INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

CARDIAC HYPERTROPHY is a compensatory process that leads to a heart better suited for the functional demands caused by myocyte dysfunction or loss. Because cardiac myocytes are terminally differentiated and nonproliferative, hypertrophic myocytes demonstrate an increase in protein content rather than an increase in myocyte number. Cardiac hypertrophy is also associated with sarcomeric reorganization and characteristic changes in gene expression and protein turnover. Cardiac hypertrophy, however, may independently lead to cardiac dysfunction and is also a common feature associated with the heart failure state. Despite advancements in the treatment and understanding of the pathogenesis of heart failure, the cellular and molecular mechanisms involved in hypertrophy induction are still largely unknown.

Recent studies (12, 17, 18) have suggested that nonreceptor protein tyrosine kinases are important in transducing mechanical stimuli to biochemical pathways that lead to cardiomyocyte hypertrophy. Proline-rich tyrosine kinase 2 (PYK2) and focal adhesion kinase (FAK) are members of the FAK family of nonreceptor protein tyrosine kinases. PYK2 and FAK have been implicated in linking G protein-coupled receptors to activation of mitogen-activated protein kinase (MAPK) cascades and cellular growth in a variety of cultured cells (6, 7, 19, 21, 30, 34, 38). PYK2 and FAK are highly homologous, sharing 45% amino acid sequence identity with 60% identity in the catalytic domain. Unlike FAK, which is predominantly localized to focal adhesions, PYK2 is largely cytosolic (2, 3, 25, 29) and its phosphotyrosine content is independent of cell adhesion when expressed in COS cells (29). The noncatalytic regions of FAK and PYK2 contain proline-rich residues (capable of binding proteins with SH3 domains), a tyrosine autophosphorylation site (Src family tyrosine kinase SH2 binding site), and other tyrosine residues, including an adapter protein Grb2 SH2-binding site.

Our laboratory (3) recently reported the first description of PYK2 expression and phosphorylation in cultured neonatal rat ventricular myocytes (NRVM). We (3) also demonstrated that PYK2 expression was increased in cardiomyocytes undergoing contraction-induced hypertrophy, and the hypertrophic agonist endothelin-1 (ET) activated PYK2 in both NRVM and freshly isolated adult rat cardiomyocytes. Similarly, previous studies (9, 10) from our laboratory have shown that FAK is activated in response to ET stimulation of NRVM and that FAK plays a critical role in ET- and contraction-induced NRVM hypertrophy. Several investigators (12, 17, 18) have also shown a role for FAK in acute pressure-overload models of hypertrophy.

In this study, we have examined PYK2 and FAK expression and phosphorylation in an in vivo model of pressure overload-induced left ventricular (LV) hypertrophy (LVH). This model of suprarenal aortic coarctation has been extensively characterized in our laboratory (5, 11, 20, 24) and consists of three stages. During the first phase, banded animals develop concentric LVH slowly over an 8-wk period, followed by an established phase of compensatory LVH. Once established, LVH progresses to heart failure (16-24 wk), with the development of diastolic dysfunction and interstitial fibrosis. Characteristic alterations in LV protein content and -myosin heavy chain (MHC), atrial natriuretic factor, and sarco(endo)plasmic reticulum Ca²⁺ ATPase-2 mRNA and protein levels are evident during disease progression (5, 11, 20, 24). Therefore, this model is ideal for examining in vivo PYK2 and FAK in relation to the induction of LVH and its transition to heart failure. Our results are consistent with a role for PYK2 in the induction of pressure overload-induced cardiomyocyte hypertrophy, and suggest that PYK2 and FAK may have distinctly different roles in LVH progression.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Animals. Male Sprague-Dawley rats (160 g) were purchased from Harlan (Indianapolis, IN). The animals used in these experiments were handled in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85-23, Revised 1985).

