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Aim: This study aimed to investigate the phenolic composition, antioxidant capacity, and toxicity of aqueous extracts of Calamintha nepeta L. leaves and their potential vasorelaxant effects., Methods: Aqueous extracts of Calamintha nepeta L. were prepared by three extraction methods: decoction, infusion, and maceration. The total phenolic contents of the extracts and their antioxidant properties were investigated. The toxicity was evaluated by Artemia salina lethality bioassay. The decoction extract was analyzed by HPLC for its chemical profile and was also used to evaluate the vasorelaxant effect on thoracic aortic rings isolated from healthy Sprague Dawley rats. Pre-contraction was induced by phenylephrine, followed by cumulative doses of the extract (0.001 up to 250 µg/ml)., Results: Aqueous extracts of Calamintha nepeta L. showed noticeable radical scavenging and chelating activities. However, the decoction extract exhibited the most powerful antioxidant capacity. No toxicity was recorded for the extracts obtained by decoction and infusion. Caffeic acid, quercetin, and rosmarinic acid were the main identified compounds. Notably, the aqueous extract obtained by decoction induced significant relaxation in endothelium-intact aortic rings at lower concentrations, and at higher concentrations in denuded aortic rings., Conclusion: This study reveals that Calamintha nepeta L. extracted with a decoction method possesses potent antioxidant capacity and has an endothelium-dependent vasorelaxant effect., Competing Interests: The authors declare that they have no financial interests or personal relationships with other people or organizations that could have influenced the work reported in this paper.

Individuals with type 2 diabetes mellitus (T2DM) are at an increased risk for heart failure, yet preventive cardiac care is suboptimal in this population. Pyridoxamine (PM), a vitamin B 6 analog, has been shown to exert protective effects in metabolic and cardiovascular diseases. In this study, we aimed to investigate whether PM limits adverse cardiac remodeling and dysfunction in rats who develop T2DM. Male rats received a standard chow diet or Western diet (WD) for 18 weeks to induce prediabetes. One WD group received additional PM (1 g/L) via drinking water. Glucose tolerance was assessed with a 1 h oral glucose tolerance test. Cardiac function was evaluated using echocardiography and hemodynamic measurements. Histology on left ventricular (LV) tissue was performed. Treatment with PM prevented the increase in fasting plasma glucose levels compared to WD-fed rats ( p < 0.05). LV cardiac dilation tended to be prevented using PM supplementation. In LV tissue, PM limited an increase in interstitial collagen deposition ( p < 0.05) seen in WD-fed rats. PM tended to decrease 3-nitrotyrosine and significantly lowered 4-hydroxynonenal content compared to WD-fed rats. We conclude that PM alleviates interstitial fibrosis and oxidative stress in the hearts of WD-induced prediabetic rats.

Microglia activity can drive excessive synaptic loss during the prodromal phase of Alzheimer's disease (AD) and is associated with lowered cyclic adenosine monophosphate (cAMP) due to cAMP phosphodiesterase 4B (PDE4B). This study aimed to investigate whether long-term inhibition of PDE4B by A33 (3 mg/kg/day) can prevent synapse loss and its associated cognitive decline in APPswe/PS1dE9 mice. This model is characterized by a chimeric mouse/human APP with the Swedish mutation and human PSEN1 lacking exon 9 (dE9), both under the control of the mouse prion protein promoter. The effects on cognitive function of prolonged A33 treatment from 20 days to 4 months of age, was assessed at 7-8 months. PDE4B inhibition significantly improved both the working and spatial memory of APPswe/PSdE9 mice after treatment ended. At the cellular level, in vitro inhibition of PDE4B induced microglial filopodia formation, suggesting that regulation of PDE4B activity can counteract microglia activation. Further research is needed to investigate if this could prevent microglia from adopting their 'disease-associated microglia (DAM)' phenotype in vivo. These findings support the possibility that PDE4B is a potential target in combating AD pathology and that early intervention using A33 may be a promising treatment strategy for AD.

Endurance exercise training is a promising cardioprotective strategy in type 2 diabetes mellitus (T2DM), but the impact of its intensity is not clear. We aimed to investigate whether and how isocaloric moderate-intensity exercise training (MIT) and high-intensity interval exercise training (HIIT) could prevent the adverse cardiac remodeling and dysfunction that develop T2DM in rats. Male rats received a Western diet (WD) to induce T2DM and underwent a sedentary lifestyle ( n = 7), MIT ( n = 7) or HIIT ( n = 8). Insulin resistance was defined as the HOMA-IR value. Cardiac function was assessed with left ventricular (LV) echocardiography and invasive hemodynamics. A qPCR and histology of LV tissue unraveled underlying mechanisms. We found that MIT and HIIT halted T2DM development compared to in sedentary WD rats ( p < 0.05). Both interventions prevented increases in LV end-systolic pressure, wall thickness and interstitial collagen content ( p < 0.05). In LV tissue, HIIT tended to upregulate the gene expression of an ROS-generating enzyme (NOX4), while both modalities increased proinflammatory macrophage markers and cytokines (CD86, TNF-α, IL-1β; p < 0.05). HIIT promoted antioxidant and dicarbonyl defense systems (SOD2, glyoxalase 1; p < 0.05) whereas MIT elevated anti-inflammatory macrophage marker expression (CD206, CD163; p < 0.01). We conclude that both MIT and HIIT limit WD-induced T2DM with diastolic dysfunction and pathological LV hypertrophy, possibly using different adaptive mechanisms., Competing Interests: The authors declare no conflicts of interest.

The use of doxorubicin (DOX) chemotherapy is restricted due to dose-dependent cardiotoxicity. Pyridoxamine (PM) is a vitamin B6 derivative with favorable effects on diverse cardiovascular diseases, suggesting a cardioprotective effect on DOX-induced cardiotoxicity. The cardioprotective nature of PM was investigated in a rat model of DOX-induced cardiotoxicity. Six-week-old female Sprague Dawley rats were treated intravenously with 2 mg/kg DOX or saline (CTRL) weekly for eight weeks. Two other groups received PM via the drinking water next to DOX (DOX+PM) or saline (CTRL+PM). Echocardiography, strain analysis, and hemodynamic measurements were performed to evaluate cardiac function. Fibrotic remodeling, myocardial inflammation, oxidative stress, apoptosis, and ferroptosis were evaluated by various in vitro techniques. PM significantly attenuated DOX-induced left ventricular (LV) dilated cardiomyopathy and limited TGF-β1-related LV fibrotic remodeling and macrophage-driven myocardial inflammation. PM protected against DOX-induced ferroptosis, as evidenced by restored DOX-induced disturbance of redox balance, improved cytosolic and mitochondrial iron regulation, and reduced mitochondrial damage at the gene level. In conclusion, PM attenuated the development of cardiac damage after DOX treatment by reducing myocardial fibrosis, inflammation, and mitochondrial damage and by restoring redox and iron regulation at the gene level, suggesting that PM may be a novel cardioprotective strategy for DOX-induced cardiomyopathy.

Doxorubicin (DOX) is commonly used in cancer treatment but associated with cardiotoxicity. Pyridoxamine (PM), a vitamin B6 derivative, could be a cardioprotectant. This study investigated the effect of PM on DOX cardiotoxicity and DOX antitumor effectiveness. Sprague Dawley rats were treated intravenously with DOX (2 mg/kg/week) or saline over eight weeks. Two other groups received PM via oral intake (1 g/L in water bottles) next to DOX or saline. Echocardiography was performed after eight weeks. PM treatment significantly attenuated the DOX-induced reduction in left ventricular ejection fraction (72 ± 2% vs. 58 ± 3% in DOX; p < 0.001) and increase in left ventricular end-systolic volume (0.24 ± 0.02 µL/cm 2 vs. 0.38 ± 0.03 µL/cm 2 in DOX; p < 0.0001). Additionally, LA7 tumor cells were exposed to DOX, PM, or DOX and PM for 24 h, 48 h, and 72 h. Cell viability, proliferation, cytotoxicity, and apoptosis were assessed. DOX significantly reduced LA7 cell viability and proliferation ( p < 0.0001) and increased cytotoxicity ( p < 0.05) and cleaved caspase-3 ( p < 0.001). Concomitant PM treatment did not alter the DOX effect on LA7 cells. In conclusion, PM attenuated DOX-induced cardiomyopathy in vivo without affecting the antitumor effect of DOX in vitro, highlighting PM as a promising cardioprotectant for DOX-induced cardiotoxicity.

