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SARS-CoV-2 infection enhances mitochondrial PTP complex activity to perturb cardiac energetics

Summary SARS-CoV-2 is a newly identified coronavirus that causes the respiratory disease called coronavirus disease 2019 (COVID-19). With an urgent need for therapeutics, we lack a full understanding of the molecular basis of SARS-CoV-2-induced cellular damage and disease progression. Here, we conducted transcriptomic analysis of human PBMCs, identified significant changes in mitochondrial, ion channel, and protein quality-control gene products. SARS-CoV-2 proteins selectively target cellular organelle compartments, including the endoplasmic reticulum and mitochondria. M-protein, NSP6, ORF3A, ORF9C, and ORF10 bind to mitochondrial PTP complex components cyclophilin D, SPG-7, ANT, ATP synthase, and a previously undescribed CCDC58 (coiled-coil domain containing protein 58). Knockdown of CCDC58 or mPTP blocker cyclosporin A pretreatment enhances mitochondrial Ca2+ retention capacity and bioenergetics. SARS-CoV-2 infection exacerbates cardiomyocyte autophagy and promotes cell death that was suppressed by cyclosporin A treatment. Our findings reveal that SARS-CoV-2 viral proteins suppress cardiomyocyte mitochondrial function that disrupts cardiomyocyte Ca2+ cycling and cell viability.


INTRODUCTION
Although several million people worldwide have been infected with coronavirus-SARS-CoV-2, the causative agent of the coronavirus disease 2019 (COVID-19) pandemic, our understanding of the fundamental mechanisms underlying the clinical manifestation of COVID-19 is still emerging. The SARS-CoV-2 entry receptor, ACE2 is highly expressed in several cell types, including myocardial cells, kidney proximal tubule cells, type II alveolar cells, and lymphocytes located in oral mucosa and other tissues. Beyond this canonical entry pathway, the mechanisms of cellular damage and tissue injury are poorly understood but are of paramount importance. Although the virus commonly causes respiratory distress, z33% of patients exhibit clinical or laboratory evidence of myocardial injury. Myocardial injury is especially common in COVID-19 patients with a history of cardiovascular disease (30%-60%). A deeper understanding of the mechanisms underlying myocardial injury is needed to develop a therapeutic strategy for such patients with co-morbidities.
Diverse viruses, including influenza M2, HIV-1, and coronavirus E, can undermine the cellular ion homeostasis machinery to allow viral entry and replication, either by co-opting the host ion channels' ability to maintain membrane potential (Fujioka et al., 2018;Hover et al., 2017) or by forming ion channels of virally encoded short membrane proteins that oligomerize to form hydrophilic pores that facilitate ion transport across host cell membranes and override the cellular homeostatic circuits (Wang et al., 2011). However, it is unknown whether SARS-CoV-2 adopts similar mechanistic routes. Among ion channels, the host Ca 2+ channels influence every process of viral infection and viruses usurp host Ca 2+ channels to disturb the homeostatic balance in such a way to benefit the virus life cycle at the cost of host survival. For example, influenza A virus infection is initiated by the interaction of the viral glycoprotein hemagglutinin with the voltage-dependent Ca 2+ channel Ca v 1.2 to trigger intracellular Ca 2+ oscillations and subsequent viral entry and replication (Fujioka et al., 2018). Of importance, in cardiac muscle, Ca 2+ influx is also mediated through the L-type Ca 2+ channel, suggesting that alterations of voltage-gated calcium channels could be an important factor in COVID-19 and myocardial injury. Inhibition of store-operated Ca 2+ channel activity reduced the viral egress, and thus infectivity, of many enveloped RNA viruses (Chen et al., 2019;Han et al., 2015). This is evidenced by recent repurposing of existing drugs to treat SARS-CoV-2 infection (Gordon et al., 2020). Specifically, pharmacological agents targeting Sigma1 receptors, known to activate IP 3 R-dependent Ca 2+ efflux (via store-operated channels) from the ER (Gordon et al., 2020), showed activity against SARS-CoV-2 infection. We have recently reported that HIV-1 Nef-mediated inhibition of cellular protein quality control is one possible mechanism involved in the development of HIV-associated cardiomyopathy and progression toward heart failure (Cheung et al., 2019;Gupta et al., 2017;Muthumani et al., 2006;Natarajaseenivasan et al., 2018;Tahrir et al., 2018). Our recent molecular characterization of the mitochondrial permeability transition pore (PTP) revealed that viral proteins, bacterial toxins, and ion dysregulation elicit PTP and induce cardiac cell death (Shanmughapriya et al., 2015b). Interesting, a new report revealed that SPG7 is upregulated upon SARS-CoV-2 infection (Bojkova et al., 2020), suggesting a role of PTP-mediated mitochondrial dysfunction in determining COVID-19 development and associated myocardial injury.
Although much work has focused on innate and adaptive immune dysregulation in the pathophysiology of COVID-19, the effect of SARS-CoV-2 infection on cellular metabolic activity is unknown. Here we deliver findings that reveal how viral proteins hijack intracellular organelles, ion channels, bioenergetics, and quality control. Our unbiased RNA-seq analysis of uninfected individuals, patients who tested positive for SARS-CoV-2 but were asymptomatic, and severely affected COVID-19 patients reveal numerous human transcripts that were either upregulated or suppressed in the severely affected COVID-19 patients when compared with counterparts. Intriguingly, mitochondrial DNA-encoded transcripts were markedly suppressed, whereas nuclear-encoded mitochondrial respiratory complex, mitochondrial permeability transition pore (mPTP), and mitophagy-related transcripts were markedly elevated in severely affected COVID-19 patients. These severely affected COVID-19 patients showed elevated levels of troponin-C and IL-6 in the plasma. Having observed the elevated cardiac injury marker in COVID-19 samples, we utilized iPSC-derived human cardiomyocytes (iPSC-CMs) to explore the host molecular pathways targeted by SARS-CoV-2 viral proteins. Although SARS-CoV-2 encodes 28 gene products, our comprehensive targeted analyses demonstrate that the viral structural protein, Membrane (M), along with NSP6, NSP10, ORF3a, and ORF9c proteins, discretely localize to mitochondria, endoplasmic reticulum (ER), or plasma membrane compartments. Reconstitution of M-protein in iPSC-CMs markedly inhibited Ca V 1.2 channel currents. Moreover, ectopic expression of M-protein in human cells resulted in mitochondrial swelling, possibly through the strong interaction with mitochondrial permeability transition pore components SPG7 and CCDC58 to enhance pore opening and causing mitochondrial dysfunction.

