bluuebunny/crossref_metadata_2025 huggingface dataset:
Angiotensin II‐preconditioning is associated with increased PKCɛ/PKCδ ratio and prosurvival kinases in mitochondria
Authors: Nuñez Rebeca , Javadov Sabzali , Escobales Nelson
Published: November 2023

Recent studies indicate that angiotensin II replicates the cardioprotective effects of ischemic preconditioning (IPC). However, the cellular mediators of angiotensin II preconditioning (APC) remain to be fully understood. In this study, Langendorff‐perfused rat hearts were subjected to IPC, APC or both (IPC/APC) followed by ischemia‐reperfusion (IR), to determine the protein expression of PKCɛ, PKCδ, Akt, Erk1/2, JNK, p38 MAPK and GSK‐3β in mitochondria, and the permeability transition pore (mPTP) opening. IPC, APC and IPC/APC increased the percent recovery of left ventricular developed pressure (LVDP), and reduced lactate dehydrogenase (LDH) release compared to controls. These effects were associated with marked increases in the mitochondrial PKCɛ/PKCδ ratio, Akt, Erk1/2 and JNK levels and mPTP inhibition. GSK‐3β levels were unaffected by the treatment protocols. Chelerythrine (5 μM), a pan‐PKC inhibitor, abolished the enhancements of PKCɛ but increased PKCδ expression, and inhibited Akt, Erk1/2, and JNK protein levels. The drug however, did not affect cardioprotection by APC and IPC/APC. Losartan (10 μM), an angiotensin II type 1 receptor (AT1‐R) blocker, abolished the APC‐stimulated LVDP increase and inhibited the protein levels of PKCɛ, Akt, Erk1/2, JNK, and p38. Both drugs reduced ischemic contracture and LDH release, and abolished the inhibition of mPTP by the preconditioning protocols. These results suggest that the cardioprotection induced by APC and IPC/APC involves an AT1‐R‐dependent, translocation of PKCɛ and downstream survival kinases to the mitochondria leading to mPTP inhibition. The observation of cardioprotection in chelerythrine‐treated hearts, suggests that reduced PKCɛ/PKCδ ratio might attenuate calcium overload and contracture‐induced damage at reperfusion.

Support or Funding Information

This study was supported by the NIH RCMI Program grant No. G12M007600, NHLBI grant SC1HL118669 (SJ), and the University of Puerto Rico.
Contractile Function of Coronary Arterioles is Impaired in Adiponectin‐deficient Mice
Authors: Bramy Jeremy , Gorman Katherine , Delp Michael , Ghosh Payal , Hotta Kazuki , Behnke Brad , Delp Judy
Published: November 2023

Increased circulating adiponectin correlates with cardioprotection whereas low levels of adiponectin have been linked to cardiovascular disease. Although adiponectin has been shown to promote differentiation towards a contractile phenotype in cultured vascular smooth muscle, a direct relationship between adiponectin and regulation of vascular smooth muscle function has not been demonstrated in vivo. Our previous work has shown that exercise training increases circulating adiponectin and reverses contractile dysfunction in coronary arterioles from aged rats. Here, we investigated the role of adiponectin in exercise training‐induced adaptations of smooth muscle contractile function in coronary arterioles. C57BL/6 wild‐type (WT) or homozygous adiponectin knockout (AdipoKO) mice were obtained at 10 weeks of age and underwent treadmill exercise (12 m/min, 5° incline, 1 hour/day, 5 days/week for 8 weeks) or remained sedentary in cages. Exercise‐trained WT mice demonstrated physiological cardiac hypertrophy, indicated by a significant increase in HW:BW ratio. In contrast, cardiac hypertrophy was absent in exercise‐trained AdipoKO mice. At the end of the training/sedentary period, coronary arterioles were isolated and contractile responses to endothelin (1e‐11 to 3e‐7M) and myogenic responsiveness to increasing intraluminal pressure (pressure increments from 10–130 cm H2O) were assessed. Myogenic responsiveness was significantly impaired in coronary arterioles from sedentary AdipoKO mice as compared to arterioles from sedentary WT mice (P<0.01), and this impairment was not mitigated by exercise training in AdipoKO mice. In coronary arterioles from WT mice, responsiveness to endothelin was enhanced by exercise training (P<0.01) but this effect of exercise training was absent in coronary arterioles from AdipoKO mice. These data indicate that adiponectin is necessary for maintenance and for exercise training‐induced adaptations of smooth muscle contractile function in coronary arterioles. These results suggest that impairment of vascular smooth muscle plasticity and contractile function of coronary arterioles contributes to the absence of physiological cardiac hypertrophy in exercise trained Adipo KO mice.
Treatment with the mitochondrial antioxidant peptide SS‐31 rescues neurovascular coupling responses and cerebrovascular endothelial function and improves cognition in aged mice
Authors: Tarantini Stefano , Valcarcel‐Ares Marta Noa , Yabluchanskiy Andriy , Fulop Gabor , Hertelendy Peter , Gautam Tripti , Farkas Eszter , Sonntag William E. , Csiszar Anna , Ungvari Zoltan
Published: November 2023

