Craig A. Lygate

Cardiac Neuronal Nitric Oxide Synthase Isoform Regulates Myocardial Contraction and Calcium Handling

Abstract— A neuronal isoform of nitric oxide synthase (nNOS) has recently been located to the cardiac sarcoplasmic reticulum (SR). Subcellular localization of a constitutive NOS in the proximity of an activating source of Ca2+ suggests that cardiac nNOS-derived NO may regulate contraction by exerting a highly specific and localized action on ion channels/transporters involved in Ca2+ cycling. To test this hypothesis, we have investigated myocardial Ca2+ handling and contractility in nNOS knockout mice (nNOS−/−) and in control mice (C) after acute nNOS inhibition with 100 &mgr;mol/L L-VNIO. nNOS gene disruption or L-VNIO increased basal contraction both in left ventricular (LV) myocytes (steady-state cell shortening 10.3±0.6% in nNOS−/− versus 8.1±0.5% in C;P <0.05) and in vivo (LV ejection fraction 53.5±2.7 in nNOS−/− versus 44.9±1.5% in C;P <0.05). nNOS disruption increased ICa density (in pA/pF, at 0 mV, −11.4±0.5 in nNOS−/− versus −9.1±0.5 in C;P <0.05) and prolonged the slow time constant of inactivation of ICa by 38% (P <0.05), leading to an increased Ca2+ influx and a greater SR load in nNOS−/− myocytes (in pC/pF, 0.78±0.04 in nNOS−/− versus 0.64±0.03 in C;P <0.05). Consistent with these data, [Ca2+]i transient (indo-1) peak amplitude was greater in nNOS−/− myocytes (410/495 ratio 0.34±0.01 in nNOS−/− versus 0.31±0.01 in C;P <0.05). These findings have uncovered a novel mechanism by which intracellular Ca2+ is regulated in LV myocytes and indicate that nNOS is an important determinant of basal contractility in the mammalian myocardium. The full text of this article is available at http://www.circresaha.org.

Abnormal sympathoadrenal development and systemic hypotension in PHD3–/– mice.

ABSTRACT Cell culture studies have implicated the oxygen-sensitive hypoxia-inducible factor (HIF) prolyl hydroxylase PHD3 in the regulation of neuronal apoptosis. To better understand this function in vivo, we have created PHD3−/− mice and analyzed the neuronal phenotype. Reduced apoptosis in superior cervical ganglion (SCG) neurons cultured from PHD3−/− mice is associated with an increase in the number of cells in the SCG, as well as in the adrenal medulla and carotid body. Genetic analysis by intercrossing PHD3−/− mice with HIF-1a+/− and HIF-2a+/− mice demonstrated an interaction with HIF-2α but not HIF-1α, supporting the nonredundant involvement of a PHD3-HIF-2α pathway in the regulation of sympathoadrenal development. Despite the increased number of cells, the sympathoadrenal system appeared hypofunctional in PHD3−/− mice, with reduced target tissue innervation, adrenal medullary secretory capacity, sympathoadrenal responses, and systemic blood pressure. These observations suggest that the role of PHD3 in sympathoadrenal development extends beyond simple control of cell survival and organ mass, with functional PHD3 being required for proper anatomical and physiological integrity of the system. Perturbation of this interface between developmental and adaptive signaling by hypoxic, metabolic, or other stresses could have important effects on key sympathoadrenal functions, such as blood pressure regulation.

Fast, high‐resolution in vivo cine magnetic resonance imaging in normal and failing mouse hearts on a vertical 11.7 T system

To establish fast, high‐resolution in vivo cine magnetic resonance imaging (cine‐MRI) on a vertical 11.7‐T MR system and to investigate the stability of normal and failing mouse hearts in the vertical position.

