During acute myocardial infarction, the composition of the extracellular matrix changes remarkably. One of the most notable changes in the extracellular matrix is in the accumulation of collagen; however, hyaluronan rivals collagen in its abundance. Yet, the extent to which specific cells and enzymes may contribute to such accumulation has been largely unexplored. Here, we hypothesized that activated cardiac fibroblasts produce hyaluronan via hyaluronan synthase 2 (HAS2). We show that hyaluronan accumulates following myocardial infarction and persists through at least 4 wk. Our analyses of failing heart RNA sequencing data suggest that fibroblasts are the cells most changed in the expression of HAS2. Given these insights, we used HAS2 gain- and loss-of-function approaches to examine the extent to which activated cardiac fibroblasts produce hyaluronan. Transforming growth factor β (TGFβ)-induced activation of fibroblasts caused a significant increase in Has2 mRNA and concomitant accumulation of hyaluronan >1 MDa in size. Deletion of Has2 abrogated TGFβ-induced production of hyaluronan. In addition, overexpression of Has2 was sufficient to cause an increase in hyaluronan accumulation in the absence of TGFβ-induced activation. Our data indicated negligible impacts of Has2 on proliferation, migration, and collagen production. Exposing fibroblasts to exogenous hyaluronan also had minimal impact on fibroblasts. We also assessed whether fibroblast-borne Hyal2 plays a role in the degradation of hyaluronan, and our data indicated little impact of Hyal2 on hyaluronan accumulation (or even any impacts on the transcriptional profile of fibroblasts). Activated fibroblasts produce high-molecular-weight hyaluronan via Has2, which occurs independent of other fibroblast functions.NEW & NOTEWORTHY Activated cardiac fibroblasts produce copious quantities of collagen, and much is known about this process. They also produce hyaluronan, which is abundant in the extracellular matrix, but less is known about hyaluronan. Here, we identify cardiac fibroblasts as major producers of hyaluronan and, specifically, that they produce high-molecular-weight hyaluronan via HAS2. This has important implications for ventricular remodeling and for metabolic regulation of activated fibroblasts, as they produce this abundant matrix component.

Introduction: Cardiac fibroblasts, known for their reactivity to pro-inflammatory stimuli, are increasingly recognized as key mediators of inflammation. TGF-β activated kinase 1 (TAK1) has been identified to integrate diverse inflammatory signals, but its role in fibroblast biology and myocardial remodeling remains unclear. The goal of this study was to determine the role of fibroblast-TAK1 signaling on acquisition of an inflammatory phenotype and in post-myocardial infarction (MI) repair and remodeling. We hypothesize that TAK1 signaling integrates inflammatory signals to promote cardiac fibroblast activation, which regulates inflammation and remodeling after MI. Methods and Results: We used a fibroblast-specific model for TAK1 knockdown, i.e. Col1a2-Cre ERT TAK1 fl/fl mice, and littermate controls to determine how TAK1 influences remodeling after permanent coronary ligation. Deletion of TAK1 in fibroblasts in vivo prior to MI improved functional outcomes and reduced fibrosis 28 days post-MI in male mice (n=9-14/group; p<0.05). Interestingly, the female cohort experienced greater rates of mortality in TAK1 deleted mice (52.0% vs 22.7%; n=22-25/group). Flow cytometric evaluation of immune cells in the hearts showed that fibroblast-TAK1 deletion reduced CD45 pos leukocytes, neutrophils, and Ly6C low patrolling monocytes acutely post-MI (n=6-13/group; p<0.05). To determine how TAK1 may regulate the pro-inflammatory phenotype, isolated cardiac fibroblasts from TAK1 fl/fl mice were pretreated with Ad-CMV-iCre and stimulated with TGFβ, TNFα, or IL-1β. Notably, TAK1 deletion suppressed TNFα and IL-1β-dependent p-p38, p-JNK, and NFκB signaling (n=5/group; p<0.05). Bulk RNAseq analysis further revealed that TAK1 inhibition significantly reduced the expression of several inflammatory and stress response genes following IL-1β activation, many of which were also decreased at the protein level (MCP-1, RANTES, G-CSF, MMP3, iNOS; n=4/group, p<0.05). Lastly, LC-MS/MS evaluation of lipid mediators in conditioned medium showed that TAK1 deletion increases production of several resolvins (RvE2, RvD1, RvD4) and lipoxins (LXA 4 , 15( R )-LXA 4 ) (n=3/group; p<0.05). Conclusion: TAK1 signaling in cardiac fibroblasts is a critical regulator of inflammation and immune cell recruitment post-MI. Fibroblast-specific TAK1 suppression improves cardiac function and structure in male mice, likely by attenuating inflammatory signaling and promoting pro-resolving lipid mediator production. Notably, the differences in outcomes observed between males and females may be attributed to previously documented sex-specific variations in inflammatory responses. NIH R01 HL168198, NIH F30 HL165813, This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

