The crosstalk between autophagy and other signaling pathways plays a crucial role in the modulation of cardiac differentiation. Zhang et al. were the first to recognize the connection between autophagy and cardiac stem-cell differentiation, finding that fibroblast growth factor (FGF) regulates cardiac development through autophagy ^[1][8]. Their study revealed that the inhibition of FGF signaling led to an upsurge in the initiation of autophagy, thereby promoting the differentiation of cardiomyocytes. These findings strongly suggest that autophagy serves as a positive regulator of both cardiac progenitor cell differentiation and cardiomyocyte maturation.

FGF signaling exhibits a biphasic function in cardiogenesis. During the early stages, FGF signaling promotes mesoderm formation and commitment to the cardiac lineage. However, in later stages, FGF signaling inhibits premature differentiation to ensure proper cardiac development and maturation. The involvement of autophagy in mediating FGF signaling during the differentiation of heart progenitor cells highlights the significance of autophagy in cardiac development. The interplay between autophagy and FGF signaling provides novel insights into the regulatory mechanisms underlying cardiac differentiation.

Additionally, other signaling pathways, such as Wnt, Notch, and bone morphogenetic proteins (BMPs), are also involved in regulating heart development ^[2][23]. Wnt and Notch signaling pathways are important in heart development, and there is evidence of a crosstalk between the two ^[3][24]. The main components of the Wnt signaling pathway include dishevelled (Dvl), AXIN, glycogen synthase kinase-3 (GSK3)-β, and β-catenin. It has been shown that Wnt signaling plays a biphasic function in cardiogenesis. This pathway stimulates cardiomyocyte differentiation in the early stages of heart development but suppresses cardiogenesis later on ^[4][25]. Autophagy can be negatively regulated by Wnt signaling by promoting Dvl degradation through the autophagy–lysosome pathway, which, in turn, induces a concomitant decrease in the nuclear accumulation of β-catenin ^[5][26]. Furthermore, it has been demonstrated that autophagy induction resulted in a considerable drop in the gene expression of Axin2, Cyclin-D1, and c-Myc, as well as a decrease in the protein expression of β-catenin and Dvl2. Based on these findings, it was determined that autophagy has a negative regulatory effect on the Wnt signaling pathway ^[6][1].

Notch signaling, a pivotal signaling pathway in cardiac differentiation, demonstrates intricate dynamics and complexity throughout the process of cardiac development. Initially, it is believed that Notch signaling suppresses cardiac differentiation to maintain a pool of undifferentiated progenitor cells. However, different studies show that Notch1 is required for cardiac progenitor cells to differentiate into cardiomyocytes by positively regulating the expression of cardiac transcription factors in mouse embryos and embryonic stem (ES) cells ^[7][8][9][32,33,34]. In the context of cardiac development, autophagy has been investigated in relation to the Wnt and Notch signaling pathways ^[10][11][35,36]. Zhuqing Jia et al. studied the involvement of autophagy in the cardiac development of P19CL6 cells, a well-established in vitro cardiomyocyte differentiation system ^[12][37]. Their findings demonstrated that autophagy is initiated during the early stages of cardiomyocyte differentiation and remains active throughout the final stages. The researchers also observed that β-catenin and Notch intracellular domain (NICD), which are effectors of the Wnt and Notch pathways, can form a complex with LC3 and P62, leading to their elimination through autophagy. Interestingly, β-catenin not only inhibits the formation of autophagosomes but also directly reduces the expression of the autophagy adaptor p62 ^[11][36].

Moreover, epigenetic factors, such as histone deacetylases (HDACs), have also been shown to play a role in regulating autophagy during cardiac differentiation ^{[13][14][15][16][17]}[27,28,29,30,31]. Specifically, studies have demonstrated that HDACs control the differentiation of P19 embryonic cancer cells into cardiac lineage cells ^[18][39]. Despite the advances in understanding the roles of autophagy and epigenetic regulation in cardiac differentiation, the exact mechanisms underlying their interaction in this process remain incompletely understood. Further investigations are required to elucidate the intricate relationship between autophagy, Wnt signaling, and cardiac differentiation, which will provide valuable insights into the development and potential therapeutic strategies for cardiac-related conditions.

