Submolecular mechanisms underlying in vitro and in vivo effect of cardiac glycosides on contractile activity of myocardial myofibrils during heart failure.

The development of severe heart failure associated with toxicoallergic myocarditis is accompanied by profound structural and conformational changes in the outer domain of actin (major protein in a thin filament of cardiomyocyte sarcomere). These changes were revealed in subdomains 1 (Cys374 and Cys10) and 2 (Lys61 and Tyr69). Structural and conformational changes in the monomer and protomer of the actin thread during heart failure were energetically forbidden. Variations in the distance between amino acid residues exceeded 0.26 nm. They were partly or completely reversible in vivo under the influence of cardiotropic drug refracterin with high antihypoxic activity, as well as in vitro after treatment with digitalis preparations optimizing the concentration of ATP.

Effect of NAD on recovery of adenine nucleotide pool, phosphorylation potential, and stimulation of apoptosis during late period of reperfusion damage to myocardium.

The system of energy supply in the myocardium of the left and right ventricles did not recover after short-term circulatory disturbances. ATP synthesis decreased in parallel with activation of poly-(ADP-ribose)-polymerase in the ischemic region of the right ventricle, extra-ischemic region, and in the left ventricle by 5.85, 5.4, and 2.2 times, respectively. Intravenous injection of NAD immediately after blood flow resumption in the subacute period of ischemia-reperfusion damage virtually completely normalized the pool of adenine nucleotides, energy change of the adenine nucleotide system, and phosphorylation potential. Exogenous NAD inhibited activity of poly-(ADP-ribose)-polymerase in the ischemic region of the right ventricle, extra-ischemic region, and in the ischemic region of the left ventricle by 2.4, 2.9, and 1.52 times, respectively. We hypothesize that NAD acts as a regulator of signal mechanism of apoptosis induction during ischemia-reperfusion damages to the myocardium.

Elastic modulus of the thin filament under normal conditions and during heart failure.

We developed an original approach to prepare samples for electron microscopy in electric field allowing calculation of the Youngs modulus of the thin filament in a direction perpendicular to the axis of the filament (longitudinal to the protomer) under normal conditions and during heart failure induced by 10-day toxic and allergic myocarditis. Electric field stretches thin filaments in the transverse direction and the increase in its diameter linearly depends on the applied voltage. The elastic modulus was in inverse proportion to the applied voltage. We found that during heart failure thin filament had an extreme conformation and to a great extent lost its mobility.

[Cardioprotective effect of energostim in toxic allergic myocarditis]. / Kardioprotektornoe deistvie énergostima pri toksiko-allergicheskom miokardite.

Energostim is a combined drug comprising a mixture of nicotinamide adenine dinucleotide (0.5 mg), cytochrome C (10 mg), and inosine (80 mg), representing antihypoxant and antioxidant of direct action in one ampule. After pretreatment and subsequent 3-day energostim therapy of animals with 3-day toxico-allergic myocarditis (3d-TAM), the ECG was free of any rhythm disorders and showed evidence of improved conduction, restoration of the normal form of T-wave and the position of ST segment, while the content of myofibrillar fraction of creatine phosphokinase and toxic products of disturbed metabolism (degree of endotoxemia) decreased to the upper normal level. Under the action of energostim, neither pressure nor the maximum rate of pressure buildup in the left ventricle are reduced (as they do upon 3d-TAM); neither systolic and diastolic functions are disturbed, nor their coordination (r = 0.79 between dP/dtmin and dP/dtmax, p < 0.01). The restoration of contractile activity and maximum rate of relaxation of myocardial microfibrils during 3d-TAM is accompanied by an increase in the content of adenyl nucleotides, in the ATP/ADP, ADP/AMP, NAD/NADH, and NADP/NADPH ratios, and in the cytosol phosphorylation potential. The energostim-induced improvement in the energy supply system are accompanied by restoration of the ability of sarcoplasmic reticulum to efflux Ca2+. Thus, it is demonstrated that the effect of energostim is related to its ability to actively participate in intracell metabolic processes in myocardium, abolish necrotic changes and endotoxicosis, and restore homeostasis in the systems responsible for the contraction--relaxation process (thus preventing from the development of dysfunction of the left ventricle and the heart failure).

[Effect of energostim on myocardial damage during coronary artery occlusion]. / Vliianie énergostima na porazhenie miokarda pri okkliuzii koronarnoi arterii.

