Simultaneous loss of phospholipase Cδ1 and phospholipase Cδ3 causes cardiomyocyte apoptosis and cardiomyopathy

Phospholipase C (PLC) is a key enzyme in phosphoinositide turnover. Among 13 PLC isozymes, PLCδ1 and PLCδ3 share high sequence homology and similar tissue distribution, and are expected to have functional redundancy in many tissues. We previously reported that the simultaneous loss of PLCδ1 and PLCδ3 caused embryonic lethality because of excessive apoptosis and impaired vascularization of the placenta. Prenatal death of PLCδ1/PLCδ3 double-knockout mice hampered our investigation of the roles of these genes in adult animals. Here, we generated PLCδ1/PLCδ3 double-knockout mice that expressed PLCδ1 in extra-embryonic tissues (cDKO mice) to escape embryonic lethality. The cDKO mice were born at the expected Mendelian ratio, which indicated that the simultaneous loss of PLCδ1 and PLCδ3 in the embryo proper did not impair embryonic development. However, half of the cDKO mice died prematurely. In addition, the surviving cDKO mice spontaneously showed cardiac abnormalities, such as increased heart weight/tibial length ratios, impaired cardiac function, cardiac fibrosis, dilation, and hypertrophy. Predating these abnormalities, excessive apoptosis of their cardiomyocytes was observed. In addition, siRNA-mediated simultaneous silencing of PLCδ1 and PLCδ3 increased apoptosis in differentiated-H9c2 cardiomyoblasts. Activation of Akt and protein kinase C (PKC) θ was impaired in the hearts of the cDKO mice. siRNA-mediated simultaneous silencing of PLCδ1 and PLCδ3 also decreased activated Akt and PKCθ in differentiated-H9c2 cardiomyoblasts. These results indicate that PLCδ1 and PLCδ3 are required for cardiomyocyte survival and normal cardiac function.

Y Nakamura 1,2 , K Kanemaru 1,2 , R Kojima 1 , Y Hashimoto 1 , T Marunouchi 3 , N Oka 1 , T Ogura 1 , K Tanonaka 3 and K Fukami* , 1,2 Phospholipase C (PLC) is a key enzyme in phosphoinositide turnover. Among 13 PLC isozymes, PLCd1 and PLCd3 share high sequence homology and similar tissue distribution, and are expected to have functional redundancy in many tissues. We previously reported that the simultaneous loss of PLCd1 and PLCd3 caused embryonic lethality because of excessive apoptosis and impaired vascularization of the placenta. Prenatal death of PLCd1/PLCd3 double-knockout mice hampered our investigation of the roles of these genes in adult animals. Here, we generated PLCd1/PLCd3 double-knockout mice that expressed PLCd1 in extra-embryonic tissues (cDKO mice) to escape embryonic lethality. The cDKO mice were born at the expected Mendelian ratio, which indicated that the simultaneous loss of PLCd1 and PLCd3 in the embryo proper did not impair embryonic development. However, half of the cDKO mice died prematurely. In addition, the surviving cDKO mice spontaneously showed cardiac abnormalities, such as increased heart weight/tibial length ratios, impaired cardiac function, cardiac fibrosis, dilation, and hypertrophy. Predating these abnormalities, excessive apoptosis of their cardiomyocytes was observed. In addition, siRNA-mediated simultaneous silencing of PLCd1 and PLCd3 increased apoptosis in differentiated-H9c2 cardiomyoblasts. Activation of Akt and protein kinase C (PKC) h was impaired in the hearts of the cDKO mice. siRNAmediated simultaneous silencing of PLCd1 and PLCd3 also decreased activated Akt and PKCh in differentiated-H9c2 cardiomyoblasts. These results indicate that PLCd1 and PLCd3 are required for cardiomyocyte survival and normal