Transaortic Constriction Research Articles

Abstract We109: AAV9-Mediated Overexpression of Mixed Lineage Kinase 3 After Transaortic Constriction Improves Left Ventricular Function

Introduction: Mixed-lineage kinase 3 (MLK3) is a cGMP-dependent protein kinase 1α (PKG1α) substrate, preserving left ventricular (LV) function without affecting blood pressure (BP). MLK3 kinase-dependent effects maintain LV function, while kinase-independent signalling regulates BP. Thus, enhancing MLK3 kinase activity may improve LV function in heart failure (HF) while circumventing PKG1-induced hypotension. No MLK3-activation strategies exist currently, thus hindering progress in MLK3 research and potential therapeutics. Hypothesis: Augmenting MLK3 after pressure overload by transaortic constriction (TAC) would prevent LV hypertrophy and dysfunction in mice. Goal: To test the effect of adeno-associated virus 9 (AAV9)-mediated MLK3 overexpression on LV function, when administered in the setting of pre-existing LV hypertrophy and dysfunction. Methods: Male mice 12 weeks of age underwent 26G TAC or sham surgery. At day 5 post-TAC we administered by retro-orbital injection AAV9 encoding MLK3 or TdTomato negative control, each under control of the CM-specific cTNT promoter (AAV-MLK3: n=12 TAC, 7 sham; AAV-TdTomato: n=10 Tac, 8 sham. At day 28 post-AAV9 injection, we measured cardiac structure and function by echocardiography, invasive LV hemodynamics, and organ weights. Results: Echocardiography 5 days post-TAC confirmed TAC-induced LV hypertrophy and reduced systolic function prior to AAV9 treatment. At day 28, AAV-MLK3-treated TAC mice demonstrated improved LV ejection fraction and fractional shortening compared with TAC-AAV-Td controls, indicating improved systolic function. MLK3-treated TAC mice maintained higher LV systolic pressure post-TAC vs. Td controls, further indicating improved cardiac performance. Diastolic indices, including ratio of mitral E/A inflow velocities, tau, and LV relaxation rate were also improved in AAV-MLK3 TAC versus AAV-Td controls. AAV9-MLK3 administration had no effect on LV wall thickness or on LV or whole heart mass normalized to tibia length. MLK3 expression was increased 3-fold in AAV9-MLK3 TAC LVs compared with AAV9-Td controls. Conclusion: AAV9-mediated MLK3 overexpression after the onset of LV hypertrophy and dysfunction improves LV function in mice.

Abstract Tu036: Adipose tissue plasticity in response to early pathological stress on the heart and mediation by adipose thermogenic activation

Background: Cardiovascular diseases (CVD) are the leading cause of death globally, accounting for approximately 17.9 million mortalities per year. Previous studies have shown that there is an early response of white and brown adipose tissue (WAT and BAT, respectively) to pathological stress on the heart prior to cardiac dysfunction. Interestingly, human subjects with detectable BAT activity (a thermogenic tissue) exhibit a reduced risk of Congestive Heart Failure, but the mechanism for this is unclear. We hypothesized that increasing the thermogenic acitivity of adipose tissue would be beneficial during the initial cardiac hypertrophic signaling prior to the development of compensatory hypertrophy. Methods: We studied both WAT and BAT in male and female C57BL/6J mice subjected to cardiac pressure overload via transaortic constriction (TAC). 24 hours post-surgery, mice were subjected to room temperature (RT) 22° C or mild cold 16°C to stimulate BAT activation for 48 hours after which tissues were collected. The study groups included: SHAM RT, TAC RT, SHAM COLD, TAC COLD. We used uncoupling protein 1 knockout (UCP1-KO) mice, with impaired thermogenic activity as a negative control. Results: At 72 hours post tac surgery, expression of thermogenic and metabolic genes remained unchanged in BAT and WAT of both sexes. However, mild cold exposure resulted in an increase in UCP1 expression in BAT of male, but not female mice while other metabolic and thermogenic gene markers’ expression was increased in a sex-specific manner. Adaptations to scWAT were more moderate than BAT, with an increase in Ucp1 after cold exposure in both sexes. Moreover, 48 hours of cold exposure attenuated expression of gene markers for hypertrophic signaling in the heart during TAC. Importantly, these adaptations were absent in UCP1-KO mice, signifying that intact thermogenic activity in adipose tissue was required for the attenuated expression of hypertrophic markers in the heart. Interestingly, cardiac fibrosis markers were downregulated with TAC-Cold in male mice only, suggesting a stronger response compared to female mice. Conclusions: Our findings demonstrate that pathological stress on the heart during TAC does not impact adipose tisse thermogenesis. Additionally, activating thermogenic gene expression of adipose tissue via cold exposure mediates the early induction of hypertrophic signaling in the heart within 72 hours of TAC. Notably, adipose tissue plasticity is sex-specific.

