Characterization of the molecular mechanisms of imidazo[1,2-a][1,8]naphthyridine derivatives in regulating ions transport and cell death in cultured cardiomyocytes

Despite considerable therapeutic advances, congestive heart failure (CHF) remains a medical and socioeconomic problem. CHF is usually treated with the plant-derived digitalis cardiac glycosides such as digoxin, which increase the force of contractions of failing cardiomyocytes and reduce cardiac conduction rate. However, the usage of digoxin in CHF has lessened considerably due to its narrow therapeutic window and high drug toxicity. This leads to a need for new drugs, particularly positive inotropic agents, which would not only improve the quality of life but also reduce mortality in CHF patients. So far, there has been a lack of desirable inotropic agent. With this aim in mind, an alternative drug namely 8-(4-methoxyphenyl)-2,4-dimethyl-9-((4-methylpiperidin-1-yl)methyl)imidazo[1,2-a][1,8]naphthyridine, abbreviated as compound [1], was chemically synthesized. Previous reports indicated that compound [1] has two significant advantages for CHF treatment. Firstly, it increases muscular contraction and decreases heart rate in anaesthetized rats. The fall in heart rate is desirable in the case of CHF because it allows the heart to function more efficiently. Secondly, it has a relaxant effect on the vascular smooth muscles of the aortic ring causing a drop in the systemic blood pressure, which is an added advantage in cardiovascular treatment. However, the molecular mechanism that underlies these effects remain unclear and the toxicity of compound [1] especially its effects on the regulation of intracellular calcium concentration [Ca2+]i homeostasis and cell death in cultured cardiomyocytes have yet to be evaluated. Therefore, both of these aspects were investigated in this study. In addition, to discover more biologically active heterocyclic compounds, new imidazo[1,2-a][1,8]naphthyridine derivative was synthesized. This compound was named GKL. The structure of GKL was identified from 1H and 13C NMR spectra and molecular weight was confirmed using the mass spectrum generated by LC-MS (NIST library). The potential of both compound [1] and GKL as positive inotropic agents were assessed via comparative analyses to digoxin using a standard in vitro experimental model, H9c2(2-1) cells, with a significant advantage of being an animal-free alternative. It was found that the mechanism of action of compound [1] and GKL might be through specific activation of L-type voltage calcium channel via G protein (β1-adrenoceptor) which was supported by the increased in cAMP (3′-5′-cyclic adenosine monophosphate) concentrations. Digoxin, besides inhibiting Na+, K+-ATPase, might activate ryanodine receptors in particular to exert positive inotropic effect. Although myocardial oxygen consumptions were increased in compound [1]- and GKL-treated cells, which reduces cardiac efficiency, this consumption did not differ significantly to that of the control. As toxicity evaluation is an important step in the early stages of drug discovery, toxicity of compound [1] and GKL was examined. Compound [1] and GKL were found to induce cell death in a time-dependent manner. The cells appeared to undergo apoptotic cell death and confirmed by Western blot analysis where compound [1] and GKL regulated both extrinsic and intrinsic apoptotic signalling cascades. The most important finding was that GKL induced an influx of [Ca2+]i with reduced toxicity in mitochondria as compared to digoxin and compound [1], suggesting its potential application in targeting CHF. In summary, the positive inotropic action of compound [1] and GKL can potentially give hope to the therapeutic promise of medicine in the field of cardiovascular disease.