cardiac function. Dilated cardiomyopathy (DCM) is the most common type of non-ischemic cardiomyopathy, and is characterized by myocardial contractile dysfunction and cardiac diameter enlargement, which leads to heart failure. DCM is often accompanied by pathological remodeling, such as cardiac fibrosis and hypertrophy. In addition, apoptotic cardiomyocytes are observed in the hearts of humans with DCM. [1][2][3] Interestingly, cardiomyocyte apoptosis was reported to be sufficient to induce adverse cardiac remodeling in an animal model. 4 Phospholipase C (PLC) is a key enzyme in phosphoinositide turnover. PLC hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP 2 ) to generate two second messengers, inositol 1,4,5-trisphosphate (IP 3 ) and diacylglycerol. 5,6 There are 13 PLC isoforms in mammals, and they are divided into six types on the basis of sequence homology and activation mechanisms. 7,8 Among them, d-type PLC has a relatively simple structure and is thought to be a primitive type of PLC isoform. 9 We previously reported that the loss of PLCd1 resulted in skin abnormalities in mice, such as hair loss, epidermal hyperplasia, and cytokine overproduction. [10][11][12][13] Disruption of the PLCd1 gene also protects mice from obesity by inhibiting lipid accumulation in adipose tissue. 14 Regarding PLCd3, siRNA-mediated silencing of PLCd3 inhibits neuronal migration and neurite outgrowth. 15 PLCd1 and PLCd3 are expressed in the heart, in addition to the skin, adipose, and neuronal tissues, and are expected to have critical roles in the cardiovascular system. The downstream effector of PLC, protein kinase C (PKC) has critical roles in cardiac structure and function. Several PKC isozymes are expressed in cardiomyocytes and regulate cardiac responses. 16 Overexpression of PKCa in cultured cardiomyocytes induces hypertrophy. 17 On the other hand, loss of PKCa prevents the transition from cardiac hypertrophy to cardiac failure. 18 In addition, overexpression of PKCb 1 leads to cardiac hypertrophy and sudden death. 19 Thus, hyperactivation or overexpression of PKCa or PKCb 1 is an inducible factor for cardiac hypertrophy and failure. In contrast, PKCe protects the heart from apoptosis induced by ischemia and reperfusion injury, 20 and PKCy is required for cardiomyocyte survival and cardiac remodeling. 21 The conventional PKC isozymes, PKCa and PKCb I are activated by calcium and diacylglycerol, whereas the novel PKC isozymes, PKCe and PKCy require diacylglycerol but do not require calcium. Their distinct activation mechanisms may be involved in the difference in physiological functions between conventional and novel PKC isozymes. Both conventional and novel PKC isozymes are activated downstream of PLC. However, mice that lack either PLCd1 or PLCd3 do not show apparent cardiac abnormalities. As PLCd1 and PLCd3 share high sequence homology, one gene product likely compensates for the lack of the product of the other gene in mice. Unfortunately, the simultaneous loss of PLCd1 and PLCd3 results in embryonic lethality because of excessive apoptosis of placental trophoblasts and impaired vascularization of the placenta, 22 which hampered our investigation into the role of the PLCd1 and PLCd3 genes in the cardiovascular systems in adult animals. In this study, we generated and analyzed mice that lack PLCd1 and PLCd3 with extra-embryonic PLCd1 expression and found that the simultaneous loss of PLCd1 and PLCd3 results in DCM-like phenotypes that are associated with excessive apoptosis of cardiomyocytes.
