Samuel Sossalla

CaMKII-Dependent Diastolic SR Ca2+ Leak and Elevated Diastolic Ca2+ Levels in Right Atrial Myocardium of Patients With Atrial Fibrillation

Rationale: Although research suggests that diastolic Ca2+ levels might be increased in atrial fibrillation (AF), this hypothesis has never been tested. Diastolic Ca2+ leak from the sarcoplasmic reticulum (SR) might increase diastolic Ca2+ levels and play a role in triggering or maintaining AF by transient inward currents through Na+/Ca2+ exchange. In ventricular myocardium, ryanodine receptor type 2 (RyR2) phosphorylation by Ca2+/calmodulin-dependent protein kinase (CaMK)II is emerging as an important mechanism for SR Ca2+ leak. Objective: We tested the hypothesis that CaMKII-dependent diastolic SR Ca2+ leak and elevated diastolic Ca2+ levels occurs in atrial myocardium of patients with AF. Methods and Results: We used isolated human right atrial myocytes from patients with AF versus sinus rhythm and found CaMKII expression to be increased by 40±14% (P<0.05), as well as CaMKII phosphorylation by 33±12% (P<0.05). This was accompanied by a significantly increased RyR2 phosphorylation at the CaMKII site (Ser2814) by 110±53%. Furthermore, cytosolic Ca2+ levels were elevated during diastole (229±20 versus 164±8 nmol/L, P<0.05). Most likely, this resulted from an increased SR Ca2+ leak in AF (P<0.05), which was not attributable to higher SR Ca2+ load. Tetracaine experiments confirmed that SR Ca2+ leak through RyR2 leads to the elevated diastolic Ca2+ level. CaMKII inhibition normalized SR Ca2+ leak and cytosolic Ca2+ levels without changes in L-type Ca2+ current. Conclusion: Increased CaMKII-dependent phosphorylation of RyR2 leads to increased SR Ca2+ leak in human AF, causing elevated cytosolic Ca2+ levels, thereby providing a potential arrhythmogenic substrate that could trigger or maintain AF.

Ranolazine improves diastolic dysfunction in isolated myocardium from failing human hearts : Role of late sodium current and intracellular ion accumulation

The goal of this study was to test the hypothesis that the novel anti-ischemic drug ranolazine, which is known to inhibit late I(Na), could reduce intracellular [Na(+)](i) and diastolic [Ca(2+)](i) overload and improve diastolic function. Contractile dysfunction in human heart failure (HF) is associated with increased [Na(+)](i) and elevated diastolic [Ca(2+)](i). Increased Na(+) influx through voltage-gated Na(+) channels (late I(Na)) has been suggested to contribute to elevated [Na(+)](i) in HF. In isometrically contracting ventricular muscle strips from end-stage failing human hearts, ranolazine (10 micromol/L) did not exert negative inotropic effects on twitch force amplitude. However, ranolazine significantly reduced frequency-dependent increase in diastolic tension (i.e., diastolic dysfunction) by approximately 30% without significantly affecting sarcoplasmic reticulum (SR) Ca(2+) loading. To investigate the mechanism of action of this beneficial effect of ranolazine on diastolic tension, Anemonia sulcata toxin II (ATX-II, 40 nmol/L) was used to increase intracellular Na(+) loading in ventricular rabbit myocytes. ATX-II caused a significant rise in [Na(+)](i) typically seen in heart failure via increased late I(Na). In parallel, ATX-II significantly increased diastolic [Ca(2+)](i). In the presence of ranolazine the increases in late I(Na), as well as [Na(+)](i) and diastolic [Ca(2+)](i) were significantly blunted at all stimulation rates without significantly decreasing Ca(2+) transient amplitudes or SR Ca(2+) content. In summary, ranolazine reduced the frequency-dependent increase in diastolic tension without having negative inotropic effects on contractility of muscles from end-stage failing human hearts. Moreover, in rabbit myocytes the increases in late I(Na), [Na(+)](i) and [Ca(2+)](i) caused by ATX-II, were significantly blunted by ranolazine. These results suggest that ranolazine may be of therapeutic benefit in conditions of diastolic dysfunction due to elevated [Na(+)](i) and diastolic [Ca(2+)](i).

