Citations

Background: Treatment of chronic lymphocytic leukemia (CLL) has been transformed with targeted therapies including inhibitors of Bruton's tyrosine kinase (BTKi). Currently three covalent BTK inhibitors (cBTKi) are approved for CLL, but most patients eventually relapse, commonly through acquisition of the C481S BTK mutation (Woyach et al. 2014). Pirtobrutinib and nemtabrutinib are non-covalent BTK inhibitors (ncBTKi) developed to target and inhibit C481S mutant BTK. However novel secondary site mutations in BTK, namely T474I and L528W, have been found to confer resistance to both cBTKi and ncBTKi (Wang et al. 2022). Regardless of these mutations, BCR signaling remains intact, suggesting that inhibition of BTK maintains its therapeutic importance. LP-168 is a novel ultra-selective 4 th generation BTKi with an active warhead capable of covalent interaction with WT BTK or non-covalent binding when a BTK C481 mutation is present. Methods: To determine the target selectivity of LP-168 we screened the compound against 468 kinases in the scanMAX Kinase Assay. For validation of covalent binding, dialysis against WT BTK was performed. Primary CLL B cells were isolated by negative selection and treated with LP-168 for all experiments. BCR signaling alterations were assessed via immunoblot to observe changes in target protein phosphorylation. Changes in CLL migration towards CXCL12 or CXCL13 were interrogated by transwell migration. Cytotoxicity towards primary CLL cells was measured via Annexin V/PI flow cytometry with and without HS-5 stromal cell support. Experiments using TMD8 cells were performed following CRISPR modification to insert WT, C481S, or T474I BTK. In vivo survival studies were performed using both the Eμ-TCL1 and Eμ-MTCP1 mouse engraftment models. Results: LP-168 demonstrated selectivity towards BTK with roughly 700-fold selectivity for BTK vs its next off-target. In an enzymatic assay, LP-168 displayed nanomolar potency towards both WT BTK (IC 50=0.11 nM) and C481S BTK (IC 50=1.0 nM). Following 2-hour drugging, LP-168 inhibited BCR signaling in primary CLL B cells at increasing concentrations, demonstrated by reduced phosphorylation of BTK (92%, p<0.0001, n=8) and its immediate downstream target PLCγ2 (41%, p=0.0013, n=8). Migration capacity of primary CLL cells towards CXCL12 and CXCL14 was also found to decrease after 2-hours of treatment with LP-168 (52%, p<0.001, n=10 and 51%, p<0.001, n=10, respectively). After 48 hours of exposure, LP-168 was able to induce cytotoxicity of primary CLL cells alone and in co-culture with HS-5 stromal cells in a dose-dependent manner, with similar or improved efficacy to other BTKi. LP-168 decreased CLL cell production of CCL3 and CCL4 chemokines (CCL3: 93%, p<0.001, n=8; CCL4: 93%, p<0.001, n=8). LP-168 also demonstrated modest cytotoxicity towards TMD8 cells harboring either C481S BTK (24%, p=0.0008 , n=3) or T474I BTK (13%, p=0.0690, n=3), as well as decreased production of CCL3 (C481S: 95%, p<0.0001, n=3; T474I: 99%, p<0.0001, n=3) and CCL4 (C481S: 97%, p<0.0001, n=3; T474I: 98%, p<0.0001, n=3) (Figure 1A). Further, we observed inhibition of downstream BCR signaling in primary CLL B cells harboring C481S via immunoblotting and cytokine production. Finally, to evaluate the in vivo efficacy of LP-168 we tested this compound in both the Eµ-TCL1 and Eµ-MTCP1 models. We treated mice daily via oral gavage and found that 50 mg/kg of LP-168 significantly improved survival in the Eµ-TCL1 model when compared to vehicle (median 51 vs 44 days; p=0.0018) or ibrutinib at 50 mg/kg (median 51 vs 45 days; p=0.0098) and the Eµ-MTCP1 model when compared with vehicle (median 122 vs 62 days; p<0.0001) or ibrutinib at 50 mg/kg (median 122 vs 96 days; p=0.0162) (Figure 1B). Conclusions: Collectively, our data demonstrate that LP-168 is a potent and selective inhibitor of BTK with activity against CLL, even in the presence of mutations that mediate resistance to cBTKi and ncBTKi. These data support the continued preclinical and clinical investigation of LP-168, which is currently being studied in the phase 1 setting of CLL (NCT04775745) and NHL (NCT04993690).

