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Biomedical subjects

Richard B Robinson

Publications and source records attributed to Richard B Robinson.

16 recordsLinked to original sources
PubMed2004

Human mesenchymal stem cells make cardiac connexins and form functional gap junctions.

Human mesenchymal stem cells (hMSCs) are a multipotent cell population with the potential to be a cellular repair or delivery system provided that they communicate with target cells such as cardiac myocytes via gap junctions. Immunostaining revealed typical punctate staining for Cx43 and Cx40 along regions of intimate cell-to-cell contact between hMSCs. The staining patterns for Cx45 rather were typified by granular cytoplasmic staining. hMSCs exhibited cell-to-cell coupling to each other, to HeLa cells transfected with Cx40, Cx43 and Cx45 and to acutely isolated canine ventricular myocytes. The junctional currents (I(j)) recorded between hMSC pairs exhibited quasi-symmetrical and asymmetrical voltage (V(j)) dependence. I(j) records from hMSC-HeLaCx43 and hMSC-HeLaCx40 cell pairs also showed symmetrical and asymmetrical V(j) dependence, while hMSC-HeLaCx45 pairs always produced asymmetrical I(j) with pronounced V(j) gating when the Cx45 side was negative. Symmetrical I(j) suggests that the dominant functional channel is homotypic, while the asymmetrical I(j) suggests the activity of another channel type (heterotypic, heteromeric or both). The hMSCs exhibited a spectrum of single channels with transition conductances (gamma(j)) of 30-80 pS. The macroscopic I(j) obtained from hMSC-cardiac myocyte cell pairs exhibited asymmetrical V(j) dependence, while single channel events revealed gamma(j) of the size range 40-100 pS. hMSC coupling via gap junctions to other cell types provides the basis for considering them as a therapeutic repair or cellular delivery system to syncytia such as the myocardium.

Animals↗
PubMed2004

Biological pacemaker implanted in canine left bundle branch provides ventricular escape rhythms that have physiologically acceptable rates.

BACKGROUND: We hypothesized that administration of the HCN2 gene to the left bundle-branch (LBB) system of intact dogs would provide pacemaker function in the physiological range of heart rates. METHODS AND RESULTS: An adenoviral construct incorporating HCN2 and green fluorescent protein (GFP) as a marker was injected via catheter under fluoroscopic control into the posterior division of the LBB. Controls were injected with an adenoviral construct of GFP alone or saline. Animals were monitored electrocardiographically for up to 7 days after surgery, at which time they were anesthetized and subjected to vagal stimulation to permit emergence of escape pacemakers. Hearts were then removed and injection sites visually identified and removed for microelectrode study of action potentials, patch clamp studies of pacemaker current, and/or immunohistochemical studies of HCN2. For 48 hours postoperatively, 7 of 7 animals subjected to 24-hour ECG monitoring showed multiple ventricular premature depolarizations and/or ventricular tachycardia attributable to injection-induced injury. Thereafter, sinus rhythm prevailed. During vagal stimulation, HCN2-injected dogs showed rhythms originating from the left ventricle, the rate of which was significantly more rapid than in the controls. Excised posterior divisions of the LBB from HCN2-injected animals manifested automatic rates significantly greater than the controls. Isolated tissues showed immunohistochemical and biophysical evidence of overexpressed HCN2. CONCLUSIONS: A gene-therapy approach for induction of biological pacemaker activity within the LBB system provides ventricular escape rhythms that have physiologically acceptable rates. Long-term stability and feasibility of the approach remain to be tested.

Action Potentials↗
PubMed2003

Neuropeptide Y is an essential in vivo developmental regulator of cardiac ICa,L.

