Autophagy in major human diseases.

Autophagy is a core molecular pathway for the preservation of cellular and organismal homeostasis. Pharmacological and genetic interventions impairing autophagy responses promote or aggravate disease in a plethora of experimental models. Consistently, mutations in autophagy-related processes cause severe human pathologies. Here, we review and discuss preclinical data linking autophagy dysfunction to the pathogenesis of major human disorders including cancer as well as cardiovascular, neurodegenerative, metabolic, pulmonary, renal, infectious, musculoskeletal, and ocular disorders.

Distinct Roles of DRP1 in Conventional and Alternative Mitophagy in Obesity Cardiomyopathy.

BACKGROUND: Obesity induces cardiomyopathy characterized by hypertrophy and diastolic dysfunction. Whereas mitophagy mediated through an Atg7 (autophagy related 7)-dependent mechanism serves as an essential mechanism to maintain mitochondrial quality during the initial development of obesity cardiomyopathy, Rab9 (Ras-related protein Rab-9A)-dependent alternative mitophagy takes over the role during the chronic phase. Although it has been postulated that DRP1 (dynamin-related protein 1)-mediated mitochondrial fission and consequent separation of the damaged portions of mitochondria are essential for mitophagy, the involvement of DRP1 in mitophagy remains controversial. We investigated whether endogenous DRP1 is essential in mediating the 2 forms of mitophagy during high-fat diet (HFD)-induced obesity cardiomyopathy and, if so, what the underlying mechanisms are. METHODS: Mice were fed either a normal diet or an HFD (60 kcal %fat). Mitophagy was evaluated using cardiac-specific Mito-Keima mice. The role of DRP1 was evaluated using tamoxifen-inducible cardiac-specific Drp1knockout (Drp1 MCM) mice. RESULTS: Mitophagy was increased after 3 weeks of HFD consumption. The induction of mitophagy by HFD consumption was completely abolished in Drp1 MCM mouse hearts, in which both diastolic and systolic dysfunction were exacerbated. The increase in LC3 (microtubule-associated protein 1 light chain 3)-dependent general autophagy and colocalization between LC3 and mitochondrial proteins was abolished in Drp1 MCM mice. Activation of alternative mitophagy was also completely abolished in Drp1 MCM mice during the chronic phase of HFD consumption. DRP1 was phosphorylated at Ser616, localized at the mitochondria-associated membranes, and associated with Rab9 and Fis1 (fission protein 1) only during the chronic, but not acute, phase of HFD consumption. CONCLUSIONS: DRP1 is an essential factor in mitochondrial quality control during obesity cardiomyopathy that controls multiple forms of mitophagy. Although DRP1 regulates conventional mitophagy through a mitochondria-associated membrane-independent mechanism during the acute phase, it acts as a component of the mitophagy machinery at the mitochondria-associated membranes in alternative mitophagy during the chronic phase of HFD consumption.

New intranasal and injectable gene therapy for healthy life extension.

As the global elderly population grows, it is socioeconomically and medically critical to provide diverse and effective means of mitigating the impact of aging on human health. Previous studies showed that the adeno-associated virus (AAV) vector induced overexpression of certain proteins, which can suppress or reverse the effects of aging in animal models. In our study, we sought to determine whether the high-capacity cytomegalovirus vector (CMV) can be an effective and safe gene delivery method for two such protective factors: telomerase reverse transcriptase (TERT) and follistatin (FST). We found that the mouse cytomegalovirus (MCMV) carrying exogenous TERT or FST (MCMVTERT or MCMVFST) extended median lifespan by 41.4% and 32.5%, respectively. We report CMV being used successfully as both an intranasal and injectable gene therapy system to extend longevity. Specifically, this treatment significantly improved glucose tolerance, physical performance, as well as preventing body mass loss and alopecia. Further, telomere shortening associated with aging was ameliorated by TERT and mitochondrial structure deterioration was halted in both treatments. Intranasal and injectable preparations performed equally well in safely and efficiently delivering gene therapy to multiple organs, with long-lasting benefits and without carcinogenicity or unwanted side effects. Translating this research to humans could have significant benefits associated with quality of life and an increased health span.

ACBP/DBI protein neutralization confers autophagy-dependent organ protection through inhibition of cell loss, inflammation, and fibrosis.

