HDAC Inhibition Improves the Sarcoendoplasmic Reticulum Ca2+-ATPase Activity in Cardiac Myocytes.

SERCA2a is the Ca2+ ATPase playing the major contribution in cardiomyocyte (CM) calcium removal. Its activity can be regulated by both modulatory proteins and several post-translational modifications. The aim of the present work was to investigate whether the function of SERCA2 can be modulated by treating CMs with the histone deacetylase (HDAC) inhibitor suberanilohydroxamic acid (SAHA). The incubation with SAHA (2.5 µM, 90 min) of CMs isolated from rat adult hearts resulted in an increase of SERCA2 acetylation level and improved ATPase activity. This was associated with a significant improvement of calcium transient recovery time and cell contractility. Previous reports have identified K464 as an acetylation site in human SERCA2. Mutants were generated where K464 was substituted with glutamine (Q) or arginine (R), mimicking constitutive acetylation or deacetylation, respectively. The K464Q mutation ameliorated ATPase activity and calcium transient recovery time, thus indicating that constitutive K464 acetylation has a positive impact on human SERCA2a (hSERCA2a) function. In conclusion, SAHA induced deacetylation inhibition had a positive impact on CM calcium handling, that, at least in part, was due to improved SERCA2 activity. This observation can provide the basis for the development of novel pharmacological approaches to ameliorate SERCA2 efficiency.

Acetylation mediates Cx43 reduction caused by electrical stimulation.

Communication between cardiomyocytes depends upon gap junctions (GJ). Previous studies have demonstrated that electrical stimulation induces GJ remodeling and modifies histone acetylase (HAT) and deacetylase (HDAC) activities, although these two results have not been linked. The aim of this work was to establish whether electrical stimulation modulates GJ-mediated cardiac cell-cell communication by acetylation-dependent mechanisms. Field stimulation of HL-1 cardiomyocytes at 0.5 Hz for 24 h significantly reduced connexin43 (Cx43) expression and cell-cell communication. HDAC activity was down-regulated whereas HAT activity was not modified resulting in increased acetylation of Cx43. Consistent with a post-translational mechanism, we did not observe a reduction in Cx43 mRNA in electrically stimulated cells, while the proteasomal inhibitor MG132 maintained Cx43 expression. Further, the treatment of paced cells with the HAT inhibitor Anacardic Acid maintained both the levels of Cx43 and cell-cell communication. Finally, we observed increased acetylation of Cx43 in the left ventricles of dogs subjected to chronic tachypacing as a model of abnormal ventricular activation. In conclusion, our findings suggest that altered electrical activity can regulate cardiomyocyte communication by influencing the acetylation status of Cx43.

Late sodium current inhibition reverses electromechanical dysfunction in human hypertrophic cardiomyopathy.

BACKGROUND: Hypertrophic cardiomyopathy (HCM), the most common mendelian heart disorder, remains an orphan of disease-specific pharmacological treatment because of the limited understanding of cellular mechanisms underlying arrhythmogenicity and diastolic dysfunction. METHODS AND RESULTS: We assessed the electromechanical profile of cardiomyocytes from 26 HCM patients undergoing myectomy compared with those from nonfailing nonhypertrophic surgical patients by performing patch-clamp and intracellular Ca(2+) (Ca(2+)(i)) studies. Compared with controls, HCM cardiomyocytes showed prolonged action potential related to increased late Na(+) (I(NaL)) and Ca(2+) (I(CaL)) currents and decreased repolarizing K(+) currents, increased occurrence of cellular arrhythmias, prolonged Ca(2+)(i) transients, and higher diastolic Ca(2+)(i). Such changes were related to enhanced Ca(2+)/calmodulin kinase II (CaMKII) activity and increased phosphorylation of its targets. Ranolazine at therapeutic concentrations partially reversed the HCM-related cellular abnormalities via I(NaL) inhibition, with negligible effects in controls. By shortening the action potential duration in HCM cardiomyocytes, ranolazine reduced the occurrence of early and delayed afterdepolarizations. Finally, as a result of the faster kinetics of Ca(2+)(i) transients and the lower diastolic Ca(2+)(i), ranolazine accelerated the contraction-relaxation cycle of HCM trabeculae, ameliorating diastolic function. CONCLUSIONS: We highlighted a specific set of functional changes in human HCM myocardium that stem from a complex remodeling process involving alterations of CaMKII-dependent signaling, rather than being a direct consequence of the causal sarcomeric mutations. Among the several ion channel and Ca(2+)(i) handling proteins changes identified, an enhanced I(NaL) seems to be a major contributor to the electrophysiological and Ca(2+)(i) dynamic abnormalities of ventricular myocytes and trabeculae from patients with HCM, suggesting potential therapeutic implications of I(NaL) inhibition.

