Transgenic Overexpression of Constitutively Active Protein Kinase C ε Causes Concentric Cardiac Hypertrophy
Abstract—To test the hypothesis that activation of the protein kinase C (PKC) ε isoform leads to cardiac hypertrophy without failure, we studied transgenic mice with cardiac-specific overexpression of a constitutively active mutant of the PKCε isoform driven by an α–myosin heavy chain promoter. In transgenic mice, the protein level of PKCε in heart tissue was increased 9-fold. There was a 6-fold increase of the membrane/cytosol ratio, and PKC activity in the membrane fraction was 4.2-fold compared with wild-type mice. The heart weight was increased by 28%, and upregulation of the mRNA for β-myosin heavy chain and α-skeletal actin was observed in transgenic mouse hearts. Echocardiography demonstrated increased anterior and posterior wall thickness with normal left ventricular function and dimensions, indicating concentric cardiac hypertrophy. Isolated cardiomyocyte mechanical function was slightly decreased, and Ca2+ signals were markedly depressed in transgenic mice, suggesting that myofilament sensitivity to Ca2+ was increased. No differences were observed in either the levels of cardiac Ca2+-handling proteins or the degree of cardiac regulatory protein phosphorylation between wild-type and transgenic mice. Unlike mice with PKCβ2 overexpression, transgenic mice with cardiac-specific overexpression of the active PKCε mutant demonstrated concentric hypertrophy with normal in vivo cardiac function. Thus, PKC isoforms may play differential functional roles in cardiac hypertrophy and failure.
Activation of the protein kinase C (PKC) signaling pathway has been implicated in the development of cardiomyocyte hypertrophy.1 2 Currently, at least 11 isoforms of this family of serine/threonine kinases have been identified, and their expression in the heart is developmentally regulated.3 Although PKC isoforms may play different functional roles in cell signaling, the exact significance of individual isoforms is not yet known. We have reported in the failing human myocardium with end-stage heart failure that the expression and activity of Ca2+-sensitive PKCα and -β isoforms are elevated.4 In isolated guinea pig hearts, oxidative stress using H2O2 induces left ventricular dysfunction associated with translocation of Ca2+-sensitive PKC isoforms.5 We have also demonstrated that postnatal cardiac-specific overexpression of the PKCβ2 isoform in transgenic mice causes a cardiomyopathy that is characterized by left ventricular hypertrophy, myocardial fibrosis, and decreased in vivo left ventricular performance.6 In these mice, PKCβ2-induced phosphorylation of the myofilament regulatory protein troponin I decreases cardiomyocyte Ca2+ sensitivity and may cause the depressed cardiomyocyte function.7 These observations have suggested a critical role of the PKCβ isoform in the genesis of contractile dysfunction.
On the other hand, the Ca2+-independent PKCε isoform has been implicated in cardiac hypertrophy and ischemic preconditioning.8 9 An in vitro study using neonatal cardiomyocytes has shown that PKC, but not tyrosine kinase or Ras, is critical for angiotensin II–induced activation of extracellular signal–regulated kinase (ERK), which promotes cardiac hypertrophy by activating transcription factors.10 Among PKC isoforms, PKCε, but not PKCα, is a mediator for ERK activation induced by endothelin-1 and phenylephrine.11 Moreover, we have demonstrated in the isolated adult guinea pig heart that pathophysiologic elevation of left ventricular diastolic pressure activates phospholipase C and accumulates inositol phosphate with resultant translocation of the PKCε isoform.12 This PKCε translocation by mechanical stretch is attenuated by an AT1 antagonist. In addition, we have shown that the PKCε isoform is essential for ERK activation in in vitro rabbit cardiomyocytes13 and in vivo mouse hearts.14 Interestingly, activation of PKCε is not observed in explanted myocardial tissue from patients with end-stage heart failure.4 On the basis of these findings, we hypothesized that activation of the PKCε isoform may lead to compensated ventricular hypertrophy. To test this hypothesis, we generated transgenic mice with cardiac-specific overexpression of a constitutively active mutant of the PKCε isoform using an α–myosin heavy chain (MHC) promoter. Cardiac-specific PKCε transgenesis made possible an in vivo evaluation of PKCε-mediated signaling pathways on cardiac hypertrophy and function without interference from phosphorylation events mediated either by other PKC isoforms or by upstream Gαq.
