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Protection against endotoxemia-induced contractile dysfunction in mice with cardiac-specific expression of slow skeletal troponin I

Cardiovascular Division, King’s College London, London, UK; and
^* University of Illinois at Chicago, Chicago, Illinois, USA

¹ Correspondence: Department of Cardiology, GKT School of Medicine, Bessemer Rd., London SE5 9PJ, UK. E-mail: ajay.shah{at}kcl.ac.uk

SPECIFIC AIMS

Cardiac contractile dysfunction occurring in gram negative endotoxemia is associated with reduced myofilament Ca²⁺ responsiveness. An increased phosphorylation of cardiac troponin I (cTnI) at protein kinase A (PKA) -sensitive sites in the N-terminal, which would be predicted to reduce myofilament Ca²⁺sensitivity, has been reported in endotoxemia. To investigate the functional significance of cTnI phosphorylation in endotoxemia, we studied the cardiac response to systemic bacterial lipopolysaccharide (LPS) treatment in transgenic mice (TG) with cardiac-specific replacement of cTnI by slow skeletal TnI (ssTnI), which lacks the PKA-sensitive phosphorylation sites.

PRINCIPAL FINDINGS

1. Contractile function is impaired in isolated perfused hearts and isolated ventricular myocytes from wild-type CD1 mice treated with LPS, due mainly to reduction in myofilament Ca²⁺ responsiveness
A murine model of systemic endotoxemia was established in wild-type CD1 mice administered a single bolus of bacterial LPS (6 mg/kg, i.p.). After 16–18 h, there was significant systolic and diastolic dysfunction in isolated Langendorff-perfused hearts. The maximum rate of left ventricular pressure rise (dP/dt_max) decreased from 4881 ± 302 to 3167 ± 257 mmHg·s^–1 (P<0.05) and dP/dt_min decreased from –3462 ± 220 to –1833 ± 233 mmHg·s^–1 (P<0.05); end diastolic pressure also increased significantly (P<0.05). In isolated ventricular myocytes, unloaded sarcomere shortening was reduced from 6.1 ± 0.2% to 3.9 ± 0.2% (P<0.05) and time from peak twitch to 50% relaxation (RT₅₀) was prolonged from 64 ± 2 ms to 87 ± 4 ms (P<0.05, n>100 cells, 6 hearts/group). Since previous work has suggested that a reduction in myofilament Ca²⁺ responsiveness causes contractile dysfunction after LPS treatment, we studied the sarcomere length: Ca²⁺ relationship in Triton-skinned myocytes exposed to activating solutions containing varying free [Ca²⁺] up to 0.65 µM. LPS treatment was associated with a rightward shift of the sarcomere length: Ca²⁺ relationship, indicating a significant reduction in myofilament Ca²⁺ responsiveness.

2. LPS-induced contractile dysfunction is markedly reduced in cardiomyocytes from TG mice expressing ssTnI compared with myocytes from nontransgenic (NTG) littermates expressing cTnI, despite comparable intracellular Ca²⁺ transients
In the TG group, LPS treatment reduced myocyte shortening by only 13% from 7.5 ± 0.2% to 6.5 ± 0.2% (P<0.05, Fig. 1 A, D) with no significant prolongation of twitch RT₅₀ (Fig. 1F ); in the NTG mice, LPS treatment reduced myocyte shortening by 42% from 6.7 ± 0.2% to 3.9 ± 0.1% (P<0.05, Fig. 1A, D ) and prolonged RT₅₀ from 69 ± 3 ms to 104 ± 5 ms (P<0.05, Fig. 1F ). To investigate the contribution of changes in Ca²⁺ handling to contractile dysfunction, intracellular Ca²⁺ transients were assessed in cells loaded with indo-1 AM. The contractile effects of LPS treatment in NTG myocytes occurred with no significant changes in the amplitude or kinetics of intracellular Ca²⁺ transients (indo-1 410:480 ratio, Fig. 1B ). The protection against LPS-induced contractile dysfunction in TG could not be explained by differences in intracellular Ca²⁺ handling since Ca²⁺ transient amplitudes were not significantly different (indo-1 410:480 ratio amplitude was 0.19±0.01 for NTG myocytes and 0.18±0.01 for TG myocytes after LPS treatment; 3 hearts and 60 cells/group, Fig. 1B ). These results suggest that the major mechanism responsible for impaired contractile function after LPS treatment in the NTG group, as well as the preserved function in the TG group, was an alteration in myofilament properties rather than in intracellular Ca²⁺ transients.

