bims-mifefi 2024-02-11 papers

bims-mifefi

Biomed News

on Mitochondria and female physiology

Issue of 2024–02–11
eleven papers selected by
Kayla Vandiver, East Carolina University

Activation of mitochondrial oxygen consumption rate by delivering coenzyme Q10 to mitochondria of rat skeletal muscle cell (L6).
High Throughput Screening Assessment of Reactive Oxygen Species (ROS) Generation using Dihydroethidium (DHE) Fluorescence Dye.
Mitochondrial dynamics as a novel treatment strategy for triple-negative breast cancer.
Ablation of skeletal muscle estrogen receptor alpha impairs contractility in male mice.
Comparison of skeletal muscle oxidative function in young, middle-aged, and older males and females with similar physical activity levels.
17β-Estradiol Effects in Skeletal Muscle: A 31P MR Spectroscopic Imaging (MRSI) Study of Young Females during Early Follicular (EF) and Peri-Ovulation (PO) Phases.
The role of GPR81-cAMP-PKA pathway in endurance training-induced intramuscular triglyceride accumulation and mitochondrial content changes in rats.
A break in mitochondrial endosymbiosis as a basis for inflammatory diseases.
Parkin is a critical player in the effects of caffeine over mitochondrial quality control pathways during skeletal muscle regeneration in mice.
AMPK phosphorylation of FNIP1 (S220) controls mitochondrial function and muscle fuel utilization during exercise.
C-peptide in diabetes: A player in a dual hormone disorder?

J Pharm Sci. 2024 Feb 05. pii: S0022-3549(24)00041-8. [Epub ahead of print]

Activation of mitochondrial oxygen consumption rate by delivering coenzyme Q10 to mitochondria of rat skeletal muscle cell (L6).

Itsumi Sato, Mitsue Hibino, Atsuhito Takeda, Hideyoshi Harashima, Yuma Yamada.

  Numerous mitochondria are present in skeletal muscle cells. Muscle disease and aging impair mitochondrial functioning in the skeletal muscle. However, there have been few reports of therapeutic intervention via drug delivery to mitochondria owing to methodological difficulties. We surmised that mitochondrial activation is associated with improved skeletal muscle function. In this study, we attempted to activate the mitochondrial respiratory capacity in rat skeletal muscle cells (L6 cells) by delivering Coenzyme Q10 (CoQ10), a mitochondrial functional activator, to mitochondria using MITO-Porter, a nanoparticle that facilitates mitochondria-targeted drug delivery. Cellular uptake was confirmed by measuring the amount of fluorescence-modified MITO-Porter taken up by cells using flow cytometry. Intracellular dynamics of MITO-Porter was observed using confocal laser scanning microscopy. Mitochondrial function was assessed by measuring the mitochondrial oxygen consumption rate using an extracellular flux analyzer. The results indicated MITO-Porter-assisted delivery of CoQ10 to the mitochondria activated mitochondrial respiratory capacity in L6 cells. We believe that our results indicate the possibility of skeletal muscle therapy using mitochondrial drug delivery.

Keywords:  Drug delivery system; Lipid Nanoparticle (LNP); Liposome; Microfluidics; Muscle; Nanoparticle; Nanotechnology

DOI:  https://doi.org/10.1016/j.xphs.2024.01.020
J Vis Exp. 2024 Jan 19.

High Throughput Screening Assessment of Reactive Oxygen Species (ROS) Generation using Dihydroethidium (DHE) Fluorescence Dye.

Rahul Kumar, Rama R Gullapalli.

