Analysis of the Mitochondrial Density and Longitudinal Distribution in Rat Live-Skeletal Muscle Fibers by Confocal Microscopy.

The mitochondrion is an organelle that can be elongated, fragmented, and renovated according to the metabolic requirements of the cells. The remodeling of the mitochondrial network allows healthy mitochondria to meet cellular demands; however, the loss of this capacity has been related to the development or progression of different pathologies. In skeletal muscle, mitochondrial density and distribution changes are observed in physiological and pathological conditions such as exercise, aging, and obesity, among others. Therefore, the study of the mitochondrial network may provide a better understanding of mechanisms related to those conditions. Here, a protocol for mitochondria imaging of live-skeletal muscle fibers from rats is described. Fibers are manually dissected in a relaxing solution and incubated with a fluorescent live-cell imaging indicator of mitochondria (tetramethylrhodamine ethyl ester, TMRE). The mitochondria signal is recorded by confocal microscopy using the XYZ scan mode to obtain confocal images of the intermyofibrillar mitochondrial (IMF) network. After that, the confocal images are processed by thresholding and binarization. The binarized confocal image accounts for the positive pixels for mitochondria, which are then counted to obtain the mitochondrial density. The mitochondrial network in skeletal muscle is characterized by a high density of IMF population, which has a periodic longitudinal distribution similar to that of T-tubules (TT). The Fast Fourier Transform (FFT) is a standard analysis technique performed to evaluate the distribution of TT that allows finding the distribution frequency and the level of their organization. In this protocol, the implementation of the FFT algorithm is described for the analysis of the longitudinal mitochondrial distribution in skeletal muscle.

Role of mitochondria-associated endoplasmic reticulum membranes in insulin sensitivity, energy metabolism, and contraction of skeletal muscle.

Skeletal muscle has a critical role in the regulation of the energy balance of the organism, particularly as the principal tissue responsible for insulin-stimulated glucose disposal and as the major site of peripheral insulin resistance (IR), which has been related to accumulation of lipid intermediates, reduced oxidative capacity of mitochondria and endoplasmic reticulum (ER) stress. These organelles form contact sites, known as mitochondria-associated ER membranes (MAMs). This interconnection seems to be involved in various cellular processes, including Ca2+ transport and energy metabolism; therefore, MAMs could play an important role in maintaining cellular homeostasis. Evidence suggests that alterations in MAMs may contribute to IR. However, the evidence does not refer to a specific subcellular location, which is of interest due to the fact that skeletal muscle is constituted by oxidative and glycolytic fibers as well as different mitochondrial populations that appear to respond differently to stimuli and pathological conditions. In this review, we show the available evidence of possible differential responses in the formation of MAMs in skeletal muscle as well as its role in insulin signaling and the beneficial effect it could have in the regulation of energetic metabolism and muscular contraction.

Relative role of T-tubules disruption and decreased SERCA2 on contractile dynamics of isolated rat ventricular myocytes.

AIMS: Ventricular myocytes (VM) depolarization activates L-type Ca2+ channels (LCC) allowing Ca2+ influx (ICa) to synchronize sarcoplasmic reticulum (SR) Ca2+ release, via Ca2+-release channels (RyR2). The resulting whole-cell Ca2+ transient triggers contraction, while cytosolic Ca2+ removal by SR Ca2+ pump (SERCA2) and sarcolemmal Na+/Ca2+ exchanger (NCX) allows relaxation. In diseased hearts, extensive VM remodeling causes heterogeneous, blunted and slow Ca2+ transients. Among remodeling changes are: A) T-tubules disorganization. B) Diminished SERCA2 and low SR Ca2+. However, those often overlap, hindering their relative contribution to contractile dysfunction (CD). Furthermore, few studies have assessed their specific impact on the spatiotemporal Ca2+ transient properties and contractile dynamics simultaneously. Therefore, we sought to perform a quantitative comparison of how heterogeneous and slow Ca2+ transients, with different underlying determinants, affect contractile performance. METHODS: We used two experimental models: A) formamide-induced acute "detubulation", where VM retain functional RyR2 and SERCA2, but lack T-tubules-associated LCC and NCX. B) Intact VM from hypothyroid rats, presenting decreased SERCA2 and SR Ca2+, but maintained T-tubules. By confocal imaging of Fluo-4-loaded VM, under field-stimulation, simultaneously acquired Ca2+ transients and shortening, allowing direct correlations. KEY FINDINGS: We found near-linear correlations among key parameters of altered Ca2+ transients, caused independently by T-tubules disruption or decreased SR Ca2+, and shortening and relaxation, SIGNIFICANCE: Unrelated structural and molecular alterations converge in similarly abnormal Ca2+ transients and CD, highlighting the importance of independently reproduce disease-specific alterations, to quantitatively assess their impact on Ca2+ signaling and contractility, which would be valuable to determine potential disease-specific therapeutic targets.

