Jessica M. Berthiaume

Myocardial insulin resistance induced by high fat feeding in heart failure is associated with preserved contractile function

Previous studies have reported that high fat feeding in mild to moderate heart failure (HF) results in the preservation of contractile function. Recent evidence has suggested that preventing the switch from fatty acid to glucose metabolism in HF may ameliorate dysfunction, and insulin resistance is one potential mechanism for regulating substrate utilization. This study was designed to determine whether peripheral and myocardial insulin resistance exists with HF and/or a high-fat diet and whether myocardial insulin signaling was altered accordingly. Rats underwent coronary artery ligation (HF) or sham surgery and were randomized to normal chow (NC; 14% kcal from fat) or a high-fat diet (SAT; 60% kcal from fat) for 8 wk. HF + SAT animals showed preserved systolic (+dP/dt and stroke work) and diastolic (-dP/dt and time constant of relaxation) function compared with HF + NC animals. Glucose tolerance tests revealed peripheral insulin resistance in sham + SAT, HF + NC, and HF + SAT animals compared with sham + NC animals. PET imaging confirmed myocardial insulin resistance only in HF + SAT animals, with an uptake ratio of 2.3 ± 0.3 versus 4.6 ± 0.7, 4.3 ± 0.4, and 4.2 ± 0.6 in sham + NC, sham + SAT, and HF + NC animals, respectively; the myocardial glucose utilization rate was similarly decreased in HF + SAT animals only. Western blot analysis of insulin signaling protein expression was indicative of cardiac insulin resistance in HF + SAT animals. Specifically, alterations in Akt and glycogen synthase kinase-3β protein expression in HF + SAT animals compared with HF + NC animals may be involved in mediating myocardial insulin resistance. In conclusion, HF animals fed a high-saturated fat exhibited preserved myocardial contractile function, peripheral and myocardial insulin resistance, decreased myocardial glucose utilization rates, and alterations in cardiac insulin signaling. These results suggest that myocardial insulin resistance may serve a cardioprotective function with high fat feeding in mild to moderate HF.

Mitochondrial complex I defect and increased fatty acid oxidation enhance protein lysine acetylation in the diabetic heart

AIMS Cardiomyopathy is a major complication of diabetes. Our study was aimed to identify the sites of mitochondrial dysfunction and delineate its consequences on mitochondrial metabolism in a model of type 1 diabetes. METHODS AND RESULTS Diabetes was induced by streptozotocin injection to male Lewis rats. We found a decrease in mitochondrial biogenesis pathway and electron transport chain complex assembly that targets Complex I. Oxidation of Complex II and long-chain fatty acid substrates support the electron leak and superoxide production. Mitochondrial defects do not limit fatty acid oxidation as the hearts preferred energy source indicating that the diabetic heart has a significant reserve in Complex I- and II-supported ATP production. Both mitochondrial fatty acid oxidation and Complex I defect are responsible for increased protein lysine acetylation despite an unchanged amount of the NAD(+)-dependent mitochondrial deacetylase sirt3. We quantitatively analysed mitochondrial lysine acetylation post-translational modifications and identified that the extent of lysine acetylation on 54 sites in 22 mitochondrial proteins is higher in diabetes compared with the same sites in the control. The increased lysine acetylation of the mitochondrial trifunctional protein subunit α may be responsible for the increased fatty acid oxidation in the diabetic heart. CONCLUSION We identified the specific defective sites in the electron transport chain responsible for the decreased mitochondrial oxidative phosphorylation in the diabetic heart. Mitochondrial protein lysine acetylation is the common consequence of both increased fatty acid oxidation and mitochondrial Complex I defect, and may be responsible for the metabolic inflexibility of the diabetic heart.

The myocardial contractile response to physiological stress improves with high saturated fat feeding in heart failure

