Cardiac fibrosis, characterized by excessive deposition of extracellular matrix proteins, is one of the causes of heart failure, and it contributes to the impairment of cardiac function. Fibrosis of various tissues, including the heart, is believed to be regulated by the signalling pathway of angiotensin II (Ang II) and transforming growth factor (TGF)‐β. Transgenic expression of inhibitory polypeptides of the heterotrimeric G12 family G protein (Gα12/13) in cardiomyocytes suppressed pressure overload‐induced fibrosis without affecting hypertrophy. The expression of fibrogenic genes (TGF‐β, connective tissue growth factor, and periostin) and Ang‐converting enzyme (ACE) was suppressed by the functional inhibition of Gα12/13. The expression of these fibrogenic genes through Gα12/13 by mechanical stretch was initiated by ATP and UDP released from cardiac myocytes through pannexin hemichannels. Inhibition of G‐protein‐coupled P2Y6 receptors suppressed the expression of ACE, fibrogenic genes, and cardiac fibrosis. These results indicate that activation of Gα12/13 in cardiomyocytes by the extracellular nucleotides‐stimulated P2Y6 receptor triggers fibrosis in pressure overload‐induced cardiac fibrosis, which works as an upstream mediator of the signalling pathway between Ang II and TGF‐β.
Heart failure is the final cardiac stage that is observed in nearly all forms of cardiovascular diseases. Structural remodelling of the heart, including myocardial hypertrophy and fibrosis, is a key determinant for the clinical outcome of heart failure (Cohn et al, 2000; Berk et al, 2007). A variety of evidence indicates that the initial phase in the development of myocardial hypertrophy involves neurohumoral factors, such as endothelin (ET)‐1, angiotensin (Ang) II and norepinephrine, and their receptors being coupled to G proteins of the Gq, G12, and Gi families (Sadoshima and Izumo, 1997; Gohla et al, 2000; Arai et al, 2003). These agonists induce hypertrophic gene expression in cardiomyocytes through the Ca2+‐dependent pathway (Onohara et al, 2006). Previous studies using transgenic or conditional knockout mice clearly revealed that the Gq family protein predominantly regulates the pathogenesis of hypertrophy (Adams et al, 1998; Wettschureck et al, 2001). Therefore, it is well recognized that the Gαq‐mediated Ca2+ signalling pathway has an important function in the development of cardiac hypertrophy.
Cardiac fibrosis is characterized by excessive deposition of extracellular matrix (ECM) proteins, such as collagen type I and type III (Brown et al, 2005). A variety of growth factors, such as Ang II, ET‐1, transforming growth factor (TGF)‐β, connective tissue growth factor (CTGF) and periostin, have been reported to promote fibrotic responses of the heart (Katsuragi et al, 2004; Zhang et al, 2006; Berk et al, 2007). Although cardiac fibrosis is accompanied by maladaptive cardiac hypertrophy that eventually results in heart failure, the mechanism of the induction of cardiac fibrosis and its pathophysiological function have yet to be understood.
The relationship between Ang II and TGF‐β for induction of fibrosis is a well‐established one (Rosenkranz, 2004; Berk et al, 2007). In many types of cells, it has been reported that Ang II stimulation regulates TGF‐β expression and activation. TGF‐β mediates some Ang II‐induced responses, and the blockade of Ang II‐mediated signalling partially suppresses TGF‐β‐induced fibrosis (Xu et al, 2008). Other signalling molecules for mediating fibrosis are Rho and Rho‐associated kinase (ROCK), which are known as downstream effectors of Ang II (Nishida et al, 2005). It has been reported that targeted deletion of ROCK suppressed the development of cardiac fibrosis induced by pathological hypertension (Rikitake et al, 2005; Zhang et al, 2006). ROCK is a downstream mediator of Rho, a small GTP‐binding protein (Amano et al, 2000), and Rho is reported to exert an effect as one of the downstream mediators of Gα12/13 (Kozasa et al, 1998). We have reported previously that α subunits of the G12 family protein (Gα12 and Gα13: Gα12/13) participate in Ang II‐, ET‐1‐, and α1‐adrenergic receptor agonist‐induced cardiomyocyte hypertrophy (Maruyama et al, 2002; Arai et al, 2003; Nishida et al, 2005). However, the pathophysiological function of Gα12/13 in cardiac hypertrophy and fibrosis in vivo is still unknown.
