PKC-ζ Aggravates Doxorubicin-Induced Cardiotoxicity by Inhibiting Wnt/β-Catenin Signaling

Doxorubicin (Dox) is a chemotherapeutic drug used to treat a wide range of cancers, but its clinical application is limited due to its cardiotoxicity. Protein kinase C-ζ (PKC-ζ) is a serine/threonine kinase belonging to atypical protein kinase C (PKC) subfamily, and is activated by its phosphorylation. We and others have reported that PKC-ζ induced cardiac hypertrophy by activating the inflammatory signaling pathway. This study focused on whether PKC-ζ played an important role in Dox-induced cardiotoxicity. We found that PKC-ζ phosphorylation was increased by Dox treatment in vivo and in vitro. PKC-ζ overexpression exacerbated Dox-induced cardiotoxicity. Conversely, knockdown of PKC-ζ by siRNA relieved Dox-induced cardiotoxicity. Similar results were observed when PKC-ζ enzyme activity was inhibited by its pseudosubstrate inhibitor, Myristoylated. PKC-ζ interacted with β-catenin and inhibited Wnt/β-catenin signaling pathway. Activation of Wnt/β-catenin signaling by LiCl protected against Dox-induced cardiotoxicity. The Wnt/β-catenin inhibitor XAV-939 aggravated Dox-caused decline of β-catenin and cardiomyocyte apoptosis and mitochondrial damage. Moreover, activation of Wnt/β-catenin suppressed aggravation of Dox-induced cardiotoxicity due to PKC-ζ overexpression. Taken together, our study revealed that inhibition of PKC-ζ activity was a potential cardioprotective approach to preventing Dox-induced cardiac injury.

INTRODUCTION Doxorubicin (Dox) is a widely used chemotherapeutic agent for treatment of leukemia, lymphoma, neuroblastoma, and other human cancers (Carvalho et al., 2014). However, the serious cardiac side effects of Dox, including cardiomyopathy, arrhythmia, and congestive heart failure, limit its cumulative therapeutic dose in clinical application (Wallace et al., 2020). These cardiotoxic effects mainly stem from mitochondrial dysfunction (Wenningmann et al., 2019), autophagy (Bartlett et al., 2017), oxidative stress (Songbo et al., 2019), apoptosis (Takemura and Fujiwara, 2007), and impairment of calcium homeostasis (Octavia et al., 2012). Previous findings of our lab have established that Wnt signaling , mitophagy , and PARylation  played important roles in Dox-induced cardiotoxicity. However, management of Dox-induced cardiotoxicity is complicated by its multifactorial nature and complicated pathogenesis. Therefore, further studies are needed to elucidate mechanisms of Dox-induced cardiotoxicity.
This study explored the role of PKC-ζ in Dox-induced cardiotoxicity and underlying mechanisms. Results of the current study indicated that PKC-ζ acted as upstream suppressor of Wnt/β-catenin signaling and aggravated Doxinduced cardiac injury. PKC-ζ-based intervention was a potential strategy for alleviating Dox-induced cardiotoxicity.

Animals
Animal experimental procedures were undertaken in compliance with the Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85-23, revised 1996) and approved by the Research Ethics Committee of Sun Yat-sen University. Male Sprague-Dawley (SD) rats (weighing 220-250 g, SPF grade, certification No. 44008500019766) were purchased from the Experimental Animal Center of Sun Yat-sen University (Guangzhou, China). Animals were randomly divided into two groups, model and control groups, and each group contained 6 rats. Rats in the model group were intraperitoneally administered with Dox on the 1st, 5th, and 9th day at a dosage of 5 mg/kg. Rats in the control group were intraperitoneally administered with the same dosage of normal saline (NS).

Echocardiographic and Morphometric Measurements
The day after the final administration of Dox, SD rats were anesthetized with 3% isoflurane, and echocardiographic measurements were taken using Technos MPX ultrasound system (ESAOTE, Italy) of two-dimensional-guided M-mode echocardiography based on previous studies . Cardiac indices including ejection fraction (EF), fractional shortening (FS), left ventricular end-systolic posterior wall thickness (LVPWs), and left ventricular end-diastolic posterior wall thickness (LVPWd) were determined. After finishing echocardiographic measurements, rats were immediately sacrificed and their heart tissues were removed after injected with 0.1 M KCl. In order to conduct morphometric measurement, hearts were transversely trimmed into two parts, one part was cut into 5-μm-thick histological cross sections and fixed in 4% paraformaldehyde, which were used for hematoxylin-eosin (HE), Picrosirius Red (PSR), and TUNEL staining. The other part of heart tissues was stored in −80°C, which was used for immunoblotting and mRNA detection.

