Regulation Of Cardiac Function Research Articles

Abstract 031: β-Adrenergic Receptor-Mediated Transactivation Of Epidermal Growth Factor Receptor Decreases Cardiomyocyte Apoptosis Through Differential Activation Of ERK1/2 and Akt

β-adrenergic receptors (βAR) are critical regulators of cardiac function whose dysregulation during heart failure are associated with diminished function. However, βAR-mediated EGFR transactivation has been shown to relay cardioprotection in a mouse model of heart failure via unknown mechanisms. We hypothesized that transactivation of EGFR promotes survival via distinct cardiomyocyte signaling responses leading to decreases in apoptosis. To test this hypothesis, C57BL/6 mice were injected with isoproterenol (Iso) in the presence or absence of the EGFR antagonist AG1478 and ERK1/2 and Akt phosphorylation and subcellular distribution were assessed. Following 10 min Iso stimulation, increases in ERK1/2 and Akt phosphorylation were observed in cytosolic, plasma membrane and nuclear fractions. Phosphorylation of ERK1/2 were AG1478 sensitive in all three fractions while Akt phosphorylation occurred through EGFR-transactivation only in plasma membrane and nuclear fractions, which was confirmed in rat neonatal cardiomyocytes (RNCM). Additionally, EGFR-transactivation by βAR decreased apoptosis, as measured via caspase 3 activation/activity and TUNEL assay, which was sensitive to inhibition of both ERK1/2 and Akt signaling pathways. Increased phosphorylation of ERK1/2 and Akt in the nucleus and the ability to inhibit Iso-mediated changes in apoptosis with the transcriptional inhibitor Actinomycin D suggested that the cardioprotective effects of Iso-mediated EGFR transactivation may be influenced by changes in gene transcription. An Apoptotsis RT2 PCR Array was used to identify changes in transcript levels of 84 apoptotic genes. Of these, 12 were found to be altered in response to EGFR inhibition in the presence of Iso. These results demonstrate that βAR-mediated EGFR transactivation in the heart induces differential subcellular activation of ERK1/2 and Akt and leads to the promotion of cell survival, in part through the modulation of apoptotic gene expression in cardiomyocytes. Further understanding the downstream consequences of these effects in response to βAR-mediated EGFR transactivation could lead to improved therapies for the treatment of heart failure.

Functional subcellular distribution of β1 - and β2 -adrenergic receptors in rat ventricular cardiac myocytes

β-adrenergic stimulation is a key regulator of cardiac function. The localization of major cardiac adrenergic receptors (β1 and β2) has been investigated using biochemical and biophysical approaches and has led to contradictory results. This study investigates the functional subcellular localization of β1- and β2-adrenergic receptors in rat ventricular myocytes using a physiological approach. Ventricular myocytes were isolated from the hearts of rat and detubulated using formamide. Physiological cardiac function was measured as Ca2+ transient using Fura-2-AM and cell shortening. Selective activation of β1- and β2-adrenergic receptors was induced with isoproterenol (0.1 μmol/L) and ICI-118,551 (0.1 μmol/L); and with salbutamol (10 μmol/L) and atenolol (1 μmol/L), respectively. β1- and β2-adrenergic stimulations induced a significant increase in Ca2+ transient amplitude and cell shortening in intact rat ventricular myocytes (i.e., surface sarcolemma and t-tubules) and in detubulated cells (depleted from t-tubules, surface sarcolemma only). Both β1- and β2-adrenergic receptors stimulation caused a greater effect on Ca2+ transient and cell shortening in detubulated myocytes than in control myocytes. Quantitative analysis indicates that β1-adrenergic stimulation is ∼3 times more effective at surface sarcolemma compared to t-tubules, whereas β2- adrenergic stimulation occurs almost exclusively at surface sarcolemma (∼100 times more effective). These physiological data demonstrate that in rat ventricular myocytes, β1-adrenergic receptors are functionally present at surface sarcolemma and t-tubules, while β2-adrenergic receptors stimulation occurs only at surface sarcolemma of cardiac cells.

