A piperidinyl amide compound enhances Ca²⁺ signaling in cardiomyocytes by increasing activity of Ca²⁺ pump

Abstract

In adult cardiomyocytes, the type 2a sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA2a) plays a vital role in intracellular Ca²⁺ regulation. Reduced SERCA2a function has been associated with decreased myocardial contraction and cardiac output in several heart diseases. Consequently, increasing SERCA2a activity is a high-priority target for treating cardiac pathologies associated with abnormal Ca²⁺ homeostasis. In our previous SERCA ATPase-based screening study, we identified several small molecules as potential activators of SERCA2a function, including Compound 9, a piperidinyl amide. With porcine cardiac sarcoplasmic reticulum (SR) preparations, we confirmed activation of both SERCA2a ATPase and Ca²⁺-uptake activities. In the current study, we analyzed the effect of Compound 9 on SERCA2a activity on intracellular Ca²⁺ dynamics in ventricular myocytes. Using FRET with human SERCA2a overexpressed in mammalian cells, we confirm that Compound 9 binds and alters SERCA structural dynamics independent of peptide regulators, including phospholamban (PLB). Confocal microscopy and in-cell Ca²⁺ imaging revealed that Compound 9 enhanced Ca²⁺ dynamics in mouse ventricular myocytes. Compound 9 (10 μM) increased the action potential-induced Ca²⁺ transients by 65% and SR Ca²⁺ load by 29%. Moreover, Compound 9 increased Ca²⁺ dynamics during adrenergic receptor stimulation and in PLB knockout cardiomyocytes, suggesting the stimulatory effect of Compound 9 is PLB independent. Overall, Compound 9 displays characteristics that can be beneficial to enhance cardiac intracellular Ca²⁺ dynamics by increasing SERCA2a function.

Why it matters

In the heart, SERCA2a Ca²⁺ pump is a central element in the intracellular Ca²⁺ signaling of myocytes. The pump plays an essential role in maintaining sarcoplasmic reticulum (SR) Ca²⁺ load by pumping Ca²⁺ from the cytosol into the SR. In this study, we characterized the effect of a recently identified piperidinyl amide compound, Compound 9, on SERCA2a function. FRET analysis revealed that Compound 9 binds and alters SERCA structural dynamics. Compound 9 enhances SR Ca²⁺ load and Ca²⁺ transients in cardiomyocytes in unstressed conditions and during adrenergic receptor stimulation. Thus, this novel compound can be used to improve intracellular Ca²⁺ dynamics in cardiac diseases associated with reduced SERCA2a function.

Introduction

The sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA) is a central component of Ca²⁺ signaling virtually in all eukaryotic cells (1). The pump is responsible for the active transport of Ca²⁺ from the cytosol into the endoplasmic reticulum (ER) to maintain low cytosolic and high ER Ca²⁺ concentration ([Ca²⁺]). This steep ER-cytosol Ca²⁺ gradient plays a key role in the robust Ca²⁺ release during activation of many cellular processes, including muscle contraction and neurotransmission (2). The pump function is tightly regulated by a variety of different mechanisms, including interaction with small peptides such as phospholamban (PLB), sarcolipin, and dwarf open reading frame (3,4,5). We have recently shown that SERCA can be also regulated by dimerization (6) and Ca²⁺ within the ER lumen (7). Due to its central role in Ca²⁺ signaling, defects in SERCA function cause significant health problems. For example, mutations in the skeletal type 1a SERCA isoform cause Brody myopathy, a disease characterized by impaired muscle relaxation (8). Alterations in the cardiac type 2a SERCA (SERCA2a) regulation or expression contribute to cardiomyopathies and heart failure (9,10,11). Mutations of the non-muscle-type 2b SERCA lead to Darier disease, an autosomal-dominant inherited disorder of skin cells (12). Therefore, it is not surprising that the Ca²⁺ pump has attracted attention as a promising target for therapeutic approaches for various diseases (13,14).

