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Cardiovascular Research 2003 57(1):5-7; doi:10.1016/S0008-6363(02)00743-5
© 2003 by European Society of Cardiology
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Copyright © 2003, European Society of Cardiology

Altered phosphorylation and Ca2+-sensitivity of myofilaments in human heart failure

Wolfgang Schillinger* and Harald Kögler

Herzzentrum Göttingen, Kardiologie und Pneumologie, Georg-August Universität Göttingen, Robert-Koch-Strasse 40, 37075 Göttingen, Germany

* Corresponding author. Tel.: +49-551-396-351; fax: +49-551-399-804. schiwolf{at}med.uni-goettingen.de

Received 21 October 2002; accepted 21 October 2002

See article by Van der Velden et al. [18] (pages 37–47) in this issue.

β-Adrenergic receptor stimulation in the heart induces a plethora of physiological and biochemical reactions that serve to elevate cardiac output in situations of increased energy demand such as strong muscular exercise, hypoxia, pain, severe emotional stress, or hypoglycemia. The signal transduction cascade after binding of the ligand to the β1-adrenergic receptor involves activation of the stimulatory G-protein (Gs) and of membrane-bound adenylyl cyclase, followed by formation of the second messenger cyclic 3',5'-adenosine monophosphate (cAMP). Phosphorylation of several intracellular target proteins by cAMP-dependent protein kinase (PKA) finally mediates the various consequences of β-adrenergic receptor stimulation on the heart.

In order to elevate cardiac output, myocyte contractility must be enhanced, which can be accomplished by two principal mechanisms: Either by increasing the availability of [Ca2+]i to activate the myofilaments during systole or by increasing the Ca2+ sensitivity of the myofilaments. The major mechanism mediating the positive inotropic response to β-adrenergic receptor stimulation acts via cAMP-dependent phosphorylation of the small regulatory protein, phospholamban (PLN), which is located in the membrane of the sarcoplasmic reticulum (SR) and in its non-phosphorylated state inhibits the Ca2+ uptake activity of the sarco-endoplasmic reticulum Ca2+-ATPase (SERCA). Phosphorylation of PLN, however, relieves the inhibitory action by enhancing the Ca2+ affinity of SERCA [17], thereby boosting Ca2+ uptake into the SR during cardiac diastole. Thus, less Ca2+ ions are extruded to the extracellular space via the sarcolemmal Na+–Ca2+ exchanger and the myocyte experiences a net Ca2+ gain, which triggers a substantial positive inotropic response. However, after β-adrenergic receptor stimulation the resulting increase in force was found to be less than should have been expected from the elevation of [Ca2+]i which was simultaneously measured [8], indicating that under these conditions the contractile apparatus was desensitized to activation by Ca2+. Ray and England [14] reported that treatment of bovine myocardium with PKA caused phosphate incorporation into the thin filament regulatory protein troponin-I (TnI), which was associated with an increase in the concentration of calcium required for activation of the actomyosin Mg-ATPase. cAMP-dependent phosphorylation of TnI in skinned cardiac muscle fibers reduced isometric tension at submaximally activating calcium concentrations, while at maximum activation force was unchanged [7]. In cardiac TnI from different species, two adjacent phosphorylatable serine residues are present [9] in the cardiac-specific amino-terminal extension that is lacking in the fast and slow skeletal isoforms of TnI [19]. In the myocardium, the dephosphorylated, monophosphorylated, and bisphosphorylated forms of TnI co-exist, their respective fractions of total TnI depending on the level of β1-adrenergic activation. Both serines (Ser23 and Ser24 in human cardiac muscle) must become phosphorylated for myofilament Ca2+ sensitivity to be decreased [3,23].