Cardiomyocyte GTP cyclohydrolase 1 and tetrahydrobiopterin increase NOS1 activity and accelerate myocardial relaxation.

RATIONALE: Tetrahydrobiopterin (BH4) is an essential cofactor of nitric oxide synthases (NOS). Oral BH4 supplementation preserves cardiac function in animal models of cardiac disease; however, the mechanisms underlying these findings are not completely understood. OBJECTIVE: To study the effect of myocardial transgenic overexpression of the rate-limiting enzyme in BH4 biosynthesis, GTP cyclohydrolase 1 (GCH1), on NOS activity, myocardial function, and Ca2+ handling. METHODS AND RESULTS: GCH1overexpression significantly increased the biopterins level in left ventricular (LV) myocytes but not in the nonmyocyte component of the LV myocardium or in plasma. The ratio between BH4 and its oxidized products was lower in mGCH1-Tg, indicating that a large proportion of the myocardial biopterin pool was oxidized; nevertheless, myocardial NOS1 activity was increased in mGCH1-Tg, and superoxide release was significantly reduced. Isolated hearts and field-stimulated LV myocytes (3 Hz, 35°C) overexpressing GCH1 showed a faster relaxation and a PKA-mediated increase in the PLB Ser16 phosphorylated fraction and in the rate of decay of the [Ca2+]i transient. RyR2 S-nitrosylation and diastolic Ca2+ leak were larger in mGCH1-Tg and ICa density was lower; nevertheless the amplitude of the [Ca2+]i transient and contraction did not differ between genotypes, because of an increase in the SR fractional release of Ca2+ in mGCH1-Tg myocytes. Xanthine oxidoreductase inhibition abolished the difference in superoxide production but did not affect myocardial function in either group. By contrast, NOS1 inhibition abolished the differences in ICa density, Ser16 PLB phosphorylation, [Ca2+]i decay, and myocardial relaxation between genotypes. CONCLUSIONS: Myocardial GCH1 activity and intracellular BH4 are a limiting factor for constitutive NOS1 and SERCA2A activity in the healthy myocardium. Our findings suggest that GCH1 may be a valuable target for the treatment of LV diastolic dysfunction.

Altered calcium regulation in isolated cardiomyocytes from Egr-1 knock-out mice.

Early growth response-1 one gene (Egr-1), one of the immediate early response genes, plays an important role in the adaptive response of the myocardium to hypertrophic stimuli. We aimed to investigate the effects of Egr-1 deletion on cardiac function. Egr-1 knock-out (Egr-1(-/-)) homozygous mice were employed to evaluate the electrophysiological and molecular properties of left ventricular cardiomyocytes (VCM) by using patch-clamp technique, intracellular calcium measurements, real-time PCR, and Western blot. Action potential was prolonged and diastolic potential was positive-shifted in VCMs isolated from Egr-1(-/-) mice, in comparison with those from their wild-type (WT) littermates. The calcium content of the sarcoplasmic reticulum was reduced and the decay time for steady-state calcium transient slowed down. Serca2, Ryr, L-type Ca(2+)-channel, and PLB mRNA expression were reduced in Egr-1(-/-) mice compared with the controls. Moreover, Serca2 protein was reduced, while the amount of Ncx1 protein was increased in Egr-1(-/-) hearts compared with those of the WT littermates. Furthermore, genes involved in heart development (GATA-4, TGF-ß) and in Egr-1 regulation (Nab1, Nab2) were down regulated in Egr-1(-/-) mice. These results suggest that Egr-1 plays a pivotal role in regulating excitation-contraction coupling in cardiac myocytes.

Heart rate reduction with ivabradine prevents the global phenotype of left ventricular remodeling.