RESULTS
Unbiased RNA profiling exhibits perturbation of mitochondrial energetics, ion dynamics, and cellular quality control upon SARS-CoV-2 infection in humans To understand the global transcriptional regulation of human mRNAs upon SARS-CoV-2 infection, we utilized PBMCs obtained from (1) uninfected individuals, (2) SARS-CoV-2-positive patients with mild symptoms, and (3) SARS-CoV-2-positive patients who were admitted to the intensive care unit at the University of Texas Southwestern Dallas (UTSW) (IRB Protocol number STU-2020-0366) ( Table S1). The RBC contamination in the blood samples was eliminated by routine RBC lysis protocol followed by PBMC purification and total RNA isolation. Total RNA was isolated using the BSL-3 facility at Texas Biomedical Research Institute (TBRI) followed by RNA quantification. Total RNA isolated from PBMCs was depleted of rRNA and globin and used for library construction (NEBNext Ultra RNA Library Prep Kit). The sequencing libraries were multiplexed and sequenced on the Illumina HiSeq instrument using a 2 3 150 paired-end configuration. Image analysis and base calling are conducted by the HiSeq Control Software. Raw sequence data (.bcl files) generated from Illumina HiSeq were converted into fastq files and de-multiplexed using Illumina's bcl2fastq 2.17 software. One mismatch is allowed for index sequence identification. Using DESeq2, a comparison of gene expression between the groups of samples was performed at GENEWIZ (GENEWIZ, South Plainfield, NJ). Using these comprehensive data, we generated a heatmap that depicts the segregation of elevated versus downregulated RNA transcripts of PBMCs ( Figure 1A). A principal component analysis (PCA) was performed using the "plot PCA" function within the DESeq2 R package (Data S1). Although iScience 25, 103722, January 21, 2022 iScience Article the sample size was small, the dynamism of RNA clustering is tight in SARS-CoV-2 infected samples indicating that host cellular transcriptome changes were controlled by the virus ( Figure 1B). These RNA transcripts were assessed, and distribution of individual subjects were plotted ( Figure 1C). The GO analysis was used to functionally cluster the set of genes based on their biological process and determine their statistical significance ( Figure 1D; Data S1). Although our data included both sexes, the severely affected human subjects were male, which possibly could have an influence on the differential gene expression among groups (Table S1 and Data S1). Using this computational analysis, we focused on the highly significantly altered RNA transcripts induced upon viral infection. We found that mitochondrial energetic complex components, ion channel subunits, and autophagy and mitophagy-regulated translational precursors  iScience Article were markedly altered and thus focused our studies on these ( Figure 1E). Intriguing, the severely affected COVID-19 patient samples showed elevated cardiac injury markers such as troponin-C, proBNP, and LDH, suggesting acute cardiac damage manifestation in these patients ( Figure 1F).
SARS-CoV-2 viral proteins localized in mitochondria, ER, and plasma membrane, which modulates organelle phenotype Having observed a cardiac stress marker elevation in severely affected COVID-19 patients, we next tested whether SARS-CoV-2 infection causes cardiomyocyte damage. We utilized human-iPSC-derived cardiomyocytes as a surrogate model system to determine the severity of SARS-CoV-2 infection. To determine the mitochondrial changes, we expressed Ad-mito-GCamp6 or stained with MitoTracker Red as mitochondrial markers. Upon SARS-CoV-2 infection, we found that mitochondrial shape change and fragmentation were evident in virus-infected cardiomyocytes indicating that the viral proteins possibly target mitochondria and impact bioenergetics (Figures 2A and 2B). Based on prediction algorithms and proteomic analysis (Gordon et al., 2020), we cloned and expressed mRFP/FLAG-tagged 13 of the 29 SARS-CoV-2 proteins in COS-7 cells to monitor their cellular distribution and localization. Since these individual candidate genes were tagged with FLAG, we transiently transfected them in COS-7 cells and high-level ectopic protein expression was confirmed by western blotting ( Figure 2C). High-resolution imaging of the SARS-CoV-2 proteins was performed using the mRFP-tag in live cells loaded with ER tracker and mitochondrial marker (DHR123) to determine the spatial overlap and intensity profiles ( Figure S1). The imaging results show that four of these proteins, Orf9C, M-protein, Nsp6, and Orf3a, were localized to either ER or mitochondrial membranes (Figures S1A, S1B and 2D-2H). In particular, M-protein was predominantly localized in the mitochondrial compartment ( Figures 2D and 2H) Remarkably, Nsp7 exhibited ER remodeling and transmigrated near the plasma membrane ( Figure 2F). While assessing their host cellular distribution, we observed that M-protein expression induced mitochondrial morphological changes ( Figures 2D and  2I). These results indicate that the viral replication and structural SARS-CoV-2 proteins targeted to host cellular organelles besides their canonical function.

