Hypertrophic cardiomyopathy dysfunction mimicked in human engineered heart tissue and improved by sodium-glucose cotransporter 2 inhibitors

Abstract

Aims: Hypertrophic cardiomyopathy (HCM) is the most common inherited cardiomyopathy, often caused by pathogenic sarcomere mutations. Early characteristics of HCM are diastolic dysfunction and hypercontractility. Treatment to prevent mutation-induced cardiac dysfunction is lacking. Sodium-glucose cotransporter 2 inhibitors (SGLT2i) are a group of antidiabetic drugs that recently showed beneficial cardiovascular outcomes in patients with acquired forms of heart failure. We here studied if SGLT2i represent a potential therapy to correct cardiomyocyte dysfunction induced by an HCM sarcomere mutation.

Methods and results: Contractility was measured of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) harbouring an HCM mutation cultured in 2D and in 3D engineered heart tissue (EHT). Mutations in the gene encoding β-myosin heavy chain (MYH7-R403Q) or cardiac troponin T (TNNT2-R92Q) were investigated. In 2D, intracellular [Ca2+], action potential and ion currents were determined. HCM mutations in hiPSC-CMs impaired relaxation or increased force, mimicking early features observed in human HCM. SGLT2i enhance the relaxation of hiPSC-CMs, to a larger extent in HCM compared to control hiPSC-CMs. Moreover, SGLT2i-effects on relaxation in R403Q EHT increased with culture duration, i.e. hiPSC-CMs maturation. Canagliflozin's effects on relaxation were more pronounced than empagliflozin and dapagliflozin. SGLT2i acutely altered Ca2+ handling in HCM hiPSC-CMs. Analyses of SGLT2i-mediated mechanisms that may underlie enhanced relaxation in mutant hiPSC-CMs excluded SGLT2, Na+/H+ exchanger, peak and late Nav1.5 currents, and L-type Ca2+ current, but indicate an important role for the Na+/Ca2+ exchanger. Indeed, electrophysiological measurements in mutant hiPSC-CM indicate that SGLT2i altered Na+/Ca2+ exchange current.

Conclusion: SGLT2i (canagliflozin > dapagliflozin > empagliflozin) acutely enhance relaxation in human EHT, especially in HCM and upon prolonged culture. SGLT2i may represent a potential therapy to correct early cardiac dysfunction in HCM.

Keywords: Ca2+ handling; Contractility; Engineered heart tissue; Human induced pluripotent stem cell-derived cardiomyocytes; Hypertrophic cardiomyopathy; Sodium–glucose cotransporter 2 inhibitors.

Graphical Abstract

Figure 1

Contractile phenotype and Empa treatment of spontaneously beating R403Q EHTs. (A) Representative picture of a human EHT as viewed by the video camera and evaluated by automated figure recognition (squares). (B) Schematic contraction peak with evaluated parameters of contractile function. (C) Examples of spontaneous beating patterns of R403Qic and R403Q EHT after 6 weeks of culture (red line, original recording; pink line, velocity). (D) Example of beating patterns of an R403Q EHT at baseline and after Empa treatment. Dotted lines allow us to compare the Empa effect on relaxation to baseline. (E) Effect of cumulative treatment with Empa for 1 h on contractility in starvation medium in R403Qic (n = 11, 39 ± 1 days) and R403Q (n = 12, 46 ± 3 days). (F) Comparison of the Empa (1 µM) effect between R403Qic and R403Q, calculated as the change in force or T2_80% as a percentage compared to baseline. Data are expressed as mean ± SEM (E, F; 2–3 independent EHT preparations). #P < 0.05, ##P < 0.01, ###P < 0.001 vs. Baseline (E) or R403Qic (F), repeated measures one-way ANOVA followed by Dunnett’s post-test (E) or unpaired Student’s t-test (F).

