Differential sex-dependent susceptibility to diastolic dysfunction and arrhythmia in cardiomyocytes from obese diabetic HFpEF model.

AIM: Sex-differences in heart failure with preserved ejection fraction (HFpEF) are important, but key mechanisms involved are incompletely understood. While animal models can inform about sex-dependent cellular and molecular changes, many previous preclinical HFpEF models have failed to recapitulate sex-dependent characteristics of human HFpEF. We tested for sex-differences in HFpEF using a two-hit mouse model (leptin receptor-deficient db/db mice plus aldosterone infusion for 4 weeks; db/db+Aldo). METHODS AND RESULTS: We performed echocardiography, electrophysiology, intracellular Ca2+ imaging, and protein analysis. Female HFpEF mice exhibited more severe diastolic dysfunction in line with increased titin N2B isoform expression and PEVK element phosphorylation, and reduced troponin-I phosphorylation. Female HFpEF mice had lower BNP levels than males despite similar comorbidity burden (obesity, diabetes) and cardiac hypertrophy in both sexes. Male HFpEF mice were more susceptible to cardiac alternans. Male HFpEF cardiomyocytes (versus female) exhibited higher diastolic [Ca2+], slower Ca2+ transient decay, reduced L-type Ca2+ current, more pronounced enhancement of the late Na+ current, and increased short-term variability of action potential duration (APD). However, male and female HFpEF myocytes showed similar downregulation of inward rectifier and transient outward K+ currents, APD prolongation, and frequency of delayed afterdepolarizations. Inhibition of Ca2+/calmodulin-dependent protein kinase II (CaMKII) reversed all pathological APD changes in HFpEF in both sexes, and empagliflozin pretreatment mimicked these effects of CaMKII inhibition. Vericiguat had only slight benefits, and these effects were larger in HFpEF females. CONCLUSION: We conclude that the db/db+Aldo preclinical HFpEF murine model recapitulates key sex-specific mechanisms in HFpEF and provides mechanistic insights into impaired excitation-contraction coupling and sex-dependent differential arrhythmia susceptibility in HFpEF with potential therapeutic implications. In male HFpEF myocytes, altered Ca2+ handling and electrophysiology aligned with diastolic dysfunction and arrhythmias, while worse diastolic dysfunction in females may depend more on altered myofilaments properties.

Cardiac Protein Kinase D1 ablation alters the myocytes ß-adrenergic response.

ß-adrenergic (ß-AR) signaling is essential for the adaptation of the heart to exercise and stress. Chronic stress leads to the activation of Ca2+/calmodulin-dependent kinase II (CaMKII) and protein kinase D (PKD). Unlike CaMKII, the effects of PKD on excitation-contraction coupling (ECC) remain unclear. To elucidate the mechanisms of PKD-dependent ECC regulation, we used hearts from cardiac-specific PKD1 knockout (PKD1 cKO) mice and wild-type (WT) littermates. We measured calcium transients (CaT), Ca2+ sparks, contraction and L-type Ca2+ current in paced cardiomyocytes under acute ß-AR stimulation with isoproterenol (ISO; 100 nM). Sarcoplasmic reticulum (SR) Ca2+ load was assessed by rapid caffeine (10 mM) induced Ca2+ release. Expression and phosphorylation of ECC proteins phospholambam (PLB), troponin I (TnI), ryanodine receptor (RyR), sarcoendoplasmic reticulum Ca2+ ATPase (SERCA) were evaluated by western blotting. At baseline, CaT amplitude and decay tau, Ca2+ spark frequency, SR Ca2+ load, L-type Ca2+ current, contractility, and expression and phosphorylation of ECC protein were all similar in PKD1 cKO vs. WT. However, PKD1 cKO cardiomyocytes presented a diminished ISO response vs. WT with less increase in CaT amplitude, slower [Ca2+]i decline, lower Ca2+ spark rate and lower RyR phosphorylation, but with similar SR Ca2+ load, L-type Ca2+ current, contraction and phosphorylation of PLB and TnI. We infer that the presence of PKD1 allows full cardiomyocyte ß-adrenergic responsiveness by allowing optimal enhancement in SR Ca2+ uptake and RyR sensitivity, but not altering L-type Ca2+ current, TnI phosphorylation or contractile response. Further studies are necessary to elucidate the specific mechanisms by which PKD1 is regulating RyR sensitivity. We conclude that the presence of basal PKD1 activity in cardiac ventricular myocytes contributes to normal ß-adrenergic responses in Ca2+ handling.

