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.

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.

Increased Mitochondrial Calcium Fluxes in Hypertrophic Right Ventricular Cardiomyocytes from a Rat Model of Pulmonary Artery Hypertension.

Pulmonary artery hypertension causes right ventricular hypertrophy which rapidly progresses to heart failure with underlying cardiac mitochondrial dysfunction. Prior to failure, there are alterations in cytosolic Ca2+ handling that might impact mitochondrial function in the compensatory phase of RV hypertrophy. Our aims, therefore, were (i) to measure beat-to-beat mitochondrial Ca2+ fluxes, and (ii) to determine mitochondrial abundance and function in non-failing, hypertrophic cardiomyocytes. Male Wistar rats were injected with either saline (CON) or monocrotaline (MCT) to induce pulmonary artery hypertension and RV hypertrophy after four weeks. Cytosolic Ca2+ ([Ca2+]cyto) transients were obtained in isolated right ventricular (RV) cardiomyocytes, and mitochondrial Ca2+ ([Ca2+]mito) was recorded in separate RV cardiomyocytes. The distribution and abundance of key proteins was determined using confocal and stimulated emission depletion (STED) microscopy. The RV mitochondrial function was also assessed in RV homogenates using oxygraphy. The MCT cardiomyocytes had increased area, larger [Ca2+]cyto transients, increased Ca2+ store content, and faster trans-sarcolemmal Ca2+ extrusion relative to CON. The MCT cardiomyocytes also had larger [Ca2+]mito transients. STED images detected increased mitochondrial protein abundance (TOM20 clusters per µm2) in MCT, yet no difference was found when comparing mitochondrial respiration and membrane potential between the groups. We suggest that the larger [Ca2+]mito transients compensate to match ATP supply to the increased energy demands of hypertrophic cardiomyocytes.

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.

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.

Mitochondrial function remains impaired in the hypertrophied right ventricle of pulmonary hypertensive rats following short duration metoprolol treatment.

Pulmonary hypertension (PH) increases the work of the right ventricle (RV) and causes right-sided heart failure. This study examined RV mitochondrial function and ADP transfer in PH animals advancing to right heart failure, and investigated a potential therapy with the specific ß1-adrenergic-blocker metoprolol. Adult Wistar rats (317 ± 4 g) were injected either with monocrotaline (MCT, 60 mg kg-1) to induce PH, or with an equivalent volume of saline for controls (CON). At three weeks post-injection the MCT rats began oral metoprolol (10 mg kg-1 day-1-) or placebo treatment until heart failure was observed in the MCT group. Mitochondrial function was then measured using high-resolution respirometry from permeabilised RV fibres. Relative to controls, MCT animals had impaired mitochondrial function but maintained coupling between myofibrillar ATPases and mitochondria, despite an increase in ADP diffusion distances. Cardiomyocytes from the RV of MCT rats were enlarged, primarily due to an increase in myofibrillar protein. The ratio of mitochondria per myofilament area was decreased in both MCT groups (p ≤ 0.05) in comparison to control (CON: 1.03 ± 0.04; MCT: 0.74 ± 0.04; MCT + BB: 0.74 ± 0.03). This not only implicates impaired energy production in PH, but also increases the diffusion distance for metabolites within the MCT cardiomyocytes, adding an additional hindrance to energy supply. Together, these changes may limit energy supply in MCT rat hearts, particularly at high cardiac workloads. Metoprolol treatment did not delay the onset of heart failure symptoms, improve mitochondrial function, or regress RV hypertrophy.

Calcium mishandling impairs contraction in right ventricular hypertrophy prior to overt heart failure.

Currently, there are no tailored therapies available for the treatment of right ventricular (RV) hypertrophy, and the cellular mechanisms that underlie the disease are poorly understood. We investigated the cellular changes that occur early in the progression of the disease, when RV hypertrophy is evident, but prior to the onset of heart failure. Intracellular Ca2+ ([Ca2+]i) handling was examined in a rat model of monocrotaline (MCT)-induced pulmonary hypertension and subsequent RV hypertrophy. [Ca2+]i and stress production were measured in isolated RV trabeculae under baseline conditions (1-Hz stimulation, 1.5 mM [Ca2+]o, 37 °C), and in response to inotropic interventions (5-Hz stimulation or 1-µM isoproterenol). Under baseline conditions, MCT trabeculae had impaired Ca2+ release in response to stimulation with a 45% delay in the time-to-peak Ca2+, but there was no difference in the amplitude and decay of the Ca2+ transient, or active stress relative to RV trabeculae from normotensive hearts (CON). Increasing stimulation frequency from 1 to 5 Hz increased stress in CON, but not MCT trabeculae. Similarly, ß-adrenergic stimulation with isoproterenol increased Ca2+ transient amplitude and active stress in CON, but not in MCT trabeculae, despite accelerating Ca2+ transient decay in trabeculae from both groups. During isoproterenol treatment, MCT trabeculae showed increased diastolic Ca2+ leak, which may explain the blunted inotropic response to ß-adrenergic stimulation. Confocal imaging of trabeculae fixed following functional measurements showed that myocytes were on average wider, and transverse-tubule organisation was disrupted in MCT which provides a mechanism to explain the observed slower release of Ca2+.

Impaired ADP channeling to mitochondria and elevated reactive oxygen species in hypertensive hearts.

Systemic hypertension initially promotes a compensatory cardiac hypertrophy, yet it progresses to heart failure (HF), and energetic deficits appear to be central to this failure. However, the transfer of energy between the mitochondria and the myofibrils is not often considered as part of the energetic equation. We compared hearts from old spontaneously hypertensive rats (SHRs) and normotensive Wistar controls. SHR hearts showed a 35% depression in mitochondrial function, yet produced at least double the amount of reactive oxygen species (ROS) in all respiration states in left ventricular (LV) homogenates. To test the connectivity between mitochondria and myofibrils, respiration was further tested in situ with LV permeabilized fibers by addition of multiple substrates and ATP, which requires hydrolysis to mediate oxidative phosphorylation. By trapping ADP using a pyruvate kinase enzyme system, we tested ADP channeling towards mitochondria, and this suppressed respiration and elevated ROS production more in the SHR fibers. The ADP-trapped state was also less relieved on creatine addition, likely reflecting the 30% depression in total CK activity in the SHR heart fibers. Confocal imaging identified a 34% longer distance between the centers of myofibril to mitochondria in the SHR hearts, which increases transverse metabolite diffusion distances (e.g., for ATP, ADP, and creatine phosphate). We propose that impaired connectivity between mitochondria and myofibrils may contribute to elevated ROS production. Impaired energy exchange could be the result of ultrastructural changes that occur with hypertrophy in this model of hypertension.