Cell culture studies demonstrate an increase in cardiac L-type Ca2+ current (ICa,L) density on sympathetic innervation in vitro and suggest the effect depends on neurally released neuropeptide Y (NPY). To determine if a similar mechanism contributes to the postnatal increase in ICa,L in vivo, we prepared isolated ventricular myocytes from neonatal and adult mice with targeted deletion of the NPY gene (Npy-/-) and matched controls (Npy+/+). Whole-cell voltage clamp demonstrates ICa,L density increases postnatally in Npy+/+ (by 56%), but is unchanged in Npy-/-. Both ICa,L density and action potential duration are significantly greater in adult Npy+/+ than Npy-/- myocytes, whereas ICa,L density is equivalent in neonatal Npy+/+ and Npy-/- myocytes. These data indicate NPY does not influence ICa,L prenatally, but the postnatal increase in ICa,L density is entirely NPY-dependent. In contrast, there is a similar postnatal negative voltage shift in the I-V relation in Npy+/+ and Npy-/-, indicating NPY does not influence the developmental change in ICa,L voltage-dependence. Immunoblot analyses and measurements of maximally activated ICa,L (in presence of forskolin or BayK 8644) show that the differences in current density between Npy+/+ and Npy-/- cannot be attributed to altered Ca2+ channel alpha1C subunit protein expression. Rather, these results suggest that the in vivo NPY-dependent postnatal increase in ICa,L density in cardiac myocytes results from regulation ICa,L properties by NPY.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed2003

Heterogeneous ventricular repolarization provides a substrate for arrhythmias in a German shepherd model of spontaneous arrhythmic death.

BACKGROUND: German shepherd dogs with inherited arrhythmias and sudden death appear to be a model for catecholamine-dependent ventricular tachycardias in human subjects. We tested the hypothesis that heterogeneity of left ventricular repolarization creates an arrhythmogenic substrate for pause-dependent ventricular tachycardia in these animals. METHODS AND RESULTS: We used microelectrode techniques to record action potentials (AP) from midmyocardial sections of anteroseptal, anterobasal, and posterobasal left ventricular (LV) wall of unafflicted and afflicted dogs. There were no differences in AP duration to 90% repolarization (APD) among LV regions in unafflicted dogs. In contrast, in afflicted dogs, there was significant heterogeneity, with the longest APD in anterobasal and shortest in anteroseptal regions. Isoproterenol did not affect repolarization in unafflicted dogs, whereas in afflicted dogs, it shortened APD anterobasally and prolonged APD anteroseptally. We studied the repolarizing currents, IKr and IKs, in single anteroseptal and anterobasal LV myocytes with the use of a whole-cell voltage clamp. There were no differences in IKr and IKs between anteroseptal and anterobasal regions in unafflicted dogs, whereas in afflicted dogs, IKr was smaller anterobasally (P<0.05). Isoproterenol produced a more prominent leftward shift in IKs voltage-dependent activation in anterobasal regions of afflicted than unafflicted dogs. CONCLUSIONS: Spatial heterogeneity in expression and catecholamine responsiveness of IKr and IKs results in heterogeneous LV repolarization in afflicted German shepherd dogs, contributing importantly to the arrhythmogenic substrate.

Action Potentials↗
PubMed2003

Developmental changes in IKr and IKs contribute to age-related expression of dofetilide effects on repolarization and proarrhythmia.

