Systemic Deletion of ARRDC4 Improves Cardiac Reserve and Exercise Capacity in Diabetes.

BACKGROUND: Exercise intolerance is an independent predictor of poor prognosis in diabetes. The underlying mechanism of the association between hyperglycemia and exercise intolerance remains undefined. We recently demonstrated that the interaction between ARRDC4 (arrestin domain-containing protein 4) and GLUT1 (glucose transporter 1) regulates cardiac metabolism. OBJECTIVE: To determine whether this mechanism broadly impacts diabetic complications, we investigated the role of ARRDC4 in the pathogenesis of diabetic cardiac and skeletal myopathy. METHODS AND RESULTS: High glucose promoted translocation of MondoA into the nucleus, which upregulated Arrdc4 transcriptional expression, increased lysosomal GLUT1 trafficking, and blocked glucose transport in cardiomyocytes, forming a feedback mechanism. This role of ARRDC4 was confirmed in human muscular cells from type 2 diabetic patients. Prolonged hyperglycemia upregulated myocardial Arrdc4 expression in multiple types of mouse models of diabetes. We then analyzed hyperglycemia-induced cardiac and skeletal muscle abnormalities in insulin-deficient mice. Hyperglycemia increased advanced glycation end-products and elicited oxidative and endoplasmic reticulum stress leading to apoptosis in the heart and peripheral muscle. However, deletion of Arrdc4 augmented tissue glucose transport and mitochondrial respiration, protecting the heart and muscle from tissue damage. Stress hemodynamic analysis and treadmill exhaustion test uncovered that Arrdc4-knockout mice had greater cardiac inotropic/chronotropic reserve with higher exercise endurance than wild-type (WT) animals under diabetes. While multiple organs were involved in the mechanism, cardiac-specific overexpression (beyond levels observed during diabetes) using adenoassociated virus suggests that high levels of myocardial ARRDC4 have the potential to contribute to exercise intolerance by interfering with cardiac metabolism through its interaction with GLUT1 in diabetes. Importantly, the ARRDC4 mutation mouse line exhibited greater exercise tolerance, showing the potential therapeutic impact on diabetic cardiomyopathy by disrupting the interaction between ARRDC4 and GLUT1. CONCLUSIONS: ARRDC4 serves as a regulator of hyperglycemia-induced toxicities toward cardiac and skeletal muscle, revealing a new molecular framework that connects hyperglycemia to cardiac/skeletal myopathy to exercise intolerance.

The Ubiquitin Ligase RBX2/SAG Regulates Mitochondrial Ubiquitination and Mitophagy.

BACKGROUND: Clearance of damaged mitochondria via mitophagy is crucial for cellular homeostasis. Apart from Parkin, little is known about additional Ub (ubiquitin) ligases that mediate mitochondrial ubiquitination and turnover, particularly in highly metabolically active organs such as the heart. METHODS: In this study, we have combined in silico analysis and biochemical assay to identify CRL (cullin-RING ligase) 5 as a mitochondrial Ub ligase. We generated cardiomyocytes and mice lacking RBX2 (RING-box protein 2; also known as SAG [sensitive to apoptosis gene]), a catalytic subunit of CRL5, to understand the effects of RBX2 depletion on mitochondrial ubiquitination, mitophagy, and cardiac function. We also performed proteomics analysis and RNA-sequencing analysis to define the impact of loss of RBX2 on the proteome and transcriptome. RESULTS: RBX2 and CUL (cullin) 5, 2 core components of CRL5, localize to mitochondria. Depletion of RBX2 inhibited mitochondrial ubiquitination and turnover, impaired mitochondrial membrane potential and respiration, increased cardiomyocyte cell death, and has a global impact on the mitochondrial proteome. In vivo, deletion of the Rbx2 gene in adult mouse hearts suppressed mitophagic activity, provoked accumulation of damaged mitochondria in the myocardium, and disrupted myocardial metabolism, leading to the rapid development of dilated cardiomyopathy and heart failure. Similarly, ablation of RBX2 in the developing heart resulted in dilated cardiomyopathy and heart failure. The action of RBX2 in mitochondria is not dependent on Parkin, and Parkin gene deletion had no impact on the onset and progression of cardiomyopathy in RBX2-deficient hearts. Furthermore, RBX2 controls the stability of PINK1 in mitochondria. CONCLUSIONS: These findings identify RBX2-CRL5 as a mitochondrial Ub ligase that regulates mitophagy and cardiac homeostasis in a Parkin-independent, PINK1-dependent manner.

