American Journal of Physiology-Heart and Circulatory Physiology

AimLeft atrial (LA) strain is emerging as a valuable metric for evaluating cardiac function, particularly under pathological conditions such as pressure overload. This preclinical study investigates the predictive utility of LA strain on cardiac function in a murine model subjected to pressure overload, mimicking pathologies such as hypertension and aortic stenosis. MethodsHigh resolution ultrasound was performed in a cohort of mice (n=16) to evaluate left atrial and left ventricular function at baseline and 2- and 4-weeks after transverse aortic constriction (TAC). Acute adaptations in cardiac function were assessed in a subgroup of mice (n=10) with 3-days post TAC imaging. ResultsWe report an increase in LA max volume from 11.0 {+/-} 4.3{micro}L at baseline to 26.7 {+/-} 16.7{micro}L at 4 weeks (p=0.002) and a decrease in LA strain from 19.6 {+/-} 4.8% at baseline to 10.1 {+/-} 6.3% at 4 weeks (p=0.006). In the acute phase, LA strain dysfunction was present at 3-days (p<0.001) prior to alterations in LA volume (p=0.856) or left ventricular (LV) ejection fraction (p=0.120). LA strain correlated with key indicators of cardiac performance including left ventricular (LV) ejection fraction (r=0.563, p<0.001), longitudinal strain (r=-0.643, p<0.001) and strain rate (r=0.387, p=0.007). Furthermore, markers of atrial structure and function including LA max volume (AUC=0.858, p<0.001), ejection fraction (AUC=0.901 p<0.001), and strain (AUC=0.878, p<0.001) all predicted LV dysfunction. ConclusionLA strain and function assessments provide a reliable, non-invasive method for early detection and prediction of cardiac dysfunction in a model of pressure overload.

AimsGenome-wide association studies have identified numerous cardiac transcription factors in association with atrial fibrillation. Amongst these transcription factors, the paired-like homeodomain transcription factor 2 (PITX2) is the strongest genetic risk variant associated with atrial fibrillation. However, the downstream mechanisms of PITX2 are not completely understood. Here, we explore the role of PITX2 in oxidative metabolism and stress as a unifying mechanism of arrhythmogenesis. Methods and resultsTo identify PITX2 mechanisms, we performed transcriptomic analysis in Pitx2c-deficient neonatal rat atrial myocytes. We identify oxidative phosphorylation as the top dysregulated pathway and direct transcriptional targets lie in mitochondrial electron transport chain complexes I and IV. Using the Seahorse Extracellular Flux Analyzer, we identified a functional decrease in oxidative metabolism in Pitx2c-deficient cardiomyocytes. As electron transport chain complexes I and IV may generate reactive oxygen species (ROS) under mitochondrial dysfunction, we quantified mitochondrial specific ROS using MitoSOX and observed an increase in mitochondrial specific ROS in Pitx2c-deficient cardiomyocytes. We additionally assessed spontaneous cardiomyocyte calcium cycling using Fluo-8AM and observed an increased frequency of pro-arrhythmogenic mechanisms including early and delayed afterdepolarizations as inferred through calcium traces. Further, we identified sarcomere disassembly including a potential role of PITX2 in regulating Titin, where Pitx2c-deficient cardiomyocytes display Titin mis-localization within the sarcomeres. To assess whether ROS drives these phenotypes, we treated neonatal rat atrial myocytes with N-acetylcysteine, a potent ROS scavenger, and observed decreased early and delayed afterdepolarizations, as well as restoration of Titin localization. ConclusionPITX2C maintains atrial metabolism and redox balance; the loss of PITX2C results in reduced oxidative metabolism and an elevation in oxidative stress that ramifies cardiomyocyte dysfunction. Treatment with antioxidant restores AF-associated phenotypes including abnormal calcium cycling and sarcomere disassembly in Pitx2c-deficient atrial cardiomyocytes. TRANSLATIONAL PERSPECTIVEGenetic variants close to the PITX2 gene associate most strongly with atrial fibrillation. This study reveals a mechanistic link between multiple AF-associated phenotypes and mitochondrial dysfunction with subsequent accumulation of reactive oxygen species downstream of PITX2. Importantly, metabolic therapies and reducing oxidative stress may present a potential clinical strategy to reverse and prevent functional and structural remodelling related to AF.

BACKGROUNDPhosphodiesterase type 1 (PDE1) inhibition exerts inodilatory effects in pre-clinical models and human heart failure patients with reduced ejection fraction (HFrEF). PDE1 hydrolyzes cyclic nucleotide cAMP in a soluble but not microsomal fraction of the human myocardium. PDE1 may exert domain-specific effects, but the mechanism whereby PDE1 compartmentalization induces an inotropic change remains unknown. We sought to elucidate PDE1 regulation of cAMP and contractility. METHODSPharmacologic modulators of PDEs or G-protein coupled receptors (GPCRs) were used to study PDE1 signaling mechanisms in healthy and failing guinea pig hearts. Tissue fractionation examined the localization of different PDE types. Live cell imaging experiments assessed cytosolic and sarcolemmal membrane cAMP level ([cAMP]) and protein kinase A (PKA) activity changes in cells transduced with Forster resonance energy transfer (FRET) biosensors. Sarcomere length and intracellular calcium changes monitored contractile changes in electrically paced cells. Coronary flow and left ventricular (LV) developed pressure were measured in ex vivo Langendorff perfusion heart studies. RESULTSPDE1 isoforms were found not only in the soluble, but also in the microsomal fractions of the guinea pig heart. PDE1 hydrolysis of cAMP was greater at the sarcolemma compared to the cytosol. PDE1 specifically regulated a pool of cAMP associated with the Gs protein coupled receptor adenosine 2A receptor (A2AR) at the sarcolemma, without activating PKA. A2AR/PDE1 regulation induced positive inotropic and lusitropic changes in healthy and failing guinea pig cardiomyocytes and in the myocardium ex vivo. CONCLUSIONSPDE1 is the major regulator of cAMP pools generated by A2AR activation at the sarcolemma. Functionally, this regulation induces inotropic and lusitropic effects in cardiomyocytes and at the whole organ level. Thus, PDE1 is compartmentalized at the membrane with A2AR, and this regulation determines cardiomyocyte contractility in healthy and failing hearts.