Abstract 311: Measurements of cAMP Dynamics in the SERCA2 Microdomain in Adult Mouse Cardiomyocytes using a Targeted FRET Biosensor

PURPOSE: cAMP is a central regulator of cardiac function and disease. This global second messenger acts in a compartmentalized fashion, and changes in cAMP dynamics are linked to cardiac diseases. In this project, we visualized cAMP signals directly in such microdomains to gain insights into the molecular mechanisms involved in cAMP compartmentation and its alterations in hypertrophy. Methods: We generated transgenic mice expressing a new Förster resonance energy transfer (FRET)-based cAMP sensor Epac1-camps-PLN to measure cAMP dynamics in the microdomain around the sarco/endoplasmic reticulum Ca2+-ATPase 2 (SERCA2). This sensor is targeted to SERCA2 via phospholamban (PLN). Results: Colocalization and cell fractionation analysis confirmed proper localization of the sensor in transgenic mouse hearts. qPCR analysis revealed a two-fold overexpression of PLN. However, no adverse cardiac phenotype could be detected by histological analysis and heart weight to body weight ratios. Local cAMP dynamics were measured using freshly isolated adult ventricular myocytes and compared to cAMP signals in the bulk cytosol using cardiomyocytes from Epac1-camps mice. We detected the predominant role of phosphodiesterases (PDEs) 4 and 3 in the SERCA2 compartment under basal conditions. These PDEs were responsible for shaping the microdomain and its segregation from the cytosolic compartment. Interestingly, beta1-adrenergic stimulation led to a stronger increase of local cAMP in the SERCA2 compartment compared to the bulk cytosol. 8 weeks after transverse aortic constriction (TAC), PDE4 activity was downregulated in the SERCA2 microdomain compared to sham cardiomyocytes. Conclusion: We successfully generated transgenic mice expressing the targeted Epac1-camps-PLN biosensor to visualize cAMP dynamics in the SERCA2 compartment. We could show distinct cAMP dynamics around the SERCA2 compartment compared to the bulk cytosol and uncovered its alterations in hypertrophied cardiomyocytes

Of GIRK channels, vitamin E transfer, and a vertebrate fluorescent protein

Of GIRK channels, vitamin E transfer, and a vertebrate fluorescent protein

Loss of ectonucleotidases from the coronary vascular bed after ischemia-reperfusion in isolated rat heart

BackgroundEctonucleotidase plays an important role in the regulation of cardiac function by controlling extracellular levels of adenine nucleotides and adenosine. To determine the influence of ischemia-reperfusion injury on ectonucleotidase activity in coronary vascular bed, we compared the metabolic profile of adenine nucleotides during the coronary circulation in pre- and post-ischemic heart.MethodsLangendorff-perfused rat hearts were used to assess the intracoronary metabolism of adenine nucleotides. The effects of ischemia on the adenine nucleotide metabolism were examined after 30 min of ischemia and 30 min of reperfusion. Adenine nucleotide metabolites were measured by high performance liquid chromatography.ResultsATP, ADP and AMP were rapidly metabolized to adenosine and inosine during the coronary circulation. After ischemia, ectonucleotidase activity of the coronary vascular bed was significantly decreased. In addition, the perfusate from the ischemic heart contained a considerable amount of enzymes degrading ATP, AMP and adenosine. Immunoblot analysis revealed that the perfusate from the ischemic heart dominantly contained ectonucleoside triphosphate diphosphohydrolase 1, and, to a lesser extent, ecto-5’-nucleotidase. The leakage of nucleotide metabolizing enzymes from the coronary vascular bed by ischemia-reperfusion was more remarkable in aged rats, in which post-ischemic cardiac dysfunction was more serious.ConclusionEctonucleotidases were liberated from the coronary vascular bed by ischemia-reperfusion, resulting in an overall decrease in ectonucleotidase activity in the post-ischemic coronary vascular bed. These results suggest that decreased ectonucleotidase activity by ischemia may exacerbate subsequent reperfusion injury, and that levels of circulating ectonucleotidase may reflect the severity of ischemic vascular injury.

