American Journal of Physiology-Heart and Circulatory Physiology

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.

AimMaternal iron deficiency (ID) during pregnancy induces cardiovascular adaptations, including reduced blood pressure and improved cardiac efficiency in hypertensive pregnancy. Iron is essential for mitochondrial function, particularly oxidative phosphorylation, where it serves as a cofactor within electron transfer complexes. Given the high metabolic demands of the maternal heart and irons central role in mitochondrial metabolism, we examined how maternal ID affects cardiac mitochondrial ultrastructure, respiration, dynamics, and redox status in pregnant spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto (WKY) rats. Methods and ResultsFemale SHR and WKY rats were fed iron-replete or iron-restricted diets before and throughout gestation. On gestational day 21, cardiac mitochondrial ultrastructure was assessed by transmission electron microscopy (TEM), respiration by high-resolution respirometry, and the expression of proteins involved in fusion, fission, autophagy, and apoptosis markers by immunoblotting. Antioxidant gene expression was quantified by RT-qPCR. Data were analyzed by two-way ANOVA with Holm-Sidaks post hoc test. Maternal iron restriction reduced hemoglobin levels in both strains. TEM revealed enlarged, morphologically heterogeneous mitochondria with reduced and disrupted cristae architecture in ID dams of both strains. Iron restriction reduced succinate-supported respiration and tended to reduce NADH-supported respiration, in both strains. SHR dams exhibited reduced fusion signalling, reflected by a lower L-OPA1:S-OPA1 ratio. MFN1 expression was reduced by ID in both strains, whereas MFN2 expression was lower in SHR and further reduced by ID. In contrast, DRP1 phosphorylation increased selectively in ID-WKY dams. Iron restriction increased LC3-II:I ratio and BNIP3 in SHR, and increased PINK1 in both strains, while Parkin and p62 were unchanged. Antioxidant gene expression increased in ID-SHR but decreased in ID-WKY dams. Despite these alterations, markers of oxidative damage and apoptosis were unchanged by iron restriction. ConclusionMaternal ID induces marked remodeling of myocardial mitochondrial ultrastructure and selectively constrains iron-dependent respiration in hypertensive pregnancy without overt oxidative damage or apoptosis. These mitochondrial alterations occur alongside previously observed reductions in blood pressure and improved cardiac efficiency, suggesting favorable hemodynamic adaptations may coexist with underlying bioenergetic constraints in the maternal heart. Translational PerspectiveMaternal iron deficiency anemia (IDA) may alter the course of hypertensive pregnancy in ways not evident from hemodynamic indices alone. Here, IDA was associated with abnormal myocardial mitochondrial ultrastructure, selective reductions in respiratory capacity and stress response pathways, despite previously observed improvements in blood pressure and cardiac efficiency. These findings suggest that favourable hemodynamic changes may reflect reduced metabolic demand rather than enhanced bioenergetic capacity. If confirmed in human pregnancy, management of ID in women with underlying hypertension may need closer attention to cardiac metabolic health, as cardiovascular adaptions could coexist with myocardial stress and may vary with anemia severity and duration.