BackgroundClinical evidence supports a greater impact of arterial stiffening in cardiovascular mortality in women versus men. Arterial stiffness increases across the menopausal transition, implicating a role of the loss of estrogens in arterial stiffening, but mediating mechanisms remain unclear. MethodsThe role of estradiol and smooth muscle cell (SMC) estrogen receptor alpha (ER) in arterial stiffening, by aortic pulse wave velocity (PWV), was assessed in 3 models: (1) the loss of estradiol in young, female mice comparing sham surgery or bilateral ovariectomy (OVEX) {+/-} estradiol, (2) the impact of sham versus OVEX surgery in young, female SMC-ER-intact and SMC-ER-knockout (KO) littermates, and (3) arterial stiffening during natural aging by comparing young and aged, female and male SMC-ER-intact and SMC-ER-KO littermates. Mechanistic pathways were assessed using histological assessment of aortic fibrosis and elastin degradation, aortic MMP expression, and atomic force microscopy. ResultsOVEX increased PWV and aortic medial fibrosis, with no impact on elastin integrity, in young female mice. Arterial stiffening and fibrosis were prevented in OVEX mice that were supplemented with estradiol. OVEX-induced arterial stiffening in SMC-ER-intact female mice was prevented in SMC-ER-KO littermates. In this model, OVEX was also associated with increased aortic medial fibrosis without changes in elastin integrity. Aging from 3 to 18 months significantly increased PWV in female and male SMC-ER-intact mice. Aging-induced stiffening was fully prevented in female and partially prevented in male SMC-ER-KO mice. SMC-ER contributes to aging-associated arterial stiffening by sex-specific mechanisms, including elastin degradation in females and phenotypic changes in SMC stiffness and probability to form cellular adhesions in males. Circulating estradiol was significantly decreased in serum from aged compared with young female mice. ConclusionsThese findings support that SMC-ER contributes to arterial stiffening in female and male mice in situations where the vasculature is exposed to low levels of estradiol.

BackgroundHypertrophic cardiomyopathy (HCM) is characterized by left ventricular hypertrophy and diastolic dysfunction and is accompanied by extensive cytoskeletal remodeling, including increased protein levels of desmin, tubulin and detyrosinated tubulin. We have previously demonstrated that tubulin detyrosination contributes to diastolic dysfunction. Microtubules are connected to the nucleus by the LINC complex, yet the role of cytoskeleton-nucleus interactions in HCM remain poorly understood. ObjectivesWe investigated whether cytoskeletal remodeling in HCM alters nuclear morphology and mechanics, and modulates hypertrophy-associated signaling pathways. MethodsNuclear morphology was assessed by electron microscopy and immunofluorescence in cardiac septal tissue from obstructive HCM patients (N=19) and non-failing donors (NF, N=8), as well as in Wild-Type (WT, N=18) and homozygous HCM-associated Mybpc3 c.2373InsG mice (Mybpc3c.2373InsG, N=19). A novel live-cell imaging approach was used to study nuclear deformation during cardiomyocyte contraction. YAP1 nuclear translocation was measured to evaluate downstream mechanosensitive signaling and detyrosination inhibitor epoY was used to test causality. ResultsNuclei were highly invaginated and enlarged in HCM in both patients and mice. Nuclear deformation during contraction was restricted in HCM cardiomyocytes, indicating altered mechanotransduction. These changes were associated with increased YAP1 nuclear localization and induction of YAP1 target and hypertrophic genes. Inhibition of microtubule detyrosination reduced nuclear invaginations, restored nuclear deformation and decreased YAP1 nuclear translocation. ConclusionCytoskeletal remodeling in HCM is associated with altered nuclear morphology and mechanotransduction, accompanied by YAP1 translocation which may contribute to hypertrophic remodeling. Targeting microtubule detyrosination rescues this phenotype, identifying nuclear mechanotransduction as a potential therapeutic target for HCM. Unstructured abstractHypertrophic cardiomyopathy (HCM) is characterized by left ventricular hypertrophy and diastolic dysfunction and is accompanied by extensive cytoskeletal remodeling, including increased desmin, tubulin, and detyrosinated tubulin levels, which we previously showed contribute to impaired relaxation. Since microtubules connect to the nucleus via the LINC complex, we investigated whether cytoskeletal remodeling alters nuclear morphology, mechanics, and hypertrophy-associated signaling. Using electron microscopy and immunofluorescence in cardiac septal tissue from obstructive HCM patients and non-failing donors, and immunofluorescence and live-cell imaging in septal tissue from Wild-Type and homozygous HCM-associated Mybpc3 c.2373InsG mice, we found that nuclei were enlarged and highly invaginated, with restricted nuclear deformation during contraction, indicating altered mechanotransduction. These changes were associated with increased YAP1 nuclear localization and increased expression of YAP1 target and hypertrophic genes. Inhibiting microtubule detyrosination reduced nuclear abnormalities, restored nuclear deformation, and decreased YAP1 nuclear translocation, identifying nuclear mechanotransduction as a potential therapeutic target in HCM. Visual abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=122 SRC="FIGDIR/small/646061v2_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@1e44e94org.highwire.dtl.DTLVardef@1e3140aorg.highwire.dtl.DTLVardef@138b40forg.highwire.dtl.DTLVardef@1b550b2_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LICytoskeletal remodeling contributes to nuclear abnormalities and impaired mechanotransduction in hypertrophic cardiomyopathy. C_LIO_LIMicrotubule detyrosination promotes nuclear invaginations and increased YAP1 signaling in hypertrophic cardiomyopathy. C_LIO_LIPharmacological reduction of detyrosinated tubulin normalizes nuclear structure and mechanosignaling. C_LIO_LITargeting nuclear-cytoskeletal coupling may provide new therapeutic strategies for hypertrophic cardiomyopathy. C_LI