Moment‐to‐moment adjustment of cerebral blood flow (CBF) via neurovascular coupling has an essential role in maintenance of healthy cognitive function. In advanced age increased oxidative stress and cerebromicrovascular endothelial dysfunction impair neurovascular coupling, likely contributing to age‐related decline of higher cortical functions. There is increasing evidence showing that mitochondrial oxidative stress plays a critical role in a range of age‐related cellular impairments, but its role in neurovascular uncoupling remains unexplored. The present study was designed to test the hypothesis that attenuation of mitochondrial oxidative stress may exert beneficial effects on neurovascular coupling responses in aging. To test this hypothesis 24 month old C57BL/6 mice were treated with a cell permeable, mitochondria‐targeted antioxidant peptide (SS‐31; 10 mg/kg/day, i.p.) or vehicle for 2 weeks. Neurovascular coupling was assessed by measuring CBF responses (laser speckle contrast imaging) evoked by contralateral whisker stimulation. We found that neurovascular coupling responses were significantly impaired in aged mice. Treatment with SS‐31 significantly improved neurovascular coupling responses by increasing NO‐mediated cerebromicrovascular dilation, which was associated with significantly improved spatial working memory, motor skill learning and gait coordination. These findings are paralleled by the protective effects of SS‐31 on mitochondrial production of reactive oxygen species and mitochondrial respiration in cultured cerebromicrovascular endothelial cells derived from aged animals. Thus, mitochondrial oxidative stress contributes to age‐related cerebromicrovascular dysfunction, exacerbating cognitive decline. We propose that the antioxidant peptide SS‐31 should be considered for pharmacological microvascular protection for the prevention/treatment of age‐related vascular cognitive impairment (VCI).

Support or Funding Information

This work was supported by grants from the American Heart Association, the National Center for Complementary and Alternative Medicine, and the National Institute on Aging
Fibulin‐4 Is Important for Maintaining Vascular Wall Integrity in Large but Not Small Vessels
Authors: Halabi Carmen M , Lin Michelle , Broekelmann Thomas J. , Chu Mon‐Li , Mecham Robert P.
Published: November 2023

Deficiency or insufficiency of Fibulin‐4 (FBLN4) in mice leads to abnormal elastic fiber formation in large vessels with consequent aneurysm formation and arterial tortuosity, while FBLN4 mutations in humans lead to autosomal recessive cutis laxa type 1B (ARCL 1B), a multisystem disorder characterized by inelastic skin, arterial tortuosity, aortic aneurysms, and pulmonary emphysema and thought to be the result of aberrant elastic fiber formation. We sought to determine the consequences of a disease‐causing mutation in FBLN4 (E57K) on the cardiovascular system and vascular elastic fibers in a mouse model of ARCL 1B. Fbln4E57K/E57K mice were hypertensive and developed arterial elongation, tortuosity and ascending aortic aneurysms. Smooth muscle cell (SMC) organization within the wall of conduit arteries was abnormal and elastic fibers were fragmented and had a spongy, moth‐eaten appearance. Surprisingly, elastin and SMCs in the mesenteric, saphenous, and renal arteries of homozygous mutant mice were normal and seemingly unaffected by the E57K mutation. Furthermore, elastin and collagen content of large and small arteries were unchanged in Fbln4E57K/E57K mice. While the E57K mutation did not affect Fbln4 mRNA expression, FBLN4 protein levels were reduced in Fbln4E57K/E57K ascending aorta, but unchanged in mesenteric arteries. These results suggest a differential role for FBLN4 in elastic fiber assembly between elastic and muscular arteries. Future studies investigating tissue‐specific elastic fiber assembly may lead to novel therapeutic interventions for ARCL 1B and other disorders of elastic fiber assembly.

Support or Funding Information

CMH is a Scholar of the Child Health Research Center at Washington University School of Medicine, National Institutes of Health (NIH) K12‐HD076224. She also received support from the NIH training grant 2T32HD043010‐11 and the Mallinckrodt Foundation Physician‐Scientist Training Program Fellow support. This work was funded by NIH grants R01‐HL53325 and R01‐HL105314 to RPM.
Anti‐Ascites Adaptation of Liver to Sinusoidal Hypertension
Authors: Dongaonkar Ranjeet M , Laine Glen A , Quick Christopher M , Stewart Randolph H
Published: November 2023

The hepatic capsule is highly permeable to water and proteins. Increases in sinusoidal pressure as low as 1 mmHg have been reported to significantly elevate transudation across the capsule into the peritoneal cavity under normal conditions. However, despite the fact that hepatic sinusoidal and interstitial fluid pressures are significantly elevated with cirrhosis, transudation across the capsule is minimal and there is a lack of ascites. Because hepatic lymphatic vessels and thoracic ducts are enlarged and lymph flows are several times higher than normal levels, lack of ascites in compensated cirrhosis has been commonly ascribed to enhanced hepatic lymph flow. However, changes in capsular properties have yet to be investigated thoroughly. Therefore, the purpose of this study was to evaluate our hypothesis that sustained sinusoidal hypertension leads to decrease in capsular permeability and transudation rate. Capsular hydraulic conductance, reflection coefficient, transudation rate and hepatic lymph flow rate were evaluated following short‐term (2–3 weeks; StSH) and long term (5–6 weeks; LtSH) exposure to sinusoidal hypertension. Although hepatic capsular transudation and lymph flow rates were elevated in both groups, capsular hydraulic conductance and the transudation rate were lower and the reflection coefficient was higher in LtSH. Furthermore, comparison of hepatic interstitial fluid pressure‐lymph flow and hepatic interstitial fluid pressure‐transudation relationships revealed decreased sensitivity of capsular transudation to changes in hepatic interstitial fluid pressure in LtSH. Taken together, these findings suggest that adaptation of hepatic capsule precedes the enlargement of the hepatic lymphatic vessels and may primarily be responsible for the lack of ascites in compensated cirrhosis.
Ileal Smooth Muscle Thickness is Greater in Young Obese Zucker Rats
Authors: Burgess Andrea Rosalia , Trujillo Andrea Nichole , Barter Micaela , Breslin Jerome W
Published: November 2023

