Atrial fibrillation (AF) is characterized by episodes of rapid and irregular heart rate. Obesity increases AF risk. The impact of high fat diet (HFD) on atrial structural and electrical remodeling in the context of AF is not clear. We hypothesized that HFD promotes AF onset and progression. Our goal was to evaluate the impact of HFD on evidence of first AF and heart rate variability in a mouse model of spontaneous onset of AF. Wild-type (WT) and cAMP responsive element modulator IbΔC-X heterozygous (TG) male and female mice were fed HFD (45 kcal% fat) or matched control diet (CD, 10 kcal% fat) beginning at 4 weeks. AF burden was assessed weekly by electrocardiography (ECG) through 15 weeks. Terminal body weight was increased (p<0.05) in WT HFD compared to WT CD in females and males but was not altered in TG HFD compared to WT HFD (Fig 1A,B). Left atrial (LA) weight was increased (p<0.0001) in TG CD and TG HFD compared to WT CD and WT HFD in both male and female mice (Fig 1C,D). LA weight was not increased in WT HFD or TG HFD compared to WT CD or TG CD. The coefficient of variation of RR intervals (CVRR), a measure of heart rate variability, was increased at 11 weeks in male TG CD (p<0.0001) and TG HFD (p<0.001) compared to WT CD and WT HFD and was maintained through 15 weeks. In female mice, CVRR was increased at 9 weeks in TG CD (p<0.008) and at 10 weeks in TG HFD (p<0.009) compared to diet controls. ECGs were evaluated to identify the timepoint that AF was first evident. AF was not evident in WT CD males and females (Fig 2A,B). AF was first observed in WT HFD males at 8 weeks and WT HFD females at 14 weeks. In TG males, AF was first evident in CD and HFD at 6 weeks (Fig 2C). Notably, all TG HFD males were in AF by 8 weeks, 2 weeks earlier than TG CD males. In TG females, AF was observed at 6 weeks (Fig 2D). All TG CD and TG HFD females were in AF by 11 weeks. Our data provide suggestive evidence of earlier AF onset in female WT, male WT, and male TG mice fed HFD. Future studies will extend the duration of HFD, evaluate AF burden, and examine the mechanisms underlying AF susceptibility in the presence of a HFD.

An impaired microcirculatory network due to injury, ischemia, or disease stimulates maladaptive remodeling and disrupts broader tissue function. Additionally, innate regenerative models in zebrafish and neonatal mice have demonstrated that supplemental vascularization occurs first in heart regeneration and is critical for subsequent cell proliferation and remuscularization, yet clinical approaches to small vessel regeneration remain elusive. In previous work, we employed an iterative fractional factorial design of experiments (DOE) approach with 10 angiogenic proteins to identify 5 high-performing factors, VEGF, bFGF, Shh, PDGF, and IGF-1. In this work, we optimized a fully defined and proangiogenic biomaterial delivery system using type 1 collagen and heparin-modified alginate to prolong co-release of multiple proteins up to 21 days in vitro . Using an in vivo subcutaneous gel plug assay in the WT immune competent rat, we have empirically evaluated 1-, 2-, and 3-protein combinations of the top five factors after 1 and 2 weeks of release. Custom, automated, high-fidelity quantification of vascular size, density, and morphology enabled identification of promising cocktails based on target tissues and desired vascular kinetics. A combination of VEGF, IGF-1, and PDGF (VIP) was identified for rapid, local vascularization, increasing perfused vascular density by 6-fold vs. the blank biomaterial at 1 week, with over 2/3 of vessels stable and persisting at 2 weeks. We employed a rat model of ischemic wound healing as a severe example of ischemic injury requiring a rapid vascular flourish for wound closure. Over the course of 2 weeks, the VIP-loaded biomaterial enhanced vascularization, increased both the rate and magnitude of wound closure, and improved skin regeneration when compared to the blank material and no treatment controls. This work has demonstrated both the promise of revascularization to facilitate regeneration in ischemic injury and the success of the VIP-loaded biomaterial in directing it. In conclusion, we identified a high-performing proangiogenic protein cocktail and optimized its controlled, local release from a defined biomaterial to develop a promising revascularization therapy for local microvascular regeneration.

