Mitochondrial haplotype modulates genome expression and mitochondrial structure/function in cardiomyocytes following volume overload.

Mitochondrial DNA (mtDNA) haplotype regulates mitochondrial structure/function and reactive oxygen species in aortocaval fistula (ACF) in mice. Here, we unravel the mitochondrial haplotype effects on cardiomyocyte mitochondrial ultrastructure and transcriptome response to ACF in vivo. Phenotypic responses and quantitative transmission electron microscopy (TEM) and RNA sequence at 3 days were determined after sham surgery or ACF in vivo in cardiomyocytes from wild-type (WT) C57BL/6J (C57n:C57mt) and C3H/HeN (C3Hn:C3Hmt) and mitochondrial nuclear exchange mice (C57n:C3Hmt or C3Hn:C57mt). Quantitative TEM of cardiomyocyte mitochondria C3HWT hearts have more electron-dense compact mitochondrial cristae compared with C57WT. In response to ACF, mitochondrial area and cristae integrity are normal in C3HWT; however, there is mitochondrial swelling, cristae lysis, and disorganization in both C57WT and MNX hearts. Tissue analysis shows that C3HWT hearts have increased autophagy, antioxidant, and glucose fatty acid oxidation-related genes compared with C57WT. Comparative transcriptomic analysis of cardiomyocytes from ACF was dependent upon mtDNA haplotype. C57mtDNA haplotype was associated with increased inflammatory/protein synthesis pathways and downregulation of bioenergetic pathways, whereas C3HmtDNA showed upregulation of autophagy genes. In conclusion, ACF in vivo shows a protective response of C3Hmt haplotype that is in large part driven by mitochondrial nuclear genome interaction.NEW & NOTEWORTHY The results of this study support the effects of mtDNA haplotype on nuclear gene expression in cardiomyocytes. Currently, there is no acceptable therapy for volume overload due to mitral regurgitation. The findings of this study could suggest that mtDNA haplotype activates different pathways after ACF warrants further investigations on human population of heart disease from different ancestry backgrounds.

Sex differences in cardiopulmonary effects of acute bromine exposure.

Accidental occupational bromine (Br>2>) exposures are common, leading to significant morbidity and mortality; however, the specific effects of Br>2> inhalation in female victims are unclear. Our studies demonstrated that acute high-concentration Br>2> inhalation is fatal, and cardiac injury and dysfunction play an important role in Br>2> toxicity in males. In this study, we exposed female Sprague Dawley rats, age-matched to those males from previously studied, to 600 ppm Br>2> for 45 min and assessed their survival, cardiopulmonary injury and cardiac function after exposure. Br>2> exposure caused serious mortality in female rats (59%) 48 h after exposure. Rats had severe clinical distress, reduced heart rates and oxygen saturation after Br>2> inhalation as was previously reported with male animals. There was significant lung injury and edema when measured 24 h after exposure. Cardiac injury biomarkers were also significantly elevated 24 h after Br>2> inhalation. Echocardiography and hemodynamic studies were also performed and revealed that the mean arterial pressure was not significantly elevated in females. Other functional cardiac parameters were also altered. Aside from the lack of elevation of blood pressure, all other changes observed in female animals were also present in male animals as reported in our previous study. These studies are important to understand the toxicity mechanisms to generate therapies and better-equip first responders to deal with these specific scenarios after bromine spill disasters.>.

Chronic cardiac structural damage, diastolic and systolic dysfunction following acute myocardial injury due to bromine exposure in rats.

