Luc H. E. H. Snoeckx

αB-crystallin and hsp25 in neonatal cardiac cells — differences in cellular localization under stress conditions

Two members of the small heat shock protein family, αB-crystallin and hsp25, occur at high levels in the mammalian heart. To try and understand any differences in functioning, we compared their properties in cultured rat neonatal cardiac myocytes. Both proteins are stress-inducible, but the level of hsp25 is only slightly increased in cultured cardiac myocytes subjected to hyperthermic stress, while αB-crystallin levels even remain unchanged. Phosphorylation of αB-crystallin and to a lesser extent also of hsp2S is induced after the heat shock. Directly after heat stress, αB-crystallin and hsp25 are partly found in detergent-insoluble fractions, representing cytoskeletal/nuclear structures. Additionally, we show by confocal laser scanning microscopy that αB-crystallin and hsp25 become associated with sarcomeric structures directly after the heat shock, indicating a cytoskeletal protective function. Four to six hours after the heat shock, both proteins reoccupy their original positions in the cytoplasm again. In contrast to αB-crystallin, hsp25 not only translocates to the cytoskeleton but also migrates to positions inside the nucleus. Despite the fact that both proteins are normally part of the same complex, their behavior in neonatal cardiac myocytes appears to be very different. The sarcomeric association of αB-crystallin occurs under milder conditions and persists for a longer period of time in comparison with hsp25. Our findings suggest that αB-crystallin and hsp25 are both involved in protection of the cytoskeleton during stress situations in the heart, although in different manners. In addition, hsp25 also plays a role inside the nucleus.

Adaptation of cardiac structure by mechanical feedback in the environment of the cell: a model study.

In the cardiac left ventricle during systole mechanical load of the myocardial fibers is distributed uniformly. A mechanism is proposed by which control of mechanical load is distributed over many individual control units acting in the environment of the cell. The mechanics of the equatorial region of the left ventricle was modeled by a thick-walled cylinder composed of 6-1500 shells of myocardial fiber material. In each shell a separate control unit was simulated. The direction of the cells was varied so that systolic fiber shortening approached a given optimum of 15%. End-diastolic sarcomere length was maintained at 2.1 microns. Regional early-systolic stretch and global contractility stimulated growth of cellular mass. If systolic shortening was more than normal the passive extracellular matrix stretched. The design of the load-controlling mechanism was derived from biological experiments showing that cellular processes are sensitive to mechanical deformation. After simulating a few hundred adaptation cycles, the macroscopic anatomical arrangement of helical pathways of the myocardial fibers formed automatically. If pump load of the ventricle was changed, wall thickness and cavity volume adapted physiologically. We propose that the cardiac anatomy may be defined and maintained by a multitude of control units for mechanical load, each acting in the cellular environment. Interestingly, feedback through fiber stress is not a compelling condition for such control.

Stretch-induced hypertrophy of isolated adult rabbit cardiomyocytes

Both mechanical and humoral triggers have been put forward to explain the hypertrophic response of the challenged cardiomyocyte. The aim of the present study was to investigate whether cyclic equibiaxial stretch is a direct stimulus for isolated adult rabbit cardiomyocytes to develop hypertrophy and to explore the potential involvement of the autocrine/paracrine factors ANG II, transforming growth factor (TGF)-beta(1), and IGF-I in this process. Isolated cardiomyocytes were exposed to 10% cyclic equibiaxial stretch (1 Hz) for up to 48 h or treated with ANG II (100 nM), TGF-beta(1) (5 ng/ml), IGF-I (100 ng/ml), ANG II type 1 (AT(1)) receptor blockers, or conditioned medium of stretched fibroblasts. Cyclic stretch significantly increased cell surface area (+3.1%), protein synthesis (+21%), and brain natriuretic peptide (BNP) mRNA expression (6-fold) in cardiomyocytes. TGF-beta(1) expression increased (+42%) transiently at 4 h, whereas cardiomyocyte IGF-I expression was not detectable under all experimental conditions. The AT(1) receptor blockers candesartan and irbesartan (100 nM) did not prevent the stretch-induced hypertrophic response. Direct exposure to ANG II, TGF-beta(1), or IGF-I did not enhance cardiomyocyte BNP expression. In cardiac fibroblasts, stretch elicited a significant approximately twofold increase in TGF-beta(1) and IGF-I expression. Conditioned medium of stretched fibroblasts increased BNP expression in cardiomyocytes ( approximately 2-fold, P = 0.07). This study clearly indicates that cyclic stretch is a strong, direct trigger to induce hypertrophy in fully differentiated rabbit cardiomyocytes. The present findings do not support the notion that stretch-mediated hypertrophy of adult rabbit cardiomyocytes involves autocrine/paracrine actions of ANG II, TGF-beta(1), or IGF-I.

