bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of TRPV1 and TRPV2 channel expression and vasomotor function in human arterioles
Authors: Korishettar Ankush M , Nishijima Yoshinori , Cao Sheng , Zhang David
Published: November 2023

Transient receptor potential vanilloid (TRPV) channels, act as sensory mediators and are activated by endogenous ligands, heat, mechanical and osmotic stress. Within the vasculature, TRPV channels are expressed in smooth muscle cells, endothelial cells and in perivascular nerves. We have previously demonstrated that Ca2+ influx through endothelial TRPV4 channels mediates flow‐induced dilation (FID) of human coronary arterioles (HCA) from subjects with coronary artery disease (CAD). Other studies have also implicated endothelial TRPV1 channels in FID, whereas a functional role for endothelial TRPV2 channels has yet to be determined. Recent unpublished studies from our lab showed that FID was not affected by TRPV4 inhibition in HCA from subjects without CAD (non‐CAD). Instead, inhibition of TRPV1 or TRPV2 reduced FID of HCA from non‐CAD subjects. Collectively, these data have led us to hypothesize that different TRPV channels contribute to FID of arteries from non‐CAD vs CAD subjects, with TRPV4 prominent in CAD vessels and TRPV1/2 more important in non‐CAD vessels. The objective of the current study was to characterize the expression and function of TRPV1 and TRPV2 channels in HCA and human adipose arterioles (HAA) from non‐CAD subjects, using molecular, immunological and vascular reactivity studies. We first examined TRPV1 and TRPV2 mRNA expression in HCA and HAA using RT‐PCR and consistently detected both transcripts in multiple samples. As a positive control, human brain tissue was found to express TRPV1 and TRPV2 transcripts. Consistent with mRNA expression, TRPV1 and TRPV2 proteins were detected in membrane fractions of HCA and HAA using western blot analysis. Antibody specificity was confirmed in TRPV1 or TRPV2 transfected versus non‐transfected HEK‐293 cells. We next examined the vasomotor effect of capsaicin, a selective TRPV1 agonist and cannabidiol, a selective TRPV2 agonist on freshly isolated HAA. HAA (100–200 μm), obtained from subjects without CAD, were cannulated under 60 mmHg and examined for diameter changes using videomicroscopy. Surprisingly, bath application of capsaicin induced constriction of HAA at concentrations of 10−7 M and 10−6 M (−46.82 ± 4.3%; n=3), with no significant effect at lower concentrations (10−10–10−8 M). In contrast, cannabidiol (10−6,10−5,3×10−5 M) induced a dose‐dependent dilation of HAA. These results suggest that TRPV1 and TRPV2 channels are expressed in human arterioles and contribute to vasomotor responses. Further studies are required to determine the specific role of the endothelium versus smooth muscle and perivascular nerves, in mediating the vascular responses to TRPV1 and TRPV2 activation.

Support or Funding Information

Support for this research was provided by the National Heart, Lung and Blood Institute Grant R01‐HL 096647
Loss of Nitric Oxide Contributes to Perturbations in Myocardial Oxygen Balance in Exercising Swine with Multiple Comorbidities
Authors: van de Wouw Jens , Sorop Oana , van Drie Ruben W.A. , Merkus Daphne , Duncker Dirk J.
Published: November 2023

Introduction

Multiple comorbidities, including diabetes mellitus (DM), hypercholesterolemia, hypertension (HT) and chronic kidney disease (CKD) are thought to cause coronary microvascular dysfunction and reduce microvascular NO‐production, leading to impaired myocardial perfusion and contributing to the development of left ventricular diastolic dysfunction. In the present study, we aimed to investigate the detrimental effects of DM, CKD and high fat diet (HFD) on microvascular function and myocardial blood flow regulation and the role of NO bioavailability herein in swine.

Methods

DM and CKD were induced in 5 female swine that were subsequently fed HFD (DM+HFD+CKD), while 6 female healthy swine on normal pig chow served as controls (CON). At 6 months follow‐up coronary flow regulation was studied at rest and during treadmill exercise with/without eNOS inhibitor, nitro‐L‐arginine in 6 CON and 5 DM+HFD+CKD chronically instrumented animals.

Results

DM+HFD+CKD animals had hyperglycemia (glucose: 18.7±1.9 vs 7.5±0.6 mmol/L), renal dysfunction (reduced glomerular filtration rate: 123±12 vs 202±8 ml/min) and hypercholesterolemia (total cholesterol: 7.3±0.7 vs 1.7±0.1 mmol/l, all P<0.05). In vivo, myocardial oxygen delivery was impaired in DM+HFD+CKD swine, forcing the myocardium to increase its oxygen extraction (MO2ex) both at rest and during exercise (Figure), and resulting in reduced coronary venous oxygen content compared to CON (all P<0.05). eNOS‐inhibition resulted in coronary microvascular constriction in CON, reflected in an increased MO2ex (P<0.05), while it had no effect in DM+HFD+CKD, suggesting that loss of NO was responsible for the increased oxygen extraction in DM+HFD+CKD. This was supported by lower myocardial levels of NO metabolites NO2−+NO3− in DM+HC+HT compared to CON (0.20±0.02 vs 0.34±0.06 μmol/mg protein, P<0.05).

Conclusion

Prolonged exposure to DM, HFD and CKD results in impaired coronary microvascular NO production, thereby hampering myocardial perfusion.

