Search Results (26)

Cardiomyocyte hypertrophy is regulated by several factors, including the ADP-ribosylation factor (Arf) family of small G proteins, among others. For instance, ArfGAP with dual pleckstrin homology domains 1 (Adap1) exerts an anti-hypertrophic effect in cultured cardiomyocytes. Its homologous protein, Adap2, is also expressed in the heart but its role remains elusive. To elucidate its function, we investigated the effects of adenoviral-mediated overexpression of Adap2 in cultured neonatal rat ventricular myocytes under both basal and pro-hypertrophic conditions, employing a range of microscopy and biochemical techniques. Despite minimal detection in neonatal rat hearts, Adap2 was found to be well expressed in adult rat hearts, being predominantly localized at the membrane fraction. In contrast to Adap1, overexpression of Adap2 provokes the robust accumulation of β1-integrin at the cellular surface of cultured cardiomyocytes. Interestingly, overexpressed Adap2 relocalizes at the sarcolemma and increases the size of cardiomyocytes upon phenylephrine stimulation, despite attenuating Erk1/2 phosphorylation and Nppa gene expression. Under these same conditions, cardiomyocytes overexpressing Adap2 also express higher level of detyrosinated tubulin, a marker of hypertrophic response. These findings provide new insights into the pro-hypertrophic function of Adap2 in cardiomyocytes. Full article

Cardioprotection against ischemia is achieved using openers of mitochondrial ATP-sensitive K⁺ (mitoKATP) channels such as diazoxide (DZX), leading to pharmacological preconditioning (PPC). We previously reported that PPC decreases the abundance of ventricular Cav1.2 channels, but PPC’s effects on other channels remain largely unexplored. In this study, we hypothesized that DZX regulates the expression of hyperpolarization-activated cyclic nucleotide potassium channel 4 (HCN4) channels in sinoatrial node cells (SANCs), the specialized cardiomyocytes that generate the heartbeat. DZX increased the heart rate in intact adult rats. Patch-clamp experiments revealed an increase in the magnitude of ionic currents through HCN4 channels, which was abolished by the reactive oxygen species (ROS) scavenger N-acetylcysteine (NAC) and the selective mitoKATP channel inhibitor 5-hydroxydecanoate (5-HD). Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) and Western blot assays showed that DZX increased HCN4 channel expression at the mRNA and protein levels. Immunofluorescence analyses revealed that PPC increased HCN4 fluorescence, which was abolished by NAC. DZX increased nuclear translocation of c-Fos and decreased protein abundance of RE1 silencing transcription factor (REST)/neuron-restrictive silencer factor (NRSF), suggesting the involvement of these factors. Our results suggest that PPC increases the heart rate by upregulating HCN4 channel expression through a mechanism involving c-Fos, REST, and ROS. Full article

17β-estradiol (E2) is the most active metabolite of estrogen with a wide range of physiological action on cardiac muscle. Previous studies have reported E2 effects predominantly for the ventricles, while the E2 impact on the atria has been less examined. In this study, we focused on the direct E2 effects on atrial and ventricular contractility at the cellular and molecular levels. Single atrial and ventricular cardiomyocytes (CM) from adult (24 weeks-old) female Wistar rats were incubated with 10 nM E2 for 15 min. Sarcomere length and cytosolic [Ca²⁺]_i transients were measured in mechanically non-loaded CM, and the tension–length relationship was studied in CM mechanically loaded by carbon fibers. The actin–myosin interaction and sarcomeric protein phosphorylation were analyzed using an in vitro motility assay and gel electrophoresis with Pro-Q Diamond phosphoprotein stain. E2 had chamber-specific effects on the contractile function of CM with a pronounced influence on ventricular CM. The characteristics of [Ca²⁺]_i transients did not change in both atrial and ventricular CM. However, in ventricular CM, E2 reduced the amplitude and maximum velocity of sarcomere shortening and decreased the slope of the passive tension–length relationship that was associated with increased TnI and cMyBP-C phosphorylation. E2 treatment accelerated the cross-bridge cycle of both atrial and ventricular myosin that was associated with increased phosphorylation of the myosin essential light chain. This study shows that E2 impairs the mechanical function of the ventricular myocardium while atrial contractility remains mostly preserved. Hormonal replacement therapy (HRT) with estrogen is by far the most effective therapy for treating climacteric symptoms experienced during menopause. Here we found a chamber specificity of myocardial contractile function to E2 that should be taken into account for the potential side effects of HRT. Full article