Obesity is a risk factor for many pathologies including heart disease, type 2 diabetes and certain types of cancers. At this time little is known about how obesity effects the morphology of the small intestine or gut. The purpose of this study was to determine what morphological differences exist in the smooth muscle of the small intestine, between lean and obese Zucker rats. We hypothesized that the thickness of the circular and longitudinal muscle layers of the small intestine from obese Zucker rats would be greater compared to their lean counterparts. Ileum and jejunum from lean and obese Zucker rats were harvested, fixed in buffered formalin overnight, processed, and embedded in paraffin. Thin cross sections of the tissues were mounted on slides, stained with H&E followed by analyses of longitudinal and circular smooth muscle layer thicknesses using digital photos and FIJI/IMageJ software (version 1.5). Smooth muscle thickness data was exported from an Excel document and statistics performed utilizing GraphPad Prism (version 6.0). In addition, ileum from lean and obese Zucker rats was harvested, bisected lengthwise with the lacteals facing up, and fixed in 4% PFA overnight and then washed with sucrose solutions. The ileum tissues were pinned onto 6‐well silicone plates and strips of villi (1‐2 thick) were cut under a dissecting microscope. The villi strips were incubated with a LYVE‐1 antibody to identify lymphatic endothelial cells of the lacteals and a PECAM‐1 antibody to label microcirculation within the villi. Longitudinal and circular smooth muscle layers, identified in the ileum with H&E staining, were significantly thicker in 8‐week old obese Zucker rats compared to lean rats. No significant differences in smooth muscle thickness were observed in 12‐week or 26–32‐week old obese versus lean rats in either the ileum or the jejunum. Histological data obtained from the small intestine partially supports the hypothesis that intestinal smooth muscle layer thickness in obese juvenile Zucker rats was greater, but only at the 8‐week time point. Preliminary immunolabeling with LYVE‐1 positively identified the structure of the lacteal, while the PECAM‐1 labeling was strong within the microvasculature of the villi. Although additional studies with human tissue, e.g., samples derived from intestinal biopsies or surgical resection, are required to understand the biomedical significance of our findings, our data raise the possibility that for humans at a young age obesity may cause structural changes in the small intestine.

Support or Funding Information

This work was supported by an APS IOSP Undergraduate Summer Research Fellowship IOS‐1238831.
Thoracic Duct Lymph Suppresses the Release of Inflammatory Mediators from Macrophages in vitro
Authors: Castillo Rudy , Hodge Lisa
Published: November 2023

The thoracic duct distributes a large pool of lymph rich in immune cells, inflammatory mediators and lipids to blood circulation. Recent literature suggests that during gastrointestinal injury, soluble factors in mesenteric lymph redistribute to the lung, via the thoracic duct, where they initiate inflammation and contribute to multiple organ dysfunction syndrome (MODS). Alternatively, normal mesenteric lymph has been shown to suppress inflammation in vivo and in vitro. Specifically, under inflammatory conditions, normal mesenteric lymph reduced the expression of cell adhesion molecules on endothelial cells and inhibited neutrophil apoptosis in vitro. However, the role of normal lymph on macrophage function remains unknown. Importantly, macrophages have been to shown to contribute to MODS. The purpose of this study was to investigate the effect of normal lymph on macrophage function. Specifically, we hypothesized that normal thoracic duct lymph (TDL) would suppress the release of inflammatory mediators by LPS‐activated macrophages.

To test this hypothesis, under anesthesia the thoracic ducts of four mongrel dogs were cannulated and lymph was collected. The TDL was centrifuged to remove cells and the TDL supernatant was frozen at −80°C. Murine RAW 264.7 macrophages were cultured in vitro with TDL at 5% total volume per well with or without of lipopolysaccharide (LPS). Twenty‐four hours after incubation at 37°C with 5% CO2, culture supernatants were assayed for nitric oxide (NO) and tumor necrosis factor‐alpha (TNF‐a) production. Macrophage viability was measured using flow cytometry with the markers Annexin V and Propidium Iodide.

TDL did not augment the production of NO, TNF‐a or alter cell viability by macrophages cultured in media alone. However, when macrophages were activated with LPS, TDL suppressed the release of NO and TNF‐a. Specifically, LPS induced the release of NO (21±0.5 uM) and TNF‐a (7185±828 pg/mL). The addition of TDL suppressed the LPS‐induced release of NO (15±0.6 uM) and TNF‐a (5016±425 pg/mL). Culture with LPS and/or TDL did not alter cell viability.

Our data suggests that during stimulation with LPS, a biological factor in lymph suppressed the release of inflammatory mediators by macrophages. Furthermore, cell viability was unaltered, suggesting that that TDL augments macrophage function. Future studies will focus on the ability of lymph to suppress the inflammatory response in disease models.

Support or Funding Information

This study was funded by the National Institutes of Health R01AT004361 (LMH).
Effect of Acute Increases in Retrograde Shear on Near‐Infrared Spectroscopy Derived Microvascular Reactivity
Authors: Ramos Macarena , Black Christopher D , Branch Patrece A , Caldwell Jacob T , Wardlow Garrett C , Ade Carl J
Published: November 2023

Periods of high vascular resistance lead to increases in retrograde shear rate, this augmented shear rate has been demonstrated to acutely decrease macrovascular endothelium‐dependent flow‐mediated dilation. However, it is unknown if similar exposures to a period of retrograde shear rate negatively impacts the microvascular response via near‐infrared spectroscopy (NIRS).

Purpose

Determine the effects of acute retrograde shear rate on microvascular responses in healthy, young humans. It was hypothesized that exposure to increased levels of retrograde shear rates would elicit a decrease in post‐occlusive microvascular reactivity measured via non‐invasive NIRS.

