Cardiomyocyte Membrane Research Articles

Biochip-based study of unidirectional mitochondrial transfer from stem cells to myocytes via tunneling nanotubes

Tunneling nanotubes (TNTs) are small membranous tubes of 50–1000 nm diameter observed to connect cells in culture. Transfer of subcellular organelles through TNTs was observed in vitro and in vivo, but the formation and significance of these structures is not well understood. A polydimethylsiloxane biochip-based coculture model was devised to constrain TNT orientation and explore both TNT-formation and TNT-mediated mitochondrial transfer. Two parallel microfluidic channels connected by an array of smaller microchannels enabled localization of stem cell and cardiomyocyte populations while allowing connections to form between them. Stem cells and cardiomyocytes were deposited in their respective microfluidic channels, and stem cell-cardiomyocyte pairs were formed via the microchannels. Formation of TNTs and transfer of stained mitochondria through TNTs was observed by 24 h real-time video recording. The data show that stem cells are 7.7 times more likely to initiate contact by initial extension of filopodia. By 24 h, 67% of nanotube connections through the microchannels are composed of cardiomyocyte membrane. Filopodial extension and retraction by stem cells draws an extension of TNTs from cardiomyocytes. MitoTracker staining shows that unidirectional transfer of mitochondria between stem cell-cardiomyocyte pairs invariably originates from stem cells. Control experiments with cardiac fibroblasts and cardiomyocytes show little nanotube formation between homotypic or mixed cell pairs and no mitochondrial transfer. These data identify a novel biological process, unidirectional mitochondrial transfer, mediated by heterotypic TNT connections. This suggests that the enhancement of cardiomyocyte function seen after stem-cell injection may be due to a bioenergetic stimulus provided by mitochondrial transfer.

Unique Localization of Phospholamban in Perinuclear Membranes of Cardiomyocytes from Several Species

Phospholamban (PLB) regulates sarcoplasmic reticulum (SR) calcium-ATPase (SERCA2a) in cardiomyocytes (CMs). In addition to their well-known location in SR membranes, only SERCA2a, but not PLB, is shown expressed in the perinuclear/nuclear membranes of CMs. Here we used confocal immunofluorescence microscopy to examine the intracellular distribution of PLB. As expected, in isolated mouse CMs, co-localized PLB and SERCA2a were detected in SR by the monoclonal antibodies 2D12 (against PLB) and 2A7-A1 (against SERCA2a), respectively. Importantly, in addition to 2A7-A1 binding signals, strong 2D12 binding signal was detected around the DAPI staining of the nuclei, indicating the expression of PLB in perinuclear/nuclear membranes. Further, we probed PLB expression in tissue sections from hearts of human, dog, or rabbit with use of a polyclonal (PLB9) and three monoclonal (2D12, 1G7, or 1F1) antibodies, which were produced against different epitope of PLB in a specie-dependent manner. In particular, 2D12 and PLB9 recognized all isoforms of PLB in these species and revealed PLB expression in SR as well as in the perinuclear/nuclear regions. In comparison, 1F1, the dog specie-specific antibody, recognized PLB expression in dog SR and in perinuclear/nuclear membranes, but is completely negative for human, mouse, or rabbit CMs. Moreover, 1G7, which is specie-specific for human, mouse, or rabbit isoform of PLB, recognized similar pattern of PLB expression in these species including in perinuclear membranes, but not in dog CMs. Finally, 2D12 binding signals were completely eliminated by co-incubation of a PLB peptide (residues 2-25). These results showed for the first time that in addition to its expression in SR, PLB is expressed in and around nuclear envelope of CMs.