Through FRET and ATPase activity-based high-throughput screening, we have identified several compounds that can regulate the cardiac SERCA2a function (15,16,17,18). Three of the compounds that increased SERCA2a-dependent ATPase and Ca²⁺ uptake measured in porcine cardiac SR vesicle preparations (15,16,17) were tested and shown to increase ER Ca²⁺ load and cytosolic Ca²⁺ dynamics in HEK293 cells expressing human SERCA2a and in mouse ventricular myocytes (17,18). In our ATPase-based screening of SERCA in skeletal SR membranes (SERCA1a isoform), we also identified three piperidinyl amide compounds that increased SERCA2a ATPase and Ca²⁺ uptake activity (16). In the present study, we used FRET and in-cell Ca²⁺ imaging to characterize effects of a representative compound (Compound 9) for the piperidinyl amide family of SERCA activators on SERCA2a function and intracellular Ca²⁺ regulation in ventricular myocytes. The results show that Compound 9 can enhance intracellular Ca²⁺ dynamics in ventricular myocytes by increasing SERCA2a-mediated Ca²⁺ transport.

Materials and methods

FRET measurements in HEK293 cells

Using a previously reported fluorescence lifetime (FLT) plate reader and FRET biosensors with mMaroon1 at the N-terminus and mCyRFP1 inserted at human SERCA2a residue 509 (19) and mMaroon1 inserted at the N-terminus of monomeric PLB (AFA mutant), we acquired the fluorescence lifetime FRET response to a range of [Compound 9]. Stable HEK293 cell lines, overexpressing either a donor-acceptor or donor-only biosensor, were cultured in phenol red-free DMEM, supplemented with 2 mM glutaMAX and 10% fetal bovine serum, and grown at 37°C with 5% CO₂. For the SERCA-PLB FRET assay, donor-only expressing cells expressing mCyRFP1-SERCA2a were seeded at 70,000-80,000/cm² the afternoon before transfection 20 hours later with 3.6 or 7.2 μg of mMaroon1-PLB DNA in pTT22 vector using Lipofectamine 3000 (Thermo Fisher Scientific). After 48 h, cells were harvested and centrifuged at 300 × g for 5 min. Cell pellets were resuspended and washed in PBS three times before being passed through a 70-μm cell strainer. Cell concentration and viability (>85%) were evaluated by a Countess automated cell counter (Invitrogen) using the Trypan Blue method. Compound 9 in DMSO or DMSO only was preloaded in 1536-well plates using a FlexDrop IQ (Revvity). Over the compounds, 5 μL of cells at a density of 2.0 × 10⁶ cells/mL was dispensed into each well using a Multidrop Combi (Thermo Fisher). Plates were sealed, incubated at room temperature for 20 min, and FLT measurements were recorded using a high-throughput FLT plate reader (Fluorescence Innovations) with 532-nm excitation and 586/20-nm emission filter.

Ventricular myocyte isolation

All animal experiments were carried out in accordance with the National Institutes of Health guide for the care and use of laboratory animals (20). C57Bl6/J mice, (seven of male and six of female animals, Jackson Laboratories) and PLB knockout mice (21) (four of male and three of female animals) were housed according to approved IACUC guidelines. Mice aged between 2 and 5 months were anesthetized using isoflurane (3%). After thoracotomy, hearts were quickly excised and mounted on a Langendorff apparatus for retrograde perfusion with solution containing collagenase, Liberase H. The ventricular myocytes were isolated as previously described (22). In brief, the left ventricle was excised from the heart, placed in stop buffer containing BSA 1 mg/mL, and cut into several pieces. After gentle breakdown of the muscle to single cells, myocytes (∼0.1 mL) were pelleted by gravity and resuspended in low-Ca²⁺ Tyrode buffer (in mM: NaCl 140; KCl 4; CaCl₂ 0.1; MgCl₂ 1; glucose 10; HEPES 10 (pH 7.4)). [Ca²⁺] was gradually adjusted to 1 mM. Isolated cardiomyocytes were stored at room temperature (20°C). All chemicals and reagents were purchased from Sigma-Aldrich (St Louis, MO, USA).