Metastatic TNBC is closely associated with a fused mitochondrial morphology and a glycolytic and lipogenic metabolism.

Mitochondria modify their function and morphology to satisfy the bioenergetic demand of the cells. Cancer cells take advantage of these features to sustain their metabolic, proliferative, metastatic, and survival necessities. Understanding the morphological changes to mitochondria in the different grades of triple-negative breast cancer (TNBC) could help to design new treatments. Consequently, this research explored mitochondrial morphology and the gene expression of some proteins related to mitochondrial dynamics, as well as proteins associated with oxidative and non-oxidative metabolism in metastatic and non-metastatic TNBC. We found that mitochondrial morphology and metabolism are different in metastatic and non-metastatic TNBC. In metastatic TNBC, there is overexpression of genes related to mitochondrial dynamics, fatty-acid metabolism, and glycolysis. These features are accompanied by a fused mitochondrial morphology. By comparison, in non-metastatic TNBC, there is a stress-associated mitochondrial morphology with hyperfragmented mitochondria, accompanied by the upregulated expression of genes associated with the biogenesis of mitochondria; both of which are characteristics related to the higher production of reactive oxygen species observed in this cell line. These differences between metastatic and non-metastatic TNBC should provide a better understanding of metastasis and contribute to the development of improved specific and personalized therapies for TNBC.

A single session of physical activity restores the mitochondrial organization disrupted by obesity in skeletal muscle fibers.

BACKGROUND: Several studies have proved that physical activity (PA) regulates energetic metabolism associated with mitochondrial dynamics through AMPK activation in healthy subjects. Obesity, a condition that induces oxidative stress, mitochondrial dysfunction, and low AMPK activity leads to mitochondrial fragmentation. However, few studies describe the effect of PA on mitochondrial dynamics regulation in obesity. AIM: The present study aimed to evaluate the effect of a single session of PA on mitochondrial dynamics regulation as well as its effect on mitochondrial function and organization in skeletal muscles of obese rats (Zucker fa/fa). MAIN METHODS: Male Zucker lean and Zucker fa/fa rats aged 12 to 13 weeks were divided into sedentary and subjected-to-PA (single session swimming) groups. Gastrocnemius muscle was dissected into isolated fibers, mitochondria, mRNA, and total proteins for their evaluation. KEY FINDINGS: The results showed that PA increased the Mfn-2 protein level in the lean and obese groups, whereas Drp1 levels decreased in the obese group. OMA1 protease levels increased in the lean group and decreased in the obese group. Additionally, AMPK analysis parameters (expression, protein level, and activity) did not increase in the obese group. These findings correlated with the partial restoration of mitochondrial function in the obese group, increasing the capacity to maintain the membrane potential after adding calcium as a stressor, and increasing the transversal organization level of the mitochondria analyzed in isolated fibers. SIGNIFICANCE: These results support the notion that obese rats subjected to PA maintain mitochondrial function through mitochondrial fusion activation by an AMPK-independent mechanism.