SARS-CoV-2 virus profoundly affects host mitochondrial bioenergetic complexes
The RNA profiling analysis showed that both nuclear and mitochondrial genome encoded transcripts were markedly altered in PBMCs from severely affected COVID-19 patients ( Figure 3A; Data S1). Since severely affected COVID-19 patient outcomes include organ failure and death, we focused on candidate genes that promote cell death and mitochondrial dysfunction. Of these transcripts, the previously uncharacterized CCDC58 was significantly elevated in PBMCs obtained from COVID-19 patients ( Figure 3B). Based on the mitoCarta and Mito-miner database, CCDC58 is predicted to target mitochondrial compartment (Pagliarini et al., 2008). To assess its localization, C-terminal FLAG-tagged CCDC58 was co-transfected with the mitochondrial marker protein COX8A-mRFP. As predicted, immunofluorescence analysis confirmed that CCDC58 localized predominantly to mitochondria and exhibited near complete overlap with COX8A ( Figures 3C, 3D, S2A, and S2B). It has been reported that high-throughput RNAi-mediated silencing of mitochondrial resident proteins, including CCDC58, results in increased mitochondrial Ca 2+ retention capacity suggesting a link between mitochondrial Ca 2+ overload and cell death (Shanmughapriya et al., 2015a). Therefore, we determined if stable knockdown of CCDC58 produces resistance to Ca 2+ -induced mitochondrial permeability transition pore opening. The stable transfected control (NegshRNA) and CCDC58 KD 293T cells were subjected to permeabilization using digitonin containing intracellular medium. Upon exposure to excessive extramitochondrial Ca 2+ , control cells exhibited rapid mitochondrial membrane potential (DJ m ) loss due to Ca 2+ -induced PTP opening ( Figures 3E and S3). In contrast, knockdown of CCDC58 markedly increased mitochondrial Ca 2+ retention capacity ( Figures 3D and 3F). In addition, CCDC58 KD cells exhibited higher oxygen consumption rate (OCR) but cellular ATP levels were lowered ( Figures S4A-S4H). CCDC58 KD cells exposed to oxidants still partially retained Calcein indicating that CCDC58 likely participates in the mPTP complex ( Figures S4I-S4J). Having observed an elevation of CCDC58 under COVID-19 disease development, we next asked whether SARS-CoV-2 M-protein targets CCDC58 protein to alter mitochondrial function. To test this, we reconstituted M-protein in CCDC58 KD cells and monitored CRC.
Remarkably, cells lacking CCDC58 were able to maintain CRC when compared with control cells, highlighting a critical role for CCDC58 in COVID-19-induced mitochondrial dysfunction ( Figures 3G and 3H).

