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Noise is present in cell biology. The capability of cells to respond to noisy environment have become essential. This study aimed to investigate whether noise can enhance the contractile response and Ca 2+ handling in cardiomyocytes from a cardiomyopathy model. Experiments were conducted in an experimental setup with Gaussian white noise, frequency, and amplitude control to stimulate myocytes. Cell shortening, maximal shortening velocity, time to peak shortening, and time to half relaxation variables were recorded to cell shortening. Ca 2+ transient amplitude and raise rate variables were registered to measure Ca 2+ transients. Our results for cell shortening, Ca 2+ transient amplitude, and raise rate suggest that cell response improve when myocytes are noise stimulated. Also, cell shortening, maximal shortening velocity, Ca 2+ transient amplitude, and raise improves in control cells. Altogether, these findings suggest novel characteristics in how cells improve their response in a noisy environment., (© 2023. Springer Nature Limited.)

Pulmonary arterial hypertension (PAH) is characterized by pulmonary vessel remodeling; however, its severity and impact on survival depend on right ventricular (RV) failure. Resveratrol (RES), a polyphenol found in red wine, exhibits cardioprotective effects on RV dysfunction in PAH. However, most literature has focused on RES protective effect on lung vasculature; recent finding indicates that RES has a cardioprotective effect independent of pulmonary arterial pressure on RV dysfunction, although the underlying mechanism in RV has not been determined. Therefore, this study is aimed at evaluating sirtuin-3 (SIRT3) modulation by RES in RV using a monocrotaline- (MC-) induced PAH rat model. Myocyte function was evaluated by confocal microscopy as cell contractility, calcium signaling, and mitochondrial membrane potential (ΔΨ m ); cell energetics was assessed by high-resolution respirometry, and western blot and immunoprecipitation evaluated posttranslational modifications. PAH significantly affects mitochondrial function in RV; PAH is prone to mitochondrial permeability transition pore (mPTP) opening, thus decreasing the mitochondrial membrane potential. The compromised cellular energetics affects cardiomyocyte function by decreasing sarco-endoplasmic reticulum Ca 2+ -ATPase (SERCA) activity and delaying myofilament unbinding, disrupting cell relaxation. RES partially protects mitochondrial integrity by deacetylating cyclophilin-D, a critical component of the mPTP, increasing SIRT3 expression and activity and preventing mPTP opening. The preserved energetic capability rescues cell relaxation by maintaining SERCA activity. Avoiding Ca 2+ transient and cell contractility mismatch by preserving mitochondrial function describes, for the first time, impairment in excitation-contraction-energetics coupling in RV failure. These results highlight the importance of mitochondrial energetics and mPTP in PAH., Competing Interests: The authors declare that there is no conflict of interest regarding the publication of this paper., (Copyright © 2021 Judith Bernal-Ramírez et al.)

Background: Thyroid hormone status in hypothyroidism (HT) downregulates key elements in Ca 2+ handling within the heart, reducing contractility, impairing the basal energetic balance, and increasing the risk of cardiovascular disease. Mitochondrial Ca 2+ transport is reduced in HT, and tolerance to reperfusion damage has been documented, but the precise mechanism is not well understood. Therefore, we aimed to determine the stoichiometry and activity of the mitochondrial Ca 2+ uniporter or uniplex in an HT model and the relevance to the opening of the mitochondrial permeability transition pores (mPTP) during ischemia/reperfusion (I/R) injury. Methods: An HT model was established in Wistar rats by treatment with 6-propylthiouracil for 28 days. Uniplex composition and activity were determined in cardiac mitochondria. Hearts were perfused ex vivo to induce I/R injury, and functional parameters related to contractility and tissue viability were evaluated. Results: The cardiac stoichiometry between two subunits of the uniplex (MICU1/MCU) increased by 25% in animals with HT. The intramitochondrial Ca 2+ content was reduced by 40% and was less prone to the mPTP opening. After I/R injury, ischemic contracture and the onset of ventricular fibrillation were delayed in animals with HT, concomitant with a reduction in oxidative damage and mitochondrial dysfunction. Conclusions: Our results suggest that HT is associated with an increase in the cardiac MICU1/MCU ratio, thereby changing the stoichiometry between these subunits to increase the threshold to cytosolic Ca 2+ and reduce mitochondrial Ca 2+ overload. Our results also demonstrate that this HT model can be used to explore the role of mitochondrial Ca 2+ transport in cardiac diseases due to its induced tolerance to cardiac damage.

