Testin protects against cardiac hypertrophy by targeting a calcineurin‐dependent signalling pathway

Abstract Multiple organs express testin (TES), including the heart. Nevertheless, current understanding of the influence of TES on cardiovascular diseases, especially on cardiac hypertrophy and its etiology, is insufficient. This study investigated the influence of TES on cardiac hypertrophy and its etiology. Murine models with excessive TES expression specific to the heart were constructed with an adeno‐associated virus expression system. Cardiac hypertrophy was stimulated through aortic banding (AB). The severity of cardiac hypertrophy was evaluated through molecular, echocardiographic, pathological, and hemodynamic examination. The findings of our study revealed that TES expression was remarkably suppressed not only in failing human hearts but also in mouse hearts with cardiac hypertrophy. It was discovered that excessive TES expression driven by an adeno‐associated viral vector noticeably inhibited hypertrophy triggered by angiotensin II (Ang II) in cultivated cardiomyocytes from newborn rats. It was also revealed that TES knockdown via AdshTES caused the reverse phenotype in cardiomyocytes. Furthermore, it was proved that excessive TES expression attenuated the ventricular dilation, cardiac hypertrophy, dysfunction, and fibrosis triggered by AB in mice. It was discovered that TES directly interacted with calcineurin and suppressed its downstream signalling pathway. Moreover, the inactivation of calcineurin with cyclosporin A greatly offset the exacerbated hypertrophic response triggered by AB in TES knockdown mice. Overall, the findings of our study suggest that TES serves as a crucial regulator of the hypertrophic reaction by hindering the calcineurin‐dependent pathway in the heart.

Elucidating the etiology of cardiac hypertrophy and its progression to congestive heart failure is necessary.
The human testin (TES) gene was originally identified in 1995 and was noted to have its highest expression in the testes, yet it is also widely expressed throughout other tissues. 6,7 In humans, the TES gene is located in the FRG7G region on chromosome 7q31.2, which is believed to contain a tumour suppressor gene because of its lack of heterozygosity in multiple malignancies. 8,9 The interest in TES mainly arises from the fact that it has been shown to be downregulated in an increasing number of human tumour types, in which its downregulation correlates with disease progression. [10][11][12][13][14] As a modular scaffold protein, TES contains a central PET (Prickle, Espinas, Testin) domain, a cysteine-rich region, and three LIM domains that compose the C-terminal half of TES. [14][15][16] The LIM domain was initially discovered in three transcription factors essential to development, Isl-1, Mec-3, and Lin-11. 17,18 Later, various proteins with LIM domains were recognized that participate in various biological processes such as differentiation, the generation of malignancies, and the organization of the cytoskeleton. [19][20][21] The LIM domain of TES resembles that of other proteins, including FLH2, CRP3, and cysteine-rich domains 1 (Lmcd1), which serve as crucial modulators of cardiovascular diseases, especially cardiac hypertrophy, suggesting the potential participation of TES in cardiac hypertrophy. [22][23][24] Consequently, we hypothesized that TES could be a promising modulator of cardiac hypertrophy.
It was discovered in this study that the concentration of TES was suppressed in the hearts of patients suffering from dilated cardiomyopathy (DCM) as well as in cardiac hypertrophy models triggered by pressure overload. With the help of an adeno-associated virus expression system, it was discovered that pressure overload-stimulated cardiac hypertrophy was suppressed in mice with excessive TES expression. TES was able to directly interact with calcineurin and inhibit downstream reactions. Furthermore, calcineurin deactivation by cyclosporin A (CsA) clearly offset the amplified hypertrophic reaction stimulated by pressure overload in TES knockdown mice.
Our research suggests that TES is essential in the regulation of cardiac hypertrophy and can be used as a reliable target to treat cardiac hypertrophy.

| Reagents
Ang II and CsA were acquired from Sigma-Aldrich (St. Louis, MO, USA). A bicinchoninic acid (BCA) protein assay kit was acquired from Pierce (Rockford, IL, USA). Secondary antibodies conjugated with peroxidase (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) were applied to observe the binding of the primary antibody (Table 1). Fetal calf serum was procured from HyClone (Waltham, MA, USA). Cell cultivation reagents and other reagents were provided by Sigma-Aldrich.

