Cardiac Hypertrophy Is Not Amplified by Deletion of Cgmp-Dependent Protein Kinase I in Cardiomyocytes

Cardiac hypertrophy is not ampliﬁed by deletion of cGMP-dependent protein kinase I in cardiomyocytes

Robert Lukowskia,b,c,1, Sergei D. Rybalkina,d,e, Florian Logaa,d, Veronika Leissa,d, Joseph A. Beavoa,d,e,1, and Franz Hofmanna,d

aForschergruppe 923 and cInstitut für Pharmakologie und Toxikologie, Technische Universität München, D-80802 Munich, Germany; dCenter for Integrated Protein Science Munich, Ludwig-Maximilians-Universität München, D-81377 Munich, Germany; bDepartment of Pharmacology, Toxicology, and Clinical Pharmacy, Institute of Pharmacy, University of Tübingen, D-72076 Tuebingen, Germany; and eDepartment of Pharmacology, University of Washington, Seattle, WA 98195-7280

Contributed by Joseph A. Beavo, February 3, 2010 (sent for review December 16, 2009) It has been suggested that cGMP kinase I (cGKI) dampens cardiac More recent results (13–15) suggest that hypertrophy could be hypertrophy. We have compared the effect of isoproterenol (ISO) associated with fibroblast “dysfunction” (16). and transverse aortic constriction (TAC) on hypertrophy in WT These studies support the hypothesis that the antihypertrophic [control (CTR)] mice, total cGKI-KO mice, and cGKIβ rescue mice and antifibrotic effects of ANP and BNP are mediated by cGMP, (βRM) lacking cGKI specifically in cardiomyocytes (CMs). Infusion probably at least in part by direct effects on the CM. However, of ISO did not change the expression of cGKI in the hearts of CTR the signaling pathway downstream of cGMP is much less clear mice or βRM but raised the heart weight by ∼20% in both. An than that leading to increased cGMP levels. It has been largely identical hypertrophic growth response was measured in CMs assumed that cGMP activation of cGMP-dependent protein from CTR mice and βRM and in isolated adult CMs cultured with kinase I (cGKI) mediates most of or all the effects of cGMP in or without 1 μM ISO. In both genotypes, ISO infusion induced the cardiac myocyte (17–22). However, cardiac myocytes are similar changes in the expression of hypertrophy-associated car- reported to contain several cGMP hydrolyzing phosphodies- diac genes and significant elevation of serum atrial natriuretic terases (PDEs) (23, 24), including PDE-1s (Ca2+/calmodulin- peptide and total cardiac cGMP. No differences in cardiac hyper- dependent), PDE-2 (cGMP-stimulated), and PDE-5 (cGMP- trophy were obtained by 7-day ISO infusion in 4- to 6-week-old specific). Because several of these PDEs are also targets of conventional cGKI-KO and CTR mice. Furthermore, TAC-induced cGMP, it is possible that cGMP may affect cardiac hypertrophy hypertrophy of CTR mice and βRM was not different and did not indirectly through modulation of the activity of these PDEs (24), result in changes of the cGMP-hydrolyzing phosphodiesterase which regulate cAMP levels and, thereby, associated aspects of activities in hypertropic hearts or CMs. These results strongly sug- cardiac contractility. For example, protein kinase A (PKA)- gest that cardiac myocyte cGKI does not affect the development of dependent modulation of the L-type calcium channel, cardiac heart hypertrophy induced by pressure overload or chronic ryanodine receptor, sarcoplasmatic reticulum Ca2+-ATPase ISO infusion. (SERCA) (25–28), and calcium has been implicated in cardiac hypertrophy induced by the stimulation of β-adrenergic receptors cyclic nucleotides | phosphodiesterase | phosphorylation | transverse aortic (β-ARs) (29–31). fi constriction | sildena l Similarly, the cGMP hydrolyzing PDE-5 has been implicated in the regulation of adrenergic-stimulated cardiac contractility by linical studies and genetically modified mice have supported NOS 3-dependent signaling (32, 33) and in murine cardiac Cthe notion that natriuretic peptides (NPs), the particulate hypertrophy induced by TAC (33, 34). A growing body of evidence guanylyl cyclase (GC)-A, and the second messenger cGMP can indicates that both hypertrophy and chamber remodeling were attenuate the development of pressure- or volume-induced car- inhibited by sildenafil (SIL) treatment in mice subjected to TAC diac hypertrophy and fibrosis (1). Disruption of the murine GC- (35), presumably through activation of cGKI. However, it has also A gene, the receptor for the cardiac “hormones” atrial natriu- been reported that α-myosin heavy chain promoter-driven over- retic peptide (ANP) and brain natriuretic peptide (BNP), results expression of the cGKI enzyme had no effects on several cardiac in salt-resistant elevation of blood pressure, cardiac fibrosis, and parameters, including weight and structure (36). Furthermore, hypertrophy (2, 3). To a great extent, hypertrophy is independent recoupling of NOS in cardiac hypertrophy was effective to protect of blood pressure elevation (4, 5) but is enhanced transverse the heart from failing, but this was independent of cGKI activity aortic constriction (TAC) in the absence of GC-A (4). Further and related to reduced oxidant stress (37). studies using cardiac muscle-specific deletion of GC-A con- In contrast to these experiments carried out with adult tissues or firmed that cardiac muscle hypertrophy is independent of animals, studies with cultured neonatal CMs that express high levels changes in blood pressure (6). ANP, nitric oxide (NO) donors, of cGKI (17) showed that cGKI mediated the antihypertrophic and 8-Br-cGMP inhibited norepinephrine-induced hypertrophy effects of NO (38). cGKI inhibited the calcineurin-nuclear factor of of cardiac cells and fibroblasts (7, 8). activated T cells hypertrophy signaling pathway (20), most likely by Although it is recognized that GC-A-deficient hearts exhibit decreasing the activity of L-type calcium channels (36, 39). Alter- marked hypertrophy with interstitial fibrosis (2, 9), deletion of native pathways for the protection of the heart that have been + + ANP did not result in obvious hypertrophy and fibrosis (10). reported include inhibition of the myocardial Na /H exchanger Other reports have indicated that ANP ablation in mice causes an accelerated susceptibility to hypertrophy induced by different stress stimuli (11, 12). Obviously, ANP and BNP secreted by the Author contributions: R.L., S.D.R., and F.H. designed research; R.L., S.D.R., F.L., and V.L. performed research; R.L., J.A.B., and F.H. analyzed data; and R.L., S.D.R., J.A.B., and F.H. cardiomyocytes (CMs) act as paracrine factors that exert anti- wrote the paper. fi brotic effects in vivo and play a role as local regulators of The authors declare no conflict of interest. ventricular remodeling. Furthermore, it has been shown that rats 1To whom correspondence may be addressed. E-mail: [email protected] expressing a dominant-negative mutant of GC-B and having an or [email protected]. attenuated cGMP response to C-natriuretic peptide and a nor- This article contains supporting information online at www.pnas.org/cgi/content/full/ mal response to ANP showed marked cardiac hypertrophy (13). 1001360107/DCSupplemental.