BackgroundDoxorubicin (DOX) causes sex-specific cardiotoxicity. Metabolic impairment is a well-established cardiotoxic effect of DOX treatment that can contribute to other detrimental effects such as increased reactive oxygen species, reduced ATP, inflammation etc. We hypothesized that preserving cardiac metabolism by exercise can attenuate DOX cardiotoxicity. MethodsMale and female C57BL/6J mice at 15 weeks of age were randomly assigned to one of four groups, 1) Control (sedentary), 2) EX (exercised, treadmill running), 3) DOX (doxorubicin at 5 mg/kg/week for 6 weeks), and 4) EXDOX (exercise + doxorubicin). Echocardiography was performed every other week during the 6-week protocol to measure cardiac mechanical function. At the end of the protocol, optical mapping and seahorse analysis were performed to measure electrophysiology and metabolism, respectively. RNA sequencing, cytokine array assay and transmission electron microscopy were also performed to determine sex-specific mechanisms of DOX cardiotoxicity. ResultsDOX reduced stroke volume and left ventricular diameter in males only and exercise did not prevent these effects of DOX. In female mice, DOX prolonged action potential duration (APD) and slowed conduction velocity (CV), and importantly, exercise prevented DOX-induced CV slowing. Exercise-induced cardioprotection against DOX in female mice was associated with preservation of aerobic metabolism and attenuation of inflammation which modulated ion channel gene expression. Specifically, Cacna1c was increased in both DOX and EXDOX females, but not in males and correlated with APD prolongation. Interestingly, despite CV slowing, Gja1 and Scn5a were increased. However, increased Kcnj8 along with metabolic impairment could cause membrane hyperpolarization and underlie CV slowing. ConclusionsDOX cardiotoxicity is sex specific. Mechanical dysfunction is more prevalent in DOX-treated males while arrhythmogenic electrical remodeling is more prevalent in DOX-treated females. Exercise therapy during DOX did not prevent DOX induced mechanical dysfunction in male hearts but attenuated electrical remodeling in females by preserving metabolism and attenuating inflammation.

Diastolic dysfunction is a key feature of the aging heart. We have shown that late-life treatment with mTOR inhibitor, rapamycin, reverses age-related diastolic dysfunction in mice but the molecular mechanisms of the reversal remain unclear. To dissect the mechanisms by which rapamycin improves diastolic function in old mice, we examined the effects of rapamycin treatment at the levels of single cardiomyocyte, myofibril and multicellular cardiac muscle. Compared to young cardiomyocytes, isolated cardiomyocytes from old control mice exhibited prolonged time to 90% relaxation (RT90) and time to 90% Ca2+ transient decay (DT90), indicating slower relaxation kinetics and calcium reuptake with age. Late-life rapamycin treatment for 10 weeks completely normalized RT90 and partially normalized DT90, suggesting improved Ca2+ handling contributes partially to the rapamycin-induced improved cardiomyocyte relaxation. In addition, rapamycin treatment in old mice enhanced the kinetics of sarcomere shortening and Ca2+ transient increase in old control cardiomyocytes. Myofibrils from old rapamycin-treated mice displayed increased rate of the fast, exponential decay phase of relaxation compared to old controls. The improved myofibrillar kinetics were accompanied by an increase in MyBP-C phosphorylation at S282 following rapamycin treatment. We also showed that late-life rapamycin treatment normalized the age-related increase in passive stiffness of demembranated cardiac trabeculae through a mechanism independent of titin isoform shift. In summary, our results showed that rapamycin treatment normalizes the age-related impairments in cardiomyocyte relaxation, which works conjointly with reduced myocardial stiffness to reverse age-related diastolic dysfunction.

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.