Support or Funding Information

Funding: This study was supported by grants from the European Commission FP7‐Health‐2010 grant MEDIA‐261409, the Netherlands CardioVascular Research Initiative: an initiative with support of the Dutch Heart Foundation [CVON2014‐11 (RECONNECT)].
Glucocorticoids modulate Kvβ1 dependent cardiac function through potassium channel (Kv) activity
Authors: Tur Jared , Badole Sachin L , Chapalamadagu Kalyan C , Tipparaju Srinivas M
Published: November 2023

Cortisone is a glucocorticoid used for treatments including arthritis, immune system disorders, allergic reactions, and eye conditions. Glucocorticoids however, have the propensity to produce multiple cardiovascular effects including hypertension, hyperglycemia and dyslipidemia. Therefore in patients with history of cardiovascular diseases corticosteroids are used with caution due to high risk of myocardial injury. In vitro research has demonstrated cortisone and its analogs alter voltage‐gated potassium channels (Kv) via the Kvβ1.1 subunit. Recently we published that both male and female Kvβ1.1 knockdown (KO) mice demonstrate significant prolongation in left ventricular action potentials in addition only females presented with significantly elevated blood pressure levels including systolic and diastolic.

Based upon glucocorticoids cardiovascular effects and cortisones high affinity to bind with Kvβ1.1 we hypothesis that glucocorticoid treatment may induce cardiovascular effects through Kvβ1.1 generating a novel role of Kvβ1.1 regulation in the heart.

Initial experiments with Left ventricular action potentials measured through monophasic action potential (MAP) recordings demonstrated perfusion with cortisone (500μmole/L) showed significantly (p<0.01) prolonged MAP durations at APD 50%, 70%, and 90% repolarization within the wild type (WT) hearts however, no significant difference was noted in Kvβ1.1 KO hearts with cortisone perfusion. KO hearts were significantly prolonged at baseline in comparison with WT baseline similar to our previous work. Results were obtained with fluticasone propionate (100nmole/L) in which left ventricular action potentials showed significantly (p<0.01) prolonged MAP durations in the WT hearts at APD 50%, 70%, and 90% repolarization however, no significant difference was noted in Kvβ1.1 knockout mice (KO). Application of a combination cortisone plus 4‐aminopyridine (4‐AP) perfused WT and KO hearts resulted in MAP durations significantly (p<0.01) prolonged at APD 20%, 50%, and 70% repolarization in both WT and KO hearts. However, prolongation observed in KO hearts was due solely to 4‐AP exposure, showing no additive effects from cortisone.

Based on the results we identified that cortisone modulates cardiac electrical activity by binding and inhibiting Kvβ1.1‐Kv channel interaction in the mouse heart. This is the first report demonstrating the modulation of electrical activity by cortisone and the role of Kvβ1.1 subunit in mouse hearts. WT hearts demonstrated significant action potential prolongation while Kvβ1.1 KO hearts showed no significant cortisone induced action potential prolongation.
Exercise Training Fails to Protect against Development of Ischemia‐Induced Ventricular Fibrillation in Isolated Rat Hearts
Authors: Clements‐Jewery Hugh , Radich Patricia , Rawson Nicholas , Hatcher Amanda Shea
Published: November 2023

Background and purpose

Sudden cardiac death continues to be a major cause of mortality in the United States and beyond, with the predominant cause of death being ventricular fibrillation in the setting of ischemic heart disease. Pharmacological therapy has proven to be a limited strategy for preventing ventricular fibrillation as a consequence of the safety and efficacy of available drugs. Exercise training has been found to be effective in reducing susceptibility to spontaneous VF in a canine in vivo model (Bonila et al., 2012), but it is not known if exercise training would be effective in reducing susceptibility to ischemia‐induced VF in isolated perfused rat hearts.

Methods

Male and female 6 week old Sprague‐Dawley rats were separated into exercise (EX) and sedentary (SED) groups (N=15 per group). Rats in the exercise group were trained for 60 min on a treadmill 4 days/week for 8 weeks. Workloads were matched for bodyweight. Rats in the SED group were given equivalent handling time. Hearts were excised under pentobarbital anesthesia after the end of the 8‐week period in a randomized and blinded manner and perfused in the Langendorff mode using modified Krebs solution (3mM K+). After a 10 min baseline perfusion period, regional ischemia was induced by occlusion of the left main coronary artery and maintained for 30 min. Hearts were subsequently reperfused for 10 min. The ECG was monitored for the occurrence of VF and other arrhythmias during ischemia and used for the measurement of electrocardiographic intervals. An intraventricular balloon was used to record left ventricular developed pressure (LVDP) and end diastolic pressure (EDP). Coronary flow was recorded digitally using a calibrated signal from a pump controller.

Results

The incidence of VF during ischemia in the EX group was 87% (13/15), which was not different from the incidence of VF in the SED group (67%; 10/15). In addition, the time to onset of VF during ischemia (950±157s vs 1035±127s in SED and EX groups respectively) as well as susceptibility to reperfusion VF (93% in the SED group vs 87% in the EX group) was similar in the two groups. There was no difference in ischemic zone size between groups. Changes in heart rate, coronary flow, EDP and LVDP with onset of ischemia were all similar in both SED and EX groups with no significant differences at any time point.

Conclusions

Exercise training did not confer protection against ischemia‐induced VF in an established in vitro model. Thus the protection afforded by exercise training in the canine in vivo model may indicate a species dependence of exercise training‐induced protection, or that protection is due to factors absent from isolated rat hearts such as an intact autonomic system or factors circulating in the blood.