Accidental bromine spills are common and its large industrial stores risk potential terrorist attacks. The mechanisms of bromine toxicity and effective therapeutic strategies are unknown. Our studies demonstrate that inhaled bromine causes deleterious cardiac manifestations. In this manuscript we describe mechanisms of delayed cardiac effects in the survivors of a single bromine exposure. Rats were exposed to bromine (600 ppm for 45 min) and the survivors were sacrificed at 14 or 28 days. Echocardiography, hemodynamic analysis, histology, transmission electron microscopy (TEM) and biochemical analysis of cardiac tissue were performed to assess functional, structural and molecular effects. Increases in right ventricular (RV) and left ventricular (LV) end-diastolic pressure and LV end-diastolic wall stress with increased LV fibrosis were observed. TEM images demonstrated myofibrillar loss, cytoskeletal breakdown and mitochondrial damage at both time points. Increases in cardiac troponin I (cTnI) and N-terminal pro brain natriuretic peptide (NT-proBNP) reflected myofibrillar damage and increased LV wall stress. LV shortening decreased as a function of increasing LV end-systolic wall stress and was accompanied by increased sarcoendoplasmic reticulum calcium ATPase (SERCA) inactivation and a striking dephosphorylation of phospholamban. NADPH oxidase 2 and protein phosphatase 1 were also increased. Increased circulating eosinophils and myocardial 4-hydroxynonenal content suggested increased oxidative stress as a key contributing factor to these effects. Thus, a continuous oxidative stress-induced chronic myocardial damage along with phospholamban dephosphorylation are critical for bromine-induced chronic cardiac dysfunction. These findings in our preclinical model will educate clinicians and public health personnel and provide important endpoints to evaluate therapies.

Maintaining Myocardial Glucose Utilization in Diabetic Cardiomyopathy Accelerates Mitochondrial Dysfunction.

Cardiac glucose uptake and oxidation are reduced in diabetes despite hyperglycemia. Mitochondrial dysfunction contributes to heart failure in diabetes. It is unclear whether these changes are adaptive or maladaptive. To directly evaluate the relationship between glucose delivery and mitochondrial dysfunction in diabetic cardiomyopathy, we generated transgenic mice with inducible cardiomyocyte-specific expression of the GLUT4. We examined mice rendered hyperglycemic following low-dose streptozotocin prior to increasing cardiomyocyte glucose uptake by transgene induction. Enhanced myocardial glucose in nondiabetic mice decreased mitochondrial ATP generation and was associated with echocardiographic evidence of diastolic dysfunction. Increasing myocardial glucose delivery after short-term diabetes onset exacerbated mitochondrial oxidative dysfunction. Transcriptomic analysis revealed that the largest changes, driven by glucose and diabetes, were in genes involved in mitochondrial function. This glucose-dependent transcriptional repression was in part mediated by O-GlcNAcylation of the transcription factor Sp1. Increased glucose uptake induced direct O-GlcNAcylation of many electron transport chain subunits and other mitochondrial proteins. These findings identify mitochondria as a major target of glucotoxicity. They also suggest that reduced glucose utilization in diabetic cardiomyopathy might defend against glucotoxicity and caution that restoring glucose delivery to the heart in the context of diabetes could accelerate mitochondrial dysfunction by disrupting protective metabolic adaptations.

Chymase uptake by cardiomyocytes results in myosin degradation in cardiac volume overload.

BACKGROUND: Volume overload (VO) of isolated mitral regurgitation (MR) or aortocaval fistula (ACF) is associated with extracellular matrix degradation and cardiomyocyte myofibrillar and desmin breakdown. Left ventricular (LV) chymase activity is increased in VO and recent studies demonstrate chymase presence within cardiomyocytes. Here we test the hypothesis that chymase within the cardiomyocyte coincides with myosin and desmin breakdown in VO. METHODS AND RESULTS: Aortocaval fistula (ACF) was induced in Sprague Dawley (SD) rats and was compared to age-matched sham-operated rats at 24 hours, 4 and 12 weeks. Immunohistochemistry (IHC) and transmission electron microscopy (TEM) immunogold of LV tissue demonstrate chymase within cardiomyocytes at all ACF time points. IHC for myosin demonstrates myofibrillar disorganization starting at 24 hours. Proteolytic presence of chymase in cardiomyocytes is verified by in situ chymotryptic tissue activity that is inhibited by pretreatment with a chymase inhibitor. Real-time PCR of isolated cardiomyocytes at all ACF time points and in situ hybridization demonstrate endothelial cells and fibroblasts as a major source of chymase mRNA in addition to mast cells. Chymase added to adult rat cardiomyocytes in vitro is taken up by a dynamin-mediated process and myosin breakdown is attenuated by dynamin inhibitor, suggesting that chymase uptake is essential for myosin breakdown. In a previous study in the dog model of chronic MR, the intracellular changes were attributed to extracellular effects. However, we now demonstrate intracellular effects of chymase in both species. CONCLUSION: In response to VO, fibroblast and endothelial cells produce chymase and subsequent cardiomyocyte chymase uptake is followed by myosin degradation. The results demonstrate a novel intracellular chymase-mediated mechanism of cardiomyocyte dysfunction and adverse remodeling in a pure VO.

