Purinergic and NO-Dependent Mechanisms of Cardioprotection and Vasoprotection

The dissertation examines purinergic regulation and the role of nitric oxide in cardioprotection and vasoprotection in chronic heart failure. It considers the cardioprotective effects of the binary A1 and A3 adenosine receptor agonist MRS3630, the ACE inhibitor spirapril, and the beta-adrenergic blocker propranolol, as well as the effects of P2 receptor stimulation, P2X4 receptor overexpression, and modulation of NO synthesis on the myocardium, vascular tone, ischemia-reperfusion injury, and cardiac remodeling.

The study encompasses normotensive and spontaneously hypertensive animals, as well as mice with a calsequestrin model and postinfarction heart failure. The reported findings address the contribution of endogenous NO to vasodilation and intravascular pressure stability and evaluate the effects of P2/P2X4 receptor system activation on contractile function, myocardial remodeling, and survival.

Relevance of the Problem. The search for methods of preventing the development and progression of chronic heart failure (CHF) remains one of the pressing problems in contemporary cardiology. CHF is characterized by systolic and/or diastolic myocardial dysfunction and circulatory insufficiency, which leads to deteriorated quality of life and disability among patients (V. Yu. Mareev, 2007). Despite advances in cardiology in the prevention and treatment of CHF, existing methods remain insufficiently effective (V. A. Lyusov, N. V. Teplitova, 2005). The most important causes of CHF development are coronary heart disease (CHD) and arterial hypertension (AH) (D. V. Preobrazhensky et al., 2004). Approaches to preventing the development of CHF in patients with CHD include protection of the myocardium from ischemic and reperfusion injury. Ischemic preconditioning (IPC) has been recognized as one of the most effective methods of myocardial protection (Kloner, Rezkalla, 2006). Nevertheless, the potential of pharmacologically modulating the protective mechanisms of IPC remains poorly studied. At present, the development of agents with dual action at A1 and A3 adenosine receptors is a promising direction, as they have demonstrated a greater degree of protection against ischemic and reperfusion injury than the separate use of selective agonists (Jacobson et al., 2000). However, due primarily to their large molecular mass, the binary agonists known to date are unsuitable for oral administration. Accordingly, it is relevant to study the cardioprotective properties of new low-molecular-weight compounds with dual action at A1 and A3 adenosine receptors.

Long-standing increased afterload and disturbances in the diurnal rhythm of arterial pressure lead to hypertrophy of the heart and blood vessels (V. B. Koshelev et al., 1991; P. A. Zevelyan et al., 2001).

Insufficient endothelial production of NO is regarded as one of the pathogenetic mechanisms of increased peripheral resistance in AH and CHF (Feletou, Vanhoutte, 2006). However, researchers do not agree on the level of endothelial NO secretion in experimental arterial hypertension (Higashino, 1995; Baumann et al., 2007). Accordingly, studying the effects of blocking NO synthesis on indicators of vascular tone in normotensive and hypertensive animals, as well as assessing the role of NO in how ACE inhibitors and beta-adrenergic receptor blockers—the first-line agents in CHF treatment—act on flow-dependent vasodilation, is of considerable interest.

Stabilization of myocardial contractile function through the use of drugs with positive inotropic effects is an important element of CHF therapy (S. N. Tereshchenko, 1999). However, the cardiac glycosides used for this purpose do not slow disease progression and do not affect prognosis (V. Yu. Mareev et al., 2007). It follows that it is highly important to study the recently discovered inotropic effects of stimulation of P2X4 purinergic receptors in a model of CHF (Ni et al., 2002).

Research objective: to investigate the cardioprotective effects of activators of various types of purinergic receptors, as well as the role of endogenous nitric oxide in the regulation of vascular tone and in the infarct-limiting effect of ischemic preconditioning.

Principal research objectives:

1. To assess the effects of inhibiting nitric oxide synthase and angiotensin-converting enzyme on the severity of myocardial ischemia-reperfusion injury and the efficacy of ischemic preconditioning.

2. To assess the cardioprotective effects of the binary A1 and A3 adenosine receptor agonist MRS3630.

3. To investigate the effects of modulation of nitric oxide synthesis, ACE inhibition, and beta-adrenergic receptor blockade on vascular tone parameters under arterial normotension and hypertension.

4. To investigate the effects of prolonged stimulation of P2 purinergic receptors and overexpression of P2X4 purinergic receptors on cardiac performance and cardiac remodeling in genetically determined heart failure.

5. To study the effects of overexpression of P2X4 purinergic receptors on cardiac function and postinfarction myocardial remodeling.