5646–5651 | PNAS | March 23, 2010 | vol. 107 | no. 12 www.pnas.org/cgi/doi/10.1073/pnas.1001360107 Downloaded by guest on September 29, 2021 (40), transmission of cardioprotective signals from the cytosol to mitochondria (21), decreased apoptosis in thepresence ofenhanced nuclear accumulation of zyxin and Akt (41), reduced cardiomyofi- brillar stiffness by titin phosphorylation (42), and decreased apoptosis by interference with the TAB1-p38 mitogen-activated PK pathway (19). At present, it is unclear which, if any, of these diverse mechanisms are involved in the antihypertrophic effect of cGMP in the intact animal. Alternatively, it is also possible that at least part of the antihypertrophic effects of cGMP is mediated independent of CM cGKI, presumably by effectors secreted by myofibroblasts in response to, for example, BNP. To determine the significance of cGKI signaling in cardiac hypertrophy in the intact animal, we used mice that express cGKIβ only in smooth muscle (SM) cells [cGKIβ rescue mice (βRM)] (43) but not in CMs. Analysis of these animals shows that loss of cGKI in CMs does not potentiate hypertrophy induced by isoproterenol (ISO) or TAC, nor does it increase the basal size of the heart or change the activity of cGMP-hydrolyzing PDEs. Results cGKI has been implicated particularly in cultured cells as a cardiac factor that reduces hypertrophy to various stimuli (44, 45). To test this hypothesis in the intact animal, we used three different models: WT control (CTR) mice; conventional cGKI-KO mice that do not express the cGKI isozymes in any tissue (46); and mice that express the cGKIβ isozyme under the control of the endogenous SM22α Fig. 1. Expression analysis and activity of the cGKI in hearts of CTR mice and promoter in all SMs, including the vasculature of the heart on a βRM. (A) By immunohistochemistry, cGKI was detected in the vasculature and β myocardium of cardiac sections from adult CTR (ctr) mice but was absent from

cGKI-negative background ( RM) (43). In agreement with the PHARMACOLOGY hypothesis, cGKIα is expressed in ventricles of CTR mice but is not the heart muscle of βRM. In these animals, cGKI protein expression was limited to detectable in the cardiac muscle of βRM (Fig. 1 A and B). These the SM layer of coronary vessels as expected by the endogenous SM22α gene μ findings obtained with immunocytochemical and Western blot promoter-driven expression of the cGKI construct. (Scale bar: 100 m.) (B) fi Western blot analysis of ventricular protein lysates using polyclonal antibodies analysis were extended to isolated CMs using speci c antibodies fi α β that speci cally detect either the cGKI isoform or all cGKI isoforms. The cardiac (47). cGKI was detectable in isolated CTR but not in RM cardiac cGKIα isoform was present in heart lysates of the ctr mice but was not detected in myocytes (Fig. 1 C–F), strongly supporting the notion that βRM hearts of conventional cGKI-KO (ko) mice or βRM. By identifying MAPK, equal lack the cGKI protein in CMs. Although the cGKI protein was not loading of the gel is demonstrated. (C) Heart homogenates of ctr mice and βRM detected in CMs by several immunological procedures, it was were subjected to immunoblotting analysis to show the strongly reduced cGKI- possible that low concentrations of the enzyme were present dependent phosphorylation of cardiac target proteins. The antibodies used to and functional. Therefore, we tested the phosphorylation of vaso- detect cGKI substrate phosphorylation were specific for the phospho-Ser239 239 dilator-stimulated phosphoprotein (VASP), a well-characterized residue of the phospho-VASP (pVASP). α-Actinin and VASP common anti- substrate for cGKI (48). Activation of cGKI increased the phos- bodies were used as loading controls. cGKI immunohistochemistry (D)and immunofluorescence (E) of adult cardiac myocytes. The cGKI protein was only phorylation of Ser-239, the cGKI target site of VASP (48), strongly β μ β D detectable in control cells but was not present in CMs of RM. (Scale bar: 20 m.) in CTR but not in RM hearts (Fig. 1 ) demonstrating the func- (F)Quantification of cGKI fluorescence intensity (FI) of adult ctr mice (black bars) tional