Deficiency of Nectin-2 Leads to Cardiac Fibrosis and Dysfunction Under Chronic Pressure Overload
The intercalated disc, a cell-cell contact site between neighboring cardiac myocytes, plays an important role in maintaining the homeostasis of the heart by transmitting electric and mechanical signals. Changes in the architecture of the intercalated disc have been observed in dilated cardiomyopathy. Among cell-cell junctions in the intercalated disc, adherens junctions are involved in anchoring myofibrils and transmitting force. Nectins are Ca2+-independent, immunoglobulin-like cell-cell adhesion molecules that exist in adherens junctions. However, the role of nectins in cardiac homeostasis and integrity of the intercalated disc are unknown. Among the isoforms of nectins, nectin-2 and -4 were expressed at the intercalated disc in the heart. Nectin-2–knockout mice showed normal cardiac structure and function under physiological conditions. Four weeks after banding of the ascending aorta, cardiac function was significantly impaired in nectin-2–knockout mice compared with wild-type mice, although both nectin-2–knockout and wild-type mice developed similar degrees of cardiac hypertrophy. Banded nectin-2–knockout mice displayed cardiac fibrosis more evidently than banded wild-type mice. The disruption of the intercalated discs and disorganized myofibrils were observed in banded nectin-2–knockout mice. Furthermore, the number of apoptotic cardiac myocytes was increased in banded nectin-2–knockout mice. In the hearts of banded nectin-2–knockout mice, Akt remained at lower phosphorylation levels until 2 weeks after banding, whereas c-Jun N-terminal kinase and p38 mitogen-activated protein kinase were highly phosphorylated compared with those of wild-type mice. These results indicate that nectin-2 is required to maintain structure and function of the intercalated disc and protects the heart from pressure-overload–induced cardiac dysfunction.
In the mature heart, the intercalated discs are located at the bipolar ends of the rod-shaped cardiac myocytes and mediate mechanical and electric coupling between adjacent cardiac myocytes.1 The intercalated disc mainly consists of 3 junctional complexes: adherens junctions (AJs), desmosomes, and gap junctions. The role of AJs in the intercalated disc is to anchor myofibrils and to transmit the force developed by the contracting myofibril to neighboring cardiac myocytes. Desmosomes provide structural support by anchoring the intermediate filament system, and gap junctions provide intercellular communication via ions and small molecules. Changes in the intercalated disc architecture have been reported in mouse models for dilated cardiomyopathy (DCM), such as muscle LIM protein knockout (KO) mice and tropomodulin-overexpressing mice.2,3⇓ On the other hand, the targeted ablation of N-cadherin, which is predominantly localized at AJs in the intercalated disc, in the murine adult was shown to result in the disassembly of the intercalated disc structure and to exhibit a DCM-like phenotype.4 There are also several reports that the conditional KO mice of other AJ proteins in the heart, such as α-E-catenin,5 mXin-α,6 and vinculin,7 showed cardiac dysfunction with the disruption of the intercalated disc. These findings suggest that AJ proteins are indispensable for the homeostasis of the intercalated disc and cardiac function.
Nectins are Ca2+-independent, immunoglobulin-like cell-cell adhesion molecules found in AJs.8,9⇓ Nectins are a family composed of 4 members, nectin-1, -2, -3, and -4. All of the nectins form homo-cis-dimers and then homo- or hetero-trans-dimers through the extracellular region in a Ca2+-independent manner, causing cell-cell adhesion. The cytoplasmic region is associated with the actin cytoskeleton through afadin, a nectin and actin filament (F-actin) binding protein. Nectins first form cell-cell adhesion and recruit cadherins to the nectin-based cell-cell adhesion sites, and this leads to the formation of AJs. Thus, nectins are important for the formation of AJs.
Among the isoforms of nectins, nectin-2 is known to exist in the heart.10 However, the role of nectin-2 in the heart remains to be elucidated. The present study shows critical roles for nectin-2 in the heart under pressure overload.
Materials and Methods
Animal care and experiments were conducted according to guidelines for animal experimentation at Kobe University. Nectin-2–KO mice were generated as described previously, but the cardiac phenotype of the mice was not assessed in detail.11 Ascending aortic constriction was performed in male wild-type (WT) or nectin-2–KO mice at the age of 11 weeks, as described previously,12 except that the aorta was ligated with a 26-gauge needle. The mice were euthanized at the indicated time points.
The following materials and methods used for this study are described in detail in the online Data Supplement (please see http://hyper.ahajournals.org): echocardiography and electrocardiography, antibodies, histological analysis, Western blotting, real-time PCR, and statistics.
