Adult Mouse Cardiomyocytes Research Articles

Superiority of focused ion beam-scanning electron microscope tomography of cardiomyocytes over standard 2D analyses highlighted by unmasking mitochondrial heterogeneity.

BackgroundCardioprotection by preventing or repairing mitochondrial damage is an unmet therapeutic need. To understand the role of cardiomyocyte mitochondria in physiopathology, the reliable characterization of the mitochondrial morphology and compartment is pivotal. Previous studies mostly relied on two‐dimensional (2D) routine transmission electron microscopy (TEM), thereby neglecting the real three‐dimensional (3D) mitochondrial organization. This study aimed to determine whether classical 2D TEM analysis of the cardiomyocyte ultrastructure is sufficient to comprehensively describe the mitochondrial compartment and to reflect mitochondrial number, size, dispersion, distribution, and morphology.MethodsSpatial distribution of the complex mitochondrial network and morphology, number, and size heterogeneity of cardiac mitochondria in isolated adult mouse cardiomyocytes and adult wild‐type left ventricular tissues (C57BL/6) were assessed using a comparative 3D imaging system based on focused ion beam‐scanning electron microscopy (FIB‐SEM) nanotomography. For comparison of 2D vs. 3D data sets, analytical strategies and mathematical comparative approaches were performed. To confirm the value of 3D data for mitochondrial changes, we compared the obtained values for number, coverage area, size heterogeneity, and complexity of wild‐type cardiomyocyte mitochondria with data sets from mice lacking the cytosolic and mitochondrial protein BNIP3 (BCL‐2/adenovirus E1B 19‐kDa interacting protein 3; Bnip3 −/− ) using FIB‐SEM. Mitochondrial respiration was assessed on isolated mitochondria using the Seahorse XF analyser. A cardiac biopsy was obtained from a male patient (48 years) suffering from myocarditis.ResultsThe FIB‐SEM nanotomographic analysis revealed that no linear relationship exists for mitochondrial number (r = 0.02; P = 0.9511), dispersion (r = −0.03; P = 0.9188), and shape (roundness: r = 0.15, P = 0.6397; elongation: r = −0.09, P = 0.7804) between 3D and 2D results. Cumulative frequency distribution analysis showed a diverse abundance of mitochondria with different sizes in 3D and 2D. Qualitatively, 2D data could not reflect mitochondrial distribution and dynamics existing in 3D tissue. 3D analyses enabled the discovery that BNIP3 deletion resulted in more smaller, less complex cardiomyocyte mitochondria (number: P < 0.01; heterogeneity: C.V. wild‐type 89% vs. Bnip3 −/− 68%; complexity: P < 0.001) forming large myofibril‐distorting clusters, as seen in human myocarditis with disturbed mitochondrial dynamics. Bnip3 −/− mice also show a higher respiration rate (P < 0.01).ConclusionsHere, we demonstrate the need of 3D analyses for the characterization of mitochondrial features in cardiac tissue samples. Hence, we observed that BNIP3 deletion physiologically acts as a molecular brake on mitochondrial number, suggesting a role in mitochondrial fusion/fission processes and thereby regulating the homeostasis of cardiac bioenergetics.

Modifications of the Langendorff Method for Simultaneous Isolation of Atrial and Ventricular Myocytes from Adult Mice

A single cardiomyocyte is a vital tool in the cellular and subcellular level studies of cardiac biology and diseases as a fundamental unit of contraction and electrical activity. Hence, isolating viable, high-quality cardiomyocytes from the heart is the initial and most crucial experimental step. Comparing the various protocols for isolating the cardiomyocytes of adult mice, the Langendorff retrograde perfusion is the most successful and reproducible method reported in the literature, especially for isolating ventricular myocytes. However, isolating quality atrial myocytes from the perfused heart remains challenging, and few successful isolation reports are available. Solving this complicated problem is extremely important because apart from ventricular disease, atrial disease accounts for a large part of heart diseases. Therefore, further investigations on the cellular level to reveal the mechanisms are warranted. In this paper, a protocol based on the Langendorff retrograde perfusion method is introduced and some modifications in the depth of aorta cannulation and the steps that may affect the digestion process to isolate atrial and ventricular myocytes were simultaneously made. Moreover, the isolated cardiomyocytes are confirmed to be amenable to patch clamp investigation.

Modifications of the Langendorff Method for Simultaneous Isolation of Atrial and Ventricular Myocytes from Adult Mice

A single cardiomyocyte is a vital tool in the cellular and subcellular level studies of cardiac biology and diseases as a fundamental unit of contraction and electrical activity. Hence, isolating viable, high-quality cardiomyocytes from the heart is the initial and most crucial experimental step. Comparing the various protocols for isolating the cardiomyocytes of adult mice, the Langendorff retrograde perfusion is the most successful and reproducible method reported in the literature, especially for isolating ventricular myocytes. However, isolating quality atrial myocytes from the perfused heart remains challenging, and few successful isolation reports are available. Solving this complicated problem is extremely important because apart from ventricular disease, atrial disease accounts for a large part of heart diseases. Therefore, further investigations on the cellular level to reveal the mechanisms are warranted. In this paper, a protocol based on the Langendorff retrograde perfusion method is introduced and some modifications in the depth of aorta cannulation and the steps that may affect the digestion process to isolate atrial and ventricular myocytes were simultaneously made. Moreover, the isolated cardiomyocytes are confirmed to be amenable to patch clamp investigation.