Reagents. PC-1 tissue culture medium was obtained from BioWhittaker (Walkersville, MD). Dulbecco's modified Eagle's medium (DMEM) was purchased from GIBCO-BRL (Grand Island, NY). Medium 199 (M199), Ca²⁺-free and Mg²⁺-free Hanks' balanced salt solution, acid-soluble calf skin collagen, and antibiotic-antimycotic solution were obtained from Sigma (St. Louis, MO). Type II collagenase was aquired from Worthington (Lakewood, NJ). DMEM/F-12 and penicillin-streptomyosin were obtained from Fisher Scientific MediaTech (Itasca, IL). Tissue culture plates were obtained from Costar (Cambridge, MA) and chamber slides from Nunc (Naperville, IL). FAK polyclonal antibody (PAb) and phosphotyrosine monoclonal antibody (MAb) were acquired from Upstate Biotechnologies (Lake Placid, NY). Phosphospecific anti-FAK Y397 (pFAK₃₉₇) PAb was obtained from Biosource (Camarillo, CA). PYK2 MAb was obtained from Transduction Laboratories (Lexington, KY). PYK2 PAb used for immunoprecipitation was obtained from Santa Cruz Biotechnology (Santa Cruz, CA), whereas PYK2 PAb used for immunohistochemistry was kindly provided by Dr. H. Avraham (Harvard Medical School, Boston, MA). MHC PAb for Western blotting was raised in guinea pigs, as previously described (28). MHC MAb used for confocal microscopy was obtained from the Developmental Studies Hybridoma Bank maintained by the Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, and the Department of Biological Sciences, University of Iowa. Immunohistochemistry was performed using Vectastain Elite ABC kit (Vector Laboratories; Burlingame, CA). Protein G Sepharose beads and horseradish peroxidase-conjugated goat anti-mouse or anti-rabbit IgG were obtained from Sigma. All other reagents were of the highest grade commercially available and were purchased from Sigma and Baxter (McGaw Park, IL).

NRVM isolation. NRVM were isolated from hearts of 2-day-old Sprague-Dawley rats by multiple collagenase digestions as previously described (27). Myocytes were preplated for 1 h in serum-free PC-1 medium to reduce nonmyocyte contamination. Myocytes in PC-1 medium were then plated at various densities onto collagen-coated dishes and left undisturbed in a 5% CO₂ incubator for 18 h. Unattached myocytes were removed, and the attached myocytes were maintained in a solution of DMEM-M199 (4:1) containing antibiotic-antimycotic solution. The medium was changed daily.

Experimental animals. Male Sprague-Dawley rats weighing 160-170 g were anesthetized with an intramuscular injection of ketamine (90 mg/kg) and xylazine (10 mg/kg). Constriction of the suprarenal abdominal aorta was produced using a tantalum hemoclip (Weck). The applicator was modified to allow passage of a 25-gauge needle through the hemoclip when completely closed. Sham-operated animals underwent dissection of the abdominal aorta without application of the suprarenal band. Postoperatively, all animals received food and water ad libitum. Before the animals were euthanized, arterial blood pressure was recorded by carotid artery catheterization. There was a minimum of seven animals in each group.

Immunoprecipitation and Western blotting. Adult rat LV tissue was homogenized in lysis buffer composed of 50 mM HEPES (pH 7.4), 150 mM NaCl, 10% glycerol, 1.5 mM MgCl₂, 1.0 mM EGTA, 1.0 mM Na₃VO₄, 10 mM Na pyrophoshate, 100 mM NaF, 1% Triton X-100, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 10 µg/ml leupeptin and aprotinin, and 1 mM Pefabloc. For Western blots, equal amounts of extracted protein were separated on 10% SDS-polyacrylamide gels with 5% stacking gels. For immunoprecipitation and Western blotting, equal amounts of extracted cell proteins were immunoprecipitated with anti-PYK2 PAb overnight at 4°C, followed by the addition of protein G Sepharose beads. Proteins were transferred to a polyvinylidene difluoride membrane using the recommended transfer buffer. Western blots were probed with an antibody specific for PYK2, FAK, the phosphorylated form of FAK at Y397, or MHC. Each immunoprecipitation blot was probed using anti-phosphotyrosine (pTyr) mAb. Primary antibody binding was detected with horseradish peroxidase-conjugated goat anti-mouse, anti-rabbit, or anti-guinea pig secondary antibody and visualized by enhanced chemiluminescence (Amersham; Arlington Heights, IL). Band intensity was quantified with the use of laser densitometry.