What is a potential physiological role for this seemingly counter-productive Ca2+ desensitization under conditions when cardiac output needs to be increased? An important consequence of decreased Ca2+ sensitivity is the lusitropic effect of TnI phosphorylation, i.e. the enhancement of relaxation rate [22]. The reduced Ca2+ sensitivity due to TnI phosphorylation reflects a decreased calcium affinity of the calcium-binding troponin subunit (TnC). If the dissociation constant of the TnC–Ca2+ complex is increased, Ca2+ will dissociate more rapidly from TnC when the free [Ca2+]i decreases to about 10–7 M during diastole. Since cAMP-dependent phosphorylation of PLN enhances the rate of Ca2+ removal, PLN phosphorylation and TnI phosphorylation act synergistically to increase the relaxation rate of cardiac muscle. This lusitropic effect is beneficial during β-adrenergic stimulation, which is associated with an increase in heart rate. If under these conditions the relaxation rate remained unaltered, the diastolic phase of the cardiac cycle would be abbreviated and coronary blood flow, which peaks during diastole, would be impaired. Ventricular relaxation indeed has been shown to be a critical determinant of coronary blood flow in canine hearts [4].

In human end-stage heart failure, the cellular responses to the effects of β-receptor agonists are diminished due to a reduction in β-adrenergic receptor density following sustained activation of the sympathetic nervous system with chronically elevated levels of endogenous catecholamines [2]. Myofilament Ca2+ sensitivity, on the other hand, has been found to be increased in heart failure. Wolff et al. [20] suggested that the increased Ca2+ sensitivity might be due to a reduction in the β-adrenergically mediated phosphorylation of TnI. In this issue, Van der Velden et al. [18] report on a study in which they, by using single skinned human left ventricular cardiomyocytes mechanically isolated from end-stage failing human hearts and non-failing donor hearts, investigate potential mechanisms mediating this increase in Ca2+ sensitivity of the contractile apparatus. In accordance with previous studies they find myofilament Ca2+ sensitivity to be enhanced in failing myocytes compared to non-failing control cells. This difference was completely abolished after treatment of the skinned preparations with PKA, which decreased Ca2+ sensitivity in failing myocytes to values observed in nonfailing preparations in the absence of PKA. These results implicate a potential role of an altered phosphorylation state of myofilament proteins in end-stage heart failure. Interestingly, both the difference in Ca2+-responsiveness between failing and non-failing samples and the shift upon PKA treatment were much more pronounced in dilated (DCM) compared to ischemic cardiomyopathy (ICM), denoting a role of the etiology of the disease. The composition of bis-, mono- and dephosphorylated Troponin I (TnI) was analyzed by isoelectric focusing gels. This demonstrated a shift from bis- and monophosphorylated to dephosphorylated TnI in failing human myocardial samples, resulting in a positive correlation between myofilament Ca2+ sensitivity and the fraction of TnI present in the dephosphorylated form. In DCM, this shift was more pronounced than in ICM. In addition, using a 2-D gel-electrophoresis technique, it was shown that the degree of ventricular myosin light chain 2 (MLC-2) phosphorylation was lower in failing compared to non-failing human myocardium with no significant differences between DCM and ICM. Reduced MLC-2 phosphorylation would be expected to cause Ca2+ desensitization of the myofilaments [10] and the authors suggest that this desensitization in failing myocardium was overruled by the effect of TnI dephosphorylation, resulting in an overall enhanced Ca2+ responsiveness of the contractile apparatus. Depression of β-adrenergic response was proposed as an underlying mechanism which may mediate its effect on myofilaments directly by decreased PKA activity or indirectly by an increased protein phosphatase 1 activity. In contrast to previous publications that investigated molecular alterations associated with increased Ca2+-responsiveness of myofilaments in human heart failure, the authors neither found changes in the isoform composition nor degradation of contractile proteins. The discrepancies between the different studies are not resolved. The merit of the study by Van der Velden et al. [18] is however, that the authors instead of focusing on a single factor investigated a combination of contractile proteins changes contributing to increased Ca2+-responsiveness.

