Cinaciguat prevents the development of pathologic hypertrophy in a rat model of left ventricular pressure overload

Pathologic myocardial hypertrophy develops when the heart is chronically pressure-overloaded. Elevated intracellular cGMP-levels have been reported to prevent the development of pathologic myocardial hypertrophy, therefore we investigated the effects of chronic activation of the cGMP producing enzyme, soluble guanylate cyclase by Cinaciguat in a rat model of pressure overload-induced cardiac hypertrophy. Abdominal aortic banding (AAB) was used to evoke pressure overload-induced cardiac hypertrophy in male Wistar rats. Sham operated animals served as controls. Experimental and control groups were treated with 10 mg/kg/day Cinaciguat (Cin) or placebo (Co) p.o. for six weeks, respectively. Pathologic myocardial hypertrophy was present in the AABCo group following 6 weeks of pressure overload of the heart, evidenced by increased relative heart weight, average cardiomyocyte diameter, collagen content and apoptosis. Cinaciguat did not significantly alter blood pressure, but effectively attenuated all features of pathologic myocardial hypertrophy, and normalized functional changes, such as the increase in contractility following AAB. Our results demonstrate that chronic enhancement of cGMP signalling by pharmacological activation of sGC might be a novel therapeutic approach in the prevention of pathologic myocardial hypertrophy.


Methods
All animals received humane care in compliance with the "Principles of Laboratory Animal week old, body weight = 220-240g) male Wistar rats (n=35) ("Toxi-Coop" Zrt., Dunakeszi, Hungary) were housed in a room with constant temperature of 22±2°C with a 12h light-dark cycle, were fed a standard laboratory rat chow ad libitum and had free access to water.

Abdominal aortic banding procedure
After acclimation, banding of the abdominal aorta (AAB, n=19) between the renal arteries and the superior mesenteric artery, or sham operation (n=16) was performed in pentobarbital sodium (60mg/kg i.p.) anaesthesia as previously described 1 . Briefly, to achieve a standard degree of stenosis in our aortic banded animals, a blunted needle with an outer diameter of 20 Gauge (G, 0.9mm) was placed in parallel with the aorta, around which a 2-0 surgical suture was tightened in all cases. Then, the needle was removed, leaving a standard 20G stenosis behind. Sham operation comprised all steps excluding the banding procedure, which was replaced by a 1 min occlusion at the same level of the aorta. After recovering from anaesthesia and on the first and second postoperative day, all animals received meloxicam (1.5mg/kg p.o.) for postoperative analgesia.

Experimental groups, chronic treatment protocol
5 days after the operations, sham and AAB animals were randomized into control or treatment groups (ShamCo, n=8; ShamCin, n=8; AABCo, n=10; AABCin, n=9). Treated animals received Cinaciguat (10mg/kg p.o.) suspended in 0.5% methylcellulose solution via oral gavage, while control rats were given only the vehicle every day for 6 weeks. The dosage was adjusted to body weight, which was measured three times a week during the whole study period.

Echocardiography
We performed echocardiographic measurements at the 3 rd and 6 th week after the operations as previously described 2 . Briefly, the rats were anesthetised with pentobarbital sodium (60mg/kg i.p.), were placed on controlled heating pads, and the core temperature, measured via rectal probe, was maintained at 37°C. After the anterior chest was shaved, transthoracic

Hemodynamic Measurements: LV Pressure-Volume (P-V) Analysis
Rats were anesthetised with pentobarbital sodium (60mg/kg i.p.), tracheotomised, intubated and ventilated with a tidal volume and frequency adjusted to body weight using an Inspira Advanced Safety Ventilator (MA1 557058, Harvard Apparatus, Holliston, MA, USA).
Animals were placed on controlled heating pads, and the core temperature, measured via rectal probe, was maintained at 37°C. A polyethylene catheter was inserted into the left external jugular vein for fluid administration. A 2-Fr micro tip pressure-conductance catheter (SPR-838, Millar Instruments, Houston, TX, USA) was inserted into the right carotid artery and advanced into the ascending aorta. After stabilization for 5 min, arterial blood pressure and heart rate (HR) were recorded. Then, the catheter was advanced into the LV under pressure control. After stabilization for 5 min, signals were continuously recorded at a sampling rate of 1,000 samples/s using a P-V conductance system (MPVS-Ultra, Millar Instruments) connected to the PowerLab 16/30 data acquisition system (AD Instruments, Colorado Springs, CO, USA), stored, and displayed on a personal computer by the LabChart5 Software System (AD Instruments). LV end-systolic pressure and volume (LVESP and LVESV), LV end-diastolic pressure and volume (LVEDP and LVEDV), the maximum (dP/dtmax) rate of LV pressure change, time constant of LV pressure decay [τ; according to the Glantz method], EF and stroke work (SW) were computed and calculated using a special P-V analysis program (PVAN, Millar Instruments). SV and cardiac output (CO) were calculated and corrected according to in vitro and in vivo volume calibrations using PVAN software. In addition to the above parameters, P-V loops recorded at different preloads can be used to derive other useful systolic function indexes that are less influenced by loading conditions and cardiac mass 5 . Therefore, LV P-V relations were measured by transiently reducing preload by compressing the inferior caval vein under the diaphragm with a cotton-tipped applicator. The slope [end-systolic elastance (Ees)] of the LV end-systolic P-V relationship (ESPVR; according to the parabolic curvilinear model 6 ), preload recruitable SW (PRSW), and the slope of the dP/dtmax-end diastolic volume relationship (dP/dtmax-EDV) were calculated as loadindependent indices of LV contractility. At the end of each experiment, 100 µl of hypertonic saline was injected intravenously, and from the shift of P-V relations, parallel conductance volume was calculated by the software and used for the correction of the cardiac mass volume. The volume calibration of the conductance system was performed as previously described 2 . Briefly, nine cylindrical holes in a block 1 cm deep and with known diameters ranging from 2 to 11 mm were filled with fresh heparinized whole rat blood. In this calibration, the linear volume-conductance regression of the absolute volume in each cylinder versus the raw signal acquired by the conductance catheter was used as the volume calibration formula. After completion of the hemodynamic measurements all animals were euthanized by exsanguination.

