Physiology of cardiomyocyte injury in COVID-19

Abstract

COVID-19 affects multiple organs. Clinical data from the Mount Sinai Health System shows that substantial numbers of COVID-19 patients without prior heart disease develop cardiac dysfunction. How COVID-19 patients develop cardiac disease is not known. We integrate cell biological and physiological analyses of human cardiomyocytes differentiated from human induced pluripotent stem cells (hiPSCs) infected with SARS-CoV-2 in the presence of interleukins, with clinical findings, to investigate plausible mechanisms of cardiac disease in COVID-19 patients. We infected hiPSC-derived cardiomyocytes, from healthy human subjects, with SARS-CoV-2 in the absence and presence of interleukins. We find that interleukin treatment and infection results in disorganization of myofibrils, extracellular release of troponin-I, and reduced and erratic beating. Although interleukins do not increase the extent, they increase the severity of viral infection of cardiomyocytes resulting in cessation of beating. Clinical data from hospitalized patients from the Mount Sinai Health system show that a significant portion of COVID-19 patients without prior history of heart disease, have elevated troponin and interleukin levels. A substantial subset of these patients showed reduced left ventricular function by echocardiography. Our laboratory observations, combined with the clinical data, indicate that direct effects on cardiomyocytes by interleukins and SARS-CoV-2 infection can underlie the heart disease in COVID-19 patients.

Figure 1.. COVID-19 patients without prior history of cardiac disease have elevated troponin I levels, and other clinical characteristics associated with cardiac dysfunction.

Clinical data of COVID-19 positive patients (with encounter data, labs and vital signs, n = 7738) downloaded from Mount Sinai Data Warehouse on July 15^th, 2020. (A) Flow chart: COVID-19 patients for whom Troponin I measurements were available (n= 4228) were further divided into patients with and without prior cardiac disease. Patients having one or more of following three comorbidities or prior histry: 1. CORONARY ARTERY DISEASE, 2. ATRIAL FIBRILLATION 3. HEART FAILURE were binned as patients as “with prior cardiac disease”. Each category was further divided into subgroups with respect to troponin levels using standard clinical cutoffs. 32.2% (1020/3163) of COVID-19 patients without prior history of heart disease have clinically significant elevated (>0.09 ng/ml) levels of troponin I. (B) Patients without prior cardiac disease were classified with respect to kidney function using eGFR values (< or ≥) 30 and binned for comorbidities (Hypertension, Obesity, Diabetes) in the three cohorts with different Troponin I levels. (C) We developed predictive machine learning models to identify clinical features that predict elevated levels of troponin I. The workflow for model development is shown in Figure 1-C-I: After preprocessing, data for patients with COVID-19 with troponin I data (n=4228) were randomly divided in an 80:20 ratios into a prediction model development dataset (n=3382) and an independent retrospective validation dataset (test dataset; n=846). For prediction model training and selection, the development dataset was further randomly split into a 75% training dataset (n=2536) and a 25% holdout dataset (n=846). We ran imputation model on the training set to obtain an optimum missing value imputation cutoff which is 35% for this model (Suppl. fig S1). We used a recursive feature elimination method to obtain the optimum number of features to reach plateau (Supplementary fig S2). We tested two classification algorithms, and the XGBoost (eXtreme Gradient Boosting) classification algorithm performed better than logistic regression (Supplementary fig S3). The final predictive model was validated on the test dataset; n= 846). C-II: Evaluation results for the test set are shown in terms of the ROC curves obtained, as well as their AUC scores, with 95% confidence interval in parentheses. C-III: The top fifteen predictive features identified using the recursive feature elimination method for XGBoost classification algorithms across the three independent sets of 100 runs used to select the most discriminative features, and train the corresponding candidate prediction models; the values in parentheses indicate the number of times the feature was selected as top ranked in the development dataset.

Figure 2. Characterization of hiPSC-derived ventricular cardiomyocytes infected by SARS-CoV-2

hiPSC cells reprogrammed from skin fibroblasts of healthy subjects were differentiated into beating ventricular cardiomyocytes (CMs), and after culturing for 30days were used in these experiments. (A) Expression of ACE 2 protein. By immunoblotting, the expression of ACE-2 at the predicted size of 120kD was identified in cardiomyocytes (cardio) compared to the standard monkey Vero cell line at two different passages (P4 & P14). (B) Immunostaining of CMs for ACE-2 (Green) and Troponin T (Cyan). Although all cells express ACE2, we find only some cells express ACE-2 on the plasma membrane, as indicated by the arrows (left-most panel). The CMs express Troponin T (Cyan) which is seen as ordered myofibrils (middle panel). Overlay of the two-stains is shown in the right panel. The scale bar in the composite image is 50μm. (C) Top panels- Mock infected cardiomyocytes express ACE-2 and Troponin T, nuclei are stained by Hoescht. Level 2 panels - CM treated with 30ng/ml each of IL-6 and IL-1β express ACE-2 and the cells appear larger, and the Troponin T appeared more disorganized. Level 3 panels - CM can be infected with SARS-CoV-2 at a MOI 0.1 as detected by positive immunostaining for viral NP (Red), all infected cells were positive for ACE-2 with some cells having notable amounts of Troponin T disruption. Level −4 panels infected with SARS-CoV-2 in the presence of ILs also show increase in cell diameter and disruption in Troponin T organization. The scale bar in the composite image is for 50μm. (D) The changes in cell diameter with ILs and/or infection were measured using ImageJ. Cell diameter for two different CM lines were measured and plotted, We observe a statistically significant increase in cell size with infection but a larger increase in cell diameter with ILs only or ILs and infection with SARS-CoV-2. (E) To confirm that CM were being productively infected and shedding SARS-CoV-2 into the culture media, we collected supernatants from 3 different CM lines 48 and 72hrs post-infection, with or without ILs and performed a TCID50 plaque assay. We observed that CM lines are infected and the level of infection, as assessed by virus release in to culture medium did not increase with the addition of IL-6 and IL-1β. (F) We counted the numbers of cells stained by N protein antibody as a measure of infectivity. Each point is representative from one well of a 96 well plate with 10,000 CM cells. The bar graph is the average of four different CM lines measuring viral NP protein positive cells over total number of cells. Counting was done in an automated fashion using the InCell Analyzer. ILs do not increase the percentage of CM cells infected by SARS-CoV-2. (G) Three different CM lines were treated with ILs and/or infection with SARS-CoV-2 at a MOI of 0.1 for 72hrs. The supernatants were collected and used for the measurement of levels of Troponin I released into the media. We found that ILs or infection with SARS-CoV-2 did not significantly release Troponin I into the media. However, infection in the presence of ILs significantly increased release of Troponin into the culture media. Comparing IL treatment or Infection only to ILs with infection, we see there was significant increase in Troponin release when we combined ILs with infection. Statistics are ANOVA with Bonferroni multiple comparison test, *p<0.05 and **p<0.01.

