An improved reporter identifies ruxolitinib as a potent and cardioprotective CaMKII inhibitor

Abstract

Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) hyperactivity causes cardiac arrhythmias, a major source of morbidity and mortality worldwide. Despite proven benefits of CaMKII inhibition in numerous preclinical models of heart disease, translation of CaMKII antagonists into humans has been stymied by low potency, toxicity, and an enduring concern for adverse effects on cognition due to an established role of CaMKII in learning and memory. To address these challenges, we asked whether any clinically approved drugs, developed for other purposes, were potent CaMKII inhibitors. For this, we engineered an improved fluorescent reporter, CaMKAR (CaMKII activity reporter), which features superior sensitivity, kinetics, and tractability for high-throughput screening. Using this tool, we carried out a drug repurposing screen (4475 compounds in clinical use) in human cells expressing constitutively active CaMKII. This yielded five previously unrecognized CaMKII inhibitors with clinically relevant potency: ruxolitinib, baricitinib, silmitasertib, crenolanib, and abemaciclib. We found that ruxolitinib, an orally bioavailable and U.S. Food and Drug Administration-approved medication, inhibited CaMKII in cultured cardiomyocytes and in mice. Ruxolitinib abolished arrhythmogenesis in mouse and patient-derived models of CaMKII-driven arrhythmias. A 10-min pretreatment in vivo was sufficient to prevent catecholaminergic polymorphic ventricular tachycardia, a congenital source of pediatric cardiac arrest, and rescue atrial fibrillation, the most common clinical arrhythmia. At cardioprotective doses, ruxolitinib-treated mice did not show any adverse effects in established cognitive assays. Our results support further clinical investigation of ruxolitinib as a potential treatment for cardiac indications.

Fig. 1.. CaMKAR is a sensitive and specific CaMKII activity reporter.

(A) Schematic depiction of CaMKII activity reporter (CaMKAR). PAABD, phosphorylated amino acid binding domain). The phospho-substrate threonine is shown in red. Upon phosphorylation by CaMKII, the substrate is bound by the PAABD, which enables conformational reconstitution of GFP fluorescence: 488-nm emission is increased, whereas 405-nm emission is unchanged. (B) Fluorescence confocal microscopy of CaMKAR-expressing HEK293T cells treated with ionomycin (5 μM). R, ratio of 488 nm–excited (green) and 405 nm–excited intensities (gray). Scale bars, 20 μm. The third row is pseudo-colored to display fold change over baseline. (C) Summary data for the CaMKAR signal in HEK293T cells over time treated with vehicle only (black, n = 2488 to 2615 cells) or ionomycin (iono; magenta; 5 μM; n = 1733 to 2220 cells), pretreatment with the CaMKII inhibitor AS100397 (purple, 10 μM; n = 1305 to 1348 cells), or posttreatment with EGTA (teal, 5 mM; n = 1474 to 1746). No exogenous CaMKII was added. R/R₀, R normalized to mean baseline. Arrows denote administration time. (D) CaMKAR signal in HEK293T cells expressing doxycycline-inducible wild type (WT), kinase dead (KD; K43M mutation), or constitutively active (CA; T287D mutation) CaMKIIδ_C compared with vehicle-treated. N = 3 wells per condition. (E) deadCaMKAR(T6A mutant) signal over time in HEK293T cells coexpressing WT (n = mean of three wells), CA (n = mean of three wells), kinase dead CaMKIIδ_C (KD; n = mean of three wells), or empty vector (n = mean of three wells) and then stimulated with ionomycin. (F) CaMKAR signal in CaMKAR-expressing HEK293T cells cotransfected with CA CaMKI (n = 1471 cells), CaMKIV (n = 1732 cells), or CaMKIIδ (n = 1816 cells). (G) CaMKAR signal in CaMKAR-expressing HEK293T cells treated with forskolin (Fsk; 50 μM)/IBMX (100 μM; n = 1834 to 1891 cells) or ionomycin (n = 1997 to 2293). (H) CaMKAR signal in CaMKAR-expressing HEK293T cells treated with PMA (100 ng/ml; n = 696 to 737 cells) or ionomycin (5 μM; n = 767 to 916). (I) CaMKAR signal in CaMKAR-expressing HEK293T cells treated with PDBu (200 nM; n = 1706 to 1736 cells) or ionomycin (5 μM; n = 1206 to 1228). For (G to I), arrows denote administration time of either ionomycin or one of Fsk/IBMX, PMA, and PDBu. Data are shown as means ± SEM (error is not displayed whenever it is smaller than the data point). All observations are taken from more than three biological replicates. ns, P > 0.05 and ****P < 0.0001; significance was determined via two-way analysis of variance (ANOVA) and Šidák’s multiple comparisons test (D and G to I), linear regression (E), and one-way ANOVA with Dunnett’s multiple comparisons test (F).

Fig. 2.. CaMKAR-based screen identifies CaMKII inhibitors among drugs in clinical use.

