Acute insulin withdrawal contributes to ischemic heart failure in spontaneously diabetic BB Wistar rats

The contribution of poor metabolic control to myocardial ischemic failure was determined in isolated working hearts from insulin-dependent BB Wistar rats. Removal of insulin treatment 24 h prior to study (uncontrolled diabetic rats) resulted in significant increases in serum glucose, serum fatty acids, and myocardial triglyceride, compared with animals in which insulin treatment was not withheld (insulin-treated diabetic rats). Isolated working hearts obtained from these two groups were subjected to a 40% reduction in coronary flow in the presence of a maintained metabolic demand (hearts were paced at 200 beats/min and perfused at an 80 mmHg (1 mmHg = 133.3 Pa) left aortic afterload, 11.5 mmHg left atrial preload). Within 15 min of ischemia, a significant deterioration of mechanical function occurred in the uncontrolled diabetic rats, whereas function was maintained in the insulin-treated diabetic rats. Oxygen consumption by the two groups of hearts was similar prior to the onset of ischemia and decreased during ischemia in parallel with the work performed by the hearts. This suggests that the accelerated failure rate in uncontrolled diabetic rat hearts is unlikely a result of an increased oxygen requirement. These data are a direct demonstration that acute changes in metabolic control of the diabetic can contribute to the severity of myocardial ischemic injury.     

High-affinity prostaglandin E receptors attenuate adenylyl cyclase activity in isolated bovine myometrial membrane

Purified bovine myometrial plasma membranes were used to characterize prostaglandin (PG) E2 binding. Two binding sites were found: a high-affinity site with a dissociation constant (KD) of 0.27 +/- 0.08 nM and maximum binding (Bmax) of 102.46 +/- 8.6 fmol/mg membrane protein, and a lower affinity site with a KD = 6.13 +/- 0.50 nM and Bmax = 467.93 +/- 51.63 fmol/mg membrane protein. Membrane characterization demonstrated that [3H]PGE2 binding was localized in the plasma membrane. In binding competition experiments, unlabelled PGE1 displaced [3H]PGE2 from its receptor at the same concentrations as did PGE2. Neither PGF2 alpha nor PGD2 effectively competed for [3H]PGE2 binding. Adenylyl cyclase activity was inhibited at concentrations of PGE2 that occupy the high-affinity receptor. These data demonstrate that two receptor sites, or states of binding within a single receptor, are present for PGE2 in purified myometrial membranes. PGE2 inhibition of adenylyl cyclase activity support the view that cAMP has a physiological role in the regulation of myometrial contractility by PGE2.     

Actin Disruption by Latrunculin B Causes Turgor-Related Changes in Tip Growth of Saprolegnia ferax Hyphae

Hyphae of Saprolegnia ferax growing under normal or low-turgor conditions were exposed to 0.1-10 &mgr;g/ml latrunculin B, an actin inhibitor. In the first 10 s of addition, hyphae with normal turgor levels accelerated while those with low turgor decelerated, consistent with the suggestion that actin restrains or protrudes tips under these respective turgor conditions. Both sets of hyphae then decelerated and eventually ceased extension within 60 s. These changes were reflected in rhodamine-phalloidin staining patterns, which showed that actin caps were disrupted progressively under both conditions in a time-dependent manner. After 60 s, normal-turgored hyphae started to swell rapidly while low-turgored hyphae showed little or no swelling. Swelling was characteristically subapical, which is best explained by tip growth models which incorporate actin-mediated exocytosis.     

Alpha-catalytic subunits of 5'AMP-activated protein kinase display fiber-specific expression and are upregulated by chronic low-frequency stimulation in rat muscle

5'-AMP-activated protein kinase (AMPK) signaling initiates adaptive changes in skeletal muscle fibers that restore homeostatic energy balance. The purpose of this investigation was to examine, in rats, the fiber-type protein expression patterns of the alpha-catalytic subunit isoforms in various skeletal muscles, and changes in their respective contents within the tibialis anterior (TA) after chronic low-frequency electrical stimulation (CLFS; 10 Hz, 10 h daily), applied for 4 +/- 1.2 or 25 +/- 4.8 days. Immunocytochemical staining of soleus (SOL) and medial gastrocnemius (MG) showed that 86 +/- 4.1 to 97 +/- 1.4% of type IIA fibers stained for both the alpha1- and alpha2-isoforms progressively decreased to 63 +/- 12.2% of type IID/X and 9 +/- 2.4% of IIB fibers. 39 +/- 11.4% of IID/X and 83 +/- 7.9% of IIB fibers expressed only the alpha2 isoform in the MG, much of which was localized within nuclei. alpha1 and alpha2 contents, assessed by immunoblot, were lowest in the white gastrocnemius [WG; 80% myosin heavy chain (MHC) IIb; 20% MHCIId/x]. Compared with the WG, alpha1 content was 1.6 +/- 0.08 (P < 0.001) and 1.8 +/- 0.04 (P < 0.0001)-fold greater in the red gastrocnemius (RG: 13%, MHCIIa) and SOL (21%, MHCIIa), respectively, and increased in proportion to MHCIIa content. Similarly, alpha2 content was 1.4 +/- 0.10 (P < 0.02) and 1.5 +/- 0.07 (P < 0.001)-fold greater in RG and SOL compared with WG. CLFS induced 1.43 +/- 0.13 (P < 0.007) and 1.33 +/- 0.08 (P < 0.009)-fold increases in the alpha1 and alpha2 contents of the TA and coincided with the transition of faster type IIB and IID/X fibers toward IIA fibers. These findings indicate that fiber types differ with regard to their capacity for AMPK signaling and that this potential is increased by CLFS.     

Targeting fatty acid and carbohydrate oxidation--a novel therapeutic intervention in the ischemic and failing heart

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.     