Methods

17 participants (22.7 ± 2.91 yrs) completed the study. Microvascular function was measured as the post‐occlusive reactive responses for tissue saturation index (TSI%) and total hemoglobin + myoglobin ([Hb] total) in the m. rectus femoris before and after a 30‐minute retrograde shear rate treatment. The retrograde shear rate in the treatment leg (T) was achieved via a cuff placed below the knee inflated to 75 mmHg, meanwhile the contralateral leg served as control (C). Shear rate was measured in the superficial femoral artery. Due to the limitations of non‐invasive 2D Doppler ultrasound interrogating smaller vessels, the evaluation of changes in shear rate in the microcirculation could not be performed.

Results

The cuff inflation significantly increased the retrograde shear in the treatment leg (71.2 ± 46.9 s−1 vs. 156.5 ± 63.5 s‐1, P<0.01) compared to the control leg (74.8 ± 58.6 s−1 vs. 66.6 ± 45.7 s−1, P=0.37). The peak post‐occlusive response for TSI% (T: 79.9 ±10.2 %; C: 76.8 ± 11.9%, P=0.15) and [Hb] total (T: 44.7± 29.7 μM C: 51.5 ± 43.3 μM, P=0.06) were not different between legs following the intervention. Similarly, the rate of change for StO2 (T: 6.0 ± 7.0 % s−1 104; C: 5.0 ± 6.0 % s−1 104) and [Hb] total (T: 4.0 ± 4.0 μM s−1 104; C: 3.0 ± 4.0 0 μM s−1 104) were not different between legs.

Conclusion

Increases in retrograde shear rate achieved via application of cuff pressure does not alter post‐occlusive NIRS derived indices of microvascular reactivity. Results may be due to different retrograde shear patterns elicited along the arterial tree, limitations of NIRS, or limited effect of retrograde shear on microvascular function.
Influence of Ischaemia‐Reperfusion Injury on Sensory Nerve Control of Vasodilation During a Subsequent Bout of Local Skin Heating in the Forearm
Authors: McGarr Gregory W. , Hodges Gary J. , Cheung Stephen S.
Published: November 2023

Cutaneous sensory nerves play a vital role in moderating microcirculatory blood flow by releasing vasodilatory neuropeptides in response to various stimuli including local heating. Ischaemia‐reperfusion (I‐R) injury is associated with altered sensory nerve conduction and depletion of sensory neuropeptide stores, which may contribute to an impaired vasodilatory response to local skin heating following the initial ischemic insult. In ten participants we evaluated the contribution of sensory nerves in driving the vasodilatory response to local heating in forearm skin before and after 20 minutes of ischaemia induced by inflating (220 mmHg) a standard blood pressure cuff around the arm. Red blood cell flux, an index of skin blood flow, was examined using laser‐Doppler probes with integrated local heating units. Flux values were then divided by mean arterial pressure and reported as cutaneous vascular conductance (CVC). Local heating was initiated prior to ischaemia and 20 minutes after the release of cuff occlusion at separate skin sites. At each site local temperature was controlled at 33°C and then increased (3°C min−1) to 42°C and held for ~30 min. Following this, local temperature was increased to 44°C for 20 min. Responses were compared between sites with sensory nerves blocked via local anaesthetic (EMLA) cream and untreated (UNTR) sites before and after I‐R. At UNTR sites, I‐R itself had no effect on CVC during the initial vasodilation (5%; P>0.05). However, relative to the UNTR site before I‐R, EMLA reduced CVC during the initial vasodilation by 45% before ischaemia and 20% after I‐R (both P<0.05). Neither I‐R nor EMLA treatment altered CVC during the secondary plateau at 42°C or at maximum heating to 44°C (all P>0.05). I‐R injury caused a significant delay in vasodilatory onset of 36% (P<0.05) at the UNTR site compared to delays of ~76% at EMLA sites before and after I‐R (both P<0.05). The time to initial vasodilatory peak between UNTR sites was unchanged by I‐R (2%; P>0.05). Conversely, it was significantly delayed by 24% (P<0.05) with EMLA application before I‐R, but not after (17%; P>0.05). We conclude that cutaneous sensory nerve impairment caused by 20 minutes of forearm I‐R injury significantly impairs the kinetics of the response to a rapid, non‐painful local heating stimulus, which may be associated with a higher temperature threshold for initiating vasodilation post‐ischaemia. This alteration of sensory nerve function may contribute to persistent tissue hypoxia and impaired thermoregulatory responses to local skin or whole body heating following an initial ischemic insult if temperature is not adequately controlled.

Support or Funding Information

Natural Sciences and Engineering Research Council of Canada: Discovery Grant (227912‐12, S.S. Cheung)
Phenotypic and Sex Differences in Microvesicles Released from Cultured Human Brain Microvascular Endothelial Cells in Response to Proinflammatory and Prothrombotic Stimuli
Authors: Hunter Larry W. , Jayachandran Muthuvel , Miller Virginia M.
Published: November 2023

Objective

Activation of brain microvascular endothelium by inflammatory and procoagulant stimuli increases expression of surface cell‐adhesion molecules (CAMs), releases endothelium‐derived microvesicles (MV) into the circulation, and increases permeability of the blood‐brain barrier (BBB). This study evaluated characteristics of MV released from human brain microvascular endothelial cells (HBMEC) following stimulation with proinflammatory or procoagulant stimuli and examined their effect on monolayer permeability.

Methods

Cultured HBMECs from age‐matched male and female donors were treated for 24 hours with medium supplemented with tumor necrosis factor alpha (TNFα, 20 ng/mL), thrombin (THR, 2 U/mL), or vehicle (control). MVs released into the medium were isolated by differential centrifugation, labeled with annexin V together with antibodies against PECAM‐1, ICAM‐1, E‐selectin, integrin aV, or MCAM, and then analyzed by digital flow cytometry. Permeability (mg/mL FITC‐dextran) of the HBMEC monolayer in response to MV treatment was quantified by fluorescence microplate assay of passage of FITC‐dextran across monolayer cells cultured on microporous supports.