Immunohistochemistry. Cardiac tissue was frozen in liquid nitrogen followed by isopentane and then cut into 16-µm-thick sections. Immunohistochemistry was performed using Vectastain Elite ABC kit and used according to the manufacturer's instruction. Sections were stained with anti-PYK2 PAb (1:2,000) or anti-FAK PAb (1:200) in a closed chamber, followed by biotinylated goat anti-rabbit secondary antibody. Sections were viewed using a Nikon Eclipse E600 Microscope with the Digital Science Microscopy Documentation System MDS120 and PhotoEnhancer software (Eastman Kodak; Rochester, NY). A standard Masson trichrome staining procedure was used to detect regions of collagen deposition on adjacent tissue sections. Confocal microscopy of similar frozen tissue sections stained with antibodies specific for PYK2, FAK, and MHC was performed as previously described (3).

Data analysis. Results were expressed as means ± SE. Normality was assessed using the Kolmogorov-Smirnov test. Data were compared using one-way blocked analysis of variance (ANOVA), followed by Student-Newman-Keuls test, one-way blocked ANOVA on signed-ranks test, followed by Dunn's test or unpaired t-test, where appropriate. Differences among means were considered significant at P < 0.05. Data were analyzed with SigmaStat statistical software (version 2.0, Jandel Scientific; San Rafael, CA).

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

PYK2 expression is increased in hypertrophied NRVM. To confirm that PYK2 expression was increased in NRVM undergoing hypertrophy in vitro, myocytes were plated at low density (100-125 cells/mm²) and cultured in serum-free medium or medium containing 100 nM ET or 200 nM phorbol 12-myristate 13-acetate (PMA) for 48 h. Both agents have been shown to induce cardiomyocyte hypertrophy in low-density NRVM cultures (8, 10). Furthermore, our laboratory (3) has shown that ET-induced NRVM hypertrophy was accompanied by an increase in the level of phosphorylated PYK2. As seen in Fig. 1A, chronic ET and PMA treatment both significantly increased the level of PYK2 expression by 2.3 ± 0.2- and 1.7 ± 0.3-fold, respectively.

View larger version (34K): [in this window] [in a new window]   Fig. 1.   Proline-rich tyrosine kinase 2 (PYK2) expression and phosphorylation are increased in hypertrophied neonatal rat ventricular myocytes (NRVM). NRVM were plated at low density and maintained in serum-free medium or in medium containing 100 nM endothelin (ET)-1 or 200 nM phorbol 12-myristate 13-acetate (PMA) for 48 h. Cell extracts (25-100 µg of total protein) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting with anti-PYK2 monoclonal antibody (MAb) (A) and anti-focal adhesion kinase (FAK) polyclonal antibody (PAb) (B). Left, representative blots are depicted. The position of molecular weight markers is indicated to the left of the Western blots. Right, quantitative analysis of 3-4 Western blotting experiments are depicted. CTL, control. Data are means ± SE. *P < 0.05 vs. untreated controls by one-way blocked analysis of variance (ANOVA), followed by Student-Newman-Keuls test.

Aortic banding produced sustained hypertension and gradually developing LVH. To examine whether the differential changes in PYK2 and FAK expression were observed in an in vivo model of hypertrophy, we examined the expression and phosphorylation of PYK2 and FAK in an adult rat model of pressure-overload-induced LVH. This suprarenal abdominal aortic coarctation model has been extensively characterized in our laboratory (5, 11, 20, 24) and consists of three relatively distinct stages. Animals develop hypertension and LVH over an 8-wk period so that animals euthanized within 1 wk after suprarenal abdominal coarctation represent a group that are in the induction phase of LVH. Animals euthanized at 8 wk represent a group that is in the established phase of compensatory LVH. These animals have normal systolic function but have increased heart weight-to-body weight ratio (HW/BW) and evidence of slowed isovolumic relaxation indicative of early diastolic dysfunction. Animals euthanized at 24 wk represent a group that is in a transition phase from compensatory LVH to heart failure. These rats have markedly elevated LV end-diastolic pressure and markedly prolonged isovolumic relaxation, indicative of reduced ventricular compliance and diastolic heart failure (24).