Materials and Methods
All procedures were performed in accordance with Case Western Reserve University animal care guidelines, which conform with the Guide for the Care and Use of Laboratory Animals, published by the National Institutes of Health.
Production of PKCε Transgenic Mice
PKCε transgenic mice were generated by P.P. and R.B.14 Briefly, a full-length PKCε cDNA was cloned from a rabbit heart cDNA library. Because the majority of the wild-type PKCε isoform resides in the cytosolic fraction and is usually self-inhibited, transgenesis with the wild-type PKCε may not lead to effective substrate phosphorylation in the membrane-particulate fraction. Thus, a constitutively active PKCε cDNA was generated through a mutation by converting A to E (amino acid 159) as previously described.13 14 This mutation prevents the pseudosubstrate domain from binding to the catalytic domain, and thus renders the molecule active. A linear 11.4-kb DNA fragment containing the entire α-MHC promoter (a gift from J. Robbins, Children’s Hospital Research Foundation, Cincinnati, Ohio), the complete PKCε cDNA with the mutation, and a polyadenylation signal was released by digestion with NotI and was used for microinjection into pronuclei of fertilized FVB mouse eggs as previously reported.6 15 16 17 The presence of the transgene was screened by Southern analysis of genomic DNA extracted from mouse tail using a 32P-labeled 1.9-kb EcoRI fragment as a probe.
The total RNA (10 μg/lane) was extracted from mouse hearts and hybridized under conditions previously described6 15 16 17 using a 32P-labeled BamHI-SalI fragment as a probe. Quantitative assessment of cardiac hypertrophic gene expression was performed using gene-specific oligonucleotides (gifts from G.J. Babu and M. Periasamy, University of Cincinnati) as previously described.6 16
The isoform-selective PKCε phosphorylation activity in the myocardium was measured as previously described.4 6 8 Briefly, proteins were immunoprecipitated with PKCε-specific antibody, and the activity was defined as phosphatidylserine- and phorbol 12-myristate 13-acetate–stimulated transfer of 32P from [γ-32P]ATP into the PKCε-specific substrate (ERMRPRKRQGSVRRRV).
Mice were anesthetized with tribromoethanol, and cardiac ultrasound studies were performed with an Acuson Sequoia ultrasonograph equipped with a 15-MHz linear array imaging transducer as previously reported.6 15
Isolated Cardiomyocyte Mechanical Properties and Ca2+ Signals
Left ventricular cardiomyocytes were isolated from mouse hearts, and cardiomyocyte mechanical properties were examined, as we previously described.7 15 17 Half of the isolated cells were used for measurements of cytosolic free Ca2+ by ratio imaging of fura-2 fluorescence, as reported previously.7 15 17 Eight to ten cardiomyocytes were analyzed for each mouse, and statistical analyses were performed on the basis of the number of hearts studied.
Phosphorylation of Membranous and Myofibrillar Proteins
Isolated cardiomyocytes were incubated with [32P]orthophosphate as previously described.7 18 PAGE of 32Pi-labeled proteins was performed using 4% to 20% gradient SDS gels, and 32Pi-labeled proteins were identified using a phosphor imager and autoradiography.
Statistical analysis was done with unpaired t tests. If data were not normally distributed or failed equal variance tests after log10 transformations, they were analyzed by nonparametric statistics (Mann-Whitney rank sum test). A P value of <0.05 was considered significant.
