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(Circulation. 1997;96:1495-1500.)
© 1997 American Heart Association, Inc.

Articles

Troponin I Phosphorylation in the Normal and Failing Adult Human Heart

Geza S. Bodor, MD; Annette E. Oakeley; Paul D. Allen, MD, PhD; Dan L. Crimmins, PhD; Jack H. Ladenson, PhD; ; Page A. W. Anderson, MD

From the Department of Laboratories (G.S.B.), Denver Health Medical Center, Denver, Colo; the Department of Pediatrics (A.E.O., P.A.W.A.), Duke University Medical Center, Durham, NC; the Department of Anesthesiology (P.D.A.), Brigham and Women's Hospital, Boston, Mass; and the Department of Pathology (G.S.B., D.L.C., J.H.L.), Division of Laboratory Medicine, Washington University, St Louis, Mo.

Correspondence to Page A.W. Anderson, MD, Department of Pediatrics, PO Box 3218, Duke University Medical Center, Durham, NC 27710. E-mail ander005{at}mc.duke.edu

	Abstract

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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.

Key Words: heart failure • calcium • myocardial contraction • myocardium • cardiomyopathy

	Introduction

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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. ß-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 heart^{1 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.

	Methods

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Tissue
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.

Reagents
The reagents for SDS-PAGE and Western blots have been described.¹⁵ PKC from rat brain containing isozymes expressed by the heart^{12 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 membranes^{15 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
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--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.

Statistical Analysis
Comparison of mean values was made by using Student's unpaired t test (independent t test) using commercially available software.

	Results

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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.

View larger version (36K): [in this window] [in a new window]   Figure 1. Western blot expression of TnI isoforms in fetal myocardium and adult failing and control myocardium. Myofibrillar proteins from fetal and adult hearts were resolved in a 7.5% SDS-PAGE gel and transferred to a PVDF membrane. TnI was detected by using TnI MAb 3C5.10, which is equally reactive with cTnI and both skeletal muscle TnI isoforms. The skeletal muscle preparations did not contain cTnI; the adult cardiac preparations contained only cTnI. Numbered lanes indicate the following myofibrillar preparations: 1, failing myocardium; 2, control myocardium; 3, human fetal heart (14 weeks of gestation); 4, fetal human skeletal muscle; and 5, adult human skeletal muscle.

View larger version (25K): [in this window] [in a new window]   Figure 2. Western blots of cTnI in myocardium from heart preparations from (A) a BWH control patient, (B) a heart failure patient, (C) a Russian control patient, and (D) a Hungarian control patient. Myofibrillar proteins were resolved in a 20-cm-long 9.5% SDS-PAGE gel and then transferred to a PVDF membrane. cTnI was detected by using the cTnI-specific MAb 2F6.6.

View larger version (94K): [in this window] [in a new window]   Figure 3. Western blots. Myofibrillar proteins from a patient with end-stage heart failure were resolved in an SDS-PAGE gel, transferred to PVDF membrane, and probed. 2F6.6 and 1E11.3 indicate membranes probed with the cTnI-specific MAb 2F6.6 and cTnI NH2-terminus–specific MAb 1E11.3, respectively.

AP Treatment
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).

View larger version (64K): [in this window] [in a new window]   Figure 4. AP treatment decreases the relative amount of cTNI1. Myofibrillar proteins from heart failure (A) and control (B) myocardium were resolved in a 7.5% SDS-PAGE gel and silver stained. 1, 2, and 3 indicate samples incubated in AP treatment buffer with no AP (negative control), AP-treated (dephosphorylated) samples, and untreated samples, respectively.

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).

View larger version (58K): [in this window] [in a new window]   Figure 5. PK treatment increases the relative amount of cTnI1. Myofibrillar proteins of heart failure cardiac myofibrils were resolved in a 7.5% SDS-PAGE gel, transferred to PVDF membrane, and probed with the cTnI-specific MAb 2F6.6. Lanes 1 through 3 show an untreated sample, a cAMP-dependent PK-treated (phosphorylated) sample, and cAMP-dependent PK treatment buffer and no enzyme (negative control).

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 cTnI_1. 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 cTnI_2. 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%.

View larger version (42K): [in this window] [in a new window]   Figure 6. Bar graph. Densitometric scans of cTnI1 and cTnI2 were obtained after SDS-PAGE; cTnI1 is expressed as percent of total cTnI (%TnI1). Myocardium from the heart failure patients (n=20) contained a significantly smaller amount of cTnI1 than did myocardium from control patients (n=24). Bar=1 SD.

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.

	Discussion

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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 PKC, 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 ß-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 ß-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 ß- or ß₁-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. ß-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 ß-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

 

AP = alkaline phosphatase 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 MAb = monoclonal antibody PK = protein kinase PKA, PKC = protein kinase A, protein kinase C PVDF = polyvinylidene fluoride ssTnI = slow-twitch skeletal muscle troponin I TnI, TnT = troponin I, troponin T

	Acknowledgments

 
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.

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