Nitric Oxide Modulates Ca2+ Leak and Arrhythmias via S-Nitrosylation of CaMKII.

BACKGROUND: Nitric oxide (NO) has been identified as a signaling molecule generated during ß-adrenergic receptor stimulation in the heart. Furthermore, a role for NO in triggering spontaneous Ca2+ release via S-nitrosylation of CaMKIIδ (Ca2+/calmodulin kinase II delta) is emerging. NO donors are routinely used clinically for their cardioprotective effects on the heart, but it is unknown how NO donors modulate the proarrhythmic CaMKII to alter cardiac arrhythmia incidence. We test the role of S-nitrosylation of CaMKIIδ at the Cysteine-273 inhibitory site and cysteine-290 activating site in cardiac Ca2+ handling and arrhythmogenesis before and during ß-adrenergic receptor stimulation. METHODS: We measured Ca2+-handling in isolated cardiomyocytes from C57BL/6J wild-type (WT) mice and mice lacking CaMKIIδ expression (CaMKIIδ-KO) or with deletion of the S-nitrosylation site on CaMKIIδ at cysteine-273 or cysteine-290 (CaMKIIδ-C273S and -C290A knock-in mice). Cardiomyocytes were exposed to NO donors, S-nitrosoglutathione (GSNO; 150 µM), sodium nitroprusside (200 µM), and ß-adrenergic agonist isoproterenol (100 nmol/L). RESULTS: Both WT and CaMKIIδ-KO cardiomyocytes responded to isoproterenol with a full inotropic and lusitropic Ca2+ transient response as well as increased Ca2+ spark frequency. However, the increase in Ca2+ spark frequency was significantly attenuated in CaMKIIδ-KO cardiomyocytes. The protection from isoproterenol-induced Ca2+ sparks and waves was mimicked by GSNO pretreatment in WT cardiomyocytes but lost in CaMKIIδ-C273S cardiomyocytes. When GSNO was applied after isoproterenol, this protection was not observed in WT or CaMKIIδ-C273S but was apparent in CaMKIIδ-C290A. In Langendorff-perfused isolated hearts, GSNO pretreatment limited isoproterenol-induced arrhythmias in WT but not CaMKIIδ-C273S hearts, while GSNO exposure after isoproterenol sustained or exacerbated arrhythmic events. CONCLUSIONS: We conclude that prior S-nitrosylation of CaMKIIδ at cysteine-273 can limit subsequent ß-adrenergic receptor-induced arrhythmias, but that S-nitrosylation at cysteine-290 might worsen or sustain ß-adrenergic receptor-induced arrhythmias. This has important implications for the administration of NO donors in the clinical setting.

Direct regulation of the cardiac ryanodine receptor (RyR2) by O-GlcNAcylation.

BACKGROUND: O-GlcNAcylation is the enzymatic addition of a sugar, O-linked ß-N-Acetylglucosamine, to the serine and threonine residues of proteins, and is abundant in diabetic conditions. We have previously shown that O-GlcNAcylation can trigger arrhythmias by indirectly increasing pathological Ca2+ leak through the cardiac ryanodine receptor (RyR2) via Ca2+/calmodulin-dependent kinase II (CaMKII). However, RyR2 is well known to be directly regulated by other forms of serine and threonine modification, therefore, this study aimed to determine whether RyR2 is directly modified by O-GlcNAcylation and if this also alters the function of RyR2 and Ca2+ leak. METHODS: O-GlcNAcylation of RyR2 in diabetic human and animal hearts was determined using western blotting. O-GlcNAcylation of RyR2 was pharmacologically controlled and the propensity for Ca2+ leak was determined using single cell imaging. The site of O-GlcNAcylation within RyR2 was determined using site-directed mutagenesis of RyR2. RESULTS: We found that RyR2 is modified by O-GlcNAcylation in human, animal and HEK293 cell models. Under hyperglycaemic conditions O-GlcNAcylation was associated with an increase in Ca2+ leak through RyR2 which persisted after CaMKII inhibition. Conversion of serine-2808 to alanine prevented an O-GlcNAcylation induced increase in Ca2+ leak. CONCLUSIONS: These data suggest that the function of RyR2 can be directly regulated by O-GlcNAcylation and requires the presence of serine-2808.

