Orai1 channel inhibition preserves left ventricular systolic function and normal Ca2+ handling after pressure overload

Abstract

Sources of Funding, see page 214 BACKGROUND: Orai1 is a critical ion channel subunit, best recognized as a mediator of store-operated Ca 2+ entry (SOCE) in nonexcitable cells. SOCE has recently emerged as a key contributor of cardiac hypertrophy and heart failure but the relevance of Orai1 is still unclear. METHODS: To test the role of these Orai1 channels in the cardiac pathophysiology, a transgenic mouse was generated with cardiomyocytespecific expression of an ion pore-disruptive Orai1 R91W mutant (C-dnO1). Synthetic chemistry and channel screening strategies were used to develop 4-(2,5-dimethoxyphenyl)-N-[(pyridin-4-yl)methyl]aniline (hereafter referred to as JPIII), a small-molecule Orai1 channel inhibitor suitable for in vivo delivery. RESULTS: Adult mice subjected to transverse aortic constriction (TAC) developed cardiac hypertrophy and reduced ventricular function associated with increased Orai1 expression and Orai1-dependent SOCE (assessed by Mn 2+ influx). C-dnO1 mice displayed normal cardiac electromechanical function and cellular excitation-contraction coupling despite reduced Orai1-dependent SOCE. Five weeks after TAC, C-dnO1 mice were protected from systolic dysfunction (assessed by preserved left ventricular fractional shortening and ejection fraction) even if increased cardiac mass and prohypertrophic markers induction were observed. This is correlated with a protection from TAC-induced cellular Ca 2+ signaling alterations (increased SOCE, decreased [Ca 2+ ] i transients amplitude and decay rate, lower SR Ca 2+ load and depressed cellular contractility) and SERCA2a downregulation in ventricular cardiomyocytes from C-dnO1 mice, associated with blunted Pyk2 signaling. There was also less fibrosis in heart sections from C-dnO1 mice after TAC. Moreover, 3 weeks treatment with JPIII following 5 weeks of TAC confirmed the translational relevance of an Orai1 inhibition strategy during hypertrophic insult. CONCLUSIONS: The findings suggest a key role of cardiac Orai1 channels and the potential for Orai1 channel inhibitors as inotropic therapies for maintaining contractility reserve after hypertrophic stress.

Orai1 Inhibitors as Potential Treatments for Pulmonary Arterial Hypertension

“…Additionally, we used another selective Orai1 blocker, JPIII, based on the scaffold of the potent and selective blocker, Synta66. 10 JPIII did not modify the function of Orai3, TRPC5, TRPC6, TRPC4/C1, or TRPC5/C1 in HEK293 cells. 10 JPIII (0.5 µmol/L) recapitulates results obtained in in vitro experiments with siOrai1 or after 1 µmol/L BTP2 application.…”

“…10 JPIII did not modify the function of Orai3, TRPC5, TRPC6, TRPC4/C1, or TRPC5/C1 in HEK293 cells. 10 JPIII (0.5 µmol/L) recapitulates results obtained in in vitro experiments with siOrai1 or after 1 µmol/L BTP2 application. Finally, we obtained similar in vivo results with 5J4 application in monocrotaline rats, for which no side effects have been reported on other Ca 2+ channels until now 17 to those obtained with BTP2 and JPIII application.…”

“…Since Orai1 may also contribute to the left ventricular (LV) remodeling, 10 we investigated the putative role of Orai1 on RV remodeling. We found unchanged RV Orai1 mRNA levels in CH rats compared with control rats (Figure 6J) and similar localization of Orai1 (Figure 6K).…”

Chronic intermittent hypoxia accelerates cardiac dysfunction and cardiac remodeling during cardiac pressure overload in mice and can be alleviated by PHD3 overexpression

“…Cardiac dysfunction caused by TAC still plays an important role. TAC results in downregulation of SERCA2a expression in the heart via multiple pathways (32,33). This is faster than the HIF-1α inhibition of SERCA2a.…”

CD38‐NADase is a new major contributor to Duchenne muscular dystrophic phenotype

“…Ca 2+ handling was evaluated using a classic protocol of cell electrical stimulation at 2 Hz. Each electrical stimulation gives rise to a cytosolic Ca 2+ elevation called Ca 2+ transient, which is visualized by confocal microscopy (Leica SP5) (“line‐scanning mode”) in cardiomyocytes loaded with the calcium‐sensitive dye Fluo‐3 AM excited at 500 nm with a white light laser and emission collected at >510 nm as previously described (Bartoli et al , 2020). The cytosolic Ca 2+ variation was normalized by dividing the peak fluorescence intensity (F) by the average resting fluorescence intensity (F0) after background subtraction.…”