absence of myocardial cGKI. Because the Iβ isoform of cGKI and βRM (white bars) CMs. FI was determined using the specific cGKI antibodies 239 is present in SMs (Fig. 1A), the minor phospho-VASP signal and fluorophore-coupled secondary antibodies to detect the primary antibody detectable in βRM hearts most likely resulted from expression in protein complexes (***P < 0.001). a.u., arbitrary units; n.s., not significant. the vasculature. We next tested whether or not the gene-targeted animals responded normally to a single injection of 0.1 mg/kg of ISO (Fig. average heart rates during either the day or night in both the S1). The basic cardiac response to ISO in βRM was without any CTR and +ISO groups (Fig. S3). pathological findings. The βRM had a normal cardiac electro- ISO infusion increased the heart weights (HWs) to the same cardiogram at rest and in the presence of ISO (Fig. S1A). ISO extent, 19.7% and 19.6% in CTR mice and βRM, respectively increased heart rate (Fig. S1B) and fractional shortening (Fig. S1 (Fig. 2). If cGKI activity was required as a normal brake on C and D) to the same extent in CTR mice and βRM. Exposure of regulation of HW under “basal” cGMP conditions, lack of cGKI isolated adult CMs for 24 h to 1 μM ISO increased the cell size by should give larger hearts. Nevertheless, calculation of the HW 7.3% and 7.9% in CTR mice and βRM, respectively (Fig. S2 A per body weight (BW) confirmed that ISO infusion did not and B). The hypertrophic response of CMs was extended by augment the cardiac weight of βRM (CTR mice, +26.5%; analyzing the gene expression of SERCA. A similar level of down- βRM, +12.7%) (Fig. 2B). As another measure of hypertrophy, in regulation was detected in the CMs of both genotypes treated situ morphometric analysis of left ventricular myocyte size (Fig. 2 with ISO (Fig. S2C). These results are consistent with previous C and D) revealed that ISO infusion raised the myocyte size to reports supporting the notion that CM cGKIα is dispensable for 133.6% and 132.6% as compared with the saline-treated groups the basic β-AR regulation of the heart (29, 30, 38, 49, 50). in CTR mice and βRM, respectively. Cardiac fibrosis also was In the next series of experiments, 10–14-week-old littermates equally accelerated in animals subjected to chronic ISO treat- of either genotype were stimulated by ISO infused at a constant ment, with no differences seen between the two genotypes. rate of 30 mg/kg per day for 7 days using an osmotic minipump. Further evidence that the lack of cGKI in cardiac myocytes Heart rate was recorded continuously by telemetry and was used does not alter ISO-induced hypertrophy came from mRNA as an indirect measure for the amount of ISO delivered to analysis of the cardiac genes associated with the hypertrophy individual animals. Heart rate increased in all mice treated with program. Genes that are normally expressed in the adult heart ISO infusion to the same extent. No difference was noted in the were down-regulated (Fig. S4B), whereas expression of fetal

Lukowski et al. PNAS | March 23, 2010 | vol. 107 | no. 12 | 5647 Downloaded by guest on September 29, 2021 Fig. 2. Heart growth induced by ISO infusion for 7 days via implanted miniosmotic pumps in CTR mice and βRM. (A) Representative H&E-stained cardiac cross-sections of ISO-treated animals. Note that the hearts of βRM appear smaller because the animals weigh less [26.4 vs. 23.1 g for CTR (ctr) mice (n = 20) and βRM (n = 16), respectively]. (B) Chronic ISO infusion caused hypertrophy of the heart as indicated by increases of the HW/BW ratios. The hypertrophy response was similar in each genotype (**P < 0.01; ***P < 0.001). (C and D) Cross-sectional areas of cardiac myocytes. (C) H&E staining Fig. 3. Cardiachypertrophy and signaling invarious gene-targetedcGKI mouse of heart sections from ctr mice and βRM at baseline (Left, −ISO) and after models. (A) Effects of 7 days of chronic ISO infusion via implanted miniosmotic chronic ISO infusion (Right, +ISO). (Scale bar: 10 μm.) (D) Summary of cell size pumps on the hearts of 4- to 6-week-old CTR (ctr) mice and littermate conven- quantified by morphometry. ISO treatment provoked a