Rat Left Ventricular Myocardium Research Articles

Naltrexone-Induced Cardiac Function Improvement is Associated With an Attenuated Inflammatory Response and Lipid Perioxidation in Volume Overloaded Rats.

In previous studies, upregulation of myocardial opioid receptors as well as the precursors of their endogenous ligands were detected in the failing heart due to chronic volume overload. Moreover, opioid receptor blockade by naltrexone improved left ventricular function. In parallel, inflammatory processes through cytokines have been confirmed to play an important role in the pathogenesis of different forms of heart failure. Thus, the present study examined the systemic and myocardial inflammatory response to chronic volume overload and its modulation by chronic naltrexone therapy. Chronic volume overload was induced in male Wistar rats by applying an infrarenal aortocaval fistula (ACF) for 28 days during which the selective opioid receptor antagonist naltrexone (n = 6) or vehicle (n = 6) were administered via a subcutaneously implanted Alzet minipump. The ultrastructural, morphometric and hemodynamic characterization of ACF animals were performed using an intraventricular conductance catheter in vivo and electron microscopy in vitro. Co-localization of mu-, delta- and kappa-opioid receptor subtypes (MOR, DOR, and KOR respectively) with the voltage gated L-type Ca2+ channel (Cav1.2), the ryanodine receptor (RyR), and mitochondria in cardiomyocytes as well as IL-6, IL-12, TNF-alpha, and Malondialdehyde (MDA) were determined using double immunofluorescence confocal microscopy, RT-PCR and ELISA, respectively. In rat left ventricular myocardium, three opioid receptor subtypes MOR, DOR, and KOR colocalized with Cav1.2, RyR and mitochondria suggesting a modulatory role of the excitation-contraction coupling. In rats with ACF-induced volume overload, signs of heart failure and myocardial ultrastructural damage, chronic naltrexone therapy improved cardiac function and reversed the systemic and myocardial inflammatory cytokine expression as well as lipid peroxidation. In conclusion, antagonism of the cardiodepressive effects of the myocardial opioid system does not only improve left ventricular function but also blunts the inflammatory response and lipid peroxidation.

Organic Cation Transporter-Mediated Accumulation of Quinolinium Salts in the LV Myocardium of Rodents

PurposeQuaternary ammonium salts have demonstrated marked accumulation in the left ventricular (LV) myocardium of rodents and swine. To investigate the mechanism underlying this uptake, the present study examined the interaction of [18F]fluoroethylquinolinium ([18F]FEtQ) with the family of organic cation transporters (OCTs).ProceduresThe cellular uptake of [18F]FEtQ into HEK293 cells, expressing human OCT1, -2, or -3 (HEK293-hOCT), and its inhibition by corticosterone was evaluated in vitro. The inhibitory effect of decynium 22 (D 22) in vivo was also studied, using PET/CT of HEK293-hOCT tumor-bearing mice. Furthermore, the distribution kinetics of [18F]FEtQ were determined in rats, with and without pre-administration of corticosterone, and following administration to a non-human primate (NHP).ResultsThe accumulation of [18F]FEtQ in HEK293-hOCT cells was 15–20-fold higher than in control cells and could be inhibited by corticosterone. in vivo, the uptake of [18F]FEtQ in the LV myocardium of corticosterone-treated rats was significantly reduced compared to that of untreated animals. Similarly, following administration of D 22 to HEK293-hOCT tumor-bearing mice, the peak tumor uptake of [18F]FEtQ was reduced by 40–45 % compared to baseline. Contrary to the distinct accumulation of [18F]FEtQ in the LV myocardium of rats, no cardiac uptake was observed following its administration to a NHP.ConclusionsThe quinolinium salt derivative [18F]FEtQ interacts with the family of OCTs, and this interaction could account, at least in part, for the increased uptake in the LV myocardium of rodents. Nonetheless, its low affinity for hOCT3 and the results of PET/CT imaging in a NHP indicate a limited clinical applicability as a radiopharmaceutical for cardiac and/or OCT imaging.