Heterogeneous distribution of fatty acid-binding protein in the hearts of Wistar Kyoto and spontaneously hypertensive rats

In the present study we investigated the concentrations of cardiac cytoplasmic fatty acid-binding protein (H-FABPc) in various regions of the left and right ventricles of both Wistar Kyoto rats (WKY) and spontaneously hypertensive rats (SHR). To this end, the ventricles of six WKY and six SHR hearts were cut in three slices, which were further dissected in one right ventricular piece and ten left ventricular pieces (five inner layer and five outer layer pieces). After homogenisation. H-FABPc was assessed using an Enzyme Linked Immuno Sorbent Assay (ELISA) of the sandwich type. It was found that, when expressed per gram wet tissue, the overall concentration of H-FABPc tended to be lower in SHR than in WKY hearts (874 +/- 53 micrograms/g and 955 +/- 51 micrograms/g, respectively; 0.1 less than P less than 0.2, means +/- S.E.M. for n = 6 animals in each group). However, due to a 30-35% higher ventricular heart mass in SHR than in WKY, the total H-FABPc content per heart turned out to be about 20% higher in SHR than in WKY rats. No concentration differences could be detected between right and left ventricles in WKY and SHR but a marked difference between the outer layer and the inner layer of the left ventricular wall was monitored in both groups. In general, the concentration in the outer layer was 5-15% higher than in the corresponding inner layer. These differences reached the level of significance (P less than 0.05) in regions close to the basis of the heart.

Effects of pyruvate on post-ischemic myocardial recovery at various workloads

In the present study the hemodynamic and metabolic effects of pyruvate (5 mM), added as cosubstrate to glucose (11 mM) perfused, transiently ischemic, isolated working rat hearts, were evaluated. During 2 h of normoxic perfusion pyruvate improved functional stability, prevented depletion of glycogen and triacylglycerol stores, and increased non-esterified fatty acid (NEFA) levels, even at relatively high workloads. The elevated NEFA levels are in line with the notion that pyruvate competes with endogenously produced fatty acids for oxidative energy production. After 45 min of global ischemia pyruvate was found (a) to affect markedly the relative contribution of ATP, ADP and AMP to the total adenine nucleotide content and (b) to stimulate the degradation of glycogen and to enhance the accumulation of lactate, suggesting enhanced anaerobic ATP rroduction. After restoration of flow pyruvate reduced the incidence of fibrillation and markedly improved recovery of cardiac output at both normal and high workload. Pyruvate did neither attenuate the release of lactate dehydrogenase, a marker for cell death, nor improve the conservation of the total adenine nucleotide and ATP content of hearts reperfused for 30 min. The latter findings indicate that hemodynamic recovery during reperfusion in the presence of pyruvate is neither related to the absolute tissue content of ATP nor to a reduction of irreversible cell damage, and suggest that pyruvate exerts its advantageous hemodynamic effects rather by improving the condition of reversibly damaged cells during reperfusion.

Proteins involved in salvage of the myocardium.

