key: cord-0964539-mg4v80nk authors: Wong, Andy On-Tik; Gurung, Bimal; Wong, Wing Sum; Mak, Suet Yee; Tse, Wan Wai; Li, Chloe M.; Lieu, Deborah K.; Costa, Kevin D.; Li, Ronald A.; Hajjar, Roger J. title: Adverse effects of hydroxychloroquine and azithromycin on contractility and arrhythmogenicity revealed by human engineered cardiac tissues date: 2020-12-27 journal: J Mol Cell Cardiol DOI: 10.1016/j.yjmcc.2020.12.014 sha: 0ddac61df38739f0e6f9ba5e5e7b309b66fe23dc doc_id: 964539 cord_uid: mg4v80nk The coronavirus disease 2019 (COVID-19) outbreak caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become a global pandemic as declared by World Health Organization (WHO). In the absence of an effective treatment in early 2020, different drugs with unknown effectiveness, including antimalarial hydroxychloroquine (HCQ), with or without concurrent administration with azithromycin (AZM), have been tested for treating COVID-19 patients with developed pneumonia. However, the efficacy and safety of HCQ and/or AZM have been questioned by recent clinical reports. Direct effects of these drugs on the human heart remain very poorly defined. To better understand the mechanisms of action of HCQ +/− AZM, we employed bioengineered human ventricular cardiac tissue strip (hvCTS) and anisotropic sheet (hvCAS) assays, made with human pluripotent stem cell (hPSC)-derived ventricular cardiomyocytes (hvCMs), which have been designed for measuring cardiac contractility and electrophysiology, respectively. Our hvCTS experiments showed that AZM induced a dose-dependent negative inotropic effect which could be aggravated by HCQ; electrophysiologically, as revealed by the hvCAS platform, AZM prolonged action potentials and induced spiral wave formations. Collectively, our data were consistent with reported clinical risks of HCQ and AZM on QTc prolongation/ventricular arrhythmias and development of heart failure. In conclusion, our study exposed the risks of HCQ/AZM administration while providing mechanistic insights for their toxicity. Our bioengineered human cardiac tissue constructs therefore provide a useful platform for screening cardiac safety and efficacy when developing therapeutics against COVID-19. 2020, different drugs with unknown effectiveness, including antimalarial hydroxychloroquine (HCQ), with or without concurrent administration with azithromycin (AZM), have been tested for treating COVID-19 patients with developed pneumonia. However, the efficacy and safety of HCQ and/or AZM have been questioned by recent clinical reports. Direct effects of these drugs on the human heart remain very poorly defined. To better understand the mechanisms of action of HCQ +/-AZM, we employed bioengineered human ventricular cardiac tissue strip (hvCTS) and anisotropic sheet (hvCAS) assays, made with human pluripotent stem cell (hPSC)-derived ventricular cardiomyocytes (hvCMs), which have been designed for measuring cardiac contractility and electrophysiology, respectively. Our hvCTS experiments showed that AZM induced a dose-dependent negative inotropic effect which could be aggravated by HCQ; electrophysiologically, as revealed by the hvCAS platform, AZM prolonged action potentials and induced spiral wave formations. Collectively, our data were consistent with reported clinical risks of HCQ and AZM on QTc prolongation/ventricular arrhythmias and development of heart failure. In conclusion, our study exposed the risks of HCQ/AZM administration while providing mechanistic insights for their toxicity. Our bioengineered human cardiac tissue constructs therefore provide a useful platform for screening cardiac safety and efficacy when developing therapeutics against COVID-19. The coronavirus disease 2019 (COVID-19) outbreak induced by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has rapidly spread worldwide. As of November 2020, COVID-19 has infected more than fifty million people around the world and claimed the lives of over a million patients. In the setting of this pandemic, unproven J o u r n a l P r e -p r o o f Journal Pre-proof drugs have been publicized to be effective in the treatment or prevention of COVID-19. Hydroxychloroquine (HCQ) or chloroquine, a medication for treating malaria, has been used alone or in combination with the antibiotic azithromycin (AZM) to treat COVID-19 patients who have developed pneumonia [1, 2] . It was reported that the prescription of HCQ or chloroquine in the United States has been increased by 80-fold from March 2019 to March 2020 [3] . However, recent reports have shown no clinical benefits to support the usage of HCQ with or without AZM in the setting of COVID-19 infection [4, 5] . In addition, it was reported that high dose of chloroquine would give rise to potential toxicities as shown by a recent randomized trial with 81 