Transgenic mice 9 to 12 weeks of age and age-matched wild-type littermate control mice were used for the present study. A transgenic line expressing intermediate levels of the transgene (No. 388) was chosen for detailed characterization. Northern blot analysis revealed that the PKCε mRNA level in transgenic hearts was 15-fold in this line. Immunoblot analysis performed with specific antibodies for PKCε showed that the protein levels of the PKCε isoform in the heart were increased by 9-fold in the transgenic mice compared with the wild-type mice (Figure 1A⇓). As shown in Figure 1B⇓, the membrane-particulate/cytosol ratio of PKCε was 0.56±0.21 in wild-type mice and 3.04±0.44 in transgenic mice (n=4, P<0.01). PKCε activity in membrane-particulate and cytosolic fractions were 4.2±0.3-fold and 1.6±0.1-fold compared with wild-type controls (n=5, P<0.01), respectively. The PKCε transgenesis did not alter the expression and subcellular distribution of any of the other PKC isoforms expressed in the mouse heart (α, β2, γ, δ, θ, and ζ). Immunoblots of PKCα are shown in Figure 1C⇓ as an example. The protein expressions of the PKCε isoform in lungs, liver, kidney, large intestine, and small intestine were similar between wild-type and transgenic mice (data not shown). Systolic blood pressure of 3 transgenic and 3 wild-type littermate mice was measured with the mice in the conscious state using the standard tail cuff method in a blinded fashion. There were no differences in systolic blood pressure between transgenic and wild-type mice (136±9 versus 139±11 mm Hg, respectively).
Heart Weight and Lung Weight
The gravimetric data of wild-type littermate and PKCε transgenic mice are summarized in Table 1⇓. The absolute heart weight and ratio of heart to body weight were increased in transgenic mice compared with wild-type mice by 28% and 21%, respectively. The lung weight and ratio of lung to body weight were the same between the wild-type and transgenic mice. There was no evidence of fibrosis on microscopic examinations of multiple histological sections from transgenic mouse hearts (data not shown).
Expression of Hypertrophic Genes
Quantitative assessment of cardiac hypertrophic gene expression, such as atrial natriuretic factor (ANF), c-fos, β-MHC, and α-skeletal actin, was performed by Northern blot analysis. Representative Northern blots and quantitative data are shown in Figure 2⇓. Each value was normalized to the mRNA expression of GAPDH. Increased transcript levels of β-MHC (4-fold) and α-skeletal actin (7-fold) were observed in transgenic mouse hearts without significant changes in levels of ANF and c-fos.
M-mode echocardiographic measurements include the left ventricular minor axis dimension at end-diastole (EDD) and end-systole (ESD) and wall thickness at end-diastole of the anterior (AWTh) and posterior (PWTh) walls. Representative M-mode echocardiograms from a wild-type mouse and a transgenic mouse are shown in Figure 3A⇓, and group data of echocardiographic measurements are summarized in Table 2⇓. There were no differences in EDD, ESD, and fractional shortening between wild-type and transgenic mice. In contrast, the AWTh, PWTh, and left ventricular mass were increased in transgenic mice compared with wild-type mice. The relative wall thickness was higher in transgenic mice than in wild-type mice, indicating the presence of concentric hypertrophy in PKCε transgenic hearts.
Isolated Cardiomyocyte Mechanical Function and Ca2+ Transients
Representative analog recordings of isolated left ventricular cardiomyocyte mechanics and Ca2+ transients for wild-type and PKCε transgenic mice are shown in Figure 3B⇑. Group data for the cardiomyocyte mechanical properties and Ca2+ transients are summarized in Table 3⇓. The percentage of cardiomyocyte shortening (P<0.05) was slightly decreased in PKCε transgenic mice compared with wild-type control mice. The baseline Ca2+ level was slightly lower, and the amplitude of the Ca2+ transient was markedly decreased in transgenic mice compared with wild-type mice (P<0.01). The times from start to 80% decay of the Ca2+ signal (T80) and 50% decay of the Ca2+ signal (T50) were prolonged in transgenic mice. The observed disparities between cardiomyocyte mechanics and Ca2+ transient data suggested that myofilament sensitivity to Ca2+ was relatively increased in transgenic mouse hearts.
