Monitoring mitochondrial calcium in cardiomyocytes during coverslip hypoxia using a fluorescent lifetime indicator.

An increase in mitochondrial calcium via the mitochondrial calcium uniporter (MCU) has been implicated in initiating cell death in the heart during ischemia-reperfusion (I/R) injury. Measurement of calcium during I/R has been challenging due to the pH sensitivity of indicators coupled with the fall in pH during I/R. The development of a pH-insensitive indicator, mitochondrial localized Turquoise Calcium fluorescence Lifetime Sensor (mito-TqFLITS), allows for quantifying mitochondrial calcium during I/R via fluorescent lifetime imaging. Mitochondrial calcium was monitored using mito-TqFLITS, in neonatal mouse ventricular myocytes (NMVM) isolated from germline MCU-KO mice and MCUfl/fl treated with CRE-recombinase to acutely knockout MCU. To simulate ischemia, a coverslip was placed on a monolayer of NMVMs to prevent access to oxygen and nutrients. Reperfusion was induced by removing the coverslip. Mitochondrial calcium increases threefold during coverslip hypoxia in MCU-WT. There is a significant increase in mitochondrial calcium during coverslip hypoxia in germline MCU-KO, but it is significantly lower than in MCU-WT. We also found that compared to WT, acute MCU-KO resulted in no difference in mitochondrial calcium during coverslip hypoxia and reoxygenation. To determine the role of mitochondrial calcium uptake via MCU in initiating cell death, we used propidium iodide to measure cell death. We found a significant increase in cell death in both the germline MCU-KO and acute MCU-KO, but this was similar to their respective WTs. These data demonstrate the utility of mito-TqFLITS to monitor mitochondrial calcium during simulated I/R and further show that germline loss of MCU attenuates the rise in mitochondrial calcium during ischemia but does not reduce cell death.

Mitochondrial permeability transition pore-dependent necrosis.

Mitochondrial permeability transition pore (mPTP)-dependent cell death is a form of necrotic cell death that is driven by mitochondrial dysfunction by the opening of the mPTP and is triggered by increases in matrix levels of Ca2+ and reactive oxygen species. This form of cell death has been implicated in ischemic injuries of the heart and brain as well as numerous degenerative diseases in the brain and skeletal muscle. This review focuses on the molecular triggers and regulators of mPTP-dependent necrosis in the context of myocardial ischemia reperfusion injury. Research over the past 50 years has led to the identity of regulators and putative pore-forming components of the mPTP. Finally, downstream consequences of activation of the mPTP as well as ongoing questions and areas of research are discussed. These questions pose a particular interest as targeting the mPTP could potentially represent an efficacious therapeutic strategy to reduce infarct size following an ischemic event.

ADH5-mediated NO bioactivity maintains metabolic homeostasis in brown adipose tissue.

Brown adipose tissue (BAT) thermogenic activity is tightly regulated by cellular redox status, but the underlying molecular mechanisms are incompletely understood. Protein S-nitrosylation, the nitric-oxide-mediated cysteine thiol protein modification, plays important roles in cellular redox regulation. Here we show that diet-induced obesity (DIO) and acute cold exposure elevate BAT protein S-nitrosylation, including UCP1. This thermogenic-induced nitric oxide bioactivity is regulated by S-nitrosoglutathione reductase (GSNOR; alcohol dehydrogenase 5 [ADH5]), a denitrosylase that balances the intracellular nitroso-redox status. Loss of ADH5 in BAT impairs cold-induced UCP1-dependent thermogenesis and worsens obesity-associated metabolic dysfunction. Mechanistically, we demonstrate that Adh5 expression is induced by the transcription factor heat shock factor 1 (HSF1), and administration of an HSF1 activator to BAT of DIO mice increases Adh5 expression and significantly improves UCP1-mediated respiration. Together, these data indicate that ADH5 controls BAT nitroso-redox homeostasis to regulate adipose thermogenesis, which may be therapeutically targeted to improve metabolic health.

Perilipin 2 downregulation in ß cells impairs insulin secretion under nutritional stress and damages mitochondria.

Perilipin 2 (PLIN2) is a lipid droplet (LD) protein in ß cells that increases under nutritional stress. Downregulation of PLIN2 is often sufficient to reduce LD accumulation. To determine whether PLIN2 positively or negatively affects ß cell function under nutritional stress, PLIN2 was downregulated in mouse ß cells, INS1 cells, and human islet cells. ß Cell-specific deletion of PLIN2 in mice on a high-fat diet reduced glucose-stimulated insulin secretion (GSIS) in vivo and in vitro. Downregulation of PLIN2 in INS1 cells blunted GSIS after 24-hour incubation with 0.2 mM palmitic acid. Downregulation of PLIN2 in human pseudoislets cultured at 5.6 mM glucose impaired both phases of GSIS, indicating that PLIN2 is critical for GSIS. Downregulation of PLIN2 decreased specific OXPHOS proteins in all 3 models and reduced oxygen consumption rates in INS1 cells and mouse islets. Moreover, we found that PLIN2-deficient INS1 cells increased the distribution of a fluorescent oleic acid analog to mitochondria and showed signs of mitochondrial stress, as indicated by susceptibility to fragmentation and alterations of acyl-carnitines and glucose metabolites. Collectively, PLIN2 in ß cells has an important role in preserving insulin secretion, ß cell metabolism, and mitochondrial function under nutritional stress.

