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The introduction of stem cells in cardiology provides new tools in understanding the regenerative processes of the normal and pathologic heart and opens new options for the treatment of cardiovascular diseases. The feasibility of adult bone marrow autologous and allogenic cell therapy of ischemic cardiomyopathies has been demonstrated in humans. However, many unresolved questions remain to link experimental with clinical observations. The demonstration that the heart is a self-renewing organ and that its cell turnover is regulated by myocardial progenitor cells offers novel pathogenetic mechanisms underlying cardiac diseases and raises the possibility to regenerate the damaged heart. Indeed, cardiac stem progenitor cells (CSPCs) have recently been isolated from the human heart by several laboratories although differences in methodology and phenotypic profile have been described. The present review points to the potential role of CSPCs in the onset and development of congestive heart failure and its reversal by regenerative approaches aimed at the preservation and expansion of the resident pool of progenitors.

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 β-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.

Wanting to explore the epigenetic basis of Duchenne cardiomyopathy, we found that global histone acetylase activity was abnormally elevated and the acetylase P300/CBP-associated factor (PCAF) coimmunoprecipitated with connexin 43 (Cx43), which was N(ε)-lysine acetylated and lateralized in mdx heart. This observation was paralleled by Cx43 dissociation from N-cadherin and zonula occludens 1, whereas pp60-c-Src association was unaltered. In vivo treatment of mdx with the pan-histone acetylase inhibitor anacardic acid significantly reduced Cx43 N(ε)-lysine acetylation and restored its association to GAP junctions (GJs) at intercalated discs. Noteworthy, in normal as well as mdx mice, the class IIa histone deacetylases 4 and 5 constitutively colocalized with Cx43 either at GJs or in the lateralized compartments. The class I histone deacetylase 3 was also part of the complex. Treatment of normal controls with the histone deacetylase pan-inhibitor suberoylanilide hydroxamic acid (MC1568) or the class IIa-selective inhibitor 3-{4-[3-(3-fluorophenyl)-3-oxo-1-propen-1-yl]-1-methyl-1H-pyrrol-2-yl}-N-hydroxy-2-propenamide (MC1568) determined Cx43 hyperacetylation, dissociation from GJs, and distribution along the long axis of ventricular cardiomyocytes. Consistently, the histone acetylase activator pentadecylidenemalonate 1b (SPV106) hyperacetylated cardiac proteins, including Cx43, which assumed a lateralized position that partly reproduced the dystrophic phenotype. In the presence of suberoylanilide hydroxamic acid, cell to cell permeability was significantly diminished, which is in agreement with a Cx43 close conformation in the consequence of hyperacetylation. Additional experiments, performed with Cx43 acetylation mutants, revealed, for the acetylated form of the molecule, a significant reduction in plasma membrane localization and a tendency to nuclear accumulation. These results suggest that Cx43 N(ε)-lysine acetylation may have physiopathological consequences for cell to cell coupling and cardiac function.

Aims: Bone marrow mesenchymal stromal cell (BMStC) transplantation into the infarcted heart improves left ventricular function and cardiac remodelling. However, it has been suggested that tissue-specific cells may be better for cardiac repair than cells from other sources. The objective of the present work has been the comparison of in vitro and in vivo properties of adult human cardiac stromal cells (CStC) to those of syngeneic BMStC., Methods and Results: Although CStC and BMStC exhibited a similar immunophenotype, their gene, microRNA, and protein expression profiles were remarkably different. Biologically, CStC, compared with BMStC, were less competent in acquiring the adipogenic and osteogenic phenotype but more efficiently expressed cardiovascular markers. When injected into the heart, in rat a model of chronic myocardial infarction, CStC persisted longer within the tissue, migrated into the scar, and differentiated into adult cardiomyocytes better than BMStC., Conclusion: Our findings demonstrate that although CStC and BMStC share a common stromal phenotype, CStC present cardiovascular-associated features and may represent an important cell source for more efficient cardiac repair.

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.

In a rat model of long-lasting pressure-overload hypertrophy, we investigated whether changes in the relative expression of myocardial actin isoforms are among the early signs of ventricular mechanical dysfunction before the transition toward decompensation. Forty-four rats with infrarenal aortic banding (AC rats) were studied. Hemodynamic parameters were measured 1 mo (AC(1) group; n = 20) or 2 mo (AC(2); n = 24) after aortic ligature. Then subgroups of AC(1) and AC(2) left ventricles (LV) were used to evaluate 1) LV anatomy and fibrosis (morphometry), 2) expression levels (immunoblotting) and spatial distribution (immunohistochemistry) of alpha-skeletal actin (alpha-SKA), alpha-cardiac actin (alpha-CA), and alpha-smooth muscle actin (alpha-SMA), and 3) cell mechanics and calcium transients in enzimatically isolated myocytes. Although the two AC groups exhibited a comparable degree of hypertrophy (+30% in LV mass; +20% in myocyte surface) and a similar increase in the amount of fibrosis compared with control animals (C group; n = 22), a worsening of LV mechanical performance was observed only in AC(2) rats at both organ and cellular levels. Conversely, AC(1) rats exhibited enhanced LV contractility and preserved cellular contractile behavior associated with increased calcium transients. Alpha-SKA expression was upregulated (+60%) in AC(1). In AC(2) ventricles, prolonged hypertension also induced a significant increase in alpha-SMA expression, mainly at the level of arterial vessels. No significant differences among groups were observed in alpha-CA expression. Our findings suggest that alpha-SKA expression regulation and wall remodeling of coronary arterioles participate in the development of impaired kinetics of contraction and relaxation in prolonged hypertension before the occurrence of marked histopathologic changes.