Protein Levels Involved in Ca2+ Homeostasis
To determine whether the observed changes in the cardiomyocyte Ca2+ signal were associated with altered expression of Ca2+-handling proteins, the relative levels of these proteins in the heart were determined by quantitative immunoblotting (Table 4⇓). No significant differences in phospholamban, sarcoplasmic reticulum Ca2+ ATPase (SERCA2a), the Na+-Ca2+ exchanger, or the Na+-H+ exchanger were found between PKCε transgenic and wild-type mice.
Phosphorylation of Cardiac Proteins
To clarify whether the suspected changes in myofilament Ca2+ sensitivity were associated with altered phosphorylation status of cardiac regulatory proteins, we examined the degree of cardiac protein phosphorylation. The incorporation of [32P]orthophosphate into a variety of cardiac proteins was studied in cardiomyocytes isolated from wild-type and transgenic mice hearts. The degree of protein phosphorylation at basal condition was expressed as a percentage of that after maximal stimulation with dibutyryl cAMP. As shown in Figure 3C⇑ and Table 5⇓, no differences were found in the degree of phosphorylation of troponin I, troponin T, phospholamban, and 15-kDa protein between wild-type and transgenic mice.
The major findings of the present study were as follows: transgenic mice with cardiac-specific overexpression of a constitutively active mutant of PKCε demonstrated (1) 4.2-fold increase of PKC activity in the membrane fraction; (2) mild concentric hypertrophy; (3) no evidence of fibrosis; (4) normal in vivo left ventricular performance; (5) slightly decreased isolated cardiomyocyte function and markedly depressed Ca2+ transients, suggesting an increase in myofilament Ca2+ sensitivity; (6) partial recapitulation of fetal gene expression; (7) unchanged abundance of Ca2+ cycling proteins; and (8) no differences in the degree of cardiac myofilament or sarcoplasmic reticulum regulatory protein phosphorylation.
Differential Roles of PKC Isoforms in Cardiac Hypertrophy and Failure
The phenotype resulting from postnatal cardiac overexpression of PKCε differs considerably from that observed in transgenic mice overexpressing PKCβ2. These mice, which had a comparable level of PKC activity (5-fold in membrane fraction), demonstrated a heart failure phenotype6 7 that was characterized by (1) in vivo systolic dysfunction by echocardiography, (2) decreased isolated cardiomyocyte function and normal Ca2+ kinetics, and (3) decreased myofilament responsiveness to Ca2+ resulting from increased phosphorylation of troponin I. Taken together, these data suggest differential functional roles for distinct PKC isoforms and support the notion that increased activity of PKCβ, but not PKCε, could depress contractile function in heart failure. However, in a separate study, a transgenic line with an extraordinarily high level of constitutively active PKCε (34-fold in protein level), demonstrated a heart failure phenotype.19 Whether this represents a “dose effect” from excessive PKCε gene expression, a nonspecific effect of very high levels of cardiomyocyte protein loading, or an insertional effect of the transgene is unclear at this time.
Despite normal in vivo cardiac performance by echocardiography, isolated cardiomyocyte mechanical function was modestly reduced in transgenic mice compared with wild-type control (9.6% versus 11.8%). This discordance might be explained by differences in experimental conditions such as partial contracture after enzymatic myocyte extraction, which has been observed in this and other studies.7 20 In addition, compensatory alterations in chamber geometry (concentric hypertrophy) and reflex control of the circulation may affect in vivo systolic left ventricular performance.
