Troponin I Phosphorylation in the Normal and Failing Adult Human Heart
Background In the failing human heart myofibrillar calcium sensitivity of tension development is greater and maximal myofibrillar ATPase activity is less than in the normal heart. Phosphorylation of the cardiac troponin I (cTnI)–specific NH2-terminus decreases myofilament sensitivity to calcium, while phosphorylation of other cTnI sites decreases maximal myofibrillar ATPase activity.
Methods and Results We examined cTnI phosphorylation in left ventricular myocardium collected from failing hearts at the time of transplant (n=20) and normal hearts from trauma victims (n=24). The relative amounts of actin, tropomyosin, and TnI did not differ between failing and normal myocardium. Using Western blot analysis with a monoclonal antibody (MAb) that recognizes the striated muscle TnI isoforms, we confirmed that the adult human heart expresses only cTnI. A cTnI-specific MAb recognized two bands of cTnI, designated cTnI1 and cTnI2, while a MAb whose epitope is located in the cTnI-specific NH2-terminus recognized only cTnI1. Alkaline phosphatase decreased the relative amount of cTnI1, while protein kinase A and protein kinase C increased cTnI1. The percentage of cTnI made up of cTnI1, the phosphorylated form of TnI, is greater in the normal than the failing human heart (P<.001).
Conclusions This phosphorylation difference could underlie the reported greater myofibrillar calcium sensitivity of failing myocardium. The functional consequence of this difference may be an adaptive or maladaptive response to the lower and longer calcium concentration transient of the failing heart, eg, enhancing force development or producing ventricular diastolic dysfunction.
The biochemical and biophysical properties of the normal and failing human heart differ. Tension development of skinned myocardium from the failing heart has been found to be more sensitive to calcium than that from the normal heart,1 while maximal myofibrillar ATPase activity is depressed in the failing heart.2 3 These differences must follow from sarcomeric protein differences. β-Myosin heavy chain, skeletal muscle actin, and cTnI are dominantly expressed in both normal and failing adult human myocardium, precluding a role of isoform switching of these proteins.4 5 6 7 Although heart failure–associated changes in the myosin light chain 2 profile and TnT isoform expression are correlated with depressed myofibrillar ATPase activity,8 9 the increased calcium sensitivity of the failing heart does not appear to follow from these changes.1
The heart failure–associated changes in function are reminiscent of the effects of cTnI phosphorylation. Specifically, phosphorylation of the cTnI-specific NH2-terminal extension decreases myofilament sensitivity to calcium,10 11 12 while phosphorylation of other cTnI sites decreases maximal ATPase activity.11 12 13 The finding that PKA treatment eliminates the calcium sensitivity difference between failing and control heart preparations1 suggests that decreased cTnI terminal extension phosphorylation causes the heart failure–associated increase in myofibril sensitivity to calcium.
In this study we found a novel difference in the level of cTnI phosphorylation in the normal and failing human heart and confirmed the findings that while both cTnI and ssTnI are expressed in the developing human heart, only cTnI is expressed in the normal and failing adult human heart.6 7 We found that cTnI is phosphorylated to a greater extent in the normal heart; the site of this difference is likely to reside in the NH2-terminal extension cTnI. The functional consequence of such a difference has been found in both the human and canine heart1 14 : tension development of failing myocardium is more sensitive to calcium. Whether decreased phosphorylation is a positive, adaptive response or a maladaptive response remains to be established.
TnI expression was examined in left ventricular myocardium from normal and failing adult and fetal human hearts. Tissue was obtained at the time of orthotopic cardiac transplantation from patients at BWH with severe heart failure (n=20). The control group of adult trauma victims (n=24) consisted of potential organ donors whose hearts could not be used for transplantation: 18 BWH patients, 17 of whom were maintained on life support systems for 24 to 72 hours; three trauma victims maintained on life support <2 hours (a gift of Dr Frank Sreter, Budapest, Hungary); and three trauma victims whose tissue was harvested 2 to 5 hours after their death (a gift of Dr Valdur Saks, Moscow, Russia). Cardiac and skeletal muscle from fetuses (14.5 to 15 weeks of gestation) were obtained at the time of therapeutic abortion. Adult skeletal muscle samples were obtained from biopsies taken at the time of surgery for unrelated problems. All samples were obtained under protocols approved by the BWH Institutional Committee for the Protection of Human Subjects. The foreign samples were collected after obtaining consent under protocols approved by the relevant institutions.