Влияние преждевременного рождения на васкуляризацию миокарда левого желудочка крыс в постнатальном периоде онтогенеза

Introduction. Preterm birth disrupts important processes of fetal growth and development and can serve as a trigger for maladaptive organ remodeling in the postnatal ontogenesis. The aim of the study was to evaluate the effect of preterm birth on the angiogenesis of the left ventricular myocardium of rats from days 1 to 21 of the postnatal ontogenesis. Materials and methods. We performed immunohistochemical (CD31) and morphometric (specific volume of blood capillaries, specific volume of cardiomyocytes, diameter of blood capillaries, trophic index and pericapillary diffusion zone) study of the left ventricular myocardium of full-term and prematurely born (12 and 24 hours preterm) Wistar rats (n=88) of both sexes on days 1, 7, 14, and 21 of the postnatal ontogenesis. Results. The birth of rats 12 and 24 hours preterm does not result in change in the specific volume of blood capillaries and the trophic index in the left ventricular myocardium on days 1, 7, 14, and 21 of the postnatal period. Preterm birth leads to a decrease in the specific volume of cardiomyocytes of the left ventricular myocardium of rats, compared to the parameters of animals in the control group, on days 7–21 of the postnatal period. Preterm birth results in a decrease in the diameter of the blood capillaries of the left ventricular myocardium in female rats on day 21 of the postnatal period. Preterm birth (24 hours preterm) is accompanied by an increase in the size of the zone of pericapillary diffusion in the left ventricular myocardium of rats on day 14 of the postnatal ontogenesis, in comparison with that of animals in the control group. Conclusion. The shorter the duration of the intrauterine period of development of rats, the greater the severity of the observed changes in the left ventricular myocardium are. It seems promising to study the functional state of endotheliocytes of myocardial blood vessels in preterm animals in the postnatal period. Keywords: preterm birth, prematurity, myocardium, angiogenesis, vascular indexes

Marker profile of myocardial remodeling in different etiopathogenetic forms of arterial hypertension in the experiment

The aim was to determine the morphodensitometric parameters of cardiomyocytes and features of the marker profile of remodeling in the left ventricular myocardium of rats with experimental hypertension of different etiopathogenetic forms.Materials and methods. The experiment was conducted on 20 male Wistar rats and 10 SHR which were divided into two groups of 10 animals each: the 1st –control; the 2nd – rats of the SHR line (EAH); the 3rd – 10 male Wistar rats with endocrine-salt arterial hypertension. Systolic and diastolic BP levels were measured in all the rats using the system of non-invasive arterial pressure measurement BP-2000. The objects of the study were myocardial sections in which the content of immunoreactive material to remodeling markers (cardiotrophin-1, titin, collagen type I, annexin V) was determined by the immunofluorescent method, titin / collagen ratio was calculated, the average linear size of cardiomyocyte nuclei, their density, RNA concentrations in the nucleus and cytoplasm were determined by morphodensitometric method.Results. Arterial hypertension, regardless of its etiopathogenetic form, is characterized by an increase in the number of cardiomyocyte nuclei with an increase in their size, decrease in the density and decrease in their RNA concentration against increase in the cytoplasm. In rats with EAH, the content of cardiotrophin-1 was higher than the control by 27 %, and in ESAH by 80 %. Titin levels were 12% higher in EAH and 46 % in ESAH. The collagen type I content of EAH was higher than the control by 49 %, while in the group with ESAH by 68 %. EAH rats had a higher annexin V content by 58 %, while ESAH – 64 %. The ratio of the content of titin and type I collagen in control rats was 1.01, in rats with EAH decreased to 0.76, while in rats ESAH it was 1.3.Conclusions. The formation of arterial hypertension, regardless of its etiopathogenetic form, leads to the development of pathological myocardial remodeling, as indicated by increased heart mass and specific gravity, changes in the cytoarchitectonics of cardiomyocytes, the development of polymorphism of their nuclei with increasing size against increasing of its number with nuclear dysfunction. The marker profile of myocardial remodeling in rats with essential hypertension is characterized by a moderate increase in cardiotrophin-1 and titin, with a significant increase in collagen type I and annexin V, which in combination with a low ratio of titin / collagen type indicates fibrosis with increased myocardial stiffness and the development of hypertrophic-fibrous type of myocardial remodeling with moderate apoptosis. In rats with endocrine-salt arterial hypertension, the marker profile of myocardial remodeling is characterized by a significant increase in all 4 immunohistochemical markers of remodeling, moderate fibrosis and an increase in the titin / collagen ratio, which is characteristic of hypertrophic-apoptotic type of pathological remodeling.

