Fractional Order Dual-Phase-Lag Model of Heat Conduction in a Composite Spherical Medium.

In the paper, a solution of the fractional dual-phase-lag heat conduction problem is presented. The considerations are related to the heat conduction in a multi-layered spherical medium with azimuthal symmetry. The final form of the analytical solution is given in a form of the double series of spherical Bessel functions and Legendre functions. Numerical calculations concern the study of the effect of the order of the Caputo derivative on the temperature distribution in a composite solid sphere, hemisphere and spherical cone.

A Fractional Single-Phase-Lag Model of Heat Conduction for Describing Propagation of the Maximum Temperature in a Finite Medium.

In this paper, an investigation of the maximum temperature propagation in a finite medium is presented. The heat conduction in the medium was modelled by using a single-phase-lag equation with fractional Caputo derivatives. The formulation and solution of the problem concern the heat conduction in a slab, a hollow cylinder, and a hollow sphere, which are subjected to a heat source represented by the Robotnov function and a harmonically varying ambient temperature. The problem with time-dependent Robin and homogenous Neumann boundary conditions has been solved by using an eigenfunction expansion method and the Laplace transform technique. The solution of the heat conduction problem was used for determination of the maximum temperature trajectories. The trajectories and propagation speeds of the temperature maxima in the medium depend on the order of fractional derivatives occurring in the heat conduction model. These dependencies for the heat conduction in the hollow cylinder have been numerically investigated.

Optogenetic targeting of cardiac myocytes and non-myocytes: Tools, challenges and utility.

In optogenetics, light-activated proteins are used to monitor and modulate cellular behaviour with light. Combining genetic targeting of distinct cellular populations with defined patterns of optical stimulation enables one to study specific cell classes in complex biological tissues. In the current study we attempted to investigate the functional relevance of heterocellular electrotonic coupling in cardiac tissue in situ. In order to do that, we used a Cre-Lox approach to express the light-gated cation channel Channelrhodopsin-2 (ChR2) specifically in either cardiac myocytes or non-myocytes. Despite high specificity when using the same Cre driver lines in a previous study in combination with a different optogenetic probe, we found patchy off-target ChR2 expression in cryo-sections and extended z-stack imaging through the ventricular wall of hearts cleared using CLARITY. Based on immunohistochemical analysis, single-cell electrophysiological recordings and whole-genome sequencing, we reason that non-specificity is caused on the Cre recombination level. Our study highlights the importance of careful design and validation of the Cre recombination targets for reliable cell class specific expression of optogenetic tools.

Transformation diffusion reconstruction of three-dimensional histology volumes from two-dimensional image stacks.

Traditional histology is the gold standard for tissue studies, but it is intrinsically reliant on two-dimensional (2D) images. Study of volumetric tissue samples such as whole hearts produces a stack of misaligned and distorted 2D images that need to be reconstructed to recover a congruent volume with the original sample's shape. In this paper, we develop a mathematical framework called Transformation Diffusion (TD) for stack alignment refinement as a solution to the heat diffusion equation. This general framework does not require contour segmentation, is independent of the registration method used, and is trivially parallelizable. After the first stack sweep, we also replace registration operations by operations in the space of transformations, several orders of magnitude faster and less memory-consuming. Implementing TD with operations in the space of transformations produces our Transformation Diffusion Reconstruction (TDR) algorithm, applicable to general transformations that are closed under inversion and composition. In particular, we provide formulas for translation and affine transformations. We also propose an Approximated TDR (ATDR) algorithm that extends the same principles to tensor-product B-spline transformations. Using TDR and ATDR, we reconstruct a full mouse heart at pixel size 0.92µm×0.92µm, cut 10µm thick, spaced 20µm (84G). Our algorithms employ only local information from transformations between neighboring slices, but the TD framework allows theoretical analysis of the refinement as applying a global Gaussian low-pass filter to the unknown stack misalignments. We also show that reconstruction without an external reference produces large shape artifacts in a cardiac specimen while still optimizing slice-to-slice alignment. To overcome this problem, we use a pre-cutting blockface imaging process previously developed by our group that takes advantage of Brewster's angle and a polarizer to capture the outline of only the topmost layer of wax in the block containing embedded tissue for histological sectioning.

Electrotonic coupling of excitable and nonexcitable cells in the heart revealed by optogenetics.

