Left Ventricular and Myocardial Function in Mice Expressing Constitutively Pseudophosphorylated Cardiac Troponin I
Rationale: Protein kinase (PK)C-induced phosphorylation of cardiac troponin (cTn)I has been shown to regulate cardiac contraction.
Objective: Characterize functional effects of increased PKC-induced cTnI phosphorylation and identify underlying mechanisms using a transgenic mouse model (cTnIPKC-P) expressing mutant cTnI (S43E, S45E, T144E).
Methods and Results: Two-dimensional gel analysis showed 7.2±0.5% replacement of endogenous cTnI with the mutant form. Experiments included: mechanical measurements (perfused isolated hearts, isolated papillary muscles, and skinned fiber preparations), biochemical and molecular biological measurements, and a mathematical model–based analysis for integrative interpretation. Compared to wild-type mice, cTnIPKC-P mice exhibited negative inotropy in isolated hearts (14% decrease in peak developed pressure), papillary muscles (53% decrease in maximum developed force), and skinned fibers (14% decrease in maximally activated force, Fmax). Additionally, cTnIPKC-P mice exhibited slowed relaxation in both isolated hearts and intact papillary muscles. The cTnIPKC-P mice showed no differences in calcium sensitivity, cooperativity, steady-state force-MgATPase relationship, calcium transient (amplitude and relaxation), or baseline phosphorylation of other myofilamental proteins. The model-based analysis revealed that experimental observations in cTnIPKC-P mice could be reproduced by 2 simultaneous perturbations: a decrease in the rate of cross-bridge formation and an increase in calcium-independent persistence of the myofilament active state.
Conclusions: A modest increase in PKC-induced cTnI phosphorylation (≈7%) can significantly alter cardiac muscle contraction: negative inotropy via decreased cross-bridge formation and negative lusitropy via persistence of myofilament active state. Based on our data and data from the literature we speculate that effects of PKC-mediated cTnI phosphorylation are site-specific (S43/S45 versus T144).
The trimeric protein cardiac troponin (cTn) is associated with the sarcomeric thin filament and is a key protein that regulates cardiac contraction. Calcium binds to cTn causing a conformational shift in tropomyosin and allowing actin and myosin to form a force generating cross-bridge. cTn is more than a simple on-off switch for contraction; it can exert more complex control through its phosphorylation. For example, the inhibitor subunit of cTn, cTnI, has shown significant modulatory capacity when phosphorylated by protein kinase (PK)A and PKC. Some of these aspects of control include filament sliding speed,1 calcium sensitivity,2 cross-bridge cycling,3 and MgATPase activity.4 These affect global force production and relaxation responses to afterload,5 frequency5 and length.6
There are at least five phosphorylatable sites on cTnI: serines at 23, 24, 43 and 45 (S23, S24, S43, S45) and a threonine at 144 (T144). The sites nearest the N-terminal (S23, S24) are primarily phosphorylated by PKA, whereas the other 3 sites (S43, S45, T144) are primarily phosphorylated by PKC. There may be other phosphorylation sites, including S1507 and S76/T778 (in human) as well as other kinases that act on cTnI, including p21-activated kinase7 and Mst1 (mammalian sterile 20-like kinase 1).9 In the present study, we have focused on PKC phosphorylation for 2 reasons: (1) PKC is upregulated during heart failure10,11; and (2) its effects are largely dominant over those of PKA.4
We previously created a transgenic (TG) mouse with mutated PKC phosphorylation sites on cTnI: S43 and S45 were replaced with alanines to mimic nonphosphorylatable sites (cTnIS43/S45-NP). Decreased phosphorylation at these sites causes a positive inotropic response,12,13 no change in relaxation13 or calcium sensitivity,14 increased MgATPase activity,14 and increased susceptibility to ischemic contracture.15 The cTnIS43/S45-NP mouse also rescued cardiac function when it was crossed with a heart failure model (PKCε overexpression mutation).16
To study increased PKC phosphorylation of cTnI, we created a new TG mouse model (cTnIPKC-P) wherein S43, S45, and T144 on cTnI were replaced with glutamic acids to simulate constitutive phosphorylation. Note this new model mutated the threonine at position 144, which the cTnIS43/S45-NP mouse did not. Authentic phosphorylation of these 3 sites by PKC produced responses that closely mimic the effect of pseudophosphorylation using glutamic acid in reconstituted fibers.1 Based on previous work with the cTnIS43/S45-NP mouse, we hypothesized that the cTnIPKC-P mouse will exhibit negative inotropy and unchanged relaxation. Our goal was to test this hypothesis and determine how activator calcium, myofilament activation, and myofilament contraction contribute to the observed functional effects.
