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Cardiac contractile impairment associated with increased phosphorylation of troponin I in endotoxemic rats

BENOIT TAVERNIER^*,, JIAN-MEI LI^*,3, MAGDI M. EL-OMAR^,3, SOPHIE LANONE, ZHAO-KANG YANG, IAN P. TRAYER^§, ALEXANDRE MEBAZAA and AJAY M. SHAH^*

^* Department of Cardiology, Guy’s King’s and St. Thomas’s School of Medicine, King’s College London, U.K.;
Department of Anesthesiology, Lariboisière Hospital, Paris, France;
Department of Cardiology, University of Wales College of Medicine, Cardiff, U.K.; and
^§ School of Biosciences, University of Birmingham, U.K.

²Correspondence: Professor A.M. Shah, Department of Cardiology, GKT School of Medicine, King’s College London (Denmark Hill Campus), Bessemer Road, London SE5 9PJ, U.K. E-mail: ajay.shah{at}kcl.ac.uk

SPECIFIC AIMS

Thisstudy aims to investigate the subcellular mechanisms responsible for intrinsic cardiac myocyte dysfunction in systemic sepsis in vivo, in particular the relative contribution of alterations in cytosolic Ca²⁺ homeostasis versus changes in myofilament responsiveness to Ca²⁺. We studied contractile function, intracellular Ca²⁺, intracellular pH, and myofilament properties in ventricular cardiac myocytes isolated from rats that had been subjected to lipopolysaccharide (LPS)-induced systemic sepsis in the conscious state.

PRINCIPAL FINDINGS

1. Reduced twitch contraction of myocytes from LPS-treated rats is attributable primarily to alteration in myofilament properties
Myocytes were isolated from rats 12 h after LPS (or saline) injection, at which stage LPS-treated animals manifested pyrexia, modest weight loss, and hypotension. Cell shortening was significantly lower in LPS compared with control cells (4.1±0.2% versus 7.8±0.3% ; P<0.001 ; 0.5 Hz), whereas peak [Ca²⁺]_i was similar (indo-1 fluorescence transient 0.16±0.01 in both groups). At higher stimulation frequency (to 2.5 Hz), there was a modest reduction in peak indo-1 fluorescence ratio in the LPS group, but cell shortening was considerably lower than could be accounted for by this change. These data suggested that the major component of contractile dysfunction during sepsis was attributable not to altered intracellular [Ca²⁺]_i but rather to a change in myofilament properties.

2. Steady-state myofilament response to Ca²⁺ is reduced in intact cardiac myocytes from septic rats
A repetitive electrical tetanization technique was used to assess ‘steady-state’ myofilament response to Ca²⁺ in single myocytes with intact sarcolemmal membranes and intact subcellular signal transduction pathways. Cells were treated with thapsigargin (0.4 µmol/L, 10-15 min) to disable the sarcoplasmic reticulum Ca²⁺ATPase and were then repetitively tetanized (10 Hz for 10–20 s) at extracellular Ca²⁺ from 0.31–3.75 mmol/L. The relationship between the peak tetanic Ca²⁺ transient and peak tetanic shortening was shifted rightwards—that is, towards higher [Ca²⁺]_{i ,} in septic myocytes compared with control cells—which confirms a significant reduction in myofilament response to Ca²⁺ (Fig. 1 ).

View larger version (18K): [in this window] [in a new window]   Figure 1. Relationship between peak indo-1 fluorescence ratio transient and peak cell shortening (% diastolic cell length) during tetanic contraction of intact single cardiac myocytes. Each point represents the mean±SEM response for the myocytes studied in each group; the extracellular [Ca2+] is indicated next to each point, and the values in parentheses are numbers of cells studied. The control group comprised 41 myocytes isolated from 7 control rats, and the septic group 30 myocytes isolated from 6 endotoxemic animals. *, P<0.001 by comparing curve intercepts.