Electrophysiological studies of excitable organs usually focus on action potential (AP)-generating cells, whereas nonexcitable cells are generally considered as barriers to electrical conduction. Whether nonexcitable cells may modulate excitable cell function or even contribute to AP conduction via direct electrotonic coupling to AP-generating cells is unresolved in the heart: such coupling is present in vitro, but conclusive evidence in situ is lacking. We used genetically encoded voltage-sensitive fluorescent protein 2.3 (VSFP2.3) to monitor transmembrane potential in either myocytes or nonmyocytes of murine hearts. We confirm that VSFP2.3 allows measurement of cell type-specific electrical activity. We show that VSFP2.3, expressed solely in nonmyocytes, can report cardiomyocyte AP-like signals at the border of healed cryoinjuries. Using EM-based tomographic reconstruction, we further discovered tunneling nanotube connections between myocytes and nonmyocytes in cardiac scar border tissue. Our results provide direct electrophysiological evidence of heterocellular electrotonic coupling in native myocardium and identify tunneling nanotubes as a possible substrate for electrical cell coupling that may be in addition to previously discovered connexins at sites of myocyte-nonmyocyte contact in the heart. These findings call for reevaluation of cardiac nonmyocyte roles in electrical connectivity of the heterocellular heart.

Three-dimensional histology: tools and application to quantitative assessment of cell-type distribution in rabbit heart.

AIMS: Cardiac histo-anatomical organization is a major determinant of function. Changes in tissue structure are a relevant factor in normal and disease development, and form targets of therapeutic interventions. The purpose of this study was to test tools aimed to allow quantitative assessment of cell-type distribution from large histology and magnetic resonance imaging- (MRI) based datasets. METHODS AND RESULTS: Rabbit heart fixation during cardioplegic arrest and MRI were followed by serial sectioning of the whole heart and light-microscopic imaging of trichrome-stained tissue. Segmentation techniques developed specifically for this project were applied to segment myocardial tissue in the MRI and histology datasets. In addition, histology slices were segmented into myocytes, connective tissue, and undefined. A bounding surface, containing the whole heart, was established for both MRI and histology. Volumes contained in the bounding surface (called 'anatomical volume'), as well as that identified as containing any of the above tissue categories (called 'morphological volume'), were calculated. The anatomical volume was 7.8 cm(3) in MRI, and this reduced to 4.9 cm(3) after histological processing, representing an 'anatomical' shrinkage by 37.2%. The morphological volume decreased by 48% between MRI and histology, highlighting the presence of additional tissue-level shrinkage (e.g. an increase in interstitial cleft space). The ratio of pixels classified as containing myocytes to pixels identified as non-myocytes was roughly 6:1 (61.6 vs. 9.8%; the remaining fraction of 28.6% was 'undefined'). CONCLUSION: Qualitative and quantitative differentiation between myocytes and connective tissue, using state-of-the-art high-resolution serial histology techniques, allows identification of cell-type distribution in whole-heart datasets. Comparison with MRI illustrates a pronounced reduction in anatomical and morphological volumes during histology processing.

Impact of combined clenbuterol and metoprolol therapy on reverse remodelling during mechanical unloading.

BACKGROUND: Clenbuterol (Cl), a ß2 agonist, is associated with enhanced myocardial recovery during left ventricular assist device (LVAD) support, and exerts beneficial remodelling effects during mechanical unloading (MU) in rodent heart failure (HF). However, the specific effects of combined Cl+ß1 blockade during MU are unknown. METHODS AND RESULTS: We studied the chronic effects (4 weeks) of ß2-adrenoceptor (AR) stimulation via Cl (2 mg/kg/day) alone, and in combination with ß1-AR blockade using metoprolol ((Met), 250 mg/kg/day), on whole heart/cell structure, function and excitation-contraction (EC) coupling in failing (induced by left coronary artery (LCA) ligation), and unloaded (induced by heterotopic abdominal heart transplantation (HATx)) failing rat hearts. Combined Cl+Met therapy displayed favourable effects in HF: Met enhanced Cl's improvement in ejection fraction (EF) whilst preventing Cl-induced hypertrophy and tachycardia. During MU combined therapy was less beneficial than either mono-therapy. Met, not Cl, prevented MU-induced myocardial atrophy, with increased atrophy occurring during combined therapy. MU-induced recovery of Ca2+ transient amplitude, speed of Ca2+ release and sarcoplasmic reticulum Ca2+ content was enhanced equally by Cl or Met mono-therapy, but these benefits, together with Cl's enhancement of sarcomeric contraction speed, and MU-induced recovery of Ca2+ spark frequency, disappeared during combined therapy. CONCLUSIONS: Combined Cl+Met therapy shows superior functional effects to mono-therapy in rodent HF, but appears inferior to either mono-therapy in enhancing MU-induced recovery of EC coupling. These results suggest that combined ß2-AR simulation +ß1-AR blockade therapy is likely to be a safe and beneficial therapeutic HF strategy, but is not as effective as mono-therapy in enhancing myocardial recovery during LVAD support.