Slo2.1 Potassium Channel Knockout Mice have an Altered Metabolic Phenotype in Cardiac Mitochondria

Mitochondrial K+ channels are important mediators of cell protection against stress. Although the existence of mitochondrial K+ channels can be demonstrated phenomenologically, intense debate surrounds their molecular identity, their role in patho-physiology, and their regulation by endogenous signals. The mammalian Slo family of K+ channels consists of Slo1, Slo2.1, Slo2.2 and Slo3. While Slo3 expression is restricted to the germline, Slo1, Slo2.1 and Slo2.2 are widely expressed. Examination of these channels has been limited to pharmacologic profiling which is hampered by overlapping sensitivities to activators and inhibitors, and off-target effects of small molecules. Work from our lab using genetic knockout mice has demonstrated that Slo1 in intrinsic cardiac neurons is required for protection against ischemia/reperfusion injury elicited by ischemic preconditioning, while Slo2.1 in cardiomyocytes is required for protection via anesthetic preconditioning. Slo2.1 is expressed in the heart and has been detected in the cell membranes of cardiomyocytes. We predict that Slo2.1 may also be a mitochondrial potassium channel. Herein we use pharmacologic activators and inhibitors of Slo2 channels in wildtype and Slo2.1-/- mice to demonstrate the presence of Slo2.1 in cardiac mitochondria. This was also confirmed directly by electophysiology (patch clamp) of isolated mitochondrial inner membranes (mitoplasts). Additional analysis of hearts, cardiomyocytes, and cardiac mitochondria from these knockout mice has revealed a metabolic phenotype, indicating a functional relationship between mitochondrial potassium channels and regulation of mitochondrial oxidative phosphorylation. These data demonstrate a role for Slo2.1 in the regulation of cardiac mitochondrial function.

NON-UNIFORMITY OF THE DISTRIBUTION OF ELECTRICAL TRANSMEMBRANE POTENTIALS IN CARDIAC DEFIBRILLATION

The need for emergency cessation of ventricular fibrillation is one of the problems of modern reanimatology. In spite of searches for novel methods, there is the only effective method — electrical cardiac defibrillation. Objective: to investigate the effect of different forms of pulses on cell membranes in a model experiment and to assess their use for effective cardiac defibrillation. Materials and methods. The Maxwell model was used for theoretical analysis of the spatial distribution of an electric field in the red blood cell membrane. The electric effect on a single cell was calculated using the experimental findings and the equivalent electrical circuit of the myocardial structure during a defibrillation procedure. The cardiomyocyte membrane potential upon exposure to defibrillator discharge was estimated. Exposure of the red blood cell membrane to single, two unipolar and two heteropolar pulses was examined. Results. There is non-additivity of speeds upon double exposure as compared to single one. Single pulse causes a lower effect of electroporation than two double pulses. Hyperpolarization and depolarization processes in the cardiomyocyte membranes occur successively during electrical cardiac defibrillation. Conclusion. Two heteropolar pulses cause an effect of biological membrane electroporation with a greater probability than two unipolar ones.

Sustained βAR Stimulation Mediates Cardiac Insulin Resistance in a PKA-Dependent Manner.

Insulin resistance is a condition in which cells are defective in response to the actions of insulin in tissue glucose uptake. Overstimulation of β-adrenergic receptors (βARs) leads to the development of heart failure and is associated with the pathogenesis of insulin resistance in the heart. However, the mechanisms by which sustained βAR stimulation affects insulin resistance in the heart are incompletely understood. In this study, we demonstrate that sustained βAR stimulation resulted in the inhibition of insulin-induced glucose uptake, and a reduction of insulin induced glucose transporter (GLUT)4 expression that were mediated by the β2AR subtype in cardiomyocytes and heart tissue. Overstimulation of β2AR inhibited the insulin-induced translocation of GLUT4 to the plasma membrane of cardiomyocytes. Additionally, βAR mediated cardiac insulin resistance by reducing glucose uptake and GLUT4 expression via the cAMP-dependent and protein kinase A-dependent pathways. Treatment with β-blockers, including propranolol and metoprolol antagonized isoproterenol-mediated insulin resistance in the heart. The data in this present study confirm a critical role for protein kinase A in βAR-mediated insulin resistance.

Calcium-Activated Potassium Channels: Potential Target for Cardiovascular Diseases.

Ca(2+)-activated K(+) channels (KCa) are classified into three subtypes: big conductance (BKCa), intermediate conductance (IKCa), and small conductance (SKCa) KCa channels. The three types of KCa channels have distinct physiological or pathological functions in cardiovascular system. BKCa channels are mainly expressed in vascular smooth muscle cells (VSMCs) and inner mitochondrial membrane of cardiomyocytes, activation of BKCa channels in these locations results in vasodilation and cardioprotection against cardiac ischemia. IKCa channels are expressed in VSMCs, endothelial cells, and cardiac fibroblasts and involved in vascular smooth muscle proliferation, migration, vessel dilation, and cardiac fibrosis. SKCa channels are widely expressed in nervous and cardiovascular system, and activation of SKCa channels mainly contributes membrane hyperpolarization. In this chapter, we summarize the physiological and pathological roles of the three types of KCa channels in cardiovascular system and put forward the possibility of KCa channels as potential target for cardiovascular diseases.

