Simulating the Hydrodynamic Conditions of the Human Ascending Colon: A Digital Twin of the Dynamic Colon Model

The performance of solid oral dosage forms targeting the colon is typically evaluated using standardised pharmacopeial dissolution apparatuses. However, these fail to replicate colonic hydrodynamics. This study develops a digital twin of the Dynamic Colon Model; a physiologically representative in vitro model of the human proximal colon. Magnetic resonance imaging of the Dynamic Colon Model verified that the digital twin robustly replicated flow patterns under different physiological conditions (media viscosity, volume, and peristaltic wave speed). During local contractile activity, antegrade flows of 0.06–0.78 cm s−1 and backflows of −2.16–−0.21 cm s−1 were measured. Mean wall shear rates were strongly time and viscosity dependent although peaks were measured between 3.05–10.12 s−1 and 5.11–20.34 s−1 in the Dynamic Colon Model and its digital twin respectively, comparable to previous estimates of the USPII with paddle speeds of 25 and 50 rpm. It is recommended that viscosity and shear rates are considered when designing future dissolution test methodologies for colon-targeted formulations. In the USPII, paddle speeds >50 rpm may not recreate physiologically relevant shear rates. These findings demonstrate how the combination of biorelevant in vitro and in silico models can provide new insights for dissolution testing beyond established pharmacopeial methods.

Calponin 1 contributes to myofibroblast differentiation of human pleural mesothelial cells

Pleural mesothelial cells (PMCs) can become myofibroblasts via mesothelial-mesenchymal transition (MesoMT) and contribute to pleural organization, fibrosis, and rind formation. However, how these transformed mesothelial cells contribute to lung fibrosis remains unclear. Here, we investigated the mechanism of contractile myofibroblast differentiation of PMCs. TGF-b induced marked upregulation of calponin 1 expression, which was correlated with notable cytoskeletal rearrangement in human PMCs (HPMCs) to produce stress fibers. Downregulation of calponin 1 expression reduced stress fiber formation. Interestingly, induced stress fibers predominantly contain αSMA associated with calponin 1 but not b-actin. Calponin 1 associated stress fibers also contained myosin II and α-actinin. Further, focal adhesions were aligned with the produced stress fibers. These results suggest that calponin 1 facilitates formation of stress fibers that resemble contractile myofibrils. Supporting this notion, TGF-b significantly increased the contractile activity of HPMCs, an effect that was abolished by downregulation of calponin 1 expression. We infer that differentiation of HPMCs to contractile myofibroblasts facilitates stiffness of scar tissue in pleura to promote pleural fibrosis and that upregulation of calponin 1 plays a central role in this process.

Layer-By-Layer Fabrication of Thicker and Larger Human Cardiac Muscle Patches for Cardiac Repair in Mice

The engineered myocardial tissues produced via most manufacturing techniques are typically just a few dozen micrometers thick, which is too thin for therapeutic applications in patients. Here, we used a modified layer-by-layer (LBL) fabrication protocol to generate thick human cardiac muscle patches (hCMPs) with thicknesses of ~3.75 mm. The LBL-hCMPs were composed of a layer of endothelial cells (ECs) sandwiched between two layers of cardiomyocytes (CMs): both cell populations were differentiated from the same human induced pluripotent stem cell line (hiPSCs) and suspended in a fibrin matrix, and the individual layers were sutured together, leaving channels that allowed the culture medium to access the internal cell layer. The LBL-hCMPs were cultured on a dynamic culture platform with electrical stimulation, and when compared to Control-hCMPs consisting of the same total number of hiPSC-ECs and -CMs suspended in a single layer of fibrin, hiPSC-CMs in the LBL-hCMPs were qualitatively more mature with significantly longer sarcomeres and expressed significantly higher levels of mRNA transcripts for proteins that participate in cardiomyocyte contractile activity and calcium handing. Apoptotic cells were also less common in LBL- than in Control-hCMPs. The thickness of fabricated LBL-hCMP gradually decreased to 0.8 mm by day 28 in dynamic culture. When the hCMP constructs were compared in a mouse model of myocardial infarction, the LBL-hCMPs were associated with significantly better measurements of engraftment, cardiac function, infarct size, hypertrophy, and vascularity. Collectively these observations indicate that our modified LBL fabrication protocol produced thicker hCMPs with no decline in cell viability, and that LBL-hCMPs were more potent than Control-hCMPs for promoting myocardial repair in mice.