Increased PYK2 expression precedes the onset of LVH. To determine whether PYK2 levels were altered in cardiomyocytes undergoing LVH progression, PYK2 expression was examined in LV tissue extracts from 1-, 8-, and 24-wk sham or banded animals by SDS-polyacrylamide gel electrophoresis (PAGE) and Western blotting with anti-PYK2 MAb. Representative blots for PYK2 are depicted in Fig. 2A, and the quantitative analysis of Western blotting experiments from 1-, 8-, and 24-wk animals are summarized in Fig. 2B. PYK2 levels were increased 1.8 ± 0.2-, 2.7 ± 0.6-, and 2.0 ± 0.2-fold in 1-, 8-, and 24-wk banded animals, respectively, compared with their time-matched sham-operated controls. Of note, the increase in PYK2 expression preceded the development of significant LVH. As expected, there was no significant correlation (R = 0.22; P = 0.42) between the elevated levels of PYK2 and HW/BW ratios in 1-wk sham and banded rats. Once established, however, there was a high degree of correlation (R = 0.81; P < 0.001) between the level of PYK2 and HW/BW ratios in the 8-wk sham and banded animals (data not shown). There was also a high degree of correlation between PYK2 and HW/BW ratios in the 24-wk sham and banded animals (R = 0.75; P < 0.001) (Fig. 2C).

View larger version (32K): [in this window] [in a new window]   Fig. 2.   Increased PYK2 expression precedes the onset of left ventricular (LV) hypertrophy (LVH). A: LV tissue extracts from 1-, 8-, or 24-wk sham or banded animals were separated by SDS-PAGE, followed by Western blotting with anti-PYK2 MAb. Representative Western blots for PYK2 (400 µg tissue protein) are depicted. The position of molecular weight markers is indicated to the left of the Western blots. B: quantitative analysis of Western blotting experiments from 1-, 8-, and 24-wk animals is depicted. Data are means ± SE for 7-16 animals in each group; *P < 0.05 vs. time-matched, sham-operated controls by unpaired t-test. C: heart weight-to-body weight ratios (HW/BW) are plotted vs. normalized PYK2 expression in 24-wk sham and banded animals. PYK2 expression was normalized to the level of expression in time-matched, sham-operated controls. Data were analyzed by linear regression for 13-16 animals in each group.

Tyrosine phosphorylated PYK2 is also increased before development of LVH. Previous studies (25) from our laboratory have demonstrated a close correlation between tyrosine phosphorylation of PYK2 and increased PYK2 tyrosine kinase activity. PYK2 tyrosine phosphorylation also accompanies NRVM hypertrophy in vitro (3) and is required for downstream signaling to MAPK cascades in other cell types (6, 7, 19, 21, 30, 34). Therefore, we evaluated the level of tyrosine phosphorylation of PYK2 in the same tissue extracts from 1-, 8-, and 24-wk sham and banded animals. LV tissue extracts were immunoprecipitated with anti-PYK2 PAb, separated by SDS-PAGE, and subjected to Western blotting with an anti-pTyr MAb. As seen in Fig. 3, A and B, the amount of tyrosine-phosphorylated PYK2 (pPYK2) was increased in the 1-wk banded animals and remained elevated in the 8- and 24-wk banded animals (1.7 ± 0.2-, 1.5 ± 0.2-, and 2.4 ± 0.4-fold in 1-, 8-, and 24-wk banded animals, respectively, compared with their time-matched sham-operated controls). As was the case for total PYK2, the increase in pPYK2 levels preceded the development of LVH. This was manifested by increased pPYK2 levels without a significant correlation with HW/BW ratio in 1-wk banded animals (R = 0.04; P = 0.88). However, there was a high degree of correlation (R = 0.76; P < 0.05) between the level of pPYK2 and HW/BW ratios in the 8-wk sham and banded animals (data not shown). There was also a high degree of correlation (R = 0.68; P < 0.001) between pPYK2 and HW/BW ratios in the 24-wk sham and banded animals (Fig. 3C). However, there was no significant difference in the ratio of pPYK2 to total PYK2 at any time point examined (Fig. 3D).

