Citations

The polymerization of globular to filamentous actin in smooth muscle occurs upon activation and is associated with cytoskeletal remodeling. Remodeling of the cortical cytoskeleton in this manner allows for tethering of contractile proteins and force transmission to the extracellular matrix. Muscle contraction involves ATP hydrolysis and cross‐bridge cycling, which are both sensitive to internal load. During isometric activation of smooth muscle, ATP hydrolysis rate peaks initially but decreases with time, reaching a steady‐state, while maintaining maximum force. Thus, tension cost, i.e., the rate of ATP hydrolysis per unit of force, decreases over time, reflecting increased efficiency of smooth muscle contraction. We hypothesized that the time‐dependent decrease in tension cost and ATP hydrolysis are associated with actin polymerization and a subsequent increase in internal loading on cross‐bridges. In permeabilized porcine airway smooth muscle (ASM) strips, ATP hydrolysis rate and isometric force were measured simultaneously after activation with maximum Ca 2+ (pCa 4.0). Two strips were tested from each animal. One strip was treated with Cytochalasin‐D (Cyto‐D; 1 μM for 30 min), an inhibitor of actin polymerization, and one was an untreated, time‐matched control. We found that peak and steady‐state isometric force decreased by ~40%, and peak and steady‐state ATP hydrolysis rate increased by ~30% and ~20%, respectively, following Cyto‐D treatment. Thus, tension cost increased by ~100% and ~60% at peak and steady‐state, respectively. These results support our hypothesis that the time‐dependent decrease in ATP hydrolysis rate relates to an increase in internal load on cross‐bridges via actin polymerization. Inhibition of actin polymerization induced by Cyto‐D in smooth muscle impairs contractile protein tethering and therefore reduces internal loading, resulting in increases in tension cost, ATP hydrolysis rate and faster cross‐bridge cycling.

The diaphragm muscle (DIAm) shortens and produces force to generate negative intra‐thoracic pressure for airflow into the lungs and positive intra‐abdominal pressure for airway clearance and expulsive behaviors. Breathing is a low force repetitive behavior that requires recruitment of fatigue resistant type I and IIa DIAm fibers. In contrast, short duration airway clearance behaviors are infrequent and associated with greater DIAm force that require the additional recruitment of more fatigable type IIx/IIb fibers. Aging is characterized by selective atrophy of type IIx/IIb DIAm fibers and decreased specific force, i.e., sarcopenia. Older individuals are able to breathe without difficulty but have trouble with some higher force airway clearance behaviors of the DIAm such as coughing and sneezing. In 6‐ and 24‐month Fischer 344 rats (n=6 per group), DIAm strips were dissected from the mid‐costal region, mounted in a tissue chamber filled with Rees‐Simpson solution, and stimulated directly to measure maximum isometric specific force (Po) and maximum shortening velocity (V max ) using a Cambridge dual‐mode length‐force servo controller (model 300B). In addition, power fatigue was assessed during repetitive isovelocity (30%V max ) contractions evoked by 75 Hz in 400‐ms‐duration stimulus trains repeated for a 2‐min period. Additional muscle segments were rapidly frozen at optimal sarcomere length in melting isopentane, and transverse muscle fiber sections were cut at 10 µm thickness for determination of muscle fiber type (based on myosin heavy chain immunoreactivity) and fiber cross‐sectional areas. The relationship between force (load) and shortening velocity was determined using the Hill equation. Aging significantly reduced maximum isometric force, maximum shortening velocity, and maximum power output of the DIAm in older rats. The reduction in power during repetitive isovelocity shortening was reduced in older rats, but the residual power output after 2 min stimulation was comparable between young and old rats. These changes in mechanical and fatigue properties were consistent with the selective atrophy of type IIx/IIb DIAm fibers and reflected the sustained output of type I and IIa DIAm fibers, which are recruited for essential breathing behaviors.