Background Chimeric antigen receptor (CAR) modified T-cells are leading to a paradigm shift in the treatment of relapsed/refractory (RR) multiple myeloma (MM). A high proportion of patients treated with CAR T-cells, however, experience prolonged cytopenia, with the mechanism remaining poorly understood. Here, we aimed to explore potential biomarkers that might correlate with cytopenia following CAR T-cell therapy in RRMM. Methods We prospectively collected peripheral blood (PB) of RRMM patients treated with idecabtagen-vicleucel (ide-cel) at the following time points: prior to lymphodepleting chemotherapy (LDC) (baseline), after ide-cel on day 4, 7, 14 and 28, and monthly thereafter. Cytopenia was determined according to the CTCAE version 5.0. Flow cytometry was performed with the following markers: CD45, CD3, CD4, CD8, CD62L, CD45RA, CD19, CD14, CD138, CD38 and a BCMA-CAR-detection marker. Results We included 222 PB samples at different sampling time points from 35 RRMM patients, who were pretreated with a median of 5 therapy lines (range 2-10). All patients were triple-class exposed and, 34 (97%) and 9 (26%) patients underwent autologous and allogeneic stem cell transplant (SCT), respectively. First, we analyzed the relationship between baseline parameters and the duration of grade ≥3 cytopenia after ide-cel (n=35). Notably, patients who received allogeneic SCT or >4 lines of therapy did not develop longer lasting cytopenia than the remaining patients. We found a correlation between the baseline hemoglobin level and the duration of grade ≥3 anemia (r=-0.55, P<0.001). Similarly, low baseline platelet count indicated long duration of grade ≥3 thrombocytopenia (r=-0.48, P=0.003). Moreover, high baseline ferritin level correlated with long duration of grade ≥3 anemia (r=0.52, P<0.001) and thrombocytopenia (r=0.51, P=0.002). Furthermore, the maximum ferritin level after ide-cel was an indicator for long lasting grade ≥3 anemia (r=0.73, P<0.001) and thrombocytopenia (r=0.71, P<0.001). Interestingly, we found significant correlations between the duration of grade ≥3 lymphopenia and baseline CD4+ T-cell frequency (r=0.41, P=0.02), CD8+ T-cell frequency (r=-0.38, P=0.04) and CD4+/CD8+ ratio (r=0.4, P=0.03). Noteworthy, patients with β2-microglobulin level >3.5 mg/l displayed longer duration of grade ≥3 anemia (median: 9 vs 0 day, P=0.007) and neutropenia (median: 43 vs 12 days, P=0.02) compared with the remaining patients, suggesting that high tumor load might be a risk factor for prolonged cytopenia after ide-cel. Second, we divided the follow-up PB samples (n=187) into two groups: early (<d60) and prolonged (≥d60) cytopenia to account for direct toxic effects related to LDC. A high ferritin level was associated with low hemoglobin level (<d60: r=-0.45, P<0.001, ≥d60: r=-0.62, P<0.001) and low platelet count (<d60: r=-0.58, P<0.001, ≥d60: r=-0.81, P<0.001) in both groups. In prolonged cytopenia (≥d60), lymphocyte count correlated with the T-cell count (r=0.7, P<0.001), suggesting that prolonged lymphopenia was mainly attributed to the reduced T-cell count after ide-cel. Of note, the frequency of naïve CD4+ T-cells (CD3+CD4+CD62L+CD45RA+) positively correlated with neutrophil count (r=0.58, P<0.001), hemoglobin level (r=0.37, P=0.03) and white blood cell count (r=0.5, P=0.002) in the ≥d60 group, suggesting that delayed immune reconstruction with reduced naïve CD4+ T-cells might contribute to prolonged cytopenia after ide-cel. Moreover, in the ≥d60 group, lymphocyte count was related with the frequency of CAR+CD4+ (r=-0.58, P<0.001), CAR+CD8+ (r=0.58, P<0.001), CAR-CD4+ (r=-0.72, P<0.001), CAR-CD8+ (r=0.75, P<0.001) T-cells, as well as the ratios of CAR+CD8+/CAR+CD4+ (r=-0.6, P<0.001) and CAR-CD8+/CAR-CD4+ (r=-0.75, P<0.001). Conclusion Here, we present one of the first prospective studies investigating the factors associated with prolonged cytopenia after CAR T-cell therapy in RRMM. High tumor load, preexisting cytopenia and high ferritin level are related with prolonged cytopenia after ide-cel. Delayed immune reconstruction, characterized by reduced naïve CD4+ T-cell count, indicates prolonged cytopenia following ide-cel therapy. Patients with high CD4+/CD8+ ratio are at risk of prolonged lymphopenia (with reduced T-cell count), which may be a risk factor for infectious complications and an issue for subsequent T-cell based immunotherapies.