3. Mechanism of reduction in myofilament Ca²⁺ response: Increased troponin I phosphorylation but no change in cytosolic pH
Two major mechanisms that reduce cardiac myofilament Ca²⁺ response are cytosolic acidification and an increase in phosphorylation of troponin I. Intracellular pH assessed with the fluorescence probe SNARF-1 was similar in septic and control myocytes (fluorescence ratio 4.00±0.08 versus 4.21±0.12 ; P not significant). To assess troponin I phosphorylation, we used phosphorylation-independent and phosphorylation-specific anti-cardiac troponin I monoclonal antibodies (mAb 19 and mAb 14, respectively). mAb 14 detects troponin I that has been phosphorylated at a specific cAMP-dependent protein kinase (PKA) site in the N-terminal. Although the total amount of myocardial troponin I detected with mAb 19 was similar in both groups, the amount of phosphorylated troponin I was significantly (2-fold) higher in the LPS group (Fig. 2 ). As a positive control, treatment of normal hearts with isoproterenol also increased troponin I phosphorylation.

View larger version (37K): [in this window] [in a new window]   Figure 2. Level of troponin I phosphorylation in left ventricular myocardium from control and LPS-treated animals. A) Western blot probed with mAb 19, which is phosphorylation-independent. B) Western blots probed with phosphorylation-specific mAb 14; right panel shows positive control experiment in which two hearts were perfused with isoproterenol (3 nmol/L) and 2 with buffer solution alone for 3 min each, prior to freeze-clamping and isolation of protein.

4. Possible role of the ß-adrenergic/cAMP pathway or the nitric oxide/cGMP pathway in reduced myofilament response to Ca²⁺
Because the major physiological pathway for troponin I phosphorylation is via PKA activation secondary to ß-adrenoceptor stimulation, we tested the response of isolated myocytes to the ß-agonist, isoproterenol, at a concentration (3 nmol/L) that was 50% maximally effective in both groups. Cell-twitch shortening and intracellular Ca²⁺ transients were increased to a similar extent by isoproterenol in control and LPS groups. The effect of isoproterenol on myofilament Ca²⁺ response was tested in intact myocytes during tetanization at varying [Ca²⁺]o. In control cells, isoproterenol shifted the tetanic [Ca²⁺]_i-shortening relation rightwards, consistent with a reduced myofilament Ca²⁺ sensitivity secondary to troponin I phosphorylation. In septic myocytes, however, where the tetanic [Ca²⁺]_i-shortening relationship was already rightward shifted, there was minimal further shift with isoproterenol. Thus, the myofilament desensitizing effect of isoproterenol was impaired selectively in septic myocytes, despite preservation of other isoproterenol-induced effects such as an increase in intracellular Ca²⁺.

A suggested alternative pathway for phosphorylation of troponin I is via activation of cGMP-dependent protein kinase (PKG), secondary to nitric oxide (NO)-stimulated increases in cGMP. In sepsis, a high-output NO synthase isoform (iNOS) is induced, which may contribute to myocardial dysfunction. Both iNOS protein and Ca²⁺-independent biochemical NO synthase activity were increased in the myocardium of LPS-treated rats compared with control rats. However, treatment of isolated septic cardiac myocytes with either of two different NOS inhibitors, L-NMMA or L-NAME, had no effect on cell shortening or indo-1 fluorescence ratio. These NOS inhibitors also failed to improve contractile function in isolated perfused hearts from LPS-treated animals.

