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In vitro Assessment of Myocardial Protection following Hypothermia-Preconditioning in a Human Cardiac Myocytes Model
Published on: October 27, 2020
SurR9C84A protects and recovers human cardiomyocytes from hypoxia induced apoptosis
Ajay Ashok1, Jagat Rakesh Kanwar2, Uma Maheswari Krishnan3
1Nanomedicine-Laboratory of Immunology and Molecular Biomedical Research (NLIMBR), School of Medicine (SoM), Faculty of Health, Centre for Molecular and Medical Research (C-MMR), Deakin University, Waurn Ponds, Victoria 3216, Australia; Department of Pathology, Case Western Reserve University, 2103 Cornell Rd. WRB 5128, Cleveland, OH 44106-7288, USA.
Abstract:
Survivin, as an anti-apoptotic protein and a cell cycle regulator, is recently gaining importance for its regenerative potential in salvaging injured hypoxic cells of vital organs such as heart. Different strategies are being employed to upregulate survivin expression in dying hypoxic cardiomyocytes. We investigated the cardioprotective potential of a cell permeable survivin mutant protein SurR9C84A, for the management of hypoxia mediated cardiomyocyte apoptosis, in a novel and clinically relevant model employing primary human cardiomyocytes (HCM). The aim of this research work was to study the efficacy and mechanism of SurR9C84A facilitated cardioprotection and regeneration in hypoxic HCM. To mimic hypoxic microenvironment in vitro, well characterized HCM were treated with 100µm (48h) cobalt chloride to induce hypoxia. Hypoxia induced (HI) HCM were further treated with SurR9C84A (1µg/mL) in order to analyse its cardioprotective efficacy. Confocal microscopy showed rapid internalization of SurR9C84A and scanning electron microscopy revealed the reinstatement of cytoskeleton projections in HI HCM. SurR9C84A treatment increased cell viability, reduced cell death via, apoptosis (Annexin-V assay), and downregulated free cardiac troponin T and MMP-9 expression. SurR9C84A also upregulated the expression of proliferation markers (PCNA and Ki-67) and downregulated mitochondrial depolarization and ROS levels thereby, impeding cell death. Human Apoptosis Array further revealed that SurR9C84A downregulated expression of pro-apoptotic markers and augmented expression of HSPs and HTRA2/Omi. SurR9C84A treatment led to enhanced levels of survivin, VEGF, PI3K and pAkt. SurR9C84A proved non-toxic to normoxic HCM, as validated through unaltered cell proliferation and other marker levels. Its pre-treatment exhibited lesser susceptibility to hypoxia/damage. SurR9C84A holds a promising clinical potential for human cardiomyocyte survival and proliferation following hypoxic injury.
Insights
A novel survivin mutant protein, SurR9C84A, effectively protects human cardiomyocytes from hypoxia-induced apoptosis. This protein promotes cell survival, proliferation, and regeneration, offering potential for treating heart injuries.
Area of Science:
- Cardiovascular Biology
- Cellular and Molecular Medicine
- Regenerative Medicine
Background:
- Survivin is an anti-apoptotic protein crucial for cell cycle regulation and organ regeneration.
- Hypoxic injury to cardiomyocytes leads to apoptosis, contributing to heart damage.
- Developing strategies to upregulate survivin expression is vital for salvaging injured cardiac cells.
Purpose of the Study:
- To investigate the cardioprotective and regenerative potential of a cell-permeable survivin mutant, SurR9C84A, in primary human cardiomyocytes (HCM) under hypoxic conditions.
- To elucidate the mechanism by which SurR9C84A facilitates cardioprotection and regeneration in hypoxic HCM.
- To assess the efficacy and safety of SurR9C84A in a clinically relevant in vitro model.
Main Methods:
- Primary human cardiomyocytes (HCM) were subjected to hypoxia using cobalt chloride (100µM, 48h).
- Hypoxic HCM were treated with SurR9C84A (1µg/mL) to evaluate its cardioprotective effects.
- Assays included confocal and scanning electron microscopy, Annexin-V assay, cardiac troponin T and MMP-9 level analysis, proliferation marker (PCNA, Ki-67) assessment, mitochondrial depolarization and ROS measurement, Human Apoptosis Array, and Western blotting for key signaling proteins (VEGF, PI3K, pAkt).
Main Results:
- SurR9C84A demonstrated rapid internalization and restored cytoskeleton integrity in hypoxic HCM.
- Treatment increased cell viability, reduced apoptosis, and decreased cardiac troponin T and MMP-9 levels.
- SurR9C84A upregulated proliferation markers, reduced mitochondrial depolarization and ROS, and modulated apoptotic and stress-response proteins.
- Key pro-survival pathways (VEGF, PI3K/pAkt) and survivin levels were enhanced.
- SurR9C84A was non-toxic to normoxic HCM and provided protection against subsequent hypoxic damage.
Conclusions:
- SurR9C84A effectively protects human cardiomyocytes from hypoxia-induced apoptosis and promotes regeneration.
- The mechanism involves restoring cellular integrity, enhancing survival pathways, and modulating apoptotic and stress responses.
- SurR9C84A exhibits promising therapeutic potential for managing hypoxic cardiac injury and improving cardiomyocyte survival and proliferation.
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