The Ubiquitin Ligase RBX2/SAG Regulates Mitochondrial Ubiquitination and Mitophagy.

Clearance of damaged mitochondria via mitophagy is crucial for cellular homeostasis. While the role of ubiquitin (Ub) ligase PARKIN in mitophagy has been extensively studied, increasing evidence suggests the existence of PARKIN-independent mitophagy in highly metabolically active organs such as the heart. Here, we identify a crucial role for Cullin-RING Ub ligase 5 (CRL5) in basal mitochondrial turnover in cardiomyocytes. CRL5 is a multi-subunit Ub ligase comprised by the catalytic RING box protein RBX2 (also known as SAG), scaffold protein Cullin 5 (CUL5), and a substrate-recognizing receptor. Analysis of the mitochondrial outer membrane-interacting proteome uncovered a robust association of CRLs with mitochondria. Subcellular fractionation, immunostaining, and immunogold electron microscopy established that RBX2 and Cul5, two core components of CRL5, localizes to mitochondria. Depletion of RBX2 inhibited mitochondrial ubiquitination and turnover, impaired mitochondrial membrane potential and respiration, and increased cell death in cardiomyocytes. In vivo , deletion of the Rbx2 gene in adult mouse hearts suppressed mitophagic activity, provoked accumulation of damaged mitochondria in the myocardium, and disrupted myocardial metabolism, leading to rapid development of dilated cardiomyopathy and heart failure. Similarly, ablation of RBX2 in the developing heart resulted in dilated cardiomyopathy and heart failure. Notably, the action of RBX2 in mitochondria is not dependent on PARKIN, and PARKIN gene deletion had no impact on the onset and progression of cardiomyopathy in RBX2-deficient hearts. Furthermore, RBX2 controls the stability of PINK1 in mitochondria. Proteomics and biochemical analyses further revealed a global impact of RBX2 deficiency on the mitochondrial proteome and identified several mitochondrial proteins as its putative substrates. These findings identify RBX2-CRL5 as a mitochondrial Ub ligase that controls mitophagy under physiological conditions in a PARKIN-independent, PINK1-dependent manner, thereby regulating cardiac homeostasis.

Lysosomal dysfunction in diabetic cardiomyopathy.

Diabetes is a major risk factor for a variety of cardiovascular complications, while diabetic cardiomyopathy, a disease specific to the myocardium independent of vascular lesions, is an important causative factor for increased risk of heart failure and mortality in diabetic populations. Lysosomes have long been recognized as intracellular trash bags and recycling facilities. However, recent studies have revealed that lysosomes are sophisticated signaling hubs that play remarkably diverse roles in adapting cell metabolism to an ever-changing environment. Despite advances in our understanding of the physiological roles of lysosomes, the events leading to lysosomal dysfunction and how they relate to the overall pathophysiology of the diabetic heart remain unclear and are under intense investigation. In this review, we summarize recent advances regarding lysosomal injury and its roles in diabetic cardiomyopathy.

Metformin Inhibits Autophagy, Mitophagy and Antagonizes Doxorubicin-Induced Cardiomyocyte Death.

The antidiabetic drug metformin has been shown to reduce cardiac injury under various pathological conditions, including anticancer drug doxorubicin (DOX)-induced cardiotoxicity, which makes metformin a prime candidate for repurposing. However, the mechanisms that mediate the cardioprotective effects of metformin remain highly controversial. In this study, we tested a prevailing hypothesis that metformin activates autophagy/mitophagy to reduce DOX cardiotoxicity. FVB/N mice and H9C2 cardiac myoblasts were treated with metformin, respectively. Autophagy/mitophagy was determined by Western blot analysis of microtubule-associated protein light chain 3, form-II (LC3-II), a well-established marker of autophagic vesicles. Although metformin had minimal effects on basal LC3-II levels, it significantly inhibited the accumulation of LC3-II levels by the lysosomal protease inhibitors pepstatin A and E64d in both total cell lysates and mitochondrial fractions. Also, dual fluorescent autophagy/mitophagy reporters demonstrated that metformin slowed the degradation rate of autophagic cargos or mitochondrial fragments in the lysosomes. These surprising results suggest that metformin inhibits rather than stimulates autophagy/mitophagy, sharply contrasting the popular belief. In addition, metformin diminished DOX-induced autophagy/mitophagy as well as cardiomyocyte death. Together, these results suggest that the cardioprotective effects of metformin against DOX cardiotoxicity may be mediated by its ability to inhibit autophagy and mitophagy, although the underlying molecular mechanisms remain to be determined.

