Colleen S. Kondo

Developmental Changes in the Delayed Rectifier K+ Channels in Mouse Heart

Expression of cardiac transient outward current and inwardly rectifying K+ current is age dependent. However, little is known about age-related changes in cardiac delayed rectifier K+ current (IK, with rapidly and slowly activating components, IKr and IKs, respectively). Accordingly, the purpose of the present study was to assess developmental changes in IK channels in fetal, neonatal, and adult mouse ventricles. Three techniques were used: conventional microelectrode to measure the action potential, voltage clamp to record macroscopic currents of IK, and radioligand assay to examine [3H]dofetilide binding sites. The extent of prolongation of action potential duration at 95% repolarization (APD95) by a selective IKr blocker, dofetilide (1 mumol/L), dramatically decreased from fetal (137% +/- 18%) to day-1 (75% +/- 29%) and day-3 (20% +/- 15%) neonatal mouse ventricular tissues (P < .01). Dofetilide did not prolong APD95 in adult myocardium. IKr is the sole component of IK in day-18 fetal mouse ventricular myocytes. However, both IKr and IKs were observed in day-1 neonatal ventricular myocytes. With further development, IKs became the dominant component of IK in day-3 neonates. In adult mouse ventricular myocytes, neither IKr nor IKs was observed. Correspondingly, a high-affinity binding site for [3H]dofetilide was present in fetal mouse ventricles but was absent in adult ventricles. The complementary data from microelectrode, voltage-clamp, and [3H]dofetilide binding studies demonstrate that expression of the IK channel is developmentally regulated in the mouse heart.

Electrophysiological Characterization of an Alternatively Processed ERG K+ Channel in Mouse and Human Hearts

Mutants of HERG, the human form of ERG (the ether-a-go-go-related K+ channel gene), are responsible for some forms of the long-QT syndrome, an abnormality of cardiac repolarization. HERG was cloned from brain and has properties similar but not identical to the rapidly activating component of the native cardiac K+ channel current (Ikr). We identified in the mouse an alternatively processed form of ERG (MERG B) that is expressed abundantly in heart but only in trace amounts in brain. MERG B has a unique 36-amino acid NH2-terminal domain that is strongly basic and considerably shorter than the 376-amino acid NH2-terminal domain of HERG. When expressed in Xenopus oocytes, the kinetics of activation and deactivation of the MERG B current were best fit by a biexponential function, with the fast components dominant over the slow components. The fast component of activation had a mean tau value of 163 +/- 16 ms at -20 mV and 8 +/- 4 ms at +20 mV (n = 4). The fast component of deactivation had a mean tau value of 145 +/- 29 ms at -20 mV and 12 +/- 4 ms at -90 mV (n = 4). The MERG B current was blocked by the selective IKr blocker, dofetilide, with an IC50 of 54 nmol/L. In addition, we isolated HERG B, the human homologue of MERG B, which has electrophysiological characteristics qualitatively similar to those of MERG B. We have identified ERG B, an alternatively processed isoform of the ERG gene, expressed selectively in heart and with electrophysiological characteristics similar to those of native cardiac IKr.

Voltage-sensitive dye mapping of activation and conduction in adult mouse hearts.

AbstractA custom-made apparatus based on a charge-coupled-device camera has been used to monitor changes in fluorescence from Langendorff-perfused adult mouse hearts stained with a voltage-sensitive dye, di-4-ANEPPS. With this approach it is possible to monitor activation of the ventricles at high temporal (375 μs/frame) and spatial resolution 72 × 78pixels,100 ×100 μm/pixel. In sinus rhythm, activation occurred with a complicated breakthrough pattern on both ventricles, and a total activation time of 3.51 ± 0.16ms (32 °C). A stimulus applied near the apex of the left ventricle resulted in a single activation wave front with a total activation time of 8.18 ± 0.25 ms. Pacing from a site near the middle of the left ventricular epicardial surface revealed anisotropic conduction, indicating that conduction occurs preferentially in the direction of the predominant fiber orientation. The total activation time in this configuration was 5.44 ± 0.24 ms. The difference in total activation time between sinus rhythm and epicardial stimulation suggests an important role for transmural conduction (the Purkinje system) in the mouse heart. These findings provide much of the necessary background needed for studying conduction abnormalities in genetically altered mice and suggest that the comparison of sinus rhythm and epicardial pacing can be used to reveal transmural conduction abnormalities.