hospitalized patients with severe COVID-19, especially when taken together with AZM or oseltamivir [6] . In another small cohort study, 90 patients with COVID-19 pneumonia who received HCQ for the treatment of pneumonia associated with COVID-19 were found to be at high risk of QTc prolongation, and concurrent treatment with AZM was associated with even greater changes in QTc [7] . Despite these clinical observations, the cardiac effects in humans as well as the mechanistic basis for the reported arrhythmias remain very poorly defined. Human cardiomyocytes (CMs) do not proliferate in culture and are difficult to obtain for practical reasons. With the advent of human pluripotent stem cells (hPSC), the derivation of CMs has been extensively used for insights into the development, physiology as well as pathophysiology of the human heart [8] [9] [10] [11] [12] [13] . Since the heart is a complex 3D organ, single-cell experiments often do not fully recapitulate cardiac physiological properties such as contractile force and conduction which are inherently J o u r n a l P r e -p r o o f Journal Pre-proof multi-cellular in nature. As such, various 3D bioengineered tissue constructs have been developed using hPSC-CMs for modeling human-specific diseases and cardiotoxicity screening. In this study, hPSCs (HES2: human embryonic stem cell; ESI, NIH code ES02. And L-EdV: human induced pluripotent stem cell; provided by the University of Hong Kong) -were specified into human ventricular cardiomyocytes (hvCMs) as we have previously reported [14] , followed by assembly into engineered tissues: human ventricular cardiac tissue strips (hvCTS) and anisotropic sheet (hvCAS) [9, 15, 16] , which have been specifically designed and engineered for measuring contractility and electrophysiology, respectively, and used to investigate the cardiac effects of HCQ and AZM individually and in combination. Hydroxychloroquine with or without azithromycin reduced hvCTS contractility By casting 100μl collagen and a cell mixture of 1.3 million hPSC-hvCMs and 0.13 million human fibroblasts into a custom-made PDMS bioreactor, contracting engineered hvCTS could be formed between the two force-sensing cantilever posts in a rectangular well. Figure 1C) . Similarly, there were significant decreases of normalized maximum +dF/dt and -dF/dt of AZM-alone and HCQ+AZM treated groups when compared to vehicle control group ( Figure 1D-E) .Qualitatively similar results were obtained with hvCTS derived from another hPSC line (L-EdV; Figure 1F -G). We (Figure 2A, F, G) . We also measured conduction velocities, in the longitudinal and the transverse axes relative to cell alignment, as the rate of action potential propagation between pairs of selected points on the hvCAS substrate. Analysis of isochronal maps showed that the conduction velocity (LCV) of HCQ group, and the transverse conduction velocity (TCV) of all treatment groups were significantly decreased ( Figure 2D , E) in comparison to vehicle control. Importantly, re-entry in the form of spiral wave formation was promoted in the presence of HCQ-or AZM-alone, or in combination ( Figure 2B , C). Collectively, these data were consistent with the findings that QTc interval and pro-arrhythmogenicity increases with HCQ +/-AZM. In this study, we report the reduction of contractile force in the presence of 10-100μM AZM in a dose-dependent manner with or without 10 μM HCQ (Figure 1 C-D) . compared to the plasma [20] . Indeed, cellular accumulation of AZM has also been J o u r n a l P r e -p r o o f Journal Pre-proof detected in the liver, spleen and heart [21] . For HCQ, the serum level in COVID-19 patients who receive 600 mg/day is 1.1-3.2 μM [2] , but its blood concentration can be 3.2-3.7-fold higher [22] . Therefore, we have made every effort to match the drug conditions tested in our in vitro engineered tissues to the clinical situation. Overall, our observations of compromised contractility and increased arrhythmogenicity by HCQ and AZM in human engineered cardiac tissue models were consistent with clinical reports. The action potential duration was significantly prolonged in hvCAS treated with 30 µM of AZM with or without 10 µM of HCQ, which was physiologically relevant to our observation in increased arrhymogenic risk in the treated group as the prolongation of the QT interval by as low as 20 ms was high risk for Torsade de Pointes under the FDA guideline. Interestingly, the arrhythmogenic risk as shown by hvCAS treated with HCQ or AZM alone was similar to that of HCQ+AZM, although the combined treatment synergistically led to a stronger reduction of contractile force and HCQ alone did not show significant reduction in hvCTS force generation ( Figure 1C and 2C). It is likely that HCQ and AZM act on multiple ion channels and pumps whose effects