Biology subjects

Garza-Lopez, E.

Publications and source records attributed to Garza-Lopez, E..

12 recordsLinked to original sources

bioRxiv2024

The MICOS Complex Regulates Mitochondrial Structure and Oxidative Stress During Age-Dependent Structural Deficits in the Kidney

Due to aging, the efficiency of kidney function begins to decrease. Dysfunction in mitochondria and their cristae is a hallmark of aging. Therefore, age-related decline in kidney function could be attributed to changes in mitochondrial ultrastructure, increased reactive oxygen species, and alterations in metabolism and lipid composition. We sought to understand how mitochondrial ultrastructure is altered over time in tubular kidney cells. A serial block facing-scanning electron microscope and manual segmentation using the Amira software were employed to visualize murine kidney samples during the aging process at 3 months (young) and 2 years (old). We found that 2-year mitochondria are more fragmented with many uniquely shaped mitochondria observed across aging, concomitant with shifts in ROS, metabolomics, and lipid homeostasis. Furthermore, we demonstrate that the mitochondrial contact site and cristae organizing system (MICOS) complex is impaired in the kidney during aging. Disruption of the MICOS complex resulted in altered mitochondrial metabolic function and increased ROS levels. We found significant, detrimental structural changes in the mitochondria of aged kidney tubules, suggesting a potential mechanism underlying the increased frequency of kidney disease with aging. We hypothesize that disruption of the MICOS complex exacerbates mitochondrial dysfunction, creating a vicious cycle of mitochondrial degradation and oxidative stress, which impacts kidney health. Impact and ImplicationsDue to aging, the efficiency of kidney function begins to decrease, and the risk of kidney diseases may increase; however, the specific regulators of mitochondrial age-related changes are poorly understood. This study demonstrates that the MICOS complex may be a target for mitigating age-related mitochondrial changes. The MICOS complex is associated with oxidative stress and calcium dysregulation, which also arise in many kidney pathologies. HighlightsO_LIAging alters the MICOS mRNA levels and disease markers. C_LIO_LIAging reduces cristae architecture, mitochondrial volume and complexity in murine kidney ultrastructure C_LIO_LIReducing MIC60 and CHCHD6 lowers Ca2+ uptake and retention and induces oxidative stress in HEK cells. C_LIO_LIMetabolomic Profiling revealed that NAD+ and amino acid metabolism were altered in aged kidneys. C_LIO_LIMICOS deficiency alters the reduced basal, ATP-linked, maximal capacity and spare capacity. C_LIO_LIDecreased modeled expression of CHCHD6 in individuals of European genetic ancestry is linked to chronic kidney disease, whereas decreased modeled expression of OPA1 in individuals of African genetic ancestry is associated with chronic kidney disease. C_LI Graphical AbstractKidney aging causes a decline in the MICOS complex, concomitant with metabolic, lipidomic, and mitochondrial structural alterations. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=142 SRC="FIGDIR/small/598108v3_ufig1.gif" ALT="Figure 1"> View larger version (53K): org.highwire.dtl.DTLVardef@1a868f0org.highwire.dtl.DTLVardef@1817bfborg.highwire.dtl.DTLVardef@1f2a1a3org.highwire.dtl.DTLVardef@520692_HPS_FORMAT_FIGEXP M_FIG C_FIG

physiology↗

bioRxiv2023

Human Heart Failure Alters Mitochondria and Fiber 3D Structure Triggering Metabolic Shifts

This study, utilizing SBF-SEM, reveals structural alterations in mitochondria and myofibrils in human heart failure (HF). Mitochondria in HF show changes in structure, while myofibrils exhibit increased cross-sectional area and branching. Metabolomic and lipidomic analyses indicate concomitant dysregulation in key pathways. The findings underscore the need for personalized treatments considering individualized structural changes in HF.

physiology↗

bioRxiv2023

Quantitative Assessment of Morphological Changes in Lipid Droplets and Lipid-Mito Interactions with Aging in Brown Adipose