Nitric Oxide modulates spontaneous Ca2+ release and ventricular arrhythmias during ß-adrenergic signalling through S-nitrosylation of Calcium/Calmodulin dependent kinase II.

Rationale: Nitric oxide (NO) has been identified as a signalling molecule generated during ß-adrenergic receptor (AR) stimulation in the heart. Furthermore, a role for NO in triggering spontaneous Ca2+ release via S-nitrosylation of Ca2+/calmodulin kinase II delta (CaMKIIδ) is emerging. NO donors are routinely used clinically for their cardioprotective effects in the heart, but it is unknown how NO donors modulate the pro-arrhythmic CaMKII to alter cardiac arrhythmia incidence. Objective: We test the role of S-nitrosylation of CaMKIIδ at the Cys-273 inhibitory site and Cys-290 activating site in cardiac Ca2+ handling and arrhythmogenesis before and during ß-AR stimulation. Methods and Results: We measured Ca2+-handling in isolated cardiomyocytes from C57BL/6J wild-type (WT) mice and mice lacking CaMKIIδ expression (CaMKIIδ-KO) or with deletion of the S-nitrosylation site on CaMKIIδ at Cys-273 or Cys-290 (CaMKIIδ-C273S and -C290A knock-in mice). Cardiomyocytes were exposed to NO donors, S-nitrosoglutathione (GSNO; 150 µM), sodium nitroprusside (SNP; 200 µM) and/or ß-adrenergic agonist isoproterenol (ISO; 100 nM). WT and CaMKIIδ-KO cardiomyocytes treated with GSNO showed no change in Ca2+ transient or spark properties under baseline conditions (0.5 Hz stimulation frequency). Both WT and CaMKIIδ-KO cardiomyocytes responded to ISO with a full inotropic and lusitropic Ca2+ transient response as well as increased Ca2+ spark frequency. However, the increase in Ca2+ spark frequency was significantly attenuated in CaMKIIδ-KO cardiomyocytes. The protection from ISO-induced Ca2+ sparks and waves was mimicked by GSNO pre-treatment in WT cardiomyocytes, but lost in CaMKIIδ-C273S cardiomyocytes that displayed a robust increase in Ca2+ waves. This observation is consistent with CaMKIIδ-C273 S-nitrosylation being critical in limiting ISO-induced arrhythmogenic sarcoplasmic reticulum Ca2+ leak. When GSNO was applied after ISO this protection was not observed in WT or CaMKIIδ-C273S but was apparent in CaMKIIδ-C290A. In Langendorff-perfused isolated hearts, GSNO pre-treatment limited ISO-induced arrhythmias in WT but not CaMKIIδ-C273S hearts, while GSNO exposure after ISO sustained or exacerbated arrhythmic events. Conclusions: We conclude that prior S-nitrosylation of CaMKIIδ at Cys-273 can limit subsequent ß-AR induced arrhythmias, but that S-nitrosylation at Cys-290 might worsen or sustain ß-AR-induced arrhythmias. This has important implications for the administration of NO donors in the clinical setting.

Photoactivation of tDodSNO induces localized vasodilation in rats: Metabolically stable S-nitrosothiols can act as targeted nitric oxide donors in vivo.

