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the Department of Cardiovascular Medicine (G.K., A.S., D.H.-K., D.O., P.S., A.Q., E.P., A.H., F.S., H.D.), Hannover Medical School, Germany
the Department of Experimental Endocrinology (M.L.), Max-Planck-Institute, Hannover, Germany.
Abstract
Overexpression and activation of protein kinase C- (PKC) results in myocardial hypertrophy. However, these observations do not establish that PKC is required for the development of myocardial hypertrophy. Thus, we subjected PKC-knockout (KO) mice to a hypertrophic stimulus by transverse aortic constriction (TAC). KO mice show normal cardiac morphology and function. TAC caused similar cardiac hypertrophy in KO and wild-type (WT) mice. However, KO mice developed more interstitial fibrosis and showed enhanced expression of collagen I1 and collagen III after TAC associated with diastolic dysfunction, as assessed by tissue Doppler echocardiography (Ea/Aa after TAC: WT 2.1±0.3 versus KO 1.0±0.2; P<0.05). To explore underlying mechanisms, we analyzed the left ventricular (LV) expression pattern of additional PKC isoforms (ie, PKC, PKC, and PKC). After TAC, expression and activation of PKC protein was increased in KO LVs. Moreover, KO LVs displayed enhanced activation of p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK), whereas p42/p44eCMAPK activation was attenuated. Under stretch, cultured KO fibroblasts showed a 2-fold increased collagen I1 (col I1) expression, which was prevented by PKC inhibitor rottlerin or by p38 MAPK inhibitor SB 203580. In conclusion, PKC is not required for the development of a pressure overloadeCinduced myocardial hypertrophy. Lack of PKC results in upregulation of PKC and promotes activation of p38 MAPK and JNK, which appears to compensate for cardiac hypertrophy, but in turn, is associated with increased collagen deposition and impaired diastolic function.
Key Words: hypertrophy protein kinase C mitogen-activated protein kinases
Introduction
Myocardial hypertrophy is the fundamental response of the heart to increasing workload, such as hypertension, valve disorders, or ischemic events. Myocardial hypertrophy also represents a principal risk factor for the development of heart failure and subsequent cardiac death.
There are various pathways mediating myocardial hypertrophy. Among these, the involvement of protein kinases C (PKCs) plays a crucial role in mediating cardiac responses to growth factors and neurotransmitters such as endothelin-1, angiotensin II, and -adrenergic receptors, all of which are signaled via Gq-coupled receptors.1,2 The PKC family is a group of serine/threonine kinases that can be divided in three subfamilies (ie, classical PKCs [PKC, PKC1, PKC2, and PKC], novel PKCs [PKC, PKC, PKC, and PKC], and the atypical PKCs [PKC and PKC/]).3,4 Gq-coupled receptors activate phospholipase C, thereby catalyzing hydrolysis of phosphotidylinositol bisphosphate into diacylglycerol and inositol triphosphate,5 which in turn can activate classical and novel PKC isoforms. Subsequently, activated PKCs are known to modulate transcription factor activation (ie, c-jun and c-fos),6,7 voltage-dependent calcium channels,8 and myofilament proteins (ie, troponin I and troponin T).9,10 However, the precise role of the different isoforms of PKC in mediating cardiac hypertrophy has not been determined yet.
Activation of myocardial PKC has been linked to a hypertrophic phenotype because hypertrophic stimuli (eg, endothelin-1) causes a rapid translocation and activation of PKC in rat cardiomyocytes in vitro.11,12 Moreover, cardiac-specific overexpression of PKC results in concentric hypertrophy with normal in vivo cardiac function;13 respectively, cardiac specific overexpression of a PKC translocation activator peptide (receptor of activated C kinase ) causes hypertrophic cardiomyopathy with preserved systolic function.14 However, these observations do not establish that activation of PKC is required for the development of myocardial hypertrophy (ie, after chronic pressure overload). In the present study, we investigated the role of PKC using mice with a systemic knockout (KO) of PKC in myocardial hypertrophy in response to pressure overload.
Materials and Methods
Animal Experiments
PKC-KO mice generation has been described previously.15 In brief, E14 embryonic stem (ES) cells (129/Ola) were used for the targeting experiment following the standard procedures of the gene-targeting approach in the mouse (bred by M. Leitges). Homologe-recombined ES cell clones were then introduced by injection into C57BL/6 blastocytes. The possible germline transmission of the injected ES cells was identified by crossing the observed chimeric males to C57BL/6 females and subsequently the presence of agouti coat color in the F1 progeny. F1 heterozygote breedings gave rise to homozygote animals, which were finally used in this study and correspond to the hybrid (C57BL/6x129/Ola) background. Male KO mice at the age of 12±2 weeks and their age-matched littermate controls were used. Absence of PKC was confirmed by immunoblot (Figure 1A).