View larger version (26K): [in this window] [in a new window]   Figure 1. A) Representative traces of percent sarcomere shortening (% SS) in myocytes from saline- or LPS-treated NTG and TG-hearts. B) Examples of intracellular Ca2+ transients (indo-1 410:480 ratio) measured in saline or LPS treated myocytes from NTG and TG hearts. C–H) Mean data comparing contractile parameters in saline-treated (open bars) and LPS-treated (solid bars) myocytes from NTG and TG hearts (>00 cells, 6 hearts/group): C) resting sarcomere length (RSL); D) % SS; E)time to peak twitch (Tpk); F)time from peak to 50% relaxation (RT50): G)maximum shortening velocity (SV); and H)maximum relengthening velocity (RV). *P < 0.05 for saline-treated compared with LPS within NTG or TG (ANOVA). #P < 0.05 for TG compared with NTG within saline- or LPS-treated groups (ANOVA).

3. Contractile response in isoproterenol-stimulated myocytes remains impaired in the LPS-treated NTG compared with the TG group, despite similar increases in Ca²⁺ transient amplitude
To investigate whether the above differences between NTG and TG myocytes from LPS-treated animals persisted in the presence of ß-adrenergic stimulation, we studied the contractile response to isoproterenol (Iso, 10 nM). During Iso stimulation, myocyte shortening in the NTG group remained lower in LPS-treated compared with saline-treated cells (10.7±0.6% vs. 12.6±0.5%; P<0.05; >35 cells, 3 hearts/group) despite similar Ca²⁺ transients (indo-1 410:480 ratio: 0.30±0.21 and 0.30±0.01 in saline and LPS group, respectively; P=NS; 60 cells, 3 hearts/group). In contrast, in the TG group there was no significant difference in myocyte shortening during Iso stimulation between saline (15.1±0.4%) and LPS-treated (14.4±0.5%) cells. Ca²⁺ transients in TG myocytes during Iso stimulation were not significantly different from those in NTG myocytes (indo-1 410:480 ratio: 0.31±0.01 and 0.28±0.01 in saline- and LPS-treated TG myocytes, respectively; >60 cells, 3 hearts/group). Thus, as with baseline contractility, the LPS-induced contractile dysfunction observed in NTG myocytes during Iso stimulation is unlikely to involve major changes in Ca²⁺ handling but is likely to result from changes in myofilament Ca²⁺ responsiveness.

4. LPS-induced reduction in myofilament Ca²⁺-responsiveness observed in NTG myocytes is significantly attenuated in TG myocytes
To more directly assess the effects of LPS treatment on myofilament Ca²⁺responsiveness, we generated sarcomere-length Ca²⁺ relationships using Triton-skinned myocytes from NTG and TG hearts. Myocytes from LPS-treated NTG mice showed a rightward shift of the sarcomere length-Ca²⁺ relationship compared with saline-treated NTG cells whether data were related to absolute sarcomere length, to percentage resting sarcomere length, or to matched resting sarcomere length. As previously observed, skinned myocytes from saline-treated TG mice expressing ssTnI showed an enhanced myofilament Ca²⁺-responsiveness compared with the NTG saline group. After LPS treatment, the rightward significant shift in the sarcomere length-Ca²⁺ relationship seen in the NTG group was markedly attenuated in TG myocytes.

CONCLUSIONS AND SIGNIFICANCE

This study provides the first definitive evidence that a cTnI-dependent reduction in myofilament Ca²⁺ responsiveness is a major causal mechanism underlying the intrinsic depression of myocyte contractile function that characterizes gram-negative endotoxemia. Prevention of cTnI phosphorylation at PKA-sensitive sites in the N-terminal by transgenic replacement of cTnI with ssTnI (which lacks these sites) provided significant protection against contractile dysfunction induced by in vivo endotoxemia. These results suggest that cTnI phosphorylation and/or myofilament Ca²⁺responsiveness may be potential therapeutic targets for the improvement of cardiac function in gram-negative endotoxemia (Fig. 2 ).

View larger version (23K): [in this window] [in a new window]   Figure 2. Potential pathways responsible for cardiomyocyte contractile dysfunction in endotoxemia. Gram negative bacterial infection triggers multiple inflammatory signaling cascades resulting in production of secondary mediators (such as cytokines) that affect multiple organs, including the heart. In the cardiac myocyte, this results in impairment of contractile function due, to a large extent, to reduction in myofilament Ca2+ responsiveness. The present study indicates that an increased phosphorylation (P) of cardiac troponin I (cTnI) in the N-terminal is likely to be a major mechanism underlying this reduction in myofilament Ca2+ responsiveness. The lack of significant change in intracellular Ca2+ transients suggests that pathways involved in Ca2+ cycling [e.g., sarcolemmal Ca2+ channels (Ica), the sarcoplasmic reticulum (SR), and the Na+-Ca2+ exchanger (NCX)] probably do not play a major role in the contractile dysfunction.

