No abstract

No abstract

Background: Significant improvements in myocardial structure and function have been reported in some patients with advanced heart failure (termed responders [R]) following left ventricular assist device (LVAD)–induced mechanical unloading. This therapeutic strategy may alter myocardial energy metabolism in a manner that reverses the deleterious metabolic adaptations of the failing heart. Specifically, our previous work demonstrated a post-LVAD dissociation of glycolysis and oxidative-phosphorylation characterized by induction of glycolysis without subsequent increase in pyruvate oxidation through the tricarboxylic acid cycle. The underlying mechanisms responsible for this dissociation are not well understood. We hypothesized that the accumulated glycolytic intermediates are channeled into cardioprotective and repair pathways, such as the pentose-phosphate pathway and 1-carbon metabolism, which may mediate myocardial recovery in R. Methods: We prospectively obtained paired left ventricular apical myocardial tissue from nonfailing donor hearts as well as R and nonresponders at LVAD implantation (pre-LVAD) and transplantation (post-LVAD). We conducted protein expression and metabolite profiling and evaluated mitochondrial structure using electron microscopy. Results: Western blot analysis shows significant increase in rate-limiting enzymes of pentose-phosphate pathway and 1-carbon metabolism in post-LVAD R (post-R) as compared with post-LVAD nonresponders (post-NR). The metabolite levels of these enzyme substrates, such as sedoheptulose-6-phosphate (pentose phosphate pathway) and serine and glycine (1-carbon metabolism) were also decreased in Post-R. Furthermore, post-R had significantly higher reduced nicotinamide adenine dinucleotide phosphate levels, reduced reactive oxygen species levels, improved mitochondrial density, and enhanced glycosylation of the extracellular matrix protein, α-dystroglycan, all consistent with enhanced pentose-phosphate pathway and 1-carbon metabolism that correlated with the observed myocardial recovery. Conclusions: The recovering heart appears to direct glycolytic metabolites into pentose-phosphate pathway and 1-carbon metabolism, which could contribute to cardioprotection by generating reduced nicotinamide adenine dinucleotide phosphate to enhance biosynthesis and by reducing oxidative stress. These findings provide further insights into mechanisms responsible for the beneficial effect of glycolysis induction during the recovery of failing human hearts after mechanical unloading.

Background Platelets are dynamic effector cells with functions that span hemostatic, thrombotic and inflammatory continua. Phosphoinositide-dependent protein kinase 1 (PDK1) regulates protease-activated receptor 4-induced platelet activation and thrombus formation through glycogen synthase kinase3β. However, whether PDK1 also signals through the ADP receptor and its functional importance in vivo remain unknown. Objective To establish the mechanism of PDK1 in ADP-induced platelet activation and thrombosis. Methods We assessed the role of PDK1 on 2MeSADP-induced platelet activation by measuring aggregation, thromboxane generation and phosphorylation events in the presence of BX-795, which inhibits PDK1, or by using platelet-specific PDK1 knockout mice and performing western blot analysis. PDK1 function in thrombus formation was assessed with an in vivo pulmonary embolism model. Results PDK1 inhibition with BX-795 reduced 2-methylthio-ADP (2MeSADP)-induced aggregation of human and murine platelets by abolishing thromboxane generation. Similar results were observed in pdk1 mice. PDK1 was also necessary for the phosphorylation of mitogen-activated protein kinase kinase 1/2 (MEK1/2), extracellular signal-regulated kinase 1/2, and cytosolic phospholipase A2, indicating that PDK1 regulates an upstream kinase in the mitogen-activated protein kinase (MAPK) pathway. We next determined that this upstream kinase is Raf-1, a serine/threonine kinase that is necessary for the phosphorylation of MEK1/2, as pharmacological inhibition and genetic ablation of PDK1 were sufficient to prevent Raf1 phosphorylation. Furthermore, in vivo inhibition or genetic ablation of PDK1 protected mice from collagen/epinephrine-induced pulmonary embolism. Conclusion PDK1 governs thromboxane generation and thrombosis in platelets that are stimulated with 2MeSADP by regulating activation of the MAPK pathway.

Platelets play a key role in the physiological hemostasis or pathological process of thrombosis. Rhodocytin, an agonist of the C-type lectin-like receptor-2 (CLEC-2), elicits powerful platelet activation signals in conjunction with Src family kinases (SFKs), spleen tyrosine kinase (Syk), and phospholipase γ2 (PLCγ2). Previous reports have shown that rhodocytin-induced platelet aggregation depends on secondary mediators such as thromboxane A2 (TxA2) and ADP, which are agonists for G-protein-coupled receptors (GPCRs) on platelets. How the secondary mediators regulate CLEC-2-mediated platelet activation in terms of signaling is not clearly defined. In this study, we report that CLEC-2-induced Syk and PLCγ2 phosphorylation is potentiated by TxA2 and that TxA2 plays a critical role in the most proximal event of CLEC-2 signaling, the CLEC-2 receptor tyrosine phosphorylation. We show that the activation of other GPCRs, such as the ADP receptors and protease-activated receptors, can also potentiate CLEC-2 signaling. By using the specific G inhibitor, UBO-QIC, or G knock-out murine platelets, we demonstrate that G signaling, but not other G-proteins, is essential for GPCR-induced potentiation of Syk phosphorylation downstream of CLEC-2. We further elucidated the signaling downstream of G and identified an important role for the PLCβ-PKCα pathway, possibly regulating activation of SFKs, which are crucial for initiation of CLEC-2 signaling. Together, these results provide evidence for novel G-PLCβ-PKCα-mediated regulation of proximal CLEC-2 signaling by G-coupled receptors.

