Sieck, Gary C.

Recent findings suggest that autophagy plays a role in the development of aging-related neuromuscular dysfunction; however, the contribution of autophagy impairment to the maintenance of diaphragm force generation in old age is unknown. This study shows that in old mice, chloroquine administration decreases maximal transdiaphragmatic pressure generation. These chloroquine effects suggest a susceptibility to impairments in autophagy in old age.

Cellular mitochondrial function can be assessed using high-resolution respirometry that measures the O2 consumption rate (OCR) across a number of cells. However, a direct measurement of cellular mitochondrial function provides valuable information and physiological insight. In the present study, we used a quantitative histochemical technique to measure the activity of succinate dehydrogenase (SDH), a key enzyme located in the inner mitochondrial membrane, which participates in both the tricarboxylic acid (TCA) cycle and electron transport chain (ETC) as Complex II. In this study, we determine the maximum velocity of the SDH reaction (SDHmax) in individual human airway smooth muscle (hASM) cells. To measure SDHmax, hASM cells were exposed to a solution containing 80 mM succinate and 1.5 mM nitroblue tetrazolium (NBT, reaction indicator). As the reaction proceeded, the change in optical density (OD) due to the reduction of NBT to its diformazan (peak absorbance wavelength of 570 nm) was measured using a confocal microscope with the pathlength for light absorbance tightly controlled. SDHmax was determined during the linear period of the SDH reaction and expressed as mmol fumarate/liter of cell/min. We determine that this technique is rigorous and reproducible, and reliable for the measurement of mitochondrial function in individual cells.

Succinate dehydrogenase (SDH) is a key enzyme in the tricarboxylic acid (TCA) cycle, as well as Complex II of the electron transport chain (ETC). In the SDH reaction, succinate is oxidized to fumarate in the TCA cycle, producing FADH2 contributing to the proton gradient that drives the reduction of ubiquinone to ubiquinol in the ETC. Thus, measurement of the maximum velocity of the SDH reaction (SDHmax) provides insight into both TCA cycle and ETC contributions to the maximum respiratory capacity of mitochondria. The goal of the present study was to use a quantitative histochemical technique, which we previously developed in skeletal muscle fibers, to quantify the SDHmax in permeabilized (45 μg/mL saponin) cardiomyocytes isolated from male Sprague-Dawley rats. In the SDH reaction, the oxidation of succinate (80 mM succinate; determined to be the maximum substrate concentration) to fumarate is coupled to the reduction of nitroblue tetrazolium (NBT; 1.5 mM) to its diformazan (NBTdfz) using 1-methoxyphenazine methosulphate (mPMS; 1 mM) as an exogenous electron carrier and azide (0.75 mM) to inhibit Complex IV. The progressive accumulation of NBTdfz as the reaction proceeds is measured by repeated densitometric (at 570 nm, the peak absorbance wavelength for NBTdfz) measurements. The optical density (OD) measurements are converted to mM fumarate using the Beer-Lambert-Bouger equation based on pathlength for light absorbance and the molar extinction coefficient for NBTdfz. Importantly, the pathlength for light absorbance must be controlled. In cross-sections of skeletal muscle fibers, pathlength was controlled by cryosectioning at 6 μm. In the present study, pathlength was controlled by optical sectioning using a confocal microscope. The isolated cardiomyocytes were imaged in 3D using a Nikon Eclipse A1 laser scanning confocal system with a ×40x/1.3 NA oil-immersion lens with an optical slice set at 0.5 μm. Changes in OD within a series of 10 optical slices were measured every 15 s over a 5 min period and expressed as mM fumarate/min using the Beer-Lambert-Bouger equation. We found that the SDH reaction was linear across 5 min, and that the change in OD was consistent throughout all 10 optical slices within a cardiomyocyte, suggesting that NBTdfz accumulates in the cytoplasm. Our results confirm that this assay of SDHmax is reproducible in cardiomyocytes and offers a single cell alternative to high-resolution respirometry. The ability to measure SDHmax in single cells is in contrast to respirometry, which only measures the average