View larger version (27K): [in this window] [in a new window]   Fig. 3.   Increased phosphotyrosine (pTyr) PYK2 (pPYK2) expression also precedes the onset of LVH. A: LV tissue extracts (2,500 µg tissue protein) were immunoprecipitated (IP) with anti-PYK2 PAb, followed by addition of protein G Sepharose beads. Immunoprecipitates were separated by SDS-PAGE, and the resulting Western blots probed with anti-pTyr MAb. Representative blots are depicted. The position of molecular weight markers is indicated to the left of the Western blots. B: quantitative analysis of immunoprecipitation and Western blotting experiments from 1-, 8-, and 24-wk animals is depicted. Data are means ± SE for 7-16 animals in each group. C: HW/BW ratios are plotted vs. normalized pPYK2 expression in 24-wk sham and banded animals. pPYK2 expression was normalized to the level of expression in time-matched sham-operated controls. Data were analyzed by linear regression for 13-16 animals in each group. D: quantitative analysis of the ratio of pPYK2 to total PYK2 in 1-, 8-, and 24-wk animals is depicted. Data are means ± SE for 7-16 animals in each group. *P < 0.05 vs. time-matched, sham-operated controls by unpaired t-test.

Increased PYK2 expression occurred predominantly in cardiomyocyte population. To ensure that the increased PYK2 expression in LV tissue extracts occurred specifically in cardiomyocytes, tissue sections were evaluated by immunohistochemistry using a PYK2-specific PAb. As shown in Fig. 4A, PYK2 was localized throughout the myocardium of sham-operated animals, including the cardiomyocytes, and the medial layer of penetrating blood vessels. Aortic banding for 8 wk induced obvious cardiomyocyte hypertrophy (Fig. 4B). The increased PYK2 expression that we noted by Western blotting in banded animals appeared to occur predominantly in the cardiomyocyte population. To further ensure that PYK2 was expressed predominantly in cardiomyocytes, confocal microscopy was used to examine colocalization of PYK2 and MHC in 8-wk banded animals. As seen in Fig. 4C, diffuse PYK2 staining was only observed in cells that also stained positively for MHC, indicating that PYK2 was indeed present in cardiomyocytes.

View larger version (70K): [in this window] [in a new window]   Fig. 4.   Increased PYK2 expression occurred predominantly in the cardiomyocyte population. Frozen sections of 8-wk sham (A) or 8-wk banded (B) LV tissue were stained with anti-PYK2 PAb (1:2,000), followed by biotinylated goat anti-rabbit secondary antibody. Sections were viewed using a Nikon Eclipse E600 microscope with Kodak Digital Science Microscopy Documentation System MDS120 and PhotoEnhancer software. C: similar frozen sections of 8-wk banded LV tissue were stained with anti-PYK2 (1:100) or myosin heavy chain (MHC) monoclonal antibody (MF20) (1:3) antibodies. Primary antibody localization was detected with fluorescein isothiocyanate (FITC)-conjugated donkey anti-rabbit IgG (green) or rhodamine-conjugated goat anti-mouse IgG (red). Sections were viewed using a Zeiss LSM 510 confocal microscopy. Right, areas of colocalization appear yellow. Middle, white bar represents 100 µm.

FAK levels increase during established phase of compensatory LVH and the transition from compensated LVH to heart failure. We next evaluated whether FAK expression was altered in myocytes undergoing LVH. Tissue extracts from 1-, 8-, and 24-wk animals were analyzed by Western blotting with an antibody specific for FAK. In contrast to the increase in PYK2 expression, FAK levels were not significantly increased in 1-wk banded animals compared with their time-matched sham-operated controls (Fig. 5, A and B). However, FAK levels were increased 2.4 ± 0.4- and 2.1 ± 0.2-fold in 8- and 24-wk banded animals, respectively. These data also revealed that during the transition from compensatory LVH to heart failure, there was a high degree of correlation between the degree of hypertrophy and FAK expression. There was no correlation between FAK levels and HW/BW ratios in 1- or 8-wk animals (data not shown). However, there was a high degree of correlation between HW/BW ratios and FAK expression in the 24-wk sham and banded animals (R = 0.68; P < 0.001) (Fig. 5C).