The aim of this study was to investigate the effect of chronic heart rate (HR) reduction with the hyperpolarization-activated current inhibitor ivabradine on the global phenotype of left ventricular (LV) remodeling in a ligated rat model. Seven days after coronary artery ligation, Wistar rats received ivabradine (10 mg · kg(-1) · day(-1) administered in drinking water) [myocardial infarction + ivabradine (MI+IVA), n = 22] or vehicle only (drinking water) (MI, n = 20) for 90 days. A sham group (n = 20) was included for model validation. MI+IVA rats had 12% lower HR (P < 0.01), improved LV volumes, 15% higher LV ejection fraction (LVEF, P < 0.01) than MI rats, and 33% reductions in both plasma atrial natriuretic peptide (ANP, P = 0.052) and cardiac hydroxyproline. Using patch-clamp, action potential duration was reduced and transient outward current density increased (P < 0.05). Cardiac energy metabolism was also improved (+33% creatine phosphate, P < 0.001; +15% ATP; and +9% energy charge, P < 0.05). Significant correlations were found between HR and parameters of cardiac metabolism, ANP, and LVEF (all P < 0.05). The HR-reducing properties of ivabradine prevent changes in the global phenotype of LV remodeling in the rat, optimize energy consumption, and avoid electrophysiological and structural remodeling.

The effect of losartan treatment on the response of diabetic cardiomyocytes to ATP depletion.

The present work aimed to investigate the effect of losartan treatment of healthy and diabetic rats on cardiomyocyte response to ATP depletion. Cells were isolated from normoglycemic (N) and streptozotocin-injected (55 mg/kg) rats (D) treated or not treated with losartan (20 mg/kg/day in the drinking water; NL and DL, respectively) for 3 weeks. In each group of cells, enzyme activities such as glucose-6-phosphate (G6PDH) and glycerol-3-phosphate dehydrogenases (G3PDH), lactate/pyruvate, glycogen levels and citrate synthase were measured as an index of glycolytic dysregulation and mitochondrial mass, respectively. Cells were then challenged with NaCN (2 mM) in glucose-free Tyrode solution (metabolic intoxication, MI), a protocol to study ischemia at cell level. Under these conditions, the time to contractile failure up to rigor-type hyper-contracture in field-stimulated cells and K(ATP) current activation by patch-clamp recordings were measured. In comparison with N and NL, D cells presented higher G6PDH and cytoplasmic G3PDH activities, lactate/pyruvate, glycogen content but similar levels of citrate synthase, and decay time of contraction. When subjected to MI, D cells showed delayed activation of the K(ATP) current (25.7±7.1 min; p<0.001 vs. N and NL), increased time to contractile failure and rigor-type hyper-contracture (p<0.001 vs. N and NL). In cells from DL rats both functional (time to rigor and to K(ATP) current activation) and metabolic parameters, approached values similar to those measured in N and NL cells. These results demonstrate that diabetic cardiomyocytes from rats treated with losartan, maintain the capacity to respond promptly to ATP depletion reaching contractile failure, rigor-type hypercontracture and K(ATP) opening with a similar timing of N cells.

Prenatal exposure to carbon monoxide delays postnatal cardiac maturation.

Prenatal exposure to toxicants, such as maternal smoking, may impair cardiovascular autonomic maturation in infants. We recently showed that exposure of pregnant rats to a mild concentration of carbon monoxide (CO), a component of cigarette smoke, delays postnatal electrophysiological maturation of ventricular myocytes from newborns rats, likely predisposing to life-threatening arrhythmias. To get a comprehensive view of developmental molecular abnormalities induced, at cardiac level, by prenatal CO exposure, we used microarray analysis approach on the rat heart at 4, 7 and 20 days postnatal life. The relationship between molecular and functional alterations was investigated by assessing the ventricular expression of f-current, an electrophysiological marker of immature cardiac phenotype. Rats were prenatally exposed to 0 (CTR) or 150 p.p.m. CO and mRNA obtained from ventricular samples. Differential analysis and biological pathway analysis of microarray data were performed by using Newton's approach and the GENMAPP/MAPPFinder, respectively. The real-time RT-PCR reactions were performed by TaqMan probe-based chemistry. Freshly isolated patch-clamped ventricular cardiomyocytes were used to measure I(f). Genes and pathways controlling cell cycle and excitation-contraction coupling were significantly modified in CO-exposed rats. The higher effect was observed in cardiomyocytes harvested from 7-day-old rats, in which mRNA expression for crucial sarcomeric proteins (myosin and actin subunits, troponin I), transporters (Ca(2+) transporting ATPase) and enzymes (aldolase) were significantly downregulated. Accordingly, the molecular and functional expression of f-channels, which represents a marker of fetal ventricular phenotype, was transiently greater in CO-exposed rats (+200%) than in control ones. In conclusion, our study provides new insights into the molecular and functional mechanisms underlying cardiac maturation and its impairment by prenatal exposure to toxic components of smoking, such as CO.