Reactive oxygen species (ROS) play a key role in the regulation of cellular metabolism in physiological and pathological processes. Physiological ROS production plays a central role in the spatial and temporal modulation of normal cellular functions such as proliferation, signaling, apoptosis, and senescence. In contrast, chronic ROS overproduction is responsible for a wide spectrum of diseases, such as cancer, cardiovascular disease, and diabetes, among others. Quantifying ROS levels in an accurate and reproducible manner is thus essential to understanding normal cellular functionality. Fluorescence imaging-based methods to characterize intra-cellular ROS species is a common approach. Many of the imaging ROS protocols in the literature use 2'-7'-dichlorodihydrofluorescein diacetate (DCFH-DA) dye. However, this dye suffers from significant limitations in its usage and interpretability. The current protocol demonstrates the use of a dihydroethidium (DHE) fluorescent probe as an alternative method to quantify total ROS production in a high-throughput setting. The high throughput imaging platform, CX7 Cellomics, was used to measure and quantify the ROS production. This study was conducted in three hepatocellular cancer cell lines - HepG2, JHH4, and HUH-7. This protocol provides an in-depth description of the various procedures involved in the assessment of ROS within the cells, including - preparation of DHE solution, incubation of cells with DHE solution, and measurement of DHE intensity necessary to characterize the ROS production. This protocol demonstrates that DHE fluorescent dye is a robust and reproducible choice to characterize intracellular ROS production in a high-throughput manner. High throughput approaches to measure ROS production are likely to be helpful in a variety of studies, such as toxicology, drug screening, and cancer biology.

DOI: https://doi.org/10.3791/66238
Cancer Med. 2024 Jan;13(2): e6987

Mitochondrial dynamics as a novel treatment strategy for triple-negative breast cancer.

Yuechen Wang, Narumi Harada-Shoji, Narufumi Kitamura, Yuto Yamazaki, Akiko Ebata, Masakazu Amari, Mika Watanabe, Minoru Miyashita, Hiroshi Tada, Takaaki Abe, Takashi Suzuki, Kohsuke Gonda, Takanori Ishida.

   INTRODUCTION: Triple-negative breast cancer (TNBC), recognized as the most heterogeneous type of breast cancer (BC), exhibits a worse prognosis than other subtypes. Mitochondria dynamics play a vital role as mediators in tumorigenesis by adjusting to the cell microenvironments. However, the relationship between mitochondrial dynamics and metabophenotype exhibits discrepancies and divergence across various research and BC models. Therefore, this study aims to explore the role of mitochondrial dynamics in TNBC drug resistance and tumorigenesis.
METHODS: The Wst-8 test was conducted to assess doxorubicin sensitivity in HCC38, MDA-MB-231 (TNBC), and MCF-7 (luminal). Confocal microscopy and FACS were used to quantify the mitochondrial membrane potential (ΔφM), mitophagy, and reactive oxygen species (ROS) production. Agilent Seahorse XF Analyzer was utilized to measure metabolic characteristics. Dynamin-related protein-1 (DRP1), Parkin, and p62 immunohistochemistry staining were performed using samples from 107 primary patients with BC before and after neoadjuvant chemotherapy (NAC).
RESULTS: MDA-MB-231, a TNBC cell line with reduced sensitivity to doxorubicin, reduced ΔφM, and enhanced mitophagy to maintain ROS production through oxidative phosphorylation (OXPHOS)-based metabolism. HCC38, a doxorubicin-sensitive cell line, exhibited no alterations in ΔφM or mitophagy. However, it demonstrated an increase in ROS production and glycolysis. Clinicopathological studies revealed that pretreatment (before NAC) expression of DRP1 was significant in TNBC, as was pretreatment expression of Parkin in the hormone receptor-negative group. Furthermore, low p62 levels seem to be a risk factor for recurrence-free survival.
CONCLUSION: Our findings indicated that the interplay between mitophagy, linked to a worse clinical prognosis, and OXPHOS metabolism promoted chemotherapy resistance in TNBC. Mitochondrial fission is prevalent in TNBC. These findings suggest that targeting the unique mitochondrial metabolism and dynamics in TNBC may offer a novel therapeutic strategy for patients with TNBC.

Keywords:  breast cancer; drug resistance; mitochondria; mitophagy

DOI:  https://doi.org/10.1002/cam4.6987
J Appl Physiol (1985). 2024 Feb 08.