Figure 2. Characterization of hiPSC-derived ventricular cardiomyocytes infected by SARS-CoV-2

hiPSC cells reprogrammed from skin fibroblasts of healthy subjects were differentiated into beating ventricular cardiomyocytes (CMs), and after culturing for 30days were used in these experiments. (A) Expression of ACE 2 protein. By immunoblotting, the expression of ACE-2 at the predicted size of 120kD was identified in cardiomyocytes (cardio) compared to the standard monkey Vero cell line at two different passages (P4 & P14). (B) Immunostaining of CMs for ACE-2 (Green) and Troponin T (Cyan). Although all cells express ACE2, we find only some cells express ACE-2 on the plasma membrane, as indicated by the arrows (left-most panel). The CMs express Troponin T (Cyan) which is seen as ordered myofibrils (middle panel). Overlay of the two-stains is shown in the right panel. The scale bar in the composite image is 50μm. (C) Top panels- Mock infected cardiomyocytes express ACE-2 and Troponin T, nuclei are stained by Hoescht. Level 2 panels - CM treated with 30ng/ml each of IL-6 and IL-1β express ACE-2 and the cells appear larger, and the Troponin T appeared more disorganized. Level 3 panels - CM can be infected with SARS-CoV-2 at a MOI 0.1 as detected by positive immunostaining for viral NP (Red), all infected cells were positive for ACE-2 with some cells having notable amounts of Troponin T disruption. Level −4 panels infected with SARS-CoV-2 in the presence of ILs also show increase in cell diameter and disruption in Troponin T organization. The scale bar in the composite image is for 50μm. (D) The changes in cell diameter with ILs and/or infection were measured using ImageJ. Cell diameter for two different CM lines were measured and plotted, We observe a statistically significant increase in cell size with infection but a larger increase in cell diameter with ILs only or ILs and infection with SARS-CoV-2. (E) To confirm that CM were being productively infected and shedding SARS-CoV-2 into the culture media, we collected supernatants from 3 different CM lines 48 and 72hrs post-infection, with or without ILs and performed a TCID50 plaque assay. We observed that CM lines are infected and the level of infection, as assessed by virus release in to culture medium did not increase with the addition of IL-6 and IL-1β. (F) We counted the numbers of cells stained by N protein antibody as a measure of infectivity. Each point is representative from one well of a 96 well plate with 10,000 CM cells. The bar graph is the average of four different CM lines measuring viral NP protein positive cells over total number of cells. Counting was done in an automated fashion using the InCell Analyzer. ILs do not increase the percentage of CM cells infected by SARS-CoV-2. (G) Three different CM lines were treated with ILs and/or infection with SARS-CoV-2 at a MOI of 0.1 for 72hrs. The supernatants were collected and used for the measurement of levels of Troponin I released into the media. We found that ILs or infection with SARS-CoV-2 did not significantly release Troponin I into the media. However, infection in the presence of ILs significantly increased release of Troponin into the culture media. Comparing IL treatment or Infection only to ILs with infection, we see there was significant increase in Troponin release when we combined ILs with infection. Statistics are ANOVA with Bonferroni multiple comparison test, *p<0.05 and **p<0.01.

Figure 2. Characterization of hiPSC-derived ventricular cardiomyocytes infected by SARS-CoV-2