(A) Schematic depiction of CaMKAR-based high-throughput screen. K562 cells were coinfected with CaMKAR- and CA CaMKIIδ^T287D–encoding lentiviruses. CaMKII expression could be induced with doxycycline. K562^{CaMKII-CaMKAR} cells were screened against the Johns Hopkins Drug Library v3.0. Primary hits were further screened using in vitro assays to eliminate false positives and narrow the list to five CaMKII inhibitors. (B) Drugs ranked according to in cellulo CaMKII inhibition in primary screen. CaMKII inhibition percentage defined by minimum-maximum normalization using means of control groups [CTRL (untreated) and AS100397; same data as fig. S4B]. A total of 118 selected hits are shown in blue. Positive control staurosporine is shown in magenta. The dashed line represents hit selection threshold (see Materials and Methods). Data shown are subsets (those scoring between 0 and 140% for visualization of hits) from the complete dataset in data file S1. (C) Hits from (B) ranked according to in vitro CaMKII inhibition as detected by CaMKAR secondary screen. CaMKII inhibition normalized against untreated control (CTRL). AS100397 was used as a positive control. Thirteen identified hits are in blue. Staurosporine is shown in magenta. Data shown are subsets from the complete dataset (see Supplementary Materials). (D) Five CaMKII inhibitory drugs and their intended targets within the human kinase homology dendrogram. Kinase families: AGC, containing PKA, PKG, and PKC families; CK1, casein kinase 1; CMGC, containing CDK, MAPK, GSK3, and CLK; STE, homologs of yeast sterile 7, sterile 11, and sterile 20; TK, tyrosine kinase; TKL, tyrosine kinase–like. (E) IC₅₀ (left) and cell viability (right) curves from 293T cells expressing CaMKAR and CaMKII^T287D and exposed to five candidate drugs and CaMKII inhibitor (AS100397, 10 mM). Measurements in (E) are done in biological triplicate; complete dataset is shown in fig. S5.

Fig. 3.. CaMKAR indicates that drugs in clinical use inhibit CaMKII in CMs.

(A) Fluorescence imaging time lapse (0, 30, and 60 s) of CaMKAR-expressing neonatal rat ventricular CMs (NRVMs) and (B) summary data during 3-Hz field pacing (n = 1065 to 1160 cells). R, ratio of 488 nm–excited and 405 nm–excited intensities. Third row of images is pseudo-colored to display fold change over baseline. R/R₀, R normalized to mean R before stimulation. Scale bars, 50 μm. (C) Effect of treatment with vehicle (n = 317 cells), AS100397 (n = 151), ruxolitinib (n = 148), crenolanib (n = 156), abemaciclib (n = 138), baricitinib (n = 136), and silmitasertib (n = 149) on pacing-induced CaMKII activity in NRVMs. CaMKII inhibition percentage defined by minimum-maximum normalization between untreated and maximally stimulated CaMKAR signal. (D) CaMKII inhibition over time in NRVMs treated with drugs adjusted to their maximum human plasma concentrations during 3-Hz pacing: ruxolitinib (1.51 μM, n = 384 cells), crenolanib (478 nM, n = 302), abemaciclib (243 nM, n = 302), baricitinib (58 nM n = 334), and silmitasertib (3.42 μM, n = 285). The arrow indicates start of pacing. (E) Summary data from (D) at 60 s after stimulation. (F) Analysis of CaMKII activity based on CaMKAR time-lapse imaging in NRVMs treated with ruxolitinib after initiation of pacing and CaMKII activation. Black arrow denotes pacing start; blue arrow denotes addition of ruxolitinib (n = 3 wells) or control vehicle (n = 3 wells). (G) IC₅₀ curves for ruxolitinib (n = 308 to 384 per data point) and DiOHF (n = 305 to 363) in NRVMs against pacing-induced CaMKII activity. (H) Cell viability after 48-hour compound incubation. All measurements were taken from more than three biological replicates. ns, P > 0.05 and ****P < 0.0001; significance determined via one-way ANOVA and Dunnett’s multiple comparisons test (C and E) and two-way ANOVA and Tukey’s multiple comparisons test (F).

Fig. 4.. Ruxolitinib inhibits CaMKII in vivo.

(A) Immunoblot and (B to C) quantitation of phospholamban (PLN; pentameric or monomer) and threonine-17–phosphorylated phospholamban in whole-heart lysates from mice treated with intraperitoneal ruxolitinib (Rux) for 10 min before isoproterenol (ISO) stimulation. P, pentameric PLN; M, monomeric PLN. Data points represent individual mice. ****P < 0.0001; significance determined via one-way ANOVA and Tukey’s multiple comparisons test.

