Adenosine kinase attenuates cardiomyocyte microtubule stabilization and protects against pressure overload-induced hypertrophy and LV dysfunction.

Adenosine exerts numerous protective actions in the heart, including attenuation of cardiac hypertrophy. Adenosine kinase (ADK) converts adenosine to adenosine monophosphate (AMP) and is the major route of myocardial adenosine metabolism, however, the impact of ADK activity on cardiac structure and function is unknown. To examine the role of ADK in cardiac homeostasis and adaptation to stress, conditional cardiomyocyte specific ADK knockout mice (cADK-/-) were produced using the MerCreMer-lox-P system. Within 4 weeks of ADK disruption, cADK-/- mice developed spontaneous hypertrophy and increased ß-Myosin Heavy Chain expression without observable LV dysfunction. In response to 6 weeks moderate left ventricular pressure overload (transverse aortic constriction;TAC), wild type mice (WT) exhibited ~60% increase in ventricular ADK expression and developed LV hypertrophy with preserved LV function. In contrast, cADK-/- mice exhibited significantly greater LV hypertrophy and cardiac stress marker expression (atrial natrurietic peptide and ß-Myosin Heavy Chain), LV dilation, reduced LV ejection fraction and increased pulmonary congestion. ADK disruption did not decrease protein methylation, inhibit AMPK, or worsen fibrosis, but was associated with persistently elevated mTORC1 and p44/42 ERK MAP kinase signaling and a striking increase in microtubule (MT) stabilization/detyrosination. In neonatal cardiomyocytes exposed to hypertrophic stress, 2-chloroadenosine (CADO) or adenosine treatment suppressed MT detyrosination, which was reversed by ADK inhibition with iodotubercidin or ABT-702. Conversely, adenoviral over-expression of ADK augmented CADO destabilization of MTs and potentiated CADO attenuation of cardiomyocyte hypertrophy. Together, these findings indicate a novel adenosine receptor-independent role for ADK-mediated adenosine metabolism in cardiomyocyte microtubule dynamics and protection against maladaptive hypertrophy.

Role of bone marrow-derived CD11c+ dendritic cells in systolic overload-induced left ventricular inflammation, fibrosis and hypertrophy.

Inflammatory responses play an important role in the development of left ventricular (LV) hypertrophy and dysfunction. Recent studies demonstrated that increased T-cell infiltration and T-cell activation contribute to LV hypertrophy and dysfunction. Dendritic cells (DCs) are professional antigen-presenting cells that orchestrate immune responses, especially by modulating T-cell function. In this study, we investigated the role of bone marrow-derived CD11c+ DCs in transverse aortic constriction (TAC)-induced LV fibrosis and hypertrophy in mice. We observed that TAC increased the number of CD11c+ cells and the percentage of CD11c+ MHCII+ (major histocompatibility complex class II molecule positive) DCs in the LV, spleen and peripheral blood in mice. Using bone marrow chimeras and an inducible CD11c+ DC ablation model, we found that depletion of bone marrow-derived CD11c+ DCs significantly attenuated LV fibrosis and hypertrophy in mice exposed to 24 weeks of moderate TAC. CD11c+ DC ablation significantly reduced TAC-induced myocardial inflammation as indicated by reduced myocardial CD45+ cells, CD11b+ cells, CD8+ T cells and activated effector CD8+CD44+ T cells in LV tissues. Moreover, pulsing of autologous DCs with LV homogenates from TAC mice promoted T-cell proliferation. These data indicate that bone marrow-derived CD11c+ DCs play a maladaptive role in hemodynamic overload-induced cardiac inflammation, hypertrophy and fibrosis through the presentation of cardiac self-antigens to T cells.

Repetitive ischemia increases myocardial dimethylarginine dimethylaminohydrolase 1 expression.

Pharmacologic inhibition of nitric oxide production inhibits growth of coronary collateral vessels. Dimethylarginine dimethylaminohydrolase 1 (DDAH1) is the major enzyme that degrades asymmetric dimethylarginine (ADMA), a potent inhibitor of nitric oxide synthase. Here we examined regulation of the ADMA-DDAH1 pathway in a canine model of recurrent myocardial ischemia during the time when coronary collateral growth is known to occur. Under basal conditions, DDAH1 expression was non-uniform across the left ventricular (LV) wall, with expression strongest in the subepicardium. In response to ischemia, DDAH1 expression was up-regulated in the midmyocardium of the ischemic zone, and this was associated with a significant reduction in myocardial interstitial fluid (MIF) ADMA. The decrease in MIF ADMA during ischemia was likely due to increased DDAH1 because myocardial protein arginine N-methyl transferase 1 (PRMT1) and the methylated arginine protein content (the source of ADMA) were unchanged or increased, respectively, at this time. The inflammatory mediators interleukin (IL-1ß) and tumor necrosis factor (TNF-α) were also elevated in the midmyocardium where DDAH1 expression was increased. Both of these factors significantly up-regulated DDAH1 expression in cultured human coronary artery endothelial cells. Taken together, these results suggest that inflammatory factors expressed in response to myocardial ischemia contributed to up-regulation of DDAH1, which was responsible for the decrease in MIF ADMA.