Nitric oxide (NO) is a key vasodilatory signalling molecule and NO releasing molecules (NO donors) are being examined as potential treatments for many pathologies. The photoresponsive NO donor tert-dodecane S-nitrosothiol (tDodSNO) has been designed to be highly resistant to metabolism; in principle photoactivation of tDodSNO should therefore enable the controlled release of NO in situ via light modulation. To investigate the therapeutic utility of tDodSNO, we tested drug efficacy in Sprague Dawley rats to assess systemic and localised hemodynamic responses under photoactivation, and to confirm drug safety. For comparison, drug action was evaluated alongside the existing NO donors sodium nitroprusside (SNP) and S-nitrosoglutathione (GSNO). Across a dosing range (0.1-3.0 mg/kg) tDodSNO exerted markedly reduced systemic hypotensive action compared to these standard NO donors, inducing a slight decrease in mean arterial pressure (maximum 14.2 ± 3.0%) without affecting heart rate. Target limb photoactivation of tDodSNO resulted in a substantial localized vasodilatory response, with increases to mean (26.0 ± 7.3%) and maximum (53.2 ± 10.4%) blood flow and decreases to vascular resistance (27.1 ± 3.9%) that were restricted to light exposed tissue. In comparison GSNO and SNP showed variable peripheral effects and were not responsive to photoactivation. tDodSNO did not induce met-Hb formation in blood, or display any signs of toxicity, and was rapidly cleared from the systemic circulation, with no hemodynamic effects detectable 5 min post administration. These data are the first demonstration that drugs based upon a metabolically stable S-nitrosothiol group can be photoactivated in vivo to release NO, and that such agents cause less systemic side effects than existing NO donors. Our data support the use of S-nitrosothiols to enable the spatiotemporal control of NO for therapeutic applications.

CaMKII Splice Variants in Vascular Smooth Muscle Cells: The Next Step or Redundancy?

Vascular smooth muscle cells (VSMCs) help to maintain the normal physiological contractility of arterial vessels to control blood pressure; they can also contribute to vascular disease such as atherosclerosis. Ca2+/calmodulin-dependent kinase II (CaMKII), a multifunctional enzyme with four isoforms and multiple alternative splice variants, contributes to numerous functions within VSMCs. The role of these isoforms has been widely studied across numerous tissue types; however, their functions are still largely unknown within the vasculature. Even more understudied is the role of the different splice variants of each isoform in such signaling pathways. This review evaluates the role of the different CaMKII splice variants in vascular pathological and physiological mechanisms, aiming to show the need for more research to highlight both the deleterious and protective functions of the various splice variants.

Treadmill running increases the calcium sensitivity of myofilaments in diabetic rats.

The cardiovascular benefits of regular exercise are unequivocal, yet patients with type 2 diabetes respond poorly to exercise due to a reduced cardiac reserve. The contractile response of diabetic cardiomyocytes to ß-adrenergic stimulation is attenuated, which may result in altered myofilament calcium sensitivity and posttranslational modifications of cardiac troponin I (cTnI). Treadmill running increases myofilament calcium sensitivity in nondiabetic rats, and thus we hypothesized that endurance training would increase calcium sensitivity of diabetic cardiomyocytes and alter site-specific phosphorylation of cTnI. Calcium sensitivity, or pCa50, was measured in Zucker diabetic fatty (ZDF), nondiabetic (nDM), and diabetic (DM) rat hearts after 8 wk of either a sedentary (SED) or progressive treadmill running (TR) intervention. Skinned cardiomyocytes were connected to a capacitance-gauge transducer and a torque motor to measure force as a function of pCa (-log[Ca2+]). Specific phospho-sites on cTnI and O-GlcNAcylation were quantified by immunoblot and total protein phosphorylation by fluorescent gel staining (ProQ Diamond). The novel finding in this study was that training increased pCa50 in both DM and nDM cardiomyocytes (P = 0.009). Phosphorylation of cTnI amino acid residues Ser23/24, a crucial protein kinase A site, and Threonine (Thr)144 was lower in DM hearts, but there was no effect of training on site-specific phosphorylation. In addition, total phosphorylation and O-GlcNAcylation levels were not different between SED and TR groups. These findings suggest that regular exercise may benefit the diabetic heart by specifically targeting myofilament contractile function.NEW & NOTEWORTHY We examined the effects of training on the myofilament calcium in diabetic rat hearts. After 8 wk of treadmill running, both nondiabetic and diabetic cardiomyocytes had increased myofilament calcium sensitivity compared with their sedentary counterparts, but there was no effect of training on the phosphorylation or O-GlcNAcylation status of myofilament proteins measured in this study. These data highlight one potential mechanism capable of reversing, in part, reduced cardiac reserve in the diabetic heart.

Fine-tuning the cardiac O-GlcNAcylation regulatory enzymes governs the functional and structural phenotype of the diabetic heart.