Although hard to predict, it is tempting to speculate about the consequences of the alterations reported by Van der Velden et al. [18] on the contractile performance of the failing human heart. The effect of Ca2+ sensitization by itself on the one hand might be beneficial in maintaining systolic contractile function of the failing myocyte but on the other hand might be detrimental for the diastolic phase of the contraction, promoting impaired relaxation. Because the function of the contractile apparatus is intimately linked to [Ca2+]i, changes in Ca2+ homeostasis of the failing myocyte will induce pronounced effects. In failing human ventricular myocytes, Ca2+-transients have been shown to be impaired with flattened rise in Ca2+ during systole and slowed decline during diastole [1]. Moreover, studies using the photoprotein aequorin have shown that the frequency potentiation of force in isolated nonfailing human myocardium was associated with a parallel increase in the Ca2+ transients, whereas in the failing human heart the frequency-dependent increase in force and Ca2+ was blunted [12]. Using rapid cooling contractures, we could demonstrate that this was a result of an impaired sarcoplasmic reticulum Ca2+ accumulation at higher stimulation rates resulting in defective SR Ca2+ load [13]. On the molecular level, decreased sarcoplasmic reticulum Ca2+-ATPase protein levels and activity as well as increased sarcolemmal Na+–Ca2+ exchanger protein levels and activity have been identified as an underlying mechanism (reviewed in Ref [16]). Taken together, at low heart rates increased TnI phosphorylation and Ca2+-responsiveness of myofilaments may increase contractility without induction of diastolic dysfunction thereby improving contractile function of the failing myocyte, because at low heart rates SR Ca2+ uptake may be sufficient despite decreased SR Ca2+ pump activity. However, at high heart rates increased Ca2+ responsiveness is expected to promote severely compromised diastolic function due to impaired SR Ca2+-ATPase activity and impaired cytoplasmic Ca2+ removal. Of note, increased transsarcolemmal Ca2+ efflux by Na+–Ca2+ exchange may partly compensate for decreased SR Ca2+ uptake and may preserve diastolic function even at high heart rates [6] (reviewed in Ref. [15]).

These considerations implicate a favorable effect of β-receptor blocker therapy on contractile function in heart failure with respect to the alterations reported by Van der Velden et al. [18]. β-receptor blocker therapy has emerged during the past decade in the treatment of heart failure and is strongly supported by consensus recommendations and clinical guidelines even in the end-stage of the disease [5]. β-Receptor blockers may improve intracellular Ca2+ handling by regulating and lowering heart rate. It has been reported that the deleterious changes associated with the compensatory increase in sympathetic activation followed by left-ventricular remodeling may be reversed by β-receptor blocker therapy, thereby improving ejection fraction and cardiac output. In addition, β-receptor blockers prevent β-adrenoceptor downregulation and desensitization [11]. It is also noteworthy that a recent study of Zeitz et al. [21] has demonstrated that the β1-selective blocker nebivolol acts as a direct Ca2+ desensitizer in skinned trabeculae from human myocardium. Of note, in the study of Van der Velden et al. [18] none of the patients was on treatment with β-receptor blockers, which may account for the observed decrease in myofilament phosphorylation and increase in Ca2+ responsiveness. Accordingly, it may be speculated that Van der Velden et al. [18] might have found less pronounced differences in Ca2+-responsiveness and phosphorylation of contractile proteins between failing and nonfailing human myocardium if patients with end-stage heart failure would have been treated with β-receptor blockers.


