventricular wall motion abnormalities (ballooning) with chest pain; (2) new electrocardiographic changes (either ST elevation or T wave inversion); (3) no significant obstructive epicardial coronary artery disease; (4) absence of recent significant head trauma, intracardial bleeding, phaeochromocytoma, myocarditis, and hypertrophic cardiomyopathy.2 Emotional or physical stress usually precedes this cardiomyopathy. A unifying mechanistic explanation responsible for this acute but rapidly reversible contractile dysfunction is still lacking. Multivessel epicardial coronary artery vasospasm, coronary microvascular dysfunction or spasm, impaired fatty acid metabolism, transient obstruction of the left ventricular outflow, and catecholamine-mediated myocardial dysfunction have been proposed as potential mechanisms.3–6 The optimal treatment of patients presenting with this syndrome depends primarily on the haemodynamic conditions and remains rather symptomatic in nature. This article primarily addresses the clinical setting of tako-tsubo cardiomyopathy and describes a broad spectrum of diagnostic tools. Moreover, currently proposed pathophysiological mechanisms are discussed in detail, providing more insight into this new cardiac entity. Tako-tsubo cardiomyopathy is characterised by acute onset of chest pain and a completely reversible regional contractile dysfunction. In left ventriculography typical wall motion abnormalities, such as apical and mid-ventricular akinesia and a hypercontractile basis, can be documented. Coronary angiography reveals no relevant epicardial coronary artery disease (fig 1). Recently, several cases of transient ballooning involving the mid-ventricular part, sparing the apical and basal segments, have also been documented.7 …

Abnormalities in intracellular Ca2+ regulation contribute to the pathomechanism of Tako-Tsubo cardiomyopathy

AIMS The Tako-Tsubo cardiomyopathy (TTC) is characterized by a transient contractile dysfunction that has been assigned to excessive catecholamine levels after episodes of severe emotional or physical stress. Several studies have indicated that beta-adrenoceptor stimulation is associated with alteration in gene expression of Ca(2+)-regulatory proteins. Thus, the present study investigated the gene expression of crucial proteins [sarcoplasmic Ca(2+) ATPase (SERCA2a), sarcolipin (SLN), phospholamban (PLN), ryanodine receptor (RyR2), and sodium-calcium exchanger (NCX)] involved in the Ca(2+)-regulating system in TTC. METHODS AND RESULTS In 10 consecutive patients, TTC was diagnosed by coronary angiography, ventriculography, and echocardiography. Endomyocardial biopsies were taken during the phase of severely impaired left ventricular (LV) function and after functional recovery. Non-diseased LV tissue from three donor hearts not used for transplantation served as healthy controls. Expression levels of Ca(2+)-regulatory proteins were analysed by means of real-time PCR, western blot, and immunohistochemistry. SLN, predominantly expressed in the atrial component, showed a remarkable ventricular expression in TTC patients. Gene expression of SERCA2a was significantly down-regulated. Conversely, PLN/SERCA2a ratio was increased. For PLN, dephosphorylation was documented using western blot and immunostaining of PLN-Ser(16) and PLN-Thr(17). No changes could be documented for NCX and RyR2. CONCLUSION In TTC, ventricular expression of SLN and dephosphorylation of PLN potentially result in a reduced SERCA2a activity and its Ca(2+) affinity. Thus, the TTC is associated with specific alteration of Ca(2+)-handling proteins, which might be crucial for contractile dysfunction.

Intracoronary infusion of bone marrow-derived mononuclear cells abrogates adverse left ventricular remodelling post-acute myocardial infarction: insights from the reinfusion of enriched progenitor cells and infarct remodelling in acute myocardial infarction (REPAIR-AMI) trial

Depressed left ventricular ejection fraction (LVEF) despite successful reperfusion therapy is the single most powerful predictor of progressive LV enlargement after acute myocardial infarction (AMI) and independently determines adverse outcome in these patients.

Myocardial preconditioning and remote renal preconditioning--identifying a protective factor using proteomic methods?