Fumarate Is Cardioprotective via Activation of the Nrf2 Antioxidant Pathway

Summary The citric acid cycle (CAC) metabolite fumarate has been proposed to be cardioprotective; however, its mechanisms of action remain to be determined. To augment cardiac fumarate levels and to assess fumarates cardioprotective properties, we generated fumarate hydratase (Fh1) cardiac knockout (KO) mice. These fumarate-replete hearts were robustly protected from ischemia-reperfusion injury (I/R). To compensate for the loss of Fh1 activity, KO hearts maintain ATP levels in part by channeling amino acids into the CAC. In addition, by stabilizing the transcriptional regulator Nrf2, Fh1 KO hearts upregulate protective antioxidant response element genes. Supporting the importance of the latter mechanism, clinically relevant doses of dimethylfumarate upregulated Nrf2 and its target genes, hence protecting control hearts, but failed to similarly protect Nrf2-KO hearts in an in vivo model of myocardial infarction. We propose that clinically established fumarate derivatives activate the Nrf2 pathway and are readily testable cytoprotective agents.

Quantitative 3-Dimensional Echocardiography for Accurate and Rapid Cardiac Phenotype Characterization in Mice

Background—Insufficient techniques exist for rapid and reliable phenotype characterization of genetically manipulated mouse models of cardiac dysfunction. We developed a new, robust, 3-dimensional echocardiography (3D-echo) technique and hypothesized that this 3D-echo technique is as accurate as magnetic resonance imaging (MRI) and histology for assessment of left ventricular (LV) volume, ejection fraction, mass, and infarct size in normal and chronically infarcted mice. Methods and Results—Using a high-frequency, 7/15-MHz, linear-array ultrasound transducer, we acquired ECG and respiratory-gated, 500-&mgr;m consecutive short-axis slices of the murine heart within 4 minutes. The short-axis movies were reassembled off-line in a 3D matrix by using the measured platform locations to position each slice in 3D. Epicardial and endocardial heart contours were manually traced, and a B-spline surface was fitted to the delineated image curves to reconstruct the heart volumes. Excellent correlations were obtained between 3D-echo and MRI for LV end-systolic volumes (r=0.99, P<0.0001), LV end-diastolic volumes (r=0.99, P<0.0001), ejection fraction (r=0.99, P<0.0001), LV mass (r=0.94, P<0.0019), and infarct size (r=0.98, P<0.0001). Also, excellent correlations were found between the 3D-echo–derived LV mass and necropsy LV mass in normal mice (r=0.99, P<0.0001), as well as for 3D-echo–derived infarct size and histologically determined infarct size (r=0.99, P<0.0001) in mice with chronic heart failure. Bland-Altman analysis showed excellent limits of agreement between techniques for all measured parameters. Conclusion—This new, fast, and highly reproducible 3D-echo technique should be of widespread applicability for high-throughput murine cardiac phenotyping studies.

Increased mitochondrial uncoupling proteins, respiratory uncoupling and decreased efficiency in the chronically infarcted rat heart.

Heart failure patients have abnormal cardiac high energy phosphate metabolism, the explanation for which is unknown. Patients with heart failure also have elevated plasma free fatty acid (FFA) concentrations. Elevated FFA levels are associated with increased cardiac mitochondrial uncoupling proteins (UCPs), which, in turn, are associated with decreased mitochondrial respiratory coupling and low cardiac efficiency. Here, we determined whether increased mitochondrial UCP levels contribute to decreased energetics in the failing heart by measuring UCPs and respiration in mitochondria isolated from the viable myocardium of chronically infarcted rat hearts and measuring efficiency (hydraulic work/O(2) consumption) in the isolated, working rat heart. Ten weeks after infarction, cardiac levels of UCP3 were increased by 53% in infarcted, failing hearts that had ejection fractions less than 45%. Cardiac UCP3 levels correlated positively with non-fasting plasma FFAs (r=0.81; p<0.01). Mitochondria from failing hearts were less coupled than those from control hearts, as demonstrated by the lower ADP/O ratio of 1.9+/-0.1 compared with 2.5+/-0.2 in controls (p<0.05). The decreased ADP/O ratio was reflected in an efficiency of 14+/-2% in the failing hearts when perfused with 1 mM palmitate, compared with 20+/-1% in controls (p<0.05). We conclude that failing hearts have increased UCP3 levels that are associated with high circulating FFA concentrations, mitochondrial uncoupling, and decreased cardiac efficiency. Thus, respiratory uncoupling may underlie the abnormal energetics and low efficiency in the failing heart, although whether this is maladaptive or adaptive would require direct investigation.

