Small molecule ligands for enhanced intracellular delivery of lipid nanoparticle formulations of siRNA.

Gene silencing activity of lipid nanoparticle (LNP) formulations of siRNA requires LNP surface factors promoting cellular uptake. This study aimed to identify small molecules that enhance cellular uptake of LNP siRNA systems, then use them as LNP-associated ligands to improve gene silencing potency. Screening the Canadian Chemical Biology Network molecules for effects on LNP uptake into HeLa cells found that cardiac glycosides like ouabain and strophanthidin caused the highest uptake. Cardiac glycosides stimulate endocytosis on binding to plasma membrane Na(+)/K(+) ATPase found in all mammalian cells, offering the potential to stimulate LNP uptake into various cell types. A PEG-lipid containing strophanthidin at the end of PEG (STR-PEG-lipid) was synthesized and incorporated into LNP. Compared to non-liganded systems, STR-PEG-lipid enhanced LNP uptake in various cell types. Furthermore, this enhanced uptake improved marker gene silencing in vitro. Addition of STR-PEG-lipid to LNP siRNA may have general utility for enhancing gene silencing potency. FROM THE CLINICAL EDITOR: In this study, the authors identified small molecules that enhance cellular uptake of lipid nanoparticle siRNA systems, then used them as LNP-associated ligands to improve gene silencing potency.

Relationship between cardiotonic effects and inhibition on cardiac sarcolemmal Na+,K+-ATPase of strophan-thidin at low concentrations.

AIM: To evaluate the effects of strophanthidin (Str) on cardiac contractile function and sarcolemmal Na+, K+-ATPase activities in isolated guinea-pig hearts. METHODS: Isolated guinea-pig hearts were perfused through aorta in a Langendorff mode. Heart rate (HR), left ventricular pressure (LVP), and first derivatives (+/-dp/dt(max)) of LVP were recorded by eight-channel physiological instrument. Cardiac sarcolemmal Na+, K+-ATPase activities were determined with colorimetry. RESULTS: Str 0.1 nmol/L stimulated the Na+, K+-ATPase activities (P<0.05), but had no effect on HR, LVP, and +/-dp/dt(max). Str 1 nmol/L increased +dp/dt(max) (P<0.05) and Na+, K+-ATPase activities (P<0.01). Str 10 and 100 nmol/L significantly increased both LVP (P<0.05) and +dp/dt(max) (P<0.05 or P<0.01), and had no significant effects on Na+, K+-ATPase activities. However, Str 1-100 micromol/L at first enhanced the LVP and +dp/dtmax (P<0.01), then reduced them resulting from irregular contraction, and effects of Str on Na+, K+-ATPase activities revealed a concentration-dependent inhibition (P<0.01). CONCLUSION: The positive inotropic effects and irregular contraction produced by Str at higher concentrations result from the inhibition of Na+, K+-ATPase activities, and the positive inotropic effects of Str at lower concentrations are not related to the inhibition of the Na+, K+-ATPase activities.

Effect of nitric oxide on strophanthidin-induced ventricular tachycardia.

Nitric oxide (NO) has been demonstrated to have several effects on the heart. Through the stimulation of guanylate cyclase, NO increases cyclic GMP and decreases intracellular calcium. The purpose of this study was to evaluate the effects of NO on ventricular arrhythmia induced by strophanthidin in guinea pigs and dogs. In experiment 1, after strophanthidin-induced ventricular tachycardia, guinea pigs received different doses of L-arginine (0, 25, 50, 100, 200, and 400 mg/kg; n = 10 for each dose), 200 mg/kg L-arginine combined with 100 mg/kg N(G)-nitro-L-arginine methylester (L-NAME, n = 10), or 200 mg/kg D-arginine (n = 10). In experiment 2, after strophanthidin-induced ventricular tachycardia, dogs (n = 7) received 200 mg/kg L-arginine. By 12-lead ECG, monophasic action potentials in left and right ventricles were recorded throughout the study. In experiment 1, guinea pigs which received 200 mg/kg or 400 mg/kg L-arginine had greater incidences of ventricular tachycardia termination (60 and 80%, respectively) than those which received 0, 25, 50, and 100 mg/kg L-arginine (0, 0, 20, and 30%, respectively), those which received L-arginine with L-NAME (0%), and those which received D-arginine (0%). In experiment 2, 5 (71%) of the dogs had successful termination of ventricular tachycardia. These findings suggest that L-arginine was effective in treating strophanthidin-induced ventricular tachycardia in vivo and that the underlying mechanism is through a NO pathway.

Effects of cardiac glycosides on atrial contractile dysfunction after short-term atrial fibrillation.