Малые белки теплового шока HSP10 и HSP27 в миокарде левого желудочка сердца крыс при сочетании артериальной гипертензии с инсулинозависимым сахарным диабетом

Малые белки теплового шока HSP10 и HSP27 в миокарде левого желудочка сердца крыс при сочетании артериальной гипертензии с инсулинозависимым сахарным диабетом

Regulation of VDAC1 contributes to the cardioprotective effects of penehyclidine hydrochloride during myocardial ischemia/reperfusion

Penehyclidine hydrochloride (PHC) preconditioning can alleviate myocardial ischemia/reperfusion (I/R) injury and inhibits the upregulation of voltage-dependent anion channel 1 (VDAC1) during I/R. To validate that VDAC1 is a bona fide target of PHC for the protection against myocardial I/R injury, VDAC1 expression construct was delivered by lentiviruses into rat left ventricular myocardium before PHC preconditioning and myocardial I/R. Overexpression of VDAC1 exacerbated cardiac dysfunction and myocardial injury following I/R, and abolished the cardioprotective effect of PHC during I/R injury. Moreover, VDAC1 overexpression with myocardial I/R further increased cytochrome c release from mitochondria to cytoplasm, elevated the levels of cleaved caspase-3 and Bax, and decreased the level of Bcl-2 as compared with I/R alone, and PHC-mediated inhibition of mitochondria-dependent apoptosis during myocardial I/R was abolished by VDAC1 overexpression. In addition, VDAC1 was overexpressed in H9c2 cardiomyocytes undergoing anoxia/reoxygenation (A/R) with or without PHC pretreatment. The in vitro results showed that overexpression of VDAC1 further reduced mitochondrial membrane potential, increased mitochondrial membrane permeability and enhanced mitochondria-dependent apoptosis in H9c2 cells after A/R, and VDAC1 overexpression abrogated the protective effect of PHC on the mitochondrial function and integrity during A/R. In conclusion, exogenous overexpression of VDAC1 during myocardial I/R inhibits the cardioprotective effects of PHC. These effects may be associated with the suppression of VDAC1 expression.