A preliminary echocardiographic study in a small number of retired breeders demonstrated that fractional shortening in 48-week-old transgenic mice was depressed compared with age-matched wild-type littermate controls. These data suggest that PKCε mice may develop an age-related impairment of systolic function, similar to that observed in other transgenic models.21
Although ANF is thought to be a marker of hypertrophy, the mRNA level of ANF was unexpectedly unchanged in PKCε transgenic mouse hearts in the present study. However, it should be recognized that hypertrophy may not always be associated with increased ventricular expression of ANF.22
Possible Mechanisms for Decreased Cardiomyocyte Ca2+ Transients
Although the amplitude of Ca2+ signals of isolated cardiomyocytes was decreased in transgenic mice compared with wild-type mice, the levels of Ca2+ handling proteins such as SERCA2a, phospholamban, the Na+-Ca2+ exchanger, and the Na+-H+ exchanger were similar between wild-type and transgenic mice. We cannot exclude the possibility that changes in the intrinsic activities of these proteins may account for the depressed Ca2+ amplitude in transgenic mouse hearts. Other possible mechanisms for reduced Ca2+ signals include (1) altered biophysical environment of the sarcoplasmic reticulum23 ; (2) altered spatial coupling between voltage-gated Ca2+ channels and the ryanodine receptor24 ; and (3) altered activity and abundance of other Ca2+-dependent phospholipid binding proteins such as annexins. For example, it has been demonstrated that transgenic overexpression of annexin VI in mice resulted in decreased basal and peak Ca2+ transients.20
Alterations in Myofilament Ca2+ Sensitivity
It is well known that altered Ca2+ kinetics modify cardiac contractility.1 Although the amplitude of Ca2+ signals of isolated cardiomyocytes was depressed in transgenic mice (53% of wild type), isolated cardiomyocyte function was relatively preserved (81% of wild type), and in vivo cardiac function assessed by echocardiography was normal in these transgenic mice. These findings suggest that increased myofilament Ca2+ sensitivity may contribute to the preserved left ventricular chamber and cardiomyocyte function that we observed. This compensatory mechanism would offset the functional results of diminished cellular Ca2+ handling.
The mechanisms by which myofibrillar Ca2+ sensitivity may be altered include (1) phosphorylation of myofibrillar proteins,25 (2) changes in regulatory contractile protein isoforms,26 and (3) regulation of intracellular pH.27 It has been reported that phosphorylation of troponin I or T by PKC reduces Ca2+ sensitivity and maximal activity of actomyosin MgATPase and thus impairs actin-myosin interactions.28 However, in the present study, unlike in mice with PKCβ2 overexpression, the degree of phosphorylation of both troponin I and T was unchanged in PKCε transgenic hearts. Phosphorylation specificities of PKC isoforms for cardiac regulatory proteins have been reported in in vitro studies.29 PKCα phosphorylates Ser43/Ser45 of troponin I and reduces Ca2+ sensitivity and maximal activity of MgATPase. In contrast, PKCζ phosphorylates 2 unknown sites of troponin T and results in a slight increase of Ca2+ sensitivity. PKC can potentially modify the regulation of intracellular pH through the activation of the Na+-H+ exchanger and secondarily alter myofibrillar Ca2+ sensitivity.30 However, we did not find a change in the protein level of the Na+-H+ exchanger in the present study. It is possible that changes in intracellular pH mediated by PKCε-induced phosphorylation and resultant activation of the Na+-H+ exchanger might contribute, at least in part, to an increase in myofilament sensitivity to Ca2+ observed in the present study. We are currently examining this possibility.
Cardiac-specific overexpression of a constitutively active mutant of PKCε causes mild concentric hypertrophy with normal in vivo cardiac performance. These and other data from our laboratory support the notion that activation of Ca2+-sensitive PKC isoforms, but not the PKCε isoform, predominates in mediating contractile dysfunction of failing myocardium. Furthermore, they suggest that distinct PKC isoforms may play differential functional roles in cell signaling pathways leading to cardiac hypertrophy and failure.
This work was supported in part by grants from NIH (HL-52318 to R.A.W., HL-58166 to P.P., and HL-43151 and HL-55757 to R.B.), the American Heart Association (9750721N to P.P. and 9650695N to B.D.H.), and the Japanese Heart Foundation (to Y.T.).
This manuscript was sent to Stephen F. Vatner, Consulting Editor, for review by expert referees, editorial decision, and final disposition.
