Can M. Sag

Calcium/Calmodulin-Dependent Protein Kinase II Contributes to Cardiac Arrhythmogenesis in Heart Failure

Background —Transgenic CaMKIIδC (TG) mice have heart failure and isoproterenol (ISO)-inducible arrhythmias. We hypothesized that CaMKII contributes to arrhythmias and to underlying cellular events and that inhibition of CaMKII reduces cardiac arrhythmogenesis in vitro and in vivo. Methods and Results —Under baseline conditions, isolated cardiac myocytes from TG mice revealed an increased incidence of early afterdepolarizations (ADs) as compared to wild-type (WT) myocytes (P<0.05). CaMKII-inhibition (AIP) completely abolished these ADs in TG cells ( P <0.05). Elevating intracellular Ca stores using ISO (10-8 M) induced a larger amount of delayed ADs and spontaneous action potentials in TG as compared to WT ( P <0.05). This seems to be due to an increased sarcoplasmic reticulum (SR) Ca leak since diastolic [Ca] rose clearly upon ISO in TG but not in WT cells (+20±5% vs. +3±4% at 10-6 M ISO, P <0.05). In parallel, SR Ca leak assessed by spontaneous SR Ca release events showed an increased Ca spark frequency (3.9±0.5 vs. 2.0±0.4 sparks per 100 μm-1*s-1, P <0.05). However, CaMKII-inhibition (either pharmacologically using KN-93 or genetically using an isoform-specific CaMKIIδ-knockout mouse model) significantly reduced SR Ca spark frequency although this rather increased SR Ca content. In parallel, ISO increased the incidence of early (54% vs. 4%, P <0.05) and late (86% vs. 43%, P <0.05) non-stimulated events (NSEs) in TG vs. WT myocytes but CaMKII-inhibition (KN-93 and KO) reduced these proarrhythmogenic events ( P <0.05). In addition, CaMKII-inhibition in TG mice (KN-93) clearly reduced ISO-induced arrhythmias in vivo ( P <0.05). Conclusions —We conclude that CaMKII contributes to cardiac arrhythmogenesis in TG CaMKIIδC mice having heart failure and suggest the increased SR Ca leak as an important mechanism. Moreover, CaMKII-inhibition reduces cardiac arrhythmias in vitro and in vivo and may therefore indicate a potential role for future antiarrhythmic therapies warranting further studies.Background—Transgenic (TG) Ca/calmodulin-dependent protein kinase II (CaMKII)&dgr;C mice have heart failure and isoproterenol (ISO)-inducible arrhythmias. We hypothesized that CaMKII contributes to arrhythmias and underlying cellular events and that inhibition of CaMKII reduces cardiac arrhythmogenesis in vitro and in vivo. Methods and Results—Under baseline conditions, isolated cardiac myocytes from TG mice showed an increased incidence of early afterdepolarizations compared with wild-type myocytes (P<0.05). CaMKII inhibition (AIP) completely abolished these afterdepolarizations in TG cells (P<0.05). Increasing intracellular Ca stores using ISO (10−8 M) induced a larger amount of delayed afterdepolarizations and spontaneous action potentials in TG compared with wild-type cells (P<0.05). This seems to be due to an increased sarcoplasmic reticulum (SR) Ca leak because diastolic [Ca]i rose clearly on ISO in TG but not in wild-type cells (+20±5% versus +3±4% at 10−6 M ISO, P<0.05). In parallel, SR Ca leak assessed by spontaneous SR Ca release events showed an increased Ca spark frequency (3.9±0.5 versus 2.0±0.4 sparks per 100 &mgr;m−1·s−1, P<0.05). However, CaMKII inhibition (either pharmacologically using KN-93 or genetically using an isoform-specific CaMKII&dgr;-knockout mouse model) significantly reduced SR Ca spark frequency, although this rather increased SR Ca content. In parallel, ISO increased the incidence of early (54% versus 4%, P<0.05) and late (86% versus 43%, P<0.05) nonstimulated events in TG versus wild-type myocytes, but CaMKII inhibition (KN-93 and KO) reduced these proarrhythmogenic events (P<0.05). In addition, CaMKII inhibition in TG mice (KN-93) clearly reduced ISO-induced arrhythmias in vivo (P<0.05). Conclusions—We conclude that CaMKII contributes to cardiac arrhythmogenesis in TG CaMKII&dgr;C mice having heart failure and suggest the increased SR Ca leak as an important mechanism. Moreover, CaMKII inhibition reduces cardiac arrhythmias in vitro and in vivo and may therefore indicate a potential role for future antiarrhythmic therapies warranting further studies.

