LIM and Cysteine-Rich Domains 1 Regulates Cardiac Hypertrophy by Targeting Calcineurin/Nuclear Factor of Activated T Cells Signaling
LIM domain proteins are important regulators in cell growth, cell fate determination, cell differentiation, and remodeling of the cell cytoskeleton. LIM and cysteine-rich domains 1 (Lmcd1) is a novel protein that contain 2 LIM domains with regular spacing in the carboxy-terminal region. However, its roles in cardiac growth remain unknown. Here, we investigated whether Lmcd1 regulates cardiac hypertrophy in vitro and in vivo and elucidated the underlying molecular mechanisms. We used primary cultured cardiac myocytes and cardiac-specific Lmcd1 transgenic mice. In wild-type mice subjected to the aortic banding, cardiac hypertrophy was evident at 8 weeks. In transgenic mice, however, cardiac hypertrophy was significantly greater than that in wild-type mice, as estimated by heart weight:body weight ratio, cardiomyocyte area, and echocardiographic measurements, as well as cardiac atrial natriuretic peptide and B-type natriuretic peptide mRNA and protein levels. Our results further showed that cardiac fibrosis observed in wild-type aortic banding mice was augmented in transgenic aortic banding mice. Importantly, calcineurin activity and nuclear factor of activated T cells activation level were increased more in transgenic mice than those in wild-type mice after 8-week aortic banding. In vitro experiments in cardiac myocytes further revealed that angiotensin II-induced calcineurin activity and nuclear factor of activated T cells activation were enhanced by overexpression but blunted by downregulation of Lmcd1. In conclusion, our results suggest that Lmcd1 plays a critical role in the development of cardiac hypertrophy via activation of calcineurin/nuclear factor of activated T cells signaling pathway.
Cardiac hypertrophy is a response of the myocardium to increased workload, characterized by an increase of myocardial mass and accumulation of extracellular matrix, leading to left ventricular (LV) dilatation, fibrosis, and impaired systolic function, and it potentates the development of ventricular arrhythmias, heart failure, and subsequent cardiovascular mortality.1,2 Although initially a beneficial adaptive response, prolonged hypertrophy may result in ventricular dilatation and heart failure, which is increasing in prevalence and is a debilitating disease with high rates of mortality and morbidity.3–6 However, antihypertensive therapies and aortic valve replacement are the only 2 treatments proven to effectively reverse both structural and functional cardiac abnormalities associated with pathological cardiac hypertrophy. Understanding the underlying processes regulating cardiac remodeling will allow us to identify specific new therapies to improve the long-term outcomes of pathological cardiac hypertrophy in patients.
LIM domain proteins are well recognized as key components of the regulatory machinery of the cell and are important regulators in cell growth, cell fate determination, cell differentiation, and remodeling of the cell cytoskeleton.7 LIM domains are composed of ≈55 amino acids with 8 highly conserved residues. The LIM consensus sequence has been defined as CX2CX16-23HX2CX2CX2CX16-21CX2(C/H/D), and all of the identified human LIM sequences show a slightly broader consensus sequence.7 A wide variety of eukaryotic organisms contain LIM domain proteins. Currently, there are 135 identifiable LIM-encoding sequences located within 58 genes in the human genome. LIM proteins have been found in both the nucleus and the cytoplasm. Therefore, most characterized LIM proteins can interact directly or indirectly with different proteins, transcriptional factors, actin cytoskeletons, and kinases.7–10 LIM proteins function in diverse biological processes through interacting or binding of target proteins.8–10 LIM and cysteine-rich domains 1 (Lmcd1) was identified by screening of the expressed sequence tag database (dbEST) with partially overlapping expressed sequence tag clones, which encodes a family of LIM domain proteins.11 The predicted Lmcd1 protein contains a novel cysteine-rich domain residing in the amino-terminal region and 2 LIM domains with regular spacing in the carboxy-terminal region.11 Northern blot analysis indicated expression of the 1.7-kb transcript in many tissues, and it was found to be highly expressed in both cardiac and skeletal muscles. The potentially significant roles of Lmcd1 and its presence in human myocardial myofibrils implicate that LIM domain proteins might be associated with cardiac growth. Therefore, the purpose of this study was to determine whether Lmcd1 can regulate cardiac growth in the condition of pathology and physiology and to elucidate the molecular mechanisms of Lmcd1 on cardiac growth in vitro and in vivo.