BackgroundDisease modifying therapies for heart diseases, including hypertrophic cardiomyopathy (HCM) are a prevailing unmet need. Human-based drug testing platforms capable of distinguishing direct myocardial and systemic effects are needed to enable targeted cardiac therapeutics. Sodium glucose cotransporter inhibitors (SGLTi) may have direct effects on cardiac contractility but have not been adequately studied in human tissues in the context of HCM. MethodsHuman myocardial tissues were procured from non-failing donor hearts or patients with HCM undergoing septal myectomy. Human living myocardial slices (LMS) were freshly prepared and mechanically tested to generate biomimetic work loops across a range of physiologic preloads and afterloads, before and after loading with drug (isoproterenol, mavacamten, sotagliflozin, or empagliflozin) or vehicle (DMSO). Mixed effects linear regression modeling accounting for clinical characteristics of patient tissue samples were generated across prespecified preloads and afterloads for baseline work loops and following drug exposure, permitting correlation between myocardial work and clinical characteristics, underlying disease, fuel source, and drug treatment. By repeating work loop analysis following drug administration, each slice acted as its own control. ResultsA total of 120 hLMS from 32 patients (16 non-failing and 16 HCM) were analyzed. Younger age, presence of hypertension, and ejection fraction was positively associated with hLMS work. Ketone supplementation augmented work and work-strain slope particularly in HCM hLMS at high afterloads. We validated our drug testing methodology by demonstrating known positive inotropy of isoproterenol, negative contractile effects of mavacamten particularly in the context of HCM, and null effect of DMSO vehicle. Finally, we tested sotagliflozin or empagliflozin and showed that SGLTis acutely and directly reduce myocardial work, with increased potency of sotagliflozin at high afterloads. ConclusionsOur findings validate a human tissue-based platform to test pharmacologic agents at predefined and physiologic preloads and afterloads, providing insight into the direct effects of SGLTi on human HCM myocardium. We demonstrate that SGLTi and ketones have distinct and discordant effects on human myocardial contractility.

RationaleNitric oxide (NO) has been identified as a signalling molecule generated during {beta}-adrenergic receptor (AR) stimulation in the heart. Furthermore, a role for NO in triggering spontaneous Ca2+ release via S-nitrosylation of Ca2+/calmodulin kinase II delta (CaMKII{delta}) is emerging. NO donors are routinely used clinically for their cardioprotective effects in the heart, but it is unknown how NO donors modulate the pro-arrhythmic CaMKII to alter cardiac arrhythmia incidence. ObjectiveWe test the role of S-nitrosylation of CaMKII{delta} at the Cys-273 inhibitory site and Cys-290 activating site in cardiac Ca2+ handling and arrhythmogenesis before and during {beta}-AR stimulation. Methods and ResultsWe measured Ca2+-handling in isolated cardiomyocytes from C57BL/6J wild-type (WT) mice and mice lacking CaMKII{delta} expression (CaMKII{delta}-KO) or with deletion of the S-nitrosylation site on CaMKII{delta} at Cys-273 or Cys-290 (CaMKII{delta}-C273S and -C290A knock-in mice). Cardiomyocytes were exposed to NO donors, S-nitrosoglutathione (GSNO; 150 M), sodium nitroprusside (SNP; 200 M) and/or {beta}-adrenergic agonist isoproterenol (ISO; 100 nM). WT and CaMKII{delta}-KO cardiomyocytes treated with GSNO showed no change in Ca2+ transient or spark properties under baseline conditions (0.5 Hz stimulation frequency). Both WT and CaMKII{delta}-KO cardiomyocytes responded to ISO with a full inotropic and lusitropic Ca2+ transient response as well as increased Ca2+ spark frequency. However, the increase in Ca2+ spark frequency was significantly attenuated in CaMKII{delta}-KO cardiomyocytes. The protection from ISO-induced Ca2+ sparks and waves was mimicked by GSNO pre-treatment in WT cardiomyocytes, but lost in CaMKII{delta}-C273S cardiomyocytes that displayed a robust increase in Ca2+ waves. This observation is consistent with CaMKII{delta}-C273 S-nitrosylation being critical in limiting ISO-induced arrhythmogenic sarcoplasmic reticulum Ca2+ leak. When GSNO was applied after ISO this protection was not observed in WT or CaMKII{delta}-C273S but was apparent in CaMKII{delta}-C290A. In Langendorff-perfused isolated hearts, GSNO pre-treatment limited ISO-induced arrhythmias in WT but not CaMKII{delta}-C273S hearts, while GSNO exposure after ISO sustained or exacerbated arrhythmic events. ConclusionsWe conclude that prior S-nitrosylation of CaMKII{delta} at Cys-273 can limit subsequent {beta}-AR induced arrhythmias, but that S-nitrosylation at Cys-290 might worsen or sustain {beta}-AR-induced arrhythmias. This has important implications for the administration of NO donors in the clinical setting.