Wound age estimation based on next-generation sequencing: Fitting the optimal index system using machine learning.

Accurate estimation of the wound age is critical in investigating intentional injury cases. Establishing objective and reliable biological indicators to estimate wound age is still a significant challenge in forensic medicine. Therefore, exploring an objective, flexible, and reliable index system selection method for wound age estimation based on next-generation sequencing gene expression profiles is necessary. We randomly divided 63 Sprague-Dawley rats into a control group, seven experimental groups (n = 7 per group), and an external validation group. After rats in the experimental and external validation groups suffered contusions, we sacrificed them at 4, 8, 12, 16, 20, 24, and 48 h after contusion, respectively. We selected 54 genes with the most significant changes between adjacent time points after contusion and defined set A. The Hub genes with time-related expression patterns were set B, C, and D through next-generation sequencing and bioinformatics analysis. Four different machine learning classification algorithms, including logistic regression, support vector machine, multi-layer perceptron, and random forest were used to compare and verify the efficiency of four index systems to estimate the wound age. The best combination for wound age estimation is the Genes ascribed to set A combined with the random forest classification algorithm. The accuracy of external verification was 85.71%. Only one rat was incorrectly classified (4 h post-injury incorrectly classified as 8 h). This study demonstrated the potential advantage of the index system selection based on next-generation sequencing and bioinformatics analysis for wound age estimation.

Demystifying the Relationship Between Metformin, AMPK, and Doxorubicin Cardiotoxicity.

Doxorubicin (DOX) is an extremely effective and wide-spectrum anticancer drug, but its long-term use can lead to heart failure, which presents a serious problem to millions of cancer survivors who have been treated with DOX. Thus, identifying agents that can reduce DOX cardiotoxicity and concurrently enhance its antitumor efficacy would be of great clinical value. In this respect, the classical antidiabetic drug metformin (MET) has stood out, appearing to have both antitumor and cardioprotective properties. MET is proposed to achieve these beneficial effects through the activation of AMP-activated protein kinase (AMPK), an essential regulator of mitochondrial homeostasis and energy metabolism. AMPK itself has been shown to protect the heart and modulate tumor growth under certain conditions. However, the role and mechanism of the hypothesized MET-AMPK axis in DOX cardiotoxicity and antitumor efficacy remain to be firmly established by in vivo studies using tumor-bearing animal models and large-scale prospective clinical trials. This review summarizes currently available literature for or against a role of AMPK in MET-mediated protection against DOX cardiotoxicity. It also highlights the emerging evidence suggesting distinct roles of the AMPK subunit isoforms in mediating the functions of unique AMPK holoenzymes composed of different combinations of isoforms. Moreover, the review provides a perspective regarding future studies that may help fully elucidate the relationship between MET, AMPK and DOX cardiotoxicity.

Mitochondrial Fission and Mitophagy Coordinately Restrict High Glucose Toxicity in Cardiomyocytes.