Whole-heart multiparametric optical imaging reveals sex-dependent heterogeneity in cAMP signaling and repolarization kinetics.

Cyclic adenosine 3',5'-monophosphate (cAMP) is a key second messenger in cardiomyocytes responsible for transducing autonomic signals into downstream electrophysiological responses. Previous studies have shown intracellular heterogeneity and compartmentalization of cAMP signaling. However, whether cAMP signaling occurs heterogeneously throughout the intact heart and how this drives sex-dependent functional responses are unknown. Here, we developed and validated a novel cardiac-specific fluorescence resonance energy transfer-based cAMP reporter mouse and a combined voltage-cAMP whole-heart imaging system. We showed that in male hearts, cAMP was uniformly activated in response to pharmacological ß-adrenergic stimulation. In contrast, female hearts showed that cAMP levels decayed faster in apical versus basal regions, which was associated with nonuniform action potential changes and notable changes in the direction of repolarization. Apical phosphodiesterase (PDE) activity was higher in female versus male hearts, and PDE inhibition prevented repolarization changes in female hearts. Thus, our imaging approach revealed sex-dependent regional breakdown of cAMP and associated electrophysiological differences.

Diabetes and Excess Aldosterone Promote Heart Failure With Preserved Ejection Fraction.

Background The pathobiology of heart failure with preserved ejection fraction (HFpEF) is still poorly understood, and effective therapies remain limited. Diabetes and mineralocorticoid excess are common and important pathophysiological factors that may synergistically promote HFpEF. The authors aimed to develop a novel animal model of HFpEF that recapitulates key aspects of the complex human phenotype with multiorgan impairments. Methods and Results The authors created a novel HFpEF model combining leptin receptor-deficient db/db mice with a 4-week period of aldosterone infusion. The HFpEF phenotype was assessed using morphometry, echocardiography, Ca2+ handling, and electrophysiology. The sodium-glucose cotransporter-2 inhibitor empagliflozin was then tested for reversing the arrhythmogenic cardiomyocyte phenotype. Continuous aldosterone infusion for 4 weeks in db/db mice induced marked diastolic dysfunction with preserved ejection fraction, cardiac hypertrophy, high levels of B-type natriuretic peptide, and significant extracardiac comorbidities (including severe obesity, diabetes with marked hyperglycemia, pulmonary edema, and vascular dysfunction). Aldosterone or db/db alone induced only a mild diastolic dysfunction without congestion. At the cellular level, cardiomyocyte hypertrophy, prolonged Ca2+ transient decay, and arrhythmogenic action potential remodeling (prolongation, increased short-term variability, delayed afterdepolarizations), and enhanced late Na+ current were observed in aldosterone-treated db/db mice. All of these arrhythmogenic changes were reversed by empagliflozin pretreatment of HFpEF cardiomyocytes. Conclusions The authors conclude that the db/db+aldosterone model may represent a distinct clinical subgroup of HFpEF that has marked hyperglycemia, obesity, and increased arrhythmia risk. This novel HFpEF model can be useful in future therapeutic testing and should provide unique opportunities to better understand disease pathobiology.

Nitrosylation of cardiac CaMKII at Cys290 mediates mechanical afterload-induced increases in Ca2+ transient and Ca2+ sparks.