Confocal microscopy and [Ca²⁺]_i measurements

Changes in the cytosolic [Ca²⁺] ([Ca²⁺]_i) and the luminal ER [Ca²⁺] ([Ca²⁺]_ER) were measured with laser-scanning confocal microscopy (Radiance 2000 MP, Bio-Rad, UK) equipped with a ×40 oil-immersion objective lens (N.A. = 1.3). To record [Ca²⁺]_i, we loaded cardiomyocytes with the high-affinity Ca²⁺ indicator Fluo-4 AM (Molecular Probes/Invitrogen, Carlsbad, CA, USA) as described previously (23,24). To load the cytosol with Fluo-4 AM, ventricular myocytes were incubated at room temperature with 10 μM Fluo-4 AM for 15 min in Tyrode solution (in mM: NaCl 140; KCl 4; CaCl₂; MgCl₂; glucose 10; HEPES 10 (pH 7.4)), followed by a 20-min wash. Fluo-4 AM was excited with the 488-nm line of an argon laser, and the emission signal was collected at wavelengths above 515 nm. Changes in [Ca²⁺]_i were expressed as changes in F/F₀, where F₀ is the Fluo-4 signal at the resting condition before electrical field stimulation. [Ca²⁺]_i transients were evoked by electrical field stimulation (2-ms supra-threshold voltage pulses applied at a frequency of 0.5 Hz). Compound 9 was purchased from ChemBridge (San Diego, CA). Stock solutions (10 mM) were made in DMSO, and the compounds were used in a final concentration of 10 μM in all experiments described here.

Statistics

Data are presented as mean ± standard deviation of the mean (SD) of numbers (n) of measurements. Statistical significance was determined by the Student’s t-test when two groups were compared. Unpaired two-way Student’s t-test was used to determine the significance between multiple groups for FRET experiments (Fig. 1). One-way ANOVA followed by a Tukey post hoc test was used to determine the significance between two groups for Ca²⁺ imaging experiments (Figs. 2, 3, and 4). p < 0.05 was considered statistically significant. Statistical analysis and graphical representation of averaged data were done with the OriginPro7.5 or Origin 2015 software (OriginLab, USA).

Figure 1.

Compound 9 chemical structure and binding to human SERCA2a using FRET. (A) Chemical structure of Compound 9. (B) Fluorescence lifetime (FLT) response of the human SERCA2a mCyRFP1-mMaroon FRET biosensor to a range of [Compound 9]. Samples were obtained from a stable HEK293 cell line expressing the FRET biosensor. Null controls containing the corresponding volume of DMSO were read at the same time. Data are represented as means ± SD (n = 3). ∗p < 0.05 versus control, using unpaired, two-way Student’s t-test.

Figure 2.

Effect of Compound 9 on Ca²⁺ signaling in mouse ventricular myocytes. (A) F/F₀ profiles of cytosolic Ca²⁺ during AP-induced and caffeine-induced Ca²⁺ transients (i.e., SR Ca²⁺ load) in control conditions and in the presence of Compound 9 (10 μM). The recordings were made from wild-type (WT) ventricular myocytes. (B) The average effects of Compound 9 (n = 12 myocytes) on AP-induced Ca²⁺ transient amplitude, SR Ca²⁺ load LTCC-induced Ca²⁺ transient amplitude, and the fractional release (FR). ∗p < 0.05 versus control.

Figure 3.

Effect of Compound 9 on Ca²⁺ signaling during adrenergic receptor activation. (A) F/F₀ profiles of cytosolic Ca²⁺ during AP-induced and caffeine-induced Ca²⁺ transients (i.e., SR Ca²⁺ load) in control conditions, in the presence of ISO (0.1 μM) with following application of Compound 9 (10 μM). The recordings were made from WT ventricular myocytes. (B) The average effects of ISO (0.1 μM) and ISO + Compound 9 (n = 10 myocytes) on AP-induced Ca²⁺ transient amplitude, SR Ca²⁺ load LTCC-induced Ca²⁺ transient amplitude, and the fractional release (FR). ∗p < 0.05 versus ISO alone.

Figure 4.