Bromine inhalation mimics ischemia-reperfusion cardiomyocyte injury and calpain activation in rats.

Halogens are widely used, highly toxic chemicals that pose a potential threat to humans because of their abundance. Halogens such as bromine (Br2) cause severe pulmonary and systemic injuries; however, the mechanisms of their toxicity are largely unknown. Here, we demonstrated that Br2 and reactive brominated species produced in the lung and released in blood reach the heart and cause acute cardiac ultrastructural damage and dysfunction in rats. Br2-induced cardiac damage was demonstrated by acute (3-24 h) increases in circulating troponin I, heart-type fatty acid-binding protein, and NH2-terminal pro-brain natriuretic peptide. Transmission electron microscopy demonstrated acute (3-24 h) cardiac contraction band necrosis, disruption of z-disks, and mitochondrial swelling and disorganization. Echocardiography and hemodynamic analysis revealed left ventricular (LV) systolic and diastolic dysfunction at 7 days. Plasma and LV tissue had increased levels of brominated fatty acids. 2-Bromohexadecanal (Br-HDA) injected into the LV cavity of a normal rat caused acute LV enlargement with extensive disruption of the sarcomeric architecture and mitochondrial damage. There was extensive infiltration of neutrophils and increased myeloperoxidase levels in the hearts of Br2- or Br2 reactant-exposed rats. Increased bromination of sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) and increased phosphalamban after Br2 inhalation decreased cardiac SERCA activity by 70%. SERCA inactivation was accompanied by increased Ca2+-sensitive LV calpain activity. The calpain-specific inhibitor MDL28170 administered within 1 h after exposure significantly decreased calpain activity and acute mortality. Bromine inhalation and formation of reactive brominated species caused acute cardiac injury and myocardial damage that can lead to heart failure. NEW & NOTEWORTHY The present study defines left ventricular systolic and diastolic dysfunction due to cardiac injury after bromine (Br2) inhalation. A calpain-dependent mechanism was identified as a potential mediator of cardiac ultrastructure damage. This study not only highlights the importance of monitoring acute cardiac symptoms in victims of Br2 exposure but also defines calpains as a potential target to treat Br2-induced toxicity.

AKT-independent activation of p38 MAP kinase promotes vascular calcification.

Vascular calcification is prevalent in patients with atherosclerosis, and oxidative stress promotes pathogenesis of atherosclerosis. We have previously reported that activation of AKT by oxidative stress induces vascular calcification. Using sodium dichloroacetate (DCA), a previously reported small molecule inhibitor of AKT, the present studies uncovered an AKT-independent mechanism in regulating vascular calcification. We found that DCA dose-dependently induced calcification of vascular smooth muscle cells (VSMC) in vitro and aortic rings ex vivo. Furthermore, DCA markedly enhanced vascular calcification in atherosclerotic ApoE knockout mice in vivo. DCA-induced VSMC calcification was associated with increased Runx2, but not via activation of AKT, a key upstream signal that upregulates Runx2 during VSMC calcification. In contrast, DCA inhibited AKT activation and induced activation of p38 MAPK in calcified atherosclerotic lesions in vivo and calcified VSMC in vitro. Using a pharmacological inhibitor and shRNA for p38 MAPK, we demonstrated that inhibition of p38 MAPK blocked DCA-induced Runx2 upregulation and VSMC calcification. Furthermore, Runx2 deletion attenuated DCA-induced VSMC calcification. Immunoprecipitation analysis revealed association of p38 MAPK with Runx2, which was enhanced by DCA treatment. Knockdown p38 MAPK inhibited DCA-induced Runx2 transactivity, supporting the function of p38 MAPK in regulating Runx2 transactivity. Our studies have uncovered a new function of DCA in regulating vascular calcification, via AKT-independent activation of p38 MAPK. Furthermore, we have identified novel interaction between p38 MAPK and Runx2 enhances Runx2 transactivity, thus promoting VSMC calcification. These results revealed a novel signaling mechanism underlying DCA-induced vascular calcification, and offer opportunities to identify new therapeutic targets.