In contrast to traditional therapy (beta-adrenoblocker and nitrates), energostim improves the systolic and diastolic functions of myocardium during 120-min occlusion of the left descending coronary artery. The energostim-induced improvement in the central hemodynamics is correlated with an adaptive increase in activity of the antioxidant system enzymes in response to the ischemic production of reactive oxygen species, which is evidence of the mobilization of reserves of the enzymatic link in the antioxidant defense system of cardiomyocytes. Analogous pattern is observed in the blood. In the control group of traditional therapy, a decrease in the superoxide dismutase (SOD) activity and the redox potential (NAD/NADH) in myocardium are correlated with a decrease in the maximum rate of pressure increase in the left ventricle (R 6.4, p < 0.01) observed 2 h after the coronary occlusion. In the energostim treated group, there is a correlation between the SOD activity and the content of cytochrome C in mitochondria (R 6.1, p < 0.01): a change in the level of cytochrome C during 2-h acute ischemia is correlated with the decrease in redox potential (NAD/NADH) and in the ratio of glutathione peroxidase to Mn-dependent SOD (r 0.64, p < 0.01). Thus, disturbances in the antioxidant defense system of both myocardium and blood plasma of the patients with acute myocardium infarction are correlated with inability of the energy supply system to utilize oxygen in the process of glycolysis and oxidative phosphorylation in mitochondria. Stable adaptive increase in activity of the antioxidant defense system enzymes and a decrease in the content of cytochrome C in the blood plasma are probably the independent indications of beneficial prognosis and high efficacy of the proposed treatment of the ischemic damage of myocardium.

Subcellular and molecular mechanisms of the effects of cardiac glycosides and angiotensin-converting enzyme inhibitors on contractile function and energy conversion in myocardial myofibrils under normal conditions and during acute cardiac insufficiency.

Experiments on skinned and hybrid myocardial fibers isolated from normal dogs and animals subjected to 120-min occlusion of the anterior interventricular branch of the coronary artery showed that in contrast to cardiac glycosides, angiotensin-converting enzyme inhibitors suppress contractile ability of myocardial myofibrils in a dose-independent manner within the concentration range of 10(-12)-10(-4)M. This effect is accompanied by a decrease in fiber relaxation rate most pronounced in the presence of captopril. Actin, the major protein of fine filaments is the target for b-acetyldigoxin, K-strophanthin, captopril, enalapril, and trandolapril in myocardial myofibrils. During coronary occlusion, the inhibitors of angiotensin-converting enzyme induce structural and conformational changes in actin that decrease efficiency of contraction. The data obtained cast doubt on advisability of therapeutic use of angiotensin-converting enzyme inhibitors in the therapy of myocardial infarction, especially in its early period.

Cardioprotective effect of energostim during occlusion of coronary artery.

Experiments on dogs showed that energostim, a directly acting antihypoxant, injected 15 min after occlusion of the upper one-third of the left descending branch of the interventricular coronary artery produced a pronounced cardioprotective effect. The effect was confirmed by electron microscopy (evaluation the necrotic focus), biochemical tests of the heart and blood, and changes in intracardiac hemodynamics (recovery of systolic and diastolic functions). The cardioprotective effect of energostim greatly surpasses that of routine therapy applied during acute myocardium infarction.

Effects of energostim on the sympathoadrenal system and contents of pyridine nucleotides during acute myocardial infarction.

The contents of myofibrillar creatine phosphokinase and cytochrome c, the key enzyme of the mitochondrial respiratory chain, increased, while the content of nicotinamide coenzymes and redox potential (NAD/NADH ratio) sharply decreased over the first 6 h of acute myocardial infarction. The contents of norepinephrine and epinephrine increased by 51 and 49%, respectively, 6 h after onset of acute myocardial infarction. By contrast, dopamine concentration decreased by 116% during this period. The increase in the content of cytochrome c directly correlated with the concentration of myofibrillar creatine phosphokinase and Peel index. No correlations were found between norepinephrine/epinephrine and dopamine/(norepinephrine+epinephrine) ratios. The antihypoxant with direct action energostim normalized the content of cytochrome c, increased NAD and dopamine concentrations and NAD/NADH ratio, and decreased the content of norepinephrine and epinephrine to the baseline level in patients with acute myocardial infarction. These results indicate that energostim possesses not only antihypoxic and antioxidant activities, but also pronounced antisympathomimetic properties.

Role of actin and myosin in the mechanism of the decrease of myocardial contractility and efficiency of energy transformation by myocardial myofibrils in chronic heart failure in humans.

Experiments with hybrid myocardial fibers showed that abnormalities of actin (basic protein of fine sarcomer threads) are responsible for reduced contraction rate, decreased developed force, and low efficiency of cardiomyocyte contraction in chronic heart failure caused by dilatation and ischemic cardiomyopathies and infective allergic myocarditis. Wastefulness of the contractile process in cardiomyocyte under conditions of pronounced energy deficit play a key role in progression of chronic heart failure. Hence, actin hypothesis of reduced contractile activity of myocardial contractile protein system in acute heart failure transforms into the actomyosin concept in chronic heart failure.

[A disorder of myocardial contractile function in acute experimental coronary failure: the submolecular mechanisms and the action of cardiac glycosides]. / Narushenie sokratitel'noi funktsii miokarda pri ostroi éksperimental'noi koronarnoi nedostatochnosti: submolekuliarnye mekhanizmy, deistvie serdechnykh glikozidov.