Materials and Methods
Antibodies for the calcineurin/nuclear factor of activated T cells (NFAT) signaling pathways were purchased from Cell Signaling Technology. The anti-Lmcd1 (reactive with mouse or human) antibody was purchased from Santa Cruz Biotechnology. Antibodies for collagen I and connective tissue growth factor were ordered from Abcam. All of the other antibodies were purchased from Santa Cruz Biotechnology. [3H]leucine was purchased from Amersham. The bicinchoninic acid protein assay kit was purchased from Pierce. Cell culture reagents and all of the other reagents were obtained from Sigma.
Transgenic Mice, Animal Models, Echocardiography, and Cardiac Catheterization
The study protocol was approved by the animal care and use committee of our hospital. The details for transgenic (TG) mice generation and the aortic banding (AB) model are given in the online Data Supplement (available at http://hyper.ahajournals.org). Echocardiography was performed using a SONOS 5500 ultrasound (Philips Electronics) with a 15-MHz linear array ultrasound transducer. Cardiac catheterization was performed using a 1.4-F Millar catheter-tip micromanometer catheter, which was inserted through the right carotid artery into the left ventricle. The detailed information is available in the online Data Supplement.
Cardiac Morphology Analysis
After blood samples were taken, hearts were cleared by perfusion with PBS, arrested in diastole with 10% KCl, weighted, fixed by perfusion with 10% paraformaldehyde, embedded in paraffin, and sectioned at 5 μm or snap-frozen in liquid nitrogen for RNA and protein analysis. Sections were stained with hematoxylin/eosin for histopathology. For myocyte cross-sectional area, sections were stained with hematoxylin/eosin. A single myocyte was measured with an image quantitative digital analysis system (Image-Pro 4.5). The outline of 120 myocytes was traced in each group. To determine collagen deposition, tissue sections were stained with picrosirius red in saturated picric acid solution. With the use of an image analysis system, these sections were analyzed morphometrically. Fibrillar collagen was identified in the picrosirius-stained sections by its red appearance.
Recombinant Adenoviral Vectors and Cell Culture
We used replication-defective adenoviral vectors expressing the full-length rat Lmcd1 gene under the control of the cytomegalovirus promoter and a control adenoviral vector expressing the green fluorescent protein. Primary cultures of cardiac myocytes were prepared as described.12–14 For details, please see the online Data Supplement.
[3H]Leucine Incorporation, Calcineurin Activity, and NFAT-Luciferase Assays
[3H]Leucine incorporation was measured as described previously.13,14 Calcineurin activity was measured as the dephosphorylation rate of a synthetic phosphopeptide substrate and was determined in lysates of whole ventricular tissues and cardiac myocytes, and NFAT-luciferase assay was performed as described previously.15 Cardiac myocytes were transfected with 0.5 μg of NFAT luciferase reporter construct (ordered from Addgene), and 10 μL of LipofectAMINE reagent (Invitrogen) was used as the control plasmid DNA, according to the manufacturer’s instructions. For details, please see the online Data Supplement.
Northern and Western Blot Analysis and Quantitative Real-Time PCR
Whole RNA was extracted from cardiac tissue with TRIzol reagent according to the manufacturer’s instructions (Invitrogen). RNA (30 μg) was run on a 1% agarose/formaldehyde gel at 120 V for 2 hours. The RNA was transferred to a nylon membrane by vacuum for 1.5 hours and cross-linked by UV wave. Protein extracts from different groups of myocardium (50 μg) were fractionated on a 10% polyacrylamide gel, transferred to nitrocellulose membranes, and probed with various antibodies. Specific protein expression levels were normalized to either the GAPDH protein for total cell lysate and cytosolic protein or the Lamin-B1 protein for nuclear protein. For real-time PCR, we quantified PCR amplifications using SYBR Green PCR Master Mix (Applied Biosystems) and normalized results against GAPDH gene expression.