Background Branched‐chain amino acids (BCAAs), which are derived from the diet, are markedly elevated in cardiac tissue following myocardial infarction (MI). Nevertheless, it remains unclear whether dietary BCAA levels influence post‐MI remodeling. Methods To investigate the impact of dietary BCAAs on cardiac remodeling and function after MI, we fed mice a low or a high BCAA diet for 2 weeks before MI and for 4 weeks after MI. Cardiac structural and functional changes were evaluated by echocardiography, gravimetry, and histopathological analyses. Immunoblotting was used to evaluate the effects of BCAAs on isolated cardiac myofibroblast differentiation. Results The low BCAA diet decreased circulating BCAA concentrations by >2‐fold when compared with the high BCAA diet. Although neither body weights nor heart masses were different in female mice fed the custom diets, male mice fed the high BCAA diet had significantly higher body and heart masses than those on the low BCAA diet. The low BCAA diet preserved stroke volume and cardiac output after MI, whereas the high BCAA diet promoted progressive decreases in cardiac function. Although BCAAs were required for myofibroblast differentiation in vitro, cardiac fibrosis, scar collagen topography, and cardiomyocyte cross‐sectional area were not different between the dietary groups; however, male mice fed the high BCAA diet had longer cardiomyocytes and higher capillary density compared with the low BCAA group. Conclusions A low BCAA diet mitigates eccentric cardiomyocyte remodeling and loss of cardiac function after MI in mice, with dietary effects more prominent in males.

Organ health and function depend on communication between cell types to coordinate tissue growth and repair. Recent studies have indicated that fibroblasts are critical to this process; however, their role in regulating inflammatory responses to injury have remained ambiguous. Here, we demonstrate that transforming growth factor β-activated kinase 1 (TAK1) is a gatekeeper of the inflammatory cardiac fibroblast phenotype. We find that TAK1 propagates IL-1β and TNF-α signaling in cardiac fibroblasts and coordinates the synthesis and secretion of chemokines as well as inflammatory and pro-resolving lipid mediators. Deletion of TAK1 in fibroblasts decreased immune cell recruitment after MI, which was associated with improved cardiac structural and functional remodeling in male mice. Nevertheless, we found the effects of TAK1 deletion to be sexually dimorphic in nature, providing support to the idea that the protected phenotype of the female sex may be based in disparate immune and inflammatory responses. Moreover, TAK1 signaling controlled the acquisition of novel markers of the inflammatory fibroblast phenotype, having a biological basis in redox stress, chemokine and lipid mediator biosynthesis, metalloproteinase activity, and damage-associated molecular pattern recognition. Collectively, these results further resolve the nature and function of inflammatory cardiac fibroblasts in cardiac responses to injury and identify TAK1 signaling in fibroblasts as a potential target for therapy.

