Frequency- and Afterload-Dependent Cardiac Modulation In Vivo by Troponin I With Constitutively Active Protein Kinase A Phosphorylation Sites

Abstract

Abstract-Acute ␤-adrenergic stimulation enhances cardiac contractility, accelerates muscle relaxation, and amplifies the inotropic and lusitropic response to increased stimulation frequency. These effects are modulated by phosphorylation of calcium handling and myofilament proteins such as troponin I (TnI) by protein kinase A (PKA). To more directly delineate the role of TnI PKA phosphorylation, transgenic mice were generated that overexpress cardiac TnI in which the serine residues normally targeted by PKA are mutated to aspartic acid to mimic constitutive phosphorylation (TnIDD 22,23 ). Native cardiac TnI was near completely replaced in one transgenic line as assessed by in vitro phosphorylation, and this led to reduced calcium sensitivity of myofibrillar MgATPase, as expected. TnIDD 22,23 mice had mildly enhanced basal systolic and diastolic function, and displayed marked augmentation of frequency-dependent inotropy and relaxation, with a peak frequency response 2-fold greater in mutants than controls (PϽ0.005). Increasing afterload prolonged relaxation more in nontransgenic than TnIDD 22,23 (PϽ0.02), whereas contractile responses to afterload were similar between these strains. Isoproterenol treatment eliminated the differential force-frequency and afterload response between TnIDD 22,23 and controls. In contrast to in vivo studies, isolated isometric trabeculae from nontransgenic and TnIDD 22,23 mice had similar basal, isoproterenol-, and frequency-stimulated function, suggesting that muscle shortening may be important to TnI PKA effects. These results support a novel role for cardiac TnI PKA phosphorylation in the rate-dependent enhancement of systolic and diastolic function in vivo and afterload sensitivity of relaxation. These results have implications for cardiac failure in which force-frequency modulation is blunted and afterload relaxation sensitivity increased in association with diminished PKA TnI phosphorylation. Key Words: troponin I Ⅲ protein kinase A Ⅲ cardiac function Ⅲ myofilament S timulation of cardiac ␤-adrenergic receptors augments myocardial contractility, accelerates the rate of force development and relaxation, and amplifies the positive inotropic and lusitropic effects of higher stimulation frequency. 1 In addition, the load dependence of cardiac relaxation is influenced by cardiac contractility and level of ␤-stimulation. 2,3 These effects are thought to be mediated by the phosphorylation of target proteins by protein kinase A (PKA), including the myofilament proteins cardiac troponin I (cTnI) and myosin binding protein-C (MyBP-C), sarcoplasmic reticulum (SR) regulatory proteins phospholamban (PLB) and ryanodine receptor channel, and the sarcolemmal calcium channel. The net result is greater availability of intracellular calcium during systole, more rapid calcium removal from the cytosol into the SR, and enhanced calcium dissociation from myofilaments during diastole (reviewed by Bers 4 ). Of these PKA target proteins, strong evidence exists for potent regulation by PLB and calcium ch...

Determinants of frequency-dependent contraction and relaxation of mammalian myocardium

“…In IDAR, this TnI phosphorylation is thought to be responsible, at least for a significant part, for the faster relaxation. A likely role for TnI in frequencydependent processes is supported by the observations of Takimoto et al [7] and Bilchick et al [50]. These studies observed an effect of TnI phosphorylation via PKA as well as PKC that affected FFR and FDAR in intact murine hearts.…”

“…This important regulatory mechanism was first described in the year 1871; Henry Bowditch noted that the contractile force of the frog heart increased when it was paced at increasing frequency [1]. Since the first description of this force frequency relationship (FFR), the underlying mechanism has been a target for experimental investigation [2][3][4][5][6][7]. Although several contributing factors and modifiers of the FFR have been identified, our current understanding of frequency dependent activation is far from complete.…”

The unique functions of cardiac troponin I in the control of cardiac muscle contraction and relaxation

“…Yasuda et al [12] concluded from their studies of hearts of mice expressing these mutant forms of cTnI that phosphorylation of cTnI is a significant factor in cardiac relaxation with an importance similar to that of phospholamban phosphorylation. Takimoto et al [13] also reported that hearts of mice expressing cTnI-S23D/ S24D had a constitutive enhancement of rate-dependent increases in systolic and diastolic function in vivo. Along these lines, Varian and Janssen [14] reported a frequency dependent decrease in myofilament sensitivity to Ca 2+ associated with increasing heart rates and most likely attributable to cTnI phosphorylation.…”

“…The role of cTnI phosphorylation in the relaxant effect of adrenergic stimulation has also been emphasized in work reported by Stelzer et al [15] in studies of mice expressing cTnI-S23D/S24D against a cTnI and myosin binding protein C null background. These studies together with others provide compelling evidence for a prominent role of cTnI phosphorylation in the maintenance of power and frequency response in ejecting ventricles [13,[16][17][18][19]. Apart from their significant effects on cardiac dynamics, cTnI phosphorylation at the PKA sites affects length dependence of activation (LDA) [20].…”

“…While the inhibitory region in both skeletal and cardiac TnI has been shown to be helical in the absence of Ca 2+ -binding to the regulatory domain [36], bioinformatics analyses supports a β-turn type conformation for this region when bound to actin [29], as mentioned. Recently, the determined structure for the cardiac N-extension bisphosphorylated Ser-23/Ser-24 was found to contain a C-terminal helix (residues 21-30) containing the phosphorylation motif, an extended poly(L-proline)II helix (residues [11][12][13][14][15][16][17][18][19], and an acidic N-terminus with some propensity for helical structure [47]. Using this structure, the X-ray crystal structure of the cTn core, and uniform density models of the cTn subunits derived from neutron contrast variation data, atomic models were built that show the conformational transition induced by PKA phosphorylation at Ser-23/Ser-24 of cTnI and suggest a molecular linkage between the cardiac N-extension and the inhibitory region of cTnI [47].…”