For transient transfection and coimmunoprecipitation experiments, mammalian expression plasmids for calcineurin A and Lmcd1 were ordered from Addgene and subsequently cloning into cytomegalovirus promoter-based vectors containing a Myc or Flag tag. Cultured primary cardiac myocytes were transfected with 1 μg of expression plasmids for full-length forms of calcineurin A and Lmcd1 using LipofectAMINE reagent (Invitrogen). The detailed information for coimmunoprecipitation is described in the online Data Supplement.
All of the values are expressed as mean±SEM. The differences in the data simply between 2 groups were determined by a Student t test. Comparisons among groups on all of the other data were assessed by 2-way ANOVA followed by the Bonferroni post hoc test. A value of P<0.05 was considered statistically significant.
Lmcd1 Expression Is Regulated in Response to AB
To determine the response of Lmcd1 to hypertrophic stresses, we analyzed the mRNA and protein levels of Lmcd1 after AB or sham surgery for different durations by Northern blot and Western blot assays. Intriguingly, both mRNA and protein expression were markedly upregulated after 4 weeks of AB but not in the sham group (Figure 1A and 1B).
Overexpression of Lmcd1 Augments Cardiac Hypertrophy In Vitro
To investigate the effects of Lmcd1 on cardiac hypertrophy, we first examined its effect in primary cultured cardiac myocytes. We generated an adenoviral vector with the Lmcd1 gene under the control of the cytomegalovirus promoter. Expression of exogenous Lmcd1 protein in myocytes was detected by Western blot (Figure S1A, available in the online Data Supplement). We also screened 3 short hairpin (sh) Lmcd1 and found that the third one markedly inhibited Lmcd1 protein expression in cardiac myocytes (Figure S1A). Therefore, the third shLmcd1 was chosen for the following experiments. Lmcdl overexpression significantly enhanced [3H]leucine incorporation and cell surface area induced by angiotensin (Ang) II (1 μmol/L) treatment compared with adenoviral (Ad) green fluorescent protein infection, whereas inhibition of Lmcdl by infection with Ad-shLmcd1 reduced these effects of Ang II (Figure 2). Western blot further demonstrated that Lmcdl overexpression led to greater Ang II-induced protein expression of atrial natriuretic peptide and B-type natriuretic peptide, which are molecular markers of cardiac hypertrophy (Figure S1B and S1C). These findings indicate that Lmcdl expression promotes cardiac hypertrophy in vitro.
Overexpression of Lmcd1 Augments Cardiac Hypertrophy in Response to AB
To further examine whether Lmcd1 is involved in the regulation of cardiac hypertrophy, we generated TG mouse lines with full-length human Lmcd1 cDNA under the control of the α-myosin heavy chain promoter (Figure S2A). Four lines of TG mice were confirmed by Western blot (Figure S2B). These lines were viable and fertile, and there were no detectable differences in cardiac size and structure between TG and wild-type (WT) mice at 10 weeks of age. We analyzed Lmcd1 protein levels in various tissues of TG mice by Western blot using anti-human Lmcd1 antibody. We found a robust expression of human Lmcd1 protein in the heart, but it was not detected in the other organs (Figure S2C). Among 4 established lines of the adult TG mice, the first, third, and fourth lines that expressed the higher levels of the human Lmcd1 protein in the heart did not show obvious difference in response to stress. Therefore, the first line was used for further experiments. We then subjected these mice to AB-induced pressure overload for 8 weeks to explore the possible role of Lmcd1 in pathological hypertrophy. The results showed that Lmcd1 TG animals displayed a marked increase in cardiac hypertrophy compared with WT littermates. AB-induced increases in the heart weight:body weight ratio and lung weight:body weight ratio were higher in TG mice compared with WT controls (Table S1). Analysis of the cardiomyocyte cross-sectional area from the histological sections revealed a significantly larger cell size in TG mice after AB than in WT controls (Table S1 and Figure 3). Echocardiographic analysis was further performed to evaluate geometric remodeling of the left ventricle in response to AB (Table S1). Although the intraventricular septum and posterior wall thickness were significantly increased in both TG and WT mice after AB, Lmcd1 TG mice displayed substantially enlarged LV chambers 8 weeks after AB (Table S1). The fractional shortening was significantly decreased in TG mice compared with WT controls after 8 weeks of AB (Table S1). In addition, pressure overload-induced hypertrophy in TG animals was accompanied by extensive fibrosis, as indicated by picrosirius red staining (6.2% of fibrosis in WT AB versus 11.8% in TG AB; Figure S3C). Furthermore, the mRNA expression and protein expression of atrial natriuretic peptide, B-type natriuretic peptide, and β-myosin heavy chain (markers of pathological hypertrophy), as well as transforming growth factor-β1, procollagen type I α1 (Col1α1), procollagen type III α1(Col3α1), and connective tissue growth factor (markers of fibrosis), were significantly higher in AB-operated TG mice compared with AB-operated WT animals (Figure S3A through S3E), suggesting that Lmcd1 efficiently augments cardiac remodeling by modulating the expression of hypertrophic and fibrotic markers.