Computational Modelling of Cardiac Trabecula Mechanics

Cardiac trabeculae are thin strips of muscle within the ventricles that can be readily excised and used to investigate contractile mechanics of cardiac muscle. Recently, the Auckland Bioengineering Institute has developed a novel cardiac myometer that simultaneously measures force, length and shape of actively contracting isolated cardiac trabeculae. Here we have developed a muscle-specific computational model based on optical coherence tomography geometric surface data that replicates passive mechanics of trabecula. We hypothesised that the muscle's surface geometry data, in addition to force-length data, would improve the fit between the model simulated mechanics and the experimental data. The trabecula model was optimised using two different objective functions (muscle length or shape) driven by a pressure boundary condition. For both objective functions, there was a region of optimal parameters the optimiser tended towards but, due to the coupling between parameters, the ability to find the true optimal parameters was hindered. Due to the limitations of the data, we found that the addition of surface data did not improve parameter estimation and that using only the force-length data provided sufficient information to produce an optimal fit. References A. Anderson. The Cardiac Myometer: Measuring Matters of the Heart. PhD thesis, University of Auckland, 2016. K. F. Augenstein, Brett R. Cowan, Ian J. LeGrice, Poul M. F. Nielsen, and Alistair A. Young. Method and apparatus for soft tissue material parameter estimation using tissue tagged Magnetic Resonance Imaging. Journal of Biomechanical Engineering, 127(1):148–157, February 2005. C. Bradley, Andy Bowery, Randall Britten, Vincent Budelmann, Oscar Camara, Richard Christie, Andrew Cookson, Alejandro F. Frangi, Thiranja Babarenda Gamage, Thomas Heidlauf, Sebastian Krittian, David Ladd, Caton Little, Kumar Mithraratne, Martyn Nash, David Nickerson, Poul Nielsen, Oyvind Nordbo, Stig Omholt, Ali Pashaei, David Paterson, Vijayaraghavan Rajagopal, Adam Reeve, Oliver Rohrle, Soroush Safaei, Rafael Sebastian, Martin Steghofer, Tim Wu, Ting Yu, Heye Zhang, and Peter Hunter. OpenCMISS: A multi-physics and multi-scale computational infrastructure for the VPH/Physiome project. Progress in Biophysics and Molecular Biology, 107(1):32–47, October 2011. doi:http://dx.doi.org/10.1016/j.pbiomolbio.2011.06.015 M. L. Cheuk, A. J. Anderson, J. C. Han, N. Lippok, F. Vanholsbeeck, B. P. Ruddy, D. S. Loiselle, P. M. F. Nielsen, and A. J. Taberner. Four-Dimensional Imaging of Cardiac Trabeculae Contracting In Vitro Using Gated OCT. IEEE Transactions on Biomedical Engineering, 64(1):218–224, January 2017. doi:http://dx.doi.org/10.1109/TBME.2016.2553154 M. L. Cheuk, N. Lippok, A. W. Dixon, B. P. Ruddy, F. Vanholsbeeck, P. M. F. Nielsen, and A. J. Taberner. Optical coherence tomography imaging of cardiac trabeculae. In 2014 36th Annual International Conference of the IEEE Engineering in Medicine and Biology Society, pages 182–185, August 2014. doi:http://dx.doi.org/10.1109/EMBC.2014.6943559 J. M Guccione, Andrew D McCulloch, and LK Waldman. Passive material properties of intact ventricular myocardium determined from a cylindrical model. J Biomech Eng, 113(1):42–55, 1991. J. C. Han, Andrew J. Taberner, Robert S. Kirton, Poul M. Nielsen, Nicholas P. Smith, and Denis S. Loiselle. A unique micromechanocalorimeter for simultaneous measurement of heat rate and force production of cardiac trabeculae carneae. Journal of Applied Physiology, 107(3):946–951, September 2009. doi:http://dx.doi.org/10.1152/japplphysiol.00549.2009 M. P. Nash and P. J. Hunter. Regional mechanics of the beating heart. In Cardiac Perfusion and Pumping Engineering, volume Volume 1 of Clinically-Oriented Biomedical Engineering, pages 83–127. WORLD SCIENTIFIC, July 2007. doi:http://dx.doi.org/10.1142/9789812775597_0004 J. H. Omens, D. A. MacKenna, and A. D. McCulloch. Measurement of strain and analysis of stress in resting rat left ventricular myocardium. Journal of Biomechanics, 26(6):665–676, June 1993. doi:http://dx.doi.org/10.1016/0021-9290(93)90030-I V. Y. Wang, H. I. Lam, Daniel B. Ennis, Brett R. Cowan, Alistair A. Young, and Martyn P. Nash. Modelling passive diastolic mechanics with quantitative MRI of cardiac structure and function. Medical Image Analysis, 13(5):773–784, October 2009. doi:http://dx.doi.org/10.1016/j.media.2009.07.006

Long Noncoding RNA Kcna2 Antisense RNA Contributes to Ventricular Arrhythmias via Silencing Kcna2 in Rats With Congestive Heart Failure.

BackgroundCongestive heart failure (CHF) is a common cardiovascular disease that is often accompanied by ventricular arrhythmias. The decrease of the slow component of the delayed rectifier potassium current (IK s) in CHF leads to action potential (AP) prolongation, and the IK s is an important contributor to the development of ventricular arrhythmias. However, the molecular mechanisms underlying ventricular arrhythmias are still unknown.Methods and ResultsKcna2 and Kcna2 antisense RNA (Kcna2 AS) transcript expression was measured in rat cardiac tissues using quantitative real‐time reverse transcription–polymerase chain reaction and Western blotting. There was a 43% reduction in Kcna2 mRNA in the left ventricular myocardium of rats with CHF. Kcna2 knockdown in the heart decreased the IKs and prolonged APs in cardiomyocytes, consistent with the changes observed in heart failure. Conversely, Kcna2 overexpression in the heart significantly attenuated the CHF‐induced decreases in the IKs, AP prolongation, and ventricular arrhythmias. Kcna2 AS was upregulated ≈1.7‐fold in rats with CHF and with phenylephrine‐induced cardiomyocyte hypertrophy. Kcna2 AS inhibition increased the CHF‐induced downregulation of Kcna2. Consequently, Kcna2 AS mitigated the decrease in the IKs and the prolongation of APs in vivo and in vitro and reduced ventricular arrhythmias, as detected using electrocardiography.ConclusionsVentricular Kcna2 AS expression increases in rats with CHF and contributes to reduced IK s, prolonged APs, and the occurrence of ventricular arrhythmias by silencing Kcna2. Thus, Kcna2 AS may be a new target for the prevention and treatment of ventricular arrhythmias in patients with CHF.