Cardiac responses to 24 hrs hyperoxia in Bmp2 and Bmp4 heterozygous mice

Background: Hyperoxia or clinical oxygen (O2) therapy is known to result in increased oxidative burden. Therefore, understanding susceptibility to hyperoxia exposure is clinically important. Bone morphogenetic proteins (BMPs) 2 and 4 are involved in cardiac development and may influence responses to hyperoxia.Methods. Bmp2+/−. Bmp4+/− and wild-type mice were exposed to hyperoxia (100% O2) for 24 hrs. Electrocardiograms (ECG) were recorded before and during exposure by radio-telemetry.Results: At baseline, a significantly higher low frequency (LF) and total power (TP) heart rate variability (HRV) were found in Bmp2+/− mice only (p < 0.05). Twenty-four hours hyperoxia-induced strain-independent reductions in heart rate, QTcB and ST-interval and increases in QRS, LF HRV and standard deviation of RR-intervals were observed. In Bmp4+/− mice only, increased PR-interval (PR-I) (24 hrs), P-wave duration (P-d; 18 and 21–24 hrs), PR-I minus P-d (PR – Pd; 24 hrs) and root of the mean squared differences of successive RR-intervals (24 hrs) were found during hyperoxia (p < 0.05).Discussion: Elevated baseline LF and TP HRV in Bmp2+/− mice suggests an altered autonomic nervous system regulation of cardiac function in these mice. However, this was not related to strain specific differences in responses to 24 hrs hyperoxia. During hyperoxia, Bmp4+/− mice were the most susceptible in terms of atrioventricular conduction changes and risk of atrial fibrillation, which may have important implications for patients treated with O2 who also harbor Bmp4 mutations. This study demonstrates significant ECG and HRV responses to 24 hrs hyperoxia in mice, which highlights the need to further work on the genetic mechanisms associated with cardiac susceptibility to hyperoxia.

Axon Guidance of Sympathetic Neurons to Cardiomyocytes by Glial Cell Line-Derived Neurotrophic Factor (GDNF)

Molecular signaling of cardiac autonomic innervation is an unresolved issue. Here, we show that glial cell line-derived neurotrophic factor (GDNF) promotes cardiac sympathetic innervation in vitro and in vivo. In vitro, ventricular myocytes (VMs) and sympathetic neurons (SNs) isolated from neonatal rat ventricles and superior cervical ganglia were cultured at a close distance. Then, morphological and functional coupling between SNs and VMs was assessed in response to GDNF (10 ng/ml) or nerve growth factor (50 ng/ml). As a result, fractions of neurofilament-M-positive axons and synapsin-I-positive area over the surface of VMs were markedly increased with GDNF by 9-fold and 25-fold, respectively, compared to control without neurotrophic factors. Pre- and post-synaptic stimulation of β1-adrenergic receptors (BAR) with nicotine and noradrenaline, respectively, resulted in an increase of the spontaneous beating rate of VMs co-cultured with SNs in the presence of GDNF. GDNF overexpressing VMs by adenovirus vector (AdGDNF-VMs) attracted more axons from SNs compared with mock-transfected VMs. In vivo, axon outgrowth toward the denervated myocardium in adult rat hearts after cryoinjury was also enhanced significantly by adenovirus-mediated GDNF overexpression. GDNF acts as a potent chemoattractant for sympathetic innervation of ventricular myocytes, and is a promising molecular target for regulation of cardiac function in diseased hearts.