Dietary potassium regulates vascular calcification and arterial stiffness.

Vascular calcification is a risk factor that predicts adverse cardiovascular complications of several diseases including atherosclerosis. Reduced dietary potassium intake has been linked to cardiovascular diseases such as hypertension and incidental stroke, although the underlying molecular mechanisms remain largely unknown. Using the ApoE-deficient mouse model, we demonstrated for the first time to our knowledge that reduced dietary potassium (0.3%) promoted atherosclerotic vascular calcification and increased aortic stiffness, compared with normal (0.7%) potassium-fed mice. In contrast, increased dietary potassium (2.1%) attenuated vascular calcification and aortic stiffness. Mechanistically, reduction in the potassium concentration to the lower limit of the physiological range increased intracellular calcium, which activated a cAMP response element-binding protein (CREB) signal that subsequently enhanced autophagy and promoted vascular smooth muscle cell (VSMC) calcification. Inhibition of calcium signals and knockdown of either CREB or ATG7, an autophagy regulator, attenuated VSMC calcification induced by low potassium. Consistently, elevated autophagy and CREB signaling were demonstrated in the calcified arteries from low potassium diet-fed mice as well as aortic arteries exposed to low potassium ex vivo. These studies established a potentially novel causative role of dietary potassium intake in regulating atherosclerotic vascular calcification and stiffness, and uncovered mechanisms that offer opportunities to develop therapeutic strategies to control vascular disease.

Mechanisms and Treatment of Halogen Inhalation-Induced Pulmonary and Systemic Injuries in Pregnant Mice.

Inhalation of oxidant gases has been implicated in adverse outcomes in pregnancy, but animal models to address mechanisms and studies to identify potential pregnancy-specific therapies are lacking. Herein, we show that inhalation of bromine at 600 parts per million for 30 minutes by pregnant mice on the 15th day of embryonic development results in significantly lower survival after 96 hours than an identical level of exposure in nonpregnant mice. On the 19th embryonic day, bromine-exposed pregnant mice have increased systemic blood pressure, abnormal placental development, severe fetal growth restriction, systemic inflammation, increased levels of circulating antiangiogenic short fms-like tyrosine kinase-1, and evidence of pulmonary and cardiac injury. Treatment with tadalafil, an inhibitor of type 5 phosphodiesterase, by oral gavage 1 hour post-exposure and then once daily thereafter, attenuated systemic blood pressures, decreased inflammation, ameliorated pulmonary and cardiac injury, and improved maternal survival (from 36% to 80%) and fetal growth. These pathological changes resemble those seen in preeclampsia. Nonpregnant mice did not exhibit any of these pathological changes and were not affected by tadalafil. These findings suggest that pregnant women exposed to bromine may require particular attention and monitoring for signs of preeclampsia-like symptoms.

Increased fibroblast chymase production mediates procollagen autophagic digestion in volume overload.

BACKGROUND: Previous work has identified mast cells as the major source of chymase largely associated with a profibrotic phenotype. We recently reported increased fibroblast autophagic procollagen degradation in a rat model of pure volume overload (VO). Here we demonstrate a connection between increased fibroblast chymase production and autophagic digestion of procollagen in the pure VO of aortocaval fistula (ACF) in the rat. METHODS AND RESULTS: Isolated LV fibroblasts taken from 4 and 12week ACF Sprague-Dawley rats have significant increases in chymase mRNA and chymase activity. Increased intracellular chymase protein is documented by immunocytochemistry in the ACF fibroblasts compared to cells obtained from age-matched sham rats. To implicate VO as a stimulus for chymase production, we show that isolated adult rat LV fibroblasts subjected to 24h of 20% cyclical stretch induces chymase mRNA and protein production. Exogenous chymase treatment of control isolated adult cardiac fibroblasts demonstrates that chymase is internalized through a dynamin-dependent mechanism. Chymase treatment leads to an increased formation of autophagic vacuoles, LC3-II production, autophagic flux, resulting in increased procollagen degradation. Chymase inhibitor treatment reduces cyclical stretch-induced autophagy in isolated cardiac fibroblasts, demonstrating chymase's role in autophagy induction. CONCLUSION: In a pure VO model, chymase produced in adult cardiac fibroblasts leads to autophagic degradation of newly synthesized intracellular procollagen I, suggesting a new role of chymase in extracellular matrix degradation.