Effect of Compound 9 on Ca²⁺ signaling in cardiomyocytes isolated from PLB knockout mice. (A) F/F₀ profiles of cytosolic Ca²⁺ during AP-induced and caffeine-induced Ca²⁺ transients (i.e., SR Ca²⁺ load) in control conditions and in the presence of Compound 9 (10 μM). The recordings were made from PLB knockout ventricular myocytes. (B) The average effects of Compound 9 (n = 13 myocytes) on AP-induced Ca²⁺ transient amplitude, SR Ca²⁺ load LTCC-induced Ca²⁺ transient amplitude, and the fractional release (FR). ∗p < 0.05 versus control.

Results

Effect of Compound 9 on binding to SERCA2a in cells

Using an enzyme-linked NADH-coupled Ca²⁺-ATPase assay of rabbit fast skeletal muscle SR, we previously carried out a high-throughput screen of 46,000 drug-like small molecules in the ChemBridge DIVERSet-CL library (16). In that screen, Compound 9 (Fig. 1A) and two other piperidinyl amides were identified as enhancers of both Ca²⁺-ATPase and Ca²⁺ uptake activities of SR from pig heart left ventricles (Fig. S1) (16). To determine whether Compound 9 directly interacts with human SERCA2a, we measured its effect on the fluorescence lifetime of the intramolecular human SERCA2a FRET biosensor. The FRET readout reflects the heterogeneous population of structural states. Our acceptor probe is attached to the N-terminus, and the donor probe is attached to a flexible loop located on the nucleotide binding domain (N-domain) of SERCA. A shift in FRET indicates a shift in the equilibrium of SERCA structural states. We observed a decrease in FRET of mCyRFP1-mMaroon1 SERCA2a (Fig. 1 B). This suggests that Compound 9 directly binds to the SERCA2a, and with this binding the population shifts more to a state with the N-domain further from the N-terminus of SERCA2a. The resolution and interpretation for structural change monitored by FRET is limited, especially given that shifts in FRET could arise from one or many structural changes, including spatial separation of the domains or different motility of domains. Of note, the compound affinity appears to be lower in these FRET experiments, in comparison to ATPase and Ca²⁺ uptake assays using isolated cardiac SR. This may be due to the ester group on Compound 9, which is metabolically unstable in HEK293 cells. Using the human SERCA2a isoform in intact mammalian cells is desirable for this study, though it comes with the caveat that the intracellular concentration may be lower due to cellular metabolism and/or membrane permeability.

To assess the effect of Compound 9 on PLB binding to SERCA2a, we monitored the effect of the compound on FRET between mCyRFP1-SERCA2a and mMaroon1-PLB recombinantly expressed in HEK293 cells. This FRET-based assay uses the monomeric version of PLB (native Cys replaced with AFA) to maximize binding PLB availability for binding to SERCA. As shown in Fig. S2, Compound 9 did not alter FRET, indicating that this compound likely does not alter SERCA-PLB binding.

Effect of Compound 9 on Ca²⁺ signaling in mouse ventricular myocytes

To characterize the effect of Compound 9 on intracellular Ca²⁺ signaling, action potential- and caffeine-induced Ca²⁺ transients were measured and analyzed in ventricular myocytes isolated from hearts of wild-type (WT) C57Bl6/J mice (Fig. 2 A). In testing of Compound 9 on SERCA2a ATPase and Ca uptake activities (Fig. S1 C) (16), we observed that the percent effect on both ATPase and Ca uptake was similar at 10 μM Compound 9. For comparison with those studies, the effect of 10 μM Compound 9 on cardiomyocytes was tested here. Cardiomyocytes were electrically stimulated at 0.5 Hz to evoke periodic action potentials and cytosolic Ca²⁺ transients. Then, electrical stimulation was suspended for 5 s, and caffeine (5 mM) was applied to activate RyR2, which resulted in the global SR Ca²⁺ release and complete intra-SR Ca²⁺ ([Ca²⁺]_SR) depletion. The amplitude of SR Ca²⁺ release during caffeine application was used as an index of SR Ca²⁺ load. Compound 9 (10 μM) increased action potential-induced Ca²⁺ transients by 65.1% ± 33% (n = 12 cells) and SR Ca²⁺ load by 32.6% ± 8.1% (n = 12 cells; Fig. 2B), without a significant effect on the resting [Ca²⁺]_i. The first AP-induced Ca²⁺ transient after complete [Ca²⁺]_SR depletion with caffeine is mainly mediated by inward Ca²⁺ current via L-type Ca²⁺ channel (LTCC) (25). Analysis of LTCC-mediated Ca²⁺ transients revealed that Compound 9 did not change significantly the amplitude of LTCC-mediated Ca²⁺ transients the fraction of SR Ca²⁺ released during APs (FR = AP Ca²⁺ transient/SR Ca²⁺ load; Fig. 2B). Similar stimulatory effects of Compound 9 on Ca²⁺ transients and SR Ca²⁺ load were observed at higher electrical pacing frequency (1.0 Hz; Fig. S3). These results indicate that Compound 9 can enhance Ca²⁺ transients during action potential by increasing SR Ca²⁺ load but not LTCC Ca²⁺.

