Is Phospholamban or Troponin I the “Prima Donna” in -Adrenergic Induced Lusitropy?

In normal myocardium, acute -adrenergic stimulation augments both systolic and diastolic performance via protein kinase A (PKA)–mediated phosphorylation of key proteins governing Ca 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 -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 Ca availability during systole is the major, though perhaps not the only, driver of enhanced inotropy with adrenergic stimulation.2–4 The effect on Ca dynamics is primarily mediated by phosphorylation of PLN, though increased ionic current through the L-type calcium channel contributes to Ca loading. When PLN is phosphorylated by PKA, the “brake” imposed by PLN on sarcoplasmic reticulum (SR) Ca ATPase is relieved, resulting in an increase in the activity of the latter that leads to a faster sequestration of Ca into the SR, enhancing cardiac relaxation and re-loading the SR with Ca to increase Ca 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 Ca , increases the off rate of Ca 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 -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 -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 Ca 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 -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 -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 -adrenergic stimulation. Similarly, there is no further decrease in pCa50 (rightward shift) with the addition of PKA during Ca 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 -agonist increased the peak Ca 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 nonphosThe opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association. 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 [email protected] (Circ Res. 2007;101:326-327.) © 2007 American Heart Association, Inc.