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Anthracyclines, such as doxorubicin (Dox), are widely used chemotherapeutic agents for the treatment of solid tumors and hematologic malignancies. However, they frequently induce cardiotoxicity leading to dilated cardiomyopathy and heart failure. This study sought to investigate the role of the exchange protein directly activated by cAMP (EPAC) in Dox-induced cardiotoxicity and the potential cardioprotective effects of EPAC inhibition. We show that Dox induces DNA damage and cardiomyocyte cell death with apoptotic features. Dox also led to an increase in both cAMP concentration and EPAC1 activity. The pharmacological inhibition of EPAC1 (with CE3F4) but not EPAC2 alleviated the whole Dox-induced pattern of alterations. When administered in vivo, Dox-treated WT mice developed a dilated cardiomyopathy which was totally prevented in EPAC1 knock-out (KO) mice. Moreover, EPAC1 inhibition potentiated Dox-induced cell death in several human cancer cell lines. Thus, EPAC1 inhibition appears as a potential therapeutic strategy to limit Dox-induced cardiomyopathy without interfering with its antitumoral activity.

Background Human cardiac biopsies are widely used in clinical and fundamental research to decipher molecular events that characterize cardiac physiological and pathophysiological states. One of the main approaches relies on the analysis of semiquantitative immunoblots that reveals alterations in protein expression levels occurring in diseased hearts. To maintain semiquantitative results, expression level of target proteins must be standardized. The expression of HKP (housekeeping proteins) is commonly used to this purpose. Methods and Results We evaluated the stability of HKP expression (actin, β‐tubulin, GAPDH, vinculin, and calsequestrin) and total protein staining within control (coefficient of variation) and comparatively with ischemic human heart biopsies (P value). All HKP exhibited a high level of intragroup (ie, actin, β‐tubulin, and GAPDH) and/or intergroup variability (ie, GAPDH, vinculin, and calsequestrin). Among all, we found total protein staining to exhibit the highest degree of stability within and between groups, which makes this reference the best to study protein expression level in human biopsies from ischemic hearts and age‐matched controls. In addition, we illustrated that using an inappropriate reference protein marker misleads interpretation on SERCA2 (sarco/endoplasmic reticulum Ca2+ ATPase) and cMyBPC (cardiac myosin binding protein‐C) expression level after myocardial infarction. Conclusions These reemphasize the need to standardize the level of protein expression with total protein staining in comparative immunoblot studies on human samples from control and diseased hearts.

In mesenteric arteries (MAs), aldosterone (ALDO) binds to the endogenous mineralocorticoid receptor (MR) and increases the expression of the voltage-gated L-type Cav1.2 channel, an essential ion channel for vascular contraction, sarcoplasmic reticulum (SR) Ca2+ store refilling, and Ca2+ spark generation. In mesenteric artery smooth muscle cells (MASMCs), Ca2+ influx through Cav1.2 is the indirect mechanism for triggering Ca2+ sparks. This process is facilitated by plasma membrane-sarcoplasmic reticulum (PM-SR) nanojunctions that drive Ca2+ from the extracellular space into the SR via Sarco/Endoplasmic Reticulum Ca2+ (SERCA) pump. Ca2+ sparks produced by clusters of Ryanodine receptors (RyRs) at PM-SR nanodomains, decrease contractility by activating large-conductance Ca2+-activated K+ channels (BKCa channels), which generate spontaneous transient outward currents (STOCs). Altogether, Cav1.2, SERCA pump, RyRs, and BKCa channels work as a functional unit at the PM-SR nanodomain, regulating intracellular Ca2+ and vascular function. However, the effect of the ALDO/MR signaling pathway on this functional unit has not been completely explored. Our results show that short-term exposure to ALDO (10 nM, 24 h) increased the expression of Cav1.2 in rat MAs. The depolarization-induced Ca2+ entry increased SR Ca2+ load, and the frequencies of both Ca2+ sparks and STOCs, while [Ca2+]cyt and vasoconstriction remained unaltered in Aldo-treated MAs. ALDO treatment significantly increased the mRNA and protein expression levels of the SERCA pump, which counterbalanced the augmented Cav1.2-mediated Ca2+ influx at the PM-SR nanodomain, increasing SR Ca2+ content, Ca2+ spark and STOC frequencies, and opposing to hyperpolarization-induced vasoconstriction while enhancing Acetylcholine-mediated vasorelaxation. This work provides novel evidence for short-term ALDO-induced upregulation of the functional unit comprising Cav1.2, SERCA2 pump, RyRs, and BKCa channels; in which the SERCA pump buffers ALDO-induced upregulation of Ca2+ entry at the superficial SR-PM nanodomain of MASMCs, preventing ALDO-triggered depolarization-induced vasoconstriction and enhancing vasodilation. Pathological conditions that lead to SERCA pump downregulation, for instance, chronic exposure to ALDO, might favor the development of ALDO/MR-mediated augmented vasoconstriction of mesenteric arteries.

