John Fassett

AMP Activated Protein Kinase-α2 Deficiency Exacerbates Pressure-Overload–Induced Left Ventricular Hypertrophy and Dysfunction in Mice

AMP activated protein kinase (AMPK) plays an important role in regulating myocardial metabolism and protein synthesis. Activation of AMPK attenuates hypertrophy in cultured cardiac myocytes, but the role of AMPK in regulating the development of myocardial hypertrophy in response to chronic pressure overload is not known. To test the hypothesis that AMPKα2 protects the heart against systolic overload–induced ventricular hypertrophy and dysfunction, we studied the response of AMPKα2 gene deficient (knockout [KO]) mice and wild-type mice subjected to 3 weeks of transverse aortic constriction (TAC). Although AMPKα2 KO had no effect on ventricular structure or function under control conditions, AMPKα2 KO significantly increased TAC-induced ventricular hypertrophy (ventricular mass increased 46% in wild-type mice compared with 65% in KO mice) while decreased left ventricular ejection fraction (ejection fraction decreased 14% in wild-type mice compared with a 43% decrease in KO mice). AMPKα2 KO also significantly exacerbated the TAC-induced increases of atrial natriuretic peptide, myocardial fibrosis, and cardiac myocyte size. AMPKα2 KO had no effect on total S6 ribosomal protein (S6), p70 S6 kinase, eukaryotic initiation factor 4E, and 4E binding protein-1 or their phosphorylation under basal conditions but significantly augmented the TAC-induced increases of p-p70 S6 kinaseThr389, p-S6Ser235, and p-eukaryotic initiation factor 4ESer209. AMPKα2 KO also enhanced the TAC-induced increase of p-4E binding protein-1Thr46 to a small degree and augmented the TAC-induced increase of p-AktSer473. These data indicate that AMPKα2 exerts a cardiac protective effect against pressure-overload–induced ventricular hypertrophy and dysfunction.

Oxidative Stress Regulates Left Ventricular PDE5 Expression in the Failing Heart

Background— Phosphodiesterase type 5 (PDE5) inhibition has been shown to exert profound beneficial effects in the failing heart, suggesting a significant role for PDE5 in the development of congestive heart failure (CHF). The purpose of this study is to test the hypothesis that oxidative stress causes increased PDE5 expression in cardiac myocytes and that increased PDE5 contributes to the development of CHF. Methods and Results— Myocardial PDE5 expression and cellular distribution were determined in left ventricular samples from patients with end-stage CHF and normal donors and from mice after transverse aortic constriction (TAC)–induced CHF. Compared with donor human hearts, myocardial PDE5 protein was increased ≈4.5-fold in CHF samples, and the increase of myocardial PDE5 expression was significantly correlated with myocardial oxidative stress markers 3′-nitrotyrosine or 4-hydroxynonenal expression (P<0.05). Histological examination demonstrated that PDE5 was mainly expressed in vascular smooth muscle in normal donor hearts, but its expression was increased in both cardiac myocytes and vascular smooth muscle of CHF hearts. Myocardial PDE5 protein content and activity also increased in mice after TAC-induced CHF (P<0.05). When the superoxide dismutase (SOD) mimetic M40401 was administered to attenuate oxidative stress, the increased PDE5 protein and activity caused by TAC was blunted, and the hearts were protected against left ventricular hypertrophy and CHF. Conversely, increased myocardial oxidative stress in superoxide dismutase 3 knockout mice caused a greater increase of PDE5 expression and CHF after TAC. In addition, administration of sildenafil to inhibit PDE5 attenuated TAC-induced myocardial oxidative stress, PDE5 expression, and CHF. Conclusions— Myocardial oxidative stress increases PDE5 expression in the failing heart. Reducing oxidative stress by treatment with M40401 attenuated cardiomyocyte PDE5 expression. This and selective inhibition of PDE5 protected the heart against pressure overload-induced left ventricular hypertrophy and CHF.