Hyperglycemia-induced mitochondrial dysfunction plays a key role in the pathogenesis of diabetic cardiomyopathy. Injured mitochondrial segments are separated by mitochondrial fission and eliminated by autophagic sequestration and subsequent degradation in the lysosome, a process termed mitophagy. However, it remains poorly understood how high glucose affects the activities of, and the relationship between, mitochondrial fission and mitophagy in cardiomyocytes. In this study, we determined the functional roles of mitochondrial fission and mitophagy in hyperglycemia-induced cardiomyocyte injury. High glucose (30 mM, HG) reduced mitochondrial connectivity and particle size and increased mitochondrial number in neonatal rat ventricular cardiomyocytes, suggesting an enhanced mitochondrial fragmentation. SiRNA knockdown of the pro-fission factor dynamin-related protein 1 (DRP1) restored mitochondrial size but did not affect HG toxicity, and Mdivi-1, a DRP1 inhibitor, even increased HG-induced cardiomyocyte injury, as shown by superoxide production, mitochondrial membrane potential and cell death. However, DRP1 overexpression triggered mitochondrial fragmentation and mitigated HG-induced cardiomyocyte injury, suggesting that the increased mitochondrial fission is beneficial, rather than detrimental, to cardiomyocytes cultured under HG conditions. This is in contrast to the prevailing hypothesis that mitochondrial fragmentation mediates or contributes to HG cardiotoxicity. Meanwhile, HG reduced mitophagy flux as determined by the difference in the levels of mitochondria-associated LC3-II or the numbers of mitophagy foci indicated by the novel dual fluorescent reporter mt-Rosella in the absence and presence of the lysosomal inhibitors. The ability of HG to induce mitochondrial fragmentation and inhibit mitophagy was reproduced in adult mouse cardiomyocytes. Overexpression of Parkin, a positive regulator of mitophagy, or treatment with CCCP, a mitochondrial uncoupler, induced mitophagy and attenuated HG-induced cardiomyocyte death, while Parkin knockdown had opposite effects, suggesting an essential role of mitophagy in cardiomyocyte survival under HG conditions. Strikingly, Parkin overexpression increased mitochondrial fragmentation, while DRP1 overexpression accelerated mitophagy flux, demonstrating a reciprocal activation loop that controls mitochondrial fission and mitophagy. Thus, strategies that promote the mutual positive interaction between mitochondrial fission and mitophagy while simultaneously maintain their levels within the physiological range would be expected to improve mitochondrial health, alleviating hyperglycemic cardiotoxicity.

Comprehensive lipidomic profiling in serum and multiple tissues from a mouse model of diabetes.

INTRODUCTION: Diabetes mellitus is a serious metabolic disorder causing multiple organ damage in human. However, the lipidomic profiles in different organs and their associations are rarely studied in either diabetic patients or animals. OBJECTIVES: To evaluate and compare the characteristics of lipid species in serum and multiple tissues in a diabetic mouse model. METHODS: Semi-quantitative profiling analyses of intact and oxidized lipids were performed in serum and multiple tissues from a diabetic mouse model fed a high fat diet and treated with streptozotocin by using LC/HRMS and MS/MS. The total content of each lipid class, and the tissue-specific lipid species in all tissue samples were determined and compared by multivariate analyses. RESULTS: The diabetic mouse model displayed characteristic differences in serum and multiple organs: the brain and heart showed the largest reduction in cardiolipin, while the kidney had more alterations in triacylglycerol. Interestingly, the lipidomic differences also existed between different regions of the same organ: cardiolipin species with highly polyunsaturated fatty acyls decreased only in atrium but not in ventricle, while renal cortex showed longer fatty acyl chains for both increased and decreased triacylglycerol species than renal medulla. Importantly, diabetes caused an accumulation of lipid hydroperoxides, suggesting that oxidative stress was induced in all organs except for the brain during the development of diabetes. CONCLUSIONS: These findings provided novel insight into the organ-specific relationship between diabetes and lipid metabolism, which might be useful for evaluating not only diabetic tissue injury but also the effectiveness of diabetic treatments.

Novel Dual-Fluorescent Mitophagy Reporter Reveals a Reduced Mitophagy Flux in Type 1 Diabetic Mouse Heart.

CONTEXT: Patients with diabetes are susceptible to heart failure. Defective mitochondria can cause cardiac damage. Mitochondrial autophagy or mitophagy is a quality control mechanism that eliminates dysfunctional mitochondria through lysosome degradation. Mitophagy is essential for maintaining a pool of healthy mitochondria for normal cardiac function. However, the effect of diabetes on the functional status of cardiac mitophagy remains unclear. OBJECTIVE: To determine and compare cardiac mitophagy flux between diabetic and nondiabetic mice. METHODS: Using a novel dual fluorescent mitophagy reporter termed mt-Rosella, we labeled and traced mitochondrial fragments that are sequestered by the autophagosome and delivered to and degraded in the lysosome. RESULTS: Mitophagic activity was reduced in high-glucose-treated cardiomyocytes and in the heart tissue of type 1 diabetic mice. CONCLUSIONS: Mitophagy was impaired in the heart of diabetic mice, suggesting that restoring or accelerating mitophagy flux may be a useful strategy to reduce cardiac injury caused by diabetes.

Hyperglycemia-induced cardiomyocyte death is mediated by lysosomal membrane injury and aberrant expression of cathepsin D.