AbstractIt is still unknown whether remote ischemic preconditioning is mediated by a humoral or a neurogenic mechanism from the preconditioning to the preconditioned tissue. The purpose of the following study was to identify a possible humoral trigger of ischemic myocardial preconditioning and remote renal preconditioning. Open chest rats were subjected to a coronary artery occlusion period of 45 min followed by 2 h of reperfusion (Control animals; n = 6). The coronary preconditioned group (IPC, n = 6) was subjected to a preceding preconditioning period of 5 min coronary artery occlusion followed by 5 min of reperfusion, repeated three times. The renal preconditioned group (IPR, n = 6) was subjected to a preceding renal artery occlusion period of 10 min followed by 20 min of reperfusion. Area at risk (AAR) and infarcted area (IA) were determined at the end of each protocol. Blood samples were taken at the end of the preconditioning protocols from parallel experiments for proteomic analysis using two–dimensional gel electrophoresis (2-DE), matrix assisted laser desorption and ionization—time of flight—mass spectrometry (MALDI–TOF–MS), and liquid chromatography—electrospray ionization—tandem mass spectrometry (nanoLC–ESI–MS/MS). IA/AAR was 87.8 ± 10.7% in the control group. IPC and IPR signi.cantly reduced IA/AAR (58.2 ± 9.3% and 56.9 ± 9.0%, p < 0.001). Proteomic analyses detected four protein spots which were either up– (n = 3) or down–regulated in the preconditioned groups vs. the control group. The three up–regulated protein spots were identi.ed as albumin fragments, whereas the downregulated spot was identified as liver regeneration–related protein (LRRG03). Interestingly, albumin modification by brief ischemia has been recently shown and evaluated for the clinical diagnosis of sublethal myocardial ischemia. However, no differentially abundant proteins which possess a known signaling function could be found. Hence, though there is a differential protein expression in blood following IPC and IPR, our data are not in favor of a humoral mediator of remote preconditioning with a molecular weight of more than 8 kDa. Our results rather suggest either a neurogenic pathway or a mediator smaller than 8 kDa.

The role of apoptosis in myocardial ischemia: a critical appraisal.

Abstract The role of apoptosis in cardiac ischemia is not clarified yet. Own data show that suicidal cell death is apparently not important in global ischemia where it only affects a small number of myocytes (8 %) while the majority of cells, i.e. 92 % die by oncosis. In acute regional ischemia it is most probably not a decisive factor. However, more solid data are needed to justify this statement. Human hibernating myocardium shows an activation of the apoptotic cascade, i.e., apoptosis might contribute to cell loss in this pathophysiological situation of multiple ischemic episodes. Manifold unresolved issues contribute to problems in determining the role of apoptosis in ischemia. These include 1) Uncertainty of the duration of the apoptotic cascade from activation of death receptors at the cellular membrane until DNA fragmentation occurs, 2) The role of the mitochondrial pathway, 3) The mode of removal of myocytes after cell death has occurred, 4) Technical problems such as specificity of the TUNEL method, detection of low abundance proteins such as activated caspases or cytochrome C, statistical considerations. These issues and many others should be clarified before any definite conclusion as to the role of apoptosis in ischemia may be drawn.

N-Terminal B-Type Natriuretic Peptide Assessment Provides Incremental Prognostic Information in Patients With Acute Coronary Syndromes and Normal Troponin T Values Upon Admission