Anti‑aging effects of melatonin on the myocardial mitochondria of rats and associated mechanisms.

The present study investigated the anti-aging effects of melatonin on the myocardial mitochondria of D-galactose-aged rats and associated mechanisms. A total of 30 male Sprague‑Dawley (SD) rats were randomly divided into three equal groups: An accelerated aging group that received 125mg/kg/day D‑galactose; a melatonin‑treated group of D‑galactose‑aged rats that received 10mg/kg/day melatonin; and a control group receiving normal saline. ATP, ADP and AMP levels in the left ventricular myocardium of rats were determined by high performance liquid chromatography and the total adenylic acid number (TAN) was subsequently calculated. Bax, Bcl‑2, and cytochrome c (cyt‑c) protein expression levels in myocardial mitochondria and cytoplasm were quantified by western blot analysis. In the melatonin‑treated group, ATP levels were significantly higher when compared with the untreated control group and the accelerated‑ageing group (0.068 vs. 0.052 and 0.058; P=0.002 and P=0.045, respectively), and TAN was significantly increased in the melatonin‑treated group when compared with controls (P=0.011). In addition, cyt‑c levels in the cytoplasm, but not in the mitochondria, were significantly higher in the accelerated‑aging group compared with the control and melatonin‑treated groups (P=0.001 and P=0.002, respectively). Bcl‑2 and Bax ratios were significantly higher in the control and melatonin‑treated groups when compared with the accelerated‑aging group (P=0.004 and P=0.032, respectively). These results suggest that melatonin exhibits a protective effect on mitochondrial function in a rat model of accelerated aging.

Exercise preconditioning-induced late phase of cardioprotection against exhaustive exercise: possible role of protein kinase C delta.

The objective of this study was to investigate the late cardiac effect of exercise preconditioning (EP) on the exhaustive exercise-induced myocardial injury in rats and the role of protein kinase C (PKC) in EP. Rats were subjected to a run on the treadmill for four periods of 10 min each at 30 m/min with intervening periods of rest of 10 min as an EP protocol. The exhaustive exercise was performed 24 h after EP. PKC inhibitor chelerythrine (CHE) was injected before EP. The results showed that EP increased the running ability of rats, and alleviated the exhaustive exercise-induced injury in cardiomyocytes, but pretreatment with PKC inhibitor CHE did not abolish the late phase cardioprotection of EP. A significant increase of PKCδ, both at the protein level and the mRNA level in the left ventricular myocardium of rats, accompanied by its activated form (phosphorylated on Thr507, p-PKCδThr507) translocated to intercalated disks and was found in the late phase of EP. This circumstance was not attenuated by CHE. These results suggested that a high level of PKCδ might be involved in cardioprotection against myocardial damage, but if activated PKCδ at reperfusion took on a key role in cardioprotection was still an outstanding question.

Transmural variation and anisotropy of microvascular flow conductivity in the rat left ventricular myocardium (675.4)

Three‐dimensional (3D) ex vivo data on microvascular anatomy in the myocardium is used to determine the transmural gradient in fluid transport properties via mathematical averaging methods. The 3D capillary network is modeled as a porous medium, whose conductivity is quantified in terms of a permeability tensor. This metric is directly computed by volume‐averaging blood flow solutions in model networks with geometrical and topological properties derived from an anatomically‐detailed data set recently extracted from the rat left ventricular myocardium using confocal microscopy, high‐precision sectioning and image processing techniques.Results showed that the permeability was strongly anisotropic: the network was approximately ten times more permeable in the principal direction of capillary alignment (the ‘longitudinal’ direction) than perpendicular to this direction. Structural properties of the network varied smoothly from subepicardium to subendocardium, with a 30% transmural increase in capillary diameter translating to a 130% rise in permeability in the longitudinal direction. This supports the hypothesis that perfusion is preferentially facilitated during ventricular diastole in the subendocardial microvasculature to compensate for severely‐reduced systolic perfusion in the subendocardium.Grant Funding Source: Supported by NIH GM094503‐1, EPSRC EP/F043929/1 and EP/G007527/2, and Award KUK‐C1‐013‐0 from KAUST.