| Human heart specimens
Samples of failing hearts were acquired from the left ventricles (LVs) of patients who suffered from DCM and had received treatments subsequent to heart transplantation ( Table 2). Control samples were acquired from the LVs of normal donors. Written informed consent was obtained from the families of the prospective heart donors. This study complied with a protocol approved by the First Affiliated Hospital of Zhengzhou University Human Research Ethics Committee, and samples were collected after informed consent. AAV9-green fluorescent protein (GFP), AAV9-TES, AAV9-shTES, and scrambled shRNA were created. Mice that were 4 weeks old were injected with 100 μL of physiological serum solution containing 3 × 10 11 genome copies per microlitre of AAV9 in the retro-orbital sinus. AB was performed if hemodynamic stabilization was reached subsequent to the transduction of the virus. 25,26 Doppler assay was utilized to evaluate whether appropriate aortic constriction had been achieved. Mice that received sham operations were treated similarly but did not undergo aortic constriction. Echocardiography was applied to evaluate the wall thickness and the internal diameter of the LV at particular time points subsequent to the operation. Mice were sacrificed, and the hearts, tibiae, and lungs were obtained. Tissues were weighed and evaluated to determine the lung weight/body weight (LW/BW, mg/g), heart weight/tibia length (HW/TL, mg/mm), and HW/BW (mg/g) ratios of the groups.

| Echocardiography and hemodynamic assessment
Echocardiography was conducted on mice anaesthetized with 1.5% isoflurane using a MyLab 30CV ultrasound machine (Biosound Esaote, Irvine, CA, USA) with a 10-MHz linear-array ultrasound transducer as described previously. [25][26][27] The LV dimensions were evaluated in the parasternal short-axis and long-axis views with a frame rate of 120 Hz. The LV end-diastolic diameter (LVEDD), ejection fraction (EF), LV end-systolic diameter (LVESD), and fractional shortening (FS) were determined through M-mode tracing with a sweeping velocity of 50 mm/s at the midpapillary muscle level.
For hemodynamic evaluation, a microtip catheter transducer (SPR-839; Millar Instruments, Houston, TX, USA) was inserted into the right carotid artery and guided into the murine LV. Subsequent to a 15-minute stabilization period, heart rate (HR) and pressure, and

| Histological examination
Hearts were resected and immediately placed in a ten percent potassium chloride solution to ensure that they were halted in diastole. A saline solution was used to wash the hearts, which were subsequently put into ten percent formalin. The hearts were transected near the apex to reveal the LVs and the right ventricles. Some slices with a thickness of 4-5 μm underwent hematoxylin-eosin (HE) staining in preparation for histopathology and picrosirius red (PSR) staining to enable evaluation of collagen deposition under a microscope. HE staining was used to examine the cross-sectional area of cardiac muscle cells. A quantitative digital image analysis system (Image-Pro Plus 6.0, Rockville, MD, USA) was used to examine the cardiomyocytes.

| Immunofluorescent staining
Immunofluorescent staining was performed in tissue slices or neonatal rat cardiomyocytes (NRCMs) with a TES antibody to evaluate TES expression or with an a-actinin antibody to evaluate cell surface area. Briefly, NRCMs underwent 24 hours of transduction with various adeno-associated viruses and were subsequently stimulated for 48 hours with Ang II. Cells were fixed in 3.7% of formaldehyde in PBS before permeabilization with 0.1% of Triton X-100 in PBS.
Staining was performed with a 1:100 dilution of a-actinin. The staining procedures were identical to those used for the NRCMs after the deparaffinization step.

| Cardiomyocyte cultivation and transduction with recombinant adeno-associated viral vectors
Cultivated NRCMs were treated as described previously, with minor changes. [25][26][27] Briefly, PBS with 0.04% collagenase type II and 0.03% trypsin was used to separate cardiomyocytes from 1-to 2-day-old Sprague Dawley rats. After fibroblasts were eliminated with a preferential attachment technique, NRCMs were seeded in To overexpress TES, we used replication-defective adeno-associated viral vectors containing the entire rat TES cDNA coding region under the control of the cytomegalovirus promoter. An adeno-associated viral vector that encoded GFP served as the control. To perform TES knockdown, three rat TES constructs were acquired from SABiosciences. We then created three Ad-shTES adeno-associated viruses and selected the one that caused noticeable endogenous TES downregulation for further experiments.
Ad-shRNA served as a non-targeting control.

| Western blotting
Heart tissues and cultivated cardiac myocytes were lysed in RIPA buffer. A BCA protein assay kit was used for protein quantification. and observed with a FluorChem E imaging system (Cell Biosciences, Lake Franklin, NJ, USA). The expression of the proteins was normalized to that of GAPDH on an identical nitrocellulose membrane.

| Reverse transcription-PCR
Total mRNA was isolated from cultivated cells or LVs with TRIzol reagent (Invitrogen) and reverse transcribed into cDNA with a Transcriptor First Strand cDNA Synthesis Kit (Roche, Diagnostics, Mannheim, Germany). The relative expression of specific genes was evaluated using quantitative real-time PCR with SYBR Green (Roche).
The expression levels were normalized to GAPDH expression.