Catecholamine dysregulation is a common feature of multiple acute and chronic cardiac conditions, including heart failure. To investigate the role of altered -adrenergic stimulation on cardiac function, we developed a short-term exposure model, administering phenylephrine subcutaneously to mice for one week. Compared to vehicle-injected controls, phenylephrine-treated animals exhibited increased ejection fraction, decreased chamber size, diastolic dysfunction and ventricular hypertrophy in the absence of hypertension. Remarkably, these animals developed extensive fibrotic remodeling of the tissue that plateaued at 24 hours and myocyte hypertrophy localized to regions of fibrotic deposition after 3 days of treatment. Transcriptome analyses of purified myocyte and fibroblast populations from these hearts revealed an unexpected role for myocytes in the production of extracellular matrix. Comparison with other models of cardiac stress, including pressure overload hypertrophy and cytokine activation of fibroblasts, identified stimulus-specific transcriptional circuits associated with cardiac pathology. Given the rapid, robust fibrotic response that preceded myocyte hypertrophy, intercellular communication analyses were conducted to investigate fibroblast to myocyte signaling, identifying potential crosstalk between these cells. These studies thoroughly describe and phenotypically characterize a new model of short-term catecholamine stress and provide an atlas of transcriptional remodeling in myocytes and fibroblasts. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=187 SRC="FIGDIR/small/617933v1_ufig1.gif" ALT="Figure 1"> View larger version (59K): org.highwire.dtl.DTLVardef@1e504faorg.highwire.dtl.DTLVardef@a9d5c1org.highwire.dtl.DTLVardef@10809aorg.highwire.dtl.DTLVardef@16b288d_HPS_FORMAT_FIGEXP M_FIG C_FIG

BackgroundCardiovascular disease (CVD) is the leading cause of global mortality, with recent increases attributed to demographic shifts in age and rising rates of obesity. Diminished contractile reserve is a hallmark of a diseased heart; assessing contractile reserve is pivotal in prognosticating and monitoring CVD progression. The Frank-Starling mechanism and sympathetic stimulation are key to enhance contractile reserve but have not been explored in vivo in old and obese mouse models of CVD. This project aims to use speckle tracking echocardiography (STE) to characterize the function of the heart at baseline, with increased preload, and with sympathetic stimulation. We hypothesize that along with blunted systolic function, diastolic function, and contractility, old and obese mice will have a blunted contractile reserve. MethodsSTE was obtained for control (4- month-old), aged (24-month-old), and obese mice (high fat diet-induced). Mice received an intravenous injection of 150L saline to increase preload to assess the Frank-Starling response, followed by injection of {beta}1adrenergic receptor agonist dobutamine to assess sympathetic response. ResultsAt baseline, aging and obese mice demonstrated blunted systolic, diastolic function, and contractility. Endocardial and epicardial wall displacement differed between aging and obese mice with contractile reserve, indicating that they have functionally distinct cardiac phenotypes. ConclusionsThis study is the first to demonstrate blunted systolic function, diastolic function, and contractility through STE in aging and obese mice. Our novel method for investigating the contractile reserve of mice demonstrated aging and obese mice have dissimilar responses when assessing contractile reserve, which could contribute to their distinct functional phenotypes.

PurposeExercise requires a rapid cardiac response to maintain cardiovascular function. CapZ is a critical stress-response protein in cardiac myocytes. While its role in the pathological stress response has been explored, its part in the physiological response to exercise is unknown. This study examined CapZ regulation during acute exercise and sought to determine its importance in the cardiac response to exercise. MethodsWildtype or cardiac CapZ-deficient transgenic mice were subjected to 20 min of swimming, or exhaustive exercise protocols. Time to exhaustion was a measurement of exercise capacity. Following submaximal exercise, cardiac myofilaments were isolated and probed for CapZ and key regulatory proteins. Myofilament function was assessed using an actomyosin MgATPase assay and total protein phosphorylation quantified with ProQ Diamond staining. Myofilament regulatory proteins following submaximal exercise were quantified by immunoblotting. ResultsTotal myofilament CapZ was unaffected by exercise but increased total CapZIP and decreased phosphorylated CapZIP indicated weakened CapZ-actin interaction. CapZ-deficient transgenic myofilaments lacked changes in CapZIP but BAG3 increased 10%. Time to exhaustion was lower in CapZ-deficient mice in both swimming and running protocols. Actomyosin MgATPase activity was maintained in wildtype mice and impaired with CapZ deficiency. Exercise increased the phosphorylation of several myofilament proteins in wildtype mice but not transgenic animals. Exercise-dependent Increases in myofilament PKC- and -{varepsilon} were mitigated in CapZ-deficient mice. Tcap levels decreased 39 {+/-} 8% in CapZ-deficient myofilaments with exercise and leiomodin 2 increased 78 {+/-} 28% in wildtype myofilaments. ConclusionsCardiac CapZ is a critical player in the physiological response to exercise. CapZ-actin binding is rapidly altered with exercise. Decreased cardiac CapZ limits exercise capacity, impairs myofilament regulation, and leads to a less stable contractile apparatus.