An expanded Methods section is available in the Online Data Supplement at http://circres.ahajournals.org.
Serine 43 and 45 and threonine 144 residues on cTnI were replaced with glutamic acid to simulate constitutive phosphorylation. Steps to create the TG mouse were similar to the creation of the cTnIS43/S45-NP mouse and have been described elsewhere.15 Here, we refer to this mouse as cTnIPKC-P, or the TG mouse.
Two-dimensional difference in gel electrophoresis (2D-DIGE) was used to determine percentage replacement of endogenous cTnI with mutant cTnI in the cTnIPKC-P mice, changes in the posttranslational modifications of cTnI (primarily phosphorylation), and other myofilament proteins.
The mice were anesthetized with an intraperitoneal injection of Avertin (2,2,2-tribromoethanol, 250 mg/kg body weight), and hearts were quickly excised. Left ventricular (LV) pressure was measured in isolated heart preparations17; wall stress (σ) was estimated using Online Equation A2.1.18 Force and intracellular calcium transients ([Ca]i) were measured simultaneously19 in intact posterior right ventricular papillary muscles. Isometric force and ATPase activity were measured simultaneously20 in skinned LV papillary muscle strips (sarcomere length set at 2.2 μm).
For the isolated heart and papillary muscle data, regression analysis with dummy variables was used to identify differences in various relationships between wild-type (WT) and TG groups. Student’s t tests were used to compare all other data. Data are expressed as means±SEM.
Characterization of the Mouse Model
The cTnIPKC-P mice showed no differences in body mass (WT: 30.7±1.6 g; cTnIPKC-P: 31.3±1.0 g), LV mass (WT: 102.4±6.7 mg; cTnIPKC-P: 103.7±4.2 mg), or their ratio (WT: 296±9; cTnIPKC-P: 303±9). There were also no overt signs of heart failure, including lethargy or differences in feeding.
Two-Dimensional Difference in Gel Electrophoresis Analysis
Proteins from WT and cTnIPKC-P myofibrils were separated using 2D-DIGE (Figure 1A). The 2D-DIGE gels were capable of separating cTnI species with posttranslational modifications and unmodified species (U: unmodified cTnI; Px: posttranslational modified cTnI, where x=number of possible modifications). In cTnIPKC-P samples, there was an additional spot near P3, indicated by an asterisk in Figure 1A, which was not present in the WT samples. We postulated this spot represented mutant cTnI.
To confirm this spot was constitutively pseudophosphorylated mutant cTnI, we treated WT and cTnIPKC-P myofibrils with PP1A and PP2A1 to dephosphorylate cTnI (Figure 1B). The WT cTnI spot profile was reduced to the U (unmodified) spot, indicating: (1) that phosphorylation was the primary posttranslational modification, (2) complete dephosphorylation, and (3) that at least 1 phosphorylation site was associated with each Px spot. In the phosphatase-treated cTnIPKC-P samples, there were 2 spots: U and mutant cTnI (indicated by an asterisk in Figure 1B). The ratio of the intensity of the mutant cTnI spot to the total intensity (ie, sum of unmodified spot [U] and mutant spot) represents the percentage replacement, which was 7.2±0.5% (n=9) (Online Table I).