View larger version (37K): [in this window] [in a new window]   Fig. 5.   FAK levels increase during the established phase of compensatory LVH and the transition from compensated LVH to heart failure. A: LV tissue extracts from 1-, 8-, or 24-wk sham or banded animals were separated by SDS-PAGE, followed by Western blotting with anti-FAK PAb. Representative Western blots for FAK (200 µg tissue protein) are depicted. The position of molecular weight markers is indicated to the left of the Western blot. B: quantitative analysis of Western blotting experiments from 1-, 8-, and 24-wk animals is depicted. Data are means ± SE vs. time-matched, sham-operated control for 7-16 animals in each group. *P < 0.05 by unpaired t-test. C: HW/BW ratios are plotted vs. normalized FAK expression in 24-wk sham and banded animals. FAK expression was normalized to the level of expression in time-matched, sham-operated controls. Data were analyzed by linear regression for 13-16 animals in each group.

Phosphorylated FAK levels also increase during established phase of compensatory LVH and transition from compensated LVH to heart failure. Previous studies from our laboratory (10, 13) have shown that FAK undergoes tyrosine phosphorylation in response to growth factor stimulation in both cardiomyocytes and smooth muscle cells, and FAK phosphorylation is necessary for activation of downstream signaling cascades leading to cytoskeletal reorganization and cellular growth. Therefore, we then examined whether the level of pFAK was altered in cardiac tissue extracts from animals during LVH progression. Representative Western blots and quantitative analysis of Western blotting experiments from 1-, 8-, and 24-wk banded animals are depicted in Fig. 6, A and B. As is evident from the figure, pFAK levels were not increased in 1-wk banded animals compared with their time-matched, sham-operated controls. However, pFAK levels were increased 3.0 ± 0.6- and 3.2 ± 0.4-fold in 8- and 24-wk banded animals, respectively. Similarly, there was no correlation between pFAK levels and HW/BW ratios in 1- or 8-wk banded animals (data not shown). However, there was a high degree of correlation between HW/BW ratios and pFAK expression in the 24-wk banded animals (R = 0.69; P < 0.001) (Fig. 6C). Interestingly, there was no difference in the ratio of pFAK to total FAK in 1- and 8-wk banded animals, but there was a significant increase in this ratio in 24-wk banded animals compared with their time-matched controls (Fig. 6D).

View larger version (30K): [in this window] [in a new window]   Fig. 6.   Phosphorylated FAK levels also increase during the established phase of compensatory LVH and the transition from compensated LVH to heart failure. A: LV tissue extracts from 1-, 8-, or 24-wk sham or banded animals were separated by SDS-PAGE, followed by Western blotting with anti-pFAK PAb. Representative Western blots for pFAK (200 µg tissue protein) are depicted. The position of molecular weight markers is indicated to the left of the Western blot. B: quantitative analysis of Western blotting experiments from 1-, 8-, and 24-wk animals is depicted. pFAK was detected using an antibody that is specific for the autophosphorylation site at Y397. Data are means ± SE for 7-16 animals in each group; *P < 0.05 vs. time-matched, sham-operated controls by unpaired t-test. C: HW/BW ratios are plotted vs. normalized pFAK expression in 24-wk sham and banded animals. pFAK expression was normalized to the level of expression in time-matched, sham-operated controls. Data were analyzed by linear regression for 13-16 animals in each group. D: quantitative analysis of the ratio of pFAK to total FAK in 1-, 8-, and 24-wk animals is depicted. Data are means ± SE for 7-16 animals in each group. *P < 0.05 vs. time-matched, sham-operated controls by unpaired t-test.