Expression and Localization of Nectins and Other Proteins in the Intercalated Disc
To examine the expression and localization of nectins in the murine heart, immunostaining of nectin-1, -2, -3, and -4 was performed. As shown in Figure 1A, nectin-2 and -4 were selectively localized to the intercalated discs of the WT mouse hearts, whereas nectin-1 or -3 was not detected. Western blotting showed no compensatory increase of nectin-1, -3, or -4 expression in the nectin-2–KO mouse hearts (Figure S1).
In the intercalated disc structure, nectin-2 was colocated with N-cadherin in the WT mouse hearts, indicating that nectin-2 exists in AJs of the intercalated discs (Figure 1B). The trans-interactions of nectins were reported to initiate and enhance the formation of cadherin-based AJs in epithelial cells.8 However, the deficiency of nectin-2 affected neither N-cadherin expression and distribution nor the structure of the intercalated disc depicted by N-cadherin fluorescence (Figure 1B). Other AJ proteins, such as α-catenin, β-catenin, and afadin, as well as connexin43 (Cx43), a gap junction protein, were also present and distributed normally in the intercalated discs in the nectin-2–KO mouse hearts (Figure S2). These findings suggest that nectin-2 might not be essential for the development of the intercalated discs or the expression of other AJ and GP proteins.
Cardiac Hypertrophy and Dysfunction Induced by Pressure Overload in Nectin-2–KO Mice
The functional phenotypes of the nectin-2–KO mouse heart were investigated under physiological conditions. The heart weights of nectin-2–KO mice were similar to those of the WT mice (Figure S3, see sham-operated groups), and normal systolic function assessed by echocardiography was observed in nectin-2–KO mice (Table and Figure 2A, see sham-operated groups), suggesting that nectin-2–KO mice exhibit normal cardiac function under physiological conditions. The function of nectin-2 was then explored in the heart under pathological conditions. A left ventricular (LV) pressure-overload model was used in nectin-2–KO and WT mice to assess cardiac dimensions and function. The heart weights of WT and nectin-2–KO mice were increased significantly, and the degrees of hypertrophy were similar between WT and nectin-2–KO mice after aortic banding (Figure S3). Histological analysis showed that the increase in the cross-sectional areas of cardiac myocytes in nectin-2–KO mice under chronic pressure overload was similar to that in WT mice (Figure 2B). Echocardiographic measurements revealed similar extents of increases in LV wall thickness in nectin-2–KO mice with aortic banding compared with corresponding WT mice (Table and Figure 2A). However, LV end-diastolic dimension tended to be larger, and LV end-systolic dimension was significantly greater in banded nectin-2–KO mice than in those in banded WT mice (LV end-systolic dimension: 2.03±0.10 versus 1.60±0.09 mm; banded nectin-2-KO mice versus banded WT mice: P<0.05). Systolic function was significantly depressed in banded nectin-2–KO mice compared with banded WT mice (percentage of fractional shortening: 33±2 versus 43±1; banded nectin-2-KO mice versus banded WT mice: P<0.05; Table and Figure 2A). Furthermore, a significant increase in lung weight was observed in banded nectin-2–KO mice, whereas the lung weight in WT mice was not changed after banding, indicating that pulmonary congestion was induced by pressure overload in nectin-2–KO mice but not in WT mice (Figure 2C). The mRNA expression of atrial natriuretic factor (ANF), a marker of cardiac stress that is elevated in the heart in pathological hypertrophy or heart failure,12,13⇓ was significantly increased in banded nectin-2–KO mice compared with banded WT mice (Figure 2D). Taken together, these results suggest that nectin-2 plays important roles in the transition from cardiac hypertrophy to progressive heart failure under chronic pressure overload, although nectin-2 is not indispensable for the development of cardiac hypertrophy.
Evident Myocardial Fibrosis Induced by Pressure Overload in Nectin-2–KO Mice
To examine whether pressure overload could induce distinct pathological differences between WT and nectin-2–KO mice, Masson trichrome staining was performed on heart sections to evaluate cardiac fibrosis. As shown in Figure 3A and 3B, the pressure-overload model used in this study displayed mild cardiac fibrosis in banded WT mice compared with sham-operated WT mice. In sham-operated nectin-2–KO mice, there was no overt cardiac fibrosis. However, pressure overload induced severe and diffuse cardiac fibrosis in banded nectin-2–KO mice compared with banded WT mice (fibrosis area: 21.7±2.5% versus 4.8±0.8%; banded nectin-2-KO mice versus banded WT mice: P<0.01).