Gα12 and Gα13 appear to be expressed ubiquitously (Simon et al, 1991), and a lack of Gα13 in mice results in embryonic lethality because of the defective organization of the vascular system (Offermanns et al, 1997). Therefore, we generated mice with a cardiomyocyte‐specific overexpression of Gα12/13‐specific inhibitory polypeptide, which mimics the tissue‐specific knockout of Gα12/13. Using these mice, we tested the hypothesis that Gα12/13 signalling contributes to pressure overload‐induced cardiac hypertrophy and associated events in vivo.
Cardiac‐specific expression of p115‐RGS attenuates pressure overload‐induced cardiac fibrosis
Previous studies using transgenic or conditional knockout mice clearly revealed that Gαq predominantly regulates the development of hypertrophy (Adams et al, 1998; Wettschureck et al, 2001). In addition, we reported previously that Gα12/13 also have an important function in agonist‐induced hypertrophic responses of cardiomyocytes using a Gα12/13‐specific inhibitor, a regulator of the G protein signalling domain of p115RhoGEF (p115‐RGS) (Maruyama et al, 2002; Arai et al, 2003; Nishida et al, 2005). We generated mice with a cardiomyocyte‐specific overexpression of p115‐RGS protein to test the hypothesis that Gα12/13 signalling contributes to pressure overload‐induced cardiac hypertrophy in vivo (Supplementary Figure 1A–C). Ang II stimulation caused Rho activation in the hearts of wild‐type (WT) mice, and the activation was completely suppressed in transgenic (p115‐Tg) mice (Supplementary Figure 1D). This result confirmed that receptor‐stimulated activation of Gα12/13 signalling is inhibited in the p115‐Tg heart. Pressure overload was induced by surgical transverse aortic constriction (TAC) in WT and p115‐Tg mice. The increase in size of the p115‐Tg heart is essentially the same as that in WT mice (Figure 1A–C). TAC of p115‐Tg mice increased left ventricular end‐systolic pressure (LVESP) to the same extent as that in WT mice (Figure 1D), indicating that pressure overload by TAC was equally performed. TAC induced a significant elevation of left ventricular end‐diastolic pressure (LVEDP) in WT mice. However, there was no alteration in p115‐Tg mice (Figure 1E). Although the LV systolic function in p115‐Tg mice was slightly reduced in sham operation compared with that in WT mice, there was no further impairment by TAC (Figure 1E and Supplementary Table 1). These results suggest that systolic and diastolic function of the p115‐Tg heart is not impaired after TAC. TAC in WT mice strongly increased the expression of messenger ribonucleic acid (mRNA) of classical markers of pathological hypertrophy in myocardium, atrial natriuretic peptide (ANP), β‐myosin heavy chain (β‐MHC), and α‐skeletal muscle actin (α‐SKA) (Figure 1F). However, the expression of these genes in p115‐Tg hearts was less than half of that in WT hearts. We have reported that Gα12/13 mediate activation of Rho and c‐Jun NH2‐terminal kinase (JNK) in cultured cardiomyocytes (Maruyama et al, 2002; Arai et al, 2003; Nishida et al, 2005). Pressure overload increased the activities of Rho and JNK in WT hearts, but the activation of Rho and JNK was significantly suppressed in TAC of p115‐Tg hearts (Figure 1G and H). However, TAC‐induced Akt activation was not suppressed in p115‐Tg hearts, suggesting that p115‐RGS specifically inhibits Gα12/13‐mediated signalling in the heart. As the TAC‐induced Rac activation was also suppressed in p115‐Tg hearts (Supplementary Figure 1E), Gα12/13 may mediate TAC‐induced JNK activation through Rho‐ and Rac‐dependent pathways in mouse hearts as well as rat cardiac myocytes.
