Marek Treiman

Exenatide reduces reperfusion injury in patients with ST-segment elevation myocardial infarction.

AIMS Exenatide, a glucagon-like-peptide-1 analogue, increases myocardial salvage in experimental settings with coronary occlusion and subsequent reperfusion. We evaluated the cardioprotective effect of exenatide at the time of reperfusion in patients with ST-segment elevation myocardial infarction (STEMI) treated with primary percutaneous coronary intervention (pPCI). METHODS AND RESULTS A total of 172 patients with STEMI and Thrombolysis in Myocardial Infarction flow 0/1 were randomly assigned to exenatide or placebo (saline) intravenously. Study treatment was commenced 15 min before intervention and maintained for 6 h after the procedure. The primary endpoint was salvage index calculated from myocardial area at risk (AAR), measured in the acute phase, and final infarct size measured 90 ± 21 days after pPCI by cardiac magnetic resonance (CMR). In 105 patients evaluated with CMR, a significantly larger salvage index was found in the exenatide group than in the placebo group (0.71 ± 0.13 vs. 0.62 ± 0.16; P= 0.003). Infarct size in relation to AAR was also smaller in the exenatide group (0.30 ± 0.15 vs. 0.39 ± 0.15; P= 0.003). In a regression analysis, there was a significant correlation between the infarct size and the AAR for both treatment groups and an analysis of covariance showed that datapoints in the exenatide group lay significantly lower than for the placebo group (P= 0.011). There was a trend towards smaller absolute infarct size in the exenatide group (13 ± 9 vs. 17 ± 14 g; P= 0.11). No difference was observed in left ventricular function or 30-day clinical events. No adverse effects of exenatide were observed. CONCLUSION In patients with STEMI undergoing pPCI, administration of exenatide at the time of reperfusion increases myocardial salvage.

Cardioprotective effects of ischemic postconditioning in patients treated with primary percutaneous coronary intervention, evaluated by magnetic resonance.

Background—Postconditioning has been suggested to reduce myocardial damage during primary percutaneous coronary intervention (PPCI) in patients with ST-segment–elevation myocardial infarction. However, because clinical experience is limited, we examined the cardioprotective effects of postconditioning, using cardiac MRI in patients treated with PPCI. Methods and Results—One hundred eighteen patients with ST-segment–elevation myocardial infarction referred for PPCI were randomly assigned to have either conventional PPCI or PPCI with postconditioning. Postconditioning was performed immediately after obtained reperfusion with 4 balloon occlusions, each lasting 30 seconds, followed by 30 seconds of reperfusion. The primary end point was myocardial salvage after 3 months as judged by delayed enhancement cardiac MRI. We found a 19% relative reduction of infarct size in the postconditioning group (51±16% of total area at risk versus 63±17%, P<0.01), corresponding to a 31% increase in salvage ratio. The number of patients developing heart failure was significantly fewer in the postconditioning group (27% versus 46%, P=0.048). No significant evidence of interaction between the impact of postconditioning and the location of the culprit lesion or size of the myocardium at risk was detected (P=0.21 and P=0.71). Conclusions—Mechanical postconditioning reduces infarct size in patients with ST-segment–elevation myocardial infarction treated with PPCI. The impact of mechanical postconditioning seems to be independent of the size of myocardium at risk. Clinical Trial Registration—URL: http://www.clinicaltrials.gov. Unique Identifier: NCT00507156.