Results

Fewer specific antigen‐positive MV were released from unstimulated male compared to female HBMECs. TNFa, but not THR induced early‐stage apoptosis; determined by fluorogenic Caspase‐3 assay in the HBMEC monolayers. TNFa induced release of all MV subtypes from male cells, whereas only integrin aV, ICAM‐1 and E‐selectin positive MVs were released from female cells. THR increased release of PECAM‐1, ICAM‐1, and MCAM positive MVs from male cells, but did not increase release of MV in female cells. After 24 hours exposure to MV (2,500 MV/mL) derived from TNFa‐treated female cells, permeability increased by 17%. Permeabiity was not increased when female cells were exposed to THR‐generated MV.

Conclusions

These results indicate that: 1) inflammatory and procoagulant stimuli release a different array of MV subtypes from male and female HBMECs; 2) specific stimuli‐derived MVs can modify HMBMEC permeability.

Support or Funding Information

(Supported by NIH P50 AG44170 and the Mayo Clinic Fnd.).
Assessment of Cutaneous Microcirculation Autoregulation with Phase and Linear Based Eulerian Video Magnification
Authors: Golden Michael , Smith Jillian , Oswald Derek , Haselby Cyrus , Murphy Robert , Heinking Kurt P , Henderson Kyle K
Published: November 2023

Introduction

The identification of low frequency variations in blood pressure recordings date back to the mid‐1800's. The mechanism for the low frequency oscillation is associated with intrinsic myogenic activity of smooth muscle cells in resistance arteries. Therefore, local metabolic demand modulates blood flow distribution. Non‐invasive assessment of the microcirculation in dermatology, diabetes, skin‐graft, and burn‐injury patients could facilitate assessment of therapeutic interventions. Eulerian Video Magnification (EVM) software was developed by the Massachusetts Institute of Technology (MIT) to magnify subtle changes in motion and color in video recordings. Using this technology, magnification of a video recording may reveal local changes in the cutaneous human microcirculation.

Hypothesis

We hypothesize that EVM can be used to assess oscillations in cutaneous blood flow velocity.

Methods

IRB approval was obtained to acquire physiological data from healthy graduate students (N = 10). Tissue Doppler was used to measure local changes in blood flow velocity on the forehead. Heart rate was assessed with an ECG and subjects breathed in time to a metronome (15 breaths/min) to normalize the effect of ventilation. Videos were processed using MIT's EVM Matlab code to magnify motion (Phase), or color & motion (Linear). All videos were magnified in the frequency range of interest (0.067–1.67 Hz; 4–100 cycles/min) to enhance human physiological frequencies. Gain was set to 50%; and a Gaussian filter was set to level 4. Phase magnification used a pyramid filtering scheme with octaves (1, ½, ¼) to specify the number of times the video is filtered; while linear magnification utilized chrominance attenuation to reduce color changes created by the magnification process. Red, green, and blue color spectrums extracted from magnified videos were analyzed with a Fast Fourier Transform (FFT) to identify magnified frequencies and power.

Results

Phase magnification (motion) and corresponding FFTs suggest strong correlations to heart rate and respiration rate but not tissue Doppler blood flow. On the other hand, linear magnification (color) and accompanying FFTs were strongly correlated to heart rate (r = 0.987; p = 0.001), respiration rate (r = 0.741; p = 0.014), and local tissue Doppler recordings (r = 0.945; p = 0.001).

Conclusion

These preliminary data demonstrate that a video recording of a human subject can be magnified with EVM to assess heart rate, respiration rate, and regional blood flow velocity oscillations in the cutaneous microcirculation. EVM may be a useful non‐invasive modality to evaluate changes in microcirculation autoregulation in human subjects.

Support or Funding Information

Midwestern University, Chicago College of Osteopathic Medicine, College of Health Sciences
An Ischemia Model on a Chip
Authors: Yin Wei , Abejar Louie , Rubenstein David A , Frame Mary D.
Published: November 2023

Ischemia refers to reduced blood flow to tissues. Coronary ischemia can cause heart attack, and cerebral ischemia can cause stroke. Ischemic heart attack caused more than 7 million deaths in 2008, counting for more than 12% of total global mortality. Short‐term fatality rate following first ischemic stroke could exceed 20%, and long‐term mortality rate following minor ischemic stroke was reported to be more than 30%. Many in vivo models have been established to study ischemia pathology and explore ways to re‐establish proper oxygen supply and blood flow to ischemic tissues. However, complicated in vivo environment often makes it difficult to identify small changes occurring at cellular/molecular level. In vitro models were also widely used to study ischemia. It is common to keep cultured cells under hypoxic conditions without flow. The major limitation of those in vitro models is the lack of physiological relevance, as ischemic tissue is usually surrounded by healthy blood vessels with normal blood supply. To investigate how hypoxia and stagnant flow conditions affect ischemia development, and how vascular cells within the ischemic area interact/communicate with surrounding blood vessels, a simple “ischemia model on a chip” was developed. Microfluidics channels (50 – 100 μm in diameter, and 50–100 μm apart) in various configurations were fabricated using PDMS. Human coronary artery endothelial cells (HCAEC) were grown to confluence within the channels. The “chip” was then isolated from ambient air using paraffin wax. Channels simulating healthy blood vessels were perfused with normal culture medium at physiological flow rate. Channels simulating ischemic blood vessels were filled with culture medium (no flow) with significantly reduced oxygen. The chip was kept at 37°C for 1 hour. A live‐dead assay was used to assess endothelial cell viability within the healthy and ischemic microfluidics channels. Immunofluorescence microscope was used to examine cell morphology. The results indicated that paraffin wax coating effectively maintained the low oxygen level within the ischemic microfluidics channels. HCAEC had high viability and normal morphology under physiological oxygen and flow conditions. Ischemic conditions induced cell death and decreased cell surface area. Overall, this “ischemia on a chip” model provided a simple and physiologically relevant solution to simulate complicated in vivo environment. Both flow conditions and oxygen level within the microfluidics channels can be controlled to mimic various physiological or pathological conditions; vascular endothelial cells can be cultured to cover the inside of the channels to simulate blood vessels. Thus, ischemia‐induced cellular responses (such as inflammation and angiogenesis) and ischemic‐healthy blood vessel communications can be investigated.
Second Hand Smoke Exposure Increases Susceptibility to Arrhythmogenic Cardiac Alternans
Authors: Wang Zhen , Wang Lianguo , Pinkerton Kent E. , Chen Chao‐Yin , Ripplinger Crystal M.
Published: November 2023