Acyl-coenzyme A (CoA)-binding protein (ACBP), also known as diazepam-binding inhibitor (DBI), is an extracellular feedback regulator of autophagy. Here, we report that injection of a monoclonal antibody neutralizing ACBP/DBI (α-DBI) protects the murine liver against ischemia/reperfusion damage, intoxication by acetaminophen and concanavalin A, and nonalcoholic steatohepatitis caused by methionine/choline-deficient diet as well as against liver fibrosis induced by bile duct ligation or carbon tetrachloride. α-DBI downregulated proinflammatory and profibrotic genes and upregulated antioxidant defenses and fatty acid oxidation in the liver. The hepatoprotective effects of α-DBI were mimicked by the induction of ACBP/DBI-specific autoantibodies, an inducible Acbp/Dbi knockout or a constitutive Gabrg2F77I mutation that abolishes ACBP/DBI binding to the GABAA receptor. Liver-protective α-DBI effects were lost when autophagy was pharmacologically blocked or genetically inhibited by knockout of Atg4b. Of note, α-DBI also reduced myocardium infarction and lung fibrosis, supporting the contention that it mediates broad organ-protective effects against multiple insults.

Metabolic control of mitophagy.

Mitochondrial dysfunction is a major hallmark of ageing and related chronic disorders. Controlled removal of damaged mitochondria by the autophagic machinery, a process known as mitophagy, is vital for mitochondrial homeostasis and cell survival. The central role of mitochondria in cellular metabolism places mitochondrial removal at the interface of key metabolic pathways affecting the biosynthesis or catabolism of acetyl-coenzyme A, nicotinamide adenine dinucleotide, polyamines, as well as fatty acids and amino acids. Molecular switches that integrate the metabolic status of the cell, like AMP-dependent protein kinase, protein kinase A, mechanistic target of rapamycin and sirtuins, have also emerged as important regulators of mitophagy. In this review, we discuss how metabolic regulation intersects with mitophagy. We place special emphasis on the metabolic regulatory circuits that may be therapeutically targeted to delay ageing and mitochondria-associated chronic diseases. Moreover, we identify outstanding knowledge gaps, such as the ill-defined distinction between basal and damage-induced mitophagy, which must be resolved to boost progress in this area.

Decoding the Impact of the Hippo Pathway on Different Cell Types in Heart Failure.

The evolutionarily conserved Hippo pathway plays a pivotal role in governing a variety of biological processes. Heart failure (HF) is a major global health problem with a significant risk of mortality. This review provides a contemporary understanding of the Hippo pathway in regulating different cell types during HF. Through a systematic analysis of each component's regulatory mechanisms within the Hippo pathway, we elucidate their specific effects on cardiomyocytes, fibroblasts, endothelial cells, and macrophages in response to various cardiac injuries. Insights gleaned from both in vitro and in vivo studies highlight the therapeutic promise of targeting the Hippo pathway to address cardiovascular diseases, particularly HF.

Infiltrating macrophages amplify doxorubicin-induced cardiac damage: role of catecholamines.

BACKGROUND: The functional contribution of non-myocyte cardiac cells, such as inflammatory cells, in the setup of heart failure in response to doxorubicin (Dox) is recently becoming of growing interest. OBJECTIVES: The study aims to evaluate the role of macrophages in cardiac damage elicited by Dox treatment. METHODS: C57BL/6 mice were treated with one intraperitoneal injection of Dox (20 mg/kg) and followed up for 5 days by cardiac ultrasounds (CUS), histological, and flow cytometry evaluations. We also tested the impact of Dox in macrophage-depleted mice. Rat cardiomyoblasts were directly treated with Dox (D-Dox) or with a conditioned medium from cultured murine macrophages treated with Dox (M-Dox). RESULTS: In response to Dox, macrophage infiltration preceded cardiac damage. Macrophage depletion prevents Dox-induced damage, suggesting a key role of these cells in promoting cardiotoxicity. To evaluate the crosstalk between macrophages and cardiac cells in response to DOX, we compared the effects of D-Dox and M-Dox in vitro. Cell vitality was lower in cardiomyoblasts and apoptosis was higher in response to M-Dox compared with D-Dox. These events were linked to p53-induced mitochondria morphology, function, and autophagy alterations. We identify a mechanistic role of catecholamines released by Dox-activated macrophages that lead to mitochondrial apoptosis of cardiac cells through ß-AR stimulation. CONCLUSIONS: Our data indicate that crosstalk between macrophages and cardiac cells participates in cardiac damage in response to Dox.

MST1 mediates doxorubicin-induced cardiomyopathy by SIRT3 downregulation.