TLR4 regulates pulmonary vascular homeostasis and remodeling via redox signaling.

Pulmonary arterial hypertension (PAH) contributes to morbidity and mortality of patients with lung and heart diseases. We demonstrated that hypoxia induced PAH and increased pulmonary arterial wall thickness in wild-type mice. Mice deficient in toll-like receptor 4 (TLR4-/-) spontaneously developed PAH, which was not further enhanced by hypoxia. Echocardiography determined right ventricular hypertrophy and decreased pulmonary arterial acceleration time were associated with the development of PAH in TLR4(-/-) mice. In pulmonary arterial smooth muscle cells (PASMC), hypoxia decreased TLR4 expression and induced reactive oxygen species (ROS) and Nox1/Nox4. Inhibition of NADPH oxidase decreased hypoxia-induced proliferation of wild-type PASMC. PASMC derived from TLR4(-/-) mice exhibited increased ROS and Nox4/Nox1 expression. Our studies demonstrate an important role of TLR4 in maintaining normal pulmonary vasculature and in hypoxia-induced PAH. Inhibition of TLR4, by genetic ablation or hypoxia, increases the expression of Nox1/Nox4 and induces PASMC proliferation and vascular remodeling. These results support a novel function of TLR4 in regulating the development of PAH and reveal a new regulatory axis contributing to TLR4 deficiency-induced vascular hypertrophy and remodeling.

Volume overload induces autophagic degradation of procollagen in cardiac fibroblasts.

In a pure volume overloaded (VO) heart, interstitial collagen loss is degraded by matrix metalloproteinases (MMPs) that leads to left ventricular (LV) dilatation and heart failure. Cardiac fibroblasts are the primary source of extracellular matrix proteins that connect cardiomyocytes. The goal of this study was to determine how VO affects intracellular procollagen in cardiac fibroblasts. Using the aortocaval fistula (ACF) model in Sprague-Dawley rats, we demonstrate that cardiac fibroblasts isolated from 4 and 12 wk ACF animals have decreased intracellular procollagen I compared to the fibroblasts from age-matched shams. The reduction of procollagen I is associated with increased autophagy as demonstrated by increased autophagic vacuoles and LC3-II expression. To test the relationship between autophagy and procollagen degradation, we treated adult cardiac fibroblasts with either an autophagy inducer, rapamycin, or an inhibitor, wortmannin, and found that procollagen I protein levels were decreased in fibroblasts treated with rapamycin and elevated in wortmannin-treated cells. In addition, we demonstrated that VO induces oxidative stresses in cardiac fibroblasts from 4 and 12 wk ACF rats. Treatment of cultured cardiac fibroblasts with an oxidative stress-inducing agent (DMNQ) induces autophagy and intracellular procollagen I and fibronectin degradation, which is reversed by wortmannin but not by the global MMP inhibitor (PD166793). Mechanical stretch of cardiac fibroblasts also induces oxidative stress and autophagic degradation of procollagen I and fibronectin. Our results suggest that in addition to the well-known effects of MMPs on extracellular collagen degradation in VO, there is a concurrent degradation of intracellular procollagen and fibronectin mediated by oxidative stress-induced autophagy in cardiac fibroblasts.

Chlorine inhalation-induced myocardial depression and failure.

Victims of chlorine (Cl2) inhalation that die demonstrate significant cardiac pathology. However, a gap exists in the understanding of Cl2-induced cardiac dysfunction. This study was performed to characterize cardiac dysfunction occurring after Cl2 exposure in rats at concentrations mimicking accidental human exposures (in the range of 500 or 600 ppm for 30 min). Inhalation of 500 ppm Cl2 for 30 min resulted in increased lactate in the coronary sinus of the rats suggesting an increase in anaerobic metabolism by the heart. There was also an attenuation of myocardial contractile force in an ex vivo (Langendorff technique) retrograde perfused heart preparation. After 20 h of return to room air, Cl2 exposure at 500 ppm was associated with a reduction in systolic and diastolic blood pressure as well echocardiographic/Doppler evidence of significant left ventricular systolic and diastolic dysfunction. Cl2 exposure at 600 ppm (30 min) was associated with biventricular failure (observed at 2 h after exposure) and death. Cardiac mechanical dysfunction persisted despite increasing the inspired oxygen fraction concentration in Cl2-exposed rats (500 ppm) to ameliorate hypoxia that occurs after Cl2 inhalation. Similarly ex vivo cardiac mechanical dysfunction was reproduced by sole exposure to chloramine (a potential circulating Cl2 reactant product). These results suggest an independent and distinctive role of Cl2 (and its reactants) in inducing cardiac toxicity and potentially contributing to mortality.