In the Western world, cardiac ischemic disease is still the most common cause of death despite significant improvements of therapeutic drugs and interventions. The fact that the heart possesses an intrinsic protection mechanism has been systematically overlooked before the 1980s. It has been clearly shown that the activation of this mechanism can reduce the infarct size after an ischemic insult. Prerequisite is the induction of the synthesis of such cardio-protective proteins as heat shock proteins (HSPs) and anti-oxidative enzymes. HSPs are involved in the maintenance of cell homeostasis by guiding the synthesis, folding and degradation of proteins. Besides, the various family members cover a broad spectrum of anti-oxidative, anti-apoptotic and anti-inflammatory activities. Although the major inducible HSP72 has received most attention, other HSPs are able to confer cardioprotection as well. In addition, it seems that there is a concerted action between the various cardio-protective proteins. One drawback is that the beneficial effects of HSPs seem to be less effective in the compromised than in the normal heart. Although clinical studies have shown that there is a therapeutic potential for HSPs in the compromised heart, major efforts are needed to fully understand the role of HSPs in these hearts and to find a safe and convenient way to activate these protective proteins.

The effects of global ischemia and reperfusion on compensated hypertrophied rat hearts

Abdominal aorta constriction was performed in 10-week-old Lewis rats (Aoband). Ten weeks later the hearts were isolated and attached to a non-recirculating perfusion apparatus. The hearts could eject against a diastolic aortic pressure of either 60 or 100 mmHg. The functional recovery was compared with that of hearts of sham-operated (Sham) rats. After 45 min of global ischemia, Sham hearts regained cardiac output up to 75% and 70% of the pre-ischemic levels at 60 and 100 mmHg, respectively. At 60 mmHg Aoband hearts showed a minor recovery of ejection function. However, at 100 mmHg the recovery of Aoband hearts was completely comparable with that of Sham hearts. At 60 mmHg but not at 100 mmHg, the pre-ischemic and post-ischemic coronary flow was lower in Aoband than in Sham hearts (P less than or equal to 0.05). During the initial reperfusion phase Sham hearts, perfused at 60 mmHg, released more degradation products of adenine nucleotides and lactate dehydrogenase (LDH) than Aoband hearts (P less than or equal to 0.05), while the Aoband hearts lost more degradation products and LDH than the Sham hearts later during the reperfusion phase (P less than or equal to 0.05). In the groups perfused at 60 mmHg, higher tissue levels of ATP were found in Sham than in Aoband hearts at the end of the reperfusion period (P less than or equal to 0.05). However, at 100 mmHg comparable levels were found in the Sham and Aoband hearts. It is concluded that the height of the coronary perfusion pressure is of critical importance for the post-ischemic functional recovery of the compensated hypertrophied heart. At sufficiently high perfusion pressure levels, the functional and biochemical recovery of the hypertrophied heart is at least as good as in the non-hypertrophied heart. However, in the hypertrophied heart a coronary perfusion pressure which is too low leads to relative underperfusion during the initial reperfusion period which is associated with severely depressed cardiac performance and delayed wash-out of metabolites and intracellular enzymes.

Recovery of hypertrophied rat hearts after global ischemia and reperfusion at different perfusion pressures

The ability to resist transient ischemia was studied in isolated hearts of 18 months old spontaneously hypertensive (SHR) and Wistar-Kyoto (WKY) rats. Both types of hearts showed optimal performance during the preischemic period when perfused at a diastolic perfusion pressure of 8.0 (WKY) and 13.3 (SHR) kPa. Hemodynamic recovery of WKY hearts during reperfusion at 8.0 kPa, following 45 min global ischemia, was satisfactory. Coronary perfusion completely normalized, contractility (dPlv/dtmax) was slightly depressed and cardiac output returned, on the average, to 40% of the preischemic values. In contrast, hemodynamic function of SHR hearts reperfused at 13.3 kPa was greatly depressed, as evidenced by almost complete abolition of cardiac output, severe reduction ofdPlv/dtmax and persistent underperfusion of the endocardial layers. In addition, the postischemic release of lactate dehydrogenase was retarded and enhanced. The release patterns of degradation products of adenine nucleotides showed a shift to the endstage produets xanthine and uric acid. The enhanced vulnerability of the hypertrophied heart to ischemia was even more expressed when the SHR hearts were reperfused at 8.0 kPa. Postischemic function was characterized by electrical instability, loss of contractility and cardiac output, and noreflow in the endocardial layers. Persistent accumulation of lactate and degradation products of adenine nucleotides in the postischemic hearts are in line with the lack of reperfusion. The present results indicate that a detailed mechanistic explanation for the reduced ability to withstand ischemia of SHR cannot be based on differences in ATP content or an altered anaerobic glycolitic activity prior and during ischemia. It is suggested that a defect on the circulatory level, probably caused by enhanced reactivity of the coronary vessels towards ischemia-elicited factors, is responsible for the higher vulnerability of hypertrophied heart to an ischemia insult.