Background

Cardiovascular morbidity and mortality are significantly increased by second hand smoke (SHS) exposure in humans. However, the impact of SHS on cardiac electrophysiology, Ca2+ handling, and subsequent arrhythmia risk has not been investigated.

Methods

To investigate the effect of SHS on susceptibility to electrophysiological remodeling and arrhythmias, C57BL/6 male mice were exposed to SHS (3 mg/m3) or filtered air (FA) for 6 hours per day, 5 days per week for 4, 8, or 12 weeks (n = 4–5/group). Following exposure, hearts were excised and Langendorff perfused for optical mapping studies using voltage‐ and Ca2+‐ sensitive dyes (RH237 and Rhod‐2 AM, respectively). Two ventricular pacing protocols were performed: 1) S1–S2 (premature stimulus) to measure action potential duration (APD) and Ca2+ transient restitution across a range of premature stimuli, and 2) continuous pacing at progressively faster frequencies to induce cardiac alternans. For data sets in which SHS and FA groups were compared across a range of pacing frequencies, data were fit with a one phase exponential and the extra sum‐of‐squares (ESS) F test was performed. P<0.05 was considered statistically significant.

Results

SHS exposure did not alter mean APD or AP rise time at any exposure time point. However, the maximal slope of the APD restitution curve was steeper for SHS hearts at longer exposure time points (SHS vs. FA: slope = 0.004 vs. 0.002 normalized APD/ms at 8 weeks, p<0.05 and 0.005 vs. 0.001 normalized APD/ms at 12 weeks, p<0.05), indicating dynamical instability and increased arrhythmia risk following SHS exposure. APD and Ca2+ alternans magnitude progressively increased with increasing pacing frequency in all groups. At 4 and 8 weeks, there were no statistical differences in APD or Ca2+ alternans magnitude between SHS and FA groups. However, following 12 weeks of SHS exposure, both APD and Ca2+ alternans magnitude were significantly increased in the SHS compared to FA group (p<0.05 SHS vs. FA, ESS F test). Alterations in intracellular Ca2+ handling can drive both Ca2+ and APD alternans; therefore, dynamics of Ca2+ release and reuptake were assessed. SHS did not change the time constant of Ca2+ transient decay (tau) at any exposure time point. However, the recovery of Ca2+ transient amplitude with premature stimuli was delayed in SHS compared to FA hearts following 12 weeks of exposure (p<0.05 SHS vs. FA, ESS F test). This result suggests that Ca2+ release via ryanodine receptors may be impaired following SHS exposure.

Conclusion

SHS exposure increases susceptibility to cardiac alternans, a known precursor to ventricular arrhythmia. Increased alternans magnitude may be due to steepened APD restitution as well as impaired Ca2+‐induced Ca2+ release.

Support or Funding Information

NIH R01ES025229 and NIH R01HL111600
N‐Acetylcysteine Influence on Oxidative Stress and Cardiac Remodeling in Rats with Persistent Pressure Overload.
Authors: Reyes David Rafael Abreu , Moreno Camila Rosa , Gomes Mariana Janini , Pagan Luana Urbano , Damatto Ricardo L. , Damatto Felipe C. , Depra Igor Carbalho , Fernandes Ana Angélica Henrique , Okoshi Katashi , Okoshi Marina Politi
Published: November 2023

The influence of antioxidants on cardiac remodeling during persistent pressure overload is not clear. In this study we evaluated the effects of N‐acetylcysteine administration on cardiac structures and function and oxidative stress in rats with aortic‐stenosis (AS)‐induced heart failure.

Methods

Eighteen weeks after inducing AS, Wistar rats were assigned into control (Sham, n=16), AS (n=22), and AS treated with N‐acetylcysteine (AS‐NAC, 120 mg/day/kg, n=15) groups. Activity of myocardial antioxidant enzymes was analyzed by spectrophotometry. Statistical analysis was performed by ANOVA and Tukey test.

Results

Serum concentration of malondialdehyde was lower in AS‐NAC than Sham and AS groups [Sham 1.30 (1.18–1.43); AS 1.20 (1.00–1.50); AS‐NAC 0.90 (0.73–1.08) μmol/ml]. Myocardial concentration of hydroperoxide lipid was higher in AS than Sham and AS‐NAC (Sham 199±48.1; AS 301±36.0; AS‐NAC 181±41.3 nmol/g tissue). NAC preserved myocardial total glutathione concentration (Sham 20.8±3.00; AS 12.6±2.92; AS‐NAC 17.6±2.45 nmol/g tissue; p<0.05 AS vs Sham). Activity of catalase, superoxide dismutase, and glutathione peroxidase did not differ between groups. In echocardiographic evaluation, AS and AS‐NAC presented left ventricular concentric hypertrophy with systolic and diastolic dysfunction. NAC did not change ventricular remodeling.

Conclusion

N‐acetylcysteine administration reduces systemic and myocardial oxidative stress without changing cardiac remodeling in rats with aortic stenosis‐induced heart failure.

