Lipotoxicity as a therapeutic target in obesity and diabetic cardiomyopathy.

Unhealthy sources of fats, ultra-processed foods with added sugars, and a sedentary lifestyle make humans more susceptible to developing overweight and obesity. While lipids constitute an integral component of the organism, excessive and abnormal lipid accumulation that exceeds the storage capacity of lipid droplets disrupts the intracellular composition of fatty acids and results in the release of deleterious lipid species, thereby giving rise to a pathological state termed lipotoxicity. This condition induces endoplasmic reticulum stress, mitochondrial dysfunction, inflammatory responses, and cell death. Recent advances in omics technologies and analytical methodologies and clinical research have provided novel insights into the mechanisms of lipotoxicity, including gut dysbiosis, epigenetic and epitranscriptomic modifications, dysfunction of lipid droplets, post-translational modifications, and altered membrane lipid composition. In this review, we discuss the recent knowledge on the mechanisms underlying the development of lipotoxicity and lipotoxic cardiometabolic disease in obesity, with a particular focus on lipotoxic and diabetic cardiomyopathy.

Ser14 phosphorylation of Bcl-xL mediates compensatory cardiac hypertrophy in male mice.

The anti-apoptotic function of Bcl-xL in the heart during ischemia/reperfusion is diminished by K-Ras-Mst1-mediated phosphorylation of Ser14, which allows dissociation of Bcl-xL from Bax and promotes cardiomyocyte death. Here we show that Ser14 phosphorylation of Bcl-xL is also promoted by hemodynamic stress in the heart, through the H-Ras-ERK pathway. Bcl-xL Ser14 phosphorylation-resistant knock-in male mice develop less cardiac hypertrophy and exhibit contractile dysfunction and increased mortality during acute pressure overload. Bcl-xL Ser14 phosphorylation enhances the Ca2+ transient by blocking the inhibitory interaction between Bcl-xL and IP3Rs, thereby promoting Ca2+ release and activation of the calcineurin-NFAT pathway, a Ca2+-dependent mechanism that promotes cardiac hypertrophy. These results suggest that phosphorylation of Bcl-xL at Ser14 in response to acute pressure overload plays an essential role in mediating compensatory hypertrophy by inducing the release of Bcl-xL from IP3Rs, alleviating the negative constraint of Bcl-xL upon the IP3R-NFAT pathway.

Bcl-x short-isoform is essential for maintaining homeostasis of multiple tissues.

BCL-2-like protein 1 (BCL2L1) is a key component of cell survival and death mechanisms. Its dysregulation and altered ratio of splicing variants associate with pathologies. However, isoform-specific loss-of-function analysis of BCL2L1 remains unexplored. Here we show the functional impact of genetically inhibiting Bcl-x short-isoform (Bcl-xS) in vivo. Bcl-xS is expressed in most tissues with predominant expression in the spleen and blood cells in mice. Bcl-xS knockout (KO) mice show no overt abnormality until 3 months of age. Thereafter, KO mice develop cardiac hypertrophy with contractile dysfunction and splenomegaly by 6 months. Cardiac fibrosis significantly increases in KO, but the frequency of apoptosis is indistinguishable despite cardiomyopathy. The Akt/mTOR and JNK/cJun signaling are upregulated in male KO heart, and the JNK/cJun is activated with increased Bax expression in KO spleen. These results suggest that Bcl-xS may be dispensable for development but is essential for maintaining the homeostasis of multiple organs.

Guidelines on models of diabetic heart disease.

Diabetes is a major risk factor for cardiovascular diseases, including diabetic cardiomyopathy, atherosclerosis, myocardial infarction, and heart failure. As cardiovascular disease represents the number one cause of death in people with diabetes, there has been a major emphasis on understanding the mechanisms by which diabetes promotes cardiovascular disease, and how antidiabetic therapies impact diabetic heart disease. With a wide array of models to study diabetes (both type 1 and type 2), the field has made major progress in answering these questions. However, each model has its own inherent limitations. Therefore, the purpose of this guidelines document is to provide the field with information on which aspects of cardiovascular disease in the human diabetic population are most accurately reproduced by the available models. This review aims to emphasize the advantages and disadvantages of each model, and to highlight the practical challenges and technical considerations involved. We will review the preclinical animal models of diabetes (based on their method of induction), appraise models of diabetes-related atherosclerosis and heart failure, and discuss in vitro models of diabetic heart disease. These guidelines will allow researchers to select the appropriate model of diabetic heart disease, depending on the specific research question being addressed.