Overexpression of Lmcd1 Accelerates the Activation of Calcineurin/NFAT Signaling
Muscle LIM protein is required for calcineurin/NFAT signaling at the sarcomeric Z disc.16 Therefore, Lmcd1 may regulate cardiac growth by activating calcineurin/NFAT signaling. To test this hypothesis, we exposed cardiomyocytes to 1 μmol/L of Ang II infected with Ad-Lmcd1 or Ad-shLmcd1. Calcineurin phosphatase activity significantly increased after treatment with Ang II, and such an increase was evidently enhanced by infection with Ad-Lmcd1 but blocked by Ad-shLmcd1 (Figure S4A). Furthermore, calcineurin activity was also elevated by 43% in WT mice and by 93% in TG mice after AB (Figure 4A). Our data further showed that infection of cardiomyocytes with Ad-Lmcd1 significantly promoted the Ang II-induced NFAT nuclear translocation, whereas infection with Ad-shLmcd1 markedly blocked NFAT nuclear translocation (Figure S4B). To examine whether the alterations in NFAT nuclear translocation affected NFAT transcriptional activity, cardiomyocytes were transfected with a luciferase reporter plasmid driven by 3 NFAT binding sites. Treatment with Ang II significantly increased NFAT transcriptional activity, and such an increase was exacerbated by infections with Ad-Lmcd1 but blocked by Ad-shLmcd1 (Figure S4C). Myocyte-enriched calcineurin interacting protein 1 (MCIP1) is known to be a direct transcriptional target of calcineurin/NFAT signaling in cardiomyocytes.17 Our studies showed that infection of Ad-shLmcd1 almost completely blocked the Ang II-induced increase of MCIP1.4, whereas infection of Ad-Lmcd1 augmented the Ang II-induced increase of MCIP1.4 (Figure S4C). The levels of nuclear NFATc4 and MCIP1.4 were significantly increased in the hearts of WT mice and elevated more in TG mice after AB (Figure 4B and 4C). To investigate whether Lmcd1 directly interacts with calcineurin protein, coimmunoprecipitation was preformed. Indeed, we found that Lmcd1 coimmunoprecipitated with calcineurin (Figure S4D). These findings suggest that overexpression of Lmcd1 directly augments the activation of calcineurin/NFAT signaling in vitro and in vivo in response to hypertrophic stimuli and then exacerbates cardiac hypertrophy.