Altered Na+Currents in Atrial Fibrillation: Effects of Ranolazine on Arrhythmias and Contractility in Human Atrial Myocardium

OBJECTIVES We investigated changes in Na(+) currents (I(Na)) in permanent (or chronic) atrial fibrillation (AF) and the effects of I(Na) inhibition using ranolazine (Ran) on arrhythmias and contractility in human atrial myocardium. BACKGROUND Electrical remodeling during AF is typically associated with alterations in Ca(2+) and K(+) currents. It remains unclear whether I(Na) is also altered. METHODS Right atrial appendages from patients with AF (n = 23) and in sinus rhythm (SR) (n = 79) were studied. RESULTS Patch-clamp experiments in isolated atrial myocytes showed significantly reduced peak I(Na) density ( approximately 16%) in AF compared with SR, which was accompanied by a 26% lower expression of Nav1.5 (p < 0.05). In contrast, late I(Na) was significantly increased in myocytes from AF atria by approximately 26%. Ran (10 mumol/l) decreased late I(Na) by approximately 60% (p < 0.05) in myocytes from patients with AF but only by approximately 18% (p < 0.05) in myocytes from SR atria. Proarrhythmic activity was elicited in atrial trabeculae exposed to high [Ca(2+)](o) or isoprenaline, which was significantly reversed by Ran (by 83% and 100%, respectively). Increasing pacing rates from 0.5 to 3.0 Hz led to an increase in diastolic tension that could be significantly decreased by Ran in atria from SR and AF patients. CONCLUSIONS Na(+) channels may contribute to arrhythmias and contractile remodeling in AF. Inhibition of I(Na) with Ran had antiarrhythmic effects and improved diastolic function. Thus, inhibition of late I(Na) may be a promising new treatment option for patients with atrial rhythm disturbances and diastolic dysfunction.

Inhibition of Elevated Ca2+/Calmodulin-Dependent Protein Kinase II Improves Contractility in Human Failing Myocardium

Rationale: Heart failure (HF) is known to be associated with increased Ca2+/calmodulin-dependent protein kinase (CaMK)II expression and activity. There is still controversial discussion about the functional role of CaMKII in HF. Moreover, CaMKII inhibition has never been investigated in human myocardium. Objective: We sought to investigate detailed CaMKII&dgr; expression in end-stage failing human hearts (dilated and ischemic cardiomyopathy) and the functional effects of CaMKII inhibition on contractility. Methods and Results: Expression analysis revealed that CaMKII&dgr;, both cytosolic &dgr;C and nuclear &dgr;B splice variants, were significantly increased in both right and left ventricles from patients with dilated or ischemic cardiomyopathy versus nonfailing. Experiments with isometrically twitching trabeculae revealed significantly improved force frequency relationships in the presence of CaMKII inhibitors (KN-93 and AIP). Increased postrest twitches after CaMKII inhibition indicated an improved sarcoplasmic reticulum (SR) Ca2+ loading. This was confirmed in isolated myocytes by a reduced SR Ca2+ spark frequency and hence SR Ca2+ leak, resulting in increased SR Ca2+ load when inhibiting CaMKII. Ryanodine receptor type 2 phosphorylation at Ser2815, which is known to be phosphorylated by CaMKII thereby contributing to SR Ca2+ leak, was found to be markedly reduced in KN-93–treated trabeculae. Interestingly, CaMKII inhibition did not influence contractility in nonfailing sheep trabeculae. Conclusions: The present study shows for the first time that CaMKII inhibition acutely improves contractility in human HF where CaMKII&dgr; expression is increased. The mechanism proposed consists of a reduced SR Ca2+ leak and consequently increased SR Ca2+ load. Thus, CaMKII inhibition appears to be a possible therapeutic option for patients with HF and merits further investigation.