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.

The recognition that the adult heart continuously renews its myocyte compartment raises the possibility that the age and lifespan of myocytes does not coincide with the age and lifespan of the organ and organism. If this were the case, myocyte turnover would result at any age in a myocardium composed by a heterogeneous population of parenchymal cells which are structurally integrated but may contribute differently to myocardial performance. To test this hypothesis, left ventricular myocytes were isolated from mice at 3 months of age and the contractile, electrical, and calcium cycling characteristics of these cells were determined together with the expression of the senescence-associated protein p16(INK4a) and telomere length. The heart was characterized by the coexistence of young, aged, and senescent myocytes. Old nonreplicating, p16(INK4a)-positive, hypertrophied myocytes with severe telomeric shortening were present together with young, dividing, p16(INK4a)-negative, small myocytes with long telomeres. A class of myocytes with intermediate properties was also found. Physiologically, evidence was obtained in favor of the critical role that action potential (AP) duration and I(CaL) play in potentiating Ca(2+) cycling and the mechanical behavior of young myocytes or in decreasing Ca(2+) transients and the performance of senescent hypertrophied cells. The characteristics of the AP appeared to be modulated by the transient outward K(+) current I(to) which was influenced by the different expression of the K(+) channels subunits. Collectively, these observations at the physiological and structural cellular level document that by necessity the heart has to constantly repopulate its myocyte compartment to replace senescent poorly contracting myocytes with younger more efficient cells. Thus, cardiac homeostasis and myocyte turnover regulate cardiac function.

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.

We have analysed alterations of alpha-skeletal actin expression and volume fraction of fibrosis in the ventricular myocardium and their functional counterpart in terms of arrhythmogenesis and haemodynamic variables, in rats with different degrees of compensated cardiac hypertrophy induced by infra-renal abdominal aortic coarctation. The following coarctation calibres were used: 1.3 (AC1.3 group), 0.7 (AC0.7) and 0.4 mm (AC0.4); age-matched rats were used as controls (C group). One month after surgery, spontaneous and sympathetic-induced ventricular arrhythmias were telemetrically recorded from conscious freely moving animals, and invasive haemodynamic measurements were performed in anaesthetized animals. After killing, subgroups of AC and C rats were used to evaluate in the left ventricle the expression and spatial distribution of alpha-skeletal actin and the amount of perivascular and interstitial fibrosis. As compared with C, all AC groups exhibited higher values of systolic pressure, ventricular weight and ventricular wall thickness. AC0.7 and AC0.4 rats also showed a larger amount of fibrosis and upregulation of alpha-skeletal actin expression associated with a higher vulnerability to ventricular arrhythmias (AC0.7 and AC0.4) and enhanced myocardial contractility (AC0.4). Our results illustrate the progressive changes in the extracellular matrix features accompanying early ventricular remodelling in response to different degrees of pressure overload that may be involved in the development of cardiac electrical instability. We also demonstrate for the first time a linear correlation between an increase in alpha-skeletal actin expression and the degree of compensated cardiac hypertrophy, possibly acting as an early compensatory mechanism to maintain normal mechanical performance.

Cardiac hypertrophy induces morpho-functional myocardial alterations favoring arrhythmogenesis, especially under specific conditions such as sympathetic stimulation. We analyzed whether the dopaminergic agent CHF-1024, given its effect in decreasing adrenergic drive and collagen deposition in hypertrophied hearts, can also reduce arrhythmia vulnerability. Eighty-one male Wistar rats with intrarenal aortic coarctation and 18 control animals were studied. Fifty-eight banded animals were treated with CHF-1024 at four different doses (6, 2, 0.67, or 0.067 mg/Kg/die). One month after aortic ligature, spontaneous and sympathetic-induced ventricular arrhythmic events (VAEs) were telemetrically recorded in conscious animals. After sacrifice, membrane capacitance (Cm) and action potential duration (APD) were measured in isolated left ventricular myocytes (patch-clamp). In all groups, spontaneous VAEs were negligible whereas they significantly increased during sympathetic activation (stress exposure). Banded untreated animals showed a higher number of stress-induced VAEs, longer action potentials, and larger values of Cm and cell width as compared with control group. The treatment with CHF-1024 exhibited an antiarrhythmic effect, abolished APD prolongation, and reduced cell width at all doses. The lowest dose also prevented Cm increase. In conclusion, we demonstrated that in this model of pressure-overload hypertrophy CHF-1024 reduces arrhythmogenesis and causes a recovery of cell excitable properties toward a normal phenotype.