Support or Funding Information

This study was supported by an intramural grant from the West Virginia School of Osteopathic Medicine.
Heterozygous Deletion of Mediator Subunit 1 Causes Heart Failure in Mice
Authors: Spitler Kathryn , Grueter Chad
Published: November 2023

Cardiac gene expression is altered during heart failure, but the mechanisms underlying these transcriptional changes remains unclear. The Mediator complex governs gene transcription by linking upstream cellular signals with transcriptional machinery. Mediator subunit 1 (Med1) plays a pivotal role in regulating gene expression by serving as a transcriptional coactivator for nuclear receptors. Our objective is to elucidate the role for Med1 in cardiac transcriptional regulation. Expression of Med1 in the heart is essential as cardiac deletion of Med1 results in rapid progression into heart failure and death within 2–3 weeks. Given this early lethality during an important developmental time period for the heart, we generated a heterozygous cardiac‐specific deletion of Med1 (cMed1+/−) using the alpha myosin heavy chain expressing cre recombinase bred into Med1 floxed mice. These mice allow us the ability to understand the role of Med1 under more physiological conditions and enables examination into gene expression changes in response to cardiac stresses that lead to heart failure. cMed1+/− resulted in a more gradual lethality phenotype starting at 10 months of age. At one year of age echocardiography, qPCR and Western blot analysis was performed. cMed1+/− male hearts had the expected ~50% reduction in Med1 mRNA and protein levels, resulting in reduced ejection fraction (47% vs. 87%) and increased heart mass (50%) compared to Med1 floxed controls. Transverse sections of cMed1+/− hearts revealed ventricular dilation. Expression of heart failure marker genes (Nppa, Nppb, and Myh7) and pro‐fibrotic genes (Tgfb2 and Col11a1) was increased in hearts from cMed1+/− mice compared to Med1 floxed controls while there was an decrease in the major adult‐associated contractile component Myh6. Taken together, these data indicate that physiological Med1 levels are essential for cardiac gene expression and function as Med1 haploinsufficiency causes cardiac dysfunction and failure.

Support or Funding Information

NIH/NHLBI R01 HL125436‐02, Fraternal Order of Eagles Diabetes Research Center, the University of Iowa Carver College of Medicine, and NIH T32 Postdoctoral Fellowship‐5T32HL007121‐38
MULTIPLE CO‐MORBIDITIES HAVE DETRIMENTAL EFFECTS ON MYOCARDIAL FUNCTION AND PERFUSION IN A LARGE ANIMAL MODEL OF DIASTOLIC DYSFUNCTION
Authors: Sorop Oana , van de Wouw Jens , van Drie Ruben W.A. , Heinonen Ilkka , de Beer Vincent J. , van den Meiracker Anton H. , van der Velden Jolanda , Joles Jaap A. , Merkus Daphne , Duncker Dirk J.
Published: November 2023

Comorbidities such as diabetes mellitus (DM), hypercholesterolemia (HC) and chronic kidney disease (CKD), are often associated with diastolic dysfunction. We tested the hypothesis that the co‐morbidities produce systemic inflammation and microvascular dysfunction resulting in impaired myocardial perfusion, structural changes in the myocardium as well as cardiac dysfunction in swine.

Methods and Results

DM (streptozotocin, 150mg/kg i.v.), HC (high fat diet) and CKD (renal artery embolization), (DM+HC+CKD) were induced in 16 female swine, which were followed for 6 months. 15 female healthy swine on normal pig‐chow served as controls. Myocardial vascular tone control and function were studied at rest and during graded treadmill exercise. The DM+HC+CKD group showed hyperglycemia (22.7±0.9mmol/l in DM+HC+CKD vs 6.1±0.7mmol/l in CON), hypercholesterolemia (16.9±3.4 vs 2.2±0.1mmol/l), hypertriglyceridemia (1.20±0.36 vs 0.28±0.05mmol/l) and impaired kidney function (Glomerular Filtration Rate: 139±19 vs 197±10 ml/min, all P<0.05), which were associated with systemic inflammation and impaired coronary small artery endothelium‐dependent vasodilation to bradykinin. These abnormalities were accompanied by increased myocardial collagen content, reduced capillary/fiber ratio and elevated passive cardiomyocyte stiffness, resulting in increased left ventricular end‐diastolic stiffness (pressure‐volume catheter at sacrifice, 0.15±0.02 vs 0.07±0.01 mmHg/ml, P<0.05), while ejection fraction was maintained (45±4 vs 54±3%, P=NS). A subgroup of animals, studied during exercise, showed reduced cardiac index (CI, P<0.05), increased levels of left atrial pressure required for similar CI, reduced stroke volume index and impaired myocardial efficiency (Figure, all P<0.05). Furthermore, the animals with the three comorbidities demonstrated perturbations in myocardial blood flow, requiring an increased myocardial oxygen extraction (Figure), resulting in lower coronary venous oxygen content levels during exercise.

Conclusion

The combination of three common comorbidities leads to systemic inflammation, coronary microvascular dysfunction, which associate with impaired vascular tone control, myocardial stiffening and LV diastolic dysfunction with preserved ejection fraction.

Support or Funding Information

Funding: This study was supported by grants from the European Commission FP7‐Health‐2010 grant MEDIA‐261409, the Netherlands CardioVascular Research Initiative: an initiative with support of the Dutch Heart Foundation [CVON2014‐11 (RECONNECT)].
How does the overexpression of DGKζ can improve the survival and rescue the heart failure associated loss of contractility?
Authors: Aslam Naveed
Published: November 2023