Introduction Primary mediastinal large B-cell lymphoma (PMLBCL) is a rare and aggressive type of non-Hodgkin B-cell lymphoma, accounting for about 3% of all non-Hodgkin lymphomas. The role of radiation therapy following immunochemotherapy is controversial and lately there has been more studies suggesting there is less benefit of adding radiation therapy. Large-scale studies describing and evaluating the effect of radiation therapy on survival outcome are lacking. Materials and Methods The National Cancer Database (NCDB) was queried for patients diagnosed with PMLBCL between 2004 and 2019. All patients aged ³18 years diagnosed with PMLBCL were included. We excluded patients with unknown chemotherapy and radiation therapy status, or received radiation prior to chemotherapy, or received radiation 6 months after chemotherapy or lost to follow-up (unknown vital status of last contact). Kaplan-Meier and multivariate Cox regression were used in the analyses. Results The total cohort consisted of 1,899 patients with PMLBCL. Out of whom 1,177 (62%) were females, 1,560 (76%) were White non-Hispanic, 113 (6%) were White Hispanic and 205 (11%) were African Americans. Among patients with known disease stage, 1,164 (61%) had early-stage disease (stage I and II) and 351 (19%) had advanced stage disease (stage III and IV). 1,443 (76%) received chemotherapy only and 456 (24%) received chemotherapy followed by radiation therapy. The median age at diagnosis was 35 years (range 18-90), 1,215 (64%) were diagnosed at younger age (40 years or less). The median follow-up time was 60 months (95% CI 57-62), the median overall survival (OS) for the whole cohort was not reached, and the 5-year OS was 91%. The 5-year OS for early and advanced stage were 93% and 83%, respectively. In patients with early-stage disease, there was no statistically significant difference in the median OS between patients who received chemotherapy only (N=824) compared to patients who received chemotherapy followed by radiation therapy (N=340) (not reached in both groups, P=0.130) with 5-year OS of 92% and 93%, respectively. In patients with advanced stage disease, there was no statistically significant difference in the median OS between patients who received chemotherapy only (N=294) and patients who received chemotherapy followed by radiation therapy (N=57) (median OS not reached in both groups, P=0.067) with 5-year OS of 82% and 88%, respectively. On multivariate analysis, older age and Charlson-Deyo score ≥3 were associated with worse OS (HR 1.062 95% CI 1.047-1.078; P<0.001 and HR 6.032 95% CI 1.853-19.634; P=0.003, respectively), early stage was associated with better OS (HR 0.508 95% CI 0.364-0.708; P<0.001), whereas adding radiation to chemotherapy did not affect the OS (HR 0.713 95% CI 0.491-1.035; P=0.075). Over the years, there was a significant down trend in using radiation therapy (Figure 1). Using univariate and multivariate logistic regression analysis, we found early-stage disease was associated with increased use of radiation therapy after chemotherapy. Whereas patients who were treated at academic/research programs were less likely to receive radiation therapy (Table 1). Conclusion This large real-world analysis shows a practice change in PMLBCL treatment with a trend in omitting radiation therapy. In our analysis, we found that adding radiation therapy to systemic therapy did not affect the survival outcome in both early and advanced stage disease.

