Metabolic Reprogramming in Cardiac Fibrosis and Its Implications for Targeting PDK1

Metabolic Reprogramming in Cardiac Fibrosis and Its Implications for Targeting PDK1 | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 12 February 2026 V1 Latest version Share on Metabolic Reprogramming in Cardiac Fibrosis and Its Implications for Targeting PDK1 Authors : Wenyu Hui , Xiaofan Lin , Ling Yang , Yingyu Wang , Yunru Peng , and Yongfang Ding [email protected] Authors Info & Affiliations https://doi.org/10.22541/au.177090156.69909573/v1 126 views 59 downloads Contents Abstract Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Cardiac fibrosis is driven by the activation of resident cardiac fibroblasts and their subsequent differentiation into myofibroblasts upon injury or stress. These activated myofibroblasts drive the fibrotic process through excessive secretion and deposition of extracellular matrix components, leading to adverse cardiac remodeling, tissue stiffening, and eventual functional impairment. Although myofibroblast formation serves as a physiological repair mechanism following acute injury, the sustained presence of these cells promotes maladaptive remodeling and gradual deterioration of cardiac function. In contrast to the well-characterized roles of transforming growth factor-β (TGF-β) and angiotensin II (AngII) signaling in fibrosis, the mitochondrial and metabolic reprogramming events essential for myofibroblast establishment and longevity remain comparatively understudied. 3-phosphoinositide-dependent protein kinase-1 (PDK1) is a master regulator of cellular energy metabolism and a key upstream activator of the AKT/mTOR axis; however, its specific role and therapeutic relevance in cardiac fibrosis remain elusive. Critically, although targeting the downstream AKT/mTOR pathway holds promise, the clinical translation of such inhibitors faces challenges including feedback activation and systemic toxicity. This review integrates current knowledge on metabolic and mitochondrial adaptations in cardiac fibrosis, with a focus on the potential involvement of PDK1. In support of this aim, our structural and computational analyses reveal a well-defined, ligandable ATP-binding pocket in PDK1 and demonstrate high-affinity binding of classical inhibitors, thereby structurally validating its potential as a druggable therapeutic target for mitigating fibrotic progression. Metabolic Reprogramming in Cardiac Fibrosis and Its Implications for Targeting PDK1 Wenyu Hui 1,2 , Xiaofan Lin 1,2 , Ling Yang 1,2 , Yingyu Wang 1,2 , Yunru Peng 1,2* , Yongfang Ding 1,2* 1 Affiliated Hospital of Integrated Traditional Chinese and Western Medicine, Nanjing University of Chinese Medicine, Nanjing 210023, China 2 Jiangsu Province Academy of Traditional Chinese Medicine, Nanjing 210028, China *Correspondence to: Yongfang Ding , Department of Pharmacology and Toxicology, Jiangsu Province Academy of Traditional Chinese Medicine, Nanjing 210028, People’s Republic of China. E-mail: [email protected] Yunru Peng, Department of Pharmacology and Toxicology, Jiangsu Province Academy of Traditional Chinese Medicine, Nanjing 210028, People’s Republic of China. E-mail: [email protected] Abstract Cardiac fibrosis is driven by the activation of resident cardiac fibroblasts and their subsequent differentiation into myofibroblasts upon injury or stress. These activated myofibroblasts drive the fibrotic process through excessive secretion and deposition of extracellular matrix components, leading to adverse cardiac remodeling, tissue stiffening, and eventual functional impairment. Although myofibroblast formation serves as a physiological repair mechanism following acute injury, the sustained presence of these cells promotes maladaptive remodeling and gradual deterioration of cardiac function. In contrast to the well-characterized roles of transforming growth factor-β (TGF-β) and angiotensin II (AngII) signaling in fibrosis, the mitochondrial and metabolic reprogramming events essential for myofibroblast establishment and longevity remain comparatively understudied. 3-phosphoinositide-dependent protein kinase-1 (PDK1) is a master regulator of cellular energy metabolism and a key upstream activator of the AKT/mTOR axis; however, its specific role and therapeutic relevance in cardiac fibrosis remain elusive. Critically, although targeting the downstream AKT/mTOR pathway holds promise, the clinical translation of such inhibitors faces challenges including feedback activation and systemic toxicity. This review integrates current knowledge on metabolic and mitochondrial adaptations in cardiac fibrosis, with a focus on the potential involvement of PDK1. In support of this aim, our structural and computational analyses reveal a well-defined, ligandable ATP-binding pocket in PDK1 and demonstrate high-affinity binding of classical inhibitors, thereby structurally validating its potential as a druggable therapeutic target for mitigating fibrotic progression. Keywords: Cardiac fibrosis, Metabolic reprogramming, Mitochondrial function, PDK1, Myofibroblast Abbreviations AngII, angiotensin II; CPT1, carnitine palmitoyltransferase 1; CPT2, carnitine palmitoyltransferase 2; DRP1, dynamin-related protein 1; ECM, extracellular matrix; ET-1, endothelin-1; FACS, fatty acyl CoA synthase; FFAR3, free fatty acid receptor 3; FAO, fatty acid oxidation; GLS, glutaminase; GDH, glutamate dehydrogenase; HK2, hexokinase 2; JMJD, JmjC-domain-containing histone demethylases; MFN1/2, mitofusins 1/2; mTOR, mammalian target of rapamycin; mtROS, mitochondrial ROS; OPA1, optic atrophy 1; OXPHOS, oxidative phosphorylation; PINK1, PTEN-induced kinase 1; PFD, pirfenidone; PDK1, 3-phosphoinositide-dependent protein kinase-1; PDK, pyruvate dehydrogenase kinase; PFKFB3, phosphofructokinase-2/fructose-2,6-bisphosphatase 3; PKM2, pyruvate kinase M2; PDH, pyruvate dehydrogenase; ROS, reactive oxygen species; S6K, S6 kinase; TAG, triacylglycerol; TGF-β, transforming growth factor-β; α-KG, α-ketoglutarate; 4E-BP1, 4E binding protein 1. Introduction Cardiac fibrosis, a common pathological hallmark of numerous cardiovascular diseases including myocardial infarction (Chen et al. 2025), heart failure (Yamada et al. 2025b), and hypertensive heart disease (Li et al. 2023b), represents a substantial clinical