CaMKII Contributes to Cardiac Arrhythmogenesis in Heart Failure

Background —Transgenic CaMKIIδC (TG) mice have heart failure and isoproterenol (ISO)-inducible arrhythmias. We hypothesized that CaMKII contributes to arrhythmias and to underlying cellular events and that inhibition of CaMKII reduces cardiac arrhythmogenesis in vitro and in vivo. Methods and Results —Under baseline conditions, isolated cardiac myocytes from TG mice revealed an increased incidence of early afterdepolarizations (ADs) as compared to wild-type (WT) myocytes (P<0.05). CaMKII-inhibition (AIP) completely abolished these ADs in TG cells ( P <0.05). Elevating intracellular Ca stores using ISO (10-8 M) induced a larger amount of delayed ADs and spontaneous action potentials in TG as compared to WT ( P <0.05). This seems to be due to an increased sarcoplasmic reticulum (SR) Ca leak since diastolic [Ca] rose clearly upon ISO in TG but not in WT cells (+20±5% vs. +3±4% at 10-6 M ISO, P <0.05). In parallel, SR Ca leak assessed by spontaneous SR Ca release events showed an increased Ca spark frequency (3.9±0.5 vs. 2.0±0.4 sparks per 100 μm-1*s-1, P <0.05). However, CaMKII-inhibition (either pharmacologically using KN-93 or genetically using an isoform-specific CaMKIIδ-knockout mouse model) significantly reduced SR Ca spark frequency although this rather increased SR Ca content. In parallel, ISO increased the incidence of early (54% vs. 4%, P <0.05) and late (86% vs. 43%, P <0.05) non-stimulated events (NSEs) in TG vs. WT myocytes but CaMKII-inhibition (KN-93 and KO) reduced these proarrhythmogenic events ( P <0.05). In addition, CaMKII-inhibition in TG mice (KN-93) clearly reduced ISO-induced arrhythmias in vivo ( P <0.05). Conclusions —We conclude that CaMKII contributes to cardiac arrhythmogenesis in TG CaMKIIδC mice having heart failure and suggest the increased SR Ca leak as an important mechanism. Moreover, CaMKII-inhibition reduces cardiac arrhythmias in vitro and in vivo and may therefore indicate a potential role for future antiarrhythmic therapies warranting further studies.Background—Transgenic (TG) Ca/calmodulin-dependent protein kinase II (CaMKII)&dgr;C mice have heart failure and isoproterenol (ISO)-inducible arrhythmias. We hypothesized that CaMKII contributes to arrhythmias and underlying cellular events and that inhibition of CaMKII reduces cardiac arrhythmogenesis in vitro and in vivo. Methods and Results—Under baseline conditions, isolated cardiac myocytes from TG mice showed an increased incidence of early afterdepolarizations compared with wild-type myocytes (P<0.05). CaMKII inhibition (AIP) completely abolished these afterdepolarizations in TG cells (P<0.05). Increasing intracellular Ca stores using ISO (10−8 M) induced a larger amount of delayed afterdepolarizations and spontaneous action potentials in TG compared with wild-type cells (P<0.05). This seems to be due to an increased sarcoplasmic reticulum (SR) Ca leak because diastolic [Ca]i rose clearly on ISO in TG but not in wild-type cells (+20±5% versus +3±4% at 10−6 M ISO, P<0.05). In parallel, SR Ca leak assessed by spontaneous SR Ca release events showed an increased Ca spark frequency (3.9±0.5 versus 2.0±0.4 sparks per 100 &mgr;m−1·s−1, P<0.05). However, CaMKII inhibition (either pharmacologically using KN-93 or genetically using an isoform-specific CaMKII&dgr;-knockout mouse model) significantly reduced SR Ca spark frequency, although this rather increased SR Ca content. In parallel, ISO increased the incidence of early (54% versus 4%, P<0.05) and late (86% versus 43%, P<0.05) nonstimulated events in TG versus wild-type myocytes, but CaMKII inhibition (KN-93 and KO) reduced these proarrhythmogenic events (P<0.05). In addition, CaMKII inhibition in TG mice (KN-93) clearly reduced ISO-induced arrhythmias in vivo (P<0.05). Conclusions—We conclude that CaMKII contributes to cardiac arrhythmogenesis in TG CaMKII&dgr;C mice having heart failure and suggest the increased SR Ca leak as an important mechanism. Moreover, CaMKII inhibition reduces cardiac arrhythmias in vitro and in vivo and may therefore indicate a potential role for future antiarrhythmic therapies warranting further studies.