Cyclin D2 Overexpression Enhances the Efficacy of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes for Myocardial Repair in a Swine Model of Myocardial Infarction.

Human induced pluripotent stem cells with normal (wild-type) or upregulated (overexpressed) levels of CCND2 (cyclin D2) expression were differentiated into cardiomyocytes (CCND2WTCMs or CCND2OECMs, respectively) and injected into infarcted pig hearts. Acute myocardial infarction was induced by a 60-minute occlusion of the left anterior descending coronary artery. Immediately after reperfusion, CCND2WTCMs or CCND2OECMs (3×107 cells each) or an equivalent volume of the delivery vehicle was injected around the infarct border zone area. The number of the engrafted CCND2OECMs exceeded that of the engrafted CCND2WTCMs from 6- to 8-fold, rising from 1 week to 4 weeks after implantation. In contrast to the treatment with the CCND2WTCMs or the delivery vehicle, the administration of CCND2OECM was associated with significantly improved left ventricular function, as revealed by magnetic resonance imaging. This correlated with reduction of infarct size, fibrosis, ventricular hypertrophy, and cardiomyocyte apoptosis, and increase of vascular density and arterial density, as per histologic analysis of the treated hearts. Expression of cell proliferation markers (eg, Ki67, phosphorylated histone 3, and Aurora B kinase) was also significantly upregulated in the recipient cardiomyocytes from the CCND2OECM-treated than from the CCND2WTCM-treated pigs. The cell proliferation rate and the hypoxia tolerance measured in cultured human induced pluripotent stem cell cardiomyocytes were significantly greater after treatment with exosomes isolated from the CCND2OECMs (CCND2OEExos) than from the CCND2WTCMs (CCND2WTExos). As demonstrated by our study, CCND2OEExos can also promote the proliferation activity of postnatal rat and adult mouse cardiomyocytes. A bulk miRNA sequencing analysis of CCND2OEExos versus CCND2WTExos identified 206 and 91 miRNAs that were significantly upregulated and downregulated, respectively. Gene ontology enrichment analysis identified significant differences in the expression profiles of miRNAs from various functional categories and pathways, including miRNAs implicated in cell-cycle checkpoints (G2/M and G1/S transitions), or the mechanism of cytokinesis. We demonstrated that enhanced potency of CCND2OECMs promoted myocyte proliferation in both grafts and recipient tissue in a large mammal acute myocardial infarction model. These results suggest that CCND2OECMs transplantation may be a potential therapeutic strategy for the repair of infarcted hearts.

Poloxamer 188 Dose‐Dependently Protects Adult Mouse Coronary Artery Endothelial Cell Layers from Reoxygenation Injury

Poloxamer (P)188 is a tri-block copolymer shown to provide a cardioprotective effect when administered at the beginning of reoxygenation in adult mouse cardiomyocyte hypoxia/reoxygenation models. It is thought that it seals cells damaged by hypoxia via membrane stabilization, thus, protecting the cells from additional injury during reoxygenation. To further explore the effects of P188 on maintaining endothelial cell layer integrity, we treated confluent adult mouse coronary artery endothelial cell layers with varying concentrations of P188 upon initiation of a 2-hr reoxygenation period immediately following hypoxic periods of 3, 12, and 48 hrs. Using trans-endothelial electrical resistance (TEER), we found that P188 dose-dependently protected cells from further damage after hypoxia, with some doses returning TEER measurements even back to control/normoxia levels. However, we also observed that similar concentrations of P188 varied in effects as hypoxia times increased. Our results indicate that P188 protects the endothelial layer in a dose-dependent manner as long as it is administered after an optimal ischemia time to effectively preserve membrane integrity in tissue barriers. More research will need to be conducted to confirm our findings and determine the optimal combination of dosage and timing of P188 administration.

