Diving and decompression performed under immersed conditions have been shown to reduce cardiac function. The mechanisms for these changes are not known. The effect of immersion before a simulated hyperbaric dive on cardiomyocyte function was studied. Twenty-three rats were assigned to four groups: control, 1 h thermoneutral immersion, dry dive, and 1 h thermoneutral immersion before a dive (preimmersion dive). Rats exposed to a dive were compressed to 700 kPa, maintained for 45 min breathing air, and decompressed linearly to the surface at a rate of 50 kPa/min. Postdive, the animals were anesthetized and the right ventricle insonated for bubble detection using ultrasound. Isolation of cardiomyocytes from the left ventricle was performed and studied using an inverted fluorescence microscope with video-based sarcomere spacing. Compared with a dry dive, preimmersion dive significantly increased bubble production and decreased the survival time (bubble grade 1 vs. 5, and survival time 60 vs. 17 min, respectively). Preimmersion dive lead to 18% decreased cardiomyocyte shortening, 20% slower diastolic relengthening, and 22% higher calcium amplitudes compared with controls. The protein levels of the sarco-endoplasmic reticulum calcium ATPase (SERCA2a), Na+/Ca2+ exchanger (NCX), and phospholamban phosphorylation in the left ventricular tissue were significantly reduced after both dry and preimmersion dive compared with control and immersed animals. The data suggest that immersion before a dive results in impaired cardiomyocyte and Ca2+ handling and may be a cellular explanation to reduced cardiac function observed in humans after a dive.
- vascular bubbles
- cardiomyocyte contractility
- calcium handling
during decompression bubbles may form in blood and tissues due to supersaturation. Bubbles are believed mainly to form on the venous side of the circulation (9) but may become arterial bubbles through shunts in the lungs or the heart. Vascular bubbles may lead to detrimental effects on the organism, both by endothelial dysfunction and ischemic effects in, for instance, brain tissue (3).
Diving performed in water has been reported to produce more vascular bubbles compared with dives performed in a dry hyperbaric chamber (7). This difference might be due to hemodynamic changes. Despite this, a majority of the studies related to diving and decompression are performed in dry hyperbaric chambers and not under immersed circumstances. During immersion, water exerts a pressure to the body resulting in a compression in the superficial veins particularly of the lower extremities and abdomen (19). The compression results in blood flow toward the thorax and the heart, and the central blood volume is estimated to increase by about 300–500 ml in humans (13) where a quarter of this is in the cardiac chambers (12). At head-out immersion, the sympathetic nerve system will be activated, resulting in increased stroke volume and cardiac output which is sustained for at least several hours during the immersion (17, 21). After water immersion a decrease in preload and cardiac output for up to 16 h (2) has been shown, indicating that the effect of immersion is not immediately abolished. Immersion is prominent during SCUBA (self-contained underwater breathing apparatus), and results obtained after open sea dives show decreased stroke volume (1, 5). Prolonged effects of SCUBA dives have also been reported by Marabotti et al. (18), who observed a right ventricular (RV) overload and reduction of both RV and left ventricular (LV) diastolic performance 2 h postdiving. An enhanced pulmonary artery pressure accompanied by an increased right ventricular end-diastolic and end-systolic volume, and a reduced ejection fraction and cardiac output (CO) have been found (6).
No studies have examined cellular mechanisms behind the cardiac changes induced by immersion and diving. Based on the findings in humans, we initiated this study to examine if the effect of immersion before a simulated dive would influence cardiomyocyte function in rats. We hypothesized that the effect of immersion before the dive would lead to reduced cardiomyocyte function.
The experimental protocol was approved by the Norwegian Council for Animal Research, and all experimental procedures conformed with the European Convention for the Protection of Vertebrate Animals used for Experimental And Other Scientific Purposes.
A total of 23 adult female (295 ± 10.5 g) Sprague-Dawley rats (Kirkeby, Sweden) were included in the study. The rats were controlled at a 12:12-h dark-light cycle with a temperature at 21 ± 2°C and humidity of 50 ± 4%. Animals were fed on a rodent diet and had free access to water. All experimental protocols were performed during the rat's dark cycle, and during the study the same person handled the rats. The rats were assigned into four groups, as described in Table 1. Group I served as a control group, whereas group II (immersion) was used to determine the effect of thermoneutral immersion on cardiomyocyte function. Group III (dry dive) was assigned to determine the impact of a simulated dive in a hyperbaric chamber on cardiomyocyte function. In group IV (preimmersion dive) we wanted to observe if 1 h thermoneutral immersion before a simulated dive would have an impact on bubble formation and cardiomyocyte function. At the end of the exposure period in each group the rats were immediately anesthetized with a mixture of haldol (5 mg/ml, Janssen-Cilag), fentanyl (0.05 mg/ml, Alpharma), and midazolam (5 mg/ml, Alpharma), 0.4 ml/100 g, before decapitation. Immediately after decapitation, blood samples were taken from the vena cava caudalis, and the heart was quickly removed and put on ice-cold buffer.