Thoracic aortic constriction was performed as described previously.16 Briefly, mice were anesthetized with enflurane (3%) and intubated and ventilated (0.5 mL tidal volume [room air], 100 cycles per minute; Harvard apparatus). The chest cavity was opened at the second intercostal space at the left upper sternal border through a small incision. The transverse section of the aorta was freed, a suture (7-0 TI-CRON; Davis and Geck) was placed around the aorta between the right innominate and left common carotid arteries, and a tight ligature was tied against a 27-gauge needle; the needle was then promptly removed. The lungs were re-expanded and the thoracotomy was closed.
Transthoracic Echocardiography and Hemodynamics
Echocardiography was performed as described previously.17 Left ventricular (LV) hemodynamics were measured with a 1.4-F micromanometer conductance catheter (SPR-719; Millar Instruments) as described.18 In brief, mice were anesthetized and mechanically ventilated with enflurane (3%), a bilateral vagotomy was performed, and the catheter was inserted in the right carotid artery.
All animal studies were in compliance with the Guide for the Care and Use of Laboratory Animals as published by the US National Institutes of Health and were approved by the local animal care committees.
Histological Analyses and Immunostaining
In situ fixed LV tissue slices were embedded in paraffin and stained with picro-Sirus red F3BA or hematoxylin/eosin (H&E), as described.19 Tissue morphometry was performed in a blinded fashion using the Quantimet 500MC digital image analyzer. Mean cardiomyocyte cross-sectional area (CSA) was determined in H&E sections.19 Apoptotic nuclei were detected by in situ terminal deoxynucleotidyl transferase-mediated digoxigenin-conjugated dUTP nick end-labeling (TUNEL) in paraformaldehyde-fixated or frozen sections, as described.18 Slides were counterstained with Hoechst 33258 (Sigma) to confirm apoptotic morphology of individual nuclei.
Interstitial collagen fraction, collagen I, and collagen III were determined in picro-Sirius red F3BA-stained sections as described recently.20 Collagen I and III were differentiated using spectral analysis polarization microscopy on Sirius redeCstained sections: collagen III appears as green fibers, whereas collagen I appears as yelloweCred fibers.21
Cell Culture
Primary fibroblasts were isolated from lungs of adult KO mice and cultured in DMEM supplemented with 10% FCS. After the first passage, cells were seeded on FlexWell plates and cultivated up to 70% confluence. Cells were switched to DMEM without FCS for 24 hours before stimulation. Inhibitors were applied 60 minutes before stretch was started. Fibroblasts were stretched with the use of a FlexerCell Strain Unit (FX-4000; FlexCell). Biaxial cyclic stretch (15%; 0.5 Hz) was applied for 24 hours. Controls were cultured on FlexWell plates without mechanical stretch.
RT-PCR, Northern Blotting, and Immunoblotting
RT-PCR, Northern blotting, and immunoblotting were performed according to standard procedures. Total RNA was isolated from tissue samples by TriFast (peqLab). Concentration and purity were determined spectrophotometrically. A total of 2 e RNA was reverse transcribed using Superscript II reverse transcriptase (Invitrogen). Real-time measurement of PCR amplification was performed using the Stratagene MX4000 multiplex QPCR System with a SYBR green dye method (Brilliant SYBR Green Mastermix-Kit; Stratagene). Specific primers used for real-time PCR were: col I1 (GenBank No. MMU50767: 5'-ACAGACGAACAACCCAAACT-3'; 5'-GGTTTTTGGTCACGTTCAGT3'); atrial natriuretic factor (ANF): 5'-GCCGGTAGAAGATGAGGTCA-3'; 5'-GGGCTCCAATCCT-GTCAATC-3';22 skeletal muscle -actin (-Sk-actin): 5'-ATCTCACGTTCAGCTGTGGTCA-3'; 5'-ACCACCGGCATCG-TGTTGGAT-3';22 and G3PDH: 5'-AACGACCCCTTCATTGAC-3; 5'-TCCACGACATACTCAGCAC-3.