Hyperglycemia is an independent risk factor for diabetic heart failure. However, the mechanisms that mediate hyperglycemia-induced cardiac damage remain poorly understood. Previous studies have shown an association between lysosomal dysfunction and diabetic heart injury. The present study examined if mimicking hyperglycemia in cultured cardiomyocytes could induce lysosomal membrane permeabilization (LMP), leading to the release of lysosome enzymes and subsequent cell death. High glucose (HG) reduced the number of lysosomes with acidic pH as shown by a fluorescent pH indicator. Also, HG induced lysosomal membrane injury as shown by an accumulation of Galectin3-RFP puncta, which was accompanied by the leakage of cathepsin D (CTSD), an aspartic protease that normally resides within the lysosomal lumen. Furthermore, CTSD expression was increased in HG-cultured cardiomyocytes and in the hearts of 2 mouse models of type 1 diabetes. Either CTSD knockdown with siRNA or inhibition of CTSD activity by pepstatin A markedly diminished HG-induced cardiomyocyte death, while CTSD overexpression exaggerated HG-induced cell death. Together, these results suggested that HG increased CTSD expression, induced LMP and triggered CTSD release from the lysosomes, which collectively contributed to HG-induced cardiomyocyte injury.

Enhanced mTOR complex 1 signaling attenuates diabetic cardiac injury in OVE26 mice.

The protein kinase mechanistic target of rapamycin (mTOR) performs diverse cellular functions through 2 distinct multiprotein complexes, mTOR complex (mTORC)1 and 2. Numerous studies using rapamycin, an mTORC1 inhibitor, have implicated a role for mTORC1 in several types of heart disease. People with diabetes are more susceptible to heart failure. mTORC1 activity is increased in the diabetic heart, but its functional significance remains controversial. To investigate the role of mTORC1 in the diabetic heart, we crossed OVE26 type 1 diabetic mice with transgenic mice expressing a constitutively active mTOR (mTORca) or kinase-dead mTOR (mTORkd) in the heart. The expression of mTORca or mTORkd affected only mTORC1 but not mTORC2 activities, with corresponding changes in the activities of autophagy, a cellular degradation pathway negatively regulated by mTORC1. Diabetic cardiac damage in OVE26 mice was dramatically reduced by mTORca but exacerbated by mTORkd expression as assessed by changes in cardiac function, oxidative stress, and myocyte apoptosis. These findings demonstrated that the enhanced mTORC1 signaling in the OVE26 diabetic heart was an adaptive response that limited cardiac dysfunction, suggesting that manipulations that enhance mTORC1 activity may reduce diabetic cardiac injury, in sharp contrast to the results previously obtained with rapamycin.-Xu, X., Kobayashi, S., Timm, D., Huang, Y., Zhao, F., Shou, W., Liang, Q. Enhanced mTOR complex 1 signaling attenuates diabetic cardiac injury in OVE26 mice.

Doxorubicin-induced cardiomyocyte death is mediated by unchecked mitochondrial fission and mitophagy.

Doxorubicin (Dox) is a widely used antineoplastic agent that can cause heart failure. Dox cardiotoxicity is closely associated with mitochondrial damage. Mitochondrial fission and mitophagy are quality control mechanisms that normally help maintain a pool of healthy mitochondria. However, unchecked mitochondrial fission and mitophagy may compromise the viability of cardiomyocytes, predisposing them to cell death. Here, we tested this possibility by using Dox-treated H9c2 cardiac myoblast cells expressing either the mitochondria-targeted fluorescent protein MitoDsRed or the novel dual-fluorescent mitophagy reporter mt-Rosella. Dox induced mitochondrial fragmentation as shown by reduced form factor, aspect ratio, and mean mitochondrial size. This effect was abolished by short interference RNA-mediated knockdown of dynamin-related protein 1 (DRP1), a major regulator of fission. Importantly, DRP1 knockdown decreased cell death as indicated by the reduced number of propidium iodide-positive cells and the cleavage of caspase-3 and poly (ADP-ribose) polymerase. Moreover, DRP1-deficient mice were protected from Dox-induced cardiac damage, strongly supporting a role for DRP1-dependent mitochondrial fragmentation in Dox cardiotoxicity. In addition, Dox accelerated mitophagy flux, which was attenuated by DRP1 knockdown, as assessed by the mitophagy reporter mt-Rosella, suggesting the necessity of mitochondrial fragmentation in Dox-induced mitophagy. Knockdown of parkin, a positive regulator of mitophagy, dramatically diminished Dox-induced cell death, whereas overexpression of parkin had the opposite effect. Together, these results suggested that Dox cardiotoxicity was mediated, at least in part, by the increased mitochondrial fragmentation and accelerated mitochondrial degradation by the lysosome. Strategies that limit mitochondrial fission and mitophagy in the physiologic range may help reduce Dox cardiotoxicity.-Catanzaro, M. P., Weiner, A., Kaminaris, A., Li, C., Cai, F., Zhao, F., Kobayashi, S., Kobayashi, T., Huang, Y., Sesaki, H., Liang, Q. Doxorubicin-induced cardiomyocyte death is mediated by unchecked mitochondrial fission and mitophagy.

