Natig Gassanov

Ischemic Preconditioning for Prevention of Contrast Medium–Induced Nephropathy Randomized Pilot RenPro Trial (Renal Protection Trial)

Background— Contrast medium–induced acute kidney injury is associated with substantial morbidity and mortality. The underlying mechanism has been attributed in part to ischemic kidney injury. The aim of this randomized, double-blind, sham-controlled trial was to assess the impact of remote ischemic preconditioning on contrast medium–induced acute kidney injury. Methods and Results— Patients with impaired renal function (serum creatinine >1.4 mg/dL or estimated glomerular filtration rate <60 mL · min−1 · 1.73 m−2) undergoing elective coronary angiography were randomized in a 1:1 ratio to standard care with (n=50) or without ischemic preconditioning (n=50; intermittent arm ischemia through 4 cycles of 5-minute inflation and 5-minute deflation of a blood pressure cuff). Overall, both study groups were at high risk of developing contrast medium–induced acute kidney injury according to the Mehran risk score. The primary end point was the incidence of contrast medium–induced kidney injury, defined as an increase in serum creatinine ≥25% or ≥0.5 mg/dL above baseline at 48 hours after contrast medium exposure. Contrast medium–induced acute kidney injury occurred in 26 patients (26%), 20 (40%) in the control group and 6 (12%) in the remote ischemic preconditioning group (odds ratio, 0.21; 95% confidence interval, 0.07–0.57; P =0.002). No major adverse events were related to remote ischemic preconditioning. Conclusions— Remote ischemic preconditioning before contrast medium use prevents contrast medium–induced acute kidney injury in high-risk patients. Our findings merit a larger trial to establish the effect of remote ischemic preconditioning on clinical outcomes. Clinical Trial Registration— URL: [http://www.germanctr.de][1]. Unique identifier: U1111-1118-8098. # Clinical Perspective {#article-title-44} [1]: http://www.germanctr.de.Background— Contrast medium–induced acute kidney injury is associated with substantial morbidity and mortality. The underlying mechanism has been attributed in part to ischemic kidney injury. The aim of this randomized, double-blind, sham-controlled trial was to assess the impact of remote ischemic preconditioning on contrast medium–induced acute kidney injury. Methods and Results— Patients with impaired renal function (serum creatinine >1.4 mg/dL or estimated glomerular filtration rate <60 mL · min−1 · 1.73 m−2) undergoing elective coronary angiography were randomized in a 1:1 ratio to standard care with (n=50) or without ischemic preconditioning (n=50; intermittent arm ischemia through 4 cycles of 5-minute inflation and 5-minute deflation of a blood pressure cuff). Overall, both study groups were at high risk of developing contrast medium–induced acute kidney injury according to the Mehran risk score. The primary end point was the incidence of contrast medium–induced kidney injury, defined as an increase in serum creatinine ≥25% or ≥0.5 mg/dL above baseline at 48 hours after contrast medium exposure. Contrast medium–induced acute kidney injury occurred in 26 patients (26%), 20 (40%) in the control group and 6 (12%) in the remote ischemic preconditioning group (odds ratio, 0.21; 95% confidence interval, 0.07–0.57; P=0.002). No major adverse events were related to remote ischemic preconditioning. Conclusions— Remote ischemic preconditioning before contrast medium use prevents contrast medium–induced acute kidney injury in high-risk patients. Our findings merit a larger trial to establish the effect of remote ischemic preconditioning on clinical outcomes. Clinical Trial Registration— URL: http://www.germanctr.de. Unique identifier: U1111-1118-8098.

Identification and characterization of embryonic stem cell-derived pacemaker and atrial cardiomyocytes

The aim of this study was to identify and functionally characterize cardiac subtypes during early stages of development. For this purpose, transgenic embryonic stem cells were generated using the α‐myosin heavy chain promoter driving the expression of the enhanced green fluorescent protein (EGFP). EGFP‐positive clusters of cells were first observed as early as 7 days of development, thus, even before the initiation of the contractile activity. Flow cytometry and single‐cell fluorescence measurements evidenced large diversities of EGFP intensity. Patch‐clamp experiments showed EGFP expression exclusively in pacemaker and atrial but not ventricular cells. The highest fluorescence intensities were detected in pacemaker‐like cardiomyocytes. In accordance, multielectrode‐array recordings of whole embryoid bodies confirmed that the pacemaker center coincided with strongly EGFP‐positive areas. The cardiac subtypes displayed already at this early stage differential characteristics of electrical activity and ion channel expression. Thus, quantitation of the α‐myosin heavy chain driven reporter gene expression allows identification and functional characterization of early cardiac subtypes.