Morphometric Study of the Role of Pulmonary Arterial Flow in Fetal Lung Growth in Sheep

ABSTRACT: Pulmonary hypoplasia has been associated with absent or hypoplastic pulmonary artery in four cases in humans. Despite these reports, the effects of decreased pulmonary arterial flow on fetal lung growth have not been adequately studied. This study defines the effects of left pulmonary artery (LPA) ligation on fetal lung growth in sheep by comparing morphometrically determined pulmonary volumes from LPA-ligated, sham-operated, and unoperated control fetuses. LPA ligation (n = 5) or sham operation (n = 4) was performed at 105- to 114-d gestation. At 140-d gestation these fetuses were delivered and the lungs were intratracheally fixed for light microscopy. At 112 d (n = 4) and at 140 d (n = 4), unoperated control fetuses were similarly delivered. Absolute pulmonary volumes were then measured using standard stereologic methods. Normal growth of the left lung from 112 to 140 d resulted in significant increases in wet and dry wt, displacement volume, and volumes of future airspace and capillary contents. LPA ligation caused significant decreases in left lung wet and dry wt, displacement volume, and in absolute volumes of fine nonparenchyma, future airspace, parenchymal tissue, and capillary contents compared to sham-operated and 140-d controls. Parenchymal tissue volume was also less than in 112-d controls. In addition, lung wt, displacement volume, and future airspace volume were significantly decreased in sham-operated fetuses compared to 140-d controls. The effects of LPA ligation on bronchial collateral circulation and factors known to affect lung growth (i.e. lung fluid volume) remain to be determined. Clearly, during the canalicular and alveolar stage of fetal lung development, pulmonary arterial flow is necessary for normal lung growth.

Influence of carotid denervation on the arousal and cardiopulmonary response to rapidly developing hypoxemia in lambs

ABSTRACT: Experiments were done on five lambs to determine if carotid denervation influences the arousal and cardiopulmonary responses to rapidly developing hypoxemia during sleep. Each lamb was anesthetized and instrumented for recordings of electrocorticogram, electrooculogram, nuchal and diaphragm electromyograms, and measurements of arterial blood pressure and arterial hemoglobin oxygen saturation. The carotid chemoreceptors and baroreceptors were denervated, a tracheostomy was done, and a fenestrated tracheostomy tube was placed in the trachea so that the inspired oxygen mixture could be changed quickly. No sooner than 3 d after surgery, measurements were made in quiet sleep and active sleep during control periods when the animal was breathing 21% oxygen and during experimental periods of rapidly developing hypoxemia when the animal was breathing 5% oxygen. Rapidly developing hypoxemia was terminated during each epoch by changing the inspired gas mixture back to 21% oxygen once the animal aroused from sleep or once the arterial Hb oxygen saturation decreased to 30%. Arousal occurred during only 4 of 11 epochs in quiet sleep and during only 3 of 14 epochs in active sleep before the arterial Hb oxygen saturation decreased to 30%. These data provide evidence that the carotid chemoreceptors and/or carotid baroreceptors play a major role in causing arousal from sleep during rapidly developing hypoxemia in lambs.

Influence of carotid denervation on the arousal and cardiopulmonary responses to upper airway obstruction in lambs.

ABSTRACT: Experiments were done on five lambs to determine if carotid denervation influences the arousal and cardiopulmonary responses to upper airway obstruction during sleep. Each lamb was anesthetized and instrumented for recordings of electrocorticogram, electro-oculogram, nuchal and diaphragm electromyograms. and measurements of arterial blood pressure and arterial Hb oxygen saturation. A tracheotomy was done and a fenestrated tracheotomy tube placed in the trachea. During the study, a 5 F balloon-tipped catheter was inserted into the tracheotomy tube so that air flow could be obstructed by inflating the balloon. No sooner than 3 d after surgery, measurements were made in quiet sleep and active sleep during control periods when the animal was breathing room air and during experimental periods of upper airway obstruction. Carotid denervation significantly affected the arousal response to upper airway obstraction. Arousal occurred during 14 of 14 epochs in quiet sleep and during 12 of 13 epochs in active sleep before the arterial Hb oxygen saturation decreased to 30%. However, the time to arousal was increased and the arterial Hb oxygen saturation at arousal was decreased in carotid-denervated lambs compared with what we have previously observed in carotid-intact lambs. These data provide evidence that the caroid chemoreceptors and/or carotid baroreceptors play a major role in causing arousal from sleep during upper airway obstruction in lambs. Our results may have implications for sudden infant death syndrome, because it is possible that alterations in the arousal response to respiratory stimuli play a role in sudden infant death.

Monocyte chemotactic protein-1 inhibits chondrogenesis of synovial mesenchymal progenitor cells: an in vitro study.