Figure 2

Contractile phenotype and Empa treatment of electrically stimulated R403Q EHTs. (A) Examples of beating patterns of an electrically stimulated (1 Hz) R403Qic and R403Q EHT (red line, original recording; pink line, and velocity). (B) EHT width, force of contraction and T2_80% of R403Qic (n = 24, 42 ± 3 days) were compared to R403Q (n = 24, 42 ± 0.2) EHTs paced at 1 Hz in starvation medium. (C) Example of beating patterns of an electrically stimulated (1 Hz) R403Q EHT at baseline and after Empa treatment. Dotted lines allow us to compare the Empa effect on relaxation to baseline. (D) Effect of Empa treatment for 1 h on contractility in starvation medium in R403Qic (n = 10, 39 ± 3 days) and R403Q (n = 9, 43 ± 0.2 days) paced at 1 Hz. (E) Comparison of the Empa (1 µM) effect on contractility between R403Qic and R403Q, calculated as the change compared to baseline. Data are expressed as mean ± SEM (B; three independent EHT preparations, D, E; two independent EHT preparations). #P < 0.05, ##P < 0.01, ###P < 0.001 vs. R403Qic (B, E) or Baseline (D), unpaired Student’s t-test (B, E) or Repeated measures one-way ANOVA followed by Dunnett’s post-test (D).

Figure 3

Comparison of SGLT2i-effects on contractility in EHT. (A) Effect of Cana treatment for 1 h on force and T2_80% in spontaneously beating R403Q (n = 9, 52 ± 5 days). At 10 µM Cana, 3 EHTs stopped beating. (B) Effect of Dapa treatment for 1 h in spontaneously beating R403Q (n = 8, 48 ± 5 days). At 50 µM Dapa, 4 EHTs stopped beating. (C) Empa, Cana, and Dapa effects on T2_80% in spontaneously beating (Spon) and in paced (1 Hz) R403Q were compared to each other, calculated as the change in T2_80% as a percentage compared to baseline (Empa data from Figures 1F and 2E). (D) The effect size on relaxation time of Empa and Cana was compared between R403Qic and R403Q, showing a significantly larger reduction in relaxation time upon Empa and Cana treatment in R403Q compared to R403Qic (Empa data from Figures 1F and 2E, Cana data in R403Qic from Supplementary material online, Figure S8B). Data are expressed as mean ± SEM (A: two independent EHT preparations, B: one EHT preparation). (A, B) #P < 0.05, ##P < 0.01, ###P < 0.001 vs. Baseline or (D) R403Qic, (A, B) a mixed-effects analysis followed by Dunnett’s post-test was performed if values are missing because EHTs stopped beating as a result of the treatment, or (C) one-way ANOVA followed by Tukey’s multiple comparisons test, or (D) unpaired Student’s t-test.

Figure 4

SGLT2i-effect on relaxation time in R403Q EHT depends on culture duration. (A) Western blots of R403Q EHTs stored on different days after EHT casting, as indicated above each lane. A human donor sample (donor) was loaded as a positive control for cardiac troponin I (cTnI), a mouse soleus sample as a positive control for slow skeletal troponin I (ssTnI), and HEK 293 cells as a negative control for TnI. cTnI expression corrected for actin increased upon prolonged culture time of R403Q EHT. This was also demonstrated with an antibody that detects both cTnI and ssTnI. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and α-actinin staining illustrate that similar amounts of protein were loaded in each lane. The cTnI blot was cut at ∼50 kDa and ∼100 kDa, and the cTnI-ssTnI blot was cut at ∼85 kDa and ∼43 kDa. (B) Spontaneously beating R403Q were treated with SGLT2i (n = 3–4 EHTs per SGLT2i) on different days after EHT casting with cumulative concentrations for 1 h. Measurements with Empa, Cana, and Dapa were performed on days 20, 28, 37, 55, and 69, and with Cana also on day 44. The effect of SGLT2i on T2_80% was expressed as SGLT2i-induced changes compared to the baseline expressed in percentage. At 10 µM Cana, 2 EHTs stopped beating at days 44, 55, and 69. At 50 µM Dapa, 1 EHT stopped beating on day 28, two EHTs on day 55, and all four EHTs on day 69. Baseline values of T2_80% before SGLT2i treatment were stable over time and were only significantly higher at day 69 compared to day 20 (n = 12 EHTs on days 20, 28, 37, and 55; n = 4 EHTs on day 44; and n = 11 EHTs on day 69). Baseline values of beating frequency (beats per minute, BPM) before SGLT2i treatment stabilized from day 44. (C) Empa, Cana, and Dapa effects on relaxation time in R403Q EHTs were also plotted together at the same concentration for a direct comparison between SGLT2i (data were plotted on the days when all three SGLT2i were tested). (D) The effect of Empa on T2_80% plotted against the culture duration of electrically stimulated (1 Hz) R403Qic EHTs (n = 4 on day 29, n = 5 on day 42, and n = 1 on day 62—data of Figure 2). Data are expressed as mean ± SEM (B, C: one EHT preparation, D: two independent EHT preparations). ###P < 0.001 vs. day 20, one-way ANOVA followed by Dunnett’s post-test (B, Baseline T2_80%), or one-way ANOVA followed by Tukey’s multiple comparisons test (B, BPM). Linear regression lines (B–D) and non-linear lines (A, one phase decay) were fitted.

