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The conserved phosphoinositide 3‐kinase pathway determines heart size in mice

The conserved phosphoinositide 3‐kinase pathway determines heart size in mice The EMBO Journal Vol. 19 No. 11 pp. 2537-2548, 2000 The conserved phosphoinositide 3-kinase pathway determines heart size in mice Among various signaling pathways, growth hormone, Tetsuo Shioi, Peter M.Kang, insulin-like growth factor (IGF)-1 and their downstream Pamela S.Douglas, James Hampe, 1 2 effectors are particularly implicated in the regulation of Claudine M.Vballe , Joel Lawitts , 1 3 body and organ size (Conlon and Raff, 1999). IGF-1 and Lewis C.Cantley and Seigo lzumo their downstream effectors, such as insulin receptor Cardiovascular Division, Division of Signal Transduction and 86K substrate (IRS)-1 and p70 S6 kinase (p70 ), play an 1'ransgenic Facility, Beth Israel Deaconess Medical Center and important role in body size determination in mammals Departments of Medicine and Cell Biology, Harvard Medical School, (DeChiara et al., 1990; Baker et al., 1993; Liu et al., 1993; Boston, MA 02215, USA Araki et al., 1994; Tamemoto et al., 1994; Shima et al., Corresponding author 1998; Withers et al., 1998). Recent genetic studies in e-mail: [email protected] Drosophila also revealed effectors of insulin-like protein play a critical role in the determination of organ size as Phosphoinositide 3-kinase (PI3K) has been shown to well as body size (Leevers et al., 1996; Bohni et al., 1999; regulate cell and organ size in Drosophila, but the role Weinkove et al., 1999). of PI3K in vertebrates in vivo is not well understood. Phosphoinositide 3-kinase (PI3K) lies downstream of To examine the role of PI3K in intact mammalian many receptor tyrosine kinases including insulin and tissue, we have created and characterized transgenic IGF-1 receptors. PI3Ks play crucial roles in many aspects mice expressing constitutively active or dominant­ of biological response, such as membrane trafficking, negative mutants of PI3K in the heart. Cardiac­ cytoskeletal organization, cell growth and apoptosis specific expression of constitutively active PI3K (Toker and Cantley, 1997; Rameh and Cantley, 1999). resulted in mice with larger hearts, while dominant­ The genes encoding PI3Ks have been isolated from a wide negative PI3K resulted in mice with smaller hearts. range of tissues and organisms. They are divided into three The increase or decrease in heart size was associated major classes based on their amino acid sequence, the with comparable increase or decrease in myocyte size. homology among their lipid-kinase domains and substrate Cardiomyopathic changes, such as myocyte necrosis, specificity (Vanhaesebroeck et al., 1997; Fruman et al., apoptosis, interstitial fibrosis or contractile dysfunc­ 1998). Class I PI3Ks phosphorylate the 3-position of the tion, were not observed in either of the transgenic inositol ring of phosphatidylinositol (Ptdlns), Ptdlns mice. Thus, the PI3K pathway is necessary and suffi­ 4-phosphate and Ptdlns 4,5-diphosphate to form Ptdlns cient to promote organ growth in mammals. 3-phosphate, Ptdlns 3,4-diphosphate and Ptdlns 3,4,5- Keywords: cell size/heart/hypertrophy/phosphoinositide trisphosphate, respectively. Class I PI3Ks can be further 3-kinase/transgenic mice divided into two subclasses. The class IA PI3Ks, which include at least three isozymes, a, � and B, are heterodimers of the catalytic 110 kDa subunit (pll0) and the regulatory subunit of 85 or 55 kDa (p85/p55). The Introduction interaction of p85/p55 and pll0 via the inter-Src One of the least understood areas in biology is how the size homology 2 (iSH2) domain of p85/p55 and the of animals and their organs is determined (Conlon and N-terminal 123 amino acids of pl 10 is critical for Raff, 1999). In most animals, determination of the organ achieving the maximal activity of this class of PI3Ks in size is achieved by controlling the number of cells. mammalian cells (Dhand et al., 1994; Klippel et al., 1994; However, in certain tissue such as cardiac or skeletal Yu et al., 1998). muscle, hypertrophy (increase in cell size without cell A serine/threonine kinase Akt, also known as protein division) plays a major role in determining the organ mass. kinase B, is the most well characterized target of PI3K In particular, the cardiac myocyte withdraws from the cell (Alessi and Cohen, 1998; Downward, 1998; Chan et al., cycle soon after birth and the postnatal growth of the heart, 1999). Akt is known to mediate cell survival signal by which is essential to meet the increasing demand for regulating several effectors such as Bad or procaspase-9 cardiac work to support animal growth, is achieved (Datta et al., 1997; Cardone et al., 1998). Another protein 86K primarily by hypertrophy (Soonpaa and Field, 1998). serine/threonine kinase downstream of PI3K is p70 , Extensive studies over the last decade have yielded which is known to be a physiological kinase for the significant information on neurohumoral growth factors ribosomal S6 protein whose phosphorylation increases the involved in cardiac hypertrophy utilizing cultured cardiac rate of initiation of translation of mRNA by ribosomes myocytes as a model system (Chien et al., 1991; (Chou and Blenis, 1995; Thomas and Hall, 1997). Sadoshima and Izumo, 1997). However, the mechanism Despite the wealth of knowledge of biochemical and of cell-size regulation in cardiac myocytes in the intact biological effects of PI3K in cultured cells, little informa­ heart is still not well understood (Sugden and Clerk, 1998). tion is available regarding its physiological roles in the © European Molecular Biology Organization 2537 T.Shioi et al. intact animal. Targeted deletion of the pl lOa catalytic subunit (Bi et al., 1999) or the p85a regulatory subunit NTg Tg -- - --0- (Fruman et al., 1999) in mice resulted in embryonic or IP Q. perinatal lethality, respectively. This makes it difficult to ◄iSH2p110 Blot; anti•p110 examine the role of PI3K in the later developmental ◄p110 process. Interestingly, a previous study utilizing tissue­ ◄ �K:ll)l 10 e:1O1: arrti-Myc specific expression of constitutively active (ea) or 1 • 1 3 dominant-negative (dn) PI3K in Drosophila suggested a cell-autonomous role of PI3K in the determination of organ size (Leevers et al., 1996). However, there is greater IP p 1 1 0 __ __ genetic diversity in vertebrates than in lower metazoans, -- - -' _ BLIJH LiK Sk and in many cases functional redundancy exists that is not Blot; antl• Myc ◄iSH2p110 revealed in less complex organisms (Miklos and Rubin, 1996). To examine the role of PI3K in organ size determination in mammals, we have made transgenic mice expressing constitutively active or dominant­ negative mutants of PI3K specifically in the heart. JP --'-p1_10 __ PIP-. Results Production of constitutively active P/3K (caP/3KJ transgenic mice iSH2pl 10 (Franke et al., 1997b) is a chimeric molecule that contains the iSH2 domain of p85 fused to the N-terminus of bovine pl lOa by a flexible glycine linker. iSH2pl 10 has been shown to function in vitro and in vivo as a constitutively active molecule (Hu et al., 1995; Martin et al., 1996). The a myosin heavy chain (MyHC) promoter drives transgenes exclusively in cardiac myocytes and has ortgin-+- t been used extensively in previous transgenic studies Blol ; ar11 1•p8S - (Wakasaki et al., 1997; Fentzke et al., 1998; Kadambi and Kranias, 1998). In the atrium this promoter is Fig. 1. Production of constitutively active PBK (caPBK) transgenic expressed in both embryonic and adult myocytes. mice. (A) Expression of transgene in the heart. Cardiac tissue lysates However, its expression in ventricular myocytes is (15 mg) from transgenic mice or non-transgenic mice were immuno­ precipitated with anti-pllOa-specific antibody, and probed with observed mainly after birth (Ng et al., 1991; Palermo anti-pllOa antibody or anti-Myc epitope tag antibody. (B) Tissue et al., 1996). The iSH2p110 gene together with the Myc specificity of transgene expression. Protein (5 mg) from brain (B), epitope tag was cloned into the aMyHC promoter lung (Lu), heart (H), liver (Li), kidney (K) or skeletal muscle (Sk) construct and transgenic mice were produced. Five tissue was immunoprecipitated using anti-pl 10a antibody. The immunoprecipitated protein was blotted and probed with anti-Myc independently derived founders for the caPI3K were antibody. (C) PBK activity in the heart. Heart tissue lysate (1 mg) produced from 16 F mice screened by Southern blot was immunoprecipitated with anti-pllOa-specific antibody and analysis. All of the founders transmitted the transgene to subjected to in vitro lipid kinase assay using phosphatidylinositol the F generation. To confirm the transgene expression, (Ptdlns) as a substrate. Part of the immunoprecipitated enzyme was 15 mg of cardiac tissue lysates from transgenic mice or subjected to western blotting and probed with anti-p85 antibody to confirm that an equal amount of enzyme was used for the assay. PI3K non-transgenic mice were immunoprecipitated with the activity in caPBK transgenic mice increased 6.5-fold compared with anti-pll0a antibody, and probed with either anti-pll0a non-transgenic mice. Data from two non-transgenic mice (NTg) and antibody or anti-Myc epitope tag antibody (Figure IA). two transgenic mice (Tg) are shown. PIP, Ptdlns 3-phosphate. Four out of five lines expressed significant amounts of the transgene product and were named as lines A to D. Line A expressed significantly lower amounts of the transgene To confirm the activity of the transgene product, 1 mg of product than the other lines on western blot analysis (data heart tissue lysate was immunoprecipitated with the anti­ not shown). Immunoprecipitation with anti-pl lOa anti­ pll0a antibody and was subjected to an in vitro lipid body followed by western blotting using the same kinase assay using Ptdlns as a substrate (Figure IC). To antibody weakly detected the endogenous p 110a molecule confirm that an equal amount of enzyme was used for the in the non-transgenic heart (Figure IA, lane 2, upper assay, part of the immunoprecipitated enzyme was panel). Expression of the iSH2p 110 transgene product was subjected to western blotting. The blot was probed with confirmed by western blotting using the anti-p 11 0a anti-p85 antibody, because we could not detect the antibody, as well as the anti-Myc epitope tag antibody endogenous pll0 molecule when we used 1 mg of protein (Figure IA, lane 4, upper and lower panels). To confirm for immunoprecipitation (Figure 1 C, lower panel). PI3K that transgene expression is specific to the heart, 5 mg of protein from several tissues were immunoprecipitated with activity in caPI3K transgenic mice was increased 6.5-fold anti-pll0a antibody followed by western blotting using compared with that of non-transgenic mice (NTg, anti-Myc antibody. As shown in Figure lB, the transgene 1.00 ::!:: 0.10 arbitrary units, n = 4; Tg, 6.54 ::!:: 1.50 product was expressed exclusively in the heart. units, n = 4; p = 0.0104). 