The effect of immersion was tested in a 22-liter plastic container. Water was filled until there was a clearance of 10 cm to the top, which was an adequate water level to prevent the rats from climbing out of the container. The rats were adapted to water twice for 30 min 7 and 3 days before the experiment. During the experiment the rats were immersed for 1 h in thermoneutral water (35.9 ± 0.6°C). A heater lamp was placed next to the container to stabilize the water temperature. Thermoneutral water was chosen since we wanted to minimize temperature-dependent effects and solely investigate prolonged effects of immersion. Additionally, a pilot study with continuous monitoring of core temperature showed that there was no significant change in core temperature in the rats during immersion, in air immediately after immersion, or during a subsequent dive. Rats performing a preimmersion dive were placed in the hyperbaric chamber immediately after the immersion protocol.
Dive protocol and bubble analysis.
Rats performing a dry (group III) and preimmersion dive (group IV) were compressed one at a time in a dry, hyberbaric chamber (Sira Engineering, Trondheim, Norway) at a rate of 200 kPa/min to a pressure of 700 kPa, maintained for 45 min breathing air and decompressed linearly to the surface at a rate of 50 kPa/min. After reaching surface, the animals were immediately anesthetized before the right ventricle was insonated using a GE Vingmed Vivid 5 ultrasonic scanner, with a 10-MHz transducer as previously described in detail (25). Images were graded according to a previously described method (10). Rats were monitored up to 60 min before decapitation.
Isolation of left ventricular myocytes.
Heparinized (0.2 ml heparin 1,000 IU/ml iv) hearts were removed from the ice-cold buffer and connected to an aortic cannula of a standard Langendorff retrograde perfusion system. Myocytes were isolated from septal plus left ventricular free wall portions of the myocardium as previously described by Wisloff et al. (26). One heart was isolated each day.
Fura-2 loading of isolated myocytes.
Isolated myocytes deposited on coverslips were allowed to rest and stick to the coverslips for 1 h in HEPES buffer (37°C, pH 7.4) consisting of (mM) 135 NaCl, 5 KCl, 1 MgCl2·6 H2O, 1.2 CaCl2, 10 HEPES, 8 glucose H2O before the cells were washed with new HEPES buffer to remove dead cells and add nutrition. After 1 h recovery, the cells were loaded in Fura-2 solution (2 μM) (26) for 20 min in a dark room at room temperature. Before examination in the microscope, the cells were washed for 10 min in HEPES buffer.
Cell shortening and Ca2+ transients.
The coverslips were placed in an inverted epifluorescence microscope (Nikon eclipse -TE 2000-S, Tokyo, Japan) and stimulated electrically by bipolar pulses (5-ms duration, 0.5–2 Hz, room temperature) using platinum electrodes on either side of the chamber. To choose healthy and viable cells, rod shaped cells without blebs or other visible morphological damages were tested before the recordings started to determine whether they could handle stimulation up to 2 Hz. In each animal, recordings of four cardiomyocytes were performed. To examine the cell shortening properties we defined the time of shortening as the duration from an electrical stimulation to maximal contraction. Contraction was calculated as percentage cell shortening and contraction time as time to 50% shortening (TTP50). Relaxation time was calculated as time from peak contraction to 50% normalization of full cell length. Ca2+ was calculated by ratiometric measurements as previously described in detail (23). To examine the time course of Ca2+, peak systolic [Ca2+]i, diastolic [Ca2+]i, and [Ca2+]i amplitude were calculated. All calculations were done on data obtained at an electrical stimulation at 2 Hz after stabilizing the cells for at least 10 consecutive contractions.
