Ribosome distribution in normal and infarcted rat hearts

Summary Distribution of ribosomes throughout the myocardium of normal and infarcted rat hearts was studied by immunofluorescence and laser confocal scanning microscopy. In addition, sections were labelled with peroxidase or immunogold particles for electron microscopic examination. Ligation of the proximal free left coronary artery produced severe myocardial ischaemia, and after 6 days of ligation most of the left ventricular wall was necrotic and partially replaced by granulation tissue. Immunofluorescence microscopy revealed the presence of ribosomes throughout the non-necrotic myocardium. Some cardiac muscle cells located in subendocardial areas and in the border areas surrounding the infarct were particularly intensely stained. Cells constituting the granulation tissue frequently exhibited strong ribosomal immunostaining. Within longitudinally sectioned cardiac muscle cells, ribosomes were organized in strands oriented along the long axis of the cell as well as in a cross-striated pattern. By double labelling of muscle cells with antibodies against ribosomes and Z-line-associated proteins (desmin or α-actinin), it was shown that the cross-striated bands of anti-ribosomal staining coincided with the I-bands along the myofibrils. Immunoelectron microscopy confirmed a wide distribution of ribosomes throughout the intermyofibrillar and subsarcolemmal sarcoplasm, and some labelling was also observed within the I-band. The present results indicate that ribosomes are distributed in a characteristic pattern throughout the sarcoplasm of cardiac muscle cells in association with the myofibrils. Furthermore, it is suggested that within viable cardiac muscle cells located adjacent to the infarct, protein synthesis is increased; this might be an important factor in regional development of compensatory hypertrophy of the surviving cardiac muscle cells.     

DITPA restores the repolarizing potassium currents Itof and Iss in cardiac ventricular myocytes of diabetic rats

Diabetes Mellitus (DM) can produce an increase in the cardiac action potential duration and QT interval that can be associated with sudden death. These cardiac effects are due to a region-specific decrease in repolarizing outward K(+) currents. Some authors have suggested that the proarrhythmic effects of diabetes can be due to diabetes-induced hypothyroidism. Thus, we have examined the effect of the thyroid hormone analog diiodothyropropionic acid (DITPA) on calcium-independent outward potassium currents in ventricular myocytes from diabetic rats. Sustained (I(ss)) and fast transient outward (I(tof)) K(+) currents were recorded using the whole-cell configuration of the patch-clamp technique. Myocytes were enzymatically isolated from the free wall of the right ventricle, and the epicardial and endocardial layers of the left ventricle of healthy, diabetic and DITPA-treated diabetic rats. Circulating thyroid hormones were measured by electrochemiluminescence. DITPA-treatment of diabetic rats restored I(tof) and I(ss) current densities in cardiac myocytes from the three regions studied, but did not alter current densities in myocytes of control rats. T(3) and T(4) levels were reduced by diabetes, and DITPA-treatment increased circulating T(3) levels. T(3)-treatment of diabetic rats also restored current densities to control values. However, direct incubation of diabetic myocytes with DITPA did not restore current densities. In summary, DITPA-treatment of diabetic rats restored the potassium current (I(tof) and I(ss)) densities in myocytes from all ventricular regions.     