2538 Organ size regulation in mammals non-transgenic mice were immunoprecipitated with the A �...!!!.... II) l;l) IP m "' anti-p85 antibody, and probed with anti-pll0a antibody, Q. Q. anti-FLAG epitope tag antibody or anti-p85 antibody ◄pl1D (Figure 2B). One of the two lines expressed the transgene. The amount of endogenous pll0a that co-immuno­ Blot; anti-4"LAG precipitated with p85 was 20% of that of non-transgenic mice (Figure 2B, compare lanes 2 and 4, upper panel), indicating that the majority of the endogenous p85 regulatory subunit was sequestered by the pll0Akinase B molecule. The pll0� protein was not detected in either IP __ _ _:ps5_· ___ _ dnPI3K mice or non-transgenic mice using the same blot B LU 1H LI K Sk - - - --- (data not shown). To confirm that transgene expression is Bl ot; antl..fLAG ◄ p110Aklnase specific to the heart, 5 mg of protein from several tissues 2 .3 A 5 S were immunoprecipitated with anti-p85 antibody followed by western blotting using anti-FLAG antibody. As shown C NTg Tg in Figure 2B, the transgene product was expressed IP 5 8 exclusively in the heart. To confirm that pl l0Akinase functioned as a dominant­ PIP_. negative molecule, 1 mg of heart tissue lysate was immunoprecipitated with the anti-p85 antibody and was subjected to an in vitro lipid kinase assay (Figure 2C). To confirm that an equal amount of enzyme was used for the assay, part of the immunoprecipitated enzyme was subjected to western blotting and probed with anti-p85 antibody (Figure 2C, lower panel). PI3K activity of the heart from dnPI3K mice was decreased by 77% compared with that of non-transgenic mice (NTg, 1.00 ± 0.21 units, n = 4; Tg, 0.23 ± 0.03 units, n = 4; p = 0.0102). Taken together, we were able to achieve a significant increase Bl ot; anti-p8S - (6.5-fold) or decrease (by 77%) in PI3K activity in the heart using caPI3K and dnPI3K transgenes, respectively. Fig. 2. Production of dominant-negative PBK (dnPBK) transgenic mice. (A) Expression of the transgene in the heart. Cardiac tissue Activation of potential downstream targets in P/3K lysates (5 mg) from dnPBK transgenic mice or non-transgenic mice transgenic mice were immunoprecipitated with anti-p85 antibody, and probed with anti­ Next, we examined the activation of potential downstream pi 10a antibody, anti-FLAG epitope tag antibody or anti-p85 antibody. (B) Tissue specificity of transgene expression. Protein (5 mg) from targets of PI3K. Representative western blots are shown in brain (B), lung (Lu), heart (H), liver (Li), kidney (K) or skeletal muscle the upper and middle panels of Figure 3 and the results of (Sk) tissue was immunoprecipitated using anti-p85 antibody. The quantitative densitometry of 6-8 animals in each group are immunoprecipitated protein was blotted and probed with anti-FLAG shown in the lower panels. Akt ( or protein kinase B) is one antibody. (C) PBK activity in the heart tissue. Heart tissue lysate of the best characterized targets of PI3K in cultured cells (1 mg) was immunoprecipitated with anti-p85-specific antibody and subjected to in vitro lipid kinase assay. Part of the immunoprecipitated (Chan et al., 1999). Activation of Akt in the heart tissue enzyme was subjected to western blotting and probed with anti-p85 was assessed by measuring the amount of Akt phos­ antibody to confirm that an equal amount of enzyme was used for the phorylated at Ser473 using a phosphospecific antibody assay. PBK activity of dnPBK mice was decreased by 77% compared (pAkt in Figure 3A, upper panel). In caPI3K mice, the with that of non-transgenic mice. Data from two non-transgenic mice (NTg) and two transgenic mice (Tg) are shown. amount of phosphorylated Akt was increased 2.2-fold, whereas the amount of unphosphorylated Akt (Akt in Figure 3A, middle panel) was decreased by 44%. In Production of dominant-negative P/3K (dnP/3K) dnPI3K mice, the amount of phosphorylated Akt was transgenic mice decreased by 74%, whereas the unphosphorylated Akt was A catalytically inactive pll0 molecule, when over­ increased by 45%. The ratio of the phosphorylated Akt to expressed in the cell, should compete with the endogenous the unphosphorylated Akt (pAkt/ Akt in Figure 3A, lower pll0 for interaction with the p85 regulatory subunit, panel) is increased 3.9-fold in caPI3K mice, but is thereby having an inhibitory effect on the function of the decreased by 77% in dnPI3K mice compared with non­ endogenous pl 10 molecule in vivo. Therefore, we made a transgenic littermates. These results are concordant with truncated pll0 mutant that has p85 binding domains, but the relative activities of PI3K in caPI3K and dnPI3K mice, lacks the kinase domain (pl lOAkinase). The pl lOAkinase thus suggesting that activation of Akt is regulated by PI3K gene, together with FLAG epitope tag, was cloned into in intact tissue. the aMyHC promoter construct and transgenic mice We also examined the activation of extracellularly were produced (Figure 2A). Four independently derived responsive kinase (ERK), which is one of the downstream ras founders were produced from 39 mice screened by targets of p21 , by measuring the amount of ERK Southern blotting. Two of them transmitted the transgene phosphorylated at Tyr204 (pERK in Figure 3B, upper to the progeny. To confirm the transgene expression, 5 mg panel). In both caPI3K and dnPI3K mice, there was no of cardiac tissue lysates from dnPI3K transgenic mice or significant difference in the amounts of phosphorylated 2539 T.Shioi et al. S6K 86K p70 phosphorylated at multiple sites (Lp70 in A B NTg caPl3K dnPl3K N'Tg c•PlJK dnPl3K 86K Figure 3C, middle panel). The total amount of p70 -4kDa pAkt pERK ◄60kDa - was estimated by adding densitometric scores of these S6K bands. The total amount of p70 was not significantly Ak.l ◄42kD1 , 86K different among three animal groups (Lp70 in Figure 3C, lower panel). The ratio of the phosphorylated (unit) (unh) S6K S6K 86K 86K 2.---- - -- p70 to the total p70 (p-p70 fLp70 in Figure 3C, 5,--- ---� lower panel) was 1.7-fold higher in caPI3K mice, but was decreased by 72% in dnPI3K mice compared with non­ transgenic littermates. These results suggest that PI3K S6K plays an important role in the regulation of p70 activity in the intact mammalian tissue. 86K To confirm the p70 activity in the heart, the amount pERK ERKJ pERK/ ERKJ of the phosphorylated form of ribosomal S6 protein was measured using antibody specific for S6 protein (Kimball Mfg CIIPl3K dnPl3K C D et al., 1999). Loading of equal amounts of protein was NTg c■P,13K dnPl3K confirmed by the Coomassie Blue staining of the mem­ p-S6 brane (data not shown). The amount of phosphorylated S6 was increased 1.8-fold in caPI3K mice, and it was ◄7llk0a decreased by 36% in dnPI3K mice (Figure 3D). (nn lt ) J,-- --- - � P/3K activity correlates with the size of the heart Heart weight/body weight (HW/BW) ratio is a well established index of cardiac hypertrophy. HW/BW ratios of lines A, B, C and D were 4.18 ± 0.10 (n = 9), 4.93 ± 0.53 (n = 6), 4.94 ± 0.13 (n = 4) and 5.12 ± 0.31 (n = 5), respectively. HW/BW of non­ transgenic mice was 4.18 ± 0.19 (n = 12). Lines B, C and D had a significant increase in HW/BW ratio QNTg ■ caP131< � dnP13K (p <0.05). In all four lines of caPI3K mice, there was no premature death or sign of heart failure after 1 year Fig. 3. Activation of Akt and p70S6K, not ERK, was regulated by PBK in the heart. (A) Activation of Akt in the heart tissue of PBK of observation. Three- to four-month-old female mice transgenic mice. In caPBK mice, the amount of phosphorylated Akt at from line B were used for the subsequent analysis. Ser473 was increased, whereas the unphosphorylated form of Akt was Male caPI3K mice showed the same degree of increase decreased. In dnPBK mice, the amount of phosphorylated Akt was in HW /BW ratio as female mice. Body weight, lung decreased and the unphosphorylated Akt was increased. (B) Activation weight and liver weight of the transgenic mice were of ERK in the heart tissue of PBK transgenic mice. The amount of phosphorylated ERK or total ERK2 was not different among non­ not different from those of non-transgenic animals 56K transgenic, caPBK and dnPBK mice. (C) Activation of p70 in the (Table I). However, as shown in Figure 4 (left panel), heart tissue of PBK transgenic mice. The ratio of the phosphorylated there was a proportional increase in the size of all 56K 56K 56K 56K p70 to the total p70 (p-p70 fLp70 ) was 1.7-fold higher in chambers and the thickness of left ventricular walls, caPBK mice, but is decreased by 72% in dnPBK mice compared with non-transgenic littermates. (D) Phosphorylation of ribosomal which resembles 'physiological hypertrophy' associated S6 protein in the heart tissue of PBK transgenic mice. The amount with normal growth of the animal. of phosphorylated S6 was increased 1.8-fold in caPBK mice and Transgenic mice that express dnPI3K did not show any decreased by 36% in dnPBK mice. Hearts from eight non-transgenic premature death or sign of heart failure after 1 year of mice, six caPBK mice and six dnPBK mice were used for the assay of observation. Interestingly, the HW/BW ratio was de­ Akt and ERK. Hearts from eight non-transgenic mice, eight caPBK 56K mice and eight dnPBK mice were used for the assay of p70 and S6 creased by 17% in dnPI3K mice (NTg, 4.18 ± 0.29, protein. *p <0.05 versus non-transgenic (NTg) mice. n = 6; Tg, 3.53 ± 0.42, n = 7; p = 0.008), even though there was no difference in body weight, lung weight, liver weight or tibial length compared with non-transgenic mice ERK or total amount of ERK2 (ERK2 in Figure 3B, (Table I). Three- to four-month-old female mice were used middle panel), suggesting that ERK does not seem to be for the detailed subsequent analysis. Male dnPI3K mice regulated by PI3K in the heart. showed the same degree of decrease in the HW/BW ratio 86K p70 phosphorylates 40S ribosomal protein S6, which as female mice. All of the chambers of the transgenic heart results in increased protein synthesis. p70 6K has been were smaller compared with those of non-transgenic litter­ shown to be downstream of PI3K (Chou and Blenis, 1995). mates, and the proportion of the chambers was maintained 86K We examined the activation of p70 by measuring the (Figure 4, right panel). The hearts of dnPI3K mice 86K 86K amount of p70 phosphorylated at Thr389 (p-p70 in remained smaller without any sign of heart failure after Figure 3C, upper panel), which is known to correlate well > 1 year's observation. 86K with p70 activity (Weng et al., 1998). The amount of We intercrossed caPI3K and dnPI3K mice. The HW/ S6K phosphorylated p70 was increased 2.2-fold in caPI3K BW ratio of double transgenic mice having both caPI3K mice, whereas it was decreased by 74% in the dnPI3K and dnPI3K transgenes was 4.83 ± 0.45 (n = 3), which is 86K mouse heart. Western blotting using p70 antibody similar to that of caPI3K mice. This is consistent with the produces broad bands at ~ 70 kDa due to the presence of expected mechanism of how this caPI3K transgene 2540 Organ size regulation in mammals Table I. Heart weight, lung weight and liver weight of PBK transgenic mice Non-transgenic caPBK• dnPBK Number of animals 6 6 7 Body weight (g) 22.9 ± 2.5 22.6 ± 1.3 22.5 ± 3.1 Tibial length (mm) 17.1 ± 0.2 17.1 ± 0.2 16.5 ± 0.5 Heart weight (mg) 94.4 ± 4.7 112.0 ± 10.3* 78.5 ± 3.6* Lung weight (mg) 141.1 ± 21.4 146.8 ± 16.0 140.2 ± 13.4 Liver weight (mg) 1054 ± 91 1127 ± 155 1026 ± 130 Heart weight/body weight (mg/g) 4.18 ± 0.29 3.53 ± 0.42* 4.93 ± 0.53* Lung weight/body weight (mg/g) 6.48 ± 0.47 6.17 ± 0.56 6.29 ± 0.80 Liver weight/body weight (mg/g) 46.7 ± 5.2 49.3 ± 3.1 45.8 ± 3.93 Heart weight/tibial length (mg/mm) 5.53 ± 0.30 6.55 ± 0.65* 4.75 ± 0.18* Lung weight/tibial length (mg/mm) 8.60 ± 0.88 8.24 ± 1.19 8.50 ± 0.73 Liver weight/tibial length (mg/mm) 61.8 ± 5.3 65.9 ± 9.0 62.0 ± 6.4 Results are presented as mean ± SE. •caPI3K, constitutively active PI3K transgenic. dnPI3K, dominant-negative PI3K transgenic. *p <0.05 versus non-transgenic. caPl3K dnPl3K NTg Tg NTg Tg Fig. 4. Perturbation of PBK activity alters heart size in transgenic mice. In caPBK, there was a proportional increase in the size of all chambers and ventricular wall thickness. In dnPI3K mice, the heart was proportionally smaller than in non-transgenic mice. Bars represent 1 mm. functions, which should be independent of p85, while the number of nuclei in individual cardiac myocytes in dnPI3K transgene should work through sequestering p85. isolated adult myocyte preparations (Figure SA). The It is shown that there is a very tight correlation between myocytes dissociated from caPI3K heart had a significant heart weight and body weight in normal mice (Goodman increase in the mean cell area compared with those from et al., 1984; Ernsberger et al., 1996). Thus, perturbation of the non-transgenic heart (NTg, 2501 ± 49 µm , PI3K activity in the myocytes selectively modulated the data from five mice; Tg, 2974 ± 107 µm , data from size of the heart without changes in the size of other organs four mice; p = 0.004). Both the long axis length (NTg, or overall body weight. 