Recent evidence has shown that nanoparticles that have been used to improve or create new functional properties for common products may pose potential risks to human health. Silicon dioxide (SiO 2 ) has emerged as a promising therapy vector for the heart. However, its potential toxicity and mechanisms of damage remain poorly understood. This study provides the first exploration of SiO 2 -induced toxicity in cultured cardiomyocytes exposed to 7- or 670-nm SiO 2 particles. We evaluated the mechanism of cell death in isolated adult cardiomyocytes exposed to 24-h incubation. The SiO 2 cell membrane association and internalization were analyzed. SiO 2 showed a dose-dependent cytotoxic effect with a half-maximal inhibitory concentration for the 7 nm (99.5 ± 12.4 µg/ml) and 670 nm (>1,500 µg/ml) particles, which indicates size-dependent toxicity. We evaluated cardiomyocyte shortening and intracellular Ca 2+ handling, which showed impaired contractility and intracellular Ca 2+ transient amplitude during β-adrenergic stimulation in SiO 2 treatment. The time to 50% Ca 2+ decay increased 39%, and the Ca 2+ spark frequency and amplitude decreased by 35 and 21%, respectively, which suggest a reduction in sarcoplasmic reticulum Ca 2+ -ATPase (SERCA) activity. Moreover, SiO 2 treatment depolarized the mitochondrial membrane potential and decreased ATP production by 55%. Notable glutathione depletion and H 2 O 2 generation were also observed. These data indicate that SiO 2 increases oxidative stress, which leads to mitochondrial dysfunction and low energy status; these underlie reduced SERCA activity, shortened Ca 2+ release, and reduced cell shortening. This mechanism of SiO 2 cardiotoxicity potentially plays an important role in the pathophysiology mechanism of heart failure, arrhythmias, and sudden death. NEW & NOTEWORTHY Silica particles are used as novel nanotechnology-based vehicles for diagnostics and therapeutics for the heart. However, their potential hazardous effects remain unknown. Here, the cardiotoxicity of silica nanoparticles in rat myocytes has been described for the first time, showing an impairment of mitochondrial function that interfered directly with Ca 2+ handling., (Copyright © 2017 the American Physiological Society.)

Intracellular Ca 2+ mishandling is an underlying mechanism in hypoxia/reoxygenation (H/R) injury that results in mitochondrial dysfunction and cardiomyocytes death. These events are mediated by mitochondrial Ca 2+ ( m Ca 2+ ) overload that is facilitated by the mitochondrial calcium uniporter (MCU) channel. Along this line, we evaluated the effect of siRNA-targeting MCU in cardiomyocytes subjected to H/R injury. First, cardiomyocytes treated with siRNA demonstrated a reduction of MCU expression by 67%, which resulted in significant decrease in mitochondrial Ca 2+ transport. siRNA treated cardiomyocytes showed decreased mitochondrial permeability pore opening and oxidative stress trigger by Ca 2+ overload. Furthermore, after H/R injury MCU silencing decreased necrosis and apoptosis levels by 30% and 50%, respectively, and resulted in reduction in caspases 3/7, 9, and 8 activity. Our findings are consistent with previous conclusions that demonstrate that MCU activity is partly responsible for cellular injury induced by H/R and support the concept of utilizing siRNA-targeting MCU as a potential therapeutic strategy., Competing Interests: The authors declare that they have no competing interests.