Introduction: Extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue (MALT lymphoma) is a low-grade B cell non-Hodgkin lymphoma that originates in lymphoid tissue of epithelial lined structures. The commonest site for this lymphoma is the stomach (gastric MALT lymphoma). MALT lymphoma in other mucosal sites, including other gastrointestinal sites, lungs, salivary glands, and lacrimal glands, has been well-described. However, MALT lymphoma in urothelial organs (kidneys, ureters, and urinary bladder) has not been well-characterized. This study aimed to investigate the clinicopathological features and therapeutic outcomes in urologic MALT lymphoma (U-MALT). Methods: We identified patients with U-MALT who were treated at the Mayo Clinic Cancer Center between March 2013 and May 2022 through electronic medical chart review. Clinical, pathologic, radiologic, therapeutic, and survival data were extracted. Kaplan-Meier analysis was used to determine the survival outcomes. Results: The characteristic features of six patients (N=6) with U-MALT are summarized in Table-1. 4/6 (67%) are Caucasian, and 3/6 (50%) are males. The median age at diagnosis was 73.5 years. Urologic sites are the urinary bladder (3/6), kidneys (3/6), and ureter (1/6, this patient had both kidney and ureter involvement). 4/6 patients had primary U-MALT. Concurrent non-urologic involvement was identified in 3/6 patients and included bone marrow, lungs, stomach, and axillary lymph nodes. Bone marrow involvement was observed in 2/6 patients.Next-generation sequencing was performed on one patient's peripheral blood, revealing an ATM mutation. Regarding the initial clinical presentation, kidney-involved U-MALT patients presented with renal failure, and those with urinary bladder/ureteric involvement, U-MALT was diagnosed incidentally upon cystoscopic evaluation and biopsy. Various management strategies were used, including observation, surgical resection, and rituximab treatment. Notably, all patients achieved complete remission (CR) except one with partial response to rituximab, who subsequently achieved CR with the Bendamustine/Rituximab regimen. Median follow-up is 6 years (range, 1-10). All six patients are alive without relapse at the time of analysis, with the flat overall survival curve at 100%, indicating excellent long-term survival. Conclusions: Our study indicates that urologic MALT lymphomas are extremely rare and predominantly affect elderly people. The most common urologic sites are the kidneys and urinary bladder. Urologic manifestations are dependent on the urologic site involved. They can be primary to the urologic structures or part of the disseminated systemic marginal zone lymphoma. They can be treated like other low-grade lymphomas with observation, surgical resection, and systemic therapy. They appear to be quite responsive to CD-20 targeted therapy with Rituximab. Prognosis appears to be excellent with long overall survival. Further research with multi-institutional studies is needed to study this rare lymphoma entity and identify optimal therapy.

Introduction: Gray zone lymphoma (GZL) is a rare lymphoid neoplasm exhibiting features intermediate between diffuse large B-cell lymphoma and Hodgkin lymphoma. Due to its rarity and relatively recent recognition by the World Health Organization in 2008, the natural history, clinical behavior, and best treatment strategies are still evolving. We present a retrospective review of GZL patients diagnosed and managed at the Mayo Clinic. This study aims to explore the clinical characteristics, treatment modalities, and outcomes of this poorly understood disease. Methods: After reviewing the electronic medical records of patients presented to the Mayo Clinic Cancer Center between March 2012 and July 2022 at the Mayo Clinic, we identified the patients who were diagnosed with GZL. Clinicopathological characteristics, disease features, treatment modalities, and outcomes were analyzed. The Kaplan-Meier method was utilized to assess survival outcomes. Results: Six patients (n=6) with GZL, all Caucasian and predominantly male 4/6 (67%), were identified. The median age at diagnosis was 37 years (range 24-57). Disease characteristics included high rates of mediastinal involvement 5/6 (83%) and bulky disease 4/6 (67%), with various stages of Ann Arbor classification represented. First-line treatment regimen varied between R-CHOP, ABVD, and DA R-EPOCH. The R-CHOP regimen was received in 3/6 patients (50%); the first patient achieved Complete Remission (CR), then managed with R-ICE, followed by consolidative autologous stem cell transplantation (auto-SCT) when relapsed. The second patient had Partial Response (PR) to R-CHOP and required consolidative radiotherapy to achieve CR, and the third R-CHOP patient achieved CR however, after multiple disease progressions that were managed with multiple regimens including R-ICE, R-CODOX-M/IVAC regimen, Nivolomab/Ipilumuab, consolidative radiotherapy, and Brentuximab vedotin. The ABVD regimen was received by 2/6 patients (33%). The first patient had CR, and the second patient had disease progression, which required treatment with R-ICE, followed by consolidative radiotherapy and auto-SCT. One patient (17%) received the DA R-EPOCH regimen and achieved CR, consolidated with auto-SCT. The median follow-up for our cohort is nine years (range, 5-13). All six GZL patients are alive and in CR (since 2019 or before) at the time of the study analysis, with an excellent flat overall survival curve. For this reason and due to the small patient sample, we could not calculate the progression-free survival data. Conclusions: Our Mayo Clinic review provides unique insights into managing Gray Zone Lymphoma (GZL), a rare disease. We reported 100% survival and remission in six GZL patients using diverse treatment strategies, contrasting with varied outcomes from other institutions. This underscores the potential of individualized treatments for GZL and the need for further, broader studies to validate our findings and refine optimal treatment approaches for this rare disease entity.

