The effect of aging on the specialized conducting system: a telemetry ECG study in rats over a 6 month period.

Advanced age alone appears to be a risk factor for increased susceptibility to cardiac arrhythmias. We previously observed in the aged rat heart that sinus rhythm ventricular activation is delayed and characterized by abnormal epicardial patterns although conduction velocity is normal. While these findings relate to an advanced stage of aging, it is not yet known when and how ventricular electrical impairment originates and which is the underlying substrate. To address these points, we performed continuous telemetry ECG recordings in freely moving rats over a six-month period to monitor ECG waveform changes, heart rate variability and the incidence of cardiac arrhythmias. At the end of the study, we performed in-vivo multiple lead epicardial recordings and histopathology of cardiac tissue. We found that the duration of ECG waves and intervals gradually increased and heart rate variability gradually decreased with age. Moreover, the incidence of cardiac arrhythmias gradually increased, with atrial arrhythmias exceeding ventricular arrhythmias. Epicardial multiple lead recordings confirmed abnormalities in ventricular activation patterns, likely attributable to distal conducting system dysfunctions. Microscopic analysis of aged heart specimens revealed multifocal connective tissue deposition and perinuclear myocytolysis in the atria. Our results demonstrate that aging gradually modifies the terminal part of the specialized cardiac conducting system, creating a substrate for increased arrhythmogenesis. These findings may open new therapeutic options in the management of cardiac arrhythmias in the elderly population.

Growth factor-induced mobilization of cardiac progenitor cells reduces the risk of arrhythmias, in a rat model of chronic myocardial infarction.

Heart repair by stem cell treatment may involve life-threatening arrhythmias. Cardiac progenitor cells (CPCs) appear best suited for reconstituting lost myocardium without posing arrhythmic risks, being commissioned towards cardiac phenotype. In this study we tested the hypothesis that mobilization of CPCs through locally delivered Hepatocyte Growth Factor and Insulin-Like Growth Factor-1 to heal chronic myocardial infarction (MI), lowers the proneness to arrhythmias. We used 133 adult male Wistar rats either with one-month old MI and treated with growth factors (GFs, n = 60) or vehicle (V, n = 55), or sham operated (n = 18). In selected groups of animals, prior to and two weeks after GF/V delivery, we evaluated stress-induced ventricular arrhythmias by telemetry-ECG, cardiac mechanics by echocardiography, and ventricular excitability, conduction velocity and refractoriness by epicardial multiple-lead recording. Invasive hemodynamic measurements were performed before sacrifice and eventually the hearts were subjected to anatomical, morphometric, immunohistochemical, and molecular biology analyses. When compared with untreated MI, GFs decreased stress-induced arrhythmias and concurrently prolonged the effective refractory period (ERP) without affecting neither the duration of ventricular repolarization, as suggested by measurements of QTc interval and mRNA levels for K-channel α-subunits Kv4.2 and Kv4.3, nor the dispersion of refractoriness. Further, markers of cardiomyocyte reactive hypertrophy, including mRNA levels for K-channel α-subunit Kv1.4 and ß-subunit KChIP2, interstitial fibrosis and negative structural remodeling were significantly reduced in peri-infarcted/remote ventricular myocardium. Finally, analyses of BrdU incorporation and distribution of connexin43 and N-cadherin indicated that cytokines generated new vessels and electromechanically-connected myocytes and abolished the correlation of infarct size with deterioration of mechanical function. In conclusion, local injection of GFs ameliorates electromechanical competence in chronic MI. Reduced arrhythmogenesis is attributable to prolongation of ERP resulting from improved intercellular coupling via increased expression of connexin43, and attenuation of unfavorable remodeling.

The histone deacetylase inhibitor suberoylanilide hydroxamic acid reduces cardiac arrhythmias in dystrophic mice.

AIMS: The effect of histone deacetylase inhibitors on dystrophic heart function is not established. To investigate this aspect, dystrophic mdx mice and wild-type (WT) animals were treated 90 days either with suberoylanilide hydroxamic acid (SAHA, 5 mg/kg/day) or with an equivalent amount of vehicle. METHODS AND RESULTS: The following parameters were evaluated: (i) number of ventricular arrhythmias in resting and stress conditions (restraint test) or after aconitine administration; (ii) cardiac excitability, conduction velocity, and refractoriness; (iii) expression and distribution of connexins (Cxs) and Na(v)1.5 sodium channel. Ventricular arrhythmias were negligible in all resting animals. During restraint, however, an increase in the number of arrhythmias was detected in vehicle-treated mdx mice (mdx-V) when compared with SAHA-treated mdx (mdx-SAHA) mice or normal control (WT-V). Interestingly, aconitine, a sodium channel pharmacologic opener, induced ventricular arrhythmias in 83% of WT-V mice, 11% of mdx-V, and in 57% of mdx-SAHA. Epicardial multiple lead recording revealed a prolongation of the QRS complex in mdx-V mice in comparison to WT-V and WT-SAHA mice, paralleled by a significant reduction in impulse propagation velocity. These alterations were efficiently counteracted by SAHA. Molecular analyses revealed that in mdx mice, SAHA determined Cx remodelling of Cx40, Cx37 and Cx32, whereas expression levels of Cx43 and Cx45 were unaltered. Remarkably, Cx43 lateralization observed in mdx control animals was reversed by SAHA treatment which also re-induced Na(v)1.5 expression. CONCLUSION: SAHA attenuates arrhythmias in mdx mice by a mechanism in which Cx remodelling and sodium channel re-expression could play an important role.