Cell Culture
Heart tissues of SD rats (1-3 days old) were isolated based on a previously described protocol (Feng et al., 2015) to obtain neonatal rat cardiomyocytes (NRCMs). NRCMs were then incubated in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS) and 0.1 mM 5bromodeoxyuridine for 24 h. NRCMs were then washed with PBS and cultured in new DMEM supplemented with 10% FBS for 12 h before treatment. NRCMs were treated with Dox for 12 h to induce cardiomyocyte injury.

Detection of Nuclear Condensation, Mitochondrial Membrane Potential, and Mitochondrial Morphology
Cardiomyocytes were seeded into 48-well plates. After corresponding treatments, cells were washed thrice with PBS and then incubated with 10 nM tetra-methylrhodamine ethyl ester (TMRE) (Invitrogen, United States) for 30 min followed by 10 μg/ml Hoechst 33,42 staining (Solarbio, China) for 10 min at 37°C. Mitochondrial membrane potential and nuclear condensation were detected using EVOS FL Auto (Life Technologies, Bothell, WA, United States).
After washing with PBS, cardiomyocytes were fixed with 4% paraformaldehyde for 15 min and incubated with 0.3% Triton X-100 for 10 min at room temperature; cardiomyocytes were then incubated with 1 μM MitoTracker Red (Invitrogen, United States) for 30 min followed by 10 μg/ml Hoechst 33342 staining for 10 min. Mitochondrial morphology was detected using an ultrahigh-resolution laser scanning microscope (Olympus, Japan).

Co-Immunoprecipitation
A total of 300-400 μg of protein was respectively incubated with anti-β-catenin (diluted 1:100) or anti-PKC-ζ antibody (diluted 1: 50) and IgG antibody (Beyotime, China) overnight at 4°C, followed by incubation with 20 μl of protein A/G beads (Pierce, United States) for 4 h at 4°C. Then, beads were washed with IP Lysis buffer and diluted in loading buffer. The interactive protein was assayed by immunoblotting.

Statistical Analyses
Data are presented as mean ± SEM. Statistical analyses between two groups were done using Student's t-tests. One-way ANOVA with Bonferroni post hoc tests were undertaken for multiple group comparisons. p < 0.05 was considered statistically significant.

PKC-ζ Phosphorylation Was Increased by Dox Treatment
To explore the role of PKC-ζ in Dox-induced cardiac injury, we examined the change of PKC-ζ phosphorylation level in vivo and in vitro. SD rats were intraperitoneally administered with Dox at a dosage of 5 mg/kg body weight on the 1st, 5th, and 9th day, leading to a cumulative dosage of 15 mg/kg body weight. The echocardiographic analysis revealed decreased ejection fraction (EF%), fractional shortening (FS%), and left ventricular posterior wall thickness (LVPW) in Dox-treated group rats compared with those of control group rats ( Figure 1A). Heart weight-to-tibia length ratio was decreased in comparison with those of control group rats ( Figure 1B). Figures 1C,D showed that hearts of rats in the Dox-treated group were smaller. HE staining as well as PSR staining indicated aggravation of cardiomyocyte disorganization and cardiac fibrosis, respectively ( Figures 1E,F,H). Moreover, TUNEL staining and protein level of Bax/Bcl-2 revealed some degree of elevated apoptosis in the Dox group ( Figures 1G,I,J). Figures 1K,L, administration of Dox increased Thr410 phosphorylation of PKC-ζ, although it had no effect on total protein and mRNA levels of PKC-ζ. Figure 2A indicated that the purity of cardiomyocytes isolated from NRCMs was more than 95%, which was observed by IF staining of Troponin T. Next, cardiomyocytes were then treated with 1 μM Dox at different times and the change of PKC-ζ was explored in vitro. It was found that Dox treatment led to nuclear condensation, decreased mitochondrial membrane potential, and mitochondrial morphological disorder (Figures 2B-D, Supplementary Figure S1A). In addition, Dox caused a timedependent increase in apoptosis indices including cleaved-PARP1/PARP1, Bax/Bcl-2, and cleaved-caspase3/caspase3 ( Figures 2E,F). Based on these results, cardiomyocytes were stimulated with 1 μM Dox for 12 h to induce cardiotoxicity in Frontiers in Pharmacology | www.frontiersin.org February 2022 | Volume 13 | Article 798436 subsequent experiments. Notably, results of in vitro levels of Thr410 phosphorylation of PKC-ζ as well as total protein and mRNA levels of PKC-ζ were parallel to findings generated in vivo ( Figures 2G-I). These results, therefore, indicated that Dox treatment increased PKC-ζ phosphorylation level and induced PKC-ζ activation.
These results revealed that PKC-ζ was an essential regulatory factor in Dox-induced cardiac injury.