Cytoplasmic overexpression of Akt in the heart results in a myopathy characterized by organ and myocyte hypertrophy. Conversely, nuclear-targeted Akt does not lead to cardiac hypertrophy, but the cellular basis of this distinct heart phenotype remains to be determined. Similarly, whether nuclear-targeted Akt affects ventricular performance and mechanics, calcium metabolism, and electrical properties of myocytes is unknown. Moreover, whether the expression and state of phosphorylation of regulatory proteins implicated in calcium cycling and myocyte contractility are altered in nuclear-targeted Akt has not been established. We report that nuclear overexpression of Akt does not modify cardiac size and shape but results in an increased number of cardiomyocytes, which are smaller in volume. Additionally, the heart possesses enhanced systolic and diastolic function, which is paralleled by increased myocyte performance. Myocyte shortening and velocity of shortening and relengthening are increased in transgenic mice and are coupled with a more efficient reuptake of calcium by the sarcoplasmic reticulum (SR). This process increases calcium loading of the SR during relengthening. The enhanced SR function appears to be mediated by an increase in SR Ca2+-ATPase2a activity sustained by a higher degree of phosphorylation of phospholamban. This posttranslational modification was associated with an increase in phospho-protein kinase A and a decrease in protein phosphatase-1. Together, these observations provide a plausible biochemical mechanism for the potentiation of myocyte and ventricular function in Akt transgenic mice. Therefore, nuclear-targeted Akt in myocytes may have important implications for the diseased heart.

Cardiac stem cells and early committed cells (CSCs-ECCs) express c-Met and insulin-like growth factor-1 (IGF-1) receptors and synthesize and secrete the corresponding ligands, hepatocyte growth factor (HGF) and IGF-1. HGF mobilizes CSCs-ECCs and IGF-1 promotes their survival and proliferation. Therefore, HGF and IGF-1 were injected in the hearts of infarcted mice to favor, respectively, the translocation of CSCs-ECCs from the surrounding myocardium to the dead tissue and the viability and growth of these cells within the damaged area. To facilitate migration and homing of CSCs-ECCs to the infarct, a growth factor gradient was introduced between the site of storage of primitive cells in the atria and the region bordering the infarct. The newly-formed myocardium contained arterioles, capillaries, and functionally competent myocytes that with time increased in size, improving ventricular performance at healing and long thereafter. The volume of regenerated myocytes was 2200 microm3 at 16 days after treatment and reached 5100 microm3 at 4 months. In this interval, nearly 20% of myocytes reached the adult phenotype, varying in size from 10,000 to 20,000 microm3. Moreover, there were 43+/-13 arterioles and 155+/-48 capillaries/mm2 myocardium at 16 days, and 31+/-6 arterioles and 390+/-56 capillaries at 4 months. Myocardial regeneration induced increased survival and rescued animals with infarcts that were up to 86% of the ventricle, which are commonly fatal. In conclusion, the heart has an endogenous reserve of CSCs-ECCs that can be activated to reconstitute dead myocardium and recover cardiac function.

An important determinant of cardiovascular stress reactivity and morbidity is the individual behavioral strategy of coping with social challenge. This review summarizes the results of a number of studies that we performed in rats, aimed at investigating the relationship between aggression and cardiovascular responsivity under social stress conditions. We show that rats belonging to the 'aggressive tail' of a population are characterized by a higher sympathetic-adrenomedullary activation during social and non-social stress episodes. Wild-type rats are characterized by a larger sympathetic dominance and a higher susceptibility to cardiac arrhythmias during defeat as compared to Wistars. Cardiovascular habituation takes place when social challenge is an intermittent victory experience, whereas no habituation is observed across repeated defeat episodes. Dominant rats whose social dominance is challenged by the aggression of another subject display long-term alterations of heart rate circadian rhythmicity. Such changes are linked to individual proness to defend social dominance: the more the animal counterattacks the aggressor, the smaller the subsequent rhythm disturbance. These data underline how important it is to carefully consider individual differences in aggression and the context in which aggression is expressed, when studying cardiovascular effects of social interactions.

Neuropeptide Y (NPY) is primarily synthesised and released by neurones, it is co-localised with noradrenaline and is involved in the regulation of cardiovascular function. In a mouse model lacking NPY Y1 receptor (KO), the ability of NPY to potentiate noradrenaline-induced vasoconstriction is abolished during stress but normal in baseline conditions, locomotor activity and metabolic rate are lowered, blood insulin levels and glucose storage activity are increased. The present study was aimed at further characterising NPY Y1 mutants, with special emphasis on: behavioural responses to novelty seeking and open-field with objects tests, heart rate responsiveness during acute social defeat, alpha2-adrenoceptor (alpha2-ARs) function in brain areas involved in cardiovascular regulation, and cardiac structure. As compared to wild-type controls (n=9), NPY Y1 KOs (n=9) showed: reduced somatomotor activation during non-social challenges, lower heart rate in baseline conditions, larger heart rate responsiveness during social defeat, increased number of alpha2-ARs in the dorsal motor nucleus of the vagus (nX) and the locus coeruleus (LC), moderately larger volume fraction of myocardial fibrosis. The remarkable increment of alpha2-adrenoceptor density in the nX and LC allows to view KO mice behavioural and anatomo-physiological peripheral characteristics as 'adaptations' to central adrenergic rearrangement due to NPY Y1 receptor deletion.