Fibroblasts are crucial for cardiac repair after myocardial infarction (MI). In response to signaling cues, they differentiate to phenotypes with robust capacities to synthesize and secrete extracellular matrix (ECM) and signaling molecules. Although activated fibroblast phenotypes are associated with pronounced changes in metabolism, it remains unclear how the metabolic network upholds the effector functions of fibroblasts in the infarcted heart. We found that two enzymes that could facilitate a phosphoenolpyruvate cycle, i.e. pyruvate kinase muscle isoform 2 (PKM2) and phosphoenolpyruvate carboxykinase 2 (PCK2), are elevated in the heart after MI. Although Pck2 deletion had no effect on post-MI remodeling, fibroblast-specific switching of Pkm2 to Pkm1 (fbPkm2 → 1) mitigated ventricular dilation, wall thinning, and losses in ejection fraction caused by MI. Despite these salutary effects, fbPkm2 → 1 switching did not alter cardiac fibrosis in vivo, nor did it affect collagen production, cytokine or chemokine secretion, myofibroblast differentiation markers, or transcriptional regulation in vitro. Nevertheless, Pkm2 → 1 splice variant switching increased myofibroblast contractile activity as well as influenced the metabolic phenotype of fibroblasts, as shown by increased pyruvate kinase activity, higher mitochondrial respiratory capacity, and elevation in glycolytic intermediate abundance. Despite these changes, Pkm2 → 1 switching had relatively minor effects on glucose carbon fate, as determined by stable isotope-resolved metabolomics. Nevertheless, these metabolic data demonstrate that cardiac fibroblasts exhibit minimal glucose-supported de novo glycine synthesis in vitro, yet possess high hexosamine and glucuronate biosynthetic pathway activity. Collectively, these findings reveal that fibroblast PKM isoforms influence post-MI remodeling, highlighting pyruvate kinase as a potential therapeutic target.

Background: Many benefits related to exercise are ascribed to reduction in cardiovascular disease risk. Yet, the impact of exercise after the event—i.e., during ventricular remodeling after myocardial infarction (MI)—is unresolved. Because physical exercise alters myocardial structure and function in the healthy heart, it is reasonable to wager that exercise can also impact the remodeling heart. Unfortunately, the mechanism and magnitude of impact of exercise on ventricular remodeling following MI is largely unexplored. Goal: Establish whether and the extent to which exercise impacts ventricular remodeling post-MI. Methods: Twenty-six adult male mice were subjected to non-reperfused MI. Five days after MI, echocardiography was performed, and the next day mice were allocated into Exercise (EXE; n=13) or Sedentary (SED; n=13) groups based on ventricular dysfunction. EXE mice were single housed and given ad libitum access to a voluntary exercise wheel for the duration of the study; SED mice had no wheel access. Mice in the EXE group ran an average of 9 km/d. An exercise capacity test and echocardiography were performed at the end of the five-week protocol and tissue was harvested. Results: Ventricular chambers were significantly larger (p<0.05) in the EXE group than the SED group. These unexpected findings, along with an observed trend towards improved survival in the EXE group led us to ponder the potential implications. Because the study design included a pre-treatment (but post-MI) echocardiogram, we assessed a potential relationship between survival and starting chamber dimensions. Of the mice with the largest chamber dimensions (i.e., >100 µL), 0% of the SED mice survived while 62% of the EXE mice survived. Conclusions: This study affirms the positive impact of exercise following MI. Furthermore, the benefits of exercise may be most noticeable in subjects with the most severe ventricular dilation.

Background: Hyaluronan (HA) accumulates after tissue injury and may regulate the wound-healing process. We found that HA is increased in and around the scar following myocardial infarction (MI). Fibroblasts are the main producers of the collagen that comprises the scar, and we have shown that they are responsible for HA accumulation. Given that these two extraordinarily high-volume synthetic processes occur in the same place and simultaneously, we queried whether alterations in Has2 -dependent HA synthesis impacted collagen secretion and other fibroblast functions. Goal: To determine the impact of HA synthesis on fibroblast function. Methods and Results: We isolated cardiac fibroblasts from Has2 fl/fl mice and treated with Cre adenovirus to delete Has2 expression or Has2 adenovirus to overexpress Has2. We assessed cell number and found no differences when Has2 was deleted or overexpressed in both naïve fibroblasts (n=6); activation with TGF-b did not change this response . We used a scratch assay to assess migration and found no change between treatments in naïve fibroblasts (n=6). Hydroxyproline assessment of deposited collagen and immunoblotting for soluble collagen secretion in the media indicated no differences between treatments in naïve fibroblasts (n=3). Conclusion: Genetic manipulation of HA production did not impact proliferation, migration, or collagen secretion in fibroblasts. Considering the enormous metabolic requirement of fibroblasts to produce HA and collagen, these results suggest that the regulation of these processes may be distinct or that the substrate is not limiting in our experimental conditions. In the intact heart, substrate could become limiting and a relationship between these two biosynthetic processes may emerge