Ablation of skeletal muscle estrogen receptor alpha impairs contractility in male mice.

Brian P Sullivan, Brittany C Collins, Shawna L McMillin, Elise Toussaint, Clara Z Stein, Espen E Spangenburg, Dawn A Lowe.

  Estradiol and estrogen receptor α (ERα) have been shown to be important for the maintenance of skeletal muscle strength in females, however little is known about the roles of estradiol and ERα in male muscle. The purpose of this study was to determine if skeletal muscle ERα is required for optimal contractility in male mice. We hypothesize that reduced ERα in skeletal muscle impairs contractility in male mice. Skeletal muscle specific knockout (skmERαKO) male mice exhibited reduced strength across multiple muscles and several contractile parameters related to force generation and kinetics compared to wildtype littermates (skmERαWT). Isolated EDL muscle specific isometric tetanic force, peak twitch force, peak concentric and peak eccentric forces, as well as the maximal rates of force development and relaxation were 11-21% lower in skmERαKO compared to skmERαWT mice. In contrast, isolated soleus muscles from skmERaKO mice were not affected. In vivo peak torque of the anterior crural muscles were 20% lower in skmERαKO compared to skmERαWT mice. Muscle masses, contractile protein contents, fiber types, phosphorylation of the myosin regulatory light chain, and caffeine-elicited force did not differ between muscles of skmERαKO and skmERαWT mice suggesting that strength deficits were not due to size, composition, or calcium release components of muscle contraction. These results indicate that in male mice reduced skeletal muscle ERα blunts contractility to a magnitude similar to that previously reported in females, however, the mechanism may be sexually dimorphic.

Keywords:  Estradiol; Strength; knockout; physiology; sex differences

DOI:  https://doi.org/10.1152/japplphysiol.00714.2023
J Appl Physiol (1985). 2024 Feb 08.

Comparison of skeletal muscle oxidative function in young, middle-aged, and older males and females with similar physical activity levels.

Rian Q Landers-Ramos, Kai Merriman, Gregory L Chartier, Jordyn Parks, Hyunjeong Park, Nicolas D Knuth.

  Optimal skeletal muscle oxidative function (microvascular reactivity and mitochondrial capacity) is an integral part of healthy aging and is related to physical function and quality of life. We aimed to extend upon the understanding of skeletal muscle oxidative function with healthy aging in males and females across the adult lifespan. Younger (N = 22; 11 males), middle-aged (N = 19; 10 males), and older (N = 21; 10 males) adults completed this study. Time spent in moderate and vigorous physical activity was self-reported and similar among groups. Near-infrared spectroscopy was used to investigate skeletal muscle microvascular reperfusion (O2Hb+Mb half time to peak hyperemia; T 1/2), mitochondrial capacity (mVO2 recovery rate constant), and walking tissue oxygen saturation (StO2) of the tibialis anterior (TA) muscle at 7 incremental walking speeds. Mitochondrial capacity was not significantly different across groups (p = 0.07). Younger adults exhibited significantly slower T 1/2 compared with older adults (p = 0.006) and middle-aged adults (p = 0.025). There were no observed sex differences for mitochondrial capacity (p = 0.442) or T 1/2 (p = 0.402). Older adults exhibited significantly lower StO2 across all walking speeds compared with younger adults (p = 0.003). Mitochondrial capacity and microvascular reperfusion are maintained in middle- and older age with no sex differences in either outcome. However, in older adults, whole-body functional movement, such as walking, may place an additional demand on the TA as a compensatory response to lower functional reserve not evident in distinct measures of mitochondrial capacity and microvascular reperfusion.

Keywords:  aging; microvascular reperfusion; mitochondrial capacity; sex differences; skeletal muscle

DOI:  https://doi.org/10.1152/japplphysiol.00545.2023
Diagnostics (Basel). 2024 Jan 23. pii: 235. [Epub ahead of print]14(3):

17β-Estradiol Effects in Skeletal Muscle: A 31P MR Spectroscopic Imaging (MRSI) Study of Young Females during Early Follicular (EF) and Peri-Ovulation (PO) Phases.