Sigmar1's Subcellular Localization and Function in the Heart

Background Sigma-1 receptor (Sigmar1) is a widely expressed molecular chaperone protein in mammalian cell systems. Accumulating research demonstrated the cardioprotective roles of Sigmar1 targeted ligands in clinical and preclinical rodent models of heart failure. However, Extensive biochemical and immuno-electron microscopic research demonstrated Sigmar1's sub-cellular localization largely depends on cell and organ types. Despite extensive studies, Sigmar1's subcellular localization and transmembrane topology in cardiomyocytes remain elusive. Objective We determined Sigmar1's subcellular localization, transmembrane topology, and functionality using complementary microscopy, biochemical, and functional assays in cardiomyocytes. Methods and Results Subcellular fractionation of heart cell lysates showed Sigmar1's localization in purified mitochondria fraction. Sigmar1's mitochondrial localization in isolated adult mouse cardiomyocytes by immunocytochemistry was confirmed by Sigmar1 co-localization with the mitochondrial indicator OXPHOS. We performed quantum dots in transmission electron microscopy and observed Sigmar1 leveled quantum dots on the mitochondrial membranes. We also confirmed Sigmar1 localization confocal microscopy using isolated mitochondria stained with Mito-Tracker red and Sigmar1 with the anti-Sigmar1 antibody. We confirmed Sigmar1 is an integral mitochondrial membrane protein by performing a series of biochemical experiments, including alkaline extraction and proteinase K treatment of purified heart mitochondria. We determined the Sigmar1's structural requirement for mitochondrial localization by expressing Sigmar1 fragments in cells. Immunostaining with anti-FLAG antibody showed that full-length Sigmar1 and Sigmar1's C terminal-deletion mutants (amino acid (aa) 1-223 and aa1-101) is localized in the mitochondrial membrane (mitochondria stained with Mito-Tracker red) whereas N- terminal deletion mutants (aa 102-223) were unable to incorporate into the mitochondria. Finally, we determined the functional role of Sigmar1 in cardiomyocytes using high-resolution Respirometry (Oroboros O2K) in intact cardiomyocytes. Genetic loss-of-function studies demonstrated that siRNA knockdown of Sigmar1 causes a significant reduction of oxygen flux in the presence of Complex I substrates and mitochondrial respiration un-coupler, under ADP supported OXPHOS state in isolated neonatal cardiomyocytes. Conclusions Overall, our studies revealed that Sigmar1 localizes to mitochondrial membranes and indispensable for maintaining mitochondrial respiratory homeostasis in cardiac myocytes.

Pharmacological and cell-specific genetic PI3Kα inhibition worsens cardiac remodeling after myocardial infarction

BackgroundPI3Kα (Phosphoinositide 3-kinase α) regulates multiple downstream signaling pathways controlling cell survival, growth, and proliferation and is an attractive therapeutic target in cancer and obesity. The clinically-approved PI3Kα inhibitor, BYL719, is in further clinical trials for cancer and overgrowth syndrome. However, the potential impact of PI3Kα inhibition on the heart and following myocardial infarction (MI) is unclear. We aim to determine whether PI3Kα inhibition affects cardiac physiology and post-MI remodeling and to elucidate the underlying molecular mechanisms. Methods and resultsWildtype (WT) 12-wk old male mice receiving BYL719 (daily, p.o.) for 10 days showed reduction in left ventricular longitudinal strain with normal ejection fraction, weight loss, mild cardiac atrophy, body composition alteration, and prolonged QTC interval. RNASeq analysis showed gene expression changes in multiple pathways including extracellular matrix remodeling and signaling complexes. After MI, both p110α and phospho-Akt protein levels were increased in human and mouse hearts. Pharmacological PI3Kα inhibition aggravated cardiac dysfunction and resulted in adverse post-MI remodeling, with increased apoptosis, elevated inflammation, suppressed hypertrophy, decreased coronary blood vessel density, and inhibited Akt/GSK3β/eNOS signaling. Selective genetic ablation of PI3Kα in endothelial cells was associated with worsened post-MI cardiac function and reduced coronary blood vessel density. In vitro, BYL719 suppressed Akt/eNOS activation, cell viability, proliferation, and angiogenic sprouting in coronary and human umbilical vein endothelial cells. Cardiomyocyte-specific genetic PI3Kα ablation resulted in mild cardiac systolic dysfunction at baseline. After MI, cardiac function markedly deteriorated with increased mortality concordant with greater apoptosis and reduced hypertrophy. In isolated adult mouse cardiomyocytes, BYL719 decreased hypoxia-associated activation of Akt/GSK3β signaling and cell survival. ConclusionsPI3Kα is required for cell survival (endothelial cells and cardiomyocytes) hypertrophic response, and angiogenesis to maintain cardiac function after MI. Therefore, PI3Kα inhibition that is used as anti-cancer treatment, can be cardiotoxic, especially after MI.

