A new approach to heart valve tissue engineering: mimicking the heart ventricle with a ventricular assist device in a novel bioreactor

The `biomimetic` approach to tissue engineering usually involves the use of a bioreactor mimicking physiological parameters whilst supplying nutrients to the developing tissue. Here we present a new heart valve bioreactor, having as its centrepiece a ventricular assist device (VAD), which exposes the cell-scaffold constructs to a wider array of mechanical forces. The pump of the VAD has two chambers: a blood and a pneumatic chamber, separated by an elastic membrane. Pulsatile air-pressure is generated by a piston-type actuator and delivered to the pneumatic chamber, ejecting the fluid in the blood chamber. Subsequently, applied vacuum to the pneumatic chamber causes the blood chamber to fill. A mechanical heart valve was placed in the VAD`s inflow position. The tissue engineered (TE) valve was placed in the outflow position. The VAD was coupled in series with a Windkessel compliance chamber, variable throttle and reservoir, connected by silicone tubings. The reservoir sat on an elevated platform, allowing adjustment of ventricular preload between 0 and 11 mmHg. To allow for sterile gaseous exchange between the circuit interior and exterior, a 0.2 mu m filter was placed at the reservoir. Pressure and flow were registered downstream of the TE valve. The circuit was filled with culture medium and fitted in a standard 5% CO(2) incubator set at 37 degrees C. Pressure and flow waveforms were similar to those obtained under physiological conditions for the pulmonary circulation. The `cardiomimetic` approach presented here represents a new perspective to conventional biomimetic approaches in TE, with potential advantages. Copyright (C) 2010 John Wiley & Sons, Ltd.

Repeatability of maximal voluntary force and of surface EMG variables during voluntary isometric contraction of quadriceps muscles in healthy subjects

The repeatability of initial values and rate of change of EMG signal mean spectral frequency (MNF), average rectified values (ARV), muscle fiber conduction velocity (CV) and maximal voluntary contraction (MVC) was investigated in the vastus medialis obliquus (VMO) and vastus lateralis (VL) muscles of both legs of nine healthy male subjects during voluntary, isometric contractions sustained for 50 s at 50% MVC. The values of MVC were recorded for both legs three times on each day and for three subsequent days, while the EMG signals have been recorded twice a day for three subsequent days. The degree of repeatability was investigated using the Fisher test based upon the ANalysis Of VAriance (ANOVA), the Standard Error of the Mean (SEM) and the Intraclass Correlation Coefficient (ICC). Data collected showed a high level of repeatability of MVC measurement (normalized SEM from 1.1% to 6.4% of the mean). MNF and ARV initial values also showed a high level of repeatability (ICC > 70% for all muscles and legs except right VMO). At 50% MVC level no relevant pattern of fatigue was observed for the VMO and VL muscles, suggesting that other portions of the quadriceps might have contributed to the generated effort. These observations seem to suggest that in the investigation of muscles belonging to a multi-muscular group at submaximal level, the more selective electrically elicited contractions should be preferred to voluntary contractions. (C) 2001 Elsevier Science Ltd. All rights reserved.

Myoarchitecture and vasculature of the heart ventricle in some freshwater teleosts

The morphological characteristics of the ventricular myocardium and of coronary vascularization were studied in three freshwater teleost species, Piaractus mesopotamicus, Colossoma macropomum and Clarias gariepinus (African catfish), by correlating their ventricular shapes and swimming habits. In Piaractus mesopotamicus and Colossoma macropomum, species with highly active swimming habits, the cardiac ventricle showed a pyramidal shape and a richly vascularized myocardium consisting of an outer compact layer and inner spongy layer. In Clarias gariepinus, a less active species, we observed a saccular ventricle with a mixed myocardium and coronary arteries, in contrast to the ventricular structure of other species described in the literature.

Morphological studies on the heart ventricle of African catfish (Clarias gariepinus)

The histological and ultrastructural characteristics of the heart ventricle in Clarias gariepinus (African catfish) has been studied by light microscopy and transmission electron microscopy. The ventricle of the heart has a saccular shape and the myocardial wall consists of an outer thin compact myocardium and an inner well-developed spongy myocardium. The myocardial layer has small myocytes, interstitial spaces and blood vessels. The myocytes are the major constituents of the ventricular wall. They are long cells, with large nuclei, and predominantly euchromatin. The sarcoplasmic reticulum of the ventricular myocytes consists of a network of tubules and subsarcolemmal cisternae oriented mainly along the longitudinal axis of the myofibrils. In contrast to the ventricular structure of other fish species described in the literature (Greer-Walker et al., 1985 Santer, 1985 Sanchez-Quintana et al., 1995, 1996), the African catfish, a freshwater sedentary fish recently introduced in neotropical climatic environments, showed a saccular ventricle that consisted of two muscle layers, a thin compact layer with large vessels and a developed spongy layer. The ultrastructure of the ventricular myocardium of C.gariepinus is similar to that of other teleosts, inclusive that of fish with other swimming habits.