burden globally due to its irreversible effects on cardiac structure and function (Li et al. 2026). This process is defined by the aberrant deposition of extracellular matrix (ECM) proteins-primarily collagens type I and III-which disrupts myocardial tissue organization, compromises ventricular compliance, and increases the risk of life-threatening arrhythmias and pump failure (Timmer et al. 2025). Despite decades of research targeting classical pathways, the clinical translation of therapies capable of halting or reversing cardiac fibrosis has stalled (Tan et al. 2025). This persistent gap underscores the urgent need to elucidate the fundamental cellular and molecular mechanisms governing the initiation and progression of cardiac fibrosis. Metabolic reprogramming, defined as the shift in cellular energy metabolism to support specific biological processes, is a fundamental hallmark of cell fate transitions, such as differentiation and activation (Zhu et al. 2025). In contrast to quiescent fibroblasts, which primarily rely on oxidative phosphorylation for energy production, activated myofibroblasts exhibit profound metabolic changes, such as increased glycolysis (Liu et al. 2025a), glutaminolysis (Bai et al. 2025b), and fatty acid oxidation (Yuan et al. 2023; Sun et al. 2025b). These metabolic shifts not only meet the high bioenergetic demands of ECM synthesis and other anabolic processes but also generate metabolic intermediates that act as signaling molecules to reinforce the pro-fibrotic phenotype (Gibb et al. 2020). Concurrently, mitochondria-central hubs of energy metabolism and redox homeostasis-play a dual role in myofibroblast biology (Tu et al. 2025): they provide ATP for cellular functions while also regulating reactive oxygen species (ROS) production, which can either promote or constrain fibrosis depending on its magnitude and localization (Lv et al. 2024; Peng et al. 2024). Dysregulation of mitochondrial dynamics (fission/fusion) (Cheng et al. 2024; Niu et al. 2025) or quality control (mitophagy) (Yu et al. 2024; Li et al. 2025a) further contributes to myofibroblast dysfunction and sustained fibrosis, highlighting mitochondria as integral players in the fibrotic cascade (He et al. 2025). The reprogramming of metabolism and mitochondrial function is orchestrated by upstream signaling hubs, among which 3-phosphoinositide-dependent protein kinase-1 (PDK1) is a pivotal node (Xiao et al. 2025). As a master kinase upstream of AKT, PDK1 converges major pro-fibrotic signals via the PDK1/AKT axis. This activation engages downstream effectors such as mammalian target of rapamycin (mTOR) and glycolytic enzymes, driving the characteristic anabolic and glycolytic shift in activated myofibroblasts (Pan et al. 2023). Moreover, PDK1 signaling directly links pro-fibrotic stimuli to the sustenance of fibrosis by regulating mitochondrial biogenesis and function (Zheng et al. 2021). While PDK1’s role in cellular energy homeostasis is well-established, its specific contributions to cardiac metabolic reprogramming and therapeutic potential remain unclear. Notably, targeting the well-characterized AKT/mTOR axis downstream of PDK1 holds therapeutic promise; however, the clinical translation of direct AKT or mTOR inhibitors in fibrosis has been hampered by issues such as feedback activation, metabolic toxicity, and lack of cell-type specificity. Upstream targeting of PDK1 itself presents a conceptually distinct strategy. As a convergent node for multiple growth factor and metabolic signals, PDK1 inhibition may offer a means to more broadly dampen the anabolic and glycolytic reprogramming of myofibroblasts while potentially avoiding some compensatory mechanisms triggered by direct downstream inhibition. This review synthesizes evidence on metabolic reprogramming in cardiac fibrosis to build a rationale for PDK1 as a novel, structurally validated, and druggable target within this network. 1. Activating Signaling Pathways in Myofibroblast Differentiation Cardiac fibrosis is closely associated with the activation of resident cardiac fibroblasts (Aguado-Alvaro et al. 2025). These quiescent, spindle-shaped cells account for approximately 10-20% of all cardiac cells (Gibb et al. 2020); their primary function is to maintain extracellular matrix homeostasis by balancing the synthesis and degradation of matrix proteins (Li et al. 2024a). Resident cardiac fibroblasts differentiate into myofibroblasts upon exposure to injury-related stimuli (e.g., ischemia-reperfusion (Huang et al. 2024), mechanical stress (Schneider et al. 2025)), profibrotic cytokines such as TGF-β (Pan et al. 2025), as well as GPCR signaling agonists including angiotensin II (AngII) (Cantrell et al. 2025) and endothelin-1 (ET-1) (Mourad et al. 2025) (Fig. 1). The activated myofibroblasts exhibits several distinct morphological features (Garside et al. 2026), including elongated and serrated nuclei, multiple dendritic processes, extensive rough endoplasmic reticulum, and a prominent stress fiber network (Fig. 1). These structural changes underpin its enhanced contractile function and elevated capacity for ECM production. A hallmark of this phenotype is the de novo expression of α-SMA (Jiang and Li 2025). Furthermore, activated myofibroblasts are characterized by the copious synthesis and secretion of ECM components, including POSTN (Hou et al. 2024), collagens (e.g., COL1 and COL3) (Yamauchi et al. 2024), and the specialized EDA splice variant of fibronectin (FN-EDA) (Dai et al. 2025). TGF-β signaling is transduced through canonical SMAD and non-canonical non-SMAD pathways (Gallardo et al. 2025). In the canonical pathway, TGF-β ligand binding to TβRII recruits and phosphorylates TβRI, forming an activated receptor complex (Wang et al. 2025b). This complex then phosphorylates SMAD2/3 (Ren et al. 2025), which forms a complex with SMAD4 and translocates to the nucleus to drive the transcription of key fibrosis-related genes, such as Col1a2 , Col3a1 , Col6a1 , Col6a3 , and Timp1 (Sarker et al. 2024; Hegedűs et al. 2025). Notably, SMAD3, but not SMAD2, directly activates the transcription of the ACTA2 gene, which encodes α-SMA (Randall et al. 2025). Non-SMAD pathways, which are co-regulated by TβRI and TβRII, activate several key axes-including PI3K/AKT/mTOR (Peng et al. 