Jorge Suarez

Adenovirus-Mediated Overexpression of O-GlcNAcase Improves Contractile Function in the Diabetic Heart

To examine whether excessive protein O-GlcNAcylation plays a role in the dysfunction of the diabetic heart, we delivered adenovirus expressing O-GlcNAcase (Adv-GCA) into the myocardium of STZ-induced diabetic mice. Our results indicated that excessive cellular O-GlcNAcylation exists in the diabetic heart, and that in vivo GCA overexpression reduces overall cellular O-GlcNAcylation. Myocytes isolated from diabetic hearts receiving Adv-GCA exhibited improved calcium transients with a significantly shortened Tdecay (P<0.01) and increased sarcoplasmic reticulum Ca2+ load (P<0.01). These myocytes also demonstrated improved contractility including a significant increase in +dL/dt and −dL/dt and greater fractional shortening as measured by edge detection (P<0.01). In isolated perfused hearts, developed pressure and −dP/dt were significantly improved in diabetic hearts receiving Adv-GCA (P<0.05). These hearts also exhibited a 40% increase in SERCA2a expression. Phospholamban protein expression was reduced 50%, but the phosphorylated form was increased 2-fold in the diabetic hearts receiving Adv-GCA. We conclude that excess O-GlcNAcylation in the diabetic heart contributes to cardiac dysfunction, and reducing this excess cellular O-GlcNAcylation has beneficial effects on calcium handling and diabetic cardiac function.

Increased Enzymatic O-GlcNAcylation of Mitochondrial Proteins Impairs Mitochondrial Function in Cardiac Myocytes Exposed to High Glucose

Increased nuclear protein O-linked β-N-acetylglucosamine glycosylation (O-GlcNAcylation) mediated by high glucose treatment or the hyperglycemia of diabetes mellitus contributes to cardiac myocyte dysfunction. However, whether mitochondrial proteins in cardiac myocytes are also submitted to O-GlcNAcylation or excessive O-GlcNAcylation alters mitochondrial function is unknown. In this study, we determined if mitochondrial proteins are O-GlcNAcylated and explored if increased O-GlcNAcylation is linked to high glucose-induced mitochondrial dysfunction in neonatal rat cardiomyocytes. By immunoprecipitation, we found that several mitochondrial proteins, which are members of complexes of the respiratory chain, like subunit NDUFA9 of complex I, subunits core 1 and core 2 of complex III, and the mitochondrial DNA-encoded subunit I of complex IV (COX I) are O-GlcNAcylated. By mass spectrometry, we identified that serine 156 on NDUFA9 is O-GlcNAcylated. High glucose treatment (30 mm glucose) increases mitochondrial protein O-GlcNAcylation, including those of COX I and NDUFA9 which are reduced by expression of O-GlcNAcase (GCA). Increased mitochondrial O-GlcNAcylation is associated with impaired activity of complex I, III, and IV in addition to lower mitochondrial calcium and cellular ATP content. When the excessive O-GlcNAc modification is reduced by GCA expression, mitochondrial function improves; the activity of complex I, III, and IV increases to normal and mitochondrial calcium and cellular ATP content are returned to control levels. From these results we conclude that specific mitochondrial proteins of cardiac myocytes are O-GlcNAcylated and that exposure to high glucose increases mitochondrial protein O-GlcNAcylation, which in turn contributes to impaired mitochondrial function.

Deletion of the Inducible 70-kDa Heat Shock Protein Genes in Mice Impairs Cardiac Contractile Function and Calcium Handling Associated With Hypertrophy