The most frequently encountered forms of heart failure in the clinical practice are diastolic heart failure (DHF) and systolic heart failure (SHF). These syndromes are characterized by a progressive loss in contractility, ejection failure, ventricular chamber dilation, ventricular wall thinning, increased peripheral vascular resistance and dysregulated fluid homeostasis. The propensity towards these forms of cardiac insufficiency could be linked to the activation of PKCα molecule in DHF & SHF condition. The PKCα molecule act as a nodal integrator of cardiac contractility and is now considered as a milder and probably more effective alternative to positive inotropes. Clinical evidence suggests the positive inotropes worsens the prognosis in individuals with somewhat stable heart failure. Data from previous studies suggests that overexpression of DGKζ molecule can improve the survival and reduce the propensity of contractile dysfunction in HF. Here, I address the question that how the overexpression of DGKζ molecule could improve the survival and reduce the propensity towards heart failure? This becomes interesting as GPCR agonists such as angiotensin II and phenylephrine not only leads to the activation of PKCα but also the braking mechanism to check the increase in PKCα activity during HF. So, one could wonder that why the PKCα activity is increased and sustained during HF? Here, through a DGKζ overexpression model I show that DGKζ modulates the PKCα activity through inducing the metabolism of its activator DAG. My results suggest that in the wild type models the expression of DGKζ & PKCα could be in same proportions therefore, once activated by DAG, the PKCα molecule could deactivate the DGKζ through phosphorylation, thus blunting the braking mechanism and sustaining its activity during DHF & SHF conditions. My results imply that in overexpression model the DGKζ proportions increases compared to PKCα thus, counteracting the negative effects of PKCα on its activity by enhancing the DAG metabolism in the plasma membrane compartment. My results also show that the DGKζ overexpression can reduce the cytosol to membrane translocation of PKCα in a dose dependent manner. These results are also consistent with previously reported dose‐dependent effects of DGKζ overexpression on the translocation characteristics & activity of PKCα molecule in HF. Results presented in this study could provide interesting possibilities for DGKζ as another potential translation target for DHF and SHF conditions.

Support or Funding Information

Privately funded
Reinstallation of Glucocorticoid Receptor (GR) in the Heart Rescues Left Ventricular Systolic Function of Cardiac GR Knockout mice
Authors: He Bo , Oakley Robert H. , Myers Page H , Cidlowski John A
Published: November 2023

Glucocorticoidsare primary stress hormones necessary for life. In recent years, stress isincreasingly associated with cardiovasculardiseases. We have previously demonstrated that adeficiencyin glucocorticoid receptor (GR) signaling in cardiomyocytes resulted in cardiachypertrophy, left ventricular (LV) systolic dysfunction, and heart failure. This suggested a crucial role for cardiomyocyte GR signaling in the function of the heart. In order to rescue the phenotypes of cardiomyocyte‐specific GR Knockout mice (cardioGRKO), we recently created an Adeno‐Associated Virus (AAV) to express mouse GR under the control of the cardiac‐specific troponin T(TnT) promoter to achieve cardiomyocytes‐specific GR expression (AAV‐GFP was used as a control). Our approach successfully achieved GR expression in the heart of 1‐month‐old CardioGRKO mice. We found that reinstallation of GR by AAV‐mediated gene transfer in cardiomyocytes completely rescued cardiovascular function in CardioGRKO mice. Specifically, ejection fraction (EF) and fractional shortening (FS) are significantly increased in the CardioGRKO treated with AAV‐mGRα compared to vector backbone (AAV‐GFP)‐treated control, suggesting the improvement of systolic function. In addition, left ventricular end‐systolic dimension (LVESD) and volume in systole (LV VolS) are significantly increased and restored to normal level compared to wild‐type controls. Further studies will be focused on the gene expression changes in genome wide expression analysis. In summary, the AAV‐mediated gene delivery system has allowed us to reinstall mGRα in cardioGRKO heart and thus to study the significance of GR signaling in heart diseases.

Support or Funding Information

NIH
Glucocorticoid Receptor Signaling is Critical for microRNA Gender‐Specific Regulation of Gene Expression in the Adult Mouse Heart.
Authors: Cruz‐Topete Diana , Oakley Robert H , Xu Xiaojiang , Cidlowski John A
Published: November 2023

Heart disease remains a leading cause of death among women in the US. Stress is considered a risk factor for heart disease. Growing evidence indicates that women are more susceptible and present higher stress levels at baseline than men, suggesting an important role for stress signaling in female cardiac health. Glucocorticoids are the primary regulators of the stress response and exert their actions by binding the glucocorticoid receptor (GR), a member of the nuclear receptor superfamily of transcription factors. GR has been shown to play a direct role in heart physiology, but its sexual dimorphic effects in cardiac gene expression are unclear. microRNAs regulate a plethora of cellular and physiological processes and dysregulation in their expression has been linked to heart disease. However, there are limited studies on gender‐specific differences in microRNAs expression and regulation in the adult heart. Our goal was to determine the contribution of GR signaling to the sexually dimorphic expression of microRNAs and their target genes in the mouse adult heart. To accomplish this objective, we performed a RNA‐seq and microRNA array on male and female hearts of the cardiomyocyte‐specific GR knockout (cardio GRKO) and their littermate controls (GR flox/flox). RNA‐seq data revealed that over 6000 genes are differentially expressed between male and female controls hearts at baseline. Data comparison between cardio GRKO and GR flox/flox showed that 3900 (65%) of the identified genes required GR for sexually dimorphic expression. In agreement with the RNA‐seq data, we found that 163 microRNAs were also influenced by sex and that GR was necessary for the gender‐specific regulation of 130 (~80%) of the identified microRNAs. Integrated analysis of microRNA and mRNA expression data revealed that 33 of the 163 identified sex‐dependent microRNAs target the expression of 4200 (70%) of the 6000 genes found differentially regulated in male and female control hearts. The expression of 25 of these 33 microRNAs depends on GR. Among the GR‐dependent microRNAs, we found miR 1–3p, miR‐29, miR‐128 and miR‐370, which are important biomarkers of heart failure in humans. Paired analysis of these 4 microRNAs and the RNA‐seq data, showed that 600 genes associated with cardiac hypertrophy, heart failure and hypoxia are targeted by these microRNAs. Among these genes, we found collagen type I alpha 1 (COL1A), epidermal growth factor receptor (EGFR), angiopoietin 2 (ANGPT2), myocardin (MYOCD) and endothelin receptor type B (EDNRB). These results suggest that GR regulation of microRNAs is a potential mechanism by which stress selectively modulates gene expression and consequently influences cardiac health in women.