AIMS: The glucose-driven enzymatic modification of myocardial proteins by the sugar moiety, ß-N-acetylglucosamine (O-GlcNAc), is increased in pre-clinical models of diabetes, implicating protein O-GlcNAc modification in diabetes-induced heart failure. Our aim was to specifically examine cardiac manipulation of the two regulatory enzymes of this process on the cardiac phenotype, in the presence and absence of diabetes, utilising cardiac-targeted recombinant-adeno-associated viral-vector-6 (rAAV6)-mediated gene delivery. METHODS AND RESULTS: In human myocardium, total protein O-GlcNAc modification was elevated in diabetic relative to non-diabetic patients, and correlated with left ventricular (LV) dysfunction. The impact of rAAV6-delivered O-GlcNAc transferase (rAAV6-OGT, facilitating protein O-GlcNAcylation), O-GlcNAcase (rAAV6-OGA, facilitating de-O-GlcNAcylation), and empty vector (null) were determined in non-diabetic and diabetic mice. In non-diabetic mice, rAAV6-OGT was sufficient to impair LV diastolic function and induce maladaptive cardiac remodelling, including cardiac fibrosis and increased Myh-7 and Nppa pro-hypertrophic gene expression, recapitulating characteristics of diabetic cardiomyopathy. In contrast, rAAV6-OGA (but not rAAV6-OGT) rescued LV diastolic function and adverse cardiac remodelling in diabetic mice. Molecular insights implicated impaired cardiac PI3K(p110α)-Akt signalling as a potential contributing mechanism to the detrimental consequences of rAAV6-OGT in vivo. In contrast, rAAV6-OGA preserved PI3K(p110α)-Akt signalling in diabetic mouse myocardium in vivo and prevented high glucose-induced impairments in mitochondrial respiration in human cardiomyocytes in vitro. CONCLUSION: Maladaptive protein O-GlcNAc modification is evident in human diabetic myocardium, and is a critical regulator of the diabetic heart phenotype. Selective targeting of cardiac protein O-GlcNAcylation to restore physiological O-GlcNAc balance may represent a novel therapeutic approach for diabetes-induced heart failure.

Increased myofilament calcium sensitivity is associated with decreased cardiac troponin I phosphorylation in the diabetic rat heart.

NEW FINDINGS: What is the central question of this study? In Zucker Diabetic Fatty rats, does cardiomyocyte myofilament function change through the time course of diabetes and what are the mechanisms behind alterations in calcium sensitivity? What is the main finding and its importance? Zucker Diabetic Fatty rats had increased myofilament calcium sensitivity and reduced phosphorylation at cardiac troponin I without differential O-GlcNAcylation. ABSTRACT: The diabetic heart has impaired systolic and diastolic function independent of other comorbidities. The availability of calcium is altered, but does not fully explain the cardiac dysfunction seen in the diabetic heart. Thus, we explored if myofilament calcium regulation of contraction is altered while also categorizing the levels of phosphorylation and O-GlcNAcylation in the myofilaments. Calcium sensitivity (pCa50 ) was measured in Zucker Diabetic Fatty (ZDF) rat hearts at the initial stage of diabetes (12 weeks old) and after 8 weeks of uncontrolled hyperglycaemia (20 weeks old) and in non-diabetic (nDM) littermates. Skinned cardiomyocytes were connected to a capacitance-gauge transducer and a torque motor to measure force as a function of pCa (-log[Ca2+ ]). Fluorescent gel stain (ProQ Diamond) was used to measure total protein phosphorylation. Specific phospho-sites on cardiac troponin I (cTnI) and total cTnI O-GlcNAcylation were quantified using immunoblot. pCa50 was greater in both 12- and 20-week-old diabetic (DM) rats compared to nDM littermates (P = 0.0001). Total cTnI and cTnI serine 23/24 phosphorylation were lower in DM rats (P = 0.003 and P = 0.01, respectively), but cTnI O-GlcNAc protein expression was not different. pCa50 is greater in DM rats and corresponds with an overall reduction in cTnI phosphorylation. These findings indicate that myofilament calcium sensitivity is increased and cTnI phosphorylation is reduced in ZDF DM rats and suggests an important role for cTnI phosphorylation in the DM heart.

