AJP - Heart Fuel your research with LabChart
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Heart Circ Physiol 292: H2475-H2484, 2007. First published January 19, 2007; doi:10.1152/ajpheart.01164.2006
0363-6135/07 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
292/5/H2475    most recent
01164.2006v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kawai, K.
Right arrow Articles by Yatani, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kawai, K.
Right arrow Articles by Yatani, A.

Cellular mechanisms of burn-related changes in contractility and its prevention by mesenteric lymph ligation

Kentaro Kawai, Tomoko Kawai, Justin T. Sambol, Da-Zhong Xu, Zhiqiang Yuan, Francis J. Caputo, Chirag D. Badami, Edwin A. Deitch, and Atsuko Yatani

Department of Surgery, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark, New Jersey

Submitted 23 October 2006 ; accepted in final form 9 January 2007


   ABSTRACT
TOP
 ABSTRACT
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Major burn injury results in impairment of left ventricular (LV) contractile function. There is strong evidence to support the involvement of gut-derived factor(s) transported in mesenteric lymph in the development of burn-related contractile dysfunction; i.e., mesenteric lymph duct ligation (LDL) prevents burn-related contractile depression. However, the cellular mechanisms for altered myocardial contractility of postburn hearts are largely unknown, and the cellular basis for the salutary effects of LDL on cardiac function have not been investigated. We examined contractility, Ca2+ transients, and L-type Ca2+ currents (ICa) in LV myocytes isolated from four groups of rats: 1) sham burn, 2) sham burn with LDL (sham + LDL), 3) burn ({approx}40% of total body surface area burn), and 4) burn with LDL (burn + LDL). Myocytes isolated from hearts at 24 h postburn had a depressed contractility ({approx}20%) at baseline and blunted responsiveness to elevation of bath Ca2+. Myocyte contractility was comparable in sham + LDL and sham burn hearts. LDL completely prevented burn-related changes in myocyte contractility. Mechanistically, the decrease in contractility in myocytes from postburn hearts occurred with a decrease in the amplitude of Ca2+ transients ({approx}20%) without changes in resting Ca2+ or Ca2+ content of the sarcoplasmic reticulum. On the other hand, ICa density was decreased ({approx}30%) in myocytes from postburn hearts, with unaltered voltage-dependent properties. Thus burn-related myocardial contractile dysfunction is linked with depressed myocyte contractility associated with a decrease in ICa density. These findings also provide strong evidence that mesenteric lymph is involved in the onset of burn-related cardiomyocyte dysfunction.

burn injury; L-type calcium current; cardiac myocyte

BURN INJURY initiates a series of pathophysiological changes, including depressed cardiac function. Clinical and experimental studies have shown that a major burn injury ({approx}40% of total body surface area burn, TBSA) results in impaired left venticular (LV) contractile function despite aggressive fluid resuscitation (1, 4, 14). However, the source(s) or the signaling pathways that trigger burn-related myocardial dysfunction are not well understood and remains an area of intense ongoing investigation.

The burn-related fall in cardiac output is associated with significant reduction in myocardial contractility, as assessed by LV pressure, LV pressure change over time (dP/dt), and blunted responses to elevated external Ca2+ (2, 14, 15, 21, 22). Of potential mechanistic significance, these changes are similar to those observed in the hypertrophied and/or failing heart (HF). Although the precise mechanisms are not established, numerous studies in cardiac hypertrophy and HF have shown that intrinsic changes in excitation-contraction (E-C) coupling due to abnormal Ca2+ handling at the cellular level are involved in the initiation and progression of myocardial contractile depression (5, 13, 16, 17). Consistent with this notion, there is clear evidence that prevention or correction of Ca2+-regulatory defects in the early stages of cardiac diseases can delay or prevent the onset of HF (9, 16).

Previous studies in hearts following burn injury have indicated that changes in cellular Ca2+ handling may contribute to burn-related myocardial contractile dysfunction (2, 14, 15, 20, 24, 32). For example, Horton's group (2, 15, 20, 24, 32) have reported that diastolic levels of Ca2+ concentration in unstimulated ventricular myocytes isolated from rat or guinea pig hearts were significantly elevated (two- to threefold relative to control value) at 24 h after burn injury. Additionally, one of their studies show significantly elevated amplitude (threefold of control value) of Ca2+ transients and blunted cell motion to isoproterenol in rat ventricular myocytes from postburn hearts (20). However, most of these studies were done in the absence of cell stimulation and did not characterize basic E-C coupling (value of cell shortening and/or kinetics of contractility) and Ca2+ flux during twitch contraction. Since cardiac E-C coupling is an intricate process involved in the interaction of a wide variety of systems (7), it is difficult to obtain meaningful data of cellular mechanisms for burn-related changes in myocardial contractility without cell stimulation.

Although the etiology of burn-related cardiac failure is likely multifactorial, there is emerging evidence that gut-derived factor(s) carried in the mesenteric lymph contribute to organ dysfunction and failure following trauma such as burn injury, hemorrhagic shock, and major elective surgical procedures (11, 23, 26, 29). For example, in rats, cardiac function, assessed by measuring LV pressure and LV dP/dt in the Langendorff isolated heart system, was significantly depressed at 24 h after burn injury, and responses to increases in coronary blood flow rate or Ca2+ were blunted (2, 15, 26, 32). When the main mesenteric lymph duct was ligated before burn injury, burn-related myocardial contractile dysfunction was totally abrogated (26). More recently, our studies have shown that the mesenteric lymph collected from rats with {approx}40% TBSA burn injury (burn lymph) exerted concentration-dependent effects on the contractility of LV myocytes isolated from healthy rats. At lower concentrations (<0.5%), burn lymph increased the amplitude of myocyte contraction (38), whereas physiologically relevant concentrations (0.1–5%) of burn lymph reduced myocyte contractility (37). Taken together, these observations strongly suggest that burn injury promotes the mobilization or release of factor(s) in mesenteric lymph, which trigger abnormal myocyte contractility and that defective cardiomyocyte contractility contributes to burn-related cardiac depression.