HEAT STRESS PRETREATMENT MITIGATES POSTISCHEMIC ARACHIDONIC ACID ACCUMULATION IN RAT HEART

Heat stress pretreatment of the heart is known to protect this organ against an ischemic/reperfusion insult 24 h later. Degradation of membrane phospholipids resulting in tissue accumulation of polyunsaturated fatty acids, such as arachidonic acid, is thought to play an important role in the multifactorial process of ischemia/reperfusion-induced damage.The present study was conducted to test the hypothesis that heat stress mitigates the postischemic accumulation of arachidonic acid in myocardial tissue, as a sign of enhanced membrane phospholipid degradation. The experiments were performed on hearts isolated from rats either 24 h after total body heat treatment (42°C for 15 min) or 24 h after sham treatment (control). Hearts were made ischemic for 45 min and reperfused for another 45 min.Heat pretreatment resulted in a significant improvement of postischemic hemodynamic performance of the isolated rat hearts. The release of creatine kinase was reduced from 30 ± 14 (control group) to 17 ± 5 units/g wet wt per 45 min (heat-pretreated group) (p < 0.05). Moreover, the tissue content of the inducible heat stress protein HSP70 was found to be increased 3-fold 24 h after heat treatment. Preischemic tissue levels of arachidonic acid did not differ between heat-pretreated and control hearts. The postischemic ventricular content of arachidonic acid was found to be significantly reduced in heat-pretreated hearts compared to sham-treated controls (6.6 ± 3.3. vs. 17.8 ± 12.0 nmol/g wet wt). The findings suggest that mitigation of membrane phospholipid degradation is a potential mechanism of heat stress-mediated protection against the deleterious effects of ischemia and reperfusion on cardiac cells.

Computer-supported collaborative learning in the medical workplace: Students' experiences on formative peer feedback of a critical appraisal of a topic paper

Background: Medical workplace learning consists largely of individual activities, since workplace settings do not lend themselves readily to group learning. An electronic Learning Management with System Computer-Supported Collaborative Learning (CSCL) could enable learners at different workplace locations to discuss personal clinical experiences at a distance to enhance learning. Aim: To explore whether CSCL-enabled structured asynchronous discussions on an authentic task has additional value for learning in the medical workplace. Methods: Between January 2008 and June 2010, we conducted an exploratory evaluation study among senior medical students that were engaged in clinical electives. Students wrote a Critical Appraisal of a Topic paper about a clinical problem they had encountered and discussed it in discipline homogeneous subgroups on an asynchronous forum in a CSCL environment. A mixed method design was used to explore students’ perceptions of the CSCL arrangement with respect to their preparation and participation, the design and knowledge gains. We analysed the messages recorded during the discussions to investigate which types of interactions occurred. Results: Students perceived knowledge improvement of their papers. The discussions were mostly task-focused. The students considered an instruction session and a manual necessary to prepare for CSCL. A high amount of sent messages and a high activity in discussion seem to influence scores on perceptions: ‘participation’ and ‘knowledge gain’ positively. Conclusion: CSCL appears to offer a suitable environment for peers to provide formative feedback on a Critical Appraisal of a Topic paper during workplace learning. The CSCL environment enabled students to collaborate in asynchronous discussions, which positively influenced their learning.