Dimethylarginine Dimethylaminohydrolase-1 Is the Critical Enzyme for Degrading the Cardiovascular Risk Factor Asymmetrical Dimethylarginine

Objective—The objective of this study was to identify the role of dimethylarginine dimethylaminohydrolase-1 (DDAH1) in degrading the endogenous nitric oxide synthase inhibitors asymmetrical dimethylarginine (ADMA) and Ng-monomethyl-L-arginine (L-NMMA). Methods and Results—We generated a global-DDAH1 gene–deficient (DDAH1−/−) mouse strain to examine the role of DDAH1 in ADMA and L-NMMA degradation and the physiological consequences of loss of DDAH1. Plasma and tissue ADMA and L-NMMA levels in DDAH1−/− mice were several folds higher than in wild-type mice, but growth and development of these DDAH1−/− mice were similar to those of their wild-type littermates. Although the expression of DDAH2 was unaffected, DDAH activity was undetectable in all tissues tested. These findings indicate that DDAH1 is the critical enzyme for ADMA and L-NMMA degradation. Blood pressure was ≈20 mm Hg higher in the DDAH1−/− mice than in wild-type mice, but no other cardiovascular phenotype was found under unstressed conditions. Crossing DDAH1+/− male with DDAH1+/− female mice yielded DDAH1+/+, DDAH1+/−, and DDAH1−/− mice at the anticipated ratio of 1:2:1, indicating that DDAH1 is not required for embryonic development in this strain. Conclusion—Our findings indicate that DDAH1 is required for metabolizing ADMA and L-NMMA in vivo, whereas DDAH2 had no detectable role for degrading ADMA and L-NMMA.

Global Dimethylarginine Dimethylaminohydrolase-1 (DDAH1) Gene–Deficient Mice Reveal That DDAH1 Is the Critical Enzyme for Degrading the Cardiovascular Risk Factor Asymmetrical Dimethylarginine

Objective—The objective of this study was to identify the role of dimethylarginine dimethylaminohydrolase-1 (DDAH1) in degrading the endogenous nitric oxide synthase inhibitors asymmetrical dimethylarginine (ADMA) and Ng-monomethyl-L-arginine (L-NMMA). Methods and Results—We generated a global-DDAH1 gene–deficient (DDAH1−/−) mouse strain to examine the role of DDAH1 in ADMA and L-NMMA degradation and the physiological consequences of loss of DDAH1. Plasma and tissue ADMA and L-NMMA levels in DDAH1−/− mice were several folds higher than in wild-type mice, but growth and development of these DDAH1−/− mice were similar to those of their wild-type littermates. Although the expression of DDAH2 was unaffected, DDAH activity was undetectable in all tissues tested. These findings indicate that DDAH1 is the critical enzyme for ADMA and L-NMMA degradation. Blood pressure was ≈20 mm Hg higher in the DDAH1−/− mice than in wild-type mice, but no other cardiovascular phenotype was found under unstressed conditions. Crossing DDAH1+/− male with DDAH1+/− female mice yielded DDAH1+/+, DDAH1+/−, and DDAH1−/− mice at the anticipated ratio of 1:2:1, indicating that DDAH1 is not required for embryonic development in this strain. Conclusion—Our findings indicate that DDAH1 is required for metabolizing ADMA and L-NMMA in vivo, whereas DDAH2 had no detectable role for degrading ADMA and L-NMMA.

Extracellular Superoxide Dismutase Deficiency Exacerbates Pressure Overload–Induced Left Ventricular Hypertrophy and Dysfunction

