The Regulatory Role Of CARD6 In Cardiac Hypertrophy And The Underlying Mechanism

BackgroundCardiac hypertrophy is a complex dynamic process that involves a variety of signaling pathways and regulators, meanwhile, which is also a common pathological process, including longstanding hypertension, valvular disease, myocardial infarction and cardiomyopathy. Initially, cardiac hypertrophy arises as an adaptive response to various stimulating factors, including hemodynamic load, angiotensin, growth factors and hormones factors, in which the workload/mass ratio is normalized and resting cardiac output is maintained. However, under conditions of prolonged overload, the initially compensatory hypertrophic response may progress to contractile dysfunction and cardiac decompensation, and is characterized by an increase in inflammation, enhanced fibrosis, and heightened abnormal gene expression changes, eventually leading to myocardial ischemia, malignant arrhythmia, chronic heart failure and even sudden death. Thus, identifying novel modulators that regulate cardiac hypertrophy and the elucidation of their regulatory mechanisms is essential for the development of new targets/strategies to treat cardiac hypertrophy and heart failure.Caspase recruitment domain protein 6(CARD6), is one of the members of CARDs that positively modulates signal transduction pathways converging on NF-κB activation triggered by Nod-like receptors(NLRs) and Toll-like receptors(TLRs) proteins, which is important for innate and adaptive immune responses and inflammatory response. Notably, northern blotting revealed that CARD6 is expressed ubiquitously in various tissues in mammals, including the heart and skeletal muscle. However, to the best of our knowledge, the potential role of CARD6 in cardiovascular diseases, especially in cardiac hypertrophy, has not been studied yet. In the present study, we observed CARD6 expression changes in cardiac hypertrophy, and used both gain- and loss-of-function approaches to thoroughly decipher the role of CARD6 in chronic pressure overload-induced cardiac remodeling, and further clarify its molecular mechanisms. MethodsPart 1: Male wild type(WT) mice(C57BL/6 background) ranging in age from 10 to 12 weeks and weight from 24 to 26 g were selected for experiments, and mice were underwent aortic binding(AB) operation to establish pressure overload-induced cadiac hypertrophy model. The WT mice were randomly divided into four groups, respectively, e.g. Sham operation group, 2 weeks post operation, 4 weeks post operation and 8 weeks post operation. Primarily, the Real-Time PCR analysis were used to determine the m RNA expression level of hypertrophy markers(ANP、BNP、β-MHC) in mouse heart tissue, and then both Real-Time PCR and Western Blot analysis was used to detect the m RNA and protein expression levels of CARD6 after AB stimulation at different time points.Part 2: Male CARD6-Flox mice and cardiac-specific CARD6 knockout(c CARD6-KO) mice with C57BL/6 background ranging in age from 10 to 12 weeks and weight from 24 to 26 g were selected for experiments. Mice were subjected to sham or aortic banding(AB) surgeries for 4 weeks, respectively, and divided into sham- and AB-operated animals groups. Firstly, echocardiography were used to evaluate cardiac function, and then the hearts and lungs of the sacrificed mice were dissected and weighed to compare the heart weight/body weight(HW/BW), lung weight/body weight(LW/BW), and heart weight/tibia length(HW/TL) ratios. H&E staining was used to measure the cross sectional area of the myocytes, and both interstitial and perivascular collagen deposition were detected by PSR staining. Real-Time PCR was used to detect the m RNA expression level of hypertrophy markers(ANP, BNP, β-MHC) and fibrosis markers(Collagen Iα、Collagen III、CTGF) markers in different groups.Part 3: Cardiac specifc CARD6 transgenic(TG) mice were constructed using microinjection techniques. The male NTG and TG with body weight ranged from 24 to 26 g and aged from 10 to 12 weeks were employed and established mouse model subjected to AB. NTG and TG mice were radomely divided into sham and AB group. After 4 weeks AB, echocardiography were employed to assess cardiac function, then the sacrificed mice were dissected and weighed to compare the HW/BW, LW/BW, and HW/TL ratios. Moreover, H&E staining was used to evaluate the cross sectional area of the myocytes, and both interstitial and