Modulation of Staurosporine-Activated Volume-Sensitive Outwardly Rectifying Cl- Channel by PI3K/Akt in Cardiomyocytes

Chloride (Cl⁻) channels participate in the regulation of cardiac function in response to stress although the underlying regulatory mechanism remains poorly understood. This study was designed to examine the impact of the pro-apoptotic stimulus staurosporine (STS) on the volume-sensitive outwardly rectifying Cl⁻ current (I(Cl,Vol)) in cardiomyocytes and possible regulatory mechanism involved with a focus on phosphatidylinositol-3 kinase (PI3K)/Akt. Primary cultured rat neonatal cardiomyocytes were subjected to hypotonic and isotonic environment in the presence or absence of STS prior to whole-cell voltage-clamp evaluation of Cl⁻ current. Whole-cell recordings revealed that STS activated an outwardly rectifying Cl⁻ current with phenotypic properties reminiscent of I(Cl,Vol). These currents were outwardly rectifying with a time-dependent inactivation at positive potentials and were sensitive to 4,4'-diisothiocya-natostilbene- 2,2'- disulfonicacid (DIDS), a non-selective Cl⁻ channel blocker, and 4-(2-butyl-6,7-dichlor-2-cyclopentyl-indan-1-on-5-yl)oxybutyric acid (DCPIB), a selective VSOR Cl⁻ channel blocker. DIDS and DCPIB inhibited I(Cl,Vol) by 92.6% ± 7.3% and 78.4% ± 5.5%, respectively. Our data further revealed that the PI3K inhibitor LY294002 facilitated the current with the peak amplitude of 19.54 ± 2.70 pA/pF. To the contrary, insulin partially inhibited the current amplitude with the peak current amplitude of 15.4 ± 2.13 pA/pF. Taken together, our data depicted staurosporine is capable of activating I(Cl,Vol) channel in cardiomyocytes via possibly a PI3K/Akt-dependent mechanism.

MiR-222 contributes to sex-dimorphic cardiac eNOS expression via ets-1

It is well recognized that there is sex-dimorphic expression of mRNA and protein in the heart; however, the underlying mechanism is poorly understood. Endothelial nitric oxide synthase (eNOS) is an important regulator of cardiac function, and the expression levels of eNOS differ between male and female hearts. The aim of this study was to examine whether expression of specific microRNA (miRNA, miR) in males and females contributes to changes in the expression of eNOS. miRNA was extracted from the myocardium of male and female C57BL/6 mice and subjected to an Affymetrix miRNA array. Decreased expression of miR-222 was discovered in females and confirmed by qRT-PCR from whole heart or isolated cardiomyocytes. The transcription factor V-ets erythroblastosis virus E26 oncogene homolog-1 (ets-1) was identified as a potential target of miR-222 using TargetScan, and fivefold increased ets-1 protein expression in females was confirmed by Western blot. Targeting of ets-1 by miR-222 was determined in HEK293 cells overexpressing luciferase under regulation of either the ets-1 3'-UTR, a null 3'-UTR control, or a scrambled ets-1 3'-UTR and treated with a small molecule miR-222 mimic or inhibitor. Additionally qRT-PCR confirmed that mRNA levels of the ets-1 transcriptional target, eNOS, were 25% higher in females. Compared with untreated myocyte controls, 50% inhibition of eNOS expression was achieved by treatment with a miR-222 mimic, compared with a 25% increase due to miR-222 inhibitor. Our findings indicate that sex-dependent miR-222 regulation alters the expression of the cardiac regulatory protein eNOS.

Circulating Porphyromonas gingivalis lipopolysaccharide induces left ventricular dysfunction through MMP‐9 regulation of inflammation

Periodontal disease (PD) has been correlated to myocardial infarction (MI) however the mechanism whereby PD influences remodeling of the left ventricle (LV) is not understood. Matrix metalloproteinase (MMP)‐9 regulates LV remodeling by coordinating the inflammatory and fibrotic responses. We hypothesized that low circulating levels of P. gingivalis‐lipopolysaccharide (Pg‐LPS) would induce an LV inflammatory response regulated by MMP‐9. Using echocardiography, we compared LV function in 4–7 month old wild type (WT) and MMP‐9 null mice (n≥6/group) exposed to 1 or 28 days of Pg‐LPS (1 μg/g body weight) or saline. LV function was not affected at d1. By d28, the ejection fraction of WT+LPS significantly decreased to 57±1% compared to 67±1% for saline (p&lt;0.05). This decrease was attenuated in null+LPS (63±1%), implicating MMP‐9 as a key regulator of cardiac function. WT+LPS LV MMP‐9 expression increased 3 fold at d1 compared to d28 and saline (both p&lt;0.05). IL‐1α plasma levels increased at d28 in WT+LPS (187±32 pg/mL) compared to saline (110±14 pg/mL, p&lt;0.05) and null+LPS mice (102±12 pg/mL; p&lt;0.05). LV genes, Ccl6, Ccl9, and C3, doubled in WT+LPS at d1 compared to saline, but returned to baseline by d28. In contrast, the inflammatory response in null mice was sustained until d28. In conclusion, Pg‐LPS induced significant changes in the inflammatory response in an MMP‐9 dependent manner causing a decrease in LV function.