The isolated working heart model in infarcted rat hearts

Congestive heart failure (CHF) is one of the most common causes of death in western countries. The aim of this study was to establish and validate the working heart model in rat hearts with CHF. In the rat model the animals show parameters and symptoms that can be extrapolated to the clinical situation of patients with end-stage heart failure. The focus of attention was the evaluation of cardiodynamics (e.g.contractility) in the isolated 'working heart' model. The geometric properties of the left ventricle were measured by planimetry (stereology). Formulae available in the past for determining certain parameters in the working heart model (e.g.external heart work) have to be fitted to the circumstances of the infarcted rat hearts with its different organ properties.CHF was induced in Wistar Kyoto (WKY/NHsd) and spontaneously hypertensive rats (SHR/NHsd) by creating a permanent (8 week) occlusion of the left coronary artery, 2 mm distal to the origin from the aorta, by a modified technique (Itter et al. 2004). This resulted in a large infarction of the free left ventricular wall.We were able to establish and adapt a new and predictive working heart model in spontaneously hypertensive rat hearts with myocardial infarction (MI) 8-12 weeks after coronary artery ligation. At this stage the WKY rat did not show any symptoms of CHF. The SHR rat represented characteristic parameters and symptoms that could be extrapolated to the clinical situation of patients with end-stage heart failure (NYHA III-IV). Upon inspection, severe clinical symptoms of CHF such as dyspnoea, subcutaneous oedema, palebluish limbs and impaired motion were prominent. On necropsy the SHR showed lung oedema, hydrothorax, large dilated left and right ventricular chambers and hypertrophy of the septum. In the working heart model the infarcted animals showed reduced heart power, diminished contractility and enhanced heart work, much more so in the SHR/NHsd than in the Wistar Kyoto rat (WKY/NHsd).The aim for the future is to find a causal therapy of heart failure treatment. At present, only palliative therapy is possible for patients with heart failure. For this reason the working heart model in CHF rat hearts should provide a valuable method for early testing of new therapeutic approaches for patients with CHF.     

Experimental aspects of isolation of myocytes from adult guinea-pig hearts

Ionic channels can now be effectively studied on enzymatically isolated cardiac myocytes by means of the patch clamp technique. Three procedures reported to give consistently high yields of Ca-tolerant myocytes were tested for applicability to calcium channel studies under our laboratory conditions. None of them was found to be suitable for direct use. Therefore, a modified method for isolation of myocytes from adult guinea-pig hearts was developed. Calcium channel currents measured in Ca-tolerant myocytes isolated by this procedure have been presented and problems of myocytes isolation and of patch-clamp measurement discussed.     

VEGF improves survival of mesenchymal stem cells in infarcted hearts

Bone marrow-derived mesenchymal stem cells (MSC) are a promising source for cell-based treatment of myocardial infarction (MI), but existing strategies are restricted by low cell survival and engraftment. We examined whether vascular endothelial growth factor (VEGF) improve MSC viability in infracted hearts. We found long-term culture increased MSC-cellular stress: expressing more cell cycle inhibitors, p16^INK, p21 and p19^ARF. VEGF treatment reduced cellular stress, increased pro-survival factors, phosphorylated-Akt and Bcl-xL expression and cell proliferation. Co-injection of MSCs with VEGF to MI hearts increased cell engraftment and resulted in better improvement of cardiac function than that injected with MSCs or VEGF alone. In conclusion, VEGF protects MSCs from culture-induce cellular stress and improves their viability in ischemic myocardium, which results in improvements of their therapeutic effect for the treatment of MI.     

Regional appearance of atrial natriuretic peptide in the ventricles of infarcted rat hearts

The appearance of atrial natriuretic peptide (ANP) in the ventricular myocardium was investigated in rat hearts subjected to severe left ventricular infarction. The left coronary artery was ligated for 1, 2, 3, 4 and 6 days and for 3 weeks, and the tissue was prepared for microscopic examination of immunoreactive ANP and for electron microscopy. In the normal and sham-operated hearts, and in hearts subjected to 1 day of coronary ligation, ANP immunoreactivity was restricted to a few ventricular myocytes of the conduction system. Following 2–3 days of coronary ligation, ANP immunoreactivity was detected in the viable myocardium of the lateral border of the infarct and in a few layers of viable cardiac myocytes located in the subendocardial areas of the ischemic left free ventricular wall. Further, during the following days and after 3 weeks of coronary ligation, a gradient of specific labeling was commonly seen across the lateral border area of the infarct. Thus, the strongest immunoreactivities were present in the cardiac myocytes located adjacent to the non-contracting myocardium. Electron microscopic examination of the immunoreactive cardiac myocytes confirmed the presence of electron-dense specific granules within these cells. The present findings suggest that the increased regional production of ANP within the ventricular myocardium is induced by increased mechanical stretch of the cardiac myocytes, and that this might contribute to the increased release of ANP in myocardial infarction.     