Introduction: Myocardial transthyretin (TTR) accumulation can cause amyloid TTR cardiomyopathy (ATTR-CM), a form of restrictive heart disease found in ~16% of patients diagnosed with heart failure with preserved ejection fraction (HFpEF). ATTR-CM can be classified as hereditary (mutation in TTR) or non-hereditary (wild-type TTR). The mechanisms underlying the pathogenesis of non-hereditary ATTR-CM (ATTRwt-CM) remain unknown. To address this knowledge gap, we performed an in-depth mass spectrometry (MS) proteomic study on myocardial tissue from ATTRwt-CM compared to non-failing (NF) donor control, heart failure with a reduced ejection fraction (HFrEF), and HFpEF. Methods: Myocardial tissue lysates from NF controls (n=7) and ATTRwt-CM (n=4) were digested with trypsin and analyzed by label-free liquid chromatography-tandem mass spectrometry using an Evosep One LC coupled to an Orbitrap Astral MS. HFrEF and HFpEF proteome data were sourced from publicly available repositories. Results: ATTRwt-CM patients and NF controls were age-matched (54.1 ± 11.5 vs 60.0 ± 17.9 years, p=0.41). A total of 539/7093 (7.6% of total) proteins were significantly altered (Q-value < 0.05), with upregulated expression occurring in 227 (42.1% of significant proteins; log 2 -fold change: 1.10 ± 1.19) and downregulated expression in 312 (57.9% of significant proteins; log 2 -fold change: -0.55 ± 0.27). Analysis of Gene Ontology Biological Processes (GO:BP) enriched pathways in downregulated proteins indicated reductions in oxidative phosphorylation, mitochondrial protein translation, and impaired transmembrane transport. Conversely, upregulated proteins correlated with enhanced endocytosis, intracellular vesicle mediated transport, and immune cell migration. Compared to other forms of heart failure, ATTRwt-CM exhibited distinct protein regulation but overlapping pathway impairment with reduced oxidative phosphorylation and a unique increase in endocytosis and inflammatory-response. Conclusion: Myocardial tissue from patients with ATTRwt-CM shows signs of impaired oxidative phosphorylation, mitochondrial dysfunction, as well as augmented endocytosis, compared to NF controls, HFrEF, and HFpEF. Targeting these pathways could offer novel therapeutic strategies for addressing ATTRwt-CM.

Embryonic tissue resident macrophages (TRMs) are a distinct innate immune cell population that contribute to cardiac vascular development, mitochondrial homeostasis, and electrical conductance. While cardiac TRM functions in the developing heart and in the setting of disease have been characterized, their roles in early postnatal cardiac maturation and physiological remodeling are not fully elucidated. Understanding substates and functions of TRMs during postnatal angiogenesis, myocyte cell cycling, and adaptive hypertrophy may open up avenues for enhancing these processes by targeting the innate immune system. A major obstacle in manipulating TRMs is marker profile heterogeneity. To overcome this, we leverage an orthogonal genetic approach targeting two genes highly expressed in TRMs, Cd169 and Cx3cr1 . We hypothesize this will allow us to more selectively interrogate the TRMs of the heart. We performed both lineage tracing and cell ablation experiments. For lineage tracing, Siglec1 Cre mice (Cre expression driven by CD169) were crossed with mice expressing both CX3CR1 GFP and Cre-responsive Rosa26 TdT alleles to characterize TRM localization and morphology during the first week of postnatal life (p1-p7). Preliminary analysis of the heart by confocal microscopy and by flow cytometry demonstrated robust GFP and TdTomato expression, with co-expression representing over 70% of labeled CD45 + cells across postnatal week one. In contrast, splenic CD45 + cells were primarily single CX3CR1-GFP + , suggesting that a dual CD169 + and CX3CR1 + targeted approach may more readily affect cardiac immune cell populations. To that end, we crossed Siglec1 Cre Rosa26 TdT mice with the CX3CR1 DTR mouse line to express the human diphtheria toxin receptor only in CD169 + CX3CR1 + cells. Mice were given IP injections of diphtheria toxin or vehicle at p1, p2, and p3 and then analyzed at p7. Preliminary results suggest a reduction in normalized heart weight (p=0.03, n=9) and slightly decreased vascular density (1.78 vs. 2.04 CD31 + vessels/myocyte; p=0.26, n=6) at p7. These genetic models suggest feasibility and a possible adverse effect of ablating CD169 + CX3CR1 + TRMs during postnatal heart maturation, and open new avenues for mapping TRM states and functions in this window.