Subtype specific β‐adrenerigic receptor‐mediated transactivation of epidermal growth factor receptor decreases apoptosis through differential activation of ERK1/2 and Akt

β‐adrenergic receptors (βAR) are critical regulators of cardiac function whose dysregulation during heart failure are associated with diminished cardiac function, however βAR‐mediated EGFR transactivation has been shown to relay cardioprotection via unknown mechanisms. While both β1AR and β2AR are capable of inducing EGFR transactivation the relative impact of each on cardiac function is unknown. We hypothesized that transactivation of EGFR will produce distinct cardiomyocyte signaling responses leading to decreases in apoptosis that are βAR subtype‐specific. To test this hypothesis, rat neonatal cardiomyocytes (RNCM) were treated with dobutamine (β1AR agonist) or salbutamol (β2AR agonist) in the presence or absence of the EGFR antagonist AG1478. Following 10 min stimulation, differences in ERK1/2 and Akt phosphorylation and localization were observed. Apoptosis was decreased which was abrogated with EGFR inhibition. EGFR‐transactivation by βAR decreased apoptosis through ERK1/2 and Akt pathways. These results demonstrate differential impact of β1AR and β2AR‐mediated EGFR transactivation on the subcellular activation of ERK1/2 and Akt and the regulation of cell death in cardiomyocytes.

Pathophysiological Consequences of TAT-HKII Peptide Administration Are Independent of Impaired Vascular Function and Ensuing Ischemia

We have shown that partial dissociation of hexokinase II (HKII) from mitochondria in the intact heart using low-dose transactivating transcriptional factor (TAT)-HKII (200 nmol/L) prevents the cardioprotective effects of ischemic preconditioning, whereas high-dose TAT-HKII (10 μmol/L) administration results in rapid myocardial dysfunction, mitochondrial depolarization, and disintegration. In this issue of Circulation Research, Pasdois et al argue that the deleterious effects of TAT-HKII administration on cardiac function are likely because of vasoconstriction and ensuing ischemia. To investigate whether altered vascular function and ensuing ischemia recapitulate the deleterious effects of TAT-HKII in intact myocardium. Using a variety of complementary techniques, including mitochondrial membrane potential (ΔΨm) imaging, high-resolution optical action potential mapping, analysis of lactate production, nicotinamide adenine dinucleotide epifluorescence, lactate dehydrogenase release, and electron microscopy, we provide direct evidence that refutes the notion that acute myocardial dysfunction by high-dose TAT-HKII peptide administration is a consequence of impaired vascular function. Moreover, we demonstrate that low-dose TAT-HKII treatment, which abrogates the protective effects of ischemic preconditioning, is not associated with ischemia or ischemic injury. Our findings challenge the notion that the effects of TAT-HKII are attributable to impaired vascular function and ensuing ischemia, thereby lending further credence to the role of mitochondria-bound HKII as a critical regulator of cardiac function, ischemia-reperfusion injury, and cardioprotection by ischemic preconditioning.