Jimin Ren, Luis Rodriguez, Talon Johnson, Anke Henning, Yasin Y Dhaher.

  The natural variation in estrogen secretion throughout the female menstrual cycle impacts various organs, including estrogen receptor (ER)-expressed skeletal muscle. Many women commonly experience increased fatigue or reduced energy levels in the days leading up to and during menstruation, when blood estrogen levels decline. Yet, it remains unclear whether endogenous 17β-estradiol, a major estrogen component, directly affects the energy metabolism in skeletal muscle due to the intricate and fluctuating nature of female hormones. In this study, we employed 2D 31P FID-MRSI at 7T to investigate phosphoryl metabolites in the soleus muscle of a cohort of young females (average age: 28 ± 6 years, n = 7) during the early follicular (EF) and peri-ovulation (PO) phases, when their blood 17β-estradiol levels differ significantly (EF: 28 ± 18 pg/mL vs. PO: 71 ± 30 pg/mL, p < 0.05), while the levels of other potentially interfering hormones remain relatively invariant. Our findings reveal a reduction in ATP-referenced phosphocreatine (PCr) levels in the EF phase compared to the PO phase for all participants (5.4 ± 4.3%). Furthermore, we observe a linear correlation between muscle PCr levels and blood 17β-estradiol concentrations (r = 0.64, p = 0.014). Conversely, inorganic phosphate Pi and phospholipid metabolite GPC levels remain independent of 17β-estradiol but display a high correlation between the EF and PO phases (p = 0.015 for Pi and p = 0.0008 for GPC). The robust association we have identified between ATP-referenced PCr and 17β-estradiol suggests that 17β-estradiol plays a modulatory role in the energy metabolism of skeletal muscle.

Keywords:  31P MRS; 7T; estrogen; magnesium; metabolism; phosphocreatine; skeletal muscle

DOI:  https://doi.org/10.3390/diagnostics14030235
J Physiol Sci. 2024 Feb 08. 74(1): 8

The role of GPR81-cAMP-PKA pathway in endurance training-induced intramuscular triglyceride accumulation and mitochondrial content changes in rats.

Lin Li, Xiangdeng Lai, Yihan Ni, Siyu Chen, Yaqian Qu, Zhiqiang Hu, Jingquan Sun.

  The athlete's paradox phenomenon involves the accumulation of intramuscular triglycerides (IMTG) in both insulin-resistant and insulin-sensitive endurance athletes. Nevertheless, a complete understanding of this phenomenon is yet to be achieved. Recent research indicates that lactate, a common byproduct of physical activity, may increase the accumulation of IMTG in skeletal muscle. This is achieved through the activation of G protein-coupled receptor 81 (GPR81) leads to the suppression of the cyclic adenosine monophosphate-protein kinase A (cAMP-PKA) pathway. The mechanism accountable for the increase in mitochondrial content in skeletal muscle triggered by lactate remains incomprehensible. Based on current research, our objective is to explore the role of the GPR81-inhibited cAMP-PKA pathway in the aggregation of IMTG and the increase in mitochondrial content as a result of prolonged exercise. The GPR81-cAMP-PKA-signaling pathway regulates the buildup of IMTG caused by extended periods of endurance training (ET). This is likely due to a decrease in proteins related to fat breakdown and an increase in proteins responsible for fat production. It is possible that the GPR81-cAMP-PKA pathway does not contribute to the long-term increase in mitochondrial biogenesis and content, which is induced by chronic ET. Additional investigation is required to explore the possible hindrance of the mitochondrial biogenesis and content process during physical activity by the GPR81-cAMP-PKA signal.