2025), TRAF/TAK1/MKK4/p38 (Takaguri et al. 2025), RHO/ROCK1 (Li et al. 2024b), and RAS/RAF/MEK/ERK (Xia et al. 2023). Among these, mTORC1 phosphorylation activates ribosomal protein S6 kinase (S6K) (Ye et al. 2023) and inhibits eukaryotic initiation factor 4E binding protein (4E-BP1) (Zou et al. 2024), thereby strongly initiating the translation synthesis of extracellular matrix components such as collagen. These pathways are critical for myofibroblast differentiation and intersect with SMAD signaling to orchestrate a comprehensive fibrotic response (Fig. 2). In addition to classical characteristics, emerging evidence now positions mitochondrial dysfunction (Song et al. 2024) and metabolic remodeling (Cho et al. 2025) as key drivers of myofibroblast differentiation. While signaling pathways like TGF-β have been extensively studied, the accompanying metabolic and mitochondrial reprogramming has only recently been recognized as a crucial regulatory axis in cardiac fibrosis. This reprogramming provides essential bioenergetic and biosynthetic support for myofibroblast activation, persistence, and function; however, its full scope and mechanisms remain incompletely elucidated. 2. Metabolic Reprogramming in Cardiac Fibroblast Activation 2.1. Glycolysis Quiescent cardiac fibroblasts primarily depend on oxidative phosphorylation for energy production (Yang et al. 2023). However, upon activation by TGF-β or pathological conditions such as stress load and ischemia, they undergo a metabolic shift toward glycolysis (Faakye et al. 2025). This transition is marked by increased expression and activity of key glycolytic enzymes, including hexokinase 2 (HK2), phosphofructokinase-2/fructose-2,6-bisphosphatase 3 (PFKFB3), and pyruvate kinase M2 (PKM2) (Luo et al. 2025) (Fig. 3). For instance, PFKFB3 inhibitors 3PO attenuated cardiac myofibroblasts activation, proliferation, and migration, while also reducing cardiac fibrosis in mouse models (Han et al. 2025a). TEPP-46, a pharmacological PKM2 activator, alleviated mitochondrial fission and cardiac fibrosis (Luo et al. 2025). TGF-β directly upregulates the gene expression of PFKFB3, PKM2 and LDHA (Han et al. 2025a). Even without initial stimulation of TGF-β, a high glucose environment can enhance TGF-β/Smad signaling (Han et al. 2025b). Glycolytic metabolites such as pyruvate and lactate (An et al. 2023) are important substrates or cofactors for histone acetylation (Shao et al. 2024) and methylation. For example, acetyl CoA is a substrate for histone acetyltransferase (Bishop et al. 2023), and lactate inhibits histone deacetylase, leading to the opening and activation of pro fibrotic gene chromatin (Liu et al. 2025b) (Fig. 4). Furthermore, under pathological conditions, actual tissue hypoxia or a “pseudohypoxia” state triggered by factors such as TGF-β can stabilize HIF-1α (Yang et al. 2025b). Once activated, HIF-1α transcriptionally activates glycolytic enzymes, upregulates pyruvate dehydrogenase kinase （PDK） (Liu et al. 2024), and inhibits pyruvate dehydrogenase (PDH) (Li et al. 2025b). These actions collectively prevent pyruvate from entering the mitochondria, effectively locking cellular metabolism into a glycolytic state. Moreover, activated HIF-1 α can directly bind to the promoter region of the α-SMA (ACTA2) gene, driving its expression and exacerbates myocardial fibrosis (Lin et al. 2024). HIF-1 α inhibitors YC-1 decreased the expression of α-SMA and diminished hepatic fibrosis (Nayak et al. 2016). 2.2. Glutaminolysis Glutaminolysis is the metabolic process that converts glutamine into glutamate via glutaminase (GLS) (Bai et al. 2025a), which is then further metabolized to α-ketoglutarate (α-KG) (Yang et al. 2025a) by glutamate dehydrogenase (GDH) or transaminases. This pathway has increasingly been recognized as a crucial source of energy and biosynthetic precursors in fibrotic progression across multiple organs, including the heart (Bai et al. 2025b), liver (Wu et al. 2024), and kidneys. In cardiac fibroblasts, TGF-β upregulates the expression of glutaminase 1 (GLS1)-the rate-limiting enzyme of glutaminolysis-through a Smad-dependent pathway, resulting in a two- to threefold increase in intracellular glutamine consumption and glutamate accumulation within 24 hours (Shan et al. 2025) (Fig. 4). Recent pivotal studies have identified that pharmacological inhibition of GLS1 with the selective inhibitor CB-839 attenuates TGF-β-induced fibroblast activation (Han et al. 2025c) and thereby suppresses the phenotypic features of cardiac fibrosis (Bai et al. 2025b). Furthermore, fibroblast GLS1 is upregulated in fibrotic mouse lungs; conversely, its genetic ablation specifically in fibroblasts protects mice from bleomycin-induced lung fibrosis (Cui et al. 2019). Beyond its role in ATP generation, glutaminolysis provides α-KG, an essential cofactor for JmjC-domain-containing histone demethylases (JMJD) (Huang et al. 2023) and TET DNA demethylases (Duvey et al. 2025), which collectively modulate the epigenetic landscape of pro-fibrotic genes to reinforce the myofibroblast phenotype (Fig. 4). 2.3. Fatty Acid Oxidation While glycolysis and glutaminolysis are markedly upregulated during cardiac fibroblast activation, fatty acid oxidation (FAO) also plays a nuanced and context-dependent role in fibrotic remodeling (Sun et al. 2025a). The adult heart normally obtains 50-70% of its ATP from fatty acid β-oxidation (Lopaschuk et al. 2010). Fatty acids enter cardiac myocytes through diffusion or specific transporters like CD36/FATP (Huang et al. 2025). Within the cytosol, fatty acid-binding proteins chaperone these fatty acids to fatty acyl CoA synthase (FACS), which activates them to fatty acyl CoA (Lopaschuk et al. 2010). This activated compound can then be directed toward two pathways: esterification into complex lipids such as triacylglycerol (TAG) (Muscella et al. 2020), or conjugation to carnitine via carnitine palmitoyltransferase 1 (CPT1) for mitochondrial import (Tang et al. 2025a). The resulting acylcarnitine is shuttled across the mitochondrial membrane, where carnitine palmitoyltransferase 2 (CPT2) converts it back to fatty acyl CoA (Guo et al. 2025). The majority mitochondrial fatty acyl CoA subsequently enters the β-oxidation cycle (Zheng et al. 2025b), yielding acetyl CoA (Wu et al. 2025), NADH, and FADH 2 (Fig. 5). CPT1 is a rate-limiting step of mitochondrial β-oxidation, responsible for transporting long-chain fatty acids into mitochondria (Pakhira and Roy 2025). Currently, no specific agonists or inhibitors targeting CPT1 or CPT2 have demonstrated proven efficacy for treating myocardial fibrosis. Restoration of FAO by overexpressing CPT1A in peritoneal dialysis (PD) mice reversed profibrotic phenotype in mesothelial cells and reduced fibrotic lesions in the peritoneum (Su et al. 2023b). Similarly, ketogenic diet attenuates unilateral ureteral obstruction-induced renal fibrosis by enhancing FAO via the free fatty acid receptor 3 (FFAR3)-dependent pathway (Qiu et al. 2023). These findings underscore the functional impact of FAO in fibrosis. 