Heart failure is a major side effect of doxorubicin (DOX) treatment in patients with cancer. However, the mechanisms underlying the development of DOX-induced heart failure need to be addressed. This study aims to test whether the serine/threonine kinase MST1, a major Hippo pathway component, contributes to the development of DOX-induced myocardial injury. C57BL/6J WT mice and mice with cardiomyocyte-specific dominant-negative MST1 (kinase-dead) overexpression received three weekly injections of DOX, reaching a final cumulative dose of 18 mg/kg. Echocardiographic, histological and biochemical analyses were performed six weeks after the first DOX administration. The effects of MST1 inhibition on DOX-induced cardiomyocyte injury were also tested in vitro. MST1 signaling was significantly activated in cardiomyocytes in response to DOX treatment in vitro and in vivo. Wild-type (WT) mice treated with DOX developed cardiac dysfunction and mitochondrial abnormalities. However, these detrimental effects were abolished in mice with cardiomyocyte-specific overexpression of dominant-negative MST1 (DN-MST1) or treated with XMU-MP-1, a specific MST1 inhibitor, indicating that MST1 inhibition attenuates DOX-induced cardiac dysfunction. DOX treatment led to a significant downregulation of cardiac levels of SIRT3, a deacetylase involved in mitochondrial protection, in WT mice, which was rescued by MST1 inhibition. Pharmacological inhibition of SIRT3 blunted the protective effects of MST1 inhibition, indicating that SIRT3 downregulation mediates the cytotoxic effects of MST1 activation in response to DOX treatment. Finally, we found a significant upregulation of MST1 and downregulation of SIRT3 levels in human myocardial tissue of cancer patients treated with DOX. In summary, MST1 contributes to DOX-induced cardiomyopathy through SIRT3 downregulation.

Suppression of myeloid YAP antagonizes adverse cardiac remodeling during pressure overload stress.

Inflammation is an integral component of cardiovascular disease and is thought to contribute to cardiac dysfunction and heart failure. While ischemia-induced inflammation has been extensively studied in the heart, relatively less is known regarding cardiac inflammation during non-ischemic stress. Recent work has implicated a role for Yes-associated protein (YAP) in modulating inflammation in response to ischemic injury; however, whether YAP influences inflammation in the heart during non-ischemic stress is not described. We hypothesized that YAP mediates a pro-inflammatory response during pressure overload (PO)-induced non-ischemic injury, and that targeted YAP inhibition in the myeloid compartment is cardioprotective. In mice, PO elicited myeloid YAP activation, and myeloid-specific YAP knockout mice (YAPF/F;LysMCre) subjected to PO stress had better systolic function, and attenuated pathological remodeling compared to control mice. Inflammatory indicators were also significantly attenuated, while pro-resolving genes including Vegfa were enhanced, in the myocardium, and in isolated macrophages, of myeloid YAP KO mice after PO. Experiments using bone marrow-derived macrophages (BMDMs) from YAP KO and control mice demonstrated that YAP suppression shifted polarization toward a resolving phenotype. We also observed attenuated NLRP3 inflammasome priming and function in YAP deficient BMDMs, as well as in myeloid YAP KO hearts following PO, indicating disruption of inflammasome induction. Finally, we leveraged nanoparticle-mediated delivery of the YAP inhibitor verteporfin and observed attenuated PO-induced pathological remodeling compared to DMSO nanoparticle control treatment. These data implicate myeloid YAP as an important molecular nodal point that facilitates cardiac inflammation and fibrosis during PO stress and suggest that selective inhibition of YAP may prove a novel therapeutic target in non-ischemic heart disease.

Mitophagy for cardioprotection.

Mitochondrial function is maintained by several strictly coordinated mechanisms, collectively termed mitochondrial quality control mechanisms, including fusion and fission, degradation, and biogenesis. As the primary source of energy in cardiomyocytes, mitochondria are the central organelle for maintaining cardiac function. Since adult cardiomyocytes in humans rarely divide, the number of dysfunctional mitochondria cannot easily be diluted through cell division. Thus, efficient degradation of dysfunctional mitochondria is crucial to maintaining cellular function. Mitophagy, a mitochondria specific form of autophagy, is a major mechanism by which damaged or unnecessary mitochondria are targeted and eliminated. Mitophagy is active in cardiomyocytes at baseline and in response to stress, and plays an essential role in maintaining the quality of mitochondria in cardiomyocytes. Mitophagy is mediated through multiple mechanisms in the heart, and each of these mechanisms can partially compensate for the loss of another mechanism. However, insufficient levels of mitophagy eventually lead to mitochondrial dysfunction and the development of heart failure. In this review, we discuss the molecular mechanisms of mitophagy in the heart and the role of mitophagy in cardiac pathophysiology, with the focus on recent findings in the field.

