doi: 10.1161/CIRCRESAHA.107.158873
© 2007 American Heart Association, Inc.
Editorials |
Is Phospholamban or Troponin I the "Prima Donna" in β-Adrenergic Induced Lusitropy?
From the Department of Pediatrics, Division of Cardiology, Johns Hopkins University School of Medicine, Baltimore Md.
Correspondence to Anne M. Murphy, Department of Pediatrics, Division of Cardiology, Johns Hopkins University School of Medicine, Ross Building 1144, 720 Rutland Avenue, Baltimore MD 21205. E-mail murphy{at}jhmi.edu
See related articles, pages 377–386
Key Words: troponin • phospholamban • adrenergic stimulation
In normal myocardium, acute β-adrenergic stimulation augments both systolic and diastolic performance via protein kinase A (PKA)–mediated phosphorylation of key proteins governing Ca2+ handling and the contractile machinery. Twenty-five years ago Kranias and Solaro identified troponin I (TnI) and phospholamban (PLN) as the 2 major cardiac proteins which were nearly simultaneously phosphorylated by β-adrenergic stimulation of the beating heart in synchrony with the agonist effect of augmenting contractility (inotropy) and rate of relaxation (lusitropy).1 Numerous subsequent studies have mechanistically examined the role of these proteins in inotropy and lusitropy. It is well established that enhanced Ca2+ availability during systole is the major, though perhaps not the only, driver of enhanced inotropy with β- adrenergic stimulation.2–4 The effect on Ca2+ dynamics is primarily mediated by phosphorylation of PLN, though increased ionic current through the L-type calcium channel contributes to Ca2+ loading. When PLN is phosphorylated by PKA, the "brake" imposed by PLN on sarcoplasmic reticulum (SR) Ca2+ ATPase is relieved, resulting in an increase in the activity of the latter that leads to a faster sequestration of Ca2+ into the SR, enhancing cardiac relaxation and re-loading the SR with Ca2+ to increase Ca2+ release in subsequent beats. Yet, PLN phosphorylation by PKA is not the sole mechanism implicated in enhancing relaxation. In particular, phosphorylation of TnI by PKA has long been proposed to have a role in diastole because it desensitizes the myofilament to Ca2+, increases the off rate of Ca2+ from troponin, and speeds cross-bridge cycling (reviewed in5), although PKA phosphorylation of myosin binding protein C may also contribute to these effects. In the past decade a more detailed understanding of how the PKA phosphorylation of PLN and TnI influence function in vivo has been advanced through the development of several genetically modified models in mice.
Studies using PLN knock-out mice demonstrated that on β-adrenergic stimulation, PLN phosphorylation had much more dominant role over cTnI to enhance lusitropy.6 However, more recently, experiments using transgenic mice which express the nonphosphorylatable slow skeletal TnI suggested that PKA phosphorylation of TnI had a more important role than initially suggested.7,8 In addition, 2 independent groups reported the development of transgenic lines which express a pseudo phosphorylated mutant of the PKA sites of TnI and demonstrated that these mice had enhanced rate of relaxation of the left ventricle under baseline conditions without alterations in PLN phosphorylation.4,9 The relative role of β-adrenergic induced cardiac TnI phosphorylation on lusitropy is mechanistically and practically important, particularly with respect to congestive heart failure (CHF) because the TnI PKA target sites (Ser 23/24) are hypophosphorylated in failing human hearts.10 However, because regulation of Ca2+ dynamics via SR is also altered in heart failure (reviewed in11), understanding the relative contribution of phosphorylation of these two proteins is crucial.