hiPSC cells reprogrammed from skin fibroblasts of healthy subjects were differentiated into beating ventricular cardiomyocytes (CMs), and after culturing for 30days were used in these experiments. (A) Expression of ACE 2 protein. By immunoblotting, the expression of ACE-2 at the predicted size of 120kD was identified in cardiomyocytes (cardio) compared to the standard monkey Vero cell line at two different passages (P4 & P14). (B) Immunostaining of CMs for ACE-2 (Green) and Troponin T (Cyan). Although all cells express ACE2, we find only some cells express ACE-2 on the plasma membrane, as indicated by the arrows (left-most panel). The CMs express Troponin T (Cyan) which is seen as ordered myofibrils (middle panel). Overlay of the two-stains is shown in the right panel. The scale bar in the composite image is 50μm. (C) Top panels- Mock infected cardiomyocytes express ACE-2 and Troponin T, nuclei are stained by Hoescht. Level 2 panels - CM treated with 30ng/ml each of IL-6 and IL-1β express ACE-2 and the cells appear larger, and the Troponin T appeared more disorganized. Level 3 panels - CM can be infected with SARS-CoV-2 at a MOI 0.1 as detected by positive immunostaining for viral NP (Red), all infected cells were positive for ACE-2 with some cells having notable amounts of Troponin T disruption. Level −4 panels infected with SARS-CoV-2 in the presence of ILs also show increase in cell diameter and disruption in Troponin T organization. The scale bar in the composite image is for 50μm. (D) The changes in cell diameter with ILs and/or infection were measured using ImageJ. Cell diameter for two different CM lines were measured and plotted, We observe a statistically significant increase in cell size with infection but a larger increase in cell diameter with ILs only or ILs and infection with SARS-CoV-2. (E) To confirm that CM were being productively infected and shedding SARS-CoV-2 into the culture media, we collected supernatants from 3 different CM lines 48 and 72hrs post-infection, with or without ILs and performed a TCID50 plaque assay. We observed that CM lines are infected and the level of infection, as assessed by virus release in to culture medium did not increase with the addition of IL-6 and IL-1β. (F) We counted the numbers of cells stained by N protein antibody as a measure of infectivity. Each point is representative from one well of a 96 well plate with 10,000 CM cells. The bar graph is the average of four different CM lines measuring viral NP protein positive cells over total number of cells. Counting was done in an automated fashion using the InCell Analyzer. ILs do not increase the percentage of CM cells infected by SARS-CoV-2. (G) Three different CM lines were treated with ILs and/or infection with SARS-CoV-2 at a MOI of 0.1 for 72hrs. The supernatants were collected and used for the measurement of levels of Troponin I released into the media. We found that ILs or infection with SARS-CoV-2 did not significantly release Troponin I into the media. However, infection in the presence of ILs significantly increased release of Troponin into the culture media. Comparing IL treatment or Infection only to ILs with infection, we see there was significant increase in Troponin release when we combined ILs with infection. Statistics are ANOVA with Bonferroni multiple comparison test, *p<0.05 and **p<0.01.

Figure 2. Characterization of hiPSC-derived ventricular cardiomyocytes infected by SARS-CoV-2

hiPSC cells reprogrammed from skin fibroblasts of healthy subjects were differentiated into beating ventricular cardiomyocytes (CMs), and after culturing for 30days were used in these experiments. (A) Expression of ACE 2 protein. By immunoblotting, the expression of ACE-2 at the predicted size of 120kD was identified in cardiomyocytes (cardio) compared to the standard monkey Vero cell line at two different passages (P4 & P14). (B) Immunostaining of CMs for ACE-2 (Green) and Troponin T (Cyan). Although all cells express ACE2, we find only some cells express ACE-2 on the plasma membrane, as indicated by the arrows (left-most panel). The CMs express Troponin T (Cyan) which is seen as ordered myofibrils (middle panel). Overlay of the two-stains is shown in the right panel. The scale bar in the composite image is 50μm. (C) Top panels- Mock infected cardiomyocytes express ACE-2 and Troponin T, nuclei are stained by Hoescht. Level 2 panels - CM treated with 30ng/ml each of IL-6 and IL-1β express ACE-2 and the cells appear larger, and the Troponin T appeared more disorganized. Level 3 panels - CM can be infected with SARS-CoV-2 at a MOI 0.1 as detected by positive immunostaining for viral NP (Red), all infected cells were positive for ACE-2 with some cells having notable amounts of Troponin T disruption. Level −4 panels infected with SARS-CoV-2 in the presence of ILs also show increase in cell diameter and disruption in Troponin T organization. The scale bar in the composite image is for 50μm. (D) The changes in cell diameter with ILs and/or infection were measured using ImageJ. Cell diameter for two different CM lines were measured and plotted, We observe a statistically significant increase in cell size with infection but a larger increase in cell diameter with ILs only or ILs and infection with SARS-CoV-2. (E) To confirm that CM were being productively infected and shedding SARS-CoV-2 into the culture media, we collected supernatants from 3 different CM lines 48 and 72hrs post-infection, with or without ILs and performed a TCID50 plaque assay. We observed that CM lines are infected and the level of infection, as assessed by virus release in to culture medium did not increase with the addition of IL-6 and IL-1β. (F) We counted the numbers of cells stained by N protein antibody as a measure of infectivity. Each point is representative from one well of a 96 well plate with 10,000 CM cells. The bar graph is the average of four different CM lines measuring viral NP protein positive cells over total number of cells. Counting was done in an automated fashion using the InCell Analyzer. ILs do not increase the percentage of CM cells infected by SARS-CoV-2. (G) Three different CM lines were treated with ILs and/or infection with SARS-CoV-2 at a MOI of 0.1 for 72hrs. The supernatants were collected and used for the measurement of levels of Troponin I released into the media. We found that ILs or infection with SARS-CoV-2 did not significantly release Troponin I into the media. However, infection in the presence of ILs significantly increased release of Troponin into the culture media. Comparing IL treatment or Infection only to ILs with infection, we see there was significant increase in Troponin release when we combined ILs with infection. Statistics are ANOVA with Bonferroni multiple comparison test, *p<0.05 and **p<0.01.