Abstract Tu094: p53 Regulates the Extent of Fibroblast Proliferation and Fibrosis in Left Ventricle Pressure Overload

Background: Cardiomyopathy is characterized by the pathological accumulation of resident cardiac fibroblasts that deposit ECM (extracellular matrix) and generate a fibrotic scar. However, the mechanisms that control the timing and extent of cardiac fibroblast proliferation and ECM production are not known, hampering the development of antifibrotic strategies to prevent heart failure. Methods: We used the Tcf21 (transcription factor 21) MerCreMer mouse line for fibroblast-specific lineage tracing and p53 (tumor protein p53) gene deletion. We characterized cardiac physiology and used single-cell RNA-sequencing and in vitro studies to investigate the p53-dependent mechanisms regulating cardiac fibroblast cell cycle and fibrosis in left ventricular pressure overload induced by transaortic constriction. Results: Cardiac fibroblast proliferation occurs primarily between days 7 and 14 following transaortic constriction in mice, correlating with alterations in p53-dependent gene expression. p53 deletion in fibroblasts led to a striking accumulation of Tcf21- lineage cardiac fibroblasts within the normal proliferative window and precipitated a robust fibrotic response to left ventricular pressure overload. However, excessive interstitial and perivascular fibrosis does not develop until after cardiac fibroblasts exit the cell cycle. Single-cell RNA sequencing revealed p53 null fibroblasts unexpectedly express lower levels of genes encoding important ECM proteins while they exhibit an inappropriately proliferative phenotype. in vitro studies establish a role for p53 in suppressing the proliferative fibroblast phenotype, which facilitates the expression and secretion of ECM proteins. Importantly, Cdkn2a (cyclin-dependent kinase inhibitor 2a) expression and the p16Ink4a-retinoblastoma cell cycle control pathway is induced in p53 null cardiac fibroblasts, which may eventually contribute to cell cycle exit and fulminant scar formation. Conclusions: This study reveals a mechanism regulating cardiac fibroblast accumulation and ECM secretion, orchestrated in part by p53-dependent cell cycle control that governs the timing and extent of fibrosis in left ventricular pressure overload.

Abstract Tu054: Deficiency or targeting inhibition of histone lysine demethylase KDM5B in vivo for potential therapeutic effects on both HFrEF and HFpEF

Heart failures with reduced ejection fraction (HFrEF) or preserved ejection fraction (HFpEF) have been recognized as the leading cause of morbidity and mortality worldwide. Histone lysine demethylase KDM5B, an important anti-tumor drug target, has been involved in several cardiovascular diseases. However, its role in heart failure remains largely unknown. In the current study, we found that the expression of KDM5B were significantly enhanced and KDM5B’s downstream substrate: the methylation level of H3K4me3 was decreased in the mouse hearts of both trans-aortic constriction (TAC)-induced HFrEF and high fat diet (HFD) with Nω-nitro-L-arginine methyl ester (L-NAME) (two-hit)-induced HFpEF. By generating a tamoxifen-induced cardiomyocyte- (CM-KO) and a myofibroblast- (MF-KO) specific KDM5B Cre-loxP conditional knockout mice, specifically, we further found that either KDM5B CM-KO or MF-KO mouse significantly ameliorated pathological ventricular remodeling, fibrosis and rescued the cardiac dysfunction phenotypes induced by TAC or the two-hit models, specifically. Furthermore, pretreatment of a KDM5B specific inhibitor TK-129, which was previously identified by our group, also significantly attenuated myocardial hypertrophy, fibrosis and improved cardiac dysfunction in the two mouse heart failure models. Our findings demonstrated, for the first time, that genetic deficiency of KDM5B in either cardiomyocytes or myofibroblasts in vivo or pharmacological inhibition of KDM5B display a similar therapeutic effect in the pathological cardiac remodeling and heart failure in both pressure overload-induced HFrEF and HFpEF mouse models, suggesting that KDM5B is a potential therapeutic target for these two different HFs.

Abstract Tu085: The β IV -spectrin/STAT3 Complex regulates the orientation of cardiac hypertrophic growth

Background: Cardiac hypertrophy is a major risk factor for adverse cardiovascular events including heart failure and arrhythmia. However, the precise orientation of cell and organ growth varies considerably depending on stress type and duration with important implications for cardiac function. Despite this, little is known regarding the mechanisms that regulate hypertrophic orientation. Here we evaluate the role of the cytoskeletal protein β IV -spectrin and signal transducer and activator of transcription (STAT3) in directing the orientation of pressure overload-induced hypertrophy. Methods: Transgenic mouse models with altered STAT3 signaling through modified interaction with its scaffolding partner β IV -spectrin, or phospho-regulation of STAT3 directly, were evaluated at baseline and transaortic constriction (TAC) for its role in promoting concentric versus eccentric morphologies and resulting impact on systolic function. Unbiased screening of gene expression from these structurally divergent states were evaluated for pathways responsible for directing myocyte length/width. These pathways were tested in vitro using primary mouse myocytes and in vivo to tune growth patterns for therapeutic intervention. Results: Loss of β IV -spectrin or direct STAT3 activation promoted a preferential increase in myocyte length over width, resulting in dilation of the left ventricular (LV) chamber (eccentric hypertrophy) and decreased systolic function. Conversely, preservation of β IV -spectrin favored an increase in myocyte width without LV dilation (concentric hypertrophy) and preserved systolic function in response to TAC. Differential expression of genes associated with microtubule dynamics were identified in concentric vs. eccentric hypertrophic states. In vitro assays revealed a relationship between β IV -spectrin/STAT3 signaling and microtubule stability which impacted spatial distribution of mRNA for the sarcomeric gene actc1 . Finally, intervention with pharmacologic STAT3 inhibition following chronic 6-week TAC successfully recovered concentric growth with improved systolic function. Conclusions: These data identify a novel and pivotal role for β IV -spectrin/STAT3 to modify microtubule dynamics and sarcomeric transcript distribution to direct myocyte geometry and therapeutically serve in the prevention or recovery from HF. These mechanisms further illustrate the unique separation of hypertrophic drivers from growth orientation as distinct pathways in cardiac remodeling.