Figure 5

Empa and Cana effects on Ca²⁺ handling and contractility in 2D MYH7-R403Q and TNNT2-R92Q hiPSC-CM. R403Qic and R403Q (∼30–32 days old) hiPSC-CM were thawed and cultured in 2D and after 4 weeks contraction kinetics and Ca²⁺ handling were measured simultaneously in Tyrode medium. Relaxation time (T2_80%), Ca²⁺ transient decay time (Ca²⁺ decay time), and Ca²⁺ transient amplitude (Ca²⁺ amplitude) were measured. (A) R403Qic and R403Q hiPSC-CM treated for 15 min with 1 or 10 µM Empa. (B) R403Q hiPSC-CM treated for 15 min with 1, 3, or 10 µM Cana. (C) Contractile parameters of isogenic control hiPSC-CM (R92Qic) were compared to hiPSC-CM harbouring a TNNT2-R92Q mutation (R92Q) 35–45 days after thawing (60 days old). (D) R92Q were treated for 15 min with 10 µM Cana. The Ca²⁺ amplitude could only be determined as a relative change compared to the baseline. Each data point represents the average value of at least three spots measured within one well. Data are expressed as mean ± SEM. #P < 0.05, ##P < 0.01, ###P < 0.001 vs. (A, B, D) Baseline or (C) R92Qic, and (A–D) unpaired Student’s t-test.

Figure 6

Potential molecular targets of SGLT2i investigated in R403Q EHT. (A) Empa treatment for 1 h lowers relaxation time of spontaneously beating R403Q (n = 12, 70 ± 10 days) in starvation medium without glucose and insulin. (B) Effect of treatment with the selective NHE-1 inhibitor cariporide (Cari) for 1 h on contractility in R403Q (n = 9, 61 ± 7 days). (C) Treatment with the late Na⁺ current inhibitor ranolazine hydrochloride (Ran) for 1 h in spontaneously beating R403Q (1–50 µM: n = 6, 54 ± 6 days). (D) Effect of treatment with the mitochondrial antioxidant elamipretide (SS-31; n = 7, 79 ± 0 days) for 1 h on contractility in R403Q. (E, F) Western blots of R403Q treated for 1 h with DMSO (D) or Empa (1 or 50 µM). As control samples a human donor sample (donor) was loaded and an untreated or isoprenalin (Iso, 100 nM) treated control EHT (Ctrl). Almost no phosphorylation of PLB at Ser16 or cMyBP-C at Ser282 was detected in R403Q, in contrast to control samples. Results were identical when GAPDH or α-actinin was used as a loading control (indicated by α-actinin/GAPDH). Blots were cut at 75 kDa and below 20 kDa. Data are expressed as mean ± SEM (A: 3 independent EHT preparations; B, C: two independent EHT preparations; D: one EHT preparation; E, F: three–five EHTs per group). #P < 0.05, ##P < 0.01, ###P < 0.001 vs. (A–D) Baseline vs. (E, F) DMSO, (A, B, D) repeated measures one-way ANOVA followed by Dunnett’s post-test, or (C) if values are missing because EHTs stopped beating as a result of the treatment a mixed-effects analysis followed by Dunnett’s post-test was performed, or (E, F) one-way ANOVA followed by Dunnett’s post-test. Abbreviation: M, marker.