Cardiomyocytes derived from human embryonic stem cells in pro-survival factors enhance function of infarcted rat hearts

Cardiomyocytes derived from human embryonic stem (hES) cells potentially offer large numbers of cells to facilitate repair of the infarcted heart. However, this approach has been limited by inefficient differentiation of hES cells into cardiomyocytes, insufficient purity of cardiomyocyte preparations and poor survival of hES cell-derived myocytes after transplantation. Seeking to overcome these challenges, we generated highly purified human cardiomyocytes using a readily scalable system for directed differentiation that relies on activin A and BMP4. We then identified a cocktail of pro-survival factors that limits cardiomyocyte death after transplantation. These techniques enabled consistent formation of myocardial grafts in the infarcted rat heart. The engrafted human myocardium attenuated ventricular dilation and preserved regional and global contractile function after myocardial infarction compared with controls receiving noncardiac hES cell derivatives or vehicle. The ability of hES cell-derived cardiomyocytes to partially remuscularize myocardial infarcts and attenuate heart failure encourages their study under conditions that closely match human disease.     

Comparison of calcium-current in isolated atrial myocytes from failing and nonfailing human hearts

To identify possible alterations of the L-type calcium currents (I_Ca,L) in cardiomyopathy, I_Ca,L were recorded in atrial myocytes dissociated from the nonfailing heart (NF) of patients undergoing corrective open-heart surgery and explanted failing heart (FH) of patients with dilated cardiomyopathy undergoing heart transplantation. The patch-clamp technique was applied in the single-electrode whole-cell mode. The electrophysiological properties of I_Ca,L, including cell capacitance and current density, were similar in atrial myocytes from both groups of patients. Further to identify possible alterations of the myocardial beta-adrenergic pathway in cardiomyopathy, we examined the effects of isoproterenol, forskolin, 8-Br-cAMP and IBMX on I_Ca,L in both groups of atrial myocytes. Perfusion of isoproterenol (1 M) significantly increased the peak I_Ca,L by 515 ± 44% in 6 atrial myocytes from NF but increased only by 135 ± 25% in 27 atrial myocytes from FH. However, forskolin (1 M) or 8-Br-cAMP (0.1 mM) increased the peak I_Ca,L to a similar extent in atrial myocytes from NF and FH. IBMX (20 M) also induced a comparable increase in the peak I_Ca,L by 213 ± 31% (n=5) and 207 ± 59% (n=4) in atrial myocytes from NF and FH, respectively. The above findings suggest that in atrial myocytes obtained from FH the beta-adrenoceptor numbers might be decreased but no impairment of the signal transduction cascade occurred beyond the GTP binding proteins level.     

Restoration of calcium handling properties of adult cardiac myocytes from hypertrophied hearts

Reductions in cardiac sarcoplasmic reticulum calcium-ATPase (Serca2a) levels are thought to underlie the prolonged calcium (Ca(2+)) transients and consequent reduced contractile performance seen in human cardiac hypertrophy and heart failure. In freshly isolated cardiac myocytes from rats with monocrotaline-induced right ventricular hypertrophy we found reduced sarcoplasmic reticulum Serca2a expression and prolonged Ca(2+)transients, characteristic of hypertrophic cardiac disease.Modulation of intracellular Ca(2+)levels, Ca(2+) kinetics or Ca(2+)sensitivity is the focus of many current therapeutic approaches to improve contractile performance in the hypertrophic or failing heart. However, the functional effects of increasing Serca2a expression on Ca(2+) handling properties in myocytes from an animal model of cardiac hypertrophy are largely unknown. Here, we describe enhancement of the deficient Ca(2+) handling properties evident in myocytes from hypertrophied hearts following adenoviral-mediated transfer of the human Serca2a gene to these myocytes.These results highlight the importance of Serca2a deficiencies in the hypertrophic phenotype of cardiac muscle and suggest a simple, effective approach for manipulation of normal cardiac function.     