The PPARgamma-activator rosiglitazone does not alter remodeling but increases mortality in rats post-myocardial infarction.

OBJECTIVE Peroxisome proliferator-activated receptor gamma (PPARgamma) activators may be beneficial in heart failure due to their metabolic and antihypertrophic effects, but these agents can cause oedema. We hypothesized that, on balance, the PPARgamma activator rosiglitazone would be beneficial in heart failure post-myocardial infarction. METHODS AND RESULTS Rosiglitazone (3 mg/kg/day p.o.) given to male Wistar rats for 14 days, caused a 31% increase in left ventricular (LV) dP/dt(max) (P<0.05 vs. placebo). A separate group of rats was subjected to sham (SH) or coronary artery ligation and randomised to: untreated (UT); rosiglitazone 3 mg/kg/day (R); captopril, 2 g/l in drinking water (C); captopril+rosiglitazone (C+R). Mean LV infarct sizes were similar for all groups at 40+/-2%. After 8 weeks, echocardiographic ejection fractions were 82+/-3, 40+/-3, 50+/-2*, 49+/-2, 50+/-3% for SH, UT, R, C and C+R groups, respectively (*P<0.05 vs. UT). Captopril prevented LV dilatation, but rosiglitazone did not. In vivo hemodynamics showed that only UT had significantly elevated LV end-diastolic pressures and reduced +dP/dt(max), with R partially, and C and C+R almost completely preventing the increase in LVEDP. Captopril, but not rosiglitazone, significantly reduced LV hypertrophy [LV/bw; 1.97+/-0.09 (SH), 2.15+/-0.04 (UT), 2.10+/-0.05 (R), 1.81+/-0.04* (C), 1.88+/-0.07 (C+R); *(P<0.05 vs. UT)]. Rosiglitazone increased 8-week mortality, which was 26% for R and 19% for C+R compared with 0% for UT and C (P=0.03 vs. UT). CONCLUSIONS Rosiglitazone did not modulate LV remodeling, but was associated with increased mortality post-myocardial infarction (MI) in rats. The mechanisms require further study, but these results caution against use of PPARgamma activators in post-MI heart failure in non-diabetics.

Reduced Inotropic Reserve and Increased Susceptibility to Cardiac Ischemia/Reperfusion Injury in Phosphocreatine-Deficient Guanidinoacetate-N-Methyltransferase–Knockout Mice

Background—The role of the creatine kinase (CK)/phosphocreatine (PCr) energy buffer and transport system in heart remains unclear. Guanidinoacetate-N-methyltransferase–knockout (GAMT−/−) mice represent a new model of profoundly altered cardiac energetics, showing undetectable levels of PCr and creatine and accumulation of the precursor (phospho-)guanidinoacetate (P-GA). To characterize the role of a substantially impaired CK/PCr system in heart, we studied the cardiac phenotype of wild-type (WT) and GAMT−/− mice. Methods and Results—GAMT−/− mice did not show cardiac hypertrophy (myocyte cross-sectional areas, hypertrophy markers atrial natriuretic factor and β-myosin heavy chain). Systolic and diastolic function, measured invasively (left ventricular conductance catheter) and noninvasively (MRI), were similar for WT and GAMT−/− mice. However, during inotropic stimulation with dobutamine, preload-recruitable stroke work failed to reach maximal levels of performance in GAMT−/− hearts (101±8 mm Hg in WT versus 59±7 mm Hg in GAMT−/−; P<0.05). 31P-MR spectroscopy experiments showed that during inotropic stimulation, isolated WT hearts utilized PCr, whereas isolated GAMT−/− hearts utilized P-GA. During ischemia/reperfusion, GAMT−/− hearts showed markedly impaired recovery of systolic (24% versus 53% rate pressure product recovery; P<0.05) and diastolic function (eg, left ventricular end-diastolic pressure 23±9 in WT and 51±5 mm Hg in GAMT−/− during reperfusion; P<0.05) and incomplete resynthesis of P-GA. Conclusions—GAMT−/− mice do not develop hypertrophy and show normal cardiac function at low workload, suggesting that a fully functional CK/PCr system is not essential under resting conditions. However, when acutely stressed by inotropic stimulation or ischemia/reperfusion, GAMT−/− mice exhibit a markedly abnormal phenotype, demonstrating that an intact, high-capacity CK/PCr system is required for situations of increased cardiac work or acute stress.