Figure 2. Characterization of hiPSC-derived ventricular cardiomyocytes infected by SARS-CoV-2

hiPSC cells reprogrammed from skin fibroblasts of healthy subjects were differentiated into beating ventricular cardiomyocytes (CMs), and after culturing for 30days were used in these experiments. (A) Expression of ACE 2 protein. By immunoblotting, the expression of ACE-2 at the predicted size of 120kD was identified in cardiomyocytes (cardio) compared to the standard monkey Vero cell line at two different passages (P4 & P14). (B) Immunostaining of CMs for ACE-2 (Green) and Troponin T (Cyan). Although all cells express ACE2, we find only some cells express ACE-2 on the plasma membrane, as indicated by the arrows (left-most panel). The CMs express Troponin T (Cyan) which is seen as ordered myofibrils (middle panel). Overlay of the two-stains is shown in the right panel. The scale bar in the composite image is 50μm. (C) Top panels- Mock infected cardiomyocytes express ACE-2 and Troponin T, nuclei are stained by Hoescht. Level 2 panels - CM treated with 30ng/ml each of IL-6 and IL-1β express ACE-2 and the cells appear larger, and the Troponin T appeared more disorganized. Level 3 panels - CM can be infected with SARS-CoV-2 at a MOI 0.1 as detected by positive immunostaining for viral NP (Red), all infected cells were positive for ACE-2 with some cells having notable amounts of Troponin T disruption. Level −4 panels infected with SARS-CoV-2 in the presence of ILs also show increase in cell diameter and disruption in Troponin T organization. The scale bar in the composite image is for 50μm. (D) The changes in cell diameter with ILs and/or infection were measured using ImageJ. Cell diameter for two different CM lines were measured and plotted, We observe a statistically significant increase in cell size with infection but a larger increase in cell diameter with ILs only or ILs and infection with SARS-CoV-2. (E) To confirm that CM were being productively infected and shedding SARS-CoV-2 into the culture media, we collected supernatants from 3 different CM lines 48 and 72hrs post-infection, with or without ILs and performed a TCID50 plaque assay. We observed that CM lines are infected and the level of infection, as assessed by virus release in to culture medium did not increase with the addition of IL-6 and IL-1β. (F) We counted the numbers of cells stained by N protein antibody as a measure of infectivity. Each point is representative from one well of a 96 well plate with 10,000 CM cells. The bar graph is the average of four different CM lines measuring viral NP protein positive cells over total number of cells. Counting was done in an automated fashion using the InCell Analyzer. ILs do not increase the percentage of CM cells infected by SARS-CoV-2. (G) Three different CM lines were treated with ILs and/or infection with SARS-CoV-2 at a MOI of 0.1 for 72hrs. The supernatants were collected and used for the measurement of levels of Troponin I released into the media. We found that ILs or infection with SARS-CoV-2 did not significantly release Troponin I into the media. However, infection in the presence of ILs significantly increased release of Troponin into the culture media. Comparing IL treatment or Infection only to ILs with infection, we see there was significant increase in Troponin release when we combined ILs with infection. Statistics are ANOVA with Bonferroni multiple comparison test, *p<0.05 and **p<0.01.

Figure 2. Characterization of hiPSC-derived ventricular cardiomyocytes infected by SARS-CoV-2

hiPSC cells reprogrammed from skin fibroblasts of healthy subjects were differentiated into beating ventricular cardiomyocytes (CMs), and after culturing for 30days were used in these experiments. (A) Expression of ACE 2 protein. By immunoblotting, the expression of ACE-2 at the predicted size of 120kD was identified in cardiomyocytes (cardio) compared to the standard monkey Vero cell line at two different passages (P4 & P14). (B) Immunostaining of CMs for ACE-2 (Green) and Troponin T (Cyan). Although all cells express ACE2, we find only some cells express ACE-2 on the plasma membrane, as indicated by the arrows (left-most panel). The CMs express Troponin T (Cyan) which is seen as ordered myofibrils (middle panel). Overlay of the two-stains is shown in the right panel. The scale bar in the composite image is 50μm. (C) Top panels- Mock infected cardiomyocytes express ACE-2 and Troponin T, nuclei are stained by Hoescht. Level 2 panels - CM treated with 30ng/ml each of IL-6 and IL-1β express ACE-2 and the cells appear larger, and the Troponin T appeared more disorganized. Level 3 panels - CM can be infected with SARS-CoV-2 at a MOI 0.1 as detected by positive immunostaining for viral NP (Red), all infected cells were positive for ACE-2 with some cells having notable amounts of Troponin T disruption. Level −4 panels infected with SARS-CoV-2 in the presence of ILs also show increase in cell diameter and disruption in Troponin T organization. The scale bar in the composite image is for 50μm. (D) The changes in cell diameter with ILs and/or infection were measured using ImageJ. Cell diameter for two different CM lines were measured and plotted, We observe a statistically significant increase in cell size with infection but a larger increase in cell diameter with ILs only or ILs and infection with SARS-CoV-2. (E) To confirm that CM were being productively infected and shedding SARS-CoV-2 into the culture media, we collected supernatants from 3 different CM lines 48 and 72hrs post-infection, with or without ILs and performed a TCID50 plaque assay. We observed that CM lines are infected and the level of infection, as assessed by virus release in to culture medium did not increase with the addition of IL-6 and IL-1β. (F) We counted the numbers of cells stained by N protein antibody as a measure of infectivity. Each point is representative from one well of a 96 well plate with 10,000 CM cells. The bar graph is the average of four different CM lines measuring viral NP protein positive cells over total number of cells. Counting was done in an automated fashion using the InCell Analyzer. ILs do not increase the percentage of CM cells infected by SARS-CoV-2. (G) Three different CM lines were treated with ILs and/or infection with SARS-CoV-2 at a MOI of 0.1 for 72hrs. The supernatants were collected and used for the measurement of levels of Troponin I released into the media. We found that ILs or infection with SARS-CoV-2 did not significantly release Troponin I into the media. However, infection in the presence of ILs significantly increased release of Troponin into the culture media. Comparing IL treatment or Infection only to ILs with infection, we see there was significant increase in Troponin release when we combined ILs with infection. Statistics are ANOVA with Bonferroni multiple comparison test, *p<0.05 and **p<0.01.