Alternative Mitophagy Protects the Heart Against Obesity-Associated Cardiomyopathy.

RATIONALE: Obesity-associated cardiomyopathy characterized by hypertrophy and mitochondrial dysfunction. Mitochondrial quality control mechanisms, including mitophagy, are essential for the maintenance of cardiac function in obesity-associated cardiomyopathy. However, autophagic flux peaks at around 6 weeks of high-fat diet (HFD) consumption and declines thereafter. OBJECTIVE: We investigated whether mitophagy is activated during the chronic phase of cardiomyopathy associated with obesity (obesity cardiomyopathy) after general autophagy is downregulated and, if so, what the underlying mechanism and the functional significance are. METHODS AND RESULTS: Mice were fed either a normal diet or a HFD (60 kcal% fat). Mitophagy, evaluated using Mito-Keima, was increased after 3 weeks of HFD consumption and continued to increase after conventional mechanisms of autophagy were inactivated, at least until 24 weeks. HFD consumption time-dependently upregulated both Ser555-phosphorylated Ulk1 (unc-51 like kinase 1) and Rab9 (Ras-related protein Rab-9) in the mitochondrial fraction. Mitochondria were sequestrated by Rab9-positive ring-like structures in cardiomyocytes isolated from mice after 20 weeks of HFD consumption, consistent with the activation of alternative mitophagy. Increases in mitophagy induced by HFD consumption for 20 weeks were abolished in cardiac-specific ulk1 knockout mouse hearts, in which both diastolic and systolic dysfunction were exacerbated. Rab9 S179A knock-in mice, in which alternative mitophagy is selectively suppressed, exhibited impaired mitophagy and more severe cardiac dysfunction than control mice following HFD consumption for 20 weeks. Overexpression of Rab9 in the heart increased mitophagy and protected against cardiac dysfunction during HFD consumption. HFD-induced activation of Rab9-dependent mitophagy was accompanied by upregulation of TFE3 (transcription factor binding to IGHM enhancer 3), which plays an essential role in transcriptional activation of mitophagy. CONCLUSIONS: Ulk1-Rab9-dependent alternative mitophagy is activated during the chronic phase of HFD consumption and serves as an essential mitochondrial quality control mechanism, thereby protecting the heart against obesity cardiomyopathy.

KLF5 Is Induced by FOXO1 and Causes Oxidative Stress and Diabetic Cardiomyopathy.

RATIONALE: Diabetic cardiomyopathy (DbCM) is a major complication in type-1 diabetes, accompanied by altered cardiac energetics, impaired mitochondrial function, and oxidative stress. Previous studies indicate that type-1 diabetes is associated with increased cardiac expression of KLF5 (Krüppel-like factor-5) and PPARα (peroxisome proliferator-activated receptor) that regulate cardiac lipid metabolism. OBJECTIVE: In this study, we investigated the involvement of KLF5 in DbCM and its transcriptional regulation. METHODS AND RESULTS: KLF5 mRNA levels were assessed in isolated cardiomyocytes from cardiovascular patients with diabetes and were higher compared with nondiabetic individuals. Analyses in human cells and diabetic mice with cardiomyocyte-specific FOXO1 (Forkhead box protein O1) deletion showed that FOXO1 bound directly on the KLF5 promoter and increased KLF5 expression. Diabetic mice with cardiomyocyte-specific FOXO1 deletion had lower cardiac KLF5 expression and were protected from DbCM. Genetic, pharmacological gain and loss of KLF5 function approaches and AAV (adeno-associated virus)-mediated Klf5 delivery in mice showed that KLF5 induces DbCM. Accordingly, the protective effect of cardiomyocyte FOXO1 ablation in DbCM was abolished when KLF5 expression was rescued. Similarly, constitutive cardiomyocyte-specific KLF5 overexpression caused cardiac dysfunction. KLF5 caused oxidative stress via direct binding on NADPH oxidase (NOX)4 promoter and induction of NOX4 (NADPH oxidase 4) expression. This was accompanied by accumulation of cardiac ceramides. Pharmacological or genetic KLF5 inhibition alleviated superoxide formation, prevented ceramide accumulation, and improved cardiac function in diabetic mice. CONCLUSIONS: Diabetes-mediated activation of cardiomyocyte FOXO1 increases KLF5 expression, which stimulates NOX4 expression, ceramide accumulation, and causes DbCM.