Osteoarthritis (OA) is a multifactorial, often progressive, painful disease. OA often progresses with an apparent irreversible loss of articular cartilage, exposing underlying bone, resulting in pain and loss of mobility. This cartilage loss is thought to be permanent due to ineffective repair and apparent lack of stem/progenitor cells in that tissue. However, the adjacent synovial lining and synovial fluid are abundant with mesenchymal progenitor/stem cells (synovial mesenchymal progenitor cells [sMPCs]) capable of differentiating into cartilage both in vitro and in vivo. Previous studies have demonstrated that MPCs can home to factors such as monocyte chemotactic protein 1 (MCP‐1/CCL2) expressed after injury. While MCP‐1 (and its corresponding receptors) appears to play a role in recruiting stem cells to the site of injury, in this study, we have demonstrated that MCP‐1 is upregulated in OA synovial fluid and that exposure to MCP‐1 activates sMPCs, while concurrently inhibiting these cells from undergoing chondrogenesis in vitro. Furthermore, exposure to physiological (OA knee joint synovial fluid) levels of MCP‐1 triggers changes in the transcriptome of sMPCs and prolonged exposure to the chemokine induces the expression of MCP‐1 in sMPCs, resulting in a positive feedback loop from which sMPCs cannot apparently escape. Therefore, we propose a model where MCP‐1 (normally expressed after joint injury) recruits sMPCs to the area of injury, but concurrently triggers changes in sMPC transcriptional regulation, leading to a blockage in the chondrogenic program. These results may open up new avenues of research into the lack of endogenous repair observed after articular cartilage injury and/or arthritis. Stem Cells 2013;31:2253–2265

Hypomagnesemia : Characterization of a model of sudden cardiac death

OBJECTIVES We sought to compare the incidence of sudden death in rats treated with magnesium-deficient and control diets and to address the electrophysiologic characteristics associated with these end points. BACKGROUND Although magnesium deficiency is associated with an increased incidence of sudden cardiac death in patients, there has been no clear cause and effect relation because of a number of covariables, including diuretic use, hypokalemia, digitalis use and left ventricular dysfunction. METHODS Hypomagnesemic rats and their paired control rats underwent in vivo electrophysiologic studies and measurements of the total calcium and magnesium content of their cardiac ventricles RESULTS Serum magnesium levels were 0.5 +/- 0.3 mEq/liter (mean +/- SD) in hypomagnesemic animals and 1.2 +/- 0.9 mEq/liter in control animals. A modest but significant prolongation of the repolarization time was seen at the apical epicardial site (83 +/- 8 ms in hypomagnesemic rats vs. 68 +/- 13 ms in control rats, p < 0.05), but not at the other sites studied. Bradyarrhythmias and tachyarrhythmias were observed in 82% of the hypomagnesemic rats during the in vivo electrophysiologic studies, compared with 0% in the control group. During these studies, sudden, unexpected asystolic deaths were observed in 4 of 11 hypomagnesemic rats and 0 of 8 control rats. Polymorphic nonsustained ventricular tachycardia was provoked by rapid pacing in 5 to 11 hypomagnesemic rats and 0 of 8 control rats. Three of six hypomagnesemic rats exposed to auditory stimuli developed seizures, followed immediately by sudden deaths-two due to asystole and one due to ventricular fibrillation-although no end points occurred in the control animals. CONCLUSIONS In this model, magnesium deficiency results in sudden cardiac death. The presence of startle induction of sudden death preceded by seizures suggests that sudden cardiac death results from a neurologic trigger.

Two-pore domain K⁺ channels regulate membrane potential of isolated human articular chondrocytes.

Non‐Technical Summary  The debilitating condition of arthritis is caused by degeneration of the cartilage, a tissue that allows almost frictionless motion between the ends of bones in articulating joints such as the knee. The integrity of the cartilage is maintained by the metabolic activity of chondrocytes, the only type of cell found within the cartilage. An important factor in regulating the rate of metabolic activity of the chondrocytes is thought to be the magnitude of the electrical potential difference across the cell membrane, i.e. the ‘membrane potential’. This study identifies a type of ion channel, a so‐called ‘two‐pore potassium channel’, which was not previously known to be expressed in human chondrocytes. This ion channel importantly contributes to controlling chondrocyte membrane potential. Elucidation of the factors that control chondrocyte membrane potential is important for understanding the normal and pathophysiology of the chondrocytes, and consequently the health of the cartilage.

Atrial natriuretic peptide production in association with nonimmune fetal hydrops

The presence and elevation of atrial natriuretic peptide in fetuses has not previously been demonstrated. This study of right atrial pacing in fetal lambs demonstrated a threefold to fourfold increase in atrial natriuretic peptide during the production of fetal hydrops. Its rate of return to a normal level paralleled the clearance of fetal hydrops. Its possible role in fetal cardiovascular hemodynamics is discussed.