Support or Funding Information

This work was supported by the Intramural Research Program of the National Institutes of Health, NIEHS.
The Uremic Metabolite, Trimethylamine‐N‐Oxide (TMAO), Directly Alters Cardiac Contractile Function
Authors: Oakley Carlee , Hendrix Matt , Grillo Michael , McCarthy Darla , Stubbs Jason R. , Wacker Michael J.
Published: November 2023

Background

TMAO is produced by intestinal microbial metabolism of phosphatidylcholine. Clearance of this uremic metabolite is dependent on urinary excretion. Therefore, as kidney function declines during chronic kidney disease (CKD), plasma levels of TMAO increase. Recently, elevated TMAO plasma levels have been reported to show concentration‐dependent associations with cardiovascular disease. However, there have been no experiments to determine the direct effect of TMAO on cardiac muscle. Therefore, our objective in this study was to determine if TMAO directly alters cardiac contractility and intracellular calcium levels.

Methods

CD1 adult mouse hearts were extracted, attached to a force transducer, oxygenated, and paced within an organ bath. Changes in contractility were measured after diffusing TMAO through the organ bath. As a follow‐up approach, mouse hearts were reverse perfused through the aorta via a modified Langendorff apparatus to facilitate TMAO tissue perfusion. To further explore a mechanism of action, embryonic cardiac myocytes were isolated and cultured to conduct epifluorescence calcium imaging experiments.

Results

Acute treatment with TMAO, in the diffusion‐based model, increased contraction amplitude of ventricular muscle 17% and 46% at 300 μM (P=0.08) and 3000 μM (P<0.05), respectively. TMAO also increased the slope and area under the curve of the contractile waveform compared to vehicle (n=4–6). Langendorff perfusion of hearts with 300 μM TMAO generated a greater response and increased average contraction height 34% (P<0.05; n=3). TMAO, at 300 and 3000 μM, also increased intracellular calcium levels in spontaneously beating cardiomyocytes.

Conclusions

TMAO directly increases cardiac contractility and intracelluar calcium levels. Acutely, these actions may be an adaptive response. Chronically, however, increases in intracellular calcium and cardiac stress promote cardiac remodeling and heart disease. Therefore, TMAO may be a novel contributor in the development of heart disease in CKD and thus an important therapeutic target for reducing morbidity and mortality.
Cardiac Hypertrophy and Myocyte Remodeling Are Exacerbated with Multiple Pregnancies
Authors: Wagner Marcus , Nelson Aaron , Flake Caela , Eaton Deborah , Bailey Beth A
Published: November 2023

Background

The female heart is adapted to support the hemodynamic load of pregnancy; it hypertrophies during pregnancy then returns to its pre‐pregnancy size relatively soon after delivery. This cardiac hypertrophy has generally been categorized as physiologic hypertrophy similar to that seen with exercise, however a reduction in cardiac function in late pregnancy has been suggested. In addition, the hemodynamic stress of pregnancy can induce a maladaptive, pathologic hypertrophy in a small number of women, and multiparity is thought to increase the risk for this maladaptive response. Because prolonged, persistent hemodynamic stress is associated with the development of pathological hypertrophy, multiple pregnancies may exacerbate any contractile changes seen in a single pregnancy. This study seeks to characterize the contractile properties of the pregnant myocardium during the first versus the third pregnancy.

Methods and Results

Late Pregnancy (LP) Female Swiss‐Webster mice were bred then studied at near term (Embryonic day 17–19). Multi‐pregnant mice (MP) were bred two times and allowed to wean each litter for 3 weeks then bred a third time and studied at embryonic day 17–19. LP and MP were compared to age‐matched, non‐pregnant (NP) controls. Significant cardiac hypertrophy occurred in LP vs NP (253 ± 7mg vs 216 ± 9mg, p<.05), and MP hearts were significantly larger than both LP and NP (339 ±18 mg, p<.01). Individual cardiac myocytes were isolated using collagenase‐based perfusion technique. Sarcomere length (light diffraction) and Ca2+ transients (fluo‐3) were measured at pacing rates of 1 Hz and at bath [Ca] of 1.5mM. Duration of twitch contraction was prolonged (p<.05) in MPM as measured by Time to 75% Recovery (.62 ± .05 vs .43 ± .04 sec in NPM). While twitch duration was prolonged in LPM (.53 ± .04 sec), the difference did not reach significance. Interestingly, the duration of the fluo‐3 calcium transients were similar in LPM (.24 ± .01 sec) and NPM (.25 ± .02 sec), but the MPM calcium transient duration (.34 ± .03 sec) was significantly prolonged (p<.05) compared to both NPM and LPM. There were no differences in other contractile parameters measured or in the fluo‐3 calcium transient properties. 10−7M Isoproterenol (ISO) was used to determine the responsiveness to adrenergic stimulation. ISO induced significantly enhanced contractility in NPM, LPM, and MPM, and the response was heightened in LPM and MPM such that the presence of ISO normalized the differences in the durations of twitch contractions between NPM, LPM, and MPM.

Conclusions

These results suggest that the cardiac hypertrophic response to pregnancy and subsequent alterations in contractile duration are exacerbated after multiple pregnancies. Enhanced response to ISO normalizes the contractile differences and suggests and increased cardiac reserve in LPM and MPM.