Зависимость глубины радиочастотного повреждения миокарда от исходного сопротивления тканей в различных температурных условиях

ObjectiveThe aim of the study is to analyze the efficacy of RF ablation at different temperature conditions of the surgical area.Methods20 isolated hearts were studied. To evaluate the effectiveness of radio frequency exposure, use was made of two temperature settings: myocardial normothermia (36.6 °C) and myocardial hypothermia (20 °C). The hearts were placed on the electrophysiology bench heated to 36.6 °C; the ablation depth and temperature of the epicardium, endocardium and myocardium at the exposed points were then measured and evaluated. Tissue impedance was measured before and after RF ablation. RF energy was applied by means of a 40 W unipolar irrigated ball electrode over a period of 7 s.ResultsIt was found out that under hypothermia the myocardium has low resistance which hinders adequate heating of its deep layers and in this connection there arises a high risk for recovery of cardiomyocyte membrane potentials and for retention of their function.Conclusion When the ablation time in both temperature settings is the same, RF exposure at low temperatures causes a lesser depth of damage.

Evidence for MOR on cell membrane, sarcoplasmatic reticulum and mitochondria in left ventricular myocardium in rats.

Cardiac function is one important determinant to maintain tissue oxygenation and is thus highly regulated. In this context, it is interesting that centrally mediated opioidergic influence on cardiac function has long been known. Only recently, KOR and DOR have been found to be expressed in healthy left ventricular myocardium in rats and colocalized with parts of the excitation-contraction-coupling system. However, several comments in literature exist doubting the existence of MOR in cardiac tissue. We, therefore, aimed to detect MOR in rat left ventricular cardiomyocytes, and to evaluate whether MOR and POMC are regulated during heart failure. After IRB approval, heart failure was induced using a modified infrarenal aortocaval fistula (ACF) in male Wistar rats. All rats of the control and ACF group were characterized by their morphometrics and hemodynamics and the existence of MOR and POMC was investigated by means of radioligand binding, double immunofluorescence confocal analysis, RT-PCR and Western blot. Membrane MOR selective binding sites were detected in the left ventricular myocardium, however, they were lower in abundance than KOR- and DOR-specific binding sites and B max of MOR could not be determined. In left ventricular cardiomyocytes, MOR colocalized with parts of the excitation-coupling mechanism, e.g., Cav1.2 of the cell membrane and invaginated T-tubules as well as the ryanodine receptor of the sarcoplasmatic reticulum. More importantly, MOR strongly colocalized with mitochondria of left ventricular cardiomyocytes. Volume overload was not associated with an altered expression of MOR and POMC on both mRNA and protein level. These findings provide evidence for the existence of MOR on the cell membrane, sarcoplasmatic reticulum and mitochondria in left ventricular cardiomyocytes in rats. However, heart failure does not result in an altered expression of the cardiac MOR-opioid system. Thus, MOR agonist treatment-commonly used in the clinical setting-might directly affect cardiac function, which needs to be evaluated in greater detail in the near future.

PPARγ Agonist Ameliorates the Impaired Fluidity of the Myocardial Cell Membrane and Cardiac Injury in Hypercholesterolemic Rats.