In normal rats, we analysed the arrhythmogenic role of intrinsic action potential duration (APD) heterogeneity. In each animal, ventricular arrhythmic events (VAEs) occurring spontaneously and during the exposure to an acute social challenge were telemetrically recorded. Action potentials were recorded from isolated left ventricular myocytes, at a pacing rate of 5 Hz (patch clamp: current-clamp mode). APDs were measured at -20 mV, -30 mV, -40 mV, -50 mV and -60 mV. The difference between the shortest and the longest APD was also computed, as an index of individual APD heterogeneity. Animals predisposed to stress-induced arrhythmias showed higher values of APD and APD heterogeneity as compared with the remaining rats. We concluded that, in the normal heart, a large intrinsic APD heterogeneity resulting from specific electrophysiological properties of ventricular myocytes is not in itself arrhythmogenic, but can predispose towards arrhythmia development under certain conditions, such as autonomic activation., (Copyright 2004 The Physiological Society)

To determine whether cellular aging leads to a cardiomyopathy and heart failure, markers of cellular senescence, cell death, telomerase activity, telomere integrity, and cell regeneration were measured in myocytes of aging wild-type mice (WT). These parameters were similarly studied in insulin-like growth factor-1 (IGF-1) transgenic mice (TG) because IGF-1 promotes cell growth and survival and may delay cellular aging. Importantly, the consequences of aging on cardiac stem cell (CSC) growth and senescence were evaluated. Gene products implicated in growth arrest and senescence, such as p27Kip1, p53, p16INK4a, and p19ARF, were detected in myocytes of young WT mice, and their expression increased with age. IGF-1 attenuated the levels of these proteins at all ages. Telomerase activity decreased in aging WT myocytes but increased in TG, paralleling the changes in Akt phosphorylation. Reduction in nuclear phospho-Akt and telomerase resulted in telomere shortening and uncapping in WT myocytes. Senescence and death of CSCs increased with age in WT impairing the growth and turnover of cells in the heart. DNA damage and myocyte death exceeded cell formation in old WT, leading to a decreased number of myocytes and heart failure. This did not occur in TG in which CSC-mediated myocyte regeneration compensated for the extent of cell death preventing ventricular dysfunction. IGF-1 enhanced nuclear phospho-Akt and telomerase delaying cellular aging and death. The differential response of TG mice to chronological age may result from preservation of functional CSCs undergoing myocyte commitment. In conclusion, senescence of CSCs and myocytes conditions the development of an aging myopathy.

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.

The notion of the adult heart as terminally differentiated organ without self-renewal potential has been undermined by the existence of a subpopulation of replicating myocytes in normal and pathological states. The origin and significance of these cells has remained obscure for lack of a proper biological context. We report the existence of Lin(-) c-kit(POS) cells with the properties of cardiac stem cells. They are self-renewing, clonogenic, and multipotent, giving rise to myocytes, smooth muscle, and endothelial cells. When injected into an ischemic heart, these cells or their clonal progeny reconstitute well-differentiated myocardium, formed by blood-carrying new vessels and myocytes with the characteristics of young cells, encompassing approximately 70% of the ventricle. Thus, the adult heart, like the brain, is mainly composed of terminally differentiated cells, but is not a terminally differentiated organ because it contains stem cells supporting its regeneration. The existence of these cells opens new opportunities for myocardial repair.

The degree of cardiovascular stress responsivity and its possible implications for the onset and progression of cardiovascular pathologies seem to be linked to the individual strategy of behavioral coping with stressors. This study was designed to investigate the relationship among cardiac autonomic, endocrine and behavioral responses to real-life stress episodes. Thirty university students were exposed to two brief social challenges (stress interviews), during which the state of sympathovagal balance (time-domain indexes of heart rate variability) and a number of non-verbal behaviors were quantified. Psychometric measurements were also obtained via SPRAS questionnaire, administered just after each stress interview. Samples of saliva were collected for cortisol determination immediately prior and after the experimental session. Subjects showing higher levels of sympathetic dominance were characterized by higher scores of submissive behavior, larger cortisol increments, and higher perception of psychophysiological arousal. A clear consistency in the individual response to the two stress interviews was found, at the behavioral, physiological and psychophysiological level. Finally, the gender of the subjects did not clearly influence their stress responsivity. These results support the hypothesis of a close relationship between the degree of physiological arousal and the style of behavioral adaptation to social stressors.

This study investigated the effects of exposure to an intermittent homotypic stressor on: (i) habituation of acute autonomic responsivity (i.e. cardiac sympathovagal balance and susceptibility to arrhythmias), and (ii) circadian rhythmicity of heart rate, body temperature, and physical activity. After implantation of a transmitter for the radiotelemetric recording of electrocardiogram (ECG), body temperature and physical activity, adult male rats (Rattus norvegicus, Wild Type Groningen strain) were repeatedly exposed (10 consecutive times, on alternate days) to either a social stressor (defeat by a con-specific, n = 15) or an open-field, control challenge (transfer to a new cage; n = 8). ECGs, body temperature and physical activity were continuously recorded in baseline, test and recovery periods (each lasting 15 min), at the 1st and 10th episodes of both defeat and open-field challenge. The circadian rhythms of heart rate, body temperature and physical activity were monitored before (5 days), during (16 days) and after (21 days) the intermittent stress protocol. This study indicates that there is no clear habituation of either acute cardiac autonomic responsivity (as estimated by means of time-domain indexes of heart rate variability) or arrhythmia occurrence to a brief, intermittent, homotypic challenge, regardless of the nature of the stressor (social or non-social). On the other hand, rats exposed to social challenge also failed to show adaptation of acute temperature and activity stress responsiveness, whereas rats facing open-field challenge developed habituation of activity and sensitization of temperature responses. Repeated social challenge produced remarkable reductions of the heart rate circadian rhythm amplitude (this effect being significantly greater than that produced by intermittent open-field), but only minor changes in the daily rhythms of body temperature and physical activity.