Congenital proximal renal tubular acidosis (pRTA) is a rare systemic disease caused by mutations in the SLC4A4 gene that encodes the electrogenic sodium bicarbonate cotransporter, NBCe1. The major NBCe1 protein variants are designated NBCe1-A, NBCe1-B, and NBCe1-C. NBCe1-A expression is kidney-specific, NBCe1-B is broadly expressed and is the only NBCe1 variant expressed in the heart, and NBCe1-C is a splice variant of NBCe1-B that is expressed in the brain. No cardiac manifestations have been reported from patients with pRTA, but studies in adult rats with virally induced reduction in cardiac NBCe1-B expression indicate that NBCe1-B loss leads to cardiac hypertrophy and prolonged QT intervals in rodents. NBCe1-null mice die shortly after weaning, so the consequence of congenital, global NBCe1 loss on the heart is unknown. To circumvent this issue, we characterized the cardiac function of NBCe1-B/C-null (KO_b/c) mice that survive up to 2 months of age and which, due to the uninterrupted expression of NBCe1-A, do not exhibit the confounding acidemia of the globally null mice. In contrast to the viral knockdown model, cardiac hypertrophy was not present in KO_b/c mice as assessed by heart-weight-to-body-weight ratios and cardiomyocyte cross-sectional area. However, echocardiographic analysis revealed reduced left ventricular ejection fraction, and intraventricular pressure–volume measurements demonstrated reduced load-independent contractility. We also observed increased QT length variation in KO_b/c mice. Finally, using the calcium indicator Fura-2 AM, we observed a significant reduction in the amplitude of Ca²⁺ transients in paced KO_b/c cardiomyocytes. These data indicate that congenital, global absence of NBCe1-B/C leads to impaired cardiac contractility and increased QT length variation in juvenile mice. It remains to be determined whether the cardiac phenotype in KO_b/c mice is influenced by the absence of NBCe1-B/C from neuronal and endocrine tissues. Full article

The developmental changes in the excitation–contraction mechanisms of the ventricular myocardium of small animals (guinea pig, rat, mouse) and their sympathetic regulation will be summarized. The action potential duration monotonically decreases during pre- and postnatal development in the rat and mouse, while in the guinea pig it decreases during the fetal stage but turns into an increase just before birth. Such changes can be attributed to changes in the repolarizing potassium currents. The T-tubule and the sarcoplasmic reticulum are scarcely present in the fetal cardiomyocyte, but increase during postnatal development. This causes a developmental shift in the Ca²⁺ handling from a sarcolemma-dependent mechanism to a sarcoplasmic reticulum-dependent mechanism. The sensitivity for beta-adrenoceptor-mediated positive inotropy decreases during early postnatal development, which parallels the increase in sympathetic nerve innervation. The alpha-adrenoceptor-mediated inotropy in the mouse changes from positive in the neonate to negative in the adult. This can be explained by the change in the excitation–contraction mechanism mentioned above. The shortening of the action potential duration enhances trans-sarcolemmal Ca²⁺ extrusion by the Na⁺-Ca²⁺ exchanger. The sarcoplasmic reticulum-dependent mechanism of contraction in the adult allows Na⁺-Ca²⁺ exchanger activity to cause negative inotropy, a mechanism not observed in neonatal myocardium. Such developmental studies would provide clues towards a more comprehensive understanding of cardiac function. Full article