Support or Funding Information

FAPESP, CNPq, and AUIP/PAEDEx.
Exercise Training Augments Neuronal Nitric Oxide Synthase Dimerization in the Paraventricular Nucleus of Rats with Heart Failure
Authors: Sharma Neeru , Liu Xuefei , Llewellyn Tamra , Patel Kaushik P
Published: November 2023

Exercise training (ExT) is an established non‐pharmacological therapy that improves the health and quality of life in patients with chronic heart failure (CHF). Exaggerated sympathetic drive characterizes CHF due to an imbalance of the autonomic nervous system, possibly due to reduced neuronal nitric oxide synthase (nNOS) within the paraventricular nucleus (PVN). Previously we have demonstrated that during CHF, the catalytically active dimeric form of nNOS is significantly decreased (~40% decrease in dimer/monomer ratio) with a simultaneous increase in PIN expression (~50%), a protein that uncouples dimeric nNOS to monomers and facilitates its degradation. Dimerization of nNOS also requires tetrahydrobiopterin (BH4) for stability and activity. We have also shown that ExT improves NO‐mediated sympathoinhibition from the PVN. However, the underlying molecular mechanism remains unclear. We hypothesized that ExT restores the sympathetic drive by normalization of the levels and catalytically active form of nNOS by abrogating changes in PIN within the PVN of rats with CHF. CHF was induced in adult male Sprague‐Dawley rats by coronary artery ligation, which reliably mimics CHF in patients with myocardial infarction. After 4‐weeks, Sham and CHF rats were subjected to 3–4 weeks of progressive treadmill exercise. ExT significantly (p < 0.05) restored PIN expression (0.68±0.05 CHF vs 0.40±0.06* ExT‐CHF), and normalized dimer/monomer ratio (0.67±0.04 CHF vs 0.90±0.07* ExT‐CHF), in the PVN of rats with CHF. Moreover, we found decreased GTP cyclohydrolase 1(GCH1) expression: a rate‐limiting enzyme for BH4 biosynthesis in the PVN of CHF rats (0.74±0.03 Sham vs. 0.48±0.02* CHF), suggesting that perhaps reduced BH4 availability may also contribute to decreased nNOS dimer formation. Interestingly, CHF induced reduction in GCH1 expression was improved with ExT (0.48±0.02 CHF vs. 0.70±0.07* ExT‐CHF). Taken together, our findings revealed that ExT‐rectified, decreased PIN and GCH1 expression and improved dimer/monomer ratio of nNOS in the PVN, resulting in increased NO which ameliorates the enhanced sympathetic drive in CHF.

Support or Funding Information

Supported by AHA‐14SDG19980007 and NIH HL62222.
The Effects of Quarky Ca2+ Release on Ca2+ Spark in Cardiac Myocytes
Authors: Chen Xudong , Huo Yunlong , Tan Wenchang
Published: November 2023

Ca2+ sparks and quarks are significant events of Ca2+ releasing from the sarcoplasmic reticulum (SR) in cardiac myocytes, but caused by different releasing mechanisms. The objective of the study is to investigate the effects of Ca2+ quarks on the incidence and properties of Ca2+ sparks theoretically and experimentally. A two‐dimensional (2D) model was developed to simulate Ca2+ sparks and quarks based on the anomalous subdiffusion of Ca2+ in the cytoplasm. The numerical model included some specific features of Ca2+ quarks, such as long duration and small currents. The model was validated against the experimental measurements. It was found that Ca2+ sparks and quarks coexisted at a single release site. Quarky Ca2+ release (QCR) events from rogue ryanodine receptors (RyRs) can activate the clustered RyRs and hence result in Ca2+ sparks with different quantal units. Moreover, the sensitivity analysis showed some variables of rogue RyRs that may exist under pathological conditions significantly affected the incidence of Ca2+ sparks. This study provided some novel insights into the mechanisms of SR Ca2+ releasing events in cardiac myocytes.

Support or Funding Information

This work was supported by the National Natural Science Foundation of China (Grant No. 11272014 and No. 11328201) and the National Key Basic Research Program of China (Grant No. 2013CB531200).
Rewarming Shock Decreases Mitochondrial Respiratory Function in Cardiomyocytes
Authors: Cody Alex , Eshaq Abdulaziz , Schaible Niccole , Han Young Soo , Tveita Torkjel , Sieck Gary
Published: November 2023

In previous studies, we found that prolonged hypothermia (2 h or greater) followed by rewarming induces cardiac contractile dysfunction (rewarming shock) mimicking the effects of accidental hypothermia followed by rapid rewarming in trauma patients. We found that rewarming shock was also presented in isolated cardiomyocytes in which reduced contractility was attributed to PKA‐mediated TnI phosphorylation and reduced Ca2+ sensitivity. During hypothermia rewarming in cardiomyocytes there is increased production of reactive oxidant species (ROS ‐ superoxide anion), which may trigger the intracellular mechanisms underlying reduced contractility. We hypothesized that in cardiomyocytes, hypothermia/rewarming‐induced ROS production is due to altered mitochondrial respiratory function. To test this hypothesis, isolated cardiomyocytes from 6 rats underwent the following hypothermia/rewarming protocol in which they were electrically stimulated every 2 s: experimental temperature was cooled from 35°C to 15°C during the initial 30 min, then maintained at 15°C for 2 h, followed by rewarming to 35°C in the final 30 min. Time‐matched control cardiomyocytes were also electrically stimulated every 2 s and maintained at 35°C for 3 h. After the 3‐h protocol, cardiomyocytes were placed into separate chambers of an Oroboros Oxygraph (high‐resolution respirometry) to measure O2 consumption rate (OCR) under varying conditions. Initially, basal OCR was measured for 5 min followed by serial exposure to oligomycin (1 μM for 5 min) to block ATP synthase activity, FCCP (0.5 μM repeated 3 times for 5 min each) an H+ ionophore to dissipate mitochondrial membrane potential; and antimycin‐A (0.5 μM for 5 min) to block the electron transport chain. This procedure allowed assessment of the key parameters of mitochondrial respiratory function: basal and maximal respiration (after FCCP exposure), ATP production and proton leak (after oligomycin), and reserve respiratory capacity (calculated after antimycin‐A). We found that compared to control cardiomyocytes, hypothermia/rewarming decreased key parameters of mitochondrial function, which suggests that altered mitochondrial function may have contributed to increased ROS production. In addition, decreased OCR following rewarming may contribute to contractile dysfunction by limiting ATP supply in response to metabolic demand, as well as by contributing to a decline in cell viability (e.g., triggering apoptosis).