Therapeutic angiogenesis of three-dimensionally cultured adipose-derived stem cells in rat infarcted hearts

Background aimsTo successfully treat myocardial infarction (MI), blood must be resupplied to the ischemic myocardium by inducing angiogenesis. Many studies report enhanced angiogenesis using stem cells; however, the therapeutic efficacy of cell transplant remains low because transplanted cells may not survive, be retained at the site of transplant, or develop into vascular tissue. In this study, we assessed the therapeutic potential of three-dimensional cell masses (3DCM) composed of human adipose-derived stem cells (hASC) in a rat MI model.MethodsFor formation of 3DCM, hASC were cultured on a substrate with immobilized fibroblast growth factor 2. The morphology and phenotypes of 3DCM were analyzed 1 day after culture. The cells (hASC and 3DCM, 5 × 10⁵ cells) were injected into ischemic regions after ligation of the left coronary artery (n = 6 in each group). Cell retention ratio, therapeutic efficacy and vascularization were evaluated 4 weeks after transplant.ResultsA spheroid-type 3DCM, which included vascular cells (CD34⁺/CD31⁺/KDR⁺/α-SMA⁺) with high production of human vascular endothelial growth factor, was obtained. Infarct size and cardiomyocyte apoptosis were reduced in the 3DCM-injected group compared with the hASC-injected group. The retention ratio of hASC was 14-fold higher in the 3DCM-injected group. Many transplanted cells differentiated into endothelial and smooth muscle cells and formed vascular networks incorporated into host vessels.ConclusionsTransplant of 3DCM may be useful for angiogenic cell therapy to treat MI.     

Bone marrow stromal cells improve cardiac performance in healed infarcted rat hearts

Postinfarct congestive heart failure is one of the leading causes of morbidity and mortality in developed and developing countries. The main purpose of this study was to investigate whether transplantation of bone marrow stromal cells (BMSC) directly into the myocardium could improve the performance of healed infarcted rat hearts. Cell culture medium with or without BMSC was injected into borders of cardiac scar tissue 4 wk after experimental infarction. Cardiac performance was evaluated 2 wk after cellular (n = 10) or medium (n = 10) injection by electro- and echocardiography. Histological study was performed 3 wk after treatment. Electrocardiography of BMSC-treated infarcted rats showed electrical and mechanical parameters more similar to those in control than in medium-treated animals: a normal frontal QRS axis in 6 of 10 BMSC-treated and all control rats and a rightward deviation of the QRS axis in all 10 medium-treated animals. BMSC treatment, assessed by echocardiography, improved fractional shortening (39.00 +/- 4.03%) compared with medium-treated hearts (18.20 +/- 0.74%) and prevented additional changes in cardiac geometry. Immunofluorescence microscopy revealed colocalization of 4',6-diamidino-2-phenylindole-labeled nuclei of transplanted cells with cytoskeletal markers for cardiomyocytes and smooth muscle cells, indicating regeneration of damaged myocardium and angiogenesis. These data provide strong evidence that BMSC implantation can improve cardiac performance in healed infarctions and open new promising therapeutic opportunities for patients with postinfarction heart failure.     

Oleate prevents palmitate-induced cytotoxic stress in cardiac myocytes

The cytotoxicity of saturated fatty acids has been implicated in the pathophysiology of cardiovascular disease, though their effects on cardiac myocytes are incompletely understood. We examined the effects of palmitate and the mono-unsaturated fatty acid oleate on neonatal rat ventricular myocyte cell biology. Palmitate (0.5mM) increased oxidative stress, as well as activation of the stress-associated protein kinases (SAPK) p38, Erk1/2, and JNK, following 18h and induced apoptosis in approximately 20% of cells after 24h. Neither antioxidants nor SAPK inhibitors prevented palmitate-induced apoptosis. Low concentrations of oleate (0.1mM) completely inhibited palmitate-induced oxidative stress, SAPK activation, and apoptosis. Increasing mitochondrial uptake of palmitate with l-carnitine decreased apoptosis, while decreasing uptake with the carnitine palmitoyl transferase-1 inhibitor perhexiline nearly doubled palmitate-induced apoptosis. These results support a model for palmitate-induced apoptosis, activation of SAPKs, and protein oxidative stress in myocytes that involves cytosolic accumulation of saturated fatty acids.     