Background and Purpose: Despite the importance of mitochondrial Ca(2+) to metabolic regulation and cell physiology, little is known about the mechanisms that regulate Ca(2+) entry into the mitochondria. Accordingly, we established a system to determine the role of the mitochondrial Ca(2+) uniporter in an isolated heart model, at baseline and during increased workload following β-adrenoceptor stimulation., Experimental Approach: Cardiac contractility, oxygen consumption and intracellular Ca(2+) transients were measured in ex vivo perfused murine hearts. Ru360 and spermine were used to modify mitochondrial Ca(2+) uniporter activity. Changes in mitochondrial Ca(2+) content and energetic phosphate metabolite levels were determined., Key Results: The addition of Ru360 , a selective inhibitor of the mitochondrial Ca(2+) uniporter, induced progressively and sustained negative inotropic effects that were dose-dependent with an EC50 of 7 μM. Treatment with spermine, a uniporter agonist, showed a positive inotropic effect that was blocked by Ru360 . Inotropic stimulation with isoprenaline elevated oxygen consumption (2.7-fold), Ca(2+) -dependent activation of pyruvate dehydrogenase (5-fold) and mitochondrial Ca(2+) content (2.5-fold). However, in Ru360 -treated hearts, this parameter was attenuated. In addition, β-adrenoceptor stimulation in the presence of Ru360 did not affect intracellular Ca(2+) handling, PKA or Ca(2+) /calmodulin-dependent PK signalling., Conclusions and Implications: Inhibition of the mitochondrial Ca(2+) uniporter decreases β-adrenoceptor response, uncoupling between workload and production of energetic metabolites. Our results support the hypothesis that the coupling of workload and energy supply is partly dependent on mitochondrial Ca(2+) uniporter activity., (© 2014 The British Pharmacological Society.)

Background and Purpose: The mitochondrial permeability transition pore (mPTP), an energy-dissipating channel activated by calcium, contributes to reperfusion damage by depolarizing the mitochondrial inner membrane potential. As mitochondrial Ca(2+) overload is a main inductor of mPTP opening, we examined the effect of Ru(360), a selective inhibitor of the mitochondrial calcium uptake system against myocardial damage induced by reperfusion in a rat model., Experimental Approach: Myocardial reperfusion injury was induced by a 5-min occlusion of the left anterior descending coronary artery, followed by a 5-min reperfusion in anaesthetized open-chest rats. We measured reperfusion-induced arrhythmias and functions indicative of unimpaired mitochondrial integrity to evaluate the effect of Ru(360) treatment., Key Results: Reperfusion elicited a high incidence of arrhythmias, haemodynamic dysfunction and loss of mitochondrial integrity. A bolus intravenous injection of Ru(360) (15-50 nmol kg(-1)), given 30-min before ischaemia, significantly improved the above mentioned variables in the ischaemic/reperfused myocardium. Calcium uptake in isolated mitochondria from Ru(360)-treated ventricles was partially diminished, suggesting an interaction of this compound with the calcium uniporter., Conclusions and Implications: We showed that Ru(360) treatment abolishes the incidence of arrhythmias and haemodynamic dysfunction elicited by reperfusion in a whole rat model. Ru(360) administration partially inhibits calcium uptake, preventing mitochondria from depolarization by the opening of the mPTP. We conclude that myocardial damage could be a consequence of failure of the mitochondrial network to maintain the membrane potential at reperfusion. Hence, it is plausible that Ru(360) could be used in reperfusion therapy to prevent the occurrence of arrhythmia.

Mitochondrial calcium overload has been implicated in the irreversible damage of reperfused heart. Accordingly, we studied the effect of an oxygen-bridged dinuclear ruthenium amine complex (Ru360), which is a selective and potent mitochondrial calcium uniporter blocker, on mitochondrial dysfunction and on the matrix free-calcium concentration in mitochondria isolated from reperfused rat hearts. The perfusion of Ru360 maintained oxidative phosphorylation and prevented opening of the mitochondrial permeability transition pore in mitochondria isolated from reperfused hearts. We found that Ru360 perfusion only partially inhibited the mitochondrial calcium uniporter, maintaining the mitochondrial matrix free-calcium concentration at basal levels, despite high concentrations of cytosolic calcium. Additionally, we observed that perfused Ru360 neither inhibited Ca2+ cycling in the sarcoplasmic reticulum nor blocked ryanodine receptors, implying that the inhibition of ryanodine receptors cannot explain the protective effect of Ru360 in isolated hearts. We conclude that the maintenance of postischemic myocardial function correlates with an incomplete inhibition of the mitochondrial calcium uniporter. Thus, the chemical inhibition by this molecule could be an approach used to prevent heart injury during reperfusion.