CD28/B7 Deficiency Attenuates Systolic Overload-Induced Congestive Heart Failure, Myocardial and Pulmonary Inflammation, and Activated T Cell Accumulation in the Heart and Lungs.

The inflammatory response regulates congestive heart failure (CHF) development. T cell activation plays an important role in tissue inflammation. We postulate that CD28 or B7 deficiency inhibits T cell activation and attenuates CHF development by reducing systemic, cardiac, and pulmonary inflammation. We demonstrated that chronic pressure overload-induced end-stage CHF in mice is characterized by profound accumulation of activated effector T cells (CD3(+)CD44(high) cells) in the lungs and a mild but significant increase of these cells in the heart. In knockout mice lacking either CD28 or B7, there was a dramatic reduction in the accumulation of activated effector T cells in both hearts and lungs of mice under control conditions and after transverse aortic constriction. CD28 or B7 knockout significantly attenuated transverse aortic constriction-induced CHF development, as indicated by less increase of heart and lung weight and less reduction of left ventricle contractility. CD28 or B7 knockout also significantly reduced transverse aortic constriction-induced CD45(+) leukocyte, T cell, and macrophage infiltration in hearts and lungs, lowered proinflammatory cytokine expression (such as tumor necrosis factor-α and interleukin-1ß) in lungs. Furthermore, CD28/B7 blockade by CTLA4-Ig treatment (250 µg/mouse every 3 days) attenuated transverse aortic constriction-induced T cell activation, left ventricle hypertrophy, and left ventricle dysfunction. Our data indicate that CD28/B7 deficiency inhibits activated effector T cell accumulation, reduces myocardial and pulmonary inflammation, and attenuates the development of CHF. Our findings suggest that strategies targeting T cell activation may be useful in treating CHF.

ATP sensitive K(+) channels are critical for maintaining myocardial perfusion and high energy phosphates in the failing heart.

Congestive heart failure (CHF) is associated with intrinsic alterations of mitochondrial oxidative phosphorylation which lead to increased myocardial cytosolic free ADP. ATP sensitive K(+) channels (KATP) act as metabolic sensors that are important for maintaining coronary blood flow (MBF) and in mediating the response of the myocardium to stress. Coronary adenosine receptors (AdR) are not normally active but cause vasodilation during myocardial ischemia. This study examined the myocardial energetic response to inhibition of KATP and AdR in CHF. CHF (as evidenced by LVEDP>20mmHg) was produced in adult mongrel dogs (n=12) by rapid ventricular pacing for 4weeks. MBF was measured with radiolabeled microspheres during baseline (BL), AdR blockade with 8-phenyltheophylline (8-PT; 5mg/kg iv), and KATP blockade with glibenclamide (GLB; 20µg/kg/min ic). High energy phosphates were examined with (31)P magnetic resonance spectroscopy (MRS) while myocardial oxygenation was assessed from the deoxymyoglobin signal (Mb-δ) using (1)H MRS. During basal conditions the phosphocreatine (PCr)/ATP ratio (1.73±0.15) was significantly lower than in previously studied normal dogs (2.42±0.11) although Mb-δ was undetectable. 8-PT caused ≈21% increase in MBF with no change in PCr/ATP. GLB caused a 33±0.1% decrease in MBF with a decrease in PCr/ATP from 1.65±0.17 to 1.11±0.11 (p<0.0001). GLB did not change the pseudo-first-order rate constant of ATP production via CK (kf), but the ATP production rate via CK was reduced by 35±0.08%; this was accompanied by an increase in Pi/PCr and appearance of a Mb-δ signal indicating tissue hypoxia. Thus, in the failing heart the balance between myocardial ATP demands and oxygen delivery is critically dependent on functioning KATP channels.