For protein detection, 100 μg of total lysate was loaded onto 4–12% Tris-glycine precasted Novex Gel (Invitogen, Carlsbad, CA). Proteins were transferred onto PVDF (Bio-Rad, Hercules, CA), and membranes were blocked with PBS-T/milk for 1 h at room temperature. Subsequently, overnight incubation was performed with the following antibodies; total phospholamban (PLN) and phosphor-Thr-17-PLN antibodies (Badrilla, Leeds, UK), phosphor-SER-16-PLN antibody (Upstate, Charlottesville, VA), SERCA2a (ABR, Rockford, IL), and NCX (Sigma Chemicals, St.Louis, MO). Horseradish peroxidase-conjugated secondary antibodies and enhanced chemiluminescence (ECL) (Thermo Fisher Scientific Inc, Rockford, IL) were used for protein detection. All protein levels were normalized to GAPDH (Cell Signalling) or total PLN and quantified using ImageJ software (NIH, Bethesda, MD).
Cardiomyocyte data and body weight are expressed as means ± SE. Scan grade and survival time are shown as median and ranges. A one-way ANOVA test using LSD as post hoc test was used to evaluate the myocyte parameters in the different groups. For evaluating the differences in bubble formation, a Mann-Whitney U-test was used, while the Gehan generalized Wilcoxon test was used to evaluate differences in survival time between dry dive and preimmersion dive. P < 0.05 was set to be statistically significant.
Data from rats exposed to only water immersion did not differ from controls in any parameters measured, and for clarity reasons these data are not presented.
Bubble formation and survival.
Rats performing a preimmersion dive to 700 kPa had significantly reduced survival time and a significant increased bubble production compared with rats only performing a simulated dive (Table 1, groups IV and III, respectively).
Cell shortening and Ca2+ transients.
Cardiomyocyte fractional shortening and shortening kinetics during systole and diastole are presented in Fig. 1 In rats exposed to a preimmersion dive, cell shortening (%) was significantly attenuated relative to controls, but there was no change after preimmersion dive compared with dry dive (Fig. 1A). Time to peak 50% shortening (ms) (TTP50) was significantly slower after preimmersion dive compared with the other groups (Fig. 1B). Accordingly, time to baseline (T50 relaxation) (ms) was significantly slower after a preimmersion dive compared with controls (Fig. 1C).
Interestingly, the preimmersion dive resulted in a significantly higher peak systolic [Ca2+] compared with all other groups (Fig. 2A), indicating greatest release of [Ca2+] from the intracellular Ca2+ stores, the sarcoplasmic reticulum (SR). No differences between groups were observed for time to peak Ca2+ (data not shown). Additionally, the highest baseline [Ca2+] values during diastole were observed after the preimmersion dive (Fig. 2B). Despite higher diastolic Ca2+, the highest Ca2+ amplitude was observed after the preimmersion dive (Fig. 2C). The velocity of [Ca2+] removal, T50 [Ca2+] decay (ms), was significantly slower in dry dive and preimmersion dive compared with control (Fig. 2D).
Protein expression and phosphorylation status.
Uptake of Ca2+ into SR by SERCA2a is an important determinant of the rate of cardiac muscle relaxation. It also determines the Ca2+ loading of the SR and thus the amount of Ca2+ available for release during cardiomyocyte contraction. SERCA2a is regulated by PLN; unphosphorylated PLN binds to SERCA2a and inhibits its activity, whereas phosphorylation removes PLN from SERCA2a and increases Ca2+ uptake rate. Mainly, PLN is phosphorylated by cAMP-dependent protein kinase A (PKA) at serine (Ser)-16 and by Ca2+/calmodulin-dependent kinase II (CaMKII) at threonine (Thr)-17 (15). We observed that the protein level of SERCA2a and the levels of PLN phosphorylation at threonine-17 and serine-16 became significantly reduced after both dives (Fig. 3, A and C). Another important regulator of intracellular Ca2+ is the Na+/Ca2+ exchanger (NCX) that during diastole exchanges Ca2+ out of the cells with Na+ into the cell. Also the protein expression level of NCX was reduced after the two dives (Fig. 3B). There was no significant difference between dry dive and preimmersion dive with respect to protein expression or phosphorylation status.
This study is the first to demonstrate that immersion before a simulated dry dive results in impaired contractility and calcium handling in isolated cardiomyocytes. The study further shows that bubble formation by decompression is increased following a dive preceded by immersion.