The effect of SN-6, a novel sodium-calcium exchange inhibitor, on contractility and calcium handling in isolated failing rat ventricular myocytes.

BACKGROUND AND PURPOSE: Specific Na(+) /Ca(2+) exchanger (NCX) inhibition is a potential strategy to correct reduced contractility and depleted sarcoplasmic reticulum (SR) Ca(2+) content in heart failure (HF). SN-6, a benzyloxyphenyl derivative and proposed selective NCX inhibitor, could be used for this purpose. This study aimed to evaluate the effects of SN-6 on contractility and Ca(2+) handling in normal and failing rat cardiomyocytes. EXPERIMENTAL APPROACH: HF was induced in rats by coronary artery ligation. Left ventricular myocytes were isolated and superfused with increasing concentrations of SN-6. KEY RESULTS: Sarcomere shortening, induced by field-stimulation, was reduced in amplitude with increasing concentrations of SN-6 compared with control solution. This effect was greater in failing cells. Kinetics of contractility (time to 90% peak and time to 50% relaxation) were significantly faster. Despite this, intracellular Ca(2+) transients demonstrated no change in the peak amplitude at low concentrations of SN-6, suggesting that SN-6 may affect myofilament sensitivity to Ca(2+) . Ten micro molar SN-6 significantly reduced peak Ca(2+) amplitude by 61.57% and 64.73% in normal and failing cells, respectively. Diastolic Ca(2+) was significantly increased at 1 µM SN-6. SR Ca(2+) content, assessed by rapid application of caffeine, was reduced in failing cells with 1 µM SN-6. Peak ICa , measured by whole-cell patch clamping, was significantly reduced in normal and failing myocytes at 1 µM SN-6. CONCLUSIONS AND IMPLICATIONS: Our data suggest that SN-6 is not a selective inhibitor of NCX and impairs contractility and Ca(2+) handling. Its use, together with similar putative NCX blockers, in correcting the contractile abnormalities of heart failure requires further studies.

Heterotopic abdominal heart transplantation in rats for functional studies of ventricular unloading.

INTRODUCTION: Chronic changes in mechanical load regulate long-term cardiac function. Chronic overload of the ventricle results in myocardial failure. Clinical use of ventricular assist devices shows that chronic reduction in load has a number of different consequences on the myocardium, including beneficial reverse remodeling as well as undesired remodeling (e.g., myocardial atrophy and fibrosis, both of which could have negative functional implications). The complex response to mechanical unloading necessitates reproducible animal models of mechanical unloading for use in the laboratory. This article aims to describe the operative technique of two animal models of mechanical unloading in detail, to enable the reproducible use of these animal models. METHODS: In 1964, Abbott et al first described the heterotopic abdominal heart transplantation technique as a means to study the biology of transplanted cardiac grafts. This involves an aorto-aortic anastomosis and a pulmonary artery to inferior vena cava anastomosis. In this model, the left ventricle is virtually completely volume unloaded, receiving only thebesian venous return, and substantially but not entirely pressure unloaded. In this report we describe two refined techniques for mechanical unloading of healthy or failing hearts based on experience with over 500 operations. RESULTS: We describe an operative technique, including cardioprotective strategies, that provides a model of mechanical unloading with no immunological rejection and allows measurements of parameters of myocardial structure and function for many months. We describe a refined technique that achieves a lesser degree of left ventricular volume unloading, involving transplantation of both heart and lungs via a single aorto-aortic anastomosis. CONCLUSIONS: This article is the first to describe these two techniques in sufficient detail to enable novices to attempt and understand these operations and the differences between them. The technique we describe provides an effective and reproducible model of complete and partial mechanical unloading.