SARS-CoV-2 infection promotes ER remodeling and exacerbates autophagy in cardiomyocytes
We again performed a reductionist approach to determine whether these mitochondrial functional changes result in aberrant organelle quality control upon SARS-CoV-2 infection. Our targeted transcriptomic analysis showed that autophagy/mitophagy markers were significantly elevated in PBMCs derived from severely affected COVID-19 patients ( Figure 5A; Data S1). We next asked whether SARS-CoV-2 viral infection reorganizes cytoskeletal apparatus and ER network in human-iPSC-derived cardiomyocytes. Cardiomyocytes were ectopically coexpressed in tubulin-GFP and Sec61b-mCherry adenoviral constructs (AdV5a), and the architecture was visualized using confocal imaging. As expected, myocytes displayed choreographed cytoskeletal and ER compartments ( Figure 5B). Upon SARS-CoV-2 infection for 24 h, both cytoskeletal and ER reticular network were collapsed ( Figure 5C). Having observed that the live SARS-CoV-2 virus causes cellular stress, we next assessed the effect of the virus on the cellular quality control mechanism. Autophagy is a dynamic phenomenon involving autophagosome formation, autophagosome/lysosome fusion, and clearance by lysosomes. To assess whether autophagy was impacted by viral infection, we monitored autophagic flux in cardiomyocytes after SARS-CoV-2 infection using adenovirus expressing a tandem LC3 (light chain 3)-GFP (green fluorescent protein)-RFP (red fluorescent protein) reporter that enables the detection of the autophagic flux process ( Figure 5D). Green puncta indicate autophagosome formation, whereas red puncta indicate autophagolysosome formation. In the acidic environment of lysosomes, the GFP signal is quenched. Under basal culture conditions, we observed nominal levels of LC3 puncta in both compartments, whereas cardiomyocytes infected with SARS-CoV-2 for 24 h elicited massive number of positive autophagic vesicles ( Figures 5D and 5E). These data suggest that viral infection exacerbates autophagic flux that possibly eliminates a large number of dysfunctional mitochondria.

mPTP blocker cyclosporin A restores mitochondrial Ca 2+ retention capacity and cardiomyocyte viability after viral infection
We next tested whether viral proteins alter mitochondrial bioenergetics and possibly induce cardiomyocyte cell death. The mitochondrial oxygen consumption rate (OCR) in HEK293 cells overexpressing viral proteins was measured. Remarkably, cells expressing M-protein or ORF3a had lower basal OCR and iScience Article minimal FCCP-induced maximal OCR ( Figure 6A). Interestingly, NSP6 and NSP7 exhibited a partial suppression of maximal OCR ( Figure 6A). However, the ECAR (extracellular acidification rate) was similar among these cell types ( Figure 6B). Although OCR was affected by these viral proteins, the cells were viable, indicating a switch from oxidative to glycolytic phenotypes. To further investigate the impact of viral proteins on mitochondrial ion homeostasis, we measured MCU channel activity as well as mitochondrial CRC in the permeabilized cell system. We found that the CRC is reduced by ectopic expression of SARS-CoV-2 proteins; however, the MCU activity remains intact ( Figures 6C-6E and S6). We next evaluated whether mPTP blocker cyclosporin A restores the CRC in viral protein expressing condition. As expected, CsA enhanced the mitochondrial CRC but the viral M-protein-mediated CRC reduction was still observed ( Figures 6C, 6D and S6). Reconstitution of these viral proteins exhibited lower CRC with the exception of NSP7 ( Figure 6E). Finally, we assessed the cardiomyocyte viability under viral infection. Twenty-four hours post infection, cardiomyocyte viability was assessed using MTT assay ( Figure 6F). Cardiomyocytes exposed to virus exhibited a significant cell death that was considerably prevented by cyclosporin A treatment suggesting that these viral particle/proteins could disrupt mitochondrial bioenergetics through mPTP opening and eventual cardiomyocyte damage ( Figure 6F).
SARS-CoV-2 proteins disrupt cardiomyocyte Ca 2+ cycling and suppress voltage-gated Ca V 1.2 Ca 2+ channel activity The RNA-seq analysis from the severely affected patients revealed a significant elevation of Ca 2+ channel component transcripts, in particular, LTCC (CACNA1C) and the CRAC channel regulator, STIM1, were upregulated ( Figure 1). Having shown that SARS-CoV2 infection alters the expression of ion channels in cardiomyocytes, we next determined the impact of SARS-CoV-2 on cardiomyocyte function using hiPSC-CMs. We first determined the effect of SARS-CoV-2 infection on RNA transcript changes in human iPSC derived cardiomyocytes (hiPSC-CMs). hiPSCs-CMs were developed as described previously    Pan et al., 2013). Conversely, the CRAC channel, Orai3, and mitochondrial Mg 2+ uptake channel Mrs2 were suppressed ( Figure 7D) . Since efficient Ca 2+ cycling is essential for cardiomyocyte contraction and relaxation, we first tested the spontaneous Ca 2+ cycling in hiPSC-CMs. A robust Ca 2+ cycling was observed in control hiPSC-CMs, but this rhythmic Ca 2+ cycling was perturbed in hiPSC-CMs expressing SARS-CoV-2 M-protein ( Figure 7E). Although the Ca 2+ amplitude was similar, the diastolic concentrations and frequencies of systolic Ca 2+ were markedly prolonged in hiPSC-CMs expressing SARS-CoV-2 M-protein ( Figures 7E and 7F). Similarly, overexpression of NSP-6 exhibited dramatic disruption in both amplitude and Ca 2+ cycling ( Figures 7E and 7F) indicating that impairment of cardiomyocytes Ca 2+ cycling by viral proteins could alter force generation. Functional characterization (using wholecell configuration at various membrane potential) further showed the presence of L-type Ca 2+ currents in control hiPSC-CMs ( Figure 7G). The hiPSC-CMs displayed current-voltage (I/V) properties and inhibition by nifedipine typical for Ca v 1.2 channels (LTCC) (Bers, 2002(Bers, , 2008(Bers, , 2014Catterall et al., 2005) ( Figure 7G). Having observed a perturbation of Ca 2+ cycling, we ectopically expressed SARS-CoV-2 M-protein, iScience Article ORF-9c, ORF-3a, NSP-6, and NSP-7 in hiPSC-CMs. These viral proteins resulted in significant suppression of Ca v 1.2 currents ( Figure 7H). Collectively, these results reveal that SARS-CoV-2 viral proteins disrupt cardiomyocyte Ca 2+ cycling and ion channel activity.