Endothelin induces differentiation of ANP-EGFP expressing embryonic stem cells towards a pacemaker phenotype

Currently, only limited insight into mechanisms promoting the differentiation and specification of the mammalian cardiac conduction system is available. Therefore, we established a murine embryonic stem (ES) cell line stably expressing the enhanced green fluorescent protein (EGFP) under the transcriptional control of the human atrial natriuretic peptide (ANP) promoter to further characterize the development of very early stages of the mammalian cardiac conduction tissue. The cardiac nature of ANP‐EGFP positive cells was confirmed by immunostaining. In ANP‐EGFP expressing ES cell‐derived cardiomyocytes, a distinct sublineage of pacemaker cells could be identified. Pacemaker cells displayed a spindle shape and exhibited a higher spontaneous beating rate, faster If current activation and larger If current densities compared with triangular atrial‐like cardiocytes. Exposure to endothelin‐1 significantly increased the percentage of pacemaker‐like cells without affecting their electrophysiological properties. These findings were corroborated by immunostaining with antibodies against connexin 40 and connexin 45, known markers for cardiac conduction tissue. Conversely, treatment of ANP‐EGFP expressing ES cells with neuregulin‐1 exhibited no effect on differentiation. These results indicate that ANP‐EGFP expression enables the identification of ES cell‐derived pacemaker cells by their fluorescence and morphology and that endothelin‐1 promotes the development of ANP‐EGFP positive cardiomyocytes to a pacemaker‐like phenotype.

Testosterone Induces Cytoprotection by Activating ATP-Sensitive K+ Channels in the Cardiac Mitochondrial Inner Membrane

Background—Whereas in the past, androgens were mainly believed to exert adverse effects on the cardiovascular system, recent experimental data postulate a benefit of testosterone for recovery of myocardial function after ischemia/reperfusion injury. Thus, we examined whether testosterone might improve myocardial tolerance to ischemia due to activation of mitochondrial (mitoKATP) and/or sarcoplasmatic (sarcKATP) KATP channels. Methods and Results—In a cellular model of ischemia, testosterone significantly decreased the rate of ischemia-induced death of cardiomyocytes that could be prevented by 5-hydroxydecainoic acid but was unaffected by the sarcKATP blocker HMR1098 and the testosterone receptor antagonist flutamide. To index mitoKATP, mitochondrial flavoprotein fluorescence was measured. Testosterone induced a highly significant increase in mitochondrial flavoprotein fluorescence in intact myocytes and isolated mitoplasts that could be abolished by 5-hydroxydecainoic acid. Testosterone-mediated flavoprotein oxidation of mitoplasts was K+ dependent and ATP sensitive. In mitoplast-attached single-channel recordings, testosterone directly activated an ATP-sensitive K+ channel of the inner mitochondrial membrane. Addition of the KATP channel opener diazoxide and pinacidil to the cytosolic solution activated the ATP-sensitive K+ current comparable to testosterone, whereas 5-hydroxydecainoic acid and glibenclamide inhibited the testosterone-induced current. Patch-clamp experiments of intact myocytes in whole-cell configuration did not demonstrate any effect of testosterone on sarcKATP channels. Conclusions—Our results provide direct evidence for the existence of cardiac mitoKATP and a link between testosterone-induced cytoprotection and activation of mitoKATP. Endogenous testosterone might play a more important role in recovery after myocardial infarction than is currently assumed.

Remote Ischemic Preconditioning and Renoprotection: From Myth to a Novel Therapeutic Option?