Altered E-C coupling in rat ventricular myocytes from failing hearts 6 wk after MI

Excitation-contraction (E-C) coupling was investigated in rat hearts 6 wk after induction of myocardial infarction (MI) by ligation of the left coronary artery. Heart weight was increased by 74% and left ventricular end-diastolic pressure was 23 +/- 2 mmHg in MI compared with 8 +/- 2 mmHg in sham-operated controls (Sham, P < 0.001). Cell shortening was measured in voltage-clamped myocytes at 36 degrees C. In solutions where Cs(+) had been replaced by K(+), the voltage dependence of contraction was sigmoidal between -20 and +100 mV in Sham and MI cells. Verapamil (20 microM) blocked L-type Ca(2+) current and reduced contraction in Sham cells by approximately 50% (P < 0.01) but did not decrease contraction significantly in MI cells at test potentials above +10 mV. Verapamil-insensitive contractions were blocked by Ni(2+) (5 mM). Na(+)/Ca(2+) exchange current was doubled in MI compared with Sham cells at test potentials between -20 and +80 mV (P < 0.05), whereas mRNA and protein expression increased by 30-40%. Finally, voltage dependence of contraction was bell shaped in Na(+)-free solutions, but contraction was significantly increased in MI cells over a wider voltage range (P < 0.05). The insensitivity to Ca(2+) channel block in MI cells may result from an increased contribution of the Na(+)/Ca(+) exchanger to triggering of E-C coupling. These results suggest significant changes in E-C coupling in the hypertrophy and failure that develop in response to extensive MI.     

Effects of amiloride on calcium uptake by myocytes isolated from adult rat hearts

Amiloride at high concentrations inhibits the uptake of Ca by rat heart myocytes containing elevated levels of intracellular Na and retards the development of Ca-dependent hypercontracture in these cells. In contrast, amiloride enhances the net uptake of Ca in Ca-tolerant myocytes containing normal levels of Na. The results suggest that amiloride may inhibit Na-Ca exchange across the sarcolemma of cardiac myocytes.     

Decreased passive stiffness of cardiac myocytes and cardiac tissue from copper-deficient rat hearts

Passive stiffness characteristics of isolated cardiac myocytes, papillary muscles, and aortic strips from male Holtzman rats fed a copper-deficient diet for approximately 5 wk were compared with those of rats fed a copper-adequate diet to determine whether alterations in these characteristics might accompany the well-documented cardiac hypertrophy and high incidence of ventricular rupture characteristic of copper deficiency. Stiffness of isolated cardiac myocytes was assessed from measurements of cellular dimensional changes to varied osmotic conditions. Stiffness of papillary muscles and aortic strips was determined from resting length-tension analyses and included steady-state characteristics, dynamic viscoelastic stiffness properties, and maximum tensile strength. The primary findings were that copper deficiency resulted in cardiac hypertrophy with increased cardiac myocyte size and fragility, decreased cardiac myocyte stiffness, and decreased papillary muscle passive stiffness, dynamic stiffness, and tensile strength and no alteration in aortic connective tissue passive stiffness or tensile strength. These findings suggest that a reduction of cardiac myocyte stiffness and increased cellular fragility could contribute to the reduced overall cardiac tissue stiffness and the high incidence of ventricular aneurysm observed in copper-deficient rats.     