Impaired myocardial contractile function is a hallmark of heart failure (HF), which may present under resting conditions and/or during physiological stress. Previous studies have reported that high fat feeding in mild to moderate HF/left ventricular (LV) dysfunction is associated with improved contractile function at baseline. The goal of this study was to determine whether myocardial function is compromised in response to physiological stress and to evaluate the global gene expression profile of rats fed high dietary fat after infarction. Male Wistar rats underwent ligation or sham surgery and were fed normal chow (NC; 10% kcal fat; Sham + NC and HF + NC groups) or high-fat chow (SAT; 60% kcal saturated fat; Sham + SAT and HF + SAT groups) for 8 wk. Myocardial contractile function was assessed using a Millar pressure-volume conductance catheter at baseline and during inferior vena caval occlusions and dobutamine stress. Steady-state indexes of systolic function, LV +dP/dt(max), stroke work, and maximal power were increased in the HF + SAT group versus the HF + NC group and reduced in the HF + NC group versus the Sham + NC group. Preload recruitable measures of contractility were decreased in HF + NC group but not in the HF + SAT group. beta-Adrenergic responsiveness [change in LV +dP/dt(max) and change in cardiac output with dobutamine (0-10 microg x kg(-1) x min(-1))] was reduced in HF, but high fat feeding did not further impact the contractile reserve in HF. The contractile reserve was reduced by the high-fat diet in the Sham + SAT group. Microarray gene expression analysis revealed that the majority of significantly altered pathways identified contained multiple gene targets correspond to cell signaling pathways and energy metabolism. These findings suggest that high saturated fat improves myocardial function at rest and during physiological stress in infarcted hearts but may negatively impact the contractile reserve under nonpathological conditions. Furthermore, high fat feeding-induced alterations in gene expression related to energy metabolism and specific signaling pathways revealed promising targets through which high saturated fat potentially mediates cardioprotection in mild to moderate HF/LV dysfunction.

Novel approach in LC-MS/MS using MRM to generate a full profile of acyl-CoAs: discovery of acyl-dephospho-CoAs

A metabolomic approach to selectively profile all acyl-CoAs was developed using a programmed multiple reaction monitoring (MRM) method in LC-MS/MS and was employed in the analysis of various rat organs. The programmed MRM method possessed 300 mass ion transitions with the mass difference of 507 between precursor ion (Q1) and product ion (Q3), and the precursor ion started from m/z 768 and progressively increased one mass unit at each step. Acyl-dephospho-CoAs resulting from the dephosphorylation of acyl-CoAs were identified by accurate MS and fragmentation. Acyl-dephospho-CoAs were also quantitatively scanned by the MRM method with the mass difference of 427 between Q1 and Q3 mass ions. Acyl-CoAs and dephospho-CoAs were assayed with limits of detection ranging from 2 to 133 nM. The accuracy of the method was demonstrated by assaying a range of concentrations of spiked acyl-CoAs with the results of 80–114%. The distribution of acyl-CoAs reflects the metabolic status of each organ. The physiological role of dephosphorylation of acyl-CoAs remains to be further characterized. The methodology described herein provides a novel strategy in metabolomic studies to quantitatively and qualitatively profile all potential acyl-CoAs and acyl-dephospho-CoAs.

Normalizing the metabolic phenotype after myocardial infarction: impact of subchronic high fat feeding.

The normal heart relies primarily on the oxidation of fatty acids (FA) for ATP production, whereas during heart failure (HF) glucose utilization increases, implying pathological changes to cardiac energy metabolism. Despite the noted lipotoxic effects of elevating FA, our work has demonstrated a cardioprotective effect of a high fat diet (SAT) when fed after myocardial infarction (MI), as compared to normal chow (NC) fed cohorts. This data has suggested a mechanistic link to energy metabolism. The goal of this study was to determine the impact of SAT on the metabolic phenotype of the heart after MI. Male Wistar rats underwent coronary ligation surgery (MI) and were evaluated after 8 weeks of SAT. Induction of MI was verified by echocardiography. LV function assessed by in vivo hemodynamic measurements revealed improvements in the MI-SAT group as compared to MI-NC. Perfused working hearts revealed a decrease in cardiac work in MI-NC that was improved in MI-SAT. Glucose oxidation was increased and FA oxidation decreased in MI-NC compared to shams suggesting an alteration in the metabolic profile that was ameliorated by SAT. (31)P NMR analysis of Langendorff perfused hearts revealed no differences in PCr:ATP indicating no overt energy deficit in MI groups. Phospho-PDH and PDK(4) were increased in MI-SAT, consistent with a shift towards fatty acid oxidation (FAO). Overall, these results support the hypothesis that SAT post-infarction promotes a normal metabolic phenotype that may serve a cardioprotective role in the injured heart.

4-Hydroxy-2(E)-nonenal (HNE) catabolism and formation of HNE adducts are modulated by β oxidation of fatty acids in the isolated rat heart.

