Porcine Heart Muscle Research Articles

Abstract 29: Biodegradable Elastomeric Polyurethane Scaffolds Mechanically Matching With Native Heart Muscle

Introduction: Biodegradable cardiac patches need to be mechanically matching with native heart muscle, in order to provide appropriate mechanical support to rapidly restore heart functions and promote tissue remodeling for myocardial infarction (MI) management. Here, we utilized chemical molecular design to develop biodegradable elastomers with low initial modulus and then process them into porous scaffolds mechanically matching with native heart muscle. Methods and Results: We synthesized various amorphous copolymers including poly (δ-valerolactone-co-ε-caprolactone) (PVCL) and poly (ether ester) triblock copolymers with various molecular weights and poly(ethylene glycol) (PEG) molecular weights (PVCL-PEG-PVCL). The polyurethanes were then synthesized from PVCL or PVCL-PEG-PVCL as a soft segment, hexamethylene diisocyanate (HDI) as a hard segment and putrescine as a chain extender. The polyurethane products were presented as PU-PEGx-VCLy, where x and y refer to molecular weights of PEG and PVCL, respectively. Five polymers including PU-VCL 2k , PU-VCL 6K , PU-PEG 1K -VCL 1K , PU-PEG 1K -VCL 6K and PU-PEG 2K -VCL 6K were obtained. All polymers gradually degraded in phosphate buffer solution and enzyme solution. The 3T3 fibroblasts can grow and proliferate on all polymer film surface within 5 day culture, indicating the polymers have good cellular compatibility. PU-VCL 6K , PU-PEG 1K -VCL 6K and PU-PEG 2K -VCL 6K were further processed into porous scaffolds using thermally induced phase separation (TIPS). The PU-PEG 2K -VCL 6K scaffold at wet state had 0.19 ± 0.08 MPa initial modulus, which has no significant difference from initial modulus (0.19 ± 0.04 MPa) of the native porcine heart muscle. But the tensile strength of this scaffold is lower than that of heart muscle, which requies to be improved in the future. Conclusions: A new family of biodegradable elastic polyurethanes was synthesized and processed into porous scaffolds. The scaffolds showed promising mechanical match with heart muscle. These biodegradable polyurethane scaffolds would find opportunities to be used as a cardiac patch for heart infarction treatment.

Minor participation of cAMP on the protein kinase phosphorylation of mitochondrial and cytosolic fractions from Ascaris suum: a comparative study with porcine heart muscle

In contrast to porcine heart muscle in which cAMP effectively activated the phosphorylation of cytosolic proteins, cAMP exerted a minor effect on the phosphorylation of proteins from the soluble fraction of Ascaris suum muscle. Similarly, cAMP did not enhance the kinase activity in the mitochondrial membranes from porcine heart and A. suum, although major differences in protein phosphorylation were observed between both fractions. However, cAMP-dependent protein kinases (PKA) were evidenced in the parasitic soluble mitochondrial fraction, since the phosphorylation of histone IIA and kemptide was augmented in this fraction, in the presence of cAMP. An increase in the phosphorylation of exogenously added A. suum phosphofructokinase was also obtained when cAMP was added to the parasite soluble mitochondrial fraction. The phosphorylation of phosphofructokinase by this fraction was inhibited when kemptide and cAMP were included in the reaction mixture, suggesting substrate competition for the same PKA. Although PKI (6–22), a reported inhibitor of the catalytic subunit of mammalian cAMP-dependent PKAs, did not affect the endogenous phosphorylation of proteins in the various A. suum fractions, an inhibition on the phosphorylation of exogenously added kemptide and phosphofructokinase was observed when PKI (6–22) was incubated with the parasite mitochondrial soluble fraction.

Mipafox differential inhibition assay for heart muscle cholinesterases: Substrate specificity and inhibition of three isoenzymes by physostigmine and quinidine