DISCUSSION
Numerous studies have shown that a large percentage of individuals diagnosed with COVID-19 manifest evidence of myocardial injury and that these individuals have a higher risk of death (Calvo-Ferná ndez et al., 2020). The SARS-CoV-2 Spike protein binds to its cellular receptor, the ACE2 protein and TMPRSS2, to enter host cells (Hoffmann et al., 2020). ACE2 is highly expressed on cardiomyocytes (Han et al., 2020), and direct infection of cardiomyocytes by SARS-CoV-2 has been documented in patients (Tavazzi et al., 2020;Tian et al., 2020) and in cultured cardiomyocytes (Wong et al., 2020). However, the mechanisms by which the virus alters cardiomyocyte function remain unclear. To better delineate the pathways perturbed during SARS-CoV-2 infection, we performed RNA-seq analysis of PBMCs from COVID-19 patients. Furthermore, we performed detailed molecular and biochemical studies of human-iPSC-derived cardiomyocytes. In addition, we also characterized cellular organelle (ER and First, we determined the global transcriptome profile of SARS-CoV-2 infection in PBMCs obtained from healthy volunteers and symptomatic and severely affected COVID-19 patients. Pathways that were significantly altered in COVID-19 patients with severe illness included transcription, mitochondrial oxidative phosphorylation, ATP biosynthesis, ion channels, autophagy, and apoptosis (Figures 1 and 3). Likewise, oxidative phosphorylation, calcium signaling, and apoptosis have been reported by other groups to be altered in COVID-19 PBMCs (Gardinassi et al., 2020;Xiong et al., 2020). This remarkable regulation suggests that SARS-CoV-2 proteins not only are essential for viral replication, assembly, and cell lysis but also potentially target host cellular compartments to affect cell and organ function. Based on predictions of SARS-CoV-2 protein structure (Gordon et al., 2020), we selected 13 viral proteins for further analysis. Of these, we found that several were localized to ER, mitochondria, and/or plasma membrane. We also observed that these viral proteins resulted in dramatic changes in mitochondrial morphology ( Figure 2) and mitochondrial function. These findings emphasize that SARS-CoV-2 proteins have a moonlighting role beside their canonical functions.
Having noted transcriptomic changes associated with oxidative phosphorylation and localization of SARS-CoV-2 proteins to mitochondria, we focused our attention on mitochondrial proteins. Of these, we found a significant elevation of a previously uncharacterized nuclear-encoded mitochondrial protein, CCDC58, in COVID-19 PBMCs ( Figures 3A and 3B). A recent report also identified an upregulation of mPTP complex components SPG7 and cyclophilin D in Caco-2 cells infected with SARS-Co-V-2 (Bojkova et al., 2020;Shanmughapriya et al., 2015a;Zhou et al., 2019). Therefore, we explored the role of CCDC58 in the mPTP. We confirmed that CCD58 is localized to the mitochondria ( Figure 3C) and showed that knockdown of CCDC58 dramatically enhanced the capacity of mitochondria to sequester calcium. Of interest, expression of the SARS-CoV-2 M protein reduced calcium sequestration in the absence of CCDC58. We then performed a systematic assessment of interactions between SARS-CoV-2 proteins and components of the mPTP. Indeed, we found that SPG7, PPIF, and CCDC58 physically interact with each other and that several SARS-CoV-2 viral proteins, including M protein, ORF3a, ORF9b and 9C, ORF10 and NSP6, bind to mPTP complex proteins (Figure 4) suggesting a likely involvement in COVID-19-induced mitochondrial dysfunction and cell death (Figure 7). Since our results suggested that SARS-CoV-2 may alter mitochondrial morphology and function, we measured the effect of specific SARS-CoV-2 proteins on oxidative phosphorylation and mitochondrial calcium sequestration. Several of the proteins resulted in a substantial inhibition of maximal mitochondrial respiration and reduced the capacity of mitochondria to sequester calcium (Figures 6A-6D). Cyclosporin A, which inhibits the mPTP, reversed these effects. These results suggest that SARS-CoV-2 proteins may alter the mPTP making it more prone to opening and triggering subsequent cell death. Consistent with this view, we found that infection of hiPSC-CMs with SARS-CoV-2 decreased cell viability and that this effect was reversed by cyclosporin A ( Figure 6F).
Proper Ca 2+ dynamics are essential to cardiomyocyte function, and any aberrations lead to cardiac pathological conditions (Bers, 2002(Bers, , 2008(Bers, , 2014. The ER, mitochondria, and plasma membranes all participate in the orchestration of calcium dynamics. In addition, to the perturbation of mitochondrial calcium sequestration ( Figure 6), we noted that several transcripts involved in calcium transport, such as STIM1 and L-type calcium channels, were upregulated in COVID-19 PBMCs. To explore the effects of SARS-CoV-2 on cardiomyocyte calcium dynamics, we performed electrophysiologic studies on hi-PSC-CMs expressing SARS-CoV-2 proteins. The results show that these proteins dramatically disrupt the baseline oscillations of cell calcium and almost completely block L-type calcium channel activity indicating the cardiomyocyte damage and the development of heart failure.
In conclusion, our results show that SARS-CoV-2 proteins localize to mitochondria, bind to the mPTP complex, alter mitochondrial morphology and function, disrupt Ca 2+ cycling, and inhibit VDCC channel activity, all indicating a perturbation of ion homeostasis and mitochondrial dysfunction (Figure 7). These observations lead us to speculate that COVID-19-induced cardiac damage may be due to mitochondrial dysfunction. Moreover, these comprehensive analyses point toward new drug targets to preserve host cells against SARS-CoV-2-induced cardiac damage. Specifically, our results suggest that the mPTP blocker and immunosuppressive drug cyclosporin A may offer an additional therapeutic option to treat COVID-19 patients.