Keywords:  Endurance training; Intramuscular triglyceride; Lactate; Mitochondrial content; Skeletal muscle; cAMP

DOI:  https://doi.org/10.1186/s12576-024-00902-x
Nature. 2024 Feb;626(7998): 271-279

A break in mitochondrial endosymbiosis as a basis for inflammatory diseases.

Michael P Murphy, Luke A J O'Neill.

Mitochondria retain bacterial traits due to their endosymbiotic origin, but host cells do not recognize them as foreign because the organelles are sequestered. However, the regulated release of mitochondrial factors into the cytosol can trigger cell death, innate immunity and inflammation. This selective breakdown in the 2-billion-year-old endosymbiotic relationship enables mitochondria to act as intracellular signalling hubs. Mitochondrial signals include proteins, nucleic acids, phospholipids, metabolites and reactive oxygen species, which have many modes of release from mitochondria, and of decoding in the cytosol and nucleus. Because these mitochondrial signals probably contribute to the homeostatic role of inflammation, dysregulation of these processes may lead to autoimmune and inflammatory diseases. A potential reason for the increased incidence of these diseases may be changes in mitochondrial function and signalling in response to such recent phenomena as obesity, dietary changes and other environmental factors. Focusing on the mixed heritage of mitochondria therefore leads to predictions for future insights, research paths and therapeutic opportunities. Thus, whereas mitochondria can be considered 'the enemy within' the cell, evolution has used this strained relationship in intriguing ways, with increasing evidence pointing to the recent failure of endosymbiosis being critical for the pathogenesis of inflammatory diseases.

DOI: https://doi.org/10.1038/s41586-023-06866-z
Acta Physiol (Oxf). 2024 Feb 05. e14111

Parkin is a critical player in the effects of caffeine over mitochondrial quality control pathways during skeletal muscle regeneration in mice.

M V Esteca, I A Divino, A L Vieira da Silva, M B Severino, R R Braga, E R Ropelle, F M Simabuco, I L Baptista.

   AIM: This study aimed to investigate the effects of caffeine on pathways associated with mitochondrial quality control and mitochondrial capacity during skeletal muscle regeneration, focusing on the role of Parkin, a key protein involved in mitophagy.
METHODS: We used in vitro C2C12 myoblast during differentiation with and without caffeine in the medium, and we evaluated several markers of mitochondrial quality control pathways and myotube growth. In vivo experiments, we used C57BL/6J (WT) and Parkintm 1Shn lineage (Parkin-/- ) mice and injured tibial anterior muscle. The mice regenerated TA muscle for 3, 10, and 21 days with or without caffeine ingestion. TA muscle was used to analyze the protein content of several markers of mitochondrial quality pathways, muscle satellite cell differentiation, and protein synthesis. Furthermore, it analyzed mtDNA, mitochondrial respiration, and myofiber growth.
RESULTS: C2C12 differentiation experiments showed that caffeine decreased Parkin content, potentially leading to increased DRP1 and PGC-1α content and altered mitochondrial population, thereby enhancing growth capacity. Using Parkin-/- mice, we found that caffeine intake during the regenerative process induces an increase in AMPKα phosphorylation and PGC-1α and TFAM content, changes that were partly Parkin-dependent. In addition, the absence of Parkin potentiates the ergogenic effect of caffeine by increasing mitochondrial capacity and myotube growth. Those effects are related to increased ATF4 content and activation of protein synthesis pathways, such as increased 4E-BP1 phosphorylation.
CONCLUSION: These findings demonstrate that caffeine ingestion changes mitochondrial quality control during skeletal muscle regeneration, and Parkin is a central player in those mechanisms.

Keywords:  AMPK; caffeine; mitochondrial respiration; muscle recovery

DOI:  https://doi.org/10.1111/apha.14111
Sci Adv. 2024 Feb 09. 10(6): eadj2752

AMPK phosphorylation of FNIP1 (S220) controls mitochondrial function and muscle fuel utilization during exercise.