Background— Hspa1a and Hspa1b genes encode stress-inducible 70-kDa heat shock proteins (Hsp70) that protect cells from insults such as ischemia. Mice with null mutations of both genes (KO) were generated, and their cardiac phenotype was explored. Methods and Results— Heart rate and blood pressures were normal in the KO mice. Hearts from KO mice were more susceptible to both functional and cellular damage by ischemia/reperfusion. Cardiac hypertrophy developed in Hsp70-KO mice. Ca2+ transients in cardiomyocytes of KO mice showed a delayed (120%) calcium decline and decreased sarcoplasmic reticulum calcium content. Cell shortening was decreased by 35%, and rates of contraction and relaxation were slower by 40%. These alterations can be attributed to the absence of Hsp70 because viral expression of Hsp70 in KO cultured cardiomyocytes restored these parameters. One mechanism underlying myocyte dysfunction could be decreased SERCA2a expression. This hypothesis was supported by a prolonged calcium decline and decreased SERCA2a protein. Viral SERCA2a expression restored contractility and Ca2+ transients. We examined the involvement of Jun N-terminal kinase (JNK), p38-mitogen–activated protein kinase (p38-MAPK), Raf-1, and extracellular signal–regulated kinase (ERK) in SERCA2a downregulation and the cardiac phenotype of KO mice. Levels of phosphorylated JNK, p38-MAPK, Raf-1, and ERK were elevated in KO hearts. Activation of the Raf-1–ERK pathway in normal cardiomyocytes resulted in decreased SERCA2a. Conclusions— Absence of Hsp70 leads to dysfunctional cardiomyocytes and impaired stress response of Hsp70-KO hearts against ischemia/reperfusion. In addition, deletion of Hsp70 genes might induce cardiac dysfunction and development of cardiac hypertrophy through the activation of JNK, p38-MAPK, Raf-1, and ERK.

Modulation of dynamin-related protein 1 (DRP1) function by increased O-linked-β-N-acetylglucosamine modification (O-GlcNAc) in cardiac myocytes

Background: DRP1 plays a significant role to control mitochondrial fission. Results: DRP1 is O-GlcNAcylated. Increased O-GlcNAcylation augments the level of the GTP-bound active form of DRP1 and induces translocation of DRP1 from cytoplasm to mitochondria. Conclusion: O-GlcNAcylation modulates DRP1 function, which has consequences for mitochondrial function. Significance: The modulation of DRP1 function by increased overall O-GlcNAcylation could play a significant role in the development of diabetic mitochondrial dysfunction. O-linked-N-acetyl-glucosamine glycosylation (O-GlcNAcylation) of the serine and threonine residues of cellular proteins is a dynamic process and affects phosphorylation. Prolonged O-GlcNAcylation has been linked to diabetes-related complications, including mitochondrial dysfunction. Mitochondria are dynamically remodeling organelles, that constantly fuse (fusion) and divide (fission). An imbalance of this process affects mitochondrial function. In this study, we found that dynamin-related protein 1 (DRP1) is O-GlcNAcylated in cardiomyocytes at threonine 585 and 586. O-GlcNAcylation was significantly enhanced by the chemical inhibition of N-acetyl-glucosaminidase. Increased O-GlcNAcylation decreases the phosphorylation of DRP1 at serine 637, which is known to regulate DRP1 function. In fact, increased O-GlcNAcylation augments the level of the GTP-bound active form of DRP1 and induces translocation of DRP1 from the cytoplasm to mitochondria. Mitochondrial fragmentation and decreased mitochondrial membrane potential also accompany the increased O-GlcNAcylation. In conclusion, this report shows, for the first time, that O-GlcNAcylation modulates DRP1 functionality in cardiac muscle cells.

Downregulation of connexin40 is associated with coronary endothelial cell dysfunction in streptozotocin-induced diabetic mice

Vascular endothelial cells (ECs) play a major role in regulating vascular tone and in revascularization. There is increasing evidence showing endothelial dysfunction in diabetes, although little is known about the contribution of connexins (Cxs) to vascular complications in the diabetic heart. This study was designed to investigate the role of Cxs in coronary endothelial dysfunction in diabetic mice. Coronary ECs isolated from diabetic mice exhibit lowered protein levels of Cx37 and Cx40 (but not Cx43) and a loss of gap junction intercellular communication (GJIC). Vasodilatation induced by the assumed contribution of EC-dependent hyperpolarization was significantly reduced in the diabetic coronary artery (CA). Cx40-specific inhibitory peptide (40)GAP27 strongly attenuated endothelium-dependent relaxation in diabetic CA at the concentration that does not affect the relaxation in control CA, suggesting that the total amount of Cx40 is lower in diabetic CA than in control CA. In diabetic mice, coronary capillary density was significantly decreased in vivo. In vitro, GJIC inhibitor attenuated the ability of EC capillary network formation. High-glucose treatment caused a decrease in Cx40 protein expression in ECs and impaired endothelial capillary network formation, which was restored by Cx40 overexpression. Furthermore, we found that the hyperglycemia-induced decrease in Cx40 was associated with inhibited protein expression of Sp1, a transcriptional factor that regulates Cx40 expression. These data suggest that downregulation of Cx40 protein expression and resultant inhibition of GJIC contribute to coronary vascular dysfunction in diabetes.