As overproduction of ECM protein causes ventricular stiffness leading to the impairment of diastolic function (Berk et al, 2007), we examined the involvement of Gα12/13 in pressure overload‐induced cardiac fibrosis. TAC increased the expression of collagen types I and III proteins in the interstitial tissue in WT mice, as determined by picrosirius red staining (Figure 2A). In contrast, TAC‐induced collagen deposition in p115‐Tg mice was less than half of that in WTs (Figure 2B). The relationship between heart weight to body weight ratio (HW/BW) and collagen expression in WT hearts reveals that the degree of hypertrophy correlates positively with severity of fibrosis (Figure 2C). In contrast, the correlation between HW/BW and collagen expression in p115‐Tg hearts also indicates that TAC‐induced cardiac fibrosis was significantly suppressed despite the development of cardiac hypertrophy. TAC increased the expression of mRNAs for procollagen type I and type III, fibrogenic factors (CTGF (Hahn et al, 2000), periostin (Katsuragi et al, 2004), and TGF‐βs (Zhang et al, 2000)) in WT hearts (Figure 2D). These increases were significantly attenuated in p115‐Tg hearts. Furthermore, TAC increased the expression of periostin, mature TGF‐βs, and angiotensin‐converting enzyme (ACE) in WT hearts, but these increases were attenuated also in p115‐Tg hearts (Figure 2E). It has been postulated that activation of the renin–angiotensin–aldosterone system (RAAS) and increased levels of active TGF‐β1 has an important function in pressure overload‐induced cardiac fibrosis (Berk et al, 2007). Pressure overload increased three TGF‐β mRNA isoforms and proteins in WT hearts, and these increases were suppressed in p115‐Tg hearts. These results suggest that Gα12/13 mediate pressure overload‐induced cardiac fibrosis by an increase in induction of CTGF, periostin, and TGF‐βs.
Activation of G13 in cardiomyocytes induces cardiac fibrosis
We also generated mice with cardiomyocyte‐specific overexpression of a constitutively active (CA) mutant of Gα13 protein (CA‐Gα13) (Supplementary Figure 2). The CA‐Gα13 heterozygous mice did not show an increase in heart size, compared with WT mice (Supplementary Figure 3A and B). However, collagen deposition was significantly increased (Supplementary Figure 3C). The expression of mRNA for CTGF was strongly increased in CA‐Gα13 mice (Supplementary Figure 3D). The expression of mRNA for ACE and protein expression were slightly increased (Supplementary Figure 3E). The expression of mRNAs for periostin and TGF‐βs was not increased, but proteins of periostin and TGF‐βs were increased in CA‐Gα13 hearts. As CA‐Gα13 increased proteins but not mRNAs for periostin and TGF‐βs, Gα13 may participate in the stabilizing of periostin and TGF‐βs proteins. These results also suggest that pressure overload‐induced expression of fibrogenic factors is mediated by Gα13. In contrast to CTGF, the expression of hypertrophy‐related genes (ANP and β‐MHC) was not increased in CA‐Gα13 heart, consistent with the inability of CA‐Gα13 to induce hypertrophy. However, strong activation of Gα13 signalling may induce cardiac hypertrophy as well as fibrosis, as CA‐Gα13 homozygous mice showed a significant increase in heart size (data not shown). The LV function of CA‐Gα13 mice was significantly decreased compared with that of WT mice (Supplementary Figure 3F and G). These results suggest that Gα12/13 mediate cardiac fibrosis and dysfunction induced by pressure overload.
Extracellular nucleotides mediate mechanical stretch‐induced G12/13 activation through purinergic receptors
As heterotrimeric G proteins are activated primarily by receptor stimulation, it is reasonable to assume that pressure overload activates Gα12/13‐coupled receptors. As mechanical stretch of cardiomyocytes is frequently used as an in vitro model of pressure overload, we examined which G protein‐coupled receptor(s) are involved in mechanical stress‐induced Gα12/13 activation. As activation of small GTP‐binding protein Rho is a sensitive marker of Gα12/13 activity (Kozasa et al, 1998), we measured Rho activity as an index of the magnitude of Gα12/13 signalling. Mechanical stretch of cardiomyocytes increased Rho activity, and this increase was sustained for 30 min (Figure 3A). Overexpression of p115‐RGS completely inhibited mechanical stretch‐induced Rho activation at early time and 30 min (Figure 3B and C). As a mutation in the RGS domain of p115RhoGEF loses the interaction with Gα12/13 (Bhattacharyya and Wedegaertner, 2003), we expressed the mutated p115‐RGS to examine whether the effects of