The physical characteristics of brown adipose tissue (BAT) are defined by the presence of multilocular lipid droplets (LD) within the brown adipocytes and a high abundance of iron-containing mitochondria, which give it its characteristic color. Normal mitochondrial function is, in part, regulated by organelle-to-organelle contacts. Particularly, the contact sites that mediate mitochondria-LD interactions are thought to have various physiological roles, such as the synthesis and metabolism of lipids. Aging is associated with mitochondrial dysfunction, and previous studies show that there are changes in mitochondrial structure and proteins that modulate organelle contact sites. However, how mitochondria-LD interactions change with aging has yet to be fully clarified. Therefore, we sought to define age-related changes in LD morphology and mitochondria-lipid interactions in BAT. We examined the three-dimensional morphology of mitochondria and LDs in young (3-month) and aged (2-year) murine BAT using serial block face-scanning electron microscopy and the Amira program for segmentation, analysis, and quantification. Analysis showed reductions in LD volume, area, and perimeter in aged samples compared to young samples. Additionally, we observed changes in LD appearance and type in aged samples compared to young samples. Notably, we found differences in mitochondrial interactions with LDs, which could implicate that these contacts may be important for energetics in aging. Upon further investigation, we also found changes in mitochondrial and cristae structure for mitochondria interacting with LD lipids. Overall, these data define the nature of LD morphology and organelle-organelle contacts during aging and provide insight into LD contact site changes that interconnect biogerontology and mitochondrial functionality, metabolism, and bioactivity in aged BAT. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=141 SRC="FIGDIR/small/559135v2_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@e5b7org.highwire.dtl.DTLVardef@e965e5org.highwire.dtl.DTLVardef@120c7e9org.highwire.dtl.DTLVardef@124bfb5_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract Workflow highlighting the process of murine interscapular BAT extraction, processing and imaging serial block facing-scanning electron microscopy and using Amira software for 3D reconstruction of LDs and mitochondrial lipid contact sites to elucidate their structure across aging. C_FIG

biophysics↗

bioRxiv2023

Alterations in Cardiovascular and Cerebral Pulse Wave Velocity in 5XFAD Murine Model of Alzheimer's Disease

Alzheimers Disease (AD) is a global health issue, affecting over 6 million in the United States, with that number expected to increase as the aging population grows. As a neurodegenerative disorder that affects memory and cognitive functions, it is well established that AD is associated with cardiovascular risk factors beyond only cerebral decline. However, the study of cerebrovascular techniques for AD is still evolving. Here, we provide reproducible methods to measure impedance-based pulse wave velocity (PWV), a marker of arterial stiffness, in the systemic vascular (aortic PWV) and in the cerebral vascular (cerebral PWV) systems. Using aortic impedance and this relatively novel technique of cerebral impedance to comprehensively describe the systemic vascular and the cerebral vascular systems, we examined the sex-dependent differences in 5x transgenic mice (5XFAD) with AD under normal and high-fat diet, and in wild-type mice under a normal diet. Additionally, we validated our method for measuring cerebrovascular impedance in a model of induced stress in 5XFAD. Together, our results show that sex and diet differences in wildtype and 5XFAD mice account for very minimal differences in cerebral impedance. Interestingly, 5XFAD, and not wildtype, male mice on a chow diet show higher cerebral impedance, suggesting pathological differences. Opposingly, when we subjected 5XFAD mice to stress, we found that females showed elevated cerebral impedance. Using this validated method of measuring impedance-based aortic and cerebral PWV, future research may explore the effects of modifying factors including age, chronic diet, and acute stress, which may mediate cardiovascular risk in AD. New and NoteworthyHere, we presented a new technique which is an application of the concept of aortic impedance to determining cerebral impedance. While aortic PWV is typically utilized to study aortic stiffness, we also developed a technique of cerebral PWV to study cerebral vascular stiffness. This method may be useful in improving the rigor of studies that seek to have a dual focus on cardiovascular and cerebral function.

physiology↗

bioRxiv2023

Optimizing In Situ Proximity Ligation Assays for Mitochondria, ER, or MERC Markers in Skeletal Muscle Tissue and Cells