122.9 ± 1.2 µm, data from five mice; Tg, 127.3 ± 1.1 µm, data from four mice; p = 0.033) and the short axis length Cell size of isolated cardiac myocytes from P/3K (NTg, 28.4 ± 0.7 µm, data from five mice; Tg, transgenic mice 32.1 ± 0.6 µm, data from four mice; p = 0.006) were To examine whether the increase in organ size was due to increased in caPI3K mice. The ratio of the long axis to the cellular growth (increase in cell size) or proliferation short axis tended to be smaller in caPI3K mice, although it (increase in cell number), we measured cell size and the was not statistically significant (NTg, 4.63 ± 0.17, data 2541 T.Shioi et al. Table II. Morphometric analysis of isolated cardiac myocytes Number of nuclei (%) Mean cell area (µm ) Long axis (µm) Short axis (µm) Long axis/short axis 2 >3 Non-transgenic (n = 5) 2501 ± 49 122.9 ± 1.2 28.4 ± 0.7 4.63 ± 0.17 9.8 ± 3.0 85.9 ± 4.2 4.4 ± 1.4 caPBK (n = 4) 2974 ± 107* 127.3 ± 1.1* 32.1 ± 0.6* 4.26 ± 0.05 9.6 ± 2.4 85.1 ± 4.0 5.3 ± 2.2 dnPBK (n = 4) 2061 ±51* 108.6 ± 1.2* 26.4 ± 0.8* 4.37 ± 0.08 11.6 ± 1.8 87.1 ± 2.0 1.3 ± 0.2 n, number of animals. Mean values from each mouse were calculated using the measurements from 100 cells isolated from an individual mouse. Next, new mean values (± SE) for each experimental group were calculated based on the data from the mean values from the individual mouse, and are presented in the table. *p <0.05 versus non-transgenic. bigger cells) for myocytes isolated from caPI3K mice, and it was shifted to the left for myocytes from dnPI3K mice. dnPl3K NTg caP l3K Cardiac myocytes withdraw from the cell cycle soon after birth in rodents (Yoshizumi et al., 1995). However, forced expression of cyclin Dl under aMyHC promoter has been shown to cause an increase in the number of multinucleated cells (Soonpaa et al., 1997). To determine whether PI3K affects the nuclear profile of cardiac mean ceU area 2061 %51 250b A9 2974:t1 07 , myocytes, we counted the number of nuclei in isolated (µm2) myocytes. Distributions of the number of nuclei in each myocyte were not different among caPI3K, dnPI3K and non-transgenic hearts (Table II). dnPl3K Absence of cardiomy opathic changes or apoptosis 2D in Pl3K transgenic mice Upon microscopic observation, necrosis or myocyte disarray was not observed in caPI3K mice or dnPI3K mice (Figure 6A, upper panels). Masson trichrome stain showed no interstitial fibrosis in caPI3K mice or dnPI3K mice (Figure 6A, lower panels). Since apoptosis has been postulated to be a critical determinant of organ size by counterbalancing cell proliferation (Conlon and Raff, 0--t"-"--,----,--���---r-'- 1000 2000 3000 4000 Pl-OOO (pm \ 1999), and PI3K has been implicated in cell survival in several systems (Chan et al., 1999), we used a DNA­ Fig. 5. PBK regulates cell size in transgenic heart. (A) The cardiac laddering assay to detect the presence of apoptosis in the myocytes were enzymatically dissociated from the mice hearts. The heart (Figure 6B). There was no evidence of DNA mean cell areas of myocytes isolated from caPBK mice hearts were laddering in dnPI3K or caPI3K hearts even after a significantly increased compared with non-transgenic controls. The mean cell areas of myocytes isolated from dnPBK mice hearts were prolonged exposure of the gel (Figure 6B, lanes 2 and decreased. (B) Distribution of the cell size in the representative animals 3). In control experiments, this assay readily detected from non-transgenic, caPBK and dnPBK mice. The measurement of increased DNA fragmentation in heart tissue taken from cell size was performed on 100 cells for each heart. mice inj ected with doxorubicin (Figure 6B, lane 4) and in the thymus of animals treated with dexamethasone (Figure 6B, lane 5). TUNEL staining of the heart sections showed no differences in TUNEL-positive cells in non­ from five mice; Tg, 4.26 ± 0.05, data from four mice; transgenic, caPI3K or dnPI3K hearts (data not shown). p = 0.098). In contrast, the mean cell area of the myocytes These results suggest that the regulation of organ size by isolated from dnPI3K transgenic heart was significantly PI3K does not involve apoptosis. smaller than that from non-transgenic heart (NTg, 2501 ± 49 µm , data from five mice; Tg, 2061 ± 51 µm , data from four mice; p = 0.004), accom­ Echocardiographic assessment of left ventricular panied by the significant decrease in both the long axis function of Pl3K transgenic mice length (NTg, 122.9 ± 1.2 µm, data from five mice; Tg, Contractile dysfunction leads to compensatory hyper­ 108.6 ± 1.2 µm, data from four mice; p <0.001) and the trophy of the heart. Therefore, we assessed left ventricular short axis length (NTg, 28.4 ± 0.7 µm, data from five (L V) dimensions and systolic function of caPI3K mice mice; Tg, 26.4 ± 0.8 µm, data from four mice; p = 0.036) using M-mode echocardiography (Table III). There was a of the cells. Figure SB shows histogram analysis demon­ significant increase in wall thickness in both the anterior strating that there were normal distributions of myocyte (NTg, 0.6 ± 0.0 mm, n = 8; Tg, 0.7 ± 0.0 mm, n = 4; size isolated from transgenic and non-transgenic mice. p = 0.009) and the posterior (NTg, 0.6 ± 0.0 mm, n = 8; Interestingly, the curve was shifted to the right (more Tg, 0.7 ± 0.0 mm, n = 4; p <0.0001) LV walls. There 2542 Organ size regulation in mammals Table III. Echocardiographic data of PI3K transgenic mice Non-transgenic caPI3K dnPI3K Number of animals 8 4 4 Body weight (g) 22.3 ± 0.7 23.3 ± 0.5 21.0 ± 0.8 Heart rate (b.p.m.) 285 ± 23 269 ± 21 254 ± 5 Diastolic anterior wall thickness (mm) 0.6 ± 0.0 0.7 ± 0.0* 0.5 ± 0.0 Diastolic posterior wall thickness (mm) 0.6 ± 0.0 0.7 ± 0.0* 0.6 ± 0.0 L V diastolic diameter (mm) 3.6 ± 0.1 3.4 ± 0.1 3.4 ± 0.1 L V systolic diameter (mm) 1.2 ± 0.1 1.1 ± 0.1 1.5 ± 0.1 Fractional shortening (%) 65 ± 2 68 ± 3 58 ± I b.p.m., beats per minute; L V, left ventricle. Results are presented as mean ± SE. *p <0.05 versus non-transgenic. A NTg caP13K dn Pl3K H&E Tr ic hr ome ;:J � � -- ('I) M 0) )( ii: Q.. ._ ta C (.) 0 ,, (bp) Fig. 6. Absence of cardiomyopathic changes or apoptosis in PI3K transgenic mice. (A) Histopathological analysis of heart tissue from PI3K transgenic mice. Myocardial necrosis, fibrosis or disarray was not 1078 - observed in caPI3K or dnPI3K transgenic mice. Upper panels show sections stained with hematoxylin and eosin, and lower panels show sections stained with Masson trichrome. Bars represent 10 µm. 503 - (B) Absence of apoptosis in caPI3K transgenic or dnPI3K transgenic 27 ' 1 lH C= mice. Low molecular weight DNA was prepared from mouse tissues 234 - and end labeled using terminal deoxynucleotidyl transferase. No 'DNA 1� laddering' was evident in the hearts from caPI3K mice or dnPI3K mice. Heart tissues from mice that received doxorubicin and thymus tissues from dexamethasone in jected mice were used as positive 2 3 4 5 controls. was no difference in L V diastolic diameter or L V systolic systolic diameter or fractional shortening compared with non-transgenic mice (Table III). diameter. Fractional shortening, an echocardiographic index of left ventricular systolic function, was not altered in these transgenic mice. Although there was a trend of ANF and pMyHC genes are differently regulated in decreased L V anterior wall thickness in dnPI3K mice Pl3K transgenic mice (NTg, 0.6 ± 0.0 mm, n = 8; Tg, 0.5 ± 0.0 mm, n = 4; Myocardial hypertrophy is typically associated with p = 0.120) , there was no significant difference in LV transcriptional activation of several 'fetal' genes, such as posterior wall thickness, L V diastolic diameter, L V atrial natriuretic factor (ANF) and pMyHC (lzumo et al., 2543 T.Shioi et al. with those caused by the caPI3K transgene, leads us to conclude that the phenotypic changes observed in these transgenic hearts are most likely due to specific modula­ tions of PI3K activity. The size of an organ is determined by the coordinate regulation of cell growth, proliferation and death (Conlon and Raff, 1999). It is very difficult, if not impossible, to 28S count accurately the cell number in a compact organ, such as the heart. However, we speculate that PI3 K regulated heart size mainly through the regulation of cell size in t . t t '.. • t ' ' •• 18S our transgenic mice, because (i) an 18% increase of the HW/BW ratio was associa ted with a comparable (19%) Fig. 7. PI3K differentially regulates 'feta!' gene expression in the transgenic heart. Expression of ANF and �MyHC gene was analyzed increase in the mean cell size in caPI3K mice, and a 17% by northern hybridization analysis. In caPI3K mice, the expression of decrease of the HW/BW ratio was associated with a MyHC mRNA shifted from the a isoform to the � isoform, whereas similar degree (1 8%) of decrease in cell size in dnPI3K ANF mRNA was not increased. In dnPI3K mice, the expression of mice; (ii) distribution of the number of nuclei in each MyHC isoforrns was not different from non-transgenic mice, whereas ANF mRNA was markedly increased. Data from two animals from myocyte was not different among caPI3K, dnPI3K and each group are shown. non-transgenic hearts; and (iii) no apoptosis was observed in either transgenic mice using a DNA-laddering assa y or TUNEL analy sis. However, we cannot exclude the potential role of PI3 K in the regulation of cell number in mitotically competent organs, because most of the increase 1988; Chien et al ., 1998). We analyzed the expression of in heart size occurs postnatally when the myocytes are aMyHC, �MyHC and ANF genes by northern hybridiza­ post-mitotic (Soonpaa et al ., 1996; Soonpaa and Field, tion analysis (Figure 7). In caPI3K mice, the expression of 1998), and because the promoter used to generate trans­ MyHC genes shifted from the a isoform to the isoform, genic mice in this study is active mainly after birth in the whereas ANF mRNA was not increased. The expression of ventricles (Ng et al ., 1991; Palermo et al ., 1996) . MyHC isoforms was not different from non-transgenic Modulation of PI3K activity in Drosophila wing alters mice, whereas ANF mRNA was markedly increased in both cell size and number in the organ (Leevers et al ., dnPI3K mice. This discordant regulation of MyHC and 1996). ANF genes suggests that PI3K differe ntially regulates the What are the downstream targets of PI3 K that are 'fe tal' gene program (see Discussion) . involved in the regulation of organ size? The available information does not provide us with the definitive answer to this question. However, Akt, a well characterized Discussion downstream target of PI3K (Alessi and Cohen, 1998; In order to determine the role of PI3 K in the adult heart, we Downward, 1998), is likely to be one of the major have perturbed the activity of PI3K specifically in cardiac mediators of this process. Akt is necessary and sufficient myocytes by expressing constitutively active or dominant­ for phosphorylation and subsequent inactivation of negative forms of PI3K in transgenic mice. The caPI3K 4E-BP1, a repressor of mRNA translation (Gingras et al ., transgene caused a larger heart associated with an increase 1998; Dufner et al ., 1999; Takata et al ., 1999). Akt can 86K in myocyte size, whereas dnPI3K expression resulted in a also activate p7 in some contexts (Burgering and S6K smaller heart associated with smaller myocytes. The Coffer, 1995), although activation of p7 might not be proportion of chamber size, architecture of myocardium solely dependent on Akt (Conus et al ., 1998; Dufner et al ., or cardiac function was not disturbed by the modulation of 1999). Recent genetic experiments in Drosophila show PI3 K activity, suggesting the specific role of PI3K in organ that Akt regulates organ growth (Verdu et al ., 1999). Our size determination. Thus, our study represents the first results showed that PI3K is necessary and sufficient for the example to show that PI3 K is necessary and sufficient to activation of Akt in in situ heart. 