Activation of calcineurin in human failing heart ventricle by endothelin-1, angiotensin II and urotensin II

1 The calcineurin (CaN) enzyme-transcriptional pathway is critically involved in hypertrophy of heart muscle in some animal models. Currently there is no information concerning the regulation of CaN activation by endogenous agonists in human heart. 2 Human right ventricular trabeculae from explanted human ( 14 male/2 female) failing hearts were set up in a tissue bath and electrically paced at 1Hz and incubated with or without 100 nM endothelin-1 (ET-1), 10 mu M, angiotensin-II (Ang II) or 20 nM human urotensin-II (hUII) for 30 min. Tissues from four patients were incubated with 200 nM tacrolimus (FK506) for 30 min and then incubated in the presence or absence of ET-1 for a further 30 min. 3 ET-1 increased contractile force in all 13 patients (P < 0.001). Ang II and hUII increased contractile force in three out of eight and four out of 10 patients but overall nonsignificantly (P > 0.1). FK506 had no effect on contractile force (P = 0.12). 4 ET-1, Ang II and hUII increased calcineurin activity by 32, 71 and 15%, respectively, while FK506 reduced activity by 34%. ET-1 in the presence of FK506 did not restore calcineurin activity (P = 0.1). 5 There was no relationship between basal CaN activity and expression levels in the right ventricle. Increased levels of free phosphate were detected in ventricular homogenates that were incubated with PKC epsilon compared to samples incubated without PKCe. 6 Endogenous cardiostimulants which activate G alpha q-coupled receptors increase the activity of calcineurin in human heart following acute (30 min) exposure. PKC may contribute to this effect by increasing levels of phosphorylated calcineurin substrate.

Mechanical properties of rat cardiac skinned fibers are altered by chronic growth hormone hypersecretion.

Chronic growth hormone (GH) hypersecretion in rats leads to increased isometric force without affecting the unloaded shortening velocity of isolated cardiac papillary muscles, despite a marked isomyosin shift toward V3. To determine if alterations occurred at the level of the contractile proteins in rats bearing a GH-secreting tumor (GH rats), we examined the mechanical properties of skinned fibers to eliminate the early steps of the excitation-contraction coupling mechanism. We found that maximal active tension and stiffness at saturating calcium concentrations (pCa 4.5) were markedly higher in GH rats than in control rats (tension, 52.9 +/- 5.2 versus 38.1 +/- 4.6 mN.mm-2, p < 0.05; stiffness, 1,105 +/- 120 versus 685 +/- 88 mN.mm-2.microns-1, p < 0.01), whereas values at low calcium concentrations (pCa 9) were unchanged. In addition, the calcium sensitivity of the contractile proteins was slightly but significantly higher in GH rats than in control rats (delta pCa 0.04, p < 0.001). The crossbridge cycling rate, reflected by the response to quick length changes, was lower in GH rats than in control rats (62.0 +/- 2.6 versus 77.4 +/- 6.6 sec-1, p < 0.05), in good agreement with a decrease in the proportion of alpha-myosin heavy chains in the corresponding papillary muscles (45.5 +/- 2.0% versus 94.6 +/- 2.4%, p < 0.001). The changes in myosin heavy chain protein phenotype were paralleled by similar changes of the corresponding mRNAs, indicating that the latter occurred mainly at a pretranslational level. These results demonstrate that during chronic GH hypersecretion in rats, alterations at the myofibrillar level contribute to the increase in myocardial contractility observed in intact muscle.

The effect of eccentric strength training on heart rate and on its variability during isometric exercise in healthy older men

The purpose of this study was to investigate if chronic eccentric strength training (ST) affects heart rate (HR) and heart rate variability (HRV) during sub-maximal isometric voluntary contractions (SIVC). The training group (TG) (9 men, 62 ± 2) was submitted to ST (12 weeks, 2 days/week, 2 - 4 sets of 8-12 repetitions at 75-80% peak torque (PT). The control group (CG) (8 men, 64 ± 4) did not perform ST. The HR and the HRV (RMSSD index) were evaluated during SIVC of the knee extension (15, 30 and 40% of PT). ST increased the eccentric torque only in TG, but did not change the isometric PT and the duration of SIVC. During SIVC, the HR response pattern and the RMSSD index were similar for both groups in pre- and post-training evaluations. Although ST increased the eccentric torque in the TG, it did not generate changes in HR or HRV. © Springer-Verlag 2008.