Early exposure to hyperoxia or hypoxia adversely impacts cardiopulmonary development.

Preterm infants are at high risk for long-term abnormalities in cardiopulmonary function. Our objectives were to determine the long-term effects of hypoxia or hyperoxia on cardiopulmonary development and function in an immature animal model. Newborn C57BL/6 mice were exposed to air, hypoxia (12% oxygen), or hyperoxia (85% oxygen) from Postnatal Day 2-14, and then returned to air for 10 weeks (n = 2 litters per condition; > 10/group). Echocardiography, blood pressure, lung function, and lung development were evaluated at 12-14 weeks of age. Lungs from hyperoxia- or hypoxia-exposed mice were larger and more compliant (compliance: air, 0.034 ± 0.001 ml/cm H2O; hypoxia, 0.049 ± 0.002 ml/cm H2O; hyperoxia, 0.053 ± 0.002 ml/cm H2O; P < 0.001 air versus others). Increased airway reactivity, reduced bronchial M2 receptor staining, and increased bronchial α-smooth muscle actin content were noted in hyperoxia-exposed mice (maximal total lung resistance with methacholine: air, 1.89 ± 0.17 cm H2O ⋅ s/ml; hypoxia, 1.52 ± 0.34 cm H2O ⋅ s/ml; hyperoxia, 4.19 ± 0.77 cm H2O ⋅ s/ml; P < 0.004 air versus hyperoxia). Hyperoxia- or hypoxia-exposed mice had larger and fewer alveoli (mean linear intercept: air, 40.2 ± 0. 0.8 µm; hypoxia, 76.4 ± 2.4 µm; hyperoxia, 95.6 ± 4.6 µm; P < 0.001 air versus others; radial alveolar count [n]: air, 11.1 ± 0.4; hypoxia, 5.7 ± 0.3; hyperoxia, 5.6 ± 0.3; P < 0.001 air versus others). Hyperoxia-exposed adult mice had left ventricular dysfunction without systemic hypertension. In conclusion, exposure of newborn mice to hyperoxia or hypoxia leads to cardiopulmonary abnormalities in adult life, similar to that described in ex-preterm infants. This animal model may help to identify underlying mechanisms and to develop therapeutic strategies for pulmonary morbidity in former preterm infants.

Whole-genome profiling highlights the molecular complexity underlying eccentric cardiac hypertrophy.

OBJECTIVES: Heart failure is typically preceded by myocardial hypertrophy and remodeling, which can be concentric due to pressure overload (PO), or eccentric because of volume overload (VO). The molecular mechanisms that underlie these differing patterns of hypertrophy are distinct and have yet to be fully elucidated. Thus, the goal of this work is to identify novel therapeutic targets for cardiovascular conditions marked by hypertrophy that have previously been resistant to medical treatment, such as a pure VO. METHODS: Concentric or eccentric hypertrophy was induced in rats for 2 weeks with transverse aortic constriction (TAC) or aortocaval fistula (ACF), respectively. Hemodynamic and echocardiographic analysis were used to assess the development of left ventricular (LV) hypertrophy and functional differences between groups. Changes in gene expression were determined by microarray and further characterized with Ingenuity Pathway Analysis. RESULTS: Both models of hypertrophy increased LV mass. Rats with TAC demonstrated concentric LV remodeling while rats with ACF exhibited eccentric LV remodeling. Microarray analysis associated eccentric remodeling with a more extensive alteration of gene expression compared with concentric remodeling. Rats with VO had a marked activation of extracellular matrix genes, promotion of cell cycle genes, downregulation of genes associated with oxidative metabolism, and dysregulation of genes critical to cardiac contractile function. Rats with PO demonstrated similar categorical changes, but with the involvement of fewer individual genes. CONCLUSIONS: Our results indicate that eccentric remodeling is a far more complex process than concentric remodeling. This study highlights the importance of several key biological functions early in the course of VO, including regulation of matrix, metabolism, cell proliferation, and contractile function. Thus, the results of this analysis will inform the ongoing search for new treatments to prevent the progression to heart failure in VO.