Store-operated calcium entry (SOCE) in cardiomyocytes may be involved in cardiac remodeling, but the underlying mechanisms remain elusive. We hypothesized that SOCE may increase nuclear calcium, which alters gene expression via calcium/calmodulin-dependent enzyme signaling, and elucidated the underlying cellular mechanisms. An experimental protocol was established in isolated adult rat cardiomyocytes to elicit SOCE by re-addition of calcium following complete depletion of sarcoplasmic reticulum (SR) calcium and to quantify SOCE in relation to the electrically stimulated calcium transient (CaT) measured in the same cell before SR depletion. Using confocal imaging, calcium changes were recorded simultaneously in the cytosol and in the nucleus of the cell. In ventricular myocytes, SOCE was observed in the cytosol and nucleus amounting to ≈15% and ≈25% of the respective CaT. There was a linear correlation between the SOCE-mediated calcium increase in the cytosol and nucleus. Inhibitors of TRPC or Orai channels reduced SOCE by ≈33–67%, whereas detubulation did not. In atrial myocytes, SOCE with similar characteristics was observed in the cytosol and nucleus. However, the SOCE amplitudes in atrial myocytes were ≈two-fold larger than in ventricular myocytes, and this was associated with ≈1.4- to 3.6-fold larger expression of putative SOCE proteins (TRPC1, 3, 6, and STIM1) in atrial tissue. The results indicated that SOCE in atrial and ventricular myocytes is able to cause robust calcium increases in the nucleus and that both TRPC and Orai channels may contribute to SOCE in adult cardiomyocytes. Full article

Background and Objectives: This experiment evaluated the preventative influence of the tomato-derived Esculeoside A (ESA) on diabetic cardiomyopathy in type 1 diabetes mellitus (T1DM) in rats induced by streptozotocin (STZ). It also examined whether the activation of Nrf2 signaling affords this protection. Materials and Methods: Adult male Wistar control nondiabetic rats and rats with T1DM (STZ-T1DM) were given either carboxymethylcellulose as a vehicle or ESA (100 mg/kg) (eight rats/group) orally daily for 12 weeks. A group of STZ-T1DM rats was also treated with 100 mg/kg ESA and co-treated i.p. with 2 mg/kg (twice/week), brusatol, and Nrf2 inhibitors for 12 weeks. Results and Conclusions: Treatment with ESA prevented the gain in heart weight and cardiomyocyte hypertrophy and improved the left ventricular (LV) systolic and diastolic function (LV) in the STZ-T1DM rat group. Likewise, it reduced their serum levels of triglycerides, cholesterol, and low-density lipoproteins (LDL-c), as well as their LV mRNA, cytoplasmic total, and nuclear total levels of NF-κB. ESA also reduced the total levels of malondialdehyde, tumor necrosis factor-α, interleukine-6 (IL-6), Bax, cytochrome-c, and caspase-3 in the LV of the STZ-T1DM rats. In parallel, ESA enhanced the nuclear and cytoplasmic levels of Nrf2 and the levels of superoxide dismutase, glutathione, and heme oxygenase-1, but decreased the mRNA and cytoplasmic levels of keap-1 in the LVs of the STZ-T1DM rats. Interestingly, ESA did not affect the fasting insulin and glucose levels of the diabetic rats. All of these beneficially protective effects of ESA were not seen in the ESA-treated rats that received brusatol. In conclusion, ESA represses diabetic cardiomyopathy in STZ-diabetic hearts by activating the Nrf2/antioxidant/NF-κB axis. Full article

