All experiments were carried out on the chinchilla rabbits weighing between 2.5 - 3.0 kg and reviewed in accordance with Principles of Laboratory Animal Care (“Guiding Principles in the Care and Use of Animals” of the American Physiology Society, NIH Publication no. 86-23, revised in 1996) and approved by the Local Ethics Committee at International Centre of Introduction of New Biomedical Technology and Kuban State Medical University, Krasnodar, Russia. The rabbits were kept in a daylight environment and fed commercial laboratory rabbit food pellet, in specially designed housed (animal room) at a mean temperature 20˚C, humidity 40% - 70%, lighting 12 hours per day for at least 1 week before the experiments.

CHF was reproduced by left descending coronary artery stenosis (banding (ligation) up to one third of the original size under sterile conditions [13] ) 3-month duration (average ratio of heart to the body mass increased from the 2.22 ± 0.24 to 4.05 ± 0.24 (p > 0.001) and did not changed in the sham operation group 2.32 ± 0.19, NS). Average survival of animals in the interval 2 - 7 days was about 78% (54 from 69 animals in the experiments) in the operation group and 100% in the sham operated group. After 8 weeks all experimental animals were randomized into three group: control II (n = 10) were treated with either vehicle (normal saline), main group I in which animals received NAD-containing positive inotropic drug Adenocin^® (20 mg/kg solved in 1 ml of water for injection intravenously, once daily) (n = 12) for 14 days and main II group in which animals received traditional therapy of CHF, containing ACE inhibitors, enalapril (Sigma Aldrich Inc., St. Louis, MO) intramuscularly (0.25 mg/kg body weight/day), furosemide 1.0 mg/kg intravenously (bolus), and pimobendan (Sigma Aldrich Inc., St. Louis, MO) 0.1 mg/kg i.v. once daily during 14 days, n = 11).

Cardiac hemodynamics and SIRS were determined as described below [7] [11] [12] [13] . The ventricles trimmed of atria and visible blood vessels. The left ventricular weight (LVW) and the ratio of LVW to body weight (LVW/BW) were obtained. The heart weight index was calculated by dividing the heart weight by the body weight in each group and as a cardiac wet-to-dry weight ratio. Wet-to- dry weight ratios were determined by drying tissue at 70˚C. The brain natriuretic peptide (BNP) was measurement using MyBioSource ELISA assay.

The average severity of clinically symptoms of endotoxemia measured by the lethargy score: temperature and behavioral disturbances (the blinded to treatment status, as follows: 0 = normal activity; 1 = quiet, spontaneous activity; 2 = no spontaneous movement, but responsive to touch; 3 = responds only to noxious stimuli; 4 = unresponsive to noxious stimuli) were 2.5 ± 0.2 in control and 2.8 ± 0.3 in main group with CHF before treatment. In control I (sham operated group) its equal 0.13 ± 0.03.

Plasma and myocardium content of TNF-α and NF-kB in nuclear fraction of myocardium from left ventricle were determined by ELISA technique using standard kits (RayBiotech, Inc., USA).

Frozen sections of hearts were mixed with 1.5 ml of physiological solution contained 0.5% NaN₃ and homogenized. Supernatants containing cytosolic protein were used to detect the intracardiac cytokines with commercially available enzyme-linked immunosorbent assay (ELISA) kits (TNF-α and IL-6; IL-1β: BioSource International Co., Camarillo, California).

The content of NAD and NADH, ratio of lactate/pyruvate in the myocardium homogenate were measurement as described below [4] [7] [11] .

The disturbances in the intrinsic defense system activities were measurement by the level of catepsin D activity with and without 0.1% triton X-100 (as a marker of lysosomal integrity of the myocardium [14] [15] ), production of superoxide anion and hydrogen peroxide in mitochondrial fraction of LV myocardium as a markers of deterioration in antioxidant defense system were described below [4] [7] [11] .

The statistical significance assessed as the mean ± standard deviation (SD). Comparison of two means was performed using the Student t test for normally distributed variables and p < 0.05 considered significant. Chi-square testing was used to analyze categorical data. All statistical calculations were performed using the Statistical Sciences (SPSS, version 23).

Survival rates up to 3 month after the 14 days Adenocin^® treatment animals with SIRS and CHF syndromes exerted beneficial effects on survival, increased by 100%, in group with traditional treatment the final survival rates were 88%, and in control group, 80%. The cardiac hypertrophic index (CHI) in animals with CHF significantly increased above the control, and averaged 2.81 ± 0.18, after treatment with pimobendan in combination with benazepril and furosemide was slightly decreased by 11% and riches control level in group treated with Adenocin^®. Water concentration in LV of myocardium was also increase and exceeded the control level by 6% (averaged 776 ± 23 mg/g in control animals and 814 ± 25 mg/g in CHF group). After treatment with combined therapy containing enalapril, furosemide and pimobendan and monotherapy with Adenocin^® water content decreased to 773 ± 16 mg/g (p < 0.05). Moreover, the recovery rates for +dp/dt_max, −dp/dt_max and left ventricle end diastolic pressure (LV EDP) were markedly higher in Adenocin^®-treated group (Table 1).