- Received February 8, 2000.
- Accepted April 28, 2000.
- © 2000 American Heart Association, Inc.
Walsh RA, Dorn GW II. Growth and hypertrophy of the heart and blood vessels. In: Hurst JW, Schlant RC, Rackley CE, Sonnenblick EH, Wenger NK, eds. The Heart. 9th ed. New York, NY: McGraw-Hill, Inc; 1997:155–168.
Walsh RA. The role of angiotensin II in stretch activated signal transduction of the normal, hypertrophied and failing adult heart. In: Dhalla NS, Zahradka P, Dixon IMC, Beamish RE, eds. Angiotensin II Blockade: Physiology and Clinical Implications. Boston, Mass: Kluwer Academic Publishers; 1998:423–434.
Bowling N, Walsh RA, Song G, Estridge T, Sandusky GE, Fouts RL, Mintze K, Pickard T, Roden R, Bristow MR, Sabbah HN, Mizrahi JL, Gromo G, King GL, Vlahos CJ. Increased protein kinase C activity and expression of Ca2+-sensitive isoforms in the failing human heart. Circulation. 1999;99:384–391.
Takeishi Y, Jalili T, Ball NA, Walsh RA. Responses of cardiac protein kinase C isoforms to distinct pathologic stimuli are differentially regulated. Circ Res. 1999;85:264–271.
Wakasaki H, Koya D, Schoen FJ, Hoit BD, Jirousek MR, Ways DK, Walsh RA, King GL. Targeted overexpression of protein kinase C β2 isoform in myocardium causes cardiomyopathy. Proc Natl Acad Sci U S A. 1997;94:9320–9325.
Ping P, Zhang J, Qui Y, Tang XL, Manchikalapudi S, Cao X, Bolli R. Ischemic preconditioning induces selective translocation of protein kinase C isoforms ε and η in the heart of conscious rabbits without subcellular redistribution of total protein kinase C activity. Circ Res. 1997;81:404–414.
Qiu Y, Ping P, Tang X, Manchikalapudi S, Rizvi A, Zhang J, Takano H, Wu W, Teschner S, Bolli R. Direct evidence that protein kinase C plays an essential role in the development of late preconditioning against myocardial stunning in conscious rabbits and that ε is the isoform involved. J Clin Invest. 1998;101:2182–2198.
Zou Y, Komuro I, Yamazaki T, Aikawa R, Kudoh S, Shiojima I, Hiroi Y, Mizuno T, Yazaki Y. Protein kinase C, but not tyrosine kinase or Ras, plays a critical role in angiotensin II-induced activation of Raf-1 kinase and extracellular signal-regulated protein kinases in cardiac myocytes. J Biol Chem. 1996;271:33592–33597.
Clerk A, Bogoyevitch MA, Andersson MB, Sugden PH. Differential activation of protein kinase C isoforms by endothelin-1 and phenylephrine and subsequent stimulation of p42 and p44 mitogen-activated protein kinases in ventricular myocytes cultured from neonatal rat hearts. J Biol Chem. 1994;269:32848–32857.
Paul K, Ball NA, Dorn GW II, Walsh RA. Left ventricular stretch stimulates angiotensin II-mediated phosphatidylinositol hydrolysis and protein kinase C ε isoform translocation in adult guinea pig hearts. Circ Res. 1997;81:643–650.
Ping P, Zhang J, Cao X, Li RCX, Kong D, Tang XL, Qiu Y, Manchikalapudi S, Auchampach JA, Black RG, Bolli R. PKC-dependent activation of p44/p42 MAPKs during myocardial ischemia-reperfusion in conscious rabbits. Am J Physiol. 1999;276:H1468–H1481.
Ping P, Li RCX, Zhang J, Kong D, Jones K, Zheng YT, Cao X, Bolli R. Identification of 2 distinct mechanisms for the activation of p44/p42 mitogen-activated protein kinases (MAPKs) in vivo by a PKC isoform epsilon dependent signaling pathway in transgenic mice. Circulation. 1998;98(suppl I):I-416. Abstract.