Exercise training is an encouraging approach to treat cardiac dysfunction in type 2 diabetes (T2DM), but the impact of its intensity is not understood. We aim to investigate whether and, if so, how moderate-intensity training (MIT) and high-intensity interval training (HIIT) alleviate adverse cardiac remodeling and dysfunction in rats with T2DM. Male rats received standard chow (n = 10) or Western diet (WD) to induce T2DM. Hereafter, WD rats were subjected to a 12-week sedentary lifestyle (n = 8), running MIT (n = 7) or HIIT (n = 7). Insulin resistance and glucose tolerance were assessed during the oral glucose tolerance test. Plasma advanced glycation end-products (AGEs) were evaluated. Echocardiography and hemodynamic measurements evaluated cardiac function. Underlying cardiac mechanisms were investigated by histology, western blot and colorimetry. We found that MIT and HIIT lowered insulin resistance and blood glucose levels compared to sedentary WD rats. MIT decreased harmful plasma AGE levels. In the heart, MIT and HIIT lowered end-diastolic pressure, left ventricular wall thickness and interstitial collagen deposition. Cardiac citrate synthase activity, mitochondrial oxidative capacity marker, raised after both exercise training modalities. We conclude that MIT and HIIT are effective in alleviating diastolic dysfunction and pathological cardiac remodeling in T2DM, by lowering fibrosis and optimizing mitochondrial capacity.

Aims: To compare the cardiac function and pulmonary vascular function during exercise between dyspnoeic and non-dyspnoeic patients with Type 2 diabetes mellitus (T2DM)., Methods and Results: Forty-seven T2DM patients with unexplained dyspnoea and 50 asymptomatic T2DM patients underwent exercise echocardiography combined with ergospirometry. Left ventricular (LV) function [stroke volume, cardiac output (CO), LV ejection fraction, systolic annular velocity (s')], estimated LV filling pressures (E/e'), mean pulmonary arterial pressures (mPAPs) and mPAP/COslope were assessed at rest, low- and high-intensity exercise with colloid contrast. Groups had similar patient characteristics, glycemic control, stroke volume, CO, LV ejection fraction, and E/e' (P > 0.05). The dyspnoeic group had significantly lower systolic LV reserve at peak exercise (s') (P = 0.021) with a significant interaction effect (P < 0.001). The dyspnoeic group also had significantly higher mPAP and mPAP/CO at rest and exercise (P < 0.001) with significant interaction for mPAP (P < 0.009) and insignificant for mPAP/CO (P = 0.385). There was no significant difference in mPAP/COslope between groups (P = 0.706). However, about 61% of dyspnoeic vs. 30% of non-dyspnoeic group had mPAP/COslope > 3 (P = 0.009). The mPAP/COslope negatively predicted V̇O2peak in dyspneic group (β = -1.86, 95% CI: -2.75, -0.98; multivariate model R2:0.54)., Conclusion: Pulmonary hypertension and less LV systolic reserve detected by exercise echocardiography with colloid contrast underlie unexplained exertional dyspnoea and reduced exercise capacity in T2DM., Competing Interests: Conflict of interest: None declared., (© The Author(s) 2022. Published by Oxford University Press on behalf of the European Society of Cardiology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Chronically increased levels of high molecular weight advanced glycation end products (HMW-AGEs) are known to induce cardiovascular dysfunction. Whether an acute increase in HMW-AGE levels affects vascular function remains unknown. In this study, we examined whether acute exposure to HMW-AGEs disturbs aortic vasomotor function. Aortae were obtained from healthy male rats and were acutely pre-treated with HMW-AGEs in organ baths. Aortic relaxation responses to cumulative doses of acetylcholine (ACh), in the presence or absence of superoxide dismutase (SOD), were measured after precontraction with phenylephrine (PE). Furthermore, levels of 3-nitrotyrosine were evaluated on aortic paraffine sections. In our study, we show that acute exposure to HMW-AGEs significantly decreases the aortic relaxation response to ACh. SOD pre-treatment prevents acute HMW-AGEs-induced impairment by limiting superoxide formation. In conclusion, our data demonstrate that acute exposure to HMW-AGEs causes adverse vascular remodelling, characterised by disturbed vasomotor function due to increased oxidative stress. These results create opportunities for future research regarding the acute role of HMW-AGEs in cardiovascular dysfunction.

Purpose: Exercise training improves exercise capacity in type 2 diabetes mellitus (T2DM). It remains to be elucidated whether such improvements result from cardiac or peripheral muscular adaptations, and whether these are intensity dependent., Methods: 27 patients with T2DM [without known cardiovascular disease (CVD)] were randomized to high-intensity interval training (HIIT, n = 15) or moderate-intensity endurance training (MIT, n = 12) for 24 weeks (3 sessions/week). Exercise echocardiography was applied to investigate cardiac output (CO) and oxygen (O 2 ) extraction during exercise, while exercise capacity [([Formula: see text] (mL/kg/min)] was examined via cardiopulmonary exercise testing at baseline and after 12 and 24 weeks of exercise training, respectively. Changes in glycaemic control (HbA1c and glucose tolerance), lipid profile and body composition were also evaluated., Results: 19 patients completed 24 weeks of HIIT (n = 10, 66 ± 11 years) or MIT (n = 9, 61 ± 5 years). HIIT and MIT similarly improved glucose tolerance (p Time  = 0.001, p Interaction  > 0.05), [Formula: see text] (mL/kg/min) (p Time  = 0.001, p Interaction  > 0.05), and exercise performance (W peak ) (p Time  < 0.001, p Interaction  > 0.05). O 2 extraction increased to a greater extent after 24 weeks of MIT (56.5%, p 1  = 0.009, p Time  = 0.001, p Interaction  = 0.007). CO and left ventricular longitudinal strain (LS) during exercise remained unchanged (p Time  > 0.05). A reduction in HbA1c was correlated with absolute changes in LS after 12 weeks of MIT (r = - 0.792, p = 0.019, LS at rest) or HIIT (r = - 0.782, p = 0.038, LS at peak exercise)., Conclusion: In patients with well-controlled T2DM, MIT and HIIT improved exercise capacity, mainly resulting from increments in O 2 extraction capacity, rather than changes in cardiac output. In particular, MIT seemed highly effective to generate these peripheral adaptations., Trial Registration: NCT03299790, initially released 09/12/2017., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Aims: Type 2 diabetes mellitus (T2DM) is associated with reduced exercise capacity and cardiovascular diseases, both increasing morbidity and risk for premature death. As exercise intolerance often relates to cardiac dysfunction, it remains to be elucidated to what extent such an interplay occurs in T2DM patients without overt cardiovascular diseases. Design: Cross-sectional study, NCT03299790., Methods and Results: Fifty-three T2DM patients underwent exercise echocardiography (semi-supine bicycle) with combined ergospirometry. Cardiac output (CO), left ventricular longitudinal strain (LS), oxygen uptake (O2), and oxygen (O2) extraction were assessed simultaneously at rest, low-intensity exercise, and high-intensity exercise. Glycaemic control and lipid profile were assessed in the fasted state. Participants were assigned according to their exercise capacity being adequate or impaired (EXadequate: O2peak <80% and EXimpaired: O2peak ≥80% of predicted O2peak) to compare O2 extraction, CO, and LS at all stages. Thirty-eight participants (EXimpaired: n = 20 and EXadequate: n = 18) were included in the analyses. Groups were similar regarding HbA1c, age, and sex (P > 0.05). At rest, CO was similar in the EXimpaired group vs. EXadequate group (5.1 ± 1 L/min vs. 4.6 ± 1.4 L/min, P > 0.05) and increased equally during exercise. EXimpaired patients displayed a 30.7% smaller increase in O2 extraction during exercise compared to the EXadequate group (P = 0.016) which resulted in a lower O2 extraction at high-intensity exercise (12.5 ± 2.8 mL/dL vs. 15.3 ± 3.9 mL/dL, P = 0.012). Left ventricular longitudinal strain was similar at rest but increased significantly less in the EXimpaired vs. EXadequate patients (1.9 ± 2.5% vs. 5.9 ± 4.1%, P = 0.004)., Conclusions: In asymptomatic T2DM patients, an impaired exercise capacity is associated with an impaired response in oxygen extraction and myocardial deformation (LS)., Trial Registry: Effect of High-intensity Interval Training on Cardiac Function and Regulation of Glycemic Control in Diabetic Cardiomyopathy (https://clinicaltrials.gov/ct2/show/NCT03299790)., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2020. For permissions, please email: journals.permissions@oup.com.)