A critical role for Telethonin in regulating t-tubule structure and function in the mammalian heart.

The transverse (t)-tubule system plays an essential role in healthy and diseased heart muscle, particularly in Ca(2+)-induced Ca(2+) release (CICR), and its structural disruption is an early event in heart failure. Both mechanical overload and unloading alter t-tubule structure, but the mechanisms mediating the normally tight regulation of the t-tubules in response to load variation are poorly understood. Telethonin (Tcap) is a stretch-sensitive Z-disc protein that binds to proteins in the t-tubule membrane. To assess its role in regulating t-tubule structure and function, we used Tcap knockout (KO) mice and investigated cardiomyocyte t-tubule and cell structure and CICR over time and following mechanical overload. In cardiomyocytes from 3-month-old KO (3mKO), there were isolated t-tubule defects and Ca(2+) transient dysynchrony without whole heart and cellular dysfunction. Ca(2+) spark frequency more than doubled in 3mKO. At 8 months of age (8mKO), cardiomyocytes showed progressive loss of t-tubules and remodelling of the cell surface, with prolonged and dysynchronous Ca(2+) transients. Ca(2+) spark frequency was elevated and the L-type Ca(2+) channel was depressed at 8 months only. After mechanical overload obtained by aortic banding constriction, the Ca(2+) transient was prolonged in both wild type and KO. Mechanical overload increased the Ca(2+) spark frequency in KO alone, where there was also significantly more t-tubule loss, with a greater deterioration in t-tubule regularity. In conjunction, Tcap KO showed severe loss of cell surface ultrastructure. These data suggest that Tcap is a critical, load-sensitive regulator of t-tubule structure and function.

Influence of ivabradine on reverse remodelling during mechanical unloading.

AIMS: Ivabradine (Iva) has shown beneficial structural and functional effects in clinical and experimental heart failure (HF), but its action in combination with mechanical unloading (MU), such as during treatment with left ventricular assist devices (LVAD), is unknown. The aim of this study was to investigate the effects of Iva during MU, in a rodent model of HF. METHODS AND RESULTS: We studied the chronic effects (4 weeks) of Iva (10 mg/kg/day) alone and in combination with MU [induced by heterotopic abdominal heart transplantation (HATx)] on whole-heart and cellular structure, function, and excitation-contraction (E-C) coupling in a rodent (rat) model of HF, 12 weeks post-left coronary artery (LCA) ligation. Effects of Iva were compared with those of ß-blockade using metoprolol [(Met), 250 mg/kg/day]. Iva, but not Met, reversed myocardial fibrosis, alone and in combination with MU. MU-induced restoration of deranged E-C coupling was enhanced by Iva to a greater extent than Met: both Iva and Met enhanced the recovery of the Ca(2+) transient amplitude and the sarcoplasmic reticulum (SR) Ca(2+) content, but Iva alone maintained MU-induced normalization of L-type Ca(2+) current and t-tubule abnormalities. Met prevented MU-induced reduction in the myocardial size (myocardial atrophy); Iva had no effect on this parameter. CONCLUSION: Iva shows beneficial structural and E-C coupling effects during MU: Iva reverses myocardial fibrosis and enhances the restoration of deranged E-C coupling, displaying more beneficial effects than that of Met. These results suggest that Iva may prove effective in enhancing functional recovery in heart failure patients receiving LVAD therapy.

Cardiomyocyte Ca2+ handling and structure is regulated by degree and duration of mechanical load variation.

