Evaluation of Long-Chain Fatty Acid Respiration in Neonatal Mouse Cardiomyocytes Using SeaHorse Instrument

Metabolic switches play a critical role in the pathophysiology of cardiac diseases, including heart failure. Here, we describe an assay for long-chain fatty acid oxidation in neonatal mouse cardiomyocytes by using a SeaHorse Flux Analyzer (Agilent). This protocol is a simplified but robust adaptation of the standard protocol that enables metabolic measurements in cells isolated from transgenic mouse models, which can be timesaving and informative. Cell isolation and culture represent a critical point that may require bench optimization. For complete details on the use and execution of this protocol, please refer to Angelini et al. (2021)

The role of BMPs in endothelial cell function and dysfunction

The bone morphogenetic protein (BMP) family of proteins has a multitude of roles throughout the body. In embryonic development, BMPs promote endothelial specification and subsequent venous differentiation. The BMP pathway also plays important roles in the adult vascular endothelium, promoting angiogenesis and mediating shear and oxidative stress. The canonical BMP pathway functions through the Smad transcription factors; however, other intracellular signaling cascades can be activated, and receptor complexes beyond the traditional type I and type II receptors add additional layers of regulation. Dysregulated BMP signaling has been linked to vascular diseases, including pulmonary hypertension and atherosclerosis. This review addresses recent advances in the roles of BMP signaling in the endothelium and how BMPs affect endothelial dysfunction and human disease

Crat Links Cholesterol Metabolism to Innate Immune Responses in the Heart

Chronic inflammation is associated with increased risk and poor prognosis of heart failure; however, the precise mechanism that provokes sustained inflammation in the failing heart remains elusive. Here we report that depletion of carnitine acetyltransferase (CRAT) promotes cholesterol catabolism through bile acid synthesis pathway in cardiomyocytes. Intracellular accumulation of bile acid or intermediate, 7α-hydroxyl-3-oxo-4-cholestenoic acid, induces mitochondrial DNA stress and triggers cGAS-STING-dependent type I interferon responses. Furthermore, type I interferon responses elicited by CRAT deficiency substantially increase AIM2 expression and AIM2-dependent inflammasome activation. Genetic deletion of cardiomyocyte CRAT in mice of both sexes results in myocardial inflammation and dilated cardiomyopathy, which can be reversed by combined depletion of caspase-1, cGAS or AIM2. Collectively, we identify a mechanism by which cardiac energy metabolism, cholesterol homeostasis and cardiomyocyte-intrinsic innate immune responses are interconnected via a CRAT-mediated bile acid synthesis pathway, which contributes to chronic myocardial inflammation and heart failure progression

A KLF2-BMPER-Smad1/5 checkpoint regulates high fluid shear stress-mediated artery remodeling

Vascular remodeling to match arterial diameter to tissue requirements commonly fails in ischemic disease. Endothelial cells sense fluid shear stress (FSS) from blood flow to maintain FSS within a narrow range in healthy vessels. Thus, high FSS induces vessel outward remodeling, but mechanisms are poorly understood. We previously reported that Smad1/5 is maximally activated at physiological FSS. Smad1/5 limits Akt activation, suggesting that inhibiting Smad1/5 may facilitate outward remodeling. Here we report that high FSS suppresses Smad1/5 by elevating KLF2, which induces the bone morphogenetic protein (BMP) pathway inhibitor, BMP-binding endothelial regulator (BMPER), thereby de-inhibiting Akt. In mice, surgically induced high FSS elevated BMPER expression, inactivated Smad1/5 and induced vessel outward remodeling. Endothelial BMPER deletion impaired blood flow recovery and vascular remodeling. Blocking endothelial cell Smad1/5 activation with BMP9/10 blocking antibodies improved vascular remodeling in mouse models of type 1 and type 2 diabetes. Suppression of Smad1/5 is thus a potential therapeutic approach for ischemic disease

The Role of Oxygen Sensors, Hydroxylases, and HIF in Cardiac Function and Disease

Ischemic heart disease is the leading cause of death worldwide. Oxygen-sensing proteins are critical components of the physiological response to hypoxia and reperfusion injury, but the role of oxygen and oxygen-mediated effects is complex in that they can be cardioprotective or deleterious to the cardiac tissue. Over 200 oxygen-sensing proteins mediate the effects of oxygen tension and use oxygen as a substrate for posttranslational modification of other proteins. Hydroxylases are an essential component of these oxygen-sensing proteins. While a major role of hydroxylases is regulating the transcription factor HIF, we investigate the increasing scope of hydroxylase substrates. This review discusses the importance of oxygen-mediated effects in the heart as well as how the field of oxygen-sensing proteins is expanding, providing a more complete picture into how these enzymes play a multifaceted role in cardiac function and disease. We also review how oxygen-sensing proteins and hydroxylase function could prove to be invaluable in drug design and therapeutic targets for heart disease

