Loss of Mitochondrial Ca2+ Uniporter Limits Inotropic Reserve and Provides Trigger and Substrate for Arrhythmias in Barth Syndrome Cardiomyopathy.

BACKGROUND: Barth syndrome (BTHS) is caused by mutations of the gene encoding tafazzin, which catalyzes maturation of mitochondrial cardiolipin and often manifests with systolic dysfunction during early infancy. Beyond the first months of life, BTHS cardiomyopathy typically transitions to a phenotype of diastolic dysfunction with preserved ejection fraction, blunted contractile reserve during exercise, and arrhythmic vulnerability. Previous studies traced BTHS cardiomyopathy to mitochondrial formation of reactive oxygen species (ROS). Because mitochondrial function and ROS formation are regulated by excitation-contraction coupling, integrated analysis of mechano-energetic coupling is required to delineate the pathomechanisms of BTHS cardiomyopathy. METHODS: We analyzed cardiac function and structure in a mouse model with global knockdown of tafazzin (Taz-KD) compared with wild-type littermates. Respiratory chain assembly and function, ROS emission, and Ca2+ uptake were determined in isolated mitochondria. Excitation-contraction coupling was integrated with mitochondrial redox state, ROS, and Ca2+ uptake in isolated, unloaded or preloaded cardiac myocytes, and cardiac hemodynamics analyzed in vivo. RESULTS: Taz-KD mice develop heart failure with preserved ejection fraction (>50%) and age-dependent progression of diastolic dysfunction in the absence of fibrosis. Increased myofilament Ca2+ affinity and slowed cross-bridge cycling caused diastolic dysfunction, in part, compensated by accelerated diastolic Ca2+ decay through preactivated sarcoplasmic reticulum Ca2+-ATPase. Taz deficiency provoked heart-specific loss of mitochondrial Ca2+ uniporter protein that prevented Ca2+-induced activation of the Krebs cycle during ß-adrenergic stimulation, oxidizing pyridine nucleotides and triggering arrhythmias in cardiac myocytes. In vivo, Taz-KD mice displayed prolonged QRS duration as a substrate for arrhythmias, and a lack of inotropic response to ß-adrenergic stimulation. Cellular arrhythmias and QRS prolongation, but not the defective inotropic reserve, were restored by inhibiting Ca2+ export through the mitochondrial Na+/Ca2+ exchanger. All alterations occurred in the absence of excess mitochondrial ROS in vitro or in vivo. CONCLUSIONS: Downregulation of mitochondrial Ca2+ uniporter, increased myofilament Ca2+ affinity, and preactivated sarcoplasmic reticulum Ca2+-ATPase provoke mechano-energetic uncoupling that explains diastolic dysfunction and the lack of inotropic reserve in BTHS cardiomyopathy. Furthermore, defective mitochondrial Ca2+ uptake provides a trigger and a substrate for ventricular arrhythmias. These insights can guide the ongoing search for a cure of this orphaned disease.

Myocardial aging as a T-cell-mediated phenomenon.

In recent years, the myocardium has been rediscovered under the lenses of immunology, and lymphocytes have been implicated in the pathogenesis of cardiomyopathies with different etiologies. Aging is an important risk factor for heart diseases, and it also has impact on the immune system. Thus, we sought to determine whether immunological activity would influence myocardial structure and function in elderly mice. Morphological, functional, and molecular analyses revealed that the age-related myocardial impairment occurs in parallel with shifts in the composition of tissue-resident leukocytes and with an accumulation of activated CD4+ Foxp3- (forkhead box P3) IFN-γ+ T cells in the heart-draining lymph nodes. A comprehensive characterization of different aged immune-deficient mouse strains revealed that T cells significantly contribute to age-related myocardial inflammation and functional decline. Upon adoptive cell transfer, the T cells isolated from the mediastinal lymph node (med-LN) of aged animals exhibited increased cardiotropism, compared with cells purified from young donors or from other irrelevant sites. Nevertheless, these cells caused rather mild effects on cardiac functionality, indicating that myocardial aging might stem from a combination of intrinsic and extrinsic (immunological) factors. Taken together, the data herein presented indicate that heart-directed immune responses may spontaneously arise in the elderly, even in the absence of a clear tissue damage or concomitant infection. These observations might shed new light on the emerging role of T cells in myocardial diseases, which primarily affect the elderly population.

SPRED2 deficiency elicits cardiac arrhythmias and premature death via impaired autophagy.

Cardiac functionality is dependent on a balanced protein turnover. Accordingly, regulated protein decay is critical to maintain cardiac function. Here we demonstrate that deficiency of SPRED2, an intracellular repressor of ERK-MAPK signaling markedly expressed in human heart, resulted in impaired autophagy, heart failure, and shortened lifespan. SPRED2-/- mice showed cardiomyocyte hypertrophy, cardiac fibrosis, impaired electrical excitability, and severe arrhythmias. Mechanistically, cardiomyocyte dysfunction resulted from ERK hyperactivation and dysregulated autophagy, observed as accumulation of vesicles, vacuolar structures, and degenerated mitochondria. The diminished autophagic flux in SPRED2-/- hearts was reflected by a reduced LC3-II/LC3-I ratio and by decreased Atg7, Atg4B and Atg16L expression. Furthermore, the autophagosomal adaptors p62/SQSTM1 and NBR1 and lysosomal Cathepsin D accumulated in SPRED2-/- hearts. In wild-type hearts, SPRED2 interacted physically with p62/SQSTM1, NBR1, and Cathepsin D, indicating that SPRED2 is required for autophagolysosome formation in regular autophagy. Restored inhibition of MAPK signaling by selumetinib led to an increase in autophagic flux in vivo. Therefore, our study identifies SPRED2 as a novel, indispensable regulator of cardiac autophagy. Vice versa, SPRED2 deficiency impairs autophagy, leading to cardiac dysfunction and life-threatening arrhythmias.