Abstract Tu099: GDF11 accelerates NF-kB-mediated inflammation prior to reduction in cardiac fibrosis in response to experimental pressure overload.

Background: Cardiac fibrosis is a common pathophysiology in a broad variety of heart diseases. GDF11, a member of the TGF-beta superfamily, can reduce cardiac hypertrophy and fibrosis in the pressure overload model of experimental Trans Aortic Constriction (TAC). However, the molecular mechanisms by which GDF11 can reduce cardiac fibrosis are incompletely described. Hypothesis: GDF11 is known to activate both NF-kB as well as SMAD2/3 signaling which are essential in cardiac fibrosis and hypertrophy processes. We hypothesized that exogenous GDF11 can change the dynamics of early inflammatory NF-kB target genes during the response to pressure overload. Methods: We used mice implanted with AngII-osmotic pumps to induce left ventricular fibrosis. The mice were harvested at different time points: 3, 7, 14, and 28 days post-pump implantation (ppi) to follow the evolution of cardiac fibrosis and cardiac hypertrophy, and to identify the role of GDF11 in modulating these processes. Results: GDF11 delivery (1mg/kg every other day) in AngII mice (AngII+GDF11) induced earlier expression of NF-kB target genes (IL-1beta, IL-6, TNF-alpha – p>0.05) compared to AngII+Veh control group at 3d ppi, revealing accelerated activation of NF-kB response pathways in AngII+GDF11 group. This is associated with faster recruitment of the M1 macrophage population observed by an increased expression of CD86 or iNOS at 3d ppi. We also observed decreased markers of fibroblast activation after GDF11 supplementation, with less collagen deposition (F-CHP) and fibrosis (Masson Trichrome) at 28 days. Conclusions: Our data suggest that exogenous GDF11 changes the dynamics of NF-kB inflammatory mechanisms while reducing later fibrosis. This new finding could suggest that the dynamics of the inflammatory response early after pressure overload is a critical factor in cardiac fibrosis.

Abstract Tu083: Oxytocin improves cardiovascular outcomes in a rat model of pressure-overload heart failure

Oxytocin (OT) is a peptide made in the hypothalamus and released into circulation by the pituitary gland. Known for its role in childbirth and lactation, recent research suggests that OT has significant cardiovascular benefits, reducing the risk of cardiovascular diseases such as hypertension and atherosclerosis. We tested if OT has beneficial effects on cardiovascular function in a rat model of pressure overload heart failure (HF). Neonatal Sprague Dawley rats underwent transaortic constriction (TAC). Pressure overload and cardiac dysfunction progressed as animals matured. Echocardiography at 5 weeks of age confirmed reduced ejection fraction. Animals were then randomly assigned to receive daily IP injections of either OT (treatment) or saline (control) for two weeks. Echocardiography was performed again before hearts were excised for optical mapping studies to test for electrophysiological differences between groups. Hearts were electromechanically uncoupled using blebbistatin and transmembrane potential (RH237) and intracellular Ca2+ (Rhod2am) were simultaneously mapped during a dynamic restitution protocol. Preliminary results indicate that rats in the treatment (OT, n=3) group had improved cardiac function compared to controls (saline, n=3), with higher ejection fraction (66.71 ± 9.44 vs. 25.83 ± 17.93, p=0.0125), higher cardiac output (62.4 ± 11.45 vs. 20.35 ± 6.12, p=0.0025), and greater stroke volume (180.2 ± 18.19 vs. 73.96 ± 37.29, p=0.0057). Preliminary optical mapping results indicate that the hearts of OT-treated rats (n=3) have lower steady state action potential duration (APD) compared to controls (n=2). APD60 decreased from 109.5 ± 10.09 to 75.79 ± 21.94 (p=.0725) and APD80 decreased from 133.1 ± 10.22 to 101.1 ± 24.5 (p=.09595), indicating preservation of repolarizing currents corresponding to healthier electrophysiological function. In summary, our preliminary results suggest exogenous IP OT administration could have a potent cardioprotective effect on chronic and severe pressure overload hearts. Future work will investigate potential mechanisms of OT mediated cardio-protection, including sites of action.

Cell size induced bias of current density in hypertrophic cardiomyocytes

ABSTRACT Alterations in ion channel expression and function known as “electrical remodeling” contribute to the development of hypertrophy and to the emergence of arrhythmias and sudden cardiac death. However, comparing current density values – an electrophysiological parameter commonly utilized to assess ion channel function – between normal and hypertrophied cells may be flawed when current amplitude does not scale with cell size. Even more, common routines to study equally sized cells or to discard measurements when large currents do not allow proper voltage-clamp control may introduce a selection bias and thereby confound direct comparison. To test a possible dependence of current density on cell size and shape, we employed whole-cell patch-clamp recording of voltage-gated sodium and calcium currents in Langendorff-isolated ventricular cardiomyocytes and Purkinje myocytes, as well as in cardiomyocytes derived from trans-aortic constriction operated mice. Here, we describe a distinct inverse relationship between voltage-gated sodium and calcium current densities and cell capacitance both in normal and hypertrophied cells. This inverse relationship was well fit by an exponential function and may be due to physiological adaptations that do not scale proportionally with cell size or may be explained by a selection bias. Our study emphasizes the need to consider cell size bias when comparing current densities in cardiomyocytes of different sizes, particularly in hypertrophic cells. Conventional comparisons based solely on mean current density may be inadequate for groups with unequal cell size or non-proportional current amplitude and cell size scaling.

