Contribution Of Impaired Myocardial Insulin Signaling To Mitochondrial Dysfunction And Oxidative Stress In The Heart

Background—Diabetes-associated cardiac dysfunction is associated with mitochondrial dysfunction and oxidative stress, which may contribute to LV dysfunction. The contribution of altered myocardial insulin action, independently of associated changes in systemic metabolism is incompletely understood. The present study tested the hypothesis that perinatal loss of insulin signaling in the heart impairs mitochondrial function. Methods and Results—In 8-week-old mice with cardiomyocyte deletion of insulin receptors (CIRKO), inotropic reserves were reduced and mitochondria manifested respiratory defects for pyruvate that was associated with proportionate reductions in catalytic subunits of pyruvate dehydrogenase. Progressive age-dependent defects in oxygen consumption and ATP synthesis with the substrates glutamate and the fatty acid derivative palmitoyl carnitine (PC) were observed. Mitochondria were also uncoupled when exposed to PC due in part to increased ROS production and oxidative stress. Although proteomic and genomic approaches revealed a reduction in subsets of genes and proteins related to oxidative phosphorylation, no reduction in maximal activities of mitochondrial electron transport chain complexes were found. However, a disproportionate reduction in TCA cycle and FA oxidation proteins in mitochondria, suggest that defects in FA and pyruvate metabolism and TCA flux may explain the mitochondrial dysfunction observed. Conclusions—Impaired myocardial insulin signaling promotes oxidative stress and mitochondrial uncoupling, which together with reduced TCA and FA oxidative capacity impairs mitochondrial energetics. This study identifies specific contributions of impaired insulin action to mitochondrial dysfunction in the heart

Medium and Large N-Heterocycle Formation via Allene Hydroamination with a Bimetallic Rh(I) Catalyst

We report the synthesis of a bimetallic Rh(I) complex containing a bridging CO ligand that facilitates Rh–Rh bond formation. This bimetallic complex enables intramolecular allene hydroamination to form seven to ten-member rings in high yield. Monometallic Rh complexes, in contrast, fail to achieve any product formation. We demonstrate a broad substrate scope for formation of a variety of N-heterocycles in good to excellent yields. Macrocyclization reactions that form eleven to fifteen-membered heterocycles are also demonstrated. Mechanistic studies show that the reaction likely proceeds via catalyst protonation by trifluoroacetic acid, followed by reversible allene insertion and C–N bond-forming reductive elimination. The difference in product selectivity observed with our bimetallic catalyst vs monometallic Rh complexes may result from cooperativity between the two metals

Glucose Transporter 4-Deficient Hearts Develop Maladaptive Hypertrophy in Response to Physiological or Pathological Stresses

Pathological cardiac hypertrophy may be associated with reduced expression of glucose transporter 4 (GLUT4) in contrast to exercise-induced cardiac hypertrophy, where GLUT4 levels are increased. However, mice with cardiac-specific deletion of GLUT4 (G4H-/-) have normal cardiac function in the unstressed state. This study tested the hypothesis that cardiac GLUT4 is required for myocardial adaptations to hemodynamic demands. G4H-/- and control littermates were subjected to either a pathological model of left ventricular pressure overload [transverse aortic constriction (TAC)] or a physiological model of endurance exercise (swim training). As predicted after TAC, G4H-/- mice developed significantly greater hypertrophy and more severe contractile dysfunction. Somewhat surprisingly, after exercise training, G4H-/- mice developed increased fibrosis and apoptosis that was associated with dephosphorylation of the prosurvival kinase Akt in concert with an increase in protein levels of the upstream phosphatase protein phosphatase 2A (PP2A). Exercise has been shown to decrease levels of ceramide; G4H-/- hearts failed to decrease myocardial ceramide in response to exercise. Furthermore, G4H-/- hearts have reduced levels of the transcriptional coactivator peroxisome proliferator-activated receptor- γ coactivator-1, lower carnitine palmitoyl-transferase activity, and reduced hydroxyacyl-CoA dehydrogenase activity. These basal changes may also contribute to the impaired ability of G4H-/- hearts to adapt to hemodynamic stresses. In conclusion, GLUT4 is required for the maintenance of cardiac structure and function in response to physiological or pathological processes that increase energy demands, in part through secondary changes in mitochondrial metabolism and cellular stress survival pathways such as Akt. NEW & NOTEWORTHY Glucose transporter 4 (GLUT4) is required for myocardial adaptations to exercise, and its absence accelerates heart dysfunction after pressure overload. The requirement for GLUT4 may extend beyond glucose uptake to include defects in mitochondrial metabolism and survival signaling pathways that develop in its absence. Therefore, GLUT4 is critical for responses to hemodynamic stresses

Mechanistic Target of Rapamycin (<em>Mtor</em>) Is Essential for Murine Embryonic Heart Development and Growth

<div><p>Mechanistic target of rapamycin (<em>Mtor)</em> is required for embryonic inner cell mass proliferation during early development. However, <em>Mtor</em> expression levels are very low in the mouse heart during embryogenesis. To determine if <em>Mtor</em> plays a role during mouse cardiac development, cardiomyocyte specific <em>Mtor</em> deletion was achieved using α myosin heavy chain (α-MHC) driven Cre recombinase. Initial mosaic expression of Cre between embryonic day (E) 10.5 and E11.5 eliminated a subset of cardiomyocytes with high Cre activity by apoptosis and reduced overall cardiac proliferative capacity. The remaining cardiomyocytes proliferated and expanded normally. However loss of 50% of cardiomyocytes defined a threshold that impairs the ability of the embryonic heart to sustain the embryo’s circulatory requirements. As a result 92% of embryos with cardiomyocyte <em>Mtor</em> deficiency died by the end of gestation. Thus <em>Mtor</em> is required for survival and proliferation of cardiomyocytes in the developing heart.</p> </div

Deletion of <i>Mtor</i> by alpha-MHC-Cre is embryonic lethal.