Decreased Autonomic Modulation of Heart Rate and Altered Cardiac Sympathovagal Balance in Patients with Cushing's Syndrome: Role of Endogenous Hypercortisolism

Background/Aims: Endogenous Cushing's syndrome is associated with a higher risk of cardiovascular morbidity and mortality. Previous literature suggested multiple possible links by which hypercortisolism may alter the autonomic control of cardiovascular functions. We investigated the impact of chronic endogenous hypercortisolism on the autonomic regulation of cardiac functions by short-term heart rate variability analysis. Methods: Eighteen patients with endogenous Cushing's syndrome and 20 age-, gender- and BMI-matched controls participated in the study. ECG signal was acquired in lead II configuration for 5 min and heart rate variability assessment was made in both time and frequency domain using the extracted RR interval data. Results: All time and frequency domain measures of heart rate variability were significantly (p < 0.05) lower in the patient group compared to the control group. The patient group had an altered sympathovagal balance with low frequency/high frequency band ratio significantly higher than the control group [1.857 (0.6747-2.610) vs. 0.8581 (0.4779-1.352); p = 0.0253]. A significant negative correlation was obtained between normalized high frequency power of heart rate variability and basal cortisol levels (r = -0.6594; p = 0.0029). Multiple linear regression analysis identified age, disease duration (in months), basal cortisol levels and systolic blood pressure as independent predictors of normalized high frequency power. Conclusion: Findings of the study clearly portrayed the diminished autonomic modulation of heart rate in endogenous Cushing's syndrome and its possible relationship with hypercortisolism as the main causative factor. Diminished heart rate variability may be an indicator of the increased risk of cardiac mortality in these patients.

The heart and the brain: an intimate and underestimated relation.

The entire field of neurocardiology can be divided into three categories: 1) the brain’s effects on the heart (such as Takotsubo cardiomyopathy), 2) neurocardiac syndromes such as hypertrophic cardiomyopathy in Friedreich ataxia and 3) the heart’s effects on the brain, such as the cardiac origin of embolic stroke [1]. The broken–heart syndrome or Takotsubo cardiomyopathy is a perfect example of the intimate relation between the brain and the heart as it mimics an acute coronary syndrome and can even result in sudden unexpected death [2]. It is an example of major cardiac pathologies seen in emotional and neurological catastrophes, such as subarachnoid bleeding and cocaine-related death, and is due to severe overactivity of the sympathetic limb of the autonomic nervous system leading to cardiac myofibrillar degeneration [1]. The central role of the autonomic nervous system in the regulation of cardiac function has also resulted in the introduction of neuromodulation to effectively improve cardiac function. A relatively underestimated example of the heart’s effects on the brain is the link between dysfunction of the heart and the brain. This may become a very important health care problem in the near future, as both cardiac dysfunction and progressive loss of cognitive functioning are prominent features of ageing. The apparent lack of appreciation of the link between cardiac and brain (dys)function is probably due to the monodisciplinary approach by cardiologists and neurologists and by the reductionist approach of modern medical research. Yet it is time for a more integrative view to the heart-brain connection as recent data indicate that cardiovascular conditions contribute to cognitive impairment [3]. Pioneers in the field recognised the importance of this link and organised the successful first international conference on the Heart & Brain in Paris in March 2012. This conference aimed to ‘consolidate the hybrid field of neurocardiology or cardioneurology: the strokologist will teach the cardiologist about the brain and the neurologist will learn how the heart is affecting the brain’ (see website Heart & Brain Conference, 1–3 March 2012, Paris). Neurologists and neuropsychologists also increasingly appreciate the importance of vascular risk factors and cardiovascular diseases on cognitive function. In a preface to a recent special issue on vascular risk factors and Alzheimer’s disease in the Journal of Alzheimer’s Disease, Jack de La Torre stated that: ‘the significant association between cardiovascular diseases and an increased risk of Alzheimer’s disease offers the possibility to markedly reduce incident dementia by early identification and appropriate medical management of cardiovascular risk factors and diseases and could be a monumental step forward in reducing the worldwide prevalence of dementia, which will rise from a current 35 million to 60 million people in 2030’ [4]. Thus this special issue of the Netherlands Heart Journal is very timely by providing different perspectives into the intriguing, intimate and clinically increasingly important relations between the brain and the heart. It describes the important role of the autonomic nervous system in ventricular arrhythmias [5, 6] and in psychological distress [7] and its potential role in the treatment of cardiovascular diseases, either by electrical neuromodulation [8], vagal stimulation [9], denervation [10] or music [11]. It also provides insights on the reduction of cognitive functions after major forms of cardiac surgery, on cardiovascular risk factors that affect cognitive functions in the brain [12] and on the effects of exercise training on cardiac and cognitive function [13]. It highlights the importance of the baroreflex in the heart-brain connection [14, 15], and potential pathophysiological and molecular mechanisms of the effects of cerebral hypoperfusion and cognitive function [16]. This special issue of the Netherlands Heart Journal thus provides some unique insights into the integrative world of cardioneurology or neurocardiology in which cardiologists, neurologists and neuropsychologists join forces to better understand, diagnose and treat cardiovascular diseases and cognitive dysfunction, acknowledging the intimate and underestimated relation between the heart and the brain and offering new possibilities to treat cardiovascular diseases by modulating the activity of the autonomic nervous system and to treat cognitive dysfunction by reducing the cardiovascular risk and disease burden.