Endoplasmic reticulum stress sensor protein kinase R-like endoplasmic reticulum kinase (PERK) protects against pressure overload-induced heart failure and lung remodeling.

Studies have reported that development of congestive heart failure is associated with increased endoplasmic reticulum stress. Double stranded RNA-activated protein kinase R-like endoplasmic reticulum kinase (PERK) is a major transducer of the endoplasmic reticulum stress response and directly phosphorylates eukaryotic initiation factor 2α, resulting in translational attenuation. However, the physiological effect of PERK on congestive heart failure development is unknown. To study the effect of PERK on ventricular structure and function, we generated inducible cardiac-specific PERK knockout mice. Under unstressed conditions, cardiac PERK knockout had no effect on left ventricular mass, or its ratio to body weight, cardiomyocyte size, fibrosis, or left ventricular function. However, in response to chronic transverse aortic constriction, PERK knockout mice exhibited decreased ejection fraction, increased left ventricular fibrosis, enhanced cardiomyocyte apoptosis, and exacerbated lung remodeling in comparison with wild-type mice. PERK knockout also dramatically attenuated cardiac sarcoplasmic reticulum Ca(2+)-ATPase expression in response to aortic constriction. Our findings suggest that PERK is required to protect the heart from pressure overload-induced congestive heart failure.

Double-stranded RNA-dependent protein kinase deficiency protects the heart from systolic overload-induced congestive heart failure.

BACKGROUND: Double-stranded RNA-dependent protein kinase (PKR) is a eukaryotic initiation factor 2α kinase that inhibits mRNA translation under stress conditions. PKR also mediates inflammatory and apoptotic signaling independently of translational regulation. Congestive heart failure is associated with cardiomyocyte hypertrophy, inflammation, and apoptosis, but the role of PKR in left ventricular hypertrophy and the development of congestive heart failure has not been examined. METHODS AND RESULTS: We observed increased myocardial PKR expression and translocation of PKR into the nucleus in humans and mice with congestive heart failure. To determine the impact of PKR on the development of congestive heart failure, PKR knockout and wild-type mice were exposed to pressure overload produced by transverse aortic constriction. Although heart size increased similarly in wild-type and PKR knockout mice after transverse aortic constriction, PKR knockout mice exhibited very little pulmonary congestion, well-preserved left ventricular ejection fraction and contractility, and significantly less myocardial fibrosis compared with wild-type mice. Bone marrow-derived cells from wild-type mice did not abolish the cardiac protective effect observed in PKR knockout mice, whereas bone marrow-derived cells from PKR knockout mice had no cardiac protective effect in wild-type mice. Mechanistically, PKR knockout attenuated transverse aortic constriction-induced tumor necrosis factor-α expression and leukocyte infiltration and lowered cardiac expression of proapoptotic factors (Bax and caspase-3), so that PKR knockout hearts were more resistant to transverse aortic constriction-induced cardiomyocyte apoptosis. PKR depletion in isolated cardiomyocytes also conferred protection against tumor necrosis factor-α- or lipopolysaccharide-induced apoptosis. CONCLUSION: PKR is a maladaptive factor upregulated in hemodynamic overload that contributes to myocardial inflammation, cardiomyocyte apoptosis, and the development of congestive heart failure.

DDAH1 deficiency attenuates endothelial cell cycle progression and angiogenesis.

Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of nitric oxide (NO) synthase (NOS). ADMA is eliminated largely by the action of dimethylarginine dimethylaminohydrolase1 (DDAH1). Decreased DDAH activity is found in several pathological conditions and is associated with increased risk of vascular disease. Overexpression of DDAH1 has been shown to augment endothelial proliferation and angiogenesis. To better understand the mechanism by which DDAH1 influences endothelial proliferation, this study examined the effect of DDAH1 deficiency on cell cycle progression and the expression of some cell cycle master regulatory proteins. DDAH1 KO decreased in vivo Matrigel angiogenesis and depressed endothelial repair in a mouse model of carotid artery wire injury. DDAH1 deficiency decreased VEGF expression in HUVEC and increased NF1 expression in both HUVEC and DDAH1 KO mice. The expression of active Ras could overcome the decreased VEGF expression caused by the DDAH1 depletion. The addition of VEGF and knockdown NF1 could both restore proliferation in cells with DDAH1 depletion. Flow cytometry analysis revealed that DDAH1 sRNAi knockdown in HUVEC caused G1 and G2/M arrest that was associated with decreased expression of CDC2, CDC25C, cyclin D1 and cyclin E. MEF cells from DDAH1 KO mice also demonstrated G2/M arrest that was associated with decreased cyclin D1 expression and Akt activity. Our findings indicate that DDAH1 exerts effects on cyclin D1 and cyclin E expression through multiple mechanisms, including VEGF, the NO/cGMP/PKG pathway, the Ras/PI3K/Akt pathway, and NF1 expression. Loss of DDAH1 effects on these pathways results in impaired endothelial cell proliferation and decreased angiogenesis. The findings provide background information that may be useful in the development of therapeutic strategies to manipulate DDAH1 expression in cardiovascular diseases or tumor angiogenesis.