O2 consumption of aggregates of cells (isolated or in tissue). Thus, a direct measurement of SDHmax can provide valuable physiological insight into variation in mitochondrial function across a population of cardiomyocytes, e.g., as might occur with ischemic injury. Supported by the Mayo Clinic This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Airway inflammation is the central driver behind the pathogenesis of respiratory diseases such as asthma, chronic obstructive pulmonary disease (COPD), chronic bronchitis, and the very recent pandemic outbreak of COVID-19. Previous studies reported that proinflammatory cytokine TNFa plays a significant role in airway inflammation by inducing hyper-reactivity of airway smooth muscle (ASM) and airway remodeling. TNFa also increases ASM force generation and ATP hydrolysis that requires energy. The excess energy demand is met by an increase in the level of PGC1a protein, mitochondrial biogenesis and subsequently increased mitochondrial volume density. Based on these previous findings, we hypothesized that TNFa promotes CREB phosphorylation at a specific serine 133 residue (pCREBS133) thereby transcriptionally activating PGC1a expression leading to mitochondrial biogenesis and increased mitochondrial volume density. In the study, human ASM cells were dissociated from bronchiolar tissue samples collected during bronchiolar surgery from female and male donors (34 to 71 years of age) with no history of respiratory diseases or current smoking. The dissociated ASM cells were serum-deprived for 48 h before experimentation. The ASM cells from each donor were separated into two groups: 1) time-matched untreated controls, and 2) TNFa treated (20 ng/ml for 6 h). Expression of CREB, pCREBS133, PCG1a, VDAC (an indicator of mitochondrial volume), and downstream signaling molecules (NRFs, TFAM), which regulate transcription and replication of the mitochondrial genome, were determined by quantitative real-time PCR (qPCR) and/or Western blot analysis. The binding of pCREBS133 to the PGC1a promoter was confirmed by bioinformatics analysis and chromatin immunoprecipitation (ChIP) assay. Mitochondria within ASM cells were labeled using MitoTracker green and imaged by 3D confocal microscopy for the assessment of mitochondrial volume density. Mitochondrial biogenesis was quantified by mitochondrial DNA (mtDNA) copy number relative to nuclear DNA using qPCR. Results were analyzed by one-way analysis of variance (ANOVA). Human ASM cells treated with TNFa showed increased pCREBS133 phosphorylation without any significant change in total CREB. The ChIP assay using a CREB-specific antibody revealed that pCREBS133 binds to the promoter of PGC1a and TNFa treatment further increased the binding affinity. Consistent with these findings, TNFa treatment increased PCG1a expression in ASM cells, and subsequently activated its downstream signaling pathway revealed by increased expression of its target genes, NRF1, NRF2 and TFAM. TNFa-induced activation of the PCG1a signaling pathway in ASM cells resulted in increased mitochondrial biogenesis as indicated by increased mitochondrial DNA copy number and increased mitochondrial volume density. Supported by NIH grant HL157984 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Autophagy is a process through which damaged cytoplasmic structures are recycled and degraded within the cell. Aging can affect autophagy regulation in different steps and lead to the accumulation of altered proteins, leading to cell damage and death. Motor neuron (MN) loss is an important contributor to sarcopenia age-dependent motor dysfunction. Understanding the regulatory mechanisms of autophagy can help elucidate the motor dysfunction that occurs with aging and provide a target for developing future therapeutic agents. Previous immunofluorescence imaging studies in phrenic MNs showed an age-dependent increase in key autophagy proteins LC3 and p62, reflecting autophagosome accumulation and autophagy impairment. Accordingly, we hypothesized that aging in lumbar MNs leads to autophagy impairment reflected in regulatory protein expression. The current study assessed autophagy in choline acetyltransferase-positive MNs from spinal levels L1-L6, focused on crural muscles (extensor digitorum longus and soleus), using confocal immunofluorescence imaging. Expression of LC3 and p62 proteins