We previously reported that a novel metabolic pathway functionally catabolizes 4-hydroxy-2(E)-nonenal (HNE) via two parallel pathways, which rely heavily on β-oxidation pathways. The hypothesis driving this report is that perturbations of β oxidation will alter the catabolic disposal of HNE, favoring an increase in the concentrations of HNE and HNE-modified proteins that may further exacerbate pathology. This study employed Langendorff perfused hearts to investigate the impact of cardiac injury modeled by ischemia/reperfusion and, in a separate set of perfusions, the effects of elevated lipid (typically observed in obesity and type II diabetes) by perfusing with increased fatty acid concentrations (1mM octanoate). During ischemia, HNE concentrations doubled and the glutathione-HNE adduct and 4-hydroxynonanoyl-CoA were increased by 7- and 10-fold, respectively. Under conditions of increased fatty acid, oxidation to 4-hydroxynonenoic acid was sustained; however, further catabolism through β oxidation was nearly abolished. The inhibition of HNE catabolism was not compensated for by other disposal pathways of HNE, rather an increase in HNE-modified proteins was observed. Taken together, this study presents a mechanistic rationale for the accumulation of HNE and HNE-modified proteins in pathological conditions that involve alterations to β oxidation, such as myocardial ischemia, obesity, and high-fat diet-induced diseases.

Changes in myofilament proteins, but not Ca2+ regulation, are associated with a high-fat diet-induced improvement in contractile function in heart failure

Pathological conditions such as diabetes, insulin resistance, and obesity are characterized by elevated plasma and myocardial lipid levels and have been reported to exacerbate the progression of heart failure (HF). Alterations in cardiomyocyte Ca(2+) regulatory properties and myofilament proteins have also been implicated in contractile dysfunction in HF. However, our prior studies reported that high saturated fat (SAT) feeding improves in vivo myocardial contractile function, thereby exerting a cardioprotective effect in HF. Therefore, we hypothesized that SAT feeding improves contractile function by altering Ca(2+) regulatory properties and myofilament protein expression in HF. Male Wistar rats underwent coronary artery ligation (HF) or sham surgery (SH) and were fed normal chow (SHNC and HFNC groups) or a SAT diet (SHSAT and HFSAT groups) for 8 wk. Contractile properties were measured in vivo [echocardiography and left ventricular (LV) cannulation] and in isolated LV cardiomyocytes. In vivo measures of contractility (peak LV +dP/dt and -dP/dt) were depressed in the HFNC versus SHNC group but improved in the HFSAT group. Isolated cardiomyocytes from both HF groups were hypertrophied and had decreased percent cell shortening and a prolonged time to half-decay of the Ca(2+) transient versus the SH group; however, SAT feeding reduced in vivo myocyte hypertrophy in the HFSAT group only. The peak velocity of cell shortening was reduced in the HFNC group but not the HFSAT group and was positively correlated with in vivo contractile function (peak LV +dP/dt). The HFNC group demonstrated a myosin heavy chain (MHC) isoform switch from fast MHC-α to slow MHC-β, which was prevented in the HFSAT group. Alterations in Ca(2+) transients, L-type Ca(2+) currents, and protein expression of sarco(endo)plasmic reticulum Ca(2+)-ATPase and phosphorylated phospholamban could not account for the changes in the in vivo contractile properties. In conclusion, the cardioprotective effects associated with SAT feeding in HF may occur at the level of the isolated cardiomyocyte, specifically involving changes in myofilament function but not sarcoplasmic reticulum Ca(2+) regulatory properties.

Role of Receptor Protein Tyrosine Phosphatase γ in Sensing Extracellular CO2 and HCO3

Regulation of blood pH-critical for virtually every facet of life-requires that the renal proximal tubule (PT) adjust its rate of H(+) secretion (nearly the same as the rate of HCO3 (-) reabsorption, JHCO3 ) in response to changes in blood [CO2] and [HCO3 (-)]. Yet CO2/HCO3 (-) sensing mechanisms remain poorly characterized. Because receptor tyrosine kinase inhibitors render JHCO3 in the PT insensitive to changes in CO2 concentration, we hypothesized that the structural features of receptor protein tyrosine phosphatase-γ (RPTPγ) that are consistent with binding of extracellular CO2 or HCO3 (-) facilitate monitoring of blood CO2/HCO3 (-) concentrations. We now report that PTs express RPTPγ on blood-facing membranes. Moreover, RPTPγ deletion in mice eliminated the CO2 and HCO3 (-) sensitivities of JHCO3 as well as the normal defense of blood pH during whole-body acidosis. Thus, RPTPγ appears to be a novel extracellular CO2/HCO3 (-) sensor critical for pH homeostasis.