Cardiac transverse (t)-tubules are altered during disease and may be regulated by stretch-sensitive molecules. The relationship between variations in the degree and duration of load and t-tubule structure remains unknown, as well as its implications for local Ca(2+)-induced Ca(2+) release (CICR). Rat hearts were studied after 4 or 8 weeks of moderate mechanical unloading [using heterotopic abdominal heart-lung transplantation (HAHLT)] and 6 or 10 weeks of pressure overloading using thoracic aortic constriction. CICR, cell and t-tubule structure were assessed using confocal-microscopy, patch-clamping and scanning ion conductance microscopy. Moderate unloading was compared with severe unloading [using heart-only transplantation (HAHT)]. Mechanical unloading reduced cardiomyocyte volume in a time-dependent manner. Ca(2+) release synchronicity was reduced at 8 weeks moderate unloading only. Ca(2+) sparks increased in frequency and duration at 8 weeks of moderate unloading, which also induced t-tubule disorganization. Overloading increased cardiomyocyte volume and disrupted t-tubule morphology at 10 weeks but not 6 weeks. Moderate mechanical unloading for 4 weeks had milder effects compared with severe mechanical unloading (37% reduction in cell volume at 4 weeks compared to 56% reduction after severe mechanical unloading) and did not cause depression and delay of the Ca(2+) transient, increased Ca(2+) spark frequency or impaired t-tubule and cell surface structure. These data suggest that variations in chronic mechanical load influence local CICR and t-tubule structure in a time- and degree-dependent manner, and that physiological states of increased and reduced cell size, without pathological changes are possible.

Reduced Na(+) and higher K(+) channel expression and function contribute to right ventricular origin of arrhythmias in Scn5a+/- mice.

Brugada syndrome (BrS) is associated with ventricular tachycardia originating particularly in the right ventricle (RV). We explore electrophysiological features predisposing to such arrhythmic tendency and their possible RV localization in a heterozygotic Scn5a+/- murine model. Na(v)1.5 mRNA and protein expression were lower in Scn5a+/- than wild-type (WT), with a further reduction in the RV compared with the left ventricle (LV). RVs showed higher expression levels of K(v)4.2, K(v)4.3 and KChIP2 in both Scn5a+/- and WT. Action potential upstroke velocity and maximum Na(+) current (I(Na)) density were correspondingly decreased in Scn5a+/-, with a further reduction in the RV. The voltage dependence of inactivation was shifted to more negative values in Scn5a+/-. These findings are predictive of a localized depolarization abnormality leading to slowed conduction. Persistent Na(+) current (I(pNa)) density was decreased in a similar pattern to I(Na). RV transient outward current (I(to)) density was greater than LV in both WT and Scn5a+/-, and had larger time constants of inactivation. These findings were also consistent with the observation that AP durations were smallest in the RV of Scn5a+/-, fulfilling predictions of an increased heterogeneity of repolarization as an additional possible electrophysiological mechanism for arrhythmogenesis in BrS.

Mechanical unloading reverses transverse tubule remodelling and normalizes local Ca(2+)-induced Ca(2+)release in a rodent model of heart failure.

AIMS: Ca(2+)-induced Ca(2+) release (CICR) is critical for contraction in cardiomyocytes. The transverse (t)-tubule system guarantees the proximity of the triggers for Ca(2+) release [L-type Ca(2+) channel, dihydropyridine receptors (DHPRs)] and the sarcoplasmic reticulum Ca(2+) release channels [ryanodine receptors (RyRs)]. Transverse tubule disruption occurs early in heart failure (HF). Clinical studies of left ventricular assist devices in HF indicate that mechanical unloading induces reverse remodelling. We hypothesize that unloading of failing hearts normalizes t-tubule structure and improves CICR. METHODS AND RESULTS: Heart failure was induced in Lewis rats by left coronary artery ligation for 12 weeks; sham-operated animals were used as controls. Failing hearts were mechanically unloaded for 4 weeks by heterotopic abdominal heart transplantation (HF-UN). HF reduced the t-tubule density measured by di-8-ANEPPS staining in isolated left ventricular myocytes, and this was reversed by unloading. The deterioration in the regularity of the t-tubule system in HF was also reversed in HF-UN. Scanning ion conductance microscopy showed the reappearance of normal surface striations in HF-UN. Electron microscopy revealed recovery of normal t-tubule microarchitecture in HF-UN. L-type Ca(2+) current density, measured using whole-cell patch clamping, was reduced in HF but unaffected by unloading. The variance of the time-to-peak of the Ca(2+) transient, an index of CICR dyssynchrony, was increased in HF and normalized by unloading. The increased Ca(2+) spark frequency observed in HF was reduced in HF-UN. These results could be explained by the recoupling of orphaned RyRs in HF, as indicated by immunofluorescence. CONCLUSIONS: Our data show that mechanical unloading of the failing heart reverses the pathological remodelling of the t-tubule system and improves CICR.

Prolonged mechanical unloading affects cardiomyocyte excitation-contraction coupling, transverse-tubule structure, and the cell surface.

