Disturbed nuclear orientation and cellular migration in A-type lamin deficient cells.

The nuclear lamina and the cytoskeleton form an integrated structure that warrants proper mechanical functioning of cells. We have studied the correlation between structural alterations and migrational behaviour in fibroblasts with and without A-type lamins. We show that loss of A-type lamins causes loss of emerin and nesprin-3 from the nuclear envelope, concurring with a disturbance in the connection between the nucleus and the cytoskeleton in A-type lamin-deficient (lmna -/-) cells. In these cells functional migration assays during in vitro wound healing revealed a delayed reorientation of the nucleus and the microtubule-organizing center during migration, as well as a loss of nuclear oscillatory rotation. These observations in fibroblasts isolated from lmna knockout mice were confirmed in a 3T3 cell line with stable reduction of lmna expression due to RNAi approach. Our results indicate that A-type lamins play a key role in maintaining directional movement governed by the cytoskeleton, and that the loss of these karyoskeletal proteins has important consequences for functioning of the cell as a mechanical entity.

Role of nuclear lamina-cytoskeleton interactions in the maintenance of cellular strength.

The response of individual cells to cellular stress is vital for cellular functioning. A large network of physically interconnected cellular components, starting from the structural components of the cells' nucleus, via cytoskeleton filaments to adhesion molecules and the extracellular matrix, constitutes an integrated matrix that functions as a scaffold allowing the cell to cope with mechanical stress. Next to a role in mechanical properties, this network also has a mechanotransductional function in the response to mechanical stress. This signaling route does not only regulate a rapid reorganization of structural components such as actin filaments, but also stimulates for example gene activation via NFkappaB and other transcription factors. The importance of an intact mechano-signaling network is illustrated by the physiological consequences of several genetic defects of cellular network components e.g. actin, dystrophin, desmin and lamins. These give rise to an impaired response of the affected cells to mechanical stress and often result in dystrophy of the affected tissue. Recently, the importance of the cell nucleus in cellular strength has been established. Several new interconnecting proteins, such as the nesprins that link the nuclear lamina to the cytoskeleton, have been identified. Furthermore, the function of nuclear lamins in determining cellular strength and nuclear stability was illustrated in lamin-knock-out cells. Absence of the A-type lamins or mutations in these structural components of the nuclear lamina lead to an impaired cellular response to mechanical stress and disturbances in cytoskeletal organization. In addition, laminopathies show clinical phenotypes comparable to those seen for diseases resulting from genetic defects in cytoskeletal components, further indicating that lamins play a central role in maintaining the mechanical properties of the cell.

On-line computation of cardiac output with the thermodilution method, using a digital minicomputer.

In the present study, several techniques for calculating cardiac output were evaluated in order to find an accurate technique suitable for on-line digital computation. The thermodilution method was evaluated by different calculation techniques and by comparing these techniques with electromagnetic flowmeter values. Comparison of thermal dilution curves, manually calculated by the log-normal and the exponential assumption, showed a correlation coefficient of 0.978 between the two methods, the former values being 11% higher. The exponential method correlated very well with the technique, using a correction factor for injection errors (r = 0.999). Since the exponential technique correlated well with the electromagnetic values (r = 0.84) and since it was less complicated than the technique, using correction factors, this technique was chosen for automation. Comparison of exponential calculated thermodilution values with a digital computer and manually, showed a correlation coefficient of 0.991. Therefore, it was concluded that on-line computation of thermodilution curves improves the applicability of the termodilution techniques as a means of measuring cardiac output.

Myocardial function in normal and spontaneously hypertensive rats during reperfusion after a period of global ischaemia.