Proximity ligation assays (PLA) use specific antibodies to detect endogenous protein-protein interactions. PLA is a highly useful biochemical technique that allows two proteins within close proximity to be visualized with fluorescent probes amplified by PCR. While this technique has gained prominence, the use of PLA in mouse skeletal muscle (SkM) is novel. In this article, we discuss how the PLA method can be used in SkM to study the protein-protein interactions within mitochondria-endoplasmic reticulum contact sites (MERCs). Tweetable AbstractProximity Ligation Assays can be used in skeletal muscle tissue and myoblasts to explore the protein-protein interactions involved in MERC sites. HighlightsO_LISkeletal muscle tissue and cells are plated on glass coverslips for evaluation by proximity ligation assay (PLA). C_LIO_LIFollowing fixation, cells are probed and stained for Mfn1, Mfn2, mitochondria, and ER and imaged using fluorescence confocal microscopy. C_LIO_LIThis method shows that PLA can be used in mouse SkM and is adaptable to other models. C_LIO_LIProtocol for detection of protein-protein interactions using PLA. C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=126 SRC="FIGDIR/small/541599v2_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@1021ce7org.highwire.dtl.DTLVardef@1b78f8borg.highwire.dtl.DTLVardef@31030corg.highwire.dtl.DTLVardef@1bf0afa_HPS_FORMAT_FIGEXP M_FIG C_FIG O_TBL View this table: org.highwire.dtl.DTLVardef@169ba8corg.highwire.dtl.DTLVardef@566b30org.highwire.dtl.DTLVardef@197330dorg.highwire.dtl.DTLVardef@198ca7borg.highwire.dtl.DTLVardef@158b80_HPS_FORMAT_FIGEXP M_TBL C_TBL

cell biology↗

bioRxiv2023

Components of Isolated Skeletal Muscle Differentiated Through Antibody Validation

Isolation of skeletal muscles allows for the exploration of many complex diseases. Fibroblasts and myoblast play important roles in skeletal muscle morphology and function. However, skeletal muscles are complex and made up of many cellular populations and validation of these populations is highly important. Therefore, in this article, we discuss a comprehensive method to isolate mice skeletal muscle, create satellite cells for tissue culture, and use immunofluorescence to validate our approach. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=196 HEIGHT=200 SRC="FIGDIR/small/541600v2_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@165b34dorg.highwire.dtl.DTLVardef@1de9976org.highwire.dtl.DTLVardef@12d3cfeorg.highwire.dtl.DTLVardef@ff660c_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2023

Creating Optimal Conditions for OPA1 isoforms by Western Blot in Muscle Tissue

OPA1 is a dynamin-related GTPase that modulates various mitochondrial functions and is involved in mitochondrial morphology. There are eight different isoforms of OPA1 in humans and five different isoforms in mice that are expressed as short or long-form isoforms. These isoforms contribute to OPA1s ability to control mitochondrial functions. However, isolating OPA1 all long and short isoforms through western blot has been a difficult task. To address this issue, we outline an optimized western blot protocol to isolate 5 different isoforms of OPA1 on the basis of different antibodies. This protocol can be used to study changes in mitochondrial structure and function. Tweetable AbstractWestern blot protocol optimization to visualize OPA1 isoforms. HighlightsO_LIProtocol for isolating OPA1 isoforms in primary skeletal muscle myoblast and myotubes C_LIO_LISteps for running isolated skeletal muscle cells from muscle tissue on a gel C_LIO_LIHow to collect samples in preparation for western blotting C_LIO_LIDetection of OPA1 isoforms C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=134 SRC="FIGDIR/small/541601v2_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@1dc254dorg.highwire.dtl.DTLVardef@150d3eorg.highwire.dtl.DTLVardef@186ed2org.highwire.dtl.DTLVardef@13cc3a_HPS_FORMAT_FIGEXP M_FIG C_FIG O_TBL View this table: org.highwire.dtl.DTLVardef@1c518ceorg.highwire.dtl.DTLVardef@df826borg.highwire.dtl.DTLVardef@1eb5bf8org.highwire.dtl.DTLVardef@e324e6org.highwire.dtl.DTLVardef@8baedc_HPS_FORMAT_FIGEXP M_TBL C_TBL Method SummarySamples for western blot analysis are isolated from lysed cells, loaded onto a gel, and ran using optimized conditions to better isolate OPA1 isoforms. Samples are transferred to a membrane for incubation and protein detection using OPA1 antibodies.

cell biology↗

bioRxiv2023

Ablation of Sam50 is associated with fragmentation and alterations in metabolism in human myotubes