Sulforaphane exposure impairs contractility and mitochondrial function in three-dimensional engineered heart tissue

Sulforaphane (SFN) is a phytochemical compound extracted from cruciferous plants, like broccoli or cauliflower. Its isothiocyanate group renders SFN reactive, thus allowing post-translational modification of cellular proteins to regulate their function with the potential for biological and therapeutic actions. SFN and stabilized variants recently received regulatory approval for clinical studies in humans for the treatment of neurological disorders and cancer. Potential unwanted side effects of SFN on heart function have not been investigated yet. The present study characterizes the impact of SFN on cardiomyocyte contractile function in cardiac preparations from neonatal rat, adult mouse and human induced-pluripotent stem cell-derived cardiomyocytes. This revealed a SFN-mediated negative inotropic effect, when administered either acutely or chronically, with an impairment of the Frank-Starling response to stretch activation. A direct effect of SFN on myofilament function was excluded in chemically permeabilized mouse trabeculae. However, SFN pretreatment increased lactate formation and enhanced the mitochondrial production of reactive oxygen species accompanied by a significant reduction in the mitochondrial membrane potential. Transmission electron microscopy revealed disturbed sarcomeric organization and inflated mitochondria with whorled membrane shape in response to SFN exposure. Interestingly, administration of the alternative energy source l-glutamine to the medium that bypasses the uptake route of pyruvate into the mitochondrial tricarboxylic acid cycle improved force development in SFN-treated EHTs, suggesting indeed mitochondrial dysfunction as a contributor of SFN-mediated contractile dysfunction. Taken together, the data from the present study suggest that SFN might impact negatively on cardiac contractility in patients with cardiovascular co-morbidities undergoing SFN supplementation therapy. Therefore, cardiac function should be monitored regularly to avoid the onset of cardiotoxic side effects.

Mitochondrial functional resilience after TFAM ablation in the adult heart.

The nuclear genome-encoded mitochondrial DNA (mtDNA) transcription factor A (TFAM) is indispensable for mitochondrial energy production in the developing and postnatal heart; a similar role for TFAM is inferred in adult heart. Here, we provide evidence that challenges this long-standing paradigm. Unexpectedly, conditional Tfam ablation in vivo in adult mouse cardiomyocytes resulted in a prolonged period of functional resilience characterized by preserved mtDNA content, mitochondrial function, and cardiac function, despite mitochondrial structural alterations and decreased transcript abundance. Remarkably, TFAM protein levels did not directly dictate mtDNA content in the adult heart, and mitochondrial translation was preserved with acute TFAM inactivation, suggesting maintenance of respiratory chain assembly/function. Long-term Tfam inactivation, however, downregulated the core mtDNA transcription and replication machinery, leading to mitochondrial dysfunction and cardiomyopathy. Collectively, in contrast to the developing heart, these data reveal a striking resilience of the differentiated adult heart to acute insults to mtDNA regulation.

Effects of Hsp90 inhibitor on the RIP1-RIP3-MLKL pathway during the development of heart failure in mice

Necroptosis is a programmed form of necrotic cell death. Necroptosis is regulated by the necroptosis-regulating proteins including receptor-interacting protein (RIP) 1, RIP3, and mixed lineage kinase domain-like (MLKL), the activities of which are modulated by the molecular chaperone heat-shock protein (Hsp) 90. Presently, to clarify the relationship between Hsp90 and necroptotic pathway proteins, RIP1, RIP3, and MLKL in the development of heart failure, we examined the effects of Hsp90 inhibitor treatment on the RIP1-RIP3-MLKL pathway in mice following transverse aortic constriction (TAC). In this study, TAC mice showed typical signs of heart failure at the 8th week after the operation. In the failing heart, the levels of these regulatory proteins and those of their phosphorylated forms were increased, suggesting that necroptosis contributed to the development of heart failure in the TAC mice. The increases in RIP1, RIP3, and MLKL after TAC were reversed by the administration of an Hsp90 inhibitor. Furthermore, the rise in the phosphorylation levels of these 3 proteins were attenuated by the Hsp90 inhibitor. Concomitantly, cardiac functions were preserved. We also found that exposure of cultured adult mouse cardiomyocytes to the Hsp90 inhibitor attenuated necrotic cell death induced by tumor necrosis factor-α via suppression of RIP1, RIP3, and MLKL activation in in vitro experiments. Taken together, our findings suggest that inhibition of Hsp90 should have therapeutic effects by reducing the activation of RIP1-RIP3-MLKL pathway in the hypertrophied heart and thus could be a new therapeutic strategy for chronic heart failure.

Development of an AAV9-RNAi-mediated silencing strategy to abrogate TRPM4 expression in the adult heart