Blocking Calcineurin/NFAT Signaling Blunts Cardiac Hypertrophy in TG Mice
To further confirm the critical role of calcineurin/NFAT signaling in vivo, the calcineurin inhibitor cyclosporin A (CSA) was used. Lmcd1 TG mice were divided into 4 groups: (1) sham/CSA (n=11); (2) sham/vehicle (n=10); (3) AB/CSA (n=14); and (4) AB/vehicle (n=15). CSA (25 mg/kg per day, SC) or vehicle was treated 1 week before surgery and then continued for 4 weeks after AB or sham. Four weeks after AB, vehicle-treated TG mice displayed classic pressure-overloaded hypertrophic responses, including fetal gene program reactivation, increases in LV diastolic posterior wall thickness, and increases in heart weight:body weight ratios, as well as the cardiac myocyte area (Figure S5A and S5B), compared with sham controls. These changes were significantly attenuated in the CSA-treated AB TG mice. The induction of fetal genes, including atrial natriuretic peptide, B-type natriuretic peptide, and β-myosin heavy chain by AB, was also significantly attenuated in the CSA-treated TG hearts (Figure S5C). Taken together, our findings indicate that CSA prevents the development of cardiac hypertrophy in TG mice by blocking the calcineurin/NFAT signaling.
In the case of pathological cardiac hypertrophy, although the increased heart size is initially a compensatory mechanism, sustained hypertrophy can ultimately lead to a decline in LV function and, thus, represents an independent risk factor for heart failure.18 Therefore, it is necessary to clarify the mechanism of cardiac hypertrophy and find the key targets for the treatment of pathological hypertrophy. In the present study, we demonstrate that Lmcd1 mediates cardiac hypertrophy, likely by promoting the activation of the calcineurin/NFAT pathway in vitro and in vivo (Figure 5). These novel findings suggest that inhibiting Lmcd1 is critically important in protecting the heart from pressure overload.
According to the current LIM domain protein classification,7–10 Lmcd1 belongs to the group 3 proteins, which contain ≥1 LIM domain at the C-terminal region. The N-terminal region of the novel predicted protein contains a cysteine-rich domain: C-X2-C-X3-C-X5-H-X5-C-X2-C-X1-C-X4-H-C. Most LIM proteins of group 3 are cytoplasmic and participate in protein-protein interactions with the cytoskeleton.9,19 Similar to other LIM domain proteins with predominant expression in the skeletal muscle, the Lmcd1 protein might be involved in DNA-protein-protein interactions during muscle development and remodeling. However, until the present study, the (patho)physiological significance of Lmcd1 in cardiac responses to hemodynamic overload had not been recognized. To examine the role of Lmcd1 on cardiac hypertrophy, we used primary cultured cardiac myocytes and cardiac-specific human Lmcd1 gene overexpression in TG mice. Here, we report that cardiac Lmcd1 protein expression can be markedly induced by pressure overload. Moreover, we have investigated the pathological role of Lmcd1 in early cardiac responses to pressure overload and in the modulation of a pivotal hypertrophic signaling pathway. To determine the role of Lmcd1 in the heart to respond to stress, we subjected the TG mice to sham and transverse aortic constriction surgery. Compared with WT mice, TG mice responded differently to these procedures. At 8 weeks after the AB surgery, TG mice displayed marked reactivation of the fetal gene program: concentric cardiac hypertrophy, as evidenced by increased LV wall thickness and heart weight:body weight ratio. Compared with the WT AB group, the TG AB group showed greater reactivation of the atrial natriuretic peptide, B-type natriuretic peptide, and β-myosin heavy chain; greater cardiac hypertrophy; lower fractional shortening; and higher heart weight:body weight and lung weight:body weight ratios. These indicate that the overexpression of Lmcd1 exacerbates pathological cardiac responses to AB-induced LV pressure overload. Taken together, our in vitro and in vivo data indicated that overexpression of Lmcd1 augments cardiac hypertrophy, whereas inhibition of Lmcd1 attenuates hypertrophic responses. Therefore, Lmcd1 appears to be an endogenous and positive regulator of hypertrophic response. Cardiac fibrosis is one of the classic features of hypertrophy and is characterized by the expansion of the extracellular matrix attributed to the accumulation of collagen, especially collagen I and connective tissue growth factor.20,21 It is important to understand the mechanism that stimulates collagen deposition in the heart and to identify approaches to block these processes. One of the findings of this study revealed that Lmcd1 augments cardiac fibrosis and promotes expression of several fibrotic mediators mediated by pressure overload. In an attempt to elucidate mechanisms underlying the effect of Lmcd1 on fibrosis, we analyzed key components of transforming growth factor-β1 signal transduction. Because transforming growth factor-β is a major activator of fibroblast differentiation and activation, it is, thus, is a key player in the fibrotic response. Activation of this signaling pathway is predicted to mediate fibrosis. In line with these notions, our results demonstrated that Lmcd1 promotes transforming growth factor-β1 expression levels in vivo and concomitantly enhances collagen synthesis in cardiac fibroblasts.