AimsHeart failure (HF) due to diastolic dysfunction (DD) but normal left ventricular (LV) ejection fraction (EF) is termed HF with preserved EF (HFpEF). Given its high prevalence in post-menopausal women, we hypothesized that 17{beta}-estradiol (E2) function is mechanistically linked to DD HF and investigated E2-deficiency in the etiology of DD in a mouse model. MethodsFemale C57BL/6J mice were divided into one group with hypertension induced by N({omega})-nitro-L-arginine methyl ester (L) and cardiac pressure overload induced by angiotensin II (A), collectively inducing DD, and one group with sham treatment. Groups were subdivided to receive an ovariectomy (OVX) to induce estrogen-deficiency and emulate menopause or sham surgery. During the next 21 days, mice were tested for cardiac function, food intake, response to E2 agonists, gene profiling, cardiomyocyte-contractility and elasticity, and cardiac mitochondrial function. ResultsOVX-associated E2-deficiency exacerbated DD in a time-dependent way without affecting EF or stroke volume, emulating a severe DD phenotype. These changes paralleled those for mitochondrial dysfunction, i.e., upregulation of genes associated with stress, energy metabolism, and fibrosis, as well as functional and structural defects in cardiomyocytes. Treatment of OVX + (L + A) with E2 or a G-protein-coupled estrogen receptor agonist normalized diastolic function, whereas estrogen receptor beta agonists did not. The OVX DD mice exhibited moderately impaired mitochondrial function, which delayed cardiomyocyte relaxation but not contraction, altered cardiac substrate utilization, reduced cardiomyocyte elasticity, increased production of reactive oxygen species, and potentiated extracellular fibrosis. ConclusionsOVX-induced E2-deficiency generates metabolic, structural, and functional changes in cardiomyocytes and the adjacent extracellular matrix, exacerbating the effects of L and A on diastolic function. This robust DD model revealed a role for E2 via ER in diastole-regulation in female mice and raised questions about similar mechanisms operative in postmenopausal women. HIGHLIGHTSO_LIOvariectomy (OVX)-induced estrogen-deficiency exacerbated diastolic dysfunction (DD) induced by hypertension and pressure overload with preserved ejection fraction in female C57BL/6J mice. C_LIO_LIIn post-OVX-treated DD mice, heart weight and fibrosis preceded other metrics of cardiac dysfunction. C_LIO_LIOVX-induced estrogen-deficiency increased the expression of genes associated with stress, energy metabolism, and especially fibrosis. C_LIO_LIMetabolic imaging of the heart by positron emission tomography revealed that DD with preserved ejection fraction was associated with mitochondrial dysfunction presenting as increased accretion of cardiac energy substrates, [18F]deoxyglucose and [11C]palmitate, effects that worsened following OVX. C_LIO_LIOVX increased cardiomyocyte stiffness and fibrosis and reduced cardiomyocyte lengthening and compliance in the context of female DD. C_LIO_LIImproved diastolic function following delivery of GPER agonists or estradiol to OVX DD female mice implicates estrogen receptor in the maintenance of normal cardiac function. C_LI

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

AimsSmoothelin-like 1 (SMTNL1), a protein kinase A/G target protein, modulates the activity and expression of myosin light chain phosphatase and thus plays an important role in regulating vasoconstriction. Increased myogenic reactivity of resistance arterioles is associated with SMTNL1 silencing, and elevated baseline vascular tone is increasingly recognized as a risk factor for development of hypertension and chronic congestive heart failure. Hence, in this study we assessed cardiac function in SMTNL1 knockout mice with and without accompanying acute cardiac stress (i.e., pressure overload by transverse aortic constriction). Methods and ResultsMale and female, global Smtnl1 knockout (KO) & wild-type (WT) mice were assessed at 10 weeks of age by echocardiography and electrocardiography to define baseline cardiac function. Gross dissection revealed distinct cardiac morphology only in male mice; hearts from KO animals were significantly smaller than WT littermates but the proportion of heart mass taken up by LV was greater. Non-invasive analyses of KO mice showed reduced resting heart rate with improved ejection fraction and fractional shortening as well as elevated aortic and pulmonary flow velocities relative to their WT counterparts, but only in the male cohort. We further investigated the impact of acute pressure overload on cardiac morphometry and hemodynamics in the absence of SMTNL1 in male cohort using echocardiography and pressure-volume (PV) loop measurements. Interestingly, PV loop analysis revealed diastolic dysfunction with significantly increased end diastolic pressure and LV relaxation time along with a steeper end diastolic pressure-volume relationship an indicator of stiffer heart, in the KO group when compared to WT Sham-operated group. Sham KO mice also showed elevated arterial elastance and total peripheral resistance. With acute pressure overload, systolic function was preserved, but diastolic dysfunction was exacerbated in KO mice with higher E/E ratio and myocardial performance index along with a prolonged isovolumetric relaxation time relative to the aortic-banded WT group. ConclusionTaken together, the findings support a novel, sex-dimorphic role for SMTNL1 in modulating cardiac structure and diastolic function. Significantly, impairment of diastolic function following pressure overload in young animals lacking SMTNL1 is mainly driven by increased systemic vascular resistance, which mimics the clinical pathophysiology of heart failure with preserved ejection fraction (HFpEF). Translational PerspectiveHeart failure with preserved ejection fraction (HFpEF) is characterized by the impairment of diastolic function and accounts for half of all heart failure cases. Unfortunately, there is as yet no proven therapy available for these patients as the pathophysiology is complicated with the presence of multiple comorbidities, microvascular dysfunction and a lack of an ideal animal model. The phenotype of Smtnl1 global deletion male mice exhibits intriguing similarities to HFpEF, with elevated microvascular resistance driving diastolic dysfunction and LV remodeling. As such the SMTNL1 KO mouse represents a novel pre-clinical model to study the molecular etiology of HFpEF.