To verify the protein identity of the spots, membrane transfers of the 2D-DIGE gels were probed with a specific pan cTnI antibody. Figure 1C illustrates multiple spots in the untreated samples, whereas only one spot is visible in the phosphatase-treated sample, a pattern similar to that in the 2D-DIGE gels (Figure 1A and 1B, second images). All 6 spots are identified in the Online Figure IB. The mutant cTnI spot was also confirmed by Western analysis (Online Figure IA).
There were no differences in the relative spot intensities (U, P1–P5) of cTnI in the WT and cTnIPKC-P samples, except the presence of the mutant cTnI spot (Figure 1A and Online Table I). This indicates that the basal pattern of actual phosphorylation of cTnI was unchanged in the TG mouse. Moreover, there were no differences in the phosphorylation levels of cTnT, tropomyosin, myosin regulatory light chain, or myosin binding protein C (Figure 1D and Online Table I; n: WT=4, cTnIPKC-P=4).
Isolated Heart Experiments
LV pressure waveforms are shown for the Frank–Starling protocol for an individual WT (Figure 2A) and cTnIPKC-P (Figure 2B) animal over the same range of chamber volumes (12 μL Vmax). For a better visual comparison, the pressure waveforms from WT and cTnIPKC-P for a single chamber volume (Vmax) are shown superimposed, both as absolute values (Figure 2C, top) and normalized values (Figure 2C, bottom). Group averaged Pdev and Ped values over the entire range of chamber volumes are illustrated in Figure 2D.
The cTnIPKC-P mice exhibited reduced systolic function. The slope of the σdev–volume relationship was reduced in cTnIPKC-P mice (WT: 2.9±0.1 mm Hg · μL−1, n=6, cTnIPKC-P: 2.5±0.1 mm Hg · μL−1, n=6, P=0.02), with no change in the intercept, indicating a 14% reduction in LV contractile state over all lengths (Figure 3A). Additionally, there was a decrease in the slope of the dσ/dtmax–volume relationship (WT: 0.084±0.004 mm Hg · ms−1 · μL−1, n=6; cTnIPKC-P: 0.074±0.003 mm Hg · ms−1 · μL−1, n=6, P=0.03), with no change in the intercept, indicating a 12% decrease in the kinetic aspects of contraction (Figure 3B).
The cTnIPKC-P mice exhibited slowed relaxation as evidenced by a parallel, upward shift of the Trelax–volume relationship (intercept values: WT: 16±1 ms, n=6; cTnIPKC-P: 21±3 ms, n=6, P=0.005), with no change in the slope, indicating a 20% increase (maximum) in the time for the LV to relax (Figure 3C). Consistent with this observation, there was a decrease in the magnitude of the slope of the dσ/dtmin–volume relationship (WT: −0.067±0.003 mm Hg · ms−1 · μL−1, n=6; cTnIPKC-P: −0.056±0.003 mm Hg · s−1 · μL−1, n=6, P=0.03), with no change in the intercept, indicating a 16% reduction in the kinetic aspects of relaxation (Figure 3D).
The cTnIPKC-P mice showed no differences in passive properties compared to WT mice. There were no statistical differences in Ped (Figure 2D, triangles) or σed (Online Figure II) over the entire range of volumes studied.
Treatment with 1 μmol/L isoproterenol increased developed pressures to the same degree in both WT and cTnIPKC-P (Table 1). Isoproterenol also shortened rise and relaxation times to the same degree in WT and cTnIPKC-P animals (Table 1).
Isolated Papillary Muscle Experiments
Figure 4A shows representative force and calcium data from one WT and one cTnIPKC-P experiment. The cTnIPKC-P mice exhibited decreased force production, indicated by a downward shift of the developed force (Fdev)–length relationship (intercept values: WT: 1.88±1.22 mN · mm−2, n=5; cTnIPKC-P: −0.51±0.35 mN · mm−2, n=6, P=0.04, Figure 4B). There was also slowed relaxation in the cTnIPKC-P mice, as evidenced by an upward shift in the dF/dtmin–length relationship (intercept values: WT: −55±16 mN · mm−2 · s−1; TG: 5±8 mN · mm−2 · s−1, P<0.001; Figure 4C). In contrast, intracellular calcium concentration transients ([Ca]i) were unaltered in cTnIPKC-P mice, exhibiting no changes in the slope or intercept of the [Ca]i amplitude (as quantified by Rsys/Red)–length relationship (probability value: for slope=0.53, for intercept=0.83, Figure 4D), or the relaxation (as quantified by dR/dtmin)–length relationship (P=0.23 for slope, P=0.28 for intercept, Figure 4C).