Limitations of the study
Although over 200 million people around the world are infected with coronavirus SARS-CoV-2, the causative agent of the COVID-19 pandemic, an understanding of the fundamental mechanisms underlying the clinical manifestation of COVID-19 is still emerging. Because of the nature of its infectivity, we were able to perform critical in vitro experiments with SARS-Co-V-2 virus. To understand the molecular nature and host cellular distribution of the viral proteins, we utilized multiple cell models. Although it is considered as a limitation, our mRNA transcriptomics of human PBMCs and cellular and mitochondrial functional data are consistent and provide new insights into how viral protein components target mitochondria that elicits bioenergetic collapse and cardiomyocyte damage.

STAR+METHODS
Detailed methods are provided in the online version of this paper and include the following:

Assessment of subcellular localization by immunofluorescence
HeLa cells were plated on 30 mm diameter, number 1.5 thickness coverslips coated with Poly-L-Lysine. Cells were co-transfected with CCDC58-Flag and Cox8a-mRFP using the FuGENE HD transfection reagent as per manufacturer instructions. After 24 h, cells were fixed with 4% paraformaldehyde for 15 min at room temperature (RT). Fixed cells were washed two-times with DPBS and permeabilized with 0.15% Triton X-100 solution for 15 min at RT. Permeabilized cells were blocked with 10% (w/v) bovine serum albumin (BSA) solution for 45 min at RT and subsequently incubated overnight with monoclonal anti-FLAG M2 antibody for the detection of CCDC58. After incubation, cells were washed three times with 10% BSA, and then the secondary anti-mouse Alexa 488 antibody was added for 1 h at RT. After incubation with secondary antibody, cells were washed three times with Tris-buffered saline with 0.05% Tween 20 detergent (TBST) and imaged at LSM 510 META Confocal Laser Scanning Microscope (Carl Zeiss, Inc.) at 488-and 594-nm excitations using a 40x oil objective. Co-localized pixels were quantified by detecting the fluorescence intensity of CCDC58-Flag and Cox8a-mRFP pixels using the line scan in ZEN blue software (Carl Zeiss, Inc.).