3. Mitochondrial Dysfunction in Cardiac Fibrosis Mitochondria are the most abundant organelles in mature cardiomyocytes, occupying up to one-third of the cell volume to sustain the substantial energy demands of the continuously contracting myocardium (Lin et al. 2023). 3.1. Mitochondrial ROS: A Double-Edged Sword in Fibrotic Signaling Mitochondria are a primary source of ROS (Zheng et al. 2025a). In cardiac fibroblasts, TGF-β promotes a pro-oxidant environment by upregulating NOX4 (Mondragon et al. 2025) and downregulating the antioxidant enzyme SOD2 (Rossi et al. 2018), thereby elevating mitochondrial ROS (mtROS) levels and facilitating myofibroblast activation (Siani et al. 2023). The subsequent increase in intracellular ROS activates p38 (Yang et al. 2024) and ERK1/2 (Feng et al. 2024) signaling, which in turn enhances the transcription of profibrotic genes (Zhang et al. 2025b) (Fig. 6). The critical role of oxidative stress is further demonstrated by the fact that SOD mimetics abolish the profibrotic effects of AngII, linking fibrotic signaling directly to impaired mtROS clearance (Lijnen et al. 2008; Dikalova et al. 2024). Furthermore, NOX4 knockdown or inhibition with GLX351322 alleviating fibrotic changes induced by AngII (Li et al. 2023a). Inhibition of the redox-sensitive p38 MAPK pathway with SB203580 similarly decreases the expression of α-SMA and collagens I/III, ultimately ameliorating myocardial fibrosis (Wang et al. 2025a). Additionally, myofibroblasts exhibit marked apoptosis resistance (Jin et al. 2024). This is achieved through upregulation of anti-apoptotic factors (BLC-2, BCL-XL) and concurrent downregulation of pro-apoptotic mediators (BAX, BAK), which collectively prevent mitochondrial cytochrome c release (McElhinney et al. 2024; Rehan et al. 2023; Fellner et al. 2021). The consequent reduction in caspase cascade activation reinforces myofibroblast survival, promoting their persistence in the injured heart. 3.2. Mitochondrial Dynamics: Fission vs. Fusion in Myofibroblast Persistence Mitochondrial dynamics which involves a balance between dynamin-related protein 1 (DRP1)-mediated fission (Abudureyimu et al. 2024) and fusion regulated by optic atrophy 1 (OPA1) (Noone et al. 2022) at the inner membrane and mitofusins 1/2 (MFN1/2) (Tang et al. 2025b) at the outer membrane-is essential for preserving mitochondrial integrity, bioenergetic function, and redox homeostasis (Adebayo et al. 2021; Beg et al. 2024). In activated cardiac fibroblasts, this balance shifts markedly toward mitochondrial fission (Spurlock et al. 2025), as evidenced by elevated DRP1 expression and phosphorylation at Ser616 (Hu et al. 2022), a modification that enhances its GTPase activity and promotes mitochondrial translocation (Fig. 6). This fission bias leads to mitochondrial fragmentation, accompanied by diminished oxidative phosphorylation (OXPHOS) capacity and elevated ROS production (Jiang et al. 2022). The resulting oxidative stress activates the NLRP3 inflammasome, which processes pro-IL-1β and pro-IL-18 into their active forms (Zeng et al. 2025; Ye et al. 2024). These cytokines in turn reinforce the pro-fibrotic phenotype through autocrine/paracrine signaling, upregulating TGF-β and collagen expression (Zhao et al. 2025). Pharmacological inhibition of DRP1-mediated mitochondrial fission with mdivi-1 significantly attenuated fibroblast activation, collagen production and fibrosis at infarcted border zone after myocardial infarction, improved impaired heart function (Ding et al. 2022). Conversely, OPA1 deficiency impairs mitochondrial fusion and promotes fragmentation, which in turn enhances myofibroblast differentiation and collagen production in vitro. In contrast, OPA1 overexpression counteracts these effects (Bao et al. 2025). Together, these findings underscore that dysregulated mitochondrial dynamics, particularly excessive fission, contribute significantly to myofibroblast persistence and the progression of cardiac fibrosis. 3.3. Mitophagy: Quality Control in Fibroblast Homeostasis Mitophagy, the selective autophagic clearance of damaged or dysfunctional mitochondria, is essential for preserving mitochondrial quality control and cellular homeostasis (Ajoolabady et al. 2022). When mitophagy is impaired, dysfunctional mitochondria accumulate (Wang et al. 2023), resulting in elevated ROS production and sustained myofibroblast activation due to the inability to remove organelles that drive pro-fibrotic signaling (Su et al. 2023a; Bi et al. 2024). A central regulator of canonical mitophagy is the E3 ubiquitin ligase PARK2 (Parkin) (Iorio et al. 2021). Under conditions of mitochondrial depolarization (decreased mitochondrial membrane potential), PTEN-induced kinase 1 (PINK1) stabilizes on the outer mitochondrial membrane, phosphorylates Parkin, and recruits it to damaged mitochondria (Yi et al. 2024). Parkin subsequently ubiquitinates outer membrane proteins such as MFN1/2 (Deng et al. 2024) and TOM20 (Eldeeb et al. 2024), which are then recognized by autophagosomal markers like LC3, leading to mitochondrial degradation (Yamada et al. 2025a). The approved anti-fibrotic drug pirfenidone (PFD) enhances PARK2-mediated mitophagy, which contributes to the reduction of myofibroblast differentiation (Ham et al. 2025). Besides, recent studies suggest a similar protective role for mitophagy in the heart. For instance, nuanxinkang could prevent cardiac mitochondrial dysfunction and improve cardiac function against MI-induced chronic heart failure by promoting Pink1/Parkin-mediated mitophagy (Guan et al. 2023). Gastrodin can promote mitophagy and preserve mitochondria through PINK1/Parkin and reduce myocardial ischemia-reperfusion injury (Chen et al. 2024). Together, these findings underscore mitophagy as a critical protective mechanism against fibroblast dysfunction and pathological fibrosis. 