CaMKII-dependent SR Ca leak contributes to doxorubicin-induced impaired Ca handling in isolated cardiac myocytes.

Doxorubicin (DOX) is one of the most effective chemotherapeutic agents, but cardiotoxicity limits DOX therapy. Although the mechanisms are not entirely understood, reactive oxygen species (ROS) appear to be involved in DOX cardiotoxicity. Ca/calmodulin dependent protein kinase II (CaMKII) can be activated by ROS through oxidation and is known to contribute to myocardial dysfunction through Ca leakage from the sarcoplasmic reticulum (SR). We hypothesized that CaMKII contributes to DOX-induced defects in intracellular Ca ([Ca](i)) handling. Cardiac myocytes were isolated from wild-type (WT) adult rat hearts and from mouse hearts lacking the predominant myocardial CaMKII isoform (CaMKIIδ(-/-), KO) vs. WT. Isolated cardiomyocytes were investigated 30 min after DOX (10 μmol/L) superfusion, using epifluorescence and confocal microscopy. Intracellular ROS-generation ([ROS](i)) and [Ca](i) handling properties were assessed. In a subset of experiments, KN-93 or AIP (each 1 μmol/L) were used to inhibit CaMKII. Melatonin (Mel, 100 μmol/L) served as ROS-scavenger. Western blots were performed to determine the amount of CaMKII phosphorylation and oxidation. DOX increased [ROS](i) and led to significant diastolic [Ca](i) overload in rat myocytes. This was associated with reduced [Ca](i) transients, a 5.8-fold increased diastolic SR Ca leak and diminished SR Ca content. ROS-scavenging partially rescued Ca handling. Western blots revealed increased CaMKII phosphorylation, but not CaMKII oxidation after DOX. Pharmacological CaMKII inhibition attenuated diastolic [Ca](i) overload after DOX superfusion and led to partially restored [Ca](i) transients and SR Ca content, presumably due to reduced Ca spark frequency. In line with this concept, isoform-specific CaMKIIδ-KO attenuated diastolic [Ca](i) overload and Ca spark frequency. DOX exposure induces CaMKII-dependent SR Ca leakage, which partially contributes to impaired cellular [Ca](i) homeostasis. Pharmacological and genetic CaMKII inhibition attenuated but did not completely abolish the effects of DOX on [Ca](i). In light of the clinical relevance of DOX, further investigations seem appropriate to determine if CaMKII inhibition could reduce DOX-induced cardiotoxicity.