Background: Emavusertib (CA-4948) is a novel oral inhibitor of interleukin-1 receptor-associated kinase 4 (IRAK4), which is essential for toll-like receptor (TLR) and interleukin-1 receptor (IL-1R) signaling in B cell proliferation. Additional inhibitory activity against FMS-like tyrosine kinase 3 (FLT3) and CDC-like kinases (CLK1/2/4) is also present in emavusertib. IRAK4 forms a Myddosome complex with MYD88 adaptor protein and drives overactivation ofnuclear factor-kappa B (NF-κB), causing inflammation and tumor growth. Emavusertib has been reported to be well tolerated and active as monotherapy in heavily pretreated patients with relapsed/refractory (R/R) non-Hodgkin lymphoma (NHL) (Nowakowski 2020). Preclinical studies demonstrated that tumor resistance and survival via IRAK4 activation could be delayed or reversed. Emavusertib crossed the blood-brain barrier in a murine PDX model of PCNS lymphoma, blocked the activated IRAK4 pathway, and induced tumor response and prolonged survival (von Roemeling 2022). In combination with Bruton tyrosine kinase (BTK) inhibitors, emavusertib showed in vitro synergy and re-sensitization of resistant cell lines, overcoming BTK resistance (Guidetti 2023a and 2023b). In vivo synergy in B-cell NHL was also demonstrated. Adaptive chemotherapy treatment resistance through IRAK4 upregulation and activation was also described in other disease models (Melgar 2019, Li 2019). This abstract presents preliminary efficacy and safety data of emavusertib + ibrutinib in R/R NHL patients. Aim:Assessment of safety and clinical activity of emavusertib in combination with ibrutinib. Methods: This is an ongoing open-label trial (NCT03328078) of emavusertib as monotherapy and in combination with ibrutinib. Part A1 - completed; dose escalation of emavusertib as monotherapy; the recommended phase 2 dose (RP2D) is 300 mg BID with continuous oral dosing (Nowakowski 2020). Part A2 - completed; dose escalation of emavusertib in combination with ibrutinib at 420 and 560 mg QD (Joffe 2022). Part B - ongoing; an expansion cohort of PCNSL patients with resistance to prior BTK inhibitors for additional safety and efficacy related to exposure: emavusertib at 100 or 200 mg BID + ibrutinib at 560 mg QD in 28-Day cycles. Results:As of 19 July 2023, 18 heavily pretreated NHL (including five PCNSL) patients received emavusertib at 100, 200, or 300 mg BID in combination with full ibrutinib doses per label. Median age was 63.5 years (range 50-92). Median number of prior lines of anti-cancer therapies was 3 (range 1-10). Eleven of 18 patients failed prior BTK inhibitors. Median treatment duration was 95.5 days (21-587 days), suggesting acceptable safety and tolerability. No DLTs were observed at the 200 mg BID dose level, and two reversible DLTs (stomatitis and syncope) were observed at the 300 mg BID dose level. The preliminary efficacy data of 16 evaluable patients in combination with ibrutinib showed 5 CRs (2 MCL, 3 PCNSL) and 1 PR (1 CLL). All 3 PCNSL patients with CR were previously treated with BTK inhibitors. Conclusion: The combination ofemavusertib plus ibrutinib (ema+ibr) is well tolerated with an acceptable long-term safety profile and promising efficacy, showing several objective responses in heavily pretreated and/or BTK inhibitor resistant patients. Emavusertib may have the potential to overcome BTK inhibitor resistance and the combination of ema+ibr has the potential to show increased anti-cancer activity compared to ibrutinib monotherapy.