Tumor necrosis factor α (TNFα) contributes to the pathophysiology of several inflammatory airway diseases such as asthma, chronic obstructive pulmonary disease (COPD), chronic bronchitis, and COVID‐19. Previously, we showed that acute (24‐h) exposure to TNFα promotes mitochondrial fragmentation in human airway smooth muscle (hASM) cells, which is associated with an increased level of cytosolic GTPase dynamin‐related protein 1 (DRP1), a major protein involved in mitochondrial fragmentation. Phosphorylation of DRP1 at Ser616 (S616) promotes its translocation and binding to the outer mitochondrial membrane and induces mitochondrial fragmentation; however, phosphorylation of DRP1 at the Ser637 (S637) residue reverses the total process and diminishes fragmentation. The balance between S616 and S637 phosphorylation of DRP1 regulates mitochondrial dynamics. In hASM cells, exposure to TNFα triggers protein unfolding and selectively activates the endoplasmic reticulum (ER) stress pathway involving the phosphorylation of inositol‐requiring enzyme 1α (pIRE1α) and subsequent splicing of X‐box binding protein 1 (XBP1s) that acts as a potent transcriptional activator for several downstream genes. We hypothesized that TNFα mediated activation of the pIRE1α/ XBP1 ER stress pathway transcriptionally activates genes that code for proteins involved in DRP1 phosphorylation at S616 leading to mitochondrial fragmentation. hASM cells were dissociated from bronchial biopsies from patients without a history of respiratory diseases and exposed to TNFα (20 ng/ ml for 6 h). Cells were also treated with Mdivi1 (50 μM for 6 h), a small‐molecule inhibitor of mitochondrial fragmentation that targets the GTPase activity of DRP1. Bioinformatic analysis revealed the presence of putative binding sites for XBP1 on the promoter region of genes that translate into kinases (cyclin dependent kinases CDK1 and CDK5, cyclin B1), which are reported to phosphorylate DRP1 at S616. Western blot and/or immunocytochemistry were employed to quantify the expression and phosphorylation status of IRE1α, DRP1, XBP1 splicing, CDKs and cyclin B1. Mitochondrial morphology was assessed by 3‐D confocal microscopy using MitoTracker green to label mitochondria. Chromatin immunoprecipitation (ChIP) and quantitative real time PCR was done to validate XBP1 target genes. Spliced XBP1 transcriptionally activates expression of CDK1, CDK5 and cyclin B1 and subsequent phosphorylation at S616 of DRP1 without significant alteration in S637 phosphorylation. As a result, TNFα induces an increase in S616/S637 phosphorylation ratio that promotes the translocation of DRP1 from cytosol to mitochondria and mediates mitochondrial fragmentation. Inhibition of DRP1 GTPase activity by Mdivi1 ameliorates DRP1 phosphorylation at S616 and significantly reduces mitochondrial fragmentation. These results reveal the mechanisms that underlie TNFα induced ER stress and downstream mitochondrial fragmentation.

Introduction: Multiple supervised machine learning (ML) models have been used to assess the outcomes in patients who underwent transcatheter aortic valve replacement (TAVR) procedure, however, supervised ML requires significant expert time for annotation and labeling. Unsupervised ML can provide insight into the dataset without this process. Hypothesis: We hypothesized that unsupervised k-means ML can identify distinct phenogroups in TAVR patients that can predict prognosis without a priori knowledge. Methods: A retrospective study of patients who underwent TAVR (Jan 2012-Dec 2017) at Mayo Clinic sites was performed. Baseline demographics, EKG, cardiac computed tomography, and echocardiographic data were abstracted. Variables with ≥ 20% missing data were excluded. Prior to the k-means clustering algorithm, principal component analysis was used for dimensionality reduction. Input variables were analyzed according to k-means determined clusters. Kaplan-Meier survival analysis was used to assess the survival of each cluster. Results: A total of 1071 consecutive patients were included in the final analysis. A total of 141 unique variables were used. The mean age was 80.7±8.6 years, 58.2% were male. Patients were divided into 2 distinct phenogroups. In the two phenogroups, cluster 0 (n=639) has significantly better survival than cluster 1 (n= 432) (log-rank p=0.0002) (Figure 1). Cluster 1 has features of low flow-low gradient (LFLG) hemodynamic profile, including lower systolic blood pressure, mean aortic valve gradient, peak aortic valve velocity, and worse LV ejection fraction. Conclusions: Unsupervised ML can identify distinct phenotype features with prognostic significance in TAVR patients. Our results suggest that patients with LFLG hemodynamic features are intrinsically different from other TAVR patients and require more attention to avoid adverse outcomes.