Paul C. Dolber

Relating extracellular potentials and their derivatives to anisotropic propagation at a microscopic level in human cardiac muscle. Evidence for electrical uncoupling of side-to-side fiber connections with increasing age.

Elucidation of the mechanisms of cardiac conduction disturbances leading to reentry will require resolution of the details of multidimensional propagation at a microscopic size scale (<200μm). In practice, this will necessitate the combined analysis of extracellular and transmembrane action potentials. The purpose of this paper is to demonstrate the relationships between the time derivatives of the extracellular waveforms and the underlying action potentials in the experimental analysis of anisotropic propagation at this small size scale, and apply these relationships to human atrial muscle at different ages. The extracellular waveforms and their derivatives changed from a smooth contour during transverse propagation in young preparations to complex polyphasic waveforms in the older preparations. The major problem was to estimate the size and location of small groups of fibers that generated the complex waveforms in the older preparations. We found dissimilarities in the derivatives that distinguished source (bundle) size from the distance of the source to the measurement site. The differences in the extracellular waveforms and their derivatives indicated that there was electrical uncoupling of the side-to-side connections between small groups of fibers with aging. These changes produced a prominent zigzag course of transverse propagation at a microscopic level which, in turn, accounted for the increased complexity of the waveforms. The waveform differences also correlated with the development of extensive collagenous septa that separated small groups of fibers. The electrophysiological consequence was an age-related decrease in the ‘effective transverse conduction velocities to the range of the very slow conduction (<0.08 m/sec) which makes it possible for reentry to occur in small regions of cardiac muscle with normal cellular electrophysiological properties’.

The functional role of structural complexities in the propagation of depolarization in the atrium of the dog. Cardiac conduction disturbances due to discontinuities of effective axial resistivity.

Structural complexities produced by the inhomogeneous distribution of the connections between cells and between muscle bundles have previously been considered to be of minor importance in the propagation of depolarization in cardiac muscle; conduction disturbances usually are attributed to changes in membrane properties along the course of the fibers. However, we observed marked effects of these connections on the velocity and the safety factor of propagation in atrial muscle under normal and abnormal conditions. First, within individual muscle bundles, intermittent connective- tissue septa were associated with localized dissociation of excitation when propagation occurred in the transverse direction, but not when it occurred in the longitudinal direction. Second, at sites where muscle bundles branch or join with other bundles, we observed abrupt slowing of normal action potentials and changes in the shape of the extracellular waveform. We also studied such a junction in a computer simulation of propagation and found that the local delay of propagation and the change in the shape of the extracellular waveform could only be accounted for by an abrupt change in the effective axial resistivity in the direction of propagation. Under normal conditions, enough depolarizing current is coupled across such discontinuities to maintain propagation. However, when the maximum membrane depolarizing current was reduced by increasing the extracellular potassium concentration or by premature stimulation, block occurred at these sites. We observed that most known cardiac conduction disturbances considered to require longer refractory periods in the direction of propagation (e.g., local conduction delay, decremental conduction, block, and reentry) can be produced by the effects on propagation of such discontinuities of effective axial resistivity. The results indicate that models of propagation that ignore the inhomogeneous and multidimensional distribution of cell-to-cell connections produce incomplete, and sometimes incorrect, descriptions of normal and abnormal propagation in cardiac muscle.

Influence of the passive anisotropic properties on directional differences in propagation following modification of the sodium conductance in human atrial muscle. A model of reentry based on anisotropic discontinuous propagation.

Available models of circus movement reentry in cardiac muscle and of drug action on reentrant arrhythmias are based on continuous medium theory, which depends solely on the membrane ionic conductances to alter propagation. The purpose of this study is to show that the anisotropic passive properties at a microscopic level highly determine the propagation response to modification of the sodium conductance by premature action potentials and by sodium channel–blocking drugs. In young, uniform anisotropic atrial bundles, propagation of progressively earlier premature action potentials continued as a smooth process until propagation ceased simultaneously in all directions. In older, nonuniform anisotropic bundles, however, premature action produced either unidirectional longitudinal conduction block or a dissociated zigzag type of longitudinal conduction (a safer type of propagation, similar to transverse propagation). Directional differences in the velocity of premature action potentials demonstrated that anisotropic propagation was necessary for a reentrant circuit to be contained within an area of 50 mm2, even with very short refractory periods. Quinidine produced Wenckebach periodicity, which disappeared after acetylcholine shortened the action potential. Quinidine also produced use-dependent dissociated zigzag longitudinal conduction in the older, nonuniform anisotropic bundles but not in the young, uniform anisotropic bundles. The electrophysiological consequence was that propagation events differed in an age-related manner in response to the same modification of the sodium conductance. The electrical events at microscopic level showed that conditions leading to obliteration of side-to-side electrical coupling between fibers (e.g., aging and chronic hypertrophy) provide a primary mechanism for reentry to occur within very small areas (1-2 mm) due to a variety of propagation phenomena that do not occur in tissues with tight electrical coupling in all directions.