Cardiomyocyte Specific MLK3 Deletion Opposes LV Dysfunction and Cardiac Fetal Gene Re-Expression

Study Objective: To determine the cardiac myocyte (CM) and blood pressure-independent effects of Mixed lineage kinase 3 (MLK3) in the regulation of left ventricular (LV) function at baseline and in response to pathological stress. Background: MLK3 functions as a direct substrate effector of cGMP-dependent protein kinase 1α (PKG1α). Whole-body MLK3 deletion in mice increases cardiac dysfunction and remodeling after pressure overload and prevents the therapeutic effect of cGMP augmentation on LV function. However, MLK3 deletion also causes hypertension, which may confound these findings. Hypothesis: MLK3 opposes LV dysfunction through a blood pressure-independent function in the cardiac myocyte (CM). Methods: We generated mice harboring LoxP sites flanking the MAP3K11 gene encoding MLK3 (MLK3fl/fl) and crossed them with αMHC-Cre transgenic mice, enabling constitutive postnatal cardiac myocyte-specific MLK3 deletion (CMKO). Male and female 3- and 6-month-old MLK3 CMKO and control MLK3 intact (MLK3fl/fl Cre-) littermates were studied in the basal state. We also performed transaortic constriction (TAC) or sham surgery for 4 weeks on 3-month-old male mice. We assessed LV structure and function by organ mass, echocardiography, pressure volume loop analysis, qPCR, and immunoblot. In basal and TAC studies, we used age-matched αMHC-Cre x MLK3+/+ (non-floxed) mice as additional controls. Results: MLK3 gene excision in CMs was confirmed by PCR. By 6 months of age, MLK3 CMKO mice displayed: progressive reduction in LV ejection fraction; increase in LV internal diameter; and LV hypertrophy, indicating LV dysfunction and pathological remodeling, compared with littermate controls or with αMHC-Cre x MLK3+/+ non-floxed mice. This effect was more prominent in females than in males. LV fetal gene expression did not differ between genotypes at 3 months. At 6 months of age, however, the fetal genes nppa, RCAN, and CTGF were significantly upregulated in both male and female MLK3 CMKO mice, compared with MLK3 intact, indicating pathological remodeling. After 4-week TAC, MLK3 CMKO LVs developed more severe reduction in LV systolic function compared with MLK3 intact littermates as assayed by echocardiographic measures of LV ejection fraction; and invasive hemodynamic indices of preload recruitable stroke work, maximum LV pressure and LV developed pressure. The MLK3 CMKO LVs also demonstrated a more eccentric remodeling phenotype in which LV mass/tibia length, LV end diastolic and end systolic diameters increased more severely in CMKO mice, with corresponding reduced LV wall thicknesses. Finally, lung mass/tibia length was elevated in the MLK3 CMKO TAC mice compared to MLK3 intact TAC littermates, indicating overt heart failure. Conclusion: (1) MLK3 functions as a tonic inhibitor of LV dysfunction and pathological remodeling through a role in the CM, possibly through sex-specific mechanisms. (2) Intact MLK3 is required for normal LV compensation and concentric remodeling in response to pressure overload. These findings have important clinical implications, as drugs which activate PKG1 in heart failure improve outcomes but are limited by excess hypotension. As a myocardial PKG1 substrate which opposes pathological LV remodeling through blood pressure-independent mechanisms, MLK3 may represent a candidate therapeutic target for heart failure. NIH 1R01HL162919. 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.

