Abstract
Objective: Since apoptosis is an important contributor to heart diseases in which ischemia and hypoxia are key elements, we tested the hypothesis that hypoxia predisposes neonatal rat ...ventricular myocytes (NRVM) to Fas-mediated apoptosis, by shifting the balance between antiapoptotic and proapoptotic proteins towards the latter. Methods: Normoxic or hypoxic (22 h, 1% O2) cultured NRVM were exposed to recombinant Fas L (rFasL) for 7 h, and apoptosis measured thereafter. Results: Whereas in normoxic NRVM, rFasL did not cause apoptosis measured by the TUNEL assay (4.8±0.5% in control versus 4.5±0.9% in rFasL), in hypoxic cultures rFasL increased the background apoptosis level by 100%. That Fas was functional in normoxic NRVM, despite its inability to mediate apoptosis, was evidenced by the finding that Fas activation increased the diastolic Ca2+i levels measured by Fura 2 fluorescence, and caused arrhythmias. In support of our working hypothesis, hypoxia increased Fas expression by 200% (measured by quantitative Western blot), and the expression of the proapoptotic proteins ARTS and FADD by 323 and 250%, respectively, and decreased the expression of the antiapoptotic proteins ARC and FLIP by 90 and 60%, respectively. Conclusion: By upregulating Fas expression and key proapoptotic proteins, and by downregulating antiapoptotic proteins, hypoxia predisposes ventricular myocytes to Fas-induced apoptosis.
It is now well established that immune effector mechanisms contribute to cardiac dysfunction in several heart diseases, including myocarditis and the associated dilated cardiomyopathy, heart ...transplant rejection and Chagas' disease. These and other pathologies, in which cellular immunity plays an important role, contribute to morbidity and mortality world-wide. As a result of numerous studies performed in this exciting field, two major mechanisms of lymphocytotoxicity have been proposed: a secretory mechanism in which perforin and granzymes are key players, and a non-secretory mechanism involving Fas/FasL activation. While the common notion is that CTL-myocyte interaction, perforin- or Fas-based, inevitably results in target cell apoptotic death, the objective of this review is to consider the concept of non-apoptotic consequences of CTL-target cell interaction. It is proposed that depending on the myocyte status as well as on the fine balance between pro- and anti-apoptotic factors, CTL-myocyte interaction may result in a non-apoptotic, potentially reversible sustained damage to the myocytes, thus contributing to immune-mediated cardiac dysfunction.
Abstract
Introduction
DMD, an X-linked muscle degenerative fatal disease, is caused by mutations in the dystrophin gene. Dilated cardiomyopathy (DCM) is a major cause of morbidity and mortality in ...DMD patients. Treatments for DCM in DMD are limited to steroids and standard heart failure medications such as β-blockers and ACE-inhibitors, and therefore novel therapeutic modalities are urgently needed.
Purpose
We hypothesized that dystrophin mutations in DMD lead to cardiomyopathy-causing bioenergetic/metabolic impairments, which can be therapeutically targeted for improving cardiac function.
Methods
Induced Pluripotent Stem Cell-derived cardiomyocytes (iPSC-CMs) were generated from healthy volunteer and 3 DMD patients: young male (YM), adult male (AM) and adult female (AF). We investigated the bioenergetics, electrophysiology, mitochondrial and metabolic features of healthy and DMD iPSC-CMs using the Seahorse Flux analyzer, patch clamp, confocal fluorescence microscopy and Liquid chromatography mass spectrometry (LC-MS) technologies, respectively.