Adipose Triglyceride Lipase Is a Key Lipase for the Mobilization of Lipid Droplets in Human ß-Cells and Critical for the Maintenance of Syntaxin 1a Levels in ß-Cells.

Lipid droplets (LDs) are frequently increased when excessive lipid accumulation leads to cellular dysfunction. Distinct from mouse ß-cells, LDs are prominent in human ß-cells. However, the regulation of LD mobilization (lipolysis) in human ß-cells remains unclear. We found that glucose increases lipolysis in nondiabetic human islets but not in islets in patients with type 2 diabetes (T2D), indicating dysregulation of lipolysis in T2D islets. Silencing adipose triglyceride lipase (ATGL) in human pseudoislets with shRNA targeting ATGL (shATGL) increased triglycerides (TGs) and the number and size of LDs, indicating that ATGL is the principal lipase in human ß-cells. In shATGL pseudoislets, biphasic glucose-stimulated insulin secretion (GSIS), and insulin secretion to 3-isobutyl-1-methylxanthine and KCl were all reduced without altering oxygen consumption rate compared with scramble control. Like human islets, INS1 cells showed visible LDs, glucose-responsive lipolysis, and impairment of GSIS after ATGL silencing. ATGL-deficient INS1 cells and human pseudoislets showed reduced SNARE protein syntaxin 1a (STX1A), a key SNARE component. Proteasomal degradation of Stx1a was accelerated likely through reduced palmitoylation in ATGL-deficient INS1 cells. Therefore, ATGL is responsible for LD mobilization in human ß-cells and supports insulin secretion by stabilizing STX1A. The dysregulated lipolysis may contribute to LD accumulation and ß-cell dysfunction in T2D islets.

Adipose Triglyceride Lipase is a Key Lipase for the Mobilization of Lipid Droplets in Human Beta Cells and Critical for the Maintenance of Syntaxin1a Level in Beta Cells.

Lipid droplets (LDs) are frequently increased when excessive lipid accumulation leads to cellular dysfunction. Distinct from mouse beta cells, LDs are prominent in human beta cells, however, the regulation of LD mobilization (lipolysis) in human beta cells remains unclear. We found that glucose increases lipolysis in non-diabetic human islets, but not in type 2 diabetic (T2D) islets, indicating dysregulation of lipolysis in T2D islets. Silencing adipose triglyceride lipase (ATGL) in human pseudoislets (shATGL) increased triglycerides, and the number and size of LDs indicating that ATGL is the principal lipase in human beta cells. In shATGL pseudoislets, biphasic glucose-stimulated insulin secretion (GSIS) and insulin secretion to IBMX and KCl were all reduced without altering oxygen consumption rate compared with scramble control. Like human islets, INS1 cells showed visible LDs, glucose responsive lipolysis, and impairment of GSIS after ATGL silencing. ATGL deficient INS1 cells and human pseudoislets showed reduced Stx1a, a key SNARE component. Proteasomal degradation of Stx1a was accelerated likely through reduced palmitoylation in ATGL deficient INS1 cells. Therefore, ATGL is responsible for LD mobilization in human beta cells and supports insulin secretion by stabilizing Stx1a. The dysregulated lipolysis may contribute to LD accumulation and beta cell dysfunction in T2D islets.

Lentiviral Mediated Gene Silencing in Human Pseudoislet Prepared in Low Attachment Plates.

Various genetic tools are available to modulate genes in pancreatic islets of rodents to dissect function of islet genes for diabetes research. However, the data obtained from rodent islets are often not fully reproduced in or applicable to human islets due to well-known differences in islet structure and function between the species. Currently, techniques that are available to manipulate gene expression of human islets are very limited. Introduction of transgene into intact islets by adenovirus, plasmid, and oligonucleotides often suffers from low efficiency and high toxicity. Low efficiency is especially problematic in gene downregulation studies in intact islets, which require high efficiency. It has been known that enzymatically-dispersed islet cells reaggregate in culture forming spheroids termed pseudoislets. Size-controlled reaggregation of human islet cells creates pseudoislets that maintain dynamic first phase insulin secretion after prolonged culture and provide a window to efficiently introduce lentiviral short hairpin RNA (shRNA) with low toxicity. Here, a detailed protocol for the creation of human pseudoislets after lentiviral transduction using two commercially available multiwell plates is described. The protocol can be easily performed and allows for efficient downregulation of genes and assessment of dynamism of insulin secretion using human islet cells. Thus, human pseudoislets with lentiviral mediated gene modulation provide a powerful and versatile model to assess gene function within human islet cells.