Myocardial infarction irreversibly destroys millions of cardiomyocytes in the ventricle, making it the leading cause of heart failure worldwide. Over the past two decades, many progenitor and stem cell types were proposed as the ideal candidate to regenerate the heart after injury. The potential of stem cell therapy has been investigated thoroughly in animal and human studies, aiming at cardiac repair by true tissue replacement, by immune modulation, or by the secretion of paracrine factors that stimulate endogenous repair processes. Despite some successful results in animal models, the outcome from clinical trials remains overall disappointing, largely due to the limited stem cell survival and retention after transplantation. Extensive interest was developed regarding the combinational use of stem cells and various priming strategies to improve the efficacy of regenerative cell therapy. In this review, we provide a critical discussion of the different stem cell types investigated in preclinical and clinical studies in the field of cardiac repair. Moreover, we give an update on the potential of stem cell combinations as well as preconditioning and explore the future promises of these novel regenerative strategies., (© 2021 Wiley Periodicals LLC.)

Objectives: Cardiovascular diseases and exercise intolerance elevate mortality in type 1 diabetes (T1D). Left ventricular systolic and diastolic function are already affected in adolescents with T1D, displaying poor glycemic control (glycated hemoglobin [A1C]>7.5%) and exercise intolerance. We investigated the extent to which left ventricular function is affected by disease severity/duration and whether this is related to exercise capacity., Methods: Transthoracic echocardiography was performed in 19 adolescents with T1D (14.8±1.9 years old, A1C 7.4±0.9%) and 19 controls (14.4±1.3 years old, A1C 5.3±0.2%), matched for age and Tanner stage. Diastolic and systolic (ejection fraction [EF]) function were assessed. Cardiopulmonary exercise testing was used to evaluate exercise capacity, as measured by peak oxygen uptake (VO 2peak )., Results: VO 2peak and left ventricular systolic and diastolic function were similar in both groups. Within the T1D group, EF was negatively associated with disease duration (r=-0.79 corrected for age, standardized body mass index, glucose variability and VO 2peak ; p=0.011). Regression analyses revealed that 37.6% of the variance in EF could be attributed to disease duration., Conclusions: Although left ventricular systolic and diastolic function are preserved in T1D with adequate exercise capacity, disease duration negatively affects EF. The detrimental effects of T1D seem to be driven by disease duration, rather than by disease severity, at least during adolescence. Young patients with T1D may, therefore, benefit from cardiovascular evaluation in order to detect cardiovascular abnormalities early in the disease course, and, therefore, improve long-term cardiovascular health., (Copyright © 2021 Canadian Diabetes Association. Published by Elsevier Inc. All rights reserved.)

Myocardial infarction (MI) occurs when the coronary blood supply is interrupted. As a consequence, cardiomyocytes are irreversibly damaged and lost. Unfortunately, current therapies for MI are unable to prevent progression towards heart failure. As the renewal rate of cardiomyocytes is minimal, the optimal treatment should achieve effective cardiac regeneration, possibly with stem cells transplantation. In that context, our research group identified the cardiac atrial appendage stem cells (CASCs) as a new cellular therapy. However, CASCs are transplanted into a hostile environment, with elevated levels of advanced glycation end products (AGEs), which may affect their regenerative potential. In this study, we hypothesize that pyridoxamine (PM), a vitamin B6 derivative, could further enhance the regenerative capacities of CASCs transplanted after MI by reducing AGEs' formation. Methods and Results: MI was induced in rats by ligation of the left anterior descending artery. Animals were assigned to either no therapy (MI), CASCs transplantation (MI + CASCs), or CASCs transplantation supplemented with PM treatment (MI + CASCs + PM). Four weeks post-surgery, global cardiac function and infarct size were improved upon CASCs transplantation. Interstitial collagen deposition, evaluated on cryosections, was decreased in the MI animals transplanted with CASCs. Contractile properties of resident left ventricular cardiomyocytes were assessed by unloaded cell shortening. CASCs transplantation prevented cardiomyocyte shortening deterioration. Even if PM significantly reduced cardiac levels of AGEs, cardiac outcome was not further improved. Conclusion: Limiting AGEs' formation with PM during an ischemic injury in vivo did not further enhance the improved cardiac phenotype obtained with CASCs transplantation. Whether AGEs play an important deleterious role in the setting of stem cell therapy after MI warrants further examination.

Konzo is a toxico-nutritional upper motor neuron disease causing a spastic paraparesis in schoolchildren and childbearing women in some African countries. Almost a century since the first description of konzo, its underlying etiopathogenic mechanisms and causative agent remain unknown. This paper aims at refreshing the current knowledge of konzo determinants and pathogenesis in order to enlighten potential new research and management perspectives. Literature research was performed in PubMed and Web of Science databases according to the PRISMA methodology. Available data show that cassava-derived cyanide poisoning and protein malnutrition constitute two well-documented risk factors of konzo. However, observational studies have failed to demonstrate the causal relationship between konzo and cyanide poisoning. Thiocyanate, the current marker of choice of cyanide exposure, may underestimate the actual level of cyanide poisoning in konzo patients as a larger amount of cyanide is detoxified via other unusual pathways in the context of protein malnutrition characterizing these patients. Furthermore, the appearance of konzo may be the consequence of the interplay of several factors including cyanide metabolites, nutritional deficiencies, psycho-emotional and geo-environmental factors, resulting in pathophysiologic phenomena such as excitotoxicity or oxidative stress, responsible for neuronal damage that takes place at sparse cellular and/or subcellular levels., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Background: During myocardial infarction (MI), billions of cardiomyocytes are lost. The optimal therapy should effectively replace damaged cardiomyocytes, possibly with stem cells able to engraft and differentiate into adult functional cardiomyocytes. As such, cardiac atrial appendage stem cells (CASCs) are suitable candidates. However, the presence of elevated levels of advanced glycation end products (AGEs) in cardiac regions where CASCs are transplanted may affect their regenerative potential. In this study, we examine whether and how AGEs alter CASCs properties in vitro., Methods and Results: CASCs in culture were exposed to ranging AGEs concentrations (50 µg/mL to 400 µg/mL). CASCs survival, proliferation, and migration capacity were significantly decreased after 72 h of AGEs exposure. Apoptosis significantly increased with rising AGEs concentration. The harmful effects of these AGEs were partially blunted by pre-incubation with a receptor for AGEs (RAGE) inhibitor (25 µM FPS-ZM1), indicating the involvement of RAGE in the observed negative effects., Conclusion: AGEs have a time- and concentration-dependent negative effect on CASCs survival, proliferation, migration, and apoptosis in vitro, partially mediated through RAGE activation. Whether anti-AGEs therapies are an effective treatment in the setting of stem cell therapy after MI warrants further examination.

Blood and/or urine levels of 27 heavy metals were determined by ICPMS in 41 patients with dilated cardiomyopathy (DCM) and 29 presumably healthy subjects from the Katanga Copperbelt (KC), in the Democratic Republic of Congo (DRC). After adjusting for age, gender, education level, and renal function, DCM probability was almost maximal for blood concentrations above 0.75 and 150 µg/dL for arsenic and copper, respectively. Urinary concentrations above 1 for chromium, 20 for copper, 600 for zinc, 30 for selenium, 2 for cadmium, 0.2 for antimony, 0.5 for thallium, and 0.05 for uranium, all in μg/g of creatinine, were also associated with increased DCM probability. Concurrent and multiple exposures to heavy metals, well beyond permissible levels, are associated with increased probability for DCM. Study findings warrant screening for metal toxicity in case of DCM and prompt public health measures to reduce exposures in the KC, DRC.

Background and Purpose: The development of myocardial fibrosis is a major complication of Type 2 diabetes mellitus (T2DM), impairing myocardial deformation and, therefore, cardiac performance. It remains to be established whether abnormalities in longitudinal strain (LS) exaggerate or only occur in well-controlled T2DM, when exposed to exercise and, therefore, cardiac stress. We therefore studied left ventricular LS at rest and during exercise in T2DM patients vs. healthy controls., Methods and Results: Exercise echocardiography was applied with combined breath-by-breath gas exchange analyses in asymptomatic, well-controlled (HbA1c: 6.9 ± 0.7%) T2DM patients (n = 36) and healthy controls (HC, n = 23). Left ventricular LS was assessed at rest and at peak exercise. Peak oxygen uptake (V̇O 2peak ) and workload (W peak ) were similar between groups (p > 0.05). Diastolic (E, e' s , E/e') and systolic function (left ventricular ejection fraction) were similar at rest and during exercise between groups (p > 0.05). LS (absolute values) was significantly lower at rest and during exercise in T2DM vs. HC (17.0 ± 2.9% vs. 19.8 ± 2% and 20.8 ± 4.0% vs. 23.3 ± 3.3%, respectively, p < 0.05). The response in myocardial deformation (the change in LS from rest up to peak exercise) was similar between groups (+ 3.8 ± 0.6% vs. + 3.6 ± 0.6%, in T2DM vs. HC, respectively, p > 0.05). Multiple regression revealed that HDL-cholesterol, fasted insulin levels and exercise tolerance accounted for 30.5% of the variance in response of myocardial deformation in the T2DM group (p = 0.002)., Conclusion: Myocardial deformation is reduced in well-controlled T2DM and despite adequate responses, such differences persist during exercise., Trial Registration: NCT03299790, initially released 09/12/2017.