Extracellular superoxide dismutase (SOD) contributes only a small fraction to total SOD activity in the normal heart but is strategically located to scavenge free radicals in the extracellular compartment. To examine the physiological significance of extracellular SOD in the response of the heart to hemodynamic stress, we studied the effect of extracellular SOD deficiency on transverse aortic constriction (TAC)–induced left ventricular remodeling. Under unstressed conditions extracellular SOD deficiency had no effect on myocardial total SOD activity, the ratio of glutathione:glutathione disulfide, nitrotyrosine content, or superoxide anion production but resulted in small but significant increases in myocardial fibrosis and ventricular mass. In response to TAC for 6 weeks, extracellular SOD-deficient mice developed more severe left ventricular hypertrophy (heart weight increased 2.56-fold in extracellular SOD-deficient mice as compared with 1.99-fold in wild-type mice) and pulmonary congestion (lung weight increased 2.92-fold in extracellular SOD-deficient mice as compared with 1.84-fold in wild-type mice). Extracellular SOD-deficient mice also had more ventricular fibrosis, dilation, and a greater reduction of left ventricular fractional shortening and rate of pressure development after TAC. TAC resulted in greater increases of ventricular collagen I, collagen III, matrix metalloproteinase-2, matrix metalloproteinase-9, nitrotyrosine, and superoxide anion production. TAC also resulted in a greater decrease of the ratio of glutathione:glutathione disulfide in extracellular SOD-deficient mice. The finding that extracellular SOD deficiency had minimal impact on myocardial overall SOD activity but exacerbated TAC induced myocardial oxidative stress, hypertrophy, fibrosis, and dysfunction indicates that the distribution of extracellular SOD in the extracellular space is critically important in protecting the heart against pressure overload.

Regulation of hepatocyte cell cycle progression and differentiation by type I collagen structure.

Cell behavior is strongly influenced by the extracellular matrix (ECM) to which cells adhere. Both chemical determinants within ECM molecules and mechanical properties of the ECM network regulate cellular response, including proliferation, differentiation, and apoptosis. Type I collagen is the most abundant ECM protein in the body with a complex structure that can be altered in vivo by proteolysis, cross-linking, and other processes. Because of collagens complex and dynamic nature, it is important to define the changes in cell response to different collagen structures and its underlying mechanisms. This chapter reviews current knowledge of potential mechanisms by which type I collagen affects cell behavior, and it presents data that elucidate specific intracellular signaling pathways by which changes in type I collagen structure differentially regulate hepatocyte cell cycle progression and differentiation. A network of polymerized fibrillar type I collagen (collagen gel) induces a highly differentiated but growth-arrested phenotype in primary hepatocytes, whereas a film of monomeric collagen adsorbed to a rigid dish promotes cell cycle progression and dedifferentiation. Studies presented here demonstrate that protein kinase A (PKA) activity is significantly elevated in hepatocytes on type I collagen gel relative to collagen film, and inhibition of this elevated PKA activity can promote hepatocyte cell cycle progression on collagen gel. Additional studies are presented that examine changes in hepatocyte cell cycle progression and differentiation in response to increased rigidity of polymerized collagen gel by fiber cross-linking. Potential mechanisms underlying these cellular responses and their implications are discussed.

Exacerbated Pulmonary Arterial Hypertension and Right Ventricular Hypertrophy in Animals With Loss of Function of Extracellular Superoxide Dismutase

Studies have demonstrated that increased oxidative stress contributes to the pathogenesis and the development of pulmonary artery hypertension (PAH). Extracellular superoxide dismutase (SOD3) is essential for removing extracellular superoxide anions, and it is highly expressed in lung tissue. However, it is not clear whether endogenous SOD3 can influence the development of PAH. Here we examined the effect of SOD3 knockout on hypoxia-induced PAH in mice and a loss-of-function SOD3 gene mutation (SOD3E124D) on monocrotaline (40 mg/kg)-induced PAH in rats. SOD3 knockout significantly exacerbated 2 weeks of hypoxia-induced right ventricular (RV) pressure and RV hypertrophy, whereas RV pressure in SOD3 knockout mice under normoxic conditions is similar to wild-type controls. In untreated control rats at age of 8 weeks, there was no significant difference between wild-type and SOD3E124D rats in RV pressure and the ratio of RV weight:left ventricular weight (0.25±0.02 in wild-type rats versus 0.25±0.01 in SOD3E124D rats). However, monocrotaline caused significantly greater increases of RV pressure in SOD3E124D rats (48.6±1.8 mm Hg in wild-type versus 57.5±3.1 mm Hg in SOD3E124D rats), of the ratio of RV weight:left ventricular weight (0.41±0.01 versus 0.50±0.09; P<0.05), and of the percentage of fully muscularized small arterioles in SOD3E124D rats (55.2±2.3% versus 69.9±2.6%; P<0.05). Together, these findings indicate that the endogenous SOD3 has no role in the development of PAH under control conditions but plays an important role in protecting the lung from the development of PAH under stress conditions.

