Protein phosphatase 2A contributes to the cardiac dysfunction induced by endotoxemia.

AIMS: Sepsis-associated cardiac dysfunction represents an intrinsic impairment of cardiomyocyte function due in part to a decrease in myofilament Ca(2+) sensitivity associated with a sustained increase in cardiac troponin I (cTnI) phosphorylation at Ser23/24. Dephosphorylation of cTnI is under regulatory control. Thus, muscarinic and adenosine A(1)-receptor agonists antagonize beta-adrenergic stimulation via activation of protein phosphatase 2A (PP2A). The aim of this study was to determine whether modulation of PP2A and thus cTnI phosphorylation could improve sepsis-induced contractile dysfunction. METHODS AND RESULTS: Cardiomyocytes were isolated from control or septic mice 16-18 h after an injection of vehicle or lipopolysaccharide (LPS; 9 mg/kg ip) respectively. Protein expression and phosphatase activity were determined in homogenates of control and septic hearts. Our data showed that LPS significantly increased cTnI phosphorylation at Ser23/24 in cardiomyocytes and reduced contraction amplitude without affecting Ca(2+)-transients. Treatment of cardiomyocytes with the A(1) agonist cyclopentyladenosine (CPA) or the protein kinase A inhibitor H89 significantly attenuated the LPS-induced contractile dysfunction without effect on Ca(2+)-transients. Co-treatment with CPA and H89 completely reversed the contractile dysfunction. Increased cTnI phosphorylation in septic hearts was associated with a significant reduction in the protein expression of both the catalytic and regulatory subunits (B56 alpha) of PP2A and a decrease in PP2A activity. CPA treatment of septic hearts increased PP2A activity. An increase in the protein expression of demethylated PP2A and a decrease in the PP2A-methyltransferase (PPMT; the methyltransferase that catalyses this reaction) were also observed. CONCLUSION: These data support the hypothesis that sustained cTnI phosphorylation underlies the contractile dysfunction seen in sepsis.

Activation of p38 mitogen-activated protein kinase contributes to the early cardiodepressant action of tumor necrosis factor.

OBJECTIVES: The purpose of this study was to determine whether p38 mitogen-activated protein kinase (p38-MAPK) contributes to tumor necrosis factor-alpha (TNFalpha)-induced contractile depression. BACKGROUND: Tumor necrosis factor has both beneficial and detrimental consequences that may result from the activation of different downstream pathways. Tumor necrosis factor activates p38-MAPK, a stress-responsive kinase implicated in contractile depression and cardiac injury. METHODS: In isolated hearts from mice lacking the p38-MAPK activator, MAPK kinase 3 (MKK3), perfused at constant coronary pressure or flow, we measured the left ventricular developed pressure (LVDP) and the relationship between end-diastolic volume and LVDP in the presence and absence of 10 ng/ml TNFalpha. RESULTS: Within 15 min at constant pressure, TNFalpha significantly reduced LVDP and coronary flow in outbred and mkk3(+/+) mice. This early negative inotropic effect was associated with a marked phosphorylation of both p38-MAPK and its indirect substrate, HSP27. In hearts lacking MKK3, TNFalpha failed to activate p38-MAPK or to cause significant contractile dysfunction. The actions of TNFalpha were similarly attenuated in MAPK-activated protein kinase 2 (MK2)-deficient hearts, which have a marked reduction in myocardial p38-MAPK protein content, and by the p38-MAPK catalytic site inhibitor SB203580 (1 micromol/l). Under conditions of constant coronary flow, the p38-MAPK activation and contractile depression induced by TNFalpha, though attenuated, remained sensitive to the absence of MKK3 or the presence of SB203580. The role of p38-MAPK in TNFalpha-induced contractile depression was confirmed in isolated murine cardiac myocytes exposed to SB203580 or lacking MKK3. CONCLUSIONS: Tumor necrosis factor activates p38-MAPK in the intact heart and in isolated cardiac myocytes through MKK3. This activation likely contributes to the early cardiodepressant action of TNFalpha.

Involvement of the nicotinamide adenosine dinucleotide phosphate oxidase isoform Nox2 in cardiac contractile dysfunction occurring in response to pressure overload.