Hypercholesterolemia can increase the risk of cardiac injury, but the underlying mechanisms are not fully understood. The present study aimed to determine whether changes in the fluidity of the cardiomyocyte membrane may contribute to the increased susceptibility to myocardial ischemia/reperfusion (MI/R) injury observed in hypercholesterolemic rats. Male Wistar rats were fed a normal (n=24) or high-cholesterol diet (n=32) for 10weeks. At the 6th week, the rats in the high-cholesterol diet group were treated with vehicle (n=16, HC+V) or pioglitazone (n=16, HC+PIO), a peroxisome proliferator-activated receptor-γ (PPARγ) agonist, and treatment lasted for the next 4weeks. Rats in HC+V group displayed less membrane fluidity, a greater membrane cholesterol-to-phospholipid ratio (C/P), less Na+-K+-ATPase activity, and less cAMP content in their myocardial cells than rats fed a normal diet. A strong positive correlation was observed between membrane fluidity and cardiac injury, i.e., the myocardial infarct size when subjected to MI/R (30min/24h). Treatment with PIO restored much of the lost hypercholesterolemia-induced myocardial cell membrane fluidity, decreased membrane C/P ratio, increased Na+-K+-ATPase activity and cardiac cell cAMP content, improved cardiac function, and reduced the sizes of myocardial infarcts. Results demonstrated that hypercholesterolemia-induced decreased myocardial cell membrane fluidity may contribute to the increased susceptibility to cardiac injury, and PPARγ agonists may have therapeutic value in patients with hypercholesterolemia.

Abstract 16791: Oral Administration of Synthetic Prostacyclin Agonist Ono-1301 Prevents Deterioration of Cardiac Performance by Preserving Cardiac Structural Proteins in the Delta-sarcoglycan-deficient Hamster: Promising Treatment for Patients With DCM

Background: Although drugs such as beta blockers are reported to be effective for dilated cardiomyopathy (DCM), they are only effective in selected patients, and new therapeutics based on different mechanisms are still needed. We hypothesized that synthetic prostacyclin agonist (ONO-1301) would preserve the structural proteins in distressed cardiomyocytes, thus attenuating the deterioration of cardiac performance in the delta sarcoglycan-deficient hamster. Methods and Results: In an in vitro study, ONO-1301 was added to the culture medium of non-myocyte cells and the expression of several cytokines including VEGF was examined by ELISA. For the in vivo study, ONO-1301 (A: 3, n=5, B: 1, n=6, C: 0.3, n=7, D: 0.1 mg/kg/dose, n=7) was orally administered to delta-sarcoglycan-deficient hamsters every day from 8 to 20 weeks after birth. The control group (n=6) was untreated. Functional performance was evaluated by US before, 2, 4, 6, and 8 weeks after administration of ONO-1301. A histological analysis focusing on basement-membrane structural proteins, such as α-sarcoglycan, β-sarcoglycan, and α-dystroglycan, fibrosis, and hypertrophy was performed. In vitro, ONO-1301 enhanced the cytokine expressions dose dependently. In vivo, systolic function (EF) was gradually increased in the ONO-1301-treated groups, while it showed significant deterioration in the control group (45.7±2.9, 42.0±2.6, 32.3±1.0, 30.3±2.5, 26.3±2.7%, in groups A, B, C, D, and control, respectively, P<0.05). ONO-1301 also significantly attenuated the LV dilatation in a dose-dependent manner. Immunostaining revealed that all the cardiac structural proteins were preserved in the basement membrane of cardiomyocytes in the 3.0 mg/kg ONO-1301 treatment group, while they disappeared in the control. The % fibrosis and cell hypertrophy were significantly attenuated in the ONO-1301-treated group compared with the control. Conclusions: Oral administration of synthetic prostacyclin agonists, which can enhance cytokine expressions in non-myocyte cells, may preserve cardiac structural proteins in the delta sarcoglycan-deficient hamster, to maintain cardiac performance. This promising drug may inhibit the progression of the disease pathology in DCM, prolonging their life.

Abstract 14952: Disruption of a Newly Identified Complex Between soluble Guanylyl Cyclase and Connexin 43 is Potentially Linked to Cardiac Contractile Dysfunction