Mechanisms of mitochondrial DNA escape and its relationship with different metabolic diseases.

It is well-known that mitochondrial DNA (mtDNA) can escape to intracellular or extracellular compartments under different stress conditions, yet understanding their escape mechanisms remains a challenge. Although Bax/Bak pores and VDAC oligomers are the strongest possibilities, other mechanisms may be involved. For example, mitochondria permeability transition, altered mitophagy, and mitochondrial dynamics are associated with intracellular mtDNA escape, while extracellular traps and extracellular vesicles can participate in extracellular mtDNA escape. The evidence suggests that mtDNA escape is a complex event with more than one mechanism involved. In addition, once the mtDNA is outside the mitochondria, the effects can be complex. Different danger signal sensors recognize the mtDNA as a damage-associated molecular pattern, triggering an innate immune inflammatory response that can be observed in multiple metabolic diseases characterized by chronic inflammation, including autoimmune diseases, diabetes, cancer, and cardiovascular disorders. For these reasons, we will review the most recent evidence regarding mtDNA escape mechanisms and their impact on different metabolic diseases.

Simultaneous assessment of calcium handling and contractility dynamics in isolated ventricular myocytes of a rat model of post-acute isoproterenol-induced cardiomyopathy.

Stress-induced cardiomyopathy (SIC) results from a profound catecholaminergic surge during strong emotional or physical stress. SIC is characterized by acute left ventricular apex hypokinesia, in the absence of coronary arteries occlusion, and can lead to arrhythmias and acute heart failure. Although, most SIC patients recover, the process could be slow, and recurrence or death may occur. Despite that the SIC common denominator is a large catecholamine discharge, the pathophysiological mechanism is incompletely understood. It is thought that catecholamines have direct cytotoxicity on apical ventricular myocytes (VM), which have the highest ß-adrenergic receptors density, and whose overstimulation might cause acute Ca2+ overload and oxidative stress, causing death in some VM and stunning others. Rodents receiving acute isoproterenol (ISO) overdose (OV) mimic SIC development, however, they have not been used to simultaneously assess Ca2+ handling and contractility status in isolated VM, which might explain ventricular hypokinesia. Therefore, treating rats with a single ISO-OV (67 mg/kg body weight), we sought out to characterize, with confocal imaging, Ca2+ and shortening dynamics in Fluo-4-loaded VM, during the early (1-5 days) and late post-acute phases (15 days). We found that ISO-OV VM showed contractile dysfunction; blunted shortening with slower force development and relaxation. These correlated with Ca2+ mishandling; blunted Ca2+ transient, with slower time to peak and SR Ca2+ recovery. SR Ca2+ content was low, nevertheless, diastolic Ca2+ sparks were more frequent, and their duration increased. Contractility and Ca2+ dysfunction aggravated or remained altered over time, explaining slow recovery. We conclude that diminished VM contractility is the main determinant of ISO-OV hypokinesia and is mostly related to Ca2+ mishandling.

Underlying mechanism of the contractile dysfunction in atrophied ventricular myocytes from a murine model of hypothyroidism.