significant increase tional cGKI-KO (ko) mice. Chronic ISO infusion caused hypertrophy of the heart in the CM (cM) cross-sectional areas in hearts of ctr mice and βRM. The areas as indicated by increases of the HW/BW ratios. The response to the hypertrophy were determined in the histological sections shown in A and as described in stimulus of each genotype indicated was similar in comparison to the respective < Materials and Methods (***P < 0.001). saline-infused groups (**P 0.01). (B) cGKI expression analysis in the hearts of CTR (ctr) mice, βRM, and ko mice that were chronically treated with ISO for 7 days. DAPI (green) was used as a nuclear counterstain. The cGKI protein (red) genes (Fig. S4A) and fibrosis markers (Fig. S4C) was induced in wasdetectedinthe vasculature and myocardiumofctr micebut was absent from the heartsofkomice. Inthe βRM,the cGKI expression pattern wasnot influenced the ISO groups. The mRNA levels of all markers analyzed (Fig. by the hypertrophy stimulus that induced a fetal gene program (Fig. S4)butwas S4) changed in the same direction and to a similar extent in both similar to baseline conditions and yet limited to the vasculature. (Scale bar: genotypes. Importantly, no differences in the absolute expression 20 μm.) Western blot (C) and quantification (D)ofGSK3β-to-phospho-GSK3β levels of the NP receptors GC-A and GC-B were detected in (pGSK3β) ratio of ventricular protein lysates from ctr mice and βRM at baseline CTR mice and βRM following 7 days of ISO (Fig. S5 A–D). and after 7 days of ISO. (E) Effect of pressure overload induced by TAC on cardiac In line with the transcriptional analysis (Fig. S4), we noticed a hypertrophy. Twenty-one days of TAC caused a significant increase in the HW/ significant increase of serum ANP and cardiac cGMP levels in BW ratios in both genotypes analyzed in A (*P < 0.01; ***P < 0.001). animals that developed cardiac hypertrophy (Fig. S5 E and F). We performed two additional sets of experiments to rule out GSK3β/GSK3β ratio after ISO treatment (Fig. 3 C and D). the possibility that cGKI might be reexpressed following the β β However, in the RM hearts, the same changes were observed chronic ISO infusion in RM CMs or that a lack of accelerated (Fig. 3 C and D). These results indicate that the Ser9 phos- heart hypertrophy in these animals was conferred by cGKI phorylation status of GSK3β is not changed by a cardiac cGKI present in the SM of these animals. At the age of 4 to 6 weeks, pathway in ISO-induced hypertrophy (34). conventional cGKI-KO mice (43, 46) and their littermate juve- We also considered the possibility that ISO-induced cardiac nile CTRs were infused for 7 days with ISO at a rate of 30 mg/kg hypertrophy is unique in that this type of hypertrophy might not be per day. Again, the HW/BW ratios increased in both genotypes modulated by myocardial cGKI activity. An alternative model to a similar extent in response to the hypertrophic stimulus widely used to induce cardiac hypertrophy is TAC (32–34). There- A (CTR, +28.7%; cGKI-KO, +22.3%) (Fig. 3 ). Immunostaining fore, littermate CTR mice and βRM were subjected to the TAC of cardiac sections of ISO-treated mice clearly indicated that the procedure. Animals were killed after 21 days, and the HWs and CTR hearts expressed cGKI in myocytes, the βRM hearts BWs were determined (Fig. 3E). TAC increased the HW of both expressed cGKI in vascular SM, and the cGKI-KO hearts did not CTR mice and βRM to the same extent (CTR mice, +40.1%; βRM, express cGKI in any heart cells (Fig. 3B). +41.8%). Again, no difference was detectable between the two We further analyzed the impact of myocardial cGKI on the genotypes when the HW was normalized to BW (CTR mice, activity of glycogen synthase kinase 3β (GSK3β) because GSK3 +71.0%; βRM, +62.1%). Taken together, these results suggest that represents a point of convergence for several hypertrophic sig- the development of murine heart hypertrophy is not augmented by nals. It has been shown that dephosphorylation of GSK3β at Ser9 the absence of endogenous cGKI in cardiac myocytes. positively regulates the ability of this enzyme to antagonize ISO- Recent evidence has suggested that the antihypertrophic effect induced hypertrophy in the adult heart (51), probably in a mode of SIL in the heart is by means of inhibition of cardiac myocyte that depends on PDE-5 activity (34). Indeed, in the presence of PDE-5 activity and subsequent activation of cGKI (33, 52). It is cardiac cGKI, we detected a significant decrease in the phospho- also known that cGKI can stabilize an activated state of PDE-5