Regulation of coronary resistance vessel tone in response to exercise.

Exercise is a primary stimulus for increased myocardial oxygen demand. The ~6-fold increase in oxygen demand of the left ventricle during heavy exercise is met principally by augmenting coronary blood flow (~5-fold), as hemoglobin concentration and oxygen extraction (which is already ~70% at rest) increase only modestly in most species. As a result, coronary blood flow is tightly coupled to myocardial oxygen consumption over a wide range of physical activity. This tight coupling has been proposed to depend on periarteriolar oxygen tension, signals released from cardiomyocytes and the endothelium as well as neurohumoral influences, but the contribution of each of these regulatory pathways, and their interactions, to exercise hyperemia in the heart remain incompletely understood. In humans, nitric oxide, adenosine and K(ATP) channels each appear to contribute to resting coronary resistance vessel tone, but evidence for a critical contribution to exercise hyperemia is lacking. In dogs K(ATP)-channel activation together with adenosine and nitric oxide contribute to exercise hyperemia in a non-linear redundant fashion. In contrast, in swine nitric oxide, adenosine and K(ATP) channels contribute to resting coronary resistance vessel tone control in a linear additive manner, but do not appear to be mandatory for exercise hyperemia. Rather, exercise hyperemia in swine appears to involve ß-adrenergic activation in conjunction with exercise-induced blunting of an endothelin-mediated vasoconstrictor influence. In view of these remarkable species differences in coronary vasomotor control during exercise, future studies are required to determine the system of vasodilator components that mediate exercise hyperemia in humans. This article is part of a Special Issue entitled "Coronary Blood Flow".

AMP activated protein kinase-α2 regulates expression of estrogen-related receptor-α, a metabolic transcription factor related to heart failure development.

The normal expression of myocardial mitochondrial enzymes is essential to maintain the cardiac energy reserve and facilitate responses to stress, but the molecular mechanisms to maintain myocardial mitochondrial enzyme expression have been elusive. Here we report that congestive heart failure is associated with a significant decrease of myocardial estrogen-related receptor-α (ERRα), but not peroxisome proliferator-activated receptor-γ coactivator 1α, in human heart failure samples. In addition, chronic pressure overload in mice caused a decrease of ERRα expression that was significantly correlated to the degree of left ventricular dysfunction, pulmonary congestion, and decreases of a group of myocardial energy metabolism-related genes. We found that the metabolic sensor AMP activated protein kinase (AMPK) regulates ERRα expression in vivo and in vitro. AMPKα2 knockout decreased myocardial ERRα (both mRNA and protein) and its downstream targets under basal conditions, with no change in myocardial peroxisome proliferator-activated receptor-γ coactivator 1α expression. Using cultured rat neonatal cardiac myocytes, we found that overexpression of constitutively active AMPKα significantly induced ERRα mRNA, protein, and promoter activity. Conversely, selective gene silencing of AMPKα2 repressed ERRα and its target gene levels, indicating that AMPKα2 is involved in the regulation of ERRα expression. In addition, overexpression of ERRα in AMPKα2 knockout neonatal cardiac myocytes partially rescued the repressed expression of some energy metabolism-related genes. These data support an important role for AMPKα2 in regulating the expression of myocardial ERRα and its downstream mitochondrial enzymes.

Adenosine kinase regulation of cardiomyocyte hypertrophy.