Cardiac mechanical afterload induces an intrinsic autoregulatory increase in myocyte Ca2+ dynamics and contractility to enhance contraction (known as the Anrep effect or slow force response). Our prior work has implicated both nitric oxide (NO) produced by NO synthase 1 (NOS1) and calcium/calmodulin-dependent protein kinase II (CaMKII) activity as required mediators of this form of mechano-chemo-transduction. To test whether a single S-nitrosylation site on CaMKIIδ (Cys290) mediates enhanced sarcoplasmic reticulum Ca2+ leak and afterload-induced increases in sarcoplasmic reticulum (SR) Ca2+ uptake and release, we created a novel CRISPR-based CaMKIIδ knock-in (KI) mouse with a Cys to Ala mutation at C290. These CaMKIIδ-C290A-KI mice exhibited normal cardiac morphometry and function, as well as basal myocyte Ca2+ transients (CaTs) and ß-adrenergic responses. However, the NO donor S-nitrosoglutathione caused an acute increased Ca2+ spark frequency in wild-type (WT) myocytes that was absent in the CaMKIIδ-C290A-KI myocytes. Using our cell-in-gel system to exert multiaxial three-dimensional mechanical afterload on myocytes during contraction, we found that WT myocytes exhibited an afterload-induced increase in Ca2+ sparks and Ca2+ transient amplitude and rate of decline. These afterload-induced effects were prevented in both cardiac-specific CaMKIIδ knockout and point mutant CaMKIIδ-C290A-KI myocytes. We conclude that CaMKIIδ activation by S-nitrosylation at the C290 site is essential in mediating the intrinsic afterload-induced enhancement of myocyte SR Ca2+ uptake, release and Ca2+ transient amplitude (the Anrep effect). The data also indicate that NOS1 activation is upstream of S-nitrosylation at C290 of CaMKII, and that this molecular mechano-chemo-transduction pathway is beneficial in allowing the heart to increase contractility to limit the reduction in stroke volume when aortic pressure (afterload) is elevated. KEY POINTS: A novel CRISPR-based CaMKIIδ knock-in mouse was created in which kinase activation by S-nitrosylation at Cys290 (C290A) is prevented. How afterload affects Ca2+ signalling was measured in cardiac myocytes that were embedded in a hydrogel that imposes a three-dimensional afterload. This mechanical afterload induced an increase in Ca2+ transient amplitude and decay in wild-type myocytes, but not in cardiac-specific CaMKIIδ knockout or C290A knock-in myocytes. The CaMKIIδ-C290 S-nitrosylation site is essential for the afterload-induced enhancement of Ca2+ transient amplitude and Ca2+ sparks.

Protein Kinase D1 Regulates Cardiac Hypertrophy, Potassium Channel Remodeling, and Arrhythmias in Heart Failure.

Background Structural and electrophysiological remodeling characterize heart failure (HF) enhancing arrhythmias. PKD1 (protein kinase D1) is upregulated in HF and mediates pathological hypertrophic signaling, but its role in K+ channel remodeling and arrhythmogenesis in HF is unknown. Methods and Results We performed echocardiography, electrophysiology, and expression analysis in wild-type and PKD1 cardiomyocyte-specific knockout (cKO) mice following transverse aortic constriction (TAC). PKD1-cKO mice exhibited significantly less cardiac hypertrophy post-TAC and were protected from early decline in cardiac contractile function (3 weeks post-TAC) but not the progression to HF at 7 weeks post-TAC. Wild-type mice exhibited ventricular action potential duration prolongation at 8 weeks post-TAC, which was attenuated in PKD1-cKO, consistent with larger K+ currents via the transient outward current, sustained current, inward rectifier K+ current, and rapid delayed rectifier K+ current and increased expression of corresponding K+ channels. Conversely, reduction of slowly inactivating K+ current was independent of PKD1 in HF. Acute PKD inhibition slightly increased transient outward current in TAC and sham wild-type myocytes but did not alter other K+ currents. Sham PKD1-cKO versus wild-type also exhibited larger transient outward current and faster early action potential repolarization. Tachypacing-induced action potential duration alternans in TAC animals was increased and independent of PKD1, but diastolic arrhythmogenic activities were reduced in PKD1-cKO. Conclusions Our data indicate an important role for PKD1 in the HF-related hypertrophic response and K+ channel downregulation. Therefore, PKD1 inhibition may represent a therapeutic strategy to reduce hypertrophy and arrhythmias; however, PKD1 inhibition may not prevent disease progression and reduced contractility in HF.

Subcellular Propagation of Cardiomyocyte ß-Adrenergic Activation of Calcium Uptake Involves Internal ß-Receptors and AKAP7.