cDKO mice had hearts with abnormal morphologies and impaired function. We next analyzed cDKO mice that survived the first 4 weeks. Although surviving cDKO mice had a normal life span, they had smaller body sizes than did the mice that lacked either PLCd1 or PLCd3. Morphological analysis revealed that Meox2 þ / þ PLCd1 fl/ À PLCd3 þ / À , Meox2 cre/ þ PLCd1 fl/ À PLCd3 þ / À , and Meox2 þ / þ PLCd1 fl/ À PLCd3 À / À mice had normal-shaped hearts, whereas the cDKO mice had irregular-shaped hearts with white foci after 6 weeks of age (arrows in Figure 2a). In addition, the heart weight/tibial length ratio was significantly increased for the cDKO mice ( Figure 2b). Although heart weights were not significantly increased, cDKO mice have smaller body and their tibial length was shorter than that of control mice, resulting in increase in heart weight/tibial length ratio. This increase in the ratio was not observed in other organs, such as liver, indicating that the increase in the heart weight/tibial length ratio was specific. Because the cDKO hearts showed abnormal morphologies, we examined their cardiac function by performing echocardiography (Figures 2c and d). The ejection fraction (EF) and fractional shortening (FS) are echocardiographic indicators of overall cardiac function. There were no significant differences in the EF and FS among all genotypes at 4 weeks of age ( Figure 2c). After 6 weeks, both the FS and EF were significantly decreased in the cDKO mice ( Figure 2c). These results suggest that the cDKO mice had impaired cardiac function, and that this impairment occurred between 4 and 6 weeks of age, which was concomitant with their morphological abnormalities ( Figure 2a). In addition to the EF and FS, the end-systolic volume and end-diastolic volume were increased in the cDKO hearts (Figure 2c), which indicated that the cDKO mice showed DCM-like phenotypes. Because the induction of natriuretic peptides ANP and BNP is a marker of cardiac failure, we measured the mRNA levels of ANP and BNP in the cDKO hearts. At 8 weeks of age, both the ANP and BNP were upregulated in the cDKO hearts ( Figure 2e). The cDKO hearts also exhibited the a-to-b isoform switch of myosin heavy chain (MHC) and an increased ratio of b-MHC/a-MHC expression, which are typical features of heart failure at 8 weeks of age ( Figure 2e). Thus, the cDKO hearts showed cardiac dysfunction and upregulation of cardiac failure markers. It is possible that the cardiac abnormalities in the cDKO mice were attributable to right ventricle cardiomyopathy. Therefore, we examined whether abnormal structure of the right ventricle and subsequent structural changes in the lungs were observed in cDKO mice. There were no apparent changes in the structure of the lungs or the right ventricle in cDKO mice, suggesting that the cardiac phenotypes were not caused by right ventricle cardiomyopathy (Supplementary Figure S1). In our mating strategy, Meox2 þ / þ PLCd1 fl/ À PLCd3 þ / À , Meox2 cre/ þ PLCd1 fl/ À PLCd3 þ / À , and Meox2 þ / þ PLCd1 fl/ À PLCd3 À / À mice were obtained as littermates of cDKO (Meox2 cre/ þ PLCd1 fl/ À PLCd3 À / À ) mice (Table 1). Any cardiac abnormalities that were observed in the cDKO mice were not observed in the littermates with other genotypes. Therefore, the littermates of the cDKO mice were used as a control in subsequent experiments, irrespective of their genotypes.
Simultaneous loss of PLCd1 and PLCd3 caused cardiac fibrosis and hypertrophy. Because the cDKO mice showed cardiac dysfunction, we examined detailed histological structures of their hearts. Hematoxylin and eosin (HE) staining revealed that the cDKO hearts had fibrotic lesions ( Figure 3a). Although these fibrotic lesions were not observed at 4 weeks of age, fibrosis was observed in the ventricular walls after 6 weeks of age ( Figure 3a). We also stained collagen fibers by performing trichrome staining and found that the ventricular walls exhibited strong positive staining after 6 weeks of age (means ± S.E.M. fibrosis-to-total ventricular area ratios, 0.0074±0.00096 in 6-week-old Meox2 þ / þ PLCd1 fl/ À PLCd3 þ / À , 0.087 ± 0.0095 in 6-weekold cDKO, 0.0071 ± 0.0011 in 12-week-old Meox2 þ / þ PLCd1 fl/ À PLCd3 þ / À , and 0.094±0.0075 in 12-week-old cDKO mice; n ¼ 3, for each) ( Figure 3b). We further confirmed the occurrence of cardiac fibrosis at the molecular level by assessing the expression of fibrosis-related genes. Real-time reverse transcriptase PCR (RT-PCR) revealed that extracellular matrix components, such as fibronectin and collagen (Col1A1 and Col3A1), were upregulated in the cDKO hearts ( Figure 3c and Supplementary Table S1). We also observed the upregulation of pro-fibrotic factors, such as connective tissue growth factor (CTGF), transforming growth factor (TGF)b2, and TGFb3 in the cDKO hearts ( Figure 3c and Supplementary Table S1). The markers for cardiac remodeling that were associated with cardiac fibrosis, such as tissue inhibitors of metalloproteinase 1 (TIMP-1) and matrix metalloproteinase 2 (MMP-2), were also upregulated in the cDKO hearts (Figure 3c and Supplementary Table S1).
