| 摘要: | Background: In the latest update, the global scenario of the Corona-virus Disease 2019 (COVID-19) pandemic continues to evolve rapidly. Recent data has shown that severe acute respiratory syndrome coro-navirus 2 (SARS-CoV-2) infection induces cardiac injury in several ways including myocarditis, arrhythmias, thromboembolism, myocar-dial damage leading to myocardiopathy and cardiac fibrosis then eventually heart failure. Spike protein plays a primary role in viral cell entry by binding to the angiotensin-converting enzyme 2 (ACE2) re-ceptors which are also highly expressed in the heart. Understanding how spike protein interacts with cardiac tissue composed cause cardiac complications is essential to unravel the mechanisms underlying these complications and potential therapeutic strategies.
Materials and methods: Human cardiomyocytes and cardiac fibro-blasts were cultured with and without S1 spike protein for 24, 48, and 72 hours. The seahorse real-time adenosine triphosphate (ATP), total ATP assays, and mito stress test were used to evaluate the impacts of S1 protein on bioenergetics. Tetramethylrhodamine, ethyl ester (TMRE) staining was used for measuring mitochondrial membrane potentials in human cardiomyocytes. Immunocytochemistry (ICC) was used to stain translocase of outer mitochondrial membrane 20 (TOMM20), ACE2, and S1 protein. The expressions of ACE2, CD147, fatty acid transport-associated proteins, and markers of myofibroblasts (collagen 1, alpha-Smooth muscle actin (α-SMA)) were detected by using im-munoblotting. Fura-2, X-rhod-1, mitosox red, and mitotracker green were used to study the effects of S1 protein on intracellular, mito-chondrial Ca2+, reactive oxygen species (ROS) production, and mito-chondrial morphology, respectively. Scratch assay was used to evaluate the fibroblast migration after being treated with S1 protein.
Results: Both ACE2 and CD147 were detected in AC16 and fibroblasts. ICC staining confirmed the presence of S1 and ACE2 in the cytosol. S1 protein treatment for 24 hours (but not for 48 hours) enhanced ATP production in cardiomyocytes. Interestingly, 72h-S1-treated cardio-myocytes had lesser mitochondrial and glycolytic ATP production, which were partly reversed by antioxidant (mitotempo), and completely abolished by ACE2 neutralizing antibody. The expressions of pACC, CD36, and pAMPK, ROS levels, and mitochondrial membrane poten-tials were increased in 24h-S1-treated cardiomyocytes but were not changed in 72h-S1-treated cardiomyocytes. S1 protein increased both mitochondrial and cytosol Ca2+ as well as Ca2+ leak from SR in in 72h-S1-treated cardiomyocytes. Moreover, mitochondrial fragmenta-tion and decreased TOMM20 on the mitochondrial membrane were found in 72h-S1-treated cardiomyocytes.
24h-S1-treated human cardiac fibroblast had greater migration, collagen production with over expressed TGF-β1 and activated its downstream target protein (Smad2/3). However, cytosol or mitochon-drial Ca2+, ROS levels and mitochondrial morphology were similar between control and 24h-S1-treated human cardiac fibroblasts. In ad-dition, 24h-S1-treated human cardiac fibroblasts had higher expres-sions of IL1-β, NF-κB, and NLRP3 than control human cardiac fibro-blasts. The effects of S1 on fibroblast activation were diminished by NLRP3 blocker (MCC 950 at 10?M), and ACE2 neutralizing antibodies (10?g/ml) but not by TLR4 blockade (100nM).
Conclusion: S1 protein induced human cardiomyocyte dysfunction via disrupting mitochondrial membrane potentials, impairing ATP pro-duction and mitochondrial fragmentation, meanwhile it activates human cardiac fibroblast via NF-kB-NLRP3 inflammasome-IL1β signaling in an ACE2-dependent manner.
Background: In the latest update, the global scenario of the Corona-virus Disease 2019 (COVID-19) pandemic continues to evolve rapidly. Recent data has shown that severe acute respiratory syndrome coro-navirus 2 (SARS-CoV-2) infection induces cardiac injury in several ways including myocarditis, arrhythmias, thromboembolism, myocar-dial damage leading to myocardiopathy and cardiac fibrosis then eventually heart failure. Spike protein plays a primary role in viral cell entry by binding to the angiotensin-converting enzyme 2 (ACE2) re-ceptors which are also highly expressed in the heart. Understanding how spike protein interacts with cardiac tissue composed cause cardiac complications is essential to unravel the mechanisms underlying these complications and potential therapeutic strategies.
Materials and methods: Human cardiomyocytes and cardiac fibro-blasts were cultured with and without S1 spike protein for 24, 48, and 72 hours. The seahorse real-time adenosine triphosphate (ATP), total ATP assays, and mito stress test were used to evaluate the impacts of S1 protein on bioenergetics. Tetramethylrhodamine, ethyl ester (TMRE) staining was used for measuring mitochondrial membrane potentials in human cardiomyocytes. Immunocytochemistry (ICC) was used to stain translocase of outer mitochondrial membrane 20 (TOMM20), ACE2, and S1 protein. The expressions of ACE2, CD147, fatty acid transport-associated proteins, and markers of myofibroblasts (collagen 1, alpha-Smooth muscle actin (α-SMA)) were detected by using im-munoblotting. Fura-2, X-rhod-1, mitosox red, and mitotracker green were used to study the effects of S1 protein on intracellular, mito-chondrial Ca2+, reactive oxygen species (ROS) production, and mito-chondrial morphology, respectively. Scratch assay was used to evaluate the fibroblast migration after being treated with S1 protein.
Results: Both ACE2 and CD147 were detected in AC16 and fibroblasts. ICC staining confirmed the presence of S1 and ACE2 in the cytosol. S1 protein treatment for 24 hours (but not for 48 hours) enhanced ATP production in cardiomyocytes. Interestingly, 72h-S1-treated cardio-myocytes had lesser mitochondrial and glycolytic ATP production, which were partly reversed by antioxidant (mitotempo), and completely abolished by ACE2 neutralizing antibody. The expressions of pACC, CD36, and pAMPK, ROS levels, and mitochondrial membrane poten-tials were increased in 24h-S1-treated cardiomyocytes but were not changed in 72h-S1-treated cardiomyocytes. S1 protein increased both mitochondrial and cytosol Ca2+ as well as Ca2+ leak from SR in in 72h-S1-treated cardiomyocytes. Moreover, mitochondrial fragmenta-tion and decreased TOMM20 on the mitochondrial membrane were found in 72h-S1-treated cardiomyocytes.
24h-S1-treated human cardiac fibroblast had greater migration, collagen production with over expressed TGF-β1 and activated its downstream target protein (Smad2/3). However, cytosol or mitochon-drial Ca2+, ROS levels and mitochondrial morphology were similar between control and 24h-S1-treated human cardiac fibroblasts. In ad-dition, 24h-S1-treated human cardiac fibroblasts had higher expres-sions of IL1-β, NF-κB, and NLRP3 than control human cardiac fibro-blasts. The effects of S1 on fibroblast activation were diminished by NLRP3 blocker (MCC 950 at 10?M), and ACE2 neutralizing antibodies (10?g/ml) but not by TLR4 blockade (100nM).
Conclusion: S1 protein induced human cardiomyocyte dysfunction via disrupting mitochondrial membrane potentials, impairing ATP pro-duction and mitochondrial fragmentation, meanwhile it activates human cardiac fibroblast via NF-kB-NLRP3 inflammasome-IL1β signaling in an ACE2-dependent manner. |