perivascular collagen deposition were detected by PSR staining. In addition, Real-Time PCR was performed to detect the m RNA expression level of hypertrophy markers(ANP, BNP, β-MHC) and fibrosis markers(Collagen Iα、Collagen III、CTGF) markers in different groups.Part 4: In vitro, cultured neonatal rat cardiomyocytes(NRCMs) were infected with Adsh CARD6 and Ad CARD6 as well as Adsh RNA and Ad GFP, subsequently stimulated with Ang II(1 μmol/L) for 48 hours, and then the cells were stained with a-actinin for surface area measurements. Real-Time PCR was also used to perceive the m RNA expression level of hypertrophy markers(ANP and β-MHC).Part 5: Western blotting was used to detect the protein levels of signaling pathway molecules related to hypertrophy in different groups. ResultsPart 1: Compared with the sham-operated hearts, the m RNA expression levels of cardiac hypertrophy markers(ie, ANP, BNP, and β-MHC) were dramatically elevated at week 2, and more pronounced from 4 to 8 weeks after AB. Meanwhile, both Real-time PCR and Western blot assays revealed that the m RNA and protein expression levels of CARD6 were significantly increased in the experimental C57BL/6 mouse hearts subjected to AB from weeks 2 to 4, and then reduced at week 8.Part 2: The c CARD6-KO mice that were subjected to AB for 4 weeks displayed exacerbation of cardiac dilation compared with the CARD6-Flox mice, as assessed by echocardiographic parameters(ie, LVEDD and LVESD). Moreover, LV contraction was significantly aggravated in c CARD6-KO hearts(FS%) compared with that in CARD6-Flox hearts. Moreover, 4 weeks after AB, CARD6-defiient hearts exhibited significant hypertrophic deterioration, as indicated by a greater gross heart size. and higher ratios of HW/BW, LW/BW, and HW/TL, compared with AB-treated CARD6-Flox hearts. In addition, the cardiomyocyte cross-sectional area(H&E and WGA staining) and the extent of fibrosis(PSR staining) were also dramatically increased in c CARD6-KO hearts subjected to chronic AB. Consistently, the expression levels of several hypertrophic markers(ANP, BNP, β-MHC) and fibrotic markers(Collagen Iα, Collagen III, CTGF), were much higher in the c CARD6-KO mice compared with the CARD6-Flox mice after a 4-week course of AB.Part 3: We successfully generated cardiac specific CARD6 transgenic(TG) mice in a C57BL/6 background. In contrast to the KO mice, TG mice showed the opposite results in AB group. Compared with NTG mice, LV contractile(FS%) was greatly improved in TG mice as measured by echocardiography analyses. Similar increases were also observed in the left ventricular(LV) chamber dimension(ie, LVEDD and LVESD). Moreover, the ratios of HW/BW, LW/BW, and HW/TL of TG mice was significantly lower than NTG mice. In paralle, the cardiomyocyte cross sectional area(H&E and WGA staining) and the extent of fibrosis(PSR staining) were also dramatically decreased in TG hearts in response to AB. Consistently, the expression levels of several hypertrophic markers(ANP, BNP, β-MHC) and fibrotic markers(Collagen Iα, Collagen III, CTGF), were much lower in the TG mice compared with the NTG mice after 4 weeks of AB.Part 4: In vitro, we performed gain-of-function and loss-of-function studies in cultured NRCMs. After Ang II(1 μmol/L) stimulation for 48 hours, CARD6-deficent cells by infection of cardiomyocytes with Adsh CARD6 showed remarkably increased cell surface area, accompanied with increased the m RNA expression of hypertrophic markers(ANP and β-MHC), compared with Adsh RNA-infected controls. However, the expression of these markers was suppressed remarkably in Ad CARD6-infected cells, compared with Ad GFP-infected cardiomyocytes.Part 5: Western blot analysis further showed that loss of CARD6 significantly increased AB-triggered activation of MEKK1-dependent MEK-ERK1/2 and JNK1/2 signaling pathway, as evidenced by higher phosphorylation of MEK1/2, ERK1/2, and JNK1/2 relative to control hearts, in sharp contrast, the increase almost completely blocked by overexpression of CARD6. ConclusionIn conclusion, the salient fiding of the current study is that the absence of CARD6 in the heart profoundly aggravates aortic banding(AB)-induced cardiac hypertrophy and fibrosis Conversely, overexpression of CARD6 blunts pathological hypertrophic response. Furthermore, CARD6 negatively regulates MEKK1-dependent MEK-ERK1/2 and JNK1/2 signaling activation in the development of cardiac hypertrophy and heart failure.