In 47 male adult Wistar rats with 4-wk aortic coarctation (AC) and 39 age-matched sham-operated rats (SO) chronically instrumented for telemetry electrocardiogram recording, we investigated the mechanisms of arrhythmogenesis in moderate cardiac hypertrophy, with an approach from "in vivo" toward the cellular level, analyzing 1) stress-induced cardiac arrhythmias in all rats and 2) myocardial fibrosis in 35 animals and action potential duration and density of hyperpolarization-activated current in 19 others at the ventricular level. Aortic banding increased arterial blood pressure, cardiac weight, and ventricular myocyte volume by 11, 25, and 14%, respectively (P < 0.001-0.05). Ventricular arrhythmias occurred at similar rates in AC and SO rats throughout the stress procedure. Action potential duration and hyperpolarization-activated current were about twice as great and myocardial fibrosis about four times greater in AC animals (P < 0.005-0.05). Electrocardiogram data also revealed more supraventricular arrhythmias in AC rats during the baseline period and after stress and fewer atrioventricular block episodes after stress (P < 0.05). Thus stress-induced supraventricular and atrioventricular nodal, but not ventricular, arrhythmias were affected in moderate cardiac hypertrophy when ventricular morphofunctional alterations were evident.

Spread and modulation of electrical activity in cardiac tissue requires intercellular transfer of current via gap junctions, specialised regions of densely packed ionic channels. Electrotonic interaction is determined not merely by intercellular electrical resistance (Rj) but rather by the interplay of Rj and sarcolemmal passive and active electrical properties (Zaniboni et al., Spitzer et al.). In this work we combined a well established protocol to measure Rj in cell pairs (Weingart e Maurer) with a stimulation protocol which allowed to simultaneously study parameters relative to action potential transfer during sequential stimulation. Current clamp experiments, performed on cardiomyocyte pairs held in double-patch configuration, allowed to simultaneously monitor, at a relatively high frequency (1 Hz), membrane resistance (Rm), resting potential (Vm), maximum depolarization rate (dv/dtmax) and time to peak of dv/dtmax in both cells as well as Rj. Spontaneous electrical uncoupling was observed in guinea pig cell pairs with little or no effect on action potential transfer. Pharmacological uncoupling with 40 microM beta-glycyrrhetinic acid reached, in one case, a much higher level of Rj and dramatically increased time delay for action potential appearance. When only Rj was measured over a short time interval after approximately two minutes from cell-attachments, values of Rj approximately 40 M omega in rat cell pairs (n = 20) and Rj approximately 15 M omega in guinea pig cell pairs (n = 24) were obtained. The possibility of monitoring simultaneously active and intercellular/cellular passive electrical properties makes this protocol particularly suitable to study dynamic changes in Rj during action potential transfer.

The purpose of this study is to report new methods for manufacturing precision electrode arrays for recording high-resolution potential distributions from epicardial surfaces of small-animal hearts. Electrode arrays of 64 leads (8 x 8) and 121 leads (11 x 11) were constructed with a tulle substrate to which insulated, fine silver wires (60-micrometer diameter) were attached by knots at mesh node intervals of 540 x 720 micrometers. Insulation was removed at the tips of the knots. Potential distributions and waveforms were recorded from saline solutions and rat heart epicardium during ventricular paced beats and during passive current injection in the diastolic interval. Electrical responses obtained from rat epicardium compared favorably with those observed in studies of larger-animal hearts, which used arrays having greater electrode spacing, and revealed the effects of myocardial anisotropy. Epicardial potentials measured early after stimulation in the region surrounding the pacing site were interpreted in terms of potentials generated by an equivalent quadrupolar source. We conclude that electrode arrays for epicardial mapping of small hearts can be constructed with sufficient ease and precision to allow detailed study of fiber structure and electrophysiology in these hearts in normal and pathological conditions.

The rapid application of caffeine to cardiac myocytes is commonly used to assess changes in the Ca2+ content of the sarcoplasmic reticulum (SR) and to study other parameters of intracellular Ca2+ regulation. Here we examined the effects of rapid caffeine application on membrane potential, intracellular Ca2+, and cell shortening in ventricular myocytes (rat, rabbit, guinea pig, dog) and atrial myocytes (rabbit) that were not voltage clamped. Conditioning pacing was used to achieve a steady-state level of SR Ca2+ loading prior to caffeine (10 mM) application. Caffeine transiently depolarized myocytes as expected from activation of forward Na+-Ca2+ exchange. However, we also found in each species (50% rat, 36% rabbit ventricular, 53% rabbit atrial, 56% guinea pig, 31% dog) that the caffeine-induced depolarization could also trigger an action potential. Caffeine-triggered potentials were completely blocked by thapsigargin (1 microM). The Ca2+ transient and contraction that accompanied caffeine-triggered action potentials had a larger magnitude and slower rate of decline (or relaxation) than occurred during caffeine-induced subthreshold depolarizations. Thus, the use of rapid caffeine application to study SR function and [Ca2+]i regulation in myocytes that are not voltage clamped can yield erroneous results.