Connexin 43 impacts on mitochondrial potassium uptake

In cardiomyocytes, connexin 43 (Cx43) forms gap junctions and unopposed hemichannels at the plasma membrane, but the protein is also present at the inner membrane of subsarcolemmal mitochondria (SSM). Both inhibition and genetic ablation of Cx43 reduce ADP-stimulated complex 1 respiration. Since mitochondrial potassium influx impacts on oxygen consumption, we investigated whether or not inhibition or ablation of mitochondrial Cx43 alters mitochondrial potassium uptake. SSM were isolated from rat left ventricular myocardium and loaded with the potassium-sensitive dye PBFI (potassium-binding benzofuran isophthalate). Intramitochondrial potassium was replaced by tetraethylammonium. Mitochondria were incubated under control conditions or treated with 250 μM Gap19, a peptide that specifically inhibits Cx43-based hemichannels at plasma membranes. Subsequently, 140 mM KCl was added and the slope of the increase in PBFI fluorescence over time was calculated. The slope of the PBFI fluorescence of the control mitochondria was set to 100%. In the presence of Gap19, the mitochondrial potassium influx was reduced from 100 ± 11.6% in control mitochondria to 65.5 ± 10.7% (n = 6, p < 0.05). In addition to the pharmacological inhibition of Cx43, potassium influx was studied in mitochondria isolated from conditional Cx43 knockout mice. Here, the ablation of Cx43 was achieved by the injection of 4-hydroxytamoxifen (4-OHT; Cx43Cre-ER(T)/fl + 4-OHT). The mitochondria of the Cx43Cre-ER(T)/fl + 4-OHT mice contained 3 ± 1% Cx43 (n = 6) of that in control mitochondria (100 ± 11%, n = 8, p < 0.05). The ablation of Cx43 (n = 5) reduced the velocity of the potassium influx from 100 ± 11.2% in control mitochondria (n = 9) to 66.6 ± 5.5% (p < 0.05). Taken together, our data indicate that both pharmacological inhibition and genetic ablation of Cx43 reduce mitochondrial potassium influx.

Anisotropic Collagen Fibrillogenesis Within Microfabricated Scaffolds: Implications For Biomimetic Tissue Engineering

Anisotropic collagen fibrillogenesis is demonstrated within the pores of an accordion-like honeycomb poly(glycerol sebacate) tissue engineering scaffold. Confocal reflectance microscopy and image analysis demonstrate increased fibril distribution order, fibril density, and alignment in accordion-like honeycomb pores compared with collagen gelled unconstrained. Finite element modeling predicts how collagen gel and scaffold mechanics couple in matching native heart muscle stiffness and anisotropy.

Quantitative N-linked Glycoproteomics of Myocardial Ischemia and Reperfusion Injury Reveals Early Remodeling in the Extracellular Environment

Extracellular and cell surface proteins are generally modified with N-linked glycans and glycopeptide enrichment is an attractive tool to analyze these proteins. The role of N-linked glycoproteins in cardiovascular disease, particularly ischemia and reperfusion injury, is poorly understood. Observation of glycopeptides by mass spectrometry is challenging due to the presence of abundant, nonglycosylated analytes, and robust methods for purification are essential. We employed digestion with multiple proteases to increase glycoproteome coverage coupled with parallel glycopeptide enrichments using hydrazide capture, titanium dioxide, and hydrophilic interaction liquid chromatography with and without an ion-pairing agent. Glycosylated peptides were treated with PNGase F and analyzed by liquid chromatography-MS/MS. This allowed the identification of 1556 nonredundant N-linked glycosylation sites, representing 972 protein groups from ex vivo rat left ventricular myocardium. False positive "glycosylations" were observed on 44 peptides containing a deamidated Asn-Asp in the N-linked sequon by analysis of samples without PNGase F treatment. We used quantitation via isobaric tags for relative and absolute quantitation (iTRAQ) and validation with dimethyl labeling to analyze changes in glycoproteins from tissue following prolonged ischemia and reperfusion (40 mins ischemia and 20 mins reperfusion) indicative of myocardial infarction. The iTRAQ approach revealed 80 of 437 glycopeptides with altered abundance, while dimethyl labeling confirmed 46 of these and revealed an additional 62 significant changes. These were mainly from predicted extracellular matrix and basement membrane proteins that are implicated in cardiac remodeling. Analysis of N-glycans released from myocardial proteins suggest that the observed changes were not due to significant alterations in N-glycan structures. Altered proteins included the collagen-laminin-integrin complexes and collagen assembly enzymes, cadherins, mast cell proteases, proliferation-associated secreted protein acidic and rich in cysteine, and microfibril-associated proteins. The data suggest that cardiac remodeling is initiated earlier during reperfusion than previously hypothesized.