CaMKII Inhibition is a Novel Therapeutic Strategy to Prevent Diabetic Cardiomyopathy.

Increasing prevalence of diabetes mellitus worldwide has pushed the complex disease state to the foreground of biomedical research, especially concerning its multifaceted impacts on the cardiovascular system. Current therapies for diabetic cardiomyopathy have had a positive impact, but with diabetic patients still suffering from a significantly greater burden of cardiac pathology compared to the general population, the need for novel therapeutic approaches is great. A new therapeutic target, calcium/calmodulin-dependent kinase II (CaMKII), has emerged as a potential treatment option for preventing cardiac dysfunction in the setting of diabetes. Within the last 10 years, new evidence has emerged describing the pathophysiological consequences of CaMKII activation in the diabetic heart, the mechanisms that underlie persistent CaMKII activation, and the protective effects of CaMKII inhibition to prevent diabetic cardiomyopathy. This review will examine recent evidence tying cardiac dysfunction in diabetes to CaMKII activation. It will then discuss the current understanding of the mechanisms by which CaMKII activity is enhanced during diabetes. Finally, it will examine the benefits of CaMKII inhibition to treat diabetic cardiomyopathy, including contractile dysfunction, heart failure with preserved ejection fraction, and arrhythmogenesis. We intend this review to serve as a critical examination of CaMKII inhibition as a therapeutic strategy, including potential drawbacks of this approach.

Protein O-GlcNAcylation in the heart.

O-GlcNAcylation is a ubiquitous post-translational modification that is extremely labile and plays a significant role in physiology, including the heart. Sustained activation of cardiac O-GlcNAcylation is frequently associated with alterations in cellular metabolism, leading to detrimental effects on cardiovascular function. This is particularly true during conditions such as diabetes, hypertension, cardiac remodelling, heart failure and arrhythmogenesis. Paradoxically, transient elevation of cardiac protein O-GlcNAcylation can also exert beneficial effects in the heart. There is compelling evidence to suggest that a complex interaction between O-GlcNAcylation and phosphorylation also exists in the heart. Beyond direct functional consequences on cardiomyocytes, O-GlcNAcylation also acts indirectly by altering the function of transcription factors that affect downstream signalling. This review focuses on the potential cardioprotective role of protein O-GlcNAcylation during ischaemia-reperfusion injury, the deleterious consequences of chronically elevated O-GlcNAc levels, the interplay between O-GlcNAcylation and phosphorylation in the cardiomyocytes and the effects of O-GlcNAcylation on other major non-myocyte cell types in the heart.

CaMKII Serine 280 O-GlcNAcylation Links Diabetic Hyperglycemia to Proarrhythmia.

[Figure: see text].

CaMKII and PKA-dependent phosphorylation co-regulate nuclear localization of HDAC4 in adult cardiomyocytes.

Nuclear histone deacetylase 4 (HDAC4) represses MEF2-mediated transcription, implicated in the development of heart failure. CaMKII-dependent phosphorylation drives nucleus-to-cytoplasm HDAC4 shuttling, but protein kinase A (PKA) is also linked to HDAC4 translocation. However, the interplay of CaMKII and PKA in regulating adult cardiomyocyte HDAC4 translocation is unclear. Here we sought to determine the interplay of PKA- and CaMKII-dependent HDAC4 phosphorylation and translocation in adult mouse, rabbit and human ventricular myocytes. Confocal imaging and protein analyses revealed that inhibition of CaMKII-but not PKA, PKC or PKD-raised nucleo-to-cytoplasmic HDAC4 fluorescence ratio (FNuc/FCyto) by ~ 50%, indicating baseline CaMKII activity that limits HDAC4 nuclear localization. Further CaMKII activation (via increased extracellular [Ca2+], high pacing frequencies, angiotensin II or overexpression of CaM or CaMKIIδC) led to significant HDAC4 nuclear export. In contrast, PKA activation by isoproterenol or forskolin drove HDAC4 into the nucleus (raising FNuc/FCyto by > 60%). These PKA-mediated effects were abolished in cells pretreated with PKA inhibitors and in cells expressing mutant HDAC4 in S265/266A mutant. In physiological conditions where both kinases are active, PKA-dependent nuclear accumulation of HDAC4 was predominant in the very early response, while CaMKII-dependent HDAC4 export prevailed upon prolonged stimuli. This orchestrated co-regulation was shifted in failing cardiomyocytes, where CaMKII-dependent effects predominated over PKA-dependent response. Importantly, human cardiomyocytes showed similar CaMKII- and PKA-dependent HDAC4 shifts. Collectively, CaMKII limits nuclear localization of HDAC4, while PKA favors HDAC4 nuclear retention and S265/266 is essential for PKA-mediated regulation. These pathways thus compete in HDAC4 nuclear localization and transcriptional regulation in cardiac signaling.

