Transgenic overexpression of GTP cyclohydrolase 1 in cardiomyocytes ameliorates post-infarction cardiac remodeling

GTP cyclohydrolase 1 (GCH1) and its product tetrahydrobiopterin play crucial roles in cardiovascular health and disease, yet the exact regulation and role of GCH1 in adverse cardiac remodeling after myocardial infarction are still enigmatic. Here we report that cardiac GCH1 is degraded in remodeled hearts after myocardial infarction, concomitant with increases in the thickness of interventricular septum, interstitial fibrosis, and phosphorylated p38 mitogen-activated protein kinase and decreases in left ventricular anterior wall thickness, cardiac contractility, tetrahydrobiopterin, the dimers of nitric oxide synthase, sarcoplasmic reticulum Ca2+ release, and the expression of sarcoplasmic reticulum Ca2+ handling proteins. Intriguingly, transgenic overexpression of GCH1 in cardiomyocytes reduces the thickness of interventricular septum and interstitial fibrosis and increases anterior wall thickness and cardiac contractility after infarction. Moreover, we show that GCH1 overexpression decreases phosphorylated p38 mitogen-activated protein kinase and elevates tetrahydrobiopterin levels, the dimerization and phosphorylation of neuronal nitric oxide synthase, sarcoplasmic reticulum Ca2+ release, and sarcoplasmic reticulum Ca2+ handling proteins in post-infarction remodeled hearts. Our results indicate that the pivotal role of GCH1 overexpression in post-infarction cardiac remodeling is attributable to preservation of neuronal nitric oxide synthase and sarcoplasmic reticulum Ca2+ handling proteins, and identify a new therapeutic target for cardiac remodeling after infarction.

. Time-dependent changes in body weight and heart rate in C57BL/6 mice after surgery. Table S2. Physiological and left ventricular hemodynamic parameters in C57BL/6 and Tg mice 4 weeks after myocardial infarction.   The Tg mice with cardiomyocyte-specific overexpression of human GCH1 gene on a C57BL/6 background were developed under the control of the -myosin heavy chain promoter, as described previously 1 . The Tg mice were identified by the presence of human GCH1 gene using polymerase chain reaction (PCR) on tail-derived genomic DNA 1 . C57BL/6 WT littermates were used as controls for the Tg mice. The animals were kept on a 12-h light-dark cycle in a temperature-controlled room and received standard rodent maintenance diet and water ad libitum.

Study approval
The animal care and all experimental procedures were performed in accordance with the NIH

Induction of myocardial infarction (MI)
Male WT and Tg mice at the ages of 8-10 weeks were anesthetized by intraperitoneal injection of 80 mg/kg sodium pentobarbital. The trachea was cannulated with a polyethylene 60 tube connected to a positive-pressure mouse respirator (MiniVent type 845; Hugo Sachs ElectroniK-Harvard Apparatus, Hugstetten, Germany) with a tidal volume of 250 µl 2 . The mice were ventilated with room air supplemented with 100% oxygen at approximately 102 breaths per minute. A left thoracotomy was performed between the 4th and 5th ribs, and the lungs retracted to expose the heart 3 . After the pericardial tissue was removed, the left anterior coronary artery was visualized under a microscope and permanently ligated with an 8-0 silk suture near its origin between the pulmonary outflow tract and the edge of the left atrium, as described 4

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Liu et al. GTP Cyclohydrolase 1 and Cardiac Remodeling Page 3 lungs were inflated by increasing positive end-respiratory pressure. The chest wall was closed in 2 layers: rib-muscle and skin. Animals were kept in a warm chamber heated by both a heating pad below the chamber and a heating lamp over the chamber until recovery. Shamoperated animals underwent the same procedure except the coronary artery ligation. Rectal temperature was monitored and maintained between 36.8 and 37.3 o C by a TC-1000 Temperature controller (CWE Inc., Ardmore, PA, USA) throughout surgical procedure. For analgesia, buprenorphine (0.05 mg/kg) was administered subcutaneously immediately after the chest closure and every 8 h for the next 48 h.