Isolated working hearts of 16 month old spontaneously hypertensive rats (SHR, n = 8) and age matched Wistar-Kyoto (WKY, n = 8) rats were exposed to 30 min global normothermic ischaemia followed by 60 min reperfusion. The hearts were routinely perfused at an afterload level of 13.3 kPa and a preload level of 1.0 kPa. The control values of left ventricular pressure, its maximal positive first derivative (dP1v/dtmax), coronary flow per gram heart tissue, and release of lactate and enzymes such as lactate dehydrogenase and aspartate aminotransferase were comparable in both groups. WKY rat hearts ejected almost twice as much perfusate per gram heart weight as the SHR hearts. In pressure-flow curves, obtained during the control period in SHR hearts, cardiac output was independent of changes in afterload, varying between 10.7 and 18.7 kPa. In contrast, in WKY rat hearts increases in afterload resulted in a progressive decrease in cardiac output. Reperfusion of the SHR hearts after 30 min of global normothermic ischaemia resulted in a poor recovery of cardiac output (13% of the control values) and dP1v/dtmax (32%) compared with the values in the WKY rat hearts (66% and 91% of the control values respectively). Reactive hyperaemia was prominent in the WKY rat hearts but completely absent in the SHR hearts. During one hour reperfusion, SHR hearts lost 3.5 times more lactate dehydrogenase and 2.5 times more aspartate aminotransferase than the WKY rat hearts. Pressure-flow curves, obtained during the reperfusion period, showed modest recovery of myocardial function of the WKY rat hearts at the lowest afterload level tested but completely depressed myocardial function of the SHR hearts at all afterload levels. Heart tissue contents of adenosine triphosphate and creatine phosphate after one hour of reperfusion were lower in the SHR than in the WKY rats, but compared with native values a comparable percentage decrease was seen in both groups of rats.

Differences in ischaemia tolerance between hypertrophied hearts of adult and aged spontaneously hypertensive rats.

OBJECTIVE: The aim was to examine differences between the postischaemic functional and biochemical recovery of adult and aged hypertrophied hearts. METHODS: Isolated hypertrophied hearts of adult and aged spontaneously hypertensive rats (SHRadult; SHRaged) and normal hearts of age matched Wistar-Kyoto rats (WKYadult; WKYaged) were perfused in an ejecting heart preparation. Haemodynamic function was monitored before and after 45 min of ischaemia. Coronary effluent samples and tissue biopsies were taken for biochemical analysis. RESULTS: After ischaemia, in SHRadult and WKYadult the maximum positive first derivative of the left ventricular pressure (dP/dtmax) was restored to 105% and 97% respectively of the preischaemic values. Left ventricular developed pressure recovered to 80% (SHRadult) and 97% (WKYadult), while cardiac output reached 71% (SHRadult) and 99% (WKYadult) of preischaemic levels. In SHRaged and WKYaged the dP/dtmax recovered to 26% and 60% respectively (both p < 0.05 compared to the preischaemic values). The left ventricular developed pressure recovered to 36% in SHRaged and to 73% in WKYaged (both p < 0.05), while cardiac output was restored to 6% in SHRaged and 38% in WKYaged (both p < 0.05). Throughout reperfusion, left ventricular end diastolic pressure remained significantly elevated in SHRaged, and was associated with a prominent subendocardial underperfusion, suggesting an impaired diastolic functional recovery. Overall haemodynamic recovery was significantly better in the WKYaged than in the SHRaged. The preischaemic total adenine nucleotides content was comparable in all groups, but creatine phosphate levels were significantly lower in both aged groups than in adult groups. In all but the WKYadult, the total adenine nucleotides were depressed upon reperfusion, while creatine phosphate normalised, except in SHRaged. SHRaged lost more lactate dehydrogenase and tended to lose more xanthine and uric acid than other groups. CONCLUSIONS: The aged hypertrophied heart shows a higher vulnerability to ischaemic damage than the adult hypertrophied heart. This phenomenon is associated with subendocardial underperfusion, increased membrane damage and inadequate recovery of creatine phosphate levels.

HSP70-mediated acceleration of translational recovery after stress is independent of ribosomal RNA synthesis.