Figure 2. Characterization of hiPSC-derived ventricular cardiomyocytes infected by SARS-CoV-2

hiPSC cells reprogrammed from skin fibroblasts of healthy subjects were differentiated into beating ventricular cardiomyocytes (CMs), and after culturing for 30days were used in these experiments. (A) Expression of ACE 2 protein. By immunoblotting, the expression of ACE-2 at the predicted size of 120kD was identified in cardiomyocytes (cardio) compared to the standard monkey Vero cell line at two different passages (P4 & P14). (B) Immunostaining of CMs for ACE-2 (Green) and Troponin T (Cyan). Although all cells express ACE2, we find only some cells express ACE-2 on the plasma membrane, as indicated by the arrows (left-most panel). The CMs express Troponin T (Cyan) which is seen as ordered myofibrils (middle panel). Overlay of the two-stains is shown in the right panel. The scale bar in the composite image is 50μm. (C) Top panels- Mock infected cardiomyocytes express ACE-2 and Troponin T, nuclei are stained by Hoescht. Level 2 panels - CM treated with 30ng/ml each of IL-6 and IL-1β express ACE-2 and the cells appear larger, and the Troponin T appeared more disorganized. Level 3 panels - CM can be infected with SARS-CoV-2 at a MOI 0.1 as detected by positive immunostaining for viral NP (Red), all infected cells were positive for ACE-2 with some cells having notable amounts of Troponin T disruption. Level −4 panels infected with SARS-CoV-2 in the presence of ILs also show increase in cell diameter and disruption in Troponin T organization. The scale bar in the composite image is for 50μm. (D) The changes in cell diameter with ILs and/or infection were measured using ImageJ. Cell diameter for two different CM lines were measured and plotted, We observe a statistically significant increase in cell size with infection but a larger increase in cell diameter with ILs only or ILs and infection with SARS-CoV-2. (E) To confirm that CM were being productively infected and shedding SARS-CoV-2 into the culture media, we collected supernatants from 3 different CM lines 48 and 72hrs post-infection, with or without ILs and performed a TCID50 plaque assay. We observed that CM lines are infected and the level of infection, as assessed by virus release in to culture medium did not increase with the addition of IL-6 and IL-1β. (F) We counted the numbers of cells stained by N protein antibody as a measure of infectivity. Each point is representative from one well of a 96 well plate with 10,000 CM cells. The bar graph is the average of four different CM lines measuring viral NP protein positive cells over total number of cells. Counting was done in an automated fashion using the InCell Analyzer. ILs do not increase the percentage of CM cells infected by SARS-CoV-2. (G) Three different CM lines were treated with ILs and/or infection with SARS-CoV-2 at a MOI of 0.1 for 72hrs. The supernatants were collected and used for the measurement of levels of Troponin I released into the media. We found that ILs or infection with SARS-CoV-2 did not significantly release Troponin I into the media. However, infection in the presence of ILs significantly increased release of Troponin into the culture media. Comparing IL treatment or Infection only to ILs with infection, we see there was significant increase in Troponin release when we combined ILs with infection. Statistics are ANOVA with Bonferroni multiple comparison test, *p<0.05 and **p<0.01.

Figure 3. Effect of SARS-CoV-2 infection on beating of ventricular cardiomyocytes in cultures, and cardiac output in COVID-19 patients.

(A) Three different CM lines were recorded for their beating in culture with ILs and/or infection in a biosafety level-3 facility. These CM lines were in culture for 45–60days. Onto the movies that we recorded, we juxtaposed a 5X5 grid to count the number of beats per min, for each cell line at 48hrs post-infection. The number in each of the 25 grids (beats per minute) is the average of two experiments for one representative CM line, for each condition. We observed that infection alone can slow down the beating, as could ILs treatment. The strongest attenuation of beating was observed with SARS- CoV-2 infection in the presence of ILs. Movie 1 shows the beating of the CMs under various conditions. (B) Summary of counts of the beating in three different 3 CM lines are shown Beat measurement in each area of the grid is represented as an open dot and mean and SEM are shown. Asterisks indicate significance at p < 0.001 by ANOVA with Bonferroni multiple comparison test. Though all conditions significantly attenuated beating, the most robust effect was combining ILs with infection with SARS-CoV-2. C) COVID-19 patients without prior cardiac disease were divided into two groups, one with normal LVEF (N=165) and those with decreased LVEF<51 (N=41). We plotted the peak Troponin I (ng/ml) using a natural log scale (LN) and peak IL-6 (pg/ml). The red line indicates where Troponin levels are at 0.1 ng/ml (LN = −2.3) and the green line at 1 ng/ml (LN = 0). For the group with LVEF<51, 20 out of 41 (50%) have Troponin levels >1ng/ml, while for those with normal LVEF it is 22/165 (13.3%). Another group of patients with LVEF<51, 6/41 (14.6%), have troponin I levels greater than 20ng/ml (LN = 3); none of the patients with normal LVEF are above 20ng/ml. For troponin I, the difference is very significant by t-test, p<0.0001, when comparing normal LVEF to the LVEF<51 group. (D-F) Echocardiogram clips showing Diastolic and Systolic states. (D) Normal demonstrates an apical 4-chamber view (after administration of an ultrasonic enhancing agent) from a transthoracic echocardiogram obtained from a COVID-19 patient. The findings are consistent with preserved left ventricular ejection fraction and no regional wall motion abnormalities (diastolic and systolic frames). The end-diastolic and end-systolic volumes are 104ml and 35ml, respectively with a left ventricular ejection fraction of 66%; (E) Regional wall motion abnormality (RWMA) demonstrates an apical 3-chamber view (after administration of an ultrasonic enhancing agent) from a transthoracic echocardiogram obtained from a COVID-19 patient. The findings are consistent with basal and mid infero-lateral wall hypokinesis despite preserved left ventricular ejection fraction (diastolic and systolic frames). The RWMA is highlighted by the red oval. The end-diastolic and end-systolic volumes are 102ml and 50ml, respectively with a left ventricular ejection fraction of 51%; (F) diffuse demonstrates an apical 4-chamber view (after administration of an ultrasonic enhancing agent) from a transthoracic echocardiogram obtained from a COVID-19 patient. The findings are consistent with diffuse left ventricular wall hypokinesis and mildly decreased left ventricular ejection fraction. The end-diastolic and end-systolic volumes are 160ml and 90ml, respectively with a left ventricular ejection fraction of 44%. (G) Summary of cardiac dysfunction effects in COVID-19 patients without prior cardiac disease. Data are from two different hospitals within the Mount Sinai Health System. Data indicate that COVID-19 patients include those with normal left ventricle ejection fraction (LVEF) and with newly reduced LVEF (LVEF<51), and we have the averaged peak of IL-6 and Troponin I for these patients. The t-test for Troponin between the two groups (Normal LVEF and newly reduced LVEF) was significantly different as determined by t-test.