The left ventricular free wall of the adult hearts were cut into small transmural pieces and frozen in liquid nitrogen within 5 minutes of excision. Fetal cardiac and skeletal muscle and adult skeletal muscle biopsies were frozen in liquid nitrogen immediately after excision. The specimens were transported on dry ice to Duke University Medical Center and kept in liquid nitrogen until they were prepared for SDS-PAGE and Western blot analysis.
The reagents for SDS-PAGE and Western blots have been described.15 PKC from rat brain containing isozymes expressed by the heart12 16 was obtained from Calbiochem. Bacterial AP was obtained from GIBCO BRL. The catalytic subunit of PKA and the other reagents used in the phosphorylation and dephosphorylation protocols were obtained from Sigma. Three MAbs that recognize TnI epitopes were used in the Western blot analysis (see below); their specificity and mode of characterization have been described.17 Briefly, MAb 3C5.10 recognizes an epitope that is shared by cTnI, ssTnI, and fsTnI; MAb 2F6.6 recognizes a cTnI-specific epitope; and MAb 1E11.3 recognizes a cardiac-specific epitope located in the NH2-terminal extension of cTnI.
SDS-PAGE and Western Blots
Muscle proteins were resolved in 7.5% and 9.5% polyacrylamide gels.18 Protein staining was performed either by using silver staining19 or, after the proteins were transferred to PVDF membrane, by using gold staining with the “Bio Cell” PR0500 Protogold kit.
TnI isoform expression and the effects of cTnI phosphorylation and dephosphorylation (see below) were examined by probing proteins transblotted onto nitrocellulose or PVDF membranes15 20 with a MAb concentration of 2 μg/mL in 50 mmol/L Tris, 150 mmol/L NaCl, and 1% BSA (pH 7.2) or in 20 mmol/L Tris, 500 mmol/L NaCl, and 1% BSA (pH 7.5). The blots were incubated with AP-conjugated rabbit anti-mouse antibody (Jackson ImmunoResearch Laboratories, Inc) diluted 1:1000 or goat anti-mouse IgG (Fc specific) AP conjugate (Sigma Chemical Co) diluted 1:2000 in 50 mmol/L Tris, 150 mmol/L NaCl, 1 mmol/L MgCl2, and 1% BSA for 2 hours at ambient temperature. After the blots were washed, TnI was detected by using AP Substrate Kit II (Vector Research). On occasion, nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate (“ProtoBlot”; Promega Corp) were used as the color reagent and provided identical results.
Silver-stained gels and Western blots were scanned by using an LKB laser densitometric scanner (Pharmacia LKB). The area under the TnI densitometric waveform was integrated, and two cTnI bands (cTnI1 and cTnI2; see “Results”) were described as a percentage of total cTnI.
Myofibril preparations were dephosphorylated by following the method of Holroyde et al.21 These proteins were compared with those of untreated preparations or those treated identically without added AP by using SDS-PAGE.
Myofibril preparations were phosphorylated with a PKA catalytic subunit.21 These proteins were compared with control preparations, untreated or treated identically without added PKA, by using SDS-PAGE and Western blot analysis.
Myofibril preparations were phosphorylated with PKC according to the methods of Bell et al22 and Edes and Kranias.23 The reaction buffer contained 20 mmol/L Tris, 10 mmol/L MgCl2, 0.18 mmol/L EGTA, 4 μmol/L phorbol 12-myristate 13-acetate, 100 μg l-α-phosphatidyl-l-serine, 1.0 mmol/L CaCl2, 10 mmol/L dithiothreitol, and 10 mmol/L NaF (pH 7.5). Controls were untreated myofibrils or myofibrils treated identically without added PKC.
