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Tissue engineers and stem cell biologists have made exciting progress toward creating simplified models of human heart muscles or aligned monolayers to help bridge a longstanding gap between experimental animals and clinical trials. However, no existing human in vitro systems provide the direct measures of cardiac performance as a pump. Here, we developed a next-generation in vitro biomimetic model of pumping human heart chamber, and demonstrated its capability for pharmaceutical testing. From human pluripotent stem cell (hPSC)-derived ventricular cardiomyocytes (hvCM) embedded in collagen-based extracellular matrix hydrogel, we engineered a three-dimensional (3D) electro-mechanically coupled, fluid-ejecting miniature human ventricle-like cardiac organoid chamber (hvCOC). Structural characterization showed organized sarcomeres with myofibrillar microstructures. Transcript and RNA-seq analyses revealed upregulation of key Ca 2+ -handling, ion channel, and cardiac-specific proteins in hvCOC compared to lower-order 2D and 3D cultures of the same constituent cells. Clinically-important, physiologically complex contractile parameters such as ejection fraction, developed pressure, and stroke work, as well as electrophysiological properties including action potential and conduction velocity were measured: hvCOC displayed key molecular and physiological characteristics of the native ventricle, and showed expected mechanical and electrophysiological responses to a range of pharmacological interventions (including positive and negative inotropes). We conclude that such "human-heart-in-a-jar" technology could facilitate the drug discovery process by providing human-specific preclinical data during early stage drug development., (Copyright © 2018. Published by Elsevier Ltd.)

Rationale: Post-ischemic contractile dysfunction is a contributor to morbidity and mortality after the surgical correction of congenital heart defects in neonatal patients. Pre-existing hypertrophy in the newborn heart can exacerbate these ischemic injuries, which may partly be due to a decreased energy supply to the heart resulting from low fatty acid β-oxidation rates., Objective: We determined whether stimulating fatty acid β-oxidation with GW7647, a peroxisome proliferator-activated receptor-α (PPARα) activator, would improve cardiac energy production and post-ischemic functional recovery in neonatal rabbit hearts subjected to volume overload-induced cardiac hypertrophy., Methods and Results: Volume-overload cardiac hypertrophy was produced in 7-day-old rabbits via an aorto-caval shunt, after which, the rabbits were treated with or without GW7647 (3 mg/kg per day) for 14 days. Biventricular working hearts were subjected to 35 minutes of aerobic perfusion, 25 minutes of global no-flow ischemia, and 30 minutes of aerobic reperfusion. GW7647 treatment did not prevent the development of cardiac hypertrophy, but did prevent the decline in left ventricular ejection fraction in vivo. GW7647 treatment increased cardiac fatty acid β-oxidation rates before and after ischemia, which resulted in a significant increase in overall ATP production and an improved in vitro post-ischemic functional recovery. A decrease in post-ischemic proton production and endoplasmic reticulum stress, as well as an activation of sarcoplasmic reticulum calcium ATPase isoform 2 and citrate synthase, was evident in GW7647-treated hearts., Conclusions: Stimulating fatty acid β-oxidation in neonatal hearts may present a novel cardioprotective intervention to limit post-ischemic contractile dysfunction., (© 2015 American Heart Association, Inc.)

Aims: During reperfusion of the ischaemic myocardium, fatty acid oxidation rates quickly recover, while glucose oxidation rates remain depressed. Direct stimulation of glucose oxidation via activation of pyruvate dehydrogenase (PDH), or secondary to an inhibition of malonyl CoA decarboxylase (MCD), improves cardiac functional recovery during reperfusion following ischaemia. However, the effects of such interventions on the evolution of myocardial infarction are unknown. The purpose of this study was to determine whether infarct size is decreased in response to increased glucose oxidation., Methods and Results: In vivo, direct stimulation of PDH in mice with the PDH kinase (PDHK) inhibitor, dichloroacetate, significantly decreased infarct size following temporary ligation of the left anterior descending coronary artery. These results were recapitulated in PDHK 4-deficient (PDHK4-/-) mice, which have enhanced myocardial PDH activity. These interventions also protected against ischaemia/reperfusion injury in the working heart, and dichloroacetate failed to protect in PDHK4-/- mice. In addition, there was a dramatic reduction in the infarct size in malonyl CoA decarboxylase-deficient (MCD-/-) mice, in which glucose oxidation rates are enhanced (secondary to an inhibition of fatty acid oxidation) relative to their wild-type littermates (10.8 ± 3.8 vs. 39.5 ± 4.7%). This cardioprotective effect in MCD-/- mice was associated with increased PDH activity in the ischaemic area at risk (1.89 ± 0.18 vs. 1.52 ± 0.05 μmol/g wet weight/min)., Conclusion: These findings demonstrate that stimulating glucose oxidation via targeting either PDH or MCD decreases the infarct size, validating the concept that optimizing myocardial metabolism is a novel therapy for ischaemic heart disease.

