It has recently been reported that impaired PI3K signaling leads to cardiac mitochondrial dysfunction and that cardiac insulin receptor deficiency impairs cardiac growth. Signaling from insulin and insulin-like growth factor 1 receptors to PI3K is mediated by insulin receptor substrates (IRS) 1 and 2. Our objective was to determine the contribution of IRS isoforms on cardiac structure, mitochondrial and contractile function. For this purpose we generated mice with cardiomyocyte-specific deletion of IRS1 (CIRS1KO) or IRS2 (CIRS2KO) or IRS1 + IRS2 (CIRS12KO). While the single KOs had normal survival, CIRS12KO mice died from heart failure within the first eleven weeks of life. At the age of 8 weeks, heart weight-to-tibia length ratios, HW/TL were reduced by 11% (p<0.05) in CIRS1KO versus WT. ADP-stimulated mitochondrial oxygen consumption (V-ADP) was decreased by 22% (p<0.05) and ATP synthesis by 16% (p<0.05) in saponin-permeabilized cardiac fibers using pyruvate as substrate but not with palmitoyl carnitine (PC) or glutamate. In contrast, HW/TL was unchanged in CIRS2KO at the age of 8 weeks and was increased at the age of 14 weeks (+7%, p<0.05). Similarly, ADP-stimulated respirations were reduced in CIRS2KO at both ages by −21% and −16% respectively, (p < 0.05) only with pyruvate as substrate. In contrast, CIRS12KO developed dilated cardiomyopathy at the age of 4 weeks (LVDs + 18.9%, LVPWd −12.7%, LVPWs −21.4%, FS −36.7%, EF −26.3%, p<0.05). Histology revealed myofibrillar loss and disarray and increased fibrosis. Using stereology, cardiomyocyte nuclei per area was reduced by 27% and cardiomyocyte cross-sectional area was increased by 33% (both, p<0.05). Using pyruvate, PC or glutamate as substrates, V-ADP and ATP-synthesis rates were reduced by 33– 41%. Transcriptional analysis revealed a coordinate downregulation (by 40 – 60%) of PDK4 and PDH subunits, FAO and OXPHOS genes that were associated with a 35% reduction in expression of PGC-1β. At the protein level, expression of the complex II subunit, succinate-ubiquinol oxidoreductase was reduced by −51% (p<0.01). These data identify a critical role for IRS-mediated signaling in the regulation mitochondrial gene and protein expression, mitochondrial function and cardiomyocyte survival.
Introduction: Snf1-related Kinase (SNRK) is a serine/threonine kinase with sequence similarity to AMP-activated kinase. The function and substrates of SNRK are unknown. We have found that SNRK is upregulated in hearts from patients with ischemic cardiomyopathy, and our previous gene array data suggested that SNRK alters metabolic gene expression. Here, we assessed the hypothesis that SNRK regulates cardiac metabolism. Results: SNRK transgenic mice with specific overexpression of SNRK in the heart displayed decreased glycolysis (1267.66 vs. 1925.60 nmol/min/g dhw, p=0.004, n=6), glucose oxidation (441.73 vs. 602.68 nmol/min/g dhw, p=0.068, n=6), and palmitate oxidation (262.39 vs. 385.07 nmol/min/g dhw, p=0.005, n=6) compared to wild type littermate controls in perfused working hearts. However, cardiac power output and function were maintained, demonstrating that SNRK transgenic mice have increased metabolic efficiency. Mitochondria isolated from SNRK transgenic mice had increased state 3 respiration and oxidative capacity, along with an increased respiratory control ratio (state 3/state 4 respiration), indicating reduced mitochondrial uncoupling. To identify the mechanism for the effects of SNRK on metabolic flux, we performed a yeast two hybrid screen with SNRK bait. Tribbles homolog 3 (Trb3), an inhibitor of Akt, was identified as a SNRK-interacting protein, which was verified by co-immunoprecipitation and mammalian two hybrid assay. Trb3 protein levels were increased in the SNRK transgenic mice, and phosphorylation of Akt and its substrate GSK3 was reduced. SNRK transgenic mice also had decreased mRNA levels of glucose transporter 1 and 4, hexokinase II, and phosphofructokinase. Additionally, mRNA levels of the fatty acid metabolism regulators peroxisome proliferator-activated receptor (PPAR) α and PPARγ were reduced, as well as their targets uncoupling protein 2 and 3. Finally, SNRK transgenic mice had decreased infarct size compared to wild type littermates (14.35% vs. 20.63% total left ventricle area, p=0.023, n=6) following myocardial infarction for seven days. Conclusions: Our results demonstrate that SNRK increases cardiac metabolic and mitochondrial efficiency and resistance to myocardial infarct cell death.
We hypothesized that PP2A activation is required for lipid‐induced, ceramide‐mediated arterial dysfunction. Mice haploinsufficient for dihydroceramide desaturase (des1+/−) and their wild‐type littermates (des1+/+) were infused (iv) for 6 h with lard‐oil (LO) or vehicle (veh). Subgroups of LO and veh mice were treated (1.5 mg/kg IP) for 3 days prior to infusion with the PP2A inhibitor LB1 (Lixte Biotechnology, NY). LO increased ceramide accrual in arteries from des1+/+ but not des1+/− mice. Palmitate (3 h × 500 uM) increased (p<0.05) PP2A activity, and impaired (p<0.05) insulin‐stimulated p‐eNOS(S)1177 to eNOS in endothelial cells, and these responses were negated by LB1 (4 uM; n=5–8). Endothelium‐dependent and –independent relaxation of femoral arteries (~ 150 um i.d.) was assessed using acetylcholine (ACh) and sodium nitroprusside (SNP), respectively (n=3 mice/group, 3 vessels/mouse). ACh‐mediated (2×10−8, 3×10−8, and 6×10−8 M) relaxation (%) was less (p<0.05) in LO des1+/+ (30±2, 41±3, and 61±4, respectively) vs. veh des1+/+ mice (48±4, 67±6, and 73±6, respectively). Endothelial dysfunction observed in LO des1+/+ mice was less severe when ceramide accrual (i.e. LO des1+/− mice) or PP2A activation (i.e., LB1 + LO des1+/+ mice) were prevented. SNP‐evoked vasorelaxation was intact among groups. LO‐induced ceramide accumulation induces endothelial dysfunction that is dependent upon PP2A activation. ADA1–12‐ BS‐208, 2R15HL091493
Summa ry: Summa ry: This protocol describes the procedure used by the DiaComp for measuring left ventricular hemodynamic parameters in intact mice. Di a be ti c Compl i ca ti on: Di a be ti c Compl i ca ti on:
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