In this issue of Circulation Research, Yasuda et al describe a detailed set of experiments to further establish that in addition to PLN phosphorylation, TnI phosphorylation is a major player in the lusitropic effects of β-adrenergic stimulation.12 The first set of these experiments takes advantage of a transgenic line in which native cardiac TnI is essentially completely replaced by a version in which the 2 Serines targeted for β-adrenergic mediated phosphorylation are mutated to Aspartic Acids (cTnI S23/24D) thus mimicking maximal phosphorylation. Yasuda et al demonstrate that in cardiac myocytes from cTnI S23/24D mice, twitch relaxation is faster at baseline with minimal further enhancement by β-adrenergic stimulation. Similarly, there is no further decrease in pCa50 (rightward shift) with the addition of PKA during Ca2+ sensitivity studies. It is worth noting that peak sarcomeric shortening and peak isometric twitch tension are increased by a similar magnitude in transgenic cTnI S23/24D and nontransgenic control mice on challenge with isoproterenol, indicating that the positive inotropic response is unchanged in the cTnI S23/24D mice. Along the same lines, stimulation with the β-agonist increased the peak Ca2+ transient and its rate of decay to the same degree in both cTnI S23/24D and the nontransgenic control groups. These studies indicate a significant contribution of TnI phosphorylation to adrenergically mediated lusitropy. To further consolidate their findings, the authors conducted a second set of experiments in which they used rat cardiac myocytes with adenoviral mediated expression of the pseudo phosphorylated TnI (cTnIS23/24D). For negative controls, they transduced expression of the nonphosphorylatable slow skeletal TnI or cTnI S23/24A and control transfection with a cTnI expressing vector was also used. They focus their observations on the lack of isoproterenol speeding of twitch relaxation after cTnI S23/24A gene transfer. They argue that the lack of enhanced twitch relaxation in response to isoproterenol in the nonphosphorylatable negative control (cTnI S23/24A) and the fact that Ca2+ transient decay rate is accelerated similarly in both groups (cTnI and cTnI S23/24A), supports the role of TnI in β-adrenergic mediated enhanced relaxation. The use of both chronic and acute manipulation of TnI phosphorylation is one of the strengths of the experimental approach in this article, for although compensatory changes in PLN have not been noted in this transgenic model,9 subtle changes can occur with such long-term models.
The authors conclude their studies with a quantitative analysis of the relative contribution of TnI and PLN phosphorylation to lusitropy. Indeed, they use a similar analytical approach as Li et al used to demonstrate the primacy of PLN phosphorylation to the lusitropic effect.6 Li et al concluded that over 80% of lusitropy was determined by PLN whereas Yasuda et al conclude that nearly 80% of the lusitropy (as measured by shortening of time to 75% relaxation), is determined by TnI. Why the discrepancy? In part this may be attributable to differences in baseline TnI phosphorylation in the PLN knockout mice as pointed out in the discussion of Yasuda and colleagues.12 However, the experiments of Li et al were also conducted at room temperature, whereas those of Yasuda et al at physiologic temperature.6 As noted by Janssen et al, myofilaments appear to be the rate limiting step in cardiac muscle relaxation at physiologic temperature.13
How are these findings relevant to cardiac disease? To date, many gene transfer based strategies aimed to treat heart failure have been based on modifying calcium handling proteins such as SR Ca2+ ATPase and PLN (reviewed in14), following the mainstream notion that defective Ca2+ handling is cause rather than consequence of failing cardiac myocytes. However, others have suggested that myofilament dysfunction is a central player in the physiopathology of heart failure.15,16 Recently, Day et al have shown that gain of function strategies by genetically engineering the myofilaments, specifically a Histidine mutant of cardiac TnI, is feasible and effective in both chronic and acute animal models.17 Would a mutant TnI S23/24D gene transfer be capable of increasing cardiac relaxation in vivo in a failing heart? Certainly this and other studies suggest this is the case. This is notable because diastolic heart failure is a particularly prevalent in heart failure syndromes (
40% to 50% of cases), yet therapies for diastolic failure are lacking.
In summary, the study of Yasuda et al reports important new information reassessing the relative contribution of PLN and TnI to β-adrenergic–induced lusitropy in loaded and unloaded cardiac myocytes at physiological temperature.12 In doing so, they suggest that TnI may indeed be the "Prima Donna" of β-adrenergic induced lusitropy, and as such TnI is a valid target for future targeted therapies of diastolic heart failure.
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Acknowledgments |
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Sources of Funding
A.M.M. receives funding from NIH HL63038 and HV-28180.
Disclosures
None.
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Footnotes |
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The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association.
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References |
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Related Article:
Circ. Res. 2007 101: 377-386.
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