Ratiometric Ca 2+ imaging for mitochondrial calcium retention capacity
Ca 2+ flux analysis for the mitochondrial calcium retention capacity was performed as described earlier (Shanmughapriya et al., 2015a;Tomar et al., 2016). Briefly, HeLa cells were trypsinized and washed in Ca 2+ and Mg 2+ free DPBS, and an equal number of cells (6x10 6 cells) were resuspended and permeabilized with 40 mg/mL digitonin in 1.5 mL of a Ca 2+ -free intracellular medium (ICM; 10 mM NaCl, 120 mM KCl, 1 mM KH 2 PO 4 , 20 mM HEPES-Tris, pH 7.2), supplemented with 2 mM thapsigargin to block the SERCA pump and 15 mM succinate. 1mM Fura-FF was added to the cell suspension for the detection of free bath Ca 2+ . Fura-FF fluorescence was monitored in a multiwavelength excitation dual-wavelength emission spectrofluorometer (Delta RAM, PTI, HORIBA). Bath Ca 2+ is shown as the [Ca 2+ ] out (mM). Ca 2+ boluses (10mM) were added until the mitochondria stopped clearing the Ca 2+ bolus, and then the mitochondrial uncoupler, FCCP (3 mM), was added to confirm mitochondrial depolarization. All Ca 2+ flux measurements were performed at 37 C with constant stirring.

Mitochondrial fractionation for CCDC58 localization
HeLa, HEK293T, and COS-7 cells were cultured in 150 mm 2 cell culture dishes and transfected with CCDC58-Flag using the FuGENE HD transfection reagent per manufacturer instructions. Mitochondria were isolated by utilizing the mitochondria isolation kit for cultured cells (Abcam Cat# ab110170), as per manufacturer instructions. Total cell lysate (TCL) and cytosol (cyto) were collected, and the mitochondrial pellet was permeabilized with digitonin (0.2 mg/mL). After permeabilization, mitochondrial pellet and supernatant (sup) is collected. Trypsinization (Trypsin 0.1% w/v) of intact and permeabilized mitochondria was performed to validate the CCDC58 localization in mitochondrial compartments. Mitochondrial compartment-specific marker antibodies, TOM20 for outer mitochondrial membrane, Cytochrome c for intermembrane space, ATP5A for inner mitochondrial membrane, and PPIF for mitochondrial matrix were used.
Evaluation of intracellular SARS-CoV-2 protein localization and colocalization with ER and mitochondria using live-cell confocal imaging system hiPSC derived cardiomyocytes or COS-7 cells transiently transfected with the mRFP-tagged SARS-CoV-2 plasmid constructs were grown on 25-mm 0.1% gelatin or collagen coated glass coverslips, respectively. 48 h post-transfection, cells were loaded with 1 mM of ER Tracker blue-white DPX (ex/em 374/430 nm) and mitochondrial marker Dihydrorhodamine (2.5 mM; ex/em 505/524 nm) in cell growth media for 30 min at 37 C (5% CO2). The cells were washed and imaged within a temperature-controlled environmental chamber set at 37 C using a Leica TCS SP8 live-cell confocal imaging system. The subcellular localization of the cells transfected with SARS-CoV-2 proteins was imaged using a 100x oil objective and ll OPEN ACCESS 20 iScience 25, 103722, January 21, 2022 iScience Article analyzed using Leica Application Suite X. The colocalization of Cox-8a mRFP and the SARS-CoV-2 proteins (Vector control, M Protein, Orf9c, Orf3a, Nsp6, Nsp10) with the ER (ER BFP) and mitochondria (DHR 123) were quantitatively measured using the Leica Application Suite X. The levels of colocalization were plotted by calculating the Pearson's co-efficient of colocalization and overlap co-efficient and the % of colocalization was determined. The values were plotted using GraphPad Prism version 8.

Evaluation of mitochondrial phenotype
The mitochondrial phenotypic changes in cells infected with SARS-CoV-2 virus was evaluated using genetically encoded mitochondrial sensors and mitochondria staining dyes. hiPSC derived cardiomyocytes were grown on 25-mm gelatin-coated glass coverslips and were infected with Ad-mito-GCamp6 (ex/em 480/ 510 nm: MOI 5). After 48 h, the cells were infected with SARS-CoV-2 virus (MOI 1) for 24 h and fixed with 10% Neutral buffered formalin. The fixed cells were imaged on a Leica TCS SP8 confocal imaging system using a 100x oil objective. The levels of mitophagy and mitochondrial phenotype was imaged, and the mitochondrial length, area and perimeter were measured using the Leica Application Suite X. The results were tabulated and plotted using GraphPad Prism version 8.
hiPSC derived cardiomyocytes were grown on 25-mm gelatin-coated glass coverslips and were infected with SARS-CoV-2 virus (MOI 1) for 24 h and were stained with Mitotracker deep red FM (ex/em 644/ 665 nm) for 30 min. The stained cells were fixed with 10% Neutral buffered formalin. The fixed cells were imaged on a Leica TCS SP8 confocal imaging system using a 100x oil objective. The levels of mitophagy mitochondrial phenotype i.e., mitochondrial length, area and perimeter were measured using the Leica Application Suite X. The results were tabulated and plotted using GraphPad Prism version 8.