OBJECTIVES: This study sought to examine the role of Nox2 in the contractile dysfunction associated with pressure-overload left ventricular hypertrophy (LVH). BACKGROUND: Reactive oxygen species (ROS) production is implicated in the pathophysiology of LVH. The nicotinamide adenosine dinucleotide phosphate oxidase isoform, Nox2, is pivotally involved in angiotensin II-induced hypertrophy but is not essential for development of pressure-overload LVH. Its possible impact on contractile function is unknown. METHODS: The effects of aortic banding or sham surgery on cardiac contractile function and interstitial fibrosis were compared in adult Nox2-/- and matched wild-type (WT) mice. RESULTS: Banding induced similar increases in left ventricular (LV) mass in both groups. Banded Nox2-/- mice had better LV function than WT by echocardiography (e.g., fractional shortening 33.6 +/- 2.5% vs. 21.4 +/- 2.2%, p < 0.05). Comprehensive LV pressure-volume analyses also showed significant contractile dysfunction in banded WT compared with sham, whereas banded Nox2-/- mice had preserved function (e.g., maximum rate of rise of LV pressure: banded WT, 4,879 +/- 213; vs. banded Nox2-/-, 5,913 +/- 259 mm Hg/s; p < 0.05). Similar preservation of function was observed in isolated cardiomyocytes. The 24-h to 36-h treatment of banded WT mice with N-acetylcysteine resulted in recovery of contractile function. Cardiac interstitial fibrosis was significantly increased in banded WT but not Nox2-/- mice, together with greater increases in procollagen I and III mRNA expression. CONCLUSIONS: The Nox2 oxidase contributes to the development of cardiac contractile dysfunction and interstitial fibrosis during pressure overload, although it is not essential for development of morphologic hypertrophy per se. These data suggest divergent downstream effects of Nox2 on different components of the overall response to pressure overload.

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

Gram negative endotoxemia is associated with an intrinsic impairment of cardiomyocyte contraction, in part due to a reduction in myofilament Ca2+ responsiveness. Endotoxemic rat hearts show increased cardiac troponin I (cTnI) phosphorylation at serines 23 and 24, residues required for the protein kinase A (PKA)-dependent reduction of myofilament Ca2+ sensitivity after beta-adrenoceptor stimulation. To investigate the functional significance of increased TnI phosphorylation in endotoxemia, we studied the contractile effects of systemic bacterial lipopolysaccharide (LPS) treatment in transgenic mice (TG) with cardiac-specific replacement of cTnI by slow skeletal TnI (ssTnI, which lacks the PKA phosphorylation sites) and matched nontransgenic littermates (NTG) on a CD1 background. In wild-type CD1 mice treated with LPS (6 mg/kg ip), after 16-18 h there was a significant reduction in the maximum rates of left ventricular pressure development and pressure decline in isolated Langendorff-perfused hearts compared with saline-treated controls and a decrease in isolated myocyte unloaded sarcomere shortening from 6.1 +/- 0.2 to 3.9 +/- 0.2% (1 Hz, 32 degrees C, P<0.05). Similarly, in NTG myocytes, endotoxemia reduced myocyte shortening by 42% from 6.7 +/- 0.2 to 3.9 +/- 0.1% (P<0.05) with no change in intracellular Ca2+ transients. However, in the TG group, LPS reduced myocyte shortening by only 13% from 7.5 +/- 0.2 to 6.5 +/- 0.2% (P<0.05). LPS treatment significantly reduced the positive inotropic effect of isoproterenol in NTG myocytes but not in TG myocytes, even though isoproterenol-induced increases in Ca2+ transient amplitude were similar in both groups. Only LPS-treated NTG hearts showed a significant increase in cTnI phosphorylation. Investigation of the sarcomere shortening-Ca2+ relationship in Triton-skinned cardiomyocytes revealed a significant reduction in myofilament Ca2+ sensitivity after LPS treatment in NTG myocytes, an effect that was substantially attenuated in TG myocytes. In conclusion, the replacement of cTnI with ssTnI in the heart provides significant protection against endotoxemia-induced cardiac contractile dysfunction, most probably by preserving myofilament Ca2+ responsiveness due to prevention of phosphorylation of TnI at PKA-sensitive sites.