The Sorting and Assembly Machinery (SAM) Complex is responsible for assembling {beta}-barrel proteins in the mitochondrial membrane. Comprising three subunits, Sam35, Sam37, and Sam50, the SAM complex connects the inner and outer mitochondrial membranes by interacting with the mitochondrial contact site and cristae organizing system (MICOS) complex. Sam50, in particular, stabilizes the mitochondrial intermembrane space bridging (MIB) complex, which is crucial for protein transport, respiratory chain complex assembly, and regulation of cristae integrity. While the role of Sam50 in mitochondrial structure and metabolism in skeletal muscle remains unclear, this study aims to investigate its impact. Serial block-face-scanning electron microscopy (SBF-SEM) and computer-assisted 3D renderings were employed to compare mitochondrial structure and networking in Sam50-deficient myotubes from mice and humans with wild-type (WT) myotubes. Furthermore, autophagosome 3D structure was assessed in human myotubes. Mitochondrial metabolic phenotypes were assessed using Gas Chromatography-Mass Spectrometry-based metabolomics to explore differential changes in WT and Sam50-deficient myotubes. The results revealed increased mitochondrial fragmentation and autophagosome formation in Sam50-deficient myotubes compared to controls. Metabolomic analysis indicated elevated metabolism of propanoate and several amino acids, including {beta}-Alanine, phenylalanine, and tyrosine, along with increased amino acid and fatty acid metabolism in Sam50-deficient myotubes. Furthermore, impairment of oxidative capacity was observed upon Sam50 ablation in both murine and human myotubes, as measured with the XF24 Seahorse Analyzer. Collectively, these findings support the critical role of Sam50 in establishing and maintaining mitochondrial integrity, cristae structure, and mitochondrial metabolism. By elucidating the impact of Sam50-deficiency, this study enhances our understanding of mitochondrial function in skeletal muscle.

physiology↗

bioRxiv2023

Defining Mitochondrial Cristae Morphology Changes Induced by Aging in Brown Adipose Tissue

Mitochondria are required for energy production and even give brown adipose tissue (BAT) its characteristic color due to their high iron content and abundance. The physiological function and bioenergetic capacity of mitochondria are connected to the structure, folding, and organization of its inner-membrane cristae. During the aging process, mitochondrial dysfunction is observed, and the regulatory balance of mitochondrial dynamics is often disrupted, leading to increased mitochondrial fragmentation in aging cells. Therefore, we hypothesized that significant morphological changes in BAT mitochondria and cristae would be present with aging. We developed a quantitative three-dimensional (3D) electron microscopy approach to map cristae network organization in mouse BAT to test this hypothesis. Using this methodology, we investigated the 3D morphology of mitochondrial cristae in adult (3-month) and aged (2-year) murine BAT tissue via serial block face-scanning electron microscopy (SBF-SEM) and 3D reconstruction software for manual segmentation, analysis, and quantification. Upon investigation, we found increases in mitochondrial volume, surface area, and complexity and decreased sphericity in aged BAT, alongside significant decreases in cristae volume, area, perimeter, and score. Overall, these data define the nature of the mitochondrial structure in murine BAT across aging. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=144 SRC="FIGDIR/small/540609v2_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@96099borg.highwire.dtl.DTLVardef@50b4f4org.highwire.dtl.DTLVardef@983092org.highwire.dtl.DTLVardef@19ceb9b_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical Abstract:C_FLOATNO Overview of serial block facing-scanning electron microscopy (SBF-SEM) workflow, data segmentation, and 3D analysis of mitochondria using Amira software for murine interscapular BAT. C_FIG

biophysics↗

bioRxiv2022

Cardiovascular Hemodynamics in Mice with Tumor Necrosis Factor Receptor - Associated Factor 2 Mediated Cytoprotection in the Heart

Many studies in mice have demonstrated that cardiac-specific innate immune signaling pathways can be reprogrammed to modulate inflammation in response to myocardial injury and improve outcomes. While the echocardiography standard parameters of left ventricular (LV) ejection fraction, fractional shortening, and end-diastolic diameter, and others, are used to assess cardiac function, their dependency on loading conditions somewhat limit their utility in completely reflecting the contractile function and global cardiovascular efficiency of the heart. A true measure of global cardiovascular efficiency should include of the interaction between the ventricle and the aorta (ventriculo-vascular coupling, VVC) as well as measures of aortic impedance and pulse wave velocity. We measured cardiac Doppler velocities, blood pressures, along with VVC, aortic impedance, and pulse wave velocity to evaluate global cardiac function in mouse model of cardiac-restricted low levels TRAF2 overexpression that conferred cytoprotection in the heart. While previous studies reported that response to myocardial infraction and reperfusion was improved in the TRAF2 overexpressed mice, we found that TRAF2 mice had significantly lower cardiac systolic velocities and accelerations, diastolic atrial velocity, lower aortic pressures and rate-pressure product, lower LV contractility and relaxation, and lower stroke work when compared to littermate control mice. Also, we found significantly longer aortic ejection time, isovolumic contraction and relaxation times, and significantly higher mitral early/atrial ratio, myocardial performance index, and ventricular vascular coupling in the TRAF2 overexpression mice compared to their littermate controls. We found no significant differences in the aortic impedance and pulse wave velocity. While the reported tolerance to ischemic insults in TRAF2 overexpression mice may suggest enhanced cardiac reserve, our results indicate a diminished cardiac function in these mice.

