Marijke Pellens

Cardiomyocyte-Specific Overexpression of Nitric Oxide Synthase 3 Improves Left Ventricular Performance and Reduces Compensatory Hypertrophy After Myocardial Infarction

Nitric oxide (NO) is an important modulator of cardiac performance and left ventricular (LV) remodeling after myocardial infarction (MI). We tested the effect of cardiomyocyte-restricted overexpression of one NO synthase isoform, NOS3, on LV remodeling after MI in mice. LV structure and function before and after permanent LAD coronary artery ligation were compared in transgenic mice with cardiomyocyte-restricted NOS3 overexpression (NOS3-TG) and their wild-type littermates (WT). Before MI, systemic hemodynamic measurements, echocardiographic assessment of LV fractional shortening (FS), heart weight, and myocyte width (as assessed histologically) did not differ in NOS3-TG and WT mice. The inotropic response to graded doses of isoproterenol was significantly reduced in NOS3-TG mice. One week after LAD ligation, the infarcted fraction of the LV did not differ in WT and NOS3-TG mice (34 ± 4% versus 36 ± 12%, respectively). Four weeks after MI, however, end-systolic LVID was greater, and fractional shortening and maximum and minimum rates of LV pressure development were less in WT than in NOS3-TG mice. LV weight/body weight ratio was greater in WT than in NOS3-TG mice (5.3 ± 0.2 versus 4.6 ± 0.5 mg/g; P < 0.01). Myocyte width in noninfarcted myocardium was greater in WT than in NOS3-TG mice (18.8 ± 2.0 versus 16.6 ± 1.6 μm; P < 0.05), whereas fibrosis in noninfarcted myocardium was similar in both genotypes. Cardiomyocyte-restricted overexpression of NOS3 limits LV dysfunction and remodeling after MI, in part by decreasing myocyte hypertrophy in noninfarcted myocardium.

Ventricular Phosphodiesterase-5 Expression Is Increased in Patients With Advanced Heart Failure and Contributes to Adverse Ventricular Remodeling After Myocardial Infarction in Mice

Background— Ventricular expression of phosphodiesterase-5 (PDE5), an enzyme responsible for cGMP catabolism, is increased in human right ventricular hypertrophy, but its role in left ventricular (LV) failure remains incompletely understood. We therefore measured LV PDE5 expression in patients with advanced systolic heart failure and characterized LV remodeling after myocardial infarction in transgenic mice with cardiomyocyte-specific overexpression of PDE5 (PDE5-TG). Methods and Results— Immunoblot and immunohistochemistry techniques revealed that PDE5 expression was greater in explanted LVs from patients with dilated and ischemic cardiomyopathy than in control hearts. To evaluate the impact of increased ventricular PDE5 levels on cardiac function, PDE5-TG mice were generated. Confocal and immunoelectron microscopy revealed increased PDE5 expression in cardiomyocytes, predominantly localized to Z-bands. At baseline, myocardial cGMP levels, cell shortening, and calcium handling in isolated cardiomyocytes and LV hemodynamic measurements were similar in PDE5-TG and wild-type littermates. Ten days after myocardial infarction, LV cGMP levels had increased to a greater extent in wild-type mice than in PDE5-TG mice (P<0.05). Ten weeks after myocardial infarction, LV end-systolic and end-diastolic volumes were larger in PDE5-TG than in wild-type mice (57±5 versus 39±4 and 65±6 versus 48±4 &mgr;L, respectively; P<0.01 for both). LV systolic dysfunction and diastolic dysfunction were more marked in PDE5-TG than in wild-type mice, associated with enhanced hypertrophy and reduced contractile function in isolated cardiomyocytes from remote myocardium. Conclusions— Increased PDE5 expression predisposes mice to adverse LV remodeling after myocardial infarction. Increased myocardial PDE5 expression in patients with advanced cardiomyopathy may contribute to the development of heart failure and represents an important therapeutic target.

Cytochrome P450 Epoxygenase Gene Function in Hypoxic Pulmonary Vasoconstriction and Pulmonary Vascular Remodeling