The traditional case study has been used as a learning tool for the past 100 years, and in our program, graduate physiology students are presented with a real-world scenario and must determine the diagnosis and treatment of the patient. We found that students defaulted to memorization of disease with treatment and bypassed gaining an understanding of the mechanistic physiology behind disease and treatment. To adjust our student's approach, we developed a novel way to enhance student learning. To accomplish this shift from memorization to physiological mastery, we created the Inverted Case Study. This approach diverges from the traditional model in that students are given the diagnosis and treatment beforehand and are tasked with explaining the actual physiology of the case. In this way, students can no longer rely on the memorization of symptoms-disease-treatment but rather gain a solid understanding of the physiological mechanisms of the disease since that is the focus of the Inverted Case Study Technique. The Inverted Case Study approach is an effective approach to apply and hone critical thinking skills. NEW & NOTEWORTHY This article presents a novel approach to century-old learning techniques that enhances students' self-reported learning and also their attitudes toward learning mechanistic physiology and increases their perception of preparedness for professional school.

Introduction: Resident cardiac fibroblasts are often defined by their extraordinary capacity to deposit extracellular matrix after cardiac injury; however, fibroblasts also react avidly to pro-inflammatory stimuli, suggesting that they may also be mediators of inflammation. Initially identified as a TGFβ responsive enzyme, TGF-β activated kinase 1 (TAK1) has been shown to be significant in regulating pro-inflammatory signaling in several disease models. The goal of this study was to understand how fibroblasts integrate pro-inflammatory signals to regulate acute inflammation following myocardial infarction (MI). We hypothesize that cardiac fibroblasts utilize a TAK1-dependent mechanism to integrate pro-inflammatory signals and orchestrate the cellular immune response following injury. Methods and Results: To create a fibroblast-specific model for TAK1 knockdown, TAK1 fl/fl mice were bred to Col1a2-CreERT mice. Following 2 weeks of tamoxifen chow feeding, both Cre− and Cre+ TAK1 fl/fl mice underwent permanent coronary ligation to induce MI. Three days following MI, immune cell populations from the heart, spleen, and peripheral blood were evaluated through flow cytometry. Deletion of TAK1 in fibroblasts in vivo diminished neutrophils and Ly6C low patrolling monocytes populations in the heart and decreased the number of CCR2neg resident macrophages after MI (n=10/group; p<0.05). Interestingly, circulating levels of neutrophils in the peripheral blood as well as those in the spleen were not significantly different between the groups. To determine how TAK1 may regulate pro-inflammatory signaling, isolated cardiac fibroblasts from TAK1 fl/fl mice were treated with Ad-CMV-iCre and were subsequently treated with IL1β. Notably, TAK1 knockdown suppressed IL1β-dependent p-JNK and NFκB signaling as well as MMP3 production. Further LC-MS/MS evaluation of lipid mediators in conditioned medium showed that deletion of TAK1 significantly increased fibroblast-derived resolvins (RvE2, RvD1, RvD4, RvE4) and lipoxins (LXA4, 15( R)-LXA4) (n= 3-5; p<0.05). Conclusion: Resident cardiac fibroblasts regulate the acute immune response following MI via a TAK1-dependent mechanism. Secreted factors derived from cardiac fibroblasts likely play a pivotal role in shaping the inflammatory and immune response after MI. NIH and AHA. 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.

With the increased attention focused on active learning, educators strive to find better and more innovative ways to engage students in the classroom. One of the hurtles that educators are presented with is that the classroom is no longer limited to a physical location but rather students and professor can meet via the internet, Before COVID-19, distance or remote learning was something that students, by and large, had the option of choosing in which whether to engage. Students had the option to take "online courses," whether those be synchronous remote learning or asynchronous online courses. Indeed, numerous studies have focused on investigating the efficacy of many different approaches to distance and online learning. Unfortunately, COVID 19 mandated a rapid transition to remote learning, and with this forced change has come what some students describe as "Zoom fatigue" (Wolf CR. Psychology Today, May 2020). Many students reported feeling exhausted, overwhelmed, and disengaged by the dramatic increase in mandated distance education required by the COVID pandemic. Video conferencing has become the "go-to" panacea for education during this time, and students are spending unprecedented amounts of time in front of a screen when normally they would be in a classroom. This heretofore singular and unique approach to education coupled with decreased peer-to-peer interaction has caused a problem with student engagement (Goodman BE, Barker MK, Cooke JE. Adv Physiol Educ 42: 417-423, 2018). Students' engagement and performance have decreased during COVID-19 because of forced online learning and lack of peer interaction. We hypothesize that creating a nongraded, fun, and relaxing physiology-focused "Trivia Night" will increase student engagement and performance on summative assessments. Using a master's level class progressing through the respiratory physiology module utilizing remote, synchronous lectures to deliver content, we introduced a voluntary Trivia Night review session with teams randomly assigned to increase interaction among peers and review respiratory physiology material.NEW & NOTEWORTHY This article presents the effectiveness of the use of the "pub Trivia Night" to facilitate learning, deconstruct misconceptions, and increase engagement during remote teaching due to the COVID-19 pandemic.