The 90 kDa heat-shock protein (Hsp90) is one of the major stress proteins whose overall structure remains unknown. In this study, we investigated the influence of divalent cations Mg(2+) and Ca(2+) on the hydrodynamic properties and quaternary structure of Hsp90. Using analytical ultracentrifugation, size-exclusion chromatography, and polyacrylamide gel electrophoresis, we showed that native Hsp90 was mostly dimeric. The Hsp90 dimer had a sedimentation coefficient, s(w,20) degrees, of 6.10 +/- 0.03 S, which slightly deviated from the hydrodynamics of a globular protein. Using chemical cross-linking and analytical ultracentrifugation, we showed that Mg(2+) and Ca(2+) induced a tertiary conformational change of Hsp90, leading to a self-association process. In the presence of divalent cations, Hsp90 existed as a mixture of monomers, dimers, and tetramers at equilibrium. Finally, to identify Hsp90 domains involved in this divalent cation-dependent self-association, we studied the oligomerization state of the N-terminal (positions 1-221) of Hsp90, the influence of an N-terminal specific ligand, geldanamycin (GA), and the effect of C-terminal truncation on the ability of Hsp90 to oligomerize in the presence of divalent cations. We previously showed that GA inhibits Hsp90 heat-induced oligomerization [Garnier, C., Protasevich, I., Gilli, R., Tsvetkov, P., Lobachov, V., Peyrot, V., Briand, C., and Makarov, A. (1998) Biochem. Biophys. Res. Commun. 249, 197-201], but now we observed that GA does not influence divalent cation-dependent oligomerization of Hsp90, suggesting another mechanism. This mechanism involved the C-terminal part of the protein since C-terminally truncated Hsp90 did not oligomerize in the presence of divalent cations.

The weight-average molecular weight of Clr, an activated serine protease subcomponent of complement Cl, was measured in the presence of widely varying concentrations of Ca2+ and the other serine protease subcomponent, Cls, by utilizing the technique of tracer sedimentation equilibrium. A quantitative model for heteroassociation between the two subcomponents, which takes into account the previously observed Ca(2+)-dependent self-association of Cls, was fit to the combined data at each Ca2+ concentration. The results indicate that Clr, which exists as a dimer under all of the conditions explored in this work, can bind up to two molecules of Cls at both low and high Ca2+ concentrations, but the association constant for binding a single molecule of Cls to dimeric Clr is estimated to increase on the order of a 1000-fold as [Ca2+] increases from 1 nM to 1.0 mM. Heteroassociation of Clr and Cls is favored over self-association of Cls at all conditions. The results clearly indicate the necessity of taking into account a multiplicity of states of association when attempting to understand the equilibrium average properties of a mixture of the two subcomponents and their binding to Clq in solution.

The weight-average molecular weight of C1s, an activated serine protease subcomponent of human complement C1, has been measured by means of sedimentation equilibrium over a wide range of both protein and calcium ion concentrations. The combined data may be accounted for quantitatively by a simple model for Ca(2+)-dependent self-association of C1s to a dimer. According to this model, the monomer contains a single Ca2+ binding site with K approximately equal to 3 x 10(5) M-1, and the dimer contains three independent Ca binding sites, two having a Ca2+ affinity lower than that of the monomer (K approximately equal to 3 x 10(4) M-1). The third binding site in the dimer, which presumably lies at the interface between the two amino-terminal alpha domains, has a higher Ca2+ affinity (K approximately equal to 1 x 10(8) M-1) and provides the driving force for C1s dimerization in the presence of calcium.