There is currently no effective prophylactic regimen available to prevent contrast-induced AKI (CI-AKI), a frequent and life-threatening complication after cardiac catheterization. Therefore, novel treatment strategies are required to decrease CI-AKI incidence and to improve clinical outcomes in these patients. Remote ischemic preconditioning (rIPC), defined as transient brief episodes of ischemia at a remote site before a subsequent prolonged ischemia/reperfusion injury of the target organ, is an adaptational response that protects against ischemic and reperfusion insult. Indeed, several studies demonstrated the tissue-protective effects of rIPC in various target organs, including the kidneys. In this regard, rIPC may offer a novel noninvasive and virtually cost-free treatment strategy for decreasing CI-AKI incidence. This review evaluates the current experimental and clinical evidence for rIPC as a potential renoprotective strategy, and discusses the underlying mechanisms and key areas for future research.

Andersen mutations of KCNJ2 suppress the native inward rectifier current IK1 in a dominant-negative fashion

OBJECTIVE The Andersens syndrome is a hereditary disease, which is characterized by cardiac arrhythmias, periodic paralysis and dysmorphic features. Recently, mutations of the KCNJ2 gene, which encodes the inward rectifying potassium channel subunit Kir2.1, have been identified in affected individuals. However, the functional effects of these mutations have not yet been fully elucidated. METHODS AND RESULTS To clarify this situation we generated known Andersen disease mutants of KCNJ2 which did not yield any measurable K(+) currents in CHO cells indicating that the Andersen mutants failed to form functional homomultimeric complexes. EGFP-tagged KCNJ2 wild-type and mutant channels distributed in a similar homogeneous pattern in the cell membrane suggesting that protein trafficking was not altered by the Andersen mutations but rather implicating that the mutations rendered the KCNJ2 channel non-functional. In heterologous coexpression experiments the Andersen mutants exerted a dominant-negative effect on wild-type KCNJ2. However, the extent of suppression varied between the different KCNJ2 mutants. Given our results in CHO cells, we expressed the disease mutant KCNJ2-S136F in neonate rat cardiomyocytes using adenoviral gene transfer to test the effect of Andersen mutants on native I(K1). I(K1) density was indeed significantly reduced in KCNJ2-S136F-infected cells (n=9) compared to control cells (n=9) over a voltage range from -70 to -150 mV (P<0.05). CONCLUSION These results support that Kir2.x channels are a critical component of native I(K1) in neonate rat cardiomyocytes and that a dominant-negative suppression of I(K1) in native cells is the pathophysiological correlate of the Andersens syndrome.

Arginine vasopressin-mediated cardiac differentiation: insights into the role of its receptors and nitric oxide signaling.

Despite the existence of a functional arginine vasopressin (AVP) system in the adult heart and evidence that AVP induces myogenesis, its significance in cardiomyogenesis is currently unknown. In the present study, we hypothesized a role for AVP in cardiac differentiation of D3 and lineage-specific embryonic stem (ES) cells expressing green fluorescent protein under the control of atrial natriuretic peptide (Anp) or myosin light chain-2V (Mlc-2V) promoters. Furthermore, we investigated the nitric oxide (NO) involvement in AVP-mediated pathways. AVP exposure increased the number of beating embryoid bodies, fluorescent cells, and expression of Gata-4 and other cardiac genes. V1a and V2 receptors (V1aR and V2R) differentially mediated these effects in transgenic ES cells, and exhibited a distinct developmentally regulated mRNA expression pattern. A NO synthase inhibitor, l-NAME, powerfully antagonized the AVP-induced effects on cardiogenic differentiation, implicating NO signaling in AVP-mediated pathways. Indeed, AVP elevated the mRNA and protein levels of endothelial NO synthase (eNOS) through V2R stimulation. Remarkably, increased beating activity was found in AVP-treated ES cells with down-regulated eNOS expression, indicating the significant involvement of additional pathways in cardiomyogenic effects of AVP. Finally, patch clamp recordings revealed specific AVP-induced changes of action potentials and increased l-type Ca2+ (ICa,L) current densities in differentiated ventricular phenotypes. Thus, AVP promotes cardiomyocyte differentiation of ES cells and involves Gata-4 and NO signaling. AVP-induced action potential prolongation appears likely to be linked to the increased ICa,L current in ventricular cells. In conclusion, this report provides new evidence for the essential role of the AVP system in ES cell-derived cardiomyogenesis.