Dihydrotestosterone regulates oxidative stress and immunosuppressive cytokines in a female BALB/c mouse model of Graves' disease.

Background: Graves' disease (GD) is an autoimmune disease that affects more women than men. In our previous study, a potent bioactive androgen, 5α-dihydrotestosterone (DHT) showed a protective effect against GD in female BALB/c mice. Evidence indicates that abnormal oxidative stress and immunosuppressive cytokines (TGF-ß, IL-35) play critical roles in the pathogenesis and development of GD. The purpose of this research is to measure these cytokines and oxidative stress markers to explore potential protective mechanisms of DHT in a BALB/c mouse model of GD. Methods: GD was induced in female BALB/c mice by intramuscular injection of an adenovirus expressing the A-subunit of the TSH receptor (Ad-TSHR289). DHT or a matching placebo was injected every 3 days. Mice were sacrificed four weeks after the third virus immunization to obtain blood, thyroid and spleen for further analysis. Results: Thyroid hormones were significantly reduced in DHT treated GD mice. In addition, DHT attenuated thyroid oxidative injuries in GD mice, as shown by decreased total antioxidation capability (TAOC), superoxide dismutase (SOD) and the level of malondialdehyde (MDA). The levels of immunosuppressive cytokines (TGF-ß, IL-35) in DHT group were significant higher compared with the GD group. Conclusions: The results demonstrated that DHT could reduce the severity of GD in female BALB/c mice by regulating oxidative stress. The upregulation of immunosuppressive cytokines might be another important protective mechanism.

Regulation of BECN1-mediated autophagy by HSPB6: Insights from a human HSPB6S10F mutant.

HSPB6/Hsp20 (heat shock protein family B [small] member 6) has emerged as a novel cardioprotector against stress-induced injury. We identified a human mutant of HSPB6 (HSPB6S10F) exclusively present in dilated cardiomyopathy (DCM) patients. Cardiac expression of this mutant in mouse hearts resulted in remodeling and dysfunction, which progressed to heart failure and early death. These detrimental effects were associated with reduced interaction of mutant HSPB6S10F with BECN1/Beclin 1, leading to BECN1 ubiquitination and its proteosomal degradation. As a result, autophagy flux was substantially inhibited and apoptosis was increased in HSPB6S10F-mutant hearts. In contrast, overexpression of wild-type HSPB6 (HSPB6 WT) not only increased BECN1 levels, but also competitively suppressed binding of BECN1 to BCL2, resulting in stimulated autophagy. Indeed, preinhibition of autophagy attenuated the cardioprotective effects of HSPB6 WT. Taken together, these findings reveal a new regulatory mechanism of HSPB6 in cell survival through its interaction with BECN1. Furthermore, Ser10 appears to be crucial for the protective effects of HSPB6 and transversion of this amino acid to Phe contributes to cardiomyopathy.

The transcription factor GATA4 promotes myocardial regeneration in neonatal mice.

Heart failure is often the consequence of insufficient cardiac regeneration. Neonatal mice retain a certain capability of myocardial regeneration until postnatal day (P)7, although the underlying transcriptional mechanisms remain largely unknown. We demonstrate here that cardiac abundance of the transcription factor GATA4 was high at P1, but became strongly reduced at P7 in parallel with loss of regenerative capacity. Reconstitution of cardiac GATA4 levels by adenoviral gene transfer markedly improved cardiac regeneration after cryoinjury at P7. In contrast, the myocardial scar was larger in cardiomyocyte-specific Gata4 knockout (CM-G4-KO) mice after cryoinjury at P0, indicative of impaired regeneration, which was accompanied by reduced cardiomyocyte proliferation and reduced myocardial angiogenesis in CM-G4-KO mice. Cardiomyocyte proliferation was also diminished in cardiac explants from CM-G4-KO mice and in isolated cardiomyocytes with reduced GATA4 expression. Mechanistically, decreased GATA4 levels caused the downregulation of several pro-regenerative genes (among them interleukin-13, Il13) in the myocardium. Interestingly, systemic administration of IL-13 rescued defective heart regeneration in CM-G4-KO mice and could be evaluated as therapeutic strategy in the future.