   References
Top
 References
 

  1. Beuckelmann D.J., Näbauer M., Erdmann E. Intracellular calcium handling in isolated ventricular myocytes from patients with terminal heart failure. Circulation (1992) 85:1046–1055.[Abstract/Free Full Text]
  2. Bristow M.R., Ginsburg R., Minobe W., et al. Decreased catecholamine sensitivity and beta-adrenergic receptor density in failing human hearts. New Engl J Med (1982) 307:205–211.[Abstract]
  3. Dohet C., Al-Hillawi E., Trayer I.P., Rüegg J.C. Reconstitution of skinned cardiac fibres with human recombinant cardiac troponin-I mutants and troponin-C. FEBS Lett (1995) 377:131–134.[CrossRef][Web of Science][Medline]
  4. Domalik-Wawrzynski L.J., Powell W.J., Guerrero L., Palacios I. Effect of changes in ventricular relaxation on early diastolic coronary blood flow in canine hearts. Circ Res (1987) 61:747–756.[Abstract/Free Full Text]
  5. Foody J.M., Farrell M.H., Krumholz H.M. β-Blocker therapy in heart failure. J Am Med Assoc (2002) 287:883–889.[Abstract/Free Full Text]
  6. Hasenfuss G., Schillinger W., Lehnart S.E., et al. Relationship between Na+–Ca2+ exchanger protein levels and diastolic function of failing human myocardium. Circulation (1999) 99:641–648.[Abstract/Free Full Text]
  7. Herzig J.W., Köhler H., Pfitzer G., Rüegg J.C., Wölffle G. Cyclic AMP inhibits contractility of detergent treated glycerol extracted cardiac muscle. Pflüger's Arch (1981) 391:208–212.[CrossRef][Web of Science][Medline]
  8. Marban E., Rink T.J., Tsien R.W., Tsien R.Y. Free calcium in heart muscle at rest and during contraction measured with Ca2+-sensitive microelectrodes. Nature (1980) 286:845–850.[CrossRef][Medline]
  9. Mittmann K., Jaquet K., Heilmeyer L.M.G. Jr. A common motif of two adjacent phosphoserines in bovine, rabbit and human cardiac troponin I. FEBS Lett (1990) 273:41–45.[CrossRef][Web of Science][Medline]
  10. Morano I. Effects of different expression and posttranslational modifications of myosin light chains on contractility of skinned human cardiac fibers. Basic Res Cardiol (1992) 87:129–141.[Web of Science][Medline]
  11. Patty R.D., Udelson J.E., Konstam M.A. Ventricular remodeling and its prevention in the treatment of heart failure. Curr Opin Cardiol (1998) 13:162–167.[Web of Science][Medline]
  12. Pieske B., Kretschmann B., Meyer M., et al. Alterations in intracellular calcium handling associated with the inverse force–frequency relation in human dilated cardiomyopathy. Circulation (1995) 92:1169–1178.[Abstract/Free Full Text]
  13. Pieske B., Maier L.S., Bers D.M., Hasenfuss G. Ca2+ handling and sarcoplasmic reticulum Ca2+ content in isolated failing and nonfailing human myocardium. Circ Res (1999) 85:38–46.[Abstract/Free Full Text]
  14. Ray K.P., England P.J. Phosphorylation of the inhibitory subunit of troponin and its effects on the calcium dependence of cardiac myofibril adenosine triphosphatase. FEBS Lett (1976) 70:11–16.[CrossRef][Web of Science][Medline]
  15. Schillinger W., Hasenfuss G. Cardiac remodeling and failure. Singal P.K., Dixon I.M.C., Kirshenbaum L.A., Dhalla N.S., eds. (2002) Boston, MA: Kluwer. in press.
  16. Schillinger W., Lehnart S.E., Prestle J., et al. Influence of SR Ca2+-ATPase and Na+–Ca2+-exchanger on the force–frequency relation. Basic Res Cardiol (1998) 93(Suppl. 1):38–45.[CrossRef][Web of Science][Medline]
  17. Tada M., Katz A.M. Phosphorylation of the sarcoplasmic reticulum and sarcolemma. Annu Rev Physiol (1982) 44:401–423.[CrossRef][Web of Science][Medline]
  18. Van der Velden J., Papp Z., Zaremba R., et al. Increased Ca2+-sensitivity of the contractile apparatus in end-stage human heart failure results from altered phosphorylation of contractile proteins. Cardiovasc Res (2003) 57:37–47.[Abstract/Free Full Text]
  19. Wilkinson J.M., Grand R.J.A. Comparison of amino acid sequence of troponin I from different striated muscles. Nature (1978) 271:31–35.[CrossRef][Medline]
  20. Wolff M.R., Buck S.H., Stoker S.W., Greaser M.L., Mentzer R.M. Myofibrillar calcium sensitivity of isometric tension is increased in human dilated cardiomyopathies. J Clin Invest (1996) 98:167–176.[Web of Science][Medline]
  21. Zeitz O., Rahman A., Hasenfuss G., Janssen P.M.L. Impact of β-adrenoceptor antagonists on myofilament calcium sensitivity of rabbit and human myocardium. J Cardiovasc Pharmacol (2000) 36:126–131.[CrossRef][Web of Science][Medline]
  22. Zhang R., Zhao J.J., Mandveno A., Potter J.D. Cardiac troponin I phosphorylation increases the rate of cardiac muscle relaxation. Circ Res (1995) 76:1028–1035.[Abstract/Free Full Text]
  23. Zhang R., Zhao J.J., Potter J.D. Phosphorylation of both serine residues in cardiac troponin I is required to decrease the Ca2+ affinity of cardiac troponin C. J Biol Chem (1995) 270:30773–30780.[Abstract/Free Full Text]

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