Prolonged mechanical unloading (UN) of the heart is associated with detrimental changes to the structure and function of cardiomyocytes. The mechanisms underlying these changes are unknown. In this study, we report the influence of UN on excitation-contraction coupling, Ca(2+)-induced Ca(2+) release (CICR) in particular, and transverse (t)-tubule structure. UN was induced in male Lewis rat hearts by heterotopic abdominal heart transplantation. Left ventricular cardiomyocytes were isolated from the transplanted hearts after 4 wk and studied using whole-cell patch clamping, confocal microscopy, and scanning ion conductance microscopy (SICM). Recipient hearts were used as control (C). UN reduced the volume of cardiomyocytes by 56.5% compared with C (UN, n=90; C, n=59; P<0.001). The variance of time-to-peak of the Ca(2+) transients was significantly increased in unloaded cardiomyocytes (UN 227.4+/-24.9 ms(2), n=42 vs. C 157.8+/-18.0 ms(2), n=40; P<0.05). UN did not alter the action potential morphology or whole-cell L-type Ca(2+) current compared with C, but caused a significantly higher Ca(2+) spark frequency (UN 3.718+/-0.85 events/100 mum/s, n=47 vs. C 0.908+/-0.186 events/100 microm/s, n=45; P<0.05). Confocal studies showed irregular distribution of the t tubules (power of the normal t-tubule frequency: UN 8.13+/-1.12x10(5), n=57 vs. C 20.60+/- 3.174x10(5), n=56; P<0.001) and SICM studies revealed a profound disruption to the openings of the t tubules and the cell surface in unloaded cardiomyocytes. We show that UN leads to a functional uncoupling of the CICR process and identify disruption of the t-tubule-sarcoplasmic reticulum interaction as a possible mechanism.

Genetic background affects function and intracellular calcium regulation of mouse hearts.

AIMS: The genetic background is currently under close scrutiny when determining cardiovascular disease progression and response to therapy. However, this factor is rarely considered in physiological studies, where it could influence the normal behaviour and adaptive responses of the heart. We aim to test the hypothesis that genetic strain variability is associated with differences in excitation-contraction coupling mechanisms, in particular those involved in cytoplasmic Ca(2+) regulation, and that they are concomitant to differences in whole-heart function and cell morphology. METHODS AND RESULTS: We studied 8- to 10-week-old male C57BL/6, BALB/C, FVB, and SV129 mice. Echocardiography and radiotelemetry were used to assess cardiac function in vivo. FVB mice had increased left ventricular ejection fraction and fractional shortening with significantly faster heart rate (HR) and lack of diurnal variation of HR. Confocal microscopy, sarcomere length tracking, and epifluorescence were used to investigate cell volume, t-tubule density, contractility, and Ca(2+) handling in isolated ventricular myocytes. Sarcomere relaxation and time-to-peak of the Ca(2+) transient were prolonged in BALB/C myocytes, with more frequent Ca(2+) sparks and significantly higher sarcoplasmic reticulum (SR) Ca(2+) leak. There were no strain differences in the contribution of different Ca(2+) extrusion mechanisms. SV129 had reduced SR Ca(2+) leak with elevated SR Ca(2+) content and smaller cell volume and t-tubule density compared with myocytes from other strains. CONCLUSION: These results demonstrate that a different genetic background is associated with physiological differences in cardiac function in vivo and differences in morphology, contractility, and Ca(2+) handling at the cellular level.

Functional and morphological maturation of implanted neonatal cardiomyocytes as a comparator for cell therapy.

Knowledge of the rate of development of immature cardiomyocytes after implantation into a host heart is important for studies using cell therapy. To assess this functionally, we have implanted rat neonatal cardiomyocytes (NCMs) in normal and infarcted rat heart and re-isolated them for functional assessment. Maturation of implanted bone marrow stromal cells (BMSCs) was compared under similar conditions. NCMs from green fluorescent protein (GFP) transgenic rats were implanted into adult normal or infarcted rat hearts and re-isolated after 1, 2, or 4 weeks by standard enzymatic digestion. BMSCs labeled with DiI and iron oxide were implanted into rats with myocardial infarction and cells re-isolated 1, 2, 5, 6, and 16 weeks later. GFP-labeled myocytes approaching the adult morphology were detected 2 weeks after implantation of NCMs, but were significantly shorter than adult host myocytes and had reduced contractility. By 4 weeks after implantation, re-isolated GFP-labeled myocytes were close to the adult phenotype in contractile characteristics, although still significantly shorter. Infarction of the host did not alter the rate of maturation of implanted cells. After implantation of BMSCs, small numbers of functional DiI-labeled myocytes were re-isolated from 4/11 animals but were more mature than expected from the NCM studies. This adds evidence that BMSC-derived cardiomyocytes were not a result of transdifferentiation. The maturation rate of implanted NCMs represents a benchmark against which to evaluate the likely rate of formation of fully functional cardiomyocytes from implanted cells.