86K promote organ growth in mammal s, similar to the Another potential candidate is p7 which is demonstrated role of PI3 K in Drosophila (Leevers et al ., upregulated in caPI3K hearts and downregulated in 1996). dnPI3K hearts. The amount of phosphorylated S6 protein 86K. The availability of the highly cardiac-specific aMyHC was correlated with the activation of p7 It was shown 86K promoter prompted the creation of many transgenic mice previously that the inhibition of the p7 pathway by that overexpress 'molecules of interest' in the heart (Izumo rapamycin at nanomolar concentrations selectively sup­ and Shioi, 1998; Kadambi and Kranias, 1998). However, it presses an increase in protein synthesis of cultured is possible that an overexpressed molecule could affect a neonatal myocytes in response to growth factors biological process in which the endogenous counterpart is (S adoshima and Izumo, 1995 ; Boluyt et al ., 1997). not normally involved. To circumvent this potential Interestingly, rapamycin did not inhibit other phenotypic problem, we created a pair of transgenic lines that express changes associa ted with myocyte hypertrophy, such as 'gain-of-function' and 'loss-of-function' mutants of the re-activation of fetal genes and sarcomere organization pl 1 catalytic subunit of PI3K, and carefully compared (S adoshima and Izumo, 1995). This raises the possibility the resultant phenotypes caused by the respective mutant that p7 6K may selectively regulate cell size via control­ transgenes. The fact that the dnPI3K transgene had exactly ling the rate of protein synthesis. It was recently observed S6K the opposite effects on the cardiac phenotype, compared that gene disruption of p7 resulted in smaller body size 1 .. "' " " .. Organ size regulation in mammals in mice (Shima et al., 1998). In Drosophila, deficiency of Materi als and methods the S6K gene is associated with a reduction in body size associated with smaller cells (Montagne et al., 1999). Mutants of Pl3K used in this study iSH2p110 is a kind gift from T.Franke. To make pl lO �ase, PI3K is shown to promote cell survival in several cell nucleotides 1-1731 (amino acids 1-577) of pl l0 a (Klippel et al., systems (Franke et al., 1997a). We did not detect evidence 1994) were cloned by PCR using the mouse pl lO cDNA as a template. of cardiomyocyte apoptosis in dnPI3K mice. This may be pl l0 �ase sense (5'-CGGGATCCACCATGGACTACAAGGACGA­ because the residual PI3K activity is sufficient to prevent CGATGACAAGCCTCCACGACCATCTTCGGGT-3') primer included a BamHl restriction site, ATG, FLAG epitope tag sequence and apoptosis, or because other survival pathways compensate nucleotides 4-24 of the coding sequence of pl l0 . pll0 Af<lnase antisense for the anti-apoptotic function of PI3K. Interestingly, no (5'-TGCTCTAGATCAGCTCAGCACGCATCTGGAATTCCACTTG­ significant apoptosis was reported in p 110 knockout mice ACAGACAGAAG) primer included nucleotides 1705-1731, the CAAX (Bi et al., 1999) or in Drosophila expressing a kinase­ box of H-ras, stop codon and an Xbal restriction site. PCR product was cloned into pcDNA3 (Invitrogen). Expression of the mutant protein was deficient pll0 transgene (Leevers et al., 1996). confirmed by transient transfection into COS? cells followed by western ERK is known to be activated by most, if not blotting using M2 anti-FLAG antibody (Eastman Kodak). all, hypertrophic factors for the cardiac myocytes (Bogoyevitch et al., 1994; Foncea et al., 1997). How­ Generation of transgenic mice ever, the activity of ERK was not changed by modulation The cDNA insert for iSH2p110 or pl l0 Af<lnase gene was cloned into of PI3K activity in transgenic animals. This is consistent Sall-digested aMyHC promoter construct (clone 26, a generous gift from I.Robbins; Gulick et al., 1991). This vector contains a 5.8 kb BamHl­ with the report in vitro which showed that a constitutively Maem fragment of the murine aMyHC gene that includes the promoter active PI3K failed to activate ERK (Klippel et al., 1996). It and exons 1-3 from the 5' untranslated region of the gene, as well as the is also reported that ERK is only affected by PI3K under human growth hormone polyadenylation site. The bacterial sequence was restrictive conditions (Duckworth and Cantley, 1997). removed by digestion with Notl and injected into the male pronucleus of fertilized single-cell FVB/N embryos. Transgenic founders were identi­ ANF and �MyHC mRNA are usually co-regulated in fied by Southern blot analysis of tail DNA using the human growth the ventricle during embryonic development and in hormone poly(A) as a probe. All aspects of animal care and cardiac hypertrophy induced by mechanical overload experimentation performed in this study were approved by the (Chien et al., 1998; Izumo et al., 1988). However, in our Institutional Animal Care and Use Committee of the Beth Israel Deaconess Medical Center. transgenic mice, expression of these two genes was independently regulated and did not correlate with the Protein preparation increase in heart size. No cardiac dysfunction was The hearts were removed after cervical dislocation and were immediately observed in either transgenic mice. IGF-1, which is frozen in liquid nitrogen. The heart lysates were obtained by known to activate PI3K activity in cardiac myocytes homogenization in ice-cold buffer [1 % NP-40, 10% glycerol, 137 mM (Foncea et al., 1997), decreases ANF gene expression in NaCl, 20 mM Tris-HCl pH 7.4, 4 µg/ml aprotinin, 4 µg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride (PMSF), 4 µg/ml pepstatin, 20 mM intact heart (Donath et al., 1997), as well as in cultured NaF, 1 mM sodium pyrophosphate, 1 mM ortho vanadate]. The lysates myocytes (Eppenberger-Eberhardt et al., 1997). The PI3K were kept on ice for 15 min and cleared by centrifugation at 15 000 g for pathway is implicated in the differentiation of skeletal 20 min at 4 C. Protein concentration was determined by the Bradford myoblasts (Coolican et al., 1997; Jiang et al., 1999) and method (Bio-Rad). smooth muscle cells (Hayashi et al., 1998). It was recently shown that a member of the Forkhead family of transcrip­Analysis of transgene expression by western blotting Protein (5-15 mg) was mixed with protein A-Sepharose for 1 ha t 4 C, tion factors is directly phosphorylated by Akt (Brunet which was subsequently removed by centrifugation. Each aliquot was et al., 1999). It is likely that PI3K regulates the activity of a then mixed with anti-p85 (5 µ1; Upstate Biotechnology) or anti-p ll 0a selective number of transcription factors that regulate the (5 µg; Santa Cruz) antibodies overnight at 4 C. Protein A-Sepharose was differentiation of cardiac muscle cells. Additional work is added for an additional 1 h at 4 C, and the beads were collected by centrifugation. The beads were washed four times with 1 ml of lysis necessary to determine the effectors of PI3K-dependent buffer and resuspended in 50 µ1 of 2X SDS/gel loading buffer (1 X buffer regulation of ANF and �MyHC gene expression in the contains 62.5 mmol/1 Tris-HCl pH 6.8, 1 % SDS, 10% glycerol, 0.005% heart. Bromophenol blue and 5% �-mercaptoethanol). Immunoprecipitates were subjected to SDS-PAGE, and proteins were transferred onto In Drosophila, overexpression of constitutively active polyvinylidene difluoride membranes (Immobilon-P, Millipore). The or dominant-negative mutants of PI3K in the eye causes an blots were probed with anti-p ll 0a (1 µg/ml), anti-Myc (1 µg/ml; Santa increase or decrease in organ size through cell size Cruz) or anti-FLAG (3 µg/ml), followed by horseradish peroxidase­ regulation (Leevers et al., 1996), which is highly remin­ conjugated protein A (1:10 000; Jackson) or anti-mouse IgG (1:10 000; Jackson). The protein probed was then visualized by the enhanced iscent of our findings in the mouse heart. Disruption of the chemiluminescence system (Amersham). Drosophila IRS-1 homolog (an upstream regulator of PI3K) results in a small fly due to a decrease in body and S6K Lipid kinase assay organ size (Bohni et al., 1999). Drosophila p70 Lipid kinase assays were performed essentially as described (Serunian determines body size exclusively through cell size regu­ et al., 1991). Briefly, 10 µ1 of a 1 mg/ml mixture of phosphatidylinositol lation (Montagne et al., 1999). In the mouse, dwarfism and phosphatidylserine (3:2) (sonicated in 20 mM HEPES pH 7.0 and 0.1 mM EGTA) were added to immunoprecipitated enzyme from 1 mg of occurs in knockout mice of IGFs or their receptors lysate (30 µ1 volume), followed by the addition of 10 µ1 of 5X ATP/ (DeChiara et al., 1990; Baker et al., 1993; Liu et al., MgC1 mixture (100 µM ATP, 25 mM MgC1 , 50 mM HEPES pH 7.0) 2 2 1993), IRS-1 (Araki et al., 1994; Tamemoto et al., 1994), containing 10 µCi of [y- P]ATP. The kinase reaction was performed at 86K IRS-2 (Withers et al., 1998) or p70 (Shima et al., 1998). room temperature for 10 min, and the reaction was stopped with 60 µl of 2 M HCI. Lipids were extracted by the addition of 160 µl of chloroform­ Our findings, together with these previous reports, suggest methanol (1: 1), and the organic phase was collected and analyzed by TLC that the conserved PI3K pathway plays a critical role in the (Merk). The TLC solvent was a mixture of propanol and acetic acid (2 M) determination of organ size in insects and mammals. (65:35). After drying, TLC plates were subjected to autoradiography. 2545 T.Shioi et al. Activation of Aid, ERK, p](JS6K and 56 according to the leading-edge method of the American Society of The amount of phosphorylated Akt was examined by western blotting Echocardiography (Sahn et al., 1978). All measurements were performed using the phosphospecific Akt antibody (Ser473; New England Biolabs). with an off-line analysis system (Cardiac Workstation, Freeland Systems) Membranes were stripped and then blotted with an anti-Akt antibody by one observer who was blinded to the genotype of the animals. raised against peptide including unphosphorylated Ser473 (New England Biolabs). The amount of phosphorylated ERK was evaluated by the Northern hybr idization analysis phosphospecific ERK antibodies (Tyr204; Santa Cruz) and the anti-ERK2 Total RNA was purified from mouse tissues using the TRizol Reagent antibody (Transduction Lab). The activation of 70 kDa ribosomal S6 (Life Technologies). A 20 µg aliquot of total RNA was electrophoresed in S6K S6K kinase (p70 ) was examined by phosphospecific p70 antibody 1.2% denaturing formaldehyde agarose gels and blotted onto Hybond N S6K (Thr389; New England Biolabs) and the anti-p70 antibody (Santa (Amersham). The radiolabeled probes were hybridized at 42 C in a Cruz). The phosphorylation of ribosomal S6 protein was examined using hybridization solution (50% deionized formamide, 6X SSC, antibody specific for the phosphorylated form of S6 (gift from 5X Denhardt's solution, 0.5% SDS, 200 µg/ml denatured salmon M.Birnbaum). sperm DNA). Blots were washed twice at room temperature in 2X SSC/ 1 % SDS for 5 min, and twice at 50 C in 2X SSC for 30 min. The probe Histological analysis for mouse ANF was cloned by RT-PCR using mouse heart cDNAs as Mice were given 500 U of heparin i.p. 15 min before anesthesia. The mice a template with the following primers (sense, 5'-ATGGGCTCC­ were anesthetized by i.p. injection of ketamine (Parke-Davis; 50 mg/kg) TTCTCCATCAC; antisense, 5'-TTATCTTCGGTACCGGAAGCTG). and xylazine (Loyd Laboratories; 10 mg/kg). The thorax was opened and Rat�MyHC 3' untranslated region cDNA and mouse a.MyHC 3' the L V was punctured by a 25 G needle. The heart was arrested in diastole untranslated region cDNA were kind gifts from M.Buckingham. by the injection of 0.15 ml of cadmium chloride (100 mM) (Li et al., 1997). The inferior vena cava was cut to allow drainage, and mice were Statistical analysis perfused first by 10 ml of phosphate-buffered saline and then 30 ml of Results are presented as mean :<:: SE. The difference between the groups 3.7% buffered formaldehyde at a speed of 2 ml/min. Mouse tissues were was compared using the two-tailed unpaired Student 's t-test; p <0.05 was dehydrated and embedded in paraffin and 6 µm sections were cut. considered as significant. Sections were stained with hematoxylin and eosin or Masson trichrome. Photomicrographs were obtained using Zeiss Axiophot microscopes. Acknowledgem ents Morphomet ric analysis of isolated cardiac myocytes The cardiac myocytes were enzymatically dissociated from the mouse We thank J.Robbins, T.Franke and M.Buckingham for cDNA clones, heart according to the previously published protocol with minor M.Birnbaum for S6 antibody, L.Zhou and K.Converso for the assistance modifications (Wolska and Solaro, 1996). The hearts from 10- to 12- in echocardiography, and D.Fruman and H.Aoki for helpful discussions. week-old female transgenic or non-transgenic mice were retrogradely This work was supported in part by GM 41890 and SCOR Gtant on perfused and enzymatically dissociated using 0.3% collagenase. The Atheroscrelosis to L.C.C., and NIH grant AG 44976 to S.I. dissociated myocytes were plated on laminin (10 µg/ml) coated dishes. After 1 h of plating, unattached cells were removed by changing the media. Photographs were taken under a phase microscope. The cell area was measured by tracing the edge of the cell and determining the area Referen ces inside the outline. The long axis and short axis of the cells were Ahmed,S.A. and Sriranganathan,N. 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The conserved phosphoinositide 3‐kinase pathway determines heart size in mice

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Copyright © European Molecular Biology Organization 2000