The scarcity of liver grafts has prompted developments in living donor liver transplantations (LDLT), with previous literature illustrating similar outcomes in recipients compared to deceased donor transplants. However, significant concerns regarding living donor morbidity and mortality have yet to be examined comprehensively. This study aims to provide estimates of the incidence of various outcomes in living liver donors. In this meta-analysis, Medline and Embase were searched from inception to July 2022 for articles assessing the incidence of outcomes in LDLT donors. Complications in the included studies were classified into respective organ systems. Analysis of incidence was conducted using a generalized linear mixed model with Clopper-Pearson intervals. Eighty-seven articles involving 60,829 living liver donors were included. The overall pooled incidence of complications in LDLT donors was 24.7% (CI: 21.6%–28.1%). The incidence of minor complications was 17.3% (CI: 14.7%–20.3%), while the incidence of major complications was lower at 5.5% (CI: 4.5%–6.7%). The overall incidence of donor mortality was 0.06% (CI: 0.0%–0.1%) in 49,027 individuals. Psychological complications (7.6%, CI: 4.9%–11.5%) were the most common among LDLT donors, followed by wound-related (5.2%, CI: 4.4%–6.2%) and respiratory complications (4.9%, CI: 3.8%–6.3%). Conversely, cardiovascular complications had the lowest incidence among the subgroups at 0.8% (CI: 0.4%–1.3%). This study presents the incidence of post-LDLT outcomes in living liver donors, illustrating significant psychological, wound-related, and respiratory complications. While significant advancements in recent decades have contributed towards decreased morbidity in living donors, our findings call for targeted measures and continued efforts to ensure the safety and quality of life of liver donors post-LDLT.

Purpose: Endovascular aortic aneurysm repair (EVAR) is the dominant treatment modality over open repair for abdominal aortic aneurysms. However, a higher rate of reinterventions remains the Achilles heel of EVAR. Although type 1A endoleak from proximal seal zone failure of EVAR remains one of the leading causes for reintervention, fenestrated branched devices suitable for proximal extension of failed EVAR are not widely available in the United States. Gore Thoracoabdominal Multibranch Endoprosthesis (TAMBE) is an off-the-shelf investigational device that provides supraceliac seal by incorporating 4 visceral and renal arteries via preloaded inner branches. Case Report: In this article, we describe 2 cases of type 1A endoleak from previous EVAR devices repaired using TAMBE. Both cases were performed under the Food and Drug Administration (FDA) compassionate use exemption. Considerations on the case planning and implantation techniques of TAMBE specific to previous EVAR devices are reviewed. Conclusions: Gore TAMBE can be utilized to repair a type 1A endoleak of a previous infrarenal EVAR device. Greater supraceliac coverage necessary for TAMBE relative to the minimal seal zone should be considered when applying this device for a type 1A endoleak. Clinical Impact This report demonstrates the feasibility of applying off-the-shelf TAMBE device to treat one of the most common failure modes of EVAR, type1A endoleak.