The cation channel transient receptor potential melastatin 4 (TRPM4) is a calcium-activated non-selective cation channel and acts in cardiomyocytes as a negative modulator of the L-type Ca2+ influx. Global deletion of TRPM4 in the mouse led to increased cardiac contractility under β-adrenergic stimulation. Consequently, cardiomyocyte-specific inactivation of the TRPM4 function appears to be a promising strategy to improve cardiac contractility in heart failure patients. The aim of this study was to develop a gene therapy approach in mice that specifically silences the expression of TRPM4 in cardiomyocytes. First, short hairpin RNAmiR30 (shRNAmiR30) sequences against the TRPM4 mRNA were screened in vitro using lentiviral transduction for a stable expression of the shRNA cassettes. Western blot analysis identified three efficient shRNAmiR30 sequences out of six, which reduced the endogenous TRPM4 protein level by up to 90 ± 6%. Subsequently, the most efficient shRNAmiR30 sequences were delivered into cardiomyocytes of adult mice using adeno-associated virus serotype 9 (AAV9)-mediated gene transfer. Initially, the AAV9 vector particles were administered via the lateral tail vein, which resulted in a downregulation of TRPM4 by 46 ± 2%. Next, various optimization steps were carried out to improve knockdown efficiency in vivo. First, the design of the expression cassette was streamlined for integration in a self-complementary AAV vector backbone for a faster expression. Compared to the application via the lateral tail vein, intravenous application via the retro-orbital sinus has the advantage that the vector solution reaches the heart directly and in a high concentration, and eventually a TRPM4 knockdown efficiency of 90 ± 7% in the heart was accomplished by this approach. By optimization of the shRNAmiR30 constructs and expression cassette as well as the route of AAV9 vector application, a 90% reduction of TRPM4 expression was achieved in the adult mouse heart. In the future, AAV9-RNAi-mediated inactivation of TRPM4 could be a promising strategy to increase cardiac contractility in preclinical animal models of acute and chronic forms of cardiac contractile failure.

The possible protective effect of aldehyde dehydrogenase 2 agonist (Alda-1) on doxorubicin induced toxicity of the left ventricular cardiomyocytes of adult male mice.

ABSTRACTBackground: Doxorubicin (DOX) is an effective antineoplastic drug. However, its cardiotoxicity is a major obstacle that limit its use. Therefore, a novel protective agent is urgently needed. Enzyme activation recently gained much interest as a new therapeutic strategy. In this context, Alda-1 is a newly emerged molecule in the field of cardioprotection through its activation of aldehyde dehydrogenase 2 enzyme.Aim of the study: To evaluate the possible protective effect of aldehyde dehydrogenase agonist Alda-1 on doxorubicin induced cardiotoxicity. Material and methods: Twenty-four adult male C57BL/6 mice were divided equally into 4 groups. Group I received intraperitoneal injection of saline. Group II received intraperitoneal injection of Alda-1 (10 mg/kg) daily for 5 days. Group III received a single intraperitoneal injection of DOX (20 mg/kg). Group IV received both DOX and Alda-1 in the same regimen as in groups II and III, Alda-1 was administered 1 day before DOX. All mice were euthanized on the 5th day following DOX administration. Blood samples were collected, hearts were excised and further processed for anatomical, histological and biochemical analysis.Results: DOX administration caused extensive cardiotoxicity as evident by the marked decrease in the heart weight and the extensive myocardial lesions shown by the light and electron microscopic examination. Moreover, biochemical serum markers of cardiac injury (CK-MB &LDH) was markedly elevated. Increased oxidative stress was also encountered as shown by the decreased serum TAC and increased tissue MDA. Furthermore, mitochondrial morphometry showed increased mitochondrial size. However, concomitant administration of Alda-1 with DOX have markedly alleviated the heart injury as shown by the restoration of the heart weight and cardiac architecture together with preservation of the mitochondrial structure. Moreover, Alda-1 has ameliorated the biochemical markers of cardiac injury and the oxidative stress.Conclusion: Alda-1 provides a valuable protective effect against doxorubicin triggered cardiotoxicity.

Isolation of Adult Mouse Cardiomyocytes Using Langendorff Perfusion Apparatus.

Heart disease is one of the leading causes of death in the United States. Isolation and culture adult cardiomyocytes are important for studying cardiomyocyte contractility, heart hypertrophy, and cardiac failure. In contrast to neonatal cardiomyocyte isolation, adult mice cardiomyocytes isolation is challenging due to firm connections among cardiomyocytes through intercalated discs. The availability of newly generated genetically modified mouse lines requires to establish protocols to isolation and culture adult mouse cardiomyocyte for in vitro studies. In this manuscript, we described a straightforward method of isolating adult mouse cardiomyocytes using Langendorff perfusion apparatus. Briefly, the hearts were harvested from adult mice and the heart was mounted to Lagendorff apparatus. After perfusion with calcium depletion and collagenase digestion, the left ventricles were minced and filtered. Lastly, the separated cardiomyocytes were treated with CaCl2. The isolated cardiac myocytes can be utilized in a broad range of experiments including screening for drugs.

Proteomic analysis reveals ginsenoside Rb1 attenuates myocardial ischemia/reperfusion injury through inhibiting ROS production from mitochondrial complex I.