in MNs were determined in male and female C57BL/6 mice at 6-, 18- and 24- months of age. Increased expression of LC3 in lumbar MNs was observed with aging across all three age groups and p62 expression in lumbar MNs was significantly increased by 24- and 18-months compared to 6-months (p<0.001). There was no sex effect observed in LC3, but p62 was significantly higher in lumbar MNs of female mice (p=0.007). Expression of both LC3 and p62 increased diffusely in ventral lumbar gray matter regions with age (p<0.001), an effect that was predominant in male and not female mice. Taken together, the results of this study provide evidence for that age-dependent changes in autophagy markers expression occurs in regions of the spinal cord (motor neurons and ventral horn gray matter). The neuronal mechanisms of aging effects include dysregulation of autophagy. Supported by NIH R01 AG057052. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

During embryonic development, the number of phrenic motor neurons (PhMNs) declines precipitously during the final trimester and continues into the early postnatal period. When BDNF/TrkB signaling is absent in utero, animals do not survive after birth, most likely due to excessive loss of vital motor neurons (e.g., PhMNs). Far less is known about the early postnatal effect of BDNF/TrkB signaling. We have shown that BDNF/TrkB signaling is important in promoting PhMN neuroplasticity in mature rodents. Taken together, we hypothesized that inhibition of BDNF/TrkB signaling during early postnatal development results in fewer PhMNs. Chemo-genetic TrkBF616A mutant mice were used to induce inhibition of TrkB kinase activity in response to oral administration (via drinking water) of a phosphoprotein phosphatase 1 inhibitor derivative (1NMPP1). 1NMPP1-induced TrkB kinase inhibition was initiated at P0 and continued until P21 and compared to untreated control mice. At P28, PhMNs were retrogradely labeled via rhodamine phrenic nerve dip. One day later, mice were euthanized, perfused with 4% paraformaldehyde, and the cervical spinal cord was excised and processed for longitudinal cryosectioning (70 μm). Labeled PhMNs were imaged by 3D high resolution confocal microscopy. The number of PhMNs were counted. PhMN somal surface area and number of primary dendrites were also determined. 1NMPP1-induced inhibition of TrkB kinase resulted in 24% fewer PhMNs (P=0.01) and 21% more primary dendrites (P=0.0002) than control mice. The distribution of PhMN somal surface areas was not affected. We conclude that BDNF/TrkB signaling is essential in promoting survival of PhMNs in the early postnatal period. BDNF/TrkB signaling during postnatal development also plays a role in paring primary dendrites. While inhibition of TrkB kinase did not significantly alter the distribution of PhMN somal surface areas at P28, it is possible that further growth of PhMNs into maturity might have been altered as larger PhMNs emerge as a result of disproportionate growth beyond this age in mice. Supported by National Institutes of Health grants R01-AG044615 (GCS), R01-HL96750 (GCS). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Mitophagy is a regulated multistep process of selective removal of dysfunctional/damaged mitochondria by autophagy and is critical for the regulation of mitochondrial quality control in many physiological processes. The best characterized pathway of mitophagy involves PTEN-induced kinase 1 (PINK1) and E3-ubiquitin ligase Parkin. Upon mitochondrial depolarization, PINK1 stabilizes on the outer mitochondrial membrane (OMM) and recruits Parkin. After recruitment, PINK1 phosphorylates Parkin at Ser65, thus activating Parkin which promotes the ubiquitination of OMM proteins resulting in the subsequent lysosomal degradation of the mitochondria. Previously, we reported that tumor necrosis factor α (TNFα), a proinflammatory cytokine produced during inflammatory airway diseases, increases force generation and ATP hydrolysis, and results in increased mitochondrial fragmentation in human airway smooth muscle (hASM) cells. We also showed that TNFα induces PGC1α signaling, thus promoting mitochondrial biogenesis and increased mitochondrial volume density. In the present study, we hypothesize that TNFα induces mitophagy to promote selective clearance of dysfunctional mitochondria, thus maintaining a healthy mitochondrial