Septic shock occurring during severe gram-negative bacterial infection is characterized by hypotension and multiple organ dysfunction and remains a major cause of mortality. An intrinsic impairment of cardiac dysfunction is a major factor in the pathophysiology of septic shock but the underlying mechanisms remain to be fully elucidated. Impaired contractility may involve changes in Ca²⁺ handling (e.g., reduced intracellular Ca²⁺ transient or L-type Ca²⁺ current) and/or abnormalities of the myofilaments. In the present study, contractile dysfunction in isolated cardiomyocytes was attributable largely to a reduction in myofilament Ca²⁺ responsiveness consistent with previous results in other experimental models of sepsis. Alterations in cardiac myofilament properties in sepsis have been associated with changes in the phosphorylation status of regulatory proteins such as troponin-I, myosin binding protein-C, and/or myosin light chain-2. In previous studies in endotoxemic rats, we reported an increase in cTnI phosphorylation at serines 23/24, which was associated with a reduction in myofilament Ca²⁺responsiveness. Phosphorylation of cTnI at serines 23/24 is a well-established mechanism for reduced myofilament Ca²⁺-sensitivity after ß-adrenergic stimulation and PKA activation. In the present study, we used transgenic mice with cardiac-specific stoichiometric replacement of cTnI by ssTnI, which lacks the N-terminal residues of cTnI that are the targets for PKA-mediated reduction in myofilament Ca²⁺ sensitivity, to investigate the functional significance of increased cTnI phosphorylation during endotoxemia. Replacement of cTnI with ssTnI in this transgenic mouse abolishes the effects of ß-adrenergic stimulation on myofilament Ca²⁺ sensitivity and cross-bridge cycling kinetics due to loss of PKA phosphorylation sites in the N-terminal. We found that ssTnI TG mice were significantly protected against LPS-induced contractile dysfunction assessed in isolated cardiomyocytes (Fig. 1) . Since there were no significant differences in Ca²⁺ transient amplitude between NTG and TG cells from LPS-treated mice despite the greater shortening in the latter group, the protection observed in the TG cells is highly likely to be due to altered myofilament properties. Consistent with this, in skinned myocytes, the rightward shift (reduced Ca²⁺ sensitivity) in sarcomere shortening-Ca²⁺ relationships that occurred in LPS-treated NTG mice was greatly reduced in the TG group. The small residual decrease in cell shortening (13%, Fig. 1D ) observed after LPS treatment in TG mice suggests that there are probably additional mechanisms that contribute to contractile dysfunction.

Experimental endotoxemia is associated with diastolic as well as systolic dysfunction as illustrated by prolonged relaxation in isolated myocytes (Fig. 1A, F ). Surprisingly, this prolongation of myocyte relaxation was not associated with a reduced rate of Ca²⁺ transient decline, suggesting that it may not result entirely from defects in Ca²⁺ removal from the cytoplasm but may involve changes in myofilament properties. Myocytes from LPS-treated ssTnI mice were protected not only against systolic dysfunction but also the impaired relaxation (Fig. 1F ). The myofilament-based mechanisms responsible for these findings in respect to relaxation require further investigation since cTnI phosphorylation at PKA-sensitive sites would normally be expected to cause acceleration (rather than delay) of relaxation.

Results of the present study strongly suggest that a cTnI-dependent reduction in myofilament Ca²⁺responsiveness is a major mechanism underlying the intrinsic depression of myocyte contractile function that characterizes gram-negative endotoxemia. Replacement of cTnI with ssTnI, which lacks PKA-sensitive phosphorylation sites that mediate reduction in myofilament Ca²⁺ sensitivity, provides substantial protection against endotoxemia-induced contractile dysfunction. Although the upstream-signaling pathways resulting in the cTnI-based reduction in myofilament Ca²⁺responsiveness in endotoxemia remain to be determined, the current results suggest that cTnI phosphorylation and myofilament Ca²⁺ responsiveness may be potential therapeutic targets for the improvement of cardiac function in gram negative sepsis.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-2519fje; doi: 10.1096/fj.04-2519fje

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