The MPTP Status During Early Reoxygenation is Critical for Cardioprotection

Previous studies have revealed that the mitochondrial permeability transition pore (MPTP) plays a critical role in necrotic and apoptotic cell death. Given the opposed roles of the MPTP in cardioprotection (transient versus sustained opening) the primary aim of this study was to determine how two structurally different MPTP inhibitors (cyclosporine A and bongkrekic acid) administered for varying time regimes influenced ischemia/reperfusion (I/R)-induced injury in myocardial slices from rat left ventricle. A second objective was to explore how pharmacologic MPTP opening (using atractyloside) at different time points during I/R modulated myocardial injury. Myocardial slices from rat left ventricle were subjected to 90 min ischemia/120min reoxygenation at 37°C. MPTP inhibitors and openers were added at various time points during the experimental regime. Tissue injury was assessed by creatine kinase (CK) released and determination of cell necrosis and apoptosis. Myocardial caspase 3 activity was also determined. The results show that the status of MPTP can dramatically influence ischemic/reoxygenation induced injury and protection of the rat left ventricular myocardium. Importantly, the status of the MPTP during first 10 min of reoxygenation is of critical importance with both opening and closing of the pore being as protective as ischemic preconditioning. The present study has shown that both formation and inhibition of the MPTP can be exploited for therapeutic purposes and that there is a defined therapeutic window, with the first few minutes of reoxygenation being a crucial period to achieve cardioprotection.

The significance of pore microarchitecture in a multi-layered elastomeric scaffold for contractile cardiac muscle constructs

Multi-layered poly(glycerol-sebacate) (PGS) scaffolds with controlled pore microarchitectures were fabricated, combined with heart cells, and cultured with perfusion to engineer contractile cardiac muscle constructs. First, one-layered (1L) scaffolds with accordion-like honeycomb shaped pores and elastomeric mechanical properties were fabricated by laser microablation of PGS membranes. Second, two-layered (2L) scaffolds with fully interconnected three dimensional pore networks were fabricated by oxygen plasma treatment of 1L scaffolds followed by stacking with off-set laminae to produce a tightly bonded composite. Third, heart cells were cultured on scaffolds with or without interstitial perfusion for 7 days. The laser-microablated PGS scaffolds exhibited ultimate tensile strength and strain-to-failure higher than normal adult rat left ventricular myocardium, and effective stiffnesses ranging from 220 to 290 kPa. The 7-day constructs contracted in response to electrical field stimulation. Excitation thresholds were unaffected by scaffold scale up from 1L to 2L. The 2L constructs exhibited reduced apoptosis, increased expression of connexin-43 (Cx-43) and matrix metalloprotease-2 (MMP-2) genes, and increased Cx-43 and cardiac troponin-I proteins when cultured with perfusion as compared to static controls. Together, these findings suggest that multi-layered, microfabricated PGS scaffolds may be applicable to myocardial repair applications requiring mechanical support, cell delivery and active implant contractility.

In Vivo Assessment of the Electrophysiological Integration and Arrhythmogenic Risk of Myocardial Cell Transplantation Strategies

Cell replacement strategies are promising interventions aiming to improve myocardial performance. Yet, the electrophysiological impact of these approaches has not been elucidated. We assessed the electrophysiological consequences of grafting of two candidate cell types, that is, skeletal myoblasts and human embryonic stem cell-derived cardiomyocytes (hESC-CMs). The fluorescently labeled (DiO) candidate cells were grafted into the rat's left ventricular myocardium. Two weeks later, optical mapping was performed using the Langendorff-perfused rat heart preparation. Images were obtained with appropriate filters to delineate the heart's anatomy, to identify the DiO-labeled cells, and to associate this information with the voltage-mapping data (using the voltage-sensitive dye PGH-I). Histological examination revealed the lack of gap junctions between grafted skeletal myotubes and host cardiomyocytes. In contrast, positive Cx43 immunostaining was observed between donor and host cardiomyocytes in the hESC-CMs-transplanted hearts. Optical mapping demonstrated either normal conduction (four of six) or minimal conduction slowing (two of six) at the hESC-CMs engraftment sites. In contrast, marked slowing of conduction or conduction block was seen (seven of eight) at the myoblast transplantation sites. Ventricular arrhythmias could not be induced in the hESC-CM hearts following programmed electrical stimulation but were inducible in 50% of the myoblast-engrafted hearts. In summary, a unique method for assessment of the electrophysiological impact of myocardial cell therapy is presented. Our results demonstrate the ability of hESC-CMs to functionally integrate with host tissue. In contrast, transplantation of cells that do not form gap junctions (skeletal myoblats) led to localized conduction disturbances and to the generation of a proarrhythmogenic substrate.