In excitation‐contraction coupling, a transient elevation of cytosolic Ca 2+ concentration ([Ca 2+ ] cyt ) triggers a force response. The Ca 2+ sensitivity of muscle reflects the relationship between [Ca 2+ ] cyt and force, and in skeletal and cardiac muscle is often defined as the [Ca 2+ ] cyt at which 50% force is generated. In smooth muscle, Ca 2+ sensitivity has been assessed as the ratio of peak force to peak cytosolic [Ca 2+ ] cyt . However, this measurement overlooks the dynamic nature of Ca 2+ regulation of force generation. In the present study, in order to provide a dynamic index of Ca 2+ sensitivity in airway smooth muscle (ASM), we simultaneously measured [Ca 2+ ] cyt and force and determined their temporal dependence using a phase loop plot. We hypothesized that [Ca 2+ ] cyt /force phase loop plot in ASM would be altered by conditions in which the extent of regulatory myosin light chain (rMLC) phosphorylation was affected. Intact ASM strips were isolated from porcine tracheas obtained from local abattoir. ASM strips, were loaded with Fura‐2 AM (Ca 2+ fluorescent indicator) and stimulated using 1 μM ACh. Isometric force and [Ca 2+ ] cyt responses were simultaneously measured using a Guth Muscle Research System. In the first set of experiments we examined the effect of Y27623, a Rho‐kinase inhibitor, and calyculin A, a protein phosphatase inhibitor that affected Ca 2+ ‐induced rMLC phosphorylation. In a second set of experiments, we examined the effect of cytochalasin D (Cyto‐D), an inhibitor of actin polymerization, which reduces force independent of Ca 2+ ‐induced rMLC phosphorylation. As expected, Y27623 decreased ACh‐induced isometric force but had no effect on evoked [Ca 2+ ] cyt transients. Cyto‐D also decreased force with no effects on evoked [Ca 2+ ] cyt . By comparison, calyculin A increased force with no effect on [Ca 2+ ] cyt . Although these results suggest changes in Ca 2+ sensitivity, the phase loop plots showed no significant shift indicative of changes in dynamic Ca 2+ sensitivity. These results do not support our hypothesis that dynamic Ca 2+ sensitivity in ASM is modulated by the extent of rMLC phosphorylation. Under these experimental conditions, it appears that isometric force generation in ASM is not entirely dependent on [Ca 2+ ] cyt . Support or Funding Information Supported by NIH grant HL126451 (GCS)

In previous studies, we found that prolonged hypothermia (2 h or greater) followed by rewarming induces cardiac contractile dysfunction (rewarming shock) mimicking the effects of accidental hypothermia followed by rapid rewarming in trauma patients. We found that rewarming shock was also presented in isolated cardiomyocytes in which reduced contractility was attributed to PKA‐mediated TnI phosphorylation and reduced Ca 2+ sensitivity. During hypothermia rewarming in cardiomyocytes there is increased production of reactive oxidant species (ROS ‐ superoxide anion), which may trigger the intracellular mechanisms underlying reduced contractility. We hypothesized that in cardiomyocytes, hypothermia/rewarming‐induced ROS production is due to altered mitochondrial respiratory function. To test this hypothesis, isolated cardiomyocytes from 6 rats underwent the following hypothermia/rewarming protocol in which they were electrically stimulated every 2 s: experimental temperature was cooled from 35°C to 15°C during the initial 30 min, then maintained at 15°C for 2 h, followed by rewarming to 35°C in the final 30 min. Time‐matched control cardiomyocytes were also electrically stimulated every 2 s and maintained at 35°C for 3 h. After the 3‐h protocol, cardiomyocytes were placed into separate chambers of an Oroboros Oxygraph (high‐resolution respirometry) to measure O 2 consumption rate (OCR) under varying conditions. Initially, basal OCR was measured for 5 min followed by serial exposure to oligomycin (1 μM for 5 min) to block ATP synthase activity, FCCP (0.5 μM repeated 3 times for 5 min each) an H + ionophore to dissipate mitochondrial membrane potential; and antimycin‐A (0.5 μM for 5 min) to block the electron transport chain. This procedure allowed assessment of the key parameters of mitochondrial respiratory function: basal and maximal respiration (after FCCP exposure), ATP production and proton leak (after oligomycin), and reserve respiratory capacity (calculated after antimycin‐A). We found that compared to control cardiomyocytes, hypothermia/rewarming decreased key parameters of mitochondrial function, which suggests that altered mitochondrial function may have contributed to increased ROS production. In addition, decreased OCR following rewarming may contribute to contractile dysfunction by limiting ATP supply in response to metabolic demand, as well as by contributing to a decline in cell viability (e.g., triggering apoptosis). Support or Funding Information 5T32HL105355 (NSS); Norwegian Air Ambulance Foundation (YSH)