The biochemical mechanisms by which Lmcd1 functions in the heart remain unclear. A number of signaling cascades, including transcription factors, kinases, and G protein-coupled receptors, are implicated in the regulation of cardiac growth.6,18 Among these, the calcineurin/NFAT pathway has been shown to be a key signaling cascade that promotes cardiac hypertrophy.22,23 Calcineurin dephosphorylates NFATs in response to increased intracellular calcium and regulates gene expression in a variety of calcium-sensitive tissues, such as brain, muscle, and lymphocytes.21,22 Transcriptional activity of NFATs depends on dephosphorylation by calcineurin, which leads to nuclear translocation.23–25 Recent studies revealed that muscle LIM protein directly associates with calcineurin, and this interaction is critical for calcineurin-NFAT signaling.16,17 Thus far, the direct effect of Lmcd1 on calcineurin/NFAT signaling has not been explored. This study demonstrates for the first time that increased Lmcd1 expression provokes a remarkable activation of calcineurin/NFAT signaling. Substrates of calcineurin phosphatase activity include most members of the NFAT family of transcription factors. Overexpression of Lmcd1 resulted in significantly upregulated NFAT activity and the nuclear translocation of NFATc4. MCIP1.4 is an endogenous modulator of calcineurin, the expression of which is controlled by calcineurin/NFAT.26,27 In cells, equilibrium is established between the abundance of MCIP1.4 protein and calcineurin activity. Therefore, MCIP1.4 protein abundance can serve as a functional surrogate for calcineurin activity. This is supported by our in vitro and in vivo results, in which treatment with Ang II or AB provoked marked increases in MCIP1.4 protein abundance and was especially much higher in the Lmcd1-overexpressed group. MCIP1.4 is involved in a variety of important physiological and pathological functions. It has been implicated in regulating cardiac hypertrophy, stress responses, angiogenesis, and neurological diseases, including Alzheimer disease and Down syndrome.26,27 More importantly, pharmacological inhibition of calcineurin signaling with cyclosporin A treatment in Lmcd1 TG mice attenuated cardiac hypertrophy and fibrosis and improved cardiac dysfunction. These findings suggest that calcineurin/NFAT signaling is the key signaling that Lmcd1 exerts its prohypertrophic effects.
In conclusion, our present work provided in vitro and in vivo evidence that cardiac Lmcd1 expression exacerbated cardiac hypertrophy via regulating calcineurin/NFAT signaling. Lmcd1 is a critical signaling molecule responsible for cardiac hypertrophy and fibrosis. These observations may have significant implications for the development of novel strategies for the treatment of cardiac hypertrophy and fibrosis through targeting of the calcineurin/NFAT signaling pathway. Additional studies are necessary to attain a better understanding of the effects of Lmcd1 on cardiac function and the effects of Lmcd1 inhibition in animal models of cardiac hypertrophy.
The current study provides a new insight into the role of the LIM domain protein Lmcd1 in the development of cardiac hypertrophy and fibrosis induced by pressure overload. Our findings suggest that Lmcd1 behaves as an endogenous and positive regulator of hypertrophic response. This finding may provide a novel therapeutic target for cardiac hypertrophy and fibrosis. Future studies are needed to investigate the molecular mechanisms in which Lmcd1 promotes calcineurin/NFAT signaling in the heart using TG and knockout mice.
Sources of Funding
This research was supported by the National Natural Science Foundation of China (grants 30900524, 30972954, and 30770733) and by the Scientific Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry (grant 2006-331).
Z.-Y.B. and H.H. are joint first authors.
- Received May 4, 2009.
- Revision received May 30, 2009.
- Accepted November 25, 2009.
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