Sieck, Gary C.

Aging disrupts multiple homeostatic processes, including autophagy, a cellular process for the recycling and degradation of defective cytoplasmic structures. Acute treatment with the autophagy inhibitor chloroquine blunts the maximal forces generated by the diaphragm muscle, but the mechanisms underlying neuromuscular dysfunction in old age remain poorly understood. We hypothesized that chloroquine treatment increases the presynaptic retention of the styryl dye FM 4-64 following high-frequency nerve stimulation, consistent with the accumulation of unprocessed bulk endosomes. Diaphragm-phrenic nerve preparations from 24-month-old male and female C57BL/6 × 129 J mice were incubated with FM 4-64 (5 µM) and either chloroquine (50 µM) or vehicle during 80 Hz phrenic nerve stimulation. Acute chloroquine treatment significantly decreased FM 4-64 intensity at diaphragm neuromuscular junctions following 80 Hz phrenic nerve stimulation, consistent with disrupted synaptic vesicle recycling. A similar reduction was evident in regions with the greatest FM 4-64 fluorescence intensity, which most likely surround synaptic vesicle release sites. In the absence of nerve stimulation, chloroquine treatment significantly increased FM 4-64 intensity at diaphragm neuromuscular junctions. These findings highlight the importance of autophagy in regulating presynaptic vesicle retrieval (including vesicle recycling and endosomal processing) and support the role of autophagy impairments in age-related neuromuscular dysfunction.

Abstract Ageing‐related neuromuscular dysfunction is associated with reduced tropomyosin‐related kinase receptor subtype B (TrkB) signalling and accumulation of damaged cytoplasmic aggregates in motor neurons. Autophagy functions to remove these damaged aggregates, and we previously reported increased cervical motor neuron expression of LC3 and p62 in old age. We hypothesized that inhibition of TrkB kinase activity results in an increase in the relative expression of both LC3 and p62 in cervical motor neurons, consistent with impaired progression of autophagy. TrkB F616A mice, which possess a mutation that renders TrkB kinase activity susceptible to rapid inhibition by 1NMPP1, were treated at 6, 18 or 24 months of age with vehicle or 1NMPP1 for 7 days. Immunofluorescence intensity was measured to determine LC3 and p62 expression in choline acetyltransferase‐positive motor neurons in the cervical spinal cord. The effect of inhibiting TrkB kinase activity on progression of autophagy was age dependent. In 6‐month‐old mice, inhibiting TrkB kinase activity increased cervical motor neuron expression of LC3 by 11% ( P < 0.001) and p62 by 8% ( P = 0.019) compared with vehicle treatment. In 18‐ and 24‐month‐old mice, there was no effect of inhibiting TrkB kinase activity on motor neuron LC3 or p62 expression. We provide evidence that inhibition of TrkB signalling impairs progression of autophagy in motor neurons of young mice, similar to the response to ageing. Accordingly, a reduction of TrkB signalling in old age might contribute to neuromuscular dysfunction by impairing progression of autophagy in motor neurons.

The neuromotor control of the diaphragm muscle (DIAm) in response to hypercapnia is not well understood. We show that both the number of motor units recruited and their discharge rates increase with hypercapnia, consistent with increased respiratory drive during hypercapnia. Potentially in response to this increased drive, the greatest effect of hypercapnia is on during the postinspiratory (descending) ramp of DIAm EMG activity, which shortens to facilitate higher respiratory rates.

AbstractAging is associated with cardiac contractile abnormalities, but the etiology of these contractile deficits is unclear. We hypothesized that cardiac contractile and regulatory protein expression is altered during aging. To investigate this possibility, left ventricular (LV) lysates were prepared from young (6 months) and old (24 months) Fischer344 rats. There are no age‐related changes in SERCA2 expression or phospholamban phosphorylation. Additionally, neither titin isoform expression nor phosphorylation differed. However, there is a significant increase in β‐isoform of the myosin heavy chain (MyHC) expression and phosphorylation of TnI and MyBP‐C during aging. In permeabilized strips of papillary muscle, force and Ca2+ sensitivity are reduced during aging, consistent with the increase in β‐MyHC expression and TnI phosphorylation. However, the increase in MyBP‐C phosphorylation during aging may represent a mechanism to compensate for age‐related contractile deficits. In isolated cardiomyocytes loaded with Fura‐2, the peak of the Ca2+ transient is reduced, but the kinetics of the Ca2+ transient are not altered. Furthermore, the extent of shortening and the rates of both sarcomere shortening and re‐lengthening are reduced. These results demonstrate that aging is associated with changes in contractile and regulatory protein expression and phosphorylation, which affect the mechanical properties of cardiac muscle.