OBJECTIVES The purpose of this study was to determine the prognostic value of N-terminal B-type natriuretic peptide (NT-proBNP) in two independent samples of patients presenting with acute coronary syndromes (ACS) and normal troponin T (TnT) values. BACKGROUND Recently assessment of NT-proBNP has been studied in patients with ACS. However, the clinical relevance in patients who present without troponin elevation is unclear. METHODS We included 2,614 patients from two independent registries, one serving as a derivation cohort comprising patients with evident ACS (Bad Nauheim ACS registry, n = 1,131) and the other serving as a validation cohort including chest pain patients (PACS [Prognosis in Acute Coronary Syndromes] registry, n = 1,483). NT-proBNP and TnT were measured upon admission. Clinical outcome has been assessed over a 6-month period. RESULTS In both cohorts, the mortality rate was significantly lower among TnT negative patients: 3.8% versus 8.2% (p = 0.009) in the Bad Nauheim ACS registry, and 2.8% versus 8.6% (p = 0.009) in the PACS registry. Among TnT negative patients, receiver-operating characteristics curve analysis yielded an optimal cutoff value of 474 pg/ml for NT-proBNP that was able to discriminate patients at higher risk in the Bad Nauheim ACS and PACS registries (mortality rate 12.3% vs. 1.3%, p < 0.001 and 8.5% vs. 1.5%, p < 0.001, respectively). By Kaplan-Meier analysis, patients with NT-proBNP values over 474 pg/ml were at higher risk for death in the Bad Nauheim ACS registry (log-rank 19.01, p < 0.001, adjusted hazard ratio [HR] 9.56 [95% confidence interval (CI) 2.42 to 37.7], p = 0.001) and in the PACS registry (log-rank 23.16, p < 0.001, adjusted HR 5.02 [95% CI 2.04 to 12.33], p < 0.001). CONCLUSIONS Among patients with suspected ACS considered to be at low risk because of normal troponin values, NT-proBNP above 474 pg/ml is able to discriminate individuals at higher risk. Because of its incremental prognostic value, NT-proBNP assessment should be considered in clinical routine for risk stratification of patients with normal troponin.

Trpv4 induces collateral vessel growth during regeneration of the arterial circulation

The development of a collateral circulation (arteriogenesis), bypassing an arterial occlusion, is important for tissue survival, but it remains functionally defective. Micro array data of growing collateral vessels, exposed to chronically elevated fluid shear stress (FSS), showed increased transcription of the transient receptor potential cation channel, subfamily V, member 4 (Trpv4). Thus, the aim of this study was to investigate the role of the shear stress sensitive Trpv4 in transmitting this physical stimulus into an active growth response. qRT‐PCR at different time points during the growth of collateral vessels after femoral artery ligature (FAL) in rats showed a strong positive correlation of Trpv4 transcription and the intensity of FSS. An increased protein expression of Trpv4 was localized in the FSS‐sensing endothelium by means of confocal immunohistochemistry. Cultured porcine endothelial cells showed a dose‐dependent expression of Trpv4 and an increased level of Ki67‐positive cells upon treatment with 4α‐Phorbol 12,13‐didecanoate (4αPDD), a specific Trpv4 activator. This was also demonstrated by flow culture experiments. These results were confirmed by in vivo application of 4αPDD in rabbit hind limb circulation via an osmotic mini‐pump after FAL. Trpv4 expression as well as Ki67‐positive staining was significantly increased in collateral vessels. Finally, 4αPDD treatment after FAL led to a 61% (215.5 ml/min/mmHg versus 350 ml/min/mmHg) recovery of conductance when compared with the non‐occluded artery. Cell culture and in vivo studies demonstrate that an FSS‐ or a 4αPDD‐induced activation of Trpv4 leads to an active proliferation of vascular cells and finally triggers collateral growth. Trpv4, a well‐known FSS‐sensitive vasodilator, has hitherto not been implicated in active growth processes of collateral arteries. This new function may lead to new therapeutic strategies for the treatment of arterial occlusive diseases.

Activated cell survival cascade protects cardiomyocytes from cell death in Tako-Tsubo cardiomyopathy

Tako‐Tsubo cardiomyopathy (TTC) is characterized by rapid regeneration of contractile dysfunction. From recent studies it is known that excessive catecholamine levels due to emotional or physical stress might play a central role. After sympathetic activation, the PIK3/AKT pathway is a key regulator of many cellular responses, including cytoprotective effects. Thus, the purpose of this study was to investigate whether the PIK3/AKT pathway plays a pivotal role in TTC.