There is evidence that extracellular adenosine can attenuate cardiac hypertrophy, but the mechanism by which this occurs is not clear. Here we investigated the role of adenosine receptors and adenosine metabolism in attenuation of cardiomyocyte hypertrophy. Phenylephrine (PE) caused hypertrophy of neonatal rat cardiomyocytes with increases of cell surface area, protein synthesis, and atrial natriuretic peptide (ANP) expression. These responses were attenuated by 5 µM 2-chloroadenosine (CADO; adenosine deaminase resistant adenosine analog) or 10 µM adenosine. While antagonism of adenosine receptors partially blocked the reduction of ANP expression produced by CADO, it did not restore cell size or protein synthesis. In support of a role for intracellular adenosine metabolism in regulating hypertrophy, the adenosine kinase (AK) inhibitors iodotubercidin and ABT-702 completely reversed the attenuation of cell size, protein synthesis, and expression of ANP by CADO or ADO. Examination of PE-induced phosphosignaling pathways revealed that CADO treatment did not reduce AKT(Ser47³) phosphorylation but did attenuate sustained phosphorylation of Raf(Ser³³8) (24-48 h), mTOR(Ser²448) (24-48 h), p70S6k(Thr³89) (2.5-48 h), and ERK(Thr²°²/Tyr²°4) (48 h). Inhibition of AK restored activation of these enzymes in the presence of CADO. Using dominant negative and constitutively active Raf adenoviruses, we found that Raf activation is necessary and sufficient for PE-induced mTORC1 signaling and cardiomyocyte hypertrophy. CADO treatment still blocked p70S6k(Thr³89) phosphorylation and hypertrophy downstream of constitutively active Raf, however, despite a high level phosphorylation of ERK(Thr202/Tyr204) and AKT(Ser47³). Reduction of Raf-induced p70S6k(Thr³89) phosphorylation and hypertrophy by CADO was reversed by inhibiting AK. Together, these results identify AK as an important mediator of adenosine attenuation of cardiomyocyte hypertrophy, which acts, at least in part, through inhibition of Raf signaling to mTOR/p70S6k.

Dimethylarginine dimethylaminohydrolase 1 modulates endothelial cell growth through nitric oxide and Akt.

OBJECTIVE: Dimethylarginine dimethylaminohydrolase 1 (DDAH1) modulates NO production by degrading the endogenous nitric oxide (NO) synthase (NOS) inhibitors asymmetrical dimethylarginine (ADMA) and L-NG-monomethyl arginine (L-NMMA). This study examined whether, in addition to degrading ADMA, DDAH1 exerts ADMA-independent effects that influence endothelial function. METHODS AND RESULTS: Using selective gene silencing of DDAH1 with small interfering RNA and overexpression of DDAH1 in human umbilical vein endothelial cells, we found that DDAH1 acts to promote endothelial cell proliferation, migration, and tube formation by Akt phosphorylation, as well as through the traditional role of degrading ADMA. Incubation of human umbilical vein endothelial cells with the NOS inhibitors l-NG-nitro-arginine methyl ester (L-NAME) or ADMA, the soluble guanylyl cyclase inhibitor 1H-(1,2,4)oxadiazolo-(4,3-2)quinoxalin-1-one, or the cGMP analog 8-(4-Chlorophenylthio)-cGMP had no effect on phosphorylated (p)-Akt(Ser473), indicating that the increase in p-Akt(Ser473) produced by DDAH1 was independent of the NO-cGMP signaling pathway. DDAH1 formed a protein complex with Ras, and DDAH1 overexpression increased Ras activity. The Ras inhibitor manumycin-A or dominant-negative Ras significantly attenuated the DDAH1-induced increase in p-Akt(Ser473). Furthermore, DDAH1 knockout impaired endothelial sprouting from cultured aortic rings, and overexpression of constitutively active Akt or DDAH1 rescued endothelial sprouting in the aortic rings from these mice. CONCLUSIONS: DDAH1 exerts a unique role in activating Akt that affects endothelial function independently of degrading endogenous NOS inhibitors.

Adenosine regulation of microtubule dynamics in cardiac hypertrophy.

There is evidence that endogenous extracellular adenosine reduces cardiac hypertrophy and heart failure in mice subjected to chronic pressure overload, but the mechanism by which adenosine exerts these protective effects is unknown. Here, we identified a novel role for adenosine in regulation of the cardiac microtubule cytoskeleton that may contribute to its beneficial effects in the overloaded heart. In neonatal cardiomyocytes, phenylephrine promoted hypertrophy and reorganization of the cytoskeleton, which included accumulation of sarcomeric proteins, microtubules, and desmin. Treatment with adenosine or the stable adenosine analog 2-chloroadenosine, which decreased hypertrophy, specifically reduced accumulation of microtubules. In hypertrophied cardiomyocytes, 2-chloroadenosine or adenosine treatment preferentially targeted stabilized microtubules (containing detyrosinated alpha-tubulin). Consistent with a role for endogenous adenosine in reducing microtubule stability, levels of detyrosinated microtubules were elevated in hearts of CD73 knockout mice (deficient in extracellular adenosine production) compared with wild-type mice (195%, P < 0.05). In response to aortic banding, microtubules increased in hearts of wild-type mice; this increase was exaggerated in CD73 knockout mice, with significantly greater amounts of tubulin partitioning into the cold-stable Triton-insoluble fractions. The levels of this stable cytoskeletal fraction of tubulin correlated strongly with the degree of heart failure. In agreement with a role for microtubule stabilization in promoting cardiac dysfunction, colchicine treatment of aortic-banded mice reduced hypertrophy and improved cardiac function compared with saline-treated controls. These results indicate that microtubules contribute to cardiac dysfunction and identify, for the first time, a role for adenosine in regulating cardiomyocyte microtubule dynamics.