Objective: The study was aimed at establishing the effect of factors involved in the expression of mechanoelectric feedback in the heart, such as R-R interval and connective tissue, on time dependent changes in ventricular recovery, as determined at the body surface by beat to beat variability of QRST integral maps (BBV-IM)., Methods: We used 15 normal 6-month-old Wistar rats. In each anesthetized animal, we performed a 3-minute continuous recording of 44. The simultaneous chest ECGs. The signals were interactively processed, 1) to determine mean R-R interval and R-R variability throughout the recording period and 2) to compute QRST integral maps from approximately 50 beats belonging to the end of expiration. Then BBV-IM was calculated and expressed as percentage of beats significantly differing from a template. At sacrifice, the amount of myocardial fibrosis was morphometrically evaluated., Results: R-R interval was 149 ms +/- 4, R-R interval variability 0.008 +/- 0.001 and BBV-IM 30.7% +/- 4.4. Myocardial fibrosis expressed as % volume of left ventricular myocardium, numerical density of fibrotic foci and average cross-sectional area of the foci was 3.0% +/- 0.4, 3.8 +/- 0.6 and 4.4 microns(2)/1000 +/- 0.1 respectively, BB-IM was positively correlated to the % volume of fibrosis (r = 0.83, P < 0.0003). Both measurements were positively correlated to R-R interval (BBV-IM: r = 0.83, P < 0.0001; % volume of fibrosis: r = 0.87, P < 0.001) and negatively correlated to cardiac weights (BBV-IM: r = -0.79, P < 0.0005; % volume of fibrosis: r = -0.75, P < 0.001)., Conclusion: Beat to beat changes in ventricular repolarization attributable to mechanoelectric transduction can be detected at the body surface by means of BBV-IM.

The aim of this study was to investigate the role played by the neuropeptide galanin (GAL) in the regulation of sympathoadrenal function. We evaluated the effects of rat GAL (rGAL) and of the putative GAL receptor antagonist galantide (GLT) on epinephrine (E) and norepinephrine (NE) plasma levels in conscious freely moving male rats, during a psychosocial stress condition. Four groups of male rats were challenged by a stress stimulus, obtained by exposing the animals to a resident conspecific fighter (intruder model), following an intravenous injection with (1) rGAL + saline (SAL), (2) GLT + SAL, (3) rGAL + GLT, or (4) SAL + SAL. Plasma levels of both E and NE were also measured in an additional group of male rats not exposed to any stressor stimulus. The results (mean+/-SEM) showed that rats exposed to the stressor stimulus (intruder rats) exhibited a significant increase above baseline in circulating levels of both E (peak values of 834.13+/-115.13 pmol/l vs. basal values of 309.31+/-32.93 pmol/l; p < 0.01) and NE (peak values of 5,299.03+/-450.62 pmol/l vs. basal values of 2,798.24+/-311.56 pmol/l; p < 0.01) in comparison to control, nonstressed rats. The comparison of the areas under the curve response (AUC) among treatments in the intruder rats revealed that rGAL + SAL injections resulted in a further increase in E levels when compared to SAL + SAL treatment (AUC values: 8.26+/-0.64 vs. 25.38+/-5.52 nmol/ 1/20 min in SAL + SAL vs. rGAL + SAL treatment, respectively; p < 0.02). No significant changes in stress-induced E plasma levels were found following GLT + SAL treatment in comparison to SAL + SAL injections. When the intruder rats were submitted to rGAL + GLT injections, the increments in E levels were found to be higher than those observed following SAL + SAL treatment (AUC values: 8.26+/-0.64 vs. 36.00+/-13.76 nmol/ 1/20 min in SAL + SAL vs. rGAL + GLT treatment, p < 0.03); however, the values were not significantly different from those observed in rGAL + SAL-injected rats. No significant changes in stress-induced NE levels were found in either treatment group when compared to SAL + SAL-injected intruder rats. The results of this study demonstrate that rGAL administration leads to an increase in the E response to the stress stimulus without any effect on NE response. Galantide does not affect either the physiological stress-induced elevation of plasma catecholamines or the effects of rGAL on E plasma levels in response to stress. Therefore, GLT does not appear to behave as a GAL receptor antagonist in the regulation of sympathoadrenal function in rats.

We describe a surgical procedure for optimizing the location of telemetry ECG leads in rats. The new location was aimed at obtaining an accurate representation of ECG features throughout the cardiac cycle by limiting the voltage instability usually observed during intense somatomotor activity and improving the signal-to-noise ratio. The two electrodes (wire loops) were fixed on the dorsal surface of the xiphoid process and in the anterior mediastinum close to the right atrium. The implantation procedure was fast, little invasive, and allowed animals to completely recover from intervention. The performance of the "improved" location (IL, n = 10) with respect to two subcutaneous (SC) positionings ("conventional positioning" CSP, n = 5; "updated location," USL, n = 5) was evaluated by comparing ECGs obtained in baseline, stress and recovery conditions and during different behavioral activities (immobility and grooming). The resident-intruder test (emotional/physical challenge) was chosen as experimental stress paradigm. The noise level of ECGs obtained from IL rats was lower than in CSP and USL animals, in all recording conditions. Percentages of correctly recognized beats (CRBs) over the total number of beats (TBs) were significantly higher in IL rats than in CSP and USL animals, both in baseline conditions (99% vs. 11% and 40%) and situations involving high somatomotor activity (stress: 97%, 5% and 16% recovery; 97%, 7% and 15%) (p < 0.01). The performance of IL as compared to CSP and USL was also better when percentages during grooming and immobility were considered (grooming: 93% vs. 4% and 23%: immobility: 97%, 6%, and 33%; p < 0.01).