| Calcineurin assay
The activity of calcineurin phosphatase was evaluated in cell extracts with a Calbiochem Calcineurin Cellular Activity Assay Kit (Ref. 207007). The calcineurin activity was evaluated by examining the free phosphate released with or without EGTA buffer. Colorimetric examination was performed with a plate reader (Dynatech MR 5000, Melville, NY, USA) at a wavelength of 620 nm.

| Expression of TES is downregulated in human dilated cardiomyopathic hearts and murine hypertrophic hearts
To explore the potential influence of TES on cardiac hypertrophy, our study evaluated whether TES expression was changed in diseased hearts. Human dilated cardiomyopathic samples were obtained, and it was discovered that TES mRNA and protein expression was remarkably suppressed, while β-MHC and ANP (two biomarkers of hypertrophic hearts) expression was markedly increased, in comparison to the expression in healthy counterparts ( Figure 1A and B). With regard to a murine model of cardiac hypertrophy triggered by AB, it was revealed that TES expression was noticeably suppressed in hypertrophic hearts after 4 or 8 weeks of AB in comparison to the expression in hearts that had received a sham operation ( Figure 1C and D). In addition, TES was mainly localized to cardiomyocytes ( Figure 1E). Moreover, it was discovered that

| TES negatively regulates Ang II-induced cardiomyocyte hypertrophy in vitro
To determine whether the changes in TES expression were in response to a hypertrophic irritant, our study examined whether TES was able to modulate the progression of cardiac hypertrophy by con- was clearly promoted in cardiomyocytes that were transduced with AdshTES ( Figure 2E). However, the expression of these two markers was noticeably inhibited noticeably in cells transduced with AdTES ( Figure 2F) in comparison to their expression in control cells. These findings confirmed that cardiac hypertrophy was suppressed by the upregulation of TES, while TES downregulation enhanced pathological cardiac hypertrophy.

| TES overexpression suppresses AB-induced cardiac hypertrophy
Our study subsequently explored whether elevated cardiac TES concentrations could inhibit the progression of cardiac hypertrophy and cardiac failure. A gene transfer approach using AAV9 was selected, as  Figure 3G). Overall, the above findings suggested that excessive TES expression was able to inhibit the development of cardiac hypertrophy triggered by AB in vivo.

| TES overexpression attenuated fibrosis in pressure-overloaded hearts
Cardiac hypertrophy features fibrosis, which is manifested by cardiac collagen deposition. To examine the influence of excessive TES expression on the maladaptive remodelling of hearts, this study examined the participation of TES in fibrosis. The severity of fibrosis was quantified based on the volume of collagen through the visualization of the total quantity of collagen in the interstitial and perivascular spaces. Our study revealed that interstitial and perivascular fibrosis was noticeably reinforced in AAV9-GFP hearts that received chronic AB. However, such fibrosis was markedly inhibited in AAV9-TES hearts ( Figure 4A and B). Next, we examined collagen generation by evaluating the transcription of fibrotic markers such as collagen I, connective tissue growth factor (CTGF) and III. It was revealed that the fibrotic reaction was impaired in AAV9-TES mice in comparison to that in AAV9-GFP mice ( Figure 4C). The TGF-β/ Smad pathway is an essential pathway participating in the progression of cardiac fibrosis. To evaluate the role of TES in the etiology of collagen generation, our study explored the modulation of Smad cascade stimulation by TES. It was revealed that the enhanced Smad 2/3 phosphorylation triggered by AB was noticeably suppressed in AAV9-TES mouse hearts in comparison to that in AAV9-GFP hearts ( Figure 4D and E). In summary, the above findings suggested that excessive TES expression was able to impair myocardial fibrosis triggered by pressure overload.

| TES inhibited the calcineurin-NFAT pathway in murine hypertrophic hearts
This study next explored the mechanisms by which TES represses hypertrophy. Previous research has proven that the calcineurin-NFAT pathway participates in cardiac hypertrophy and that the muscle LIM protein is required for calcineurin-NFAT signalling at the sarcomeric Z disc. 28,29 Consequently, our study examined whether TES modulates cardiac hypertrophy via the calcineurin-NFAT axis. It has previously been proven that stimulated calcineurin directly links with NFAT transcription factors, bringing about nuclear translocation and dephosphorylation of NFAT. Thus, our study evaluated cardiac NFAT translation triggered by pressure overload. It was discovered that the AB-mediated decrease in p-NFATc3 expression in the cytoplasm was noticeably promoted in the AVV9-TES group in comparison to that in the AVV9-GFP group. However, our study failed to observe any differences in p-NFATc1, p-NFATc2, and p-NFATc4 between the groups ( Figure 5A, C and D). Despite the fact that the AKT pathway and the MAPK pathway both modulate cardiac remodelling, our study failed to detect any differences in AKT or MAPK stimulation between the groups ( Figure 5B). Overall, the above findings indicated that TESmodulated pathological cardiac hypertrophy relied, at least in part, on the modulation of the calcineurin-NFAT axis.