Next, we analyzed the effect of Compound 9 on Ca²⁺ dynamics during activation of β-adrenergic receptor with isoproterenol (ISO; Fig. 3 A). ISO (0.1 μM) increased the action potential-induced Ca²⁺ transient amplitude by 81.3% ± 23.2% (n = 10 cells), SR Ca²⁺ load by 21.3% ± 12.2% (n = 10 cells), and LTCC-induced Ca²⁺ transient by 146.3% ± 55.9% (n = 10 cells; Fig. 3 B). These effects were mainly mediated by an increase in SR Ca²⁺ uptake and sarcolemmal Ca²⁺ influx due to PLB and LTCC phosphorylation by protein kinase A (26). The addition of Compound 9 (10 μM) in the presence of ISO produced further increase of action potential-induced Ca²⁺ transients by 129.4% ± 30.3% (n = 10 cells) and SR Ca²⁺ load by 47.4% ± 6.4% (n = 10 cells; Fig. 3 B). These results suggest that Compound 9 activates the pump directly without interacting with PLB. Moreover, the analysis of Ca²⁺ dynamic in the presence of Compound 9 or ISO or Compound 9 and ISO did not reveal any spontaneous Ca²⁺ transients due to early afterdepolarization and Ca²⁺ waves that can trigger delay afterdepolarization.

To further confirm that Compound 9 directly interacts with the pump, we studied the effect of Compound 9 on Ca²⁺ dynamics in ventricular myocytes isolated from hearts of PLB knockout mice (Fig. 4 A). As expected for cells with highly active SERCA2a due to the lack of PLB inhibition, action potential-induced Ca²⁺ transient amplitude and SR Ca²⁺ load was higher in PLB knockout myocytes compare to WT myocytes. In WT myocytes, Ca²⁺ transient amplitude was 1.45% ± 0.2% (n = 12 cells) versus 1.72% ± 0.18% (n = 13 cells) in PLB knockout myocytes, and SR Ca²⁺ load was 3.81% ± 0.32% (n = 12 cells) versus 4.63% ± 0.21% (n = 13 cells), correspondently. Compound 9 (10 μM) increased action potential-induced Ca²⁺ transients by 33.3% ± 13.1% (n = 13 cells; Fig. 4 B) and SR Ca²⁺ load by 17.3% ± 6.5% (n = 13 cells; Fig. 4 C) in PLB knockout myocytes.

Discussion

In this study, we characterized the effect of the recently identified Ca²⁺ pump modulator Compound 9 on intracellular Ca²⁺ dynamics in ventricular myocytes. Using FRET, we confirmed that Compound 9 binds and structurally alters human SERCA2a. An analysis of cytosolic Ca²⁺ dynamics in mouse ventricular myocytes revealed that Compound 9 increased the SR Ca²⁺ load and action potential-induced Ca²⁺ transient amplitude in WT ventricular myocytes (Fig. 2). Additionally, Compound 9 did not change the LTCC-mediated Ca²⁺ transient amplitude. These results suggest that Compound 9 might stimulate SERCA2a-mediated Ca²⁺ transport in cardiomyocytes without affecting RyR2 and LTCC activity.