Rationale: Reactive oxygen species (ROS) burst from mitochondrial complex I is considered the critical cause of ischemia/reperfusion (I/R) injury. Ginsenoside Rb1 has been reported to protect the heart against I/R injury; however, the underlying mechanism remains unclear. This work aimed to investigate if ginsenoside Rb1 attenuates cardiac I/R injury by inhibiting ROS production from mitochondrial complex I.Methods: In in vivo experiments, mice were given ginsenoside Rb1 and then subjected to I/R injury. Mitochondrial ROS levels in the heart were determined using the mitochondrial-targeted probe MitoB. Mitochondrial proteins were used for TMT-based quantitative proteomic analysis. In in vitro experiments, adult mouse cardiomyocytes were pretreated with ginsenoside Rb1 and then subjected to hypoxia and reoxygenation insult. Mitochondrial ROS, NADH dehydrogenase activity, and conformational changes of mitochondrial complex I were analyzed.Results: Ginsenoside Rb1 decreased mitochondrial ROS production, reduced myocardial infarct size, preserved cardiac function, and limited cardiac fibrosis. Proteomic analysis showed that subunits of NADH dehydrogenase in mitochondrial complex I might be the effector proteins regulated by ginsenoside Rb1. Ginsenoside Rb1 inhibited complex I- but not complex II- or IV-dependent O2 consumption and enzyme activity. The inhibitory effects of ginsenoside Rb1 on mitochondrial I-dependent respiration and reperfusion-induced ROS production were rescued by bypassing complex I using yeast NADH dehydrogenase. Molecular docking and surface plasmon resonance experiments indicated that ginsenoside Rb1 reduced NADH dehydrogenase activity, probably via binding to the ND3 subunit to trap mitochondrial complex I in a deactive form upon reperfusion.Conclusion: Inhibition of mitochondrial complex I-mediated ROS burst elucidated the probable underlying mechanism of ginsenoside Rb1 in alleviating cardiac I/R injury.

Mitochondrial Fission and Mitophagy Coordinately Restrict High Glucose Toxicity in Cardiomyocytes.

Hyperglycemia-induced mitochondrial dysfunction plays a key role in the pathogenesis of diabetic cardiomyopathy. Injured mitochondrial segments are separated by mitochondrial fission and eliminated by autophagic sequestration and subsequent degradation in the lysosome, a process termed mitophagy. However, it remains poorly understood how high glucose affects the activities of, and the relationship between, mitochondrial fission and mitophagy in cardiomyocytes. In this study, we determined the functional roles of mitochondrial fission and mitophagy in hyperglycemia-induced cardiomyocyte injury. High glucose (30 mM, HG) reduced mitochondrial connectivity and particle size and increased mitochondrial number in neonatal rat ventricular cardiomyocytes, suggesting an enhanced mitochondrial fragmentation. SiRNA knockdown of the pro-fission factor dynamin-related protein 1 (DRP1) restored mitochondrial size but did not affect HG toxicity, and Mdivi-1, a DRP1 inhibitor, even increased HG-induced cardiomyocyte injury, as shown by superoxide production, mitochondrial membrane potential and cell death. However, DRP1 overexpression triggered mitochondrial fragmentation and mitigated HG-induced cardiomyocyte injury, suggesting that the increased mitochondrial fission is beneficial, rather than detrimental, to cardiomyocytes cultured under HG conditions. This is in contrast to the prevailing hypothesis that mitochondrial fragmentation mediates or contributes to HG cardiotoxicity. Meanwhile, HG reduced mitophagy flux as determined by the difference in the levels of mitochondria-associated LC3-II or the numbers of mitophagy foci indicated by the novel dual fluorescent reporter mt-Rosella in the absence and presence of the lysosomal inhibitors. The ability of HG to induce mitochondrial fragmentation and inhibit mitophagy was reproduced in adult mouse cardiomyocytes. Overexpression of Parkin, a positive regulator of mitophagy, or treatment with CCCP, a mitochondrial uncoupler, induced mitophagy and attenuated HG-induced cardiomyocyte death, while Parkin knockdown had opposite effects, suggesting an essential role of mitophagy in cardiomyocyte survival under HG conditions. Strikingly, Parkin overexpression increased mitochondrial fragmentation, while DRP1 overexpression accelerated mitophagy flux, demonstrating a reciprocal activation loop that controls mitochondrial fission and mitophagy. Thus, strategies that promote the mutual positive interaction between mitochondrial fission and mitophagy while simultaneously maintain their levels within the physiological range would be expected to improve mitochondrial health, alleviating hyperglycemic cardiotoxicity.