CONCLUSION

Systemic sepsis syndrome is a multi-organ disorder characterized by hypotension, vascular hyporeactivity, and cardiac dysfunction. Myocardial contractile impairment is a major determinant of morbidity and mortality in the condition. The underlying mechanisms of cardiac impairment remain unclear, but a major component of the defect is intrinsic to the cardiac myocyte. Previous in vitro studies, in which isolated tissues were exposed to LPS or selected cytokines, suggested variable mechanisms for the contractile dysfunction, including alterations in cytosolic Ca²⁺ homeostasis or changes in myofilament Ca²⁺ response. Such studies do not, however, reproduce the complex patterns of cytokine activation and other abnormalities found in systemic sepsis in vivo. In the present investigation, we studied an experimental model of systemic endotoxemia that more closely resembles the analogous clinical situation, and studied myocardial tissues ex vivo in order to assess intrinsic contractile dysfunction. By measuring both twitch and tetanic contraction in intact indo-1-loaded cells with preserved sarcolemmal membranes and subcellular signaling pathways (i.e., non-‘skinned’ preparations), relative role of altered [Ca²⁺]_i transients versus altered myofilament Ca²⁺ response could be reliably assessed in the same cell population. The results a) demonstrate that the intrinsic myocardial depression that occurs with in vivo LPS-induced sepsis in conscious rats is attributable primarily to a decrease in myofilament Ca²⁺ responsiveness, and b) suggest for the first time that this defect may result from an increased level of troponin I phosphorylation.

The troponin complex is centrally involved in Ca²⁺-dependent regulation of the cardiac contractility. An important mechanism that exerts fine control over this process is PKA-dependent phosphorylation of troponin I, resulting in a reduced ability of Ca²⁺ to activate the myofilaments. Using mAb 14, which specifically recognizes troponin I phosphorylated at the PKA-sensitive Ser24 in the N-terminal, we found a significant twofold increase in troponin I phosphorylation in septic myocardium. This result, with the similar effect obtained with isoproterenol treatment of control hearts (which reduces myofilament Ca²⁺ sensitivity via PKA activation), suggests that increased troponin I phosphorylation may significantly contribute to the reduced myofilament Ca²⁺ response of septic myocytes. PKA-dependent troponin I phosphorylation can involve either Ser23 or Ser24, or both sites. Our results do not establish the precise state of troponin I phosphorylation in septic myocardium, but they do indicate that Ser 24, at least, is phosphorylated to a greater extent. Alterations in troponin I function recently have been implicated in the pathophysiology of several conditions. Decreased troponin I phosphorylation reportedly accounts for increased myofilament Ca²⁺ sensitivity in failing human myocardium. Proteolysis of troponin I, resulting in reduced myofilament Ca²⁺ sensitivity, is postulated to account for the reversible contractile dysfunction of myocardial stunning. No evidence of troponin I proteolysis was found in the present study, and total troponin I levels were similar in both groups.

The precise reasons for increased troponin I phosphorylation in septic myocardium require further study. An obvious possibility is an increase in PKA-dependent phosphorylation associated with ß-adrenergic activation (Fig. 3 ). Consistent with this theory, the myofilament desensitizing effect of isoproterenol was markedly blunted in septic cells. This was not attributable to a reduction in ß-adrenergic responsiveness per se, since isoproterenol-induced increases in [Ca²⁺]_i transient were well-preserved both during twitch and tetanic contraction. Thus, if the increased troponin I phosphorylation is related to PKA-mediated effects, our results imply a selective effect on the myofilaments without concomitant increase in phosphorylation of other cellular targets such as L-type Ca²⁺ channels. Such a selective action could also account for the fact that overall contractile function was impaired in the LPS-treated group, whereas generalized PKA activation results in positive inotropic effects, because PKA-dependent increases in Ca²⁺ transient normally override the reduction in myofilament response to Ca²⁺.

View larger version (26K): [in this window] [in a new window]   Figure 3. Schematic diagram of abnormalities underlying cardiac myocyte contractile dysfunction in systemic sepsis. The [Ca2+]i transient, and therefore the processes involved in its generation are minimally altered, whereas myofilament Ca2+ responsiveness is depressed due to increased troponin I (TnI) phosphorylation at a PKA-dependent site(s).

The present results support a hitherto unrecognized mechanism for cardiac contractile dysfunction in systemic sepsis, namely increased troponin I phosphorylation, and may have therapeutic implications. It is feasible that Ca²⁺-sensitizing agents might prove efficacious for augmenting cardiac function.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0433fje To cite this article, use (December 8, 2000) FASEB J. 10.1096/fj.00-0433fje

³ These authors contributed equally to this work.

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