Role of nitric oxide in heart function: A transgenic mouse model with overexpression of nitric oxide synthase in the heart

Nitric oxide (NO) plays a pivotal role in human health and disease, particularly in the area of cardiovascular regulation. The importance of NO as a vasodilator, and inhibitor of platelet aggregation, endothelial leukocyte adhesion, and smooth muscle cell proliferation, is supported by the association of diseases such as hypertension, hypercholesterolemia and heart failure with disruption of NO production or action. However, little is understood about the role of this important mediator in the heart. NO is synthesised by the enzyme NO synthase (NOS), of which there are three isoforms: neuronal (nNOS), endothelial (eNOS), and inducible (iNOS). In the heart, eNOS is present not only in all forms of the cardiac endothelium, but also in cardiomyocytes, which can in addition express iNOS during immune or inflammatory reactions. The heart is also innervated by nitrergic neurons containing nNOS. Evidence concerning the role of NO in the heart is contradictory. NO mediates the inotropic responses of rat ventricular cells to adrenergic agonists and the chronotropic effects of beta-adrenergic and muscarinic cholinergic agents in neonatal rat cells. In isolated papillary muscle, NO induced a concentration-dependent biphasic contractile response (positive inotropic at low and negative inotropic at high concentrations). The effect of NO on ischemia-reperfusion injury is unclear, since both inhibition and stimulation of NO production have been shown to be protective. Roles in the regulation of heart rate and the modulation of cardiac ion conductances have also been suggested. In order to clarify the role of NO in heart function and at the same time avoid the problems associated with NO donors and NOS inhibitors, a transgenic mouse model which overexpresses human eNOS in cardiomyocytes (under the control of the murine myosin heavy chain gene promoter) has been generated. These mice, as well as mice expressing eNOS exclusively in cardiornyocytes (which will be obtained by crossing eNOS knockout mice with the transgenic mice), will be characterised in terms of the (subcellular-) location and amount of transgene protein expression as well as the histopathology of the heart and other tissues. Hemodynamic parameters such as blood pressure and heart rate will be determined, as well as the metabolic, electrical and mechanical function of isolated perfused hearts under both normoxic and ischemic conditions. Metabolic parameters to be measured will include NO, cyclic GMP, and endothelin. Finally, the basic properties of cardiac membrane conductances as well as the effects of NO on the function of cardiac L-type Ca2+ channels will be examined using single channel patchclamp techniques on isolated transgenic cardiomyocytes. This transgenic mouse model should provide answers to the continuing debate on the beneficial versus deleterious functions of NO in the heart, particularly under ischemic conditions, as well as making a major contribution to knowledge about the role of NO in cardiac Ca2+ channel function.

Nitric oxide (NO) is a wide-spread signalling molecule that affects a variety of biological processes including regulation of blood pressure, platelet aggregation, neurotransmission in the brain, and immune response against bacterial pathogens. Besides these important physiological functions, NO also may have deleterious effects, mostly when the molecule is being overproduced which may occur in the course of inflammatory diseases and cardiac pathologies. Therefore, both supplementation of NO using NO donor drugs, as well as approaches to limit NO formation by inhibiting NO synthase, the enzyme responsible for cellular NO synthesis, have been advocated as potential therapeutic strategies. The primary aim of this project was to better understand the role of NO in heart function under physiological and pathological conditions. In view of the contradictory results obtained with previous approaches, we chose a wholly novel experimental model: we generated transgenic mice which overexpress human endothelial NO synthase (the physiological myocardial NO synthase isoform) solely in cardiomyocytes, whereas all other cells would have normal enzyme and thus NO levels. This approach allows the dissection of specific cardiac functions of NO, without compromizing NO-mediated effects in other organs which is frequently the case in gene knockout approaches. Heart function was studied in normal oxygen milieu and in simulated oxygen deprivation (ischaemia/reperfusion condition) which is of particular pathophysiological relevance. A particular feature of the present project was the generation of mice with 3 different levels of NO synthase protein overexpression, which would allow graded effects on heart function, similar to different doses of an exogenous NO-donor, but without the disadavantages of the latter. Consequently, the transgenic lines synthesized some 20-, 40- or 60-fold more NO in their hearts than normal control (nontransgenic) littermates. This level of overexpression of an important functional cellular protein is quite unique and the basis for further studies regarding other (patho)physiological aspects of cardiac NO function, for instance with respect to the cardiac complications of diabetes and heart failure. A salient finding of the project was that high levels of NO depress basal heart contraction, whereas normal levels of NO re-enforce pump function. The depression by NO is a protective mechanism, largely mediated by a reduced responsiveness of myofilaments to cellular calcium, and particularly important in states of oxygen deprivation. On the other hand, the study also provided a solid argument against the importance of NO as a modulator of neurohormonal (vagal) control of myocardial function. In the course of follow-up projects we will attempt to clarify the role of NO in cardiac complications of several diseases, including diabetes mellitus and an experimental form of myocarditis.