Myocardial redox status, mitophagy and cardioprotection: a potential way to amend diabetic heart?

Diabetic cardiomyopathy (DCM) is one of the major cardiovascular complications in diabetes that increase the mortality of diabetic patients. Mechanisms underlying DCM have not been fully elucidated, hindering targeted design of effective strategies to delay or treat DCM. Mitochondrial dysfunction is recognized as the driving force for the pathogenesis of DCM; therefore, maintaining cardiac mitochondrial quality is crucial for DCM prevention. Mitophagy is the process by which cells degrade abnormal or superfluous mitochondria in order to correct mitochondrial dysfunction, improve mitochondrial quality and maintain cardiac homoeostasis. Although the roles of mitophagy in various cardiomyopathies have been suggested, it remains largely unknown how the process is regulated and whether it is altered in the diabetic heart. In this review, we summarize currently available studies that investigate mitophagy in the heart, including its pathways, features and protective roles in several situations, including DCM. Due to limited data about mitophagy in diabetic hearts, future studies are required to gain a deeper understanding of the regulatory mechanisms of mitophagy in the heart and to develop mitophagy-based strategies for protecting the heart from diabetic injury.

Mitochondrial quality control in the diabetic heart.

Diabetes is a well-known risk factor for heart failure. Diabetic heart damage is closely related to mitochondrial dysfunction and increased ROS generation. However, clinical trials have shown no effects of antioxidant therapies on heart failure in diabetic patients, suggesting that simply antagonizing existing ROS by antioxidants is not sufficient to reduce diabetic cardiac injury. A potentially more effective treatment strategy may be to enhance the overall capacity of mitochondrial quality control to maintain a pool of healthy mitochondria that are needed for supporting cardiac contractile function in diabetic patients. Mitochondrial quality is controlled by a number of coordinated mechanisms including mitochondrial fission and fusion, mitophagy and biogenesis. The mitochondrial damage consistently observed in the diabetic hearts indicates a failure of the mitochondrial quality control mechanisms. Recent studies have demonstrated a crucial role for each of these mechanisms in cardiac homeostasis and have begun to interrogate the relative contribution of insufficient mitochondrial quality control to diabetic cardiac injury. In this review, we will present currently available literature that links diabetic heart disease to the dysregulation of major mitochondrial quality control mechanisms. We will discuss the functional roles of these mechanisms in the pathogenesis of diabetic heart disease and their potentials for targeted therapeutical manipulation.

Smad3 Couples Pak1 With the Antihypertrophic Pathway Through the E3 Ubiquitin Ligase, Fbxo32.

Pathological cardiac hypertrophy is regarded as a critical intermediate step toward the development of heart failure. Many signal transduction cascades are demonstrated to dictate the induction and progression of pathological hypertrophy; however, our understanding in regulatory mechanisms responsible for the suppression of hypertrophy remains limited. In this study, we showed that exacerbated hypertrophy induced by pressure overload in cardiac-deleted Pak1 mice was attributable to a failure to upregulate the antihypertrophic E3 ligase, Fbxo32, responsible for targeting proteins for the ubiquitin-degradation pathway. Under pressure overload, cardiac overexpression of constitutively active Pak1 mice manifested strong resilience against pathological hypertrophic remodeling. Mechanistic studies demonstrated that subsequent to Pak1 activation, the binding of Smad3 on a critical singular AGAC(-286)-binding site on the FBXO32 promoter was crucial for its transcriptional regulation. Pharmacological upregulation of Fbxo32 by Berberine ameliorated hypertrophic remodeling and improved cardiac performance in cardiac-deficient Pak1 mice under pressure overload. Our findings discover Smad3 and Fbxo32 as novel downstream components of the Pak1-dependent signaling pathway for the suppression of hypertrophy. This discovery opens a new venue for opportunities to identify novel targets for the management of cardiac hypertrophy.