PHDs/CPT1B/VDAC1 Axis Regulates Long-Chain Fatty Acid Oxidation in Cardiomyocytes

Cardiac metabolism is a high-oxygen-consuming process, showing a preference for long-chain fatty acid (LCFA) as the fuel source under physiological conditions. However, a metabolic switch (favoring glucose instead of LCFA) is commonly reported in ischemic or late-stage failing hearts. The mechanism regulating this metabolic switch remains poorly understood. Here, we report that loss of PHD2/3, the cellular oxygen sensors, blocks LCFA mitochondria uptake and β-oxidation in cardiomyocytes. In high-fat-fed mice, PHD2/3 deficiency improves glucose metabolism but exacerbates the cardiac defects. Mechanistically, we find that PHD2/3 bind to CPT1B, a key enzyme of mitochondrial LCFA uptake, promoting CPT1B-P295 hydroxylation. Further, we show that CPT1B-P295 hydroxylation is indispensable for its interaction with VDAC1 and LCFA β-oxidation. Finally, we demonstrate that a CPT1B-P295A mutant constitutively binds to VDAC1 and rescues LCFA metabolism in PHD2/3-deficient cardiomyocytes. Together, our data identify an oxygen-sensitive regulatory axis involved in cardiac metabolism

LRP1 Regulates Retinal Angiogenesis by Inhibiting PARP-1 Activity and Endothelial Cell ProliferationSignificance

We recently demonstrated that low-density lipoprotein receptor related protein 1 (LRP1) is required for cardiovascular development in zebrafish. However, what role LRP1 plays in angiogenesis remains to be determined. To better understand the role of LRP1 in endothelial cell function, we investigated how LRP1 regulates mouse retinal angiogenesis

PHD3-dependent hydroxylation of HCLK2 promotes the DNA damage response

The DNA damage response (DDR) is a complex regulatory network that is critical for maintaining genome integrity. Posttranslational modifications are widely used to ensure strict spatiotemporal control of signal flow, but how the DDR responds to environmental cues, such as changes in ambient oxygen tension, remains poorly understood. We found that an essential component of the ATR/CHK1 signaling pathway, the human homolog of the Caenorhabditis elegans biological clock protein CLK-2 (HCLK2), associated with and was hydroxylated by prolyl hydroxylase domain protein 3 (PHD3). HCLK2 hydroxylation was necessary for its interaction with ATR and the subsequent activation of ATR/CHK1/p53. Inhibiting PHD3, either with the pan-hydroxylase inhibitor dimethyloxaloylglycine (DMOG) or through hypoxia, prevented activation of the ATR/CHK1/p53 pathway and decreased apoptosis induced by DNA damage. Consistent with these observations, we found that mice lacking PHD3 were resistant to the effects of ionizing radiation and had decreased thymic apoptosis, a biomarker of genomic integrity. Our identification of HCLK2 as a substrate of PHD3 reveals the mechanism through which hypoxia inhibits the DDR, suggesting hydroxylation of HCLK2 is a potential therapeutic target for regulating the ATR/CHK1/p53 pathway

Sequential roles for myosin-X in BMP6-dependent filopodial extension, migration, and activation of BMP receptors

Endothelial cell migration is an important step during angiogenesis, and its dysregulation contributes to aberrant neovascularization. The bone morphogenetic proteins (BMPs) are potent stimulators of cell migration and angiogenesis. Using microarray analyses, we find that myosin-X (Myo10) is a BMP target gene. In endothelial cells, BMP6-induced Myo10 localizes in filopodia, and BMP-dependent filopodial assembly decreases when Myo10 expression is reduced. Likewise, cellular alignment and directional migration induced by BMP6 are Myo10 dependent. Surprisingly, we find that Myo10 and BMP6 receptor ALK6 colocalize in a BMP6-dependent fashion. ALK6 translocates into filopodia after BMP6 stimulation, and both ALK6 and Myo10 possess intrafilopodial motility. Additionally, Myo10 is required for BMP6-dependent Smad activation, indicating that in addition to its function in filopodial assembly, Myo10 also participates in a requisite amplification loop for BMP signaling. Our data indicate that Myo10 is required to guide endothelial migration toward BMP6 gradients via the regulation of filopodial function and amplification of BMP signals