PCR was performed with denaturation at 95°C for 1 minute, annealing at 57°C for 1 minute, and elongation at 72°C for 30 seconds (40 cycles). Data acquisition was accomplished at 80°C to avoid measurement of nonspecific products. PCR efficiency was >95% as revealed by standard curve slope calculation. Melting curve analysis showed no nonspecific amplification products or primer. Semiquantitative PCR was performed to quantify sarcoplasmic reticulum Ca(2+) ATPase (SERCA2a) and PKC mRNA levels using the primers: SERCA2a: 5'-TGACTTTCGTCGGCTGTG-3'; 5'-AGACCACTTCCCCCACG-3'; and PKC: 5'-GGAATT-CAGTGACATCCTAGACAACAA-3'; 5'-GCGTCGACCGGA-GCAAACCAGGGCAGACG-3'. cDNA has been tested for equal expression of G3PDH.
Immunoblotting was performed according to standard procedures.23 For immunoblots, equally loaded cell lysates (50 e/lane) of each sample were fractionated in 10% sodium dodecyl sulfateeCpolyacrylamide gels. Proteins were transferred electrophoretically to a polyvinylidene difluoride membrane. Membranes were blocked using 5% BSA in TTBS (20 mmol/L Tris, 150 mmol/L NaCl, and 0.05% Tween, pH 6.8). Membranes were then incubated with antibodies directed against PKC (C terminus), PKC, phospho-PKC (Thr-505), PKC, phospho-PKC, PKCII, phospho-PKCII, p42/p44, phospho-p42/p44, SERCA2a, col I1, col III (Santa Cruz), JNK and phospho-JNK, p38, phospho-p38, and activated caspase3 (Cell Signaling). Blots were visualized by enhanced chemiluminescence (ECL; Amersham) and densitometrically analyzed.
Statistical Analysis
Data are presented as means±SEM. Differences between groups were analyzed by one-way ANOVA. P value <0.05 was considered statistically significant.
Results
Cardiac Phenotype of KO Mice
Mice with a systemic KO of PKC are born at the expected mendelian ratio and do not show morphological abnormalities in the heart. Myocardial expression of PKC was absent in KO mice, whereas heterozygous KO mice express half of the amount of PKC compared with wild-type (WT) mice (Figure 1A). KO mice have normal heart weight, body weight, and heart-to-body weight ratio compared with WT mice. Echocardiographic analysis showed normal cardiac function (Table 1) and morphometric analysis of in situeCfixed and H&E-stained myocardial cross-sections, showed normal cardiac dimensions, cardiomyocyte CSA, and interstital fibrosis in KO mice (Table 1; Figure 1B).
Pressure Overload Induced by Thoracic Aortic Constriction Triggered a Similar Hypertrophic Response in KO and WT Mice
Four weeks after transverse aortic constriction (TAC), KO and WT mice showed a similar rise in prestenotic blood pressure and a similar myocardial hypertrophy assessed by LV-to-body weight ratio, echocardiography, and morphometric measurements (Table 1). Molecular markers of hypertrophy (ie, ANF and -Sk-actin) increased significantly in LVs of both groups (Figure 1B). Induction of ANF was comparable between WT and KO mice after TAC (NS). SERCA2a mRNA and protein levels were not different between WT and KO and not altered by TAC (Figure 1E).
KO Mice Show Increased Fibrosis and Elevated Expression of Collagen After Pressure Overload
The degree of interstitial fibrosis (collagen I and III) as determined by picro-Sirius red polarization method was significantly higher in LVs of KO mice 4 weeks after TAC (Figure 2A and 2B). Furthermore, immunoblots showed that protein expression of col I1 and collagen III was increased in KO mice 4 weeks after TAC compared with WT (Figure 2C). Real-time PCR showed increased mRNA levels of col I1, indicating that upregulation of col I1 takes place at the transcriptional level in KO mice after TAC (Figure 2D).
KO Mice Display Diastolic Dysfunction After TAC
Echocardiographic analysis showed preserved systolic function in KO and WT mice 4 weeks after TAC (Table 1). However, E/A ratios (transmitral flow pattern) and Ea/Aa ratios (tissue Doppler imaging) were significantly reduced in KO mice compared with WT mice (Table 2; Figure 3), reflecting the development of diastolic dysfunction in KO mice.
