Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) are susceptible to SARS-CoV-2 infection

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Researchers from Indiana University School of Medicine have in a new study discovered that SARS-CoV-2 Nsp6, Nsp8 and M genes compromise the transcriptome of human pluripotent stem cell-derived cardiomyocytes.

The study findings on the whole uncovered the detrimental impacts of SARS-CoV-2 genes Nsp6, Nsp8 and M on the whole transcriptome and interactome of hPSC-CMs, defined the crucial role of ATP level reduced by SARS-CoV-2 genes in CM death and functional abnormalities, and explored the potentially pharmaceutical approaches to ameliorate SARS-CoV-2 genes-induced CM injury and abnormalities.
 
The study findings were published on a preprint server are currently being peer reviewed. https://www.biorxiv.org/content/10.1101/2022.01.20.477147v1

SARS-CoV-2 virus infects and enters human cells through binding to three membrane proteins, including the Angiotensin Converting Enzyme 2 (ACE2), Transmembrane Serine Protease 2 (TMPRSS2)(10, 11) and Transmembrane Serine Protease 4 (TMPRSS4)(12).

The high affinity of SARS-CoV-2 spike (S) protein with ACE2 makes human organs/tissues with high level of ACE2 expression as the primary targets attacked by SARS-CoV-2, such as the lung, small intestine, testis, kidney and heart (13). Recently, human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) were naturally utilized as an in vitro platform to evaluate the pathological effects of SARS-CoV-2 on human heart muscle cells (14).

SARS-CoV-2 infects human CMs through ACE2. SARS-CoV-2- infected hPSC-CMs exhibited prominently increased cell death (15, 16), fractionated sarcomeres (17), abnormal electrical and mechanical functions (18) and inflammation (15), which recapitulated the CM injuries in COVID-19 patients with myocarditis (9).

SARS-CoV-2 genome encodes up to 27 genes. Currently, the molecular mechanisms, by which SARS-CoV-2 genes interact with host protein networks to influence survival and functions of human CMs, still largely remain elusive.

In this study, three SARS-CoV-2 viral coding genes, Nsp6, Nsp8 and M, were overexpressed in hPSCs (human ESCs and iPSCs) derived CMs. The global impacts of SARS-CoV-2 viral genes on the transcriptome of hPSC-CMs were determined by whole mRNA-seq. We found Nsp6, Nsp8 or M overexpression in hPSC-CMs and SARS-CoV-2 viral infection concordantly affected the transcriptome of hPSC-CMs, with activated cellular injury and immune signaling and reduced cardiac function pathways.

Nsp6, Nsp8 or M overexpression in hPSC-CMs prominently increased apoptosis, and compromised calcium handling and electrical properties. Genome-wide interactome analysis found Nsp6, Nsp8 and M could all interact with proteins of the host hPSC-CMs, particularly ATPase subunits, which led to significantly reduced ATP level.

Given that reduced ATP level could impair intracellular Ca2+ signaling and contractility of CMs, we then tested pharmaceutical strategies to enhance the cellular ATP levels of hPSC-CMs overexpressing Nsp6, Nsp8 or M. Two FDA-approved drugs, ivermectin and meclizine, significantly reduced cell death and dysfunctions of hPSC-CMs with overexpression of SARS-CoV-2 genes.

Overall, SARS-CoV-2 genes Nsp6, Nsp8 and M exhibited detrimental effects on the whole transcriptome and interactome of hPSC-CMs, and the reduced cellular ATP level plays a key role in SARS-CoV-2 genes-induced CM death and abnormalities.

Discussion

Understanding the cardiac manifestations in patients with SARS-CoV-2 infection is critical for health care of acute and post-acute COVID-19 patients (39, 40). In this study, we reveal the genome-wide responses of host hPSC-CMs to SARS-CoV-2 viral genes Nsp6, Nsp8 and M in whole transcriptome and interactome. Our findings uncover the compromised cellular ATP level of hPSC-CMs which overexpress Nsp6, Nsp8 or M.

FDA-approved drugs, ivermectin and meclizine, could sustain the cellular ATP level to attenuate apoptosis and dysfunctions of hPSC-CMs upon Nsp6, Nsp8 or M overexpression. Our results suggest that the impaired ATP hemostasis might play a vital role in SARS-CoV-2 gene-induced CM injury in the heart, and possibly in other organs/tissues that are targets of SARS-CoV-2 as well.

Notably, although both Nsp6OE, Nsp8OE and MOE and SARS-CoV-2 infection led to concordant transcriptomic changes of hPSC-CMs (Figures 3B-C), ∼70% DEGs were solely induced by SARS-CoV-2 virus (Figure 3G-H), suggesting that the other SARS-CoV-2 genes might also affect the transcriptome of host human CMs via different targets or under different mechanisms.

Since the unique pathways induced by SARS-CoV-2 virus were related to apoptosis, gene transcription and multiple metabolic processes, these results imply that at least some of other SARS-CoV-2 genes might also contribute to CM injury and affect the metabolism of host human CMs.