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.

Dietary carbohydrates restriction inhibits the development of cardiac hypertrophy and heart failure.

AIMS: A diet with modified components, such as a ketogenic low-carbohydrate (LC) diet, potentially extends longevity and healthspan. However, how an LC diet impacts on cardiac pathology during haemodynamic stress remains elusive. This study evaluated the effects of an LC diet high in either fat (Fat-LC) or protein (Pro-LC) in a mouse model of chronic hypertensive cardiac remodelling. METHODS AND RESULTS: Wild-type mice were subjected to transverse aortic constriction, followed by feeding with the Fat-LC, the Pro-LC, or a high-carbohydrate control diet. After 4 weeks, echocardiographic, haemodynamic, histological, and biochemical analyses were performed. LC diet consumption after pressure overload inhibited the development of pathological hypertrophy and systolic dysfunction compared to the control diet. An anti-hypertrophic serine/threonine kinase, GSK-3ß, was re-activated by both LC diets; however, the Fat-LC, but not the Pro-LC, diet exerted cardioprotection in GSK-3ß cardiac-specific knockout mice. ß-hydroxybutyrate, a major ketone body in mammals, was increased in the hearts of mice fed the Fat-LC, but not the Pro-LC, diet. In cardiomyocytes, ketone body supplementation inhibited phenylephrine-induced hypertrophy, in part by suppressing mTOR signalling. CONCLUSION: Strict carbohydrate restriction suppresses pathological cardiac growth and heart failure after pressure overload through distinct anti-hypertrophic mechanisms elicited by supplemented macronutrients.

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.

Cardiomyopathy in obesity, insulin resistance and diabetes.

The prevalence of obesity, insulin resistance and diabetes is increasing rapidly. Most patients with these disorders have hypertriglyceridaemia and increased plasma levels of fatty acids, which are taken up and stored in lipid droplets in the heart. Intramyocardial lipids that exceed the capacity for storage and oxidation can be lipotoxic and induce non-ischaemic and non-hypertensive cardiomyopathy, termed diabetic or lipotoxic cardiomyopathy. The clinical features of diabetic cardiomyopathy are cardiac hypertrophy and diastolic dysfunction, which lead to heart failure, especially heart failure with preserved ejection fraction. Although the pathogenesis of the cardiomyopathy is multifactorial, diabetic dyslipidaemia and intramyocardial lipid accumulation are the key pathological features, triggering cellular signalling and modifications of proteins and lipids via generation of toxic metabolic intermediates. Most clinical studies have shown no beneficial effect of anti-diabetic agents and statins on outcomes in heart failure patients without atherosclerotic diseases, indicating the importance of identifying underlying mechanisms and early interventions for diabetic cardiomyopathy. Here, we summarize the molecular mechanisms of diabetic cardiomyopathy, with a special emphasis on cardiac lipotoxicity, and discuss the role of peroxisome proliferator-activated receptor α and dysregulated fatty acid metabolism as potential therapeutic targets.

Yes-Associated Protein (YAP) Facilitates Pressure Overload-Induced Dysfunction in the Diabetic Heart.

Patients with diabetes are more prone to developing heart failure in the presence of high blood pressure than those without diabetes. Yes-associated protein (YAP), a key effector of the Hippo signaling pathway, is persistently activated in diabetic hearts, and YAP plays an essential role in mediating the exacerbation of heart failure in response to pressure overload in the hearts of mice fed a high-fat diet. YAP induced dedifferentiation of cardiomyocytes through activation of transcriptional enhancer factor 1 (TEAD1), a transcription factor. Thus, YAP and TEAD1 are promising therapeutic targets for diabetic patients with high blood pressure to prevent the development of heart failure.

Glycogen Synthase Kinase-3α Promotes Fatty Acid Uptake and Lipotoxic Cardiomyopathy.