Background: Loss of SCN5A promotes fibrogenesis and disrupts metabolic balance, accelerating heart failure progression. MiR-452-5p is downregulated in heart failure and is associated with SCN5A deficiency, where it regulates fibrosis. However, whether miR-452-5p modulates metabolic reprogramming in SCN5A-deficient cardiac fibroblasts remains unclear. Methods: SCN5A knockdown (Skd) was induced in human cardiac fibroblasts using shRNA, followed by treatment with or without miR-452-5p agomir (10 nM, 24 hr). Untargeted metabolomics, ATP levels, ROS production, and metabolic signatures were evaluated. In vivo, myocardial injury was induced in 10-week-old Wistar rats (n=8) using isoproterenol (100 mg/kg). A subgroup received AAV9-miR-452-5p or vehicle via tail vein, and myocardial tissues were analyzed after 4 weeks. Results: SCN5A knockdown markedly decreased miR-452-5p expression in Skd compared to control (4.29 ± 0.7 vs 10.7 ± 1.2, p=0.001). Metabolomics identified 48 differentially regulated metabolites in Skd/Cont, significantly perturbed glucose metabolism (p=1.2E-05), and lowered ATP levels (Skd: 10.8 ± 0.9 vs. Cont: 16.4 ± 1.0, p=0.02), while ROS production increased (Skd: 41.6 ± 1.3 vs. Cont: 21.7 ± 1.0, p=0.002). MiR-452-5p attenuated glucose uptake (p=1.1E-06), and increased FAO (p=1.9E-03) as reflected by the expression of proteins involved in cardiac energy metabolism. Moreover, ATP levels were increased (22.2 ± 0.8, p=0.001) while reduced ROS (32.9 ± 0.7, p=0.005) in miR-452-5p treated cells. Myocardial tissues from the LV apex exhibited decreased Nav1.5 (44%) and miR-452-5p (60%) expression (p<0.01) with metabolic instability post W4 myocardial injury. MiR-452-5p restored the metabolic balance in the fibrotic apex by decreasing glucose transporters. However, it had no effect in the non-fibrotic remote zone, maintaining metabolic homeostasis. Conclusion: SCN5A deficiency disrupts cardiac metabolism, exacerbating disease progression. miR-452-5p restores metabolic balance, potentially offering a therapeutic avenue for heart failure.

Introduction: Tissue elasticity is essential to proper cell biology and organ function including the heart. Cell culture models on rigid polystyrene dishes are limited in studying the impact of tissue elasticity on specific cardiac cell types such as the cardiac conduction system cells. Hypothesis: We hypothesized that gelatin hydrogel at varying stiffness can uncover tissue elasticity-dependent changes to cardiac pacemaker cell physiology. Methods: Neonatal rat ventricular myocytes (NRVMs) were cultured as monolayers on polystyrene culture plates or on 1, 6, and 14 kPa gelatin hydrogels. All culture surface was coated with fibronectin. NRVMs were transduced with Adv-TBX18 or Adv-GFP at day 0 (d0). Results: The rates of rhythmic contractions were faster in TBX18-NRVMs than in GFP-NRVMs on the gelatin hydrogels from d1 to d7 (n≥4, p <0.05). TBX18-NRVMs cultured on the 14 kPa gelatin hydrogels showed faster contractions than those on 1 kPa or on plastic plates at d3 and d7 (d3: 54±12, 9±5, 0±0 bpm, d7: 31±10, 4±7, 24±10 bpm, n≥4, p <0.05). TBX18-NRVMs exhibited faster Ca 2+ transients on 14 kPa than on 1 and 6 kPa gelatin hydrogel or on plastic at d8 (33±2 vs. 9±2, 19±8, 11±5 transients/min, respectively, n=3, p<0.05). Hcn4 protein expression was higher in TBX18-NRVMs cultured on 14 kPa than on 1 kPa gelatin hydrogels or plastic plates (n=4/group, p <0.05). Cx43 protein expression was lower in TBX18-NRVMs compared to control at d8 regardless of the basement elasticity (n=4/group, p <0.05). However, Ca 2+ transient propagation velocity of TBX18-NRVMs was slower on 14 kPa than on 1 kPa hydrogels at d9 (5±1 vs. 8±1 cm/s, n≥3, p <0.05). Vimentin+ fibroblasts were more numerous in TBX18-NRVMs on 14 kPa than on 1 or 6 kPa hydrogels at d6 (40±2 vs. 28±4 or 27±2%, n=4, p <0.05), suggesting that the slower propagation velocity in TBX18-NRVMs on 14 kPa was due to increase in fibroblast content. Conclusions: The data demonstrate that automaticity of pacemaker cells is augmented by stiffer ECM substrates within the elasticity range of the healthy myocardium. This approach presents an easy-to-deploy in vitro model to study effects of tissue elasticity on cardiac conduction system cell physiology.