DISCUSSION
Dox is a widely prescribed chemotherapeutic drug, which treats cancer by intercalating with DNA and inhibiting topoisomerase Ⅱ (Renu et al., 2018). However, cardiotoxicity of Dox is among the severe side effects in clinical application. Although several research efforts have explored pathogenesis of Dox-induced cardiotoxicity, its exact mechanism is still obscure. Therefore, more studies are needed to help to develop preventive and cardioprotective drugs. The current study successfully replicated the Dox-induced cardiotoxicity model both in vitro and in vivo. Our results showed that Dox treatment caused cardiomyocyte apoptosis, nuclear condensation, decline of mitochondrial membrane potential, and mitochondrial morphology disorder. Notably, Dox-treated rats showed decreased left ventricular function and increased cardiac injury.
PKCs are serine/threonine kinases, which are divided into three subfamilies based on their activation mechanisms: classical PKC isoforms (α, βI, βII, and γ), which are regulated by calcium, DAG, and phospholipids; novel PKC isoforms (δ, ε, η, and θ), which are activated by DAG and phospholipids; and atypical PKC isoforms (ζ and λ or ι), whose activation depends on protein-protein interaction and lipids such as ceramide rather than calcium and DAG (Steinberg, 2008). Findings of previous studies confirm that the PKC family played key roles in diseases such as cancer (Reina-Campos et al., 2019), heart failure (Singh et al., 2017), and Alzheimer's disease (de Barry et al., 2010). PKCζ belongs to an atypical PKC subfamily and is considered an important regulatory factor in cardiovascular diseases. For example, oleanonic acid inhibits PKC-ζ-induced nuclear factor-kappa B (NF-κB) activation and protects against phenylephrine (PE)-induced cardiac hypertrophy (Gao et al., 2018). PKC-ζ activity is also inhibited by PICOT and increases cardiac contractility (Oh et al., 2012). Our previous findings established that Sirtuin1 negatively regulated PKC-ζ phosphorylation level and protected cardiomyocytes from PKC-ζ-induced hypertrophy . However, function of PKC-ζ in Dox-induced cardiotoxicity has not been explored. The current study showed that Dox treatment upregulated PKC-ζ Thr410 phosphorylation, and we speculated that this may partly be due to the ceramide generation induced by Dox (Delpy et al., 1999). Then, we found that overexpression of PKC-ζ aggravated Dox-induced cardiotoxicity, which was reflected by decreased mitochondrial membrane potential, elevated level of apoptosis of cardiomyocytes, and nuclear condensation. Conversely, knockdown or inhibition of PKC-ζ showed alleviated cardiomyocyte injury following Dox treatment.
In summary, the current study established that loss of function of PKC-ζ is a potential therapeutic approach to alleviate Dox-induced cardiac injury. In addition, the current study indicated that the deleterious effect of PKC-ζ is partly elucidated by inhibiting Wnt/βcatenin signaling. Further studies on cardiac-specific overexpression or knockdown of PKC-ζ in animal models are needed to further validate all the observed cellular findings in the current study. Moreover, specific regulatory mechanisms underlying PKC-ζ regulation of β-catenin should be explored further.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding authors.

ETHICS STATEMENT
The animal study was reviewed and approved by the Research Ethics Committee of Sun Yat-sen University.