5648 | www.pnas.org/cgi/doi/10.1073/pnas.1001360107 Lukowski et al. Downloaded by guest on September 29, 2021 by phosphorylating it (23). Therefore, we studied the expression of cGMP-hydrolyzing PDEs, such as PDE-1, PDE-2, and PDE-5, and the total cGMP-hydrolytic activities in the heart of CTR mice and βRM. We and others (53) have hypothesized that these PDEs might affect specific pools of cardiac cGMP, and thereby the response of the heart to hypertrophic stimuli. First, we tested the specificity of our PDE-5 antibodies by using protein extracts from lung and SM cells as positive controls (Fig. 4 A and B). We were unable to detect PDE-5 in isolated CMs by several different biochemical methods, including after TAC (Fig. 4 A–E). How- ever, cardiac myofibroblasts that were identified by SM α-actin antibodies in vitro do contain the PDE-5 protein (Fig. 4D). Again, in adult CMs isolated from both CTR mice and βRM, no PDE-5 expression was detectable, and by 21 days of TAC, we also did not detect PDE-5 (Fig. 4E). In CTR samples of total heart homogenates, the small expression of PDE-5 is consistent with the presence of PDE-5 in blood vessels and fibroblasts (Fig. 4F and Fig. S6). PDE-1C was found to be a major cGMP- hydrolyzing PDE expressed only in CMs but not in fibroblasts (Fig. 4D). Ca2+/calmodulin-activated PDE-1C can hydrolyze both cAMP and cGMP equally well and is sensitive to SIL inhibition in the high nanomolar range (Fig. 4H). In vivo, neither TAC nor chronic ISO treatment changed the levels of PDE-1C, PDE-2, or PDE-5 in both genotypes (Fig. 4F). A small but reproducible increase in the total PDE-5 protein content was apparent in βRM hearts, but there was no difference noticed between healthy and hypertrophic hearts. Therefore, we

analyzed SIL sensitivity of the cGMP-hydrolytic activity from PHARMACOLOGY CTR mice and βRM, but did not detect any significant inhibition at low concentrations of SIL (10–50 nM) (Fig. 4G). Importantly, the range of SIL concentrations that inhibited the cardiac cGMP-hydrolytic activity was similar for both genotypes and did not change with hypertrophy induced by ISO treatment or TAC. When we measured PDE activity in the presence of Ca2+/calmodulin, the inhibitory curve shifted to the left, indi- cating that the predominant PDE is PDE-1C (about 90% of the hydrolytic activity) under these conditions. At concentrations of SIL that are specific for the inhibition of PDE-5 (≤10 nM), we did not detect any inhibition of cGMP-hydrolytic activity. In fact, the IC50 for SIL inhibition was ≈400 nM, corresponding to the concentrations of SIL at which it inhibits PDE-1C (Fig. 4H). Discussion The results presented suggest the following conclusions that appear to be valid for the intact adult animal: (i) The βRM do not express cGKI in cardiac myocytes, whereas the same cells from CTRs express cGKI. (ii) In the intact animal, many physiological heart functions are not affected by the absence of cGKI in CMs and loss of cGKI does not influence the basic regulation of the heart by β-AR stimulation under basal conditions of cGMP. (iii) ISO-induced cardiac hypertrophy was not affected by the Fig. 4. Expression and activity of cGMP-hydrolyzing PDEs in the hearts and isolated cardiac cells of CTR mice and βRM. The specificity of the PDE-5 anti- absence of cGKI in two different transgenic mouse lines bodies used was verified by detecting the PDE-5 protein in the lung (A) and that lacked cGKI in the heart. SM cells (SMCs) (B). Using the same antibodies, no PDE-5 protein was (iv) The degree of cardiac hypertrophy induced by TAC was not fi detectable in CMs. (C) In adult CMs from CTR mice, which were identi ed by changed in animals that lacked cGKI in cardiac myocytes. antibodies specific for the cardiac marker protein