Consequently, the goal of this study was to improve our understanding of the cellular mechanisms underlying burn-related myocardial dysfunction. To achieve this, we examined contractility and Ca2+ transients in LV myocytes isolated from four groups of rats: 1) sham, 2) sham + lymph duct ligation (LDL), 3) burn, and 4) burn + LDL at 24 h postburn. Since Ca2+ influx through the L-type Ca2+ current (ICa) is a key determinant of cardiac contractility, we also examined ICa activity in myocytes. Three specific questions were addressed: 1) What are the changes in myocytes contractility after burn injury? 2) Does LDL prevent changes in myocyte function? 3) What are the cellular mechanisms of altered contractility?

Our data indicate that LV myocyte contractility was depressed following burn injury. LDL completely prevented burn-related changes in myocyte contraction, whereas myocyte contractility from sham + LDL hearts remained unchanged. Myocytes form postburn hearts had decreased Ca2+ transients that were not associated with changes in the rate of Ca2+ transient decay or Ca2+ storage of the sarcoplasmic reticulum (SR). On the other hand, ICa density was significantly decreased in myocytes from postburn hearts without changes in voltage-dependent characteristics. Since fractional SR Ca2+ release is regulated by trigger Ca2+ in cardiac myocytes (3, 6), the decrease in ICa density may contribute to depressed postburn myocyte function.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Burn injury model and mesenteric lymph ligation. Adult male Sprague-Dawley rats (250–350 g) were used in this study. The New Jersey Medical School Animal Care Committee approved all experiments. A total of 55 rats were used and at least 6–20 rats were examined for each group (n represents the number of myocytes examined per protocol).

The procedures used to induce burn injury were similar to those described by Walker and Mason (31). Briefly, the rats were anesthetized with pentobarbital sodium (25 mg/kg) and buprenorphine hydrochloride (0.3 mg/kg). The hair was shaved from the back and abdomen, and both areas were treated with a depilatory agent. About 40% TBSA scald burn was induced by immersing the back of the animal through a template into boiling water (100°C) for 10 s following which an abdominal burn was induced by immersion for 5 s. During the first 24 h after burn injury, the rats were resuscitated with Ringer lactate solution given via the jugular vein catheter using the Parkland formula (4 ml·kg–1·%burn–1). Total volume of Ringer solution given over the first 24 h was ~50–56 ml/rat. These conditions produce a uniform third-degree burn, thereby destroying all cutaneous nerves generating insensate wounds. The burned rats did not display discomfort or pain, moved freely in the cage, and consumed food and water within 20–30 min after burn procedure. The sham-burned rats were anesthetized, placed in the plastic template, and immersed in room temperature water. Rectal temperature was monitored periodically throughout the recovery period and maintained at around 37°C.

Mesenteric LDL was performed on anesthetized rats immediately before sham or burn injury as previously described (26). Briefly, a midline celiotomy was performed, and the main mesenteric lymphatic vessels were bluntly dissected free, following which they were ligated.

Myocyte isolation, measurements of contraction, and Ca2+ transients. LV myocytes were isolated from four groups of rats: 1) sham burn (sham), 2) sham burn with LDL (sham + LDL), 3) {approx}40% TBSA burn (burn), and 4) burn with LDL (burn + LDL). In some experiments, LV myocytes were isolated from control rats.

Rats were deeply anesthetized (60 mg/kg pentobarbital sodium), and the heart from each rat was quickly removed as previously described (37, 38). The heart was cannulated and perfused in a retrograde fashion via the aorta by the Langendorff method for about 3 min with a modified Ca2+-free Krebs-Henseleit solution containing (in mmol/l): 110 NaCl, 2.6 KCl; 1.2 MgCl2, 1.2 KH2PO4, 25 NaH2PO4, 11 glucose, 30 taurine, and 10 HEPES (pH 7.4), which was oxygenated by bubbling with 95% O2-5% CO2. The heart was then digested in the same solution containing collagenase (Type II, 0.5 mg/ml, Worthington), hyaluronidase (0.3 mg/ml, Sigma), and bovine serum albumin (1 mg/ml, Sigma) for 20 min at 37°C. At the end of the digestion, the enzyme solution was washed out with enzyme-free solution containing 0.1 mM Ca2+. The LV was cut into small pieces, and the dispersed cells were filtered through a nylon mesh (200 µm). The myocytes were centrifuged at 50 g for 1 min and resuspended in a Tyrode solution (in mmol/l): 120 NaCl, 2.6 KCl, 1.0 CaCl2, 1.0 MgCl2, 11 glucose, and 5 HEPES (pH 7.3). Cells were selected for recording only if they showed normal, rod-shaped morphology with clear striation and were quiescent in the absence of stimulation.

Myocytes contraction (%cell shortening) was measured by video edge detection, as previously described (35, 37, 38). Briefly, isolated LV myocytes were perfused with Tyrode solution at 32°C. All myocytes were field stimulated at 1.0 Hz. Myocytes were then exposed to solutions with different Ca2+ concentrations using a Y-tube method, which allows complete solution changes within 100 ms (33). To obtain frequency-dependent relations, different frequencies were tested in random to minimize time-dependent changes. To minimize the processes that tend to damage myocytes; e.g., SR Ca2+ overload (spontaneous cell motion) and cell damage (shrunken myocytes with incomplete cell relaxation), cells were allowed 15 s rest following each change in frequency. Cell shortening was then analyzed at quasisteady level.