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.

Lysosomal function is essential for cardiac proteostasis and cellular health, yet its regulation during ageing remains poorly defined. We hypothesised that ageing alters both the abundance of acidic organelles and the machinery supporting their acidification. Using fluorescence-based In Vivo Imaging Systems (IVIS) with Lysotracker Red in young (2-4 months) and aged (18 months) mouse hearts, we quantified whole-heart acidic-vesicle signals and assessed expression of lysosomal and autophagy-related genes (Lamp2, Atp6v1a, Sqstm1, Cd63, Atg12, Nfe2l2, M6pr) by RT-qPCR. Whole-heart labelled Lysotracker fluorescence did not differ significantly between age groups, indicating preservation of the total acidic-vesicle pool. No changes in Atp6v1a and Lamp2 expression suggest acidification capacity and structural stability are maintained, whereas the minor, upregulation of Sqstm1 might indicate increased autophagic demand and altered vesicle trafficking, which warrants further investigation. No statistical significant changes in M6pr, Atg12, or Nfe2l2 were detected, suggesting transcriptional stability in enzyme trafficking, core autophagy, and oxidative stress pathways. Regionally, atria showed higher Lysotracker signal than ventricles, consistent with known enrichment of acidic vesicular stores in atrial physiology. These findings highlight the utility of IVIS imaging of Lysotracker-labelled hearts, providing rapid whole-organ assessment of acidic vesicle distribution, albeit with limited depth resolution. Complementary techniques such as RT-qPCR analysis is essential to interpret IVIS findings, enabling insight into underlying molecular changes in lysosomal and autophagy pathways during cardiac ageing.

BackgroundHypertension prevalence increases with age, reaching over 70% of people over age 65. The underlying mechanisms are poorly understood. This study interrogates a new signaling pathway in vascular smooth muscle of aged mice driven by p90 ribosomal S6 kinase, RSK2, and its role in increasing peripheral vascular resistance and blood pressure (BP). MethodsBasal BP measurements were taken at 26-29 month (812-892 day) old mice with global deletion of RSK2 (Rsk2-/-) prior to and following treatment with L-NAME. Cardiac function, vessel stiffness, myogenic responses, Ca2+events, contractility, immuno-staining, histology studies and western blotting were performed. ResultsResting BP and myogenic vasoconstriction were normal in aged global Rsk2-/- mice and elevated in wild type (WT) littermates. L-NAME treatment increased BP in aged Rsk2+/+ but not aged Rsk2-/-. Vessel stiffness and glycation collagen crosslinking increased in both aged Rsk2+/+ and Rsk2-/- compared to young vessels with no remodeling or increase in collagen content, even though BP in aged Rsk2-/- arterioles was normal. Increased vessel stiffness was dissociated from increased BP. Ca2+ transients increased and sensitivity to NO-induced relaxation decreased in aged Rsk2+/+ compared to young WT arterioles. IEL structures, eNOS and Hb distribution at myoendothelial junctions were disturbed impairing vasorelaxation in aged Rsk2+/+ but not aged Rsk2-/- arterioles. ConclusionsRSK2 plays a significant role in hypertension associated with aging by downregulating prorelaxant signaling and promoting procontractile events in the vasculature, offering potential new therapeutic targets.