Neutrophils orchestrate early cardiac remodeling after cardiac pressure overload

Cardiac pressure overload is a chronic condition where the left ventricle undergoes pathological hypertrophy and fibrosis ultimately causing heart failure. Increased myocardial stretch in left ventricular pressure overload initiates activation of an inflammatory and fibrotic response within resident cells leading to infiltration of immune cells. Neutrophils and monocytes are among the first responders during cardiac injury. While the role of monocytes and macrophages in pressure overload has been somewhat elucidated by multiple groups, including our lab, the role of neutrophils in chronic cardiac injury such as pressure overload is still unclear. We hypothesized that, like acute ischemic injury, neutrophils infiltrate to the heart early in pressure overload and initiate an inflammatory cascade which worsens cardiac dysfunction after TAC and that depletion of neutrophils in the first week of TAC would improve cardiac function. Our published single cell RNAseq dataset of immune cells (CD45+) cells from sham and TAC heart at day (D) 7 shows a distinct clustering of neutrophils with S100a9 (34-fold) , S100a8 (31-fold) , Retnlg (9-fold) , Il1b (5-fold) , and Slpi (5-fold) as top 5 differentially expressed genes (DEGs). Geneset enrichment analysis of top 58 DEGs (≥1.5-folds), shows significant association with pathways such as innate immune system, neutrophil degranulation, and signaling by interleukins by Reactome 2022 pathway. Using a murine model of transaortic constriction (TAC) to induce left ventricular pressure overload, we evaluated cardiac neutrophil counts by flow cytometry over the first week of TAC at D 2, 4 and 7. We observed that neutrophils (CD45+Ly6G+CD11b+) numbers consistently increase until D4, with a 7-fold increase at D2 to a 17-fold increase at D4 before returning to sham levels by D7. Using antibody (Anti Gr-1, 4 ug/g) mediated depletion of neutrophils, we observed that neutrophil depletion showed a protective effect in females compared to isotype controls with decreased cardiac hypertrophy and improved ejection fraction; while in male mice no change was observed between isotype and antibody treated groups. In conclusion, our data show that neutrophils peak early in pressure overload and orchestrate early cardiac remodeling with a potential for a sexually dimorphic role. We acknowledge funding from National Institute of Health under the awards numbers HL155993 and HL160665. 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.

Cardiomyocyte-specific deletion of adhesion G protein-coupled receptor F5 (ADGRF5) increases susceptibility to stress and arrhythmia

G-Protein Coupled Receptors (GPCR) represent the largest family of cell surface receptors in the human body and serve as targets for more than 30% of all approved drugs. Adhesion GPCRs (AGPCR) are an understudied GPCR family containing numerous extracellular domains that interact with extracellular proteins, including many known mediators of cardiac remodeling responses to pathologic stress. However, their relative expression in the heart and impact on cardiac function normally or during disease has been largely unstudied. To address this, we evaluated RNASeq-attained AGPCR expression from left ventricular (LV) samples of adult mice, of which Adgrf5 was one of the most highly expressed. Notably, decreased ADGRF5 expression is observed across numerous failing human LV tissue GEO datasets, and C57BL6/J mice subjected to transaortic constriction (TAC) surgery to induce pressure overload-induced HF also displayed reduced LV Adgrf5 expression by 12 weeks post-TAC. Analysis of published single cell (sc)-RNASeq datasets confirmed Adgrf5 expression in cardiomyocytes (CM), which also decreased following TAC in mice and in CM from failing human hearts. To investigate the impact of CM-specific ADGRF5 on cardiac function and remodeling normally or during disease progression, we crossed floxed ADGRF5 (ADGRF5f/f) and αMHC-Cre mice to generate constitutive, CM-specific ADGRF5 knockout mice (F5cmKO). These mice display normal cardiac structure and function at 12 weeks of age versus αMHC-Cre mice, as assessed via echocardiography, gravimetrics and immunohistochemistry. However, F5cmKO mice develop cardiac dysfunction, maladaptive remodeling, and increased mortality over time, even in the absence of pathologic insult, suggesting a homeostatic function of CM-specific ADGRF5. Following TAC surgery, F5cmKO mice display similar outcomes including enhanced cardiac dysfunction with accelerated remodeling and increased mortality. To attain mechanistic insight into these outcomes, we performed RNA-sequencing on LV tissue of 12-week-old F5cmKO mice, identifying and subsequently validating Scn1b as significantly upregulated in F5cmKO CM. Scn1b encodes for the β subunits of voltage-gated sodium channels (Scnβ1/β1B), which regulate gating and kinetics of Nav1.5 as well as adhesive CM-CM interactions, disruption of which could lead to arrhythmias. Indeed, conscious electrocardiogram (ECG) readings via telemetry indicated F5cmKO mice had a high burden of premature ventricular contractions (PVC), including ventricular tachycardia associated with cardiac enlargement. Using a combination of luciferase and fluorescence resonance energy transfer (FRET) reporters and gene expression analyses in neonatal rat ventricular myocytes (NRVM), we confirmed that ADGRF5 controls Scn1b expression in a Gαq/11 protein-dependent manner. In all, we have discovered that loss of CM-specific ADGRF5 leads to increased Scn1b expression with enhanced susceptibility to stress, arrythmias and sudden death, thus may provide a novel GPCR target to promote the maintenance of cardiac rhythm. AHA Predoctoral Fellowship 834906 to J.M.E. P01 HL147841 to D.T. 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.

Protein Kinase A Is a Master Regulator of Physiological and Pathological Cardiac Hypertrophy.