These results indicate that the cDKO hearts showed cardiac fibrosis at both the histological and molecular levels.
Because elevated blood pressure may be a cause for cardiac failure and fibrosis, we measured the blood pressures of the cDKO mice. The cDKO mice did not show remarkable changes in their systolic blood pressure when compared with the control mice (Figure 3d and  Supplementary Table S2), which indicated that their cardiac fibrosis was not caused by hypertension. Apart from cardiac fibrosis, the cDKO cardiomyocytes were hypertrophic.
No. with genotype 70 (64) 64 (64) 62 (64) 62 (64)  258 The numbers in parentheses were calculated from the expected Mendelian frequencies Cardiac functions of PLCd1 and PLCd3 Y Nakamura et al , and Meox2 cre/ þ PLCd1 fl/ À PLCd3 À / À (cDKO) mice at 4, 6, 8, and 12 weeks of age. Scale bar, 2 mm. The boxed areas in the cDKO hearts at 6, 8, and 12 weeks are magnified in the right panels. The arrows indicate white foci. (b) Heart (upper panel) or liver (lower panel) weight-to-tibial length ratios of Meox2 Although the cell size remained unchanged at 2 and 4 weeks of age, the cardiomyocyte cross-sectional areas increased at 12 weeks of age ( Figure 3e). Thus, cDKO hearts showed signs of pathological remodeling, such as cardiac fibrosis and hypertrophy, which is often associated with DCM.
Simultaneous depletion of PLCd1 and PLCd3 caused cardiomyocyte apoptosis. Because apoptotic loss of cardiomyocytes leads to cardiac dysfunction and remodeling, 4 we determined the number of apoptotic cells in the heart ventricles of cDKO mice that were 4 weeks of age before the onset of these cardiac abnormalities. A terminal transferase dUTP nick end labeling (TUNEL) assay revealed that apoptotic cardiomyocytes were easily found in the cDKO ventricles, whereas apoptotic cardiomyocytes were rarely observed in control ventricles (means±S.E.M. for TUNEL-positive cells, 0.051 ± 0.021% in Meox2 þ / þ PLCd1 fl/ À PLCd3 þ / À , 0.050±0.0031% in Meox2 cre/ þ PLCd1 fl/ À PLCd3 þ / À , 0.057±0.0047% in Meox2 þ / þ PLCd1 fl/ À PLCd3 À / À , and 0.14 ± 0.046% in cDKO mice; n ¼ 3 for each) (Figures 4a and b). There were neither apparent structural abnormalities nor an increase in the number of TUNELpositive cells in the hearts of E17.5 embryos, compared with those in the hearts of control embryos, strongly suggesting that PLCd1 and PLCd3 were not required for cardiac protection during normal development (Supplementary Figure S2). We also confirmed that mRNA expression of the proapoptotic gene Bax was significantly increased in the cDKO hearts at 4 weeks of age (Figure 4c). In addition, western blotting showed that levels of proapoptotic proteins, Bax and Bad were increased in cDKO hearts, whereas that of antiapoptotic protein, Bcl-2, were decreased in the cDKO hearts ( Figure 4d). Thus, the cDKO mice showed excessive apoptosis of cardiomyocytes. We further carried out siRNA-mediated simultaneous knockdown of PLCd1 and PLCd3 in H9c2 rat cardiomyoblasts with two distinct siRNA that target PLCd1 and PLCd3, and induced the differentiation of H9c2 cells into cardiomyocytes with low-serum differentiation-promoting medium. Western blotting revealed that the amounts of PLCd1 and PLCd3 proteins were decreased in the differentiated-H9c2 cells by the introduction of the siRNA (Figure 5a), which indicated the effective knockdown of PLCd1 and PLCd3. The simultaneous knockdown of PLCd1 and PLCd3 resulted in cell spreading (Figure 5b), which suggests that PLCd1 and PLCd3 regulate the morphology of cardiomyocytes. Unexpectedly, similar morphological changes were observed by the single knockdown of PLCd3 (Figure 5b), which suggests that PLCd3 (c) mRNA expression of the proapoptotic gene Bax in the hearts of control (Meox2 þ / þ PLCd1 fl/ À PLCd3 À / À , n ¼ 2; Meox2 cre/ þ PLC fl/ À PLCd3 þ / À , n ¼ 2; Meox2 þ / þ PLCd1 fl/ À PLCd3 þ / À n ¼ 3) and cDKO mice at 4 weeks of age. The results are listed in arbitrary units (expression in control mice ¼ 1). Mean þ S.E.M. (control, n ¼ 7; cDKO, n ¼ 4). Statistical significance was assessed using Student's t-test. *Po0.05 and **Po0.01. (d) Immunoblotting of Bax, Bad, Bcl-2, and GAPDH in Meox2 þ / þ PLCd1 fl/ À PLCd3 þ / À (hetero), Meox2 cre/ þ PLC fl/ À PLCd3 þ / À (PLCd1KO), Meox2 þ / þ PLCd1 fl/ À PLCd3 À / À (PLCd3KO), and Meox2 cre/ þ PLCd1 fl/ À PLCd3 À / À (cDKO) hearts at 8 weeks of age downregulation is, in contrast to in vivo case, sufficient for the induction of abnormal morphology of cardiomyocytes in vitro. In addition to morphological changes, simultaneous downregulation of PLCd1 and PLCd3 induces nuclear shrinking, which is a typical feature of apoptosis under normal culture conditions (Figures 5c and d) and oxidative stress conditions (Figures 5e and f). We further confirmed the apoptosis of cardiomyocytes by TUNEL staining and found that simultaneous silencing of PLCd1 and PLCd3 increased the number of TUNEL-positive cells under normal culture conditions (Figures 5g and h) and oxidative stress conditions (Figures 5i and j). Although the extent was milder than the Cardiac functions of PLCd1 and PLCd3 Y Nakamura et al extent in double-knockdown cells, single knockdown of PLCd3 also induced apoptosis. Taken together, these findings suggest that simultaneous depletion of PLCd1 and PLCd3 increased the apoptosis of cardiomyocytes in a cell-intrinsic manner.
Simultaneous depletion of PLCd1 and PLCd3 impaired the activation of PKCh. Increased apoptosis in the cDKO heart could be caused by impairment of survival signals and activation of proapoptotic signals. Western blotting revealed that activation of Akt was impaired in cDKO hearts and in H9c2 cells under oxidative stress conditions, indicating that the survival signal was impaired by a combined loss of PLCd1 and PLCd3 (Figures 6a and b). In contrast, the combined loss of PLCd1 and PLCd3 did not affect activation of ERK. In addition, the combined loss of PLCd1 and PLCd3 increased the level of activated caspase 9 (Figure 6a), indicating that a proapoptotic signal was activated in the cDKO heart. PKC isozymes have critical roles in the maintenance of cardiac structure and functions. As active PKC isozymes are reported to be present in the particulate fraction of the heart, 21 we examined the amounts of PKC isozymes in particulate fractions of the heart at 6 weeks of age. The amount of PKCy was decreased in the particulate fraction of the cDKO heart compared with the control heart ( Figure 6c and Supplementary Figure S3). In contrast, simultaneous loss of PLCd1 and PLCd3 did not cause remarkable changes in the levels of PKCa, PKCb 1 , and PKCe in the particulate fraction (Figure 6c). Impaired activation of PKCy was also observed in PLCd1/PLCd3 double-knockdown H9c2 cells by examining the amount of phosphorylated PKCy (Figure 6d). Consistent with mild induction of apoptosis by single knockdown of PLCd3 (Figures 5g-j), the amount of phosphorylated PKCy was slightly decreased in PLCd3 single-knockdown H9c2 cells (Figure 6d). These results indicate that simultaneous depletion of PLCd1 and PLCd3 selectively impaired the activation of PKCy. Interestingly, siRNA-mediated silencing of PKCy in H9c2 cells resulted in morphological changes and increased apoptosis, in a manner similar to the combined silencing of PLCd1 and PLCd3 (Figures 6e-h). Furthermore, the numbers of cells with shrunken nuclei were decreased by treating PLCd1/PLCd3 double-knockdown H9c2 cells with a PKC activator, phorbol 12-myristate 13-acetate (PMA) (Figure 6i). These results strongly suggest that PKCy is involved in the morphological changes and high apoptosis caused by the simultaneous knockdown of PLCd1 and PLCd3.