Increased FAK expression occurred in both cardiomyocyte population and cardiac interstitium. Our laboratory (11) has shown that suprarenal abdominal aortic coarctation is associated with fibroblast hyperplasia and late interstitial and perivascular fibrosis occurring within 16 wk of sustained hypertension. Because FAK is highly expressed in both cardiomyocytes and fibroblasts, we examined whether the increased FAK expression occurred predominantly in either the cardiomyocyte or interstitial fibroblast population. Tissue sections from 24-wk sham and banded animals were evaluated by immunohistochemistry with a FAK-specific PAb. Although FAK was detected in cardiomyocytes of both sham and banded animals, much of the increased FAK expression noted by Western blotting in the 24-wk banded extracts appeared to occur in the cardiac interstitium compared with the sham-operated controls (Fig. 7A). To determine whether the increased FAK staining was present in areas of active collagen deposition, adjacent tissue sections were stained with Masson trichrome to examine collagen deposition as a marker for interstitial fibrosis. As shown in Fig. 7B, and in agreement with our previous results (11), there were numerous areas of focal interstitial, as well as perivascular fibrosis in 24-wk banded animals (Fig 7B, right). These areas of fibrosis corresponded to regions of enhanced FAK staining, suggesting that the increased FAK expression in the cardiac interstitium was derived from proliferating cardiac fibroblasts (Fig. 7A, asterisks). However, there also appeared to be increased FAK expression in some cardiomyocytes adjacent to areas of fibrosis (Fig. 7A, right, arrows). To further characterize these areas of enhanced FAK staining, confocal microscopy was used to examine colocalization of FAK and MHC in 24-wk banded animals. As seen in Fig. 7C, FAK colocalized with MHC, indicating that FAK was present in cardiomyocytes. However, there were areas of FAK staining within the cardiac interstitium that did not stain positively with anti-MHC antibody (Fig. 7C, right, white arrowheads). These areas appeared as linear densities near penetrating blood vessels, which were similar to the enhanced staining observed by immunohistochemistry, as in Fig. 7A. Similar frozen sections of 8-wk banded animals were examined for the presence of FAK staining in cardiomyocytes and in the interstitial space around penetrating blood vessels. In contrast to the 24-wk banded animals, FAK staining was only observed in the cardiomyocytes (data not shown).

View larger version (98K): [in this window] [in a new window]   Fig. 7.   Increased FAK expression occurred in both the cardiomyocyte population and cardiac interstitium. A: frozen sections of 24-wk sham or banded LV tissue were stained with anti-FAK PAb (1:200), followed by biotinylated goat anti-rabbit secondary antibody. The asterisks refer to FAK staining in the cardiac interstitium and the arrows indicate enhanced FAK staining in the cardiomyocyte population. B: adjacent sections were stained for interstitial collagen using the Masson trichrome method. Sections were viewed using a Nikon Eclipse E600 Microscope with Kodak Digital Science Microscopy Documentation System MDS120 and PhotoEnhancer software. C: similar frozen sections of 24-wk banded LV tissue were stained with anti-FAK (1:100) or MF20 (1:3) antibodies. Primary antibody localization was detected with FITC-conjugated, donkey anti-rabbit IgG (green) or rhodamine-conjugated, goat anti-mouse IgG (red). Sections were viewed using a Zeiss LSM 510 confocal microscopy. Areas of colocalization appear yellow. Middle, white bar represents 100 µm. Right, white arrowheads indicate FAK staining in the cardiac interstitium.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

In this study, we examined PYK2 and FAK expression and phosphorylation in both in vitro and in vivo models of cardiac hypertrophy. We recently published the first description that PYK2 expression was increased in NRVM undergoing contraction-induced hypertrophy. Furthermore, the hypertrophic agonist ET activated PYK2 in both NRVM and freshly isolated, adult rat cardiomyocytes (3). Similarly, our laboratory and others (10, 16, 22, 33) have demonstrated that FAK phosphorylation is necessary for induction of NRVM hypertrophy. The in vitro studies in the present study revealed that PYK2 expression was increased in ET- and PMA-induced NRVM hypertrophy, whereas FAK expression and phosphorylation remained unchanged. Similarly, our laboratory has previously shown that ET-induced NRVM hypertrophy was associated with a chronic increase in pPYK2, but not tyrosine-phosphorylated FAK (3, 10). Taken together, these data suggest that PYK2 is involved in the induction of NRVM hypertrophy and that PYK2 and FAK have distinctly different roles in these cell culture models of cardiomyocyte growth.