The present study was initiated due to several reports emphasizing the hematological and cardiac differences observed between dry chamber dives and open sea dives (1, 5, 6, 20). During both wet dives and dry dives at the same depth the body is exposed to the same increase in ambient pressure, but due to the antigravity effects of water, immersion significantly alters the hemodynamics. The antigravity effects offset pooling of blood in the lower parts of the body and significantly more blood is allocated to the heart and thorax (4, 22). This will stretch the chambers of the heart, and immersion-induced changes seem to last for a substantial time after getting out of the water (2). Although this study investigated the physiological effects of immersion before a simulated dry dive, our findings can be related to the studies evaluating the effects of immersion during SCUBA diving. Previously it has been demonstrated that an immersed dive resulted in a significantly increased bubble production compared with dry dives (7), and the data in the present study show an increased bubble production in rats exposed to a preimmersion dive. The mechanisms involved are unclear, but since inert gas uptake and elimination are dependent on blood flow during a dive, immersion-induced circulatory changes may effect the bubble production.
An additional challenge during open sea dive may be body cooling due to cold water exposure. It is believed that cold dives increase the susceptibility for decompression sickness (DCS) (24). Cold water may lead to peripheral vasoconstriction and thus influence uptake and elimination of gas, but until now there are no studies showing a causal relationship between thermal status and DCS risk (16, 24). In the preimmersed animals, a drop in core temperature in the period after the immersion and before onset of the dive could have influenced our results. However, since a pilot study showed that preimmersion dives did not significantly reduce core temperature compared with dry dives, our results indicate that immersion-induced hemodynamic changes are the main contributor for the increased venous gas bubble production.
In this study we observed that rats exposed to a preimmersion dive had a significant decreased cardiomyocyte shortening, which may be the cellular explanation for the reduced stroke volumes and ejection fraction observed in humans after a single sea dive (1, 5). This reduction is observable up to 48 h after the dive (6). Stroke volume is influenced by preload, afterload, and the inotropic state of the myocardium. In open-sea scuba dives impairment in diastolic performance has been observed 1 h after and 2 h after a dive (1, 18). Changes in preload may be a plausible explanation for the decreased contractility since a decreased contractility was not present in the rats exposed to only immersion or only dry compression. Diuresis is prominent both under immersion (11) and under dry hyperbaric exposures (14). The results in this study indicate that each stressor alone will not result in adverse cardiomyocyte changes; only a combination of immersion and dry dive will result in impairment of cardiac function. In addition, preimmersion dive resulted in a slower time to 50% relengthening phase during diastole, which implies that the cardiac muscle needs an increased recovery period between each contraction.
The contraction and relengthening properties are mainly determined by the Ca2+ fluctuations in cytosol through ion channels both in the sarcolemma and the SR. This study showed an increased [Ca2+] in both diastole and systole in the rats exposed to a preimmersion dive. Increased Ca2+ leak out of SR in diastole may explain increased levels of diastolic Ca2+ after a preimmersion dive. However, increased diastolic Ca2+ leak is normally followed by reduced peak systolic Ca2+ release due to reduced SR-Ca2+ content (23). Therefore, additional studies are needed to explore these observations.
SERCA2a, NCX, and PLN phosphorylation were significantly decreased in rats exposed to both dry and preimmersion dive compared with controls, and this may explain the significantly slower relengthening and Ca2+ decay. Reduced SERCA2a activity as indicated by these parameters will normally lead to reduced reuptake of Ca2+ into SR in diastole and therefore a lower Ca2+ SR content and a lowering of Ca2+ amplitude, which was not the case in this study and needs to be studied in deeper detail in future studies.
This is the first study to show that immersion followed by a dive in a hyperbaric chamber leads to a reduction in cardiomyocyte function and an increase in mortality, which may be related to an increase in vascular bubble formation. Thus these data together with previous data both from animals and humans support the argument for recommending regular physical exercise in divers (8). The study also indicates that diving may have significant influence on cardiomyocyte calcium regulation and expression of calcium handling proteins. Future studies are needed to elucidate the mechanisms behind our observations.
The study was supported by grants from the Norwegian Petroleum Directorate, Norsk Hydro, Esso Norge, and Statoil under the “dive contingency contract” (no. 4600002328) with Norwegian Underwater Intervention (NUI), Norwegian Council on Cardiovascular Disease and Norwegian Research Council Funding for Outstanding Young Investigators (U. Wisløff) and Foundation for Cardiovascular Research at St. Olav's Hospital.
No conflicts of interest, financial or otherwise, are declared by the author(s).
- Copyright © 2010 the American Physiological Society