Cardiovascular-kidney-metabolic (CKM) syndrome, affecting over 30% of adults, drives significant morbidity through interconnected cardiovascular, renal, and metabolic dysfunction. Renal proximal tubular mitochondria play a pivotal role in CKM by succumbing to dysfunction under metabolic stress, leading to oxidative damage, tubular injury, and systemic effects that exacerbate cardiac dysfunction. We investigated whether enhancing renal tubular mitochondrial AKT1 could improve cardiac outcomes. We used KMioxCAKT mice, a novel transgenic strain harboring inducible, mitochondria-targeting, constitutively active AKT1 (mcaAKT1) driven by a renal proximal tubule-specific promoter, treated with tamoxifen or corn oil and fed a high-fat diet for 16 weeks. With unchanged BUN or creatinine, tamoxifen-induced mice had significantly reduced proteinuria, urinary KIM-1, glomerulosclerosis, and fibrosis. Metabolic dysfunction was markedly alleviated, with significant reductions in fasting glucose levels, improved glucose tolerance across 15-120 minutes post-glucose load, decreased fasting insulin levels, and enhanced insulin sensitivity as reflected by a reduced HOMA-IR. Echocardiography revealed improved cardiac function in tamoxifen-induced mice, with elevated ejection fraction (72.2%-73.6% vs. 45.1%-66.4%), increased fractional shortening (35.8%-37.1% vs. 30.4%-46.9%), and reduced left ventricular wall thickness (0.08-0.13 cm vs. 0.12-0.14 cm), collectively indicating attenuated cardiac hypertrophy. These findings suggest that enhancing renal tubular mitochondrial AKT1 could ameliorate the pathophysiology of CKM syndrome through improved cardiac systolic function, highlighting a potential therapeutic target for cardiorenal protection.

Myocardial infarction (MI) leads to cardiomyocyte (CM) loss and contractile dysfunction. We previously reported that genetic knockout (KO) or pharmacological inhibition of Tip60 ( T at- i nteractive p rotein, 60 kD), an acetyltransferase encoded by the Kat5 gene, reduced scarring and improved functional recovery following MI. Transcriptomic analysis indicates the involvement of Tip60 in CM excitation-contraction-metabolism coupling. Here we investigate the role of Tip60 in CM Ca 2+ homeostasis, contractility, mitochondria function, and metabolic properties. In this study, we isolated CMs from adult mice carrying LoxP sites in the Kat5 gene, enabling its specific KO in CMs via tamoxifen-activated Myh6-merCremer recombinase. The extent and kinetics of Ca 2+ transients were studied with rhod-2 AM and fluo-4 AM, their respective localization in mitochondria and cytosol was achieved by a two-step cold/warm loading protocol. Simultaneous measurements of sarcomere shortening were obtained at increasing rates of electrical pulses in the presence of beta-adrenergic agonist to mimic stress conditions. When CMs were challenged with sympathetic stimulation, Tip60 KO improved cytosolic Ca 2+ handling, maintained mitochondria Ca 2+ uptake and buffering, and promoted contractile reserve. Tip60-dependent changes were also confirmed by Ca 2+ retention capacity and membrane potential assessments in isolated mitochondria energized with different substrates. Because Ca 2+ is a key regulator of oxidative metabolism, we further examined CM oxygen consumption and cytosolic glycolysis. In agreement with the changes observed in mitochondria Ca 2+ signaling, Tip60 KO induced a metabolic transition from fatty acid oxidation to anaerobic glycolysis reflected by repressed maximal respiration and elevated glycolytic capacity. The effects of pharmacological inhibition of Tip60, via small MW drugs, on Ca 2+ handling and contraction, mitochondria function, and metabolic reprogramming are being assessed in human induced pluripotent stem cell (hiPSC)-derived CMs. Collectively, these findings suggest that Tip60 modulates CM biomechanical and metabolic pathways, supporting its potential as a therapeutic target for improving functional recovery following MI.