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10.1093/emboj/19.11.2537
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Abstract

The EMBO Journal Vol. 19 No. 11 pp. 2537-2548, 2000 The conserved phosphoinositide 3-kinase pathway determines heart size in mice Among various signaling pathways, growth hormone, Tetsuo Shioi, Peter M.Kang, insulin-like growth factor (IGF)-1 and their downstream Pamela S.Douglas, James Hampe, 1 2 effectors are particularly implicated in the regulation of Claudine M.Vballe , Joel Lawitts , 1 3 body and organ size (Conlon and Raff, 1999). IGF-1 and Lewis C.Cantley and Seigo lzumo their downstream effectors, such as insulin receptor Cardiovascular Division, Division of Signal Transduction and 86K substrate (IRS)-1 and p70 S6 kinase (p70 ), play an 1'ransgenic Facility, Beth Israel Deaconess Medical Center and important role in body size determination in mammals Departments of Medicine and Cell Biology, Harvard Medical School, (DeChiara et al., 1990; Baker et al., 1993; Liu et al., 1993; Boston, MA 02215, USA Araki et al., 1994; Tamemoto et al., 1994; Shima et al., Corresponding author 1998; Withers et al., 1998). Recent genetic studies in e-mail: [email protected] Drosophila also revealed effectors of insulin-like protein play a critical role in the determination of organ size as Phosphoinositide 3-kinase (PI3K) has been shown to well as body size (Leevers et al., 1996; Bohni et al., 1999; regulate cell and organ size in Drosophila, but the role Weinkove et al., 1999). of PI3K in vertebrates in vivo is not well understood. Phosphoinositide 3-kinase (PI3K) lies downstream of To examine the role of PI3K in intact mammalian many receptor tyrosine kinases including insulin and tissue, we have created and characterized transgenic IGF-1 receptors. PI3Ks play crucial roles in many aspects mice expressing constitutively active or dominant­ of biological response, such as membrane trafficking, negative mutants of PI3K in the heart. Cardiac­ cytoskeletal organization, cell growth and apoptosis specific expression of constitutively active PI3K (Toker and Cantley, 1997; Rameh and Cantley, 1999). resulted in mice with larger hearts, while dominant­ The genes encoding PI3Ks have been isolated from a wide negative PI3K resulted in mice with smaller hearts. range of tissues and organisms. They are divided into three The increase or decrease in heart size was associated major classes based on their amino acid sequence, the with comparable increase or decrease in myocyte size. homology among their lipid-kinase domains and substrate Cardiomyopathic changes, such as myocyte necrosis, specificity (Vanhaesebroeck et al., 1997; Fruman et al., apoptosis, interstitial fibrosis or contractile dysfunc­ 1998). Class I PI3Ks phosphorylate the 3-position of the tion, were not observed in either of the transgenic inositol ring of phosphatidylinositol (Ptdlns), Ptdlns mice. Thus, the PI3K pathway is necessary and suffi­ 4-phosphate and Ptdlns 4,5-diphosphate to form Ptdlns cient to promote organ growth in mammals. 3-phosphate, Ptdlns 3,4-diphosphate and Ptdlns 3,4,5- Keywords: cell size/heart/hypertrophy/phosphoinositide trisphosphate, respectively. Class I PI3Ks can be further 3-kinase/transgenic mice divided into two subclasses. The class IA PI3Ks, which include at least three isozymes, a, � and B, are heterodimers of the catalytic 110 kDa subunit (pll0) and the regulatory subunit of 85 or 55 kDa (p85/p55). The Introduction interaction of p85/p55 and pll0 via the inter-Src One of the least understood areas in biology is how the size homology 2 (iSH2) domain of p85/p55 and the of animals and their organs is determined (Conlon and N-terminal 123 amino acids of pl 10 is critical for Raff, 1999). In most animals, determination of the organ achieving the maximal activity of this class of PI3Ks in size is achieved by controlling the number of cells. mammalian cells (Dhand et al., 1994; Klippel et al., 1994; However, in certain tissue such as cardiac or skeletal Yu et al., 1998). muscle, hypertrophy (increase in cell size without cell A serine/threonine kinase Akt, also known as protein division) plays a major role in determining the organ mass. kinase B, is the most well characterized target of PI3K In particular, the cardiac myocyte withdraws from the cell (Alessi and Cohen, 1998; Downward, 1998; Chan et al., cycle soon after birth and the postnatal growth of the heart, 1999). Akt is known to mediate cell survival signal by which is essential to meet the increasing demand for regulating several effectors such as Bad or procaspase-9 cardiac work to support animal growth, is achieved (Datta et al., 1997; Cardone et al., 1998). Another protein 86K primarily by hypertrophy (Soonpaa and Field, 1998). serine/threonine kinase downstream of PI3K is p70 , Extensive studies over the last decade have yielded which is known to be a physiological kinase for the significant information on neurohumoral growth factors ribosomal S6 protein whose phosphorylation increases the involved in cardiac hypertrophy utilizing cultured cardiac rate of initiation of translation of mRNA by ribosomes myocytes as a model system (Chien et al., 1991; (Chou and Blenis, 1995; Thomas and Hall, 1997). Sadoshima and Izumo, 1997). However, the mechanism Despite the wealth of knowledge of biochemical and of cell-size regulation in cardiac myocytes in the intact biological effects of PI3K in cultured cells, little informa­ heart is still not well understood (Sugden and Clerk, 1998). tion is available regarding its physiological roles in the © European Molecular Biology Organization 2537 T.Shioi et al. intact animal. Targeted deletion of the pl lOa catalytic subunit (Bi et al., 1999) or the p85a regulatory subunit NTg Tg -- - --0- (Fruman et al., 1999) in mice resulted in embryonic or IP Q. perinatal lethality, respectively. This makes it difficult to ◄iSH2p110 Blot; anti•p110 examine the role of PI3K in the later developmental ◄p110 process. Interestingly, a previous study utilizing tissue­ ◄ �K:ll)l 10 e:1O1: arrti-Myc specific expression of constitutively active (ea) or 1 • 1 3 dominant-negative (dn) PI3K in Drosophila suggested a cell-autonomous role of PI3K in the determination of organ size (Leevers et al., 1996). However, there is greater IP p 1 1 0 __ __ genetic diversity in vertebrates than in lower metazoans, -- - -' _ BLIJH LiK Sk and in many cases functional redundancy exists that is not Blot; antl• Myc ◄iSH2p110 revealed in less complex organisms (Miklos and Rubin, 1996). To examine the role of PI3K in organ size determination in mammals, we have made transgenic mice expressing constitutively active or dominant­ negative mutants of PI3K specifically in the heart. JP --'-p1_10 __ PIP-. Results Production of constitutively active P/3K (caP/3KJ transgenic mice iSH2pl 10 (Franke et al., 1997b) is a chimeric molecule that contains the iSH2 domain of p85 fused to the N-terminus of bovine pl lOa by a flexible glycine linker. iSH2pl 10 has been shown to function in vitro and in vivo as a constitutively active molecule (Hu et al., 1995; Martin et al., 1996). The a myosin heavy chain (MyHC) promoter drives transgenes exclusively in cardiac myocytes and has ortgin-+- t been used extensively in previous transgenic studies Blol ; ar11 1•p8S - (Wakasaki et al., 1997; Fentzke et al., 1998; Kadambi and Kranias, 1998). In the atrium this promoter is Fig. 1. Production of constitutively active PBK (caPBK) transgenic expressed in both embryonic and adult myocytes. mice. (A) Expression of transgene in the heart. Cardiac tissue lysates However, its expression in ventricular myocytes is (15 mg) from transgenic mice or non-transgenic mice were immuno­ precipitated with anti-pllOa-specific antibody, and probed with observed mainly after birth (Ng et al., 1991; Palermo anti-pllOa antibody or anti-Myc epitope tag antibody. (B) Tissue et al., 1996). The iSH2p110 gene together with the Myc specificity of transgene expression. Protein (5 mg) from brain (B), epitope tag was cloned into the aMyHC promoter lung (Lu), heart (H), liver (Li), kidney (K) or skeletal muscle (Sk) construct and transgenic mice were produced. Five tissue was immunoprecipitated using anti-pl 10a antibody. The immunoprecipitated protein was blotted and probed with anti-Myc independently derived founders for the caPI3K were antibody. (C) PBK activity in the heart. Heart tissue lysate (1 mg) produced from 16 F mice screened by Southern blot was immunoprecipitated with anti-pllOa-specific antibody and analysis. All of the founders transmitted the transgene to subjected to in vitro lipid kinase assay using phosphatidylinositol the F generation. To confirm the transgene expression, (Ptdlns) as a substrate. Part of the immunoprecipitated enzyme was 15 mg of cardiac tissue lysates from transgenic mice or subjected to western blotting and probed with anti-p85 antibody to confirm that an equal amount of enzyme was used for the assay. PI3K non-transgenic mice were immunoprecipitated with the activity in caPBK transgenic mice increased 6.5-fold compared with anti-pll0a antibody, and probed with either anti-pll0a non-transgenic mice. Data from two non-transgenic mice (NTg) and antibody or anti-Myc epitope tag antibody (Figure IA). two transgenic mice (Tg) are shown. PIP, Ptdlns 3-phosphate. Four out of five lines expressed significant amounts of the transgene product and were named as lines A to D. Line A expressed significantly lower amounts of the transgene To confirm the activity of the transgene product, 1 mg of product than the other lines on western blot analysis (data heart tissue lysate was immunoprecipitated with the anti­ not shown). Immunoprecipitation with anti-pl lOa anti­ pll0a antibody and was subjected to an in vitro lipid body followed by western blotting using the same kinase assay using Ptdlns as a substrate (Figure IC). To antibody weakly detected the endogenous p 110a molecule confirm that an equal amount of enzyme was used for the in the non-transgenic heart (Figure IA, lane 2, upper assay, part of the immunoprecipitated enzyme was panel). Expression of the iSH2p 110 transgene product was subjected to western blotting. The blot was probed with confirmed by western blotting using the anti-p 11 0a anti-p85 antibody, because we could not detect the antibody, as well as the anti-Myc epitope tag antibody endogenous pll0 molecule when we used 1 mg of protein (Figure IA, lane 4, upper and lower panels). To confirm for immunoprecipitation (Figure 1 C, lower panel). PI3K that transgene expression is specific to the heart, 5 mg of protein from several tissues were immunoprecipitated with activity in caPI3K transgenic mice was increased 6.5-fold anti-pll0a antibody followed by western blotting using compared with that of non-transgenic mice (NTg, anti-Myc antibody. As shown in Figure lB, the transgene 1.00 ::!:: 0.10 arbitrary units, n = 4; Tg, 6.54 ::!:: 1.50 product was expressed exclusively in the heart. units, n = 4; p = 0.0104). 