PKC Is Upregulated in KO Mice After TAC
Main PKC isoforms expressed in the heart are PKC, PKCII, PKC, and PKC. To test whether the lack of PKC alters the expression of other PKC isoforms, we analyzed protein levels and activation patterns of PKC, PKCII, and PKC in the myocardium of KO and WT mice. Protein levels and activation of PKC and PKCII were not different in WT and KO before and after TAC (Figure 4A and 4B). PKC protein levels and levels of phosphorylated PKC protein (phosphorylation at Thr-505) were increased to a similar extent (2-fold) compared with WT (Figure 4C), indicating that the higher activation of PKC results mainly from an upregulation of PKC protein.
Differential Activation of Mitogen-Activated Protein Kinase Signaling Pathways in KO Mice After Pressure Overload
PKC and PKC activate mitogen-activated protein kinase (MAPK) signaling pathways in cardiomyocytes.24 We did not observe activation of p42/44, p38 MAPKs, and JNK in LVs of KO and WT mice at baseline (Figure 4D through 4F). However, KO mice showed a higher increase in activation of p38 MAPK and JNK1/2 after TAC compared with WT mice (Figure 4E and 4F). In contrast, activation of p42/p44 MAPK was significantly diminished in KO mice after TAC (Figure 4D).
KO Mice Do Not Show Enhanced Apoptosis After TAC
Activation of PKC has been associated with proapoptotic pathways in the heart.24 However, there was no evidence for increased LV apoptosis as assessed by TUNEL assay on LV sections and by immunoblots detecting activated caspase 3 in KO or WT mice at baseline or after TAC (Figure 5B).
Mechanical Stretch Promotes Col I1 Expression in KO Fibroblasts in Vitro via PKC and p38 MAPK Signaling Pathways
Fibroblasts are the major source for collagen expression. To analyze the impact of mechanical load, respectively, the role of PKC and p38 MAPK signaling for col I1 expression in fibroblasts lacking PKC, we exposed primary cultures of adult lung fibroblasts from KO mice to biomechanical stretch in the presence or absence of rottlerin, an inhibitor of PKC (0.3 and 3 eol/L), or of SB 203580 (10 eol/L), a p38 MAPK inhibitor. As depicted in Figure 6A, col I 1 expression was increased by stretch in KO but not in WT fibroblasts. The increase of collagen gene expression was completely blocked by rottlerin (0.3 and 3eol/L) and by SB 203580 (10 eol/L) (Figure 6B). SB 202190 (10 eol/L), a second p38 MAPK inhibitor, also inhibited stretch-induced collagen gene expression in KO fibroblasts (eC80%; P<0.05). Moreover, we analyzed PKC gene expression in WT and KO fibroblasts after stretch by RT-PCR. We observed that PKC gene expression was 1.5-fold (P<0.05) increased in KO fibroblasts after stretch, whereas we did not observe an increase of PKC gene expression in WT fibroblasts.
Discussion
The present study shows that mice lacking PKC develop increased myocardial fibrosis after chronic pressure overload associated with diastolic dysfunction. However, lack of PKC did not prevent the development of a pressure overloadeCinduced cardiac hypertrophy. Interestingly, the lack of PKC is associated with upregulation of PKC and activation of p38 MAPK and JNK after pressure overload, whereas activation of downstream targets of PKC (ie, p42/p44 MAPK) is diminished.
Murine models with cardiac overexpression of PKC or a translocator activator of PKC (RACK) develop myocardial hypertrophy with preserved systolic function, indicating an important role of PKC for the development of myocardial hypertrophy.13,14 Yet PKC-KO mice in our study developed myocardial hypertrophy with preserved systolic LV function in response to pressure overload, clearly indicating that PKC is not required for pressure overloadeCinduced myocardial hypertrophy. Notably, PKC-KO LVs showed increased interstitial fibrosis after TAC compared with WT LVs, mainly because of an increase in collagen I and III expression. Interestingly, the enhanced interstitial myocardial fibrosis in pressure-overloaded KO mice was clearly associated with a pronounced diastolic dysfunction, whereas systolic function was preserved. Diastolic dysfunction may be related to either impaired active early relaxation or diminished passive distensibility.17 Because Doppler indices cannot distinguish between these two components of diastolic function, we cannot exclude that lack of PKC may affect both components. However, increased interstitial fibrosis and preserved expression of SERCA2a in our KO mice are consistent with the notion that diminished passive distensibility causes diastolic dysfunction in KO mice after pressure overload.25
Increased collagen deposition in KO mice after TAC could result from impaired collagen metabolisms because of an imbalance in expression of collagen-degrading enzymes (matrix metalloproteinases ) or their inhibitors (tissue inhibitors of metalloproteinases ). However, we did not observe alterations in the expression of MMP3, MMP9, MMP13, and TIMP1 (data not shown). Alternatively, increased collagen deposition could also result from higher collagen gene expression. Indeed, KO mice contained higher mRNA and protein levels of col I1 and collagen III, suggesting that collagen is upregulated at the transcriptional level in KO mice after pressure overload.