Cardiac-specific mindin overexpression attenuates cardiac hypertrophy via blocking AKT/GSK3β and TGF-β1–Smad signalling

AIMS Mindin is a secreted extracellular matrix protein, an integrin ligand, and an angiogenesis inhibitor, other examples of which are all key players in the progression of cardiac hypertrophy. However, its function during cardiac hypertrophy remains unclear. This study was aimed to identify the effect of mindin on cardiac hypertrophy and the underlying mechanisms. METHODS AND RESULTS A significant down-regulation of mindin expression was observed in human failing hearts. To further investigate the role of mindin in cardiac hypertrophy, we used cultured neonatal rat cardiomyocytes with gain and loss of mindin function and cardiac-specific Mindin-overexpressing transgenic (TG) mice. In cultured cardiomyocytes, mindin negatively regulated angiotensin II (Ang II)-mediated hypertrophic growth, as detected by [(3)H]-Leucine incorporation, cardiac myocyte area, and hypertrophic marker protein levels. Cardiac hypertrophy in vivo was produced by aortic banding (AB) or Ang II infusion in TG mice and their wild-type controls. The extent of cardiac hypertrophy was evaluated by echocardiography as well as by pathological and molecular analyses of heart samples. Mindin overexpression in the heart markedly attenuated cardiac hypertrophy, fibrosis, and left ventricular dysfunction in mice in response to AB or Ang II. Further analysis of the signalling events in vitro and in vivo indicated that these beneficial effects of mindin were associated with the interruption of AKT/glycogen synthase kinase 3β (GSK3β) and transforming growth factor (TGF)-β1-Smad signalling. CONCLUSION The present study demonstrates for the first time that mindin serves as a novel mediator that protects against cardiac hypertrophy and the transition to heart failure by blocking AKT/GSK3β and TGF-β1-Smad signalling.

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 A1 receptors (A1R) and A3 receptors (A3R) is not known. Methods and Results— To test the hypothesis that A1R and A3R can protect the heart against systolic overload, we exposed A3R gene–deficient (A3R knockout [KO]) mice and A1R KO mice to transverse aortic constriction (TAC). Contrary to our hypothesis, A3R 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). A3R KO also attenuated the TAC-induced increases of myocardial atrial natriuretic peptide and the oxidative stress markers 3′-nitrotyrosine and 4-hydroxynonenal. In contrast, A1R 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 A3R significantly potentiated the antihypertrophic effects of 2-chloroadenosine. Conclusions— Adenosine exerts protective effects on the overloaded heart, but the A3R acts counter to the protective effect of adenosine. The data suggest that selective attenuation of A3R activity might be a novel approach to treat pressure overload–induced left ventricular hypertrophy and dysfunction.

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-&agr; (ERR&agr;), but not peroxisome proliferator-activated receptor-&ggr; coactivator 1&agr;, in human heart failure samples. In addition, chronic pressure overload in mice caused a decrease of ERR&agr; 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 ERR&agr; expression in vivo and in vitro. AMPK&agr;2 knockout decreased myocardial ERR&agr; (both mRNA and protein) and its downstream targets under basal conditions, with no change in myocardial peroxisome proliferator-activated receptor-&ggr; coactivator 1&agr; expression. Using cultured rat neonatal cardiac myocytes, we found that overexpression of constitutively active AMPK&agr; significantly induced ERR&agr; mRNA, protein, and promoter activity. Conversely, selective gene silencing of AMPK&agr;2 repressed ERR&agr; and its target gene levels, indicating that AMPK&agr;2 is involved in the regulation of ERR&agr; expression. In addition, overexpression of ERR&agr; in AMPK&agr;2 knockout neonatal cardiac myocytes partially rescued the repressed expression of some energy metabolism-related genes. These data support an important role for AMPK&agr;2 in regulating the expression of myocardial ERR&agr; and its downstream mitochondrial enzymes.