The RyR2-R2474S Mutation Sensitizes Cardiomyocytes and Hearts to Catecholaminergic Stress-Induced Oxidation of the Mitochondrial Glutathione Pool

Missense mutations in the cardiac ryanodine receptor type 2 (RyR2) characteristically cause catecholaminergic arrhythmias. Reminiscent of the phenotype in patients, RyR2-R2474S knockin mice develop exercise-induced ventricular tachyarrhythmias. In cardiomyocytes, increased mitochondrial matrix Ca2+ uptake was recently linked to non-linearly enhanced ATP synthesis with important implications for cardiac redox metabolism. We hypothesize that catecholaminergic stimulation and contractile activity amplify mitochondrial oxidation pathologically in RyR2-R2474S cardiomyocytes. To investigate this question, we generated double transgenic RyR2-R2474S mice expressing a mitochondria-restricted fluorescent biosensor to monitor the glutathione redox potential (EGSH). Electrical field pacing-evoked RyR2-WT and RyR2-R2474S cardiomyocyte contractions resulted in a small but significant baseline EGSH increase. Importantly, β-adrenergic stimulation resulted in excessive EGSH oxidization of the mitochondrial matrix in RyR2-R2474S cardiomyocytes compared to baseline and RyR2-WT control. Physiologically β-adrenergic stimulation significantly increased mitochondrial EGSH further in intact beating RyR2-R2474S but not in RyR2-WT control Langendorff perfused hearts. Finally, this catecholaminergic EGSH increase was significantly attenuated following treatment with the RyR2 channel blocker dantrolene. Together, catecholaminergic stimulation and increased diastolic Ca2+ leak induce a strong, but dantrolene-inhibited mitochondrial EGSH oxidization in RyR2-R2474S cardiomyocytes.

Isolation and phenotyping of cardiac-derived progenitor cells from neonatal mice

Dysfunctions of resident progenitor cells play a significant role in the pathogenesis of decreased myocardial contractility in heart failure, so the most promising approaches for the treatment of heart disease are cardiac-derived stem/progenitor cells (CSCs). Materials and methods. Protocols for progenitor cell cultures from different parts of the heart of newborn FVB/N mice have been developed and their proliferative potential has been characterized. Comparative analysis of the expression of CD31, CD34, CD44, CD45, CD73, CD90, CD105, CD117, CD309 and troponin I by cells from native myocardial biopsies and in the obtained cultures was performed by flow cytometric immunophenotyping. Results. The expression of mesenchymal markers CD44 and CD90 in the absence of the hematopoietic marker CD45 was demonstrated in early passages in mouse myocardial progenitor cell cultures. Relatively high expression of CD34 and CD31 was found. The presence of a minor population of CD44+117+ cells which correspond to the phenotype of cardiac progenitor cells, was detected. Expression of troponin I as one of the key markers of cardiomyocytes as well as the vascular endothelial growth factor receptor has been confirmed in terminally differentiated cultures of cells with contractile activity. Conclusions. It was found that newborn mice in the myocardial tissue contain more cells with the expression of markers of cardiac progenitors than in adult animals. The relative content of such cells is higher in the atria than in the ventricles. Cardiac progenitor cells in neonatal mice derived from the atrial appendages have better proliferative potential than cell cultures isolated from the ventricles.

Preterm Labor and Treatment Efficacy-Safety

With medical sciences on the verge of advancement, preterm labor still remains a bothersome issue in modern obstetrics. A few therapeutic agents that suppress uterine contractile activity have gained success up to some extent. Tocolytics are medications used to suppress premature labor. These drugs can decrease the strength and frequency of uterine contractions and help in delay the onset of labor but are not able to prolong pregnancy to full-term. Presently, the choice of a best tocolytic drug remains debatable. This review discusses efficacy and safety of various useful agents which have been used so far. Further clinical trials are required to select an effective, and most importantly, safe therapy for the threatened preterm labor.

HDAC4 Is Indispensable for Reduced Slow Myosin Expression at the Early Stage of Hindlimb Unloading in Rat Soleus Muscle

It is well known that reduced contractile activity of the main postural soleus muscle during long-term bedrest, immobilization, hindlimb unloading, and space flight leads to increased expression of fast isoforms and decreased expression of the slow isoform of myosin heavy chain (MyHC). The signaling cascade such as HDAC4/MEF2-D pathway is well-known to take part in regulating MyHC I gene expression. Earlier, we found a significant increase of HDAC4 in myonuclei due to AMPK dephosphorylation during 24 h of hindlimb unloading via hindlimb suspension (HU) and it had a significant impact on the expression of MyHC isoforms in rat soleus causing a decrease in MyHC I(β) pre-mRNA and mRNA expression as well as MyHC IIa mRNA expression. We hypothesized that dephosphorylated HDAC4 translocates into the nuclei and can lead to a reduced expression of slow MyHC. To test this hypothesis, Wistar rats were treated with HDAC4 inhibitor (Tasquinimod) for 7 days before HU as well as during 24 h of HU. We discovered that Tasquinimod treatment prevented a decrease in pre-mRNA expression of MyHC I. Furthermore, 24 h of hindlimb suspension resulted in HDAC4 nuclear accumulation of rat soleus but Tasquinimod pretreatment prevented this accumulation. The results of the study indicate that HDAC4 after 24 h of HU had a significant impact on the precursor MyHC I mRNA expression in rat soleus.