Fig. 5.. Ruxolitinib suppresses abnormal calcium signaling in a cellular model of CPVT.

iPSCs with the dominant RYR2 mutation (p.S404R) were generated from a patient with a clinical diagnosis of CPVT (RYR2^S404R/WT) and differentiated into functional CMs (iPSC-CMs). After plating on glass-bottom dishes, RYR2^S404R/WT and WT iPSC-CMs were loaded with the fluorescent Ca²⁺ indicator (Fluo-4) and electrically paced at 1 Hz for 10 s. (A) Representative tracings of calcium transients in WT and RYR2^S404R/WT cells. The appearance of aCREs after the cessation of pacing is indicative of deranged Ca²⁺ signaling in CPVT iPSC-CMs (right). Raw data are in blue, and filtered data are in orange overlay. (B) Quantification of aCREs over 20 s in WT and RYR2^S404R/WT iPSC-CMs with preincubation of 2 μM ruxolitinib compared with vehicle alone (DMSO). (C to F) Automated analysis of Ca²⁺ transient parameters during 1-Hz pacing of iPSC-CMs for mean amplitude (C), Ca²⁺ transient duration at 50% of peak amplitude (TCa₅₀) (D), upstroke velocity (E), and downstroke velocity (F). The numbers of analyzed cells (n) are annotated on each graph and were from at least two independent differentiations and four separate culture wells. Statistics were performed by one-way Kruskal-Wallis with Dunn’s multiple comparisons test: ns, P > 0.1; *P < 0.05; **P < 0.01; ***P < 0.005; and ****P < 0.0001.

Fig. 6.. Ruxolitinib prevents arrhythmias in a murine model of CPVT.

Isolated adult murine CMs from mice with the pathogenic CPVT variant Ryr2^R176Q/WT were loaded with the calcium indicator Rhod-2 and paced for 30 s at 1 Hz. (A) Representative tracings of calcium transients in WT and Ryr2^R176Q/WT CMs treated with DMSO or ruxolitinib. (B) Quantification of aCRE frequency over 20 s for WT and Ryr2^R176Q/WT CMs after preincubation with 2 μM ruxolitinib (or DMSO). (C) Animals were treated with ruxolitinib (75 mg/kg) or vehicle by intraperitoneal injection and then subjected to programmed ventricular extrastimulus testing. Representative traces of induced ventricular arrhythmias Ryr2^R176Q/WT animals with vehicle- but not in ruxolitinib-treated mice. (D) Quantification of animals with ventricular arrhythmias (VF/VT) in WT and Ryr2^R176Q/WT animals with vehicle or ruxolitinib treatment after concurrent stimulation with isoproterenol (2 mg/kg) and epinephrine (4 mg/kg). (E) Quantification of the duration of arrhythmias induced by ventricular pacing in WT and Ryr2^R176Q/WT animals with vehicle or ruxolitinib treatment. Number of animals in each group (N): N = 13 (WT + DMSO), N = 10 (WT + ruxolitinib), N = 13 (Ryr2^R176Q/WT + DMSO), and N = 15 (Ryr2^R176Q/WT + ruxolitinib). Statistics were performed by one-way Kruskal-Wallis with Dunn’s multiple comparisons test, for continuous variables or chi-squared for discrete variables: ns, P > 0.1; *P < 0.05; **P < 0.01; ***P < 0.005; and ****P < 0.0001.

Fig. 7.. Ruxolitinib prevents and rescues AF in mice.

(A) Representative type 1 diabetic mouse tracings immediately after atrial burst pacing. DMSO-treated mice demonstrate AF (top), whereas ruxolitinib (Rux)–treated mice (75 mg/kg; single dose 10 min before, intraperitoneal) retain sinus rhythm (bottom). (B) AF inducibility percentage from mice in (A). The total number of mice analyzed per group is shown in each column: Nine of 16 vehicle-treated mice had AF versus 2 of 15 Rux-treated mice. (C) Sequential AF (AF) inducibility in OGT-transgenic mice. Mice were treated with vehicle immediately before AF induction; the same mice were then treated with ruxolitinib (75 mg/kg) before pacing for a second time. The number of mice analyzed per group is shown in each column (vehicle, five of five had AF; Rux, one of five had AF). (D) Percentage of time in AF or sinus rhythm (for 10 to 25 min after treatment administration) in CREM-IbΔC-X mice when treated with vehicle (baseline) or when treated with ruxolitinib (75 mg/kg) 24 hours later (n = 9, paired). (E) Individual trajectories of percentage of time in AF (for 10 to 25 min after treatment) in same mice as in (D) (n = 9, paired). Statistical comparisons were performed using two-tailed Fischer’s exact test (B and C), two-way ANOVA with Šidák’s multiple comparison’s test (D), and paired Student’s t test (E); *P < 0.05 and ****P < 0.0001.

Fig. 8.. Ruxolitinib does not impair short-term or spatial memory in mice.

(A) WT C57BL/6J mice were treated with a single dose (1 hour before) or multiple doses (7 days, twice daily) of ruxolitinib before behavioral testing via the NORT and the Y maze spatial recognition memory test. (B and C) Percentage of time spent with novel object. (D and E) Total distance traveled while in the testing chamber. (F and G) Percentage of time spent in novel arm of Y maze. Each data point represents a mouse (n = 7 to 10 mice for all conditions). ns, P > 0.05 and *P < 0.05; significance was determined by one-way ANOVA and Tukey’s multiple comparisons test.