Nampt Potentiates Antioxidant Defense in Diabetic Cardiomyopathy.

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A redox-dependent pathway for regulating class II HDACs and cardiac hypertrophy.

Thioredoxin 1 (Trx1) facilitates the reduction of signaling molecules and transcription factors by cysteine thiol-disulfide exchange, thereby regulating cell growth and death. Here we studied the molecular mechanism by which Trx1 attenuates cardiac hypertrophy. Trx1 upregulates DnaJb5, a heat shock protein 40, and forms a multiple-protein complex with DnaJb5 and class II histone deacetylases (HDACs), master negative regulators of cardiac hypertrophy. Both Cys-274/Cys-276 in DnaJb5 and Cys-667/Cys-669 in HDAC4 are oxidized and form intramolecular disulfide bonds in response to reactive oxygen species (ROS)-generating hypertrophic stimuli, such as phenylephrine, whereas they are reduced by Trx1. Whereas reduction of Cys-274/Cys-276 in DnaJb5 is essential for interaction between DnaJb5 and HDAC4, reduction of Cys-667/Cys-669 in HDAC4 inhibits its nuclear export, independently of its phosphorylation status. Our study reveals a novel regulatory mechanism of cardiac hypertrophy through which the nucleocytoplasmic shuttling of class II HDACs is modulated by their redox modification in a Trx1-sensitive manner.

The role of autophagy in death of cardiomyocytes.

Autophagy mediates cellular quality control mechanisms and energy homeostasis through lysosomal degradation. Autophagy is typically viewed as an adaptive process that allows cells to survive against stress, such as nutrient deprivation and hypoxia. However, autophagy also mediates cell death during development and in response to stress. Cell death accompanied by autophagy activation and accumulation of autophagosomes has been classified as type II programmed cell death. Compared to the wealth of knowledge regarding the adaptive role of autophagy, however, the molecular mechanisms through which autophagy induces cell death and its functional significance are poorly understood. Autophagy is activated excessively under some conditions, causing uncontrolled degradation of cellular materials and cell death. An imbalance between autophagosome formation and lysosomal degradation causes a massive accumulation of autophagosomes, which subsequently causes cellular dysfunction and death. Dysregulation of autophagy induces a unique form of cell death, termed autosis, with defined morphological and biochemical features distinct from other forms of programmed cell death, such as apoptosis and necrosis. In the heart, dysregulated autophagy induces death of cardiomyocytes and actively mediates cardiac injury and dysfunction in some conditions, including reperfusion injury, doxorubicin cardiomyopathy, and lysosomal storage disorders. The goal in this review is to introduce the concept of autophagic cell death and discuss its functional significance in various cardiac conditions.

The role of autophagic cell death in cardiac disease.

Cardiomyocytes undergo various forms of cell death during heart disease such as myocardial infarction and heart failure. Understanding the mechanisms of cell death in cardiomyocytes is one of the most fundamental issues in the treatment of heart failure. Among the several kinds of cell death mechanisms, this review will focus on autophagy-related cardiomyocyte cell death. Although autophagy plays an essential role in mediating cellular quality control mechanisms for cell survival, dysregulation of autophagy can cause cell death, referred to as autophagy-dependent cell death or type II programmed cell death. The recent discovery of autosis as a modality of autophagy-dependent cell death with unique morphological and biochemical features has allowed us to broaden our understanding of the mechanistic role of autophagy in cell death. Here, we discuss autophagy-dependent cardiomyocyte cell death, including autosis, in pathophysiological conditions of the heart.

Mst1 promotes cardiac myocyte apoptosis through phosphorylation and inhibition of Bcl-xL.

The Hippo pathway, evolutionarily conserved from flies to mammals, promotes cell death and inhibits cell proliferation to regulate organ size. The core component of this cascade, Mst1 in mammalian cells, is sufficient to promote apoptosis. However, the mechanisms underlying both its activation and its ability to elicit cell death remain largely undefined. We here identify a signaling cassette in cardiac myocytes consisting of K-Ras, the scaffold RASSF1A, and Mst1 that is localized to mitochondria and promotes Mst1 activation in response to oxidative stress. Activated Mst1 phosphorylates Bcl-xL at Ser14, which resides in the BH4 domain, thereby antagonizing Bcl-xL-Bax binding. This, in turn, causes activation of Bax and subsequent mitochondria-mediated apoptotic death. Our findings demonstrate mitochondrial localization of Hippo signaling and identify Bcl-xL as a target that is directly modified to promote apoptosis.