Figure 3. Effect of SARS-CoV-2 infection on beating of ventricular cardiomyocytes in cultures, and cardiac output in COVID-19 patients.

(A) Three different CM lines were recorded for their beating in culture with ILs and/or infection in a biosafety level-3 facility. These CM lines were in culture for 45–60days. Onto the movies that we recorded, we juxtaposed a 5X5 grid to count the number of beats per min, for each cell line at 48hrs post-infection. The number in each of the 25 grids (beats per minute) is the average of two experiments for one representative CM line, for each condition. We observed that infection alone can slow down the beating, as could ILs treatment. The strongest attenuation of beating was observed with SARS- CoV-2 infection in the presence of ILs. Movie 1 shows the beating of the CMs under various conditions. (B) Summary of counts of the beating in three different 3 CM lines are shown Beat measurement in each area of the grid is represented as an open dot and mean and SEM are shown. Asterisks indicate significance at p < 0.001 by ANOVA with Bonferroni multiple comparison test. Though all conditions significantly attenuated beating, the most robust effect was combining ILs with infection with SARS-CoV-2. C) COVID-19 patients without prior cardiac disease were divided into two groups, one with normal LVEF (N=165) and those with decreased LVEF<51 (N=41). We plotted the peak Troponin I (ng/ml) using a natural log scale (LN) and peak IL-6 (pg/ml). The red line indicates where Troponin levels are at 0.1 ng/ml (LN = −2.3) and the green line at 1 ng/ml (LN = 0). For the group with LVEF<51, 20 out of 41 (50%) have Troponin levels >1ng/ml, while for those with normal LVEF it is 22/165 (13.3%). Another group of patients with LVEF<51, 6/41 (14.6%), have troponin I levels greater than 20ng/ml (LN = 3); none of the patients with normal LVEF are above 20ng/ml. For troponin I, the difference is very significant by t-test, p<0.0001, when comparing normal LVEF to the LVEF<51 group. (D-F) Echocardiogram clips showing Diastolic and Systolic states. (D) Normal demonstrates an apical 4-chamber view (after administration of an ultrasonic enhancing agent) from a transthoracic echocardiogram obtained from a COVID-19 patient. The findings are consistent with preserved left ventricular ejection fraction and no regional wall motion abnormalities (diastolic and systolic frames). The end-diastolic and end-systolic volumes are 104ml and 35ml, respectively with a left ventricular ejection fraction of 66%; (E) Regional wall motion abnormality (RWMA) demonstrates an apical 3-chamber view (after administration of an ultrasonic enhancing agent) from a transthoracic echocardiogram obtained from a COVID-19 patient. The findings are consistent with basal and mid infero-lateral wall hypokinesis despite preserved left ventricular ejection fraction (diastolic and systolic frames). The RWMA is highlighted by the red oval. The end-diastolic and end-systolic volumes are 102ml and 50ml, respectively with a left ventricular ejection fraction of 51%; (F) diffuse demonstrates an apical 4-chamber view (after administration of an ultrasonic enhancing agent) from a transthoracic echocardiogram obtained from a COVID-19 patient. The findings are consistent with diffuse left ventricular wall hypokinesis and mildly decreased left ventricular ejection fraction. The end-diastolic and end-systolic volumes are 160ml and 90ml, respectively with a left ventricular ejection fraction of 44%. (G) Summary of cardiac dysfunction effects in COVID-19 patients without prior cardiac disease. Data are from two different hospitals within the Mount Sinai Health System. Data indicate that COVID-19 patients include those with normal left ventricle ejection fraction (LVEF) and with newly reduced LVEF (LVEF<51), and we have the averaged peak of IL-6 and Troponin I for these patients. The t-test for Troponin between the two groups (Normal LVEF and newly reduced LVEF) was significantly different as determined by t-test.

Figure 3. Effect of SARS-CoV-2 infection on beating of ventricular cardiomyocytes in cultures, and cardiac output in COVID-19 patients.