ß-adrenergic receptor (ß-AR) signaling in cardiac myocytes is central to cardiac function, but spatiotemporal activation within myocytes is unresolved. In rabbit ventricular myocytes, ß-AR agonists or high extracellular [Ca] were applied locally at one end, to measure ß-AR signal propagation as Ca-transient (CaT) amplitude and sarcoplasmic reticulum (SR) Ca uptake. High local [Ca]o, increased CaT amplitude under the pipette faster than did ISO, but was also more spatially restricted. Local isoproterenol (ISO) or norepinephrine (NE) increased CaT amplitude and SR Ca uptake, that spread along the myocyte to the unexposed end. Thus, local [Ca]i decline kinetics reflect spatio-temporal progression of ß-AR end-effects in myocytes. To test whether intracellular ß-ARs contribute to this response, we used ß-AR-blockers that are membrane permeant (propranolol) or not (sotalol). Propranolol completely blocked NE-dependent CaT effects. However, blocking surface ß-ARs only (sotalol) suppressed only ∼50% of the NE-induced increase in CaT peak and rate of [Ca]i decline, but these changes spread more gradually than NE alone. We also tested whether A-kinase anchoring protein 7γ (AKAP7γ; that interacts with phospholamban) is mobile, such that it might contribute to intracellular spatial propagation of ß-AR signaling. We found AKAP7γ to be highly mobile using fluorescence recovery after photobleach of GFP tagged AKAP7γ, and that PKA activation accelerated AKAP7γ-GFP wash-out upon myocyte saponin-permeabilization, suggesting increased AKAP7γ mobility. We conclude that local ß-AR activation can activate SR Ca uptake at remote myocyte sites, and that intracellular ß-AR and AKAP7γ mobility may play a role in this spread of activation.

Fluorescence lifetime imaging microscopy reveals sodium pump dimers in live cells.

The sodium-potassium ATPase (Na/K-ATPase, NKA) establishes ion gradients that facilitate many physiological functions including action potentials and secondary transport processes. NKA comprises a catalytic subunit (alpha) that interacts closely with an essential subunit (beta) and regulatory transmembrane micropeptides called FXYD proteins. In the heart, a key modulatory partner is the FXYD protein phospholemman (PLM, FXYD1), but the stoichiometry of the alpha-beta-PLM regulatory complex is unknown. Here, we used fluorescence lifetime imaging and spectroscopy to investigate the structure, stoichiometry, and affinity of the NKA-regulatory complex. We observed a concentration-dependent binding of the subunits of NKA-PLM regulatory complex, with avid association of the alpha subunit with the essential beta subunit as well as lower affinity alpha-alpha and alpha-PLM interactions. These data provide the first evidence that, in intact live cells, the regulatory complex is composed of two alpha subunits associated with two beta subunits, decorated with two PLM regulatory subunits. Docking and molecular dynamics (MD) simulations generated a structural model of the complex that is consistent with our experimental observations. We propose that alpha-alpha subunit interactions support conformational coupling of the catalytic subunits, which may enhance NKA turnover rate. These observations provide insight into the pathophysiology of heart failure, wherein low NKA expression may be insufficient to support formation of the complete regulatory complex with the stoichiometry (alpha-beta-PLM)2.

Beat-to-beat dynamic regulation of intracellular pH in cardiomyocytes.

The mammalian heart beats incessantly with rhythmic mechanical activities generating acids that need to be buffered to maintain a stable intracellular pH (pHi) for normal cardiac function. Even though spatial pHi non-uniformity in cardiomyocytes has been documented, it remains unknown how pHi is regulated to match the dynamic cardiac contractions. Here, we demonstrated beat-to-beat intracellular acidification, termed pHi transients, in synchrony with cardiomyocyte contractions. The pHi transients are regulated by pacing rate, Cl-/HCO3 - transporters, pHi buffering capacity, and ß-adrenergic signaling. Mitochondrial electron-transport chain inhibition attenuates the pHi transients, implicating mitochondrial activity in sculpting the pHi regulation. The pHi transients provide dynamic alterations of H+ transport required for ATP synthesis, and a decrease in pHi may serve as a negative feedback to cardiac contractions. Current findings dovetail with the prevailing three known dynamic systems, namely electrical, Ca2+, and mechanical systems, and may reveal broader features of pHi handling in excitable cells.

Deciphering cellular signals in adult mouse sinoatrial node cells.

Sinoatrial node (SAN) cells are the pacemakers of the heart. This study describes a method for culturing and infection of adult mouse SAN cells with FRET-based biosensors that can be exploited to examine signaling events. SAN cells cultured in media with blebbistatin or (S)-nitro-blebbistatin retain their morphology, protein distribution, action potential (AP) waveform, and cAMP dynamics for at least 40 h. SAN cells expressing targeted cAMP sensors show distinct ß-adrenergic-mediated cAMP pools. Cyclic GMP, protein kinase A, Ca2+/CaM kinase II, and protein kinase D in SAN cells also show unique dynamics to different stimuli. Heart failure SAN cells show a decrease in cAMP and cGMP levels. In summary, a reliable method for maintaining adult mouse SAN cells in culture is presented, which facilitates studies of signaling networks and regulatory mechanisms during physiological and pathological conditions.