Adenosine A3 receptor deficiency exerts unanticipated protective effects on the pressure-overloaded left ventricle.

BACKGROUND: Endogenous adenosine can protect the overloaded heart against the development of hypertrophy and heart failure, but the contribution of A(1) receptors (A(1)R) and A(3) receptors (A(3)R) is not known. METHODS AND RESULTS: To test the hypothesis that A(1)R and A(3)R can protect the heart against systolic overload, we exposed A(3)R gene-deficient (A(3)R knockout [KO]) mice and A(1)R KO mice to transverse aortic constriction (TAC). Contrary to our hypothesis, A(3)R KO attenuated 5-week TAC-induced left ventricular hypertrophy (ratio of ventricular mass/body weight increased to 7.6+/-0.3 mg/g in wild-type mice compared with 6.3+/-0.4 mg/g in KO mice), fibrosis, and dysfunction (left ventricular ejection fraction decreased to 43+/-2.5% and 55+/-4.2% in wild-type and KO mice, respectively). A(3)R KO also attenuated the TAC-induced increases of myocardial atrial natriuretic peptide and the oxidative stress markers 3'-nitrotyrosine and 4-hydroxynonenal. In contrast, A(1)R KO increased TAC-induced mortality but did not alter ventricular hypertrophy or dysfunction compared with wild-type mice. In mice in which extracellular adenosine production was impaired by CD73 KO, TAC caused greater hypertrophy and dysfunction and increased myocardial 3'-nitrotyrosine. In neonatal rat cardiomyocytes induced to hypertrophy with phenylephrine, the adenosine analogue 2-chloroadenosine reduced cell area, protein synthesis, atrial natriuretic peptide, and 3'-nitrotyrosine. Antagonism of A(3)R significantly potentiated the antihypertrophic effects of 2-chloroadenosine. CONCLUSIONS: Adenosine exerts protective effects on the overloaded heart, but the A(3)R acts counter to the protective effect of adenosine. The data suggest that selective attenuation of A(3)R activity might be a novel approach to treat pressure overload-induced left ventricular hypertrophy and dysfunction.

Disruption of sarcolemmal ATP-sensitive potassium channel activity impairs the cardiac response to systolic overload.

Sarcolemmal ATP-sensitive potassium channels (K(ATP)) act as metabolic sensors that facilitate adaptation of the left ventricle to changes in energy requirements. This study examined the mechanism by which K(ATP) dysfunction impairs the left ventricular response to stress using transgenic mouse strains with cardiac-specific disruption of K(ATP) activity (SUR1-tg mice) or Kir6.2 gene deficiency (Kir6.2 KO). Both SUR1-tg and Kir6.2 KO mice had normal left ventricular mass and function under unstressed conditions. Following chronic transverse aortic constriction, both SUR1-tg and Kir6.2 KO mice developed more severe left ventricular hypertrophy and dysfunction as compared with their corresponding WT controls. Both SUR1-tg and Kir6.2 KO mice had significantly decreased expression of peroxisome proliferator-activated receptor gamma coactivator (PGC)-1alpha and a group of energy metabolism related genes at both protein and mRNA levels. Furthermore, disruption of K(ATP) repressed expression and promoter activity of PGC-1alpha in cultured rat neonatal cardiac myocytes in response to hypoxia, indicating that K(ATP) activity is required to maintain PGC-1alpha expression under stress conditions. PGC-1alpha gene deficiency also exacerbated chronic transverse aortic constriction-induced ventricular hypertrophy and dysfunction, suggesting that depletion of PGC-1alpha can worsen systolic overload induced ventricular dysfunction. Both SUR1-tg and Kir6.2 KO mice had decreased FOXO1 after transverse aortic constriction, in agreement with the reports that a decrease of FOXO1 can repress PGC-1alpha expression. Furthermore, inhibition of K(ATP) caused a decrease of FOXO1 associated with PGC-1alpha promoter. These data indicate that K(ATP) channels facilitate the cardiac response to stress by regulating PGC-1alpha and its target genes, at least partially through the FOXO1 pathway.