Obesity is associated with an elevated risk of pathological cardiac hypertrophy, whereas exercise confers cardioprotective effects; however, the cellular mechanisms underlying these opposing influences remain incompletely defined, particularly in females. We investigated how obesity and exercise affect cardiomyocyte ultrastructure, Ca{superscript 2} release, and contractility in female Zucker Diabetic Fatty rats and their lean littermates. Animals were assigned at 12 weeks to sedentary or aerobic exercise-trained groups and maintained on a standard diet. By 18 weeks, obese rats exhibited increased body mass and myocardial hypertrophy in the absence of diabetes. Sedentary obese animals showed a reduced fraction of compact dyads and diminished stimulated and caffeine-induced Ca{superscript 2} release, while contractility remained preserved. In lean rats, exercise increased dyad density but reduced Ca{superscript 2} release, whereas in obese rats, exercise enhanced both dyad compactness and Ca{superscript 2} release. Across all groups, global cardiomyocyte ultrastructure and contractile function were similar. Type III ANOVA revealed a significant obesity x exercise interaction for dyadic structure and Ca{superscript 2} release. These findings demonstrate that obesity itself, independent of diabetes, triggers early dyadic remodeling and altered Ca{superscript 2} handling in female myocardium before detectable impairment of global cardiomyocyte structure or contractile function. Furthermore, exercise exerts beneficial effects on dyadic ultrastructure and Ca{superscript 2} signaling in obese animals. New & NoteworthyUsing a female rat model of obesity without diabetes, we demonstrate that obesity induces early remodeling of the dyadic system and impairs Ca{superscript 2} release in cardiac myocytes. We further show that the effects of aerobic exercise on dyadic structure and function are obesity-dependent, improving both dyad organization and Ca{superscript 2} signaling. These findings identify the dyadic microdomain as a vulnerable cellular site in obesity and a potential target for exercise-induced recovery.

Cardiac adaptation to exercise is a fundamental physiological process, but its regression and the underlying molecular mechanisms, particularly in relation to age, remain poorly understood. This study investigated the age-dependent differences in cardiac remodeling and molecular signaling during exercise training and detraining in young (5-week-old) and adult (24-week-old) female mice, focusing specifically on how cardiac plasticity changes with adulthood rather than senescence. While both age groups exhibited significant cardiac hypertrophy after the exercise period, young mice displayed significantly more hypertrophic growth (23% increase in left ventricular mass versus 15% in adults). During detraining, cardiac mass regression occurred more rapidly in young mice. Transcriptomic analysis revealed distinct gene expression profiles between age groups, with changes in metabolic and autophagy pathways. Notably, ERK1/2 phosphorylation increased significantly during exercise in young but not adult hearts, correlating with elevated expression of well-known genes associated with exercise, namely CITED4 and SOD2. Furthermore, increased LC3-II/LC3-I ratio and AMPK phosphorylation were observed exclusively in young mice during detraining, indicating age-specific activation of autophagy-mediated cardiac remodeling. These findings demonstrate that cardiac adaptability to exercise and detraining follows distinct molecular pathways in young versus adult mice, with the younger heart exhibiting greater plasticity through enhanced ERK signaling during hypertrophy and autophagy during regression. This age-dependent cardiac plasticity may have important implications for understanding the cardiovascular benefits of exercise across the lifespan and developing age-appropriate exercise recommendations.