Activation of AKT by O-linked N-acetylglucosamine induces vascular calcification in diabetes mellitus.

RATIONALE: Vascular calcification is a serious cardiovascular complication that contributes to the increased morbidity and mortality of patients with diabetes mellitus. Hyperglycemia, a hallmark of diabetes mellitus, is associated with increased vascular calcification and increased modification of proteins by O-linked N-acetylglucosamine (O-GlcNAcylation). OBJECTIVE: We sought to determine the role of protein O-GlcNAcylation in regulating vascular calcification and the underlying mechanisms. METHODS AND RESULTS: Low-dose streptozotocin-induced diabetic mice exhibited increased aortic O-GlcNAcylation and vascular calcification, which was also associated with impaired aortic compliance in mice. Elevation of O-GlcNAcylation by administration of Thiamet-G, a potent inhibitor for O-GlcNAcase that removes O-GlcNAcylation, further accelerated vascular calcification and worsened aortic compliance of diabetic mice in vivo. Increased O-GlcNAcylation, either by Thiamet-G or O-GlcNAcase knockdown, promoted calcification of primary mouse vascular smooth muscle cells. Increased O-GlcNAcylation in diabetic arteries or in the O-GlcNAcase knockdown vascular smooth muscle cell upregulated expression of the osteogenic transcription factor Runx2 and enhanced activation of AKT. O-GlcNAcylation of AKT at two new sites, T430 and T479, promoted AKT phosphorylation, which in turn enhanced vascular smooth muscle cell calcification. Site-directed mutation of AKT at T430 and T479 decreased O-GlcNAcylation, inhibited phosphorylation of AKT at S473 and binding of mammalian target of rapamycin complex 2 to AKT, and subsequently blocked Runx2 transactivity and vascular smooth muscle cell calcification. CONCLUSIONS: O-GlcNAcylation of AKT at 2 new sites enhanced AKT phosphorylation and activation, thus promoting vascular calcification. Our studies have identified a novel causative effect of O-GlcNAcylation in regulating vascular calcification in diabetes mellitus and uncovered a key molecular mechanism underlying O-GlcNAcylation-mediated activation of AKT.

Quercetin prevents left ventricular hypertrophy in the Apo E knockout mouse.

Hypercholesterolemia is a risk factor for the development of hypertrophic cardiomyopathy. Nevertheless, there are few studies aimed at determining the effects of dietary compounds on early or mild cardiac hypertrophy associated with dyslipidemia. Here we describe left ventricular (LV) hypertrophy in 12 week-old Apo E(-/-) hypercholesterolemic mice. The LV end diastolic posterior wall thickness and overall LV mass were significantly increased in Apo E(-/-) mice compared with wild type (WT) controls. Fractional shortening, LV end diastolic diameter, and hemodynamic parameters were unchanged from WT mice. Oral low dose quercetin (QCN; 0.1 µmol QCN/kg body weight for 6 weeks) significantly reduced total cholesterol and very low density lipoprotein in the plasma of Apo E(-/-) mice. QCN treatment also significantly decreased LV posterior wall thickness and LV mass in Apo E(-/-) mice. Myocardial geometry and function were unaffected in WT mice by QCN treatment. These data suggest that dietary polyphenolic compounds such as QCN may be effective modulators of plasma cholesterol and could prevent maladaptive myocardial remodeling.

Mitochondrial genetic background modulates bioenergetics and susceptibility to acute cardiac volume overload.

Dysfunctional bioenergetics has emerged as a key feature in many chronic pathologies such as diabetes and cardiovascular disease. This has led to the mitochondrial paradigm in which it has been proposed that mtDNA sequence variation contributes to disease susceptibility. In the present study we show a novel animal model of mtDNA polymorphisms, the MNX (mitochondrial-nuclear exchange) mouse, in which the mtDNA from the C3H/HeN mouse has been inserted on to the C57/BL6 nuclear background and vice versa to test this concept. Our data show a major contribution of the C57/BL6 mtDNA to the susceptibility to the pathological stress of cardiac volume overload which is independent of the nuclear background. Mitochondria harbouring the C57/BL6J mtDNA generate more ROS (reactive oxygen species) and have a higher mitochondrial membrane potential relative to those with C3H/HeN mtDNA, independent of nuclear background. We propose this is the primary mechanism associated with increased bioenergetic dysfunction in response to volume overload. In summary, these studies support the 'mitochondrial paradigm' for the development of disease susceptibility, and show that the mtDNA modulates cellular bioenergetics, mitochondrial ROS generation and susceptibility to cardiac stress.

