FRET-based biosensor experiments in adult cardiomyocytes are a powerful way of dissecting the spatiotemporal dynamics of the complicated signaling networks that regulate cardiac health and disease. However, although much information has been gleaned from FRET studies on cardiomyocytes from larger species, experiments on adult cardiomyocytes from mice have been difficult at best. Thus the large variety of genetic mouse models cannot be easily used for this type of study. Here we develop cell culture conditions for adult mouse cardiomyocytes that permit robust expression of adenoviral FRET biosensors and reproducible FRET experimentation. We find that addition of 6.25 µM blebbistatin or 20 µM (S)-nitro-blebbistatin to a minimal essential medium containing 10 mM HEPES and 0.2% BSA maintains morphology of cardiomyocytes from physiological, pathological, and transgenic mouse models for up to 50 h after adenoviral infection. This provides a 10–15-h time window to perform reproducible FRET readings using a variety of CFP/YFP sensors between 30 and 50 h postinfection. The culture is applicable to cardiomyocytes isolated from transgenic mouse models as well as models with cardiac diseases. Therefore, this study helps scientists to disentangle complicated signaling networks important in health and disease of cardiomyocytes.

Isolation of high quality cardiomyocytes is critically important for achieving successful experiments in many cellular and molecular cardiology studies. Methods for isolating cardiomyocytes from the murine heart generally are time-sensitive and experience-dependent, and often fail to produce high quality cells. Major technical difficulties can be related to the surgical procedures needed to explant the heart and to cannulate the vessel to mount onto the Langendorff system before in vitro reperfusion can begin. During this period, transient hypoxia and ischemia may damage the heart, resulting in low yield and poor quality of cells, especially for heart disease models that have fragile cells. We have developed novel in vivo cannulation methods to minimize hypoxia and ischemia, and fine-tuned the entire protocol to produce high quality ventricular myocytes. The high cell quality has been confirmed using important structural and functional criteria such as morphology, t-tubule structure, action potential morphology, Ca²⁺ signaling, responsiveness to beta-adrenergic agonist, and ability to have robust contraction under mechanically loaded condition. Together these assessments show the preservation of the cardiac excitation–contraction machinery in cells isolated using this technique. The in vivo cannulation method enables consistent isolation of high-quality cardiomyocytes, even from heart disease models that were notoriously difficult for cell isolation using traditional methods.

Malignant pleural mesothelioma is a rare disease with an annual incidence of around 3000 cases a year in the United States. Most cases are caused by asbestos exposure, with a latency period of up to 40 years. Pleural mesothelioma is an aggressive disease process with overall survival of roughly 6–12 months after the time of diagnosis. It is divided into three subtypes: epithelioid, mixed type, and sarcomatoid type, with the epithelioid subtype having the best overall survival. Often, the treatment is multimodality with surgery, chemotherapy, and radiation. The survival benefit is improved but remains marginal. New treatment options involving targeted immune therapies appear to offer some promise. The tumor microenvironment is the ecosystem within the tumor that interacts and influences the host immune system. Understanding this complex interaction and how the host immune system is involved in the progression of the disease process is important to define and guide potential treatment options for this devastating and rare disease.

Introduction: Hemorrhagic early death (HED) remains a major cause of treatment failure among patients with acute promyelocytic leukemia (APL). We aimed to investigate the prognostic potential of rotational thromboelastometry (ROTEM) for bleeding in patients with APL. Materials and Methods: 31 newly-diagnosed APL patients (median age of 40 years; 14 female/17 male) that underwent treatment at the Clinic of Hematology UCCS from 2016-2020 with all-trans retinoic acid and anthracyclines were recruited. CBCs (complete blood count), conventional coagulation tests (CCTs), and ROTEM parameters obtained before treatment initiation were evaluated. Results: All patients demonstrated at least one ROTEM parameter out of the reference range. ROTEM parameters associated with significant hemorrhage were EXTEM clotting time (CT) (P = 0.041) and INTEM amplitude 10 (A10) (P = 0.039), however, only EXTEM CT (P = 0.036) was associated with HED. Among CBCs and CCTs, only platelets were associated with significant bleeding (P = 0.015), while D-dimer was associated with both bleeding and HED (P = 0.001 and P = 0.002, respectively). Conclusion: Our results indicate that ROTEM parameters may reveal hypocoagulability in APL patients and have the potential to improve current hemorrhage prognostic methods. Additionally, these results suggest the combination of ROTEM and CCTs might be useful in identifying patients at risk for HED.