The mammalian target of rapamycin (mTOR) is a crucial serine/threonine kinase involved in cellular survival and growth. It operates through two distinct complexes, mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2), which are predominantly made up of Raptor and Rictor as their core components, respectively. Reports from different laboratories have demonstrated that mTOR complex 1 acts as a master regulator of autophagic processes since inhibition of mTORC1 was shown to be required to start autophagy. Subsequent studies have also demonstrated that mTORC1 directly regulates the subsequent phases, such as autophagosomes formation, maturation, and autophagy termination ^[19][40]. Raptor/mTORC1 and Rictor/mTORC2 play distinct and significant roles in the differentiation of cardiomyocytes derived from mouse embryonic stem cells (mESCs). Numerous molecular elements and phosphorylation processes mediate the intricate interaction between these two signaling pathways ^[20][41].

AKT, also known as protein kinase B, is a key protein and acts as a bridge between the two complexes. AKT can be phosphorylated by mTORC2 or 3-phosphoinositide-dependent kinase 1 (PDK1), thereby regulating downstream signaling ^[21][42]. Specifically, Raptor knockdown reduced the phosphorylation of Rictor at Thr1135 by p70S6K, leading to mTORC2 activation ^[20][41].

Raptor/mTORC1 primarily regulates metabolism, cell growth, and protein synthesis in response to nutrient availability, and growth factors ^[22][43]. It controls the phosphorylation of S6 kinase 1 (S6K1) and eukaryotic translation initiation factor 4E-binding protein (eIF4E), thereby promoting translation and protein synthesis ^[23][44]. In the context of cardiomyocyte differentiation, Raptor knockdown enhances the process by activating Rictor/mTORC2 signaling, which, in turn, facilitates the differentiation of mESCs into cardiomyocytes. This is accompanied by elevated expression levels of brachyury (a mesoderm protein), Nkx2.5 (a cardiac progenitor cell protein), and α-actinin (a cardiomyocyte marker) in Raptor knockdown cells ^[20][41].

On the other hand, Rictor/mTORC2 exerts its impact on cytoskeletal organization, cell survival, and cell polarization. It has been implicated in the regulation of cellular processes such as cell adhesion, migration, and differentiation ^[24][45]. Rictor knockout in cardiac-specific cells leads to cardiac dysfunction and impaired adaptation to pressure overload. In the context of mESCs differentiation into cardiomyocytes, Rictor knockdown suppresses cardiogenesis, indicating the critical role of Rictor/mTORC2 in this process ^[20][41].

Upstream of mTOR, various signals, including insulin signaling through phosphatidylinositol-3-kinase (PI3K) and AKT, and energy/stress response signaling via AMP-activated protein kinase (AMPK), converge to regulate the activity of the tuberous sclerosis complexes (TSC1-TSC2), which act as GTPase-activating proteins for Rheb. When Rheb is in its GTP-bound state, it directly activates mTOR. The convergence of these signals ensures precise control over mTOR activation and downstream cellular processes ^[23][44].

The regulation of cardiomyocyte differentiation can be better understood by gaining valuable insights from the complex relationship between these signaling pathways, which may also have implications for potential therapeutic applications using induced pluripotent stem cells.

Aside from the main autophagy pathway, other types of autophagy, such as mitophagy, play a crucial role in cardiac differentiation. Mitophagy, a form of autophagy, is a vital process in cardiac differentiation. It helps in the removal of dysfunctional mitochondria and facilitates metabolic remodeling, which is crucial for successful differentiation. Regulated mitophagy plays a critical role in maintaining the cell’s glycolytic status and is believed to contribute to the metabolic remodeling necessary for successful differentiation ^{[25][26][27][28]}[46,47,48,49].