Differential Cardiac Remodeling in Preload Versus Afterload

Background— Hemodynamic load regulates myocardial function and gene expression. We tested the hypothesis that afterload and preload, despite similar average load, result in different phenotypes. Methods and Results— Afterload and preload were compared in mice with transverse aortic constriction (TAC) and aortocaval shunt (shunt). Compared with sham mice, 6 hours after surgery, systolic wall stress (afterload) was increased in TAC mice (+40%; P<0.05), diastolic wall stress (preload) was increased in shunt (+277%; P<0.05) and TAC mice (+74%; P<0.05), and mean total wall stress was similarly increased in TAC (69%) and shunt mice (67%) (P=NS, TAC versus shunt; each P<0.05 versus sham). At 1 week, left ventricular weight/tibia length was significantly increased by 22% in TAC and 29% in shunt mice (P=NS, TAC versus shunt). After 24 hours and 1 week, calcium/calmodulin-dependent protein kinase II signaling was increased in TAC. This resulted in altered calcium cycling, including increased L-type calcium current, calcium transients, fractional sarcoplasmic reticulum calcium release, and calcium spark frequency. In shunt mice, Akt phosphorylation was increased. TAC was associated with inflammation, fibrosis, and cardiomyocyte apoptosis. The latter was significantly reduced in calcium/calmodulin-dependent protein kinase IIΔ-knockout TAC mice. A total of 157 mRNAs and 13 microRNAs were differentially regulated in TAC versus shunt mice. After 8 weeks, fractional shortening was lower and mortality was higher in TAC versus shunt mice. Conclusions— Afterload results in maladaptive fibrotic hypertrophy with calcium/calmodulin-dependent protein kinase II–dependent altered calcium cycling and apoptosis. Preload is associated with Akt activation without fibrosis, little apoptosis, better function, and lower mortality. This indicates that different loads result in distinct phenotype differences that may require specific pharmacological interventions.

Diabetes increases mortality after myocardial infarction by oxidizing CaMKII

Diabetes increases oxidant stress and doubles the risk of dying after myocardial infarction, but the mechanisms underlying increased mortality are unknown. Mice with streptozotocin-induced diabetes developed profound heart rate slowing and doubled mortality compared with controls after myocardial infarction. Oxidized Ca(2+)/calmodulin-dependent protein kinase II (ox-CaMKII) was significantly increased in pacemaker tissues from diabetic patients compared with that in nondiabetic patients after myocardial infarction. Streptozotocin-treated mice had increased pacemaker cell ox-CaMKII and apoptosis, which were further enhanced by myocardial infarction. We developed a knockin mouse model of oxidation-resistant CaMKIIδ (MM-VV), the isoform associated with cardiovascular disease. Streptozotocin-treated MM-VV mice and WT mice infused with MitoTEMPO, a mitochondrial targeted antioxidant, expressed significantly less ox-CaMKII, exhibited increased pacemaker cell survival, maintained normal heart rates, and were resistant to diabetes-attributable mortality after myocardial infarction. Our findings suggest that activation of a mitochondrial/ox-CaMKII pathway contributes to increased sudden death in diabetic patients after myocardial infarction.