For the Ca2+ transient measurements, cells were loaded with 2 µM fura-2 AM at room temperature for 60 min. Intracellular free Ca2+ was monitored as the ratio of 340–380 nm fluorescence of fura-2 using the Photoscan dual-beam spectrofluorophotometer (Photon Technology) as described previously (35, 37, 38). Cells were stimuated at 0.5 Hz, and the changes in Ca2+ in the cells were depicted as changes in the ratio of 340/380 signal. Fluorescent data were acquired at a 100-Hz sampling rate. We chose not to calibrate signals because we are primarily interested in relative changes in Ca2+ in cells between sham and postburn myocytes, and because the experimental conditions for recording fura-2 signal in both groups of myocytes were identical.

SR Ca2+ content was evaluated by a caffeine pulse protocol as previously reported (3, 30, 38). Briefly, cells were stimulated by a train of 10 stimulations (at 1.0 Hz) to load SR Ca2+. Once cells were loaded, electrical stimulation was stopped before being switched to Tyrode solution containing caffeine (10 mM).

Ca2+ current measurements. Whole cell ICa were measured as previously described (37, 38) using Na+- and K+-free external solution (in mmol/l) 2 CaCl2, 1 MgCl2, 135 tetraethyl ammonium chloride, 5 4-aminopyridine, 10 glucose, and 5 HEPES (pH 7.3). The pipette solution contained (in mmol/l) 100 Cs-aspartate, 20 CsCl, 1 MgCl2, 2 MgATP, 0.5 GTP, 5 EGTA, and HEPES 5 (pH 7.3). Cell capacitance was measured using voltage ramps of 0.8 V/s from a holding potential of –50 mV. Experiments were performed at room temperature (20–22°C).

Statistical analysis. Data are reported as mean values ± SE. Between-groups/conditions analyses were conducted by using a Student's t-test, with significance imparted at the P < 0.05 level.


   RESULTS
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
In the present study, LV myocyte function was measured at 24 h after sham or burn injury. This time point was selected because previous whole heart studies have shown that contractile defects were evident in all hearts 24 h after burn injury regardless of species (2, 14, 20, 22, 24). In addition, we have previously shown that mesenteric lymph diversion by LDL prevented burn-related myocardial contractile depression assessed at 24 h after burn injury (26).

Since the burn procedure uses anesthesia and opioids (buprenorphine hydrochloride), we wanted to examine whether these drugs affect LV myocyte morphology. To address this, we determined myocyte size isolated from control, sham, sham + LDL, burn, and burn + LDL hearts by measuring cell length and cell capacitance by patch-clamp technique (Table 1). The cell length and the surface area estimated by the cell capacitance were comparable in cells isolated from all groups. In addition, as shown in the data below, twitch contraction (% cell shortening) was similar between the cells from control and sham rats.


View this table:
[in this window]
[in a new window]

  		Table 1. Myocyte size from control, sham, sham+LDL, burn, and burn+LDL rats

 
Cardiomyocyte contractile and relaxation function. Figure 1 shows representative cell shortening recorded in LV myocytes from sham, sham + LDL, burn, and burn + LDL hearts. Cell shortening was significantly reduced, and relaxation velocity (+dL/dt) was slower in myocytes from the burn group compared with other groups (Fig. 1C). Twitch amplitude and the rate of relaxation in myocytes from burn + LDL hearts were similar to those observed in myocytes from control or sham hearts (Fig. 1, D–F). These results are consistent with data previously obtained at the whole heart levels (26) and support the hypothesis that depressed myocyte contractility is centrally involved in burn-related myocardial contractile dysfunction and that preburn LDL can prevent the development of myocyte contractile alterations.


Figure 1
View larger version (28K):
[in this window]
[in a new window]

  		Fig. 1. Examples of cell shortening in representative myocytes isolated from sham (A), sham + lymph duct ligation (LDL) (B), burn (C), and burn + LDL (D) hearts. Contractions were recorded during field stimulation at a frequency of 1.0 Hz. Pooled data for cell shortening (%) and rate of relaxation (+dL/dt) are shown in E and F, respectively. Compared with myocytes from sham or sham + LDL, myocytes from postburn hearts had lower magnitude of cell shortening and slower rate of relaxation. There was no significant change in contractility in myocytes from burn + LDL hearts. Numbers correspond to total number of cells measured. Data are means ± SE. *P < 0.01 vs. sham.

 
Since it is generally thought that slow contraction and relaxation rates are associated with reduced SR Ca2+-ATPase activity, the changes in contraction observed in postburn myocytes could be related to depressed SR function. For example, contractile dysfunction in cardiac hypertrophy and failure is associated with significantly prolonged myocyte contraction duration due to changes in SR function (16, 17, 25). On the other hand, the slower rate of relaxation could have been secondary to smaller amplitude of contraction. We therefore normalized traces and compared the kinetics of contraction between myocytes from sham and postburn hearts (Fig. 2, A and B). Comparison of the normalized and superimposed traces (Fig. 2C) showed no significant difference in either the time to peak cell shortening or the relaxation time course. We also compared the relaxation time by measuring the time for 50% decay (T50) of cell shortening in myocytes from sham, burn, and burn + LDL hearts (Fig. 2D). The data show no significant differences in relaxation time among the groups. The results suggest that the cellular basis for burn-related contractile dysfunction is significantly different from the common pattern of changes observed in cardiac hypertrophy and heart failure.