Post mortem measurements
After euthanasia, the heart, the lung and the liver of the animals were immediately placed into cold saline and were measured on a scale. This was followed by the sampling of the organs, as described below. To exclude the natural variability between the weights of the animals, the right tibia of every rat was also prepared and its length measured 7 .

Histology and immunohistochemistry
Hearts were harvested immediately after euthanasia, and samples were placed in 4% buffered paraformaldehyde solution. Transverse transmural slices of the ventricles were sectioned (5 µm) and conventionally processed for histological examination. Heart sections were stained with hematoxylin and eosin and Picrosirius red. Light microscopic examination was performed with a Zeiss microscope (Axio Observer.Z1, Carl Zeiss, Jena, Germany), and digital images were captured using an imaging software (QCapture Pro 6.0, QImaging, Surrey, BC, Canada). The transverse transnuclear widths of randomly selected, longitudinally oriented cardiomyocytes were measured by a single investigator after calibrating the system. According to the method previously described 7 , immunohistochemical analysis for cGMP was performed on paraffin sections of the heart by using the avidin-biotin method (anti-cGMP mouse monoclonal antibody 1:2000, Abcam, Cambridge, UK). Immunohistochemical reactivity was examined with light microscopy at a magnification of 400x. Semiquantitative scoring (scores 0-4; 0: no staining, 1: weak, 2: mild, 3: strong, 4: very strong staining) was performed by two people blinded to the groups as described elsewhere 8 .

Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay
Paraffin embedded, 5 µm thick heart tissue sections were used to detect DNA strand breaks in LV myocardium. TUNEL assay was performed using a commercially available kit (DeadEnd™ Colorimetric TUNEL System, Promega, Mannheim, Germany) according to the manufacturer's protocol. TUNEL positive cell nuclei were counted by two blinded observers in 10 fields of each section at 200x magnification. Data were normalized to the mean value of the ShamCo group and were used to perform statistical analysis.

Biochemical Measurements
After hemodynamic measurements were completed, blood samples from the inferior caval vein were collected in tubes prerinsed with EDTA. The blood samples were centrifuged at 3,000 RPM for 15 min at 4°C, then separated plasma was stored in aliquots at -80°C. Plasma level of cGMP was determined using an enzyme immunoassay kit as per manufacturer's protocol (Amersham cGMP EIA Biotrak System, GE Healthcare, Little Chalfont, Buckinghamshire, UK).

Drugs
All drugs listed were purchased from Sigma-Aldrich (St. Louis, MO, USA) except for Cinaciguat, which is a kind gift of Bayer AG (Leverkusen, Germany).

Statistical Analysis
Statistical analysis was performed on a personal computer with a commercially available software (GraphPad Prism 6, La Jolla, CA, USA). All data are expressed as means ± standard error of the mean. After testing normal distribution of the data using the Shapiro-Wilk test, two-factorial analysis of variance (ANOVA) (with 'aortic banding' and 'Cinaciguat treatment' as factors) was carried out to detect independent effects of the factors (pband, ptreat) and significant banding×treatment interactions (pint). Tukey's post hoc testing was performed to evaluate differences between the groups. Data that did not show normal distribution were transformed logarithmically before performing two-factorial ANOVA. A paired Student's ttest was performed for comparing data of the echocardiographic measurements at 2 time points within a group. Differences were considered statistically significant when p<0.05.