We determined whether high fatty acid oxidation rates during aerobic reperfusion of ischemic hearts could be explained by a decrease in malonyl-CoA levels, which would relieve inhibition of carnitine palmitoyl-transferase 1, the rate-limiting enzyme involved in mitochondrial uptake of fatty acids. Isolated working rat hearts perfused with 1.2 mM palmitate were subjected to 30 min of global ischemia, followed by 60 min of aerobic reperfusion. Fatty acid oxidation rates during reperfusion were 136% higher than rates seen in aerobically perfused control hearts, despite the fact that cardiac work recovered to only 16% of pre-ischemic values. Neither the activity of carnitine palmitoyltransferase 1, or the IC50 value of malonyl-CoA for carnitine palmitoyl-transferase 1 were altered in mitochondria isolated from aerobic, ischemic, or reperfused ischemic hearts. Levels of malonyl-CoA were extremely low at the end of reperfusion compared to levels seen in aerobic controls, as was the activity of acetyl-CoA carboxylase, the enzyme which produces malonyl-CoA. The activity of 5'-AMP-activated protein kinase, which has been shown to phosphorylate and inactivate acetyl-CoA carboxylase in other tissues, was significantly increased at the end of ischemia, and remained elevated throughout reperfusion. These results suggest that accumulation of 5'-AMP during ischemia results in an activation of AMP-activated protein kinase, which phosphorylates and inactivates ACC during reperfusion. The subsequent decrease in malonyl-CoA levels wil result in accelerated fatty acid oxidation rates during reperfusion of ischemic hearts.

In the heart, insulin stimulates a variety of kinase cascades and controls glucose utilization. Because insulin is able to activate Akt and inactivate AMP-activated protein kinase (AMPK) in the heart, we hypothesized that Akt can regulate the activity of AMPK. To address the potential existence of this novel signaling pathway, we used a number of experimental protocols to activate Akt in cardiac myocytes and monitored the activation status of AMPK. Mouse hearts perfused in the presence of insulin demonstrated accelerated glycolysis and glucose oxidation rates as compared with non-insulin-perfused hearts. In addition, insulin caused an increase in Akt phosphorylation and a decrease in AMPK phosphorylation at its major regulatory site (threonine 172 of the ␣ catalytic subunit). Transgenic mice overexpressing a constitutively active mutant form of Akt1 displayed decreased phosphorylation of cardiac ␣-AMPK. Isolated neonatal cardiac myocytes infected with an adenovirus expressing constitutively active mutant forms of either Akt1 or Akt2 also suppressed AMPK phosphorylation. However, Akt-dependent depression of ␣-AMPK phosphorylation could be overcome in the presence of the AMPK activator, metformin, suggesting that an override mechanism exists that can restore AMPK activity. Taken together, this study suggests that there is crosstalk between the AMPK and Akt pathways and that Akt activation can lead to decreased AMPK activity. In addition, our data suggest that the ability of insulin to inhibit AMPK may be controlled via an Akt-mediated mechanism.The insulin signaling cascade is an important signaling pathway responsible for controlling substrate preference in the heart. Indeed, insulin has been shown to stimulate myocardial glucose uptake (1, 2) and accelerate glycolysis (2, 3) and glucose oxidation rates (2, 4) as well as promote glycogen synthesis (5, 6). These effects of insulin on glucose metabolism are particularly important because a significant amount of glucose-derived ATP is used for maintaining proper cardiac function. In addition, the stimulation of myocardial glucose utilization and subsequent decrease in fatty acid oxidation has been proven to be efficacious in reducing ischemic injury (7-11). Moreover, insulin-stimulated glucose utilization may be a central component of the beneficial effects of glucose-insulin-potassium therapy on the ischemic heart (12). Two kinases that can be regulated by insulin are Akt and AMPK. 1 Akt is a serine/threonine protein kinase that can be activated by insulin via a multistep pathway involving a phosphatidylinositol 3-kinase-dependent mechanism (see Ref. 13 for review). Once Akt is phosphorylated and activated, it can promote glucose uptake and subsequent metabolism via translocation of glucose transporter (GLUT) 4 to the plasma membrane (14 -18). In addition, the phosphorylation and inhibition of glycogen synthase kinase (GSK) 3 by Akt activates glycogen synthase and thereby promotes glycogen synthesis (19). Recent evidence in heart indicates that the presence of...