The mRNA expression levels of type I collagen in nectin-2–KO mouse hearts were boosted to 4-fold of those in WT mouse hearts at 4 days after banding (Figure 3C). We also examined the mRNA expression levels of transforming growth factor-β, because this cytokine is known to be increased in the myocardium of hypertrophic cardiomyopathy, DCM, and myocardial infarction and is considered to induce cardiac fibrosis.14,15⇓ The levels of transforming growth factor-β1 mRNA in the banded nectin-2–KO mouse hearts were increased twice as much as those in the banded WT mouse hearts (Figure 3C).
Furthermore, to analyze the pathological features of diffuse and multifocal fibrosis in banded nectin-2–KO mice, cell death was assessed by TUNEL staining of cardiac sections. As shown in Figure 3D, the number of TUNEL-positive nuclei was more prominently increased in the banded nectin-2–KO mouse hearts than in the banded WT mouse hearts. These results suggest that nectin-2 has a protective role in cardiac myocytes against chronic pressure overload.
Disruption of Intercalated Discs in Nectin-2–KO Mice by Pressure Overload
Immunoblotting and immunofluorescence confocal microscopy were performed to examine the expression of component proteins and structural changes of the intercalated disc after banding. N-cadherin is well known to be present in the transmembrane part of AJs. On the cytoplasmic side of AJs, β-catenin binds to N-cadherin via the cytoplasmic domain, whereas it interacts with α-catenin at the other end. Then, α-catenin contacts the actin cytoskeleton. Deficiency of nectin-2 did not affect N-cadherin expression or distribution, even after banding (Figures 4A and S4). The notable change observed in the banded nectin-2–KO mouse hearts was the broader fluorescent line of the intercalated disc (Figure 4B). Such phenomena were reported in the hearts of DCM mice because of the higher degree of convolution of the plasma membrane in the intercalated disc.3 For other AJ proteins, the expression and distribution of afadin, β1-integrin, and α-catenin in the heart did not differ between WT mice and nectin-2–KO mice after banding, whereas broad intercalated discs were observed similarly (Figure 4A and 4⇓B). On the other hand, a gap junction protein (Cx43) expression in the banded hearts was reduced in both WT and nectin-2–KO mice (Figures S4 and 4A), whereas the distribution of Cx43 was not changed (Figure 4B). The expression of Cx43 in banded nectin-2–KO mouse hearts was significantly lower than that in banded WT mouse hearts (fold difference of sham-operated WT mice: 0.760±0.045 versus 0.438±0.040; banded WT mice versus banded nectin-2–KO mice after 4 weeks: P<0.05). To elucidate whether such a reduction in Cx43 causes electrocardiographic abnormality in nectin-2–KO mouse hearts, we performed electrocardiography (Figure S5). After banding, both WT and nectin-2–KO mice showed a prolonged QT interval to the same extent (Table S1), but no arrhythmia was observed in both WT and nectin-2–KO mice.
Transmission electron microscopy was performed to explore changes in the structure of the intercalated disc. As shown in Figure 4C, no significant change was observed in the structure of the intercalated disc in both sham-operated and banded WT mice. In sham-operated nectin-2–KO mice, the intercalated discs appeared intact. In banded nectin-2–KO mice, the intercalated discs were severely disrupted at only 2 weeks after the operation by the higher degree of membrane convolution that resembled the features of DCM, disarray of myofibrils, and disconnection from adjacent intercalated discs. These results suggest that nectin-2 is indispensable for maintaining the intercalated disc structure under chronic pressure overload.
Impaired Cellular Signalings in Nectin-2–KO Mice
Cell-cell junctions have important roles not only in cell adhesion but also in cell polarization, survival, movement, and proliferation via regulating signal transduction.16 Because the activation of signaling molecules, such as extracellular signal–regulated kinase, c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein kinase (MAPK), and Akt, has been reported to be associated with the development of the pressure-overload–induced hypertrophy and heart failure,13,17⇓ the activity of these molecules was examined after banding using Western blotting. The activation of extracellular signal–regulated kinase did not differ between WT and nectin-2–KO mice (Figure 5A). On the other hand, activation of JNK and p38 MAPK was more significant in nectin-2–KO mice than in WT mice after banding (Figure 5B and 5C). It is noteworthy that the activation of Akt was reduced more significantly in sham-operated nectin-2–KO mice than in sham-operated WT mice, and at 2 weeks after banding it decreased further in the nectin-2–KO mouse hearts (Figure 5D). However, Akt activation rose again and was increased further at 4 weeks after banding, although the significance of this finding was not further examined. These results indicate that nectin-2 is involved in intracellular signaling in the heart in response to chronic pressure overload.