Hypothyroidism (Hypo) is a risk factor for cardiovascular diseases, including heart failure. Hypo rapidly induces Ca2+ mishandling and contractile dysfunction (CD), as well as atrophy and ventricular myocytes (VM) remodeling. Hypo decreases SERCA-to-phospholamban ratio (SERCA/PLB), and thereby contributes to CD. Nevertheless, detailed spatial and temporal Ca2+ cycling characterization in VM is missing, and contribution of other structural and functional changes to the mechanism underlying Ca2+ mishandling and CD, as transverse tubules (T-T) remodeling, mitochondrial density (Dmit) and energy availability, is unclear. Therefore, in a rat model of Hypo, we aimed to characterize systolic and diastolic Ca2+ signaling, T-T remodeling, Dmit, citrate synthase (CS) activity and high-energy phosphate metabolites (ATP and phosphocreatine). We confirmed a decrease in SERCA/PLB (59%), which slowed SERCA activity (48%), reduced SR Ca2+ (19%) and blunted Ca2+ transient amplitude (41%). Moreover, assessing the rate of SR Ca2+ release (dRel/dt), we found that early and maximum dRel/dt decreased, and this correlated with staggered Ca2+ transients. However, dRel/dt persisted during Ca2+ transient relaxation due to abundant late Ca2+ sparks. Isoproterenol significantly up-regulated systolic Ca2+ cycling. T-T were unchanged, hence, cannot explain staggered Ca2+ transients and altered dRel/dt. Therefore, we suggest that these might be caused by RyR2 clusters desynchronization, due to diminished Ca2+-dependent sensitivity of RyR2, which also caused a decrease in diastolic SR Ca2+ leak. Furthermore, Dmit was unchanged and CS activity slightly decreased (14%), however, the ratio phosphocreatine/ATP did not change, therefore, energy deficiency cannot account for Ca2+ and contractility dysregulation. We conclude that decreased SR Ca2+, due to slower SERCA, disrupts systolic RyR2 synchronization, and this underlies CD.

3D Imaging Detection of HER2 Based in the Use of Novel Affibody-Quantum Dots Probes and Ratiometric Analysis.

Patients with breast cancer (BC) overexpressing HER2 (HER2+) are selected for Trastuzumab treatment, which blocks HER2 and improves cancer prognosis. However, HER2+ diagnosis, by the gold standard, immunohistochemistry, could lead to errors, associated to: a) variability in sample manipulation (thin 2D sections), b) use of subjective algorithms, and c) heterogeneity of HER2 expression within the tissue. Therefore, we explored HER2 3D detection by multiplexed imaging of Affibody-Quantum Dots conjugates (Aff-QD), ratiometric analysis (RMAFI) and thresholding, using BC multicellular tumor spheroids (BC-MTS) (~120 µm of diameter) as 3D model of BC. HER2+, HER2- and hybrid HER2+/- BC-MTS (mimicking heterogeneous tissue) were incubated simultaneously with two Aff-QD probes (anti-HER2 and negative control (NC), respectively, (1:1)). Confocal XY sections were recorded along the Z distance, and processed by automatized RMAFI (anti-HER2 Aff-QD/ NC). Quantifying the NC fluorescence allowed to predict the fraction of non-specific accumulation of the anti-HER2 probe within the thick sample, and resolve the specific HER2 level. HER2 was detected up to 30µm within intact BC-MTS, however, permeabilization improved detection up to 70µm. Specific HER2 signal was objectively quantified, and HER2 3D-density of 9.2, 48.3 and 30.8% were obtained in HER2-, HER2+ and hybrid HER2+/- permeabilized BC-MTS, respectively. Therefore, by combining the multiplexing capacity of Aff-QD probes and RMAFI, we overcame the challenge of non-specific probe accumulation in 3D samples with minimal processing, yielding a fast, specific spatial HER2 detection and objective quantification.

The 8-oxo-deoxyguanosine glycosylase increases its migration to mitochondria in compensated cardiac hypertrophy.

Cardiac hypertrophy is a compensatory mechanism maladapted because it presents an increase in the oxidative stress which could be associated with the development of the heart failure. A mechanism proposed is by mitochondrial DNA (mtDNA) oxidation, which evolved to a vicious cycle because of the synthesis of proteins encoded in the genome is committed. Therefore, the aim of the present work was to evaluate the mtDNA damage and enzyme repairing the 8-oxo-deoxyguanosine glycosylase mitochondrial isoform 1-2a (OGG1-2a) in the early stage of compensated cardiac hypertrophy induced by abdominal aortic constriction (AAC). Results showed that after 6 weeks of AAC, hearts presented a compensated hypertrophy (22%), with an increase in the cell volume (35%), mitochondrial mass (12%), and mitochondrial membrane potential (94%). However, the increase of oxidative stress did not affect mtDNA most probably because OGG1-2a was found to increase 3.2 times in the mitochondrial fraction. Besides, mitochondrial function was not altered by the cardiac hypertrophy condition but in vitro mitochondria from AAC heart showed an increased sensibility to stress induced by the high Ca2+ concentration. The increase in the oxidative stress in compensated cardiac hypertrophy induced the OGG1-2a migration to mitochondria to repair mtDNA oxidation, as a mechanism that allows maintaining the cardiac function in the compensatory stage.