Background: We previously showed that cardiomyocyte Krüppel-like factor (KLF)-5 regulates cardiac fatty acid oxidation. As heart failure has been associated with altered fatty acid oxidation, we investigated the role of cardiomyocyte KLF5 in lipid metabolism and pathophysiology of ischemic heart failure. Methods: Using rtPCR and Western Blot, we investigated the KLF5 expression changes in a myocardial infarction (MI) mouse model and heart tissue from patients with ischemic heart failure. Using 2D-echocardiography, we evaluated the effect of KLF5 inhibition after MI using pharmacological KLF5 inhibitor ML264 and mice with cardiomyocyte specific KLF5 deletion (αMHC-KLF5 -/- ). We identified the involvement of KLF5 in regulating lipid metabolism and ceramide accumulation after MI using liquid-chromatography-tandem-mass-spectrometry, and Western Blot and rtPCR analysis of ceramide-metabolism-related genes. We lastly evaluated the effect of cardiomyocyte-specific KLF5 overexpression (αMHC-rtTA-KLF5) on cardiac function and ceramide metabolism, and rescued the phenotype using myriocin to inhibit ceramide biosynthesis. Results: KLF5 mRNA and protein levels were higher in human ischemic heart failure samples and in rodent models 24h, 2- and 4-weeks post-permanent left coronary artery ligation. αMHC-KLF5 -/- mice and mice treated with ML264 had higher ejection fraction and lower ventricular volume and heart weight after MI. Lipidomic analysis showed that αMHC-KLF5 -/- mice with MI had lower myocardial ceramide levels compared with littermate control mice with MI although basal ceramide content of αMHC-KLF5 -/- mice was not different from control mice. KLF5 ablation suppressed the expression of serine-palmitoyl-transferase-long-chain-base-subunit (SPTLC)1 and SPTLC2, which regulate de novo ceramide biosynthesis. We confirmed our previous findings that myocardial SPTLC1 and SPTLC2 levels are increased in heart failure patients. Consistently, αMHC-rtTA-KLF5 mice showed increased SPTLC1 and SPTLC2 expression, higher myocardial ceramide levels, and systolic dysfunction beginning 2-weeks after KLF5 induction. Treatment of αMHC-rtTA-KLF5 mice with myriocin that inhibits SPT, suppressed myocardial ceramide levels and alleviated systolic dysfunction. Conclusions: KLF5 is induced during the development of ischemic heart failure in humans and mice and stimulates ceramide biosynthesis. Genetic or pharmacological inhibition of KLF5 in mice with MI prevents ceramide accumulation, alleviates eccentric remodeling, and increases ejection fraction. Thus, KLF5 emerges as a novel therapeutic target for the treatment of ischemic heart failure.

Voltage dependent anion channel 2 (VDAC2) is an outer mitochondrial membrane porin known to play a significant role in apoptosis and calcium signaling. Abnormalities in calcium homeostasis often leads to electrical and contractile dysfunction and can cause dilated cardiomyopathy and heart failure. However, the specific role of VDAC2 in intracellular calcium dynamics and cardiac function is not well understood. To elucidate the role of VDAC2 in calcium homeostasis, we generated a cardiac ventricular myocyte-specific developmental deletion of Vdac2 in mice. Our results indicate that loss of VDAC2 in the myocardium causes severe impairment in excitation-contraction coupling by altering both intracellular and mitochondrial calcium signaling. We also observed adverse cardiac remodeling which progressed to severe cardiomyopathy and death. Reintroduction of VDAC2 in 6-week-old knock-out mice partially rescued the cardiomyopathy phenotype. Activation of VDAC2 by efsevin increased cardiac contractile force in a mouse model of pressure-overload induced heart failure. In conclusion, our findings demonstrate that VDAC2 plays a crucial role in cardiac function by influencing cellular calcium signaling. Through this unique role in cellular calcium dynamics and excitation-contraction coupling VDAC2 emerges as a plausible therapeutic target for heart failure.

Phosphatidylinositol 3-kinase is an important signaling molecule that, once activated, leads to the generation of phosphatidylinositol (3,4,5)-trisphosphate (PIP3). We performed a proteomic screen to identify PIP3-interacting proteins in human platelets. Among these proteins, we found engulfment and cell motility 1 (ELMO1), a scaffold protein with no catalytic activity. ELMO1 is expressed in platelets and interacts with active RhoG. However, the function of ELMO1 in platelets is not known. The focus of this study was to determine the function of ELMO1 in platelets utilizing ELMO1−/− mice. Platelet aggregation, granule secretion, integrin αIIbβ3 activation, and thromboxane generation were enhanced in ELMO1−/− platelets in response to glycoprotein VI (GPVI) agonists but unaltered when a protease-activated receptor 4 agonist was used. The kinetics of spreading on immobilized fibrinogen was enhanced in ELMO1−/− platelets compared with wild-type (WT) littermate controls. This suggests that ELMO1 plays a role downstream of the GPVI and integrin αIIbβ3 pathway. Furthermore, whole blood from ELMO1−/− mice perfused over collagen exhibited enhanced thrombus formation compared with WT littermate controls. ELMO1−/− mice showed reduced survival compared with control following pulmonary embolism. ELMO1−/− mice also exhibited a shorter time to occlusion using the ferric-chloride injury model and reduced bleeding times compared with WT littermate controls. These results indicate that ELMO1 plays an important role in hemostasis and thrombosis in vivo. RhoG activity was enhanced in ELMO1−/− murine platelets compared with WT littermate controls in response to GPVI agonist. Together, these data suggest that ELMO1 negatively regulates GPVI-mediated thrombus formation via RhoG.