Figure 2
View larger version (14K):
[in this window]
[in a new window]

  		Fig. 2. Cell shortening recorded from cells isolated from sham (A) and burn (B) hearts. Traces were also normalized and shown in C and pooled data for time for 50% decay (T50) are shown D. Numbers correspond to total number of cells measured. Data are means ± SE.

 
Under normal physiological conditions, there is an increase in myocardial contraction as extracellular Ca2+ is acutely elevated. Since previous whole heart studies reported that responsiveness of the LV contractile function to Ca2+ (0.5–3 mM) was depressed in postburn hearts (15, 26), we examined Ca2+ dependence of myocyte contraction (Fig. 3). A representative experiment is shown in Fig. 3 (top trace). In every myocyte studied, increasing external Ca2+ increased the magnitude of cell shortening and the rate of relaxation (Fig. 3, A and B). However, the amplitude and rate of shortening were significantly lower in myocytes from burn hearts than sham burn hearts at all Ca2+ concentrations tested.


Figure 3
View larger version (21K):
[in this window]
[in a new window]

  		Fig. 3. Effects of changes in bath Ca2+ on the magnitude of cell shortening (A) and rate of relaxation (B) in myocytes from sham and postburn hearts. Top trace shows an example of continuous cell shortening record from sham myocytes in response to different Ca2+. Arrowheads indicate quasisteady state where analyses were made. Cell shortening was plotted vs. bath Ca2+ concentrations at the time reached steady level as indicated by arrows. Data are means ± SE from 41 to 146 cells from sham and 30 to 104 cells from postburn hearts. *P < 0.01 vs. sham.

 
Because the change in contractile force in response to a change in frequency stimulation is a general property of cardiac muscle (16, 25), we compared frequency dependence of cell shortening between myocytes from sham and burn hearts. Average data are shown in Fig. 4. For both groups, the amplitude of cell shortening decreased with increasing frequency. The amplitude of cell shortening was depressed in postburn compared with sham burn hearts at all frequencies studied. Note that the relative change of the amplitude with frequency was not significantly different between the two groups.


Figure 4
View larger version (15K):
[in this window]
[in a new window]

  		Fig. 4. Frequency-dependent changes in cell shortening in myocytes from sham and postburn hearts. Example of cell shortening record measured in sham (A) and burn (B) myocytes at indicated stimuration rates. Both groups showed negative frequency response without change in resting cell length, and the relative changes from the basal value at 1.0 Hz are similar between the two groups. Data are means ± SE from 75 to 146 cells from sham and 71 to 104 cells from postburn hearts. *P < 0.01 vs. sham.

 
Cardiomyocyte Ca2+ transients and caffeine-induced Ca2+ transients. To further understand the cellular mechanisms of depressed contractility in postburn hearts, we measured whole cell Ca2+ transients (Fig. 5) and SR Ca2+ content assessed by caffeine-induced Ca2+ transients (Fig. 6). Myocytes from postburn hearts had lower amplitude of Ca2+ transients compared with that of myocytes from sham hearts (Fig. 5, A, B, and D); however, there was no significant difference in diastolic (baseline, resting) Ca2+ signal (Fig. 5C). Since the rate of Ca2+ decline during twitch reflects primarily Ca2+ removal via the SR Ca2+ uptake (13, 16, 25), we compared the time course of Ca2+ transients. The time course of Ca2+ decline as analyzed by the T50 of Ca2+ transient (Fig. 5E), which was not significantly different between the two groups.


Figure 5
View larger version (14K):
[in this window]
[in a new window]

  		Fig. 5. Ca2+ transients recorded in myocytes from sham (A) and postburn (B) hearts. Pooled data of 340/380 ratio for baseline value (C), difference between peak and baseline (D) and T50 (E). Numbers correspond to total number of cells measured. Data are means ± SE. *P < 0.01 vs. sham.

 

Figure 6
View larger version (16K):
[in this window]
[in a new window]

  		Fig. 6. Caffeine-induced Ca2+ transients recorded in myocytes from sham (A) and postburn (B) hearts. Pooled data of 340/380 ratio for maximal Ca2+ transients upon application of caffeine expressed as difference between peak and baseline (C) and T50 (D). Numbers correspond to total number of cells measured. Data are means ± SE.

 
The amplitude of 10 mM caffeine-induced Ca2+ transients was slightly lower in myocytes from postburn hearts compared with sham myocytes (Fig. 6, A–C). However, the difference was not statistically significant. Similarly, T50 of Ca2+ decay was not significantly altered in cells from postburn hearts (Fig. 6D). These cellular Ca2+ measurements suggest that depressed myocyte contraction in postburn hearts result from decreased systolic Ca2+ transients without significant change in SR Ca2+ uptake or content.

ICa density. One of the main determinants of myocyte contraction is ICa, e.g., Ca2+ influx though the L-type Ca2+ channel triggers Ca2+ release from the SR and maintains SR Ca2+ load. We therefore, examined basal ICa characteristics in myocytes from sham, burn, and burn + LDL hearts (Fig. 7). The traces show ICa activated at different membrane potentials. Peak amplitude of ICa normalized relative to cell size, as determined from cell capacitance (in pA/pF), was plotted as a function of voltage (bottom of the current traces). Peak ICa density was significantly (P < 0.05) lower in myocytes from postburn hearts (5.4 ± 0.5 pA/pF, n = 39) compared with myocytes from sham burn hearts (7.5 ± 0.5 pA/pF, n = 56). Peak ICa density was comparable in myocytes from burn + LDL (7.0 ± 0.4 pA/pF, n = 30) and sham burn hearts. Importantly, ICa density in sham + LDL myocytes (7.2 ± 0.2 pA/pF, n = 18) was similar to myocytes from sham burn hearts. The voltage dependence of ICa activation was not significantly different in any of the three groups, i.e., ICa activated around –30 mV and reached its maximum value near +10 mV. The voltage dependence of inactivation of ICa was also analyzed by applying 5-s depolarizing prepulses from a holding potential of –80 mV (data not shown). No significant differences in the midpotential or slope factor were observed among the groups, indicating that decreased ICa density in myocytes from postburn hearts was not caused by abnormal voltage dependence of the Ca2+ channel.