Results
To test the hypothesis, we measured respiration and glycolytic rates of healthy and DMD iPSC-CMs. Compared to healthy iPSC-CMs, in both AM and AF DMD, but not in YM DMD cardiomyocytes, there was a 75% decrease in ATP production, and 80% and 45% decrease in basal respiration, respectively. In agreement with the healthy-like bioenergetic status of YM, the iPSC-CMs showed no arrhythmias, in contrast to the prominent arrhythmias in AM and AF cardiomyocytes. To determine whether the impairment in the phosphorylation pathway (OXPHOS) affects glycolysis, we measured the cardiomyocytes' response to glycolytic stress test. These experiments showed that the glycolytic rates were similar in healthy and DMD iPSC-CMs. In agreement with impaired OXPHOS, mitochondrial activity measured by 3D life confocal microscopy was attenuated in the DMD male by 35%, compared to healthy cardiomyocytes. Furthermore, the metabolomic LC-MS analyses demonstrated significant differences in metabolite levels in YM, AM and AF DMD iPSC-CMs relative to healthy iPSC-CMs. For example, compared to healthy iPSC-CMs, there was a dramatic fall to undetected levels in phosphocreatine in both AM and AF, but not in YM DMD, indicating a dysfunctional phosphocreatine energy system.
Conclusions
DMD iPSC-CMs exhibit bioenergetic/metabolic impairments, which constitute novel targets for alleviating the cardiomyopathy in DMD patients.
Funding Acknowledgement
Type of funding source: Public grant(s) – National budget only. Main funding source(s): ISF - Israel Science Foundation
Endothelin‐1 (ET‐1) is an important contributor to ventricular hypertrophy and failure, which are associated with arrhythmogenesis and sudden death. To elucidate the mechanism(s) underlying the ...arrhythmogenic effects of ET‐1 we tested the hypothesis that long‐term (24 hrs) exposure to ET‐1 impairs impulse conduction in cultures of neonatal rat ventricular myocytes (NRVM). NRVM were seeded on micro‐electrode‐arrays (MEAs, Multi Channel Systems, Reutlingen, Germany) and exposed to 50 nM ET‐1 for 24 hrs. Hypertrophy was assessed by morphological and molecular methods. Consecutive recordings of paced activation times from the same cultures were conducted at baseline and after 3, 6 and 24 hrs, and activation maps for each time period constructed. Gap junctional Cx43 expression was assessed using Western blot and confocal microscopy of immunofluorescence staining using anti‐Cx43 antibodies. ET‐1 caused hypertrophy as indicated by a 70% increase in mRNA for atrial natriuretic peptide (P < 0.05), and increased cell areas (P < 0.05) compared to control. ET‐1 also caused a time‐dependent decrease in conduction velocity that was evident after 3 hrs of exposure to ET‐1, and was augmented at 24 hrs, compared to controls (P < 0.01). ET‐1 increased total Cx43 protein by ∼40% (P < 0.05) without affecting non‐ phosphorylated Cx43 (NP‐Cx43) protein expression. Quantitative confocal microscopy showed a ∼30% decrease in the Cx43 immunofluorescence per field in the ET‐1 group (P < 0.05) and a reduced field stain intensity (P < 0.05), compared to controls. ET‐1‐induced hypertrophy was accompanied by reduction in conduction velocity and gap junctional remodelling. The reduction in conduction velocity may play a role in ET‐1 induced susceptibility to arrhythmogenesis.
Numerous studies have demonstrated that immune effector mechanisms cause serious heart diseases, among which are heart transplant rejection, myocarditis, and the resulting dilated cardiomyopathy, as ...well as Chagasʼ disease. Whereas different effectors of the immune system can affect cardiac function, this review primarily focuses on the immune damage caused by cytotoxic T lymphocytes. The immune attack staged by cytotoxic T lymphocytes is carried out by one of two distinct modes of lymphocytotoxicity(a) secretion of lytic granules containing the pore-forming protein perforin and a family of serine proteases (i.e., granzymes) and (b) interaction between the lymphocyte Fas ligand and the target cell Fas receptor. Ventricular myocytes challenged by the immune system sustain diverse intracellular changes, among which the rise in intracellular calcium (Cai) constitutes an important contributor to myocyte dysfunction. Hence, this Cai rise, which does not necessarily result in apoptosis, can affect cardiac function directly and indirectly. Importantly, the final outcomes of these perturbations vary markedly and depend on intracellular circumstances such as the magnitude of the absolute rise in Cai and its temporal and spatial determinants, the metabolic status of the myocyte, as well as a fine balance between pro-apoptotic and anti-apoptotic factors. In view of the central role of Cai rise in immune-mediated myocyte dysfunction and possibly cell death, this review addresses three topics related to the immune assault on the heart(a) Cai rise in affected myocytes; (b) the source for the Cai rise; and (c) pharmacologic modification of the immune-mediated Cai rise.
