Comparison of the Reverse-Remodeling Effect of Pharmacological Soluble Guanylate Cyclase Activation With Pressure Unloading in Pathological Myocardial Left Ventricular Hypertrophy

FIGURE 1. Experimental design. After a 1-week-long acclimatization period (at week 0), the rats underwent abdominal aortic banding (AB; n = 39) or a sham operation (n = 33). Six weeks later, the animals were randomized into seven experimental groups. A group of AB (n = 10) and sham-operated (n = 11) animals were measured after week 6 to prove that pressure overload-induced myocardial alterations were already present at this time point. The other five groups were followed up until week 12 and they were treated from the 7th to the 12th experimental week with 0.5% methylcellulose vehicle or with the sGC activator, cinaciguat in suspension (10 mg/kg/day via an oral gavage). Accordingly, these groups involved the followings: vehicle-treated control group (Sham-Co, n = 11), cinaciguat-treated control group (Sham-Cin, n = 11), vehicle-treated AB group (AB-Co, n = 11), cinaciguat-treated AB group (AB-Cin, n = 9) and vehicle-treated debanded group (Debanded, n = 9). Comparison of Sham-Co, AB-Co, AB-Cin, and Debanded groups were performed to detect the potential reverse remodeling effects of pressure unloading and chronic cinaciguat treatment. Furthremore, the Sham-Cin group was compared to the…

FIGURE 2. Effect of pressure unloading and soluble guanylate cyclase activation on pre-established left ventricular hypertrophy. Representative cross-sectional whole heart images, M-mode echocardiographic recordings at the mid-papillary level and hematoxylin-eosin stained sections (Magnification, ×400; scale bar, 50 μm) are shown in the study groups. (A) Aortic banding (AB) induced the development of left ventricular (LV) myocardial hypertrophy (LVH) already at week 6, as detected by increased heart weight-to-tibial length ratio (HW/TL), LV mass, cardiomyocyte diameter (CD) and beta/alpha myosin heavy chain ratio (β/α-MHC). (B) Pressure unloading was associated with the regression of LVH, as observed in decreased HW/TL, LV mass, CD and β/α-MHC. In contrast, treatment with cinaciguat only slightly influenced the pre-established LVH and only CD and β/α-MHC showed decrement from the non-treated AB group. ∗P < 0.05 vs. age-matched sham. #P < 0.05 vs. AB-Co. $P < 0.05 vs. AB-Cin.

FIGURE 3. Protective effect of pressure unloading and cinaciguat treatment against myocardial fibrosis. Representative microphotographs of the left ventricular myocardium after Masson’s trichrome staining are presented. (A) After week 6, the pro-fibrotic genes (transforming growth factor-β [TGF-β]; matrix metalloproteinase-2 [MMP-2], tissue inhibitor of matrix metallopeptidase-2 [TIMP-2], collagen type 1 α1 [Col1a1] and collagen type 3 α1 [Col3a1]) were activated in the aortic banded (AB) group. However, at this time point Masson’s trichrome staining revealed physiologically low levels of interstitial fibrosis. (B) In contrast, after week 12, the AB-Co group was associated with increased collagen accumulation. Both pressure unloading and cinaciguat therapy effectively inhibited the increment in myocardial fibrosis. ∗P < 0.05 vs. age-matched sham. #P < 0.05 vs. AB-Co.

FIGURE 4. Pressure unloading as well as cinaciguat administration reduces myocardial nitro-oxidative stress. Representative images of 3-nitrotyrosine (3-NT) stained sections are shown. (A,B) In the aortic banded (AB) groups after week 6 and 12, increment in 3-NT content of the cardiomyocytes and increased mRNA level of NAPDH oxidase family member 2 (NOX2) indicated increased oxidative stress in the myocardium. Furthermore, after week 6 the mRNA level of glutathione peroxidase 4 (GPX4) was also significantly elevated. In contrast, both pressure unloading and cinaciguat evoked potent anti-oxidant effects and decreased 3-NT content and NOX2 expression and increased mRNA level of GPX4. ∗P < 0.05 vs. age-matched sham. #P < 0.05 vs. AB-Co.

FIGURE 5. Apoptotis is inhibited by pressure unloading and cinaciguat treatment. Representative images of Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) stainings are shown. (A) At week 6, none of the apoptotic markers showed changes in the aortic banded (AB) group. (B) In contrast, after week 12 in the AB-Co group the TUNEL positive nuclei increased, while expression of B cell lymphoma-2 (Bcl-2) decreased significantly compared to the Sham-Co group. These alterations were successfully prevented either by cinaciguat treatment or pressure unloading. ∗P < 0.05 vs. age-matched sham. #P < 0.05 vs. AB-Co.

FIGURE 6. Effect of pressure unloading and cinaciguat on hemodynamic parameters. Representative pressure–volume (P–V) loops are shown. (A) At week 6, in the aortic banded group, increased mean arterial pressure (MAP) and arterial elastance (Ea) (MAP, Ea), increased contractility (slope of the end-systolic pressure–volume relationship, ESPVR) maintained ejection fraction (EF) and impaired active relaxation (TauGlantz) and stiffness (slope of the end-diastolic pressure-volume relationship) were detected. (B) At week 12 in the AB-Co group, increased arterial load and and diastolic dysfunction were also present. Furthermore, systolic performance was impaired as well, as detected by the reduced ESPVR and EF values. Pressure unloading decreased MAP and Ea, restored diastolic dysfunction and prevented the reduction in EF. In contrast, cinaciguat did not influence the increased afterload but increased ESPVR and maintained adequate EF values. Furhtermore, the impairment in EDPVR was also inhibited by cinaciguat, although the prolonged active relaxation (TauGlantz) did differ from the AB-Co group. ∗P < 0.05 vs. age-matched sham. #P < 0.05 vs. AB-Co. $P < 0.05 vs. AB-Cin.

FIGURE 7. Modulation of the cGMP signaling in the myocardium by pressure unloading and cinaciguat treatment. (A) Aortic banding (AB) induced increased myocardial expression of atrial natriuretic peptide (ANP) at week 6, resulting in enhanced plasma/myocardial cyclic guanosin monophosphate (cGMP) levels. Expression and activity of protein kinase G (PKG) remained unchanged at this time point. (B) At week 12, mRNA levels of ANP and plasma/myocardial cGMP levels increased in the AB-Co group. In addition, in this group PKG expression tended to increase on the mRNA and on the protein level as well, however, no alterations could be detected in its activity. Pressure unloading resulted in the deactivation of the cGMP pathway. In contrast, cinaciguat treatment led to increased PKG activity (as detected by increased p-VASP/VASP ratio) despite the fact, that myocardial cGMP levels did not differ from that of the AB-Co group. ∗P < 0.05 vs. age-matched sham. #P < 0.05 vs. AB-Co.