Background: The role of nitric oxide (NO) in regulation of excitation-contraction (EC) coupling remains controversial. NO activates soluble Guanylyl Cyclase (sGC), a heme-containing heterodimer (α/ß subunits), to increase cGMP production and regulate downstream targets such as Protein Kinase G (PKG) and phosphodiesterases (PDE). Intercalated discs (ID) are essential for electro-mechanical coupling between cardiomyocytes. Connexin 43 (Cx43) via formation of gap junctions at the ID mediate the electrical coupling. Cx43 targeting and assembly at the ID involved phosphorylation by many protein kinases, including cAMP-activated PKA. In various cardiomyopathies such as arrhythmia, Cx43 is relocated from ID to the lateral membrane of cardiomyocytes. Recently, we discovered that sGCβ is localized at the ID with Cx43 and PDE3A, using immunofluorescence (IF) and Triton/Urea heart fractionation. Importantly, we showed by co-immunoprecipitation (co-IP) that sGC physically interacts with Cx43 in the heart. Hypothesis: Cx43 assembly and function at the ID through PKA-dependent Cx43 phosphorylation depends on NO-sGC-cGMP microdomain inducing inhibition of PDE3A. Methods and Results: To assess the role and relevance of the sGC-Cx43 complex, we used Angiotensin II (AngII) to induce cardiac hypertrophy and arrhythmia in wild-type (WT) mice and sGCα-KO mice, which are depleted for sGC activity. Co-IP indicates that sGC-Cx43 complex is decreased in AngII-treated WT (n=5), independently of decreased Cx43 expression and IF showed Cx43 lateralization. NO-dependent cGMP in membrane fraction of AngII-hearts was decreased by 50%. Conversely, in sGCα-KO mice Cx43-sGC complex was reduced and even more so with AngII treatment. In addition to a significant increased cardiac hypertrophy in sGCα-KO mice (heart to body weight ratio KO: 5.16±0.19 vs WT: 4.45±0.04, n=4-8, P<0.05), preliminary EKG (n=4) indicates that sGCα-KO mice suffer from cardiac arrhythmia with, intriguingly, alteration in P waves. Conclusion: Altogether, our data suggest that sGC via modulation of Cx43 location at ID is potentially involved in EC regulation. We currently explore the mechanism underlying these observations with in vivo, biochemical approaches and imaging.

Abstract 15441: MURC/Cavin-4 Requires the Coiled-coil Domain for Membrane Trafficking of Caveolin-3 in Cardiomyocytes

[Background] Muscle-restricted coiled-coil protein (MURC), also referred to as Cavin-4, is a member of the cavin family that is cooperated with caveolins in caveola formation and function. Cavins are cytoplasmic proteins that have coiled-coil domains and form heteromeric complexes, which are recruited to caveolae in cells expressing caveolins. Among caveolins, caveolin-3 (Cav3) is exclusively expressed in muscle cells, similar to MURC/Cavin-4. In the heart, Cav3 overexpression contributes to cardiac protection, while its deficiency leads to progressive cardiomyopathy. Mutations in the MURC/Cavin-4 gene have been identified in patients with dilated cardiomyopathy. MURC/Cavin-4 has the coiled-coil domain; however, its functional significance remains unknown. [Methods and Results] MURC/Cavin-4 was localized at the plasma membrane in H9c2 cells, while a MURC/Cavin-4 mutant lacking the coiled-coil domain (ΔCC) was primarily localized to the cytoplasm. ΔCC bound to Cav3 and impaired membrane localization of Cav3 in cardiomyocytes. In addition, although ΔCC did not influence Cav3 mRNA expression, it decreased the Cav3 protein level in cardiomyocytes. MURC/Cavin-4 and ΔCC similarly induced cardiomyocytes hypertrophy; however, ΔCC showed higher hypertrophy-related fetal gene expression, such as ANP, BNP, and skeletal α-actin (SkA) mRNA expression and the βMHC/αMHC ratio, than MURC/Cavin-4. ΔCC induced ERK activation in cardiomyocytes. Cardiac function assessed by echocardiography was significantly impaired in transgenic mice expressing ΔCC in the heart (ΔCC-Tg mice) compared with non-transgenic (NTg) mice (% FS; 24.6 ± 2.4 % in ΔCC-Tg mice vs. 48.8 ± 2.5 % in NTg mice, p<0.01), which was accompanied by cardiomyocyte hypertrophy, increased interstitial fibrosis, and the increase of lung weight-to-tibial-length ratio (LW/TL; 18.04 ± 2.41 in ΔCC-Tg mice vs. 7.51 ± 0.81 in NTg mice, p<0.01). ΔCC-Tg hearts showed reduction of the Cav3 protein level and the activation of ERK. [Conclusions] These results reveal that MURC/Cavin-4 requires its coiled-coil domain to target the plasma membrane and Cav3 trafficking to the plasma membrane of cardiomyocytes, and that MURC/Cavin-4 functions as a crucial caveolar component to regulate cardiac function.