Heart Failure (HF) is defined as the inability of the heart to efficiently pump out enough blood to maintain the body's needs, first at exercise and then also at rest. Alterations in Ca2+ handling contributes to the diminished contraction and relaxation of the failing heart. While most Ca2+ handling protein expression and/or function has been shown to be altered in many models of experimental HF, in this review, we focus in the sarcoplasmic reticulum (SR) Ca2+ release channel, the type 2 ryanodine receptor (RyR2). Various modifications of this channel inducing alterations in its function have been reported. The first was the fact that RyR2 is less responsive to activation by Ca2+ entry through the L-Type calcium channel, which is the functional result of an ultrastructural remodeling of the ventricular cardiomyocyte, with fewer and disorganized transverse (T) tubules. HF is associated with an elevated sympathetic tone and in an oxidant environment. In this line, enhanced RyR2 phosphorylation and oxidation have been shown in human and experimental HF. After several controversies, it is now generally accepted that phosphorylation of RyR2 at the Calmodulin Kinase II site (S2814) is involved in both the depressed contractile function and the enhanced arrhythmic susceptibility of the failing heart. Diminished expression of the FK506 binding protein, FKBP12.6, may also contribute. While these alterations have been mostly studied in the left ventricle of HF with reduced ejection fraction, recent studies are looking at HF with preserved ejection fraction. Moreover, alterations in the RyR2 in HF may also contribute to supraventricular defects associated with HF such as sinus node dysfunction and atrial fibrillation.

Right ventricular failure (RVF) is the most important prognostic factor for morbidity and mortality in pulmonary arterial hypertension (PAH) or pulmonary hypertension (PH) caused by left heart diseases. However, right ventricle (RV) remodeling is understudied and not targeted by specific therapies. This can be partly explained by the lack of basic knowledge of RV remodeling. Since the physiology and hemodynamic function of the RV differ from those of the left ventricle (LV), the mechanisms of LV dysfunction cannot be generalized to that of the RV, albeit a knowledge of these being helpful to understanding RV remodeling and dysfunction. Store-operated Ca2+ entry (SOCE) has recently emerged to participate in the LV cardiomyocyte Ca2+ homeostasis and as a critical player in Ca2+ mishandling in a pathological context. In this paper, we highlight the current knowledge on the SOCE contribution to the LV and RV dysfunctions, as SOCE molecules are present in both compartments. he relative lack of studies on RV dysfunction indicates the necessity of further investigations, a significant challenge over the coming years.

The archetypal store-operated Ca2+ channels (SOCs), Orai1, which are stimulated by the endo/sarcoplasmic reticulum (ER/SR) Ca2+ sensor stromal interaction molecule 1 (STIM1) upon Ca2+ store depletion is traditionally viewed as instrumental for the function of non-excitable cells. In the recent years, expression and function of Orai1 have gained recognition in excitable cardiomyocytes, albeit controversial. Even if its cardiac physiological role in adult is still elusive and needs to be clarified, Orai1 contribution in cardiac diseases such as cardiac hypertrophy and heart failure (HF) is increasingly recognized. The present review surveys our current arising knowledge on the new role of Orai1 channels in the heart and debates on its participation to cardiac hypertrophy and HF.