The heterogeneity of diaphragm muscle (DIAm) activity in awake animals reflects real motor behavior diversity but makes assessments of eupneic breathing challenging. The present article uses an unsupervised machine learning model to identify eupneic breathing amidst a deluge of different DIAm electromyography (EMG) burst patterns in awake rats. This technique offers a scalable and reliable tool that improves efficiency of DIAm EMG analysis and minimizes potential sources of bias.

AbstractSuccinate dehydrogenase (SDH) is a key mitochondrial enzyme involved in the tricarboxylic acid cycle, where it facilitates the oxidation of succinate to fumarate, and is coupled to the reduction of ubiquinone in the electron transport chain as Complex II. Previously, we developed a confocal‐based quantitative histochemical technique to determine the maximum velocity of the SDH reaction (SDHmax) in single cells and observed that SDHmax corresponds with mitochondrial volume density. In addition, mitochondrial volume and motility varied within different compartments of human airway smooth muscle (hASM) cells. Therefore, we hypothesize that the SDH activity varies relative to the intracellular mitochondrial volume within hASM cells. Using 3D confocal imaging of labeled mitochondria and a concentric shell method for analysis, we quantified mitochondrial volume density, mitochondrial complexity index, and SDHmax relative to the distance from the nuclear membrane. The mitochondria within individual hASM cells were more filamentous in the immediate perinuclear region and were more fragmented in the distal parts of the cell. Within each shell, SDHmax also corresponded to mitochondrial volume density, where both peaked in the perinuclear region and decreased in more distal parts of the cell. Additionally, when normalized to mitochondrial volume, SDHmax was lower in the perinuclear region when compared to the distal parts of the cell. In summary, our results demonstrate that SDHmax measures differences in SDH activity within different cellular compartments. Importantly, our data indicate that mitochondria within individual cells are morphologically heterogeneous, and their distribution varies substantially within different cellular compartments, with distinct functional properties.

The diaphragm muscle (DIAm) as the major inspiratory pump is highly active. During breathing, fatigue resistant DIAm motor units are recruited that comprise type I and IIa fibers. Accordingly, these fibers have substantially higher mitochondrial volume density (MVD) and mitochondrial respiratory capacity (SDHmax) compared to more fatigable type IIx/IIb fibers. Cervical spinal cord injury disrupts descending inspiratory drive and markedly reduces eupneic activity of the DIAm. With C2 spinal cord hemisection (C2SH) DIAm activity on the ipsilateral side is reduced. Following a two-week period of C2SH, we found reduced MVD and SDHmax in type I and IIa DIAm fibers. As a result, we hypothesize that endurance of the DIAm is reduced. Female and male Sprague Dawley rats were used. EMG electrodes were placed in the left and right DIAm 3 days prior to C2SH surgery, and baseline recordings of eupneic DIAm activity were obtained in awake animals. The right C2 spinal cord was hemisected, and the extent of injury was confirmed by the absence of eupneic DIAm activity on the ipsilateral side while animals were anesthetized. Subsequently, DIAm EMG activity was recorded at 3, 7 and 14 days post C2SH. After two weeks, rats were anesthetized and the DIAm was excised. DIAm strips were cut and suspended in a tissue chamber containing Rees-Simpson solution maintained at 26oC, with thecostal margin clamped and the central tendon attached to a Cambridge Dual-Mode force/length transducer. DIAm strips were stimulated using platinum plate electrodes with supramaximal current (~250 mA) pulses (1 ms), and maximum specific force was determined at 75 Hz. The force-velocity relationship was then determined by systematically changing load, and maximum velocity (Vmax) was estimated based on curve fitting with the Hill-equation. Based on the force-velocity measurements peak power of the DIAm was calculated. Subsequently, the DIAm was repetitively stimulated and allowed to shorten at 30% maximum velocity (isovelocity approximating peak power output). Endurance of the DIAm was then determined as the duration that peak power output was sustained. Finally, the DIAm was rapidly frozen, and cross-sections were cut for fiber type and cross-sectional area (CSA) measurements. C2SH resulted in a marked decrease in eupneic DIAm EMG activity across the two-week period, which disproportionately affected type I and IIa DIAm fibers. However, there was no impact of C2SH on DIAm fiber type proportions or CSA. Following two-weeks C2SH, maximum specific force, Vmax, and peak power output of the DIAm were comparable between intact and injured sides. However, endurance of the DIAm was reduced on the injured side compared to the intact side. These results support our hypothesis that reduced activity of DIAm, reduces oxidative capacity of type I and IIa fibers and thereby reduces endurance. In clinical conditions such as mechanical ventilation DIAm activity is reduced and if maintained for longer periods of time this may affect DIAm endurance post weaning. Research supported by NIH grant: HL146114 (GCS). This is the full abstract presented at the American Physiology Summit 2024 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 serves a dual role in mitochondrial respiration: catalyzing the oxidation of succinate to fumarate in the tricarboxylic acid (TCA) cycle and transferring electrons from succinate to ubiquinone as complex II in the electron transport chain (ETC). SDH is composed of four subunits (SDHA-SDHD) and the loss of SDHB subunit, specifically, has been shown to increase oxidative stress. As a part of the catalytic core of the SDH complex, the SDHB subunit mediates electron transfer to ubiquinone. In human airway smooth muscle (hASM) cells with siRNA-mediated knockdown of SDHB, the maximum velocity of the SDH reaction (SDHmax) is decreased. Moreover, SDHB-knockdown leads to an increase in mitochondrial volume density, indicating a role of SDH in mitochondrial remodeling. However, the impact of SDHB-knockdown on mitochondrial dynamics has not been explored. Therefore, we hypothesize that SDHB-knockdown promotes mitochondrial fragmentation as part of mitochondrial remodeling in hASM cells. To investigate this hypothesis, bronchiolar tissue samples were collected in female and male patients undergoing lung surgery with no current history of smoking or respiratory diseases, the smooth muscle layer was dissected and hASM cells were dissociated. The targeted knockdown of SDHB was achieved by siRNA-mediated knockdown. Mitochondria in hASM cells were labeled with MitoTracker Green (200 nM) and imaged in 3D with a 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. Optical slices were deconvolved to enhance contrast and the MitoTracker labeling was thresholded to create binary images, which were then reconstructed in 3D. From these images, the mitochondrial complexity index (MCI) was calculated to quantify the extent of fragmentation. To identify the signaling pathway involved in mitochondrial fragmentation, the expression and phosphorylation of dynamin-related protein 1 (DRP1) was quantified by Western blot. Mitochondrial fragmentation is associated with increased phosphorylation of DRP1 at Ser616 (pDRP1s616). Furthermore, mitophagy was assessed by determining the mitochondrial expression of PINK1 and the phosphorylation of Parkin at Ser65 (pParkinS65). Mitochondrial biogenesis was also evaluated through the expression of PGC1α, NRF1/2 and TFAM. Our results show that SDHB-knockdown increased mitochondrial fragmentation (reduced MCI) in hASM cells, and increased pDRP1s616 phosphorylation and its translocation to mitochondria. SDHB-knockdown also increased mitochondrial expression of PINK1 and pParkins65 phosphorylation indicating activation of mitophagy. We also confirmed that SDHB-knockdown increased expression of PGC1α, NRF1/2 and TFAM consistent with mitochondrial biogenesis. These findings unveil the mechanisms underlying SDHB-knockdown-mediated mitochondrial remodeling. Supported by NIH grant R01-HL157984 (GCS). This is the full abstract presented at the American Physiology Summit 2024 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.