Comparison of mean values was made by using Student’s unpaired t test (independent t test) using commercially available software.
The group with severe heart failure contained 14 men and 6 women (age 38±12 years, mean±1 SD), 16 of whom had idiopathic dilated cardiomyopathy. The remaining patients had coronary artery disease (n=1), restrictive cardiomyopathy (n=1), idiopathic hypertrophic subaortic stenosis (n=1), and ventricular septal defect (n=1). All patients had severely compromised ventricular function3 and were on a broad range of drugs, including digoxin, calcium-channel blockers, milrinone, and amiodarone.
Control myocardium was from hearts of BWH patients (30±8 years; 15 men and 3 women) that could not be used for transplantation; 17 had been maintained on life support systems and a renal dose of dopamine and 1 had not. Additionally, there were hearts from 6 European men aged 23±1 years (see “Methods”); 3 had been maintained on life support <3 hours and 3 had not.
Myofibrillar Proteins of Failing and Control Left Ventricular Myocardium
The relative amounts of actin, tropomyosin, and TnI were compared among the groups by using SDS-PAGE and densitometric waveform analysis. The relative amounts did not differ among myocardium from failing (actin 60±5%, tropomyosin 28±3%, and TnI 12±2%) and control (BWH 61±2%, 26±3%, and 13±2% [n=18] and Hungary and Russia 59±1.5%, 26±2%, and 15±2% [n=6], respectively; mean±1 SD) hearts.
TnI Isoform Expression
Western blot analysis of myocardial proteins from control and failing adult and fetal hearts was performed with MAb 3C5.10, which recognizes cTnI, ssTnI, and fsTnI, and MAb 2F6.6, which recognizes a cTnI-specific epitope. The adult hearts expressed only cTnI, while the fetal heart predominantly expressed ssTnI (Fig 1⇓). MAb 3C5.10 and MAb 2F6.6 recognize in the adult heart two bands of cTnI (Figs 1⇓ and 2⇓). The protein with the slower electrophoretic mobility was named cTnI1 and the faster one cTnI2. By using Western blot analysis with a panel of anti-cTnI MAbs,17 we demonstrated that cTnI1 and cTnI2 are recognized by all the MAbs except MAb 1E11.3, whose epitope is located in the cTnI-specific NH2-terminus. MAb 1E11.3 recognized only cTnI1 (Fig 3⇓), suggesting NH2-terminus sequence diversity or a posttranslational modification of its epitope.
To determine if the electrophoretic mobility difference between cTnI1 and cTnI2 is secondary to phosphorylation, myofibrils were treated with AP. This treatment decreased cTnI1 while increasing cTnI2 (Fig 4⇓). In myocardium from control hearts, which contain only cTnI1, the phosphatase treatment resulted in the appearance of cTnI2 (Fig 4⇓).
PKA and PKC Treatments
To test the basis of the phosphorylation difference between cTnI1 and cTnI2, myofibril preparations were treated with cAMP-dependent PKA. cTnI2 was converted into cTnI1 by this treatment (Fig 5⇓).
To further assess the basis of the phosphorylation difference, cardiac myofibril preparations were treated with PKC. Like PKA, but to a lesser extent, PKC decreased cTnI2 and increased cTnI1. The relatively different effects of PKA and PKC are not surprising; Noland et al11 had to exhaustively phosphorylate their preparations with PKC to achieve only modest NH2-terminal extension phosphorylation.
cTnI1 and cTnI2 in Control and Failing Adult Hearts
Preparations from failing and control hearts had significantly different relative amounts of cTnI1 and cTnI2. The percentage of total TnI made up of the phosphorylated form of cTnI (cTnI1) was greater in control myocardium (BWH patients [n=18], 87±17%; European patients [n=6], 78±16%; combined group [n=24], 84±17%; Fig 6⇓) than in myocardium from the failing hearts (56±14%, n=20, P<.001; Fig 6⇓). The relative amount of phosphorylated cTnI did not differ among control groups. In considering the effect of life support, myocardium from the BWH patient who did not receive life support contained 100% cTnI1; myocardium from the other 17 patients in this group contained 85±17% cTnI1. In comparison, the mean cTnI1 percentage in myocardium from the Hungarian and Russian patients who received life support was 68%, while that of myocardium from patients who did not was 88%.