Aging is a major risk factor for cardiac diseases, including heart failure, myocardial infarction, and arrhythmias. Activation of p38 MAPKs regulates cardiac remodeling and contributes to age-related cardiac dysfunction. However, the isoform-specific roles of p38 kinases in the aging heart remain poorly understood. Although p38{beta} has been reported to exert cardioprotective effects in models of doxorubicin-induced cardiotoxicity and ischemia-reperfusion, its role in cardiac aging remains unclear. Here, we investigated the role of p38{beta} using p38{beta} germline knockout (p38{beta}-/-) mice. Aged p38{beta}-/- mice exhibited increased LV hypertrophy, QT prolongation, calcium mishandling, heightened susceptibility to arrhythmias, increased myocardial fibrosis, and an altered inflammatory microenvironment, compared with age-matched wild-type controls. Transcriptomic profiling revealed that p38{beta} deletion reprograms the cardiac transcriptome in aged mice, suppressing innate immune and proteostasis-related pathways while promoting adaptive immune activation, developmental, extracellular vesicle-mediated, and ion-transport pathways. Collectively, these findings identify p38{beta} as a critical regulator of structural, electrophysiological, and immune homeostasis in the aging heart and demonstrate that its loss promotes maladaptive remodeling and arrhythmogenic vulnerability. NEW AND NOTEWORTHYWe identify p38{beta} as a previously unrecognized regulator of cardiac aging. Systemic loss of p38{beta} disrupts structural, electrophysiological, and immune homeostasis in the aging heart, revealing its protective role in maintaining cardiac function with age. These findings underscore the importance of isoform-specific p38 signaling and suggest that broadly targeting p38 MAPKs may have unintended consequences in age-related cardiovascular diseases.

IntroductionAtrial Fibrillation (AF) and atrial flutter (AFL) are the two most common cardiac arrhythmias in the United States. While advanced age has been correlated to AF/AFL, the lack of an appropriate animal model has hindered progress on better understanding the pathophysiology of atrial arrhythmogenesis. Both hypokalemic conditions and hemodynamic stretch have been associated with atrial tachyarrhythmias in patient populations. The purpose of this study was to examine the incidence of atrial tachyarrhythmias in an ex vivo aging C57BL/6 mouse model following hypokalemia and stretch challenges. MethodsHearts were isolated with combined cannulation of the aorta and superior vena cava in a modified right-sided working heart perfusion technique. Isolated hearts of Aged (26-29 month) male (n=14) and female (n=14) mice were subjected to normokalemic and hypokalemic conditions {+/-} atrial preload elevation to 12 cmH20 to induce atrial stretch. Heart rate, right ventricular (RV) pressure development, and incidence of atrial tachyarrhythmias were monitored using a pressure catheter and intracardiac electrocardiogram. ResultsIn response to hypokalemia, there were no changes in mean heart rate, RV pressure development, or RV Rate-Pressure Product (Rate x RV peak pressure). Atrial tachyarrhythmias were not observed under baseline conditions, and only 1 of 8 hearts exhibited atrial tachycardia following the hypokalemia challenge. In response to atrial preload elevation, there was an increase in heart rate (P=0.0006 versus baseline) with no change in RV pressure development. RV Rate-Pressure Product was significantly elevated (P=0.013 versus baseline) with atrial preload due to the increase in heart rate. Atrial tachyarrhythmias were not observed under both baseline conditions and following atrial preload elevation. In response to the combined hypokalemia and preload challenges, there was an increase in heart rate (P=0.008 versus baseline) with no change in RV pressure development or RV Rate Pressure product. Atrial tachyarrhythmias were not observed under baseline conditions, yet after the combined challenges 50% of aged hearts exhibited atrial tachycardia or AF/AFL. During bouts of AF/AFL, the AF/AFL led to a variable ventricular response and concomitant contractile dysfunction in the form of variable RV pressure development. ConclusionEx vivo aged mouse hearts exhibit atrial tachyarrhythmias in response to combined hypokalemia and right atrial stretch conditions. The aged C57BL/6 mouse model is therefore useful for pre-clinical studies of atrial arrhythmogenesis.