A key role of TRPC channels in the regulation of electromechanical activity of the developing heart.

Aims It is well established that dysfunction of voltage-dependent ion channels results in arrhythmias and conduction disturbances in the foetal and adult heart. However, the involvement of voltage-insensitive cationic TRPC (transient receptor potential canonical) channels remains unclear. We assessed the hypothesis that TRPC channels play a crucial role in the spontaneous activity of the developing heart.Methods and results TRPC isoforms were investigated in isolated hearts obtained from 4-day-old chick embryos. Using RT-PCR, western blotting and co-immunoprecipitation, we report for the first time that TRPC1, 3, 4, 5, 6, and 7 isoforms are expressed at the mRNA and protein levels and that they can form a macromolecular complex with the alpha 1C subunit of the L-type voltage-gated calcium channel (Cav1.2) in atria and ventricle. Using ex vivo electrocardiograms, electrograms of isolated atria and ventricle and ventricular mechanograms, we found that inhibition of TRPC channels by SKF-96365 leads to negative chrono-, dromo-, and inotropic effects, prolongs the QT interval, and provokes first-and second-degree atrioventricular blocks. Pyr3, a specific antagonist of TRPC3, affected essentially atrioventricular conduction. On the other hand, specific blockade of the L-type calcium channel with nifedipine rapidly stopped ventricular contractile activity without affecting rhythmic electrical activity.Conclusions These results give new insights into the key role that TRPC channels, via interaction with the Cav1.2 channel, play in regulation of cardiac pacemaking, conduction, ventricular activity, and contractility during cardiogenesis.

CaMKIIdelta overexpression in hypertrophy and heart failure: cellular consequences for excitation-contraction coupling

Ca/calmodulin-dependent protein kinase IIdelta (CaMKIIdelta) is the predominant isoform in the heart. During excitation-contraction coupling (ECC) CaMKII phosphorylates several Ca-handling proteins including ryanodine receptors (RyR), phospholamban, and L-type Ca channels. CaMKII expression and activity have been shown to correlate positively with impaired ejection fraction in the myocardium of patients with heart failure and CaMKII has been proposed to be a possible compensatory mechanism to keep hearts from complete failure. However, in addition to these acute effects on ECC, CaMKII was shown to be involved in hypertrophic signaling, termed excitation-transcription coupling (ETC). Thus, animal models have shown that overexpression of nuclear isoform CaMKIIdeltaB can induce myocyte hypertrophy. Recent study from our laboratory has suggested that transgenic overexpression of the cytosolic isoform CaMKIIdeltaC in mice causes severe heart failure with altered intracellular Ca handling and protein expression leading to reduced sarcoplasmic reticulum (SR) Ca content. Interestingly, the frequency of diastolic spontaneous SR Ca release events (or opening of RyR) was greatly enhanced, demonstrating increased diastolic SR Ca leak. This was attributed to increased CaMKII-dependent RyR phosphorylation, resulting in increased and prolonged openings of RyR since Ca spark frequency could be reduced back to normal levels by CaMKII inhibition. This review focuses on acute and chronic effects of CaMKII in ECC and ETC. In summary, CaMKII overexpression can lead to heart failure and CaMKII-dependent RyR hyperphosphorylation seems to be a novel and important mechanism in ECC due to SR Ca leak which may be important in the pathogenesis of heart failure.

Myocardial contractile dysfunction contributes to the development of heart failure in rats with aortic stenosis