Delivery of shRNA via lentivirus in human pseudoislets provides a model to test dynamic regulation of insulin secretion and gene function in human islets.

Rodent islets are widely used to study the pathophysiology of beta cells and islet function, however, structural and functional differences exist between human and rodent islets, highlighting the need for human islet studies. Human islets are highly variable, deteriorate during culture, and are difficult to genetically modify, making mechanistic studies difficult to conduct and reproduce. To overcome these limitations, we tested whether pseudoislets, created by dissociation and reaggregation of islet cell suspensions, allow for assessment of dynamic islet function after genetic modulation. Characterization of pseudoislets cultured for 1 week revealed better preservation of first-phase glucose-stimulated insulin secretion (GSIS) compared with cultured-intact islets and insulin secretion profiles similar to fresh islets when challenged by glibenclamide and KCl. qPCR indicated that pseudoislets are similar to the original islets for the expression of markers for cell types, beta cell function, and cellular stress with the exception of reduced proinflammatory cytokine genes (IL1B, CCL2, CXCL8). The expression of extracellular matrix markers (ASPN, COL1A1, COL4A1) was also altered in pseudoislets compared with intact islets. Compared with intact islets transduced by adenovirus, pseudoislets transduced by lentivirus showed uniform transduction and better first-phase GSIS. Lastly, the lentiviral-mediated delivery of short hairpin RNA targeting glucokinase (GCK) achieved significant reduction of GCK expression in pseudoislets as well as marked reduction of both first and second phase GSIS without affecting the insulin secretion in response to KCl. Thus, pseudoislets are a tool that enables efficient genetic modulation of human islet cells while preserving insulin secretion.

Electron microscopic observation of human auricular chondrocytes transplanted into peritoneal cavity of nude mice for cartilage regeneration.

Restoration of damaged cartilage tissue has been deemed futile with current treatments. Although there have been many studies on cartilage regeneration thus far, there is no report that chondrocytes were completely re-differentiated in vitro. The clarification of cellular composition and matrix production during cartilage regeneration must be elucidated to fabricate viable mature cartilage in vitro. In order to achieve this aim, the chondrocytes cultured on coverslips were transplanted into the peritoneal cavities of mice. At different time points post-transplantation, the cartilage maturation progression and cells composing the regeneration were examined. Cartilage regeneration was confirmed by hematoxylin & eosin (HE) and toluidine blue staining. The maturation progression was carefully examined further by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). At the first and second week time points, various cell shapes were observed using SEM. Chronologically, by the third week, the number of fibers increased, suggesting the progression of extracellular matrix (ECM) maturation. Observation through TEM revealed the chondrocytes located in close proximity to various cells including macrophage-like cells. On the second week, infiltration of lymphocytes and capillary vessels were observed, and after the third week, the chondrocytes had matured and were abundantly releasing extracellular matrix. Chronological observation of transplanted chondrocytes by electron microscopy revealed maturation of chondrocytes and accumulation of matrix during the re-differentiation process. Emerging patterns of host-derived cells such as macrophage-like cells and subsequent appearance of lymphocytes-like cells and angiogenesis were documented, providing crucial context for the identification of the cells responsible for in vivo cartilage regeneration.

Improving chondrocyte harvests with poly(2-hydroxyethyl methacrylate) coated materials in the preparation for cartilage tissue engineering.

Remarkable advances have been made in cartilage regenerative medicine to cure congenital anomalies including microtia, tissue defects caused by craniofacial injuries, and geriatric diseases such as osteoarthritis. However, those procedures require a substantial quantity of chondrocytes for tissue engineering. Previous studies have required several passages to obtain sufficient cell numbers for three-dimensional and monolayer cultures. Thus, our objective was to improve the quantity of chondrocytes that can be obtained by examining an anti-fouling polyhydrophilic chemical called poly(2-hydroxyethyl methacrylate) (pHEMA). To determine the effectiveness of the chemical, pHEMA solution was applied via dip-coating to centrifuge tubes, serological pipettes, and pipette tips. The cell quantity obtained during standard cell culturing and passaging procedures was measured alongside non-coated materials as a control. A significant 2.2-fold increase of chondrocyte yield was observed after 2 passages when pHEMA was applied to the tubes compared to when non-coated tubes were utilized. The 3-dimensional chondrocyte pellets prepared from the respective cell populations and transplanted into nude mice were histologically and biochemically analyzed. No evidence of difference in matrix production for in vitro and in vivo cultures was found as well as similar proliferation rates and colony formation abilities. The use of pHEMA provides a powerful alternative method for expanding the quantity of chondrocytes harvested and handled during cell isolation and passaging to enhance cartilage tissue engineering.