p115‐RGS are specific for inhibition of interaction with Gα12/13. Expression of the interaction‐deficient mutant of p115‐RGS did not affect mechanical stretch‐induced Rho activation. In addition, treatment with Pertussis toxin, an uncoupler of receptor‐Gi interaction, did not suppress mechanical stretch‐induced Rho activation. These results suggest that mechanical stretch activates Rho through Gα12/13. It has been reported that Ang type 1 receptor (AT1R) is activated by mechanical stretch without the involvement of Ang II, and AT1R antagonist blocks mechanical stretch‐induced Gq activation and hypertrophic responses (Zou et al, 2004). However, mechanical stretch‐induced Rho activation through Gα12/13 was not attenuated by treatment with not only CV11974 (AT1R antagonist) but also PD123319 (AT2R antagonist), propranolol (β adrenergic receptor (AR) antagonist), prazosin (α1AR antagonist), BQ123 (ET type A receptor antagonist), BQ788 (ET type B receptor antagonist) and CGP20712A (selective β1AR antagonist) (Figure 3C and Supplementary Figure 4A and B). Mechanical stretch increases intracellular Ca2+ concentration through mechanosensitive cation channels (Christensen and Corey, 2007). However, treatment with an inhibitor of stretch‐sensitive channels GsMTx4, intracellular Ca2+ chelator BAPTA‐AM, and l‐type Ca2+ channel blocker nitrendipine did not suppress mechanical stretch‐induced Rho activation (Figure 3C and D). The Src family kinase substrate p130Cas has been reported to function as a mechanosensor (Sawada et al, 2006), but an Src inhibitor, PP2, did not affect mechanical stretch‐induced Rho activation (data not shown). In contrast, treatment with apyrase, an ATP/ADP scavenging enzyme, completely blocked mechanical stretch‐induced Rho activation (Figure 3D and Supplementary Figure 4C). Treatment with another ATP scavenging enzyme, hexokinase II, or purinergic receptor antagonists, suramin and PPADS, also suppressed mechanical stretch‐induced Rho activation. Furthermore, extracellular treatment with ATP, ADP, UTP, and UDP, but not adenosine, increased Rho activity (Supplementary Figure 4D–F). The extracellular nucleotide‐stimulated Rho activation was completely suppressed by the expression of p115‐RGS (Supplementary Figure 4G). Mechanical stretch actually activated Gα12 and Gα13, which were completely suppressed by treatment with suramin (Figure 3E and F). These results suggest that extracellular nucleotides mediate mechanical stretch‐induced Gα12/13 activation through purinergic receptors in rat cardiomyocytes.
Pannexin‐1 mediates mechanical stretch‐induced release of nucleotides
Extracellular ATP in the cardiovascular system may originate from different cellular sources, such as perivascular sympathetic nerve endings (Burnstock, 1972), activated platelets, endothelial cells, and inflammatory cells. It has also been postulated that connexin and pannexin hemichannels are involved in ATP release caused by mechanical stimulation in cardiac myocytes (Suadicani et al, 2000; Shestopalov and Panchin, 2008). Mechanical stretch of cardiomyocytes increased extracellular ATP concentration (Figure 4A). Treatment with hemichannel inhibitors, carbenoxolone, and 1‐heptanol, suppressed both mechanical stretch‐induced Rho activation and the increase in extracellular ATP concentration (Figures 3D and 4B). As the increase in extracellular ATP was not affected by p115‐RGS and P2 receptor antagonists (PPADS and suramin), Gα12/13 do not participate in ATP release, but rather mediate mechanical stretch‐induced Rho activation. The function of all connexins as gap junction channels or hemichannels is strongly dependent on Ca2+, but the function of pannexin‐1 is independent of Ca2+ (Shestopalov and Panchin, 2008). As mechanical stretch‐induced Rho activation was independent of Ca2+ (Figure 3C) and a low concentration of carbenoxolone (but not 1‐heptanol) inhibited mechanical stretch‐induced ATP release (Figure 4B), pannexin‐1 appears to be a prime candidate for an ATP release channel. Pannexin‐1 and pannexin‐2 mRNAs, but not pannexin‐3 mRNA, were expressed in mouse hearts and rat cardiomyocytes (Supplementary Figure 5). The expression of pannexin‐1 mRNA was increased by pressure overload (Supplementary Figure 5A). Treatment with siRNAs for pannexin‐1 induced a 50% decrease in pannexin‐1 mRNA levels (Supplementary Figure 5B). The mechanical stretch‐induced ATP release was decreased by about 50% in pannexin‐1 siRNA‐treated cardiomyocytes (Figure 4C). These results suggest that pannexin‐1 mediates ATP release by mechanical stretch in rat cardiomyocytes.