Introduction: Our lab has previously demonstrated that mice treated with recombinant Interleukin-13 (IL13) post myocardial infarction (MI) demonstrate improved cardiac function through IL13 directly signaling to macrophages. While treatment with IL13 improves heart function, it has no effect on cardiac fibrosis post MI. Macrophages respond to IL13 through a receptor comprised of IL-4Rα and IL13Ra1. Paradoxically, when IL-4Rα is knocked out from macrophages, treatment with IL13 post MI no longer improves cardiac function but instead increases scar area of the heart by almost 50% compared to vehicle control. This suggests that IL13 is also signaling to myofibroblasts to promote fibrosis and that macrophages are suppressing this activity. Hypothesis: We hypothesize that IL13 directly signals to cardiac myofibroblasts to aggravate scarring, as these cells express the IL13 receptor and largely contribute to fibrotic scarring in the post MI setting. Methods: To test this hypothesis, we are utilizing a Cre-lox system to knock out IL-4Rα from myofibroblasts and macrophages in mice and then subject mice to MI and IL13 administration. Scar size and heart function are assessed over a 28-day period following MI. We’ve compared this double knockout genotype to mice that only have IL-4Rα knocked out of myofibroblasts, mice that only have IL-4Rα knocked out of macrophages, and wildtype control mice. Results: Our data shows that when IL-4Rα is knocked out of both myofibroblasts and macrophages, treatment with IL13 results in less fibrosis compared to the macrophage IL-4Rα knockout group. Conclusion: Our data suggests that, knocking out the IL-4Rα receptor from both myofibroblasts and macrophages indeed rescues the scar phenotype. Findings from this study will lead to novel mechanisms by which macrophages interact with cardiac cell types to influence post MI healing.

Introduction: A recent study identified a rare variant in the MRC2 gene in individuals with familial reentrant supraventricular tachycardia, a Wolff-Parkinson-White (WPW) ECG pattern, and structurally normal hearts. WPW syndrome is associated with atrial fibrillation (AF), and MRC2 was recently proposed as a protective gene for AF. Hypothesis/Aim: We aimed to assess the hypothesis that the E990G-heterozygous (het) loss-of-function variant in MRC2 increases AF susceptibility and aberrant atrial cardiofibroblast (ACF) function in mice. Methods: Programmed electrical stimulation (PES) was performed to determine AF susceptibility in E990G-het mice and wild-type (WT) controls. ACFs were isolated from these mice and cultured, and their collagen deposition were quantified. Finally, transcriptomic profiling by RNA sequencing and secretomic/proteomic profiling by mass spectrometry were performed on ACFs and whole atrial tissue. Results: E990G-het mice (n=15) exhibited increased susceptibility to pacing-induced AF (60% vs. 20%, P =0.030) compared with WT controls (n=15) and had decreased atrioventricular effective refractory periods (mean ± SEM: 44.6 ms ± 1.8 vs. 56.7 ± 1.8, P <0.001) . ACFs isolated from E990G-het mice (n=5) deposited greater amounts of acid-soluble collagen in 2D cultures (median ± IQR: 15.5 µg/3000 ACFs ± 1.3 vs. 10.7 ± 2.6, P =0.009) compared with WT controls (n=5). Transcriptomic (n=4 WT ACF lines + 5 het; 8 WT atrial tissue + 7 het), secretomic (n=4 WT ACF lines + 4 het), and proteomic (n=4 WT atrial tissue + 4 het) profiling of cultured ACFs and whole-atrial tissue revealed differential expression of several fibrotic regulators in E990G-het versus WT mice, including decreased ACF expression of matrix metalloproteinase 13 (MMP-13, P =0.012), which degrades collagen types I, II, and III; decreased ACF expression ( P =0.002) and secretion ( P =0.041) of matrix metalloproteinase 12 (MMP-12), which degrades collagen types I, III, IV, elastin, and fibronectin; and increased tissue levels of cellular communication network factor 2/connective tissue growth factor (CCN2/CTGF, P =0.004), a profibrotic regulator. Conclusions: MRC2 E990G-het mice exhibit increased AF susceptibility, altered ACF collagen deposition, and differentially regulated fibrotic genes and proteins. Together, these findings suggest that the E990G loss-of-function MRC2 variant leads to an AF-prone substrate characterized by excess collagen deposition and reduced MMP-mediated collagen removal.