Sixty-one patients had operative repair of a massive rotator cuff tear and were followed an average of 7 years (range 3 to 13 years). The mean age of the patients was 62 years; there were 40 male and 21 female patients. All patients had significant pain, 31 patients had insidious onset of pain, and 30 patients recalled a traumatic event. Preoperatively, average active forward elevation was 88°, and average active external rotation was 27°. Forty-nine (80%) patients had marked atrophy of the supraspinatus and infraspinatus muscles. All patients had subacromial decompression as well as mobilization and transposition of rotator cuff tendons with tendon repair to bone. Fifty-two (85%) patients had satisfactory results, with 32 (52%) rated excellent and 20 (33%) rated good. Nine (15%) patients had unsatisfactory results, with four (7%) rated fair and five (8%) rated poor. Ninety-two percent of patients had satisfactory pain relief. The mean gain in forward elevation was 76%, and in active external rotation it was 30°. Adequate pain relief and the ability to raise the arm above the horizontal plane were achieved in 56 (92%) patients. Two patients tore their rotator cuff again after significant trauma., (Copyright © 1992 Journal of Shoulder and Elbow Surgery Board of Trustees. Published by Mosby, Inc. All rights reserved.)

Body surface maps recorded from 35 ischemic patients with normal resting 12-lead electrocardiograms were compared with those obtained from 36 age- and sex-matched normal subjects. From instantaneous maps of each subject 187 variables were derived relating to the configuration (80 variables) and magnitude (104 variables) of the potential distribution and duration of the electrocardiographic intervals (3 variables). By using stepwise discriminant analysis we selected 3 variables whose linear combination enabled us to correctly allocate 91% of the study population (jacknife procedure; specificity 92%, sensitivity 91%). To substantiate the validity of the results the discriminant function was tested on a new independent population consisting of 27 ischemic patients and 54 normal subjects from another laboratory. A proper allocation was obtained in 86% of the cases (specificity 87%, sensitivity 85%). The large number of correctly classified ischemic patients and the repeatability of the results indicate that the adopted criteria are good markers of ischemic heart disease.

The effect of theophylline on potassium uptake of cardiac Purkinje fibers was studied under different conditions. The following results were obtained: (1) theophylline decreases K uptake and enhances Purkinje fiber automaticity; (2) norepinephrine and high [Ca]0 counteract the decrease in K uptake induced by theophylline; (3) adrenergic receptor blockers do not modify the effect of theophylline on K uptake; (4) norepinephrine increases K uptake and this effect is potentiated by theophylline and is little affected by imidazole and acetylcholine. It is concluded that theophylline may depress K uptake by releasing intracellular calcium and thereby inhibiting the sodium-potassium pump. However, theophylline potentiates the effect of norepinephrine on K uptake probably by enhancing the intracellular levels of cyclic AMP.

Ceftriaxone effectively inhibited 332 out of 452 (73.45%) bacterial strains in vitro tests. 291 out of 365 (79.69%) gram negative and 41 out of 87 (47.12%) gram positive strains were inhibited. The tests showed ceftriaxone to be more effective than cephalothin, cephotaxime, cephuroxime, cephamandol and cephoxitin. Kinetic tests showed that cephtriaxone has a plasmatic half life of 7.25 hrs. 24 hours after administration of a 1000 mg venous bolus the drug was still present in the blood. Urinary elimination over a 24 hr. period amounted to means 486.8 mg (48.68%). The drug has liquor transfer capacity. 37 of the 38 patients treated showed complete clinical or clinicobacteriological cure. Improvement was noted in the 38th.

Microbiological, kinetic and clinical studies were conducted on a new cephamycin, cephotetan. In vitro the antibiotic was found to be very effective against all strains tested. It had a particularly strong action against Gram negative bacteria too. Kinetically speaking, an intravenous bolus produced a high plasmatic concentration with a half life of about 4 hours. Elimination via the kidneys was fastest in the first 3 hours after administration (49.82%) and the slowed down. 82.76% of the dose administered was excreted within 24 hours. This antibiotics is particularly indicated in cases of urinary, respiratory and biliary infections.

A mean plasma concentration curve, with peaks within the first hour and moderate individual variations, was obtained after a single administration of 800 mg of bacampicillin to 5 healthy volunteers. An adequate antibiotic transfer from the circulation to the bronchial apparatus was demonstrated. Treatment of 34 patients suffering from ampicillin-sensitive bacterial diseases permitted clinical cure of all patients, without onset of side-effects. It is concluded that bacampicillin is preferable to oral ampicillin because of its kinetic and tolerance features.

Kinetic and clinical evaluation of cefotiam, a new cephalosporin, is reported. It was found that the drug is rapidly distributed to the tissues. Equilibrium between tissues and plasma is reached in about an hour. Some 90-91% of the dose administered is excreted in the urine, and accumulation does not occur. A clinical cure was obtained in 27 of a series of 35 patients (77.1%). Improvement was observed in 7 cases (20%). The antibiotic proved ineffective in the remaining cases (2.8%). Tolerance was excellent and there were no side-effects worthy of note.