Stem cell-based regenerative therapies hold great promises to treat a wide spectrum of diseases. However, stem cell engraftment and survival are still challenging due to an unfavorable transplantation environment. Advanced glycation end-products (AGEs) can contribute to the generation of these harmful conditions. AGEs are a heterogeneous group of glycated products, nonenzymatically formed when proteins and/or lipids become glycated and oxidized. Our typical Western diet as well as cigarettes contain high AGEs content. AGEs are also endogenously formed in our body and accumulate with senescence and in pathological situations. Whether AGEs have an impact on stem cell viability in regenerative medicine remains unclear, and research on the effect of AGEs on stem cell proliferation and apoptosis is still ongoing. Therefore, this systematic review provides a clear overview of the effects of glycated proteins on cell viability in various types of primary isolated stem cells used in regenerative medicine., Competing Interests: The authors declare that they have no conflict of interest., (Copyright © 2020 Lize Evens et al.)

Background/aims: High-molecular-weight advanced glycation end-products (HMW-AGEs) are abundantly present in our Western diet. There is growing evidence reporting that HMW-AGEs contribute to the development of cardiovascular dysfunction in vivo, next to the well-known low-molecular-weight AGEs. The goal of our study is to assess the ultrastructure and function of cardiomyocytes after chronic exposure to HMW-AGEs. A better understanding of underlying mechanisms is essential to create new opportunities for further research on the specific role of HMW-AGEs in the development and progression of cardiovascular diseases., Methods: Adult male rats were randomly assigned to daily intraperitoneal injection for six weeks with either HMW-AGEs (20 mg/kg/day) or a control solution. Hemodynamic measurements were performed at sacrifice. Single cardiomyocytes from the left ventricle were obtained by enzymatic dissociation through retrograde perfusion of the aorta. Unloaded cell shortening, time to peak and time to 50% relaxation were measured during field stimulation and normalized to diastolic length. L-type Ca 2+ current density (I CaL ) and steady-state inactivation of I CaL were measured during whole-cell ruptured patch clamp. Myofilament functional properties were measured in membrane-permeabilized cardiomyocytes. Ultrastructural examination of cardiac tissue was performed using electron microscopy., Results: Rats injected with HMW-AGEs displayed in vivo cardiac dysfunction, characterized by significant changes in left ventricular peak rate pressure rise and decline accompanied with an increased heart mass. Single cardiomyocytes isolated from the left ventricle revealed concentric hypertrophy, indicated by the increase in cellular width. Unloaded fractional cell shortening was significantly reduced in cells derived from the HMW-AGEs group and was associated with slower kinetics. Peak L-type Ca 2+ current density was significantly decreased in the HMW-AGEs group.
L-type Ca 2+ channel availability was significantly shifted towards more negative potentials after HMW-AGEs injection. The impact of HMW-AGEs on myofilament function was measured in membrane-permeabilized cardiomyocytes showing a reduction in passive force, maximal Ca 2+ activated force and rate of force development. Ultrastructural examination of cardiac tissue demonstrated adverse structural remodeling in HMW-AGEs group characterized by a disruption of the cyto-architecture, a decreased mitochondrial density and altered mitochondrial function., Conclusion: Our data indicate that HMW-AGEs induce structural and functional cellular remodeling via a different working mechanism as the well-known LMW-AGEs. Results of our research open the door for new strategies targeting HMW-AGEs to improve cardiac outcome., Competing Interests: The authors have no conflicts of interest to declare., (© Copyright by the Author(s). Published by Cell Physiol Biochem Press.)

Growing evidence supports the role of advanced glycation end products (AGEs) in the development of diabetic vascular complications and cardiovascular diseases (CVDs). We have shown that high-molecular-weight AGEs (HMW-AGEs), present in our Western diet, impair cardiac function. Whether HMW-AGEs affect vascular function remains unknown. In this study, we aimed to investigate the impact of chronic HMW-AGEs exposure on vascular function and structure. Adult male Sprague Dawley rats were daily injected with HMW-AGEs or control solution for 6 weeks. HMW-AGEs animals showed intracardiac pressure overload, characterized by increased systolic and mean pressures. The contraction response to PE was increased in aortic rings from the HMW-AGEs group. Relaxation in response to ACh, but not SNP, was impaired by HMW-AGEs. This was associated with reduced plasma cyclic GMP levels. SOD restored ACh-induced relaxation of HMW-AGEs animals to control levels, accompanied by a reduced half-maximal effective dose (EC 50 ). Finally, collagen deposition and intima-media thickness of the aortic vessel wall were increased with HMW-AGEs. Our data demonstrate that chronic HMW-AGEs exposure causes adverse vascular remodelling. This is characterised by disturbed vasomotor function due to increased oxidative stress and structural changes in the aorta, suggesting an important contribution of HMW-AGEs in the development of CVDs.

Human cardiac stem cells isolated from atrial appendages based on aldehyde dehydrogenase activity (CASCs) can be expanded in vitro and differentiate into mature cardiomyocytes. In this study, we assess whether Wnt activation stimulates human CASC proliferation, whereas Wnt inhibition induces cardiac maturation. CASCs were cultured as described before. Conventional PCR confirmed the presence of the Frizzled receptors. Small-molecule inhibitors (IWP2, C59, XAV939, and IWR1-endo) and activator (CHIR99021) of the Wnt/β -catenin signaling pathway were applied, and the effect on β-catenin and target genes for proliferation and differentiation was assessed by Western blot and RT-qPCR. CASCs express multiple early cardiac differentiation markers and are committed toward myocardial differentiation. They express several Frizzled receptors, suggesting a role for Wnt signaling in clonogenicity, proliferation, and differentiation. Wnt activation increases total and active β-catenin levels. However, this does not affect CASC proliferation or clonogenicity. Wnt inhibition upregulated early cardiac markers but could not induce mature myocardial differentiation. When CASCs are committed toward myocardial differentiation, the Wnt pathway is active and can be modulated. However, despite its role in cardiogenesis and myocardial differentiation of pluripotent stem-cell populations, our data indicate that Wnt signaling has limited effects on CASC clonogenicity, proliferation, and differentiation., Competing Interests: The authors declare no conflicts of interest.

New Findings: What is the central question of this study? Does acute exposure to high molecular weight advanced glycation end products (HMW-AGEs) alter cardiomyocyte contractile function? What is the main finding and its importance? Ventricular cardiomyocytes display reduced Ca 2+ influx, resulting in reduced contractile capacity, after acute exposure to HMW-AGEs, independent of activation of their receptor. Given that HMW-AGEs are abundantly present in our Western diet, a better understanding of underlying mechanisms, especially in patients already displaying altered cardiac function, should be gained for these compounds., Abstract: Sustained elevated levels of high molecular weight advanced glycation end products (HMW-AGEs) are known to promote cardiac dysfunction. Recent data suggest that acutely elevated levels of AGEs occur in situations of increased oxidative stress. Whether this increase might have detrimental effects on cardiac function remains unknown. In this study, we investigated whether acute exposure to HMW-AGEs affects cardiomyocyte function via activation of their receptor (RAGE) signalling pathway. Single cardiomyocytes from the left ventricle of adult male rats were obtained by enzymatic dissociation through retrograde perfusion of the aorta. Functional experiments were performed in cardiomyocytes pre-incubated with or without an anti-RAGE antibody. Unloaded cell shortening and L-type Ca 2+ current amplitude were evaluated in the presence or absence of HMW-AGEs (200 μg ml -1 ). Expression of RAGE, c-Jun N-terminal kinase (JNK) and phosphorylated JNK (pJNK) were assessed by western blot. Experiments were performed at room temperature. After 4 min application of HMW-AGEs, unloaded cell shortening was significantly reduced. This impaired contractile function was related to reduced Ca 2+ influx. These alterations were also observed in cardiomyocytes pre-incubated with anti-RAGE antibody. Our study demonstrates that acute exposure to elevated levels of HMW-AGEs leads to direct and irreversible cardiomyocyte dysfunction, independent of RAGE activation., (© 2019 The Authors. Experimental Physiology © 2019 The Physiological Society.)