We assessed pulmonary cytochrome P450 (CYP) epoxygenase expression and activity during hypoxia and explored the effects of modulating epoxygenase activity on pulmonary hypertension. The acute hypoxic vasoconstrictor response was studied in Swiss Webster mice, who express CYP2C29 in their lungs. Animals were pretreated with vehicle, the epoxygenase inhibitor (N-methylsulfonyl-6-[2-propargyloxyphenyl] hexanamide) or an inhibitor of the soluble epoxide hydrolase. Whereas the epoxygenase inhibitor attenuated hypoxic pulmonary constriction (by 52%), the soluble epoxide hydrolase inhibitor enhanced the response (by 39%), indicating that CYP epoxygenase–derived epoxyeicosatrienoic acids elicit pulmonary vasoconstriction. Aerosol gene transfer of recombinant adenovirus containing the human CYP2C9 significantly elevated mean pulmonary artery pressure and total pulmonary resistance indices, both of which were sensitive to the inhibitor sulfaphenazole. The prolonged exposure of mice to hypoxia increased CYP2C29 expression, and transcript levels increased 5-fold after exposure to normobaric hypoxia (FIO2 0.07) for 2 hours. This was followed by a 2-fold increase in protein expression and by a significant increase in epoxyeicosatrienoic acid production after 24 hours. Chronic hypoxia (7 days) elicited pulmonary hypertension and pulmonary vascular remodeling, effects that were significantly attenuated in animals continually treated with N-methylsulfonyl-6-[2-propargyloxyphenyl] hexanamide (−46% and −55%, respectively). Our results indicate that endogenously generated epoxygenase products are associated with hypoxic pulmonary hypertension in mice and that selective epoxygenase inhibition significantly reduces acute hypoxic pulmonary vasoconstriction and chronic hypoxia-induced pulmonary vascular remodeling. These observations indicate potential novel targets for the treatment of pulmonary hypertension and highlight a pivotal role for CYP epoxygenases in pulmonary responses to hypoxia.

The relative value of strain and strain rate for defining intrinsic myocardial function

It is well accepted that strain and strain rate deformation parameters are not only a measure of intrinsic myocardial contractility but are also influenced by changes in cardiac load and structure. To date, no information is available on the relative importance of these confounders. This study was designed to investigate how strain and strain rate, measured by Doppler echocardiography, relate to the individual factors that determine cardiac performance. Echocardiographic and conductance measurements were simultaneously performed in mice in which individual determinants of cardiac performance were mechanically and/or pharmacologically modulated. A multivariable analysis was performed with radial and circumferential strains and peak systolic radial and circumferential strain rates as dependent parameters and preload recruitable stroke work (PRSW), arterial elastance (E(a)), end-diastolic pressure, and left ventricular myocardial volume (LVMV) as independent factors representing myocardial contractility, afterload, preload, and myocardial volume, respectively. Radial strain was most influenced by E(a) (β = -0.58, R(2) = 0.34), whereas circumferential strain was strongly associated with E(a) and moderately with LVMV (β = 0.79 and -0.52, respectively, R(2) = 0.54). Radial strain rate was related to both PRSW and LVMV (β = 0.79 and -0.62, respectively, R(2) = 0.50), whereas circumferential strain rate showed a prominent correlation only with PRSW (β = -0.61, R(2) = 0.51). In conclusion, strain (both radial and circumferential) is not a good surrogate measure of intrinsic myocardial contractility unless the strong confounding influence of afterload is considered. Strain rate is a more robust measure of contractility that is less influenced by changes in cardiac load and structure. Thus, peak systolic strain rate is the more relevant parameter to assess myocardial contractile function noninvasively.

Soluble Guanylate Cyclase-α1 Deficiency Selectively Inhibits the Pulmonary Vasodilator Response to Nitric Oxide and Increases the Pulmonary Vascular Remodeling Response to Chronic Hypoxia

Background— Nitric oxide (NO) activates soluble guanylate cyclase (sGC), a heterodimer composed of &agr;- and &bgr;-subunits, to produce cGMP. NO reduces pulmonary vascular remodeling, but the role of sGC in vascular responses to acute and chronic hypoxia remains incompletely elucidated. We therefore studied pulmonary vascular responses to acute and chronic hypoxia in wild-type (WT) mice and mice with a nonfunctional &agr;1-subunit (sGC&agr;1−/−). Methods and Results— sGC&agr;1−/− mice had significantly reduced lung sGC activity and vasodilator-stimulated phosphoprotein phosphorylation. Right ventricular systolic pressure did not differ between genotypes at baseline and increased similarly in WT (22±2 to 34±2 mm Hg) and sGC&agr;1−/− (23±2 to 34±1 mm Hg) mice in response to acute hypoxia. Inhaled NO (40 ppm) blunted the increase in right ventricular systolic pressure in WT mice (22±2 to 24±2 mm Hg, P<0.01 versus hypoxia without NO) but not in sGC&agr;1−/− mice (22±1 to 33±1 mm Hg) and was accompanied by a significant rise in lung cGMP content only in WT mice. In contrast, the NO-donor sodium nitroprusside (1.5 mg/kg) decreased systemic blood pressure similarly in awake WT and sGC&agr;1−/− mice as measured by telemetry (−37±2 versus −42±4 mm Hg). After 3 weeks of hypoxia, the increases in right ventricular systolic pressure, right ventricular hypertrophy, and muscularization of intra-acinar pulmonary vessels were 43%, 135%, and 46% greater, respectively, in sGC&agr;1−/− than in WT mice (P<0.01). Increased remodeling in sGC&agr;1−/− mice was associated with an increased frequency of 5′-bromo-deoxyuridine–positive vessels after 1 and 3 weeks (P<0.01 versus WT). Conclusions— Deficiency of sGC&agr;1 does not alter hypoxic pulmonary vasoconstriction. sGC&agr;1 is essential for NO-mediated pulmonary vasodilation and limits chronic hypoxia-induced pulmonary vascular remodeling.