OBJECTIVE: Clinical and experimental studies suggest that immature hearts are as or more sensitive than adult hearts to adverse effects of I(Kr) blocking drugs. We hypothesized that age-dependent changes in I(Kr) and I(Ks) contribute to the different repolarization reserves and proarrhythmic effects of I(Kr) blockers in the young and adult heart. METHODS: Dogs aged 1-150 days and adults were used to study (1) proarrhythmic effects in situ of the I(Kr) blocker dofetilide; (2) dofetilide effects on action potential duration (APD) recorded with microelectrodes from left ventricular (LV) slabs; (3) I(Kr) and I(Ks) in single LV myocytes using whole-cell voltage clamp. RESULTS: In situ, dofetilide-induced proarrhythmia occurred in 40% of adults, 86% of young (20-150 day) dogs and 0% of neonatal (1-19 day) dogs (P<0.05). Isolated tissue experiments showed no transmural gradient for repolarization from neonate through 3 months of age, after which the gradient increased through adulthood. In the presence of dofetilide, the greatest APD prolongation occurred in neonates. Yet, transmural dispersion did not increase in neonates but significantly increased in young and adults. Dofetilide-induced early after depolarization (EAD) incidence was 23% in adults, 59% in young and 8% in neonates (P<0.05). I(Kr) but not I(Ks) was expressed at <30 days, whereas both currents were present in adult myocardium. CONCLUSIONS: Our data suggest that a lack of I(Ks) results in a greater dependence on I(Kr) for repolarization in neonates and is associated with exaggerated effects of I(Kr)-blockade on APD. However, APD prolongation alone is insufficient for expression of proarrhythmia, which also requires transmural dispersion of repolarization and EADs. The extent to which APD prolongation, transmural dispersion and EADs are manifested at various ages in the absence and presence of I(Kr) blocking drugs appears to be the ultimate determinant of proarrhythmia.

Action Potentials↗
PubMed2003

Expression and function of a biological pacemaker in canine heart.

BACKGROUND: We hypothesized that localized overexpression of the hyperpolarization-activated, cyclic nucleotide-gated (HCN2) pacemaker current isoform in canine left atrium (LA) would constitute a novel biological pacemaker. METHODS AND RESULTS: Adenoviral constructs of mouse HCN2 and green fluorescent protein (GFP) or GFP alone were injected into LA, terminal studies performed 3 to 4 days later, hearts removed, and myocytes examined for native and expressed pacemaker current (I(f)). Spontaneous LA rhythms occurred after vagal stimulation-induced sinus arrest in 4 of 4 HCN2+GFP dogs and 0 of 3 GFP dogs (P<0.05). Native I(f) in nonexpressed atrial myocytes was 7+/-4 pA at -130 mV (n=5), whereas HCN2+GFP LA had expressed pacemaker current (I(HCN2)) of 3823+/-713 pA at -125 mV (n=10) and 768+/-365 pA at -85 mV. CONCLUSIONS: HCN2 overexpression provides an I(f)-based pacemaker sufficient to drive the heart when injected into a localized region of atrium, offering a promising gene therapy for pacemaker disease.

Adenoviridae↗
PubMed2003

I(f)-dependent modulation of pacemaker rate mediated by cAMP in the presence of ryanodine in rabbit sino-atrial node cells.

I(f) contributes to generation and autonomic control of spontaneous activity of cardiac pacemaker cells through a cAMP-dependent, Ca(2+)-independent mechanism of rate regulation. However, disruption of Ca(2+) release from sarcoplasmic reticulum (SR) by ryanodine (Ry) has been recently shown to slow spontaneous rate and inhibit beta-adrenergic receptor (betaAR)-induced rate acceleration, leading to the suggestion that the target of betaAR modulation of pacemaking is the intracellular Ca(2+)-regulatory process. We have investigated whether the Ry-induced decrease of betaAR rate modulation alternatively involves disruption of the betaAR-adenylate-cyclase-cAMP-I(f) mechanism. Prolonged exposure to Ry (3 microM, >2 min) slowed spontaneous rate of pacemaker cells by 29.8% via a depolarizing shift of take-off potential (TOP) without significantly changing early diastolic depolarization rate. Ry depressed rate acceleration caused by isoproterenol (Iso) (1 microM, 23.6% in control vs. 8.0%), but did not modify that caused by two membrane-permeable cAMP analogs, CPT-cAMP (300 microM, 17.7% vs. 17.3%) and Rp-cAMPs (50 microM, 18.0% vs. 20.6%). Consistent with the rate effect, exposure to Ry decreased the shift induced by Iso, but not that induced by either cAMP analog on the I(f)-activation curve. We conclude that disruption of Ry receptor function and SR Ca(2+) release depresses betaAR-induced modulation of heart rate, but does not impair cAMP-dependent rate acceleration mediated by I(f). However, abolishment of normal Ca(2+) homeostasis may result in the failure of betaAR agonists to sufficiently elevate cAMP near f-channels. The molecular basis for Ca(2+)-dependent interference in beta-adrenergic signaling remains to be determined.