Our previous studies revealed the protection of stachydrine hydrochloride (STA) against cardiopathological remodeling. One of the underlying mechanisms involves the calcium/calmodulin-dependent protein kinase Ⅱ (CaMKII). However, the way STA influences CaMKII needs to be further investigated. The nicotinamide adenine dinucleotide phosphate oxidase 2 (NOX2)-coupled reactive oxygen species (ROS) overproduction putatively induces the oxidative activation of CaMKII, resulting in the occurrence of pathological cardiac remodeling and dysfunction in experimental models of mice. Thus, in this study, we assessed the role of the NOX2-ROS signal axis in STA cardioprotection. The transverse aortic constriction (TAC)-induced heart failure model of mice, the phenylephrine-induced hypertrophic model of neonatal rat primary cardiomyocytes, and the H₂O₂-induced oxidative stress models of adult mouse primary cardiomyocytes and H9c2 cells were employed. The echocardiography and histological staining were applied to assess the cardiac effect of STA (6 mg/kg/d or 12 mg/kg/d), which was given by gavage. NOX2, ROS, and excitation-contraction (EC) coupling were detected by Western blotting, immunofluorescence, and calcium transient-contraction synchronous recordings. ROS and ROS-dependent cardiac fibrosis were alleviated in STA-treated TAC mice, demonstrating improved left ventricular ejection fraction and hypertrophy. In the heart failure model of mice and the hypertrophic model of cardiomyocytes, STA depressed NOX2 protein expression and activation, as shown by inhibited translocation of its phosphorylation, p67phox and p47phox, from the cytoplasm to the cell membrane. Furthermore, in cardiomyocytes under oxidative stress, STA suppressed NOX2-related cytosolic Ca²⁺ overload, enhanced cell contractility, and decreased Ca²⁺-dependent regulatory protein expression, including CaMKⅡ and Ryanodine receptor calcium release channels. Cardioprotection of STA against pressure overload-induced pathological cardiac remodeling correlates with the NOX2-coupled ROS signaling cascade. Full article

Serotonin effects on cardiac hypertrophy, senescence, and failure are dependent either on activation of specific receptors or serotonin uptake and serotonin degradation by monoamine oxidases (MAOs). Receptor-dependent effects are specific for serotonin, but MAO-dependent effects are nonspecific as MAOs also metabolize other substrates such as catecholamines. Our study evaluates the role of MAO-A in serotonin- and norepinephrine-dependent cell damage. Experiments were performed in vivo to study the regulation of MAOA and MAOB expression and in vitro on isolated cultured adult rat ventricular cardiomyocytes (cultured for 24 h) to study the function of MAO-A. MAOA but not MAOB expression increased in maladaptive hypertrophic stages. Serotonin and norepinephrine induced morphologic cell damage (loss of rod-shaped cell structure). However, MAO-A inhibition suppressed serotonin-dependent but not norepinephrine-dependent damages. Serotonin but not norepinephrine caused a reduction in cell shortening in nondamaged cells. Serotonin induced mitochondria-dependent oxidative stress. In vivo, MAOA was induced during aging and hypertension but the expression of the corresponding serotonin uptake receptor (SLC6A4) was reduced and enzymes that reduce either oxidative stress (CAT) or accumulation of 5-hydroxyindolacetaldehyde (ALDH2) were induced. In summary, the data show that MAO-A potentially affects cardiomyocytes’ function but that serotonin is not necessarily the native substrate. Full article

Cyclic nucleotide phosphodiesterases 2A (PDE2A) and PDE3A play an important role in the regulation of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP)-to-cAMP crosstalk. Each of these PDEs has up to three distinct isoforms. However, their specific contributions to cAMP dynamics are difficult to explore because it has been challenging to generate isoform-specific knock-out mice or cells using conventional methods. Here, we studied whether the CRISPR/Cas9 approach for precise genome editing can be used to knock out Pde2a and Pde3a genes and their distinct isoforms using adenoviral gene transfer in neonatal and adult rat cardiomyocytes. Cas9 and several specific gRNA constructs were cloned and introduced into adenoviral vectors. Primary adult and neonatal rat ventricular cardiomyocytes were transduced with different amounts of Cas9 adenovirus in combination with PDE2A or PDE3A gRNA constructs and cultured for up to 6 (adult) or 14 (neonatal) days to analyze PDE expression and live cell cAMP dynamics. A decline in mRNA expression for PDE2A (~80%) and PDE3A (~45%) was detected as soon as 3 days post transduction, with both PDEs being reduced at the protein level by >50–60% in neonatal cardiomyocytes (after 14 days) and >95% in adult cardiomyocytes (after 6 days). This correlated with the abrogated effects of selective PDE inhibitors in the live cell imaging experiments based on using cAMP biosensor measurements. Reverse transcription PCR analysis revealed that only the PDE2A2 isoform was expressed in neonatal myocytes, while adult cardiomyocytes expressed all three PDE2A isoforms (A1, A2, and A3) which contributed to the regulation of cAMP dynamics as detected by live cell imaging. In conclusion, CRISPR/Cas9 is an effective tool for the in vitro knock-out of PDEs and their specific isoforms in primary somatic cells. This novel approach suggests distinct regulation of live cell cAMP dynamics by various PDE2A and PDE3A isoforms in neonatal vs. adult cardiomyocytes. Full article