Enhancement of cardiac function after adenoviral-mediated in vivo intracoronary β2-adrenergic receptor gene delivery

Exogenous gene delivery to alter the function of the heart is a potential novel therapeutic strategy for treatment of cardiovascular diseases such as heart failure (HF). Before gene therapy approaches to alter cardiac function can be realized, efficient and reproducible in vivo gene techniques must be established to efficiently transfer transgenes globally to the myocardium. We have been testing the hypothesis that genetic manipulation of the myocardial beta-adrenergic receptor (beta-AR) system, which is impaired in HF, can enhance cardiac function. We have delivered adenoviral transgenes, including the human beta2-AR (Adeno-beta2AR), to the myocardium of rabbits using an intracoronary approach. Catheter-mediated Adeno-beta2AR delivery produced diffuse multichamber myocardial expression, peaking 1 week after gene transfer. A total of 5 x 10(11) viral particles of Adeno-beta2AR reproducibly produced 5- to 10-fold beta-AR overexpression in the heart, which, at 7 and 21 days after delivery, resulted in increased in vivo hemodynamic function compared with control rabbits that received an empty adenovirus. Several physiological parameters, including dP/dtmax as a measure of contractility, were significantly enhanced basally and showed increased responsiveness to the beta-agonist isoproterenol. Our results demonstrate that global myocardial in vivo gene delivery is possible and that genetic manipulation of beta-AR density can result in enhanced cardiac performance. Thus, replacement of lost receptors seen in HF may represent novel inotropic therapy.

Electrophysiological Effects of Remodeling Cardiac Gap Junctions and Cell Size Experimental and Model Studies of Normal Cardiac Growth

The increased incidence of arrhythmias in structural heart disease is accompanied by remodeling of the cellular distribution of gap junctions to a diffuse pattern like that of neonatal cardiomyocytes. Accordingly, it has become important to know how remodeling of gap junctions due to normal growth hypertrophy alters anisotropic propagation at a cellular level (V(max)) in relation to conduction velocities measured at a macroscopic level. To this end, morphological studies of gap junctions (connexin43) and in vitro electrical measurements were performed in neonatal and adult canine ventricular muscle. When cells enlarged, gap junctions shifted from the sides to the ends of ventricular myocytes. Electrically, normal growth produced different patterns of change at a macroscopic and microscopic level. Although the longitudinal and transverse conduction velocities were greater in adult than neonatal muscle, the anisotropic velocity ratios were the same. In the neonate, mean V(max) was not different during longitudinal (LP) and transverse (TP) propagation. However, growth hypertrophy produced a selective increase in mean TP V(max) (P<0.001), with no significant change in mean LP V(max). Two-dimensional neonatal and adult cellular computational models show that the observed increases in cell size and changes in the distribution of gap junctions are sufficient to account for the experimental results. Unexpectedly, the results show that cellular scaling (cell size) is as important (or more so) as changes in gap junction distribution in determining TP properties. As the cells enlarged, both mean TP V(max) and lateral cell-to-cell delay increased. V(max) increased because increases in cell-to-cell delay reduced the electric current flowing downstream up to the time of V(max), thus enhancing V(max). The results suggest that in pathological substrates that are arrhythmogenic, maintaining cell size during remodeling of gap junctions is important in sustaining a maximum rate of depolarization.

Multiple regional differences in cellular properties that regulate repolarization and contraction in the right atrium of adult and newborn dogs.

Recent studies of isolated cardiac myocytes have generated the need for detailed information about regional electrophysiological differences in the atrium. We measured the spatial distribution of action potentials in adult and newborn canine right atria. Multiple regional differences in action potential shape and duration were found. The multiple regional differences produced an overall simple pattern: the longest action potentials occurred in the area of the sinus node, and the action potential duration decreased with increasing distance from the sinus node area. To account for the overall pattern, we tested factors considered important in causing atrial action potential shape differences (e.g., electronic interactions). None of the factors tested accounted for the regional differences. We then found regional differences in the responses to pauses, which suggested that differences in the properties of individual cells accounted for the regional repolarization differences. If so, genetic regulation of the regional differences may produce the overall pattern on a developmental basis. Experiments in newborn atria showed that only in the upper crista was the spatial pattern similar to that of the adult; there was little variability in action potential shape and duration in the other areas. As a further test for associated regional differences in cell properties, we examined for differences in the isoform expression of troponin T (TnT1, TnT2, TnT3, and TnT4), a protein important in excitation-contraction coupling. In adults, the greatest proportion of TnT1 occurred in the area of the sinus node, and its proportion decreased with increasing distance from the sinus node area in association with a relative increase in the proportion of TnT2. In newborn atria the relative amount of TnT1 was greatest in the upper crista (similar to adult), but little difference was found in the distribution of the isoforms in the other regions. The correspondence between the regional differences in repolarization and in the expression of the troponin T isoforms in adult and newborn atria suggests that 1) cellular ionic mechanisms vary regionally to coordinate differences in action potential configuration with differences in cell properties that regulate contractility and 2) genetic expression of the systems that regulate repolarization and mechanical cellular properties are under similar developmental and regional control.