Exercise training is a low cost and safe approach for reducing the risk of cardiovascular disease development. Currently, moderate-intensity training (MIT) is the most preferred exercise type. However, high-intensity interval training (HIIT) is gaining interest especially among athletes and healthy individuals. In this study, we examined cardiac remodeling resulting from MIT and HIIT in healthy rats. Healthy male Sprague-Dawley rats were randomly assigned to MIT or HIIT for 13 weeks. Animals kept sedentary (SED) were used as control. Cardiac function was evaluated with echocardiography and hemodynamic measurements. Heart tissue was stained for capillary density and fibrosis. After 13 weeks of training, only HIIT induced beneficial cardiac hypertrophy. Overall global cardiac parameters (such as ejection fraction, cardiac output and volumes) were improved similarly between both training modalities. At tissue level, collagen content was significantly and similarly reduced in both exercise groups. Finally, only HIIT increased significantly capillary density. Our data indicate that even if very different in design, HIIT and MIT appear to be equally effective in improving cardiac function in healthy rats. Furthermore, HIIT provides additional benefits through improved capillary density and should therefore be considered as a preferred training modality for athletes and for patients.

Background: The effect of exercise on cardiac function/structure in type 2 diabetes mellitus (T2DM) with or without diabetic cardiomyopathy (DCM) is not yet completely understood. To date, results of studies have been controversial with variable outcomes due to the variety of exercise modalities., Objectives: The aim of the present review was to examine the impact of exercise intervention, and different types of exercise, on cardiac function and structure in T2DM through a systematic literature review, combining both pre-clinical and clinical studies., Methods: A systematic literature search was performed on PubMed, Web of Science, and PEDro to identify studies up to 2 April 2018. Articles were included when well-defined exercise protocols were provided, and cardiac function in T2DM patients or validated animal models was examined., Results: In diabetic animals, improvements in both diastolic and systolic function through exercise therapy were mainly attributed to reduced collagen deposition. In T2DM patients, improvements were observed in diastolic function, but not consistently in systolic function, after endurance (and combined resistance) exercise training. Different exercise intervention modalities and exercise types seemed equally effective in improving cardiac structure and function., Conclusion: Exercise training elicits significant improvements in diastolic function and beneficial remodeling in T2DM and DCM animal models, but not necessarily improvements in systolic function and left ventricular structure, regardless of exercise type. Therefore, exercise intervention should be a cornerstone in the treatment of T2DM patients not only to improve glycemic control but also to specifically enhance cardiac function.

Diabetes mellitus (DM) is a major problem worldwide. Within this patient group, cardiovascular diseases are the biggest cause of morbidity and mortality. Diabetic cardiomyopathy (DCM) is defined as diabetes-associated structural and functional changes in the myocardium, not directly attributable to other confounding factors such as coronary artery disease or hypertension. Pathophysiology of DCM remains unclear due to a lack of adequate animal models reflecting the current pandemic of diabetes, associated with a high increased sugar intake and the 'Western' lifestyle. The aim of this study was to develop an animal model mimicking this 'Western' lifestyle causing a human-like phenotype of DCM. Twenty-four Sprague-Dawley rats were randomly assigned into a normal or a 'Western' diet group for 18 weeks. Glucose and insulin levels were measured with an OGTT. Heart function was assessed by echocardiography and hemodynamic measurements in vivo. Cardiac fibrosis and inflammation were investigated in vitro. 'Western' diet given to healthy rats for 18 weeks induced hyperglycemia together with increased AGEs levels, insulin levels and hypertriglyceridemia. Heart function was altered with increased end-diastolic pressure, left ventricle hypertrophy. Changes in vivo were associated with increased collagen deposition and increased PAI-1 levels in the heart. High-sugar diet or 'Western' diet causes T2DM and the hallmarks of DCM in rats, reflecting the phenotype of the disease seen in patients. Using this new model of T2DM with DCM might open new insight in understanding the pathophysiology of DCM and on a long term, test targeted therapies for T2DM with DCM patients., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Advanced glycation end products (AGEs) play a key role in the progression of heart failure. Whether treatments limiting AGEs formation would prevent adverse left ventricular remodeling after myocardial infarction (MI) remain unknown. We investigated whether pyridoxamine (PM) could limit adverse cardiac outcome in MI. Rats were divided into MI, MI + PM and Sham. Echocardiography and hemodynamic parameters were used to assess cardiac function 8 weeks post-surgery. Total interstitial collagen, collagen I and collagen III were quantified using Sirius Red and polarized light microscopy. PM improved survival following LAD occlusion. Pre-treatment with PM significantly decreased the plasma AGEs levels. MI rats treated with PM displayed reduced left ventricular end-diastolic pressure and tau compared to untreated MI rats. Deformation parameters were also improved with PM. The preserved diastolic function was related to the reduced collagen content, in particular in the highly cross-linked collagen type I, mainly in the peri-infarct region, although not via TGF-β1 pathway. Our data indicate that PM treatment prevents the increase in AGEs levels and reduces collagen levels in a rat model of MI, resulting in an improved cardiac phenotype. As such, therapies targeting formation of AGEs might be beneficial in the prevention and/or treatment of maladaptive remodeling following MI.

Advanced glycation end products (AGEs) are a group of proteins and lipids becoming glycated and oxidized after persistent contact with reducing sugars or short-chain aldehydes with amino group and/or high degree of oxidative stress. The accumulation of AGEs in the body is a natural process that occurs with senescence, when the turnover rate of proteins is reduced. However, increased circulating AGEs have been described to arise at early lifetime and are associated with adverse outcome and survival, in particular in settings of cardiovascular diseases. AGEs contribute to the development of cardiac dysfunction by two major mechanisms: cross-linking of proteins or binding to their cell surface receptor. Recently, growing evidence shows that high-molecular weight AGEs (HMW-AGEs) might be as important as the characterized low-molecular weight AGEs (LMW-AGEs). Here, we point out the targets of AGEs in the heart and the mechanisms that lead to heart failure with focus on the difference between LMW-AGEs and the less characterized HMW-AGEs. As such, this review is a compilation of relevant papers in the form of a useful resource tool for researchers who want to further investigate the role of HMW-AGEs on cardiac disorders and need a solid base to start on this specific topic.

Scarring and remodeling of the left ventricle (LV) after myocardial infarction (MI) results in ischemic cardiomyopathy with reduced contractile function. Regional differences related to persisting ischemia may exist. We investigated the hypothesis that mitochondrial function and structure is altered in the myocardium adjacent to MI with reduced perfusion (MI adjacent ) and less so in the remote, nonischemic myocardium (MI remote ). We used a pig model of chronic coronary stenosis and MI (n = 13). Functional and perfusion MR imaging 6 wk after intervention showed reduced ejection fraction and increased global wall stress compared with sham-operated animals (Sham; n = 14). Regional strain in MI adjacent was reduced with reduced contractile reserve; in MI remote strain was also reduced but responsive to dobutamine and perfusion was normal compared with Sham. Capillary density was unchanged. Cardiac myocytes isolated from both regions had reduced basal and maximal oxygen consumption rate, as well as through complex I and II, but complex IV activity was unchanged. Reduced respiration was not associated with detectable reduction of mitochondrial density. There was no significant change in AMPK or glucose transporter expression levels, but glycogen content was significantly increased in both MI adjacent and MI remote Glycogen accumulation was predominantly perinuclear; mitochondria in this area were smaller but only in MI adjacent where also subsarcolemmal mitochondria were smaller. In conclusion, after MI reduction of mitochondrial respiration and glycogen accumulation occur in all LV regions suggesting that reduced perfusion does not lead to additional specific changes and that increased hemodynamic load is the major driver for changes in mitochondrial function., (Copyright © 2016 the American Physiological Society.)