2538 Organ size regulation in mammals non-transgenic mice were immunoprecipitated with the A �...!!!.... II) l;l) IP m "' anti-p85 antibody, and probed with anti-pll0a antibody, Q. Q. anti-FLAG epitope tag antibody or anti-p85 antibody ◄pl1D (Figure 2B). One of the two lines expressed the transgene. The amount of endogenous pll0a that co-immuno­ Blot; anti-4"LAG precipitated with p85 was 20% of that of non-transgenic mice (Figure 2B, compare lanes 2 and 4, upper panel), indicating that the majority of the endogenous p85 regulatory subunit was sequestered by the pll0Akinase B molecule. The pll0� protein was not detected in either IP __ _ _:ps5_· ___ _ dnPI3K mice or non-transgenic mice using the same blot B LU 1H LI K Sk - - - --- (data not shown). To confirm that transgene expression is Bl ot; antl..fLAG ◄ p110Aklnase specific to the heart, 5 mg of protein from several tissues 2 .3 A 5 S were immunoprecipitated with anti-p85 antibody followed by western blotting using anti-FLAG antibody. As shown C NTg Tg in Figure 2B, the transgene product was expressed IP 5 8 exclusively in the heart. To confirm that pl l0Akinase functioned as a dominant­ PIP_. negative molecule, 1 mg of heart tissue lysate was immunoprecipitated with the anti-p85 antibody and was subjected to an in vitro lipid kinase assay (Figure 2C). To confirm that an equal amount of enzyme was used for the assay, part of the immunoprecipitated enzyme was subjected to western blotting and probed with anti-p85 antibody (Figure 2C, lower panel). PI3K activity of the heart from dnPI3K mice was decreased by 77% compared with that of non-transgenic mice (NTg, 1.00 ± 0.21 units, n = 4; Tg, 0.23 ± 0.03 units, n = 4; p = 0.0102). Taken together, we were able to achieve a significant increase Bl ot; anti-p8S - (6.5-fold) or decrease (by 77%) in PI3K activity in the heart using caPI3K and dnPI3K transgenes, respectively. Fig. 2. Production of dominant-negative PBK (dnPBK) transgenic mice. (A) Expression of the transgene in the heart. Cardiac tissue Activation of potential downstream targets in P/3K lysates (5 mg) from dnPBK transgenic mice or non-transgenic mice transgenic mice were immunoprecipitated with anti-p85 antibody, and probed with anti­ Next, we examined the activation of potential downstream pi 10a antibody, anti-FLAG epitope tag antibody or anti-p85 antibody. (B) Tissue specificity of transgene expression. Protein (5 mg) from targets of PI3K. Representative western blots are shown in brain (B), lung (Lu), heart (H), liver (Li), kidney (K) or skeletal muscle the upper and middle panels of Figure 3 and the results of (Sk) tissue was immunoprecipitated using anti-p85 antibody. The quantitative densitometry of 6-8 animals in each group are immunoprecipitated protein was blotted and probed with anti-FLAG shown in the lower panels. Akt ( or protein kinase B) is one antibody. (C) PBK activity in the heart tissue. Heart tissue lysate of the best characterized targets of PI3K in cultured cells (1 mg) was immunoprecipitated with anti-p85-specific antibody and subjected to in vitro lipid kinase assay. Part of the immunoprecipitated (Chan et al., 1999). Activation of Akt in the heart tissue enzyme was subjected to western blotting and probed with anti-p85 was assessed by measuring the amount of Akt phos­ antibody to confirm that an equal amount of enzyme was used for the phorylated at Ser473 using a phosphospecific antibody assay. PBK activity of dnPBK mice was decreased by 77% compared (pAkt in Figure 3A, upper panel). In caPI3K mice, the with that of non-transgenic mice. Data from two non-transgenic mice (NTg) and two transgenic mice (Tg) are shown. amount of phosphorylated Akt was increased 2.2-fold, whereas the amount of unphosphorylated Akt (Akt in Figure 3A, middle panel) was decreased by 44%. In Production of dominant-negative P/3K (dnP/3K) dnPI3K mice, the amount of phosphorylated Akt was transgenic mice decreased by 74%, whereas the unphosphorylated Akt was A catalytically inactive pll0 molecule, when over­ increased by 45%. The ratio of the phosphorylated Akt to expressed in the cell, should compete with the endogenous the unphosphorylated Akt (pAkt/ Akt in Figure 3A, lower pll0 for interaction with the p85 regulatory subunit, panel) is increased 3.9-fold in caPI3K mice, but is thereby having an inhibitory effect on the function of the decreased by 77% in dnPI3K mice compared with non­ endogenous pl 10 molecule in vivo. Therefore, we made a transgenic littermates. These results are concordant with truncated pll0 mutant that has p85 binding domains, but the relative activities of PI3K in caPI3K and dnPI3K mice, lacks the kinase domain (pl lOAkinase). The pl lOAkinase thus suggesting that activation of Akt is regulated by PI3K gene, together with FLAG epitope tag, was cloned into in intact tissue. the aMyHC promoter construct and transgenic mice We also examined the activation of extracellularly were produced (Figure 2A). Four independently derived responsive kinase (ERK), which is one of the downstream ras founders were produced from 39 mice screened by targets of p21 , by measuring the amount of ERK Southern blotting. Two of them transmitted the transgene phosphorylated at Tyr204 (pERK in Figure 3B, upper to the progeny. To confirm the transgene expression, 5 mg panel). In both caPI3K and dnPI3K mice, there was no of cardiac tissue lysates from dnPI3K transgenic mice or significant difference in the amounts of phosphorylated 2539 T.Shioi et al. S6K 86K p70 phosphorylated at multiple sites (Lp70 in A B NTg caPl3K dnPl3K N'Tg c•PlJK dnPl3K 86K Figure 3C, middle panel). The total amount of p70 -4kDa pAkt pERK ◄60kDa - was estimated by adding densitometric scores of these S6K bands. The total amount of p70 was not significantly Ak.l ◄42kD1 , 86K different among three animal groups (Lp70 in Figure 3C, lower panel). The ratio of the phosphorylated (unit) (unh) S6K S6K 86K 86K 2.---- - -- p70 to the total p70 (p-p70 fLp70 in Figure 3C, 5,--- ---� lower panel) was 1.7-fold higher in caPI3K mice, but was decreased by 72% in dnPI3K mice compared with non­ transgenic littermates. These results suggest that PI3K S6K plays an important role in the regulation of p70 activity in the intact mammalian tissue. 86K To confirm the p70 activity in the heart, the amount pERK ERKJ pERK/ ERKJ of the phosphorylated form of ribosomal S6 protein was measured using antibody specific for S6 protein (Kimball Mfg CIIPl3K dnPl3K C D et al., 1999). Loading of equal amounts of protein was NTg c■P,13K dnPl3K confirmed by the Coomassie Blue staining of the mem­ p-S6 brane (data not shown). The amount of phosphorylated S6 was increased 1.8-fold in caPI3K mice, and it was ◄7llk0a decreased by 36% in dnPI3K mice (Figure 3D). (nn lt ) J,-- --- - � P/3K activity correlates with the size of the heart Heart weight/body weight (HW/BW) ratio is a well established index of cardiac hypertrophy. HW/BW ratios of lines A, B, C and D were 4.18 ± 0.10 (n = 9), 4.93 ± 0.53 (n = 6), 4.94 ± 0.13 (n = 4) and 5.12 ± 0.31 (n = 5), respectively. HW/BW of non­ transgenic mice was 4.18 ± 0.19 (n = 12). Lines B, C and D had a significant increase in HW/BW ratio QNTg ■ caP131< � dnP13K (p <0.05). In all four lines of caPI3K mice, there was no premature death or sign of heart failure after 1 year Fig. 3. Activation of Akt and p70S6K, not ERK, was regulated by PBK in the heart. (A) Activation of Akt in the heart tissue of PBK of observation. Three- to four-month-old female mice transgenic mice. In caPBK mice, the amount of phosphorylated Akt at from line B were used for the subsequent analysis. Ser473 was increased, whereas the unphosphorylated form of Akt was Male caPI3K mice showed the same degree of increase decreased. In dnPBK mice, the amount of phosphorylated Akt was in HW /BW ratio as female mice. Body weight, lung decreased and the unphosphorylated Akt was increased. (B) Activation weight and liver weight of the transgenic mice were of ERK in the heart tissue of PBK transgenic mice. The amount of phosphorylated ERK or total ERK2 was not different among non­ not different from those of non-transgenic animals 56K transgenic, caPBK and dnPBK mice. (C) Activation of p70 in the (Table I). However, as shown in Figure 4 (left panel), heart tissue of PBK transgenic mice. The ratio of the phosphorylated there was a proportional increase in the size of all 56K 56K 56K 56K p70 to the total p70 (p-p70 fLp70 ) was 1.7-fold higher in chambers and the thickness of left ventricular walls, caPBK mice, but is decreased by 72% in dnPBK mice compared with non-transgenic littermates. (D) Phosphorylation of ribosomal which resembles 'physiological hypertrophy' associated S6 protein in the heart tissue of PBK transgenic mice. The amount with normal growth of the animal. of phosphorylated S6 was increased 1.8-fold in caPBK mice and Transgenic mice that express dnPI3K did not show any decreased by 36% in dnPBK mice. Hearts from eight non-transgenic premature death or sign of heart failure after 1 year of mice, six caPBK mice and six dnPBK mice were used for the assay of observation. Interestingly, the HW/BW ratio was de­ Akt and ERK. Hearts from eight non-transgenic mice, eight caPBK 56K mice and eight dnPBK mice were used for the assay of p70 and S6 creased by 17% in dnPI3K mice (NTg, 4.18 ± 0.29, protein. *p <0.05 versus non-transgenic (NTg) mice. n = 6; Tg, 3.53 ± 0.42, n = 7; p = 0.008), even though there was no difference in body weight, lung weight, liver weight or tibial length compared with non-transgenic mice ERK or total amount of ERK2 (ERK2 in Figure 3B, (Table I). Three- to four-month-old female mice were used middle panel), suggesting that ERK does not seem to be for the detailed subsequent analysis. Male dnPI3K mice regulated by PI3K in the heart. showed the same degree of decrease in the HW/BW ratio 86K p70 phosphorylates 40S ribosomal protein S6, which as female mice. All of the chambers of the transgenic heart results in increased protein synthesis. p70 6K has been were smaller compared with those of non-transgenic litter­ shown to be downstream of PI3K (Chou and Blenis, 1995). mates, and the proportion of the chambers was maintained 86K We examined the activation of p70 by measuring the (Figure 4, right panel). The hearts of dnPI3K mice 86K 86K amount of p70 phosphorylated at Thr389 (p-p70 in remained smaller without any sign of heart failure after Figure 3C, upper panel), which is known to correlate well > 1 year's observation. 86K with p70 activity (Weng et al., 1998). The amount of We intercrossed caPI3K and dnPI3K mice. The HW/ S6K phosphorylated p70 was increased 2.2-fold in caPI3K BW ratio of double transgenic mice having both caPI3K mice, whereas it was decreased by 74% in the dnPI3K and dnPI3K transgenes was 4.83 ± 0.45 (n = 3), which is 86K mouse heart. Western blotting using p70 antibody similar to that of caPI3K mice. This is consistent with the produces broad bands at ~ 70 kDa due to the presence of expected mechanism of how this caPI3K transgene 2540 Organ size regulation in mammals Table I. Heart weight, lung weight and liver weight of PBK transgenic mice Non-transgenic caPBK• dnPBK Number of animals 6 6 7 Body weight (g) 22.9 ± 2.5 22.6 ± 1.3 22.5 ± 3.1 Tibial length (mm) 17.1 ± 0.2 17.1 ± 0.2 16.5 ± 0.5 Heart weight (mg) 94.4 ± 4.7 112.0 ± 10.3* 78.5 ± 3.6* Lung weight (mg) 141.1 ± 21.4 146.8 ± 16.0 140.2 ± 13.4 Liver weight (mg) 1054 ± 91 1127 ± 155 1026 ± 130 Heart weight/body weight (mg/g) 4.18 ± 0.29 3.53 ± 0.42* 4.93 ± 0.53* Lung weight/body weight (mg/g) 6.48 ± 0.47 6.17 ± 0.56 6.29 ± 0.80 Liver weight/body weight (mg/g) 46.7 ± 5.2 49.3 ± 3.1 45.8 ± 3.93 Heart weight/tibial length (mg/mm) 5.53 ± 0.30 6.55 ± 0.65* 4.75 ± 0.18* Lung weight/tibial length (mg/mm) 8.60 ± 0.88 8.24 ± 1.19 8.50 ± 0.73 Liver weight/tibial length (mg/mm) 61.8 ± 5.3 65.9 ± 9.0 62.0 ± 6.4 Results are presented as mean ± SE. •caPI3K, constitutively active PI3K transgenic. dnPI3K, dominant-negative PI3K transgenic. *p <0.05 versus non-transgenic. caPl3K dnPl3K NTg Tg NTg Tg Fig. 4. Perturbation of PBK activity alters heart size in transgenic mice. In caPBK, there was a proportional increase in the size of all chambers and ventricular wall thickness. In dnPI3K mice, the heart was proportionally smaller than in non-transgenic mice. Bars represent 1 mm. functions, which should be independent of p85, while the number of nuclei in individual cardiac myocytes in dnPI3K transgene should work through sequestering p85. isolated adult myocyte preparations (Figure SA). The It is shown that there is a very tight correlation between myocytes dissociated from caPI3K heart had a significant heart weight and body weight in normal mice (Goodman increase in the mean cell area compared with those from et al., 1984; Ernsberger et al., 1996). Thus, perturbation of the non-transgenic heart (NTg, 2501 ± 49 µm , PI3K activity in the myocytes selectively modulated the data from five mice; Tg, 2974 ± 107 µm , data from size of the heart without changes in the size of other organs four mice; p = 0.004). Both the long axis length (NTg, or overall body weight. 