Leonardo Bocchi

Correlation of α‐skeletal actin expression, ventricular fibrosis and heart function with the degree of pressure overload cardiac hypertrophy in rats

We have analysed alterations of α‐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 α‐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 α‐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 α‐skeletal actin expression and the degree of compensated cardiac hypertrophy, possibly acting as an early compensatory mechanism to maintain normal mechanical performance.

Resveratrol treatment reduces cardiac progenitor cell dysfunction and prevents morpho-functional ventricular remodeling in type-1 diabetic rats

Emerging evidence suggests that both adult cardiac cell and the cardiac stem/progenitor cell (CSPC) compartments are involved in the patho-physiology of diabetic cardiomyopathy (DCM). We evaluated whether early administration of Resveratrol, a natural antioxidant polyphenolic compound, in addition to improving cardiomyocyte function, exerts a protective role on (i) the progenitor cell pool, and (ii) the myocardial environment and its impact on CSPCs, positively interfering with the onset of DCM phenotype. Adult Wistar rats (n = 128) with streptozotocin-induced type-1 diabetes were either untreated (D group; n = 54) or subjected to administration of trans-Resveratrol (i.p. injection: 2.5 mg/Kg/day; DR group; n = 64). Twenty-five rats constituted the control group (C). After 1, 3 or 8 weeks of hyperglycemia, we evaluated cardiac hemodynamic performance, and cardiomyocyte contractile properties and intracellular calcium dynamics. Myocardial remodeling and tissue inflammation were also assessed by morphometry, immunohistochemistry and immunoblotting. Eventually, the impact of the diabetic “milieu” on CSPC turnover was analyzed in co-cultures of healthy CSPCs and cardiomyocytes isolated from D and DR diabetic hearts. In untreated animals, cardiac function was maintained during the first 3 weeks of hyperglycemia, although a definite ventricular remodeling was already present, mainly characterized by a marked loss of CSPCs and adult cardiac cells. Relevant signs of ventricular dysfunction appeared after 8 weeks of diabetes, and included: 1) a significant reduction in ±dP/dt in comparison with C group, 2) a prolongation of isovolumic contraction/relaxation times, 3) an impaired contraction of isolated cardiomyocytes associated with altered intracellular calcium dynamics. Resveratrol administration reduced atrial CSPC loss, succeeded in preserving the functional abilities of CSPCs and mature cardiac cells, improved cardiac environment by reducing inflammatory state and decreased unfavorable ventricular remodeling of the diabetic heart, leading to a marked recovery of ventricular function. These findings indicate that RSV can constitute an adjuvant therapeutic option in DCM prevention.

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.

High-resolution cry analysis in preterm newborn infants

Infant monitoring is a common procedure in clinical practice in neonatal critical care units. A number of vital functions are monitored, such as heart beat, breathing, blood flow, etc. Specifically, preterm and/or low-birth-weight infants often present respiratory problems that require monitoring. These may range from insufficient ventilation to apnoea. One of the most common events that may affect the respiratory flow is crying, a physiological action made by the infant to communicate and draw attention, but, for a preterm infant, this action requires great effort, which may cause distress and even may have an adverse impact on blood oxygenation. Acoustic analysis of newborn infant cry is thus of importance, since it is related to other basic neuro-physiological parameters. Being easy to perform, cheap and completely non-invasive, it can be successfully applied in many circumstances. The newborn infant cry is characterised by very high fundamental frequency (F(0)) and resonance frequency (RFs) values, with abrupt changes and voiced/unvoiced features of very short duration in a single utterance. To deal with such signals, a new user-friendly software tool has been developed, that allows robust tracking of main acoustic parameters on very short and time-varying signal frames. The software developed provides the user with a high-resolution picture of the cry signal characteristics.

Modulation of actin isoform expression before the transition from experimental compensated pressure-overload cardiac hypertrophy to decompensation

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.

Tissue characterization from X-ray images

The study of the fine-scale structure of biological tissues is crucial for diagnosing a wide number of different diseases. In X-ray images, fine structures usually induce a correlation among image gray levels and are commonly perceived as textures. In this paper, we report on a Computer Vision approach to the characterization of biological tissues as imaged by standard X-ray techniques. In particular, using features derived from co-occurrence matrices, we have assessed spatial gray-level dependence of bone tissue and lung parenchyma images. A hybrid neural network was adopted to distinguish pathological tissues from normal ones and to classify different pathologies.

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.

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 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.

Objective vocal fold vibration assessment from videokymographic images

Abstract Vocal folds oscillation crucially influences all the basic qualities of voice, such as pitch and loudness, as well as the spectrum. Stroboscopy provides the standard view of the larynx. However, a two-dimensional high-speed imaging system currently cannot provide enough image resolution to evaluate irregular vocal fold vibrations, due to the limitation of transmission speed and storage volume. Videokymography is a new diagnostic tool developed to overcome specific limitations of stroboscopy in severely dysphonic patients with an aperiodic signal. It registers the movements of the vocal folds with a high time resolution on a line perpendicular to the glottis. The technique, being independent from the periodicity and intensity of the vocal signal, allows an objective evaluation of vocal folds function. However, due to its novelty, no established clinical evaluation protocol, or validity and reliability data are available for videokymography. Moreover, few results concerning objective parameter estimation from videokymographic images are available. The main focus of this paper is on measuring and tracking quantitative parameters for objective vocal fold functional assessment, from videokymographic (VKG) examinations of subjects with normal and pathological laryngeal function, based on active contour search implemented with a properly adjusted robust snake algorithm. The method is designed to deliver to the clinician the essential objective information of VKG in an effective way, as an aid to its subjective and intuitive skilfulness. A set of VKG images has been analysed, coming both from healthy and dysphonic subjects, recorded at the Otolaryngology Department, University of Milan, Italy, showing the robustness and reliability of the proposed technique.

Validity of jitter measures in non-quasi-periodic voices. Part II: The effect of noise

Abstract In this paper the effect of noise on both perceptual and automatic evaluation of the glottal cycle length in irregular voice signals (sustained vowels) is studied. The reliability of four tools for voice analysis (MDVP, Praat, AMPEX, and BioVoice) is compared to visual inspection made by trained clinicians using two measures of voice signal irregularity: the jitter (J) and the coefficient of variation of the fundamental frequency (F0CV). The purpose is also to test to what extent of irregularity trained raters are capable of determining visually the glottal cycle length as compared to dedicated software tools. For a perfect control of the amount of jitter and noise put in, data consist of synthesized sustained vowels corrupted by increasing jitter and noise. Both jitter and noise can be varied to the desired extent according to built-in functions. All the tools give almost reliable measurements up to 15% of jitter, for low or moderate noise, while only few of them are reliable for higher jitter and noise levels and would thus be suited for perturbation measures in strongly irregular voice signals. As shown in Part I of this work, for low noise levels the results obtained by visual inspection from expert raters are comparable or better than those obtained with the tools presented here, at the expense of a larger amount of time devoted to searching visually for the glottal cycle lengths in the signal waveform. In this paper it is shown that results rapidly deteriorate with increasing noise. Hence, the use of a robust tool for voice analysis can give valid support to clinicians in term of reliability, reproducibility of results, and time-saving.