Background : Lipid peroxidation products, such as acrolein, are highly toxic aldehydes generated during pathological remodeling and associated with heart failure. Until now, no translatable therapy has been tested to remove these toxic products from heart and examine the subsequent effects on failing hearts. The heart contains small histidyl dipeptides, such as carnosine (β-alanine-histidine), which bind lipid peroxidation products. Carnosine is decreased in failing hearts and its levels can be increased by supplementing the precursor, β-alanine. This study investigates the cardioprotective and translational potential of β-alanine supplementation in a transaortic constriction (TAC) model of heart failure. Hypothesis : Increasing myocardial carnosine via β-alanine supplementation will improve cardiac function during heart failure. Approach : Male wild-type C57BL/6J mice were treated either (a) water alone, (b) β-alanine 1 week prior to TAC, or (c) β-alanine (20g/L) in water 4 weeks post TAC. Supplementation of β-alanine was continued for 8 weeks, followed by serial echocardiography, biochemical, and mass spectrometry analysis. Results : Myocardial carnosine increased ~3-4-fold after 1 week of β-alanine feeding compared with water alone (p < 0.05). β-alanine pre-feeding compared with water alone, after 8 weeks of TAC, decreased left ventricular (LV) mass (β-alanine: 174 ± 29 vs water: 241 ± 17 mg, p < 0.05) and LV inner diameter at systole (β-alanine: 2.2 ± 0.6 vs water: 3.5 ± 0.4 mm, p < 0.05) and diastole (β-alanine: 3.5 ± 0.4 vs water: 4.4 ± 0.3 mm, p < 0.05), and increased ejection fraction (β-alanine: 67 ± 14% vs water: 42 ± 9%, p < 0.05) and cardiac output (β-alanine:13.6 ± 2.6 vs water: 10.7 ± 1.0 mL/min, p < 0.05). Post-TAC β-alanine intervention decreased LV mass (β-alanine :177 ± 8 vs water: 214 ± 11 mg, p < 0.05), hypertrophic markers Nppa and Myh7 , and increasingly removed acrolein from heart by conjugation (β-alanine: carnosine-propanal: 12.2 ± 0.4 vs water: 0.7 ± 0.4 pmoles/mg protein, p < 0.0001). Conclusion : Increasing myocardial carnosine via oral supplementation alleviates pathological remodeling, possibly through quenching reactive aldehydes. These findings lay foundation to test the feasibility of β-alanine in heart failure patients.

Clinical studies show that chitinase 3-like-1 (CHI3L1) is a biomarker and predictor of all-cause mortality in heart failure. Studies from our lab demonstrated that CHI3L1 is elevated in hearts post-MI, and CHI3L1 deficiency in mice results in preserved ventricular structure and function post-MI. Furthermore, we found that CHI3L1 is secreted from activated neutrophils but has no effect on neutrophil recruitment in hearts after MI. The goal of the current study was to elucidate mechanisms whereby CHI3L1 contributes to exacerbated cardiac dysfunction after MI. Post-MI neutrophil recruitment is followed by the sequential programming of macrophages to pro-inflammatory or pro-reparative phenotypes. We sought to determine the impact of neutrophil-secreted CHI3L1 on macrophage polarization. Activated neutrophils isolated from Chil1 +/+ and Chil1 -/- mice were co-cultured with bone marrow-derived macrophages. We found that macrophages directly co-cultured with Chil1 -/- but not Chil1 +/+ neutrophils showed upregulation of the pro-reparative markers, Chil4 and Arg1 (n=5/group, p<0.05). Additionally, direct co-culture of Chil1 -/- neutrophils and macrophages showed trends towards downregulation of the pro-inflammatory polarization markers, Il1b and Tnfa relative to macrophages co-cultured with Chil +/+ neutrophils. These data suggest that neutrophil-derived CHI3L1 may antagonize signaling to polarize macrophages towards a pro-reparative phenotype that may explain delayed scar formation and exacerbated ventricular dysfunction after MI. Future efforts will elucidate the signaling role of CHI3L1 associated with neutrophil extracellular traps (NETs) on macrophage function.