Malonyl-CoA is a potent inhibitor of fatty acid uptake into the mitochondria. Although the synthesis of malonyl-CoA in the heart by acetyl-CoA carboxylase (ACC) has been well characterized, no information is available as to how malonyl-CoA is degraded. We demonstrate that malonyl-CoA decarboxylase (MCD) activity is present in the heart. Partial purification revealed a protein of ∼50 kDa. The role of MCD in regulating fatty acid oxidation was also studied using isolated, perfused hearts from newborn rabbits and adult rats. Fatty acid oxidation in rabbit hearts increased dramatically between 1 day and 7 days after birth, which was accompanied by a decrease in both ACC activity and malonyl-CoA levels and a parallel increase in MCD activity. When adult rat hearts were aerobically reperfused after a 30-min period of no-flow ischemia, levels of malonyl-CoA decreased dramatically, which was accompanied by a decrease in ACC activity, a maintained MCD activity, and an increase in fatty acid oxidation rates. Taken together, our data suggest that the heart has an active MCD that has an important role in regulating fatty acid oxidation rates.

Background: Sodium/glucose co-transporter 2 (SGLT2) inhibitors exert robust cardioprotective effects against heart failure in diabetes patients and there is intense interest to identify the underlying molecular mechanisms that afford this protection. As the induction of the late component of the cardiac sodium channel current (late-I Na ) is involved in the etiology of heart failure, we investigated whether these drugs inhibit late-I Na . Methods: Electrophysiological, in silico molecular docking, molecular, calcium imaging and whole heart perfusion techniques were employed to address this question. Results: The SGLT2 inhibitor empagliflozin reduced late-I Na in cardiomyocytes from mice with heart failure and in cardiac Nav1.5 sodium channels containing the LQT3 mutations R1623Q or ∆KPQ. Empagliflozin, dapagliflozin and canagliflozin are all potent and selective inhibitors of H 2 O 2 -induced late-I Na (IC 50s = 0.79, 0.58 and 1.26 µM respectively) with little effect on peak-I Na . In mouse cardiomyocytes, empagliflozin reduced the incidence of spontaneous calcium transients induced by the late-I Na activator veratridine in a similar manner to tetrodotoxin, ranolazine and lidocaine. The putative binding sites for empagliflozin within Nav1.5 were investigated by simulations of empagliflozin docking to a 3D homology model of human Nav1.5 and point mutagenic approaches. Our results indicate that empagliflozin binds to Nav1.5 in the same region as local anaesthetics and ranolazine. In an acute model of myocardial injury, perfusion of isolated mouse hearts with empagliflozin or tetrodotoxin prevented activation of the cardiac NLRP3 inflammasome and improved functional recovery after ischemia. Conclusions: Our results provide evidence that late-I Na may be an important molecular target in the heart for the SGLT2 inhibitors, contributing to their unexpected cardioprotective effects.

Acetyl-CoA carboxylase (ACC) is regarded in liver and adipose tissue to be the rate-limiting enzyme for fatty acid biosynthesis; however, in heart tissue it functions as a regulator of fatty acid oxidation. Because the control of fatty acid oxidation is important to the functioning myocardium, the regulation of ACC is a key issue. Two cardiac isoforms of ACC exist, with molecular masses of 265 kDa and 280 kDa (ACC265 and ACC280). In this study, these proteins were purified from rat heart and used in subsequent phosphorylation and immunoprecipitation experiments. Our results demonstrate that 5 H AMP-activated protein kinase (AMPK) is able to phosphorylate both ACC265 and ACC280, resulting in an almost complete loss of ACC activity. Although cAMP-dependent protein kinase phosphorylated only ACC280, a dramatic loss of ACC activity was still observed, suggesting that ACC280 contributes most, if not all, of the total heart ACC activity. ACC280 and ACC265 copurified under all experimental conditions, and purification of heart ACC also resulted in the specific copurification of the a 2 isoform of the catalytic subunit of AMPK. Although both catalytic subunits of AMPK were expressed in crude heart homogenates, our results suggest that a 2 , and not a 1 , is the dominant isoform of AMPK catalytic subunit regulating ACC in the heart. Immunoprecipitation studies demonstrated that specific antibodies for both ACC265 and ACC280 were able to coimmunoprecipitate the alternate isoform along with the a 2 isoform of AMPK. Taken together, the immunoprecipitation and the purification studies suggest that the two isoforms of ACC in the heart exist in a heterodimeric structure, and that this structure is tightly associated with the a 2 subunit of AMPK.