Objectives: To analyze the potential contribution of contractility state and ventricular geometry to the development of heart failure in rats with aortic stenosis.Methods: Rats were divided into three groups: compensated aortic stenosis (AS, n = 11), heart failure AS (n = 12) and control rats (C, n = 13).Results: After 21 weeks, failing AS rats presented higher systolic (C = 36.6 +/- 3.1, AS-78.6 +/- 4.8*, failing AS = 104.6 +/- 7.8*) and diastolic meridian stress (C = 6.9 +/- 0.4, AS = 20.1 +/- 1.1*, failing AS = 43.2 +/- 3.2*(dagger)), hydroxyproline (C = 3.6 +/- 0.7 mg/g, AS = 6.6 +/- 0.6* mg/g, failing AS = 9.2 +/- 1.4*(dagger) mg/g) and cross-sectional area (C = 338 +/- 25 mu m(2), AS = 451 +/- 32* mu m(2), failing AS = 508 +/- 36*(dagger) mu m(2)), in comparison with control and compensated AS animals (*p < 0.05 vs. control, (dagger)p < 0.05 vs. AS). In the isometric contraction study, considering the time from peak tension to 50% relaxation (RT50), the relative variation responses, following post-rest contraction and increase in Ca2+ concentration, were higher in failing AS than compensated AS animals. In contrast, following post-rest contraction, compensated AS group presented higher values of the peak developed tension (DT) than failing AS group. Following beta-adrenergic stimulation, control animals presented higher values of +dT/dt and -dT/dt than AS animals. In addition, failing AS animals presented higher TPT values than compensated AS animals.Conclusion: Myocardial contractile dysfunction contributes to the development of heart failure in rats with aortic stenosis. (c) 2006 Elsevier B.V.. All rights reserved.

Comparative mechanical study of isolated papillary muscle from Wistar-Kyoto and Wistar rats

Isolated papillary muscles have often been used in myocardial mechanical function studies. The objective of the present study was to compare the mechanical function of papillary muscle isolated from left ventricle between Wistar (W) and Wistar-Kyoto (WKY) rats of different ages (1, 3, 6 and 12 months), in order to examine whether there is a difference in intrinsic mechanical properties of muscle between the two rat strains. Muscles were perfused with Krebs-Henseleit solution at 28°C and studied isometrically and isotonically at a stimulation rate of 0.2 Hz. The W and WKY showed statistically significant differences during both isometric and isotonic contractions. During isometric contraction? (l) the peak developed tension (DT) and + dT/dt were lower in WKY rats in the 1 mo groups, (2) the resting tension (RT) was greater in WKY at 3, 6 and 12 mo. (3) time to peak tension (TPT) was greater in WKY at 3 and 12 mo, (4) time for tension to fall from peak to 50% of peak tension (RT 1/2) was greater in WKY at 3 mo and (5) - dT/dt was lower in WKY at 1 and 3 mo. During isotonic contraction, (1) the peak shortening (PS) and -dL/dt were lower in WKY at 12 mo, (2) the time to peak shortening (TPS) was greater in WKY at 3 and 12 mo; (3) + dL/dt was lower in WKY at 3, 6, and 12 mo and (4) the relative variation of length (Lmax-PS)/Lmax was greater in WKY at 6 and 12 mo. These data showed a difference in mechanical behaviour of the papillary muscle between Wistar and Wistar-Kyoto rats of different age.

Improved systolic ventricular function with normal myocardial mechanics in compensated cardiac hypertrophy

There still controversy about the relation between changes in myocardial contractile function and global left ventricular (LV) performance during stable concentric hypertrophy. To clarify this, we analyzed LV function in vivo and myocardial mechanics in vitro in rats with pressure overload-induced cardiac hypertrophy. Male Wistar rats (70 g) underwent ascending aorta stenosis for 8 weeks (group AAS, n=9). LV performance was assessed by transthoracic echocardiography under light anesthesia. Myocardial function was studied in isolated papillary muscle preparation during isometric contraction. The data were compared with age- and sex-matched sham-operated rats (group C, n=9). LV weight-to-body weight ratio (C: 2.0 ± 0.5 mg/g; AAS: 3.3 ± 0.7 mg/g), LV relative wall thickness (C: 0.19 ± 0.02; AAS; 0.34 ± 0.10), and LV fractional shortening (C: 54 ± 5%; AAS: 70 ± 8%) were increased in the group AAS (p<0.05). Echocardiographic analysis also indicated a significant association (r=0.74; p<0.001) between percent fractional shortening and LV relative wall thickness. The performance of AAS isolated muscle revealed that active tension (C: 6.6 ± 1.7 g/mm 2; AAS: 6.5 ± 1.5 g/mm 2) and maximum rate of tension development (C: 69 ± 21 g/mm 2/s; AAS: 69 ± 18 g/mm 2) were not significantly different from group C (p>0.05). In conclusion: 1) Compensated pressure-overload myocardial hypertrophy is associated with preserved myocardial function and increased ventricular performance; 2) The improved LV function might be due to the ventricular remodeling characterized by an increased relative wall thickness. Copyright © 2002 By PJD Publications Limited.