Cardiomyocyte Na+ and Ca2+ mishandling drives vicious cycle involving CaMKII, ROS, and ryanodine receptors.

Cardiomyocyte Na+ and Ca2+ mishandling, upregulated Ca2+/calmodulin-dependent kinase II (CaMKII), and increased reactive oxygen species (ROS) are characteristics of various heart diseases, including heart failure (HF), long QT (LQT) syndrome, and catecholaminergic polymorphic ventricular tachycardia (CPVT). These changes may form a vicious cycle of positive feedback to promote cardiac dysfunction and arrhythmias. In HF rabbit cardiomyocytes investigated in this study, the inhibition of CaMKII, late Na+ current (INaL), and leaky ryanodine receptors (RyRs) all attenuated the prolongation and increased short-term variability (STV) of action potential duration (APD), but in age-matched controls these inhibitors had no or minimal effects. In control cardiomyocytes, we enhanced RyR leak (by low [caffeine] plus isoproterenol mimicking CPVT) which markedly increased STV and delayed afterdepolarizations (DADs). These proarrhythmic changes were significantly attenuated by both CaMKII inhibition and mitochondrial ROS scavenging, with a slight synergy with INaL inhibition. Inducing LQT by elevating INaL (by Anemone toxin II, ATX-II) caused markedly prolonged APD, increased STV, and early afterdepolarizations (EADs). Those proarrhythmic ATX-II effects were largely attenuated by mitochondrial ROS scavenging, and partially reduced by inhibition of CaMKII and pathological leaky RyRs using dantrolene. In human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) bearing LQT3 mutation SCN5A N406K, dantrolene significantly attenuated cell arrhythmias and APD prolongation. Targeting critical components of the Na+-Ca2+-CaMKII-ROS-INaL arrhythmogenic vicious cycle may exhibit important on-target and also trans-target effects (e.g., INaL and RyR inhibition can alter INaL-mediated LQT3 effects). Incorporating this vicious cycle into therapeutic strategies provides novel integrated insight for treating cardiac arrhythmias and diseases.

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.

Two-hit mechanism of cardiac arrhythmias in diabetic hyperglycaemia: reduced repolarization reserve, neurohormonal stimulation, and heart failure exacerbate susceptibility.

AIMS: Diabetic hyperglycaemia is associated with increased arrhythmia risk. We aimed to investigate whether hyperglycaemia alone can be accountable for arrhythmias or whether it requires the presence of additional pathological factors. METHODS AND RESULTS: Action potentials (APs) and arrhythmogenic spontaneous diastolic activities were measured in isolated murine ventricular, rabbit atrial, and ventricular myocytes acutely exposed to high glucose. Acute hyperglycaemia increased the short-term variability (STV) of action potential duration (APD), enhanced delayed afterdepolarizations, and the inducibility of APD alternans during tachypacing in both murine and rabbit atrial and ventricular myocytes. Hyperglycaemia also prolonged APD in mice and rabbit atrial cells but not in rabbit ventricular myocytes. However, rabbit ventricular APD was more strongly depressed by block of late Na+ current (INaL) during hyperglycaemia, consistent with elevated INaL in hyperglycaemia. All the above proarrhythmic glucose effects were Ca2+-dependent and abolished by CaMKII inhibition. Importantly, when the repolarization reserve was reduced by pharmacological inhibition of K+ channels (either Ito, IKr, IKs, or IK1) or hypokalaemia, acute hyperglycaemia further prolonged APD and further increased STV and alternans in rabbit ventricular myocytes. Likewise, when rabbit ventricular myocytes were pretreated with isoproterenol or angiotensin II, hyperglycaemia significantly prolonged APD, increased STV and promoted alternans. Moreover, acute hyperglycaemia markedly prolonged APD and further enhanced STV in failing rabbit ventricular myocytes. CONCLUSION: We conclude that even though hyperglycaemia alone can enhance cellular proarrhythmic mechanisms, a second hit which reduces the repolarization reserve or stimulates G protein-coupled receptor signalling greatly exacerbates cardiac arrhythmogenesis in diabetic hyperglycaemia.

Intracellular ß1-Adrenergic Receptors and Organic Cation Transporter 3 Mediate Phospholamban Phosphorylation to Enhance Cardiac Contractility.