Cardiolipin (CL), the major mitochondrial phospholipid, regulates the activity of many mitochondrial membrane proteins. CL composition is shifted in heart failure with decreases in linoleate and increases in oleate side chains, but whether cardiolipin composition directly regulates metabolism is unknown. This study defines cardiolipin composition in rat heart and liver at three distinct ages to determine the influence of CL composition on beta-oxidation (ß-OX). CL species, expression of ß-OX and glycolytic genes, and carnitine palmitoyltransferase (CPT) activity were characterized in heart and liver from neonatal, juvenile, and adult rats. Ventricular myocytes were cultured from neonatal, juvenile, and adult rats and cardiolipin composition and CPT activity were measured. Cardiolipin composition in neonatal rat ventricular cardiomyocytes (NRVMs) was experimentally altered and mitochondrial respiration was assessed. Linoleate-enrichment of CL was observed in rat heart, but not liver, with increasing age. ß-OX genes and CPT activity were generally higher in adult heart and glycolytic genes lower, as a function of age, in contrast to liver. Palmitate oxidation increased in NRVMs when CL was enriched with linoleate. Our results indicate (1) CL is developmentally regulated, (2) linoleate-enrichment is associated with increased ß-OX and a more oxidative mitochondrial phenotype, and (3) experimentally induced linoleate-enriched CL in ventricular myocytes promotes a shift from pyruvate metabolism to fatty acid ß-OX. Full article

Hyperuricemia is a risk factor for heart disease. Cardiomyocytes produce uric acid via xanthine oxidase. The enzymatic reaction leads to oxidative stress in uric-acid-producing cells. However, extracellular uric acid is the largest scavenger of reactive oxygen species, specifically to nitrosative stress, which can directly affect cells. Here, the effect of plasma-relevant concentrations of uric acid on adult rat ventricular cardiomyocytes is analyzed. A concentration- and time-dependent reduction of load-free cell shortening is found. This is accompanied by an increased protein expression of ornithine decarboxylase, the rate-limiting enzyme of the polyamine metabolism, suggesting a higher arginine turnover. Subsequently, the effect of uric acid was attenuated if other arginine consumers, such as nitric oxide synthase, are blocked or arginine is added. In the presence of uric acid, calcium transients are increased in cardiomyocytes irrespective of the reduced cell shortening, indicating calcium desensitization. Supplementation of extracellular calcium or stimulation of intracellular calcium release by β-adrenergic receptor stimulation attenuates the uric-acid-dependent effect. The effects of uric acid are attenuated in the presence of a protein kinase C inhibitor, suggesting that the PKC-dependent phosphorylation of troponin triggers the desensitizing effect. In conclusion, high levels of uric acid stress cardiomyocytes by accelerating the arginine metabolism via the upregulation of ornithine decarboxylase. Full article

Background. Green tea catechins are known to promote mitochondrial function, and to modulate gene expression and signalling pathways that are altered in the diabetic heart. We thus evaluated the effectiveness of the in vivo administration of a standardized green tea extract (GTE) in restoring cardiac performance, in a rat model of early streptozotocin-induced diabetes, with a focus on the underlying mechanisms. Methods. Twenty-five male adult Wistar rats were studied: the control (n = 9), untreated diabetic animals (n = 7) and diabetic rats subjected to daily GTE administration for 28 days (n = 9). Isolated ventricular cardiomyocytes were used for ex vivo measurements of cell mechanics and calcium transients, and molecular assays, including the analysis of functional protein and specific miRNA expression. Results. GTE treatment induced an almost complete recovery of cardiomyocyte contractility that was markedly impaired in the diabetic cells, by preserving mitochondrial function and energy availability, and modulating the expression of the sarcoplasmic reticulum calcium ATPase and phospholamban. Increased Sirtuin 1 (SIRT1) expression and activity substantially contributed to the observed cardioprotective effects. Conclusions. The data supported the hypothesis that green tea dietary polyphenols, by targeting SIRT1, can constitute an adjuvant strategy for counteracting the initial damage of the diabetic heart, before the occurrence of diabetic cardiomyopathy. Full article