The Role of Autophagy in the Heart.

Autophagy is an evolutionarily conserved mechanism by which cytoplasmic elements are degraded intracellularly. Autophagy has also emerged as a major regulator of cardiac homeostasis and function. Autophagy preserves cardiac structure and function under baseline conditions and is activated during stress, limiting damage under most conditions. It reduces injury and preserves cardiac function during ischemia. It also reduces chronic ischemic remodeling and mediates the cardiac adaptation to pressure overload by restricting misfolded protein accumulation, mitochondrial dysfunction, and oxidative stress. Impairment of autophagy is involved in the development of diabetes and aging-induced cardiac abnormalities. Autophagy defects contribute to the development of cardiac proteinopathy and doxorubicin-induced cardiomyopathy. However, massive activation of autophagy may be detrimental for the heart in certain stress conditions, such as reperfusion injury. In this review, we discuss recent evidence supporting the important role of autophagy and mitophagy in the regulation of cardiac homeostasis and adaptation to stress.

A postprandial FGF19-SHP-LSD1 regulatory axis mediates epigenetic repression of hepatic autophagy.

Lysosome-mediated autophagy is essential for cellular survival and homeostasis upon nutrient deprivation, but is repressed after feeding. Despite the emerging importance of transcriptional regulation of autophagy by nutrient-sensing factors, the role for epigenetic control is largely unexplored. Here, we show that Small Heterodimer Partner (SHP) mediates postprandial epigenetic repression of hepatic autophagy by recruiting histone demethylase LSD1 in response to a late fed-state hormone, FGF19 (hFGF19, mFGF15). FGF19 treatment or feeding inhibits macroautophagy, including lipophagy, but these effects are blunted in SHP-null mice or LSD1-depleted mice. In addition, feeding-mediated autophagy inhibition is attenuated in FGF15-null mice. Upon FGF19 treatment or feeding, SHP recruits LSD1 to CREB-bound autophagy genes, including Tfeb, resulting in dissociation of CRTC2, LSD1-mediated demethylation of gene-activation histone marks H3K4-me2/3, and subsequent accumulation of repressive histone modifications. Both FXR and SHP inhibit hepatic autophagy interdependently, but while FXR acts early, SHP acts relatively late after feeding, which effectively sustains postprandial inhibition of autophagy. This study demonstrates that the FGF19-SHP-LSD1 axis maintains homeostasis by suppressing unnecessary autophagic breakdown of cellular components, including lipids, under nutrient-rich postprandial conditions.

Proteomic analysis of mitochondrial biogenesis in cardiomyocytes differentiated from human induced pluripotent stem cells.

Mitochondria play key roles in the differentiation and maturation of human cardiomyocytes (CMs). As human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) hold potential in the treatment of heart diseases, we sought to identify key mitochondrial pathways and regulators, which may provide targets for improving cardiac differentiation and maturation. Proteomic analysis was performed on enriched mitochondrial protein extracts isolated from hiPSC-CMs differentiated from dermal fibroblasts (dFCM) and cardiac fibroblasts (cFCM) at time points between 12 and 115 days of differentiation, and from adult and neonatal mouse hearts. Mitochondrial proteins with a twofold change at time points up to 120 days relative to 12 days were subjected to ingenuity pathway analysis (IPA). The highest upregulation was in metabolic pathways for fatty acid oxidation (FAO), the tricarboxylic acid (TCA) cycle, oxidative phosphorylation (OXPHOS), and branched chain amino acid (BCAA) degradation. The top upstream regulators predicted to be activated were peroxisome proliferator-activated receptor γ coactivator 1 α (PGC1-α), the insulin receptor (IR), and the retinoblastoma protein (Rb1) transcriptional repressor. IPA and immunoblotting showed upregulation of the mitochondrial LonP1 protease-a regulator of mitochondrial proteostasis, energetics, and metabolism. LonP1 knockdown increased FAO in neonatal rat ventricular cardiomyocytes (nRVMs). Our results support the notion that LonP1 upregulation negatively regulates FAO in cardiomyocytes to calibrate the flux between glucose and fatty acid oxidation. We discuss potential mechanisms by which IR, Rb1, and LonP1 regulate the metabolic shift from glycolysis to OXPHOS and FAO. These newly identified factors and pathways may help in optimizing the maturation of iPSC-CMs.