BACKGROUND: Despite a long history of use in the treatment of paroxysmal atrial fibrillation (AF), the efficacy of cardiac glycosides has not been established. If such drugs are beneficial in this condition, the general view is that the benefit must be related to their inotropic actions. METHODS AND RESULTS: To assess the effects of the rapid-acting cardiac glycoside, acetylstrophanthidin (AS), on AF and AF-induced right atrial (RA) "stunning," RA wall motion (with ultrasonic crystals), RA pressure, and peak first derivative of pressure (dp/dt) (with microtip transducers) were measured before and after 5 min of high-intensity rapid atrial stimulation (10 Hz; 10 mA; 1 ms) and after the cessation of poststimulation AF. Measurements were made in neurally intact and autonomically blockaded dogs both before and after the administration of AS (0.01 mg/kg IV bolus and 0.015 mg/kg/h IV infusion). AS prevented the post-AF reduction in RA peak dp/dt under neurally intact and autonomically blockaded conditions, and it prevented the post-AF increase in the RA end-systolic dimension and the decrease in the percentage of RA systolic shortening with autonomic blockade. AS was beneficial whether or not baseline inotropy was enhanced by AS. The duration of AF following atrial stimulation was the same before and after AS, but when compared to controls, AS treatment appeared to prolong AF. CONCLUSIONS: Cardiac glycosides exert a favorable effect on AF-induced RA stunning, but this action is unrelated to its effects on the duration of AF.

Decreased inotropic but relatively preserved relaxation response to cyclic adenosine monophosphate-dependent agents in myopathic human myocardium.

BACKGROUND: Increased intracellular concentrations of cyclic adenosine monophosphate (AMP) stimulate a positive inotropic and lusitropic response in healthy myocardial tissue. Heart failure results in an attenuated inotropic response to cyclic AMP-dependent agents. This study compares the inotropic versus relaxation response to cyclic AMP-dependent and cyclic AMP-independent agents in myocardium from patients with end-stage heart failure and control patients without heart failure. METHODS AND RESULTS: Fifty-four control and 59 myopathic human ventricular trabeculae, 1 mm or less in diameter were placed in physiologic saline, 2.5 mM Ca2+, and stretched to the length at which maximal isometric force developed at 30 degrees C, 0.33 Hz. Dose-response curves plotted as percentage change from baseline versus concentration of drug were determined for acetylstrophanthidin, isoproterenol, isobutylmethylxanthine, and milrinone. Acetylstrophanthidin, a cyclic AMP-independent agent, showed similar increases in peak tension relative to baseline over the entire dose range tested for both control and myopathic heart muscle; its effect on relaxation of control and failing cardiac muscle was equivalent over the dose range tested. In contrast, the inotropic actions of the cyclic AMP-dependent agents, isoproterenol and the phosphodiesterase inhibitors, were significantly decreased in myopathic muscle compared with control muscle, but effects on relaxation in the control and myopathic groups remained relatively preserved. CONCLUSIONS: A relatively preserved relaxant effect is associated with the cyclic AMP-dependent agents, despite significant diminution of their inotropic effects. Thus, in advanced heart failure, patients may continue to benefit from the lusitropic effects of the cyclic AMP-dependent agents, even when the inotropic effects of these agents are severely attenuated.

Reflex cardiovascular effects of intracoronary acetylstrophanthidin in the conscious dog.

The present experiments were designed to examine the reflex cardiovascular effects of intracoronary administration of acetylstrophanthidin in the conscious dog. Administration of 4 micrograms/kg of this agent into the left circumflex coronary artery increased left ventricular dP/dtmax but had no effect on mean arterial pressure, heart rate, renal resistance, or iliac resistance. The positive inotropic effects of acetylstrophanthidin were less under control conditions (+599 mmHg/s) than during bilateral cervical vagal cold block (+850 mmHg/s, P less than 0.05); however, interruption of vagal efferent influences (atropine) alone did not alter the contractile effects of acetylstrophanthidin. Interruption of sympathetic efferent influences on the heart with either the nicotinic ganglionic receptor antagonist, hexamethonium, or the beta 1-adrenergic receptor antagonist, metoprolol, also augmented the inotropic effects of acetylstrophanthidin to a degree similar to that observed with vagal cold block. In contrast to the effects observed with acetylstrophanthidin, the inotropic effects of intracoronary administration of calcium gluconate were not altered by vagal cold block or any other conditions examined in this study. We conclude that interruption of vagal afferents results in an augmentation of the positive inotropic actions of acetylstrophanthidin and that this augmented inotropic effect can be accounted for by interruption of cardiac vagal afferent-mediated restraint on sympathetic outflow to the heart.

Chronotropic effect of acetylstrophanthidin infusion into the canine sinus nodal artery.