Discussion
In this study, we demonstrated that the simultaneous loss of PLCd1 and PLCd3 induces cardiac fibrosis, hypertrophy of cardiomyocytes, so-called pathological remodeling, and cardiomyopathy. In addition, we found that double silencing of PLCd1 and PLCd3 resulted in morphological changes in H9c2 cells, which suggests that these enzymes contribute to the maintenance of the shape of cardiomyocytes. Therefore, deletion of these enzymes likely contributes to the induction of cardiomyocyte hypertrophy. Furthermore, we found that the absence of both PLCd1 and PLCd3 causes enlargement of the left ventricular cavity, that is, a DCM-like phenotype. Given that the simultaneous loss of PLCd1 and PLCd3 in the embryo proper did not result in embryonic lethality and that the number of apoptotic cells did not apparently increase in the cDKO embryos, PLCd1 and PLCd3 are specifically required by the adult heart and are dispensable during cardiac development in the embryo. Cardiomyocyte dropout was often followed by cardiac fibrosis. Predating other cardiac abnormalities, excessive apoptosis was observed in cDKO cardiomyocytes as early as 4 weeks of age, which suggests that excessive apoptosis seems to be a cause for cardiac fibrosis, at least partially. Interestingly, we have reported that simultaneous loss of PLCd1 and PLCd3 results in excessive apoptosis in placental trophoblasts, 22 which indicates that PLCd1 and PLCd3 regulate cell survival in both cardiomyocytes and trophoblasts. Unexpectedly, the silencing of merely PLCd3 leads to a modest increase in apoptotic cells in H9c2 cells despite no apparent apoptotic phenotypes in the PLCd3 KO heart in vivo. Cardiomyocytes may be protected from apoptosis in an in vivo environment by unknown mechanisms, and the loss of PLCd3 is insufficient to induce apoptosis.