α-actinin, PDE-5 was not detectable. (Scale bar: 20 μm.) (D) Western blot analysis of adult CMs and cardiac myofibroblasts (MFs) from CTR (ctr) mice and βRM. PDE-5 was absent from CMs but was detectable in MFs. SM α-actin was used to discriminate 2+ between MFs and CMs. The Ca /calmodulin-dependent cGMP- and cAMP- basal level and after ISO treatment or TAC. The IC50 of SIL on the cGMP- hydrolyzing PDE-1C was present only in CMs. (E) Western blot analysis of hydrolyzing PDE activity was similar for both genotypes and did not change fi adult CMs puri ed previous to (basal) and after 21 days of TAC. (F) Expression as a result of the hypertrophic stimuli. Importantly, the IC50 determined was of PDE-1/2/5 and the cGKI protein in different models of cardiac hypertrophy. about 450 nM, and was therefore in the range of PDE-1C inhibition. (H) No significant changes were detectable in the heart after TAC and/or ISO cGMP-hydrolyzing activity of different recombinant PDEs in the presence of β treatment between ctr mice and RM as compared with the respective basal SIL (100 pM to 100 μM). The IC50s for the recombinant enzymes were 1.9 nM, expression. (G) SIL inhibition of cGMP-hydrolytic activity in heart lysates at 422 nM, and 15 μM for PDE-5, PDE-1C, and PDE-2, respectively.

Lukowski et al. PNAS | March 23, 2010 | vol. 107 | no. 12 | 5649 Downloaded by guest on September 29, 2021 (v) cGMP-hydrolytic activity is not affected by the absence of PDE-5 in CMs of CTR mice or βRM in isolated myocytes and we do cGKI in CMs and does not change in response to hyper- not observe an increase in PDE-5 protein or activity during trophic growth signals to the heart. hypertrophy; furthermore, we do not find any changes in the total cGMP-hydrolytic activity in the myocardium of the two genotypes. Thus, it would seem quite possible that the antihypertrophic effects Overall, these data suggest that ablation of cGKI in the CM of SIL could be mediated by some enzyme other the PDE-5, per- does not greatly affect several different hypertrophic stimuli that haps PDE-1C, which is abundant in CMs. Whether or not inhibition lead to hypertrophy under normal developmental drive. of PDE-1C can stimulate cAMP-dependent pathways in discrete These conclusions appear to be in contradiction to many of cellular compartments of the CM, and thereby influence hyper- those reached in several previous reports, most of which suggest trophy, remains to be determined and likely will require either that cGMP acting via cGKI in CMs attenuates cardiac hypertrophy (1–3, 5–7, 32–34, 54). How can the present results be PDE-1C knockout animals or highly selective PDE-1C inhibitors. Although clearly being beyond the scope of this report, studies of an reconciled with these previous reports? Inspection of the pre- fi vious studies indicates that in most of them, cardiac growth was inducible CM-speci c PDE-5 gene KO in the adult animal should stimulated either by unknown hormonal factors (1–3, 5–7, 12) or be a valuable tool to answer what are the molecular mode(s) of by hormones such as norepinephrine (7) that are not selective for action of PDE-5 inhibitors on cardiac cGMP and remodeling. one receptor type. It therefore seems possible that cGKI affects Finally, one simple straightforward explanation is that if cGKI is primarily cardiac hypertrophy induced by receptors that signal involved in mediating the antihypertrophic effect of cGMP in the heart, it is attributable to its expression in cell types other than the through the G proteins αq and α11 (55) but is largely dispensable CM (14). Similarly, if the effects of SIL on cardiac hypertrophy are for factors that signal through Gαs and cAMP (29). More experiments will be needed to determine if this is true. However, mediated by inhibition of PDE-5, it could also be mediated indi- even if this is true, it does not resolve the apparent discrepancy rectly by another non-CM cell type. Cardiac fibroblasts have little or with respect to the lack of effect of cGKI ablation on TAC- no cGKI in the βRM (Fig. 3B). However, we noticed that cGKI was induced hypertrophy, because this model is commonly thought to easily detectable in cardiomyofibroblasts