Skinned and hybrid myocardial fibers were studied by methods of tensometry, determination of the ATP hydrolysis intensity, and resonance fluorescent energy transfer between highly selective labels bound to various amino acid residues. It was established that development of the early stage of heart failure in the case of acute myocardial ischemia caused by 15-min coronary artery occlusion (CAO) is related to a reversible damage or adaptive (functional) depression of the contractile protein system. As a result, the system features isolated submolecular post-translational variation in the properties of major proteins in a thin actin filament (myosin is not significantly damaged). This leads to a decrease in the force developed by the hybrid fibers (reconstructed using ghost myocardial fibers taken from ischemic area and normal myosin) and in the ATPase activity of actomyosin (ATP hydrolysis intensity) without any significant change in the Ca-sensitivity, cooperativity of the Ca-response of the actomyosin ensemble, and efficiency of the contractile process. In actin of the ischemic area, CAO results in a serious damage of the Lys61 and Cys374 regions and in a less pronounced damage of the Tyr69 and Cys10 regions. These results suggest that the Lys61 and, probably, Cys374-Lys61 regions are included in the actin monomer as a protomer, without adequate prepolymerization structural-conformational changes necessary to provide for the normal functioning of the filament. In the CAO-induced early stage of heart failure, cardiac glycosides (beta-acetyldigoxin, beta-methyldigoxin, and strophanthin K) produce a direct effect upon the intramolecular structure of myocardial actin, restore the generated force level, and increase the intensity of ATP hydrolysis by actomyosin ensemble. This is achieved by improving or normalizing the structural-conformational state and conformational mobility of the Lys61 and Cys374 region of actin.

[The action of a number of cardiac glycosides on an isolated system of myocardial contractile proteins in heart failure due to toxic-allergic myocarditis. The molecular mechanism]. / Deistvie riada serdechnykh glikozidov na izolirovannuiu sistemu kontraktil'nykh belkov miokarda pri nedostatochnosti serdtsa, obuslovlennoi toksiko-allergicheskim miokarditom. Molekuliarnyi mekhanizm.

Experiments conducted on an isolated contractile apparatus, myocardial fibers (MF) in cardiac insufficiency (CI) caused by toxico-allergic myocarditis of 10 days duration (TAM10dd) showed that cardiac glycosides (CG), beta-acetyldigoxin (beta AD), beta-methyldigoxin, and strophanthin K (SK) increase the capacity of the actomyosin ensemble (AME) for generation of force, hydrolization, and economic use of the free energy of ATP hydrolysis. The mechanism of the effect of these CG in the phase of contraction differs from that of their effect on the AE of a normal myocardium. For instance, in severe CI induced by TAM10dd beta AD, in distinction from its action on the AME of a normal myocardium, can increase contractility economy, particularly in the phase of highest energy capacity, the phase of force generation, and exceed the level encountered in normal conditions, it also increases significantly the rate and reduces the time of MF relaxation as in the case of MF of a normal heart.

[The subcellular pathophysiology of heart failure due to toxic-allergic myocarditis and the action of refracterin on intracardiac hemodynamics and the functional state of the 3 subcellular cardiomyocyte systems responsible for the act of contraction-relaxation]. / Subkletochnaia patofiziologiia nedostatochnosti serdtsa, obuslovennoi toksiko-allergicheskim miokarditom, i deistvie refrakterina na vnutriserdechnuiu gemodinamiku i funktsional'noe sostoianie trekh subkletochnykh sistem kardiomiotsita otvetstvennykh za akt sokrashchenie--rasslablenie.

It is shown that cardiotropic drug refracterin promotes recovery of cardiac contraction and relaxation, their coordination destroyed in cardiac failure (CF) caused by 10-day toxico-allergic myocarditis (TAM). Pumping capacity of the heart returns to normal after normalization of functional activity of three systems of cardiomyocyte responsible for contraction-relaxation: contractile proteins, energy supply and calcium transport. The key process is refracterin-related reestablishment of normal content and proportion of adenyl nucleotides and creatininephosphate and regulation role of phosphorylation and energy of metabolic processes in the cells and their interaction. Thus, refracterin effectiveness lies in its ability to interfere in intracellular metabolic processes in the myocardium, to reestablish normal homeostasis of the systems responsible for contraction-relaxation function and eventually to remove left ventricular cardiac dysfunction.

[The positive action of refracterin on the reserve potentials and metabolism of the myocardium during its overload in toxic-allergic myocarditis]. / Polozhitel'noe deistvie refrakterina na rezervnye vozmozhnosti i metabolizm miokarda pri ego peregruzke na fone toksiko-allergicheskogo miokardita.

Heart overloading due to pressure as a result of 8 periodic full aortic constriction in heart failure (HF) caused by 10-day toxic-allergic myocarditis (TAM) leads to deterioration of heart contractility (pump function). This is explained by additional decline in functional activity of all three systems of cardiomyocyte responsible for contraction-relaxation. In particular, by a sharp fall of ATP and CP content in the myocardium, a 400% decrease in myofibril power, 200% reduction in efficiency of contraction and marked deterioration of calcium transport. The resultant exhaustion of myocardial reserve brought 70% lethality among the animals. Under the above conditions coordination between the systolic and diastolic cardiac functions, correlation between myocardial functional activity and subcellular systems of cardiomyocyte are impaired. In pressure heart overloading refracterin initiates profound metabolic rearrangements improving metabolism, remodelling of the system of energy supply, reestablishment of systemic homeostasis, normalization of cardiomyocyte and cardiac reserves.