Support or Funding Information

Howard Hughes Medical Institute American Physiological Society
Endurance Exercise is Associated with Decreased Calpain to Calpastatin Ratios in Aged Rat Hearts
Authors: Sepe Joseph J , Hacker Timothy A , Diffee Gary M
Published: November 2023

The Ca2+‐activated protease calpain has been shown to play a deleterious role in the heart during the aging process, which ultimately impacts cardiac function. Calpastatin is an endogenous inhibitor of calpain, and the ratio of calpain to calpastatin in cardiac myocytes is physiologically important because this ratio greatly impacts the ability of Ca2+ to activate calpain. It has been suggested that activation of calpain may lead to proteolytic degradation of sarcomeric proteins, which ultimately contributes to impaired contractile function in the aging heart. Exercise training has been shown to mitigate and even reverse many aspects of the aging process in the heart. However, little is known about the effects of endurance exercise training on cardiac levels of calpain I, calpain II, or calpastatin in senescent hearts. To address this question, we conducted a 3‐month endurance treadmill training study in aged rats. Exercise capacity (as measured by time to exhaustion, EX‐TIME) and VO2max were measured in 22 month old rats and then animals were divided into Sedentary (SED) vs. Exercise (EX) groups. EX animals were trained 5 days a week for 3 months, with speed and duration gradually increased to the point that animals were running at 14 m/min for 45 min. At the end of the 3‐month training period, exercise capacity and VO2max were again measured in all animals. Mean EX‐TIME and VO2max values significantly (p <0.05) declined in SED animals from Pre‐training (PRE) to Post‐training (POST). EX‐TIME declined by 58% in POST compared to PRE, while VO2max declined by 21% in POST vs. PRE. In the EX animals there were significant (p <0.05) increases in EX‐TIME (POST values 172% of PRE) and VO2max (POST values 141% of PRE). To assess cardiac function, echocardiography was used to measure Ejection Fraction (EF%) and Fractional Shortening (FS%). EX training significantly (p <0.05) improved EF% and FS% compared to SED animals (EF% was 36% greater in EX compared to SED; FS% was 26% higher in EX compared to SED). Following sacrifice, rat hearts were quickly excised, and the left ventricle was dissected and flash frozen for Western blot analysis. Endurance exercise training was associated with decreased calpain to calpastatin ratios in aged hearts compared to sedentary counterparts (p <0.05). There was no significant difference in calpain II levels between EX and SED hearts. The improved calpain/calpastatin ratio exhibited by EX animals appears to be due to a significant decrease in calpain I levels compared to SED animals (p <0.05). These results show that endurance exercise increased cardiac function in senescent rats, and that this increase was associated with a decrease in the ratio of calpain to calpastatin. Changes in the cardiac levels of calpain I appear to underlie the decrease in this ratio.

Support or Funding Information

Marsh Center for Research in Exercise and Movement, University of Wisconsin‐Madison
5‐Methoxytryptophan Promotes Endothelial Cell Regeneration After Vascular Injury
Authors: Ho Yen‐Chun , Chen Chung‐Huang , Wu Meng‐Ling , Ho Hua‐Hui , Liang Li‐Yu , Yet Shaw‐Fang
Published: November 2023

5‐Methoxytryptophan (5‐MTP) is a metabolite of tryptophan metabolism. We recently demonstrated that 5‐MTP protects against intimal hyperplasia in a mouse neointima formation model of left common carotid artery ligation. The novel protective function of 5‐MTP is in part through reducing vascular smooth muscle cell proliferation and migration. In this study, we investigated the effect of 5‐MTP on endothelial cell (EC) function in response to vascular injury. We subjected approximately 12 weeks old C57BL/6 mice to a femoral artery guide wire injury model to denude endothelium. Following surgery, mice were treated with vehicle (PBS) or 25 mg/kg of 5‐MTP by intraperitoneal injection 3 times a week. Femoral arteries were harvested at early time points after injury for histological analysis to evaluate endothelium. CD31 staining revealed that endothelium was partially regenerated in the injured femoral arteries of vehicle‐treated mice. In comparison, 5‐MTP significantly increased endothelial regeneration in the injured arteries. Given that proinflammatory cytokine IL‐1β and TNF‐α are increased in the injured vessel wall, we next examined whether 5‐MTP could protect against EC dysfunction induced by IL‐1β or TNF‐α. Interestingly, 5‐MTP did not affect IL‐1β or TNF‐α‐reduced EC proliferation. In contrast, 5‐MTP ameliorates IL‐1β‐induced inflammatory cell adhesion and TNF‐α‐reduced EC migration. Taken together, our results suggest an important role of 5‐MTP on EC regeneration after vascular injury.

Support or Funding Information

This work was supported by the Ministry of Science and Technology of Taiwan grants MOST 104‐2321‐B‐400‐002 and 104‐2320‐B‐400‐006‐MY3, and National Health Research Institutes of Taiwan grant CS‐106‐PP‐05
Microparticle Uptake by Pulmonary Microvascular Endothelial Cells
Authors: Scruggs April K , Rich Thomas C , Bauer Natalie N
Published: November 2023

Microparticles (MPs) are important for the exchange of information between cells. MPs are extracellular vesicles that range in size from 0.1 to 1 μm in diameter. They are constitutively released from many cell types and are thought to play a role in vascular homeostasis. Furthermore the number and content of MPs are altered in pathologies involving inflammation and vascular cell activation. MPs carry proteins and RNAs from parent cells and are extracellular packets of information that affect target cell function. However, the mechanisms of targeting and delivery of the message are unknown. Understanding the message delivery system between MPs and target cells is the focus of this project.

We first tested whether MPs are internalized by rat pulmonary microvascular endothelial cells (PMVECs) in culture. Cells labeled with Hoechst nuclear stain were incubated with calcein green and PKH26 labeled MPs for four hours. The cells were imaged using an environmental chamber on a confocal microscope (Nikon A1R). An image stack (Z‐stack) was acquired every 15 minutes. MPs were observed within cells and the number of MPs within cells increased over the first 90 minutes of the experiment. Maximum MP‐cell association was observed at 1 hour and few MPs were observed inside cells after 3 hours. These results indicate that MPs are taken up by target PMVECs in a time‐dependent manner.