MuRF1 mono-ubiquitinates TRα to inhibit T3-induced cardiac hypertrophy in vivo

Thyroid hormone (TH) is recognized for its role in cellular metabolism and growth and participates in homeostasis of the heart. T3 activates pro-survival pathways including Akt and mTOR. Treatment with T3 after myocardial infarction is cardioprotective and promotes elements of physiological hypertrophic response after cardiac injury. Although T3 is known to benefit the heart, very little about its regulation at the molecular level has been described to date. The ubiquitin proteasome system (UPS) regulates nuclear hormone receptors such as estrogen, progesterone, androgen, and glucocorticoid receptors by both degradatory and non-degradatory mechanisms. However, how the UPS regulates T3-mediated activity is not well understood. In this study, we aim to determine the role of the muscle-specific ubiquitin ligase muscle ring finger-1 (MuRF1) in regulating T3-induced cardiomyocyte growth. An increase in MuRF1 expression inhibits T3-induced physiological cardiac hypertrophy, whereas a decrease in MuRF1 expression enhances T3s activity both in vitro and in cardiomyocytes in vivo MuRF1 interacts directly with TRα to inhibit its activity by posttranslational ubiquitination in a non-canonical manner. We then demonstrated that a nuclear localization apparatus that regulates/inhibits nuclear receptors by sequestering them within a subcompartment of the nucleus was necessary for MuRF1 to inhibit T3 activity. This work implicates a novel mechanism that enhances the beneficial T3 activity specifically within the heart, thereby offering a potential target to enhance cardiac T3 activity in an organ-specific manner.

Catabolism of (2E)-4-Hydroxy-2-nonenal via ω- and ω-1-Oxidation Stimulated by Ketogenic Diet

Background: (2E)-4-hydroxy-2-nonenal (HNE) catabolism and its regulation remain to be fully investigated. Results: New catabolic pathways of HNE via ω-/ω-1-oxidation are elucidated and up-regulated in a ketogenic diet. Conclusion: HNE catabolism plays a major role in HNE disposal in certain organs. Significance: This work will further enhance our understanding of the metabolic fate of HNE and the roles of ω- and ω-1-oxidations in HNE disposal. Oxidative stress triggers the peroxidation of ω-6-polyunsaturated fatty acids to reactive lipid fragments, including (2E)-4-hydroxy-2-nonenal (HNE). We previously reported two parallel catabolic pathways of HNE. In this study, we report a novel metabolite that accumulates in rat liver perfused with HNE or 4-hydroxynonanoic acid (HNA), identified as 3-(5-oxotetrahydro-2-furanyl)propanoyl-CoA. In experiments using a combination of isotopic analysis and metabolomics studies, three catabolic pathways of HNE were delineated following HNE conversion to HNA. (i) HNA is ω-hydroxylated to 4,9-dihydroxynonanoic acid, which is subsequently oxidized to 4-hydroxynonanedioic acid. This is followed by the degradation of 4-hydroxynonanedioic acid via β-oxidation originating from C-9 of HNA breaking down to 4-hydroxynonanedioyl-CoA, 4-hydroxyheptanedioyl-CoA, or its lactone, 2-hydroxyglutaryl-CoA, and 2-ketoglutaric acid entering the citric acid cycle. (ii) ω-1-hydroxylation of HNA leads to 4,8-dihydroxynonanoic acid (4,8-DHNA), which is subsequently catabolized via two parallel pathways we previously reported. In catabolic pathway A, 4,8-DHNA is catabolized to 4-phospho-8-hydroxynonanoyl-CoA, 3,8-dihydroxynonanoyl-CoA, 6-hydroxyheptanoyl-CoA, 4-hydroxypentanoyl-CoA, propionyl-CoA, and acetyl-CoA. (iii) The catabolic pathway B of 4,8-DHNA leads to 2,6-dihydroxyheptanoyl-CoA, 5-hydroxyhexanoyl-CoA, 3-hydroxybutyryl-CoA, and acetyl-CoA. Both in vivo and in vitro experiments showed that HNE can be catabolically disposed via ω- and ω-1-oxidation in rat liver and kidney, with little activity in brain and heart. Dietary experiments showed that ω- and ω-1-hydroxylation of HNA in rat liver were dramatically up-regulated by a ketogenic diet, which lowered HNE basal level. HET0016 inhibition and mRNA expression level suggested that the cytochrome P450 4A are main enzymes responsible for the NADPH-dependent ω- and ω-1-hydroxylation of HNA/HNE.