Calcium Handling In Cardiac Myocytes Research Articles

Muscle-Specific Microrna miR-208 Regulates Calcium Handling Release in Cardiomyocytes by Targeting Phosphodiesterase 4D and Calcineurin

MicroRNAs are small non-coding RNAs which act as endogenous regulators of gene expression, have gained much attention in the past several years due to increasing evidence of their involvement in numerous pathological processes including cardiac disease. Several microRNAs have been implicated in heart failure (HF) and sudden cardiac death (SCD) via regulation of calcium handling in cardiac myocytes. Upregulation of muscle-specific miRNA miR-208 has been associated with adverse clinical outcomes including acute failure and SCD in patients with dilated cardiomyopathy, while therapeutic inhibition of miR-208 was demonstrated to improve survival and cardiac function in the rat model of HF. However, the molecular mechanisms underlying such functional effects of miR-208 dysregulation remain largely unexplored. We hypothesize that miR-208 is an important regulator of calcium handling by targeting components of phosphorylation-dephosphorylation system in cardiac myocytes. An experimental model of miR-208 overexpression in rat ventricular myocytes was generated via adenoviral infection. Its effects on Ca2+ handling and pro-arrhythmic activity was investigated using confocal microscopy for Ca2+ imaging, western blot analysis, and luciferase reporter assays. Under conditions of beta-adrenergic stimulation we observed decreased Ca2+ transient amplitude, reduced SR Ca2+ content, faster onset of pro-arrhythmic spontaneous Ca2+ waves, and increased spark frequency in miR-208 overexpressing cells. Additionally, there was increased phosphorylation of a negative regulator of SERCA, phospholamban, and ryanodine receptors as well as decreased levels of putative targets phosphodiesterase 4D and calcineurin in miR-208 overexpressed myocytes. We can conclude that miR-208 affects Ca2+ handling by regulating the expression of proteins involved in phosphorylation-dephosphorylation of ryanodine receptors and phospholamban. This mechanism may contribute to the increased risk of arrhythmia and SCD in cardiac disease states accompanied by miR-208 upregulation including HF.

Instabilities of the resting state in a mathematical model of calcium handling in cardiac myocytes

We analyze a recently published model of calcium handling in cardiac myocytes in order to find conditions for the presence of instabilities in the resting state of the model. Such instabilities can create calcium waves which in turn may be able to initiate cardiac arrhythmias. The model was developed by Swietach, Spitzer and Vaughan-Jones [1] in order to study the effect, on calcium waves, of varying ryanodine receptor (RyR)-permeability, sarco/endoplasmic reticulum calcium ATPase (SERCA) and calcium diffusion. We study the model using the extracellular calcium concentration ce and the maximal velocity of the SERCA-pump vSERCA as control parameters. In the (ce,vSERCA)-domain we derive an explicit function v∗=v∗(ce), and we claim that any resting state based on parameters that lie above the curve (i.e. any pair (ce,vSERCA) such that with vSERCA>v∗(ce)) is unstable in the sense that small perturbations will grow and can eventually turn into a calcium wave. And conversely; any pair (ce,vSERCA) below the curve is stable in the sense that small perturbations to the resting state will decay to rest. This claim is supported by analyzing the stability of the system in terms of computing the eigenmodes of the linearized model. Furthermore, the claim is supported by direct simulations based on the non-linear model.Since the curve separating stable from unstable states is given as an explicit function, we can show how stability depends on other parameters of the model.

SGCα1 mediates the negative inotropic effects of NO in cardiac myocytes independent of changes in calcium handling

