New N-aryl-N-alkyl-thiophene-2-carboxamide compound enhances intracellular Ca2+ dynamics by increasing SERCA2a Ca2+ pumping.

The type 2a sarco/endoplasmic reticulum Ca2+-ATPase (SERCA2a) plays a central role in the intracellular Ca2+ homeostasis of cardiac myocytes, pumping Ca2+ from the cytoplasm into the sarcoplasmic reticulum (SR) lumen to maintain relaxation (diastole) and prepare for contraction (systole). Diminished SERCA2a function has been reported in several pathological conditions, including heart failure. Therefore, development of new drugs that improve SERCA2a Ca2+ transport is of great clinical significance. In this study, we characterized the effect of a recently identified N-aryl-N-alkyl-thiophene-2-carboxamide (or compound 1) on SERCA2a Ca2+-ATPase and Ca2+ transport activities in cardiac SR vesicles, and on Ca2+ regulation in a HEK293 cell expression system and in mouse ventricular myocytes. We found that compound 1 enhances SERCA2a Ca2+-ATPase and Ca2+ transport in SR vesicles. Fluorescence lifetime measurements of fluorescence resonance energy transfer between SERCA2a and phospholamban indicated that compound 1 interacts with the SERCA-phospholamban complex. Measurement of endoplasmic reticulum Ca2+ dynamics in HEK293 cells expressing human SERCA2a showed that compound 1 increases endoplasmic reticulum Ca2+ load by enhancing SERCA2a-mediated Ca2+ transport. Analysis of cytosolic Ca2+ dynamics in mouse ventricular myocytes revealed that compound 1 increases the action potential-induced Ca2+ transients and SR Ca2+ load, with negligible effects on L-type Ca2+ channels and Na+/Ca2+ exchanger. However, during adrenergic receptor activation, compound 1 did not further increase Ca2+ transients and SR Ca2+ load, but it decreased the propensity toward Ca2+ waves. Suggestive of concurrent desirable effects of compound 1 on RyR2, [3H]-ryanodine binding to cardiac SR vesicles shows a small decrease in nM Ca2+ and a small increase in µM Ca2+. Accordingly, compound 1 slightly decreased Ca2+ sparks in permeabilized myocytes. Thus, this novel compound shows promising characteristics to improve intracellular Ca2+ dynamics in cardiomyocytes that exhibit reduced SERCA2a Ca2+ uptake, as found in failing hearts.

Cardiac ryanodine receptor N-terminal region biosensors identify novel inhibitors via FRET-based high-throughput screening.

The N-terminal region (NTR) of ryanodine receptor (RyR) channels is critical for the regulation of Ca2+ release during excitation-contraction (EC) coupling in muscle. The NTR hosts numerous mutations linked to skeletal (RyR1) and cardiac (RyR2) myopathies, highlighting its potential as a therapeutic target. Here, we constructed two biosensors by labeling the mouse RyR2 NTR at domains A, B, and C with FRET pairs. Using fluorescence lifetime (FLT) detection of intramolecular FRET signal, we developed high-throughput screening (HTS) assays with these biosensors to identify small-molecule RyR modulators. We then screened a small validation library and identified several hits. Hits with saturable FRET dose-response profiles and previously unreported effects on RyR were further tested using [3H]ryanodine binding to isolated sarcoplasmic reticulum vesicles to determine effects on intact RyR opening in its natural membrane. We identified three novel inhibitors of both RyR1 and RyR2 and two RyR1-selective inhibitors effective at nanomolar Ca2+. Two of these hits activated RyR1 only at micromolar Ca2+, highlighting them as potential enhancers of excitation-contraction coupling. To determine whether such hits can inhibit RyR leak in muscle, we further focused on one, an FDA-approved natural antibiotic, fusidic acid (FA). In skinned skeletal myofibers and permeabilized cardiomyocytes, FA inhibited RyR leak with no detrimental effect on skeletal myofiber excitation-contraction coupling. However, in intact cardiomyocytes, FA induced arrhythmogenic Ca2+ transients, a cautionary observation for a compound with an otherwise solid safety record. These results indicate that HTS campaigns using the NTR biosensor can identify compounds with therapeutic potential.