We next examined MP localization with organelles within cells. Organelle markers included monoclonal antibodies against early endosomes (Rab5 and EEA1), lysosomes (LAMP1), cis‐golgi (GRASP65) and trans‐golgi (TGN65), as well as labels for mitochondria, microtubules and cell nuclei. MPs were isolated from cell culture media via serial centrifugation and stained with a lipophilic membrane stain (PKH26). PMVECs were treated with stained MPs for 1, 2, 4 and 6 hours before organelle labeling. At one hour MPs colocalized with early endosomes (EEA1) and a small percentage colocalized with lysosomes (LAMP1). At six hours MPs colocalized with trans‐golgi. No overlap between MP‐staining and cis‐golgi, mitochondria, microtubules, or nuclei was observed at any time point. Taken together, these data indicate that MPs are internalized by target PMVECs. After internalization by target cells, most MPs are trafficked to the trans‐golgi for possible recycling of MP‐contents. Future studies will investigate the mechanisms responsible for internalization, whether payloads of MPs are delivered to specific organelles, and whether MP‐delivered proteins alter target cell function.

Support or Funding Information

This work was supported by NIH T32HL076125, P01HL066299, S10RR027535, and AHA 11SDG7390037.
Endothelium‐Independent Vasorelaxation Induced by Nitric Oxide Generation from the Novel Organic Nitrate NDOP Is Partially Mediated by Inward‐Rectifier Potassium Channels
Authors: de Paula Ricardo B , Montenegro Marcelo F , França‐Silva Maria S , Brandão Maria C , Diniz Thiago F , Lemos Virginia S , Athayde‐Filho Petrônio F , Magnani Marciane , Lundberg Jon O , Carlström Mattias , Braga Valdir A
Published: November 2023

Nitric oxide (NO) deficiency is crucially linked to the development and progression of cardiovascular diseases, including arterial hypertension. Here, the mechanisms underlying the vascular effects of the new organic nitrate 2‐nitrate‐1,3‐di(octanoxy)propane (NDOP), a putative nitric oxide (NO) donor, were investigated. A combination of in vitro and ex vivo approaches was used to assess the effects of NDOP on vascular reactivity and NO release in normotensive C57BL/6J mice. The intracellular fluorescent probe DAF‐2 DA revealed NDOP‐mediated NO formation in isolated vascular smooth muscle cells from mice aortae (53.2 ± 1.6 vs. 10.7 ± 0.9 a.u., n = 13, P < 0.05). The increased NO signal by NDOP was not affected by inhibition of endothelial nitric oxide synthase (eNOS). In isolated phenylephrine‐preconstricted aortic ring preparations, NDOP‐mediated vasorelaxation (10−8–10−3 M) was not reduced by previous inhibition of eNOS (112.5 ± 7.4 vs. 107.3 ± 7.5%, n = 7). However, inhibition of soluble guanylyl cyclase with ODQ (22.2 ± 6.7, n = 6, P < 0.05) and treatment with the NO scavengers PTIO (75.7 ± 5.6, n=7, P < 0.05) and PTIO+hydroxocobalamin (38.8 ± 4.6, n = 4, P < 0.05) all reduced NDOP‐mediated endothelium‐independent vasorelaxation in aortae compared with control. When aortic rings were contracted with modified Krebs solution by KCl (60 mM), NDOP‐induced vasorelaxation was attenuated (80.4 ± 5.2 n = 6, P < 0.05). Non‐selective inhibition of potassium (K+) channels using Krebs depolarizing solution (KCl 20 mM) reduced relaxation induced by NDOP (72.8 ± 3.4; n = 6, P < 0.05) suggesting the involvement of K+ channels. Selective blockade of large conductance calcium‐activated potassium channels (BKca) by tetraethylammonium (1 mM), voltage‐gated potassium channel (Kv) by 4‐aminopyridine (1 mM) or ATP‐sensitive potassium channels (KATP) by glibenclamide (10 μM) did not affect relaxation induced by NDOP. However, pre‐incubation with barium chloride (30 μM), an inward‐rectifier potassium channels (KIR) blocker reduced NDOP‐mediated vasorelaxation (73.7 ± 5.7, n = 6, P < 0.05) compared with control. Direct measurements of NO gas by chemiluminescence demonstrated dose‐dependent NO generation by NDOP (0.1 mM = 1.8 ± 0.2 ppb; 1 mM = 9.0 ± 0.8 ppb; 10 mM = 17.2 ± 2.3 ppb, n = 3), which was dependent on xanthine oxidoreductase activity. In conclusion, NDOP‐mediated vasorelaxation involves increased NO formation, which can stimulate sGC‐cGMP signaling and alter the function of inward‐rectifier potassium channels.

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Capes, CNPq and STINT
Phosphorylation and redistribution of Vangl2, a planar cell polarity protein, in endothelial cells in response to laminar shear stress
Authors: Curtis‐Whitchurch Logan , Rekapally Harish , Hoying Jay
Published: November 2023

In response to fluid flow, vascular endothelial cells (ECs) reorganize intracellular compartments to orchestrated regional signaling responses related to shear‐dependent EC function. While it is known that the EC cytoskeleton is involved in shear‐dependent EC reorganization, less is known about the complex dynamics integrating cytoskeleton reorganization and signaling. Using cell culture and ex vivo systems, we have evidence that human ECs express Vangl2, a highly conserved, transmembrane protein that organizes signaling in epithelial cells via a planar cell polarity mechanism in a phosphorylation‐dependent manner. Given its function as a cell signaling organizer, we hypothesized that Vangl2 plays a similar role in EC, but responds to unidirectional laminar fluid flow, a unique endothelial cell stimulus. Laminar flow (~15 dynes/cm2 for 2 to 48 hours) induced phosphorylation of Vangl2 and segregation into the detergent insoluble fraction in cultured human ECs as compared to static conditions. phospho‐Vangl2 co‐immunoprecipitated with vimentin, a cytoskeleton element associated with shear‐dependent EC polarization, and displayed colocalization in a time‐dependent manner following exposure to laminar flow. Knockdown of either vimentin or Vangl2 via siRNA revealed an interdependent dynamic between the two proteins. Finally, Vangl2 was asymmetrically located in the ECs of the intact mouse aorta, but uniformly distributed throughout the cells in areas of disturbed flow, similar to what we observed in cultured ECs. We report that the highly conserved, cell‐organizing protein, Vangl2 is expressed in ECs and that it differentially regulated by laminar flow. Our findings suggest a role for Vangl2 in organizing signaling systems within ECs in response to shear stress.