<p>(A) Schematic showing conditional <i>Mtor</i> allele and location of the loxP sites. The position of AC11, AC14 and AC16 primers and the size of the DNA segments amplified by AC primer pairs are illustrated. (B) Representative picture of an 8-week old (surviving) <i>CMtorKO</i> heart and an <i>Mtor^fl/fl</i> control. (C) Histological analysis of 8-week old <i>Mtor^fl/fl</i> and <i>CMtorKO</i> hearts: upper panel is H&E staining; lower panel is Masson’s trichrome staining. (D) Fractional shortening (FS) measured by echocardiography, black line represents an average of 5 <i>Mtor^fl/fl</i> control mice, and each colored line represents a single <i>CMtorKO</i> mouse. (E) Agarose gel electrophoresis of AC11, AC14 and AC16 PCR products using DNA isolated from cardiomyocytes (CM) obtained from a <i>CMtorKO</i> heart, <i>CMtorKO</i> heart tissue, <i>Mtor^fl/fl</i> heart tissue and wild type (WT) heart tissue respectively. (F) Survival curve of <i>CMtorKO</i> embryos.</p

Development of cardiac dysfunction and the death of <i>CMtorKO</i> embryos.

<p>(A) Fetal echocardiography measurements of E14.5 embryonic hearts (n = 9–17). LVIDd: left ventricular interior dimension-diastole; LVIDs: left ventricular interior dimension-systole; LV vol d: left ventricular volume-diastole; LV vol s: left ventricular volume-systole. (B) Representative cardiac echocardiogram of a control embryonic heart (left) and a <i>CMtorKO</i> embryonic heart (right). The arrow indicates pericardial fluid in the <i>CMtorKO</i> embryo. (C) <i>ANP</i> and <i>BNP</i> mRNA levels in E15.5 <i>CMtorKO</i> hearts from live embryos (n = 8). (D) A summary of cardiac wall volume and cardiac nuclei number from E12.5 to E15.5. (E). <i>Cre</i> recombinase transcripts levels in <i>CMtorHet</i> and <i>CMtorKO</i> hearts at E11.5 and E15.5 (n = 8). (F). Western blots of Cre recombinase in E12.5 and E14.5 embryonic hearts. (G). Agarose gel electrophoresis of AC11, AC14 and AC16 PCR products using DNA isolated from E14.5 <i>Mtor^fl/fl</i> hearts, <i>CMtorHet</i> hearts and <i>CMtorKO</i> hearts. (H). Western blots of Mtor, raptor, rictor and Mtor downstream signaling molecules in 6–9 week old (adult) failing <i>CMtorKO</i> hearts. (I). Western blot of Mtor protein from adult, doxycycline-induced Mtor deficient hearts (<i>iCMtorKO</i>) (left) and densitometric quantification (right) (n = 4–6). (J). Western blot of Cre recombinase protein from 8-week old <i>CMtorHet</i>, <i>CMtorKO</i> hearts (α-MHC-Cre) and 10-week old <i>iCMtorHet</i>, <i>iCMtorKO</i> hearts (TetO-Cre). (K). A summary of cellular and physiological events in <i>CMtorKO</i> embryos and suggested model of how artificial selection by expressing α-MHC-Cre in mouse heart leads to embryonic lethality. “^__“ indicates no change, blank means not measured at the time point. *: p≤0.05 vs. control, #: p≤0.01 vs. control.</p

Fetal echocardiographic evaluation of embryonic cardiac function at E14.5.

<p>Abbreviations: IVSd: interventricular septum thickness-diastole; LVIDd: left ventricular interior dimension-diastole; LVPWd: left-ventricular posterior wall thickness at diastole; LVIDs: left ventricular interior dimension-systole; RVd: right ventricular dimension-diastole; RVs: right ventricular dimension-systole. Data shown are mean ± SEM.</p>#<p>p<0.01 VS Control,</p>*<p>p<0.05 VS Control,</p>%<p>p<0.10 VS Control,</p>&<p>p<0.05 VS CMtorKO,</p>$<p>p<0.10 VS CMtorKO.</p

Restored proliferation and normal apoptosis rate in E14.5 <i>CMtorKO</i> hearts.

<p>(A) A representative picture of E14.5 <i>Mtor^fl/fl</i> (CTR) and <i>CMtorKO</i> (KO) embryonic hearts (left), and calculated cardiac volume (right) (n = 3–4). (B) Quantification of cardiac wall volume and cardiac nuclei number of E15.5 control and <i>CMtorKO</i> (KO) embryonic hearts from live embryos (n = 3–4). (C) Representative EM pictures of control and <i>CMtorKO</i> embryonic heart from live embryo at E15.5. (D) Relative gene expression levels of E15.5 <i>CMtorKO</i> embryonic hearts from live embryos compared to their littermate <i>Mtor^fl/fl</i> controls (n = 8). (E) Western blots of Mtor and Mtor downstream signaling molecules in E14.5 embryonic hearts. (F) Representative TUNEL staining for E14.5 embryonic hearts (right) (n = 3–4), quantification is shown on the left. Arrows indicate TUNEL positive nuclei. (G). Representative Edu staining for E14.5 embryonic hearts (right) (n = 3), quantification is shown on the left. *: p≤0.05 vs. control, #: p≤0.01 vs. control.</p