Involvement of P2Y6 receptor in mechanical stretch‐induced fibrotic responses
We also examined which receptor subtype(s) is involved in mechanical stretch‐induced Gα12/13 activation. RT–PCR analysis showed that mouse hearts express mRNAs coding P2Y1, P2Y2, P2Y4, P2Y6, and P2Y12 receptors (Supplementary Figure 6). Among them, mRNA levels of P2Y2 and P2Y6 receptors were upregulated in TAC hearts and CA‐Gα13 hearts. We also found that neonatal cardiomyocytes express mRNAs coding P2Y1, P2Y2, P2Y6, and P2Y12 receptors (data not shown). Treatment with MRS2578, a selective P2Y6 receptor antagonist, suppressed mechanical stretch‐induced Rho activation in a concentration‐dependent manner, with an IC50 value of about 0.1 μM (Figure 5A and B). In contrast, treatment with MRS2179 (a P2Y1 receptor antagonist), AR‐C67719MX (a P2Y12 receptor antagonist), and 8‐SPT (an adenosine receptor antagonist) did not suppress mechanical stretch‐induced Rho activation. As Rho is reported to regulate the expression levels of CTGF (Hahn et al, 2000) and periostin (Butcher et al, 2007), we examined the effects of P2Y receptor antagonists on the expression of these fibrogenic factors. Mechanical stretch increased expression of CTGF mRNA, which had been completely suppressed by the expression of p115‐RGS, and by treatment with suramin, PPADS, and MRS2578 (Figure 5C and D). Mechanical stretch increased the expression of TGF‐β2 mRNA but did not affect the expression of TGF‐β1 and ‐β3 mRNAs, and the induction of TGF‐β2 mRNA was also suppressed by suramin, PPADS, and MRS2578. In addition, mechanical stretch increased periostin proteins two‐fold, which had been completely suppressed by the expression of p115‐RGS, and by treatment with suramin, PPADS, and MRS2578 (Figure 5E). These increases were not affected by the expression of G protein‐coupled receptor kinase 2–RGS, a Gαq‐specific RGS domain (Nishida et al, 2005; Onohara et al, 2006), nor by the treatment with P2Y1 receptor antagonist (MRS2179), P2Y12 receptor antagonist (AR‐C67719MX), and 8‐SPT (an adenosine receptor antagonist). As P2Y2 receptor‐selective antagonist is not commercially available, we examined the involvement of the P2Y2 receptor in mechanical stretch‐induced Rho activation with siRNAs. The treatment with P2Y2‐specific siRNAs decreased the mRNA by about 50% but did not suppress Rho activity (Figure 5F). In contrast, the treatment with P2Y6‐specific siRNAs decreased the mRNA by about 70% and significantly suppressed mechanical stretch‐induced Rho activation in cardiomyocytes. These results suggest that the P2Y6 receptor predominantly regulates the mechanical stretch‐induced activation of fibrotic signalling in cardiomyocytes.
As the P2Y6 receptor is mainly activated by UDP (Vassort, 2001), and uridine nucleotides are known to be released by mechanical stretch (Lazarowski and Boucher, 2001), we also examined whether UDP is released by mechanical stretch. Mechanical stretch of cardiomyocytes increased extracellular UDP concentration three‐fold (Figure 4D). In addition, treatment of P2Y6 receptor‐overexpressing HEK293 cells with supernatant from mechanically stretched rat cardiomyocytes significantly increased intracellular Ca2+ concentrations ([Ca2+]i) (Figure 4E). The magnitude of maximal increase in [Ca2+]i induced by the supernatant was equivalent to the peak [Ca2+]i increase induced by 30 nM of extracellular UDP (Figure 4F and G). As H9c2 myofibroblasts do not express P2Y1 and P2Y2 receptors, we further examined the effects of nucleotides on [Ca2+]i increase using H9c2 cells. Treatment of vector‐expressing H9c2 cells with UDP, ATP, or the supernatant of stretch‐activated cardiomyocytes did not show any significant increases in [Ca2+]i, but the treatment with the supernatant significantly increased [Ca2+]i in P2Y6 receptor‐overexpressing H9c2 cells (Figure 4H). This [Ca2+]i increase was completely suppressed by the treatment of cardiomyocytes with carbenoxolone, suggesting that pannexin‐1 mediates mechanical stretch‐induced UDP release. Furthermore, treatment of cardiomyocytes with 3‐phenacyl‐UDP, a highly selective P2Y6 receptor agonist, increased Rho activity in a concentration‐dependent manner (Supplementary Figure 4H). These results suggest that extracellular UDP predominantly mediates mechanical stretch‐induced P2Y6 receptor activation in cardiomyocytes.