Regulation of cardiac contractile function by troponin I phosphorylation.

Cardiac troponin I (cTnI) is a key regulatory protein in cardiac muscle contraction and relaxation, linking Ca(2+)-troponin C binding with activation of crossbridge reactions with the thin filament. In recent years, it has become increasingly apparent that myofilament properties as well as changes in intracellular Ca(2+) have a major role in the dynamic modulation of contractile function. The phosphorylation of specific serine and threonine residues on cTnI by several different kinases represents a major physiological mechanism for alteration of myofilament properties. Furthermore, altered thin filament function plays an important role in the contractile dysfunction associated with heart failure. Modification of cTnI by protein kinases A and C has been extensively studied with especially useful information deriving from (a) in vitro studies in reconstituted detergent-skinned fibre bundles in which endogenous cTnI was replaced with various targeted cTnI mutants and (b) transgenic animals in which endogenous cTnI was similarly manipulated through overexpression of cardiomyocyte-targeted cTnI mutants. cTnI may also be specifically modified by protein kinase G, p21-activated kinases and by dephosphorylation. This review focuses on recent advances in understanding the mechanisms of cTnI modification by these kinases and the consequent functional effects both under physiological conditions and in pathophysiological settings.

Analysis of ex vivo left ventricular pressure-volume relations in the isolated murine ejecting heart.

The development of microconductance technology to study cardiac pressure-volume relations in mice in vivo has significantly advanced the haemodynamic assessment of gene-modified models of cardiovascular disease. In this study, we describe the application of microconductance analysis of cardiac function to the isolated murine ejecting heart. This ex vivo model is complementary to the previously described in vivo preparation, allows assessment without confounding effects of anaesthetic or neurohumoral influences and enables careful control of cardiac loading (particularly preload). Ex vivo pressure-volume relations in the isolated murine heart are sensitive to changes in myocardial contractility induced by beta-adrenoceptor stimulation or beta-adrenoceptor blockade, as well as the effects of chronic pressure overload induced by aortic banding. We present data for both steady-state analyses of the Frank-Starling relation and for assessment of the left ventricular pressure-volume relation over variably loaded beats, which allows investigation of the end-systolic and end-diastolic pressure-volume relations. The measurement of ventricular volume in addition to pressure under carefully controlled loading conditions in the isolated ejecting heart allows a comprehensive analysis of cardiac contractile function, and provides a useful complementary model for the assessment of cardiac performance in murine models of heart disease.

Essential role of troponin I in the positive inotropic response to isoprenaline in mouse hearts contracting auxotonically.

PKA-dependent phosphorylation of cardiac troponin I (cTnI) contributes significantly to beta-adrenergic agonist-induced acceleration of myocardial relaxation (lusitropy). However, the role of PKA-dependent cTnI phosphorylation in the positive inotropic response to beta-adrenergic stimulation is unclear. We studied the contractile response to isoprenaline (10 nm) in isolated hearts and isolated cardiomyocytes from transgenic mice with cardiac-specific expression of slow skeletal TnI (ssTnI, which lacks the N-terminal protein extension containing PKA-sensitive phosphorylation sites in cTnI) and matched wild-type littermate controls. As expected, the lusitropic effect of isoprenaline was significantly blunted in ssTnI hearts. However, the positive inotropic response to isoprenaline was also blunted in ssTnI hearts. This effect was especially prominent for ejection-phase indices in isolated auxotonically loaded ssTnI hearts whereas the positive inotropic response of isovolumic hearts or unloaded isolated myocytes was much less affected. Isoprenaline decreased left ventricular end-systolic volume in wild-type hearts (10.6 +/- 1.6 to 6.2 +/- 0.4 microl at a preload of 20 cmH(2)O; P < 0.05) but not transgenic hearts (11.4 +/- 1.3 to 10.9 +/- 1.3 microl; P= n.s.). Likewise, isoprenaline increased stroke work in control hearts (14.5 +/- 1.0 to 22.5 +/- 1.8 mmHg microl mg(-1); P < 0.05) but not transgenic hearts (15.4 +/- 1.3 to 18.3 +/- 1.2 mmHg microl mg(-1); P= n.s.). The end-systolic pressure-volume relation was increased by isoprenaline to a greater extent in control than transgenic hearts. However, isoprenaline induced a similar rise in intracellular Ca(2+) transients in transgenic and non-transgenic cardiomyocytes. These results indicate that cTnI has a pivotal role in the positive inotropic response of the murine heart to beta-adrenergic stimulation, an effect that is highly dependent on loading conditions and is most evident in the auxotonically loaded ejecting heart.