Obesity induces lipotoxic cardiomyopathy, a condition in which lipid accumulation in cardiomyocytes causes cardiac dysfunction. Here, we show that glycogen synthase kinase-3α (GSK-3α) mediates lipid accumulation in the heart. Fatty acids (FAs) upregulate GSK-3α, which phosphorylates PPARα at Ser280 in the ligand-binding domain (LBD). This modification ligand independently enhances transcription of a subset of PPARα targets, selectively stimulating FA uptake and storage, but not oxidation, thereby promoting lipid accumulation. Constitutively active GSK-3α, but not GSK-3ß, was sufficient to drive PPARα signaling, while cardiac-specific knockdown of GSK-3α, but not GSK-3ß, or replacement of PPARα Ser280 with Ala conferred resistance to lipotoxicity in the heart. Fibrates, PPARα ligands, inhibited phosphorylation of PPARα at Ser280 by inhibiting the interaction of GSK-3α with the LBD of PPARα, thereby reversing lipotoxic cardiomyopathy. These results suggest that GSK-3α promotes lipid anabolism through PPARα-Ser280 phosphorylation, which underlies the development of lipotoxic cardiomyopathy in the context of obesity.

Mitophagy Is Essential for Maintaining Cardiac Function During High Fat Diet-Induced Diabetic Cardiomyopathy.

RATIONALE: Diabetic patients develop cardiomyopathy characterized by hypertrophy, diastolic dysfunction, and intracellular lipid accumulation, termed lipotoxicity. Diabetic hearts utilize fatty acids as a major energy source, which produces high levels of oxidative stress, thereby inducing mitochondrial dysfunction. OBJECTIVE: To elucidate how mitochondrial function is regulated in diabetic cardiomyopathy. METHODS AND RESULTS: Mice were fed either a normal diet or high-fat diet (HFD, 60 kcal % fat). Although autophagic flux was activated by HFD consumption, peaking at 6 weeks ( P<0.05), it was attenuated thereafter. Mitophagy, evaluated with Mito-Keima, was increased after 3 weeks of HFD feeding (mitophagy area: 8.3% per cell with normal diet and 12.4% with HFD) and continued to increase even after 2 months ( P<0.05). By isolating adult cardiomyocytes from GFP-LC3 mice fed HFD, we confirmed that mitochondria were sequestrated by LC3-positive autophagosomes during mitophagy. In wild-type mice, cardiac hypertrophy, diastolic dysfunction (end diastolic pressure-volume relationship =0.051±0.009 in normal diet and 0.11±0.004 in HFD) and lipid accumulation occurred within 2 months of HFD feeding ( P<0.05). Deletion of atg7 impaired mitophagy, increased lipid accumulation, exacerbated diastolic dysfunction (end diastolic pressure-volume relationship =0.11±0.004 in wild type and 0.152±0.019 in atg7 cKO; P<0.05) and induced systolic dysfunction (end systolic pressure-volume relationship =24.86±2.46 in wild type and 15.93±1.76 in atg7 cKO; P<0.05) during HFD feeding. Deletion of Parkin partially inhibited mitophagy, increased lipid accumulation and exacerbated diastolic dysfunction (end diastolic pressure-volume relationship =0.124±0.005 in wild type and 0.176±0.018 in Parkin KO, P<0.05) in response to HFD feeding. Injection of TB1 (Tat-Beclin1) activated mitophagy, attenuated mitochondrial dysfunction, decreased lipid accumulation, and protected against cardiac diastolic dysfunction (end diastolic pressure-volume relationship =0.110±0.009 in Control peptide and 0.078±0.015 in TB1, P<0.05) during HFD feeding. CONCLUSIONS: Mitophagy serves as an essential quality control mechanism for mitochondria in the heart during HFD consumption. Impairment of mitophagy induces mitochondrial dysfunction and lipid accumulation, thereby exacerbating diabetic cardiomyopathy. Conversely, activation of mitophagy protects against HFD-induced diabetic cardiomyopathy.

Hippo Deficiency Leads to Cardiac Dysfunction Accompanied by Cardiomyocyte Dedifferentiation During Pressure Overload.