Abstract
Introduction
Familial hypertrophic cardiomyopathy (HCM) is caused by over 400 mutations affecting mostly key sarcomere components, such as titin and myosin. However, HCM also results from ...mutations in non-structural genes such as PRKAG2 which is involved directly in a variety of bioenergetic and metabolic pathways. When the metabolic processes fail to work properly or effectively, structural and functional aberrations resulting in cardiac dysfunction can occur. Thus, mutations in the human PRKAG2 gene lead to HCM, autosomal dominant ventricular pre-excitation - Wolff-Parkinson-White syndrome (WPW), a progressive conduction system disease and vacuolar glycogen accumulation in cardiomyocytes.
Purpose
To investigate the hypothesis that intervening with the bioenergetic and metabolic consequences of the R302Q mutation in the PRKAG2 gene causing inherited cardiomyopathy, will attenuate the cardiac impairments.
Methods
We generated mutated and isogenic induced Pluripotent Stem Cell-derived cardiomyocytes (iPSC-CMs) from a WPW patient carrying the R302Q mutation. As additional control, we used healthy volunteers' iPSC-CMs. Bioenergetic (Oxygen Consumption Rate, OCR) and metabolic status were measured using the Seahorse Flux Analyzer and LC-MS, respectively. To decipher the molecular basis underlying the bioenergetic and metabolic deficits, RNA-seq analysis was performed.
Results
The OCR results demonstrated in PRKAG2-mutated compared to isogenic and healthy iPSC-CMs, a 2-fold increase in maximal respiration rate and a 3.75-fold increase in spare respiratory capacity, while basal OCR parameters were similar in all groups. Importantly, when treated with the AMPK activator metformin (2.5 mM), all the abovementioned OCR parameters were similar in the three groups. RNA-seq analysis demonstrated that of the 553 differently expressed genes (DEGs), and of the 99 DEGs mutually differentially expressed, compared to isogenic and healthy cells, the most relevant altered pathways were glycolysis, carbon metabolism, biosynthesis of amino acids, HIF-1 signaling and fructose and mannose metabolism. These findings are consistent with the LC-MS results demonstrating in PRKAG2-mutated versus isogenic and healthy iPSC-CMs, at least a 3-fold decrease in metabolites linked to the abovementioned pathways: butyryl-carnitine, creatine, docosahexaenoic acid, GMP, IMP, myristoyl-carnitine, palmitoyl-carnitine, succinyl-Cys, UMP, UTP, UDP-GlcNAc.
Conclusion
PRKAG2-mutated iPSC-CMs exhibit bioenergetic and metabolic aberrations, which contribute to the cardiac pathological aspects of WPW syndrome. Importantly, treatment with the AMPK activator metformin eliminated the bioenergetic abnormalities in the mutated cells, while isogenic and healthy control cells remained unaffected. Based on these novel findings, a new therapeutic modality in WPW patients may be considered.
Funding Acknowledgement
Type of funding source: Public grant(s) – National budget only. Main funding source(s): Israel Science Foundation (ISF)
The study of human cardiac tissue development is hampered by the lack of a suitable in vitro model. We describe the phenotypic properties of cardiomyocytes derived from human embryonic stem (ES) ...cells. Human ES cells were cultivated in suspension and plated to form aggregates termed embryoid bodies (EBs). Spontaneously contracting areas appeared in 8.1% of the EBs. Cells from the spontaneously contracting areas within EBs were stained positively with anti-cardiac myosin heavy chain, anti--alpha-actinin, anti-desmin, anti--cardiac troponin I (anti-cTnI), and anti-ANP antibodies. Electron microscopy revealed varying degrees of myofibrillar organization, consistent with early-stage cardiomyocytes. RT-PCR studies demonstrated the expression of several cardiac-specific genes and transcription factors. Extracellular electrograms were characterized by a sharp component lasting 30 +/- 25 milliseconds, followed by a slow component of 347 +/- 120 milliseconds. Intracellular Ca(2+) transients displayed a sharp rise lasting 130 +/- 27 milliseconds and a relaxation component lasting 200--300 milliseconds. Positive and negative chronotropic effects were induced by application of isoproterenol and carbamylcholine, respectively. In conclusion, the human ES cell--derived cardiomyocytes displayed structural and functional properties of early-stage cardiomyocytes. Establishment of this unique differentiation system may have significant impact on the study of early human cardiac differentiation, functional genomics, pharmacological testing, cell therapy, and tissue engineering.