4. The Therapeutic Promise of PDK1 Inhibition in Cardiac Fibrosis 4.1 The Promise of Targeting PDK1 in Cardiac Fibrosis PDK1 is a central Ser/Thr kinase and a pivotal node within the PI3K signaling pathway. It transduces key mitogenic and metabolic signals by phosphorylating and activating AGC family kinases, most notably AKT at Threonine 308 (Thr308) (Fig. 7). Studies indicate that Akt/mTOR signaling acts as a central driver of fibrosis, with aberrant activation of this axis observed across nearly all forms of organ fibrosis-including hepatic, renal, pulmonary, and cardiac fibrosis. The AKT/mTOR axis orchestrates a profound metabolic shift towards aerobic glycolysis in activated fibroblasts (Meng et al. 2025). Furthermore, AKT promotes mitochondrial fission by phosphorylating DRP1 (Xu et al. 2025). This signaling axis alters mitochondrial metabolism, which can suppress oxidative phosphorylation and increase the generation of mtROS (Zhang et al. 2025a). The elevated ROS levels not only cause cellular damage but also act as signaling molecules that further activate pro-fibrotic pathways. While targeting the AKT-mTOR axis theoretically holds promise for treating cardiac fibrosis, its clinical translation faces several substantial challenges. First, feedback activation and compensatory pathways limit the efficacy of directly inhibiting downstream nodes. For instance, inhibiting mTORC1 with rapamycin unexpectedly enhances AKT signaling through the loss of S6K1-mediated negative feedback on IRS1 (O’Reilly et al. 2006). This compensatory response may undermine the anti-fibrotic effect and potentially promote cell survival pathways. Second, systemic metabolic toxicities represent a major clinical hurdle for such agents. Chronic administration of rapamycin substantially impairs glucose tolerance and insulin action (Lamming et al. 2012). Besides, the risk of all grade and grade 3-4, hyperglycemia, hypercholesterolemia, and hypertriglyceridemia, are increase in patients treated with mTOR inhibitors compared with control (Sivendran et al. 2014). These side effects not only compromise patient tolerability but may also exacerbate metabolic disorders during long-term therapy, particularly in cardiovascular patients with pre-existing metabolic syndrome. Third, mTOR inhibitors may also exert undesirable immune modulating effects, and ATP competitive inhibitors in particular may be largely dose-limited by toxicity (Wood and Gutkind 2022). Therefore, targeting upstream regulatory nodes like PDK1 in the AKT-mTOR cascade represents a promising therapeutic strategy. PDK1 acts as the essential kinase for AKT activation, serving as a rate-limiting step within the PI3K pathway. Theoretically, its inhibition could more proximally suppress the entire AKT-mTOR axis, potentially circumventing compensatory feedback mechanisms and providing a superior therapeutic window owing to its unique network position. As a master regulator of cellular metabolism, proliferation, and survival, PDK1 is a well-established driver of metabolic reprogramming in cancer. However, its specific function and therapeutic potential in the pathogenesis of cardiac fibrosis remain largely unexplored. In idiopathic pulmonary fibrosis, both cells and tissues exhibit enhanced PDK1 activity, and its inhibition can augment fibroblast apoptosis, identifying PDK1 as a critical node sustaining the survival of fibrotic cells (Jia et al. 2018). Similarly, in glomerular mesangial cells, Ang II activates PDK1 by inducing phosphorylation at its tyrosine residues (9 and 373/376). Introduction into the cells of PDK1, mutated on these tyrosine residues or kinase-inactive, attenuates Ang II-induced hypertrophy and fibronectin accumulation (Block et al. 2008). Within the heart, PDK1 is required for exercise-induced cardiac hypertrophy (Noh et al. 2015). However, its activation requires precise spatiotemporal control, as evidenced in pathological cardiac hypertrophy promoted by the 5-hydroxytryptamine receptor 2A (HTR2A), where inhibition of PDK1 suppresses the hypertrophic process (Gao et al. 2020). Furthermore, long noncoding RNA LNC_000898 also alleviates cardiomyocyte apoptosis and promotes cardiac repair after myocardial infarction through modulating the miR-375/PDK1 axis (Yan et al. 2020). Collectively, these findings across pulmonary, renal, and cardiac pathologies underscore PDK1’s role as a master regulatory node in fibrogenic and remodeling processes. Given its position as a critical hub governing energy metabolism, we hypothesized that PDK1 could be a novel, mechanistically grounded target for interfering with the fibrotic cascade. To evaluate this hypothesis, we elucidated its structural architecture and computationally assess its “druggability”. 4.2 Structural Architecture of PDK1 The three-dimensional structure of human PDK1 was resolved to delineate its domain organization. As shown in Fig. 8A, the overall molecular surface of the protein is presented. The two primary functional domains are highlighted: the N-terminal kinase domain (colored pink) and the C-terminal pleckstrin homology (PH) domain (colored blue). This spatial separation underlies the protein’s functional duality: the PH domain is responsible for membrane recruitment by binding to phosphatidylinositol (3,4,5)-trisphosphate (PIP3) generated by PI3K, while the kinase domain directly phosphorylates and activates downstream substrates such as AKT. Fig. 8B provides a complementary linear schematic that maps the precise amino acid boundaries of these domains (kinase domain: residues 78-359; PH domain: residues 459-550) onto the full-length sequence. 