While systolic cardiomyocyte function is preserved, diastolic myocyte function and recovery from acidosis are impaired in CaMKIIδ-KO mice

OBJECTIVE CaMKII contributes to impaired contractility in heart failure by inducing SR Ca(2+)-leak. CaMKII-inhibition in the heart was suggested to be a novel therapeutic principle. Different CaMKII isoforms exist. Specifically targeting CaMKIIδ, the dominant isoform in the heart, could be of therapeutic potential without impairing other CaMKII isoforms. RATIONALE We investigated whether cardiomyocyte function is affected by isoform-specific knockout (KO) of CaMKIIδ under basal conditions and upon stress, i.e. upon ß-adrenergic stimulation and during acidosis. RESULTS Systolic cardiac function was largely preserved in the KO in vivo (echocardiography) corresponding to unchanged Ca(2+)-transient amplitudes and isolated myocyte contractility in vitro. CaMKII activity was dramatically reduced while phosphatase-1 inhibitor-1 was significantly increased. Surprisingly, while diastolic Ca(2+)-elimination was slower in KO most likely due to decreased phospholamban Thr-17 phosphorylation, frequency-dependent acceleration of relaxation was still present. Despite decreased SR Ca(2+)-reuptake at lower frequencies, SR Ca(2+)-content was not diminished, which might be due to reduced diastolic SR Ca(2+)-loss in the KO as a consequence of lower RyR Ser-2815 phosphorylation. Challenging KO myocytes with isoproterenol showed intact inotropic and lusitropic responses. During acidosis, SR Ca(2+)-reuptake and SR Ca(2+)-loading were significantly impaired in KO, resulting in an inability to maintain systolic Ca(2+)-transients during acidosis and impaired recovery. CONCLUSIONS Inhibition of CaMKIIδ appears to be safe under basal physiologic conditions. Specific conditions exist (e.g. during acidosis) under which CaMKII-inhibition might not be helpful or even detrimental. These conditions will have to be more clearly defined before CaMKII inhibition is used therapeutically.

Hemopexin counteracts systolic dysfunction induced by heme-driven oxidative stress

Abstract Heart failure is a leading cause of morbidity and mortality in patients affected by different disorders associated to intravascular hemolysis. The leading factor is the presence of pathologic amount of pro‐oxidant free heme in the bloodstream, due to the exhaustion of the natural heme scavenger Hemopexin (Hx). Here, we evaluated whether free heme directly affects cardiac function, and tested the therapeutic potential of replenishing serum Hx for increasing serum heme buffering capacity. The effect of heme on cardiac function was assessed in vitro, on primary cardiomyocytes and H9c2 myoblast cell line, and in vivo, in Hx‐/‐ mice and in genetic and acquired mouse models of intravascular hemolysis. Purified Hx or anti‐oxidants N‐Acetyl‐L‐cysteine and &agr;‐tocopherol were used to counteract heme cardiotoxicity. In mice, Hx loss/depletion resulted in heme accumulation and enhanced reactive oxygen species (ROS) production in the heart, which ultimately led to severe systolic dysfunction. Similarly, high ROS reduced systolic Ca2+ transient amplitudes and fractional shortening in primary cardiomyocytes exposed to free heme. In keeping with these Ca2+ handling alterations, oxidation and CaMKII‐dependent phosphorylation of Ryanodine Receptor 2 were higher in Hx‐/‐ hearts than in controls. Administration of anti‐oxidants prevented systolic failure both in vitro and in vivo. Intriguingly, Hx rescued contraction defects of heme‐treated cardiomyocytes and preserved cardiac function in hemolytic mice. We show that heme‐mediated oxidative stress perturbs cardiac Ca2+ homeostasis and promotes contractile dysfunction. Scavenging heme, Hx counteracts cardiac heme toxicity and preserves left ventricular function. Our data generate the rationale to consider the therapeutic use of Hx to limit the cardiotoxicity of free heme in hemolytic disorders. Graphical abstract Figure. No Caption available. HighlightsIn hemolytic conditions, non‐hemopexin bound heme (NHBH) accumulates in the heart.In the heart, heme‐driven ROS affect Ca2+ homeostasis and impair systolic function.Hemopexin, by binding extracellular heme, prevents/limits heme cardiotoxicity.Hemolytic patients could have benefits from a therapy with heme chelators.