A Timing Effect of 17-ß Estradiol on Atherosclerotic Lesion Development in Female ApoE-/- Mice.

Differences in size or composition of existing plaques at the initiation of estrogen (E2) therapy may underpin evidence of increased risk of atherosclerosis-associated clinical sequelae. We investigated whether E2 had divergent effects on actively-growing versus established-advanced atherosclerotic lesions. Eight weeks of subcutaneous bi-weekly injections of 3 µg/g 17ß-estradiol (n = 18) or vehicle control (n = 22) were administered to female Apolipoprotein null-mice aged 25- or 45 weeks old. Histological assessment of lesion size within the brachiocephalic artery was conducted. Lesion composition was also assessed with acellular, calcification and fibrosis areas measured and other cellular features (intimal thickening, foam cells, lipid pools and cholesterol) scored (0-3) for severity. The comparison showed increased lesion size and calcified area with advancing age but no effect of E2. However, subtle changes in composition were observed following E2. Within the younger group, E2 increased intima thickening and acceleration of calcification. In the older group, E2 increased the thickness of the lesion cap. Therefore, this study shows different effects of E2 depending on the underlying stage of lesion development at the time of initiation of treatment. These divergent changes help explain the controversy of the adverse effects of E2 treatment in cardiovascular disease.

Carvedilol and metoprolol are both able to preserve myocardial function in type 2 diabetes.

PURPOSE: Increasing cohorts of patients present with diabetic cardiomyopathy, and with no targeted options, treatment often rely on generic pharmaceuticals such as ß-blockers. ß-blocker efficacy is heterogenous, with second generation ß-blocker metoprolol selectively inhibiting ß1 -AR, while third generation ß-blocker carvedilol has α1 -AR inhibition, antioxidant, and anti-apoptotic actions alongside nonselective ß-AR inhibition. These additional properties have led to the hypothesis that carvedilol may improve cardiac contractility in the diabetic heart to a greater extent than metoprolol. The present study aimed to compare the efficacy of metoprolol and carvedilol on myocardial function in animal models and cardiac tissue from patients with type 2 diabetes and preserved ejection fraction. METHODS: Echocardiographic examination of cardiac function and assessment of myocardial function in isolated trabeculae was carried out in patients with and without diabetes undergoing coronary artery bypass grafting (CABG) who were prescribed metoprolol or carvedilol. Equivalent measures were undertaken in Zucker Diabetic Fatty (ZDF) rats following 4 weeks treatment with metoprolol or carvedilol. RESULTS: Patients receiving carvedilol compared to metoprolol had no difference in cardiac function, and no difference was apparent in myocardial function between ß-blockers. Both ß-blockers similarly improved myocardial function in diabetic ZDF rats treated for 4 weeks, without significantly affecting in vivo cardiac function. CONCLUSIONS: Metoprolol and carvedilol were found to have no effect on cardiac function in type 2 diabetes with preserved ejection fraction, and were similarly effective in preventing myocardial dysfunction in ZDF rats.

Lower sarcoplasmic reticulum Ca2+ threshold for triggering afterdepolarizations in diabetic rat hearts.