pool to meet the increased energy demand in hASM cells. Experiments were performed in hASM cells dissociated from bronchiolar tissue samples collected during bronchiolar surgery from female and male donors with no current history of smoking or respiratory diseases. The hASM cells were serum deprived for 48 h and the phenotype was confirmed prior to experimentation. hASM cells were separated into three 6-h treatment groups: 1) TNFα (20 ng/mL), 2) FCCP (5 μM; positive control), and 3) untreated controls. The mitochondria and lysosomes in hASM cells were labelled with MitoTracker green (200 nM) and LysoTracker red (100 nM), respectively and imaged in 3D (Z optical slice of 0.5 μm) using an oil-immersion ×60/1.4 NA objective on a Nikon Eclipse A1 laser scanning confocal system. The 3D images were used to determine the coefficient for colocalization of mitochondria with lysosomes, lysosomal volume density, mitochondrial volume density, and mitochondrial complexity index (MCI). Protein expression and phosphorylation status of PINK1, Parkin, phospho-Parkin, VDAC (voltage-dependent anion channel), and LAMP1 (Lysosomal-associated membrane protein 1) were quantified by western blot. We observed that TNFα and FCCP treatment increased lysosomal volume density and mitochondrial colocalization with lysosomes in hASM cells, indicating the formation of mitophagosomes, characteristic of mitophagy. Consistent with previous findings, TNFα treatment increased mitochondrial volume density and decreased MCI, indicative of increased mitochondrial fragmentation. In addition, TNFα treatment increased the expression and mitochondrial translocation of PINK1 and Parkin. The results suggest that TNFα activates PINK1/Parkin-mediated mitophagy in hASM cells. Supported by NIH grants HL157984 (GCS) and HL105355 (OARR) This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Succinate dehydrogenase (SDH) is a mitochondrial enzyme that participates in both the tricarboxylic acid (TCA) cycle and the electron transport chain (ETC) as Complex II. In the TCA cycle, SDH catalyzes the oxidation of succinate to fumarate, which is coupled to the reduction of ubiquinone to ubiquinol in the ETC. Previously we developed and validated a quantitative histochemical technique to determine the maximum velocity of the SDH (SDHmax) reaction as a direct measurement of mitochondrial respiratory capacity in individual human airway smooth muscle (hASM) cells. The measurement of SDHmax was shown to provide a reliable estimate of the maximum respiratory capacity of an individual cell within a heterogeneous population of cells when compared to respirometry-based OCR measurements. In the present study we further assessed the specificity of this method by performing a targeted knockdown of the SDHB subunit in hASM cells. As a part of the catalytic core of the SDH complex, the SDHB subunit mediates electron transfer to ubiquinone and thereby regulates electron transfer by SDH. Therefore, we hypothesize that Sdhb-knockdown decreases SDHmax in hASM cells. This study was performed in hASM cells dissociated from bronchiolar tissue samples collected during bronchiolar surgery from female and male donors with no current history of smoking or respiratory diseases. Targeted Sdhb-knockdown was generated by siRNA mediated knockdown. hASM cells were also treated with malonate (1 mM for 24 h), which is a competitive inhibitor of SDH. To measure SDHmax, the cells were exposed to a solution containing 1-methoxyphenazine methosulphate (mPMS; exogenous electron carrier), azide (cytochrome oxidase inhibitor), 80 mM succinate (maximum substrate concentration) and 1.5 mM nitro blue tetrazolium (NBT; reaction indicator). As the SDH reaction proceeded, the hASM cells were imaged in 3D (Z optical slice of 0.5 μm) using an oil-immersion ×60/1.4 NA objective on a Nikon Eclipse A1 laser scanning confocal system. In the quantitative histochemical procedure, the change in optical density (OD), reflecting the progressive accumulation of NBT diformazan (NBTdfz; peak absorbance of 570 nm) in the cell, was measured every 15 s over an 8-min period (a linear reaction). SDHmax was determined from the OD using the Beer-Lambert equation and expressed as mM fumarate/L cell/min. In addition, we used MitoTracker Green (200 nM) to label and image mitochondria in hASM cells to determine mitochondrial volume density