Skinned Fiber Experiments
There was a 14% decrease (n: WT, 10 fibers from 4 mice; cTnIPKC-P, 10 fibers from 3 mice) in maximally activated force (Fmax) in the cTnIPKC-P mice (Figure 5A and Table 2). Although there was a similar decrease (9%) in maximal MgATPase activity (Figure 5C and Table 2), it was not statistically significant. The skinned fiber data were fit to the modified Hill equation (Online Equation A4.1). There were no differences in pCa50 (calcium sensitivity) or Hill coefficient (nH, cooperativity) for either the pCa-force (Figure 5B and Table 2) or pCa-MgATPase (Figure 5D) relationships. The force-MgATPase activity relationship was linear (Figure 5E), and its slope is defined as the tension cost. There were no differences in the tension cost between the WT (7.33±0.13, R2=0.97) and cTnIPKC-P (7.41±0.10, R2=0.98) mice (Table 2).
We created a new TG mouse model (cTnIPKC-P) wherein the 3 PKC phosphorylation sites on cTnI were mutated to glutamic acid to simulate constitutive pseudophosphorylation. Despite low integration of mutant protein (≈7%), TG mice show significant functional changes, indicating high sensitivity of cardiac contraction to PKC cTnI phosphorylation. There are 3 main experimental findings of the present study. Compared to WT mice, cTnIPKC-P mice exhibited: (1) decreased active contraction and slowed relaxation, (2) a preserved response to β-adrenergic stimulation, and (3) decreased maximally activated force without changes in calcium sensitivity or tension cost. We discuss each of these observations individually below, followed by an integrative interpretation that reconciles these experimental findings.
TG Mouse Model
The cTnIPKC-P mouse showed ≈7% replacement of endogenous cTnI with mutant cTnI. There were no differences in the basal actual phosphorylation pattern of cTnI. Therefore, there is an increase in total phosphorylation of cTnI at the PKC sites: the summation of basal actual phosphorylation (unchanged in cTnIPKC-P mice) and pseudophosphorylation (increased by ≈7% in cTnIPKC-P mice). In spite of the relatively low level of replacement, there were significant functional effects. This is an unexpected and remarkable finding, suggesting a high sensitivity of cardiac contraction to PKC-mediated cTnI phosphorylation and potentially important physiological and pathophysiologic roles for this posttranslational regulatory process.
Recent evidence indicates that basal in vivo cTnI phosphorylation at the PKC sites is very low.8,21 Thus, the percentage replacement in our TG mice, although small, may represent a physiologically relevant level of increased PKC cTnI phosphorylation. Our previous mouse model, cTnIS43/S45-NP (serine 43 and 45 mutated to alanine), showed ≈50% replacement of endogenous cTnI by the mutant cTnI.14 However, given the low basal phosphorylation state, a higher replacement of nonphosphorylatable sites would be required to observe functional effects.
There were also no alterations in phosphorylation of tropomyosin, TnT, essential light chain, or myosin binding protein-C, indicating that the observed effects were from cTnI PKC pseudophosphorylation alone.
There was evidence of reduced contraction in the cTnIPKC-P mice compared to WT mice at all 3 levels studied: (1) isolated heart experiments showed depressed developed pressures, (2) intact papillary muscles exhibited lower developed force, and (3) skinned fibers showed significantly lower Fmax. In reconstituted fibers where the cTnI PKC phosphorylation sites were rendered constitutively pseudophosphorylated, a decrease in Fmax was also seen.1 The cTnIS43/S45-NP mouse model showed an increase in developed pressures at high extracellular calcium levels (3.5 mmol/L [Ca]).13 Together with previous data, the new TG model supports the hypothesis that phosphorylation of cTnI by PKC lowers the ability of the myocardium to generate active force, both under dynamic and steady-state activations.