Cardiomyopathy caused by A-type lamins gene (LMNA) mutations (LMNA cardiomyopathy) is associated with dysfunction of the heart, often leading to heart failure. LMNA cardiomyopathy is highly penetrant with bad prognosis with no specific therapy available. Searching for alternative ways to halt the progression of LMNA cardiomyopathy, we studied the role of calcium homeostasis in the evolution of this disease. We showed that sarcolipin, an inhibitor of the sarco/endoplasmic reticulum (SR) Ca2+ ATPase (SERCA) was abnormally elevated in the ventricular cardiomyocytes of mutated mice compared with wild type mice, leading to an alteration of calcium handling. This occurs early in the progression of the disease, when the left ventricular function was not altered. We further demonstrated that down regulation of sarcolipin using adeno-associated virus (AAV) 9-mediated RNA interference delays cardiac dysfunction in mouse model of LMNA cardiomyopathy. These results showed a novel role for sarcolipin on calcium homeostasis in heart and open perspectives for future therapeutic interventions to LMNA cardiomyopathy.

Aims: Urocortin-2 (Ucn-2) is a potent cardioprotector against Ischemia and Reperfusion (I/R) injuries. However, little is known about its role in the regulation of intracellular Ca2+ concentration ([Ca2+]i) under I/R. Here, we examined whether the addition of Ucn-2 in reperfusion promotes cardioprotection focusing on ([Ca2+]i handling.Methods and Results: Cardiac Wistar rat model of I/R was induced by transient ligation of the left coronary artery and experiments were conducted 1 week after surgery in tissue and adult cardiomyocytes isolated from risk and remote zones. We observed that I/R promoted significant alteration in cardiac contractility as well as an increase in hypertrophy and fibrosis in both zones. The study of confocal [Ca2+]i imaging in adult cardiomyocytes revealed that I/R decreased the amplitude of [Ca2+]i transient and cardiomyocytes contraction in risk and remote zones. Interestingly, intravenous infusion of Ucn-2 before heart’s reperfusion recovered significantly cardiac contractility and prevented fibrosis, but it didn’t affect cardiac hypertrophy. Moreover, Ucn-2 recovered the amplitude of [Ca2+]i transient and modulated the expression of several proteins related to [Ca2+]i homeostasis, such as TRPC5 and Orai1 channels. Using Neonatal Rat Ventricular Myocytes (NRVM) we demonstrated that Ucn-2 blunted I/R-induced Store Operated Ca2+ Entry (SOCE), decreased the expression of TRPC5 and Orai1 as well as their interaction in reperfusion.Conclusion: Our study provides the first evidences demonstrating that Ucn-2 addition at the onset of reperfusion attenuates I/R-induced adverse cardiac remodeling, involving the [Ca2+]i handling and inhibiting the expression and interaction between TRPC5 and Orai1.

Whereas cardiac TRPC (transient receptor potential canonical) channels and the associated store-operated Ca2+ entry (SOCE) are abnormally elevated during cardiac hypertrophy and heart failure, the mechanism of this upregulation is not fully elucidated but might be related to the activation of the mineralocorticoid pathway. Using a combination of biochemical, Ca2+ imaging, and electrophysiological techniques, we determined the effect of 24-h aldosterone treatment on the TRPCs/Orai-dependent SOCE in adult rat ventricular cardiomyocytes (ARVMs). The 24-h aldosterone treatment (from 100 nM to 1 µM) enhanced depletion-induced Ca2+ entry in ARVMs, as assessed by a faster reduction of Fura-2 fluorescence decay upon the addition of Mn2+ and increased Fluo-4/AM fluorescence following Ca2+ store depletion. These effects were prevented by co-treatment with a specific mineralocorticoid receptor (MR) antagonist, RU-28318, and they are associated with the enhanced depletion-induced N-[4-[3,5-Bis(trifluoromethyl)-1H-pyrazol-1-yl]phenyl]-4-methyl-1,2,3-thiadiazole-5-carboxamide (BTP2)-sensitive macroscopic current recorded by patch-clamp experiments. Molecular screening by qRT-PCR and Western blot showed a specific upregulation of TRPC1, TRPC5, and STIM1 expression at the messenger RNA (mRNA) and protein levels upon 24-h aldosterone treatment of ARVMs, corroborated by immunostaining. Our study provides evidence that the mineralocorticoid pathway specifically promotes TRPC1/TRPC5-mediated SOCE in adult rat cardiomyocytes.