A specific process of mitophagy is initiated by the PINK1/Parkin system, where Parkin, an E3 ubiquitin ligase, is recruited to dysfunctional mitochondria. Parkin ubiquitinates outer mitochondrial proteins, labeling them for proteasome degradation ^[29][50]. In mice, the deletion of Parkin during early postnatal stages impedes cardiomyocyte maturation, affects mitochondrial biogenesis, and disrupts fatty acid oxidation, highlighting the importance of PINK1/Parkin-mediated mitophagy in cardiac development ^[25][46]. These findings highlight the important role of PINK1/Parkin-mediated mitophagy in promoting proper cardiac development.

The regulation of mitophagy in cardiac differentiation is influenced by nutrient-sensing pathways, such as Akt/mTOR, which is upregulated in high-nutrient states, and AMPK, which is upregulated during fasting. Inhibiting mTOR signaling increases mitophagy, providing cardioprotection after myocardial infarction. Conversely, AMPKα2 phosphorylates PINK1, enhancing mitophagy and protecting against pressure overload in the heart. AMPK also influences the opening of the mitochondrial permeability transition pore (mPTP), which regulates mitochondrial depolarization and subsequent mitophagy ^[30][31][18,51]. Increasing Parkin-mediated mitophagy through enhanced fatty acid oxidation, achieved by deleting acetyl coenzyme A carboxylase 2 (ACC2), prevents myocardial dysfunction resulting from a high-fat diet ^[32][52]. These interconnected mechanisms underscore the significance of metabolic shifts, mitochondrial quality control, and mitophagy in maintaining proper cardiac function.

Recent advances in stem-cell research have utilized embryoid body suspension and monolayer attachment methods to increase differentiation efficiency, but further improvements are required to optimize this process ^[33][34][35][22,57,58]. Adding growth factors such as BMP2, BMP4, FGFs, activin A, b-fibroblast growth factor (bFGF), FGF2, FGF10, and Wnt family member 3A (Wnt3a) during cardiac differentiation can improve induction efficiency, but the high cost of these reagents limits their practicality ^[6][36][37][1,59,60]. Thus, the use of small chemical cocktails that target the autophagy pathway is a promising alternative. However, in order to boost differentiation efficacy even further, the induction strategy that improves differentiation efficiency, as well as the detailed mechanisms, must be defined.

Methods that have been developed to manipulate the autophagy pathway to promote cardiac differentiation are discussed below. Rapamycin is an inhibitor of mTOR that has been found to promote cardiomyocyte differentiation in a stage-dependent manner. It works similarly to Ku0063794, another mTOR kinase inhibitor ^[6][1]. While rapamycin inhibits mTORC1 activity, its effect on mTORC2 is limited ^[38][61]. Nevertheless, rapamycin has been found to induce autophagy, upregulate Fgf8 and Nodal expression, alleviate stressors during cardiac differentiation, reduce hPSC apoptosis, promote BMP signaling, and suppress the activation of Wnt/β-catenin and Notch signaling during cardiac induction ^{[33][39][40][41][42]}[21,22,38,62,63]. The efficiency of cardiac differentiation can be affected by different rapamycin concentrations and the presence of CHIR99021, a small molecule inhibitor that targets the GSK-3β enzyme. In addition to reducing hPSC apoptosis and boosting hESC survival, rapamycin has been shown to have other benefits in cardiac differentiation. It has been found to reduce medium usage by around 50% and overcome variability in differentiation efficiency among different hESC lines and repetitions, resulting in a greater efficiency, ranging from 87% to 99.06% ^[33][22].

Moreover, combining rapamycin and ascorbic acid has also been suggested as an effective regimen for cardiac induction. This combination has been found to increase collagen IV synthesis as well as a number of molecular signals triggered by collagen, such as the extracellular signal-regulated kinase (ERK), c- JNKs, and STAT1/3 pathways, which promote cardiac differentiation through various molecular signaling pathways ^[43][66]. These findings provide insight into the diverse and complex mechanisms underlying the regulation of cellular differentiation and suggest the potential therapeutic applications of rapamycin in promoting cardiac regeneration.