Transthoracic echocardiography
Non-invasive transthoracic echocardiography was used to evaluate left ventricular geometry and function in WT and Tg mice at 0 (baseline), 1, 2, 4, 8, and 12 weeks after MI or sham surgery.
Animals were sedated by the inhalation of oxygen with 1.5 % isoflurane.
Echocardiography was performed with a VisualSonics Vevo 770 High-resolution Imaging System (Toronto, Canada) equipped with a 30 MHz transducer (Scanhead RMV 707), as described previously 5,6 . M-mode images were recorded from the short axis 2-chamber view at the papillary muscle level. Heart rate, the thickness of anterior wall and interventricular septum of the left ventricle (LV), and LV end-diastolic and systolic diameters were measured. Fractional shortening was calculated using the following equation: % factional shortening = (LV enddiastolic diameter-LV end-systolic diameter)/LV end-diastolic diameter100.

Polymerase chain reaction (PCR) analysis of GCH1 mRNA
To examine time-dependent changes in GCH1 mRNA levels, C57BL/6 mouse hearts were excised at 0 (baseline), 1, 2, 4, 8, and 12 weeks after surgery and snap frozen in liquid nitrogen immediately after excision. The LV was homogenized at 4 o C for PCR analysis of GCH1 mRNA, as described. 7 Briefly, total RNA was extracted from snap frozen tissue in 1 ml of

Measurements of blood pressure and intracardiac hemodynamics
Pentobarbital-anesthetized mice were connected to a mouse ventilator after endotracheal intubation, as described in the section of "Induction of MI". After bilateral vagotomy, the right carotid artery was cannulated with a micromanometer-tipped mouse pressure catheter (SPR- ventricle (LV) for recording of LV pressure 8 . Steady-state LV pressure was used to analyze offline for the derivation of traditional parameters of LV systolic (peak LV pressure and +dP/dt) and diastolic (end-diastolic pressure, -dP/dt, and Tau (τ), as described. 9 In the sunsets of animals from each group, intravenous isoproterenol (a  1 -and  2 -adrenergic agonist) at the concentrations of 0.1, 1.0, 10.0, and 100 pg/g body weight was administered to assess systolic and diastolic function of the LV under -adrenergic stimulation. At the end of the experiment, animals were euthanized, and heart, LV, and lung were weighed. Heart weight, left ventricular weight, and lung weight were normalized to body weight.

Histopathological examination of mouse hearts
Pentobarbital-anesthetized mice received intracardiac saturated KCl (0.1 ml) to arrest the heart in diastole. The hearts were washed with cold phosphate-buffered saline, fixed with 4% paraformaldehyde, dehydrated, and embedded in paraffin. The hearts were sliced transversely from the apex to the basal part of the LV at 6 μm-thickness for measurements of LV morphology and interstitial fibrosis or 4 μm-thickness for measurements of myocyte cross-sectional area with the interval of 300 μm between each section 7 . All sections were mounted on glass slides and stained with Masson's trichrome. Images were captured with a SPOT Insight TM camera with the use of SPOT imaging software (Diagnostic Instruments, Inc., Sterling, MI, USA) 1,5 . SPOT software was used to measure the circumference of the infarcted region and the LV. Total LV circumference was calculated as the sum of endocardial and pericardial segment lengths from all sections. Infarct size was expressed as total infarct circumference divided by total LV circumference. The LV internal diameters were measured with SPOT software at midventricular level. Fibrosis areas within Masson's trichrome-stained sections were measured by visualizing blue-stained areas, exclusive of staining that colocalized with perivascular or intramural vascular structures, the endocardium, or LV trabeculae using SPOT software. The percentage of total fibrosis area was calculated as the summed blue-stained areas divided by total ventricular area. To determine myocyte cross-sectional area, the remote zone and border zone of Masson's trichrome-stained sections were imaged with a Nikon microscope using a 20 objective. A minimum of 100 myocytes from five different animals was quantified for each experimental group.

Real-time reverse transcriptional-polymerase chain reaction analysis of microRNA-21
Heart tissues were collected from both the septum and the LV free wall in sham WT or Tg mice and from non-infarct myocardium in MI mice 4 weeks after surgery. Homogenized tissues were used in real-time quantitative reverse transcriptional-polymerase chain reaction (qRT-PCR) analysis of microRNA-21 6 .
Total RNA from heart tissues was extracted using Qiazol reagent according to the protocol of the manufacturer (Qiagen, Valencia, CA, USA). Chloroform was added and samples were centrifuged to facilitate phase separation. The aqueous phase was extracted and combined with ethanol in miRNeasy Mini spin columns (Qiagen). Total RNA was eluted in RNase-free water.
The concentration of extracted total RNA was quantified by the Epoch spectrophotometer (Biotek, Winooski, VT, USA). Samples were considered pure if the A260/280 ratio was between 1.9 and 2.0. One µg of total RNA from each sample was used to generate cDNA using miScript Reverse transcriptase mix, nucleic mix, and HiFlex Buffer The LV pressure signal was monitored to obtain LV end-systolic and end-diastolic pressure.