Calcium Pumps of Plasma Membrane and Cell Interior

Calcium entering the cell from the outside or from intracellular organelles eventually must be returned to the extracellular milieu or to intracellular storage organelles. The two major systems capable of pumping Ca2+ against its large concentration gradient out of the cell or into the sarco/endoplasmatic reticulum are the plasma membrane Ca2+ ATPases (PMCAs) and the sarco/endoplasmic reticulum Ca2+ ATPases (SERCAs), respectively. In mammals, multigene families code for these Ca2+ pumps and additional isoform subtypes are generated via alternative splicing. PMCA and SERCA isoforms show developmental-, tissue- and cell type-specific patterns of expression. Different PMCA and SERCA isoforms are characterized by different regulatory and kinetic properties that likely are optimized for the distinct functional tasks fulfilled by each pump in setting resting cytosolic or intra-organellar Ca2+ levels, and in shaping intracellular Ca2+ signals with spatial and temporal resolution. The loss or malfunction of specific Ca2+ pump isoforms is associated with defects such as deafness, ataxia or heart failure. Understanding the involvement of different Ca2+ pump isoforms in the pathogenesis of disease allows their identification as therapeutic targets for the development of selective strategies to prevent or combat the progression of these disorders.

Exenatide Reduces Final Infarct Size in Patients With ST-Segment–Elevation Myocardial Infarction and Short-Duration of Ischemia

Background— Exenatide has been demonstrated to be cardioprotective as an adjunct to primary percutaneous coronary intervention in patients with ST-segment–elevation myocardial infarction (STEMI). The aim of the post hoc analysis study was to evaluate the effect of exenatide in relation to system delay, defined as time from first medical contact to first balloon. Methods and Results— Patients with STEMI and Thrombolysis In Myocardial Infarction flow 0/1 were randomly assigned to intravenous exenatide or placebo continuous infusion. Study treatment was commenced 15 minutes before intervention and maintained for 6 hours after the procedure. The patients were stratified according to median system delay (132 minutes). Final infarct size and myocardial area at risk were measured by cardiovascular magnetic resonance. Among patients with a system delay ⩽132 minutes (n=74), treatment with exenatide resulted in a smaller infarct size (9 grams [interquartile range (IQR), 4–13] versus 13 grams [IQR, 8–24], P=0.008, corresponding to 8% [IQR, 4–12] versus 11% [IQR, 7–17] of the left ventricle, P=0.015). In a regression analysis adjusting for myocardial area at risk the data points of the exenatide group lay significantly lower than for the placebo group (P=0.006). In the patients with system delay >132 minutes (n=74) no difference was observed in infarct size expressed as grams (P=0.49) or percentage (P=0.46). There was significant interaction between system delay (less than or equal to median versus greater than median) and treatment allocation in terms of infarct size (P=0.018). Conclusions— In this post hoc analysis, exenatide treatment was associated with a 30% decrease in final infarct size in patients with short system delay, whereas no cardioprotective effect in patients with long system delay was seen. However, this finding must be confirmed in larger studies. Clinical Trial Registration— URL: http://www.clinicaltrials.gov. Unique identifier: NCT00835848.

The sarco/endoplasmic reticulum calcium-ATPase 2b is an endoplasmic reticulum stress-inducible protein.

The sarco/endoplasmic reticulum calcium-ATPase (SERCA) translocates Ca2+ from the cytosol to the lumen of the endoplasmic reticulum. This Ca2+ storage is important for cellular processes such as calcium signaling and endoplasmic reticulum (ER)-associated posttranslational protein modifications. We investigated the expression of the SERCA2 and SERCA3 isozymes in PC12 cells exposed to agents interfering with different aspects of the posttranslational protein processing within the ER, thereby activating the ER stress-induced unfolded protein response (UPR). All agents increased the SERCA2b mRNA level 3–4-fold, in parallel with increasing mRNA levels for the ER stress marker proteins BiP/GRP78 and CHOP/GADD153. In contrast, SERCA3 mRNA levels did not change. SERCA2b mRNA stability was not changed, indicating that the mechanism of its up-regulation was transcriptional, in accordance with the presence of ER stress response elements in the promoter region of the SERCA2 gene. SERCA2b was also increased at the protein level upon ER stress treatments. Induction of ER stress by tunicamycin, dithiothreitol, or l-azetidine 2-carboxylic acid did not result in depletion of ER calcium, showing that such depletion was not necessary for up-regulation of SERCA2b expression or UPR activation in general. We conclude that the SERCA2b expression can be controlled by the UPR pathway independently of ER Ca2+depletion.