A microbiological and clinical study of the action of piperacillin is presented. The drug showed an excellent in vitro antibacterial action on gram positive and gram negative microorganisms. Its in vitro action on Ps. aeruginosa (70.8% of strains inhibited) was also extremely interesting, making it the second most effective of the antibiotics tested after polymyxin B and colistin, polypeptides unsuitable for clinical use. In vivo, the administration of piperacillin achieved a clinical and bacteriological cure in 35 out of 40 patients and a clinical cure in 2 out of 40. It was only therapeutically unsuccessful in 3 cases. It is concluded that its microbiological, kinetic and tolerance features make piperacillin suitable for a wide range of therapeutic purposes.

This retrospective review of 50 osteonecrotic femoral heads in 36 young patients demonstrates the general futility of conservative treatment in slowing progression to total hip arthroplasty (THA). Etiologic factors, i.e., alcoholism, steroids, and idiopathic, did not appear to modify the rate or pattern of clinical deterioration. Pathogenetic stages, i.e., Ficat II to IV, seemed unrelated to the degree of disability. Despite modified weight-bearing, analgesics, and antiinflammatory agents, only three of 50 hips (6%) remained clinically stable and none improved. Thirty-four of the 50 osteonecrotic hips (68%) had been treated by THA after a mean interval of 16 months. In an additional four hips (8%) THA had been recommended, but not carried out. Progressive femoral head collapse was documented in 67% of the hips, the remainder, having reached an end stage (Ficat IV). This study confirms previous reports of the inability of bed rest, crutches, or other ambulatory aids to arrest the progression of femoral head collapse and increasing disability in young individuals with osteonecrosis of the hip.

Quantitative data on the normal variability of body surface maps (BSM) are scarce in the literature. This is one of the reasons why BSM are not yet widely used in clinical practice despite their superior information contents. In this study we determined the average value and variability of a number of parameters derived from BSM in a group of 36 normal adult males, ages 22 to 60. Forty to 60 homogeneous beats were averaged for each subject. This enabled us to extend our study to the low voltage intervals (P,PQ,ST,U) which encompass more than 60% of the entire P-U duration and to contribute new data to controversial issues, such as the presence of two simultaneous maxima during atrial excitation. The following parameters were measured: a) the coordinates of the absolute potential maximum and minimum on the chest surface during the entire cardiac cycle; b) the time course of four voltage-related functions, namely: highest instantaneous potential value on the chest surface, lowest (most negative) potential, highest potential difference, and surface integral of the absolute value of the potential function. In recent studies these parameters were shown to be of considerable value in discriminating normal subjects from different categories of cardiac patients.

The sinus node of the guinea pig was perfused in vitro and potassium uptake was studied in the absence and in the presence of several nicotinic agonists and antagonists. The following results were obtained: (1) the decrease in K uptake caused by a low (10(-9)M) concentration of acetylcholine was changed to an increase when acetylcholine was given in the presence of nicotinic blockade; (2) a small concentration (10(-10)M) of atropine potentiated the decrease in K uptake induced by a low concentration of acetylcholine and 10(-8)M atropine abolished it: (3) a small concentration of nicotine (10(-10)M) decreased K uptake; (4) 1,1-dimethyl-4-phenylpiperazinium (DMPP) did not modify K uptake; (5) the lack of effect of DMPP was not affected by hexamethonium; (6) trimethylphenylammonium (TMPA) decreased K uptake; and (7) the inhibitory effect of TMPA was abolished by decamethonium. It is concluded that a nicotinic receptor in the sinus node mediates the inhibitory action of a low concentration of acetylcholine on K uptake.

Previous work on the spread of excitation on the dog's ventricular surface enabled us to locate up to 30 breakthrough points (BKTPs) where excitation reaches the ventricular surface. In particular the equipotential contour maps enabled us to detect 3 to 5 BKTPs on the anterior right ventricular surface, near the a-v groove when a large part of ventricular surface was still at rest. With a view to investigating the mechanism underlying the early excitation of these basal regions, we stimulated the heart at several right ventricular BKTPs and in other points located at a distance from the BKTPs. The instantaneous equipotential maps showed that after stimulation most right ventricular BKTPs remained in the same position as observed the normal beats. The early appearance of epicardial wavefronts in the basal region and generally in other areas of the right ventricle was attributed to the rapid propagation of excitation waves through the Purkinje network, probably associated to a short transmural crossing time, due to a local thinness of the ventricular wall.

Patients with ischemic heart disease frequently have a normal 12-lead electrocardiogram. We recorded body surface maps from 14 ischemic patients with normal (group A) and 5 with abnormal (group B) resting electrocardiograms. ST-T map data were compared with those of 36 normal subjects. In ischemic patients the following abnormalities were found: an anomalous location and/or trajectory of the potential minimum (lowest potential) on the chest in some; in others the instantaneous values of the time functions: Mxi (highest potential on the chest), delta Vi (highest potential difference) and integral of s/Vi/dS (integral of the absolute value of the potential function extended to the entire chest surface) were lower. In some ischemic patients, both abnormalities were observed. All changes were detectable during the first 200 msec of ST-T. The anomalous potential patterns were similar in group A and B patients, suggesting an ischemic origin of group A abnormalities. By submitting 10 properly selected variables, obtained from body surface maps, to Fisher's discriminant analysis, we succeeded in correctly classifying more than 90% of the cases. The efficacy of the method was validated by using one third of the cases as a test set, with correct allocation in 80.9% of the cases. We conclude that body surface maps at rest can reveal an altered cardiac electrogenesis induced by myocardial ischemia, not apparent in the 12-lead electrocardiogram.