LV contractile performance
LV end-diastolic pressure was set to 5 mmHg by adjusting the volume of the intracardiac balloon. After 30 min of stabilization, LV systolic pressure and +dP/dt were determined 5 .

LV end-diastolic pressure-volume relationship
The intracardiac balloon volume was set at zero volume, and the heart was stabilized for 30 min. The balloon volume was inflated to 20 µl and subsequently increased in 5-µl intervals using an air-tight Hamilton syringe until 70 µl 11 . LV functional measurements were obtained 2 min after each increment in volume when a new steady state was reached.

Isolation of cardiomyocytes
Cardiomyocytes were isolated from adult C57BL/6 or Tg mice 4 weeks after MI or sham surgery, as described 7  Experiments were conducted at room temperature within 6 h after isolation using Tyrode solution.

Measurements of SR Ca 2+ release
Cardiomyocytes were loaded with Fura-2 AM with Pluronic F-127 (0.04%) to aid dispersion followed by 30 min of de-esterization at 22 o C before recordings. Resting Ca 2+ measurements were taken from the quiescent cells for 3-4 min prior to the application of any agonists. SR Ca 2+ release was assessed by rapid application of 10 mM caffeine to the cells to induce SR Ca 2+ release in the presence of 0 Na + and 0 Ca 2+ Tyrode buffer to inhibit Na + -Ca 2+ exchange, as Sigma-Aldrich). Immunoblots were performed using standard techniques, as described 6,14 .
Briefly, tissue homogenates that contained 50 µg of protein were applied to 7.

Treatment of Tg mice with 7-nitroindazole
Tg mice were subjected to permanent ligation of the left coronary artery to produce infarction, and sham-operated mice underwent the same surgical procedure except coronary artery ligation. At 3 days after surgery, Tg mice were given 10 mg/kg/day 7-nitroindazole (7-NI) analyzed in Langendorff-perfused mouse hearts, as described in the section of "Determination of cardiac function in isolated hearts".

Statistics
All data are expressed as mean ± S.E.M. Statistical analysis was performed with one-way ANOVA followed by Bonferroni post-hoc test for multiple comparisons of multiple group means or with Student's t-test for comparisons between two group means. Repeated-measures ANOVA was used to compare the differences in heart surface area, echocardiographic parameters, GCH1 mRNA, GCH1 proteins, and LV end-diastolic pressure-volume relationships at different time points. A value of P<0.05 was considered statistically different.

Body weight and heart rate were not changed in WT mice after MI
Body weight and heart rate were comparable between MI WT and sham WT groups throughout the experiment (P>0.05, n=8-10 mice/group) (Table S1).

GCH1 overexpression improved myocardial responsiveness to -adrenergic stimulation after MI in vivo
Abnormalities in myocardial -adrenoceptor signaling are implicated in cardiac remodeling and dysfunction after MI 12,19,20 . We determined myocardial responsiveness to the 1 and 2adrenergic agonist isoproterenol in vivo in Tg and WT mice 4 weeks after MI or sham surgery.
Blood pressure and intracardiac hemodynamics were measured with a pressure conductance catheter. There were no significant differences in LV systolic pressure, LV diastolic pressure, the rate of LV pressure rise (+dP/dt), the rate of LV pressure decrease (-dP/dt), and time constant of left ventricular relaxation Tau (τ) values among 4 experimental groups without intravenous administration of isoproterenol (P>0.0083, n=8-10 mice/group) (Table S2)     Based on previous studies and our current findings, the possible signaling pathways linking GCH1 to cardiac remodeling and dysfunction after MI are summarized in Figure S4. GCH1 plays a predominant role in cardiac BH 4 , a key co-factor for NOS to produce the cardioprotective mediator, NO 21,22 . However, GCH1 proteins are degraded in post-infarction remodeled hearts