Cardiac ischemia and its consequences including heart failure, which itself has emerged as the leading cause of morbidity and mortality in developed countries are accompanied by complex alterations in myocardial energy substrate metabolism. In contrast to the normal heart, where fatty acid and glucose metabolism are tightly regulated, the dynamic relationship between fatty acid β-oxidation and glucose oxidation is perturbed in ischemic and ischemic-reperfused hearts, as well as in the failing heart. These metabolic alterations negatively impact both cardiac efficiency and function. Specifically there is an increased reliance on glycolysis during ischemia and fatty acid β-oxidation during reperfusion following ischemia as sources of adenosine triphosphate (ATP) production. Depending on the severity of heart failure, the contribution of overall myocardial oxidative metabolism (fatty acid β-oxidation and glucose oxidation) to adenosine triphosphate production can be depressed, while that of glycolysis can be increased. Nonetheless, the balance between fatty acid β-oxidation and glucose oxidation is amenable to pharmacological intervention at multiple levels of each metabolic pathway. This review will focus on the pathways of cardiac fatty acid and glucose metabolism, and the metabolic phenotypes of ischemic and ischemic/reperfused hearts, as well as the metabolic phenotype of the failing heart. Furthermore, as energy substrate metabolism has emerged as a novel therapeutic intervention in these cardiac pathologies, this review will describe the mechanistic bases and rationale for the use of pharmacological agents that modify energy substrate metabolism to improve cardiac function in the ischemic and failing heart. This article is part of a Special Issue entitled: Mitochondria and Cardioprotection., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Leptin directly acts on peripheral tissues and alters energy metabolism in obese mice. It also has acute beneficial effects on these tissues via its hypothalamic action. However, it is not clear what effect chronic intracerebroventrical (ICV) leptin administration has on cardiac energy metabolism. We examined the effects of chronic ICV leptin on glucose and fatty acid metabolism in isolated working hearts from high-fat-fed and low-fat-fed mice. Mice were fed a high-fat (60% calories from fat) or low-fat (10% calories from fat) diet for 8 weeks before ICV leptin (5 [mu]g/d) for 7 days. In low-fat-fed mice, leptin increased glucose oxidation rates in isolated working hearts when compared with control [203 +/- 21 vs. 793 +/- 93 nmol[middle dot](g dry weight)-1[middle dot]min-1]. In high-fat-fed mice leptin inhibited fatty acid oxidation [476 +/- 73 vs. 251 +/- 38 nmol[middle dot](g[middle dot]dry[middle dot]wt)-1[middle dot]min-1]. The increase in glucose oxidation in low-fat-fed mice was accompanied by increased pyruvate dehydrogenase activity. In high-fat-fed mice, leptin increased cardiac malonyl coenzyme A levels, secondary to a decrease in malonyl coenzyme A decarboxylase expression. These results suggest that ICV leptin alters cardiac energy metabolism opposite to its peripheral effects and that these effects differ depending on energy substrate supply to the mice.