To assess the effects of potential differences in acquisition timing on cTnI phosphorylation, samples from control and failing hearts were left at room temperature for 6 hours. The relative amounts of cTnI1 and cTnI2 were unaffected. Similarly, increasing the dithiothreitol concentration in the sample buffer by 10-fold did not affect the relative amounts of cTnI1 and cTnI2.
The differences in myofibril function between normal and failing human hearts has lead to careful examinations of contractile protein expression in these two groups. No differences in TnI, actin, or myosin heavy chain isoform expression have been found.4 5 6 7 Alterations in TnT isoform expression9 and the myosin light chain 2 profile8 have been observed in human heart failure, while altered isoform expression of the essential myosin light chain has been observed with human cardiac hypertrophy.24 Correlations between maximal ATPase activity and cTnT isoform expression have been found.9 Wolff et al,1 however, who also found altered cTnT isoform expression in the failing human heart, found no correlation between this altered expression and the enhanced calcium sensitivity of tension development in failing human heart preparations. The basis for this increased calcium sensitivity is unknown.
We identified two bands of cTnI, cTnI1 and cTnI2, by using high-resolution SDS-PAGE and Western blots. Using MAbs that recognize epitopes in different regions of cTnI17 allowed us to confirm that these two bands were cTnI and that only cTnI was expressed in the normal and failing heart, while ssTnI was coexpressed with cTnI in the developing human heart.6 7
cTnI is a substrate for both PKA and PKC.10 11 12 13 25 It is generally recognized that PKA phosphorylates the serines in the NH2-terminal extension of cTnI,10 11 13 a peptide absent in ssTnI and fsTnI.26 Although in vitro phosphorylation of cTnI by PKC is well recognized, opinion differs as to whether cTnI serves as a PKC substrate in vivo; recent evidence supports in vivo phosphorylation.13 23 PKC has been shown to phosphorylate the cTnI NH2-terminal extension, which was previously thought to be the target only of PKA,12 in addition to sites outside the NH2-terminal peptide.13 The phosphorylation of the NH2-terminus is PKC isozyme dependent, with PKCδ, an isoform expressed in the heart,12 16 being most effective.12
The recognition of only cTnI1 by a MAb whose epitope is located in the cTnI NH2-terminal extension focused our attention on whether phosphorylation is the basis of the differences between cTnI1 and cTnI2. Phosphatase and kinase treatment and Western blot analysis together demonstrated that cTnI amino-terminal phosphorylation is the basis of this difference, and cTnI is more phosphorylated in the normal heart. For example, AP converted cTnI1 to cTnI2; PKA and PKC converted cTnI2 to cTnI1; MAb 1E11.3, whose epitope resides in the NH2-terminal extension, recognized only cTnI1; and normal myocardium contained a greater amount of cTnI1 (the phosphorylated form). These results demonstrated that cTnI1 and cTnI2 are not products of hydrolysis or alternative splicing but rather differ by phosphorylation states. In our study of cTnT isoform expression in the failing and normal adult human heart, we sought posttranslational differences in cTnT among normal and failing human hearts and were unable to find any.27
Phosphorylation of cTnI by PKA has been defined as the basis for the decrease in myofilament sensitivity to calcium that follows β-adrenoreceptor stimulation.28 Wattanapermpool et al10 have demonstrated that phosphorylation of the NH2-terminal region is necessary and sufficient for this decrease and that it does not affect maximal ATPase activity. In vitro phosphorylation of this cTnI peptide by PKC decreases myofilament sensitivity to calcium.11 12
Our finding that the NH2-terminal region of cTnI was more phosphorylated in control than failing myocardium should result in control heart preparations being less sensitive to calcium, but this functional difference has been variably found.1 29 30 31 Wolff et al1 found that myocyte-sized preparations harvested from normal human hearts are less sensitive to calcium than those from the failing heart. They also found that PKA treatment had a smaller effect on normal heart preparations than those from the failing heart, resulting in pCa50 of normal and failing heart preparations being similar. We suspect that if they had used our SDS-PAGE protocol, they would have identified cTnI1 and cTnI2, and the normal myocardium would have contained more (phosphorylated) cTnI1.