BACKGROUND: Type 2 diabetes (T2D) increases arrhythmia risk through incompletely elucidated mechanisms. Ventricular arrhythmias could be initiated by delayed afterdepolarizations (DADs) resulting from elevated spontaneous sarcoplasmic reticulum (SR) Ca2+ release (SR Ca2+ leak). OBJECTIVE: The purpose of this study was to test the role of DADs and SR Ca2+ leak in triggering arrhythmias in T2D hearts. METHODS: We compared rats with late-onset T2D that display pancreatic and cardiac phenotypes similar to those in humans with T2D (HIP rats) and their nondiabetic littermates (wild type [WT]). RESULTS: HIP rats showed higher propensity for premature ventricular complexes and ventricular tachyarrhythmias, whereas HIP myocytes displayed more frequent DADs and had lower SR Ca2+ content than WT. However, the threshold SR Ca2+ at which depolarizing transient inward currents (Itis) are generated was also significantly decreased in HIP myocytes and was below the actual SR Ca2+ load, which explains the increased DAD incidence despite reduced Ca2+ in SR. In agreement with these findings, Ca2+ spark frequency was augmented in myocytes from HIP vs WT rats, which suggests activation of ryanodine receptors (RyRs) in HIP hearts. Indeed, RyR phosphorylation (by CaMKII and protein kinase A) and oxidation are enhanced in HIP hearts, whereas there is no RyR O-GlcNAcylation in either HIP or control hearts. CaMKII inhibition dissipated the difference in Ca2+ spark frequency between HIP and WT myocytes. CONCLUSION: The threshold SR Ca2+ for generating depolarizing Itis is lower in T2D because of RyR activation after hyperphosphorylation and oxidation, which favors the occurrence of DADs despite low SR Ca2+ loads.

Cardiomyocyte Functional Etiology in Heart Failure With Preserved Ejection Fraction Is Distinctive-A New Preclinical Model.

BACKGROUND: Among the growing numbers of patients with heart failure, up to one half have heart failure with preserved ejection fraction (HFpEF). The lack of effective treatments for HFpEF is a substantial and escalating unmet clinical need-and the lack of HFpEF-specific animal models represents a major preclinical barrier in advancing understanding of HFpEF. As established treatments for heart failure with reduced ejection fraction (HFrEF) have proven ineffective for HFpEF, the contention that the intrinsic cardiomyocyte phenotype is distinct in these 2 conditions requires consideration. Our goal was to validate and characterize a new rodent model of HFpEF, undertaking longitudinal investigations to delineate the associated cardiac and cardiomyocyte pathophysiology. METHODS AND RESULTS: The selectively inbred Hypertrophic Heart Rat (HHR) strain exhibits adult cardiac enlargement (without hypertension) and premature death (40% mortality at 50 weeks) compared to its control strain, the normal heart rat. Hypertrophy was characterized in vivo by maintained systolic parameters (ejection fraction at 85%-90% control) with marked diastolic dysfunction (increased E/E'). Surprisingly, HHR cardiomyocytes were hypercontractile, exhibiting high Ca2+ operational levels and markedly increased L-type Ca2+ channel current. In HHR, prominent regions of reparative fibrosis in the left ventricle free wall adjacent to the interventricular septum were observed. CONCLUSIONS: Thus, the cardiomyocyte remodeling process in the etiology of this HFpEF model contrasts dramatically with the suppressed Ca2+ cycling state that typifies heart failure with reduced ejection fraction. These findings may explain clinical observations, that treatments considered appropriate for heart failure with reduced ejection fraction are of little benefit for HFpEF-and suggest a basis for new therapeutic strategies.

Inhibition of calcium/calmodulin-dependent kinase II restores contraction and relaxation in isolated cardiac muscle from type 2 diabetic rats.