PYK2 expression and phosphorylation has not been previously examined in cardiomyocytes undergoing LVH in vivo. However, several investigators have shown that FAK is activated in response to an acute pressure overload (12, 17, 18), but its expression and phosphorylation during cardiac hypertrophy progression has not been previously described. Suprarenal abdominal aortic coarctation was used in the present study to examine PYK2 and FAK in an in vivo model of pressure-overload-induced hypertrophy. The use of this model was ideal for several reasons. First, this model resembles the pathological progression of cardiac hypertrophy from its induction, until its transition to heart failure. Second, this model allowed us to examine PYK2 and FAK expression and phosphorylation at different phases of disease progression to ascertain at which phases these kinases may be involved. Third, this model has been extensively characterized in our laboratory with respect to organ and cellular function, as well as to changes in gene expression associated with the hypertrophic phenotype (5, 11, 20, 24). Our results indicate that the alterations in PYK2 expression and phosphorylation observed in the cell culture models of hypertrophy were indeed similar to this in vivo model of pressure-overload-induced LVH.

In addition to its role in the induction of cardiomyocyte hypertrophy, our data suggest that PYK2 may be involved in the signaling pathways that are responsible for the transition to heart failure. Other reports have indicated that PYK2 and FAK may function differently within the same cell type. Zhao et al. (39) demonstrated that PYK2 and FAK differentially regulate cell cycle progression in NIH3T3 cells. These differences were in part due to their opposing effects on c-jun NH₂-terminal kinases (JNK) and extracellular signal-regulated kinase (ERK) activation and the ability of FAK and PYK2 to compete for binding to Src-family kinases. Similarly, PYK2 overexpression has been shown to induce apoptosis (36), whereas FAK overexpression promotes cell survival (4). It is conceivable that the increased expression and phosphorylation of both PYK2 and FAK in cardiomyocytes during the transition to heart failure results in differential activation of downstream signaling to individual MAPK cascades. Of note, we (14) have recently shown that differential activation of ERKs and JNKs in cultured NRVM is associated with cardiomyocyte hypertrophy and apoptosis, respectively. However, proof of this speculation will require in vivo experimental models that incorporate either loss- or gain-of-function mutations in FAK and PYK2.

There are several signaling components that have been associated with the induction of load-induced hypertrophy, including G proteins, Src family kinases, MAPKs, and protein kinase C (PKC) (1, 15, 17, 26, 31, 35, 37). However, involvement of PYK2 in these signaling pathways has not been well characterized in cardiomyocytes. Ping et al. (23) have recently shown that PYK2 is part of a multicomponent signaling complex that contains PKC- and Src, phosphatidylinositol 3-kinase, and all three members of the MAPK family. Transgenic overexpression of constitutively active PKC- induced cardiomyocyte hypertrophy (32) and increased expression of PYK2 as well as other signaling components that bind to PKC- (23). These data as well as our own lead us to speculate that PYK2 may indeed be a key regulator of at least some of the signaling pathways leading to the induction of cardiac hypertrophy and its progression to heart failure.

As with PYK2, FAK contains several tyrosine residues and proline-rich domains that are also capable of binding Src family kinases and adapter proteins. FAK has been shown in vitro and in vivo to form multicomponent complexes with Src, Shc, Grb2, and/or p130Cas in cardiomyocytes (12, 16-18). Further studies are necessary to elucidate the specific roles that PYK2 and FAK have in cardiac hypertrophy progression. An understanding of these pathways would provide valuable insight into how these signaling cascades are regulated and how they lead to a switch from compensatory hypertrophy to heart failure.