Support or Funding Information

Supported by the AHA and KSCIRC Neuroscience core facilities that are supported by the NIH/NIGMS P30 GM102507 grant.
Use of Phase Loop Plots to Assess Ca2+ Sensitivity of Force Generation in Airway Smooth Muscle
Authors: Han Young Soo , Sieck Gary
Published: November 2023

In excitation‐contraction coupling, a transient elevation of cytosolic Ca2+ concentration ([Ca2+]cyt) triggers a force response. The Ca2+ sensitivity of muscle reflects the relationship between [Ca2+]cyt and force, and in skeletal and cardiac muscle is often defined as the [Ca2+]cyt at which 50% force is generated. In smooth muscle, Ca2+ sensitivity has been assessed as the ratio of peak force to peak cytosolic [Ca2+]cyt. However, this measurement overlooks the dynamic nature of Ca2+ regulation of force generation. In the present study, in order to provide a dynamic index of Ca2+ sensitivity in airway smooth muscle (ASM), we simultaneously measured [Ca2+]cyt and force and determined their temporal dependence using a phase loop plot. We hypothesized that [Ca2+]cyt/force phase loop plot in ASM would be altered by conditions in which the extent of regulatory myosin light chain (rMLC) phosphorylation was affected.

Intact ASM strips were isolated from porcine tracheas obtained from local abattoir. ASM strips, were loaded with Fura‐2 AM (Ca2+ fluorescent indicator) and stimulated using 1 μM ACh. Isometric force and [Ca2+]cyt responses were simultaneously measured using a Guth Muscle Research System. In the first set of experiments we examined the effect of Y27623, a Rho‐kinase inhibitor, and calyculin A, a protein phosphatase inhibitor that affected Ca2+‐induced rMLC phosphorylation. In a second set of experiments, we examined the effect of cytochalasin D (Cyto‐D), an inhibitor of actin polymerization, which reduces force independent of Ca2+‐induced rMLC phosphorylation.

As expected, Y27623 decreased ACh‐induced isometric force but had no effect on evoked [Ca2+]cyt transients. Cyto‐D also decreased force with no effects on evoked [Ca2+]cyt. By comparison, calyculin A increased force with no effect on [Ca2+]cyt. Although these results suggest changes in Ca2+ sensitivity, the phase loop plots showed no significant shift indicative of changes in dynamic Ca2+ sensitivity. These results do not support our hypothesis that dynamic Ca2+ sensitivity in ASM is modulated by the extent of rMLC phosphorylation. Under these experimental conditions, it appears that isometric force generation in ASM is not entirely dependent on [Ca2+]cyt.

Support or Funding Information

Supported by NIH grant HL126451 (GCS)
In Mice, KCNQ Channel Modulators Affect Afferent Nerve Activity and not Urinary Bladder Smooth Muscle Contractility Directly
Authors: Tykocki Nathan R. , Heppner Thomas J. , Bonev Adrian D. , Nelson Mark T.
Published: November 2023

KCNQ channels are a family of voltage‐dependent K+ channels, which open in response to membrane depolarization to regulate cell excitability. KCNQ activators, such as retigabine, have been used successfully to treat epilepsy; interestingly, these drugs also caused urinary retention. Using ex vivo measurements of bladder pressure and afferent nerve activity, isometric contractility of bladder strips, and electrophysiological recordings from freshly isolated urinary bladder smooth muscle (UBSM) cells, we explored the role of KCNQ channels as regulators of murine urinary bladder function. In the ex vivo bladder, both the KCNQ activator retigabine and the voltage‐gated Ca2+ channel inhibitor diltiazem blocked transient contractions (TCs) and associated bursts of afferent nerve activity. In addition, retigabine reduced baseline afferent nerve activity in the absence of TCs. Simulating TCs by mechanical deformation of the bladder wall also caused bursts of afferent activity; retigabine, but not diltiazem, inhibited these afferent bursts as well. This suggests that the effects of retigabine were not limited to detrusor muscle, and also included inhibition of afferent nerve activity. In bladder strips, neither retigabine (10 μM) nor the KCNQ antagonist XE‐991 (10 μM) altered contractions in response to electrical field stimulation (0.5 – 50 Hz). Retigabine did, however, significantly reduce 60 mM KCl‐induced contractions, possibly indicative of voltage‐dependent Ca2+ channel inhibition and not KCNQ activation. Consistent with contractility data, retigabine inhibited currents through voltage‐dependent Ca2+ channels and had no effect on K+ currents, as measured in UBSM cells using the patch‐clamp technique. Lastly, retigabine had no effect on resting membrane potential in UBSM cells, using the current‐clamp configuration of the patch clamp technique. Taken together, these data indicate that UBSM cells lack functional KCNQ channels, and the effects of retigabine on detrusor contractility may be due instead to non‐specific inhibition of voltage‐gated Ca2+ channels. Alternatively, urinary retention caused by retigabine may be related to inhibition of afferent nerve activity and not diminished detrusor contractility.

Support or Funding Information

Supported by NIH K01DK103840 to NRT and NIH R37DK053832 to MTN.