Picrosirius Red Staining of Cardiac Muscle Following Phosphomolybdic Acid Treatment

When the picrosirius red technique was applied to cardiac muscle sections, intense yellow myocyte staining sometimes obscured thin collagenous septa. The picrosirius red technique was modified to include treatment of the sections in 0.2% (w/v) aqueous phosphomolybdic acid prior to staining. With 1-5 min treatment, cytoplasmic staining was eradicated; diminution of collagen staining occurred only with long treatments at much higher concentrations of phosphomolybdic acid. Using this phosphomolybdic acid-picrosirius red technique, collagenous septa as thin as 0.2-0.5 micron and fine collagen fibers making up the septa were clearly discernible. The technique also worked well on sections stained by other techniques and then destained. The phosphomolybdic acid-picrosirius red technique should be useful in experiments designed to investigate the effects of collagen distribution on the electrical and mechanical behavior of cardiac muscle.

β-Adrenergic Receptor Kinase-1 Levels in Catecholamine-Induced Myocardial Hypertrophy Regulation by β- but not α1-Adrenergic Stimulation

Abstract —Pressure overload ventricular hypertrophy is accompanied by dysfunctional β-adrenergic receptor signaling due to increased levels of the β-adrenergic receptor kinase-1, which phosphorylates and desensitizes β-adrenergic receptors. In this study, we examined whether increased β-adrenergic receptor kinase 1 expression is associated with myocardial hypertrophy induced by adrenergic stimulation. With use of implanted mini-osmotic pumps, we treated mice with isoproterenol, phenylephrine, or vehicle to distinguish between α 1 - and β-adrenergic stimulation. Both treatments resulted in cardiac hypertrophy, but only isoproterenol induced significant increases in β-adrenergic receptor kinase-1 protein levels and activity. Similarly, in isolated adult rat cardiac myocytes, 24 hours of isoproterenol stimulation resulted in a significant 2.8-fold increase in β-adrenergic receptor kinase-1 protein levels, whereas 24 hours of phenylephrine treatment did not alter β-adrenergic receptor kinase-1 expression. Our results indicate that increased β-adrenergic receptor kinase-1 is not invariably associated with myocardial hypertrophy but apparently is controlled by the state of β-adrenergic receptor activation.

Adenovirus-mediated gene transfer of the β2-adrenergic receptor to donor hearts enhances cardiac function

Gene transfer to modify donor heart function during transplantation has significant therapeutic implications. Recent studies by our laboratory in transgenic mice have shown that overexpression of β2-adrenergic receptors (β2-ARs) leads to significantly enhanced cardiac function. Thus, we investigated the functional consequences of adenovirus-mediated gene transfer of the human β2-AR in a rat heterotopic heart transplant model. Donor hearts received 1 ml of solution containing 1 × 1010 p.f.u. of adenovirus encoding the β2-AR or an empty adenovirus as a control. Five days after transplantation, basal left ventricular (LV) pressure was measured using an isolated, isovolumic heart perfusion apparatus. A subset of hearts was stimulated with the β2-AR agonist, zinterol. Treatment with the β2-AR virus resulted in global myocardial gene transfer with a six-fold increase in mean β-AR density which corresponded to a significant increase in basal contractility (LV + dP/dtmax, control: 3152.1 ± 286 versus β2-AR, 6250.6* ± 432.5 mmHg/s; n = 10, *P < 0.02). β2-AR overexpressing hearts also had higher contractility after zinterol administration compared with control hearts. Our results indicate that myocardial function of the transplanted heart can be enhanced by the adenovirus-mediated delivery of β2-ARs. Thus, genetic manipulation may offer a novel therapeutic strategy to improve donor heart function in the post- operative setting.

Propagating depolarization in anisotropic human and canine cardiac muscle: apparent directional differences in membrane capacitance. A simplified model for selective directional effects of modifying the sodium conductance on Vmax, tau foot, and the propagation safety factor.

As yet there is no model or simulation that accounts for the anisotropic difference in the shape of the upstroke and safety factor of propagating cardiac action potentials: fast upstrokes occur with slow transverse propagation and slow upstrokes occur with fast longitudinal propagation. The purpose of this paper is to demonstrate, however, that a simplified cable model based on directional differences in the effective membrane capacitance predicts in detail the experimentally measured directionally dependent behavior of the upstroke in response to modification of the sodium conductance. Quinidine and lidocaine produced greater relative decreases in Vmax and conduction velocity with longitudinal propagation than with transverse propagation, as predicted on the basis that the shape differences should produce an anisotropic distribution in the membrane uptake of sodium channel binding drugs. The simulation predictions of the effects of positive shifts of the take-off potential due to premature action potentials were also confirmed experimentally: there was a greater relative decrease in conduction velocity, Vmax, and Vamp with a greater increase in tau foot during longitudinal propagation than with transverse propagation. The major anisotropic differences in shape occurred when the take-off potential approached the least negative value that produced a propagated response. The extensive experimental verification of the results of a simplified model based on directional differences of effective membrane capacitance, combined with directional differences in effective axial resistivity, provides an initial quantitative basis for the anisotropic behavior of propagating depolarization in response to modification of the sodium conductance in cardiac muscle.