Cirrhotic Cardiomyopathy; Pathophysiology and Clinical Approach

Cardiac dysfunction is frequently observed in patients with cirrhosis. Cirrhotic cardiomyopathy (CCM) has recently been identified as an entity independent of the cirrhosis etiology. Increased cardiac output due to hyperdynamic circulation, systolic and diastolic left ventricular dysfunction and certain electrophysiological abnormalities are the pathophysiological features of the disease. The main underlying mechanisms are complex, including the impaired β-receptor and calcium signaling, altered cardiomyocyte membrane physiology, elevated sympathetic nervous tone and increased activity of vasodilatory pathways. CCM is characterized by impaired cardiac response to physical, physiological and pharmacological stress. Currently, no specific therapy has proved effective yet. Echocardiography and electrocardiography are the corner stones for diagnosis. In this review, we discuss in brief the pathophysiological bachground and clinical features of cirrhotic cardiomyopathy, diagnosis and the currently available treatment options.

The coiled-coil domain of MURC/cavin-4 is involved in membrane trafficking of caveolin-3 in cardiomyocytes.

Muscle-restricted coiled-coil protein (MURC), also referred to as cavin-4, is a member of the cavin family that works cooperatively with caveolins in caveola formation and function. Cavins are cytoplasmic proteins with coiled-coil domains and form heteromeric complexes, which are recruited to caveolae in cells expressing caveolins. Among caveolins, caveolin-3 (Cav3) is exclusively expressed in muscle cells, similar to MURC/cavin-4. In the heart, Cav3 overexpression contributes to cardiac protection, and its deficiency leads to progressive cardiomyopathy. Mutations in the MURC/cavin-4 gene have been identified in patients with dilated cardiomyopathy. In the present study, we show the role of MURC/cavin-4 as a caveolar component in the heart. In H9c2 cells, MURC/cavin-4 was localized at the plasma membrane, whereas a MURC/cavin-4 mutant lacking the coiled-coil domain (ΔCC) was primarily localized to the cytoplasm. ΔCC bound to Cav3 and impaired membrane localization of Cav3 in cardiomyocytes. Additionally, although ΔCC did not alter Cav3 mRNA expression, ΔCC decreased the Cav3 protein level. MURC/cavin-4 and ΔCC similarly induced cardiomyocyte hypertrophy; however, ΔCC showed higher hypertrophy-related fetal gene expression than MURC/cavin-4. ΔCC induced ERK activation in cardiomyocytes. Transgenic mice expressing ΔCC in the heart (ΔCC-Tg mice) showed impaired cardiac function accompanied by cardiomyocyte hypertrophy and marked interstitial fibrosis. Hearts from ΔCC-Tg mice showed a reduction of the Cav3 protein level and activation of ERK. These results suggest that MURC/cavin-4 requires its coiled-coil domain to target the plasma membrane and to stabilize Cav3 at the plasma membrane of cardiomyocytes and that MURC/cavin-4 functions as a crucial caveolar component to regulate cardiac function.

Effect of L-arginine and carnitine on cathepsin L and H activity and lysosomal membranes permeability in myocardium in expressed hyperhomocysteinemia

Aim. To study the activity of lysosomal cysteine proteases (cathepsins L, H) and acid phosphatase, changing of permeability, stability of myocardial lysosomal membranes in rats in experimental expressed hyperhomocysteinemia model, and while administering L-arginine and carnitine. Methods. The study was performed on male Wistar rats kept on standard vivarium conditions divided into three control and three experimental groups of 8 animals each. Experimental samples were administered methionine, or combination of L-arginine and carnitine with methionine. The level of serum homocysteine was measured by ELISA. Cathepsin L and H activity was detected by spectrofluorimetric method. Acid phosphatase activity was recorded using the «end point» method. Results. In the model of expressed hyperhomocysteinemia the increase of cathepsin H total activity due to both lysosomal and nonlysosomal fractions was found. These changes were observed along with the general increase of lysosomal membranes permeability. When correcting hyperhomocysteinemia with L-arginine and carnitine a decrease of cathepsin L and H levels was noted as well as positive effect on the myocardial lysosomal membranes stability. Conclusion. Expressed hyperhomocysteinemia is accompanied by statistically significant increase of both lysosomal and cytoplasmic fractions of the cathepsin H activity, indicating the lysosomal membranes permeabilisation phenomenon; L-carnitine and arginine correct hyperhomocysteinaemia effects, leading to cathepsin L and H reduced activity and having a stabilizing effect on the lysosomal membranes of cardiomyocytes.