and mitochondrial complexity index (MCI). Our results show that SDHmax was markedly decreased in both Sdhb-knockdown and malonate treated hASM cells indicative of loss of function of SDH. Sdhb-knockdown increased mitochondrial volume density and decreased MCI in hASM cells, indicative of increased mitochondrial fragmentation and a role of SDH in the maintenance of mitochondrial morphology. The results confirm that the measurement of SDHmax is specific to SDH. Supported by NIH grant HL157984 (GCS) This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Transdiaphragmatic pressure (Pdi) measurements are used to determine diaphragm force generation across various motor behaviors. Previous studies in the diaphragm muscle found selective age-related changes at fast-twitch fatigable motor units including increased susceptibility to neuromuscular transmission failure and denervation. Accordingly, age-related changes at diaphragm motor units predominantly impair the ability to accomplish higher force behaviors, and these aging effects are evident prior to the onset of muscle atrophy. Recent findings suggest that autophagy plays a critical role in the development of age-related neuromuscular dysfunction; however, the possible contribution of autophagy impairment to the maintenance of diaphragm force generation in old age is unknown. We hypothesized that inhibition of autophagy (using chloroquine) decreases diaphragm force-generating capacity (Pdi) in mice susceptible to age-related neuromuscular dysfunction. Adult male and female C57BL/6 mice at 16-28 months of age were randomized to receive a single intraperitoneal injection of 50 mg/kg of chloroquine or vehicle (n= 9 per treatment group) four hours before Pdi measurements, with the experimenter blinded. Pdi was measured during eupnea (room air breathing), exposure to hypoxia/hypercapnia (10% O2/5% CO2), tracheal occlusion, spontaneous deep breaths (“sighs”) and during maximal activation elicited by bilateral phrenic nerve stimulation. Consistent with our hypothesis, there was an ~25% reduction in maximal Pdi in the chloroquine group (46 ± 12 cm H2O) compared to the vehicle control (61 ± 16 cm H2O; p < 0.05). There was no effect of chloroquine on Pdi during eupnea, hypoxia/hypercapnia, tracheal occlusion, or sigh behaviors. Acute inhibition of autophagy results in reduced maximal Pdi, consistent with that evident in old mice. Thus, we propose that autophagy impairment may be an important contributor to the reduced diaphragm force generation associated with aging. These findings help us further understand the mechanisms regulating age-related neuromuscular dysfunction and may serve as a foundation to develop novel treatments to mitigate aging effects. Support: NIH R01 AG057052. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Airway inflammation and pro-inflammatory cytokines such as tumor necrosis factor alpha (TNFα) underlie the pathophysiology of respiratory diseases, including asthma. Previously, we showed that TNFα selectively activates the inositol-requiring enzyme 1α (IRE1α)/X-box binding protein 1 spliced (XBP1s) ER stress pathway in human airway smooth muscle (hASM) cells. The ER stress pathway is activated by the accumulation of unfolded proteins in the ER. Accordingly, chemical chaperones such as 4-phenylbutyrate (4-PBA) may ameliorate ER stress activation. In the present study, we hypothesize that 4-PBA mitigates TNFα induced ER stress in hASM cells. hASM cells were isolated from bronchiolar tissue obtained from 5 donors with no history of current smoking or respiratory diseases. hASM cells were serum-deprived for 48 h, and phenotype confirmed by expression of smooth muscle actin and fusiform morphology. hASM cells from the same donor were then separated into 4 12-h treatment groups: 1) TNFα (20 ng/mL), 2) TNFα + 4-PBA (30 min pretreatment), and 3) untreated control. Protein expression and phosphorylation of IRE1α and XBP1s were determined by Western blot. Gene expression of XBP1s was analyzed by PCR. We found that TNFα induced IRE1α phosphorylation and XBP1 splicing, which were significantly reduced by pretreatment with 4-PBA. These results support our hypothesis and suggest that, in the presence of chemical chaperone 4-PBA, TNFα induced ER stress is significantly decreased. R01HL 157984 (GCS) This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.