Liwei Xiao, Yujing Yin, Zongchao Sun, Jing Liu, Yuhuan Jia, Likun Yang, Yan Mao, Shujun Peng, Zhifu Xie, Lei Fang, Jingya Li, Xiaoduo Xie, Zhenji Gan.

Exercise-induced activation of adenosine monophosphate-activated protein kinase (AMPK) and substrate phosphorylation modulate the metabolic capacity of mitochondria in skeletal muscle. However, the key effector(s) of AMPK and the regulatory mechanisms remain unclear. Here, we showed that AMPK phosphorylation of the folliculin interacting protein 1 (FNIP1) serine-220 (S220) controls mitochondrial function and muscle fuel utilization during exercise. Loss of FNIP1 in skeletal muscle resulted in increased mitochondrial content and augmented metabolic capacity, leading to enhanced exercise endurance in mice. Using skeletal muscle-specific nonphosphorylatable FNIP1 (S220A) and phosphomimic (S220D) transgenic mouse models as well as biochemical analysis in primary skeletal muscle cells, we demonstrated that exercise-induced FNIP1 (S220) phosphorylation by AMPK in muscle regulates mitochondrial electron transfer chain complex assembly, fuel utilization, and exercise performance without affecting mechanistic target of rapamycin complex 1-transcription factor EB signaling. Therefore, FNIP1 is a multifunctional AMPK effector for mitochondrial adaptation to exercise, implicating a mechanism for exercise tolerance in health and disease.

DOI: https://doi.org/10.1126/sciadv.adj2752
J Cell Physiol. 2024 Feb 03.

C-peptide in diabetes: A player in a dual hormone disorder?

Ali Dakroub, Ali Dbouk, Aref Asfour, Suzanne A Nasser, Ahmed F El-Yazbi, Amirhossein Sahebkar, Assaad A Eid, Rabah Iratni, Ali H Eid.

  C-peptide, a byproduct of insulin synthesis believed to be biologically inert, is emerging as a multifunctional molecule. C-peptide serves an anti-inflammatory and anti-atherogenic role in type 1 diabetes mellitus (T1DM) and early T2DM. C-peptide protects endothelial cells by activating AMP-activated protein kinase α, thus suppressing the activity of NAD(P)H oxidase activity and reducing reactive oxygen species (ROS) generation. It also prevents apoptosis by regulating hyperglycemia-induced p53 upregulation and mitochondrial adaptor p66shc overactivation, as well as reducing caspase-3 activity and promoting expression of B-cell lymphoma-2. Additionally, C-peptide suppresses platelet-derived growth factor (PDGF)-beta receptor and p44/p42 mitogen-activated protein (MAP) kinase phosphorylation to inhibit vascular smooth muscle cells (VSMC) proliferation. It also diminishes leukocyte adhesion by virtue of its capacity to abolish nuclear factor kappa B (NF-kB) signaling, a major pro-inflammatory cascade. Consequently, it is envisaged that supplementation of C-peptide in T1DM might ameliorate or even prevent end-organ damage. In marked contrast, C-peptide increases monocyte recruitment and migration through phosphoinositide 3-kinase (PI-3 kinase)-mediated pathways, induces lipid accumulation via peroxisome proliferator-activated receptor γ upregulation, and stimulates VSMC proliferation and CD4+ lymphocyte migration through Src-kinase and PI-3K dependent pathways. Thus, it promotes atherosclerosis and microvascular damage in late T2DM. Indeed, C-peptide is now contemplated as a potential biomarker for insulin resistance in T2DM and linked to increased coronary artery disease risk. This shift in the understanding of the pathophysiology of diabetes from being a single hormone deficiency to a dual hormone disorder warrants a careful consideration of the role of C-peptide as a unique molecule with promising diagnostic, prognostic, and therapeutic applications.

Keywords:  atherosclerosis; cardiovascular disease; insulin resistance; metabolic disease; vascular smooth muscle

DOI:  https://doi.org/10.1002/jcp.31212