Concerning cardiac collagen metabolism, Liao et al have shown that activation of p38 MAPK leads to marked cardiac interstitial fibrosis, diastolic restriction, and cardiac failure,26 a cardiac phenotype very similar to the one seen in our pressure-overloaded PKC-KO mice. Increased activation of p38 MAPK in LVs of KO mice after TAC further supports the notion that activation of p38 MAPK is responsible for the higher collagen gene expression in KO mice. Besides p38 MAPK, various studies indicate that PKC activation is associated with a profibrotic stage. In this respect, Jimenez et al demonstrated that cultured fibroblasts from patients with systemic sclerosis contain increased PKC amounts.27 Inhibition of PKC by rottlerin diminished collagen I and III synthesis at the transcriptional level mediated through a rottlerin-sensitive 129-bp gene promoter segment.27 This is supported by observations showing that transforming growth factor-1 stimulation and extracellular matrix deposition of human lung fibroblasts, dermal fibroblasts, and hepatic stellate cells requires PKC and p38 MAPK.28eC30 In line with these studies, we demonstrated that KO fibroblasts subjected to stretch showed a 2-fold upregulation of collagen I expression in vitro, which could be completely blocked by addition of the specific PKC inhibitor rottlerin or the specific p38 MAPK inhibitor SB 203580. These data strongly support the notion that myocardial fibrosis in KO mice after chronic pressure overload is likely to be mediated through activation of PKC and p38 MAPK.
Differential activation of PKC isoforms might also be responsible for the preserved ability to develop pressure overloadeCinduced cardiac hypertrophy in KO mice. In fact, for various reasons, it is not unexpected that PKC might compensate for the absence of PKC after chronic pressure overload: (1) both PKCs are activated by the same receptor (Gq), which is stimulated by endothelin-1 and phenylephrine, both of which are hypertrophyeCmediating agonists; (2) in vitro studies showed PKC as well as PKC translocation and activation after stimulation of ventricular cardiomyocytes with endothelin-1 or phenylephrine;12 and (3) Heidkamp et al showed an interdependence between expression levels of PKC and PKC in neonatal rat ventricular myocytes, indicating that PKC and PKC influence their mutual expression levels.24 However, it also has been shown that downstream signaling pathways of PKC and PKC are differentially regulated in neonatal rat ventricular cardiomyocytes, human thyroid cells, and Jurkat T cells.24,31eC33 In this respect, our data are consistent with previous studies in neonatal rat ventricular cardiomyocytes, where PKC preferentially activates p42/44 MAPK,4,24,33,34 whereas JNK and p38 MAPK are preferentially activated by PKC.4,24,33,35 Although these studies support our hypothesis that PKC upregulation leads to p38 MAPK and JNK activation, we cannot exclude that the lack of PKC per se is responsible for the differential MAPK activation. Moreover, we do not know whether increased protein expression of PKC is of cardiomyocyte or nonmyocyte origin. Variability of cell-specific PKC isozymeeCdependent MAPK regulation could account for differential MAPK activation in our KO mice. Future studies will be needed to address these questions.
Despite the fact that overexpression of PKC induces cardiac hypertrophy,13,14,36 our data demonstrate that PKC is not required for pressure overloadeCinduced myocardial hypertrophy. However, PKC activation appears to be a critical rescue event in the transition of the compensated to decompensated stage in heart failure, as indicated by Wu et al,36 showing that PKC activation rescues heart failure in Gq transgenic mice and by our finding that lack of PKC promotes diastolic dysfunction in the pressure-overloaded heart.
In summary, PKC is not required for the development of pressure overloadeCinduced myocardial hypertrophy. However, lack of PKC results in upregulation of PKC and promotes activation of p38 MAPK and JNK, which appear to compensate for cardiac hypertrophy but, in turn, are associated with increased collagen deposition and impaired diastolic function.
Acknowledgments
This study was supported by a grant of the Deutsche Forschungsgemeinschaft (DFG), GK 705, to D.O. and P.S. We thank Silvia Gutzke, Faikah Geer and Birgit Brandt for excellent technical assistance.
Both authors contributed equally to the study.
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