Figure 7
View larger version (14K):
[in this window]
[in a new window]

  		Fig. 7. Whole cell L-type Ca2+ current (ICa) characteristics in myocytes from sham (A), burn (B), and burn + LDL (C) hearts. Currents were elicited from a holding potential of –50 mV to the indicated test potentials at 0.1 Hz. Current-voltage (I–V) relationships were plotted on the bottom of original current traces. ICa was normalized to the cell capacitance to give current densities (pA/pF). Data are means ± SE from sham (n = 56), postburn (n = 39), and burn + LDL (n = 30) myocytes.

 
The whole cell ICa density (I) is determined by the product of the channel number (N), open probability (Po), and single channel current (i), i.e., I = i·N·Po (34, 36). To investigate whether the decrease in ICa density is due to a downregulation of L-type Ca2+ channels (e.g., less expression of proteins and fewer numbers of channels) or decreased channel openings (less channel openings), we examined the effects of BAY K 8644, a dihydropyridine (DHP) agonist, which increases channel opening independent of protein kinase A-mediated signaling pathways (34, 35). BAY K 8644 (0.1 µM) increased ICa a similar magnitude ({approx}3 times) in all myocytes (Fig. 8, A and B). However, the peak ICa density was still significantly smaller in myocytes from burn versus sham hearts (Fig. 8C). These results indicate that the number of L-type Ca2+ channel is reduced in postburn hearts, which may contribute to depressed myocyte contractility.


Figure 8
View larger version (10K):
[in this window]
[in a new window]

  		Fig. 8. Effects of the Ca2+ agonist BAY K 8644 (0.1 µM) on ICa recorded in myocytes from sham (A) and postburn (B) hearts. Traces show currents recorded from a holding potential of –50 mV to 0 mV in the absence (open circles) and presence of the drugs (filled circles). C: comparison of average peak ICa density in the presence of BAY K 8644 in sham and burn myocytes. Data are means ± SE from four each myocytes isolated from sham and postburn hearts. *P < 0.01 vs. sham.

 

   DISCUSSION
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Myocardial contractile dysfunction is a prominent feature in experimental burn injury as well as clinically (10, 14). We have investigated the cellular basis of impaired myocardial contractility in postburn hearts and the effects of LDL. The general approach was to compare baseline myocyte contractility in LV myocytes isolated from rats subjected to 1) sham burn, 2) sham-burn with LDL, 3) burn, and 4) burn with LDL at 24 h postburn. By focusing on cellular functions in this study, we identified cellular mechanisms responsible for the early stages of burn-related contractile depression (14, 22).

The major findings of this study were that myocytes from postburn hearts have decreased contractility. This change in myocyte contraction was prevented by LDL. The decrease in myocyte contraction was associated with depressed Ca2+ transients, without significant changes in the rate of Ca2+ transient decay or SR Ca2+ content. On the other hand, the density of the L-type Ca2+ channel was reduced by {approx}30% in myocytes from postburn hearts, whereas the Ca2+ channel density was unchanged in myocytes from burn + LDL hearts. Taken together, these results suggest that under our experimental conditions, the depressed basal myocyte contractility observed in postburn hearts most likely involves a decrease in SR Ca2+ release (Ca2+ transients) due to a significant decrease in the Ca2+ trigger for SR Ca2+ release (ICa).

Myocyte contractility and SR Ca2+ transport. Defective Ca2+ regulation at the cellular level has been proposed to be a primary cause of abnormal contractility in failing hearts (5, 17, 25). However, it is also possible that depressed postburn heart function could result from other factors such as changes in cardiac structure, altered vascular structure, cytokines, and reactivity (16, 18). In the present study, we found that LV myocytes isolated from postburn hearts exhibit depressed contraction and impaired responses to Ca2+, which is consistent with previous findings observed in whole heart experiments (14, 15, 26). The failure of high extracellular Ca2+ to normalize burn myocytes contractility suggests an impairment of Ca2+ handling at the cellular level.

Slow contraction and relaxation rates associated with reduced peak systolic Ca2+ transient and slow decay of the Ca2+ transient are common features of failing hearts (5, 13, 16, 17). Interestingly, our results demonstrated that myocytes from postburn hearts exhibit depressed myocyte contractility with unaltered rate of contraction and relaxation (Fig. 2). This decrease in contraction was associated with reduced peak systolic Ca2+ transients without changes in the rate of Ca2+ transient decay. Thus the contractile phenotype of postburn myocytes appears to be significantly different from those observed in myocytes from hypertrophied and/or failing hearts.

The change in contractile force in response to change in frequency stimulation is a general property of cardiac muscle and is often used for identifying contractile states associated with SR Ca2+ uptake and release. For example, in mammalian hearts, including healthy humans, force-frequency relation is positive. In failing myocardium, the magnitude of developed force decreases or remains unchanged with an increase in stimulation frequencies (25). Changes in this relationship have been implicated in the diminished capacity of the SR to increase its Ca2+ content, secondary to depressed SR function (13, 25). In rat myocytes, the relationship is negative in the lower range of frequencies (<2.0 Hz), i.e., the magnitude of contraction decreases with increasing frequency of stimulation (37). This negative staircase is thought to reflect the fact that resting SR Ca2+ content in the rats is high and cannot be increased in this range of frequencies. As shown in Fig. 4, we found that there was no significant alteration in the force-frequency relationships in myocytes from postburn hearts. These results are consistent with the idea that depressed myocyte contractility of postburn heart is not primarily caused by defective SR Ca2+ uptake rate and load.