In isolated heart and intact papillary muscle experiments, cTnIPKC-P mice also showed negative lusitropy when compared to control mice. This is consistent with the results of Pi et al, who treated WT mice with the PKC activator endothelin-1 and observed an increase in relaxation time (negative lusitropy).22 Furthermore, in their TG animal, in which all 5 cTnI phosphorylation sites were replaced with alanines, the effects of endothelin-1 were severely blunted. These results, combined with the results presented here, suggest that phosphorylation of the PKC sites on cTnI has a negative lusitropic effect. By that same logic, the cTnIS43/S45-NP mouse would be expected to exhibit positive lusitropy, but it did not.13 However, in the cTnIS43/S45-NP mouse, the T144 is not mutated to an nonphosphorylatable alanine. Thus, phosphorylation of T144 on cTnI may be primarily responsible for PKC-induced slowing of relaxation.
Response to β-Adrenergic Stimulation
The cTnIPKC-P and WT mice responded similarly to isoproterenol (1 μmol/L) infusion: increasing developed pressure and decreasing rise and relaxation times. β-Adrenergic stimulation acts through PKA-activated pathways, specifically the phosphorylation of PKA sites on cTnI (S23 and S24) and cellular calcium–handling proteins (eg, sarcoplasmic reticulum ATPase pump, L-type calcium channels). These results show that the mutation we introduced in cTnI did not affect these PKA-dependent pathways.
Intracellular Calcium Transients
There were no differences in intracellular calcium amplitude or relaxation between WT and cTnIPKC-P mice. We have previously reported that cTnIS43/S45-NP mice do not exhibit any differences in intracellular calcium at normal extracellular calcium levels.15 Others have also shown that there are no changes in intracellular calcium with PKC phosphorylation of cTnI.22,23 Normalized fluorescence values (R/Red or Rsys/Red), instead of calibrated data, were used in analyzing calcium transients. The reasons for using this approach and its validity are discussed in Online Appendix 3.
cTnIPKC-P mice did not exhibit differences in calcium sensitivity compared to WT mice. Burkart et al conducted experiments on detergent-extracted cardiac fibers reconstituted with 3 forms of mutant cTnI that were pseudo phosphorylated at the PKC sites: S43E/S45E, S43E/S45E/T144E, and T144E.1 They showed that S43E/S45E and S43E/S45E/T144E fibers exhibited similar decreases in calcium sensitivity. However, there was no change in calcium sensitivity in T144E fibers, suggesting that phosphorylation at this site plays no role. This is inconsistent with data from Wang et al in which chemical phosphorylation of T144 by PKC-βΙΙ resulted in increased calcium sensitivity.24
The reconstituted fibers studied by Burkart et al showed a much higher replacement of endogenous cTnI with the mutant form (70% to 97%) than was present in our cTnIPKC-P mouse, although it is unclear whether it is appropriate to directly compare the extent of replacement in the TG mouse and reconstituted system. Wang et al found that PKC-βΙΙ caused a 20% to 50% incorporation of radiolabeled phosphate, which was much closer to the level of phosphorylation simulated by our mutant cTnI. This suggests calcium sensitivity may be affected differently at very high, possibly nonphysiologic, levels of PKC phosphorylation of cTnI.
Because our model simulates phosphorylation at all 3 PKC sites, it is possible there are 2 offsetting effects on calcium sensitivity: a decrease in calcium sensitivity attributable to the (pseudo)phosphorylation at S43/S45 and an increase in calcium sensitivity in response to the (pseudo)phosphorylation at T144. If PKC phosphorylates all 3 positions, what is the purpose of mutually offsetting effects at the different sites? One possibility is that certain PKC isoforms only phosphorylate or preferentially phosphorylate a given residue, resulting in unequal phosphorylation on the 3 sites. For example, PKC-βΙΙ24 and tyrosine-phosphorylated PKCδ25 preferentially phosphorylate T144.