Ventricular activation is impaired in aged rat hearts.

Ventricular arrhythmias are frequently observed in the elderly population secondary to alterations of electrophysiological properties that occur with the normal aging process of the heart. However, the underlying mechanisms remain poorly understood. The aim of the present study was to determine specific age-related changes in electrophysiological properties and myocardial structure in the ventricles that can be related to a structural-functional arrhythmogenic substrate. Multiple unipolar electrograms were recorded in vivo on the anterior ventricular surface of four control and seven aged rats during normal sinus rhythm and ventricular pacing. Electrical data were related to morphometric and immunohistochemical parameters of the underlying ventricular myocardium. In aged hearts total ventricular activation time was significantly delayed (QRS duration: +69%), while ventricular conduction velocity did not change significantly compared with control hearts. Moreover, ventricular activation patterns displayed variable numbers of epicardial breakthrough points whose appearance could change with time. Morphological analysis in aged rats revealed that heart weight and myocyte transverse diameter increased significantly, scattered microfoci of interstitial fibrosis were mostly present in the ventricular subendocardium, and gap junction connexin expression decreased significantly in ventricular myocardium compared with control rats. Our results show that in aged hearts delayed total ventricular activation time and abnormal activation patterns are not due to delayed myocardial conduction and suggest the occurrence of impaired impulse propagation through the conduction system leading to uncoordinated myocardial excitation. Impaired interaction between the conduction system and ventricular myocardium might create a potential reentry substrate, contributing to a higher incidence of ventricular arrhythmias in the elderly population.

Susceptibility to ventricular arrhythmias in aged hearts.

Cardiac arrhythmias are frequent in the elderly population, perhaps secondary to an increased prevalence of hypertension and coronary artery disease as well as aging related changes resulting in loss of pacemaker cells and degenerative alteration of the conduction system. Independent from underlying structural heart disease, advanced age alone appears to be a risk factor for increased susceptibility to ventricular arrhythmia. However, the electrophysiological basis of this phenomenon is still unclear. Thus, it is important to assess and to define the underlying arrhythmogenic substrate. The aim of the present study was to identify a likely structural-functional ventricular arrhythmogenic substrate in aged hearts. For this purpose ventricular activation patterns were measured in control (n=4) and aged (n=10) in vivo rat hearts by recording unipolar electrograms with an epicardial, 1 mm resolution, 8x8 electrode array, during pacing and spontaneous or induced ventricular ectopic beats. Our results in aged hearts suggest that peripheral conduction system might be involved in perpetuating sequences of ventricular ectopic beats, regardless of their origin.

Preservation of ventricular performance at early stages of diabetic cardiomyopathy involves changes in myocyte size, number and intercellular coupling.

In a rat model of diabetic cardiomyopathy, we tested whether specific changes in myocyte turnover and intercellular coupling contribute to preserving ventricular performance after a short period of hyperglycemia. In 41 rats with streptozotocin-induced diabetes and 24 control animals, cardiac electromechanical properties were assessed by telemetry ECG, epicardial potential mapping, and hemodynamic measurements to document normal ventricular function. Myocardial remodeling, expression of gap-junction proteins and myocyte regeneration were evaluated by tissue morphometry, immunohistochemistry and immunoblotting. Ventricular myocyte number and volume were also determined. In diabetic hearts, after 3 weeks of hyperglycemia, left ventricular mass was lowered by 23%, while left ventricular wall thickness and chamber volume were maintained, in the absence of fibrosis and myocyte hypertrophy. In the presence of a marked DNA oxidative damage, an increased rate of DNA replication and mitotic divisions associated with generation of new myocytes were detected. The number of cells expressing the receptor for Stem Cell Factor (c-kit) and their rate of proliferation were preserved in the left ventricle while the atrial storage of these primitive cells was severely reduced by diabetes-induced oxidative stress. Despite a down-regulation of Connexin43 and over-expression of both Connexin40 and Connexin45, the junctional proteins were normally distributed in diabetic ventricular myocardium,justifying the preserved tissue excitability and conduction velocity. In conclusion, before the appearance of the diabetic cardiomyopathic phenotype,myocardial cell proliferation associated with gap junction protein remodeling may contribute to prevent marked alterations of cardiac structure and electrophysiological properties, preserving ventricular performance.

Does cardiac pacing reproduce the mechanism of focal impulse initiation?