Myofilament-based relaxant effect of isoprenaline revealed during work-loop contractions in rat cardiac trabeculae.

In cardiac muscle, beta-adrenergic stimulation increases contractile force and accelerates relaxation. The relaxant effect is thought to be due primarily to stimulation of Ca(2+) uptake into the sarcoplasmic reticulum (SR), although changes in myofilament properties may also contribute. The present study investigated the contribution of the myofilaments to the beta-adrenergic response in isolated rat cardiac trabeculae undergoing either isometric or work-loop contractions (involving simultaneous force generation and shortening) at different stimulation frequencies (range 0.25-4.5 Hz). SR-dependent effects were eliminated by treatment with ryanodine (1 microM) and cyclopiazonic acid (30 microM). In isometric contractions during SR inhibition, isoprenaline increased the force but did not alter the time course of the twitch. In contrast, in work-loop contractions, the positive inotropic effect was accompanied by a reduced diastolic force between beats, most apparent at higher frequencies (e.g. diastolic stress fell from 58.6 +/- 5.5 to 28.8 +/- 5.8 mN mm(-2) at 1.5 Hz). This relaxant effect contributed to a beta-adrenoceptor-mediated increase in net work and power output at higher frequencies, by reducing the amount of work required to re-lengthen the muscle. Consequently, the frequency for maximum power output increased from 1.1 +/- 0.1 to 1.6 +/- 0.1 Hz. We conclude that the contribution of myofilament properties to the relaxant effect of beta-stimulation may be of greater significance when force and length are changing simultaneously (as occurs in the heart) than during force development under isometric conditions.

Role of cyclic GMP-dependent protein kinase in the contractile response to exogenous nitric oxide in rat cardiac myocytes.

Nitric oxide (NO) can directly modulate cardiac contractility by accelerating relaxation and reducing diastolic tone. The intracellular mechanisms underlying these contractile effects are poorly understood. Here we investigate the role of cyclic GMP-dependent protein kinase (PKG) in the contractile response to exogenous NO in rat ventricular myocytes. Isolated ventricular myocytes were stimulated electrically and contractility was assessed by measuring cell shortening. Some cells were loaded with the fluorescent Ca(2+) probe indo-1 AM for simultaneous assessment of the intracellular Ca(2+) transient. The NO donor diethylamine NONOate (DEA/NO, 10 microM) significantly increased resting cell length, reduced twitch amplitude and accelerated time to 50 % relaxation (to 100.8 +/- 0.2, 83.7 +/- 3.0 and 88.9 +/- 3.7 % of control values, respectively). The contractile effects of DEA/NO occurred without significant changes in the amplitude or kinetics of the intracellular Ca(2+) transient, suggesting that the myofilament response to Ca(2+) was reduced. These effects were abolished by inhibition of either guanylyl cyclase (with 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one; ODQ, 10 microM) or PKG (with Rp-8-Br-cGMPs, 10 microM) suggesting that, at the concentration investigated, the effects of DEA/NO were mediated exclusively by PKG, following activation of guanylyl cyclase and elevation of cGMP. Direct activation of PKG with 8-pCPT-cGMP (10 microM) mimicked the effects of DEA/NO (resting cell length and time to 50 % relaxation were 100.6 +/- 0.1 and 90.5 +/- 1.5 % of control values, respectively).The reduced myofilament Ca(2+) responsiveness was not attributable to an intracellular acidosis since the small reduction in pH(i) induced by DEA/NO was found to be uncoupled from its contractile effects. However, hearts treated with DEA/NO (10 microM) showed a significant increase (1.4-fold; P < 0.01) in troponin I phosphorylation compared to control, untreated hearts. These results suggest that the reduction in myofilament Ca(2+) responsiveness produced by DEA/NO results from phosphorylation of troponin I by PKG.