4.3 Molecular Docking Reveals High-Affinity Binding of PDK1 Inhibitors Based on the resolved structure of PDK1, molecular docking was employed to evaluate its binding potential with two classical inhibitors, BX-795 and GSK2334470. Both compounds demonstrated high-affinity binding, with calculated binding energies of -6.981 kcal/mol and -9.706 kcal/mol, respectively. As shown in Fig. 9A, BX-795 docked securely into the canonical ATP-binding pocket of the kinase domain. Its binding pose was stabilized by interactions with key residues lining the pocket, including PHE184, VAL345, THR346, and the hinge region residue GLN353-a pattern consistent with its known mechanism as an ATP-competitive inhibitor. In Fig. 9B, GSK2334470 also docked into the ATP-binding region. The predicted pose suggests potential interactions with residues such as LEU379, PHE383, and LYS173 within the catalytic cleft, alongside interactions with residues ALA471 and MET526 in the PH domain, indicating a potentially unique binding interface. Collectively, the successful docking of both inhibitors into the well-defined pocket of the PDK1 kinase domain provides computational evidence that this region is amenable to high-affinity small-molecule binding, structurally reinforcing PDK1’s relevance as a druggable therapeutic target. 5. Conclusion Cardiac fibrosis represents a complex and multifactorial pathological process, orchestrated by the activation, differentiation, and persistence of myofibroblasts, ultimately leading to compromised cardiac function and poor clinical outcomes. Recent research has established that metabolic reprogramming-marked by enhanced glycolysis glutaminolysis, and fatty acid oxidation-alongside mitochondrial dysfunction, including disrupted dynamics, defective mitophagy, and elevated ROS production, are not merely bystander effects but active drivers of the fibrotic cascade. While the downstream AKT/mTOR axis is a central driver, its direct pharmacologic inhibition faces translational challenges. Targeting its upstream activator, PDK1, presents a strategically distinct approach. As a pivotal hub that orchestrates both metabolic and mitochondrial reprogramming, PDK1 inhibition offers the potential to more broadly suppress the pro-fibrotic network while potentially avoiding limitations such as compensatory feedback activation. Our structural analyses, which confirm a well-defined, ligandable ATP-binding pocket in the PDK1 kinase domain, provide direct evidence for its druggability and solidify its promise as a novel therapeutic target for mitigating cardiac fibrotic progression. Future studies investigating PDK1 modulation in preclinical models will be crucial to validate this therapeutic hypothesis. CRediT authorship contribution statement Wenyu Hui: Writing - original draft, Conceptualization, Funding acquisition. Xiaofan Lin : Investigation, Conceptualization. Ling Yang: Investigation, Conceptualization. Yingyu Wang: Investigation, Conceptualization. Yunru Peng: Supervision, Writing - review & editing. Yongfang Ding: Supervision, Funding acquisition. Funding The work was supported by Jiangsu Provincial TCM Science and Technology Development Program Project (No. QN202512; MS2025033). Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements We acknowledge the use of BioRender.com for the preparation of graphical figures and DeepSeek for English language polishing. The authors are solely responsible for the final content and interpretations. References Abudureyimu, M., X. Luo, L. Jiang, X. Jin, C. Pan, W. Yu, J. Ge, Y. Zhang, J. Ren. 2024. FBXL4 protects against HFpEF through Drp1-Mediated regulation of mitochondrial dynamics and the downstream SERCA2a. Redox Biol 70:103081.Adebayo, M., S. Singh, A. P. Singh, S. Dasgupta. 2021. Mitochondrial fusion and fission: The fine-tune balance for cellular homeostasis. Faseb j 35 (6):e21620.Aguado-Alvaro, L. P., N. Garitano, W. 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The primary triggers are grouped as follows: (1) DNA damage, which disrupts cellular integrity and activates damage-response pathways in cardiomyocytes and fibroblasts; (2) Injury-related stimuli (e.g., ischemia, pressure overload, or myocardial infarction), serving as the foundational physical or ischemic events that trigger the fibrotic cascade; (3) Pro-fibrotic cytokines, with TGF-β being the master regulator that directly stimulates fibroblast activation and ECM production; (4) GPCR agonists, such as AngII and ET-1, which activate potent intracellular signaling cascades leading to fibroblast proliferation and fibrosis; (5) Inflammatory mediators, notably members of the interleukins family, which sustain a pro-inflammatory microenvironment conducive to fibrotic progression; (6) Oxidative stress, resulting from an imbalance between ROS production and clearance, which modifies cellular signaling and promotes a pro-fibrotic phenotype; (7) Dysregulated ECM, where abnormal ECM composition and stiffness themselves become a pathogenic signal, further driving fibroblast activation in a self-perpetuating cycle. Fig. 2. TGF-β transduces signaling through SMAD or non-SMAD signaling pathways. This schematic diagram delineates the canonical and non-canonical signaling cascades activated by TGF-β binding to its receptors TβRII/TβRI, which coordinately drive the differentiation of cardiac fibroblasts into pro-fibrotic myofibroblasts. Canonical SMAD Pathway: Upon ligand binding, TβRII phosphorylates and activates TβRI, which then phosphorylates the receptor-regulated SMADs (R-SMADs), SMAD2 and SMAD3. Phosphorylated SMAD2/3 forms a complex with the common mediator SMAD4. This complex translocates into the nucleus, where it functions as a transcription factor to directly induce the expression of key fibrosis-related genes, including those encoding collagens (e.g., COL1A1, COL3A1) and α-SMA. Notably, SMAD3 is specifically responsible for the direct transactivation of the ACTA2 gene encoding α-SMA. Non-Canonical (Non-SMAD) Pathways: TGF-β receptors also initiate several pivotal SMAD-independent signaling axes that synergize with SMAD signaling: (1) The PI3K/PDK1/AKT/mTOR axis: This pathway is critically involved in promoting cellular growth, protein synthesis (e.g., of ECM components), and the metabolic reprogramming (e.g., enhanced glycolysis) that supports the high biosynthetic demands of activated myofibroblasts; (2) The JAK/STAT axis: Contributes to inflammatory and proliferative responses; (3) MAPK pathways: Including the p38, JNK, and ERK branches, which regulate cell stress responses, apoptosis, and proliferation; (4) The RHO/ROCK axis: Primarily modulates cytoskeletal reorganization and cell