Thapsigargin analogues for targeting programmed death of androgen-Independent prostate cancer cells

A number of analogues of thapsigargin, a selective inhibitor of the sarco-endoplasmic reticulum Ca2+-ATPases have been synthesized. In all of the prepared analogues the butanoyl residue at O-8 has been replaced with a residue containing an aromatic amine. The amine can be used as an anchoring point for attaching a peptide group sensitive to the proteolytic enzyme, prostate specific antigen, secreted by prostate cancer cells. Like thapsigargin, the analogues are capable of elevating the cytoplasmic Ca2+ concentration approximately sevenfold when tested at effective cytotoxic doses. The analogues in which the 8-O-butanoyl group has been replaced with 3-(4-aminophenyl)propanoyl or 4-aminocinnamoyl were found potently to induce programmed cell death of the prostate cancer cells.

The cardioprotective and inotropic components of the postconditioning effects of GLP-1 and GLP-1(9-36)a in an isolated rat heart.

GLP-1 and its metabolite GLP-1(9-36)a have been shown to exert cardiotropic effects, and were demonstrated to be cardioprotective agents in isolated, postischemic rat or mouse hearts. An agents total effect on myocardial performance in a postconditioning paradigm is a sum of its myocyte-preserving (cardioprotective) and contractility-affecting (negative or positive inotropic) action components. These components may not always be explicitly separated by the experimental protocol. We propose an analytical approach to identify and quantify the cardioprotective and inotropic components in a postconditioning protocol, as exemplified by use of GLP-1 and GLP-1(9-36)a following a global ischemia in isolated rat hearts. Peptides were administered during the first 15min of 120min reperfusion. GLP-1 0.3nM reduced infarct size from 23.2+/-2.4% to 14.1+/-2.3% of area-at-risk (n=15, P=0.0223), an effect abolished by the GLP-1 receptor antagonist, exendin(9-39) 5nM. GLP-1 showed only a small, non-significant tendency to increase mechanical performance (increase of LVDP by 26.7%, P=0.1621; RPP 33.5%, P=0.0858; dP/dt(max) 28.5%, P=0.1609). This could be accounted for by the cardioprotective component of GLP-1 action, rather than any true inotropic effect. In contrast, GLP-1(9-36)a did not reduce infarct size significantly, but acted as a strong negative inotrope in postischemic hearts, causing a contractility deficit (LVDP 58.8%, P=0.0004; RPP 58.2%, P=0.0007; dP/dt(max)=58.2%, P=0.0012), quantifiable by an analysis of infarct size-mechanical performance plots. These results help resolve certain apparent discrepancies between some of the published effects of GLP-1 and GLP-1(9-36)a.

Derivatives of thapsigargin as probes of its binding site on endoplasmic reticulum Ca2+ ATPase: Stereoselectivity and important functional groups

The naturally occurring sesquiterpene lactone thapsigargin is a potent and selective inhibitor of SERCA ATPases, a family of Ca2+‐pumping ATPases present in the endoplasmic reticulum of all mammalian cells. We have studied some of the molecular features of thapsigargin responsible for its inhibitory action towards these Ca2+ ATPases. A series of thapsigargin analogues were synthesised and their inhibitory potencies determined using the uptake of 45Ca2+ in bovine cerebellar microsomes as a sensitive marker of Ca2+ ATPase activity. An attenuation of the inhibitory potency relative to the parent compound was found ranging from slight to over 3 orders of magnitude. The inhibitory activity showed a very strong configuration dependence, a major contribution from the ester groups at C3 and C10, and an apparently minor contribution from the lactone ring substituents. The data are consistent with thapsigargin fitting to a sterically discriminating cleft involving the hydrophobic transmembrane region of the ATPase, and is compatible with available kinetic evidence of thapsigargin‐mediated inhibition.