Figure 7

Molecular targets of SGLT2i that enhance relaxation in R403Q EHT. (A) Treatment with the L-type Ca²⁺ channel blocker diltiazem hydrochloride (Dilt) for 1 h in spontaneously beating R403Q (n = 10, 90 ± 1 days) and Ctrl (n = 8, 82 ± 3 days). R403Q EHTs stopped beating at 1 µM Dilt (three EHTs) and 3 µM Dilt (six EHTs). (B) The binding affinity of Cana for the dihydropyridine site of the L-type Ca²⁺ channel was determined (two replicates) in the rat cerebral cortex via a radioligand binding assay. Log(inhibitor) vs. response variable slope (four parameters) was fitted with the constraint top is 100. The concentration causing a half-maximal inhibition of control specific binding (IC₅₀) was 37 µM. (C) Treatment with the potent NCX inhibitor SEA0400 for 1 h lowered relaxation time of spontaneously beating R403Q (n = 6, 56 ± 8 days), whereas SEA0400 increased relaxation time in Ctrl (n = 7, 81 ± 2 days). Treatment with SEA0400 resulted in some EHTs in irregular beating patterns (R403Q: 1 EHT at 33 nM; Ctrl: 1 EHT at 100 nM), which were excluded from data analyses. (D) Examples of beating patterns of a Ctrl and R403Q EHT with the same beating frequency, before treatment (baseline) and after SEA0400 (100 nM) treatment (red line, original recording; pink line, velocity). (E) R403Q EHTs (75 ± 1 days) were cumulatively treated for 45 min with Empa (n = 6), or with SEA0400 (100 nM; n = 6), or with both SEA0400 (100 nM) and Empa (n = 8). At 50 µM Empa two EHTs stopped beating, whereas six EHTs stopped beating at 50 µM Empa + SEA0400. Data are expressed as mean ± SEM. (A, C: 2 independent EHT preparations of R403Q and Ctrl; E: 1 EHT preparation). #P < 0.05, ##P < 0.01, ###P < 0.001 vs. Baseline (A, C, E: Empa) or vs. SEA0400 (E: SEA0400 + Empa), repeated measures one-way ANOVA followed by Dunnett’s post-test (A: Ctrl, C), or if values are missing because EHTs stopped beating as a result of the treatment a mixed-effects analysis followed by Dunnett’s post-test was performed (A: R403Q, E: Empa vs. baseline), or one-way ANOVA followed by Tukey’s multiple comparisons tests (E: SEA0400 vs. SEA0400 + Empa).

Figure 8

Effects of Cana on APs and membrane currents in TNNT2-R92Q hiPSC-CM. (A) Typical APs in the absence and the presence of 10 µmol/L Cana and average effects (depicted as dot plots) of 1, 3, and 10 µmol/L Cana on AP parameters. MDP = maximum diastolic potential, APA = maximal AP amplitude, V_max = maximal AP upstroke velocity, and APD₂₀, APD₅₀, APD₉₀ = AP duration at 20, 50, and 90% of repolarization. (B) Typical sodium currents (I_Na), evoked by 300 ms depolarizing pulses from −120 to −20 mV, in the absence and the presence of 10 µmol/L Cana. The dot plots show the average effects on peak and late I_Na. (C) Current–voltage (I–V) relationships of the Na⁺/Ca²⁺ exchange current (I_NCX) in the absence and the presence of 10 µmol/L Cana. I_NCX was measured during a descending ramp as a NiCl₂-sensitive current. The dot plots indicate that I_NCX was significantly affected by Cana, with a more pronounced reduction at +70 mV, i.e. reverse-mode of I_NCX, than at −120 mV, i.e. the forward mode (D). Typical L-type Ca²⁺ current (I_Ca,L), evoked by 500 ms depolarizing pulses from −60 to 0 mV, in the absence and the presence of 10 µmol/L Cana. Neither the amplitude nor the time constant (τ) of current inactivation was significantly affected. I_Ca,L was also not affected at other potential, as shown in the average I–V relationships, and the voltage dependency of (in)activation was similar in the absence and presence of Cana. Voltage dependence of activation and inactivation curves were fitted with the Boltzmann function y = [1 + exp{(V-V_1/2)/k}]⁻¹ to determine half-maximal voltage (V_1/2; in mV) of (in) activation and the slope factor (k; in mV). *P < 0.05 (paired t-test).