The Ca2+ transient results agree with myocyte contraction measurements (Fig. 5). Ca2+ transient amplitude of myocytes from postburn hearts was lower compared with myocytes from sham burn hearts, but relaxation kinetics remained unchanged. Additionally, we found that there was no significant change in baseline (diastolic) Ca2+ levels, and that SR Ca2+ content assessed by caffeine pulse (Fig. 6) was not significantly different from myocytes from sham burn hearts. In contrast, previous studies from Horton's group (2, 15, 24, 32) have shown that myocytes from hearts 24 h after burn injury develop significantly elevated diastolic levels of Ca2+ (two- to threefold), with one study in burn myocytes reporting a threefold increase in Ca2+ transients without changes in SR Ca2+ content (20). The disparity of intracellular Ca2+ measurement results between these studies and the present one is unclear but may be explained by different experimental approaches. Horton's group used unstimulated myocytes and did not measure cellular Ca2+ during twitch contraction. In our study, contractions and Ca2+ transients were measured under field stimulation. During twitch contraction, there are extremely complex factors, including ionic currents, transporters, and exchangers, which simultaneously affect membrane potential and intracellular Ca2+ dynamics (7, 12, 30). Thus changes in cellular Ca2+ levels accompanying myocyte contraction under the relatively physiological conditions of twitch contraction may not be predicted in the absence of cell stimulation.

L-type Ca2+ channel density. In the present study, basal contractility of postburn myocytes was depressed and was prevented by LDL. The depressed contraction was associated with blunted contractile responses to Ca2+, but the frequency dependence of contraction remained unchanged. Ca2+ transient amplitude was reduced without changes in SR Ca2+ content. An interesting finding of the present study was that ICa density was significantly reduced in myocytes from postburn hearts. There was no change in voltage-dependent characteristics. Furthermore, BAY K 8644 increased ICa in postburn myocytes; however, the amplitude of ICa density in the presence of the drug was significantly smaller compared with myocytes from sham burn hearts (Fig. 8). This result is consistent with the hypothesis that the number of L-type Ca2+ channels is reduced in burn myocytes. Importantly, no such changes were observed in myocytes from sham + LDL or burn + LDL hearts. Thus these data suggest that the decreased contractility observed with myocytes from postburn hearts most likely involved a decrease in Ca2+ influx due to reduced ICa density. This notion is supported by the fact that the fraction of Ca2+ release is strongly influenced by both the level of Ca2+ influx and the SR Ca2+content (3, 7).

Since Ca2+ influx via ICa also replenishes the SR with Ca2+, we expected to find a decrease in SR Ca2+ content during a stable train of contraction. However, we found that decreases in ICa did not lead to a decrease in SR Ca2+ content. This observation is consistent with a series of experimental studies and modeling analysis in which changes in Ca2+ influx (ICa) does not necessarily lead to a parallel change of SR Ca2+ content. For example, it has been reported that increasing external Ca2+ concentration (from 1 to 2 mM) increased ICa amplitude and systolic Ca2+ transient with no effect on SR Ca2+ content (12, 30). The relative constancy of SR Ca2+ content in our study can be explained by the coordinated control of cell Ca2+; a large Ca2+ transient activates a larger Ca2+ efflux from the cell via Na+/Ca2+ exchange. Thus our observation that postburn myocytes had decreased Ca2+ entry (ICa) and systolic Ca2+ transient with little change of SR Ca2+ content can be explained by concerted regulation of cell Ca2+. In this light, the decreased trigger will decrease the amount of Ca2+ released from the SR and therefore the amount pumped out of the cell.

A great deal is understood about the function and acute modulation of L-type Ca2+ channels, but little is known about control of channel expression. However, accumulating evidence does indicate that inflammatory cytokines play a role not only in the pathogenesis of atherosclerosis and in the cardiac dysfunction that accompanies systemic sepsis, burn injury, and viral myocaditis but also in advanced HF syndromes resulting from diverse pathogenic insults (14, 19). These inflammatory mediators can induce generation of specific cytokines and expression of "inducible" and "Ca2+-insensitive" nitric oxide (NO) synthase (iNOS) in cardiac myocytes (19). NO, in turn, can interact with proteins involved in E-C coupling, and NO can be a positive or negative ionotropic agent (39, 40). Recent studies also indicate that both endothelial and neuronal NOS can be involved in the regulation of L-type Ca2+ channels via nitrosylation (28). From this literature, it is possible that NO-mediated signaling pathways play a role in downregulation of ICa in postburn hearts. However, further studies will be needed to validate this possibility.

The present study is the first to show that ICa density is reduced in myocytes from postburn hearts. In a previous biochemical study, Horton's group (2) reported no change in L-type Ca2+ channel expression at 24 h postburn cardiac tissue. However, this study did not measure protein levels of Ca2+ channel pore-forming, DHP receptor-{alpha}1 or its regulator of beta2 subunit (27) but measured an accessory subunit, {alpha}2 subunit. In our study, we used a DHP agonist, BAY K 8644, which increases Ca2+ channel opening (8, 34), and we found that the amplitude of ICa in the presence of the drug was significantly smaller compared with myocytes from sham burn hearts. The results are consistent with the idea that the number of functional L-type Ca2+ channel is decreased in postburn hearts.