HSP70 is known to protect cells against stressful events. In the present study, the hypothesis was investigated that elevated HSP70 levels protect RNA polymerase I during stress, leading to decreased inhibition of ribosomal RNA (rRNA) synthesis and accelerated recovery of protein translation after stress. To this end, transcriptional and translational activity was studied in H9c2 cells during recovery after a severe heat treatment (SHT, 1 h 45 degrees C) in the presence of elevated HSP70 levels. The latter was achieved by heat pretreatment or by adenovirus-mediated hsp70 gene transfer. Rates of transcription and translation were determined by measuring cellular 3H-labelled uridine and leucine incorporation, respectively. The two types of pretreatment did not affect basal rates of transcription and translation, immediately before SHT. During SHT, both transcriptional and translational rates dropped to less than 10% of basal levels in pretreated as well as non-pretreated cells. Two and four h after SHT, both transcriptional and translational rates were significantly higher in HSP70-overexpressing cells compared to non-pretreated cells. However, immediately after SHT, transcription rates were similarly depressed in non-pretreated and pretreated cells, showing that increased levels of HSP70 did not protect RNA polymerase I activity during SHT. Thus, the HSP70-mediated acceleration of translational recovery is not preceded in time by an enhanced recovery of rRNA synthesis. Therefore, the HSP70-mediated early recovery of protein synthesis after heat stress is independent of rRNA synthesis.

alpha B-crystallin and hsp25 in neonatal cardiac cells--differences in cellular localization under stress conditions.

Two members of the small heat shock protein family, alpha B-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 alpha B-crystallin levels even remain unchanged. Phosphorylation of alpha B-crystallin and to a lesser extent also of hsp25 is induced after the heat shock. Directly after heat stress, alpha B-crystallin and hsp25 are partly found in detergent-insoluble fractions, representing cytoskeletal/nuclear structures. Additionally, we show by confocal laser scanning microscopy that alpha B-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 alpha B-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 alpha B-crystallin occurs under milder conditions and persists for a longer period of time in comparison with hsp25. Our findings suggest that alpha B-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.

Echocardiographic dimensions in athletes in relation to their training programs.

The cardiac dimensions of long-distance runners (LDR), cycle racers (CR), and weight lifters (WL) were determined echocardiographically and were compared with those of control subjects (CS). Left ventricular hypertrophy (LVH) was also assessed from the electrocardiogram. Training information was obtained through a questionnaire. The maximal aerobic performance was assessed on a cycle ergometer. Comparison of the cardiac dimensions revealed that left ventricular mass (LVmass) was significantly increased in LDR and CR as compared to CS. This resulted from thickening of the interventricular septum and left ventricular posterior wall as well as from enlargement of the left ventricular internal diameter. The existence of LVH was confirmed by electrocardiographic investigation. Although the left ventricular wall was enlarged in WL, their LVmass was not significantly increased as compared with CS. These results are in agreement with the training program followed. Weight lifters almost exclusively performed strength training, while LDR and CR were mainly involved in endurance training. The LDR and CR reached significantly higher maximal aerobic performance levels than WL. The present results suggest a close relationship between the type of cardiac enlargement and the training program followed by the athletes.

A model approach to the adaptation of cardiac structure by mechanical feedback in the environment of the cell.

The uniformity of the mechanical load of the cardiac fibers in the wall is maintained by continuous remodeling. In this proposed model the myocyte changes direction in optimizing systolic sarcomere shortening. Early systolic stretch and contractility increases the mass of contractile proteins. Cyclic strain of the myocardial tissue diminishes passive stiffness, resulting in the control of ventricular end-diastolic volume. Utilizing these rules of remodeling in our mathematical model yields that the natural helical pathways of the myocardial fibers in the wall are formed automatically.

Mechanoperception and mechanotransduction in cardiac adaptation: mechanical and molecular aspects.

Cardiomyocytes grow in hypertrophy due to a net increase in the synthesis of proteins, especially contractile proteins, in the cell. There is abundant information about the molecular and biochemical changes involved in this process, but it is not completely understood how cells sense mechanical stimuli and how these stimuli are transferred into a biochemical signal inducing the growth response. This mechanotransduction most likely takes place at the cellular membrane. The resulting signal is transferred to the nucleus, where it can initiate alterations in gene expression.

A microcomputer system for haemodynamic measurements in isolated, working rat hearts.

This study describes the application of an Apple IIe microcomputer in combination with a pre-processor in the on-line calculation of haemodynamic variables of the isolated working rat heart and of relative rapid changes in these variables, induced by variations in left atrial filling pressure (preload) and end-diastolic aortic pressure (afterload). Variables such as heart rate, systolic and diastolic left ventricular pressure, the maximal positive and negative first derivative of the left ventricular pressure, systolic and diastolic aortic pressure and aortic flow were continuously calculated and printed at minimal intervals of 6 s. A newly designed procedure to detect the activation of the electrogram, which was necessary to start the detection of a new cardiac cycle, is described.