Visualization of autophagy using confocal microscope
To evaluate the suicidal effect of cells in response to the SARS-CoV-2 infection, hiPSC derived cardiomyocytes were infected with LC3 GFP/RFP tandem adenovirus (GFP ex/em 488/510 nm; RFP ex/em 552/ 584 nm; MOI 5). After 24 h, the cells were infected with SARS-CoV-2 (MOI 1) for 24 h and fixed with 10% neutral buffered formalin. The fixed cells were imaged on a Leica TCS SP8 imaging system using a 100x oil objective. The number of vesicular puncta were counted and plotted using GraphPad Prism version 8.

Assessment of SARS-CoV-2 protein localization
To visualize the ectopic localization of the SARS-CoV-2 viral proteins, hiPSC derived cardiomyocytes were infected with adenoviruses encoding tubulin-GFP (ex/em 484/507 nm; MOI 5) and Sec61 mCherry (Vector BioLabs, ex/em 587/610 nm; MOI 5). After 24 h, the cells were infected with SARS-CoV-2 virus (MOI 1) for 24 h and fixed with 10% neutral buffered formalin and imaged on a Leica TCS SP8 imaging system using a 100X oil objective. The phenotypic implications of the SARS-CoV-2 were analyzed using the Line Scan analysis, Leica Application Suite X.
Measurement of mitochondrial Ca 2+ uptake in a permeabilized cell system HEK293T cells stably expressing negative shRNA, CCDC58 KD, or CCDC58-pBSD; as well as CCDC58 KD HEK293T cells stably expressing SARS-CoV-2 proteins (M Protein, Orf9c, Orf3a, Nsp6, Nsp7) or only expression vector, were used for the measurement of mitochondrial Ca 2+ uptake. Briefly, the cells were washed with Ca 2+ and Mg 2+ free DPBS, pH 7.4. An equal number of cells ($4-5x10 6 cells) were resuspended and permeabilized with 40 mg/mL digitonin in 1.5 mL ICM. Mitochondrial calcium uptake was measured in the presence of 1.5 mM Mg 2+ /ATP and 5 mM succinate, with or without 5 mM cyclosporin A (CsA). The permeabilized cells were loaded with Fura-2 FF (1 mM; ex 340/380 nm and em 510 nm). The measurement of extramitochondrial Ca 2+ clearance was used as an indicator of [Ca 2+ ] m uptake. Fluorescence was measured in a multi-wavelength excitation and dual-wavelength emission spectrofluorometer (Delta RAM, PTI, HORIBA). JC-1 (800 nM) was used simultaneous measurement of mitochondrial membrane potential. iScience Article Total cellular ATP levels Cellular ATP levels were measured using CellTiter-Glo luminescent kit, and luminescence intensity was detected using the Infinite M1000 PRO plate reader (Tecan).

SARS-CoV-2 protein expression and Western analysis
hiPSC-CMs were transiently transfected with mRFP/Flag-tagged SARS-CoV-2 plasmids construct individually. 48 h post-transfection, cell lysates were prepared for Western blot analysis and SARS-CoV-2 viral protein expression was probed using FLAG antibody.

Immunoprecipitation
For immunoprecipitation, cell extracts were prepared using IP lysis buffer (ThermoFisher; 87788) containing protease and phosphatase inhibitor cocktail (ThermoFisher 78440). To assess the interaction of SARS-CoV-2 proteins with PPIF, SPG7 or CCDC58, FLAG-tagged SARS-CoV-2 plasmid constructs were co-transfected with HA-tagged PPIF or HA-tagged SPG7 or Myc-tagged CCDC58 plasmid in COS7 cells. Cell lysates were immunoprecipitated with HA for PPIF or SPG7 and with Myc for CCDC58. To determine the protein-protein interaction between SARS-CoV-2 proteins and PTP complex components, immunoprecipitated samples were probed with anti-FLAG antibody. To confirm the PPIF or SPG7 interaction with SARS-CoV-2 proteins, reverse immunoprecipitation was performed using anti-FLAG antibody and immunoprecipitated samples were probed with anti-HA antibody. To assess the CCDC58 binding with PPIF or SPG7, co-transfected lysates were immunoprecipitated with Myc. The whole-cell lysate and the eluate were probed with anti-Myc or anti-HA antibodies, respectively. To assess the interaction between SARS-CoV-2 proteins and endogenous SPG7 or ANT or ATP Synthase, FLAG-tagged SARS-CoV-2 plasmids transfected cell lysates were immunoprecipitated with anti-FLAG antibody and eluates were probed with either endogenous SPG7 or or ANT or ATP Synthase.

Statistical analysis
All data are expressed as mean G SEM unless otherwise indicated. Statistical significance was evaluated using a Student's unpaired t-test for one or multiple samples, and one-way ANOVA followed by Tukey's multiple comparison test as needed with p < 0.05 considered to be statistically significant. Data from cells was obtained using built-in Leica software and were analyzed and plotted using GraphPad Prism version 8.