| TES directly interacts with calcineurin
Subsequently, we analysed the mechanism by which TES regulates calcineurin activity. In this study, we proved that NFAT nuclear   (Figure 6D). This interaction was further confirmed with a Co-IP assay ( Figure 6E). Moreover, an interaction between TES and CaN in vivo was detected. Our results showed that TES could interact with CaN in vivo ( Figure 6G). Then, we detected an interaction between TES and CaN after cells were treated with AngII. The results showed that the interaction between TES and CaN was attenuated when H9c2 cells were treated with AngII ( Figure 6H).
Furthermore, our study investigated the localization of calcineurin and TES using fluorescent confocal microscopy. It was discovered that both calcineurin and TES were mainly located in the cytoplasm and fused rather well ( Figure 6F). The interaction between the calcineurin-NFAT axis and TES was verified via cotransduction of AdTES or AdshTES with NFAT reaction elements conjugated to a luciferase gene (NFAT-Luc) in NRVMs. It was discovered that NFAT-Luc transcription triggered by Ang II was noticeably promoted in cells that underwent transduction with AdshTES in comparison to the AngII-triggered transcription in control AdshRNA cells. In contrast, the promotion of NFAT-Luc transcription triggered by Ang II was markedly suppressed in AdTES cardiomyocytes ( Figure 6I).
Overall, the above findings indicated that TES inhibited the NFAT pathway via interaction with the stimulated calcineurin pathway.

| Blocking calcineurin-NFAT signaling blunts cardiac hypertrophy in TES knockdown mice
To better verify the essential influence of the calcineurin-NFAT axis in vivo, the calcineurin suppressor CsA was used. Mice received an injection of an AAV9 encoding shTES or the control (shRNA) in the retro-orbital sinus ( Figure 7A  Lmcd1 participates in the development of cardiac hypertrophy. 23 Hojayev and his colleagues proved that FHL2 was able to inhibit the pathological proliferation of the heart. 28 The above findings reliably indicate that TES is an essential modulatory of cardiac hypertrophy.
However, current understanding of the modulation of TES expression, particularly during the transition from cardiac hypertrophy to cardiac failure, is insufficient. Our study is the first to prove that TES is a reverse modulator of cardiac hypertrophy in cardiomyocytes, and it did so with an adeno-associated virus expression system. Nevertheless, further research is still needed elucidate the mechanism by which TES acts on the heart.
Multiple signalling pathways that include transcription factors, kinases, and G-protein coupled receptors participate in the modulation of heart growth. It has been proven that the calcineurin-NFAT axis is essential in regulating cardiac hypertrophy. 28,29 NFATs are dephosphorylated by calcineurin in response to elevated intracellular calcium.
Calcineurin modulates the expression of genes in various tissues sensitive to calcium, including muscle, brain, and lymphocytes. NFAT transcription relies on dephosphorylation by calcineurin, which brings about nuclear translocation and the stimulation of targeted genes. 32,33 It has recently been proven that the LIM protein in muscles is directly related to calcineurin and is crucial to the calcineurin-NFAT axis. 23,28 However, little research has been conducted on the direct influence of TES on the calcineurin-NFAT axis. Our study is the first to prove that In all, our study revealed a novel role for TES in the modulation of pathological cardiac hypertrophy through suppression of the calcineurin-NFAT axis. Our research is the first of its kind to prove the participation of TES in cardiac hypertrophy and to suggest that proteins consisting of LIM domains can act as a reservoir of signalling agents related to the preservation of cardiac homeostasis. We have discovered in this study that TES defends the heart in response to pressure overload, indicating that TES can serve as a promising diagnostic and therapeutic target for cardiac illness. However, further verification of the influence of TES on cardiac hypertrophy is necessary.

| CLINICAL PERSPECTIVES
This research offers in vitro and in vivo proof that excessive TES expression attenuates cardiac hypertrophy through suppression of the calcineurin-NFAT axis. We have verified that TES has an influence on cardiac hypertrophy in response to pressure overload and that TES is related to the calcineurin-NFAT axis in cardiac hypertrophy. The above findings are essential to elucidate the etiology of cardiac hypertrophy and to develop innovative strategies to treat cardiac hypertrophy by targeting TES.

CONFLI CT OF INTEREST
The authors declare no conflicts of interest.