The structure of the rat left ventricular myocardium in models of microgravity and artificial gravitation

The structure of the rat left ventricular myocardium in models of microgravity and artificial gravitation

Postnatal development of phospholipids and their fatty acid profile in rat heart

The aim of this study was to determine the concentration of phospholipids (PL), plasmalogen components of choline (PC) and ethanolamine (PE) phosphoglycerides (PLPC, PLPE) and fatty acid profile of PL and triacylglycerols (TAG) in developing rat left ventricular myocardium between postnatal day (d) 2 and 100. The steepest increase of total PL (TPL) concentration occurs between d2 and d5, followed by a further slower increase between d20 and d40. Similar developmental changes were observed in PC and PE. The PLPE concentration rises by d10, whereas PLPC does not change during the whole period investigated, except for the transient decline on d5. The concentration of diphosphatidylglycerol (DPG) increases by d60; the steepest rise occurs between d20 and d40. Phosphatidylinositol (PI) concentration rises only by d5. The concentration of phosphatidylserine (PS) decreases between d5 and d10 and then it does not change. Sphingomyelin (SM) concentration is maintained till d10, it declines on d20 and does not change thereafter. The proportion of saturated fatty acids (SFA) increases by d5 in PC, PE, PS and TAG, and by d10 in DPG and PI. After d20 the SFA proportion gradually decline in all lipids. Monounsaturated FA (MUFA) proportion decreases in PC, PE, PI and PS from d2 till d10, and in the weaning period it tends to rise again. In contrast, in DPG and TAG the proportion of MUFA declines during the whole postnatal period. N-6 polyunsaturated FA (PUFA) decrease in all PL by d20 and rise again thereafter; in TAG they decline between d2 and d10 and return to the initial level by d100. N-3 PUFA increase in all PL during the suckling period and decline after weaning; in TAG they increase only by d5 and then they decline. This remodeling of myocardial PL and TAG composition during postnatal development may affect membrane properties and contribute to developmental changes in the function of membrane proteins and cell signaling.

Myocardial IL-6 regulation by neurohormones—an in vitro superfusion study

Background: Interleukin-6 (IL-6) is expressed in the myocardium and has been implicated in cell proliferation, negative inotropic effects and myocardial hypertrophy. To determine whether myocardial IL-6 is modified by neurohumoral and immunoregulatory stimuli, we studied the effects of lipopolysaccharide (LPS), corticosterone (CS), isoproterenol and angiotensin II on myocardial IL-6 secretion in superfused myocardium. Methods: Slices of rat left ventricular myocardium were superfused in 80 μl chambers for up to 5 h. LPS (1, 50, and 100 μg/ml), CS (10 −7, 10 −6, and 10 −5 M, DSMO as vehicle), isoproterenol (10 −6, 10 −7, and 10 −8 M) and angiotensin II (10 −5, 10 −7, and 10 −9 M) were added to the culture medium at hour 2. IL-6 was measured in the perfusate by ELISA. Results: Physiological corticosterone concentrations (10 −7 M) resulted in an increase in IL-6 concentration (142%) while high doses of steroid decreased IL-6 significantly (CS 10 −6 M: 88±14%, p<.05 ; CS 10 −5: 91±9%, p<.05 ) after 5 h. Left ventricular IL-6 secretion was significantly stimulated by LPS 50 μg/ml (3262±1684% vs. CTRL: 116±34%, p<.01). Isoproterenol treatment increased in IL-6 secretion compared to controls with and without CS, while angiotensin II reduced IL-6 concentration only in combination with CS. Conclusion: Myocardial IL-6 secretion is modulated by physiological concentrations of corticosterone or angiotensin II and can be induced by LPS or isoproterenol, indicating a tight regulation of this cytokine. Suppression of cytokine expression within the heart might be a potential therapeutic goal in the treatment of various cardiovascular diseases.