RATIONALE: The Hippo pathway plays an important role in determining organ size through regulation of cell proliferation and apoptosis. Hippo inactivation and consequent activation of YAP (Yes-associated protein), a transcription cofactor, have been proposed as a strategy to promote myocardial regeneration after myocardial infarction. However, the long-term effects of Hippo deficiency on cardiac function under stress remain unknown. OBJECTIVE: We investigated the long-term effect of Hippo deficiency on cardiac function in the presence of pressure overload (PO). METHODS AND RESULTS: We used mice with cardiac-specific homozygous knockout of WW45 (WW45cKO), in which activation of Mst1 (Mammalian sterile 20-like 1) and Lats2 (large tumor suppressor kinase 2), the upstream kinases of the Hippo pathway, is effectively suppressed because of the absence of the scaffolding protein. We used male mice at 3 to 4 month of age in all animal experiments. We subjected WW45cKO mice to transverse aortic constriction for up to 12 weeks. WW45cKO mice exhibited higher levels of nuclear YAP in cardiomyocytes during PO. Unexpectedly, the progression of cardiac dysfunction induced by PO was exacerbated in WW45cKO mice, despite decreased apoptosis and activated cardiomyocyte cell cycle reentry. WW45cKO mice exhibited cardiomyocyte sarcomere disarray and upregulation of TEAD1 (transcriptional enhancer factor) target genes involved in cardiomyocyte dedifferentiation during PO. Genetic and pharmacological inactivation of the YAP-TEAD1 pathway reduced the PO-induced cardiac dysfunction in WW45cKO mice and attenuated cardiomyocyte dedifferentiation. Furthermore, the YAP-TEAD1 pathway upregulated OSM (oncostatin M) and OSM receptors, which played an essential role in mediating cardiomyocyte dedifferentiation. OSM also upregulated YAP and TEAD1 and promoted cardiomyocyte dedifferentiation, indicating the existence of a positive feedback mechanism consisting of YAP, TEAD1, and OSM. CONCLUSIONS: Although activation of YAP promotes cardiomyocyte regeneration after cardiac injury, it induces cardiomyocyte dedifferentiation and heart failure in the long-term in the presence of PO through activation of the YAP-TEAD1-OSM positive feedback mechanism.

Ankle-brachial index measured by oscillometry is predictive for cardiovascular disease and premature death in the Japanese population: An individual participant data meta-analysis.

BACKGROUND AND AIMS: The ankle-brachial index (ABI) is a predictor of cardiovascular disease (CVD) and premature death. However, few studies on this marker are available in the general Asian populations. This study aimed to investigate the association between ABI measured with oscillometry and the risk of these outcomes. METHODS: We conducted an individual participant data meta-analysis in 10,679 community-dwelling Japanese individuals without a history of CVD. The primary outcome was a composite of CVD events and all-cause mortality. RESULTS: During an average of 7.8 years of follow-up, 720 participants experienced the primary outcome. The multivariable-adjusted hazard ratios (HRs) of the primary outcome significantly increased with a lower ABI. The HRs were 1.07 (95% confidence interval [CI] 0.91-1.27) for ABI of 1.00-1.09, HR 1.37 (95% CI 1.04-1.81) for ABI of 0.91-0.99, and HR 1.60 (95% CI 1.06-2.41) for ABI of ≤0.90, compared with ABI of 1.10-1.19. Furthermore, a high ABI (≥1.30) was associated with a greater risk of outcome (HR 2.42 [95% CI 1.14-5.13]). Similar tendencies were observed for CVD events alone and all-cause mortality alone. Addition of ABI to a model with the Framingham risk score marginally improved the c-statistics (p = 0.08) and integrated discrimination improvement (p < 0.05) for the primary outcome. CONCLUSIONS: The present study suggests that lower and higher ABI are significantly associated with an increased risk of CVD and all-cause mortality in the Japanese population. The ABI, which is easily measured by oscillometry, may be incorporated into daily clinical practice to identify high-risk populations.

Simultaneously Measured Interarm Blood Pressure Difference and Stroke: An Individual Participants Data Meta-Analysis.