Adrenergic beta-Agonists↗
PubMed2002

Hyperpolarization-activated cation currents: from molecules to physiological function.

Hyperpolarization-activated cation currents, termed If, Ih, or Iq, were initially discovered in heart and nerve cells over 20 years ago. These currents contribute to a wide range of physiological functions, including cardiac and neuronal pacemaker activity, the setting of resting potentials, input conductance and length constants, and dendritic integration. The hyperpolarization-activated, cation nonselective (HCN) gene family encodes the channels that underlie Ih. Here we review the relation between the biophysical properties of recombinant HCN channels and the pattern of HCN mRNA expression with the properties of native Ih in neurons and cardiac muscle. Moreover, we consider selected examples of the expanding physiological functions of Ih with a view toward understanding how the properties of HCN channels contribute to these diverse functional roles.

Animals↗
PubMed2002

Functional comparison of HCN isoforms expressed in ventricular and HEK 293 cells.

Pacemaker current (I(f)) encoded by the HCN gene family contributes importantly to cardiac rhythm. That contribution depends on the biophysical characteristics of I(f), such as voltage dependence, which vary markedly with cardiac region, development and disease. Heterologous expression studies of individual HCN isoforms have failed to account for the diverse functionality of the native current. To investigate the influence of cellular environment on the gating of HCN channels, we compared the functional characteristics of HCN2 and HCN4, the two major ventricular isoforms, when over-expressed in a normal context (neonatal myocytes) and in a heterologous context (HEK 293 cells). Independent of cell type, HCN4 activates substantially slower than HCN2 and with a half-maximum activation voltage approximately equal 10 mV less negative. However, both isoforms activate more positively in myocytes than in HEK 293 cells. The latter result suggests a context dependence (i.e. cell-type specificity) to HCN voltage dependence that exerts a comparable influence on these two isoforms. This is distinct from the inherent difference in the biophysical properties of HCN2 and HCN4.

Animals↗
PubMed2002

Effects of gonadal steroids on gender-related differences in transmural dispersion of L-type calcium current.

OBJECTIVES: Repolarization-prolonging drugs induce torsades de pointes (TdP) in females more than males. The action potential plateau and the early afterdepolarizations that induce TdP are determined, in part, by L-type calcium current (I(Ca,L)). Therefore, we studied gender- and hormone-related differences in I(Ca,L) in age-, and weight-matched normal male, female and hormonally-treated, castrated rabbits. METHODS: Oophorectomized (OVX) or orchiectomized (ORCH) 50- to 60-day-old rabbits were subcutaneously implanted with pellets impregnated with placebo (PLA), 5 alpha-dihydroxytestosterone (DHT), or 17 beta-estradiol (EST). Four to five weeks later, epicardial and endocardial myocytes were isolated from the left ventricle. Patch clamp technique was performed to assess I(Ca,L). RESULTS: I(Ca,L) density (measured as peak current density [pA/pF] at +15 mV, V(h)= -40 mV), was greater in female epicardium (-7.4 +/- 0.9) than endocardium (-5.6 +/- 0.7, P<0.05), while male epicardial I(Ca,L) density (-6.5 +/- 0.7) did not differ from endocardial (-5.9 +/- 1.0, P>0.05). OVX-female, DHT and EST-treated groups had epicardial I(Ca,L) density (-5.6 +/- 0.6, and -5.9 +/- 0.7, respectively) greater than endocardial (-4.3 +/- 0.3, and -3.6 +/- 0.4, P<0.05). However, OVX-females had hormone levels not significantly different from female controls and EST-treated females had non-physiological levels of estradiol. There were no differences between endocardial and epicardial I(Ca,L) activation and inactivation. In contrast, epicardial-endocardial differences in I(Ca,L) density in EST-treated OVX-females were associated with epicardial-endocardial differences in I(Ca,L) activation and conductance; in DHT-treated OVX-females only epicardial-endocardial activation differed. The other groups, showed no I(Ca,L) transmural gradient, or differences in activation, inactivation or conductance. CONCLUSIONS: The greater dispersion in I(Ca,L) density of OVX-DHT and OVX-EST than OVX-PLA suggests both hormones can modulate I(Ca,L) density in females. That gonadal steroids had no effect on I(Ca,L) dispersion in males suggests gender differences in mechanism of action of both hormones. The greater I(Ca,L) dispersion in females may contribute to gender differences in repolarization.