Studies were performed to determine the chronotropic effect of acetylstrophanthidin during constant infusion through cannulation of the sinus nodal artery. Ten mongrel dogs weighing 13.5 to 18 kg were studied under sodium thiamylal anesthesia. Epicardial atrial and ventricular electrograms were recorded. The sinus nodal artery was cannulated and infused for 20 minutes at a rate of 2 cc/min with a solution containing acetylstrophanthidin, 0.5 microng/cc. Mean results for the group of 10 animals were determined. There was a gradual acceleration of the atrial rate of 45 beats/min after 6 to 8 minutes of infusion. The peak atrial rate of 175 beats/min was achieved by 10 to 12 minutes. This tachycardia persisted for 2 to 4 minutes without atrioventricular block or premature beats. By 12 to 14 minutes, there was a gradual slowing of atrial rate followed by bradycardia, sinus pauses and atrial arrest. Sinus nodal arterial infusion of acetylstrophanthidin produces an initial positive chronotropic effect and, if maintained, a depression of atrial rate and, terminally, atrial arrest. The gradual time course of development and decline of the tachycardia suggests that the "paroxysmal" atrial tachycardia caused by digitalis excess is the result of enhanced pacemaker automatically rather than reentry, and thus is not truly paroxysmal.

Effect of a digitalis drug on ventricular premature beats.

To determine the efficacy of digitalis drugs in suppressing ventricular ectopic activity, 142 patients with frequent (greater than 1 per minute) ventricular premature beats underwent acetyl strophanthidin tolerance testing. In 65 patients (46 per cent), frequency and grade were reduced during testing. In 37 (26 per cent), the ectopic activity remained unaltered; frequency increased during testing in the remaining 40 patients (28 per cent). In the group with a suppressive effect, ventricular premature beats decreased by 82 per cent, with complete abolition of arrhythmias in 46 per cent. The three groups were not distinguishable clinically by either the type or the extent of heart disease. The antiarrhythmic action of acetyl strophanthidin did not appear to depend upon its positive inotropic action. In some patients it appears to be due to an indirect reduction of Purkinje-fiber automaticity resulting from enhanced vagus-nerve activity that thereby lessens adrenergic tone on the heart.

Dissociation of the inotropic effect of digitalis from its effect on atrioventricular conduction.

The relation between sequential changes in left ventricular contractility and atrioventricular (A-V) nodal conduction and refractoriness was assessed in open chest dogs during intravenous administration of acetylstrophanthidin (5 mug/kg) at 5 minute intervals until toxic arrhythmias developed. At each time interval, high fidelity left ventricular pressure, its electronic derivative (dP/dt) and a His bundle electrogram were simultaneously recorded and the A-V nodal refractory period was measured by graded trains of stimuli. Animals were studied with an intact autonomic state (Group I), with pharmacologic blockade of both the beta adrenergic and parasympathetic system (Group II) and with parasympathetic blockade (Group III). Whereas contractility increased in response to small doses of digitalis, displaying a linear dose-response relation independent of autonomic tone, A-V nodal transmission indexes responded minimally to less than 50 percent of the toxic dose of digitalis, and the response was dependent upon autonomic tone. These results indicate a dissociation between the effects of digitalis on contractility and A-V transmission in that the major drug action on the ventricular contractile mechanism is a direct, linear one in contrast to the nonlinear response of A-V nodal transmission, which is predominantly mediated through the autonomic system. Clinically, these observations imply that the optimal dose and serum level of digitalis required to treat congestive heart failure may differ significantly from those required to treat supraventricular tachycardias, the therapeutic response of the latter being largely determined by the underlying autonomic tone.

Ischemic heart failure: sustained inotropic response to small doses of I-epinephrine without toxicity.

As a prelude to a study of severe ischemic heart failure, the therapeutic response of the ischemic ventricle to epinephrine and acetylstrophanthidin in nontoxic doses was determined in 24 intact anesthetized dogs undergoing a first episode of acute regional ischemia. A thrombotic obstruction was produced in the left ventricular dysfunction. The elevation of end-diastolic pressure and reduced stroke volume in control dogs were not significantly altered by administration of strophanthidin. Epinephrine (0.05 mug/kg per min) elicited a significant reduction in end-diastolic pressure and increase in stroke volume. The latter was not attended by an increased incidence of ventricular fibrillation, whereas fibrillation occurred in half of the group given strophantihidin. Thus, the catecholamine was selected to study pump failure. Severe ischemic heart failure was assessed in two groups with scar from previous infarction for up to 4 hours. By 60 minutes of ischemia the increase in end-diastolic pressure and volume and decrease in stroke volume and ejection fraction were comparable in both groups. Thereafter, alternate animals received small doses of epinephrine (0.05 to 0.15 mug/kg per min) with graded increments at 60 minute intervals to counter tachyphylaxis and findings were compared with those in control dogs. Over the subsequent 3 hours, there was progressive deterioration of left anterior descending coronary artery, affecting ventricular function in the untreated group with an increase in end-diastolic pressure from 10 plus or minus 1 to 33 plus or minus 2.4 mm Hg. End-diastolic volume increased by 63 percent; stroke volume and ejection fraction decreased by 48 and 66 percent, respectively. The infusion of epinephrine was attended by a significantly lower end-diastolic pressure of 20 plus or minus 2.5 mm Hg, whereas end-diastolic volume, stroke volume and ejection fraction were restored to control levels after 4 hours of ischemia. Mortality in the untreated group was 62 percent by 4 hours; all seven animals in the treated group survived.