HIGHLIGHTS:Over-the-needle (OTN) PIVC devices are at inherent risk of insertion related skin contamination. Through-the-needle (TTN) catheter deployment resulted in no measurable contamination in this study. OTN catheters were 1.67 times more likely to be contaminated than TTN in this study.AIM:To compare a traditional over-the-needle peripheral intravenous catheter device to a through-the-needle (TTN) peripheral intravenous catheter device for early bacterial contamination during insertion.METHODS:Five TTN test devices (OspreyIV 20 g SkyDance Vascular, Inc) and 5 OTN comparative devices (Insyte Autoguard 20 g Becton Dickinson) were aseptically inserted through targeted zones inoculated with 1 mL aliquot suspension of approximately 1 × 10 CFU of Staphylococcus aureus among 3 healthy sheep. Immediately after insertion, each study catheter was surgically removed from the surrounding tissue and cultured for the presence of Staphylococcus aureus inoculum that may have been transferred to the catheter during insertion.RESULTS:Final culture results of the 5 test articles found no bacterial colonies. Final culture results of the 5 comparative articles revealed 2 of 5 were contaminated with bacterial colonies. The absolute risk reduction is 40%, or a 40% rate of contamination drops to a 0% rate of contamination when the TTN catheter deployment was used. The risk ratio achieved was 1.67, indicating catheters placed using the OTN deployment were 1.67 times more like to be contaminated than the TTN deployed catheters.CONCLUSION:In this present ovine study, the data revealed that use of a novel TTN approach resulted in less contamination than the more traditional OTN approach. Traditional OTN devices, developed over 70 years ago, are at inherent risk of insertion-related contact contamination. The results of this research, as well as previously published studies, point toward considering physical catheter protection strategies such as TTN devices as a potential alternative to OTN devices.

Introduction: In universities, a traditional lecture style has shown to be less effective in helping students learn compared to a more active approach to teaching and learning. Previous research has stated that passive learning triggers a shift to a more superficial style of learning that is associated with a lower level of cognitive skill. Active learning is defined as activities that students do to construct knowledge and understanding. An active learning concept known as deep reading has been suggested to improve academic performance. In recent studies, deep reading was utilized in graduate science courses and was shown to enhance reading and comprehension skills as well as increasing overall academic ability. Objective: In this study, the aim was to assess how deep reading impacted a student’s self-perception of their understanding of course material in a graduate-level physiology course. Methods: Over a six-week period, 23 graduate physiology students participated in weekly deep reading assignments that correlated with the material taught in class that week. At the end of each week, students filled out an evaluation form indicating how they felt their assigned reading impacted their understanding of the material taught in class. Results: Results indicated that almost 70% of the class agreed that deep reading helped in understanding the course material. Student feedback data showed frequent use of the words “understanding,” “enjoyed,” “relevant,” and “helped.” Students who spent between 0-2 hours per week deep reading gave positive feedback. Individuals who spent more than 2 hours per week indicated negative feedback and perceived that deep reading counteracted their understanding of course material. Conclusion: The data implies that student understanding of material was improved with 0-2 hours of deep reading per week. More than 2 hours of deep reading per week had a countereffect. Excess time spent reading could have caused students to lose sight of the important concepts from the articles and thereby eliciting a negative effect and more confusion. Some, 1-2hrs, seemed to be helpful, however, more had a detrimental effect on learning. Future studies should aim to have more consistency in the difficulty of reading materials between students. Addition of comprehensive reading questions could also be implemented to assess student understanding. Overall, we found in this study that deep reading, when used in moderation, was effective in helping students learn and see real-world application of concepts learned in class This is the full abstract presented at the American Physiology Summit 2023 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.

