Background: The major membrane currents responsible for sinoatrial (SA) rhythm generation are generally studied in isolated cardiac cells using electrophysiological tools. Such studies are resource and labor-intensive. Materials and Methods: Here, we have studied four major currents in isolated rat heart preparations, perfused in Langendorff mode, and demonstrate that this is a good preparation for such studies. Heart rates of isolated perfused rat hearts were recorded using surface electrocardiogram before and after perfusion with drugs and solutions that affect the four major currents responsible for SA rhythm generation. Results: The rates of whole isolated hearts beating with SA rhythm decreased with cesium and decreased by about half with ivabradine, both blockers of the funny current (I f ). Importantly, the rhythm was not abolished even with a high dose of ivabradine at which total blockade of I f is expected. The rate was not affected by nickel, a blocker of T-type calcium current. The SA rhythm was abolished by the reduction or removal of sodium from the perfusate (interventions that inhibit the calcium-extrusive mode of the sodium-calcium exchanger) or by nifedipine, the L-type calcium channel blocker. Discussion: The inferences made based on these observations are (a) I f contributes significantly to pacemaking, (b) I CaT does not play a role and (c) I NCX and I CaL are obligatory rhythm-generating currents in the SA node. Cyclical calcium release from SR during diastole (the calcium clock), responsible for driving I NCX in its forward mode is probably a phenomenon independent of membrane events, as total I f blockade did not abolish rhythm generation. These results corroborate with published literature where most studies were done on single cells.

The major membrane currents responsible for sinoatrial and idioventricular rhythm-generation were studied in isolated rat heart preparations, perfused in Langendorff mode. The rates of whole isolated hearts beating with sinoatrial rhythm decreased with cesium and ivabradine, both blockers of the funny current, and were not affected by nickel, at a dose which blocks T-type calcium current. The sinoatrial rhythm was completely abolished by reduction or removal of sodium from the perfusate (interventions that inhibit calcium-extrusive mode of the sodium/calcium exchanger), or by nifedipine, an L-type calcium channel blocker. Idioventricular rhythm, however, was arrested only by reduction of sodium in the perfusate. Ivabradine reduced the idioventricular rate, nickel did not cause any change, while nifedipine in some cases increased it. The inferences made based on these observations are that INCX and ICaL are obligatory rhythm-generating currents in the sinoatrial node, while INCX is the only obligatory mechanism for an idioventricular rhythm. The funny current is not an obligatory requirement for sinoatrial as well as idioventricular rhythm-generation. However, it enhances the frequency of LCRs. Our results in the isolated whole heart are in corroboration with results from isolated cells.

Cleistanthus collinus leaf extracts are consumed for suicidal purposes in southern India. The boiled decoction is known to be more toxic than the fresh leaf juice. Although several compounds have been isolated and their toxicity tested, controversy remains as to which compounds are responsible for the high level of toxicity of C. collinus. We report herein that cleistanthoside A is the major toxin in the boiled aqueous extract of fresh leaves and causes death in rats in small doses. The toxicity of the boiled extract prepared in the manner described can be attributed entirely to cleistanthoside A. Cleistanthin A could also be isolated from the boiled extract, albeit in trace amounts. As hypotension not responding to vasoconstrictors is the cause of death in patients who have consumed the boiled extract, effects of cleistanthoside A on the determinants of blood pressure, namely, force of cardiac contraction and vascular resistance, were tested in isolated organ experiments. Cleistanthoside A has a direct vasoconstrictor effect; however, it inhibits ventricular contractility. Therefore, the notion that the shock in C. collinus poisoning is of vascular origin must be considered carefully, and the possibility of cardiogenic shock must be studied. We present the crystal structure of cleistanthin A and show the potency of fast NMR methods (NOAH4-BSCN-NUS) in the full spectral assignment of cleistanthoside A as a real-world sample of a natural product. We also compare the results of the NOAH4-BSCN-NUS NMR experiments with conventional NMR methods.