122.9 ± 1.2 µm, data from five mice; Tg, 127.3 ± 1.1 µm, data from four mice; p = 0.033) and the short axis length Cell size of isolated cardiac myocytes from P/3K (NTg, 28.4 ± 0.7 µm, data from five mice; Tg, transgenic mice 32.1 ± 0.6 µm, data from four mice; p = 0.006) were To examine whether the increase in organ size was due to increased in caPI3K mice. The ratio of the long axis to the cellular growth (increase in cell size) or proliferation short axis tended to be smaller in caPI3K mice, although it (increase in cell number), we measured cell size and the was not statistically significant (NTg, 4.63 ± 0.17, data 2541 T.Shioi et al. Table II. Morphometric analysis of isolated cardiac myocytes Number of nuclei (%) Mean cell area (µm ) Long axis (µm) Short axis (µm) Long axis/short axis 2 >3 Non-transgenic (n = 5) 2501 ± 49 122.9 ± 1.2 28.4 ± 0.7 4.63 ± 0.17 9.8 ± 3.0 85.9 ± 4.2 4.4 ± 1.4 caPBK (n = 4) 2974 ± 107* 127.3 ± 1.1* 32.1 ± 0.6* 4.26 ± 0.05 9.6 ± 2.4 85.1 ± 4.0 5.3 ± 2.2 dnPBK (n = 4) 2061 ±51* 108.6 ± 1.2* 26.4 ± 0.8* 4.37 ± 0.08 11.6 ± 1.8 87.1 ± 2.0 1.3 ± 0.2 n, number of animals. Mean values from each mouse were calculated using the measurements from 100 cells isolated from an individual mouse. Next, new mean values (± SE) for each experimental group were calculated based on the data from the mean values from the individual mouse, and are presented in the table. *p <0.05 versus non-transgenic. bigger cells) for myocytes isolated from caPI3K mice, and it was shifted to the left for myocytes from dnPI3K mice. dnPl3K NTg caP l3K Cardiac myocytes withdraw from the cell cycle soon after birth in rodents (Yoshizumi et al., 1995). However, forced expression of cyclin Dl under aMyHC promoter has been shown to cause an increase in the number of multinucleated cells (Soonpaa et al., 1997). To determine whether PI3K affects the nuclear profile of cardiac mean ceU area 2061 %51 250b A9 2974:t1 07 , myocytes, we counted the number of nuclei in isolated (µm2) myocytes. Distributions of the number of nuclei in each myocyte were not different among caPI3K, dnPI3K and non-transgenic hearts (Table II). dnPl3K Absence of cardiomy opathic changes or apoptosis 2D in Pl3K transgenic mice Upon microscopic observation, necrosis or myocyte disarray was not observed in caPI3K mice or dnPI3K mice (Figure 6A, upper panels). Masson trichrome stain showed no interstitial fibrosis in caPI3K mice or dnPI3K mice (Figure 6A, lower panels). Since apoptosis has been postulated to be a critical determinant of organ size by counterbalancing cell proliferation (Conlon and Raff, 0--t"-"--,----,--���---r-'- 1000 2000 3000 4000 Pl-OOO (pm \ 1999), and PI3K has been implicated in cell survival in several systems (Chan et al., 1999), we used a DNA­ Fig. 5. PBK regulates cell size in transgenic heart. (A) The cardiac laddering assay to detect the presence of apoptosis in the myocytes were enzymatically dissociated from the mice hearts. The heart (Figure 6B). There was no evidence of DNA mean cell areas of myocytes isolated from caPBK mice hearts were laddering in dnPI3K or caPI3K hearts even after a significantly increased compared with non-transgenic controls. The mean cell areas of myocytes isolated from dnPBK mice hearts were prolonged exposure of the gel (Figure 6B, lanes 2 and decreased. (B) Distribution of the cell size in the representative animals 3). In control experiments, this assay readily detected from non-transgenic, caPBK and dnPBK mice. The measurement of increased DNA fragmentation in heart tissue taken from cell size was performed on 100 cells for each heart. mice inj ected with doxorubicin (Figure 6B, lane 4) and in the thymus of animals treated with dexamethasone (Figure 6B, lane 5). TUNEL staining of the heart sections showed no differences in TUNEL-positive cells in non­ from five mice; Tg, 4.26 ± 0.05, data from four mice; transgenic, caPI3K or dnPI3K hearts (data not shown). p = 0.098). In contrast, the mean cell area of the myocytes These results suggest that the regulation of organ size by isolated from dnPI3K transgenic heart was significantly PI3K does not involve apoptosis. smaller than that from non-transgenic heart (NTg, 2501 ± 49 µm , data from five mice; Tg, 2061 ± 51 µm , data from four mice; p = 0.004), accom­ Echocardiographic assessment of left ventricular panied by the significant decrease in both the long axis function of Pl3K transgenic mice length (NTg, 122.9 ± 1.2 µm, data from five mice; Tg, Contractile dysfunction leads to compensatory hyper­ 108.6 ± 1.2 µm, data from four mice; p <0.001) and the trophy of the heart. Therefore, we assessed left ventricular short axis length (NTg, 28.4 ± 0.7 µm, data from five (L V) dimensions and systolic function of caPI3K mice mice; Tg, 26.4 ± 0.8 µm, data from four mice; p = 0.036) using M-mode echocardiography (Table III). There was a of the cells. Figure SB shows histogram analysis demon­ significant increase in wall thickness in both the anterior strating that there were normal distributions of myocyte (NTg, 0.6 ± 0.0 mm, n = 8; Tg, 0.7 ± 0.0 mm, n = 4; size isolated from transgenic and non-transgenic mice. p = 0.009) and the posterior (NTg, 0.6 ± 0.0 mm, n = 8; Interestingly, the curve was shifted to the right (more Tg, 0.7 ± 0.0 mm, n = 4; p <0.0001) LV walls. There 2542 Organ size regulation in mammals Table III. Echocardiographic data of PI3K transgenic mice Non-transgenic caPI3K dnPI3K Number of animals 8 4 4 Body weight (g) 22.3 ± 0.7 23.3 ± 0.5 21.0 ± 0.8 Heart rate (b.p.m.) 285 ± 23 269 ± 21 254 ± 5 Diastolic anterior wall thickness (mm) 0.6 ± 0.0 0.7 ± 0.0* 0.5 ± 0.0 Diastolic posterior wall thickness (mm) 0.6 ± 0.0 0.7 ± 0.0* 0.6 ± 0.0 L V diastolic diameter (mm) 3.6 ± 0.1 3.4 ± 0.1 3.4 ± 0.1 L V systolic diameter (mm) 1.2 ± 0.1 1.1 ± 0.1 1.5 ± 0.1 Fractional shortening (%) 65 ± 2 68 ± 3 58 ± I b.p.m., beats per minute; L V, left ventricle. Results are presented as mean ± SE. *p <0.05 versus non-transgenic. A NTg caP13K dn Pl3K H&E Tr ic hr ome ;:J � � -- ('I) M 0) )( ii: Q.. ._ ta C (.) 0 ,, (bp) Fig. 6. Absence of cardiomyopathic changes or apoptosis in PI3K transgenic mice. (A) Histopathological analysis of heart tissue from PI3K transgenic mice. Myocardial necrosis, fibrosis or disarray was not 1078 - observed in caPI3K or dnPI3K transgenic mice. Upper panels show sections stained with hematoxylin and eosin, and lower panels show sections stained with Masson trichrome. Bars represent 10 µm. 503 - (B) Absence of apoptosis in caPI3K transgenic or dnPI3K transgenic 27 ' 1 lH C= mice. Low molecular weight DNA was prepared from mouse tissues 234 - and end labeled using terminal deoxynucleotidyl transferase. No 'DNA 1� laddering' was evident in the hearts from caPI3K mice or dnPI3K mice. Heart tissues from mice that received doxorubicin and thymus tissues from dexamethasone in jected mice were used as positive 2 3 4 5 controls. was no difference in L V diastolic diameter or L V systolic systolic diameter or fractional shortening compared with non-transgenic mice (Table III). diameter. Fractional shortening, an echocardiographic index of left ventricular systolic function, was not altered in these transgenic mice. Although there was a trend of ANF and pMyHC genes are differently regulated in decreased L V anterior wall thickness in dnPI3K mice Pl3K transgenic mice (NTg, 0.6 ± 0.0 mm, n = 8; Tg, 0.5 ± 0.0 mm, n = 4; Myocardial hypertrophy is typically associated with p = 0.120) , there was no significant difference in LV transcriptional activation of several 'fetal' genes, such as posterior wall thickness, L V diastolic diameter, L V atrial natriuretic factor (ANF) and pMyHC (lzumo et al., 2543 T.Shioi et al. with those caused by the caPI3K transgene, leads us to conclude that the phenotypic changes observed in these transgenic hearts are most likely due to specific modula­ tions of PI3K activity. The size of an organ is determined by the coordinate regulation of cell growth, proliferation and death (Conlon and Raff, 1999). It is very difficult, if not impossible, to 28S count accurately the cell number in a compact organ, such as the heart. However, we speculate that PI3 K regulated heart size mainly through the regulation of cell size in t . t t '.. • t ' ' •• 18S our transgenic mice, because (i) an 18% increase of the HW/BW ratio was associa ted with a comparable (19%) Fig. 7. PI3K differentially regulates 'feta!' gene expression in the transgenic heart. Expression of ANF and �MyHC gene was analyzed increase in the mean cell size in caPI3K mice, and a 17% by northern hybridization analysis. In caPI3K mice, the expression of decrease of the HW/BW ratio was associated with a MyHC mRNA shifted from the a isoform to the � isoform, whereas similar degree (1 8%) of decrease in cell size in dnPI3K ANF mRNA was not increased. In dnPI3K mice, the expression of mice; (ii) distribution of the number of nuclei in each MyHC isoforrns was not different from non-transgenic mice, whereas ANF mRNA was markedly increased. Data from two animals from myocyte was not different among caPI3K, dnPI3K and each group are shown. non-transgenic hearts; and (iii) no apoptosis was observed in either transgenic mice using a DNA-laddering assa y or TUNEL analy sis. However, we cannot exclude the potential role of PI3 K in the regulation of cell number in mitotically competent organs, because most of the increase 1988; Chien et al ., 1998). We analyzed the expression of in heart size occurs postnatally when the myocytes are aMyHC, �MyHC and ANF genes by northern hybridiza­ post-mitotic (Soonpaa et al ., 1996; Soonpaa and Field, tion analysis (Figure 7). In caPI3K mice, the expression of 1998), and because the promoter used to generate trans­ MyHC genes shifted from the a isoform to the isoform, genic mice in this study is active mainly after birth in the whereas ANF mRNA was not increased. The expression of ventricles (Ng et al ., 1991; Palermo et al ., 1996) . MyHC isoforms was not different from non-transgenic Modulation of PI3K activity in Drosophila wing alters mice, whereas ANF mRNA was markedly increased in both cell size and number in the organ (Leevers et al ., dnPI3K mice. This discordant regulation of MyHC and 1996). ANF genes suggests that PI3K differe ntially regulates the What are the downstream targets of PI3 K that are 'fe tal' gene program (see Discussion) . involved in the regulation of organ size? The available information does not provide us with the definitive answer to this question. However, Akt, a well characterized Discussion downstream target of PI3K (Alessi and Cohen, 1998; In order to determine the role of PI3 K in the adult heart, we Downward, 1998), is likely to be one of the major have perturbed the activity of PI3K specifically in cardiac mediators of this process. Akt is necessary and sufficient myocytes by expressing constitutively active or dominant­ for phosphorylation and subsequent inactivation of negative forms of PI3K in transgenic mice. The caPI3K 4E-BP1, a repressor of mRNA translation (Gingras et al ., transgene caused a larger heart associated with an increase 1998; Dufner et al ., 1999; Takata et al ., 1999). Akt can 86K in myocyte size, whereas dnPI3K expression resulted in a also activate p7 in some contexts (Burgering and S6K smaller heart associated with smaller myocytes. The Coffer, 1995), although activation of p7 might not be proportion of chamber size, architecture of myocardium solely dependent on Akt (Conus et al ., 1998; Dufner et al ., or cardiac function was not disturbed by the modulation of 1999). Recent genetic experiments in Drosophila show PI3 K activity, suggesting the specific role of PI3K in organ that Akt regulates organ growth (Verdu et al ., 1999). Our size determination. Thus, our study represents the first results showed that PI3K is necessary and sufficient for the example to show that PI3 K is necessary and sufficient to activation of Akt in in situ heart. 