The sympathoadrenergic system and its major effector PKA (protein kinase A) are activated to maintain cardiac output coping with physiological or pathological stressors. If and how PKA plays a role in physiological cardiac hypertrophy (PhCH) and pathological CH (PaCH) are not clear. Transgenic mouse models expressing the PKA inhibition domain (PKAi) of PKA inhibition peptide alpha (PKIalpha)-green fluorescence protein (GFP) fusion protein (PKAi-GFP) in a cardiac-specific and inducible manner (cPKAi) were used to determine the roles of PKA in physiological CH during postnatal growth or induced by swimming, and in PaCH induced by transaortic constriction (TAC) or augmented Ca2+ influx. Kinase profiling was used to determine cPKAi specificity. Echocardiography was used to determine cardiac morphology and function. Western blotting and immunostaining were used to measure protein abundance and phosphorylation. Protein synthesis was assessed by puromycin incorporation and protein degradation by measuring protein ubiquitination and proteasome activity. Neonatal rat cardiomyocytes (NRCMs) infected with AdGFP (GFP adenovirus) or AdPKAi-GFP (PKAi-GFP adenovirus) were used to determine the effects and mechanisms of cPKAi on myocyte hypertrophy. rAAV9.PKAi-GFP was used to treat TAC mice. (1) cPKAi delayed postnatal cardiac growth and blunted exercise-induced PhCH; (2) PKA was activated in hearts after TAC due to activated sympathoadrenergic system, the loss of endogenous PKIα (PKA inhibition peptide α), and the stimulation by noncanonical PKA activators; (3) cPKAi ameliorated PaCH induced by TAC and increased Ca2+ influxes and blunted neonatal rat cardiomyocyte hypertrophy by isoproterenol and phenylephrine; (4) cPKAi prevented TAC-induced protein synthesis by inhibiting mTOR (mammalian target of rapamycin) signaling through reducing Akt (protein kinase B) activity, but enhancing inhibitory GSK-3α (glycogen synthase kinase-3α) and GSK-3β signals; (5) cPKAi reduced protein degradation by the ubiquitin-proteasome system via decreasing RPN6 phosphorylation; (6) cPKAi increased the expression of antihypertrophic atrial natriuretic peptide (ANP); (7) cPKAi ameliorated established PaCH and improved animal survival. Cardiomyocyte PKA is a master regulator of PhCH and PaCH through regulating protein synthesis and degradation. cPKAi can be a novel approach to treat PaCH.

Zonisamide attenuates pressure overload-induced myocardial hypertrophy in mice through proteasome inhibition.

Myocardial hypertrophy is a pathological thickening of the myocardium which ultimately results in heart failure. We previously reported that zonisamide, an antiepileptic drug, attenuated pressure overload-caused myocardial hypertrophy and diabetic cardiomyopathy in murine models. In addition, we have found that the inhibition of proteasome activates glycogen synthesis kinase 3 (GSK-3) thus alleviates myocardial hypertrophy, which is an important anti-hypertrophic strategy. In this study, we investigated whether zonisamide prevented pressure overload-caused myocardial hypertrophy through suppressing proteasome. Pressureoverload-caused myocardial hypertrophy was induced in mice by trans-aortic constriction (TAC) surgery. Two days after the surgery, the mice were administered zonisamide (10, 20, 40 mg·kg-1·d-1, i.g.) for four weeks. We showed that zonisamide administration significantly mitigated impaired cardiac function. Furthermore, zonisamide administration significantly inhibited proteasome activity as well as the expression levels of proteasome subunit beta types (PSMB) of the 20 S proteasome (PSMB1, PSMB2 and PSMB5) and proteasome-regulated particles (RPT) of the 19 S proteasome (RPT1, RPT4) in heart tissues of TAC mice. In primary neonatal rat cardiomyocytes (NRCMs), zonisamide (0.3 μM) prevented myocardial hypertrophy triggered by angiotensin II (Ang II), and significantly inhibited proteasome activity, proteasome subunits and proteasome-regulated particles. In Ang II-treated NRCMs, we found that 18α-glycyrrhetinic acid (18α-GA, 2 mg/ml), a proteasome inducer, eliminated the protective effects of zonisamide against myocardial hypertrophy and proteasome. Moreover, zonisamide treatment activated GSK-3 through inhibiting the phosphorylated AKT (protein kinase B, PKB) and phosphorylated liver kinase B1/AMP-activated protein kinase (LKB1/AMPKα), the upstream of GSK-3. Zonisamide treatment also inhibited GSK-3's downstreamsignaling proteins, including extracellular signal-regulated kinase (ERK) and GATA binding protein 4 (GATA4), both being the hypertrophic factors. Collectively, this study highlights the potential of zonisamide as a new therapeutic agent for myocardial hypertrophy, as it shows potent anti-hypertrophic potential through the suppression of proteasome.

HS135, a novel activin and GDF trap, is highly efficacious in preclinical models of pulmonary hypertension and obesity-associated heart failure with preserved ejection fraction