ST-Segment resolution and clinical outcome with ischemic postconditioning and comparison to magnetic resonance

BACKGROUND Ischemic postconditioning (IPost) during primary percutaneous coronary intervention (PPCI) is suggested to reduce myocardial damage. However, the association with ST-segment resolution (STR) and clinical outcome is not determined. The primary aim of this study was to evaluate the association of IPost with STR and clinical outcome. Secondly, we sought to determine the relationship between STR and cardiac magnetic resonance (CMR) parameters in these patients. METHODS One hundred eighteen patients referred for PPCI were randomly assigned to either conventional PPCI or PPCI with IPost. In a single electrocardiographic lead, STR was determined. Treatment modalities were compared as regards STR, ST-segment elevation, and the number of patients achieving complete-STR (≥70%), incomplete-STR (30%-70%), and no-STR (<30%). Patients were evaluated for clinical outcome after 15 months. Furthermore, patients with and without complete-STR were compared as regards CMR parameters. RESULTS There was a tendency toward a better outcome with IPost for the number of patients achieving complete-STR (55% vs 63%; P=.09), ST-segment elevation (1.41 vs 1.12 mm; P=.07), and New York Heart Association class (P=.06). No difference in other cardiac events was observed. Furthermore, data determine that patients with complete-STR have smaller infarct size (12.9% vs 21.1%; P<.01) and a better ejection fraction (55.7% vs 47.7%; P<.01). CONCLUSIONS Patients treated with IPost are suggested to have improved STR and New York Heart Association classification. Infarct size and the functional CMR parameters were better in the patients with complete-STR; as to this, single-lead STR remains an important predictor for successful treatment in patients treated with IPost.

Calcium and neurosecretion.

Neuroendocrine cells are excitable; i.e., a stimulation of them produces a plasma membrane polarization change of the type that is characteristic of nerve cells. However, they are very similar to pancreatic beta-cells in certain other aspects of their function since they release a polypeptide and since the release requires extracellular calcium. The studies by our group have centered on the neurohypophysis that releases vasopressin and oxytocin, two nonapeptide hormones. Dreifuss and his coworkers’ have also done a number of experiments on these nerve cells including studies of calcium movements over cell membranes. The hypothalamo-neurohypophyseal tract is a classical example of a neurosecretory system and most studies on the role of calcium in neurosecretion have been made on this system. The limited number of investigations reported from other neurosecretory systems seem to show that the phenomena that go on at hormone release from the neurohypophysis are typical for these systems in general. For example, it was demonstrated by Bennett & Edwardson’ that depolarizing concentrations of potassium release a corticotropin-releasing factor and a prolactin-release-inhibiting factor from isolated nerve endings (secretosomes) from rat hypothalamus in a calciumdependent manner. Warberg et al. showed that luteinizing-hormone-releasing hormone, thyrotropin-releasing-hormone, and a-MSH behave in a similar way. Neurosecretion has certain similarities to release of adrenalin and noradrenalin from the adrenal medulla. Poisner4 has given a review of the role of calcium mainly in that system, to which the reader is referred. A substantial amount of evidence supports the hypothesis that release of neurohypophyseal hormones occurs by e x o c y t ~ s i s . ~ ~ ~ Recently it has been demonstrated by freeze-cleaving electron microscopy that severely stimulated isolated neurohypophyses show exocytosis figures.’ This comprised fusion of secretory vesicles with the cell membrane. In addition, fusion of secretory vesicles with other vesicles undergoing exocytosis was observed (as well as fusion of secretory vesicles with each other) where no connection between vesicles and cell membrane was evident. In the experiment of Gratzl et al.’ it was demonstrated that strongly stimulated isolated neurohypophyseal nerve endings (secretosomes) show membrane alterations typical for exocytosis (“necks” and “holes”). It should, however, be mentioned that previously it has been stressed that certain findings could