Our goal was to identify the underlying changes in myofilamental processes that can simultaneously explain all of our results. We can group the contraction/relaxation processes into 3 main categories: (1) activator calcium, (2) myofilament activation, and (3) myofilament contraction.
There were no differences in intracellular calcium transients in the WT and cTnIPKC-P mice (Figure 4). This suggests there are no differences in the activator calcium.
The slope of the force–MgATPase activity relationship is the tension cost and represents an estimate of the cross-bridge detachment rate constant (g).26,27 The cTnIPKC-P mice exhibited no change in tension cost compared to WT mice, implying unaltered g. Because the decrease in Fmax cannot be attributed to calcium activation or cross-bridge detachment, it is most likely attributable to a decrease in cross-bridge formation rate constant (f).
We used a mathematical model (Figure 6B) to interpret experimental results and determine what myofilamental processes were altered in the cTnIPKC-P mouse (see Online Appendix 5). This model has been used for data analysis before.17,28,29
Decreasing the value of f (23%) predicted the observed decrease in maximum twitch force and Fmax, but also showed decreased calcium sensitivity and no change in relaxation, contrary to our experimental observations. Therefore, there must be another altered process that slows relaxation. What parameter (or set of parameters), altered simultaneously with f, would produce decreased magnitude of contraction (both under twitching and steady-state conditions), increased relaxation time, and no change in calcium sensitivity or tension cost? We focused on parameters that would maintain the activation state (Figure 6A): (1) increased calcium binding (increased k1) and (2) decreased cross-bridge dissolution in the absence of cTnC-bound calcium (decreased d). Although the first perturbation can produce slowed relaxation, it was unable to reconcile all of the experimental observations.
The second perturbation (a decrease in f by 23% and a decrease in d by 35%) reproduced the experimental observations (compare Figures 5E with 6B, 2C with 6C and 6D, 5A with 6E, and 5B with 6⇑⇑F). The magnitude of these changes closely mirrored the observations in the isolated heart (14% negative inotropy, 20% negative lusitropy) and skinned fiber (14% decrease in Fmax, less than 2% change in pCa50, nH, and tension cost). These magnitudes acted as a guide, the goal was not to identify exact parameter values, but identify a plausible solution which explained the major findings of our experiments. Our experimental data and the model-based analysis suggest that the TG mouse has 2 altered myofilament processes: decreased rate constant of cross-bridge formation and a calcium-independent persistence of the active state.
The model also indicated that an isolated decrease in d was associated with an increase in calcium sensitivity and slowed relaxation. Experimental data suggests T144 phosphorylation causes an increase in calcium sensitivity24 and is primarily responsible for slowed relaxation. It is therefore tempting to attribute the calcium-independent persistence of myofilament active sate (ie, decreased d) to T144 phosphorylation. Likewise, the model indicated that an isolated decrease in f was associated with a decrease in calcium sensitivity, no change in relaxation, and negative inotropy. Experimental data suggest that phosphorylation of S43 and S45 results in decreased calcium sensitivity,1 no change in relaxation,13 and negative inotropy.13 Thus, it is possible that the decrease in rate constant of cross-bridge formation (f) is a result of S43 and S45 phosphorylation. The precise biophysical mechanisms underlying the regulation of myofilament contractile properties by PKC-mediated phosphorylation of cTnI are not presently known.