Background: Several clinical and experimental studies have demonstrated that advanced glycation end products (AGEs) are associated with adverse cardiac outcome. Growing evidence shows that high molecular weight AGEs (HMW-AGEs) might be as important as the characterized low molecular weight AGEs. To date, the role of HMW-AGEs in the pathogenesis of cardiac remodeling remains unknown. In this study, we investigated whether HMW-AGEs are involved in cardiac dysfunction., Methods: Healthy rats were daily ip injected with 20mg/kg BSA-derived HMW-AGEs or, as a control, unmodified BSA, during 6 weeks. Cardiac function was assessed with echocardiography. Plasma levels of glucose, AGEs and soluble RAGE (sRAGE) were measured. AGEs, RAGE and lysyl oxidase (LOX) expression were determined by western blot., Results: After 6 weeks, animals displayed a sustained increase in circulating total AGEs without hyperglycemia. HMW-AGEs injections induced cardiac dysfunction characterized by wall hypertrophy, increased heart sphericity, reduced strain and strain rate with preserved ejection fraction. Plasma sRAGE levels were significantly higher compared to control and correlated significantly with decreased strain. RAGE expression, TNF-α and IL-6 remained unchanged. Finally, HMW-AGEs induced prominent cardiac fibrosis associated with an increased LOX expression., Conclusion: Our data demonstrate that rather than via a specific activation of RAGE, the deleterious effects of HMW-AGEs are likely mediated via an increased collagen cross-linking responsible for the observed cardiac stiffness. Additionally, we show that in the setting of elevated HMW-AGEs, increased sRAGE levels are markers of altered cardiac function., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Traditionally the heart is considered a terminally differentiated organ. However, at the beginning of this century increased mitotic activity was reported in ischemic and idiopathic dilated cardiomyopathy hearts, compared to healthy controls, underscoring the potential of regeneration after injury. Due to the presence of adult stem cells in bone marrow and their purported ability to differentiate into other cell lineages, this cell population was soon estimated to be the most suited candidate for cardiac regeneration. Clinical trials with autologous bone marrow-derived mononuclear cells, using either an intracoronary or direct intramyocardial injection approach consistently showed only minor improvement in global left ventricular ejection fraction. This was explained by their limited cardiomyogenic differentiation potential. To obtain more convincing improvement in cardiac function, based on true myocardial regeneration, the focus of research has shifted towards resident cardiac progenitor cells. Several isolation procedures have been described: the c-kit surface marker was the first to be used, however experimental research has clearly shown that c-kit+ cells only marginally contribute to regeneration post myocardial infarction. Sphere formation was used to isolate the so-called cardiosphere derived cells (CDC), and also in this cell population cardiomyogenic differentiation is a rare event. Recently a new type of stem cells derived from atrial tissue (cardiac atrial stem cells - CASCs) was identified, based on the presence of the enzyme aldehyde dehydrogenase (ALDH). Those cells significantly improve both regional and global LV ejection fraction, based on substantial engraftment and consistent differentiation into mature cardiomyocytes (98%).

Background: This study assessed whether autologous transplantation of cardiac atrial appendage stem cells (CASCs) preserves cardiac function after myocardial infarction (MI) in a minipig model., Methods and Results: CASCs were isolated from right atrial appendages of Göttingen minipigs based on high aldehyde dehydrogenase activity and expanded. MI was induced by a 2h snare ligation of the left anterior descending coronary artery. Upon reperfusion, CASCs were intramyocardially injected under NOGA guidance (MI-CASC, n=10). Non-transplanted pigs (MI, n=8) received sham treatment. 3D electromechanical mapping (EMM) and cardiac MRI were performed to assess left ventricular (LV) function. MI pigs developed LV dilatation at 2 months (2M), while in the MI-CASC group volumes remained stable. Global LV ejection fraction decreased by 16 ± 8% in MI animals vs 3 ± 10% in MI-CASC animals and regional wall thickening in border areas was better preserved in the MI-CASC group. EMM showed decreased viability and wall motion in the LV for both groups POST-MI, whereas at 2M these parameters only improved in the MI-CASC. Substantial cell retention was accompanied by cardiomyogenic differentiation in 98±1% of the transplanted CASCs, which functionally integrated. Second harmonic generation microscopy confirmed the formation of mature sarcomeres in transplanted CASCs. Absence of cardiac arrhythmias indicated the safety of CASC transplantation., Conclusion: CASCs preserve cardiac function by extensive engraftment and cardiomyogenic differentiation. Our data indicate the enormous potential of CASCs in myocardial repair., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Recently, a supramolecular model was developed for predicting striated skeletal muscle intensity profiles obtained by label-free second harmonic generation (SHG) microscopy. This model allows for a quantitative determination of the length of the thick filament antiparallel range or M-band (M ), and results in M=0.12  μm for single-band intensity profiles when fixing the A-band length (A ) to A=1.6  μm , a value originating from electron microscopy (EM) observations. Using simulations and experimental data sets, we showed that the objective numerical aperture (NA) and the refractive index (RI) mismatch (Δn=n 2ω −n ω ) between the illumination wave (ω ) and the second harmonic wave (2ω ) severely affect the simulated sarcomere intensity profiles. Therefore, our recovered filament lengths did not match with those observed by EM. For an RI mismatch of Δn=0.02 and a moderate illumination NA of 0.8, analysis of single-band SHG intensity profiles with freely adjustable A- and M-band sizes yielded A=1.40±0.04  μm and M=0.07±0.05  μm for skeletal muscle. These lower than expected values were rationalized in terms of the myosin density distribution along the myosin thick filament axis. Our data provided new and practical insights for the application of the supramolecular model to study SHG intensity profiles in striated muscl

This protocol describes the measurement of Ca(2+) sparks in intact myocytes by using a Ca(2+)-sensitive dye and imaging using laser scanning confocal microscopy. It takes advantage of spontaneous Ca(2+)-release events-sparks-using them as a measure of the activity of ryanodine receptors (RyRs). Two methodologies are described: One requires that cardiomyocytes be stimulated, preferably under voltage clamp by depolarizing pulses, until steady-state is reached, and then stimulation is stopped and Ca(2+) sparks are recorded. The second requires that cells be permeabilized and bathed in a solution to load the cell with Ca(2+) sufficient to elicit Ca(2+) sparks, but not Ca(2+) waves. These are then analyzed offline to quantify spark frequency and morphology. The advantages and disadvantages of each approach are discussed., (© 2015 Cold Spring Harbor Laboratory Press.)

The decline of an intracellular calcium ([Ca(2+)]i) transient during a single excitation-contraction coupling (ECC) cycle reflects the combined activity of the sarcoplasmic reticulum Ca(2+)-ATPase (SERCA) pump and the sarcolemmal Na(+)-Ca(2+) exchanger (NCX), along with minor contributions of the plasma membrane Ca(2+)-ATPase and mitochondrial Ca(2+) uniporter, in removing Ca(2+) from the cytosol. A traditional approach for assessing the individual components is to fit the decline of the [Ca(2+)]i transient evoked during electrical stimulation with an exponential. This reflects mostly the SERCA-dependent rate of uptake, which can be properly deduced after correcting for a component of NCX removal. As NCX function is an important determinant of the membrane potential as well as the Ca(2+) balance, we present here several detailed protocols for assessing NCX function. As the reversal potential and the amplitudes of the current are highly dependent on the prevailing concentrations of Na(+) and Ca(2+), we show how NCX function can be assessed under highly controlled conditions, with Ca(2+) and Na(+) clamped, as well as under more physiological conditions, with freely changing Ca(2+) and Na(+)., (© 2015 Cold Spring Harbor Laboratory Press.)

In cardiac myocytes, Ca(2+) release from the sarcoplasmic reticulum (SR) Ca(2+) store through the opening of ryanodine receptors (RyRs) is the major source of Ca(2+) for activation of myofilaments and contraction. Over the past 20 years, tools have become available to study this release process in detail, allowing new insights into the regulation of SR Ca(2+) release and RyR function. To assess these processes, we recommend and here review a systematic approach that evaluates the essential transport mechanisms and Ca(2+) fluxes in isolated single cardiac myocytes by using fluorescent Ca(2+) indicators and whole-cell recording of membrane voltage and ionic currents under voltage clamp. The approach includes an assessment of the L-type Ca(2+) current as a trigger for opening of RyRs and release of SR Ca(2+), of the SR Ca(2+) content, of intrinsic properties of RyRs, and of Ca(2+)-removal systems., (© 2015 Cold Spring Harbor Laboratory Press.)

Here, we describe a method for characterizing the L-type Ca(2+) current, ICaL, which is a major trigger for Ca(2+) release from the sarcoplasmic reticulum (SR). The protocol includes measuring ICaL amplitude and voltage dependence and the elicited SR Ca(2+) release. The procedure for measuring ICaL activity is performed using solutions (internal and external) and voltage control such that other ionic currents are eliminated. The resultant relationship between the Ca(2+) current and the associated internal [Ca(2+)]i transient is a first approach for evaluating coupling gain. We discuss which parameters are most appropriate for this analysis and how an evaluation of gain needs to be further explored by measuring the SR Ca(2+) content., (© 2015 Cold Spring Harbor Laboratory Press.)