Background Ischemic heart failure (IHF) has been shown to impair contractility and disrupt sarcomere function in the left ventricle. Left ventricular failure can cause left atrial dysfunction, which is associated with a greater risk of patient mortality. Despite this, the biochemical and biomechanical characteristics of the left atrium in IHF remain obscure. Methods Myocardial mechanical properties were measured using permeabilized muscle isolated from the left ventricle (LV) and left atrium (LA) of donors and patients with IHF. Tissue homogenates from these samples were used to measure titin and myosin isoforms as well as the phosphorylation of sarcomeric regulatory proteins. Histology was used to quantify fibrosis in the patients' left ventricle and left atrium. Results Length-dependent changes in Ca2+-sensitivity were blunted in LV myocardium from patients with IHF. LA myocardium did not show robust length-dependence of Ca2+-dependent force. The calcium sensitivity of both LA and LV myocardium was increased in IHF. The maximum force generated by LV but not LA myocardium was decreased in IHF. LA myocardial samples exhibited faster contractile kinetics than LV samples, irrespective of disease. Troponin I phosphorylation decreased in both chambers with IHF. Conclusions Left atrial IHF myocardium maintained contractile force and displayed increases in calcium sensitivity, which may allow for increased LA contraction under pathological conditions. The increases in calcium sensitivity observed in ischemic myocardium of both chambers are likely driven by decreased phosphorylation of troponin I, which alters thin filament regulation. Conversely, thick filament properties of the left ventricle, such as thick filament protein isoforms and phosphorylation of myosin binding protein-C, displayed chamber-specific differences independent of disease state. These biochemical changes may explain the chamber-specific differences in kinetics and length-dependent properties. Collectively, these biophysical and biochemical data suggest LA remodeling in IHF may assist in increasing LV end-diastolic volume to maintain adequate cardiac output.

AimsCaveolae are plasmalemmal microdomains regulating stretch-dependent, nitric oxide (NO), and other signalling pathways governing myocardial structure, function and resilience. We have reported that global deletion of the scaffold protein cavin-1 disrupts caveolar biogenesis and impairs ventricular compliance and tolerance to ischaemic injury. However, cardiomyocyte-specific and sex-dependent roles of cavin-1 and caveolar complexes remain unresolved. Methods and ResultsWe generated a floxed Cavin-1 transgenic mouse, enabling cardiomyocyte-specific knockdown via adeno-associated virus (AAV) mediated expression of iCre recombinase driven by a cardiac-specific troponin T promoter. Knockdown was confirmed by RNA, protein, and immunofluorescence analyses, and cardiac function was assessed via echocardiography, left ventricular pressure-volume (PV) catheterisation, and ex vivo PV analysis of perfused hearts. Conditionally deleted hearts and myocytes exhibited up to 50% knockdown of Cavin-1 mRNA together with 15% deficiency in muscle-specific Caveolin-3, 70% depletion of caveolae, and mislocalisation of NO synthase (NOS) within cardiomyocytes. This was associated with elevated heart rate and shortened PR interval; reduced intraventricular and systolic blood pressures and peripheral resistance; and sex-dependent impairment of ventricular filling (females only). Diastolic dysfunction was detectable ex vivo, to a greater extent in male vs. female hearts. Mechanisms were sex-dependent, linked to interstitial fibrosis in females and NOS overactivity (inhibited by 100 {micro}M L-NAME) in males. Female hearts also exhibited increased susceptibility to ischaemia-reperfusion injury. Coronary function appeared preserved in both sexes, with intact reactive hyperaemic responses. ConclusionThis model identifies cardiomyocyte caveolae and cavin-1 as key determinants of myocardial function and compliance, involving sex-dependent remodelling and NOS signalling. By linking cardiomyocyte disruption to whole-organ and -body dysfunction, this model provides mechanistic insight into impaired function in heart failure and ageing. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=117 SRC="FIGDIR/small/717104v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@51bfe4org.highwire.dtl.DTLVardef@10d4323org.highwire.dtl.DTLVardef@1b2baa7org.highwire.dtl.DTLVardef@fc5f21_HPS_FORMAT_FIGEXP M_FIG C_FIG

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.