Phosphate metabolite concentrations and ATP hydrolysis potential in normal and ischaemic hearts.

To understand how cardiac ATP and CrP remain stable with changes in work rate - a phenomenon that has eluded mechanistic explanation for decades - data from (31)phosphate-magnetic resonance spectroscopy ((31)P-MRS) are analysed to estimate cytoplasmic and mitochondrial phosphate metabolite concentrations in the normal state, during high cardiac workstates, during acute ischaemia and reactive hyperaemic recovery. Analysis is based on simulating distributed heterogeneous oxygen transport in the myocardium integrated with a detailed model of cardiac energy metabolism. The model predicts that baseline myocardial free inorganic phosphate (P(i)) concentration in the canine myocyte cytoplasm - a variable not accessible to direct non-invasive measurement - is approximately 0.29 mm and increases to 2.3 mm near maximal cardiac oxygen consumption. During acute ischaemia (from ligation of the left anterior descending artery) P(i) increases to approximately 3.1 mm and ATP consumption in the ischaemic tissue is reduced quickly to less than half its baseline value before the creatine phosphate (CrP) pool is 18% depleted. It is determined from these experiments that the maximal rate of oxygen consumption of the heart is an emergent property and is limited not simply by the maximal rate of ATP synthesis, but by the maximal rate at which ATP can be synthesized at a potential at which it can be utilized. The critical free energy of ATP hydrolysis for cardiac contraction that is consistent with these findings is approximately -63.5 kJ mol(-1). Based on theoretical findings, we hypothesize that inorganic phosphate is both the primary feedback signal for stimulating oxidative phosphorylation in vivo and also the most significant product of ATP hydrolysis in limiting the capacity of the heart to hydrolyse ATP in vivo. Due to the lack of precise quantification of P(i) in vivo, these hypotheses and associated model predictions remain to be carefully tested experimentally.

Regulation of coronary blood flow during exercise.