Multiple clinical studies reported that myocardial dysfunctions and injuries were commonly found in COVID-19 patients, and positively contributed to the overall mortality (3-5,22,41). Recently, Bailey et al.(9) reported that SARS-CoV-2 can directly and specifically infect CMs within the hearts of COVID-19 patients. Myocardial tissues from 4 autopsy of COVID-19 patients with clinical myocarditis were examined. In all autopsies, CM infection was identified by intramyocyte expressions of viral RNA and protein, and macrophage infiltration associated with areas of myocyte cell death. This study provided direct evidence for CM infection in the COVID-19 patient hearts.

Additionally, several research groups reported that SARS-CoV-2 virus could infect human ESC/iPSC-derived CMs, and induce apoptosis (15, 16), sarcomere fragmentations (17) and electrical and mechanical dysfunctions (18) compared with mock control. After infection, the intramyocyte viral particles were observed by using transmission electron microscopy. All these studies indicate that hPSC-CMs could be utilized as an in vitro model for conducting mechanistic studies of SARS-CoV-2 viral genes in human CMs.

In this study, we identified the susceptibility of hPSC-CMs to individual SARS-CoV-2 genes. Nsp6 and Nsp8 are the non-structural proteins of SARS-CoV-2. M is the structural protein of SARS-CoV-2, which is the most abundant structural protein in the virus particle. The mechanisms of CM death induced by those genes have not been previously described.

Interestingly, we found that enforced expression of Nsp6, Nsp8 or M was sufficient to induce apoptosis and dysfunction of hPSC-CMs (Figures 4 and 7A,B,E), which phenocopied SARS-CoV-2 infected hPSC-CMs from other reports (9,15,17,42). Whole mRNA-seq revealed global transcriptional changes of Nsp6OE, Nsp8OE and MOE hESC-CMs compared to control hESC-CMs (Figures 2C-E), particularly with differentially expressed genes enriched into activated cellular injury and immune responses whereas reduced calcium/gap junction signaling (Figure 3H).

Notably, these global transcriptome changes are consistent with the previous published whole mRNA-seq results from SARS-CoV-2 virus directly infected hPSC-CMs (9). These results thus indicate that expression of the exogenous SARS-CoV-2 viral genes could profoundly disrupt the gene expression programs of human CMs, which might subsequently cause CM abnormalities in COVID-19 patients.

The interactions of SARS-CoV-2 viral genes with host cell proteins also play a critical role in CM abnormalities. In this study, by studying the interactome of Nsp6, Nsp8 or M within hESC-CMs, we found that they all interacted with ATPase subunits and compromised the cellular ATP level in hPSC-CMs (Figures 5A,K).

Bailey et al. also found downregulated ATP metabolic process in SARS-CoV-2 infected hPSC-CMs compared to control using whole mRNA-seq (9). Therefore, our findings suggest a possible central role of ATP homeostasis in SARS-CoV-2-induced tissue injuries in CMs, and highly likely in other SARS-CoV-2 vulnerable tissue cells in lung or kidney, although the mechanisms how Nsp6, Nsp8 or M could highjack ATPase remain to be elucidated.

Due to the consistent contractions, heart muscle cells consume a large amount of energy, which makes CMs one of the most vulnerable cell types to insufficient ATP supply. Therefore, we explored the pharmaceutical strategies to enhance cellular ATP levels, and found two FDA-approved drugs ivermectin and meclizine significantly reduced SARS-CoV-2 genes-induced cell death and electrical dysfunctions in human CMs.

Ivermectin belongs to the group of avermectins (AVM), which is a series of 16-membered macrocyclic lactone compounds discovered in 1967. FDA approved it for human use in 1987. Although ivermectin is used for treating parasitic infection, it was identified as a mitochondrial ATP protector in CMs and increased mitochondrial ATP production in human CMs (33), which was verified in our study as well.

Interestingly, a study reported that ivermectin was an inhibitor of SARS-CoV-2 in vitro, and a single treatment for 48 hrs. led to ∼5000-fold reduction of virus in cell culture (34). Notably, a recent clinical study reported that administration of ivermectin was associated with significantly lower mortality in hospitalized COVID-19 patients(43), although it is unclear whether ivermectin could reduce mortality via preserving ATP levels in SARS-CoV-2 vulnerable heart muscle and/or other tissue cells.

Meclizine, an over-the-counter anti-nausea and -dizziness drug, was identified via a ’nutrient-sensitized’ chemical screen. Meclizine is primarily used as an antihistamine. However, it has been reported that meclizine had cardio-protection effect through promoting glycolysis of CMs (35), which increased ATP synthesis.

Other study also reported that meclizine stimulated glycolysis, mitigated ATP depletion and protected mitochondria function (44). Those studies support our findings that meclizine might increase ATP level to mitigate SARS-CoV-2 gene-induced cell death of hPSC-CMs. Currently, whether administration of meclizine is associated with reduced mortality in hospitalized COVID-19 patients is still unknown and requires further clinical investigations.

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