Ca/Calmodulin Kinase II Differentially Modulates Potassium Currents

Background—Potassium currents contribute to action potential duration (APD) and arrhythmogenesis. In heart failure, Ca/calmodulin-dependent protein kinase II (CaMKII) is upregulated and can alter ion channel regulation and expression. Methods and Results—We examine the influence of overexpressing cytoplasmic CaMKII&dgr;C, both acutely in rabbit ventricular myocytes (24-hour adenoviral gene transfer) and chronically in CaMKII&dgr;C-transgenic mice, on transient outward potassium current (Ito), and inward rectifying current (IK1). Acute and chronic CaMKII overexpression increases Ito,slow amplitude and expression of the underlying channel protein KV1.4. Chronic but not acute CaMKII overexpression causes downregulation of Ito,fast, as well as KV4.2 and KChIP2, suggesting that KV1.4 expression responds faster and oppositely to KV4.2 on CaMKII activation. These amplitude changes were not reversed by CaMKII inhibition, consistent with CaMKII-dependent regulation of channel expression and/or trafficking. CaMKII (acute and chronic) greatly accelerated recovery from inactivation for both Ito components, but these effects were acutely reversed by AIP (CaMKII inhibitor), suggesting that CaMKII activity directly accelerates Ito recovery. Expression levels of IK1 and Kir2.1 mRNA were downregulated by CaMKII overexpression. CaMKII acutely increased IK1, based on inhibition by AIP (in both models). CaMKII overexpression in mouse prolonged APD (consistent with reduced Ito,fast and IK1), whereas CaMKII overexpression in rabbit shortened APD (consistent with enhanced IK1 and Ito,slow and faster Ito recovery). Computational models allowed discrimination of contributions of different channel effects on APD. Conclusion—CaMKII has both acute regulatory effects and chronic expression level effects on Ito and IK1 with complex consequences on APD.

Ca2+/Calmodulin-Dependent Protein Kinase II and Protein Kinase A Differentially Regulate Sarcoplasmic Reticulum Ca2+ Leak in Human Cardiac Pathology

Background —Sarcoplasmatic reticulum (SR) Ca2+-leak through ryanodine receptor type 2 (RyR2) dysfunction is of major pathophysiological relevance in human heart failure (HF). However, mechanisms underlying progressive RyR2 dysregulation from cardiac hypertrophy (Hy) to HF are still controversial. Methods and Results —We investigated healthy control myocardium (NF, n=5) as well as myocardium from patients with compensated Hy (n=25) and HF (n=32). In Hy, Ca2+/calmodulin-dependent protein kinase II (CaMKII) and protein kinase A (PKA) both phosphorylate RyR2 at levels which are not different from NF. Accordingly, inhibitors of these kinases reduce the SR Ca2+-leak. In HF, however, the SR Ca2+-leak is nearly doubled compared to Hy leading to reduced systolic Ca2+-transients, a depletion of SR Ca2+-storage and elevated diastolic Ca2+-levels. This is accompanied by a significantly increased CaMKII-dependent phosphorylation of RyR2. In contrast, PKA-dependent RyR2 phosphorylation is not increased in HF and is independent of previous β-blocker treatment. In HF CaMKII inhibition but not inhibition of PKA yields a reduction of the SR Ca2+-leak. Moreover, PKA inhibition further reduces SR Ca2+-load and systolic Ca2+-transients. Conclusions —In human Hy CaMKII as well as PKA functionally regulate RyR2 and may induce SR Ca2+-leak. In the transition from Hy to HF the diastolic Ca2+-leak increases and disturbed Ca2+-cycling occurs. This is associated with an increase in CaMKII- but not PKA-dependent RyR2-phosphorylation. CaMKII inhibition may thus reflect a promising therapeutic target for the treatment of arrhythmias and contractile dysfunction.Background— Sarcoplasmic reticulum (SR) Ca2+ leak through ryanodine receptor type 2 (RyR2) dysfunction is of major pathophysiological relevance in human heart failure (HF); however, mechanisms underlying progressive RyR2 dysregulation from cardiac hypertrophy to HF are still controversial. Methods and Results— We investigated healthy control myocardium (n=5) and myocardium from patients with compensated hypertrophy (n=25) and HF (n=32). In hypertrophy, Ca2+/calmodulin-dependent protein kinase II (CaMKII) and protein kinase A (PKA) both phosphorylated RyR2 at levels that were not different from healthy myocardium. Accordingly, inhibitors of these kinases reduced the SR Ca2+ leak. In HF, however, the SR Ca2+ leak was nearly doubled compared with hypertrophy, which led to reduced systolic Ca2+ transients, a depletion of SR Ca2+ storage and elevated diastolic Ca2+ levels. This was accompanied by a significantly increased CaMKII-dependent phosphorylation of RyR2. In contrast, PKA-dependent RyR2 phosphorylation was not increased in HF and was independent of previous &bgr;-blocker treatment. In HF, CaMKII inhibition but not inhibition of PKA yielded a reduction of the SR Ca2+ leak. Moreover, PKA inhibition further reduced SR Ca2+ load and systolic Ca2+ transients. Conclusions— In human hypertrophy, both CaMKII and PKA functionally regulate RyR2 and may induce SR Ca2+ leak. In the transition from hypertrophy to HF, the diastolic Ca2+ leak increases and disturbed Ca2+ cycling occurs. This is associated with an increase in CaMKII- but not PKA-dependent RyR2 phosphorylation. CaMKII inhibition may thus reflect a promising therapeutic target for the treatment of arrhythmias and contractile dysfunction.