Our data cannot answer the question of whether preparations from brain-dead patients would be more phosphorylated as a result of trauma and the resulting abnormal state. Other approaches would be required to answer this question, but it is unethical to harvest cardiac tissue from the healthy human; moreover, such harvesting would alter the autonomic state and cTnI phosphorylation.
Results from the canine model in which tachycardia was used to induce heart failure14 argue that the cTnI phosphorylation differences between failing and control human hearts are not the result of life support and traumatic death. Importantly, the canine preparations were obtained from the same dogs under identical conditions using the same biopsy technique before and after the induction of heart failure. Similar to the study of the failing human heart in which myocardium from brain-dead patients was used as a control,1 failing canine heart preparations are more sensitive to calcium than normal myocardium.14 Also, as in the human study, PKA has less effect on control preparations, so that after this treatment the pCa50’s of control and failing canine heart preparations are similar. These results in dogs suggest that the functional and phosphorylation differences between failing and control human myocardium reflect the effects of heart failure and not an abnormal control state.
Myocardial β-adrenergic receptor function and the associated signaling system are impaired in human heart failure. The range of findings include decreased norepinephrine content of sympathetic terminals, total β- or β1-receptor downregulation, and subsensitivity of adenylate cyclase stimulation.32 33 34 35 36 37 38 Similar effects are observed in the canine model of tachycardia-induced heart failure.39 40 41 42 One would anticipate that this sympathetic impairment in the failing heart would produce a lower level of cTnI phosphorylation similar to that which we found. β-Adrenergic receptor stimulation does cause acceleration of left ventricular isovolumic relaxation in patients with severe heart failure, consistent with a reserve of nonphosphorylated cTnI and phospholamban in the failing heart.43
The biochemical and biophysical effects of cTnI phosphorylation differences between the normal and failing heart could be adaptive or maladaptive. The cytosolic calcium concentration transient in failing human heart myocytes is lower and more prolonged than that of control myocytes.44 The relatively greater calcium sensitivity of myofilaments in the failing heart compared with the normal heart will result in greater myofilament activation in response to a comparable increase in calcium concentration, ie, a positive effect. On the other hand, the combined effects of the prolonged calcium transient and greater calcium sensitivity could slow myocardial relaxation and produce diastolic dysfunction. The positive effect that β-block therapy can have on the survival of the heart failure patient45 underlines our incomplete understanding of the interrelationship of these processes.
In summary, we have found that cTnI phosphorylation is greater in myocardium from control hearts than in preparations from the failing heart and that this difference resides in the phosphorylation state of the cardiac-specific cTnI NH2-terminal extension. The expected effect on myofibril function, a greater calcium sensitivity of failing heart preparations, has been found in the human heart.
Selected Abbreviations and Acronyms
|BWH||=||Brigham and Women’s Hospital|
|cTnI1, cTnI2||=||slower and faster migrating cardiac troponin I isoforms|
|cTnI, cTnT||=||cardiac troponin I or T|
|fsTnI||=||fast-twitch skeletal muscle troponin I|
|PKA, PKC||=||protein kinase A, protein kinase C|
|ssTnI||=||slow-twitch skeletal muscle troponin I|
|TnI, TnT||=||troponin I, troponin T|
This work was supported in part by the Gustavus and Louise Pfeiffer Research Foundation and the National Institutes of Health (grants HL-42250 and HL-20749).
- Received January 23, 1997.
- Revision received April 3, 1997.
- Accepted April 13, 1997.
- Copyright © 1997 by American Heart Association
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