1. 1. A differential inhibition assay was developed for the quantitative determination of cholinesterase isoenzymes acetylcholinesterase (ACNE; EC 3.1.1.7), cholinesterase (BChE; EC 3.1.1.8), and atypical cholinesterase in small samples of left ventricular porcine heart muscle. 2. 2. The assay is based on kinetic analysis of irreversible cholinesterase inhibition by the organophosphorus compound N,N′-di-isopropylphosphorodiamidic fluoride (mipafox). With acetylthiocholine (ASCh) as substrate (1.25 mM), hydrolytic activities (A) of cholinesterase isoenzymes were determined after preincubation (60 min, 25°C) of heart muscle samples with either saline (total activity, A T), 7μM mipafox (A M1), or 0.8 mM mipafox (A M2): ( BChE)= A T - A M1 , ( AChE)= A M1 - A M2 , (Atypical ChE)=A M2. 3. 3. The mipafox differential inhibition assay was used to determine the substrate hydrolysis patterns of myocardial cholinesterases with ASCh, acetyl-β-methylthiocholine (AβMSCh), propionylthiocholine (PSCh), and butyrylthiocholine (BSCh). The substrate specificities of myocardial AChE and BChE resemble those of erythrocyte AChE and serum BChE, respectively. Michaelis constants K M with ASCh were determined to be 0.15 mM for AChE and 1.4 mM for BChE. 4. 4. Atypical cholinesterase, in respect to both substrate specificity and inhibition kinetics, differs from cholinesterase activities of vertebrate tissue and, up to now, could be identified exclusively in heart muscle. The enzyme's Michaelis constant with ASCh was determined to be 4.0 mM. 5. 5. The reversible inhibitory effects of physostigmine (eserine) and quinidine on heart muscle cholinesterases were investigated using the differential inhibition assay. With all three isoenzymes, the inhibition kinetics of both substances were strictly competitive. The physostigmine inhibition of AChE was most pronounced ( K i =0.22 μM). Quinidine most potently inhibited myocardial BChE ( K i =35 μM).

Inhomogeneities in wall stress measured by microergometry in the heart muscle in situ

Microergometry is a method which we have developed as a tool to measure local mesh-tension within the myocardial weave at any measuring site of both ventricles and the septum on the beating heart in situ. In a mapping procedure on pig and dog hearts, both in control conditions and in the hypertrophied state after aortic banding, local mesh-tension was measured in several areas and in up to eight depths proceeding from the epicardium to the endocardium: Probe-to-fibre coupling is definitely more stable in the canine myocardium than in the porcine heart muscle, probably due to a more effective connective tissue fettering of the canine myocardial weave. The observed longitudinal gradient, with the highest tension in the base, of control dog hearts was levelled out in the hypertrophied hearts. Furthermore, in control dog hearts mesh-tension in the subepi- and subendocardial layers was higher than in the midlayers. This pronounced midlayerhypotension was smoothed in the hypertrophied hearts. Further studies will be dedicated to the question of whether the impact of ventricular size and shape on intersegmental stress transmission is determined by the Frank-Starling mechanism alone or whether protracted remodelling processes on the level of the local fibre weave cause slow coupling alterations.

Rapid preparation of subsarcolemmal and interfibrillar mitochondrial subpopulations from cardiac muscle

1. 1. Subsarcolemmal and interfibrillar mitochondria were prepared with complete recovery from rabbit and porcine heart muscle by upward-flotation during 60 sec of Percollö density gradient centrifugation. 2. 2. Mitochondrial subpopulations were identified and characterized according to buoyant density, electron-microscopy, marker enzyme activities and respiratory performance. 3. 3. ADP-induced state 3-respiration related to latent citrate synthase activity as a marker for structurally intact mitochondria was not significantly different in both mitochondrial subtypes.

Proteinase-catalyzed activation of porcine heart muscle pyruvate dehydrogenase and identification of its cleavage site

Porcine heart muscle pyruvate dehydrogenase (PDH, EC 1.2.4.1) with subunit composition α2ß2 catalyzes the initial decarboxylation step of an oxidative decarboxylation sequence of pyruvate. Highly purified PDH, was further activated several-fold by limited digestion with trypsin, Staphylococcus aureus V8 proteinase (V8) or papain. The activation with these proteinase required about 10 min to attain a maximal level, lasted 12-2 h and thereafter decreased gradually. Addition of an inhibitor of each proteinase resulted in an immediate cessation of any further changes in the enzymatic activity. The optimal pH of the proteinase-activated PDH was not affected. Proteinases increased the maximum velocity and the apparent Km values for pyruvate, but the Hill coefficients for pyruvate were unchanged. Proteinase-activated PDH was capable of associating two other component enzymes to produce large unit resembling the native complex. The Coomassie brilliant blue stained gels after SDS-PAGE showed that the PDHα subunit (41 kDa) was cleaved by trypsin or V8 into two major fragments (31 and 10 kDa), whereas PDHß was unaffected. By amino-terminal sequence analyses of these fragments the trypsin cleavage sites were identified as Arg-273 and Arg-282 and the V8 cleavage sites were Glu-277 and Glu-280.