A Phenyl-Pyrrolidine Derivative Reveals a Dual Inhibition Mechanism of Myocardial Mitochondrial Permeability Transition Pore, Which Is Limited by Its Myocardial Distribution

Mitochondrial permeability transition pore (mPTP) opening is a key event in cell death during myocardial ischemia reperfusion. Inhibition of its modulator cyclophilin D (CypD) by cyclosporine A (CsA) reduces ischemia-reperfusion injury. The use of cyclosporine A in this indication is debated; however, targeting mPTP remains a major goal to achieve. We investigated the protective effects of a new original small-molecule cyclophilin inhibitor C31, which was specifically designed to target CypD. CypD peptidylprolyl cis-trans isomerase (PPIase) activity was assessed by the standard chemotrypsin-coupled assay. The effects of C31 on mPTP opening were investigated in isolated mouse cardiac mitochondria by measuring mitochondrial swelling and calcium retention capacity (CRC) in rat H9C2 cardiomyoblasts and in adult mouse cardiomyocytes by fluorescence microscopy in isolated perfused mouse hearts and ex vivo after drug infusion in mice. C31 potently inhibited CypD PPIase activity and mitochondrial swelling. C31 was more effective at increasing mitochondrial CRC than CsA and was still able to increase CRC in Ppif -/- (CypD-inactivated) cardiac mitochondria. C31 delayed both mPTP opening and cell death in cardiomyocytes subjected to hypoxia reoxygenation. However, high concentrations of both drugs were necessary to reduce mPTP opening in isolated perfused hearts, and neither CsA nor C31 inhibited mPTP opening in heart after in vivo infusion, underlying the importance of myocardial drug distribution for cardioprotection. C31 is an original inhibitor of mPTP opening involving both CypD-dependent and -independent mechanisms. It constitutes a promising new cytoprotective agent. Optimization of its pharmacokinetic properties is now required prior to its use against cardiac ischemia-reperfusion injury. SIGNIFICANCE STATEMENT: This study demonstrates that the new cyclophilin inhibitor C31 potently inhibits cardiac mitochondrial permeability transition pore (mPTP) opening in vitro and ex vivo. The dual mechanism of action of C31 allows the prevention of mPTP opening beyond cyclophilin D inhibition. Further development of the compound might bring promising drug candidates for cardioprotection. However, the lack of effect of both C31 and cyclosporine A after systemic administration demonstrates the difficulties of targeting myocardial mitochondria in vivo and should be taken into account in cardioprotective strategies.

Abstract 17414: Combination Therapy of Sildenafil and Rapamycin Alleviates Doxorubicin Induced Cardiotoxicity With Improvement of Skeletal Muscle Function

Background: Doxorubicin (DOX) is a chemotherapeutic agent for multiple cancers, however, it causes systemic inflammation with multi-organ injury, including irreversible skeletal muscle (SM) dysfunction and cardiotoxicity. Treatment either with phosphodiesterase 5 inhibitor, sildenafil (SILD) or mammalian target of rapamycin (mTOR) inhibitor, Rapamycin (RAPA) protects against myocardial infarction (MI), heart failure and also has anti-tumor effects. We contemplated that unique combination of SILD and RAPA may enhance protective effect against DOX-induced SM dysfunction and cardiotoxicity with attenuation of inflammation. Methods and Results: Adult female C57 mice were treated with DOX (5 mg/kg/week; i.p.) with/without RAPA (0.25 mg/kg/d, i.p.) and/or SILD (1.4 mg/kg/day; in drinking water) for 4 weeks. Cardiac function (ejection fraction) and SM grip strength were significantly decreased in DOX-treated mice ( Fig. A, B ), which were improved by co-treatment with SILD+RAPA. Myostatin which inhibits myogenesis, was significantly upregulated upon DOX treatment, but was reduced by SILD+RAPA in SM ( Fig. C) . Adult mouse cardiomyocytes were treated with 1 μM DOX with/without 100 nM RAPA and/or 10 μM SILD for 24 hours. SILD+RAPA reduced DOX-induced apoptosis (assessed by TUNEL assay, Fig. D ) and ROS-generation (assessed by H2DCFDA, Fig. E ) while maintaining mitochondrial membrane potential (Δψ m ; measured by JC-1 staining, Fig. F ). Immunofluorescence staining revealed enhanced aggregation of ASC (adaptor molecule of inflammasome) following DOX treatment, which was inhibited by SILD+RAPA in cardiomyoblast (H9C2; Fig. G) and SM sol 8 cells ( Fig. H). Conclusion: Combination therapy with SILD+RAPA protects against DOX-induced cardiac and SM injury. Dual strategy of mTOR-PDE5 inhibition may be a novel approach to alleviate DOX toxicity during cancer treatment.

Abstract 16495: Activation of Notch Signaling in the Heart Promotes Electrical Disturbances