biophysics↗

bioRxiv2022

DRP1-mediated mitochondrial fission is essential to maintain cristae morphology and bioenergetics

Mitochondria and peroxisomes are both dynamic signaling organelles that constantly undergo fission. While mitochondrial fission is known to coordinate cellular metabolism, proliferation, and apoptosis, the physiological relevance of peroxisome dynamics and the implications for cell fate are not fully understood. DRP1 (dynamin-related protein 1) is an essential GTPase that executes both mitochondrial and peroxisomal fission. Patients with de novo heterozygous missense mutations in the gene that encodes DRP1, DNM1L (Dynamin 1 Like), present with encephalopathy due to defective mitochondrial and peroxisomal fission (EMPF1). EMPF1 is a devastating neurodevelopmental disease with no effective treatment. To interrogate the mechanisms by which DRP1 mutations cause cellular dysfunction, we used human-derived fibroblasts from patients with mutations in DRP1 who present with EMPF1. As expected, patient cells display elongated mitochondrial morphology and lack of fission. Patient cells display a lower coupling efficiency of the electron transport chain, increased proton leak, and upregulation of glycolysis. In addition to these metabolic abnormalities, mitochondrial hyperfusion results in aberrant cristae structure and hyperpolarized mitochondrial membrane potential, both of which are tightly linked to the changes in metabolism. Peroxisome structure is also severely elongated in patient cells and results in a potential functional compensation of fatty acid oxidation. Understanding the mechanism by which DRP1 mutations cause these metabolic changes will give insight into the role of mitochondrial dynamics in cristae maintenance and the metabolic capacity of the cell, as well as the disease mechanism underlying EMPF1.

cell biology↗

bioRxiv2019

CaV3.1 channel pore pseudo-symmetry revealed by selectivity filter mutations in their domains I/II

There is growing evidence indicating that the pore structure of voltage-gated ion channels (VGICs) influences gating besides their conductance. Regarding low voltage-activated (LVA) Ca2+ channels, it has been demonstrated that substitutions of the pore aspartate (D) by a glutamate (D-to-E substitution) in domains III and IV alter channel gating properties such as a positive shift in the channel activation voltage dependence. In the present report, we evaluated the effects of E-to-D substitution in domains I and II on the CaV3.1 channel gating properties. Our results indicate that substitutions in these two domains differentially modify the gating properties of CaV3.1 channels. The channel with a single mutation in domain I (DEDD) presented slower activation and faster inactivation kinetics and a slower recovery from inactivation, as compared with the WT channel. In contrast, the single mutant in domain II (EDDD) presented a small but significant negative shift of activation voltage dependence with faster activation and slower deactivation kinetics. Finally, the double mutant channel (DDDD) presented intermediate properties with respect to the two single mutants but with fastest deactivation kinetics. Overall, our results indicate that single amino acid modification of the selectivity filter of LVA Ca2+ channels in distinct domains differentially influence their gating properties, suggesting a pore pseudo-symmetry.\n\nStatement of significancePrevious reports of low voltage-activated (LVA) Ca2+ channels have demonstrated that pore aspartate (D) in domains III and IV equally modulates the channel gating properties, supporting a hypothesis of pore symmetry in LVA Ca2+ channels. In the present report, we evaluated the effects of glutamate (E)-to-D pore substitution in domains I and II on the CaV3.1 channel gating properties. Our results indicate that substitutions in these two domains differentially modify the gating properties of CaV3.1 channels, therefore suggesting a pore pseudo-symmetry in them. Interestingly, our pore mutations affect inactivation of CaV3.1 Ca2+ channels indicating a selectivity filter contribution to this process similar to the recent proposed paradigm for high voltage-activated Ca2+ channels.

biophysics↗