(1) Although the 2D-DIGE data clearly indicate unchanged overall pattern of actual phosphorylation of cTnI in TG mice (Figure 1A and Online Table I), we cannot definitely say that cTnI phosphorylation at PKA sites was unchanged. Additional data using mass spectrometry will be needed to quantify cTnI phosphorylation at individual sites. (2) Because a single TG mouse line was used in the present study (ie, a single level of transgene expression), it is not possible to determine how the observed effects scale with the level of cTnI phosphorylation at PKC sites. Additional TG mouse lines and/or experiments using reconstituted fibers will be necessary to address this issue. (3) Although it is a commonly used technique, pseudophosphorylation by glutamate replacement may not fully recapitulate actual phosphorylation. Furthermore, the simultaneous (pseudo)phosphorylation of all 3 PKC sites may or may not be a physiologically relevant pattern of cTnI phosphorylation. However, our TG mouse data, together with data from the literature, provide new insights into the effects of PKC-mediated cTnI phosphorylation.
Our data show a small increase in cTnI phosphorylation at PKC sites produces significant functional changes, indicating high sensitivity of cardiac contraction to PKC-mediated cTnI phosphorylation. Model-based analysis predicts that these functional changes are brought about by specific changes in myofilament contractile properties: decreased rate of cross-bridge formation and calcium-independent persistence of the active state. Based on our data and data from the literature, we speculate that the effects of PKC-mediated cTnI phosphorylation are site-specific (S43/S45 versus T144).
We thank Kelly Clause and Dr Partha Roy for assistance with molecular biology–related issues and Dr Kenneth Campbell for illuminating discussions regarding the mathematical model–based analysis.
Sources of Funding
This work was supported by NIH grants R01-HL75643 (to M.C.), P01-HL62426 (Project 1) (to R.J.S.), R01-HL64035 (to R.J.S.), and R01-HL77788 (to S.G.S.) and McGinnis Endowed Chair research funds (to S.G.S.). J.A.K. was supported by NIH predoctoral training grant T32-HL76124 (to S.G.S.).
Original received January 16, 2009; resubmission received July 20, 2009; revised resubmission received September 28, 2009; accepted October 8, 2009.
Burkart EM, Sumandea MP, Kobayashi T, Nili M, Martin AF, Homsher E, Solaro RJ. Phosphorylation or glutamic acid substitution at protein kinase C sites on cardiac troponin I differentially depress myofilament tension and shortening velocity. J Biol Chem. 2003; 278: 11265–11272.
Noland TA Jr, Guo X, Raynor RL, Jideama NM, Averyhart-Fullard V, Solaro RJ, Kuo JF. Cardiac troponin I mutants. Phosphorylation by protein kinases C and A and regulation of Ca2+-stimulated MgATPase of reconstituted actomyosin S-1. J Biol Chem. 1995; 270: 25445–25454.
Sakthivel S, Finley NL, Rosevear PR, Lorenz JN, Gulick J, Kim S, VanBuren P, Martin LA, Robbins J. In vivo and in vitro analysis of cardiac troponin I phosphorylation. J Biol Chem. 2005; 280: 703–714.
Bilchick KC, Duncan JG, Ravi R, Takimoto E, Champion HC, Gao WD, Stull LB, Kass DA, Murphy AM. Heart failure-associated alterations in troponin I phosphorylation impair ventricular relaxation-afterload and force-frequency responses and systolic function. Am J Physiol Heart Circ Physiol. 2007; 292: H318–H325.
Tachampa K, Wang H, Farman GP, de Tombe PP. Cardiac troponin I threonine 144: role in myofilament length dependent activation. Circ Res. 2007; 101: 1081–1083.
Buscemi N, Foster DB, Neverova I, Van Eyk JE. p21-activated kinase increases the calcium sensitivity of rat triton-skinned cardiac muscle fiber bundles via a mechanism potentially involving novel phosphorylation of troponin I. Circ Res. 2002; 91: 509–516.
Zabrouskov V, Ge Y, Schwartz J, Walker JW. Unraveling molecular complexity of phosphorylated human cardiac troponin I by top down electron capture dissociation/electron transfer dissociation mass spectrometry. Mol Cell Proteomics. 2008; 7: 1838–1849.