BACKGROUND: Calcium/calmodulin-dependent kinase II-delta (CaMKIIδ) activity is enhanced during hyperglycemia and has been shown to alter intracellular calcium handling in cardiomyocytes, ultimately leading to reduced cardiac performance. However, the effects of CaMKIIδ on cardiac contractility during type 2 diabetes are undefined. METHODS: We examined the expression and activation of CaMKIIδ in right atrial appendages from non-diabetic and type 2 diabetic patients (n = 7 patients per group) with preserved ejection fraction, and also in right ventricular tissue from Zucker Diabetic Fatty rats (ZDF) (n = 5-10 animals per group) during early diabetic cardiac dysfunction, using immunoblot. We also measured whole heart function of ZDF and control rats using echocardiography. Then we measured contraction and relaxation parameters of isolated trabeculae from ZDF to control rats in the presence and absence of CaMKII inhibitors. RESULTS: CaMKIIδ phosphorylation (at Thr287) was increased in both the diabetic human and animal tissue, indicating increased CaMKIIδ activation in the type 2 diabetic heart. Basal cardiac contractility and relaxation were impaired in the cardiac muscles from the diabetic rats, and CaMKII inhibition with KN93 partially restored contractility and relaxation. Autocamtide-2-related-inhibitor peptide (AIP), another CaMKII inhibitor that acts via a different mechanism than KN93, fully restored cardiac contractility and relaxation. CONCLUSIONS: Our results indicate that CaMKIIδ plays a key role in modulating performance of the diabetic heart, and moreover, suggest a potential therapeutic role for CaMKII inhibitors in improving myocardial function during type 2 diabetes.

CaMKII in Vascular Signalling: "Friend or Foe"?

Signalling mechanisms within and between cells of the vasculature enable function and maintain homeostasis. However, a number of these mechanisms also contribute to the pathophysiology of vascular disease states. The multifunctional signalling molecule calcium/calmodulin-dependent kinase II (CaMKII) has been shown to have critical functional effects in many tissue types. For example, CaMKII is known to have a dual role in cardiac physiology and pathology. The function of CaMKII within the vasculature is incompletely understood, but emerging evidence points to potential physiological and pathological roles. This review discusses the evidence for CaMKII signalling within the vasculature, with the aim to better understand both positive and potentially deleterious effects of CaMKII activation in vascular tissue.

S-Nitrosylation Induces Both Autonomous Activation and Inhibition of Calcium/Calmodulin-dependent Protein Kinase II δ.

NO is known to modulate calcium handling and cellular signaling in the myocardium, but key targets for NO in the heart remain unidentified. Recent reports have implied that NO can activate calcium/calmodulin (Ca(2+)/CaM)-dependent protein kinase II (CaMKII) in neurons and the heart. Here we use our novel sensor of CaMKII activation, Camui, to monitor changes in the conformation and activation of cardiac CaMKII (CaMKIIδ) activity after treatment with the NO donor S-nitrosoglutathione (GSNO). We demonstrate that exposure to NO after Ca(2+)/CaM binding to CaMKIIδ results in autonomous kinase activation, which is abolished by mutation of the Cys-290 site. However, exposure of CaMKIIδ to GSNO prior to Ca(2+)/CaM exposure strongly suppresses kinase activation and conformational change by Ca(2+)/CaM. This NO-induced inhibition was ablated by mutation of the Cys-273 site. We found parallel effects of GSNO on CaM/CaMKIIδ binding and CaMKIIδ-dependent ryanodine receptor activation in adult cardiac myocytes. We conclude that NO can play a dual role in regulating cardiac CaMKIIδ activity.

The role of CaMKII in diabetic heart dysfunction.

Diabetes mellitus (DM) is an increasing epidemic that places a significant burden on health services worldwide. The incidence of heart failure (HF) is significantly higher in diabetic patients compared to non-diabetic patients. One underlying mechanism proposed for the link between DM and HF is activation of calmodulin-dependent protein kinase (CaMKIIδ). CaMKIIδ mediates ion channel function and Ca(2+) handling during excitation-contraction and excitation-transcription coupling in the myocardium. CaMKIIδ activity is up-regulated in the myocardium of diabetic patients and mouse models of diabetes, where it promotes pathological signaling that includes hypertrophy, fibrosis and apoptosis. Pharmacological inhibition and knockout models of CaMKIIδ have shown some promise of a potential therapeutic benefit of CaMKIIδ inhibition, with protection against cardiac hypertrophy and apoptosis reported. This review will highlight the pathological role of CaMKIIδ in diabetes and discuss CaMKIIδ as a therapeutic target in DM, and also the effects of exercise on CaMKIIδ.