The physiological importance of NCX in human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) is not well characterized but may depend on the relative strength of the current, compared to adult cardiomyocytes, and on the exact spatial arrangement of proteins involved in Ca²⁺ extrusion. Here, we determined NCX currents and its contribution to action potential and force in hiPSC-CMs cultured in engineered heart tissue (EHT). The results were compared with data from rat and human left ventricular tissue. The NCX currents in hiPSC-CMs were larger than in ventricular cardiomyocytes isolated from human left ventricles (1.3 ± 0.2 pA/pF and 3.2 ± 0.2 pA/pF for human ventricle and EHT, respectively, p < 0.05). SEA0400 (10 µM) markedly shortened the APD₉₀ in EHT (by 26.6 ± 5%, p < 0.05) and, to a lesser extent, in rat ventricular tissue (by 10.7 ± 1.6%, p < 0.05). Shortening in human left ventricular preparations was small and not different from time-matched controls (TMCs; p > 0.05). Force was increased by the NCX block in rat ventricle (by 31 ± 5.4%, p < 0.05) and EHT (by 20.8 ± 3.9%, p < 0.05), but not in human left ventricular preparations. In conclusion, hiPSC-CMs possess NCX currents not smaller than human left ventricular tissue. Robust NCX block-induced APD shortening and inotropy makes EHT an attractive pharmacological model. Full article

Reperfusion injury after extended ischemia accounts for approximately 50% of myocardial infarct size, and there is no standard therapy. HDAC inhibition reduces infarct size and enhances cardiomyocyte autophagy and PGC1α-mediated mitochondrial biogenesis when administered at the time of reperfusion. Furthermore, a specific autophagy-inducing peptide, Tat-Beclin 1 (TB), reduces infarct size when administered at the time of reperfusion. However, since SAHA affects multiple pathways in addition to inducing autophagy, whether autophagic flux induced by TB maintains mitochondrial homeostasis during ischemia/reperfusion (I/R) injury is unknown. We tested whether the augmentation of autophagic flux by TB has cardioprotection by preserving mitochondrial homeostasis both in vitro and in vivo. Wild-type mice were randomized into two groups: Tat-Scrambled (TS) peptide as the control and TB as the experimental group. Mice were subjected to I/R surgery (45 min coronary ligation, 24 h reperfusion). Autophagic flux, mitochondrial DNA (mtDNA), mitochondrial morphology, and mitochondrial dynamic genes were assayed. Cultured neonatal rat ventricular myocytes (NRVMs) were treated with a simulated I/R injury to verify cardiomyocyte specificity. The essential autophagy gene, ATG7, conditional cardiomyocyte-specific knockout (ATG7 cKO) mice, and isolated adult mouse ventricular myocytes (AMVMs) were used to evaluate the dependency of autophagy in adult cardiomyocytes. In NRVMs subjected to I/R, TB increased autophagic flux, mtDNA content, mitochondrial function, reduced reactive oxygen species (ROS), and mtDNA damage. Similarly, in the infarct border zone of the mouse heart, TB induced autophagy, increased mitochondrial size and mtDNA content, and promoted the expression of PGC1α and mitochondrial dynamic genes. Conversely, loss of ATG7 in AMVMs and in the myocardium of ATG7 cKO mice abolished the beneficial effects of TB on mitochondrial homeostasis. Thus, autophagic flux is a sufficient and essential process to mitigate myocardial reperfusion injury by maintaining mitochondrial homeostasis and partly by inducing PGC1α-mediated mitochondrial biogenesis. Full article