Genetic variation in SCN10A influences cardiac conduction.

To identify genetic factors influencing cardiac conduction, we carried out a genome-wide association study of electrocardiographic time intervals in 6,543 Indian Asians. We identified association of a nonsynonymous SNP, rs6795970, in SCN10A (P = 2.8 x 10(-15)) with PR interval, a marker of cardiac atrioventricular conduction. Replication testing among 6,243 Indian Asians and 5,370 Europeans confirmed that rs6795970 (G>A) is associated with prolonged cardiac conduction (longer P-wave duration, PR interval and QRS duration, P = 10(-5) to 10(-20)). SCN10A encodes Na(V)1.8, a sodium channel. We show that SCN10A is expressed in mouse and human heart tissue and that PR interval is shorter in Scn10a(-/-) mice than in wild-type mice. We also find that rs6795970 is associated with a higher risk of heart block (P < 0.05) and a lower risk of ventricular fibrillation (P = 0.01). Our findings provide new insight into the pathogenesis of cardiac conduction, heart block and ventricular fibrillation.

Adult progenitor cell transplantation influences contractile performance and calcium handling of recipient cardiomyocytes.

Adult progenitor cell transplantation has been proposed for the treatment of heart failure, but the mechanisms effecting functional improvements remain unknown. The aim of this study was to test the hypothesis that, in failing hearts treated with cell transplantation, the mechanical properties and excitation-contraction coupling of recipient cardiomyocytes are altered. Adult rats underwent coronary artery ligation, leading to myocardial infarction and chronic heart failure. After 3 wk, they received intramyocardial injections of either 10(7) green fluorescence protein (GFP)-positive bone marrow mononuclear cells or 5 x 10(6) GFP-positive skeletal myoblasts. Four weeks after injection, both cell types increased ejection fraction and reduced cardiomyocyte size. The contractility of isolated GFP-negative cardiomyocytes was monitored by sarcomere shortening assessment, Ca(2+) handling by indo-1 and fluo-4 fluorescence, and electrophysiology by patch-clamping techniques. Injection of either bone marrow cells or skeletal myoblasts normalized the impaired contractile performance and the prolonged time to peak of the Ca(2+) transient observed in failing cardiomyocytes. The smaller and slower L-type Ca(2+) current observed in heart failure normalized after skeletal myoblast, but not bone marrow cell, transplantation. Measurement of Ca(2+) sparks suggested a normalization of sarcoplasmic reticulum Ca(2+) leak after skeletal myoblast transplantation. The increased Ca(2+) wave frequency observed in failing myocytes was reduced by either bone marrow cells or skeletal myoblasts. In conclusion, the morphology, contractile performance, and excitation-contraction coupling of individual recipient cardiomyocytes are altered in failing hearts treated with adult progenitor cell transplantation.

Identification of cell-specific soluble mediators and cellular targets during cell therapy for the treatment of heart failure.

Cell therapy, the transplantation of progenitor cells into the myocardium, has been proposed as a possible treatment strategy for heart failure. Despite the lack of repopulation of the heart with progenitor cells, cell therapy induces a modest but well-documented functional improvement in patients. It is thought that paracrine mechanisms may account for the observed changes in heart function. However, there is little evidence that directly supports this hypothesis. We discuss the current views in the literature and present some preliminary data proposing that adult progenitor cells influence contractility and Ca2+ handling in neighboring failing cardiomyocytes by soluble mediators. This can be tested using a co-culture system. Our results suggest that soluble mediators from adult progenitor cells can enhance failing cardiomyocyte function, supporting the paracrine hypothesis. This co-culture strategy can be employed to identify cell-specific soluble mediators and their cellular targets during cell therapy for the treatment of heart disease.