Cardiac‐Specific Disruption of Bin1 in Mice Enables a Model of Stress‐ and Age‐Associated Dilated Cardiomyopathy

Non-compensated dilated cardiomyopathy (DCM) leading to death from heart failure is rising rapidly in developed countries due to aging demographics, and there is a need for informative preclinical models to guide the development of effective therapeutic strategies to prevent or delay disease onset. In this study, we describe a novel model of heart failure based on cardiac-specific deletion of the prototypical mammalian BAR adapter-encoding gene Bin1, a modifier of age-associated disease. Bin1 deletion during embryonic development causes hypertrophic cardiomyopathy and neonatal lethality, but there is little information on how Bin1 affects cardiac function in adult animals. Here we report that cardiomyocyte-specific loss of Bin1 causes age-associated dilated cardiomyopathy (DCM) beginning by 8-10 months of age. Echocardiographic analysis showed that Bin1 loss caused a 45% reduction in ejection fraction during aging. Younger animals rapidly developed DCM if cardiac pressure overload was created by transverse aortic constriction. Heterozygotes exhibited an intermediate phenotype indicating Bin1 is haplo-insufficient to sustain normal heart function. Bin1 loss increased left ventricle (LV) volume and diameter during aging, but it did not alter LV volume or diameter in hearts from heterozygous mice nor did it affect LV mass. Bin1 loss increased interstitial fibrosis and mislocalization of the voltage-dependent calcium channel Cav 1.2, and the lipid raft scaffold protein caveolin-3, which normally complexes with Bin1 and Cav 1.2 in cardiomyocyte membranes. Our findings show how cardiac deficiency in Bin1 function causes age- and stress-associated heart failure, and they establish a new preclinical model of this terminal cardiac disease.

Dimensions of compartments and membrane surfaces in the intact rabbit heart of importance in studies on intramyocardial transfer of blood-borne substances.

Cardiac studies on the uptake, storage and intramyocardial transfer of blood-borne substances require detailed information on the geometric ultrastructural dimensions of myocardial compartments and parts thereof, and the membranes separating these compartments. Such a specific ultrastructural set of data of the heart is yet lacking. In the present study, we quantitatively assessed these dimensions in glutaraldehyde-perfusion fixed rabbit hearts by means of histological and tailored mathematical techniques. We showed the true ellipsoid nature of the myocardial capillary cross section and estimated the mean capillary diameter dcap. After correction for the ellipsoid shape, dcap was found to be 5.21±1.41 µm. Effective widths of the endothelial cell and the pericapillary interstitium (is1), dimensions of importance in diffusion, amounted to 187±7 and 160±10 nm, respectively. The fractional volume of the large vessels (arteries and veins larger than 10 µm), capillaries, endothelium, is1, cardiomyocytes, non-pericapillary interstitium is2, t-tubular compartment and interstitial cells amounted on average to 5.92%, 9.36%, 1.83%, 1.94%, 73.07%, 5.97%, 0.95% and 0.96%, respectively, of total myocardial volume, defined as the cardiac tissue volume, the large blood vessels included. Normalized to total myocardial volume, the surface area of the luminal and abluminal endothelial membranes and of the cardiomyocyte membrane opposing the endothelial cells amounted to 75.2±5.5·10³, 82.2±6.0·10³ and 89.1±6.5·10³ m²/m³, respectively. The present study provides quantitative information about ultrastructural dimensions of the adult rabbit heart, among others, of importance for studies on cardiac uptake, and intramyocardial transfer and storage of blood-supplied substances.

Effects of rotigaptide (ZP123) on connexin43 remodeling in canine ventricular fibrillation.