Support or Funding Information

5T32HL105355 (NSS); Norwegian Air Ambulance Foundation (YSH)
Acid Sensing Ion Channel 1 Contributes to Ca2+ Influx in Endothelial Tubes from Small Mesenteric Resistance Arteries
Authors: Garcia Selina M. , Herbert Lindsay M. , Naik Jay S. , Resta Thomas C , Jernigan Nikki L.
Published: November 2023

Our laboratory has previously shown that acid sensing ion channel 1 (ASIC1) is expressed in pulmonary arterial smooth muscle and contributes to agonist‐induced Ca2+ influx and vasoconstriction secondary to depletion of intracellular Ca2+ stores. In contrast to the vasoconstrictor role of ASIC1 in the pulmonary circulation; in mesenteric arteries, ASIC1 participates in acetylcholine (ACh)‐induced vasodilation. We hypothesized that ACh activates ASIC1‐mediated mesenteric endothelial cell Ca2+ influx through a store‐operated mechanism. To test this hypothesis, we freshly isolated endothelial tubes from small mesenteric resistance arteries (100–200 mm inner diameter). To specifically examine ASIC1‐mediated endothelial cell Ca2+ influx, independent of Ca2+ release from stores, we assessed Mn2+‐quenching of fura‐2 fluorescence in the presence or absence of the specific ASIC1 antagonist, psalmotoxin 1 (PcTX1; 20 nM). Whereas PcTX1 significantly diminished ACh (1 mM)‐induced Ca2+ influx, ASIC1 inhibition was without effect on Ca2+ entry in response to store depletion with cyclopiazonic acid (CPA; 10 μM). We conclude that ASIC1 contributes to ACh‐induced Ca2+ influx in mesenteric endothelial cells; however, this response does not appear to be mediated through a store‐operated mechanism. Future studies will examine the mechanism(s) of receptor‐mediated ASIC1 activation in endothelial cells, as well as potential downstream targets leading to endothelium‐dependent vasodilation.

Support or Funding Information

Supported by National Heart, Lung, and Blood Institute Grants R01 HL‐111084 (to N.L. Jernigan), T32 HL007736 (to T.C. Resta), R01 HL132883 (to T.C. Resta), and AHA 16GRNT27700010 (to T.C. Resta).
Endothelial Cell Mechanical Responses Are Dependent on Both Fluid Shear Stress and Tensile Strain
Authors: Steadman Elisabeth , Meza Daphne , Rubenstein David A , Yin Wei
Published: November 2023

Vascular endothelial cells are sensitive to their mechanical environment. They are regularly subject to both fluid shear stress as a result of blood flow, as well as cyclic tensile (or compression) strain due to blood vessel dilation (or contraction). The purpose of this study was to characterize physiological responses and morphological changes occurring in vascular endothelial cells when they were exposed to altered mechanical loadings, i.e., fluid shear stress and/or tensile strain. Human coronary artery endothelial cells (HCAEC) were grown to confluence in a 6‐well Flexcell plate coated with fibronectin. Cells were then placed in a programmable shearing and stretching device, where they were exposed to dynamic shear stress and cyclic tensile strain simultaneously at physiological or pathological levels. Three shear stress‐tensile strain conditions were used: 1) shear stress at 1 Pa and cyclic tensile strain at 7%, mimicking normal mechanical loading conditions in a healthy coronary artery; 2) shear stress at 3.7 Pa and tensile strain at 3%, mimicking pathological shear stress/strain conditions occurring at the stenosis (60%) throat in a diseased coronary artery; 3) shear stress at 0.7 Pa and tensile strain at 5%, mimicking pathological shear stress/strain conditions in the recirculation zones downstream of a stenosis. Following 1‐hour shear stress‐strain treatment, cells were fixed and cytoskeletal F‐actin filaments were stained using FITC conjugated phalloidin. Cell morphology was visualized using immunofluorescence microscope; cell area and elongation (ratio of long axis over short axis) were quantified using the Image J software. Under all conditions, cells that were exposed to shear stress and cyclic tensile strain concurrently were much larger compared to untreated cells or cells that were exposed to either shear stress or tensile strain alone. Stenosis conditions caused a significant increase in cell area compared to the other two conditions (normal and recirculation). In contrast, cell elongation decreased significantly under concurrent shear stress and tensile strain, with the lowest values observed under recirculation conditions. PECAM‐1 phosphorylation, an indicator of mechanotransduction pathway activation, was also measured, using a solid phase ELISA approach. The results demonstrated that the simultaneous stimulation from shear stress and tensile strain induced significant changes in endothelial cell surface phosphorylated PECAM‐1 expression, compared to when cells were exposed to only shear stress or tensile strain. These findings indicated the complex interplay between altered flow‐induced shear stress and tensile strain could significantly affect vascular endothelial cell responses. Therefore, to better understand how mechanical conditions affect endothelial cell mechanotransduction and functions, both shear stress and tensile strain need to be considered.