Data demonstrating the anti-oxidant role of hemopexin in the heart

The data presented in this article are related to the research article entitled Hemopexin counteracts systolic dysfunction induced by heme-driven oxidative stress (G. Ingoglia, C. M. Sag, N. Rex, L. De Franceschi, F. Vinchi, J. Cimino, S. Petrillo, S. Wagner, K. Kreitmeier, L. Silengo, F. Altruda, L. S. Maier, E. Hirsch, A. Ghigo and E. Tolosano, 2017) [1]. Data show that heme induces reactive oxygen species (ROS) production in primary cardiomyocytes. H9c2 myoblastic cells treated with heme bound to human Hemopexin (Hx) are protected from heme accumulation and oxidative stress. Similarly, the heme-driven oxidative response is reduced in primary cardiomyocytes treated with Hx-heme compared to heme alone. Our in vivo data show that mouse models of hemolytic disorders, β-thalassemic mice and phenylhydrazine-treated mice, have low serum Hx associated to enhanced expression of heme- and oxidative stress responsive genes in the heart. Hx-/- mice do not show signs of heart fibrosis or overt inflammation. For interpretation and discussion of these data, refer to the research article referenced above.

Empagliflozin reduces Ca/calmodulin-dependent kinase II activity in isolated ventricular cardiomyocytes: Empagliflozin reduces CaMKII activity

The EMPA‐REG OUTCOME study showed reduced mortality and hospitalization due to heart failure (HF) in diabetic patients treated with empagliflozin. Overexpression and Ca2+‐dependent activation of Ca2+/calmodulin‐dependent kinase II (CaMKII) are hallmarks of HF, leading to contractile dysfunction and arrhythmias. We tested whether empagliflozin reduces CaMKII‐ activity and improves Ca2+‐handling in human and murine ventricular myocytes.

Irradiation Leads to Disturbed Excitation-Contraction Coupling in Cardiac Myocytes Through ROS-Dependent CaMKII Activation

Introduction: Chest radiotherapy is part of therapeutic concepts for malignant diseases. However, radiation-induced cardiotoxic effects have become clinically relevant. The underlying pathomechanisms are poorly understood.Hypothesis: We investigated whether excitation-contraction coupling in cardiac myocytes, which depends on intact intracellular Ca cycling, may be directly affected by irradiation (IR).Methods: Isolated ventricular mouse myocytes were exposed to graded IR (0 Gy sham IR, 4 Gy and 20 Gy). Ca and Na handling properties and intracellular reactive oxygen species (ROS) levels were measured using epifluorescence microscopy (Fura-2, SBFI and CMH2-DCFDA, respectively) and confocal Ca (Fluo-4) imaging. Trypan blue staining indicated cellular injury. Western Blots revealed protein phosphorylation levels.Results: IR increased systolic sarcoplasmic reticulum (SR) Ca release leading to an acute positive-inotropic effect with Ca transients of 0.21±0.01 ratio units (r.u., F340nm/F380nm) at 20 Gy (N=89) vs. 0.15±0.01 r.u. in untreated sham control myocytes (N=98, P<0.05 using one-way ANOVA). Although SR Ca content was unaltered, an increased diastolic SR Ca leak measured as Ca sparks (752±113 Ca sparks∗pL−1∗s−1 at 20 Gy, N=32 vs. 208±34 sparks∗pL−1∗s−1 at 0 Gy, N=12, P<0.05 using one-way ANOVA) was accompanied by diastolic Ca overload and increased cellular injury. Furthermore, a rise in intracellular Na from 0.76± 0.01 r.u. (N=27) at 0 Gy to 0.79± 0.01 (N=39, P<0.05 using one-way ANOVA) at 20 Gy was observed after IR.IR-dependent elevation of ROS levels (by ∼667% at 20 Gy) contributed to Ca/calmodulin-dependent protein kinase II (CaMKII) activation. CaMKII-inhibition and ROS-scavenging prevented IR-dependent Ca overload, cellular dysfunction, and cell death.Conclusions: IR severely disturbs cardiac Ca handling and decreases myocyte viability. As underlying pathomechanism, a ROS/CaMKII signaling pathway was identified.