AimsWe recently found that the nuclear receptor retinoic acid-related orphan nuclear receptor alpha (ROR) protects against angiotensin II-induced cardiac hypertrophy and promotes cardiomyocyte mitophagy. The underlying molecular basis for these salutary effects remains unclear. MethodsWe used RNA microarrays to profile the cardiac transcriptomes of "staggerer" (RORsg/sg) mice that carry a naturally occurring mutation in the ligand-binding domain of ROR, resulting in a global loss-of-function genetic model. We then used genetic and pharmacologic loss-and-gain of function studies in cultured cardiomyocytes to ascertain whether ROR regulates transcription of Adra1a, the gene that encodes the alpha-1A-adrenergic receptor (1A-AR). ResultsThe absence of functional ROR results in broad transcriptional changes in the heart providing a likely molecular basis for the RORsg/sg cardiac phenotype. In vivo and in vitro studies confirmed that ROR directly regulates Adra1a transcription. This effect is enhanced by hypoxia. ConclusionsCollectively these findings position ROR as a previously unrecognized central regulator of the cardiac myogenic transcriptome and the first recognized transcriptional regulator of Adra1a in cardiomyocytes. Future studies will probe the contribution of ROR-mediated transcriptional regulation of Adra1a to both the response to cardiomyocyte injury and maintenance of circadian biology.

BackgroundEndothelial dysfunction, hypertension, and multi-organ injury remain central drivers of cardiovascular disease. Avocado (Persea americana) is rich in monounsaturated fatty acids, phytosterols, and antioxidants, yet its integrative impact on hypertension-induced systemic injury has not been fully explored. MethodsMale Wistar rats (n = 4/group) were randomized into six groups: control, avocado, L-NAME, L-NAME+losartan+metaprolol succinate, L-NAME+avocado, and L-NAME+metaprolol succinate+avocado. Avocado pulp was incorporated into diet at 80% w/w. Endpoints included blood pressure indices, hematological parameters, liver enzymes, renal function tests, and correlation analyses of systolic-diastolic coupling. One-way ANOVA with Tukeys post hoc tests evaluated group differences, while forest plots and scatter analyses visualized treatment effects. ResultsL-NAME significantly elevated systolic blood pressure ({Delta}+18 mmHg), diastolic pressure ({Delta}+20 mmHg), and mean arterial pressure ({Delta}+17 mmHg; all p < 0.01) compared with controls. Avocado supplementation reduced these elevations by ~15-18 mmHg, restoring values near baseline. L-NAME increased platelet counts (p = 0.031) and trended toward leukocytosis, both of which were attenuated by avocado. ALT levels were higher in L-NAME rats (p = 0.044), while AST and ALP trended upward; avocado-fed groups maintained near-control enzyme levels. Renal markers were most affected: urea (+25 mg/dl) and creatinine (+0.8 mg/dl) rose significantly in L-NAME rats (p < 0.01), but were reduced by 20-30% with avocado supplementation. Electrolytes remained unchanged. Correlation analyses revealed pathological SBP-DBP coupling in L-NAME rats (r = 0.78), abolished by avocado (r = 0.00). ConclusionAvocado supplementation mitigates L-NAME-induced hypertension and systemic injury by stabilizing blood pressure, reducing thrombocytosis, and preserving hepatic and renal function. These findings support avocado as a pleiotropic nutraceutical adjunct for cardiometabolic protection. HighlightsO_LIAvocado pulp supplementation incorporated into chow (80% w/w) mitigates L-NAME-induced hypertension in rats. C_LIO_LIDietary avocado preserves hepatic and renal biochemical function while reducing thrombocytosis under cardiovascular stress. C_LIO_LIAvocado demonstrates pleiotropic nutraceutical potential, stabilizing systemic physiology beyond conventional pharmacological therapy. C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=106 SRC="FIGDIR/small/681766v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@101b300org.highwire.dtl.DTLVardef@8f0624org.highwire.dtl.DTLVardef@1dc4ab3org.highwire.dtl.DTLVardef@1ac4130_HPS_FORMAT_FIGEXP M_FIG C_FIG