Engineered heart tissue grafts improve systolic and diastolic function in infarcted rat hearts

The concept of regenerating diseased myocardium by implantation of tissue-engineered heart muscle is intriguing, but convincing evidence is lacking that heart tissues can be generated at a size and with contractile properties that would lend considerable support to failing hearts. Here we created large (thickness/diameter, 1-4 mm/15 mm), force-generating engineered heart tissue from neonatal rat heart cells. Engineered heart tissue formed thick cardiac muscle layers when implanted on myocardial infarcts in immune-suppressed rats. When evaluated 28 d later, engineered heart tissue showed undelayed electrical coupling to the native myocardium without evidence of arrhythmia induction. Moreover, engineered heart tissue prevented further dilation, induced systolic wall thickening of infarcted myocardial segments and improved fractional area shortening of infarcted hearts compared to controls (sham operation and noncontractile constructs). Thus, our study provides evidence that large contractile cardiac tissue grafts can be constructed in vitro, can survive after implantation and can support contractile function of infarcted hearts.     

Transplantation of embryonic stem cell-derived cardiomyocytes improves cardiac function in infarcted rat hearts

BACKGROUND: Post-infarct congestive heart failure is one of the leading causes of morbidity and mortality in industrialized countries. The main purpose of this study was to investigate whether transplantation of embryonic stem cell-derived cardiomyocytes (ESCM) directly into the infarcted myocardium could improve cardiac function in rats. METHODS: Cell culture medium with or without ESCM was injected into the borders of cardiac scar tissue 1 week after experimental infarction. Cardiac performance was evaluated 4 weeks later by means of echocardiography after ESCM (n=16) or medium (n=12) injection. RESULTS: ESCM implantation significantly improved fractional shortening (31.5+/-3. 8%) compared with medium-treated hearts (21.3+/-5.2%; P<0.05) and preserved left ventricular structure. Co-localization of 4',6-diamidino-2-phenylindole-labeled nuclei of transplanted cells with cardiomyocyte markers for cardiac troponin T and connexin-43, as detected by immunofluorescent microscopy, indicated the regeneration of damaged myocardium and the formation of gap junctions between grafted and host cells. However, intra-myocardial teratomas were observed in the hearts of two of the 16 grafted animals, at the fourth week after ESCM transplantation. DISCUSSION: Our results suggest that, although ESCM implantation can improve the function of infarcted myocardium, strategies to prevent tumorigenesis should be developed.     

Different signalling in infarcted and non‐infarcted areas of rat failing hearts: A role of necroptosis and inflammation

Necroptosis has been recognized in heart failure (HF). In this study, we investigated detailed necroptotic signalling in infarcted and non‐infarcted areas separately and its mechanistic link with main features of HF. Post‐infarction HF in rats was induced by left coronary occlusion (60 minutes) followed by 42‐day reperfusion. Heart function was assessed echocardiographically. Molecular signalling and proposed mechanisms (oxidative stress, collagen deposition and inflammation) were investigated in whole hearts and in subcellular fractions when appropriate. In post‐infarction failing hearts, TNF and pSer229‐RIP3 levels were comparably increased in both infarcted and non‐infarcted areas. Its cytotoxic downstream molecule p‐MLKL, indicating necroptosis execution, was detected in infarcted area. In non‐infarcted area, despite increased pSer229‐RIP3, p‐MLKL was present in neither whole cells nor the cell membrane known to be associated with necroptosis execution. Likewise, increased membrane lipoperoxidation and NOX2 levels unlikely promoted pro‐necroptotic environment in non‐infarcted area. Collagen deposition and the inflammatory csp‐1‐IL‐1β axis were active in both areas of failing hearts, while being more pronounced in infarcted tissue. Although apoptotic proteins were differently expressed in infarcted and non‐infarcted tissue, apoptosis was found to play an insignificant role. p‐MLKL‐driven necroptosis and inflammation while inflammation only (without necroptotic cell death) seem to underlie fibrotic healing and progressive injury in infarcted and non‐infarcted areas of failing hearts, respectively. Upregulation of pSer229‐RIP3 in both HF areas suggests that this kinase, associated with both necroptosis and inflammation, is likely to play a dual role in HF progression.