Inhibition of P2Y6 receptors attenuates pressure overload‐induced cardiac fibrosis in vivo
We next examined whether purinergic receptors actually participate in pressure overload‐induced cardiac fibrosis in vivo. Treatment with MRS2578 after TAC significantly suppressed pressure overload‐induced collagen deposition without affecting cardiomyocyte hypertrophy (Figure 6A–C). Treatment with MRS2578 significantly suppressed LV dysfunction induced by pressure overload (Figure 6D and E and Supplementary Table 3). Furthermore, the treatment with MRS2578 suppressed the increases in mRNA expressions of ANP, β‐MHC, procollagen type I, periostin, and TGF‐β2 by pressure overload (Figure 6F). We also found that MRS2578 inhibited pressure overload‐induced Rho activation and TAC‐induced increases in expression of periostin, mature TGF‐βs, and ACE proteins (Figure 6G and H). Furthermore, we found that treatment with suramin also suppressed pressure overload‐induced collagen deposition and LV dysfunction (Supplementary Figure 7 and Supplementary Table 4). These results suggest that inhibition of P2Y6 receptors actually attenuates pressure overload‐induced cardiac fibrosis and LV dysfunction.
Remodelling of the heart, including accumulation of ECM and an associated change in ventricular geometry, is a common feature of heart failure. In this study, we found that Gα12/13 mediate cardiac fibrosis without the development of hypertrophy induced by pressure overload. We reported previously that Gα12/13 mediate agonist‐induced hypertrophic responses of cardiomyocytes. However, we also found that mechanical stretch‐induced increases in NFAT‐ and BNP‐dependent transcriptional activities are not suppressed in p115‐RGS‐expressing myocytes (Supplementary Figure 8). These results suggest that activation of Gα12/13 is not involved in mechanical stress‐induced NFAT and BNP expression. Gα12/13 are activated by extracellular ATP and UDP that are released by mechanical stretch. The nucleotides released through pannexin‐1 hemichannels activate Gα12/13‐mediated Rho activation leading to the induction of fibrogenic factors, such as CTGF and periostin. Furthermore, inhibition of purinergic receptors attenuates the TAC‐induced cardiac fibrosis and LV dysfunction. These results indicate that activation of G12/13‐coupled purinergic receptors in cardiomyocytes by extracellular nucleotides stimulate the secretion of fibrogenic factors and trigger pressure overload‐induced cardiac fibrosis (Figure 7). Purinergic receptors are classified into two families: P2X and P2Y. P2X receptors are transmitter‐gated channels and consist of 7 subtypes. P2Y receptors are G protein‐coupled receptors and are divided into eight subtypes. We found that the P2Y6 receptor predominantly regulates mechanical stretch‐induced Rho activation and the expression of fibrogenic factors in rat cardiac myocytes (Figure 5). We also found that inhibition of P2Y6 receptors suppressed cardiac fibrosis and diastolic dysfunction induced by pressure overload (Figure 6). These results suggest that P2Y6 receptors in cardiomyocytes have an important function in pressure overload‐induced cardiac fibrosis.
It has been reported that CTGF has an important function in cardiac fibrosis. In contrast to CTGF, the function of periostin remains to be determined. Extracellular application of periostin induced re‐entry of cardiomyocytes into the cell cycle, and reduced fibrosis whereas improving cardiac functions (Kühn et al, 2007). However, analysis of knockout and transgenic mice reveals that periostin is involved in myocardial infarction‐induced fibrosis and impairment of ventricular functions (Oka et al, 2007; Shimazaki et al, 2008). They also demonstrated that pressure overload‐induced hypertrophic responses and fibrosis are regulated by periostin. The present results are consistent with the findings that periostin is involved in pressure‐overload‐induced cardiac fibrosis.
It is interesting to note that the G12/13‐mediated pathway regulates fibrosis, and the Gq/11‐mediated pathway regulates hypertrophy. Two different G proteins regulate two distinct responses: fibrosis and hypertrophy. Many groups using transgenic and knockout mice have reported that suppression of hypertrophy leads to the inhibition of fibrosis. However, we demonstrated that fibrosis and hypertrophy are independent processes, as revealed by expressing p115‐RGS to block Gα12/13 functions. Therefore, Gα12/13‐mediated signalling leading to cardiac fibrosis may turn on after hypertrophy is already developed. This speculation is supported by the finding that pannexin‐1 mRNA in the heart is upregulated by pressure overload (Supplementary Figure 5A). Thus, the process of the hypertrophied heart depositing ECM proteins in vivo may be triggered by the release of ATP and UDP from myocytes during transition from hypertrophy to heart failure.