Elevated oxidized low-density lipoprotein (oxLDL) is a risk factor and component that worsens cardiovascular disease states. OxLDL can elicit its detrimental action, via lectin-like oxLDL receptor 1 (LOX-1) and has been shown to disrupt vascular function. In this study, we determined whether oxLDL, via LOX-1, alters aortic vascular reactivity and determined if sex differences exist. Thoracic aortic endothelium-intact or -denuded ring segments were isolated from intact C57BL/6J female and male mice and incubated with oxLDL ex vivo (50ug/dL; 2h). Using wire myography, cumulative concentration-response curves to phenylephrine (PE) were generated to determine contractile responses. From these curves, the EC50 was determined and used to contract rings to assess acetylcholine (ACh) dependent relaxation. Calculated aortic stiffness and remodeling, as well as mRNA expression of vasoactive and pro-inflammatory mediators were assessed. BI-0115 (10M; selective LOX-1 inhibitor) was used to determine LOX-1 dependence. We observed differential sex, age, endothelial cell, and LOX-1 dependent alterations to the efficacy of PE-induced contractile responses and ACh-mediated vasorelaxation in the thoracic aortic rings following oxLDL exposure. Additionally, we observed a distinct sex and age effect on thoracic aortic stiffness following exposure to oxLDL. There was also a sex effect on calculated vessel diameter, as well as an age effect on oxLDL-mediated inward remodeling that was LOX-1 dependent. Thus, LOX-1 inhibition and the resulting attenuation of oxLDL/endothelial-mediated alterations in aortic function suggests that there are differential sex differences in the role of oxLDL/LOX-1 in the thoracic aorta of male and female mice. NEW & NOTEWORTHYWe investigated the effects of oxidized low-density lipoprotein (oxLDL) via the LOX-1 receptor on murine thoracic aortic vasoreactivity, stiffness, and remodeling across age and sex. Acute exposure to oxLDL led to altered vasoreactivity, endothelial dysfunction, and changes in aortic stiffness and remodeling. These effects were in-part age, sex, endothelial, and LOX-1 dependent. This study reveals potential complex interactions in oxLDL/LOX-1-mediated vascular responses that could serve as potential therapeutic intervention for vascular diseases such as atherosclerosis. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=134 SRC="FIGDIR/small/556764v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@1bffb67org.highwire.dtl.DTLVardef@1122c31org.highwire.dtl.DTLVardef@1352d9org.highwire.dtl.DTLVardef@a44880_HPS_FORMAT_FIGEXP M_FIG C_FIG

The anti-cancer MEK inhibitor trametinib (alone or with the RAF inhibitor dabrafenib) causes cardiac dysfunction in some patients. Our hypothesis is that cardiotoxicity is exacerbated by underlying co-morbidities such as hypertension, and this causes cardiac dysfunction detectable on echocardiograms. The objective was to determine how trametinib/dabrafenib affect cardiac function in a mouse model of hypertension-induced cardiac hypertrophy. Male mice were treated with vehicle, trametinib or dabrafenib/trametinib without/with angiotensin II (AngII) to increase blood pressure. Echocardiography was used to assess changes in cardiac function and dimensions, applying statistical analysis combined with machine-learning to dissect the effects on different segments of the left ventricle (LV). The inhibitors alone had a limited effect on mouse hearts. Trametinib or trametinib/dabrafenib inhibited cardiac hypertrophy induced by AngII over 7 d, reducing LV wall thickness and mass. AngII did not significantly affect cardiac function, but the inhibitors caused significant functional deterioration. Segmental analysis revealed variation of contraction around the LV, with selective effects of AngII and trametinib or dabrafenib/trametinib in basal/mid-regional segments. Frame-by-frame analysis of radial (not longitudinal) displacement of the LV endocardial wall demonstrated variation between consecutive cardiac cycles that enabled a high degree of classification according to treatment. In conclusion, trametinib inhibits AngII-induced cardiac hypertrophy in mice but is detrimental to cardiac function, effects that are not moderated by dabrafenib. AngII and MEK/RAF inhibition have regional effects around the LV with greater effects on radial displacement in basal/mid-regional segments. Assessment of such changes may facilitate early identification of developing cardiotoxicity. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=166 SRC="FIGDIR/small/654397v2_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@1da15b5org.highwire.dtl.DTLVardef@c72634org.highwire.dtl.DTLVardef@9d3c04org.highwire.dtl.DTLVardef@cf51e7_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIAnti-cancer drugs trametinib and dabrafenib alone had little effect on mouse hearts C_LIO_LITrametinib/dabrafenib suppressed cardiac hypertrophy in mice with hypertension C_LIO_LITrametinib/dabrafenib were detrimental to cardiac function in hypertensive mice C_LIO_LISegments of the left ventricle were affected differently by hypertension/inhibitors C_LIO_LIResponses to hypertension/inhibitors varied between consecutive cardiac cycles C_LI

Colchicine, a microtubule depolymerizing agent, is an effective therapy for the secondary prevention of cardiovascular disease and has been approved recently as a novel treatment for atherosclerosis associated with coronary artery disease. Hypertension is a leading cause of cardiovascular disease, yet the impact of colchicine on hypertension has not been studied. We hypothesized that low-dose colchicine could be used to treat hypertension to reduce cardiovascular disease risk. The aim of this study was to administer daily, low dose (0.05 mg/kg/day) oral colchicine for 4 weeks to spontaneously hypertensive rats (SHR) and normotensive controls (WKY) and determine the effect on blood pressure, vascular reactivity, remodeling and inflammation, and left ventricular hypertrophy. Daily blood pressure measurements recorded by telemetry in conscious rats showed colchicine prevented increases in mean arterial pressure observed in the SHRs receiving vehicle over the 4-week treatment period. After the 4-weeks of treatment, 3rd order mesenteric artery vasorelaxations to isoproterenol, sodium nitroprusside and the Kv7.2-5 channel activator, ML213, were enhanced in the SHRs receiving colchicine compared to vehicle. The improved isoproterenol-mediated relaxation was also observed in WKY rats receiving colchicine, and in both the SHR and WKY, this improved effect was attenuated by the {beta}2 adrenoceptor antagonist, ICI118,551. Proteomic analysis of the mesenteric arteries by mass spectrometry revealed that colchicine treatment prevented changes observed when comparing the SHR vehicle group with the WKY vehicle group in proteins associated with extracellular matrix pathways. Immunostaining of 3rd order mesenteric arteries with Sirius red found that colchicine treatment attenuated the increased media thickness of the artery wall observed in SHRs receiving vehicle. Multiplex immunoassay and Western blots revealed colchicine reduced certain inflammatory mediators in the wall of the SHR mesenteric arteries, particularly the nucleotide-binding domain and leucine-rich repeat pyrin containing protein-3 (NLRP3), IL-18, CXCL10, and CXCL2, as well as reducing phosphorylated STAT3. Finally, in the left ventricle of the SHR, colchicine treatment attenuated a number of inflammatory mediators, including NLRP3, IL-1{beta}, and IL-18, and reduced fibrosis and cell size, which are indicative of left ventricular hypertrophy. Overall, we show colchicine has the potential to elicit cardiovascular protective effects in hypertension by targeting multiple cell types.

