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. 2023 May 5:14:1127388.
doi: 10.3389/fphar.2023.1127388. eCollection 2023.

Hydroxychloroquine and azithromycin alter the contractility of living porcine heart slices

Qin Wu  1   2 , Abigail J Ross  2 , Tugce Ipek  2 , Georgina H Thompson  2 , Robert D Johnson  2 , Changhao Wu  2 , Patrizia Camelliti  2
Affiliations

Hydroxychloroquine and azithromycin alter the contractility of living porcine heart slices

Qin Wu et al. Front Pharmacol. .

Abstract

The cardiotoxicity risk of hydroxychloroquine (HCQ) and azithromycin (AZM) has been the subject of intensive research triggered by safety concerns in COVID-19 patients. HCQ and AZM have been associated with QT interval prolongation and drug-induced arrhythmias, however other cardiotoxicity mechanisms remain largely unexplored. Our group has pioneered the living heart slice preparation, an ex-vivo platform that maintains native cardiac tissue architecture and physiological electrical and contractile properties. Here, we evaluated the cardiotoxic effect of HCQ and AZM applied alone or in combination on cardiac contractility by measuring contractile force and contraction kinetics in heart slices prepared from porcine hearts. Our results show that clinically relevant concentrations of HCQ monotherapy (1-10 µM) reduced contractile force and contraction kinetics in porcine slices in a dose-dependent manner. However, AZM monotherapy decreased contractile force and contraction kinetics only at higher concentrations (30 µM). Combination of HCQ and AZM induced a dose-dependent effect similar to HCQ alone. Furthermore, pre-treating porcine heart slices with the L-type calcium channel agonist Bay K8644 prevented the effect of both drugs, while administration of Bay K8644 after drugs interventions largely reversed the effects, suggesting a mechanism involving inhibition of L-type calcium channels. These findings indicate that HCQ and AZM alter cardiac function beyond QT prolongation with significant contractile dysfunction in intact cardiac tissue. Our porcine heart slices provide a powerful platform to investigate mechanisms of drug cardiotoxicity.