In failing hearts, the hemodynamic alterations are associated with a common pattern of E-C coupling changes at the cellular levels. The changes occur with alterations in Ca2+ regulatory proteins, including sarcoplasmic(endo)reticulum Ca2+-ATPase (SERCA), phospholamban (PLB), Na+/Ca2+ exchanger, L-type Ca2+ channel {alpha}1 protein, and ryanodine receptors (5, 13, 17). An earlier report (2) from Horton's group showed decreased SERCA protein in postburn hearts; however, no data on the Ca2+-ATPase regulatory protein PLB has been reported. Since SR function is controlled by a relative ratio of SERCA2a and PLB (13), future studies that examine expression levels of Ca2+ regulatory proteins and cellular function simultaneously in postburn hearts will help to delineate which cellular processes become altered during the onset of burn-related cardiac dysfunction.


   GRANTS
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This research was supported by National Institutes of Health Grants HL-77480, GM-59841, and T32 GM-069330.


   ACKNOWLEDGMENTS
 
The authors thank K. Sano for technical help.


   FOOTNOTES
 

Address for reprint requests and other correspondence: A. Yatani, Dept. of Surgery, UMDNJ-New Jersey Medical School, 185 South Orange Ave., C-506, Newark, NJ 07103 (e-mail:yataniat{at}umdnj.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Adams HR, Baxter CR, Parker JL. Contractile function of heart muscle from burned guinea pigs. Circ Shock 9: 63–73, 1982.[ISI][Medline]
  2. Ballard-Croft C, Carlson D, Maass DL, Horton JW. Burn trauma alters calcium transporter protein expression in the heart. J Appl Physiol 97: 1470–1476, 2004.[Abstract/Free Full Text]
  3. Bassani JW, Yuan W, Bers DM. Fractional SR Ca release is regulated by trigger Ca and SR Ca content in cardiac myocytes. Am J Physiol Cell Physiol 268: C1313–C1319, 1995.[Abstract/Free Full Text]
  4. Baxter CR, Shires T. Physiological response to crystalloid resuscitation of severe burns. Ann NY Acad Sci 150: 874–894, 1968.[ISI][Medline]
  5. Bers DM. Calcium and cardiac rhythms: physiological and pathophysiological. Circ Res 90: 14–17, 2002.[Free Full Text]
  6. Bers DM. Cardiac excitation-contraction coupling. Nature 415: 198–205, 2002.[CrossRef][Medline]
  7. Bers DM. Excitation-Contraction Coupling and Cardiac Contractile Force. Dordrecht, The Netherlands: Kluwer Academic Publishers, 2001.
  8. Brown AM, Kunze DL, Yatani A. The agonist effect of dihydropyridines on Ca channels. Nature 311: 570–572, 1984.[CrossRef][Medline]
  9. Chen X, Piacentino V, 3rd Furukawa S, Goldman B, Margulies KB, Houser SR. L-type Ca2+ channel density and regulation are altered in failing human ventricular myocytes and recover after support with mechanical assist devices. Circ Res 91: 517–524, 2002.[Abstract/Free Full Text]
  10. Deitch EA. The management of burns. N Engl J Med 323: 1249–1253, 1990.[ISI][Medline]
  11. Deitch EA, Adams C, Lu Q, Xu DZ. A time course study of the protective effect of mesenteric lymph duct ligation on hemorrhagic shock-induced pulmonary injury and the toxic effects of lymph from shocked rats on endothelial cell monolayer permeability. Surgery 129: 39–47, 2001.[CrossRef][ISI][Medline]
  12. Eisner DA, Choi HS, Diaz ME, O'Neill SC, Trafford AW. Integrative analysis of calcium cycling in cardiac muscle. Circ Res 87: 1087–1094, 2000.[Abstract/Free Full Text]
  13. Hasenfuss G, Pieske B. Calcium cycling in congestive heart failure. J Mol Cell Cardiol 34: 951–969, 2002.[CrossRef][ISI][Medline]
  14. Horton JW. Left ventricular contractile dysfunction as a complication of thermal injury. Shock 22: 495–507, 2004.[ISI][Medline]
  15. Horton JW, White DJ, Maass D, Sanders B, Thompson M, Giroir B. Calcium antagonists improve cardiac mechanical performance after thermal trauma. J Surg Res 87: 39–50, 1999.[CrossRef][ISI][Medline]
  16. Houser SR, Margulies KB. Is depressed myocyte contractility centrally involved in heart failure? Circ Res 92: 350–358, 2003.[Abstract/Free Full Text]
  17. Houser SR, Piacentino V, 3rd, Weisser J. Abnormalities of calcium cycling in the hypertrophied and failing heart. J Mol Cell Cardiol 32: 1595–1607, 2000.[CrossRef][ISI][Medline]
  18. Jacob R, Gulch RW. The functional significance of ventricular geometry for the transition from hypertrophy to cardiac failure. Does a critical degree of structural dilatation exist? Basic Res Cardiol 93: 423–429, 1998.[CrossRef][ISI][Medline]
  19. Kelly RA, Smith TW. Cytokines and cardiac contractile function. Circulation 95: 778–781, 1997.[Free Full Text]
  20. Koshy US, Burton KP, Le TH, Horton JW. Altered ionic calcium and cell motion in ventricular myocytes after cutaneous thermal injury. J Surg Res 68: 133–138, 1997.[CrossRef][ISI][Medline]