The prolonged stenosis of coronary artery characterized by simultaneously developing of two syndromes: CHF, as was diagnosed by developing of cardiac hypertrophy, myocardial edema and increasing of level of BNP (Table 1) and SIRS, as diagnosed by increased the markers and symptoms of endotoxemia, tissue dysoxia and decreasing reserve ability of intrinsic defense systems such as antioxidant and lysosomal (Table 2).

Inflammatory processes underlie many diseases and syndromes associated with injury of the heart muscle, and acutely, subacutely, and chronically cause structural, functional, and molecular deficits and defects. Many sterile stimuli that can induce inflammation―specifically dead cells and a variety of irritant particles, including crystals, minerals, and protein aggregates. Although this subset of stimuli is structurally very diverse and might appear to be unrelated, there is accumulating evidence that the innate immune system may recognize them in similar ways and stimulate the sterile inflammatory response via common pathways.

Note: 1- Cardiac hypertrophic index was calculated as a ratio = Heart wet weight (mg): body weight (mg) × 10⁻³; each value remains mean ± standard deviation, symbols―significance of difference between the group, *―with control, #―with CHF, +―with traditional therapy groups; one symbol―p < 0.05, two―p < 0.01; three―p < 0.001.

Note: each value remains mean ± standard deviation, symbols―significance of difference between the group, *―with control, #―with CHF, +―with traditional therapy groups; one symbol―p < 0.05, two―p < 0.01; three―p < 0.001.

The SIRS caused by chronic ischemic heart damage 3 month duration characterized by small leucocytosis ≥ 12 × 10⁹ cells/L, stable temperature 37.2˚C ± 2.2˚C, tachycardia 278 ± 12 beats/min. Blood medium-mass molecules (MMM) concentration was revealed reflecting the condition severity, pattern, and manifestation of endogenous intoxication as a key part of SIRS under CHF (Table 2). Changes in spectrograms of plasma and erythrocytes together with the more than 2-fold increase of VCAM-1c confirmed the persistent release of ischemic- induced structural deterioration molecules from the myocardium and their absorption on the glycocalyx or in erythrocytes. This accompanied by elevated level of proinflammatory cytokine, TNF-α in 10-fold in plasma and in 5.8-fold in LV of myocardium in comparison to practically healthy rabbits (Table 2). NF-kB (p65) activity in LV also increases in 2.5 fold in nuclear fraction of LV myocardium. Combined treatment with inhibitor of ACE, pimobendan and diuretics decreased the optical density of erythrocytes and has not changed the content of MMM in plasma, while the content of VCAM1c decreased by 33% (but it exceeds by 78% the normal level). This combined therapy has not shown the beneficial inhibitory effects on the TNFα production due to inhibition of activation of NF-κB in myocardium, but decrease the level of TNFα in plasma in 1.7-fold. Treatment with Adenocin^® normalized the spectrograms of plasma and erythrocytes, content of VCAMc and decrease the level of TNFα in myocardium and plasma (in 2.9 and 7-fold respectively). Decreasing TNFα in myocardium couples with the normalization of activity of NF-kB in myocardium (decreasing in more than 2 times in comparison with CHF group, p < 0.01).

Under prolonged ischemic heart damage the decreasing the redox-potential NAD/NADH by 30% as a marker of the mitochondrial complex 1 functioning disturbances is occurred which leads to an increasing of generation of superoxide anion. The ratio of lactate/pyruvate increases about 3-fold that strongly confirm that metabolism shift to an anaerobic processes and energy supply deterioration. The decreasing of redox-potential at this stage of CHF has not coupled with reduced of total pool of pyridine nucleotide (including NADP(H) pool our unpublished dates). The strong negative correlation between increasing level of TNF-α production and NF-kB (p65) activity and redox-potential NAD/NADH (r = −0.73, p < 0.001 and r = −0.67, p < 0.01 respectively) was established. Therapy with of Adenocin^® intravenously once daily, unlike traditional treatment, was capable to restore the normal level of NAD⁺/NADH ratio and to decrease the activation of NF-kB and content of TNF-alpha in myocardium and blood and as a result blocking the myocardial hypertrophy progression.

Superoxide anion production in the ischemic injuring myocardium significantly increases (p < 0.001). Combined treatment with pimobendan has not prevented the increase in superoxide anion production in the myocardium after ischemia (NS). Adenocin reduced superoxide anion production in CHF and SIRS induced by chronic ischemic coronary arteries (p < 0.001). At the same time, influence of the traditional therapy results to the decrease of the hydrogen peroxide production in CHF ischemic genesis myocardium from 198 ± 28 nmol/min/mg to 126 ± 23 nmol/min/mg (p < 0.01; 89 ± 11 nmol/min/mg in practically healthy animals myocardium). Hyperproduction of hydrogen peroxide in ischemic genesis CHF myocardium fully diminished under course treatment with Adenocin^® (Table 2).

The free and total catepsin D activities increased in hypertrophied myocardium by 115% (p < 0.001) and 31% (p < 0.01), respectively. As a result, the ratio of total activity/free activity of catepsin D decreased by 39% in CHF myocardium. Following treatment with combined therapy with pimobendan the free activity of catepsin D has not been decrease in comparison with CHF group while the total activity of enzyme significantly has not been changed from normal level. While treatment with Adenocin pronounced decrease, the free catepsin D activity and ratio of total activity/free activity of this lysosomal enzyme, which indicated that such treatment restores the integrity of lysosomal membrane.