Background: Age is the most important risk factor for coronary artery disease (CAD) independent of traditional risk factors. Aging induces classic pro-inflammatory and prothrombotic vascular phenotypic changes whose molecular mediators remain poorly understood. Medin is a common cleavage product protein that accumulates in vasculature with aging and shown to cause endothelial dysfunction. Its role in CAD is unknown. The study aimed to evaluate the effects of medin on human coronary artery endothelial cell (HCAEC) pro-inflammatory and prothrombotic activation and establish the relationship between medin and coronary atherosclerosis in human decedents. Methods: HCAECs were exposed to physiologic dose of medin (5 M) for 20 hours and ribonucleic acid sequencing (RNAseq) with signaling pathway analyses and reverse transcription polymerase chain reaction of select pro-inflammatory and prothrombotic genes performed. Corresponding protein expression was measured by Western blot or enzyme linked immunosorbent assay in HCAECs exposed to medin (5 M) without or with nuclear factor-{kappa}B (NF{kappa}B) inhibitor RO106-9920 (10 M). Coronary arteries from 40 deceased individuals underwent immunohistochemistry and medin and plaque burden were quantified and their relationship evaluated. Results: RNAseq showed predominant pro-inflammatory gene expression changes induced by medin. HCAECs treated with medin showed increased phosphorylated NF{kappa}B, elevated protein expression of interleukin (IL)-6, IL-8, monocyte chemotactic protein (MCP)-1, intercellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule (VCAM)-1 and plasminogen activator inhibitor (PAI)-1 and reduced protein expression of thrombomodulin; these changes were reversed by RO106-9920 co-treatment. In human tissues, coronary artery medin strongly correlated with plaque burden (R=0.76, p<0.0001) and coronary macrophage content (R=0.72, p<0.0001). Coronary arteries from decedents with myocardial infarction had higher medin than those without (5.53{+/-}2.67% versus 0.02{+/-}0.02%, p=0.0005). Conclusions: Medin induced NF{kappa}B-mediated endothelial cell pro-inflammatory and prothrombotic activation and was strongly associated with coronary plaque burden and inflammation. Medin is a novel candidate mediator linking aging and coronary atherosclerosis.

In individuals with Marfan Syndrome (MFS), fibrillin-1 gene (FBN1) mutations can lead to vascular wall weakening and dysfunction. The experimental mouse model of MFS (FBN1C1041G/+) has been advantageous in investigating MFS-associated life-threatening aortic aneurysms. Although the MFS mouse model presents an accelerated-aging phenotype in elastic organs (e.g., lung, skin), the impact of FBN1 mutations on other central and peripheral arteries function and structure with the consideration of the impact of sex remains underexplored. In this study, we investigate if FBN1 mutation contributes to sex-dependent alterations in central and cerebral vascular function similar to phenotypic changes associated with normal aging in healthy control mice. In vivo ultrasound imaging of central and cerebral vasculature was performed in 6-month-old male and female MFS and C57BL/6 mice and sex-matched 12-month-old (middle-aged) healthy control mice. Our findings confirm aortic enlargement (aneurysm) and wall stiffness in MFS mice, but with exacerbation in male diameters. Coronary artery blood flow velocity (BFV) in diastole was not different but left pulmonary artery BFV was decreased in MFS and 12-month-old control mice regardless of sex. At 6 months of age, MFS male mice show decreased posterior cerebral artery BFV as compared to age-matched control males, with no difference observed between female cohorts. Reduced mitral valve early-filling velocities were indicated in MFS mice regardless of sex. Male MFS mice also demonstrated left ventricular hypertrophy. Overall, these results underscore the significance of biological sex in vascular function and structure in MFS mice, while highlighting a trend of pre-mature vascular aging phenotype in MFS mice that is comparable to phenotypes observed in older healthy controls.