RATIONALE: ß1ARs (ß1-adrenoceptors) exist at intracellular membranes and OCT3 (organic cation transporter 3) mediates norepinephrine entry into cardiomyocytes. However, the functional role of intracellular ß1AR in cardiac contractility remains to be elucidated. OBJECTIVE: Test localization and function of intracellular ß1AR on cardiac contractility. METHODS AND RESULTS: Membrane fractionation, super-resolution imaging, proximity ligation, coimmunoprecipitation, and single-molecule pull-down demonstrated a pool of ß1ARs in mouse hearts that were associated with sarco/endoplasmic reticulum Ca2+-ATPase at the sarcoplasmic reticulum (SR). Local PKA (protein kinase A) activation was measured using a PKA biosensor targeted at either the plasma membrane (PM) or SR. Compared with wild-type, myocytes lacking OCT3 (OCT3-KO [OCT3 knockout]) responded identically to the membrane-permeant ßAR agonist isoproterenol in PKA activation at both PM and SR. The same was true at the PM for membrane-impermeant norepinephrine, but the SR response to norepinephrine was suppressed in OCT3-KO myocytes. This differential effect was recapitulated in phosphorylation of the SR-pump regulator phospholamban. Similarly, OCT3-KO selectively suppressed calcium transients and contraction responses to norepinephrine but not isoproterenol. Furthermore, sotalol, a membrane-impermeant ßAR-blocker, suppressed isoproterenol-induced PKA activation at the PM but permitted PKA activation at the SR, phospholamban phosphorylation, and contractility. Moreover, pretreatment with sotalol in OCT3-KO myocytes prevented norepinephrine-induced PKA activation at both PM and the SR and contractility. CONCLUSIONS: Functional ß1ARs exists at the SR and is critical for PKA-mediated phosphorylation of phospholamban and cardiac contractility upon catecholamine stimulation. Activation of these intracellular ß1ARs requires catecholamine transport via OCT3.

Hyperglycemia regulates cardiac K+ channels via O-GlcNAc-CaMKII and NOX2-ROS-PKC pathways.

Chronic hyperglycemia and diabetes lead to impaired cardiac repolarization, K+ channel remodeling and increased arrhythmia risk. However, the exact signaling mechanism by which diabetic hyperglycemia regulates cardiac K+ channels remains elusive. Here, we show that acute hyperglycemia increases inward rectifier K+ current (IK1), but reduces the amplitude and inactivation recovery time of the transient outward K+ current (Ito) in mouse, rat, and rabbit myocytes. These changes were all critically dependent on intracellular O-GlcNAcylation. Additionally, IK1 amplitude and Ito recovery effects (but not Ito amplitude) were prevented by the Ca2+/calmodulin-dependent kinase II (CaMKII) inhibitor autocamtide-2-related inhibitory peptide, CaMKIIδ-knockout, and O-GlcNAc-resistant CaMKIIδ-S280A knock-in. Ito reduction was prevented by inhibition of protein kinase C (PKC) and NADPH oxidase 2 (NOX2)-derived reactive oxygen species (ROS). In mouse models of chronic diabetes (streptozotocin, db/db, and high-fat diet), heart failure, and CaMKIIδ overexpression, both Ito and IK1 were reduced in line with the downregulated K+ channel expression. However, IK1 downregulation in diabetes was markedly attenuated in CaMKIIδ-S280A. We conclude that acute hyperglycemia enhances IK1 and Ito recovery via CaMKIIδ-S280 O-GlcNAcylation, but reduces Ito amplitude via a NOX2-ROS-PKC pathway. Moreover, chronic hyperglycemia during diabetes and CaMKII activation downregulate K+ channel expression and function, which may further increase arrhythmia susceptibility.

CaMKIIδC Drives Early Adaptive Ca2+ Change and Late Eccentric Cardiac Hypertrophy.