Abstract Introduction Activins and growth differentiation factors (GDFs) signal via the Activin Type II receptor (ActRII) and are genetically and clinically validated drivers of heart failure (HF), pulmonary hypertension (PH) and obesity. Despite initial clinical success in PH, current inhibitors of Activin mediated signalling (eg, sotatercept, an ActRIIA receptor ectodomain Fc-fusion protein) cannot be dosed to full biological activity. HS135 is a rationally designed ActRII-based fusion protein that acts as a ligand trap for pathological Activins and GDFs while sparing BMP-9 to treat PH and obesity-associated HF. We previously demonstrated that HS135 achieves best-in-class in vitro potency against Activin and GDF targets, which translated into full in vivo target engagement (not observed with ActRIIA-Fc). Here, the differentiated efficacy profile of HS135 was explored in preclinical models of PH and HF with preserved Ejection Fraction (HFpEF). Methods HS135 PH efficacy was assessed using the rat monocrotaline (MCT) and the mouse transaortic constriction (TAC) models, while the murine High Fat Diet (HFD) and L-NAME model was utilized to explore the potential of HS135 in obesity-associated HFpEF. In the MCT model, rats were injected twice weekly for four weeks with HS135 or ActRIIA-Fc starting the day following MCT induction. In the TAC model, mice were injected twice weekly for four weeks with HS135 or ActRIIA-Fc two weeks following surgical intervention to establish TAC. HFpEF was established by feeding mice HFD in combination with L-NAME supplied in drinking water ad libitum for 5 weeks before commencing twice weekly treatment with HS135 or empagliflozin for three weeks. In each case, tissue remodelling in the heart and lungs was assessed by IHC and RNA-seq. In addition, changes in body composition as well as markers of adiposity were evaluated. Results Right ventricles (RV) of MCT treated animals showed a distinct gene expression profile by RNAseq particularly related to pathways of inflammation and energy balance. This profile was only modestly improved by ActRIIA-Fc whereas RVs of HS135-treated animals were nearly indistinguishable by RNAseq from naïve mice. Similarly, in the lung, HS135 was more efficacious than ActRIIA-Fc at improving inflammation markers and rebalancing Activin and BMP signalling. In the TAC and HFD/L-NAME models, HS135 was efficacious at modulating the left ventricle phenotype. Across all models of PH and HF, HS135 led to profound increases in lean mass and a more metabolically favourable muscle gene expression profile. Furthermore, in the HFD / L-NAME model, HS135 was able to improve fat mass and markers of adiposity. Conclusion HS135 best-in-class target engagement profile uniquely improves PH, HF, and metabolism across in vivo models. Collectively, these data support the development of HS135 as a novel agent in cardiopulmonary and cardiometabolic disease, including PH and obesity-associated HF.

Abstract 15581: Increasing Myocardial Carnosine Levels Reduces Adverse Remodeling During Heart Failure

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.

Abstract 18443: Early Inflammation in βII-Spectrin Deficient Hearts Promote Cardiac Remodeling and Heart Failure

Background: Dysregulation of cardiac cytoskeletal proteins such as βII-spectrin has been recognized to promote accelerated heart failure (HF) and arrhythmias in human and mouse models. However, the mechanism by which βII-spectrin dysregulation underlies HF is poorly understood. Hypothesis: We hypothesized that cardiomyocyte loss of βII-spectrin leads to recruitment of inflammatory macrophages that augment cardiac remodeling by promoting arrhythmogenesis and increased susceptibility to HF. Methods: Cardiomyocyte-specific βII-spectrin knockout (βII-cKO) and control mice (βII-flox) were generated and harvested for RNA-sequencing (RNA-seq), qRT-PCR (to validate candidate inflammatory genes), immunohistochemistry, immunoblotting, and flow cytometry. siRNA was also used to knockdown βII-spectrin in cultured cells to validate that direct loss of βII-spectrin contributes to immune cell recruitment. Transaortic constriction (TAC) was used to induce pressure overload. Results: RNA-seq demonstrated an early significant increase of inflammatory macrophages and T cell markers following βII-spectrin loss. Consistent with RNA-seq and qRT-PCR results, immunohistochemistry demonstrated an increase in CD45 positive cells in juvenile βII-cKO mouse hearts compared to control. siRNA targeted depletion of βII-spectrin in cultured cardiomyocyte led to increased expression and secretion of TGFβ-1 protein, which can promote infiltrating of immune cells. Notably, accelerated HF at two weeks was observed in βII-cKO mice compared to controls after TAC. Conclusions: These data suggest that βII-spectrin loss may mediate inflammation via the TGFβ-1 pathway, which in part contributes to βII-spectrin-mediated accelerated HF. Furthermore, these are the first data implicating βII-spectrin in the TGFβ1-immune signaling pathway in cardiomyocytes.

Abstract 13255: Cardiac and Generalized SGLT2 Deficiency Does Not Protect Against Development of Heart Failure With Reduced Ejection Fraction in Mice

Sodium-glucose cotransporter 2 inhibitors (SGLT2i), originally designed for the treatment of diabetes, exert profound cardiovascular benefit and are now the standard-of-care in all forms of heart failure (HF). The mechanism of their benefit in HF is unknown. We hypothesized SGLT2i act via an on-target, extra-cardiac mechanism to promote ketosis and improve myocardial energetics. Generalized- (gKO) and cardiac-specific (csKO) SGLT2 knockout mice were generated by breeding SGLT2 floxed mice with EIIa or αMHC Cre recombinase lines, respectively. While csKO grew and developed normally, gKO mice were slightly smaller compared to WT littermates. There was no baseline cardiac phenotype in either group. The gKO mice exhibited profound glucosuria, decreased fasting serum glucose and augmented fasting ketosis compared to controls, and comparable in magnitude to the metabolic effects of 14 days of treatment with empagliflozin (Empa, 50mg/kg/day orally) in WT mice. Targeted metabolomic analysis of gKO and Empa-treated WT mice identified increased circulating and myocardial levels of the ketone body 3-hydroxybutyrate (1.9-2.3x, p<0.01), along with an increase in a subset of tricarboxylic acid cycle intermediates and C4-OH, a ketone oxidation by-product. Empa treatment in WT mice following trans-aortic constriction with apical myocardial infarction (TAC/MI) improved LV dysfunction and pathological remodeling at 6 weeks (left ventricular ejection fraction (EF) 36±6 vs 28±9, p<0.03; tibia length-normalized biventricular weight 9.6±0.4 vs 11.3±1.6, p<0.001). In contrast, csKO and gKO mice subjected to TAC/MI had no difference in EF, strain, ventricular hypertrophy or dilation compared to WT controls. In conclusion, generalized SGLT2 deficiency phenocopies the metabolic effects of SGLT2i treatment but neither gKO nor csKO afford cardiac protection in an established HF model. These results suggest an off-target mechanism for beneficial effects of SGLT2i in HF.