Isoproterenol increases phosphorylation of LKB, 5'-AMP-activated protein kinase (AMPK), and acetyl-CoA carboxylase (ACC), enzymes involved in regulating fatty acid oxidation. However, inotropic stimulation selectively increases glucose oxidation in adult hearts. In the neonatal heart, fatty acid oxidation becomes a major energy source, while glucose oxidation remains low. This study tested the hypothesis that increased energy demand imposed by isoproterenol originates from fatty acid oxidation, secondary to increased LKB, AMPK, and ACC phosphorylation. Isolated working hearts from 7-day-old rabbits were perfused with Krebs solution (0.4 mM palmitate, 11 mM glucose, 0.5 mM lactate, and 100 mU/l insulin) with or without isoproterenol (300 nM). Isoproterenol increased myocardial O(2) consumption (in J·g dry wt(-1)·min(-1); 11.0 ± 1.4, n = 8 vs. 7.5 ± 0.8, n = 6, P < 0.05), and the phosphorylation of LKB (in arbitrary density units; 0.87 ± 0.09, n = 6 vs. 0.59 ± 0.08, n = 6, P < 0.05), AMPK (0.82 ± 0.08, n = 6 vs. 0.51 ± 0.06, n = 6, P < 0.05), and ACC-β (1.47 ± 0.14, n = 6 vs. 0.97 ± 0.07, n = 6, P < 0.05), with a concomitant decrease in malonyl-CoA levels (in nmol/g dry wt; 0.9 ± 0.9, n = 8 vs. 7.5 ± 1.3, n = 8, P < 0.05) and increase in palmitate oxidation (in nmol·g dry wt(-1)·min(-1); 272 ± 45, n = 8 vs. 114 ± 9, n = 6, P < 0.05). Glucose and lactate oxidation were increased (in nmol·g dry wt(-1)·min(-1); 253 ± 75, n = 8 vs. 63 ± 15, n = 9, P < 0.05 and 246 ± 43, n = 8 vs. 82 ± 11, n = 6, P < 0.05, respectively), independent of alterations in pyruvate dehydrogenase phosphorylation, but occurred secondary to a decrease in acetyl-CoA content and acetyl-CoA-to-free CoA ratio. As acetyl-CoA levels decrease in response to isoproterenol, despite an acceleration of the rates of palmitate and carbohydrate oxidation, these data suggest net rates of acetyl-CoA utilization exceed the net rates of acetyl-CoA generation.

Fatty acids are a major fuel source used to sustain contractile function in heart and oxidative skeletal muscle. To meet the energy demands of these muscles, the uptake and beta-oxidation of fatty acids must be coordinately regulated in order to ensure an adequate, but not excessive, supply for mitochondrial beta-oxidation. However, imbalance between fatty acid uptake and beta-oxidation has the potential to contribute to muscle insulin resistance. The action of insulin is initiated by binding to its receptor and activation of the intrinsic protein tyrosine kinase activity of the receptor, resulting in the initiation of an intracellular signaling cascade that eventually leads to insulin-mediated alterations in a number of cellular processes, including an increase in glucose transport. Accumulation of fatty acids and lipid metabolites (such as long chain acyl CoA, diacylglycerol, triacylglycerol, and/or ceramide) can lead to alterations in this insulin signaling pathway. An imbalance between fatty acid uptake and oxidation is believed to be responsible for this lipid accumulation, and is thought to be a major cause of insulin resistance in obesity and diabetes, due to lipid accumulation and inhibition of one or more steps in the insulin-signaling cascade. As a result, decreasing muscle fatty acid uptake can improve insulin sensitivity. However, the potential role of increasing fatty acid beta-oxidation in the heart or skeletal muscle in order to prevent cytoplasmic lipid accumulation and decrease insulin resistance is controversial. While increased fatty acid beta-oxidation may lower cytoplasmic lipid accumulation, increasing fatty acid beta-oxidation can decrease muscle glucose metabolism, and incomplete fatty acid oxidation has the potential to also contribute to insulin resistance. In this review, we discuss the proposed mechanisms by which alterations in fatty acid uptake and oxidation contribute to insulin resistance, and how targeting fatty acid uptake and oxidation is a potential therapeutic approach to treat insulin resistance.