There are three structurally distinct TGF‐βs (Bujak and Frangogiannis, 2007). TGF‐β1 is a prevalent isoform, and TGF‐β2 and ‐β3 are expressed in limited tissues. As these three isoforms do not compensate for functions of other isoforms, each TGF‐β has specific and independent roles in vivo. Among these three isoforms, it has been reported that TGF‐β1 mediates Ang II‐induced hypertrophic responses in vivo (Schultz Jel et al, 2002). Myocardial infarction increases the expression of these three TGF‐β isoforms, which participate in inflammation at an early phase and cardiac remodelling at a later phase. We found that TGF‐β2 mRNA was most responsive to TAC, which induces cardiac fibrosis (Figure 1). We also demonstrated that p115‐RGS and the P2Y6 receptor antagonist inhibit the expression of TGF‐β2 mRNA by pressure overload (Figure 2 and Figure 6). TGF‐β2 may be the predominant form of TGF‐β for the promotion of fibrosis in the heart.
Our results also indicate that Gα13 mediates pressure overload‐induced expression of ACE proteins (Figure 1 and Supplementary Figure 3). Although inhibition of ACE expression has been reported to inhibit pressure overload‐induced cardiac hypertrophy in rats (Baker et al, 1990; Zierhut et al, 1991), inhibition of Gα12/13, upstream of ACE, did not suppress cardiac hypertrophy in mice (Figure 1). We do not have any data to explain this discrepancy. However, our data are consistent with the results of Xiao et al. (2008), which show that an increase in ACE expression does not augment pressure overload‐induced cardiac hypertrophy in mice. In addition, pressure overload induces cardiac hypertrophy in angiotensinogen‐knockout mice (Zou et al, 2004). Crowley et al (2006) have reported that Ang II induces cardiac hypertrophy in mice through stimulation of AT1 receptors in the kidney. It has been reported that the expression of a gain‐of‐function mutant of Ang II type 1A receptor in the heart causes cardiac fibrosis but not hypertrophy (Billet et al, 2007). Thus, increase in cardiac ACE activity induced by pressure overload may not contribute to the development of cardiac hypertrophy in mice. Using rat cardiomyocytes, we found that mechanical stretch‐induced activation of JNK and p38 MAPK, but not ERK, was suppressed by p115‐RGS (Supplementary Figure 8). As ERK, but not JNK and p38 MAPK, participates in cardiac hypertrophy (Liang and Molkentin, 2003), Gα12/13‐mediated ACE expression may participate in mechanical stress (pressure overload)‐induced hypertrophy in rats but not mice.
Cardiac fibrosis is considered one of the inflammatory responses of the heart (Brown et al, 2005). A variety of evidence supports the idea that extracellular nucleotides function as a mediator of inflammatory responses, such as chemotaxis and phagocytosis (Chen et al, 2006; Idzko et al, 2007; Koizumi et al, 2007). Our data suggest that extracellular nucleotides function as a priming factor in the development of cardiac fibrosis induced by pressure overload. It is generally thought that activation of the RAAS system and increased levels of active TGF‐β stimulate cardiac fibroblasts and induce ECM deposition, leading to perivascular fibrosis. CTGF, periostin, and TGF‐β2 mRNAs were upregulated by pressure overload, and the increased expression of three genes were suppressed in p115‐Tg mice. Furthermore, a P2Y6 receptor antagonist MRS2578 suppressed the stress‐induced expression of periostin and TGF‐β mRNAs in vitro and in vivo. In addition to TGF‐β, we demonstrate that CTGF and periostin are also involved in pressure overload‐induced cardiac fibrosis. As Gα12/13 mediate cardiac fibrosis, which is associated with pressure overload‐induced hypertrophy, the development of drugs to block P2Y6 receptors‐Gα12/13 signalling may be a novel strategy for heart failure.
The interrelationship between Ang II and TGF‐β is well established. The blockade of TGF‐β by an antibody and a mutated TGF‐receptor suppressed some of Ang II‐induced responses (Bujak and Frangogiannis, 2007). Therefore, it is reasonable to assume that Ang II stimulates TGF‐β expression, which leads to ECM deposition. As the blockade of the P2Y6 receptor with MRS2578 suppressed the expression of ACE mRNA, and the blockade of Gα12/13 suppressed the expression of TGF‐β mRNAs, ATP and UDP work as an upstream regulator of the Ang II‐TGF‐β system. This also suggests that extracellular nucleotide‐stimulated Gα12/13 activity regulates the Ang II‐TGF‐β pathway through upregulation of ACE.