The present study investigated the effects of rotigaptide (ZP123) on the expression, distribution and phosphorylation of connexin43 (Cx43) in myocardial cell membranes in cardioversion of ventricular fibrillation (VF). A model of prolonged VF (8, 12 and 30 min) was established in mongrel dogs (n=8/group), following treatment with ZP123 or normal saline (NS control). A sham control was included. Cardiopulmonary resuscitation was begun at the start of VF followed by defibrillation. Animals received a maximum of three defibrillations of increasing energy (70, 100 and 150 J biphasic shock) as required. The average defibrillation energy, defibrillation success rate, return of spontaneous circulation and survival rate were recorded. Cx43 and phosphorylated (p-) Cx43 expression in cardiomyocyte membranes was detected by western blot and immunofluorescence analyses. Compared with the NS-treated control groups, the success defibrillation rate in the 8-min and 12-min ZP123 groups was significantly higher (P<0.05), while the average defibrillation energy was significantly lower (P<0.05). Cx43 expression in the VF groups was significantly lower than that in the sham control group (P<0.05). Cx43 expression was higher in the 12-min and 30-min ZP123 groups than that in the NS control group (P<0.05), while p-Cx43 expression decreased, although the levels were significantly higher than those in the control groups (P<0.05). Cx43 expression was positively correlated with the defibrillation success rate (r=0.91; P<0.01) and negatively with the mean defibrillation energy (r=−0.854; P<0.01), while p-Cx43 expression was positively correlated with the success rate of the previous three defibrillations (r=0.926; P<0.01). In conclusion, ZP123 reduced Cx43 remodeling through regulating the expression, distribution and phosphorylation of Cx43, thereby reducing the defibrillation energy required for successful cardioversion.

Abstract 203: Cardiomyocyte T-tubule Membrane Turns Over, Releasing BIN1 Containing Microparticles into Blood

Bridging integrator 1 (BIN1) is a cardiac muscle protein that folds cardiomyocyte T-tubule membrane. BIN1 is intrinsic to cardiac health, and is reduced in acquired heart failure. Interestingly, we have found that BIN1 is also blood available, and that plasma BIN1 correlates with cardiac function, suggesting cardiac origin of plasma BIN1. We found that low plasma BIN1 correlates with failing muscle and predicts ventricular arrhythmia. However, the paradigm does not exist for an intracellular membrane associate cardiomyocyte protein to be homeostatically turned over into blood. In this study, using a mouse model with cardiac specific deletion of Bin1 gene, we identified with biochemical techniques that plasma BIN1 levels directly correlate with cardiac tissue BIN1 levels, indicating cardiac origin. Furthermore, investigations using both super-resolution fluorescent imaging and flow cytometry analysis revealed that adult ventricular cardiomyocytes constantly release BIN1 into blood via membrane microparticle production. Microparticles are small membrane vesicles shed from plasma membrane of a variety of cell types including platelets, leukocytes, and endothelial cells. Using super-resolution three-dimensional stochastic optical reconstruction microscopy (3D-STORM), we found similar to the blood cells, isolated adult mouse cardiomyocytes release Annexin V positive microparticles with diameters ranging between 0.1 to 1.0 μm. These microparticles also carry BIN1 protein. Flow cytometry was also used to detect and quantify microparticles &lt;1.0 μm in size from medium bathing a pure population of adult mouse cardiomyocytes. BIN1 microparticle release is proportional to actin stability and amount of T-tubule membrane folds. Compared to wild type cardiomyocytes, microparticle release is significantly reduced from myocytes with heterozygous deletion of Bin1 gene. These data indicate that cardiomyocyte membrane undergoes dynamic turnover, releasing T-tubule folds into blood as microparticles. Furthermore, plasma BIN1 can be used as a direct measure of cardiomyocyte health and reserve.

A high ratio of arachidonic acid to eicosapentaenoic acid in red blood cells is associated with worse long-term ventricular function after acute myocardial infarction

Aim: It is believed that regular fish intake promotes cardiac health through its n-3 fatty acid (eicosapentaenoic [EPA] and docosahexaenoic [DHA]) content. EPA and DHA are readily incorporated into phospholipids of cardiomyocyte membranes, partially replacing arachidonic acid (AA). A decreased AA/EPA ratio is suggested to generate a cardioprotective eicosanoid profile upon ischemia, but clinical data are scarce.