In the present study, we tested the hypothesis that similar to other mechanical loads, notably cyclic stretch (simulating pre‐load), glass microspheres simulating afterload will stimulate the ...secretion of angiogenic factors. Hence, we employed glass microspheres (average diameter 15.7 μm, average mass 5.2 ng) as a new method for imposing mechanical load on neonatal rat ventricular myocytes (NRVM) in culture. The collagen‐coated microspheres were spread over the cultures at an estimated density of 3000 microspheres/mm2, they adhered strongly to the myocytes, and acted as small weights carried by the cells during their contraction. NRVM were exposed to either glass microspheres or to cyclic stretch, and several key angiogenic factors were measured by RT‐PCR. The major findings were: (1) In contrast to other mechanical loads, such as cyclic stretch, microspheres (at 24 hrs) did not cause hypertrophy. (2) Further, in contrast to cyclic stretch, glass microspheres did not affect Cx43 expression, or the conduction velocity measured by means of the Micro‐Electrode‐Array system. (3) At 24 hrs, glass microspheres caused arrhythmias, probably resulting from early afterdepolarizations. (4) Glass microspheres caused the release of angiogenic factors as indicated by an increase in mRNA levels of vascular endothelial growth factor (80%), angiopoietin‐2 (60%), transforming growth factor‐β (40%) and basic fibroblast growth factor (15%); these effects were comparable to those of cyclic stretch. (5) As compared with control cultures, conditioned media from cultures exposed to microspheres increased endothelial cell migration by 15% (P<0.05) and endothelial cell tube formation by 120% (P<0.05), both common assays for angiogenesis. In conclusion, based on these findings we propose that loading cardiomyocytes with glass microspheres may serve as a new in vitro model for investigating the role of mechanical forces in angiogenesis and arrhythmias.
ABSTRACT—A major function of haptoglobin (Hp) is to bind hemoglobin (Hb) to form a stable Hp-Hb complex and thereby prevent Hb-induced oxidative tissue damage. Clearance of the Hp-Hb complex can be ...mediated by the monocyte/macrophage scavenger receptor CD163. We recently demonstrated that diabetic individuals homozygous for the Hp 2 allele (Hp 2–2) were at 500% greater risk of cardiovascular disease (CVD) compared with diabetic individuals homozygous for the Hp 1 allele (Hp 1–1). No differences in risk by Hp type were seen in individuals without diabetes. To understand the relationship between the Hp polymorphism and diabetic CVD, we sought to identify differences in antioxidant and scavenging functions between the Hp types and to determine how these functions were modified in diabetes. The scavenging function of Hp was assessed using rhodamine-tagged and I-Hp in cell lines stably transfected with CD163 and in macrophages expressing endogenous CD163. We found that the rate of clearance of Hp 1–1-Hb by CD163 is markedly greater than that of Hp 2–2-Hb. Diabetes is associated with an increase in the nonenzymatic glycosylation of serum proteins, including Hb. The antioxidant function of Hp was assessed with glycosylated and nonglycosylated Hb. We identified a severe impairment in the ability of Hp to prevent oxidation mediated by glycosylated Hb. We propose that the specific interaction between diabetes, CVD, and Hp genotype is the result of the heightened urgency of rapidly clearing glycosylated Hb-Hp complexes from the subendothelial space before they can oxidatively modify low-density lipoprotein to atherogenic oxidized low-density lipoprotein. (Circ Res. 2003;92:1193–1200.)
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