86K promote organ growth in mammal s, similar to the Another potential candidate is p7 which is demonstrated role of PI3 K in Drosophila (Leevers et al ., upregulated in caPI3K hearts and downregulated in 1996). dnPI3K hearts. The amount of phosphorylated S6 protein 86K. The availability of the highly cardiac-specific aMyHC was correlated with the activation of p7 It was shown 86K promoter prompted the creation of many transgenic mice previously that the inhibition of the p7 pathway by that overexpress 'molecules of interest' in the heart (Izumo rapamycin at nanomolar concentrations selectively sup­ and Shioi, 1998; Kadambi and Kranias, 1998). However, it presses an increase in protein synthesis of cultured is possible that an overexpressed molecule could affect a neonatal myocytes in response to growth factors biological process in which the endogenous counterpart is (S adoshima and Izumo, 1995 ; Boluyt et al ., 1997). not normally involved. To circumvent this potential Interestingly, rapamycin did not inhibit other phenotypic problem, we created a pair of transgenic lines that express changes associa ted with myocyte hypertrophy, such as 'gain-of-function' and 'loss-of-function' mutants of the re-activation of fetal genes and sarcomere organization pl 1 catalytic subunit of PI3K, and carefully compared (S adoshima and Izumo, 1995). This raises the possibility the resultant phenotypes caused by the respective mutant that p7 6K may selectively regulate cell size via control­ transgenes. The fact that the dnPI3K transgene had exactly ling the rate of protein synthesis. It was recently observed S6K the opposite effects on the cardiac phenotype, compared that gene disruption of p7 resulted in smaller body size 1 .. "' " " .. Organ size regulation in mammals in mice (Shima et al., 1998). In Drosophila, deficiency of Materi als and methods the S6K gene is associated with a reduction in body size associated with smaller cells (Montagne et al., 1999). Mutants of Pl3K used in this study iSH2p110 is a kind gift from T.Franke. To make pl lO �ase, PI3K is shown to promote cell survival in several cell nucleotides 1-1731 (amino acids 1-577) of pl l0 a (Klippel et al., systems (Franke et al., 1997a). We did not detect evidence 1994) were cloned by PCR using the mouse pl lO cDNA as a template. of cardiomyocyte apoptosis in dnPI3K mice. This may be pl l0 �ase sense (5'-CGGGATCCACCATGGACTACAAGGACGA­ because the residual PI3K activity is sufficient to prevent CGATGACAAGCCTCCACGACCATCTTCGGGT-3') primer included a BamHl restriction site, ATG, FLAG epitope tag sequence and apoptosis, or because other survival pathways compensate nucleotides 4-24 of the coding sequence of pl l0 . pll0 Af<lnase antisense for the anti-apoptotic function of PI3K. Interestingly, no (5'-TGCTCTAGATCAGCTCAGCACGCATCTGGAATTCCACTTG­ significant apoptosis was reported in p 110 knockout mice ACAGACAGAAG) primer included nucleotides 1705-1731, the CAAX (Bi et al., 1999) or in Drosophila expressing a kinase­ box of H-ras, stop codon and an Xbal restriction site. PCR product was cloned into pcDNA3 (Invitrogen). Expression of the mutant protein was deficient pll0 transgene (Leevers et al., 1996). confirmed by transient transfection into COS? cells followed by western ERK is known to be activated by most, if not blotting using M2 anti-FLAG antibody (Eastman Kodak). all, hypertrophic factors for the cardiac myocytes (Bogoyevitch et al., 1994; Foncea et al., 1997). How­ Generation of transgenic mice ever, the activity of ERK was not changed by modulation The cDNA insert for iSH2p110 or pl l0 Af<lnase gene was cloned into of PI3K activity in transgenic animals. This is consistent Sall-digested aMyHC promoter construct (clone 26, a generous gift from I.Robbins; Gulick et al., 1991). This vector contains a 5.8 kb BamHl­ with the report in vitro which showed that a constitutively Maem fragment of the murine aMyHC gene that includes the promoter active PI3K failed to activate ERK (Klippel et al., 1996). It and exons 1-3 from the 5' untranslated region of the gene, as well as the is also reported that ERK is only affected by PI3K under human growth hormone polyadenylation site. The bacterial sequence was restrictive conditions (Duckworth and Cantley, 1997). removed by digestion with Notl and injected into the male pronucleus of fertilized single-cell FVB/N embryos. Transgenic founders were identi­ ANF and �MyHC mRNA are usually co-regulated in fied by Southern blot analysis of tail DNA using the human growth the ventricle during embryonic development and in hormone poly(A) as a probe. All aspects of animal care and cardiac hypertrophy induced by mechanical overload experimentation performed in this study were approved by the (Chien et al., 1998; Izumo et al., 1988). However, in our Institutional Animal Care and Use Committee of the Beth Israel Deaconess Medical Center. transgenic mice, expression of these two genes was independently regulated and did not correlate with the Protein preparation increase in heart size. No cardiac dysfunction was The hearts were removed after cervical dislocation and were immediately observed in either transgenic mice. IGF-1, which is frozen in liquid nitrogen. The heart lysates were obtained by known to activate PI3K activity in cardiac myocytes homogenization in ice-cold buffer [1 % NP-40, 10% glycerol, 137 mM (Foncea et al., 1997), decreases ANF gene expression in NaCl, 20 mM Tris-HCl pH 7.4, 4 µg/ml aprotinin, 4 µg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride (PMSF), 4 µg/ml pepstatin, 20 mM intact heart (Donath et al., 1997), as well as in cultured NaF, 1 mM sodium pyrophosphate, 1 mM ortho vanadate]. The lysates myocytes (Eppenberger-Eberhardt et al., 1997). The PI3K were kept on ice for 15 min and cleared by centrifugation at 15 000 g for pathway is implicated in the differentiation of skeletal 20 min at 4 C. Protein concentration was determined by the Bradford myoblasts (Coolican et al., 1997; Jiang et al., 1999) and method (Bio-Rad). smooth muscle cells (Hayashi et al., 1998). It was recently shown that a member of the Forkhead family of transcrip­Analysis of transgene expression by western blotting Protein (5-15 mg) was mixed with protein A-Sepharose for 1 ha t 4 C, tion factors is directly phosphorylated by Akt (Brunet which was subsequently removed by centrifugation. Each aliquot was et al., 1999). It is likely that PI3K regulates the activity of a then mixed with anti-p85 (5 µ1; Upstate Biotechnology) or anti-p ll 0a selective number of transcription factors that regulate the (5 µg; Santa Cruz) antibodies overnight at 4 C. Protein A-Sepharose was differentiation of cardiac muscle cells. Additional work is added for an additional 1 h at 4 C, and the beads were collected by centrifugation. The beads were washed four times with 1 ml of lysis necessary to determine the effectors of PI3K-dependent buffer and resuspended in 50 µ1 of 2X SDS/gel loading buffer (1 X buffer regulation of ANF and �MyHC gene expression in the contains 62.5 mmol/1 Tris-HCl pH 6.8, 1 % SDS, 10% glycerol, 0.005% heart. Bromophenol blue and 5% �-mercaptoethanol). Immunoprecipitates were subjected to SDS-PAGE, and proteins were transferred onto In Drosophila, overexpression of constitutively active polyvinylidene difluoride membranes (Immobilon-P, Millipore). The or dominant-negative mutants of PI3K in the eye causes an blots were probed with anti-p ll 0a (1 µg/ml), anti-Myc (1 µg/ml; Santa increase or decrease in organ size through cell size Cruz) or anti-FLAG (3 µg/ml), followed by horseradish peroxidase­ regulation (Leevers et al., 1996), which is highly remin­ conjugated protein A (1:10 000; Jackson) or anti-mouse IgG (1:10 000; Jackson). The protein probed was then visualized by the enhanced iscent of our findings in the mouse heart. Disruption of the chemiluminescence system (Amersham). Drosophila IRS-1 homolog (an upstream regulator of PI3K) results in a small fly due to a decrease in body and S6K Lipid kinase assay organ size (Bohni et al., 1999). Drosophila p70 Lipid kinase assays were performed essentially as described (Serunian determines body size exclusively through cell size regu­ et al., 1991). Briefly, 10 µ1 of a 1 mg/ml mixture of phosphatidylinositol lation (Montagne et al., 1999). In the mouse, dwarfism and phosphatidylserine (3:2) (sonicated in 20 mM HEPES pH 7.0 and 0.1 mM EGTA) were added to immunoprecipitated enzyme from 1 mg of occurs in knockout mice of IGFs or their receptors lysate (30 µ1 volume), followed by the addition of 10 µ1 of 5X ATP/ (DeChiara et al., 1990; Baker et al., 1993; Liu et al., MgC1 mixture (100 µM ATP, 25 mM MgC1 , 50 mM HEPES pH 7.0) 2 2 1993), IRS-1 (Araki et al., 1994; Tamemoto et al., 1994), containing 10 µCi of [y- P]ATP. The kinase reaction was performed at 86K IRS-2 (Withers et al., 1998) or p70 (Shima et al., 1998). room temperature for 10 min, and the reaction was stopped with 60 µl of 2 M HCI. Lipids were extracted by the addition of 160 µl of chloroform­ Our findings, together with these previous reports, suggest methanol (1: 1), and the organic phase was collected and analyzed by TLC that the conserved PI3K pathway plays a critical role in the (Merk). The TLC solvent was a mixture of propanol and acetic acid (2 M) determination of organ size in insects and mammals. (65:35). After drying, TLC plates were subjected to autoradiography. 2545 T.Shioi et al. Activation of Aid, ERK, p](JS6K and 56 according to the leading-edge method of the American Society of The amount of phosphorylated Akt was examined by western blotting Echocardiography (Sahn et al., 1978). All measurements were performed using the phosphospecific Akt antibody (Ser473; New England Biolabs). with an off-line analysis system (Cardiac Workstation, Freeland Systems) Membranes were stripped and then blotted with an anti-Akt antibody by one observer who was blinded to the genotype of the animals. raised against peptide including unphosphorylated Ser473 (New England Biolabs). The amount of phosphorylated ERK was evaluated by the Northern hybr idization analysis phosphospecific ERK antibodies (Tyr204; Santa Cruz) and the anti-ERK2 Total RNA was purified from mouse tissues using the TRizol Reagent antibody (Transduction Lab). The activation of 70 kDa ribosomal S6 (Life Technologies). A 20 µg aliquot of total RNA was electrophoresed in S6K S6K kinase (p70 ) was examined by phosphospecific p70 antibody 1.2% denaturing formaldehyde agarose gels and blotted onto Hybond N S6K (Thr389; New England Biolabs) and the anti-p70 antibody (Santa (Amersham). The radiolabeled probes were hybridized at 42 C in a Cruz). The phosphorylation of ribosomal S6 protein was examined using hybridization solution (50% deionized formamide, 6X SSC, antibody specific for the phosphorylated form of S6 (gift from 5X Denhardt's solution, 0.5% SDS, 200 µg/ml denatured salmon M.Birnbaum). sperm DNA). Blots were washed twice at room temperature in 2X SSC/ 1 % SDS for 5 min, and twice at 50 C in 2X SSC for 30 min. The probe Histological analysis for mouse ANF was cloned by RT-PCR using mouse heart cDNAs as Mice were given 500 U of heparin i.p. 15 min before anesthesia. The mice a template with the following primers (sense, 5'-ATGGGCTCC­ were anesthetized by i.p. injection of ketamine (Parke-Davis; 50 mg/kg) TTCTCCATCAC; antisense, 5'-TTATCTTCGGTACCGGAAGCTG). and xylazine (Loyd Laboratories; 10 mg/kg). The thorax was opened and Rat�MyHC 3' untranslated region cDNA and mouse a.MyHC 3' the L V was punctured by a 25 G needle. The heart was arrested in diastole untranslated region cDNA were kind gifts from M.Buckingham. by the injection of 0.15 ml of cadmium chloride (100 mM) (Li et al., 1997). The inferior vena cava was cut to allow drainage, and mice were Statistical analysis perfused first by 10 ml of phosphate-buffered saline and then 30 ml of Results are presented as mean :<:: SE. The difference between the groups 3.7% buffered formaldehyde at a speed of 2 ml/min. Mouse tissues were was compared using the two-tailed unpaired Student 's t-test; p <0.05 was dehydrated and embedded in paraffin and 6 µm sections were cut. considered as significant. Sections were stained with hematoxylin and eosin or Masson trichrome. Photomicrographs were obtained using Zeiss Axiophot microscopes. Acknowledgem ents Morphomet ric analysis of isolated cardiac myocytes The cardiac myocytes were enzymatically dissociated from the mouse We thank J.Robbins, T.Franke and M.Buckingham for cDNA clones, heart according to the previously published protocol with minor M.Birnbaum for S6 antibody, L.Zhou and K.Converso for the assistance modifications (Wolska and Solaro, 1996). The hearts from 10- to 12- in echocardiography, and D.Fruman and H.Aoki for helpful discussions. week-old female transgenic or non-transgenic mice were retrogradely This work was supported in part by GM 41890 and SCOR Gtant on perfused and enzymatically dissociated using 0.3% collagenase. The Atheroscrelosis to L.C.C., and NIH grant AG 44976 to S.I. dissociated myocytes were plated on laminin (10 µg/ml) coated dishes. After 1 h of plating, unattached cells were removed by changing the media. Photographs were taken under a phase microscope. The cell area was measured by tracing the edge of the cell and determining the area Referen ces inside the outline. The long axis and short axis of the cells were Ahmed,S.A. and Sriranganathan,N. 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Journal

The EMBO Journal – Springer Journals

Published: Jun 1, 2000

Keywords: cell size; heart; hypertrophy; phosphoinositide 3‐kinase; transgenic mice

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