RATIONALE: CaMKII (Ca2+-Calmodulin dependent protein kinase) δC activation is implicated in pathological progression of heart failure (HF) and CaMKIIδC transgenic mice rapidly develop HF and arrhythmias. However, little is known about early spatio-temporal Ca2+ handling and CaMKII activation in hypertrophy and HF. OBJECTIVE: To measure time- and location-dependent activation of CaMKIIδC signaling in adult ventricular cardiomyocytes, during transaortic constriction (TAC) and in CaMKIIδC transgenic mice. METHODS AND RESULTS: We used human tissue from nonfailing and HF hearts, 4 mouse lines: wild-type, KO (CaMKIIδ-knockout), CaMKIIδC transgenic in wild-type (TG), or KO background, and wild-type mice exposed to TAC. Confocal imaging and biochemistry revealed disproportional CaMKIIδC activation and accumulation in nuclear and perinuclear versus cytosolic regions at 5 days post-TAC. This CaMKIIδ activation caused a compensatory increase in sarcoplasmic reticulum Ca2+ content, Ca2+ transient amplitude, and [Ca2+] decline rates, with reduced phospholamban expression, all of which were most prominent near and in the nucleus. These early adaptive effects in TAC were entirely mimicked in young CaMKIIδ TG mice (6-8 weeks) where no overt cardiac dysfunction was present. The (peri)nuclear CaMKII accumulation also correlated with enhanced HDAC4 (histone deacetylase) nuclear export, creating a microdomain for transcriptional regulation. At longer times both TAC and TG mice progressed to overt HF (at 45 days and 11-13 weeks, respectively), during which time the compensatory Ca2+ transient effects reversed, but further increases in nuclear and time-averaged [Ca2+] and CaMKII activation occurred. CaMKIIδ TG mice lacking δB exhibited more severe HF, eccentric myocyte growth, and nuclear changes. Patient HF samples also showed greatly increased CaMKIIδ expression, especially for CaMKIIδC in nuclear fractions. CONCLUSIONS: We conclude that in early TAC perinuclear CaMKIIδC activation promotes adaptive increases in myocyte Ca2+ transients and nuclear transcriptional responses but that chronic progression of this nuclear Ca2+-CaMKIIδC axis contributes to eccentric hypertrophy and HF.

Aging Disrupts Normal Time-of-Day Variation in Cardiac Electrophysiology.

BACKGROUND: Cardiac gene expression and arrhythmia occurrence have time-of-day variation; however, daily changes in cardiac electrophysiology, arrhythmia susceptibility, and Ca2+ handling have not been characterized. Furthermore, how these patterns change with age is unknown. METHODS: Hearts were isolated during the light (zeitgeber time [ZT] 4 and ZT9) and dark cycle (ZT14 and ZT21) from adult (12-18 weeks) male mice. Hearts from aged (18-20 months) male mice were isolated at ZT4 and ZT14. All hearts were Langendorff-perfused for optical mapping with voltage- and Ca2+-sensitive dyes (n=4-7/group). Cardiac gene and protein expression were assessed with real-time polymerase chain reaction (n=4-6/group) and Western blot (n=3-4/group). RESULTS: Adult hearts had the shortest action potential duration (APD) and Ca2+ transient duration (CaTD) at ZT14 (APD80: ZT4: 45.4±4.1 ms; ZT9: 45.1±8.6 ms; ZT14: 34.7±4.2 ms; ZT21: 49.2±7.6 ms, P<0.05 versus ZT4 and ZT21; and CaTD80: ZT4: 70.1±3.3 ms; ZT9: 72.7±2.7 ms; ZT14: 64.3±3.3 ms; ZT21: 74.4±1.2 ms, P<0.05 versus other time points). The pacing frequency at which CaT alternans emerged was faster, and average CaT alternans magnitude was significantly reduced at ZT14 compared with the other time points. There was a trend for decreased spontaneous premature ventricular complexes and pacing-induced ventricular arrhythmias at ZT14, and the hearts at ZT14 had diminished responses to isoproterenol compared with ZT4 (ZT4: 49.5.0±5.6% versus ZT14: 22.7±9.5% decrease in APD, P<0.01). In contrast, aged hearts exhibited no difference between ZT14 and ZT4 in nearly every parameter assessed (except APD80: ZT4: 39.7±1.9 ms versus ZT14: 33.8±3.1 ms, P<0.01). Gene expression of KCNA5 (potassium voltage-gated channel subfamily A member 5; encoding Kv1.5) was increased, whereas gene expression of ADRB1 (encoding ß1-adrenergic receptors) was decreased at ZT14 versus ZT4 in adult hearts. No time-of-day changes in expression or phosphorylation of Ca2+ handling proteins (SERCA2 [sarco/endoplasmic reticulum Ca2+-ATPase], RyR2 [ryanodine receptor 2], and PLB [phospholamban]) was found in ex vivo perfused adult isolated hearts. CONCLUSIONS: Isolated adult hearts have strong time-of-day variation in cardiac electrophysiology, Ca2+ handling, and adrenergic responsiveness, which is disrupted with age.