(A) Three different CM lines were recorded for their beating in culture with ILs and/or infection in a biosafety level-3 facility. These CM lines were in culture for 45–60days. Onto the movies that we recorded, we juxtaposed a 5X5 grid to count the number of beats per min, for each cell line at 48hrs post-infection. The number in each of the 25 grids (beats per minute) is the average of two experiments for one representative CM line, for each condition. We observed that infection alone can slow down the beating, as could ILs treatment. The strongest attenuation of beating was observed with SARS- CoV-2 infection in the presence of ILs. Movie 1 shows the beating of the CMs under various conditions. (B) Summary of counts of the beating in three different 3 CM lines are shown Beat measurement in each area of the grid is represented as an open dot and mean and SEM are shown. Asterisks indicate significance at p < 0.001 by ANOVA with Bonferroni multiple comparison test. Though all conditions significantly attenuated beating, the most robust effect was combining ILs with infection with SARS-CoV-2. C) COVID-19 patients without prior cardiac disease were divided into two groups, one with normal LVEF (N=165) and those with decreased LVEF<51 (N=41). We plotted the peak Troponin I (ng/ml) using a natural log scale (LN) and peak IL-6 (pg/ml). The red line indicates where Troponin levels are at 0.1 ng/ml (LN = −2.3) and the green line at 1 ng/ml (LN = 0). For the group with LVEF<51, 20 out of 41 (50%) have Troponin levels >1ng/ml, while for those with normal LVEF it is 22/165 (13.3%). Another group of patients with LVEF<51, 6/41 (14.6%), have troponin I levels greater than 20ng/ml (LN = 3); none of the patients with normal LVEF are above 20ng/ml. For troponin I, the difference is very significant by t-test, p<0.0001, when comparing normal LVEF to the LVEF<51 group. (D-F) Echocardiogram clips showing Diastolic and Systolic states. (D) Normal demonstrates an apical 4-chamber view (after administration of an ultrasonic enhancing agent) from a transthoracic echocardiogram obtained from a COVID-19 patient. The findings are consistent with preserved left ventricular ejection fraction and no regional wall motion abnormalities (diastolic and systolic frames). The end-diastolic and end-systolic volumes are 104ml and 35ml, respectively with a left ventricular ejection fraction of 66%; (E) Regional wall motion abnormality (RWMA) demonstrates an apical 3-chamber view (after administration of an ultrasonic enhancing agent) from a transthoracic echocardiogram obtained from a COVID-19 patient. The findings are consistent with basal and mid infero-lateral wall hypokinesis despite preserved left ventricular ejection fraction (diastolic and systolic frames). The RWMA is highlighted by the red oval. The end-diastolic and end-systolic volumes are 102ml and 50ml, respectively with a left ventricular ejection fraction of 51%; (F) diffuse demonstrates an apical 4-chamber view (after administration of an ultrasonic enhancing agent) from a transthoracic echocardiogram obtained from a COVID-19 patient. The findings are consistent with diffuse left ventricular wall hypokinesis and mildly decreased left ventricular ejection fraction. The end-diastolic and end-systolic volumes are 160ml and 90ml, respectively with a left ventricular ejection fraction of 44%. (G) Summary of cardiac dysfunction effects in COVID-19 patients without prior cardiac disease. Data are from two different hospitals within the Mount Sinai Health System. Data indicate that COVID-19 patients include those with normal left ventricle ejection fraction (LVEF) and with newly reduced LVEF (LVEF<51), and we have the averaged peak of IL-6 and Troponin I for these patients. The t-test for Troponin between the two groups (Normal LVEF and newly reduced LVEF) was significantly different as determined by t-test.

Figure 3. Effect of SARS-CoV-2 infection on beating of ventricular cardiomyocytes in cultures, and cardiac output in COVID-19 patients.