contractility, essential for myofibroblast function. These interconnected pathways collectively establish a robust signaling network that ensures sustained fibroblast activation, ECM production, and tissue remodeling, culminating in cardiac fibrosis. Fig. 3. A metabolic shift toward glycolysis during myofibroblast differentiation. This schematic illustrates the fundamental metabolic reprogramming wherein quiescent cardiac fibroblasts, which primarily rely on oxidative phosphorylation for energy, transition to an activated state. Upon stimulation by TGF-β or under pathological conditions like stress and ischemia, these cells undergo a definitive shift toward aerobic glycolysis. This transition is molecularly driven by the upregulated expression and activity of key glycolytic enzymes-hexokinase 2 (HK2), phosphofructokinase-2/fructose-2,6-bisphosphatase 3 (PFKFB3), and pyruvate kinase M2 (PKM2). Mechanistically, TGF-β signaling directly promotes the transcription of these glycolytic genes, including PFKFB3, PKM2, and LDHA, through activation of the downstream PDK1/AKT/mTOR pathway. Notably, a high-glucose environment can further enhance this shift by potentiating the TGF-β/Smad pathway. Pharmacological interventions in key glycolytic nodes demonstrate therapeutic potential: PFKFB3 inhibitor 3PO attenuated cardiac myofibroblast activation, proliferation, migration, and fibrosis in mice; PKM2 activator TEPP-46 alleviated mitochondrial fission and cardiac fibrosis; and HIF-1α inhibitor YC-1 reduced α-SMA expression and hepatic fibrosis. Collectively, this glycolytic reprogramming supplies the rapid ATP and biosynthetic precursors necessary to fuel the high energetic and anabolic demands of activated myofibroblasts for extracellular matrix synthesis. Fig. 4. Glutaminolysis fuels fibrosis through metabolic and epigenetic reprogramming. This diagram delineates how the metabolic pathway of glutaminolysis is co-opted during TGF-β-induced cardiac fibroblast activation, serving dual roles in both energy metabolism and epigenetic regulation. (1) Metabolic Rewiring via GLS1 Upregulation: Upon TGF-β stimulation, the canonical Smad-dependent signaling pathway directly upregulates the expression of glutaminase 1 (GLS1), the rate-limiting enzyme that catalyzes the conversion of glutamine to glutamate. This elevation in GLS1 significantly boosts intracellular glutamine flux, channeling carbon skeletons into the tricarboxylic acid (TCA) cycle for energy (ATP) production and biosynthetic precursor supply. Pharmacological inhibition of GLS1 with the selective inhibitor CB-839 attenuates TGF-β-induced fibroblast activation and thereby suppresses the phenotypic features of cardiac fibrosis; (2) Generation of a Key Epigenetic Co-factor: Glutaminolysis proceeds to convert glutamate to α-ketoglutarate (α-KG). Beyond its metabolic role, α-KG serves as an essential cofactor for two major classes of epigenetic modifying enzymes: Jumonji C-domain-containing histone demethylases (JMJD) and Ten-eleven translocation methylcytosine dioxygenases (TET DNA demethylases); (3) Epigenetic Remodeling of Profibrotic Genes: The glutaminolysis-derived α-KG enables the activity of these enzymes, leading to: Histone demethylation (by JMJD enzymes) and DNA demethylation (by TET enzymes) at the regulatory regions of key profibrotic genes. This collective activity remodels the epigenetic landscape, resulting in a more open chromatin state that facilitates the sustained transcriptional activation of genes essential for the myofibroblast phenotype, such as those encoding α-SMA and collagens. In summary, TGF-β-induced glutaminolysis not only meets the heightened bioenergetic and anabolic demands of activated fibroblasts but also, through α-KG, directly links metabolic flux to epigenetic reprogramming, thereby cementing the persistent profibrotic cellular state. Fig. 5. The role of fatty acid oxidation in fibrotic remodeling. This diagram illustrates the complete pathway of mitochondrial fatty acid β-oxidation and its contextual relevance in the fibrotic heart. Fatty Acid Uptake and Activation: Long-chain fatty acids enter cardiac cells (including cardiomyocytes and fibroblasts) primarily via specific transporters (e.g., CD36/FATP) or by diffusion. Within the cytosol, they are activated to fatty acyl-CoA by fatty acyl-CoA synthase (FACS). Mitochondrial Import - The Rate-Limiting Step: The fate of cytosolic fatty acyl-CoA bifurcates: It can be esterified into neutral lipids (e.g., triacylglycerol, TAG) for storage. For energy production, it is conjugated to carnitine by carnitine palmitoyltransferase 1 (CPT1), located on the outer mitochondrial membrane. This step is the major rate-limiting commitment for mitochondrial β-oxidation. The resulting acylcarnitine is shuttled across the inner membrane by a translocase, then reconverted to fatty acyl-CoA by carnitine palmitoyltransferase 2 (CPT2). β-Oxidation and Energy Yield: Inside the mitochondrial matrix, the fatty acyl-CoA undergoes a series of cyclic reactions (β-oxidation), each cycle cleaving a two-carbon unit in the form of acetyl-CoA. This process also generates reducing equivalents (NADH and FADH₂). Acetyl-CoA enters the tricarboxylic acid (TCA) cycle for complete oxidation, while NADH and FADH₂ drive the electron transport chain to produce substantial ATP. Currently, no specific agonists or inhibitors targeting CPT1 or CPT2 have demonstrated proven efficacy for treating myocardial fibrosis. Fig. 6. Mitochondrial mechanisms in myofibroblast differentiation and persistence. This diagram integrates key mitochondrial alterations-encompassing ROS signaling, dynamics, and quality control-that are orchestrated by TGF-β to promote and sustain the pro-fibrotic myofibroblast phenotype. Mitochondrial ROS (mtROS) as a Signaling Nexus: TGF-β disrupts the mitochondrial redox balance, primarily by upregulating NADPH oxidase 4 (NOX4) and downregulating antioxidant defenses like superoxide dismutase 2 (SOD2). The resulting elevation in mtROS acts not merely as a damaging agent but as a critical secondary messenger. It activates multiple downstream pro-fibrotic signaling pathways, including the p38 MAPK and ERK1/2 axes, which in turn enhance the transcription of fibrosis-related genes (e.g., collagen, α-SMA). Concurrently, mtROS contributes