  21. Maass DL, Hybki DP, White J, Horton JW. The time course of cardiac NF-kappaB activation and TNF-alpha secretion by cardiac myocytes after burn injury: contribution to burn-related cardiac contractile dysfunction. Shock 17: 293–299, 2002.[CrossRef][ISI][Medline]
  22. Maass DL, Naseem RH, Garry M, Horton JW. Echocardiography assessment of myocardial function after burn injury. Shock 25: 363–369, 2006.[CrossRef][ISI][Medline]
  23. Magnotti LJ, Upperman JS, Xu DZ, Lu Q, Deitch EA. Gut-derived mesenteric lymph but not portal blood increases endothelial cell permeability and promotes lung injury after hemorrhagic shock. Ann Surg 228: 518–527, 1998.[CrossRef][ISI][Medline]
  24. Murphy JT, Giroir B, Horton JW. Thermal injury alters myocardial sarcoplasmic reticulum calcium channel function. J Surg Res 82: 244–252, 1999.[CrossRef][ISI][Medline]
  25. Pieske B, Maier LS, Bers DM, Hasenfuss G. Ca2+ handling and sarcoplasmic reticulum Ca2+ content in isolated failing and nonfailing human myocardium. Circ Res 85: 38–46, 1999.[Abstract/Free Full Text]
  26. Sambol JT, White J, Horton JW, Deitch EA. Burn-induced impairment of cardiac contractile function is due to gut-derived factors transported in mesenteric lymph. Shock 18: 272–276, 2002.[CrossRef][ISI][Medline]
  27. Sather WA, McCleskey EW. Permeation and selectivity in calcium channels. Annu Rev Physiol 65: 133–159, 2003.[CrossRef][ISI][Medline]
  28. Sun J, Picht E, Ginsburg KS, Bers DM, Steenbergen C, Murphy E. Hypercontractile female hearts exhibit increased S-nitrosylation of the L-type Ca2+ channel {alpha}1 subunit and reduced ischemia/reperfusion injury. Circ Res 98: 403–411, 2006.[Abstract/Free Full Text]
  29. Swank GM, Deitch EA. Role of the gut in multiple organ failure: bacterial translocation and permeability changes. World J Surg 20: 411–417, 1996.[CrossRef][ISI][Medline]
  30. Trafford AW, Diaz ME, Eisner DA. Coordinated control of cell Ca2+ loading and triggered release from the sarcoplasmic reticulum underlies the rapid inotropic response to increased L-type Ca2+ current. Circ Res 88: 195–201, 2001.[Abstract/Free Full Text]
  31. Walker HL, Mason AD Jr. A standard animal burn. J Trauma 8: 1049–1051, 1968.[ISI][Medline]
  32. White DJ, Maass DL, Sanders B, Horton JW. Cardiomyocyte intracellular calcium and cardiac dysfunction after burn trauma. Crit Care Med 30: 14–22, 2002.[CrossRef][ISI][Medline]
  33. Yamamoto S, Kuntzweiler TA, Wallick ET, Sperelakis N, Yatani A. Amino acid substitutions in the rat Na+, K(+)-ATPase alpha 2-subunit alter the cation regulation of pump current expressed in HeLa cells. J Physiol 495: 733–742, 1996.[Abstract/Free Full Text]
  34. Yatani A, Codina J, Imoto Y, Reeves JP, Birnbaumer L, Brown AM. A G protein directly regulates mammalian cardiac calcium channels. Science 238: 1288–1292, 1987.[Abstract/Free Full Text]
  35. Yatani A, Frank K, Sako H, Kranias EG, Dorn GW, 2nd. Cardiac-specific overexpression of Galphaq alters excitation-contraction coupling in isolated cardiac myocytes. J Mol Cell Cardiol 31: 1327–1336, 1999.[CrossRef][ISI][Medline]
  36. Yatani A, Honda R, Tymitz KM, Lalli MJ, Molkentin JD. Enhanced Ca2+ channel currents in cardiac hypertrophy induced by activation of calcineurin-dependent pathway. J Mol Cell Cardiol 33: 249–259, 2001.[CrossRef][ISI][Medline]
  37. Yatani A, Xu DZ, Irie K, Sano K, Jidarian A, Vatner SF, Deitch EA. Dual effects of mesenteric lymph isolated from rats with burn injury on contractile function in rat ventricular myocytes. Am J Physiol Heart Circ Physiol 290: H778–H785, 2006.[Abstract/Free Full Text]
  38. Yatani A, Xu DZ, Kim SJ, Vatner SF, Deitch EA. Mesenteric lymph from rats with thermal injury prolongs the action potential and increases Ca2+ transient in rat ventricular myocytes. Shock 20: 458–464, 2003.[CrossRef][ISI][Medline]
  39. Ziolo MT, Katoh H, Bers DM. Positive and negative effects of nitric oxide on Ca2+ sparks: influence of beta-adrenergic stimulation. Am J Physiol Heart Circ Physiol 281: H2295–H2303, 2001.[Abstract/Free Full Text]
  40. Ziolo MT, Maier LS, Piacentino V, 3rd, Bossuyt J, Houser SR, Bers DM. Myocyte nitric oxide synthase 2 contributes to blunted beta-adrenergic response in failing human hearts by decreasing Ca2+ transients. Circulation 109: 1886–1891, 2004.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 J. Appl. Physiol.Home page
J. T. Sambol, M. A. Lee, F. J. Caputo, K. Kawai, C. Badami, T. Kawai, E. A. Deitch, and A. Yatani
Mesenteric lymph duct ligation prevents trauma/hemorrhage shock-induced cardiac contractile dysfunction
J Appl Physiol, January 1, 2009; 106(1): 57 - 65.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
292/5/H2475    most recent
01164.2006v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kawai, K.
Right arrow Articles by Yatani, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kawai, K.
Right arrow Articles by Yatani, A.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2007 by the American Physiological Society.