Abstract 16825: Brahma Related Gene-1 (BRG1) Protein Mediates Helpful Compensatory Response to Retard Development of Heart Failure

Background: Chromatin remodeling can modulate transcription by changing gene accessibility. Brahma Related Gene-1 (Brg1) protein, which is an adenosine triphosphate catalytic subunit that supports chromatin remodeling, has been implicated in development of hypertrophic cardiomyopathy. However, contribution of Brg1 to the development of heart failure (HF) has not been clearly elucidated. Hypothesis: We hypothesize that Brg1 mediates compensatory response to cardiac stress; therefore, loss of Brg1 function will accelerate development of HF. Methods: The myosin light chain-2 Cre/loxP system was used to produce cardiac myocyte specific deletion of Brg1(-/-) mouse model with floxed littermates as control. Results: At baseline, Brg1(-/-) hearts showed mildly reduced left ventricular ejection fraction (LVEF). Aging to 12 months caused LV dilation and further reduction of LVEF in Brg1(-/-). We used trans-aortic constriction (TAC) for pressure overload challenge and chose similarly severe TAC induced pressure gradient as indicated by similarly fast peak flow velocities across the constriction. At 2-weeks post-TAC, Brg1(-/-) hearts exhibited severely reduced LVEF, severely dilated LV, and decreased ability to thicken LV wall during systole. In contrast, control hearts did not dilate and maintained its LVEF with increased LV wall thickness. Thus, Brg1 deletion greatly diminished the heart’s ability to compensate for aging and pressure challenge, thereby accelerating development of heart failure with reduced ejection fraction (HFrEF). Separately, RNA sequence analyses of 12-months aged hearts showed Brg1 deletion caused upregulation of 810 genes including HF associated long noncoding RNA XIST and down regulation of 50 genes, while 3-months old hearts were not significantly different. Conclusion: Brg1 mediates compensatory responses that are helpful to the heart to prevent development of HF; therefore, it is a potential treatment mechanism for HF.

Sphingosylphosphorylcholine alleviates pressure overload-induced myocardial remodeling in mice via inhibiting CaM-JNK/p38 signaling pathway.

Apoptosis plays a critical role in the development of heart failure, and sphingosylphosphorylcholine (SPC) is a bioactive sphingolipid naturally occurring in blood plasma. Some studies have shown that SPC inhibits hypoxia-induced apoptosis in myofibroblasts, the crucial non-muscle cells in the heart. Calmodulin (CaM) is a known SPC receptor. In this study we investigated the role of CaM in cardiomyocyte apoptosis in heart failure and the associated signaling pathways. Pressure overload was induced in mice by trans-aortic constriction (TAC) surgery. TAC mice were administered SPC (10 μM·kg-1·d-1) for 4 weeks post-surgery. We showed that SPC administration significantly improved survival rate and cardiac hypertrophy, and inhibited cardiac fibrosis in TAC mice. In neonatal mouse cardiomyocytes, treatment with SPC (10 μM) significantly inhibited Ang II-induced cardiomyocyte hypertrophy, fibroblast-to-myofibroblast transition and cell apoptosis accompanied by reduced Bax andphosphorylation levels of CaM, JNK and p38, as well as upregulated Bcl-2, a cardiomyocyte-protective protein. Thapsigargin (TG) could enhance CaM functions by increasing Ca2+ levels in cytoplasm. TG (3 μM) annulled the protective effect of SPC against Ang II-induced cardiomyocyte apoptosis. Furthermore, we demonstrated that SPC-mediated inhibition of cardiomyocyte apoptosis involved the regulation of p38 and JNK phosphorylation, which was downstream of CaM. These results offer new evidence for SPC regulation of cardiomyocyte apoptosis, potentially providing a new therapeutic target for cardiac remodeling following stress overload.

CD9 exacerbates pathological cardiac hypertrophy through regulating GP130/STAT3 signaling pathway

CD9 is a member of the tetraspanin protein family, which has been widely studied in inflammation and cancer, but not in pathological cardiac hypertrophy. In this study, we found that the expression of CD9 was increased in transaortic constriction (TAC) myocardial tissue. Knockdown of CD9 alleviated damage to cardiac function in the TAC model and reduced heart weight, cardiomyocyte size, and degree of fibrosis, and vice versa. Mechanistically, co-immunoprecipitation results showed that CD9 and GP130 can bind to each other in cardiomyocytes, and knockdown of CD9 can reduce the protein level of GP130 and the phosphorylation of STAT3 invivo and invitro, and vice versa. GP130 knockdown reversed the aggravating effects of CD9 on pathological cardiac hypertrophy. Therefore, we conclude that CD9 exacerbates pathological cardiac hypertrophy by regulating the GP130/STAT3 signaling pathway and may serve as a therapeutic target for pathological cardiac hypertrophy.