Diabetes is a major risk factor for stroke. However, the molecular mechanisms involved in cerebral artery dysfunction found in the diabetic patients are not completely elucidated. In cerebral artery smooth muscle cells (CASMCs), spontaneous and local increases of intracellular Ca2+ due to the opening of ryanodine receptors (Ca2+ sparks) activate large conductance Ca2+-activated K+ (BK) channels that generate spontaneous transient outward currents (STOCs). STOCs have a key participation in the control of vascular myogenic tone and blood pressure. Our goal was to investigate whether alterations in Ca(2+) spark and STOC activities, measured by confocal microscopy and patch-clamp technique, respectively, occur in isolated CASMCs of an experimental model of type-2 diabetes (db/db mouse). We found that mean Ca(2+) spark amplitude, duration, size and rate-of-rise were significantly smaller in Fluo-3 loaded db/db compared to control CASMCs, with a subsequent decrease in the total amount of Ca(2+) released through Ca(2+) sparks in db/db CASMCs, though Ca(2+) spark frequency remained. Interestingly, the frequency of large-amplitude Ca(2+) sparks was also significantly reduced in db/db cells. In addition, the frequency and amplitude of STOCs were markedly reduced at all voltages tested (from -50 to 0 mV) in db/db CASMCs. The latter correlates with decreased BK channel β1/α subunit ratio found in db/db vascular tissues. Taken together, Ca(2+) spark alterations lead to inappropriate BK channels activation in CASMCs of db/db mice and this condition is aggravated by the decrease in the BK β1 subunit/α subunit ratio which underlies the significant reduction of Ca(2+) spark/STOC coupling in CASMCs of diabetic animals.

Ca(2+) mediates the functional coupling between L-type Ca(2+) channel (LTCC) and sarcoplasmic reticulum (SR) Ca(2+) release channel (ryanodine receptor, RyR), participating in key pathophysiological processes. This crosstalk manifests as the orthograde Ca(2+)-induced Ca(2+)-release (CICR) mechanism triggered by Ca(2+) influx, but also as the retrograde Ca(2+)-dependent inactivation (CDI) of LTCC, which depends on both Ca(2+) permeating through the LTCC itself and on SR Ca(2+) release through the RyR. This latter effect has been suggested to rely on local rather than global Ca(2+) signaling, which might parallel the nanodomain control of CDI carried out through calmodulin (CaM). Analyzing the CICR in catecholaminergic polymorphic ventricular tachycardia (CPVT) mice as a model of RyR-generated Ca(2+) leak, we evidence here that increased occurrence of the discrete local SR Ca(2+) releases through the RyRs (Ca(2+) sparks) cause a depolarizing shift in activation and a hyperpolarizing shift in isochronic inactivation of cardiac LTCC current resulting in the reduction of window current. Both increasing fast [Ca(2+)](i) buffer capacity or depleting SR Ca(2+) store blunted these changes, which could be reproduced in WT cells by RyRCa(2+) leak induced with Ryanodol and CaM inhibition.Our results unveiled a new paradigm for CaM-dependent effect on LTCC gating and further the nanodomain Ca(2+) control of LTCC, emphasizing the importance of spatio-temporal relationships between Ca(2+) signals and CaM function.