(A) Three different CM lines were recorded for their beating in culture with ILs and/or infection in a biosafety level-3 facility. These CM lines were in culture for 45–60days. Onto the movies that we recorded, we juxtaposed a 5X5 grid to count the number of beats per min, for each cell line at 48hrs post-infection. The number in each of the 25 grids (beats per minute) is the average of two experiments for one representative CM line, for each condition. We observed that infection alone can slow down the beating, as could ILs treatment. The strongest attenuation of beating was observed with SARS- CoV-2 infection in the presence of ILs. Movie 1 shows the beating of the CMs under various conditions. (B) Summary of counts of the beating in three different 3 CM lines are shown Beat measurement in each area of the grid is represented as an open dot and mean and SEM are shown. Asterisks indicate significance at p < 0.001 by ANOVA with Bonferroni multiple comparison test. Though all conditions significantly attenuated beating, the most robust effect was combining ILs with infection with SARS-CoV-2. C) COVID-19 patients without prior cardiac disease were divided into two groups, one with normal LVEF (N=165) and those with decreased LVEF<51 (N=41). We plotted the peak Troponin I (ng/ml) using a natural log scale (LN) and peak IL-6 (pg/ml). The red line indicates where Troponin levels are at 0.1 ng/ml (LN = −2.3) and the green line at 1 ng/ml (LN = 0). For the group with LVEF<51, 20 out of 41 (50%) have Troponin levels >1ng/ml, while for those with normal LVEF it is 22/165 (13.3%). Another group of patients with LVEF<51, 6/41 (14.6%), have troponin I levels greater than 20ng/ml (LN = 3); none of the patients with normal LVEF are above 20ng/ml. For troponin I, the difference is very significant by t-test, p<0.0001, when comparing normal LVEF to the LVEF<51 group. (D-F) Echocardiogram clips showing Diastolic and Systolic states. (D) Normal demonstrates an apical 4-chamber view (after administration of an ultrasonic enhancing agent) from a transthoracic echocardiogram obtained from a COVID-19 patient. The findings are consistent with preserved left ventricular ejection fraction and no regional wall motion abnormalities (diastolic and systolic frames). The end-diastolic and end-systolic volumes are 104ml and 35ml, respectively with a left ventricular ejection fraction of 66%; (E) Regional wall motion abnormality (RWMA) demonstrates an apical 3-chamber view (after administration of an ultrasonic enhancing agent) from a transthoracic echocardiogram obtained from a COVID-19 patient. The findings are consistent with basal and mid infero-lateral wall hypokinesis despite preserved left ventricular ejection fraction (diastolic and systolic frames). The RWMA is highlighted by the red oval. The end-diastolic and end-systolic volumes are 102ml and 50ml, respectively with a left ventricular ejection fraction of 51%; (F) diffuse demonstrates an apical 4-chamber view (after administration of an ultrasonic enhancing agent) from a transthoracic echocardiogram obtained from a COVID-19 patient. The findings are consistent with diffuse left ventricular wall hypokinesis and mildly decreased left ventricular ejection fraction. The end-diastolic and end-systolic volumes are 160ml and 90ml, respectively with a left ventricular ejection fraction of 44%. (G) Summary of cardiac dysfunction effects in COVID-19 patients without prior cardiac disease. Data are from two different hospitals within the Mount Sinai Health System. Data indicate that COVID-19 patients include those with normal left ventricle ejection fraction (LVEF) and with newly reduced LVEF (LVEF<51), and we have the averaged peak of IL-6 and Troponin I for these patients. The t-test for Troponin between the two groups (Normal LVEF and newly reduced LVEF) was significantly different as determined by t-test.

Figure 3. Effect of SARS-CoV-2 infection on beating of ventricular cardiomyocytes in cultures, and cardiac output in COVID-19 patients.

(A) Three different CM lines were recorded for their beating in culture with ILs and/or infection in a biosafety level-3 facility. These CM lines were in culture for 45–60days. Onto the movies that we recorded, we juxtaposed a 5X5 grid to count the number of beats per min, for each cell line at 48hrs post-infection. The number in each of the 25 grids (beats per minute) is the average of two experiments for one representative CM line, for each condition. We observed that infection alone can slow down the beating, as could ILs treatment. The strongest attenuation of beating was observed with SARS- CoV-2 infection in the presence of ILs. Movie 1 shows the beating of the CMs under various conditions. (B) Summary of counts of the beating in three different 3 CM lines are shown Beat measurement in each area of the grid is represented as an open dot and mean and SEM are shown. Asterisks indicate significance at p < 0.001 by ANOVA with Bonferroni multiple comparison test. Though all conditions significantly attenuated beating, the most robust effect was combining ILs with infection with SARS-CoV-2. C) COVID-19 patients without prior cardiac disease were divided into two groups, one with normal LVEF (N=165) and those with decreased LVEF<51 (N=41). We plotted the peak Troponin I (ng/ml) using a natural log scale (LN) and peak IL-6 (pg/ml). The red line indicates where Troponin levels are at 0.1 ng/ml (LN = −2.3) and the green line at 1 ng/ml (LN = 0). For the group with LVEF<51, 20 out of 41 (50%) have Troponin levels >1ng/ml, while for those with normal LVEF it is 22/165 (13.3%). Another group of patients with LVEF<51, 6/41 (14.6%), have troponin I levels greater than 20ng/ml (LN = 3); none of the patients with normal LVEF are above 20ng/ml. For troponin I, the difference is very significant by t-test, p<0.0001, when comparing normal LVEF to the LVEF<51 group. (D-F) Echocardiogram clips showing Diastolic and Systolic states. (D) Normal demonstrates an apical 4-chamber view (after administration of an ultrasonic enhancing agent) from a transthoracic echocardiogram obtained from a COVID-19 patient. The findings are consistent with preserved left ventricular ejection fraction and no regional wall motion abnormalities (diastolic and systolic frames). The end-diastolic and end-systolic volumes are 104ml and 35ml, respectively with a left ventricular ejection fraction of 66%; (E) Regional wall motion abnormality (RWMA) demonstrates an apical 3-chamber view (after administration of an ultrasonic enhancing agent) from a transthoracic echocardiogram obtained from a COVID-19 patient. The findings are consistent with basal and mid infero-lateral wall hypokinesis despite preserved left ventricular ejection fraction (diastolic and systolic frames). The RWMA is highlighted by the red oval. The end-diastolic and end-systolic volumes are 102ml and 50ml, respectively with a left ventricular ejection fraction of 51%; (F) diffuse demonstrates an apical 4-chamber view (after administration of an ultrasonic enhancing agent) from a transthoracic echocardiogram obtained from a COVID-19 patient. The findings are consistent with diffuse left ventricular wall hypokinesis and mildly decreased left ventricular ejection fraction. The end-diastolic and end-systolic volumes are 160ml and 90ml, respectively with a left ventricular ejection fraction of 44%. (G) Summary of cardiac dysfunction effects in COVID-19 patients without prior cardiac disease. Data are from two different hospitals within the Mount Sinai Health System. Data indicate that COVID-19 patients include those with normal left ventricle ejection fraction (LVEF) and with newly reduced LVEF (LVEF<51), and we have the averaged peak of IL-6 and Troponin I for these patients. The t-test for Troponin between the two groups (Normal LVEF and newly reduced LVEF) was significantly different as determined by t-test.