In the heart, nitric oxide (NO) modulates contractile function; however, the mechanisms responsible for this effect are incompletely understood. NO can elicit effects via a variety of mechanisms including S-nitrosylation and stimulation of cGMP synthesis by soluble guanylate cyclase (sGC). sGC is a heterodimer comprised of a β(1)- and an α(1)- or α(2)-subunit. sGCα(1)β(1) is the predominant isoform in the heart. To characterize the role of sGC in the regulation of cardiac contractile function by NO, we compared left ventricular cardiac myocytes (CM) isolated from adult mice deficient in the sGC α(1)-subunit (sGCα(1)(-/-)) and from wild-type (WT) mice. Sarcomere shortening under basal conditions was less in sGCα(1)(-/-) CM than in WT CM. To activate endogenous NO synthesis from NO synthase 3, CM were incubated with the β(3)-adrenergic receptor (β(3)-AR) agonist BRL 37344. BRL 37344 decreased cardiac contractility in WT CM but not in sGCα(1)(-/-) myocytes. Administration of spermine NONOate, an NO donor compound, did not affect sarcomeric shortening in CM of either genotype; however, in the presence of isoproterenol, addition of spermine NONOate reduced sarcomere shortening in WT but not in sGCα(1)(-/-) CM. Neither BRL 37344 nor spermine NONOate altered calcium handling in CM of either genotype. These findings suggest that sGCα(1) exerts a positive inotropic effect under basal conditions, as well as mediates the negative inotropic effect of β(3)-AR signaling. Additionally, our work demonstrates that sGCα(1)β(1) is required for NO to depress β(1)/β(2)-AR-stimulated cardiac contractility and that this modulation is independent of changes in calcium handling.

The participation of the Na+–Ca2+ exchanger in primary cardiac myofibroblast migration, contraction, and proliferation

Cardiac ventricular myofibroblast motility, proliferation, and contraction contribute to post-myocardial infarct wound healing, infarct scar formation, and remodeling of the ventricle remote to the site of infarction. The Na+-Ca2+ exchanger (NCX1) is involved in altered calcium handling in cardiac myocytes during cardiac remodeling associated with heart failure, however, its role in cardiac myofibroblast cell function is unexplored. In this study we investigated the involvement of NCX1 as well as the role of non-selective-cation channels (NSCC) in cardiac myofibroblast cell function in vitro. Immunofluorescence and Western blots revealed that P1 cells upregulate alpha-smooth muscle actin (alphaSMA) and embryonic smooth muscle myosin heavy chain (SMemb) expression. NCX1 mRNA and proteins as well as Ca(v)1.2a protein are also expressed in P1 myofibroblasts. Myofibroblast motility in the presence of 50 ng/ml PDGF-BB was blocked with AG1296. Myofibroblast motility, contraction, and proliferation were sensitive to KB-R7943, a specific NCX1 reverse-mode inhibitor. In contrast, only proliferation and contraction, but not motility were sensitive to nifedipine, while gadolinium (NSCC blocker) was only associated with decreased motility. ML-7 treatment was associated with inhibition of the chemotactic response and contraction. Thus cardiac myofibroblast chemotaxis, contraction, and proliferation were sensitive to different pharmacologic treatments suggesting that regulation of transplasmalemmal calcium movements may be important in growth factor receptor-mediated processes. NCX1 may represent an important moiety in suppression of myofibroblast functions.

Suppression of calcium sparks in rat ventricular myocytes and direct inhibition of sheep cardiac RyR channels by EPA, DHA and oleic acid.

The anti-arrhythmic effects of long-chain polyunsaturated fatty acids (PUFAs) may be related to their ability to alter calcium handling in cardiac myocytes. We investigated the effect of eicosapentanoic acid (EPA) and docosahexaenoic acid (DHA) on calcium sparks in rat cardiac myocytes and the effects of these PUFAs and the monounsaturated oleic acid on cardiac calcium release channels (RyRs). Visualization of subcellular calcium concentrations in single rat ventricular myocytes showed that intensity of calcium sparks was reduced in the presence of EPA and DHA (15 micro M). It was also found that calcium sparks decayed more quickly in the presence of EPA but not DHA. Sarcoplasmic vesicles containing RyRs were prepared from sheep hearts and RyR activity was determined by either [(3)H]ryanodine binding or by single-channel recording. Bilayers were formed from phosphatidylethanolamine and phosphatidylcholine dissolved in either n-decane or n-tetradecane. EPA inhibited [(3)H]ryanodine binding to RyRs in SR vesicles with K(I) = 40 micro M. Poly- and mono-unsaturated free fatty acids inhibited RyR activity in lipid bilayers. EPA (cytosolic or luminal) inhibited RyRs with K(I) =32 micro M and Hill coefficient, n(1) = 3.8. Inhibition was independent of the n-alkane solvent and whether RyRs were activated by ATP or Ca(2+). DHA and oleic acid also inhibited RyRs, suggesting that free fatty acids generally inhibit RyRs at micromolar concentrations.