D’Angelo DD, Sakata Y, Lorenz JN, Boivin GP, Walsh RA, Liggett SB, Dorn GW II. Transgenic Gαq overexpression induces cardiac contractile failure in mice. Proc Natl Acad Sci U S A. 1997;94:8121–8126.
Loukianov E, Ji Y, Grupp IL, Kirkpatrick DL, Baker DL, Loukianova T, Grupp G, Lytton J, Walsh RA, Periasamy M. Enhanced myocardial contractility and increased Ca2+ transport function in transgenic hearts expressing the fast-twitch skeletal muscle sarcoplasmic reticulum Ca2+ ATPase. Circ Res. 1998;83:889–897.
Luo W, Chu G, Sato Y, Zhou Z, Kadambi VJ, Kranias EG. Transgenic approaches to define the functional role of dual site phospholamban phosphorylation. J Biol Chem. 1998;273:4734–4739.
Zhang J, Wead WB, Jones WK, Wu X, Gao J, Kong D, Li RCX, Zheng YT, Ping P. Activation of PKCε induces hypertrophy and heart failure in a dose-dependent fashion in mice. J Mol Cell Cardiol. 1999;31:A18. Abstract.
Gunteski-Hamblin A, Song G, Walsh RA, Frenzke M, Boivin GP, Dorn GW II, Kaetzel MA, Horseman ND, Dedman JR. Annexin VI overexpression targeted to heart alters cardiomyocyte function in transgenic mice. Am J Physiol. 1996;270:H1091–H1100.
Iwase M, Bishop SP, Uechi M, Vatner DE, Shannon RP, Kudej RK, Wight DC, Wagner TE, Ishikawa Y, Homcy CJ, Vatner SF. Adverse effects of chronic endogenous sympathetic drive induced by cardiac GSα overexpression. Circ Res. 1996;78:517–524.
Vikstrom KL, Bohlmeyer T, Factor SM, Leinwand LA. Hypertrophy, pathology, and molecular markers of cardiac pathogenesis. Circ Res. 1998;82:773–778.
Kiss E, Ball NA, Kranias EG, Walsh RA. Differential changes in cardiac phospholamban and sarcoplasmic reticular Ca2+-ATPase protein levels: effects on Ca2+ transport and mechanics in compensated pressure-overload hypertrophy and congestive heart failure. Circ Res. 1995;77:759–764.
Gomez AM, Valdivia HH, Cheng H, Lederer MR, Santana LF, Cannell MB, McCune SA, Altschuld RA, Lederer WJ. Defective excitation-contraction coupling in experimental cardiac hypertrophy and heart failure. Science. 1997;276:800–806.
Mope L, McClellan GB, Winegrad S. Calcium sensitivity of the contractile system and phosphorylation of troponin in hyperpermeable cardiac cells. J Gen Physiol. 1980;75:271–281.
Reiser PJ, Westfall MV, Schiaffino S, Solaro RJ. Tension production and thin-filament protein isoforms in developing rat myocardium. Am J Physiol. 1994;267:H1589–H1596.
Noland TA Jr, Kuo JF. Protein kinase C phosphorylation of cardiac troponin I or troponin T inhibits Ca2+-stimulated actomyosin MgATPase activity. J Biol Chem. 1991;266:4974–4978.
Jideama NM, Noland TA Jr, Raynor RL, Blobe GC, Fabbro D, Kazanietz MG, Blumberg PM, Hannun YA, Kuo JF. Phosphorylation specificities of protein kinase C isozymes for bovine cardiac troponin I and troponin T and sites within these proteins and regulation of myofilament properties. J Biol Chem. 1996;271:23277–23283.
Kramer BK, Smith TW, Kelly RA. Endothelin and increased contractility in adult rat myocytes: role of intracellular alkalosis induced by activation of the protein kinase C-dependent Na+-H+ exchanger. Circ Res. 1991;68:269–279.