Increased cardiac myocyte PDE5 levels in human and murine pressure overload hypertrophy contribute to adverse LV remodeling.

Background The intracellular second messenger cGMP protects the heart under pathological conditions. We examined expression of phosphodiesterase 5 (PDE5), an enzyme that hydrolyzes cGMP, in human and mouse hearts subjected to sustained left ventricular (LV) pressure overload. We also determined the role of cardiac myocyte-specific PDE5 expression in adverse LV remodeling in mice after transverse aortic constriction (TAC). Methodology/Principal Findings In patients with severe aortic stenosis (AS) undergoing valve replacement, we detected greater myocardial PDE5 expression than in control hearts. We observed robust expression in scattered cardiac myocytes of those AS patients with higher LV filling pressures and BNP serum levels. Following TAC, we detected similar, focal PDE5 expression in cardiac myocytes of C57BL/6NTac mice exhibiting the most pronounced LV remodeling. To examine the effect of cell-specific PDE5 expression, we subjected transgenic mice with cardiac myocyte-specific PDE5 overexpression (PDE5-TG) to TAC. LV hypertrophy and fibrosis were similar as in WT, but PDE5-TG had increased cardiac dimensions, and decreased dP/dtmax and dP/dtmin with prolonged tau (P<0.05 for all). Greater cardiac dysfunction in PDE5-TG was associated with reduced myocardial cGMP and SERCA2 levels, and higher passive force in cardiac myocytes in vitro. Conclusions/Significance Myocardial PDE5 expression is increased in the hearts of humans and mice with chronic pressure overload. Increased cardiac myocyte-specific PDE5 expression is a molecular hallmark in hypertrophic hearts with contractile failure, and represents an important therapeutic target.

Preexisting antiadenoviral immunity and regional myocardial gene transfer: modulation by nitric oxide.

The utility of adenoviral vectors, currently used in cardiovascular gene transfer protocols, is limited by the brevity of transgene expression and by antiadenoviral immune responses. The effect of preexisting antiadenoviral immunity on intracardiac gene transfer or its modulation by nitric oxide is unknown. Adenoviral vectors, expressing the firefly luciferase gene (AdLuc) or the human nitric oxide synthase 3 (NOS3) gene (AdNOS3), were infused into the great cardiac vein of naive pigs or immunized pigs. Pigs were immunized by intravenous injection of control virus AdRR5 and the resulting neutralizing antibody titers (median, 1:178; p < 0.0001 vs. baseline) were similar to preexisting titers in 54% of randomly selected coronary artery bypass graft patients. In naive animals distribution of transgene expression in the left ventricular free wall was focal. In immunized pigs myocardial luciferase expression 3 days after AdLuc gene transfer was more than 1000-fold lower than in naive pigs, whereas no change in NOS3 transcript levels was detected after AdNOS3 gene transfer. Severe, grade III-IV mononuclear cell infiltration and myocyte apoptosis were observed in four of five AdLuc-infected, immunized animals, compared with low-level inflammation and apoptosis in five of six AdNOS3-infected pigs. Coinfusion of AdLuc and AdNOS3 in immunized pigs resulted in spatially colocalized transgene expression, reduced T cell-mediated inflammation, and myocyte apoptosis and was associated with 200-fold greater median reporter transgene expression levels in the subendocardium (1.0 x 10(3) light units [LU]/mg protein, n = 8, vs. 4.5 x 10(1) LU/mg protein in AdLuc- and AdRR5-coinfected pigs, n = 7, p = 0.02). Preexisting antiadenoviral immunity abrogates myocardial gene expression in pigs and is associated with severe inflammation and myocyte apoptosis. Intracardiac NOS3 gene transfer may reduce these barriers to adenovirus-mediated myocardial gene transfer.

Response to Letter Regarding Article, “Ventricular Phosphodiesterase-5 Expression Is Increased in Patients With Advanced Heart Failure and Contributes to Adverse Ventricular Remodeling After Myocardial Infarction in Mice”

We appreciate the comments by Cingolani et al on a possible involvement of Na+/H+ exchanger (NHE-1) in signaling mechanisms downstream of cGMP that can mediate cardioprotection after myocardial infarction. These authors previously reported that NHE-1 expression and activity are increased after myocardial infarction in rats (as a result of myocardial acidosis) and that sustained …