of βRM when cultured for have a major angiotensin II component to it. Angiotensin sig- 5days(Fig.4D). This was expected (61), because in the βRM, β α naling is largely via AT1 receptors, which activate Gαq/11 (33, 34, cGKI is under the control of the endogenous SM22 promotor, fi 52). However, recently, it has been reported that the cardiac AT1 which is known to be active in broblast-like cells. Interestingly, we receptor is not essential for the development of TAC-induced detected PDE-5 in the same cells by Western blot analysis (Fig. 4D). hypertrophy (56, 57), so perhaps the effects of angiotensin II are PDE-5 activity contributed about 50% of the total cGMP-hydro- not mediated via G11. lyzing activity of myofibroblasts. However, we did not notice any It is also possible that the cGKI pathway is only antihyper- changes of cGKI or PDE-5 protein levels in total hearts, suggesting trophic when cGMP levels are raised rather high. Many models of that the amounts of cGKI and PDE-5 in myofibroblasts were a hypertrophy in which increased cGMP pathway components show relatively small fraction of the total heart samples. antihypertrophic responses are best seen when there is either a During the past decade, the NO/cGKI pathway has been high level of GC activation or PDE inhibition present. For implicated as being protective to the heart during ischemia/ example, when used at high nanomolar concentrations, SIL, a reperfusion (reviewed in 44, 45). A common mechanism sug- PDE-5 inhibitor, is reported to oppose TAC-induced hypertrophy gested by several researchers is that NO/cGKI transmits a (34). This is presumed to be attributable to increased cGMP in “protective signal” to the mitochondria (21). The mitochondrial one or more compartments in the heart. It has been shown that target(s) of cGKI is unclear (45); therefore, it is still quite pos- these same concentrations of SIL also inhibit PDE-1C activity by sible that NO effects on the mitochondria are mediated by ∼ 20% in heart extracts (58). It is also reported that SIL treatment mechanisms independent of cGKI. Considering the discrepant fi increases cardiac cGKI activity ef ciently, although it is not clear results presented in this report, we would like to raise the pos- that this occurs in the CM (34). Nevertheless, taking all these sibility that some of the antihypertrophic effects of cGMP are observations into account, we found it very unexpected that there mediated by a target protein that is independent of cGKI. would be no effect of deletion of cardiac cGKI on cardiac hypertrophy, particularly because our data also show that chronic infu- Materials and Methods sion of ISO significantly induced the transcript level of cardiac ANP A E Detailed materials and methods are described in SI Materials and Methods, (Fig. S4 ) as well as the serum level of ANP (Fig. S5 ). In addition, including chronic ISO administration; transverse aortic constriction surgery; direct measurement of cardiac cGMP concentration showed a 3- to echocardiography; histological, immunohistochemical, and gene expression 4-fold increase in response to ISO (59, 60) (Fig. S5F). analyses; Western blot analysis; and PDE assays. The data were subjected to Taken together, the data suggest that caution should be applied statistical analysis using OriginPro software, version 6.1 (OriginLab). in interpreting the effects of SIL on cGKI and cGMP PDE activity in the heart. It was recently reported that PDE-5 is up-regulated in ACKNOWLEDGMENTS. We thank Sabine Brummer, Teodora Kennel, and patients with end-stage heart failure; however, 2 μM SIL was used Astrid Vens for expert technical assistance. We acknowledge the excellent help of Nicolas Jäger at the cardiac myocyte planimetry facility. This research for cGMP PDE assays as a test for PDE-5 activity (54). At this was supported by grants from the Deutsche Forschungsgemeinschaft (to R.L. concentration, SIL can inhibit most of the cardiac PDE-1C activity and F.H.), the National Institutes of Health (Grant GM083926) (to J.B.), and (Fig. 4H). Moreover, in our study, we cannot detect endogenous the Leducq Foundation (to J.B.).

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