A mutation in the mitochondrial fission gene Dnm1l leads to cardiomyopathy

Mutations in a number of genes have been linked to inherited dilated cardiomyopathy (DCM). However, such mutations account for only a small proportion of the clinical cases emphasising the need for alternative discovery approaches to uncovering novel pathogenic mutations in hitherto unidentified pathways. Accordingly, as part of a large-scale N-ethyl-N-nitrosourea mutagenesis screen, we identified a mouse mutant, Python, which develops DCM. We demonstrate that the Python phenotype is attributable to a dominant fully penetrant mutation in the dynamin-1-like (Dnm1l) gene, which has been shown to be critical for mitochondrial fission. The C452F mutation is in a highly conserved region of the M domain of Dnm1l that alters protein interactions in a yeast two-hybrid system, suggesting that the mutation might alter intramolecular interactions within the Dnm1l monomer. Heterozygous Python fibroblasts exhibit abnormal mitochondria and peroxisomes. Homozygosity for the mutation results in the death of embryos midway though gestation. Heterozygous Python hearts show reduced levels of mitochondria enzyme complexes and suffer from cardiac ATP depletion. The resulting energy deficiency may contribute to cardiomyopathy. This is the first demonstration that a defect in a gene involved in mitochondrial remodelling can result in cardiomyopathy, showing that the function of this gene is needed for the maintenance of normal cellular function in a relatively tissue-specific manner. This disease model attests to the importance of mitochondrial remodelling in the heart; similar defects might underlie human heart muscle disease.

Supranormal Myocardial Creatine and Phosphocreatine Concentrations Lead to Cardiac Hypertrophy and Heart Failure Insights From Creatine Transporter–Overexpressing Transgenic Mice

Background— Heart failure is associated with deranged cardiac energy metabolism, including reductions of creatine and phosphocreatine. Interventions that increase myocardial high-energy phosphate stores have been proposed as a strategy for treatment of heart failure. Previously, it has not been possible to increase myocardial creatine and phosphocreatine concentrations to supranormal levels because they are subject to tight regulation by the sarcolemmal creatine transporter (CrT). Methods and Results— We therefore created 2 transgenic mouse lines overexpressing the myocardial creatine transporter (CrT-OE). Compared with wild-type (WT) littermate controls, total creatine (by high-performance liquid chromatography) was increased in CrT-OE hearts (66±6 nmol/mg protein in WT versus 133±52 nmol/mg protein in CrT-OE). Phosphocreatine levels (by 31P magnetic resonance spectroscopy) were also increased but to a lesser extent. Surprisingly, CrT-OE mice developed left ventricular (LV) dilatation (LV end-diastolic volume: 21.5±4.3 &mgr;L in WT versus 33.1±9.6 &mgr;L in CrT-OE; P=0.002), substantial LV dysfunction (ejection fraction: 64±9% in WT versus 49±13% in CrT-OE; range, 22% to 70%; P=0.003), and LV hypertrophy (by 3-dimensional echocardiography and magnetic resonance imaging). Myocardial creatine content correlated closely with LV end-diastolic volume (r=0.51, P=0.02), ejection fraction (r=−0.74, P=0.0002), LV weight (r=0.59, P=0.006), LV end-diastolic pressure (r=0.52, P=0.02), and dP/dtmax (r=−0.69, P=0.0008). Despite increased creatine and phosphocreatine levels, CrT-OE hearts showed energetic impairment, with increased free ADP concentrations and reduced free-energy change levels. Conclusions— Overexpression of the CrT leads to supranormal levels of myocardial creatine and phosphocreatine, but the heart is incapable of keeping the augmented creatine pool adequately phosphorylated, resulting in increased free ADP levels, LV hypertrophy, and dysfunction. Our data demonstrate that a disturbance of the CrT-mediated tight regulation of cardiac energy metabolism has deleterious functional consequences. These findings caution against the uncritical use of creatine as a therapeutic agent in heart disease.