During metabolic stress, phosphorylation and activation of 5'-AMP-activated protein kinase (AMPK) becomes a major regulator of cellular energy metabolism in heart and skeletal muscle. Despite this, the upstream regulation of AMPK in both heart and muscle is poorly understood. Recent work has implicated the atypical protein kinase Czeta (PKCzeta) as a regulator of AMPK in endothelial cells via phosphorylation of LKB1, an upstream AMPK kinase (AMPKK). Our goal was to determine the potential role PKCzeta plays in regulating AMPK in cardiac and skeletal muscle. Cultures of H9c2 myocytes (cardiac) and C(2)C(12) myotubes (skeletal muscle) were pretreated with a selective PKCzeta pseudosubstrate peptide inhibitor and treated with various AMPK activating agents to determine whether PKCzeta regulates AMPK. PKCzeta activity was also examined in isolated working rat hearts subjected to ischemia. We show that PKCzeta is not involved in regulating threonine 172 AMPK phosphorylation induced by metformin or phenformin in either cardiac or skeletal muscle cells but is involved in 5-aminoimidazole-4-carboxamine-1-beta-D-ribofuranoside (AICAR)-induced AMPK phosphorylation in cardiac muscle cells. Activation of PKCzeta with high palmitate concentrations is also insufficient to increase AMPK phosphorylation. Furthermore, we show that the ischemia-induced activation of AMPK is not accompanied by increased PKCzeta activity. Finally, we show that PKCzeta may actually be a downstream target of AMPK in skeletal muscle, since adenoviral expression of a dominant-negative mutant of AMPK prevented metformin- and AICAR-induced phosphorylation of PKCzeta. We conclude that PKCzeta plays a very minor role in the regulation of AMPK in cardiac and skeletal muscle and may actually be a downstream target of AMPK in skeletal muscle.

Inhibition of myocardial fatty acid oxidation can improve left ventricular (LV) mechanical efficiency by increasing LV power for a given rate of myocardial energy expenditure. This phenomenon has not been assessed at high workloads in nonischemic myocardium; therefore, we subjected in vivo pig hearts to a high workload for 5 min and assessed whether blocking mitochondrial fatty acid oxidation with the carnitine palmitoyltransferase-I inhibitor oxfenicine would improve LV mechanical efficiency. In addition, the cardiac content of malonyl-CoA (an endogenous inhibitor of carnitine palmitoyltransferase-I) and activity of acetyl-CoA carboxylase (which synthesizes malonyl-CoA) were assessed. Increased workload was induced by aortic constriction and dobutamine infusion, and LV efficiency was calculated from the LV pressure-volume loop and LV energy expenditure. In untreated pigs, the increase in LV power resulted in a 2.5-fold increase in fatty acid oxidation and cardiac malonyl-CoA content but did not affect the activation state of acetyl-CoA carboxylase. The activation state of the acetyl-CoA carboxylase inhibitory kinase AMP-activated protein kinase decreased by 40% with increased cardiac workload. Pretreatment with oxfenicine inhibited fatty acid oxidation by 75% and had no effect on cardiac energy expenditure but significantly increased LV power and LV efficiency (37 +/- 5% vs. 26 +/- 5%, P < 0.05) at high workload. In conclusion, 1) myocardial fatty acid oxidation increases with a short-term increase in cardiac workload, despite an increase in malonyl-CoA concentration, and 2) inhibition of fatty acid oxidation improves LV mechanical efficiency by increasing LV power without affecting cardiac energy expenditure.

Alpha-lipoic acid (ALA) is a naturally occurring enantiomer of lipoic acid and is a cofactor of key metabolic enzyme complexes catalyzing the decarboxylation of alpha-keto acids. It was recently shown that ALA increases insulin sensitivity by activating AMP-activated protein kinase (AMPK) in skeletal muscle. Also, administration of ALA to obese rats increases insulin-stimulated glucose uptake in the whole body. We investigated the metabolic effects of ALA on isolated working rat hearts. ALA (500 microM) stimulated glucose oxidation (157+/-31 nmol.dry wt(-1).min(-1) in control vs 315+/-63 nmol.dry wt(-1).min(-1) in ALA-treated, p<0.05) without affecting glycolysis, lactate oxidation, or palmitate oxidation. Cardiac work was not affected by ALA treatment. The effect of ALA on glucose oxidation was not associated with an activation of AMPK. AMPK activity was 190+/-14 pmol.mg protein(-1).min(-1) in control vs 190+/-16 pmol.mg protein(-1).min(-1) in ALA-treated hearts. This study shows that ALA stimulates glucose oxidation in isolated working rat hearts independent of AMPK activation. The beneficial effects of ALA treatment in diabetic patients may be at least in part related to its effect on glucose metabolism.