Scientific Novelty. It was demonstrated that endogenously formed NO does not affect myocardial infarct size or the severity of ischemic arrhythmias in rats. In addition, blockade of NO synthesis does not affect the antiarrhythmic or infarct-limiting effects of myocardial ischemic preconditioning.

The case for using the binary A1 and A3 adenosine receptor agonist MRS3630 as a cardioprotective agent was established.

The data obtained indicate a key role for NO in supporting flow-dependent vasodilation and arterial pressure stability in normotensive rats. Flow-dependent vasodilation in most spontaneously hypertensive rats, however, is not determined by NO synthesis. NO was shown to be an important factor supporting intravascular pressure stability in hypertensive animals.

It was established that, under conditions of NO synthesis blockade in spontaneously hypertensive rats, the ACE inhibitor spirapril lowers arterial pressure and increases vascular compliance and intravascular pressure stability. Conversely, it was shown that the beta-adrenergic blocker propranolol increases vascular tone and enhances flow-dependent vasodilation at high blood-flow velocities under arterial normotension and hypertension. Inhibition of nitric oxide synthesis attenuates flow-dependent vasodilation induced by the beta-adrenergic blocker propranolol.

It was established for the first time that overexpression of P2X4 purinergic receptors improves myocardial contractile function, increases myocardial sensitivity to catecholamines, slows remodeling processes, and prolongs the life span of mice with a calsequestrin model of heart failure.

Experimental evidence shows that long-term administration of a P2 purinergic receptor agonist slows remodeling processes and increases the life span of mice with a calsequestrin model of heart failure.

It was demonstrated that overexpression of P2X4 purinergic receptors improves cardiac function and increases survival in mice with postinfarction heart failure.

Theoretical and Practical Significance. The cardioprotective properties of the ACE inhibitor spirapril and the binary A1 and A3 adenosine receptor agonist MRS3630 were determined, as was spirapril's ability to lower the threshold for the manifestation of early IPC. The use of MRS3630 may enhance cardioprotection during cardiac surgical procedures.

The role of NO as the principal factor supporting flow-dependent vasodilation and intravascular pressure stability under normotensive conditions was demonstrated, as was the important role of NO in mediating the effects of spirapril and propranolol in normotensive animals.

Experimental data on the effects of blocking NO synthesis in spontaneously hypertensive animals suggest the existence of another factor/mechanism that determines flow-dependent vasodilation under hypertensive conditions.

The experimentally observed slowing of myocardial remodeling under the action of P2 purinergic receptor agonists may provide a basis for more comprehensive study of the effects of this group of drugs in CHF.

Provisions Put Forward for Defense:

1. Endogenously formed nitric oxide does not participate in the pathogenesis of myocardial ischemic injury and is not necessary for the manifestation of the protective effect of early ischemic preconditioning.

2. Under impaired nitric oxide formation, marked changes occur in the pattern of flow-dependent vasodilation induced by angiotensin-converting enzyme inhibition and beta-adrenergic receptor blockade.

3. The degree of involvement of endogenously formed nitric oxide in supporting flow-dependent vasodilation differs between normotensive and hypertensive rats.

4. Stimulation of P2 purinergic receptors and overexpression of P2X4 purinergic receptors improve myocardial contractile function, slow cardiac remodeling, and increase the life span of mice with various forms of chronic heart failure.

Approval of the Study. The main provisions of the study were presented at the Scientific Conference of Young Scientists, “Current Problems in Pathophysiology” (Saint Petersburg, 2001); at the Fifth All-Russian Medical and Biological Conference of Young Researchers (Saint Petersburg, 2002); at the Third All-Russian Scientific and Practical Conference, “Arterial Hypertension among Other Cardiovascular Risk Factors” (Moscow, 2002); at the II International Scientific and Practical Conference, “Endothelial Dysfunction” (Vitebsk, 2002); at the Russian National Congress of Cardiologists, “From Research to Clinical Practice” (Saint Petersburg, 2002); at the annual “Scientific Sessions 2006” conference (Chicago, United States); at the annual “Experimental Biology 2007” conference (Washington, United States); at the annual “Experimental Biology 2008” conference (San Diego, United States); and at the 15th Annual Scientific and Practical Conference with International Participation, “Current Issues in Cardiology” (Tyumen, 2008).

Implementation of the Study Results. A total of 22 scientific publications based on the dissertation were published. Seven certificates for the official registration of computer programs and two certificates for rationalization proposals were obtained.

Volume and Structure of the Dissertation. The dissertation comprises 243 pages of typewritten text and consists of an introduction, literature review, description of the research materials and methods, results of the author's research, discussion, conclusions, and list of references. The work is illustrated by 23 tables and 53 figures. The literature index contains 440 entries (37 domestic and 403 foreign authors).