Stimulation of myocardium by either a native pacemaker or an artificial stimulus requires the initiation of a self-propagating wave of depolarization originating from the site of initial activation. In the present study we perform artificial stimulation at a site of focal discharge with the aim to compare the two mechanisms of impulse formation. High resolution epicardial mapping in senescent rat hearts provided examples of focal discharge during sinus rhythm at a single epicardial breakthrough (BKT) point emerging from an isolated Purkinje-ventricular muscle junction (PMJ) site. Stimulation was also performed at the same BKT point and potential distributions recorded during spontaneous and artificial stimulation were compared. During excitation latency, the negative potential pattern was elongated perpendicularly to fiber direction at both pacing and BKT point, in agreement with virtual cathode membrane polarization predicted by the bidomain model during point stimulation. During impulse initiation, activation wave fronts were initially circular around pacing site or BKT point and then elongated along local fiber direction. The similarity between impulse initiation during focal discharge and point stimulation in cardiac muscle suggests that high resolution pace mapping studies can help to elucidate the mechanism of abnormal impulse formation.

Basal nonselective cation permeability in rat cardiac microvascular endothelial cells.

The presence of a basal nonselective cation permeability was mainly investigated in primary cultures of rat cardiac microvascular endothelial cells (CMEC) by applying both the patch-clamp technique and Fura-2 microfluorimetry. With low EGTA in the pipette solution, the resting membrane potential of CMEC was -21.2 +/- 1.1 mV, and a Ca(2+)-activated Cl(-) conductance was present. When the intracellular Ca(2+) was buffered with high EGTA, the membrane potential decreased to 5.5 +/- 1.2 mV. In this condition, full or partial substitution of external Na(+) by NMDG(+) proportionally reduced the inward component of the basal I-V relationship. This current was dependent on extracellular monovalent cations with a permeability sequence of K(+) > Cs(+) > Na(+) > Li(+) and was inhibited by Ca(2+), La(3+), Gd(3+), and amiloride. The K(+)/Na(+) permeability ratio, determined using the Goldman-Hodgkin-Katz equation, was 2.01. The outward component of the basal I-V relationship was reduced when intracellular K(+) was replaced by NMDG(+), but was not sensitive to substitution by Cs(+). Finally, microfluorimetric experiments indicated the existence of a basal Ca(2+) entry pathway, inhibited by La(3+) and Gd(3+). The basal nonselective cation permeability in CMEC could be involved both in the control of myocardial ionic homeostasis, according to the model of the blood-heart barrier, and in the modulation of Ca(2+)-dependent processes.

Ca2+ uptake by the endoplasmic reticulum Ca2+-ATPase in rat microvascular endothelial cells.

In non-excitable cells, many agonists increase the intracellular Ca(2+) concentration ([Ca(2+)](i)) by inducing an inositol 1,4,5-trisphosphate (IP(3))-mediated Ca(2+) release from the intracellular stores. Ca(2+) influx from the extracellular medium may then sustain the Ca(2+) signal. [Ca(2+)](i) recovers its resting level as a consequence of Ca(2+)-removing mechanisms, i.e. plasma-membrane Ca(2+)-ATPase (PMCA) pump, Na(+)/Ca(2+) exchanger (NCX) and sarco-endoplasmic reticulum Ca(2+)-ATPase (SERCA) pump. In a study performed in pancreatic acinar cells, evidence has been provided suggesting that, during the decay phase of the agonist-evoked Ca(2+) transients, the Ca(2+) concentration within the intracellular stores remains essentially constant [Mogami, Tepikin and Petersen (1998) EMBO J. 17, 435-442]. It was therefore hypothesized that, in such a situation, intracellular Ca(2+) is not only picked up by the SERCA pump, but is also newly released through IP(3)-sensitive Ca(2+) channels, with the balance between these two processes being approximately null. The main aim of the present work was to test this hypothesis by a different experimental approach. Using cardiac microvascular endothelial cells, we found that inhibition of the SERCA pump has no effect on the time course of agonist-evoked Ca(2+) transients. This result was not due to a low capacity of the SERCA pump since, after agonist removal, this pump proved to be very powerful in clearing the excess of intracellular Ca(2+). We showed further that: (i) in order to avoid a rapid removal of Ca(2+) by the SERCA pump, continuous IP(3) production appears to be required throughout all of the decay phase of the Ca(2+) transient; and (ii) Ca(2+) picked up by the SERCA pump can be fully and immediately released by agonist application. All these results support the model of Mogami, Tepikin and Petersen [(1998) EMBO J. 17, 435-442]. Since the SERCA pump did not appear to be involved in shaping the decay phase of the agonist-evoked Ca(2+) transient, we inhibited the PMCA pump with carboxyeosin, and NCX with benzamil and by removing extracellular Na(+). The results indicate that, during the decay phase of the agonist-evoked Ca(2+) transient, the intracellular Ca(2+) is removed by both the PMCA pump and NCX. Finally, we provide evidence indicating that mitochondria have no role in clearing intracellular Ca(2+) during agonist-evoked Ca(2+) transients.