Effects of an isotonic beverage on autonomic regulation during and after exercise.

BackgroundWith prolonged physical activity, it is important to maintain adequate fluid balance. The impact of consuming isotonic drinks during and after exercise on the autonomic regulation of cardiac function is unclear. This study aimed to analyze the effects of consuming an isotonic drink on heart rate variability (HRV) during and after prolonged exercise.MethodsThirty-one young males (21.55 ± 1.89 yr) performed three different protocols (48 h interval between each stage): I) maximal exercise test to determine the load for the protocols; II) Control protocol (CP) and; III). Experimental protocol (EP). The protocols consisted of 10 min at rest with the subject in the supine position, 90 min of treadmill exercise (60% of VO2 peak) and 60 min of rest placed in the dorsal decubitus position. No rehydration beverage consumption was allowed during CP. During EP, however, the subjects were given an isotonic solution (Gatorade, Brazil) containing carbohydrate (30 g), sodium (225 mg), chloride (210 mg) and potassium (60 mg) per 500 ml of the drink. For analysis of HRV data, time and frequency domain indices were investigated. HRV was recorded at rest (5–10 min), during exercise (25–30 min, 55–60 min and 85–90 min) and post-exercise (5–10 min, 15–20 min, 25–30 min, 40–45 min and 55–60 min).ResultsRegardless of hydration, alterations in the SNS and PSNS were observed, revealing an increase in the former and a decrease in the latter. Hydrating with isotonic solution during recovery induced significant changes in cardiac autonomic modulation, promoting faster recovery of linear HRV indices.ConclusionHydration with isotonic solution did not significantly influence HRV during exercise; however, after exercise it promoted faster recovery of linear indices.

Induction of MicroRNA-24 by HIF-1 Protects Against Ischemic Injury in Rat Cardiomyocytes

MicroRNAs are emerging as important regulators of cardiac function. This study investigated the role of microRNA-24 (miR-24) in ischemic cardiomyocytes, based on the observation that miR-24 expression was significantly enhanced in the ischemic myocardium of rats. Using primary cultured rat cardiomyocytes, cell injury was induced by ischemic conditions, and the cells were evaluated for changes in lactate dehydrogenase (LDH) release, cell viability, apoptosis and necrosis. The results showed that miR-24 was increased in myocytes exposed to ischemia. When miR-24 was further overexpressed in ischemic myocytes using the mimic RNA sequence, LDH release was reduced, cell viability was enhanced, and apoptosis and necrosis rates were both decreased. By contrast, a deficiency in miR-24 resulted in the largest LDH release, lowest cell viability and highest apoptosis and necrosis rates in normal and ischemic myocytes, with significant changes compared to that of non-transfected myocytes. Additionally, the mRNA and protein levels of the pro-apoptotic gene, BCL2L11, were down-regulated by miR-24 overexpression and up-regulated by miR-24 deficiency. The luciferase reporter assay confirmed BCL2L11 to be a target of miR-24. Overall, this study showed a protective role for miR-24 against myocardial ischemia by inhibiting BCL2L11, and may represent a potential novel treatment for ischemic heart disease.