Effect of training specificity on maximal treadmill and bicycle ergometer exercise.

Sixteen competitive athletes in long-distance running (n = 8) and cycle racing (n = 8) performed three maximal exercise tests on the treadmill as well as on the bicycle ergometer. The test protocol for both ergometers was similar in work intensity and duration. The results obtained at the maximum work load were compared between and within the two groups of athletes. Comparing treadmill to bicycle exercise, the oxygen uptake was 14% higher on treadmill in the long-distance runners, but equal in the cycle racers. The work efficiency on both types of ergometers showed a clear relationship with the training activity: the cycle racers worked more efficiently on the bicycle ergometer while the results of the long-distance runners were better on the treadmill. Heart rate and minute ventilation reached similar values in the cycle racers on both ergometers, but treadmill exercise induced higher values in the long-distance runners as compared to cycling. Opposite results were found in the variables of the acid-base balance: lactate concentration and base excess reached similar values (13 and -14 mmol l-1, respectively) in the long-distance runners on both ergometers, but in the cycle racers bicycle exercise induced higher values than running. From these results it can be concluded that congruence between the mode of ergometer exercise and the sport activity improves the validity of the test result.

Expression and cellular distribution of heat-shock and nuclear oncogene proteins in rat hearts.

An early, transient accumulation of mRNAs of the protooncogenes c-fos and c-myc and the heat-shock protein HSP70 has been described in hypertrophying rat hearts. It is unclear 1) in which cardiac cell type-these gene activations occur and 2) whether the corresponding proteins are translated. We studied protein expression in rat hearts during ontogenic development and under stress conditions associated with pressure overload with the use of immunofluorescent techniques. During cardiac development no HSP70 could be detected. c-Fos was expressed consistently after birth but only in coronary smooth muscle cells, and c-Myc was found exclusively in adult coronary endothelium and myocardial nonmuscle cells. In adult rats, HSP70 and, to a lesser extent, c-Fos were induced in myocardial muscle and some nonmuscle cells within 3 h following methohexital sodium anesthesia. A similar, more intense immunolabeling of these peptides was observed after thoracotomy and/or aortic stenosis. The coronary c-Fos and c-Myc labeling remained unchanged in these conditions. Thus the expression in cardiac muscle and nonmuscle cells of the three peptides differs and depends on different triggers.

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.

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 degrees 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 < or = 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.

Heat pretreatment differentially affects cardiac fatty acid accumulation during ischemia and postischemic reperfusion.

We investigated whether the cardioprotection induced by heat stress (HS) pretreatment is associated with mitigation of phospholipid degradation during the ischemic and/or postischemic period. The hearts, isolated from control rats and from heat-pretreated rats (42 degrees C for 15 min) either 30 min (HS0.5-h) or 24 h (HS24-h) earlier, were subjected to 45 min of no-flow ischemia, followed by 45 min of reperfusion. Unesterified arachidonic acid (AA) accumulation was taken as a measure for phospholipid degradation. Significantly improved postischemic ventricular functional recovery was only found in the HS24-h group. During ischemia, AA accumulated comparably in control and both HS groups. During reperfusion in control and HS0.5-h hearts, AA further accumulated (control hearts from 82 +/- 33 to 109 +/- 51 nmol/g dry wt, not significant; HS-0.5h hearts from 52 +/- 22 to 120 +/- 53 nmol/g dry wt; P < 0.05). In contrast, AA was lower at the end of the reperfusion phase in HS24-h hearts than at the end of the preceding ischemic period (74 +/- 18 vs. 46 +/- 23 nmol/g dry wt; P < 0.05). Thus accelerated reperfusion-induced degradation of phospholipids in control hearts is completely absent in HS24-h hearts. Furthermore, the lack of functional improvement in HS0.5-h hearts is also associated with a lack of beneficial effect on lipid homeostasis. Therefore, it is proposed that enhanced membrane stability during reperfusion is a key mediator in the heat-induced cardioprotection.