Resveratrol prevents hypertension and cardiac hypertrophy in hypertensive rats and mice

Resveratrol (RESV) is a polyphenol with pleiotropic effects that include reduction of oxidative stress and increased vascular nitric oxide (NO) production. However, whether or not RESV can prevent rises in blood pressure (BP) is controversial and remains to be firmly established. The purpose of this study was to determine whether RESV attenuates elevated BP and subsequent adaptive cardiac hypertrophy and to better understand the mechanisms involved. The spontaneously hypertensive rat (SHR) and the angiotensin (Ang)-II infused mouse were used as hypertensive models. Compared to a standard control diet, consumption of diets containing RESV by SHRs and Ang-II hypertensive mice, markedly prevented rises in systolic BP. In addition, flow-mediated vasodilation was significantly improved by RESV in SHRs. RESV also reduced serum and cardiac levels of the lipid peroxidation by-product, 4-hydroxy-2-nonenal in the hypertensive rodents and inhibited the production of superoxide in human-derived endothelial cells. Analysis of mesenteric arteries from SHRs and Ang-II infused mice demonstrated that RESV increased endothelial NO synthase (eNOS) phosphorylation by enhancing the LKB1/adenosine monophosphate (AMP)-activated protein kinase (AMPK) signal transduction pathway. Moreover, RESV reduced hypertrophic growth of the myocardium through reduced hemodynamic load and inhibition of the p70 S6 kinase pro-hypertrophic signaling cascade. Overall, we show that high dose RESV reduces oxidative stress, improves vascular function, attenuates high BP and prevents cardiac hypertrophy through the preservation of the LKB1–AMPK–eNOS signaling axis.     

Mechanisms responsible for enhanced fatty acid utilization by perfused hearts from type 2 diabetic db/db mice

The aim of this study was to determine the biochemical mechanism(s) responsible for enhanced FA utilization (oxidation and esterification) by perfused hearts from type 2 diabetic db/db mice. The plasma membrane content of fatty acid transporters FAT/CD36 and FABPpm was elevated in db/db hearts. Mitochondrial mechanisms that could contribute to elevated rates of FA oxidation were also examined. Carnitine palmitoyl transferase-1 activity was unchanged in mitochondria from db/db hearts, and sensitivity to inhibition by malonyl-CoA was unchanged. Malonyl-CoA content was elevated and AMP kinase activity was decreased in db/db hearts, opposite to what would be expected in hearts exhibiting elevated rates of FA oxidation. Uncoupling protein-3 expression was unchanged in mitochondria from db/db hearts. Therefore, enhanced FA utilization in db/db hearts is most likely due to increased FA uptake caused by increased plasma membrane content of FA transporters; the mitochondrial mechanisms examined do not contribute to elevated FA oxidation observed in db/db hearts.     

Na+/H+ Exchanger Isoform 1-Induced Osteopontin Expression Facilitates Cardiomyocyte Hypertrophy

Enhanced expression and activity of the Na⁺/H⁺ exchanger isoform 1 (NHE1) has been implicated in cardiomyocyte hypertrophy in various experimental models. The upregulation of NHE1 was correlated with an increase in osteopontin (OPN) expression in models of cardiac hypertrophy (CH), and the mechanism for this remains to be delineated. To determine whether the expression of active NHE1-induces OPN and contributes to the hypertrophic response in vitro, cardiomyocytes were infected with the active form of the NHE1 adenovirus or transfected with OPN silencing RNA (siRNA-OPN) and characterized for cardiomyocyte hypertrophy. Expression of NHE1 in cardiomyocytes resulted in a significant increase in cardiomyocyte hypertrophy markers: cell surface area, protein content, ANP mRNA and expression of phosphorylated-GATA4. NHE1 activity was also significantly increased in cardiomyocytes expressing active NHE1. Interestingly, transfection of cardiomyocytes with siRNA-OPN significantly abolished the NHE1-induced cardiomyocyte hypertrophy. siRNA-OPN also significantly reduced the activity of NHE1 in cardiomyocytes expressing NHE1 (68.5±0.24%; P<0.05), confirming the role of OPN in the NHE1-induced hypertrophic response. The hypertrophic response facilitated by NHE1-induced OPN occurred independent of the extracellular-signal-regulated kinases and Akt, but required p90-ribosomal S6 kinase (RSK). The ability of OPN to facilitate the NHE1-induced hypertrophic response identifies OPN as a potential therapeutic target to reverse the hypertrophic effect induced by the expression of active NHE1.     

Active muscle and whole body lactate kinetics after endurance training in men.

We evaluated the hypotheses that endurance training decreases arterial lactate concentration ([lactate](a)) during continuous exercise by decreasing net lactate release () and appearance rates (R(a)) and increasing metabolic clearance rate (MCR). Measurements were made at two intensities before [45 and 65% peak O(2) consumption (VO(2 peak))] and after training [65% pretraining VO(2 peak), same absolute workload (ABT), and 65% posttraining VO(2 peak), same relative intensity (RLT)]. Nine men (27.4 +/- 2.0 yr) trained for 9 wk on a cycle ergometer, 5 times/wk at 75% VO(2 peak). Compared with the 65% VO(2 peak) pretraining condition (4.75 +/- 0.4 mM), [lactate](a) decreased at ABT (41%) and RLT (21%) (P < 0.05). decreased at ABT but not at RLT. Leg lactate uptake and oxidation were unchanged at ABT but increased at RLT. MCR was unchanged at ABT but increased at RLT. We conclude that 1) active skeletal muscle is not solely responsible for elevated [lactate](a); and 2) training increases leg lactate clearance, decreases whole body and leg lactate production at a given moderate-intensity power output, and increases both whole body and leg lactate clearance at a high relative power output.