Here, we describe a protocol for the reliable measurement of the amount of Ca(2+) in the sarcoplasmic reticulum (SR) Ca(2+) store of cardiac myocytes. The whole-cell patch-clamp method is used to provide controlled loading of the SR during conditioning depolarizing pulses, followed by rapid application of a high dose of caffeine to release all SR Ca(2+) and to prevent Ca(2+) reuptake by the SR. Simultaneous measurement of membrane currents records Ca(2+) extruded through the Na(+)-Ca(2+) exchanger. The integral of the caffeine-induced Na(+)-Ca(2+) exchange current is then used as a measure of the SR Ca(2+). Derived measurements include the Ca(2+) buffering capacity and measurement of fractional release as an indicator of coupling gain. Caveats, advantages, and disadvantages of this method and alternative methods are discussed., (© 2015 Cold Spring Harbor Laboratory Press.)

In cardiac myocytes, the physiological increase of intracellular calcium, the [Ca(2+)]i transient, elicited during excitation-contraction coupling typically reaches a peak amplitude of up to 1 µm, from a resting value of ∼100 nm, within 50-100 msec, depending on the species. Various conditions will affect the amplitude and rise time of the [Ca(2+)]i transient and, depending on the nature of the Ca(2+) signals under study, a variety of different probes are available for monitoring changes in intracellular Ca(2+). In this protocol, we focus on Fluo-3, which exists in the cytosol in its salt form K5Fluo-3. This form is practically nonfluorescent in the absence of Ca(2+), but the fluorescence increases dramatically on Ca(2+) binding. Although Fluo-3 is a single excitation-emission dye, it has a number of advantages for investigators, including an ideal dissociation constant (Kd) value and high quantum yield, meaning that it can be used at low concentrations that introduce minimal buffering. Here, we describe the basic setup and methodology for recording the global cytosolic [Ca(2+)]i transient with this probe during simultaneous patch-clamp and whole-cell recording of membrane voltage or of ionic currents under voltage clamp., (© 2015 Cold Spring Harbor Laboratory Press.)

Background: The development of heart failure (HF) secondary to hypertension is a complex process related to a series of physiological and molecular factors including glucose dysregulation. The overall objective of this study was to investigate whether exercise training could improve cardiac function and insulin resistance in a rat model of hypertensive HF., Methods: Seven week old Dahl salt-sensitive rats received either 8% NaCl (n = 30) or 0.3% NaCl (n = 18) diet. After a 5-week diet, animals were randomly assigned to exercise training (treadmill running at 18 m/min, 5% inclination for 60 min, 5 days/week) or kept sedentary for 6 additional weeks. 2D echocardiography was used to calculate left ventricular (LV) dimensions, volumes and global functional parameters. LV global deformation parameters were measured with speckle tracking echocardiography. Insulin resistance was assessed using 1h oral glucose tolerance testing., Results: High salt diet led to cardiac hypertrophy and HF, characterized by increased wall thicknesses and LV volumes as well as reduced deformation parameters. In addition, high salt diet was associated with the development of insulin resistance. Exercise training improved cardiac function, reduced the extent of interstitial fibrosis and reduced insulin levels 60 min post-glucose administration., Conclusions: Even if not fully reversed, exercise training in HF animals improved cardiac function and insulin resistance. Adjusted modalities of exercise training might offer new insights not only as a preventive strategy, but also as a treatment for HF patients., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Aims: Melusin is a muscle-specific chaperone protein whose expression is required for a compensatory hypertrophy response to pressure overload. Here, we evaluated the consequences of melusin overexpression in the setting of myocardial infarction (MI) using a comprehensive multicentre approach., Methods and Results: Mice overexpressing melusin in the heart (TG) and wild-type controls (WT) were subjected to permanent LAD ligation and both the acute response (Day 3) and subsequent remodelling (2 weeks) were examined. Mortality in wild-type mice was significant between Days 3 and 7, primarily due to cardiac rupture, but melusin's overexpression strongly reduced mortality (43.2% in wild-type vs. 27.3% in melusin-TG, P = 0.005). At Day 3 after MI, a time point preceding the mortality peak, TG hearts had increased heat shock protein 70 expression, increased ERK1/2 signalling, reduced cardiomyocyte hyper-contractility and inflammatory cell infiltrates, and increased matricellular protein expression in the infarcted area. At 2 weeks after MI, melusin overexpression conferred a favourable adaptive remodelling characterized by reduced left ventricle dilatation and better preserved contractility in the presence of a comparable degree of hypertrophy. Adaptive remodelling in melusin TG mice was characterized by reduced apoptosis and fibrosis as well as increased cardiomyocyte contractility., Conclusions: Consistent with its function as a chaperone protein, melusin overexpression exerts a dual protective action following MI reducing an array of maladaptive processes. In the early phase after MI, reduced inflammation and myocyte remodelling protect against cardiac rupture. Chronically, reduced myocyte loss and matrix remodelling, with preserved myocyte contractility, confer adaptive LV remodelling.

Rationale: In ventricular myocytes of large mammals with low T-tubule density, a significant number of ryanodine receptors (RyRs) are not coupled to the sarcolemma; cardiac remodeling increases noncoupled RyRs., Objective: Our aim was to test the hypothesis that coupled and noncoupled RyRs have distinct microdomain-dependent modulation., Methods and Results: We studied single myocytes from pig left ventricle. The T-tubule network was analyzed in 3-dimension (3D) to measure distance to membrane of release sites. The rising phase of the Ca(2+) transient was correlated with proximity to the membrane (confocal imaging, whole-cell voltage-clamp, K5fluo-4 as Ca(2+) indicator). Ca(2+) sparks after stimulation were thus identified as resulting from coupled or noncoupled RyRs. We used high-frequency stimulation as a known activator of Ca(2+)/calmodulin-dependent kinase II. Spark frequency increased significantly more in coupled than in noncoupled RyRs. This specific modulation of coupled RyRs was abolished by the Ca(2+)/calmodulin-dependent kinase II blockers autocamtide-2-related inhibitory peptide and KN-93, but not by KN-92. Colocalization of Ca(2+)/calmodulin-dependent kinase II and RyR was not detectably different for coupled and noncoupled sites, but the F-actin disruptor cytochalasin D prevented the specific modulation of coupled RyRs. NADPH oxidase 2 inhibition by diphenyleneiodonium or apocynin, or global reactive oxygen species scavenging, also prevented coupled RyR modulation. During stimulated Ca(2+) transients, frequency-dependent increase of the rate of Ca(2+) rise was seen in coupled RyR regions only and abolished by autocamtide-2-related inhibitory peptide. After myocardial infarction, selective modulation of coupled RyR was lost., Conclusions: Coupled RyRs have a distinct modulation by Ca(2+)/calmodulin-dependent kinase II and reactive oxygen species, dependent on an intact cytoskeleton and consistent with a local Ca(2+)/reactive oxygen species microdomain, and subject to modification with disease.

Background: Diabetic cardiomyopathy is characterized by systolic and early diastolic ventricular dysfunction. In the metabolic syndrome (MS), ventricular stiffness is additionally increased in a later stage. It is unknown whether this is related to intrinsic cardiomyocyte dysfunction, extrinsic factors influencing cardiomyocyte contractility and/or cardiac function, or a combination of both. A first aim was to study cardiomyocyte contractility and Ca2+ handling in vitro in a mouse model of MS. A second aim was to investigate whether in vivo hypocaloric diet or ACE-inhibition (ACE-I) improved cardiomyocyte contractility in vitro, contractile reserve and Ca2+ handling., Methods: This study was performed in LDL-receptor (LDLR-/-) and leptin-deficient (ob/ob), double knock-out mice (DKO), featuring obesity, type II diabetes, atherogenic dyslipidemia and hypertension. Single knock-out LDLR-/-, ob/ob and wild type mice were used as controls. Cellular contractility, Ca2+ handling and their response to in vivo treatment with diet or ACE-I were studied in isolated cardiomyocytes at baseline, during β-adrenergic stimulation or increased extracellular Ca2+, using field stimulation and patch-clamp., Results: In untreated conditions, prolongation of contraction-relaxation cycle and altered Ca2+ handling are observed in MS. Response to increased extracellular Ca2+ and β-adrenergic stimulation is impaired and could not be rescued by weight loss. ACE-I restored impaired response to β-adrenergic stimulation in MS, but not the decreased response to increased extracellular Ca2+., Conclusions: Cardiomyocyte contractility and β-adrenergic response are impaired in MS, due to alterations in cellular Ca2+ handling. ACE-I, but not weight loss, is able to restore cardiomyocyte response to β-adrenergic stimulation in MS.