Modulation of cardiac ionic homeostasis by 3-iodothyronamine.

3-iodothyronamine (T(1)AM) is a novel endogenous relative of thyroid hormone, able to interact with trace amine-associated receptors, a class of plasma membrane G protein-coupled receptors, and to produce a negative inotropic and chronotropic effect. In the isolated rat heart 20-25 microM T(1)AM decreased cardiac contractility, but oxygen consumption and glucose uptake were either unchanged or disproportionately high when compared to mechanical work. In adult rat cardiomyocytes acute exposure to 20 microM T(1)AM decreased the amplitude and duration of the calcium transient. In patch clamped cardiomyocytes sarcolemmal calcium current density was unchanged while current facilitation by membrane depolarization was abolished consistent with reduced sarcoplasmic reticulum (SR) calcium release. In addition, T(1)AM decreased transient outward current (I(to)) and I(K1) background current. SR studies involving 20 microM T(1)AM revealed a significant decrease in ryanodine binding due to reduced B(max), no significant change in the rate constant of calcium-induced calcium release, a significant increase in calcium leak measured under conditions promoting channel closure, and no effect on oxalate-supported calcium uptake. Based on these observations we conclude T(1)AM affects calcium and potassium homeostasis and suggest its negative inotropic action is due to a diminished pool of SR calcium as a result of increased diastolic leak through the ryanodine receptor, while increased action potential duration is accounted for by inhibition of I(to) and I(K1) currents.

Electrophysiological characterization of isolated human atrial myocytes exposed to tegaserod.

Tegaserod (Teg), a 5-hydroxytryptamine type-4 (5-HT(4)) receptor partial agonist, represents a novel treatment for irritable bowel syndrome with constipation and chronic constipation. Cardiovascular safety data from pooled clinical studies showed a signal suggestive of increased occurrence of ischaemic cardiovascular events in patients exposed to Teg versus placebo. Thereafter, marketing of Teg was suspended in the USA and other countries. The clinical data did not demonstrate a causative effect but raised questions of whether a non-recognized effect on the heart was present. Our aim was to evaluate for arrhythmogenic potential of Teg on human cardiomyocytes. Cells isolated from human atrial specimens during cardiac surgery were used to assess the effects of Teg (1, 10, 30 and 100 nM) on action potential and I(f) (funny current) by patch-clamp technique. Results showed that Teg (at all concentrations tested) did not significantly affect action potential characteristics of atrial myocytes when driven at different rates (0.2, 0.5 and 1 Hz). In contrast, 5HT significantly prolonged action potential duration (1 and 10 nM) and caused cell un-excitability (100 nM). Teg, at the highest concentration tested (100 nM, corresponding to 10 times C(max), produced by the recommended dose of 6 mg b.i.d.) increased the I(f) amplitude and caused a shift of its activation curve. This effect of a high concentration of Teg is not considered clinically relevant. When evaluated on single human atrial cells, Teg does not appear to exhibit arrhythmogenic properties, as it did not affect the action potential profile.

I(f) channels as a therapeutic target in heart disease.

In the normal heart, impulses are generated from the sinoatrial node. It is generally accepted that the pacemaker current, I(f), plays a major role in the spontaneous rhythmic activity. Recently, several electrophysiological and molecular data demonstrate that I(f) channels are present in embryonic and post-natal ventricular myocytes and undergo a downregulation during maturation. Interestingly, the I(f) current is re-expressed in some pathological conditions such as cardiac hypertrophy and heart failure. In these conditions, the overexpression of f-channels is a consequence of electrophysiological remodeling and may represent an arrhythmogenic mechanism in heart failure, a condition associated with high risk for sudden cardiac death. For its physiological and pathophysiological role and the availability of selective f-channel blockers, I(f) may be a suitable therapeutic target in heart failure.