to a state of apoptosis resistance in myofibroblasts, promoting their pathological persistence. NOX4 knockdown or inhibition with GLX351322 alleviating fibrotic changes induced by AngII. Besides, inhibition of the redox-sensitive p38 MAPK pathway with SB203580 similarly decreases the expression of α-SMA and collagens I/III, ultimately ameliorating myocardial fibrosis. Imbalance in Mitochondrial Dynamics (Fission/Fusion): TGF-β signaling induces a profound shift in mitochondrial dynamics toward excessive fission. This is characterized by increased expression and activity of the pro-fission protein dynamin-related protein 1 (DRP1), notably through its phosphorylation at Ser616, which promotes its translocation to mitochondria. The consequent mitochondrial fragmentation leads to dysfunctional, punctate mitochondria with impaired oxidative phosphorylation (OXPHOS) capacity. This metabolic inefficiency further exacerbates ROS production, creating a vicious cycle. Pharmacological inhibition of DRP1-mediated mitochondrial fission with mdivi-1 significantly attenuated fibroblast activation, collagen production and fibrosis at infarcted border zone after myocardial infarction, improved impaired heart function. Fig. 7. PDK1: a potential therapeutic target in fibrosis. This schematic illustrates the pivotal role of PDK1 in integrating upstream signals to activate a major pro-fibrotic pathway. Following its recruitment to the plasma membrane via the binding of its PH domain to PIP3, PDK1 phosphorylates AKT at the critical Thr308 residue. This step is essential for the full activation of AKT. The activated AKT subsequently drives a profound anabolic program, most notably by activating the mammalian target of rapamycin complex 1 (mTORC1), which strongly promotes protein synthesis and cellular growth-processes fundamental to myofibroblast activation and excessive extracellular matrix production in fibrosis. Therefore, pharmacological inhibition of PDK1 presents a promising upstream therapeutic strategy aimed at disrupting the PDK1/AKT/mTOR axis, offering a novel mechanistic approach to mitigate cardiac fibrotic remodeling. Fig. 8. Structural architecture of human PDK1. A. Molecular surface representation. The three-dimensional structure of full-length human PDK1 illustrates the overall topology of the protein. This representation highlights the compact, globular nature of the kinase domain (colored in pink) and the distinct structural lobe of the pleckstrin homology (PH) domain (colored in blue). B. Linear domain architecture. The schematic delineates the primary sequence organization of PDK1, pinpointing the location and extent of its two critical functional modules: Kinase domain (78-359): this N-terminal catalytic domain contains the ATP-binding pocket and is responsible for phosphorylating downstream substrates, most notably AKT at Thr308. Its structural integrity is essential for propagating the PDK1/AKT/mTOR signaling axis; PH domain (459-550): this C-terminal domain specifically binds to phosphatidylinositol (3,4,5)-trisphosphate (PIP3) generated by PI3K activity. This binding recruits PDK1 to the plasma membrane, facilitating its access to membrane-localized substrates and positioning it as a key node in growth factor signaling. The defined segregation of the catalytic kinase domain and the membrane-targeting PH domain within a single polypeptide enables PDK1 to act as a crucial signaling hub, integrating upstream lipid second messenger signals with downstream metabolic and growth pathways. Fig. 9. Molecular docking of classical inhibitors reveals distinct binding modes within the PDK1 kinase domain. A. Docking pose of BX-795. The model predicts that BX-795 binds snugly within the canonical ATP-binding pocket of the PDK1 kinase domain, with a calculated binding energy of -6.981 kcal/mol. The inhibitor forms stabilizing interactions with key residues lining the pocket, including PHE184, VAL345, THR346, and the hinge region residue GLN353. This binding pose is consistent with its pharmacological characterization as an ATP-competitive inhibitor and validates the kinase domain as a viable site for direct pharmacological intervention. B. Docking pose of GSK2334470. GSK2334470 also exhibits high-affinity binding to PDK1, with a calculated binding energy of -9.706 kcal/mol. The predicted docking pose including LEU379 and PHE383 within the kinase domain, and LYS169, ARG172, LYS173 in the N-lobe, as well as ALA471 and MET526 which are located in the C-terminal PH domain. The successful in silico docking of both classical inhibitors provides computational evidence that the PDK1 kinase domain possesses a well-defined and ligandable binding cavity. 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Authors Affiliations Wenyu Hui Nanjing Integrated Chinese and Western Medicine Hospital Affiliated to Nanjing University of Chinese Medicine View all articles by this author Xiaofan Lin Nanjing Integrated Chinese and Western Medicine Hospital Affiliated to Nanjing University of Chinese Medicine View all articles by this author Ling Yang Nanjing Integrated Chinese and Western Medicine Hospital Affiliated to Nanjing University of Chinese Medicine View all articles by this author Yingyu Wang Nanjing Integrated Chinese and Western Medicine Hospital Affiliated to Nanjing University of Chinese Medicine View all articles by this author Yunru Peng Nanjing Integrated Chinese and Western Medicine Hospital Affiliated to Nanjing University of Chinese Medicine View all articles by this author Yongfang Ding [email protected] Nanjing Integrated Chinese and Western Medicine Hospital Affiliated to Nanjing University of Chinese Medicine View all articles by this author Metrics & Citations Metrics Article Usage 126 views 59 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Wenyu Hui, Xiaofan Lin, Ling Yang, et al. Metabolic Reprogramming in Cardiac Fibrosis and Its Implications for Targeting PDK1. Authorea . 12 February 2026. DOI: https://doi.org/10.22541/au.177090156.69909573/v1 If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Simply select your manager software from the list below and click Download. For more information or tips please see 'Downloading to a citation manager' in the Help menu . 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