In the nectin-2–KO mouse hearts, the distribution and expression of the proteins localized in AJs, such as N-cadherin, α-catenin, β-catenin, afadin, and nectin-4, were unaffected. Because nectins have important roles in the recruitment of cadherins to the nectin-based cell-to-cell adhesion sites and the formation of AJs in epithelial cells,8,9⇓ nectin-4 in the nectin-2–KO mouse hearts may contribute to the recruitment and expression of AJ proteins. However, nectin-2–KO mice showed the disruption of the intercalated discs and the myofibril disarray after pressure overload, suggesting that nectin-4 alone does not compensate for the function of nectin-2 in the pressure-overloaded hearts. The digitation and convolution of the intercalated disc membrane in banded nectin-2–KO mice resembled the morphology observed in the DCM models.2,3,18⇓⇓ Such changes in the intercalated-disc membrane between neighboring cardiac myocytes lead to a decrease in flexibility of the contractile tissue and an increased stiffness.1 Therefore, these changes are in part attributable to cardiac dysfunction in banded nectin-2–KO mice. Expression of connexins in the failing myocardium is reduced in both animal models and human diseases.19 We showed the reduction in Cx43 expression in the banded hearts of both WT and nectin-2–KO mice. The expression of Cx43 was significantly reduced in banded nectin-2–KO mouse hearts compared with banded WT mouse hearts. However, no arrhythmia was observed in nectin-2–KO mice even after banding. In selectively bred, cardiac-restricted Cx43-KO mice, reduction of Cx43 expression to 59% of matched controls does not induce susceptibility to arrhythmia, but when the Cx43 reduction reaches 18% of control levels, 80% of the mice are inducible into lethal ventricular arrhythmias,20 implying that the reduction in Cx43 expression in banded nectin-2–KO mice might not be sufficient to induce arrhythmia.
Increased myocardial apoptosis, the resultant loss of cardiac myocytes, and consequential fibrosis are other mechanisms of cardiac dysfunction in banded nectin-2–KO mice. We considered that signaling impairment in nectin-2–KO mice heart provides some explanation for the distinguishing cell death and fibrosis. Extracellular signal–regulated kinase, JNK, p38 MAPK, and Akt have been reported to be activated under pressure overload.12,21–23⇓⇓⇓ The phosphorylation levels of JNK and p38 MAPK in the nectin-2–KO mouse hearts were notably increased after banding and were much higher compared with those in the WT mouse hearts at each corresponding time point. Recent in vivo experiments using genetically modified animals have provided insights into important aspects of MAPK activity in the heart. That is, JNK or p38 MAPK does not positively regulate cardiac hypertrophy in vivo but facilitates apoptosis and fibrosis.24,25⇓ Therefore, the JNK and p38 MAPK activation in nectin-2–KO mice may contribute to apoptosis of cardiac myocytes and the process of fibrotic changes in the heart under pressure overload. The nectin-3–afadin system regulates phosphatidylinositol 3-kinase-Akt signaling induced by the platelet-derived growth factor.26 We showed that the phosphorylation level of Akt was significantly lower in the nectin-2–KO mouse hearts than in the WT mouse hearts even before the operation, which continued until 2 weeks after the operation (Figure 5D), suggesting that nectin-2 in the heart regulates Akt signaling. Akt is involved in the survival of various types of cells,27–29⇓⇓ and Akt1-KO mice and transgenic mice expressing dominant-negative phosphatidylinositol 3-kinase with the reduced Akt activity under basal conditions display an accelerated heart failure phenotype to pressure overload.30,31⇓ Impaired activation of Akt in the nectin-2–KO mouse hearts may be involved in the increased susceptibility of cardiac myocytes to apoptosis after banding. We also showed that the phosphorylation level of Akt at 4 weeks after banding was even greater in the nectin-2–KO mouse hearts than in the WT mouse hearts. A possible explanation is provided with the evidence that Akt is usually activated in a failing myocardium, which is regarded as a compensatory reaction.32 Because Akt is regulated by various upstream molecules because of its critical role in cell survival, signaling molecules regulating Akt activation, conceivably from the costameres, are likely to be upregulated at the stage of heart failure.
The present results indicate that nectin-2 plays a protective role in the heart against cardiac dysfunction induced by pressure overload. The effect of nectin-2 is considered to be unique and independent from other AJ proteins. Additional analysis of the roles of nectin-2 and other proteins in the intercalated disc may contribute to deepen our understanding of the complicated mechanisms of heart failure.
We thank Toshiko Kojima for technical assistance.
Sources of Funding
This work was supported in part by grants from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (to S.S-K., T.U., and R.T.); Grants-in-Aid for Scientific Research and for Cancer Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (to Y.T.); the Takeda Science Foundation (to T.U.), the Mitsubishi Pharma Research Foundation (to T.U. and Y.R.), and the Mochida Memorial Foundation for Medical and Pharmaceutical Research (to Y.R.).
- Received February 15, 2009.
- Revision received March 9, 2009.
- Accepted July 20, 2009.
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