We conducted individual participant data meta-analysis to examine the validity of interarm blood pressure difference in simultaneous measurement as a marker to identify subjects with ankle-brachial pressure index <0.90 and to predict future cardiovascular events. We collected individual participant data on 13 317 Japanese subjects from 10 cohorts (general population-based cohorts, cohorts of patients with past history of cardiovascular events, and those with cardiovascular risk factors). Binary logistic regression analysis with adjustments identified interarm blood pressure difference >5 mm Hg as being associated with a significant odds ratio for the presence of ankle-brachial pressure index <0.90 (odds ratio, 2.19; 95% confidence interval, 1.60-3.03; P<0.01). Among 11 726 subjects without a past history of cardiovascular disease, 249 developed stroke during the average follow-up period of 7.4 years. Interarm blood pressure difference >15 mm Hg was associated with a significant Cox stratified adjusted hazard ratio for subsequent stroke (hazard ratio, 2.42; 95% confidence interval, 1.27-4.60; P<0.01). Therefore, interarm blood pressure differences, measured simultaneously in both arms, may be associated with vascular damage in the systemic arterial tree. These differences may be useful for identifying subjects with an ankle-brachial pressure index of <0.90 in the overall study population, and also a reliable predictor of future stroke in subjects without a past history of cardiovascular disease. These findings support the recommendation to measure blood pressure in both arms at the first visit.

Mechanisms of physiological and pathological cardiac hypertrophy.

Cardiomyocytes exit the cell cycle and become terminally differentiated soon after birth. Therefore, in the adult heart, instead of an increase in cardiomyocyte number, individual cardiomyocytes increase in size, and the heart develops hypertrophy to reduce ventricular wall stress and maintain function and efficiency in response to an increased workload. There are two types of hypertrophy: physiological and pathological. Hypertrophy initially develops as an adaptive response to physiological and pathological stimuli, but pathological hypertrophy generally progresses to heart failure. Each form of hypertrophy is regulated by distinct cellular signalling pathways. In the past decade, a growing number of studies have suggested that previously unrecognized mechanisms, including cellular metabolism, proliferation, non-coding RNAs, immune responses, translational regulation, and epigenetic modifications, positively or negatively regulate cardiac hypertrophy. In this Review, we summarize the underlying molecular mechanisms of physiological and pathological hypertrophy, with a particular emphasis on the role of metabolic remodelling in both forms of cardiac hypertrophy, and we discuss how the current knowledge on cardiac hypertrophy can be applied to develop novel therapeutic strategies to prevent or reverse pathological hypertrophy.

Proposed Cutoff Value of Brachial-Ankle Pulse Wave Velocity for the Management of Hypertension.

BACKGROUND: The optimal cutoff values of the brachial-ankle pulse wave velocity (baPWV) for predicting cardiovascular disease (CVD) were examined in patients with hypertension.Methods and Results:A total of 7,656 participants were followed prospectively. The hazard ratio for the development of CVD increased significantly as the baPWV increased, independent of conventional risk factors. The receiver-operating characteristic curve analysis showed that the optimal cutoff values for predicting CVD was 18.3 m/s. This cutoff value significantly predicted THE incidence of CVD. CONCLUSIONS: The present analysis suggests that the optimal cutoff value for CVD in patients with hypertension is 18.3 m/s.

Brachial-Ankle Pulse Wave Velocity and the Risk Prediction of Cardiovascular Disease: An Individual Participant Data Meta-Analysis.

An individual participant data meta-analysis was conducted in the data of 14 673 Japanese participants without a history of cardiovascular disease (CVD) to examine the association of the brachial-ankle pulse wave velocity (baPWV) with the risk of development of CVD. During the average 6.4-year follow-up period, 687 participants died and 735 developed cardiovascular events. A higher baPWV was significantly associated with a higher risk of CVD, even after adjustments for conventional risk factors (P for trend <0.001). When the baPWV values were classified into quintiles, the multivariable-adjusted hazard ratio for CVD increased significantly as the baPWV quintile increased. The hazard ratio in the subjects with baPWV values in quintile 5 versus that in those with the values in quintile 1 was 3.50 (2.14-5.74; P<0.001). Every 1 SD increase of the baPWV was associated with a 1.19-fold (1.10-1.29; P<0.001) increase in the risk of CVD. Moreover, addition of baPWV to a model incorporating the Framingham risk score significantly increased the C statistics from 0.8026 to 0.8131 (P<0.001) and also improved the category-free net reclassification (0.247; P<0.001). The present meta-analysis clearly established baPWV as an independent predictor of the risk of development of CVD in Japanese subjects without preexisting CVD. Thus, measurement of the baPWV could enhance the efficacy of prediction of the risk of development of CVD over that of the Framingham risk score, which is based on the traditional cardiovascular risk factors.