The mechanisms by which loss of PLCd1 and PLCd3 causes cardiac abnormalities remain to be fully elucidated. Loss of PKCy in mice resulted in cardiac abnormalities, including a reduction in contractile performance, increased end-systolic volume, cardiac fibrosis, hypertrophy of cardiomyocytes, and apoptosis of cardiomyocytes. 21 All of these cardiac abnormalities were observed in the cDKO mice. In addition, loss of PKCy leads to inhibition of Akt and activation of caspase 9, as does the combined loss of PLCd1 and PLCd3. 21 PLCd1 and PLCd3 generate diacylglycerol, which is an activator for PKCy and simultaneous depletion of PLCd1 and PLCd3 impaired the activation of PKCy. Therefore, insufficient activation of PKCy is likely to be a cause for cardiac abnormalities in cDKO mice. Accordingly, the Figure 6 Simultaneous depletion of PLCd1 and PLCd3 impaired activation of Akt and PKCy. (a) Immunoblotting of the phosphorylated form of Akt ( Ser473 p-Akt), total Akt (Akt), phosphorylated form of ERK1/2 (p-ERK1/2), total ERK1/2 (ERK1/2), cleaved Caspase 9, and GAPDH in control (Meox2 þ / þ PLCd1 fl/ À PLCd3 þ / À ) or cDKO hearts at 8 weeks of age. (b) Immunoblotting of the phosphorylated form of Akt ( Ser473 p-Akt), total Akt (Akt), phosphorylated form of ERK1/2 (p-ERK1/2), total ERK1/2 (ERK1/2) and b-actin in differentiated-H9c2 cells under oxidative stress conditions that were transfected with scrambled, PLCd1-targeting (#1 and #2), or PLCd3-targeting (#1 and #2) siRNA. (c) Immunoblotting of PKCa, PKCb 1 , PKCe, and PKCy in control (Meox2 þ / þ PLCd1 fl/ À PLCd3 þ / À ) or cDKO hearts at 6 weeks of age. Twenty-five micrograms of protein was loaded per lane. GAPDH and Caveolin1 were used as markers for cytosolic and membrane-containing particulate fractions, respectively. silencing of PKCy leads to a partial phenocopy of the combined silencing of PLCd1 and PLCd3 in H9c2 cells. In addition to PKCy, lack of PKCe resulted in interstitial fibrosis when the mice were subjected to pressure overload by transverse aortic constriction. 24 In addition, PKCe has a protective role against cardiomyocyte apoptosis during cardiac ischemia/reperfusion injury. 20 However, simultaneous loss of PLCd1 and PLCd3 did not affect the activation status of PKCe. PLCd1 and PLCd3 may specifically regulate the activation of PKCy isozymes by unknown mechanisms. In addition to PKC, PLC activation results in the elevation of the intracellular calcium ion concentration ([Ca 2 þ ] i ) and activates calcium-dependent downstream molecules. In cardiomyocytes, cardiac excitation-contraction coupling (ECC) occurs through Ca 2 þ -induced Ca 2 þ release (CICR). Although ryanodine receptors are the primary Ca 2 þ release channel that mediates CICR during cardiac ECC, the IP 3 receptor (IP 3 R) Ca 2 þ release channel is also expressed in cardiomyocytes. 25,26 Recent evidence suggests that the activation of IP 3 R may modulate ECC. 27,28 Therefore, simultaneous loss of PLCd1 and PLCd3 may result in abnormal Ca 2 þ handling in cardiomyocytes, thereby leading to cardiac abnormalities in cDKO mice. Loss of calcineurin Ab, which is a calciumdependent serine-threonine phosphatase, increases the number of apoptotic cardiomyocytes and cardiac fibrosis in mice with DCM. 29,30 Given that our cDKO mice showed a DCM-like phenotype, calcineurin Ab may also be involved in cardiac abnormalities in cDKO mice. In addition to PKC and [Ca 2 þ ] i , the PLC substrate PIP 2 has roles in cardiac muscle relaxation between contractions by positively regulating a Na þ /Ca 2 þ exchanger, which removes Ca 2 þ into the extracellular space. 31 Therefore, impaired hydrolysis of PIP 2 may also contribute to cardiac abnormalities in cDKO mice.
The findings of this study suggest that PLCd1 and PLCd3 are possible therapeutic targets for DCM and cardiac remodeling that leads to heart failure. Future work will determine whether the expression and/or activity of PLCd1 and PLCd3 in patients with DCM and/or heart failure are decreased.
Real-time RT-PCR. Total RNA was isolated using an RNeasy Mini kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol. Template complementary DNA was synthesized from total RNA by using a ReverTra Ace qPCR RT kit (Toyobo, Osaka, Japan). Real-time PCR was performed using THUNDERBIRD SYBR qPCR Mix (Toyobo) in a CFX96 thermocycler (Bio-Rad, München, Germany). The primer sequences are listed in Table 2. The relative amounts of mRNA were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA levels.