Background: L-type calcium channel (LTCC) and Na(+)/Ca(2+) exchanger (NCX) have been implicated in repolarization-dependent arrhythmias, but also modulate calcium and contractility. Although LTCC inhibition is negative inotropic, NCX inhibition has the opposite effect. Combined block may, therefore, offer an advantage for hemodynamics and antiarrhythmic efficiency, particularly in diseased hearts. In a model of proarrhythmia, the dog with chronic atrioventricular block, we investigated whether combined inhibition of NCX and LTCC with SEA-0400 is effective against dofetilide-induced torsade de pointes arrhythmias (TdP), while maintaining calcium homeostasis and hemodynamics., Methods and Results: Left ventricular pressure (LVP) and ECG were monitored during infusion of SEA-0400 and verapamil in anesthetized dogs. Different doses were tested against dofetilide-induced TdP in chronic atrioventricular block dogs. In ventricular myocytes, effects of SEA-0400 were tested on action potentials, calcium transients, and early afterdepolarizations. In cardiomyocytes, SEA-0400 (1 μmol/L) blocked 66±3% of outward NCX, 50±2% of inward NCX, and 33±9% of LTCC current. SEA-0400 had no effect on systolic calcium, but slowed relaxation, despite action potential shortening, and increased diastolic calcium. SEA-0400 stabilized dofetilide-induced lability of repolarization and suppressed early afterdepolarizations. In vivo, SEA-0400 (0.4 and 0.8 mg/kg) had no effect on left ventricular pressure and suppressed dofetilide-induced TdPs dose dependently. Verapamil (0.3 mg/kg) also inhibited TdP, but caused a 15±8% drop of left ventricular pressure. A lower dose of verapamil without effects on left ventricular pressure (0.06 mg/kg) was not antiarrhythmic., Conclusions: In chronic atrioventricular block dogs, SEA-0400 treatment is effective against TdP. Unlike specific inhibition of LTCC, combined NCX and LTCC inhibition has no negative effects on cardiac hemodynamics.

Reducing the open probability of the ryanodine receptor (RyR) has been proposed to have beneficial effects in heart failure. We investigated whether conditional FKBP12.6 overexpression at the time of myocardial infarction (MI) could improve cardiac remodelling and cell Ca(2+) handling. Wild-type (WT) mice and mice overexpressing FKBP12.6 (Tg) were studied on average 7.5 ± 0.2 weeks after MI and compared with sham-operated mice for in vivo, myocyte function and remodelling. At baseline, unloaded cell shortening in Tg was not different from WT. The [Ca(2+)](i) transient amplitude was similar, but sarcoplasmic reticulum (SR) Ca(2+) content was larger in Tg, suggesting reduced fractional release. Spontaneous spark frequency was similar despite the increased SR Ca(2+) content, consistent with a reduced RyR channel open probability in Tg. After MI, left ventricular dilatation and myocyte hypertrophy were present in both groups, but more pronounced in Tg. Cell shortening amplitude was unchanged with MI in WT, but increased with MI in Tg. The amplitude of the [Ca(2+)](i) transient was not affected by MI in either genotype, but time to peak was increased; this was most pronounced in Tg. The SR Ca(2+) content and Na(+)- Ca(2+) exchanger function were not affected by MI. Spontaneous spark frequency was increased significantly after MI in Tg, and larger than in WT (at 4 Hz, 2.6 ± 0.4 sparks (100 μm)(-1) s(-1) in Tg MI versus 1.6 ± 0.2 sparks (100 μm)(-1) s(-1) in WT MI; P < 0.05). We conclude that FKPB12.6 overexpression can effectively reduce RyR open probability with maintained cardiomyocyte contraction. However, this approach appears insufficient to prevent and reduce post-MI remodelling, indicating that additional pathways may need to be targeted.

The Na(+)/Ca(2+) exchanger (NCX) is a key regulator of intracellular Ca(2+) in cardiac myocytes, predominantly contributing to Ca(2+) removal during the diastolic relaxation process but also modulating excitation-contraction coupling. NCX is preferentially located in the T-tubules and can be close to or within the dyad, where L-type Ca(2+) channels face ryanodine receptors (RyRs), the Ca(2+) release channels of the sarcoplasmic reticulum. However, especially in larger animals, not all RyRs are in dyads or adjacent to T-tubules, and a substantial fraction of Ca(2+) release from the sarcoplasmic reticulum thus occurs at distance from NCX. This chapter deals with the functional consequences of NCX location and how NCX can modulate diastolic and systolic Ca(2+) events. The loss of T-tubules and the effects on RyR function and NCX modulation are explored, as well as quantitative measurement of local Ca(2+) gradients at the level of the dyadic space.

Cardiomyocytes from failing hearts exhibit reduced levels of the sarcoplasmic reticulum (SR) Ca(2+)-ATPase (SERCA) and/or increased activity of the endogenous SERCA inhibitor phospholamban. The resulting reduction in the Ca(2+) affinity of SERCA impairs SR Ca(2+) cycling in this condition. We have previously investigated the physiological impact of increasing the Ca(2+) affinity of SERCA by substituting SERCA2a with the higher affinity SERCA2b pump. When phospholamban was also ablated, these double knockouts (DKO) exhibited a dramatic reduction in total SERCA levels, severe hypertrophy, and diastolic dysfunction. We presently examined the role of cardiomyocyte Ca(2+) homeostasis in both functional and structural remodeling in these hearts. Despite the low SERCA levels in DKO, we observed near-normal Ca(2+) homeostasis with rapid Ca(2+) reuptake even at high Ca(2+) loads and stimulation frequencies. Well-preserved global Ca(2+) homeostasis in DKO was paradoxically associated with marked activation of the Ca(2+)-dependent nuclear factor of activated T-cell-calcineurin pathway known to trigger hypertrophy. No activation of the MAP kinase signaling pathway was detected. These findings suggest that local changes in Ca(2+) homeostasis may play an important signaling role in DKO, perhaps due to reduced microdomain Ca(2+) buffering by SERCA2b. Furthermore, alterations in global Ca(2+) homeostasis can also not explain impaired in vivo diastolic function in DKO. Taken together, our results suggest that normalizing global cardiomyocyte Ca(2+) homeostasis does not necessarily protect against hypertrophy and heart failure development and that excessively increasing SERCA Ca(2+) affinity may be detrimental.

Alterations in RyR2 function have been proposed as a major pathophysiological mechanism of arrhythmias and heart failure (HF). Cardiac FKBP12.6 overexpression protects against myocardial infarction-induced HF and catecholamine-promoted ventricular arrhythmias. We tested the hypothesis that FKBP12.6 overexpression protects against maladaptive LVH and triggered ventricular arrhythmias following transverse aorta constriction (TAC) in the mouse. The TAC-associated mortality rate was significantly lower in male transgenic (DT) than in Ctr mice (p < 0.05). TAC-associated maladaptive hypertrophy was blunted in DT mice especially 1 month post-TAC and their SERCA2a/PLB ratio remained unchanged 1 and 2 months post-TAC. Two months after TAC, trains of 30 stimuli (burst pacing) performed following isoproterenol injection (0.2 mg/kg, ip), induced VT in 50% of the TAC-Ctr and in none of the TAC-DT mice (p = 0.022). The increase in myocyte shortening and Ca(2+) spark frequency observed in sham-operated Ctr mice in response to 50 nM isoproterenol was reduced in DT mice, and abolished in TAC-DT mice. NCX1 function was reduced in Sham-DT and TAC-DT compared with Sham-Ctr and TAC-Ctr mice, respectively (p < 0.05 for the 2 comparisons). In mice killed after isoproterenol injection and burst pacing, RyR2 S2814 phosphorylation was decreased by 50% in TAC-DT versus TAC-Ctr mice (p < 0.05), with no change in RyR2 S2808 and PLB S16 and T17 phosphorylation. Cardiac FKBP12.6 overexpression in the mouse blunts pressure overload-induced maladaptive LV remodelling and protects against catecholamine-promoted burst pacing-induced ventricular tachycardia by decreasing cardiac sensitivity to adrenergic stress and RyR2 S2814 phosphorylation, and decreasing NCX1 activity.