Arginine vasopressin (AVP) and treatment with arginine vasopressin receptor antagonists (vaptans) in congestive heart failure, liver cirrhosis and syndrome of inappropriate antidiuretic hormone secretion (SIADH).

Arginine vasopressin (AVP) is the major physiological regulator of renal water excretion and blood volume. The AVP pathways of V1aR-mediated vasoconstriction and V2R-induced water retention represent a potentially attractive target of therapy for edematous diseases. Experimental and clinical evidence suggests beneficial effects of AVP receptor antagonists by increasing free water excretion and serum sodium levels. This review provides an update on the therapeutic implication of newly developed AVP receptor antagonists in respective disorders, such as chronic heart failure, liver cirrhosis and syndrome of inappropriate antidiuretic hormone secretion.

Functional Activity of the Carboxyl‐Terminally Extended Oxytocin Precursor Peptide During Cardiac Differentiation of Embryonic Stem Cells

The hypothalamic post‐translational processing of oxytocin (OT)‐neurophysin precursor involves the formation of C‐terminally extended OT forms (OT‐X) that serve as intermediate prohormones. Despite abundant expression of the entire functional OT system in the developing heart, the biosynthesis and implication of OT prohormones in cardiomyogenesis remain unknown. In the present work, we investigated the involvement of OT‐X in cardiac differentiation of embryonic stem (ES) cells. Functional studies revealed the OT receptor‐mediated cardiomyogenic action of OT‐Gly‐Lys‐Arg (OT‐GKR). To obtain further insight into the mechanisms of OT‐GKR‐induced cardiac effects, we generated ES cell lines overexpressing the OT‐GKR gene and enhanced green fluorescent protein (EGFP). The functionality of the OT‐GKR/EGFP construct was assessed by fluorescence microscopy and flow cytometry, with further confirmation by radioimmunoassay and immunostaining. Increased spontaneously beating activity of OT‐GKR/EGFP‐expressing embryoid bodies and elevated expression of GATA‐4 and myosin light chain 2v cardiac genes indicated an inductive effect of endogenous OT‐GKR on ES cell‐derived cardiomyogenesis. Furthermore, patch‐clamp experiments demonstrated induction of ventricular phenotypes in OT‐GKR/EGFP‐transfected and in OT‐GKR‐treated cardiomyocytes. Increased connexin 43 protein in OT‐GKR/EGFP‐expressing cells further substantiated the evidence that OT‐GKR modifies cardiac differentiation toward the ventricular sublineage. In conclusion, this report provides new evidence of the biological activity of OT‐X, notably OT‐GKR, during cardiomyogenic differentiation.

K+ channel regulator KCR1 suppresses heart rhythm by modulating the pacemaker current If.

Hyperpolarization-activated, cyclic nucleotide sensitive (HCN) channels underlie the pacemaker current If, which plays an essential role in spontaneous cardiac activity. HCN channel subunits (HCN1-4) are believed to be modulated by additional regulatory proteins, which still have to be identified. Using biochemistry, molecularbiology and electrophysiology methods we demonstrate a protein-protein interaction between HCN2 and the K+ channel regulator protein 1, named KCR1. In coimmunoprecipitation experiments we show that KCR1 and HCN2 proteins are able to associate. Heterologously expressed HCN2 whole-cell current density was significantly decreased by KCR1. KCR1 profoundly suppressed IHCN2 single-channel activity, indicating a functional interaction between KCR1 and the HCN2 channel subunit. Endogenous KCR1 expression could be detected in adult and neonatal rat ventriculocytes. Adenoviral-mediated overexpression of KCR1 in rat cardiomyocytes (i) reduced If whole-cell currents, (ii) suppressed most single-channel gating parameters, (iii) altered the activation kinetics, (iv) suppressed spontaneous action potential activity, and (v) the beating rate. More importantly, siRNA-based knock-down of endogenous KCR1 increased the native If current size and single-channel activity and accelerated spontaneous beating rate, supporting an inhibitory action of endogenous KCR1 on native If. Our observations demonstrate for the first time that KCR1 modulates IHCN2/If channel gating and indicate that KCR1 serves as a regulator of cardiac automaticity.