BackgroundAngiotensin II (Ang II) and milk fat globule-epidermal growth factor VIII (MFG-E8) are involved in age-associated arterial remodeling; however, the inflammatory role of MFG-E8 in Ang II associated arterial remodeling with aging remains unknown. Methods and ResultsIn this study, 30-week-old MFG-E8 knock out (KO) and age-matched wild-type (WT) mice were infused with Ang II or saline. After infusion the with Ang II, the aortic molecular, cellular, and structural remodeling were observed in mice and compared to those infused with saline, but these effects were dependent on the expression of MFG-E8: (1) In the WT mice, Ang II infusion substantially increased intimal-medial thickness, elastic lamina degradation, collagen deposition, and the proliferation of VSMCs; in contrast, in the KO mice, these effects were significantly reduced; (2) In the WT mice, Ang II treatment significantly increased the activation and expression of MMP2, TGF-{beta}1, and its downstream signaling molecule p-SMAD2, and collagen type I production, however, in the KO mice, these molecular effects were significantly reduced; (3) In the WT mice, Ang II treatment increased inflammatory p-NF-{kappa}B p65, MCP1, TNF-, ICAM1, and VCAM1 molecular expression, while conversely, in the KO mice, no significant inflammatory changes were found; (4) Importantly, compared to untreated control mice with a wide range of age from 4-96 weeks, Ang II infused "younger" mice produced an "older" arterial inflammatory phenotype, which was alleviated by MFG-E8 deficiency. ConclusionsMFG-E8 mediates Ang II associated arterial inflammatory remodeling. Targeting MFG-E8 is a novel molecular approach to curb adverse arterial remodeling during aging and hypertension. CLINICAL PERSPECTIVEO_ST_ABSWhat Is New?C_ST_ABS* Both Ang II and MFG-E8 increases are involved in proinflammatory arterial remodeling mediating the molecular, cellular and tissue events in aging and hypertension. * MFG-E8 is essential for Ang II induced and age-associated adverse arterial remodeling via the increase of proinflammation, intimal medial thickening, elastin fragmentation, collagen deposition, and VSMC proliferation. What Are the Clinical Implications?Since MFG-E8 mediates Ang II induced proinflammation in arterial wall remodeling in aging and hypertension, targeting MFG-E8 is a potential molecular approach to curb inflammatory arterial remodeling, maintaining the health of the vascular system during aging and hypertension.

BackgroundHFpEF is a leading cause of death worldwide and clinically relevant preclinical models are required to identify new therapeutic targets. The most clinically representative murine models of heart failure with preserved ejection fraction (HFpEF) in common use include a "2-hit" model combining metabolic stress with hypertension (high-fat diet [HFD] + N(gamma)-nitro-L-arginine methyl ester [L-NAME]) and a "3-hit" model that includes age as an additional "hit" (age + HFD + deoxycorticosterone pivalate [DOCP]). However, both models have reproducibility challenges, and sub-strain and sex dependency. Here we optimize both preclinical models to overcome these challenges. MethodsIn this study we optimized both models: (1) The 2-hit model was optimised to reproduce HFpEF (defined as the induction and maintenance of obesity, hypertension, diastolic dysfunction, left ventricular hypertrophy, lung congestion, and exercise intolerance) in both C57BL/6N and 6J mice using increasing L-NAME doses (0.5 g/L to 1.75 g/L) and protocol lengths (7 weeks to 13 weeks); and (2) The 3-hit model used 12-week-old C57BL/6N and 6J mice and two aging protocols were compared: HFD for 7 months, or healthy chow for 5 months then high fat diet for 7 months. After HFD, mice received an intraperitoneal injection of DOCP to induce hypertension via sodium retention. To enhance and prolong the effect of DOCP, mice received 1% NaCl drinking water at the time of injection until sacrifice, henceforth called "4-hit". To ensure the phenotype was maintained, a second bolus of DOCP was administered 8 weeks after the first. ResultsFor the 2-hit protocol, HFpEF was successfully induced in C57BL/6J mice when exposed to a 13-week L-NAME protocol with gradually increasing dosage from 1.0 g/L to 1.75 g/L. C57BL/6N mice showed the desired parameters after 7-weeks of 0.5 g/L L-NAME, which were not augmented by increased dosage or time administered. For the 4-hit mice, after addition of 1% NaCl drinking water following DOCP administration, a clear HFpEF phenotype was observed in C57BL/6N and 6J mice in both male and females, and maintained for up to 12 weeks. ConclusionsOur modifications ensure the 2-hit model is equally effective in both commonly used J and N substrains of C57BL/6 mice. Our 4-hit model overcomes the challenges of the 3-hit model, enhances reproducibility and robustness, which we demonstrate across sexes and substrains. Both of these new protocols will enhance clinically relevant mechanistic studies on HFpEF.