Calcium metabolism and depressed contractility in isolated human and porcine heart muscle

Contractility is often depressed in isolated heart muscle. To analyze this phenomenon, we measured the derivative of left ventricular pressure (dP/dt) in intact and in isolated, blood perfused pig hearts, and peak force (F) or stress (F/mm2) in ventricular trabeculae of man and pig. When the heart was in the steady state at a priming frequency of 2 Hz an extrasystolic interval of 0.3 s was interposed, followed by four postextrasystolic intervals of 0.8 s. In the case of isolated trabeculae the priming frequency was 0.2 Hz, the extra interval 0.4 s, and the post-extrasystolic intervals were 5 s. The exponential decay of potentiation is characterized by the constant D: a low value of D indicates a rapid decay of potentiation. DP/dt was about 1000 mm Hg/s in the intact hearts, but within 1 h after isolation dP/dt decreased to about 700 mm Hg/s, and this was associated with a decrease in D from 0.63 to 0.40. Developed stress in the isolated trabeculae was about 2 mN/mm2 and D was about 0.20 under standard, in vitro conditions (a.o. 1.5 mM Ca2+. 0.2 Hz stimulus frequency). This stress is only 10% of the calculated stress in the intact heart. An increase of priming frequency, or of [Ca2+], or addition of 30 nM isoproterenol to the perfusate caused a marked increase in F and D. Properties of human and porcine trabeculae were quantitatively similar. The strong correlation between dP/dt, or F, and D suggests a causal relationship. This is consistent with the current model of e-c coupling in heart muscle, in which the activity of the Ca2+ pump of the sarcoplasmic reticulum determines the decay of potentiation and the amount of releasable Ca2+ in the reticulum determines force of contraction. Since isoproterenol stimulates the Ca2+ pump in the reticulum, the increase in D and F induced by this drug is consistent with the model. We conclude, that the decreased dP/dt, F, and D in isolated preparations was due to impaired sarcoplasmic reticulum function. The role of this phenomenon in the stunned heart syndrome, species differences and possible causes are discussed.

Stability and conformation of porcine phosphofrucktokinase M and L

1. 1. The inactivation of porcine liver enzyme in the presence of urea proceeded more rapidly than that of porcine heart muscle enzyme. 2. 2. The inactivation of both enzymes by urea was protected by allosteric activators, but inhibitors had no effect. 3. 3. The circular dichroism spectrum of liver enzyme in the near ultraviolet region was markedly affected by urea, whreas that of hear muscle enzyme was not, except for the band at 255 nm.

In Vitro Investigations on a New Positive Inotropic and Vasodilating Agent (BM 14.478) That Increases Myocardial Cyclic AMP Content and Myofibrillar Calcium Sensitivity

BM 14.478 (7,7-dimethyl-2-(4-pyridyl)-6,7-dihydro-3H,5H pyrrolo[2,3-f]benz-imidazol-6-one) was investigated in several in vitro experiments to elucidate its positive inotropic and vasodilating efficacy and its mode of action. A direct positive inotropic action was achieved in papillary muscles (10(-6) to 5 X 10(-4) M) and electrically driven atria (10(-8) to 5 X 10(-4) M) from guinea pig hearts. The effect was not affected by propranolol, cimetidine, or tetrodotoxin, but diminished by carbachol. The effect of isoprenaline was amplified by threshold concentrations of BM 14.478 (10(-6) M). There was only a slight intrinsic chronotropic activity in spontaneously beating guinea pig atria. Atrial cyclic AMP (cAMP) was increased from 1.46 +/- 0.06 to 1.97 +/- 0.03 pmol/mg wet wt, at 3 X 10(-6) M. This might be due to an inhibition of cardiac phosphodiesterase(s) (PDE). IC50 of bovine PDE was 7.2 X 10(-5) M (5.4 X 10(-5) M to 9.7 X 10(-5) M). BM 14.478 shortened the duration of transmembrane action potential (90% repol.) by 8% and increased the Vmax of slow action potentials by 32% at 3 X 10(-4) M. In skinned porcine heart muscle fibers an increase in calcium-activated force up to 43 +/- 7% was observed (10(-7) to 10(-4) M). Rat aortas were relaxed by about 75% maximally (10(-7) to 10(-4) M). It is concluded that BM 14.478 is a potent inotropic drug which acts via an increase in myocardial cAMP content and in calcium sensitivity of contractile proteins.

Purification of new calcium activated protease (low calcium requiring form) and comparison to high calcium requiring form

A new calcium activated protease which requries low concentration of calcium was purified to almost homogeneity from porcine heart muscle. The protease was composed of two polypeptide chains of approximately 90 K and 30 K. The 90 K subunit was larger than the large subunit of the high calcium requring form of calcium activated protease, therefore we concluded that the low calcium requiring form is different from the high calcium requring form and its auto-digested protease. The low calcium reqiring form of calcium activated protease was also activated by manganese and balium, and was very stable even at pH 9.0