Background: After a myocardial infarction (MI), macrophages are recruited to the site of injury to begin the process of wound healing. Macrophages are important because they clear cellular debris via phagocytosis. This period of acute inflammation in the heart coincides with extracellular matrix (ECM) remodeling. Hyaluronan (HA), one of the most abundant ECM components, accumulates in a high molecular weight form (HA HMW ). The relationship between HA HMW accumulation and macrophage function in is not fully understood. Goal: To determine the extent to which hyaluronan impacts phagocytosis in polarized macrophages. Methods and Results: Tissue sections were collected from mice one-week post-MI and stained for hyaluronan binding protein (HABP) to assess HA accumulation. A significant increase in HA was seen in both male (n=4, p=0.0031) and female (n=4, p=0.0210) MI hearts compared to sham hearts. Separately, bone marrow was isolated from naïve mice and cells were treated in culture with macrophage colony stimulating factor (M-CSF) for 7-10 days. The resultant macrophages were then polarized into a pro-inflammatory (M1) phenotype using LPS and INF-γ, or pro-reparative phenotype (M2) using IL-4 and IL-13. Macrophage polarization was confirmed via qRT-PCR. In additional experiments, macrophages were polarized for 24 hours and then incubated for 30 min with FITC-labeled, IgG-coated latex beads in the presence or absence of 1 μM HA HMW . Flow cytometry was used to assess phagocytosis of the beads. HA HMW treatment resulted in a significant reduction in the percent of FITC positive cells in M1 female (n=6, p=0.005), M2 female (n=5, p=0.0406), and M2 male (n=5, p=0.0314) macrophage subsets indicating a decrease in phagocytosis. No significant changes were seen in the M0 and M1 male macrophage subsets. Additionally, the mean fluorescence intensity (MFI) measured in the FITC channel was significantly decreased in all the female subsets as well as the M2 male macrophages (n=5-6, p<0.05). No significant changes were seen in the M0 and M1 male subsets. Conclusion: HA HMW impairs opsonin-induced phagocytosis in macrophages. Given that HA HMW accumulates post-MI, these findings may partially explain the incomplete immune response in the failing heart.

Background: Following myocardial infarction (MI), immune cells are recruited to clear apoptotic cells and facilitate replacement fibrosis. Neutrophils are the first immune cells recruited, and neutrophilia predicts death and adverse outcomes post-MI. Increased demand for neutrophils is met through emergency granulopoiesis. Methods and Results: Our prior investigations stimulating emergency granulopoiesis with injections of exogenous granulocyte-colony stimulating factor (G-CSF; 100 μg/kg/d) indicated neutrophil immunophenotypic diversity with the emergence of CD101 Neg neutrophils in peripheral blood and spleen. In this study, we tested our hypothesis that MI induced emergency granulopoiesis increases neutrophil phenotypic diversity. We used a non-reperfused MI model in C57BL/6J mice (females, 14-20 wk). At 1, 2, 3, and 7 d post-MI, we harvested bone marrow, peripheral blood, spleens, and hearts. Neutrophil immunophenotypes were quantified in dissociated tissues via flow cytometry. Agreeing with previous findings, neutrophil infiltration of the heart was observed at 1-3 d, reaching a climax at 2 d. Our data demonstrates novel CD101 Neg and CD117 Pos neutrophils present in the heart 1-3 d post-MI (n=3-6, p< 0.05). Additionally, elevated numbers of neutrophils and CD101 Neg neutrophil subtypes were observed in bone marrow, peripheral blood, and spleen relative to naïve mice (n=3-10, p<0.05). Conclusion: We conclude that post-MI production of neutrophils results in the recruitment of diverse neutrophil subtypes to the heart. Future studies will seek to understand how post-MI activation of emergency granulopoiesis instigates neutrophil diversity and how CD101 Neg and CD117 Pos neutrophils are involved in replacement fibrosis. Figure 1. Presence of CD101 Neg and CD117 Pos Neutrophils in the Heart in post-MI. Gating (A-C) and quantification (E-G) of neutrophil immunophenotypes. (n=6-9, Mixed Affects Two-way ANOVA with Multiple Comparisons).