Chronic fluoxetine reduces autonomic control of cardiac rhythms in rats with congestive heart failure

Up to 40% of patients with heart failure develop depression, and depression is an independent risk factor for cardiovascular mortality in this patient population. Consequently, increasing numbers of patients with heart failure are treated with antidepressants. Selective serotonin reuptake inhibitors are typically the antidepressant of choice since this drug class has limited cardiovascular toxicity. However, little is known about the effects of selective serotonin reuptake inhibitors on autonomic cardiac regulation in congestive heart failure (CHF). Here, indexes of cardiac autonomic control were evaluated before and during chronic fluoxetine (FLX) treatment (20 mg·kg(-1)·day(-1), 5 wk) in rats that developed CHF after coronary artery ligation. FLX reduced the low-frequency (LF) component of heart rate variability (HRV; P < 0.01) as well as the sympathetic contribution to LF HRV (P < 0.01) in both CHF and sham-operated rats. Both FLX and CHF reduced high-frequency HRV (P < 0.01). Spontaneous baroreflex gain was decreased in CHF rats 8 wk after ligation (P < 0.01). Cross-spectral coherence between the interbeat interval and mean arterial pressure was reduced in the LF domain 3 wk after ligation in CHF rats (P < 0.01) and was further reduced after chronic FLX treatment (P < 0.01). Plasma catecholamines and LF blood pressure variability were not affected by FLX. Chronotropic responses to both efferent vagal nerve stimulation and isoproterenol administration were reduced in CHF rats and by FLX (P < 0.01), whereas inotropic responses to isoproterenol were reduced only in CHF rats (P < 0.01). These data indicate that chronic FLX reduces the responsiveness to autonomic output controlling cardiac rhythm and may further compromise autonomic regulation of cardiac function in CHF.

The multifunctional Ca2 +/calmodulin-dependent protein kinase II delta (CaMKIIδ) phosphorylates cardiac titin's spring elements

Titin-based passive stiffness is post-translationally regulated by several kinases that phosphorylate specific spring elements located within titin's elastic I-band region. Whether titin is phosphorylated by calcium/calmodulin dependent protein kinase II (CaMKII), an important regulator of cardiac function and disease, has not been addressed. The aim of this work was to determine whether CaMKIIδ, the predominant CaMKII isoform in the heart, phosphorylates titin, and to use phosphorylation assays and mass spectrometry to study which of titin's spring elements might be targeted by CaMKIIδ. It was found that CaMKIIδ phosphorylates titin in mouse LV skinned fibers, that the CaMKIIδ sites can be dephosphorylated by protein phosphatase 1 (PP1), and that under baseline conditions, in both intact isolated hearts and skinned myocardium, about half of the CaMKIIδ sites are phosphorylated. Mass spectrometry revealed that both the N2B and PEVK segments are targeted by CaMKIIδ at several conserved serine residues. Whether phosphorylation of titin by CaMKIIδ occurs in vivo, was tested in several conditions using back phosphorylation assays and phospho-specific antibodies to CaMKIIδ sites. Reperfusion following global ischemia increased the phosphorylation level of CaMKIIδ sites on titin and this effect was abolished by the CaMKII inhibitor KN-93. No changes in the phosphorylation level of the PEVK element were found suggesting that the increased phosphorylation level of titin in IR (ischemia reperfusion) might be due to phosphorylation of the N2B element. The findings of these studies show for the first time that titin can be phosphoryalated by CaMKIIδ, both in vitro and in vivo, and that titin's molecular spring region that determines diastolic stiffness is a target of CaMKIIδ.

Highlights from the Literature

Highlights from the Literature

Circulation: Cardiovascular Interventions Editors’ Picks

<i>Circulation: Cardiovascular Interventions</i> Editors’ Picks