Alterations in mitochondrial function and cytosolic calcium induced by hyperglycemia are restored by mitochondrial transcription factor A in cardiomyocytes

Mitochondrial transcription factor A (TFAM) is essential for mitochondrial DNA transcription and replication. TFAM transcriptional activity is decreased in diabetic cardiomyopathy; however, the functional implications are unknown. We hypothesized that a reduced TFAM activity may be responsible for some of the alterations caused by hyperglycemia. Therefore, we investigated the effect of TFAM overexpression on hyperglycemia-induced cytosolic calcium handling and mitochondrial abnormalities. Neonatal rat cardiomyocytes were exposed to high glucose (30 mM) for 48 h, and we examined whether TFAM overexpression, by protecting mitochondrial DNA, could reestablish calcium fluxes and mitochondrial alterations toward normal. Our results shown that TFAM overexpression increased to more than twofold mitochondria copy number in cells treated either with normal (5.5 mM) or high glucose. ATP content was reduced by 30% and mitochondrial calcium decreased by 40% after high glucose. TFAM overexpression returned these parameters to even higher than control values. Calcium transients were prolonged by 70% after high glucose, which was associated with diminished sarco(endo)plasmic reticulum Ca(2+)-ATPase 2a and cytochrome-c oxidase subunit 1 expression. These parameters were returned to control values after TFAM overexpression. High glucose-induced protein oxidation was reduced by TFAM overexpression, indicating a reduction of the high glucose-induced oxidative stress. In addition, we found that TFAM activity can be modulated by O-linked beta-N-acetylglucosamine glycosylation. In conclusion, TFAM overexpression protected cell function against the damage induced by high glucose in cardiomyocytes.

Regulation of mitochondrial morphology and function by O-GlcNAcylation in neonatal cardiac myocytes.

Mitochondria are crucial organelles in cell life serving as a source of energy production and as regulators of Ca(2+) homeostasis, apoptosis, and development. Mitochondria frequently change their shape by fusion and fission, and recent research on these morphological dynamics of mitochondria has highlighted their role in normal cell physiology and disease. In this study, we investigated the effect of high glucose on mitochondrial dynamics in neonatal cardiac myocytes (NCMs). High-glucose treatment of NCMs significantly decreased the level of optical atrophy 1 (OPA1) (mitochondrial fusion-related protein) protein expression. NCMs exhibit two different kinds of mitochondrial structure: round shape around the nuclear area and elongated tubular structures in the pseudopod area. High-glucose-treated NCMs exhibited augmented mitochondrial fragmentation in the pseudopod area. This effect was significantly decreased by OPA1 overexpression. High-glucose exposure also led to increased O-GlcNAcylation of OPA1 in NCMs. GlcNAcase (GCA) overexpression in high-glucose-treated NCMs decreased OPA1 protein O-GlcNAcylation and significantly increased mitochondrial elongation. In addition to the morphological change caused by high glucose, we observed that high glucose decreased mitochondrial membrane potential and complex IV activity and that OPA1 overexpression increased both levels to the control level. These data suggest that decreased OPA1 protein level and increased O-GlcNAcylation of OPA1 protein by high glucose lead to mitochondrial dysfunction by increasing mitochondrial fragmentation, decreasing mitochondrial membrane potential, and attenuating the activity of mitochondrial complex IV, and that overexpression of OPA1 and GCA in cardiac myocytes may help improve the cardiac dysfunction in diabetes.