Notch1 receptor signaling is active in the heart during embryonic development, is progressively silenced after birth, and it is partly restored in the adult myocardium following ischemic damage, a condition characterized by occurrence of arrhythmias. We previously reported that inducible overexpression of Notch1 intracellular domain (gain-of-function, NGoF) in adult mouse cardiomyocytes alters ionic currents and action potential profile. But the consequences of Notch1 signaling activation on electrical properties and arrhythmogenicity of the entire heart remain to be defined. For this purpose, electrocardiograms were obtained in NGoF (n=25) and corresponding control (Ctrl, n=13) mice in the conscious state, over a 10 min period. Electrical signals were obtained at baseline and following administration of an arrhythmogenic cocktail (120 mg/kg BW caffeine and 2 mg/kg BW epinephrine) to test the propensity of these animals to develop electrical disturbances. Moreover, information was also obtained in mice with inducible Notch1 gene deletion for complete silencing of Notch1 signaling (Notch1 loss-of-function, NLoF, n=5). At baseline, heart rate and QT interval duration were comparable in NGoF (689±30 bpm; 49±2 ms), Ctrl (703±29 bpm; 48±1 ms), and NLoF (697±50 bpm; 48±1 ms) animals. Only a small fraction (~8%) of NGoF mice presented isolated premature ventricular complexes (PVCs) or junctional rhythm, but these events were not observed in Ctrl and NLoF mice. Administration of the arrhythmogenic cocktail induced PVCs in the majority of Ctrl (~56%) and NLoF (~60%) mice and in almost all NGoF animals (~92%). Also recurrent PVCs (≥10/10 min) were 1.7-2.1-fold higher in NGoF mice, with respect to NLoF and Ctrl. Importantly, 77% of NGoF mice developed ventricular tachycardia (VT), whereas VT occurrence was attenuated in Ctrl (56%) and NLoF (~40%) animals. Incidence of sustained VT was 1.7-fold higher in NGoF mice, with respect to Ctrl. Similarly, bidirectional VT was higher in NGoF (~62%), with respect to Ctrl (~52%) and NLoF (~40%). Thus, Notch signaling activation in the heart of experimental animals tends to enhance the occurrence of electrical disturbances, a factor that may be involved in the arrhythmogenic behavior of the post-infarcted myocardium.

Poloxamer 188 Protects Isolated Adult Mouse Cardiomyocytes from Reoxygenation Injury.

Reperfusion injury is a complex pathological event involving processes that can lead to further disruption of the cell membrane and function following an ischemic event. Return of blood flow allows for the needed reperfusion; however, for a period of time before remaining viable cells stabilize, reperfusion results in additional cellular injury. In cardiomyocytes, loss of membrane integrity allows abnormal influx of extracellular calcium, leading to hyper‐contracture and cell death. Methods to improve the membrane integrity of cardiomyocytes overwhelmed by pathological disruptions, such as reperfusion injury, are needed to prevent cell death, because of the myocardium's limited ability to regenerate. Research has shown administration of the copolymer P(oloxamer) 188 before ischemia/reperfusion can protect cardiomyocytes through membrane stabilization. This study sought to determine whether the administration of P188 at the beginning of the clinically more relevant time of reperfusion after ischemia will attenuate any additional damage to cardiomyocytes by stabilizing membrane integrity to allow the cells to maintain function. Using an in‐vitro cardiomyocyte model subjected to hypoxia/reoxygenation to simulate ischemia/reperfusion injury, we show that reoxygenation significantly potentiates the injury caused by hypoxia itself. P188, with its unique combination of hydrophobic and hydrophilic chemical properties, and only delivered at the beginning of reoxygenation, dose‐dependently protected cardiomyocytes from injury due to reoxygenation by repairing cell membranes, decreasing calcium influx, and maintaining cellular morphology. Our study also shows the hydrophobic portion of P188 is necessary for the stabilization of cell membrane integrity in providing protection to cardiomyocytes against reoxygenation injury.

Isolation, Transfection, and Long-Term Culture of Adult Mouse and Rat Cardiomyocytes.

Ex vivo culture of the adult mammalian cardiomyocytes (CMs) presents the most relevant experimental system for the in vitro study of cardiac biology. Adult mammalian CMs are terminally differentiated cells with minimal proliferative capacity. The post-mitotic state of adult CMs not only restricts cardiomyocyte cell cycle progression but also limits the efficient culture of CMs. Moreover, the long-term culture of adult CMs is necessary for many studies, such as CM proliferation and analysis of gene expression. The mouse and the rat are the two most preferred laboratory animals to be used for cardiomyocyte isolation. While the long-term culture of rat CMs is possible, adult mouse CMs are susceptible to death and cannot be cultured more than five days under normal conditions. Therefore, there is a critical need to optimize the cell isolation and long-term culture protocol for adult murine CMs. With this modified protocol, it is possible to successfully isolate and culture both adult mouse and rat CMs for more than 20 days. Moreover, the siRNA transfection efficiency of isolated CM is significantly increased compared to previous reports. For adult mouse CM isolation, the Langendorff perfusion method is utilized with an optimal enzyme solution and sufficient time for complete extracellular matrix dissociation. In order to obtain pure ventricular CMs, both atria were dissected and discarded before proceeding with the disassociation and plating. Cells were dispersed on a laminin coated plate, which allowed for efficient and rapid attachment. CMs were allowed to settle for 4-6 h before siRNA transfection. Culture media was refreshed every 24 h for 20 days, and subsequently, CMs were fixed and stained for cardiac-specific markers such as Troponin and markers of cell cycle such as KI67.