It has been reported that CTGF mediates some TGF‐β‐induced fibrogenic responses. Inhibition of CTGF synthesis or activity suppressed TGF‐β‐induced collagen synthesis (Perbal, 2004). It is also reported that CTGF synergizes fibrogenic responses with TGF‐β by the mechanisms on the basis of the binding of CTGF to TGF‐β or transcriptional suppression of Smad7 (Ruiz‐Ortega et al, 2007). As CTGF expression was increased in a CA‐Gα13‐Tg heart without affecting TGF‐β expression, extracellular ATP and UDP may directly increase the expression of CTGF through P2Y6 and Gα12/13, with CTGF then promoting the production of TGF‐β. Thus, extracellular nucleotides have an important function in fibrogenic responses of the heart.
Diastolic dysfunction associated with preserved systolic function is increasingly recognized as a critical cause of heart failure. As the cardiac ECM is the major determinant of myocardial stiffness during diastole, cardiac fibrosis contributes to diastolic dysfunction. We found that 6 weeks of TAC induces impairment of LV diastolic functions, which were attenuated by the inhibition of Gα12/13 signalling or purinergic P2Y6 receptors. As cardiac fibrosis associated with maladaptive hypertrophy is thought as a cause of impairment of cardiac function, purinergic receptors may be promising targets for the treatment of heart failure.
Materials and methods
Animals and TAC surgery
All protocols using mice and rats conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85–23, revised 1996) and were approved by the guidelines of Kyushu University. Transgenic C57BL/6J mice expressing p115‐RGS were tried to generate three times. We obtained only one line that was used in this study. Two lines of transgenic mice expressing CA‐Gα13 were generated (lines 1 and 5). Heterozygote of line 5 was used in this study. Age‐matched male WT C57BL/6J mice were used as control. TAC surgery was performed on 8‐ to 10‐week‐old male p115‐Tg and WT C57BL/6J mice. A mini‐osmotic pump (Alzet) filled with saline, MRS2578, or suramin was implanted intraperitoneally 3 days after TAC into 6‐week‐old male C57BL/6J mice. Details can be found in Supplementary methods at The EMBO Journal Online (http://embojournal.org).
Haemodynamic measurements and histological analyses
Transthoratic echocardiography was performed using ALOKA ultrasonic image analysing system (SSD‐5500) equipped with 7.5 MHz imaging transducer. Blood pressure was monitored using tail‐cuff system (BP‐98A, Softron). LV pressure and heart rate were measured with a micronanometer catheter (Millar 1.4F, SPR 671, Millar Instruments). Histological analyses can be found in Supplementary methods at The EMBO Journal Online (http://embojournal.org).
Isolation of cardiomyocytes and transfection
Cultures of neonatal rat cardiac myocytes and adenoviral infection were performed as described previously (Nishida et al, 2000). Details can be found in Supplementary methods at The EMBO Journal Online (http://embojournal.org).
Pulldown assay and western blot analysis
Methods for pulldown assay and western blot analysis can be found in Supplementary methods at The EMBO Journal Online (http://embojournal.org).
Measurement of extracellular nucleotides concentration
The determination of extracellular ATP concentration (2 × 105 cells per well) was performed using ATP Bioluminescence Assay Kit CLSII (Roche). The concentration of extracellular UDP in the supernatant of culture medium was analysed with an HPLC system (Jasco) as described previously (Koizumi et al, 2007). Details can be found in Supplementary methods at The EMBO Journal Online (http://embojournal.org).
Measurement of mRNA expressions
Real‐time RT–PCR was performed as described (Nagamatsu et al, 2006; Nishida et al, 2007). Sequences for PCR primers and Taqman probes were described in Supplementary information (Supplementary Table 5). The PCR primers used for expression analysis of P2Y receptors are described in Supplementary Table 6. Details can be found in Supplementary methods at The EMBO Journal Online (http://embojournal.org).
Data were shown as means±s.e.m. Statistical comparisons were made with two‐tailed Student's t‐test or analysis of variance followed by Student–Newman–Keuls procedure, with significance imparted at P‐values <0.05.
Supplementary data are available at The EMBO Journal Online (http://www.embojournal.org).
We thank Naoki Nishihara for TAC surgery and measurement of cardiac functions during the early stage of this study. We also thank Drs Makoto Tsuda (Kyushu University) and Isao Matsuoka (Takasaki University of Health and Welfare) for helpful discussion and Dr Jeffery Robbins (Cincinnati Children's Hospital Medical Center) for α‐MHC promoter. This study was supported by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan (to MN and HK); from the Ministry of Health, Labour, and Welfare of Japan and the Japan Health Sciences Foundation (to YS); and from the Program for Promotion of Fundamental Studies in Health Sciences of the National Institute of Biomedical Innovation (to MN).
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