Aging is a significant risk factor for cardiovascular disease and is known to be associated with an increase in vascular stiffness and decrease in sensitivity to NO mediated vasodilatation. However, the underlying mechanism for these changes in vascular function is unknown. Smooth muscle tone is known to be regulated by the expression and phosphorylation of both nonmuscle (NM) and smooth muscle (SM) myosin. NM and SM phosphorylation is regulated by the activity of myosin light chain kinase (MLCK) and MLC phosphatase. MLC phosphatase is a trimeric enzyme consisting of enzymatic, myosin targeting (MYPT1) and a 20kDa subunit of unknown function. Two isoforms of MYPT1 are produced by alternative mRNA splicing, which differ by the presence or absence of a COOH-terminal leucine zipper (LZ+/-), and the relative expression of LZ+/LZ- MYPT1 determines the sensitivity to NO mediated vasodilatation. We defined NM myosin expression using two-dimensional SDS PAGE and LZ+/LZ- MYPT1 expression using immunoblotting in aortic and tertiary mesenteric smooth muscle from 6mo and 24mo old Fischer344 rats. The data demonstrate that aging is associated with a significant decrease in the expression of both LZ+ MYPT1 and NM myosin. The reduction in LZ+ MYPT1 expression would both decrease the sensitivity to NO mediated smooth muscle dilatation and increase vascular tone, which is a hallmark of the vasculature in aging. On the other hand, the reduction in NM myosin would result in a decrease in vascular tone and could represent a compensatory mechanism to normalize blood pressure during aging. These data demonstrate that there are changes in the expression of smooth muscle regulatory and contractile proteins during aging and these changes in protein expression contribute to molecular mechanism for the decrease in sensitivity to NO mediated vasodilatation as well as the increase in vascular tone or the vascular dysfunction that occurs with aging. Supported by a Mayo CV Prospective Grant (FVB). This is the full abstract presented at the American Physiology Summit 2024 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.