Role of SERCA and the sarcoplasmic reticulum calcium content on calcium waves propagation in rat ventricular myocytes.

In Ca(2+)-overloaded ventricular myocytes, SERCA is crucial to steadily achieve the critical sarcoplasmic reticulum (SR) Ca(2+) level to trigger and sustain Ca(2+) waves, that propagate at constant rate (ʋwave). High luminal Ca(2+) sensitizes RyR2, thereby increasing Ca(2+) sparks frequency, and the larger RyR2-mediated SR Ca(2+) flux (dF/dt) sequentially activates adjacent RyR2 clusters. Recently, it was proposed that rapid SERCA Ca(2+) reuptake, ahead of the wave front, further sensitizes RyR2, increasing ʋwave. Nevertheless, this is controversial because rapid cytosolic Ca(2+) removal could instead impair RyR2 activation. We assessed whether rapid SR Ca(2+) uptake enhances ʋwave by changing SERCA activity (ҡDecay) over a large range (∼175%). We used normal (Ctrl) and hyperthyroid rat (HT; reduced phospholamban by ∼80%) myocytes treated with thapsigargin or isoproterenol (ISO). We found that ʋwave and dF/dt had a non-linear dependency with ҡDecay, while Ca(2+) waves amplitude was largely unaffected. Furthermore, SR Ca(2+) also showed a non-linear dependency with ҡDecay, however, the relationships ʋwave vs. SR Ca(2+) and ʋwave vs. dF/dt were linear, suggesting that high steady state SR Ca(2+) determines ʋwave, while rapid SERCA Ca(2+) uptake does not. Finally, ISO did not increase ʋwave in HT cells, therefore, ISO-enhanced ʋwave in Ctrl depended on high SR Ca(2+).

Changes in T-Tubules and Sarcoplasmic Reticulum in Ventricular Myocytes in Early Cardiac Hypertrophy in a Pressure Overload Rat Model.

BACKGROUND/AIMS: Pressure-overload (PO) causes cardiac hypertrophy (CH), and eventually leads to heart failure (HF). HF ventricular myocytes present transverse-tubules (TT) loss or disarrangement and decreased sarcoplasmic reticulum (SR) density, and both contribute to altered Ca2+ signaling and heart dysfunction. It has been shown that TT remodeling precedes HF, however, it is unknown whether SR structural and functional remodeling also starts early in CH. METHODS: Using confocal microscopy, we assessed TT (with Di-8-ANNEPS) and SR (with SR-trapped Mag-Fluo-4) densities, as well as SR fluorophore diffusion (fluorescence recovery after photobleach; FRAP), cytosolic Ca2+ signaling and ex vivo cardiac performance in a PO rat hypertrophy model induced by abdominal aortic constriction (at 6 weeks). RESULTS: Rats developed CH, while cardiac performance, basal and upon ß-adrenergic stimulation, remained unaltered. TT density decreased by ∼14%, without spatial disarrangement, while SR density decreased by ∼7%. More important, FRAP was ∼30% slower, but with similar maximum recovery, suggesting decreased SR interconnectivity. Systolic and diastolic Ca2+ signaling and SR Ca2+ content were unaltered. CONCLUSION: SR remodeling is an early CH event, similar to TT remodeling, appearing during compensated hypertrophy. Nevertheless, myocytes can withstand those moderate structural changes in SR and TT, preserving normal Ca2+ signaling and contractility.