Cardiac kallikrein-kinin system is upregulated in chronic volume overload and mediates an inflammatory induced collagen loss.

BACKGROUND: The clinical problem of a "pure volume overload" as in isolated mitral or aortic regurgitation currently has no documented medical therapy that attenuates collagen loss and the resultant left ventricular (LV) dilatation and failure. Here, we identify a potential mechanism related to upregulation of the kallikrein-kinin system in the volume overload of aortocaval fistula (ACF) in the rat. METHODOLOGY/PRINCIPAL FINDINGS: LV interstitial fluid (ISF) collection, hemodynamics, and echocardiography were performed in age-matched shams and 4 and 15 wk ACF rats. ACF rats had LV dilatation and a 2-fold increase in LV end-diastolic pressure, along with increases in LV ISF bradykinin, myocardial kallikrein and bradykinin type-2 receptor (BK(2)R) mRNA expression. Mast cell numbers were increased and interstitial collagen was decreased at 4 and 15 wk ACF, despite increases in LV ACE and chymase activities. Treatment with the kallikrein inhibitor aprotinin preserved interstitial collagen, prevented the increase in mast cells, and improved LV systolic function at 4 wk ACF. To establish a cause and effect between ISF bradykinin and mast cell-mediated collagen loss, direct LV interstitial bradykinin infusion in vivo for 24 hrs produced a 2-fold increase in mast cell numbers and a 30% decrease in interstitial collagen, which were prevented by BK(2)R antagonist. To further connect myocardial stretch with cellular kallikrein-kinin system upregulation, 24 hrs cyclic stretch of adult cardiomyocytes and fibroblasts produced increased kallikrein, BK(2)R mRNA expressions, bradykinin protein and gelatinase activity, which were all decreased by the kallikrein inhibitor-aprotinin. CONCLUSIONS/SIGNIFICANCE: A pure volume overload is associated with upregulation of the kallikrein-kinin system and ISF bradykinin, which mediates mast cell infiltration, extracellular matrix loss, and LV dysfunction-all of which are improved by kallikrein inhibition. The current investigation provides important new insights into future potential medical therapies for the volume overload of aortic and mitral regurgitation.

c-kit is required for cardiomyocyte terminal differentiation.

c-kit, the transmembrane tyrosine kinase receptor for stem cell factor, is required for melanocyte and mast cell development, hematopoiesis, and differentiation of spermatogonial stem cells. We show here that in the heart, c-kit is expressed not only by cardiac stem cells but also by cardiomyocytes, commencing immediately after birth and terminating a few days later, coincident with the onset of cardiomyocyte terminal differentiation. To examine the function of c-kit in cardiomyocyte terminal differentiation, we used compound heterozygous mice carrying the W (null) and W(v) (dominant negative) mutations of c-kit. In vivo, adult W/W(v) cardiomyocytes are phenotypically indistinguishable from their wild-type counterparts. After acute pressure overload adult W/W(v) cardiomyocytes reenter the cell cycle and proliferate, leading to left ventricular growth; furthermore in transgenic mice with cardiomyocyte-restricted overexpression of the dominant negative W(v) mutant, pressure overload causes cardiomyocytes to reenter the cell cycle. In contrast, in wild-type mice left ventricular growth after pressure overload results mainly from cardiomyocyte hypertrophy. Importantly, W/W(v) mice with pressure overload-induced cardiomyocyte hyperplasia had improved left ventricular function and survival. In W/W(v) mice, c-kit dysfunction also resulted in an approximately 14-fold decrease (P<0.01) in the number of c-kit(+)/GATA4(+) cardiac progenitors. These findings identify novel functions for c-kit: promotion of cardiac stem cell differentiation and regulation of cardiomyocyte terminal differentiation.