Keywords: Bay K8644; COVID-19; calcium channels; cardiotoxicity; myocardial slices; organotypic ex-vivo models; safety pharmacology.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Contractile force generated by porcine heart slices. (A) Representative contraction traces from porcine slice stretched in a stepwise manner to maximum force generation. (B) Systolic and diastolic forces produced by porcine slices under stepwise stretch and 1 Hz stimulation (n = 9 slices/4 hearts). *p < 0.05, ****p < 0.0001 vs. 0 mm stretch (two-way ANOVA with Dunnett’s multiple comparison test). (C) Representative contraction traces in response to β-adrenergic stimulation (10−7 M isoproterenol—ISO). (D) Contractile force in the presence or absence of ISO (n = 8 slices/4 hearts). ****p < 0.0001 vs. control (paired t-test). Bars represent means ± SEM. (E–G) Maximum slope of contraction and relaxation in the presence or absence of ISO (n = 8 slices/4 hearts). ***p < 0.001 vs. control (paired t-test). Bars represent means ± SEM.
FIGURE 2
FIGURE 2
Effect of hydroxychloroquine (HCQ) on the contractility of porcine heart slices. (A) Stability of contractility recordings over experimental time in the presence of vehicle control. Left: representative contraction traces. Right: averaged data (n = 6 slices/4 hearts; means ± SEM). (B) Representative contraction traces from porcine slice in the presence of vehicle control (baseline) and after exposure to increasing concentrations of HCQ (1, 3, and 10 μM). (C) Bar graph showing dose-dependent effect of HCQ on contractile force of porcine heart slices (n = 9 slices/4 hearts; means ± SEM). (D,E) Bar graphs showing dose-dependent effect of HCQ on maximum slope of contraction and relaxation (n = 9 slices/4 hearts; means ± SEM). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (one-way ANOVA with Tukey’s multiple comparisons test).
FIGURE 3
FIGURE 3
Effect of azithromycin (AZM) on the contractility of porcine heart slices. (A) Representative contraction traces in the presence of vehicle control (baseline) and after exposure to increasing concentrations of AZM (3, 10, and 30 μM). (B) Bar graph showing the average effect of AZM on contractile force of porcine heart slices (n = 5–7 slices/4 hearts; means ± SEM). (C,D) Bar graphs showing effect of AZM on maximum slope of contraction and relaxation (n = 5–7 slices/4 hearts; means ± SEM). *p < 0.05, **p < 0.01, ***p < 0.001 (one-way ANOVA with Tukey’s multiple comparisons test).
FIGURE 4
FIGURE 4
Effect of HCQ and AZM administered in combination on contractility of porcine heart slices. (A,B) Representative contraction traces from porcine slices treated with 3 μM HCQ + 10 μM AZM (A) or 10 μM HCQ + 30 μM AZM (B). (C,D) Paired dot plots showing changes in contractile force from baseline for each individual slice treated with HCQ and AZM combination (n = 6 slices/4 hearts). (E) Bar graph showing the average effect of 3 μM HCQ + 10 μM AZM and 10 μM HCQ + 30 μM AZM on contractile force of porcine heart slices (n = 6 slices/4 hearts; means ± SEM). ****p < 0.0001 (one-way ANOVA with Tukey’s multiple comparisons test). (F) Time-dependent effect of 3 μM HCQ + 10 μM AZM and 10 μM HCQ + 30 μM AZM on contractility over the first 5 min exposure (n = 6 slices/4 hearts; means ± SEM). **p < 0.01, ***p < 0.001 and ****p < 0.0001 versus vehicle control. ^^^p < 0.001 and  ^^^^p< 0.0001 versus 3 μM HCQ + 10 μM AZM (two-way ANOVA with Tukey’s multiple comparisons test). (G,H) Bar graphs showing effect of HCQ and AZM combination on maximum slope of contraction and relaxation (n = 6 slices/4 hearts; means ± SEM). *p < 0.05, ***p < 0.001, ****p < 0.0001 (one-way ANOVA with Tukey’s multiple comparisons test).
FIGURE 5
FIGURE 5
Pre-treatment of porcine heart slices with Bay K8644 prevents the effect of HCQ and AZM on cardiac contractility. (A) Representative contraction traces and bar graphs showing the effect of 1 μM (±)-Bay K8644 (BK) followed by 10 μM HCQ on contractile force, maximum slope of contraction and relaxation of porcine heart slices. (B) Representative contraction traces and bar graphs showing the effect of 1 μM BK followed by 30 μM AZM on contractile force, maximum slope of contraction and relaxation of porcine heart slices. (C) Representative contraction traces and bar graphs showing the effect of 1 μM BK followed by 10 μM HCQ + 30 μM AZM on contractile force, maximum slope of contraction and relaxation of porcine heart slices. (n = 7 slices/4 hearts; means ± SEM). *p < 0.05; **p < 0.01 (one-way ANOVA with Tukey’s multiple comparisons test).
FIGURE 6
FIGURE 6
Bay K8644 reverses the effect of HCQ and AZM on contractility of porcine heart slices. (A) Representative contraction traces and bar graphs showing the effect of 1 μM (±)-Bay K8644 (BK) administered after 10 μM HCQ treatment on contractile force, maximum slope of contraction and relaxation of porcine heart slices. (n = 7 slices/4 hearts; means ± SEM). (B) Representative contraction traces and bar graphs showing the effect of 1 μM BK following 30 μM AZM on contractile force, maximum slope of contraction and relaxation of porcine heart slices. (n = 7 slices/4 hearts; means ± SEM). (C) Representative contraction traces and bar graphs showing the effect of 1 μM BK following 10 μM HCQ + 30 μM AZM on contractile force, maximum slope of contraction and relaxation of porcine heart slices. (n = 6 slices/4 hearts; means ± SEM). *p < 0.05; **p < 0.01, ***p < 0.001, ****p < 0.0001 (one-way ANOVA with Tukey’s multiple comparisons test).
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