Exercise is the most important physiological stimulus for increased myocardial oxygen demand. The requirement of exercising muscle for increased blood flow necessitates an increase in cardiac output that results in increases in the three main determinants of myocardial oxygen demand: heart rate, myocardial contractility, and ventricular work. The approximately sixfold increase in oxygen demands of the left ventricle during heavy exercise is met principally by augmenting coronary blood flow (~5-fold), as hemoglobin concentration and oxygen extraction (which is already 70-80% at rest) increase only modestly in most species. In contrast, in the right ventricle, oxygen extraction is lower at rest and increases substantially during exercise, similar to skeletal muscle, suggesting fundamental differences in blood flow regulation between these two cardiac chambers. The increase in heart rate also increases the relative time spent in systole, thereby increasing the net extravascular compressive forces acting on the microvasculature within the wall of the left ventricle, in particular in its subendocardial layers. Hence, appropriate adjustment of coronary vascular resistance is critical for the cardiac response to exercise. Coronary resistance vessel tone results from the culmination of myriad vasodilator and vasoconstrictors influences, including neurohormones and endothelial and myocardial factors. Unraveling of the integrative mechanisms controlling coronary vasodilation in response to exercise has been difficult, in part due to the redundancies in coronary vasomotor control and differences between animal species. Exercise training is associated with adaptations in the coronary microvasculature including increased arteriolar densities and/or diameters, which provide a morphometric basis for the observed increase in peak coronary blood flow rates in exercise-trained animals. In larger animals trained by treadmill exercise, the formation of new capillaries maintains capillary density at a level commensurate with the degree of exercise-induced physiological myocardial hypertrophy. Nevertheless, training alters the distribution of coronary vascular resistance so that more capillaries are recruited, resulting in an increase in the permeability-surface area product without a change in capillary numerical density. Maintenance of alpha- and ss-adrenergic tone in the presence of lower circulating catecholamine levels appears to be due to increased receptor responsiveness to adrenergic stimulation. Exercise training also alters local control of coronary resistance vessels. Thus arterioles exhibit increased myogenic tone, likely due to a calcium-dependent protein kinase C signaling-mediated alteration in voltage-gated calcium channel activity in response to stretch. Conversely, training augments endothelium-dependent vasodilation throughout the coronary microcirculation. This enhanced responsiveness appears to result principally from an increased expression of nitric oxide (NO) synthase. Finally, physical conditioning decreases extravascular compressive forces at rest and at comparable levels of exercise, mainly because of a decrease in heart rate. Impedance to coronary inflow due to an epicardial coronary artery stenosis results in marked redistribution of myocardial blood flow during exercise away from the subendocardium towards the subepicardium. However, in contrast to the traditional view that myocardial ischemia causes maximal microvascular dilation, more recent studies have shown that the coronary microvessels retain some degree of vasodilator reserve during exercise-induced ischemia and remain responsive to vasoconstrictor stimuli. These observations have required reassessment of the principal sites of resistance to blood flow in the microcirculation. A significant fraction of resistance is located in small arteries that are outside the metabolic control of the myocardium but are sensitive to shear and nitrovasodilators. The coronary collateral system embodies a dynamic network of interarterial vessels that can undergo both long- and short-term adjustments that can modulate blood flow to the dependent myocardium. Long-term adjustments including recruitment and growth of collateral vessels in response to arterial occlusion are time dependent and determine the maximum blood flow rates available to the collateral-dependent vascular bed during exercise. Rapid short-term adjustments result from active vasomotor activity of the collateral vessels. Mature coronary collateral vessels are responsive to vasodilators such as nitroglycerin and atrial natriuretic peptide, and to vasoconstrictors such as vasopressin, angiotensin II, and the platelet products serotonin and thromboxane A(2). During exercise, ss-adrenergic activity and endothelium-derived NO and prostanoids exert vasodilator influences on coronary collateral vessels. Importantly, alterations in collateral vasomotor tone, e.g., by exogenous vasopressin, inhibition of endogenous NO or prostanoid production, or increasing local adenosine production can modify collateral conductance, thereby influencing the blood supply to the dependent myocardium. In addition, vasomotor activity in the resistance vessels of the collateral perfused vascular bed can influence the volume and distribution of blood flow within the collateral zone. Finally, there is evidence that vasomotor control of resistance vessels in the normally perfused regions of collateralized hearts is altered, indicating that the vascular adaptations in hearts with a flow-limiting coronary obstruction occur at a global as well as a regional level. Exercise training does not stimulate growth of coronary collateral vessels in the normal heart. However, if exercise produces ischemia, which would be absent or minimal under resting conditions, there is evidence that collateral growth can be enhanced. In addition to ischemia, the pressure gradient between vascular beds, which is a determinant of the flow rate and therefore the shear stress on the collateral vessel endothelium, may also be important in stimulating growth of collateral vessels.

Ecto-5'-nucleotidase deficiency exacerbates pressure-overload-induced left ventricular hypertrophy and dysfunction.

This study examined whether endogenous extracellular adenosine acts to facilitate the adaptive response of the heart to chronic systolic overload. To examine whether endogenous extracellular adenosine can protect the heart against pressure-overload-induced heart failure, transverse aortic constriction was performed on mice deficient in extracellular adenosine production as the result of genetic deletion of CD73. Although there was no difference in left ventricular size or function between CD73-deficient mice (knockout [KO] mice) and wild-type mice under unstressed conditions, aortic constriction for 2 or 4 weeks induced significantly more myocardial hypertrophy, left ventricular dilation, and left ventricular dysfunction in KO mice compared with wild-type mice. Thus, after 2 weeks of transverse aortic constriction, left ventricular fractional shortening decreased to 27.4+/-2.5% and 21.9+/-1.7% in wild-type and KO mice, respectively (P<0.05). Consistent with a role of adenosine in reducing tissue remodeling, KO mice displayed increased myocardial fibrosis and myocyte hypertrophy compared with wild-type mice. Furthermore, adenosine treatment reduced phenylephrine-induced cardiac myocyte hypertrophy and collagen production in cultured neonatal rat cardiac myocytes and cardiac fibroblasts, respectively. Consistent with a role for adenosine in modulating cardiomyocyte hypertrophy, KO mice demonstrated increased activation of mammalian target of rapamycin signaling, accompanied by higher expression of the hypertrophy marker atrial natriuretic peptide. Conversely, the adenosine analogue 2-chloro-adenosine significantly reduced cell size, mammalian target of rapamycin/p70 ribosomal S6 kinase activation, and atrial natriuretic peptide expression in cultured neonatal cardiomyocytes. These data demonstrate that CD73 helps to preserve cardiac function during chronic systolic overload by preventing maladaptive tissue remodeling.