You B, Yan G, Zhang Z, Yan L, Li J, Ge Q, Jin JP, Sun J. Phosphorylation of cardiac troponin I by mammalian sterile 20-like kinase 1. Biochem J. 2009; 418: 93–101.
van der Velden J, Narolska NA, Lamberts RR, Boontje NM, Borbely A, Zaremba R, Bronzwaer JG, Papp Z, Jaquet K, Paulus WJ, Stienen GJ. Functional effects of protein kinase C-mediated myofilament phosphorylation in human myocardium. Cardiovasc Res. 2006; 69: 876–887.
Roman BB, Goldspink PH, Spaite E, Urboniene D, McKinney R, Geenen DL, Solaro RJ, Buttrick PM. Inhibition of PKC phosphorylation of cTnI improves cardiac performance in vivo. Am J Physiol Heart Circ Physiol. 2004; 286: H2089–H2095.
MacGowan GA, Evans C, Hu TC, Debrah D, Mullet S, Chen HH, McTiernan CF, Stewart AF, Koretsky AP, Shroff SG. Troponin I protein kinase C phosphorylation sites and ventricular function. Cardiovasc Res. 2004; 63: 245–255.
Pyle WG, Sumandea MP, Solaro RJ, De Tombe PP. Troponin I serines 43/45 and regulation of cardiac myofilament function. Am J Physiol Heart Circ Physiol. 2002; 283: H1215–H1224.
MacGowan GA, Du C, Cowan DB, Stamm C, McGowan FX, Solaro RJ, Koretsky AP, Del Nido PJ. Ischemic dysfunction in transgenic mice expressing troponin I lacking protein kinase C phosphorylation sites. Am J Physiol Heart Circ Physiol. 2001; 280: H835–H843.
Scruggs SB, Walker LA, Lyu T, Geenen DL, Solaro RJ, Buttrick PM, Goldspink PH. Partial replacement of cardiac troponin I with a non-phosphorylatable mutant at serines 43/45 attenuates the contractile dysfunction associated with PKCε phosphorylation. J Mol Cell Cardiol. 2006; 40: 465–473.
Macgowan GA, Kirk JA, Evans C, Shroff SG. Pressure-calcium relationships in perfused mouse hearts. Am J Physiol Heart Circ Physiol. 2006; 290: H2614–H2624.
Chandra M, Tschirgi ML, Ford SJ, Slinker BK, Campbell KB. Interaction between myosin heavy chain and troponin isoforms modulate cardiac myofiber contractile dynamics. Am J Physiol Regul Integr Comp Physiol. 2007; 293: R1595–R1607.
Pi Y, Kemnitz KR, Zhang D, Kranias EG, Walker JW. Phosphorylation of troponin I controls cardiac twitch dynamics: evidence from phosphorylation site mutants expressed on a troponin I-null background in mice. Circ Res. 2002; 90: 649–656.
Sumandea MP, Rybin VO, Hinken AC, Wang C, Kobayashi T, Harleton E, Sievert G, Balke CW, Feinmark SJ, Solaro RJ, Steinberg SF. Tyrosine phosphorylation modifies protein kinase C δ-dependent phosphorylation of cardiac troponin I. J Biol Chem. 2008; 283: 22680–22689.
Brenner B. Effect of Ca2+ on cross-bridge turnover kinetics in skinned single rabbit psoas fibers: implications for regulation of muscle contraction. Proc Natl Acad Sci U S A. 1988; 85: 3265–3269.
de Tombe PP, Belus A, Piroddi N, Scellini B, Walker JS, Martin AF, Tesi C, Poggesi C. Myofilament calcium sensitivity does not affect cross-bridge activation-relaxation kinetics. Am J Physiol Regul Integr Comp Physiol. 2007; 292: R1129–R1136.
Rhodes SS, Ropella KM, Audi SH, Camara AK, Kevin LG, Pagel PS, Stowe DF. Cross-bridge kinetics modeled from myoplasmic [Ca2+] and LV pressure at 17°C and after 37°C and 17°C ischemia. Am J Physiol Heart Circ Physiol. 2003; 284: H1217–H1229.