Platelet plasma membrane glycoproteins IIb (GPIIb) and IIIa (GPIIIa) form a Ca(2+)-dependent heterodimer. GPIIb/IIIa, which serves as the receptor for fibrinogen and other adhesive proteins at the surface of activated platelets. Using equilibrium dialysis measurements, it was established that both GPIIb and GPIIIa in solution have low-affinity Ca(2-)-binding sites (Kd0.2-0.3 mM), five in GPIIb and two in GPIIIa, and it was confirmed that only the alpha-chain of GPIIb (GPIIb alpha) binds Ca2+. Furthermore, Ca2+ binding was found with two CNBr fragments of GPIIb, GPIIb alpha-(1-285) and GPIIb alpha-(314-489), which carry three out of the four putative Ca(2+)-binding sites. GPIIb/IIIa in solution has a single high-affinity Ca(2+)-binding site (Kd1 80 +/- 30 nM at 21 degrees C), whose degree of saturation regulates the state of association of GPIIb and GPIIIa in the GPIIb/IIIa heterodimer at room temperature, and 3-4 medium-affinity Ca(2+)-binding sites (Kd2 40 +/- 15 microM at 21 degrees C). When GPIIb/IIIa was incorporated into liposomes, Kd1 decreased by an order of magnitude (9 +/- 3 nM at 21 degrees C) and reached the dissociation constant estimated for the high-affinity Ca(2+)-binding sites at the platelet surface [Brass & Shattil (1982) J. Biol. Chem. 257, 1400-1405], whereas Kd2 remained unchanged. The high-affinity Ca(2+)-binding site of GPIIb/IIIa in solution at 4 degrees C has almost the same affinity (Kd1 65 +/- 20 nM) as at 21 degrees C; however, at 37 degrees C, either its affinity decreases enough so as to become experimentally indistinguishable from the medium-affinity Ca(2+)-binding sites determined at this temperature (number of binding sites 3.9 +/- 1.2 mol of Ca2+/mol of GP, Kd 25 +/- 11 microM), or vanishes altogether. Studies on Ca(2+)-dependent dissociation of GPIIIb/IIIa at 37 degrees C in solution seem to support the former interpretation. Further work will be necessary to decide whether the dissociation of GPIIb/IIIa in the platelet membrane at 37 degrees C is regulated by the degree of saturation of the high-affinity Ca(2+)-binding site, as occurs in solution. It is suggested that the high-affinity Ca(2+)-binding site could be related to the putative GPIIIa-binding region in GPIIb (residues 558-747 of the alpha chain).

The human platelet integrin GPIIb/IIIa (228 kDa), a Ca-dependent heterodimer formed by the alpha IIb subunit (GPIIb, 136 kDa) and the beta 3 subunit (GPIIIa, 92 kDa), serves as the fibrinogen receptor at the surface of activated platelets. The degree of dissociation of the GPIIb/IIIa heterodimer (s degrees 20*, 8.9 S) into its constituent glycoproteins (GPIIb, 5.8 S; and GPIIIa, 3.9 S) has been assessed by analytical ultracentrifugation in Triton X100 buffers, and its Ca(2+)- and temperature-dependence correlated with Ca(2+)-binding to GPIIb/IIIa and its temperature dependence. At 21 degrees C half-maximal dissociation of GPIIb/IIIa occurs at 5.5 +/- 2.5 x 10(-8) M Ca2+, very close to the dissociation constant of the high affinity Ca-binding site of GPIIb/IIIa (Kd1 8 +/- 3 x 10(-8) M) (Rivas and González-Rodriguez, 1991) and much lower than the Kd of the 3.4 medium affinity Ca-binding sites (Kd2 4 +/- 1.5 x 10(-5) M), which seems to demonstrate that the stability of the heterodimer in solution at room temperature is regulated by the degree of saturation of the high-affinity Ca-binding site. At 4 degrees C, the stability of the heterodimer is apparently Ca(2+)-independent, while at room and physiological temperatures (15-37 degrees C) the degree of dissociation of the heterodimer is regulated by the degree of dissociation of the high- and medium-affinity Ca-binding sites, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)