Histochemistry. For histological analyses, placenta or hearts were fixed in 4% paraformaldehyde, dehydrated, and embedded in paraffin. Sections (5 mm thick) were stained with HE. Trichrome staining was performed with a Trichrome Stain (Masson) Kit (Sigma, St Louis, MO, USA) according to the manufacturer's instructions. Sections were examined under a BX51 microscope (Olympus, Tokyo, Japan).
TUNEL assay. TUNEL assay was performed on paraffin sections with an In Situ Cell Death Detection Kit, TMRred (Roche, Basel, Switzerland). Counterstaining was performed with Hoechst 33258 (Life Technologies, Carlsbad, CA, USA). Sections were observed under a BZ-8000 microscope (Keyence, Osaka, Japan).
Culturing and siRNA-mediated gene silencing of H9c2 cells. The H9c2 rat cardiomyoblast cell line was purchased from DS Pharma Biomedical (Osaka, Japan). The cells were maintained in a proliferative state by culturing them in Dulbecco's modified Eagle's medium (DMEM) that was supplemented with 10% fetal bovine serum at 37 1C in a humidified atmosphere with 5% CO 2 . siRNA against rat PLCd1, rat PLCd3, and rat PKCy was purchased from Invitrogen (Stealth RNAi) and transfected with Lipofectamine RNAiMAX (Life Technologies) at 40 nM. Two individual non-overlapping Stealth RNAi duplexes per target were used for all experiments and closely similar results were obtained with these Stealth RNAi duplexes. Differentiation into cardiomyocytes was induced by changing the medium to DMEM with 1% FBS for 72 h. For PMA treatment, H9c2 cells were treated with 160 nM PMA for 48 h. In some experiments, H9c2 cells were treated with 100 mM H 2 O 2 for 1 h. After a further 24 h of culturing with DMEM with 1% FBS, cell death was determined by staining with Hoechst 33342 (DOJINDO, Japan) or by using an In Situ Cell Death Detection Kit, TMRred (Roche). The cells were observed under a BZ-8000 microscope (Keyence).
Echocardiography. Echocardiography was performed as described previously. 32 Mice were anesthetized with an intraperitoneal injection of 25 mg/kg pentobarbital (Sigma). After anesthesia, the left hemithorax of each mouse was shaved. The animals were pre-warmed with a panel heater to maintain their rectal temperature at 37 1C during the determination of cardiac parameters by echocardiography. Transthoracic echocardiography was performed using ProSound 5500R (Aloka, Tokyo, Japan) with a 13-MHz linear transducer for mice in a phased array format, which offered a lateral resolution of 0.35 mm and an axial resolution of 0.25 mm, real-time digital acquisition, storage, and review capabilities. Each cardiac parameter was calculated from the echocardiogram as described previously. 32  Measurement of blood pressure. Systolic blood pressure of nonanesthetized mice was measured by tail-cuff blood pressure measurement using a computerized CODA high-throughput noninvasive BP acquisition system (Kent Scientific Corp., Torrington, CT, USA), in accordance with the manufacturer's instructions. Briefly, the mice were placed in a warmed chamber. Cuffs were placed around the mouse tail to measure arterial systolic pressure. Three to five readings were recorded for each animal. Measurements were repeated in the event of animal movement or weak pressure/flow recordings.
Preparation of subcellular protein fractions from hearts. Preparation of subcellular protein fractions from hearts was performed as described. 21 Briefly, hearts were homogenized in homogenization buffer (20 mM Tris at pH 7.5, 2 mM EGTA, 2 mM EDTA, 250 mM sucrose, 5 mM DTT, and Complete Protease Inhibitor Cocktail tablets (Roche)) and incubated for 30 min on ice. The samples were then spun at 100 000 Â g for 30 min at 4 1C. The supernatant was stored as the cytosolic fraction, whereas the remaining pellet was further suspended in homogenization buffer containing 0.1% Triton X-100 and incubated for 30 min on ice. Then, the samples were spun at 100 000 Â g for 30 min at 4 1C and the remaining supernatant was stored as the particulate fraction. An equal amount of each sample was subjected to immunoblotting.