Analysis of Variance↗
PubMed2002

beta(1)-Receptors increase cAMP and induce abnormal Ca(i) cycling in the German shepherd sudden death model.

We studied the role of beta-adrenergic receptor subtype signaling to cAMP and calcium in the genesis of catecholamine-dependent arrhythmias in German shepherd dogs that develop lethal arrhythmias at ~5 mo of age. There were three major findings in this study: 1) isoproterenol induces similar increases in cAMP in afflicted and control dogs exclusively through beta(1)-receptors (not beta(2)), 2) cells from afflicted dogs display prolonged relaxation kinetics at long cycle lengths and large frequent spontaneous calcium oscillations (and aftercontractions) with little increase in calcium transient amplitude in response to beta(1)-receptor agonists, and 3) beta(2)-receptor agonists induce a similar marked increases in calcium transient and twitch amplitude, with only rare spontaneous calcium oscillations in afflicted and control cells. These results indicate that catecholamines provide inotropic support to canine cardiomyocytes through distinct beta(1)- and beta(2)-receptor pathways with differing requirements for cAMP. The propensity to develop arrhythmias is not induced by beta(2)-receptors (or a rise in calcium alone), but rather occurs in the context of beta(1)-receptor activation of the cAMP-dependent pathway.

Adrenergic beta-Agonists↗
PubMed2002

Block of the background K(+) channel TASK-1 contributes to arrhythmogenic effects of platelet-activating factor.

Platelet-activating factor (PAF), an inflammatory phospholipid, induces ventricular arrhythmia via an unknown ionic mechanism. We can now link PAF-mediated cardiac electrophysiological effects to inhibition of a two-pore domain K(+) channel [TWIK-related acid-sensitive K(+) background channel (TASK-1)]. Superfusion of carbamyl-PAF (C-PAF), a stable analog of PAF, over murine ventricular myocytes causes abnormal automaticity, plateau phase arrest of the action potential, and early afterdepolarizations in paced and quiescent cells from wild-type but not PAF receptor knockout mice. C-PAF-dependent currents are insensitive to Cs(+) and are outwardly rectifying with biophysical properties consistent with a K(+)-selective channel. The current is blocked by TASK-1 inhibitors, including protons, Ba(2+), Zn(2+), and methanandamide, a stable analog of the endogenous lipid ligand of cannabinoid receptors. In addition, when TASK-1 is expressed in CHO cells that express an endogenous PAF receptor, superfusion of C-PAF decreases the expressed current. Like C-PAF, methanandamide evoked spontaneous activity in quiescent myocytes. C-PAF- and methanandamide-sensitive currents are blocked by a specific protein kinase C (PKC) inhibitor, implying overlapping signaling pathways. In conclusion, C-PAF blocks TASK-1 or a closely related channel, the effect is PKC dependent, and the inhibition alters the electrical activity of myocytes in ways that would be arrhythmogenic in the intact heart.

Action Potentials↗