AMPK is an energy-sensing protein kinase involved in the regulation of autophagy. In addition to its direct role in regulating autophagy, AMPK can also influence other cellular processes, such as mitochondrial function, posttranslational acetylation, cardiomyocyte metabolism, mitochondrial autophagy, endoplasmic reticulum stress, and apoptosis. As AMPK is involved in the control of various cellular processes, it can influence the health and survival of cardiomyocytes ^[44][45][20,67]. AMPK activation increases fatty acid oxidation in human induced pluripotent stem cell (hiPSC)-cardiomyocytes (CMs) by upregulating the expression of carnitine palmitoyl transferase (CPT)-1, fatty acid transport protein (FATP), and fatty acid-binding protein (FABP) ^[44][46][20,68]. Moreover, AMPK has a distinct regulatory impact on numerous areas of mitochondrial biology and homeostasis, such as controlling the number of mitochondria via stimulating mitochondrial synthesis, regulating the architecture of the mitochondrial network, and controlling the quality of mitochondria via autophagy and mitosis ^[47][69].

Understanding the significance of AMPK in cardiomyocyte differentiation, Ye et al. employed 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR) to induce AMPK activation in hiPSC-CMs ^[48][19]. AICAR dramatically raised the oxygen consumption rate (OCR) related to baseline respiration, adenosine 5′-triphosphate (ATP) generation, maximum respiration, and reserve capacity, indicating that cardiomyocytes were in a more mature metabolic state. Furthermore, AMPK activation enhances mitochondrial fusion in hiPSC-CMs ^[48][19]. In times of cellular stress, mitochondrial respiratory reserve capacity is employed to sustain an increase in energy demand, assisting in the maintenance of cell and organ functions, cell repair, or active drug detoxification ^[49][70]. AICAR treatment boosted respiratory reserve capacity in hiPSC-CMs, implying that the treated cells may perform better in the face of increasing energy demand. In addition, AICAR-treated hiPSC-CMs exhibited a larger cell perimeter, a lower circularity index, and longer sarcomeres than the control group, making them more similar to adult cardiomyocytes ^[48][19]. AICAR also increased the expression of cardiac maturation-related genes. Although the AICAR activation of AMPK promotes the morphological and metabolic maturity of hiPSC-CMs, the effect of AMPK on the electrophysiological maturation of hiPSC-CMs remains unknown ^[48][19].

Mitophagy, the selective removal of damaged or depolarized mitochondria, plays a crucial role in maintaining mitochondrial health as a quality control mechanism ^[50][74]. During stem cell differentiation, changes in metabolism occur to increase oxidative phosphorylation and mitochondrial ATP production ^[51][75]. This metabolic shift has been shown to drive cell differentiation, implying a positive feedback loop between differentiation and metabolic rewiring ^[52][76].

The involvement of mitochondrial biogenesis and the β-catenin pathway in the differentiation of endothelial progenitor cells is supported by scientific evidence. An increase in mitochondrial biogenesis, associated with the upregulation of PGC-1α, has been observed during differentiation towards the endothelial progenitor cell stage ^[53][54][65,77]. PGAM5, a mitochondrial phosphatase released during mitophagy, activates the β-catenin pathway by dephosphorylating β-catenin, allowing it to translocate to the nucleus and transcribe Wnt/β-catenin pathway genes and promoting mitophagy and mitochondrial biogenesis ^[55][56][78,79]. This interplay between PGAM5 and β-catenin is crucial for the differentiation of hiPSCs into endothelial cells ^[53][65].

While the downstream impact of mitophagy on iPSC differentiation has been studied, the upstream initiators of mitophagy during cell differentiation remain unknown. Krantz et al. investigated the role of mitophagy in iPSC differentiation and the initiation of compensatory mitochondrial biogenesis via the PGAM5 pathway but did not identify the upstream initiators of mitophagy ^[53][65]. In the review article authored by Garbern in 2021, a comprehensive examination of the role of mitophagy in cardiomyocyte differentiation is presented ^[30][18].