can counterbalance each other. For instance, HCQ has been reported to inhibit the hyperpolarization-activated c-AMP-modulated pacemaker current in an in vitro guinea pig cardiomyocyte model [23] , but it is generally accepted that the inotropic action of HCQ/chloroquine is unspecified due to the mixed action of ion channels in cardiomyocytes and complicated by non-cardiac toxicity [24] . Further well as other therapeutic regimes for COVID-19, it will be imperative to consider both the contractile and electrophysiological properties. In this study, we show that human engineered tissues, hvCTS and hvCAS, constructed from hvCMs predict the arrhythmogenic potential of administering HCQ alone or in combination with AZM. Re-entry is a substrate for ventricular arrhythmias and Torsades de Pointes which have been described in patients on HCQ [19] . Our data also showed that AZM can induce a dose-dependent negative inotropic effect which is exacerbated Remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-nCoV) in vitro Hydroxychloroquine and azithromycin as a treatment of COVID-19: results of an open-label non-randomized clinical trial Hydroxychloroquine and Chloroquine Prescribing Patterns by Provider Specialty Following Initial Reports of Potential Benefit for COVID-19 Treatment -United States No evidence of rapid antiviral clearance or clinical benefit with the combination of hydroxychloroquine and azithromycin in patients with severe COVID-19 infection Chloroquine and hydroxychloroquine in coronavirus disease 2019 (COVID-19). Facts, fiction and the hype: a critical appraisal Effect of High vs Low Doses of Chloroquine Diphosphate as Adjunctive Therapy for Patients Hospitalized With Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Infection: A Randomized Clinical Trial Risk of QT Interval Prolongation Associated With Use of Hydroxychloroquine With or Without Concomitant Azithromycin Among Hospitalized Patients Testing Positive for Coronavirus Disease Bioengineering an electro-mechanically functional miniature ventricular heart chamber from human pluripotent stem cells Human Cardiac Ventricular-Like Organoid Chambers and Tissue Strips From Pluripotent Stem Cells as a Two-Tiered Assay for Inotropic Responses Correlation between frataxin expression and contractility revealed by in vitro Friedreich's ataxia cardiac tissue models engineered from human pluripotent stem cells A Micropatterned Human Pluripotent Stem Cell-Based Ventricular Cardiac Anisotropic Sheet for Visualizing Drug-Induced Arrhythmogenicity Cardiovascular regeneration Human pluripotent stem cell-derived cardiomyocytes for heart regeneration, drug discovery and disease modeling: from the genetic, epigenetic, and tissue modeling perspectives A simple, costeffective but highly efficient system for deriving ventricular cardiomyocytes from human pluripotent stem cells Advancing functional engineered cardiac tissues toward a preclinical model of human myocardium Effect of engineered anisotropy on the susceptibility of human pluripotent stem cell-derived ventricular cardiomyocytes to arrhythmias Clinical efficacy of hydroxychloroquine in patients with covid-19 pneumonia who require oxygen: observational comparative study using routine care data Hydroxychloroquine in patients with mainly mild to moderate coronavirus disease 2019: open label QT Interval Prolongation and Torsade De Pointes in Patients with COVID-19 treated with Hydroxychloroquine/Azithromycin. Heart Rhythm Azithromycin Causes a Novel Proarrhythmic Syndrome In vivo activity of the macrolide antibiotics azithromycin, roxithromycin and spiramycin against Toxoplasma gondii Population Pharmacokinetics of Hydroxychloroquine in Japanese Patients With Cutaneous or Systemic Lupus Erythematosus Hydroxychloroquine reduces heart rate by modulating the hyperpolarization-activated current If: Novel electrophysiological insights and therapeutic potential Cardiac effects and toxicity of chloroquine: a short update Human iPSC-Derived Cardiomyocytes are Susceptible to SARS-CoV-2 Infection B: Paired t-test between treated group and baseline; unpaired t-test between treated groups. C-H: : Two-way ANOVA test, followed by Holm-Sidak's multiple comparison to control group 30 µM azithromycin (AZM) or the combined (HCQ+AZM) treatment. (C-E) Comparisons of re-entry percentage measured by number of observed re-entry phenomena divided by number of total experiments, % change in longitudinal (LCV) and transverse (TCV) conduction velocities of hvCAS (n=5-6). (F-G) The change in APD 90 and the comparison of APD 90 at baseline or treated condition of hvCAS with different treatments (n=5-6). D-F: Oneway ANOVA test, followed by Holm-Sidak's multiple comparison to control. G: Two-way ANOVA test, followed by Holm-Sidak's multiple comparison to baseline