Excess protein O-GlcNAcylation and the progression of diabetic cardiomyopathy

We examined the role that enzymatic protein O-GlcNAcylation plays in the development of diabetic cardiomyopathy in a mouse model of Type 2 diabetes mellitus (DM2). Mice injected with low-dose streptozotocin and fed a high-fat diet developed mild hyperglycemia and obesity consistent with DM2. Studies were performed from 1 to 6 mo after initiating the DM2 protocol. After 1 mo, DM2 mice showed increased body weight, impaired fasting blood glucose, and hyperinsulinemia. Echocardiographic evaluation revealed left ventricular diastolic dysfunction by 2 mo and O-GlcNAcylation of several cardiac proteins and of nuclear transcription factor Sp1. By 4 mo, systolic dysfunction was observed and sarcoplasmic reticulum Ca(2+) ATPase expression decreased by 50%. Fibrosis was not observed at any timepoint in DM2 mice. Levels of the rate-limiting enzyme of the hexosamine biosynthetic pathway, glutamine:fructose-6-phosphate amidotransferase (GFAT) were increased as early as 2 mo. Fatty acids, which are elevated in DM2 mice, can possibly be linked to excessive protein O-GlcNAcylation levels, as cultured cardiac myocytes in normal glucose treated with oleic acid showed increased O-GlcNAcylation and GFAT levels. These data indicate that the early onset of diastolic dysfunction followed by the loss of systolic function, in the absence of cardiac hypertrophy or fibrosis, is associated with increased cardiac protein O-GlcNAcylation and increased O-GlcNAcylation levels of key calcium-handling proteins. A link between excessive protein O-GlcNAcylation and cardiac dysfunction is further supported by results showing that reducing O-GlcNAcylation by O-GlcNAcase overexpression improved cardiac function in the diabetic mouse. In addition, fatty acids play a role in stimulating excess O-GlcNAcylation. The nature and time course of changes observed in cardiac function suggest that protein O-GlcNAcylation plays a mechanistic role in the triggering of diabetic cardiomyopathy in DM2.

Thyroid Hormone Receptor-β Is Associated with Coronary Angiogenesis during Pathological Cardiac Hypertrophy

Insufficient angiogenesis is one of the causes leading to tissue ischemia and dysfunction. In heart failure, there is increasing evidence showing decreased capillary density in the left ventricle (LV) myocardium, although the detailed mechanisms contributing to it are not clear. The goal of this study was to investigate the role of thyroid hormone receptors (TRs) in the coronary microvascular rarefaction under pathological cardiac hypertrophy. The LV from hypertrophied/failing hearts induced by ascending aortic constriction (AAC) exhibited severe microvascular rarefaction, and this phenomenon was restored by chronic T(3) administration. Coronary endothelial cells (ECs) isolated from AAC hearts expressed lower TRbeta mRNA than control ECs, and chronic T(3) administration restored TRbeta mRNA expression level in AAC hearts to the control level. Among different TR subtype-specific knockout mice, TRbeta knockout and TRalpha/TRbeta double-knockout mice both exhibited significantly less capillary density in LV compared with wild-type mice. In vitro, coronary ECs isolated from TRbeta knockout mice lacked the ability to form capillary networks. In addition, we identified that kinase insert domain protein receptor/fetal liver kinase-1 (vascular endothelial growth factor-2 receptor) was one of the angiogenic mediators controlled by T(3) administration in the AAC heart. These data suggest that TRbeta in the coronary ECs regulates capillary density during cardiac development, and down-regulation of TRbeta results in coronary microvascular rarefaction during pathological hypertrophy.

Conditional increase in SERCA2a protein is able to reverse contractile dysfunction and abnormal calcium flux in established diabetic cardiomyopathy

Diabetic cardiomyopathy is characterized by reduced cardiac contractility independent of vascular disease. A contributor to contractile dysfunction in the diabetic heart is impaired sarcoplasmic reticulum function with reduced sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA2a) pump activity, leading to disturbed intracellular calcium handling. It is currently unclear whether increasing SERCA2a activity in hearts with existing diabetic cardiomyopathy could still improve calcium flux and contractile performance. To test this hypothesis, we generated a cardiac-specific tetracycline-inducible double transgenic mouse, which allows for doxycycline (DOX)-based inducible SERCA2a expression in which DOX exposure turns on SERCA2a expression. Isolated cardiomyocytes and Langendorff perfused hearts from streptozotocin-induced diabetic mice were studied. Our results show that total SERCA2a protein levels were decreased in the diabetic mice by 60% compared with control. SERCA2a increased above control values in the diabetic mice after DOX. Dysfunctional contractility in the diabetic cardiomyocyte was restored to normal by induction of SERCA2a expression. Calcium transients from diabetic cardiomyocytes showed a delayed rate of diastolic calcium decay of 66%, which was reverted toward normal after SERCA2a expression induced by DOX. Global cardiac function assessed in the diabetic perfused heart showed diminished left ventricular pressure, rate of contraction, and relaxation. These parameters were returned to control values by SERCA2a expression. In conclusion, we have used mice allowing for inducible expression of SERCA2a and could demonstrate that increased expression of SERCA2a leads to improved cardiac function in mice with an already established diabetic cardiomyopathy in absence of detrimental effects.