In cardiomyocytes, the main mechanism of SERCA2a regulation is mediated by an interaction with the small transmembrane peptide PLB. Under unstressed conditions, PLB binding to SERCA2a inhibits SR Ca²⁺ transport by decreasing pump affinity for cytosolic Ca²⁺ (27). During adrenergic receptor stimulation, PLB phosphorylation by protein kinase A relieves SERCA2a inhibition and increases SR Ca²⁺ transport (28,29). Such cellular response to adrenergic stress increases myocardial contraction and relaxation, causing positive inotropic and lusitropic effects. We analyzed whether the effect of Compound 9 on cardiac Ca²⁺ dynamics depends on SERCA2a regulation by PLB. As expected, activation of β-adrenergic receptors with ISO significantly increased SR Ca²⁺ load and Ca²⁺ transients during action potentials. The application of Compound 9 during adrenergic receptor activation produced a further increase of action potential-induced Ca²⁺ transients and SR Ca²⁺ load (Fig. 3). These results suggest that Compound 9 activates the pump without interacting with PLB, which aligns with the FRET response of SERCA without PLB. These findings were directly confirmed by experiments with cardiomyocytes isolated from PLB knockout mice (Fig. 4). As expected from the lack of SERCA2a inhibition by PLB, these myocytes exhibited an increased Ca²⁺ load. The effects of Compound 9 on Ca²⁺ dynamics in PLB knockout myocytes were similar to those observed in WT myocytes during adrenergic receptor stimulation.

In conclusion, the activation of SERCA2a by Compound 9 improves intracellular Ca²⁺ regulation in ventricular myocytes by increasing SR Ca²⁺ loading and SR Ca²⁺ release during the action potential. These effects of Compound 9 would be particularly beneficial to treat pathological conditions associated with abnormal SERCA2a function and Ca²⁺ mishandling, as found in heart failure due to myocardial infarction (13) or the PLB pathogenic gene variant PLN-R14del (30).

In our continued research, we will assess the physicochemical and ADME (absorption, distribution, metabolism, and excretion) properties of Compound 9 and further refine its structure-activity relationship to increase its efficacy with improved properties. To address the potential liabilities associated with the furan ring and ester group, we will implement a range of strategies, including bioisosteric replacements and scaffold modifications. Furans are known to be chemically reactive and can be metabolized by cytochrome P450 enzymes through epoxidation, potentially forming harmful reactive intermediates. To mitigate this risk, we plan to replace the furan ring with alternative heterocycles or aromatic rings that retain similar physicochemical characteristics but offer improved metabolic stability and reduced toxicity—such as pyrrole, isoxazole, pyridine, or phenyl groups. Additionally, to prevent ester hydrolysis by esterases, the ester group will be replaced with more stable functional groups like primary or secondary amides, or substituted with heterocycles such as oxazole, triazole, or oxadiazole. These modifications aim to generate a series of analogs with enhanced metabolic stability, improved solubility, and potentially superior pharmacological profiles.

Data and code availability

The data sets used and/or analyzed in the current study are available from the corresponding author upon reasonable request.

Acknowledgments

This work was supported by the National Institutes of Health grants R01HL151990 and R01HL176568 (to A.V.Z.), R01HL139065 and R01AR082533 (to D.D.T. and R.T.R.), T32AR007612 (to M.B. and D.D.T.), R01HL092097 (to R.T.R.) and R01HL171221 and R01HL160569 (to C.A.M.).

Author contributions

D.D.T., R.T.R. C.A.M., and A.V.Z. conceived and supervised the study. E.B., R.T.R., D.D.T., and A.V.Z. designed experiments. E.B., S.L.Y., and R.N. performed experiments and analysis. D.D.T., R.T.R., M.B., and A.V.Z. wrote the manuscript. R.T.R., D.D.T. C.A.M., and A.V.Z. edited the manuscript. All the authors read and approved the manuscript version to be published.

Declaration of interests

D.D.T. holds equity in and serves as an executive officer for Photonic Pharma LLC, which had no role in this study. These relationships have been reviewed and managed by the University of Minnesota.