Role of ranolazine in angina, heart failure, arrhythmias, and diabetes

Ranolazine which is currently approved as an antianginal agent reduces the Na-dependent Ca overload via inhibition of the late sodium current (late I(Na)) and thus improves diastolic tone and oxygen handling during myocardial ischemia. According to accumulating evidence ranolazine also exerts beneficial effects on diastolic and systolic heart failure where late I(Na) was also found to be elevated. Moreover, late I(Na) plays a crucial role as an arrhythmic substrate. Ranolazine has been described to have antiarrhythmic effects on ventricular as well as atrial arrhythmias without any proarrythmia or severe organ toxicity as it is common for several antiarrhythmic drugs. In patients with diabetes, treatment with ranolazine led to a significant improvement of glycemic control. In this article possible new clinical indications of the late I(Na)-inhibitor ranolazine are reviewed. We summarize novel experimental and clinical studies and discuss the significance of the available data.

Phosphodiesterase-2 Is Up-Regulated in Human Failing Hearts and Blunts β-Adrenergic Responses in Cardiomyocytes

OBJECTIVES This study investigated whether myocardial phosphodiesterase-2 (PDE2) is altered in heart failure (HF) and determined PDE2-mediated effects on beta-adrenergic receptor (β-AR) signaling in healthy and diseased cardiomyocytes. BACKGROUND Diminished cyclic adenosine monophosphate (cAMP) and augmented cyclic guanosine monophosphate (cGMP) signaling is characteristic for failing hearts. Among the PDE superfamily, PDE2 has the unique property of being able to be stimulated by cGMP, thus leading to a remarkable increase in cAMP hydrolysis mediating a negative cross talk between cGMP and cAMP signaling. However, the role of PDE2 in HF is poorly understood. METHODS Immunoblotting, radioenzymatic- and fluorescence resonance energy transfer-based assays, video edge detection, epifluorescence microscopy, and L-type Ca2(+) current measurements were performed in myocardial tissues and/or isolated cardiomyocytes from human and/or experimental HF, respectively. RESULTS Myocardial PDE2 expression and activity were ~2-fold higher in advanced human HF. Chronic β-AR stimulation via catecholamine infusions in rats enhanced PDE2 expression ~2-fold and cAMP hydrolytic activity ~4-fold, which correlated with blunted cardiac β-AR responsiveness. In diseased cardiomyocytes, higher PDE2 activity could be further enhanced by stimulation of cGMP synthesis via nitric oxide donors, whereas specific PDE2 inhibition partially restored β-AR responsiveness. Accordingly, PDE2 overexpression in healthy cardiomyocytes reduced the rise in cAMP levels and L-type Ca2(+) current amplitude, and abolished the inotropic effect following acute β-AR stimulation, without affecting basal contractility. Importantly, PDE2-overexpressing cardiomyocytes showed marked protection from norepinephrine-induced hypertrophic responses. CONCLUSIONS PDE2 is markedly up-regulated in failing hearts and desensitizes against acute β-AR stimulation. This may constitute an important defense mechanism during cardiac stress, for example, by antagonizing excessive β-AR drive. Thus, activating myocardial PDE2 may represent a novel intracellular antiadrenergic therapeutic strategy in HF.