Heart weight is a critical parameter in cardiology and mouse research, reflecting structural and functional changes linked to cardiac size or hypertrophy and pathophysiological conditions. Normalizing heart weight (HW) to body weight (BW) or tibia length (TL) is a common practice; however, the validity of these ratios has been questioned due to non-proportional relationships between parameters, and this becomes particularly problematic when comparing distinct populations based on such normalized values. Using data from over 25,000 C57BL/6N wildtype mice provided by the International Mouse Phenotyping Consortium (IMPC), we investigated the limitations of ratio-based normalization when comparing different groups, aiming to propose a robust framework for HW analysis. Our findings reveal negligible to weak correlations between HW, BW, and TL across age and sex groups, undermining the validity of ratio-based methods. A modelling study using simulated data demonstrated that ratios could produce misleading results, including reversed or false group differences, when scaling assumptions are violated. Ratios yield accurate and interpretable results only when a truly proportional relationship exists between the variables--specifically, when the regression line passes through the origin--conditions under which ratio-based normalization aligns with outcomes obtained from more robust modelling approaches. These results underscore the superiority of linear models with covariate adjustment and allometric scaling for organ weight analysis, as they more accurately capture biologically relevant scaling relationships. By leveraging the IMPCs large-scale wildtype dataset, we establish the necessity of reassessing normalization practices in quantitative biology traits and propose that ratios should be avoided when comparing normalized values across distinct populations unless key mathematical assumptions are met. This study advances the analytical rigor in phenotyping research, enabling more accurate interpretations of organ mass and function across biological contexts.

Hypertension-mediated left ventricular hypertrophy and cardiac fibrosis often precede heart failure. Recent studies indicate that cytoglobin (Cygb), a globin expressed in the vasculature, increases systemic blood pressure. The present work aims to determine the role of Cygb in angiotensin II (Ang II)-induced cardiac hypertrophy and fibrosis in the mouse. MethodsMales and females global Cygb knockout (Cygb-/-), and wildtype (Cygb+/+) mice were treated with Ang II (1.5 {micro}g/kg/day) for two weeks via subcutaneous osmotic minipumps. Cardiac function was assessed through echocardiography, and hearts were analyzed for changes in hypertrophy, fibrosis, and gene expression. Functional studies were also performed in isolated cardiac fibroblasts. ResultsCygb-/- mice from both sexes showed an increase in cardiac hypertrophy over Cygb+/+ mice. Cardiac functions were also depressed in Cygb-/- males with no changes in females. Importantly, genetic deletion of Cygb did not affect systemic blood pressure in mice, at baseline or after Ang II treatment. We established that Cygb was expressed in fibroblasts and pericytes in humans and mice hearts. Finally, we found that Cygb-/- cardiac fibroblast did not upregulate the expression of genes associated with myofibroblasts following treatment with Ang II. This was reversed following expression of human cytoglobin. ConclusionsOur findings indicate that Cygb plays a protective role in the mouse heart during Ang II-induced cardiac stress. This is the first study detailing the function of Cygb in the heart as a regulator of cardiac hypertrophy. This study also reveals a role for Cygb in regulating cardiac fibroblast activation by Ang II. NEW & NOTEWORTHYWe identified cytoglobin as an important globin in cardiac pathophysiology. Genetic deletion of cytoglobin led to exacerbation of angiotensin II-mediated cardiac hypertrophy in the absence of any effect on systemic blood pressure. Cytoglobin is expressed in cardiac fibroblasts and pericytes and is required for cardiac fibroblast activation to myofibroblast. The present study reveals for the first time a role for cytoglobin in regulating angiotensin II signaling.

RationaleImpaired cardiac energetics in hypertrophic cardiomyopathy (HCM) is thought to result from increased ATP utilization at the sarcomere and is believed to be central to pathophysiology. However, the precise defects in cardiac metabolism and substrate availability in human HCM have not been defined. ObjectiveThe purpose of this study is to define major disease pathways and determine the pool sizes of intermediary metabolites in human HCM. Methods and ResultsWe conducted paired proteomic and metabolomic analyses of septal myectomy samples from patients with HCM and compared results to non-failing control human hearts. Increased abundance of extracellular matrix and intermediate filament / Z-disc proteins, and decreased abundance of proteins involved in fatty acid oxidation and cardiac energetics was evident in HCM compared to controls. Acyl carnitines, byproducts of fatty acid oxidation, were markedly depleted in HCM samples. Conversely, the ketone body 3-hydroxybutyrate, lactate, and the 3 branched chain amino acids, were all significantly increased in HCM hearts, suggesting that they may serve as alternate fuel sources for the production of ATP. ATP, nicotinamide adenine dinucleotide (NADH), NADP and NADPH, and acetyl CoA were also severely depleted in HCM hearts. Based on measurements from human skinned muscle fibers, the magnitude of observed reduction in ATP content in the HCM hearts would be expected to decrease the rate of cross-bridge detachment, implying a direct effect of energy depletion on myofilament function that could contribute to diastolic dysfunction. ConclusionsHCM hearts display profound deficits in cardiac energetics, marked by depletion of fatty acid derivatives and compensatory increases in other metabolites that could serve as alternate fuel sources. These results lend support to the paradigm that energy depletion contributes to the pathophysiology of HCM and also have important therapeutic implications for the future design of metabolic modulators to treat HCM.