Microdomain switch of cGMP-regulated phosphodiesterases leads to ANP-induced augmentation of ß-adrenoceptor-stimulated contractility in early cardiac hypertrophy.

RATIONALE: Cyclic nucleotides are second messengers that regulate cardiomyocyte function through compartmentalized signaling in discrete subcellular microdomains. However, the role of different microdomains and their changes in cardiac disease are not well understood. OBJECTIVE: To directly visualize alterations in ß-adrenergic receptor-associated cAMP and cGMP microdomain signaling in early cardiac disease. METHODS AND RESULTS: Unexpectedly, measurements of cell shortening revealed augmented ß-adrenergic receptor-stimulated cardiomyocyte contractility by atrial natriuretic peptide/cGMP signaling in early cardiac hypertrophy after transverse aortic constriction, which was in sharp contrast to well-documented ß-adrenergic and natriuretic peptide signaling desensitization during chronic disease. Real-time cAMP analysis in ß1- and ß2-adrenergic receptor-associated membrane microdomains using a novel membrane-targeted Förster resonance energy transfer-based biosensor transgenically expressed in mice revealed that this unexpected atrial natriuretic peptide effect is brought about by spatial redistribution of cGMP-sensitive phosphodiesterases 2 and 3 between both receptor compartments. Functionally, this led to a significant shift in cGMP/cAMP cross-talk and, in particular, to cGMP-driven augmentation of contractility in vitro and in vivo. CONCLUSIONS: Redistribution of cGMP-regulated phosphodiesterases and functional reorganization of receptor-associated microdomains occurs in early cardiac hypertrophy, affects cGMP-mediated contractility, and might represent a previously not recognized therapeutically relevant compensatory mechanism to sustain normal heart function.

Endothelial actions of atrial natriuretic peptide prevent pulmonary hypertension in mice.

The cardiac hormone atrial natriuretic peptide (ANP) regulates systemic and pulmonary arterial blood pressure by activation of its cyclic GMP-producing guanylyl cyclase-A (GC-A) receptor. In the lung, these hypotensive effects were mainly attributed to smooth muscle-mediated vasodilatation. It is unknown whether pulmonary endothelial cells participate in the homeostatic actions of ANP. Therefore, we analyzed GC-A/cGMP signalling in lung endothelial cells and the cause and functional impact of lung endothelial GC-A dysfunction. Western blot and cGMP determinations showed that cultured human and murine pulmonary endothelial cells exhibit prominent GC-A expression and activity which were markedly blunted by hypoxia, a condition known to trigger pulmonary hypertension (PH). To elucidate the consequences of impaired endothelial ANP signalling, we studied mice with genetic endothelial cell-restricted ablation of the GC-A receptor (EC GC-A KO). Notably, EC GC-A KO mice exhibit PH already under resting, normoxic conditions, with enhanced muscularization of small arteries and perivascular infiltration of inflammatory cells. These alterations were aggravated on exposure of mice to chronic hypoxia. Lung endothelial GC-A dysfunction was associated with enhanced expression of angiotensin converting enzyme (ACE) and increased pulmonary levels of Angiotensin II. Angiotensin II/AT1-blockade with losartan reversed pulmonary vascular remodelling and perivascular inflammation of EC GC-A KO mice, and prevented their increment by chronic hypoxia. This experimental study indicates that endothelial effects of ANP are critical to prevent pulmonary vascular remodelling and PH. Chronic endothelial ANP/GC-A dysfunction, e.g. provoked by hypoxia, is associated with activation of the ACE-angiotensin pathway in the lung and PH.

Stress-dependent dilated cardiomyopathy in mice with cardiomyocyte-restricted inactivation of cyclic GMP-dependent protein kinase I.

AIMS: Cardiac hypertrophy is a common and often lethal complication of arterial hypertension. Elevation of myocyte cyclic GMP levels by local actions of endogenous atrial natriuretic peptide (ANP) and C-type natriuretic peptide (CNP) or by pharmacological inhibition of phosphodiesterase-5 was shown to counter-regulate pathological hypertrophy. It was suggested that cGMP-dependent protein kinase I (cGKI) mediates this protective effect, although the role in vivo is under debate. Here, we investigated whether cGKI modulates myocyte growth and/or function in the intact organism. METHODS AND RESULTS: To circumvent the systemic phenotype associated with germline ablation of cGKI, we inactivated the murine cGKI gene selectively in cardiomyocytes by Cre/loxP-mediated recombination. Mice with cardiomyocyte-restricted cGKI deletion exhibited unaltered cardiac morphology and function under resting conditions. Also, cardiac hypertrophic and contractile responses to ß-adrenoreceptor stimulation by isoprenaline (at 40 mg/kg/day during 1 week) were unaltered. However, angiotensin II (Ang II, at 1000 ng/kg/min for 2 weeks) or transverse aortic constriction (for 3 weeks) provoked dilated cardiomyopathy with marked deterioration of cardiac function. This was accompanied by diminished expression of the [Ca(2+)]i-regulating proteins SERCA2a and phospholamban (PLB) and a reduction in PLB phosphorylation at Ser16, the specific target site for cGKI, resulting in altered myocyte Ca(2+)i homeostasis. In isolated adult myocytes, CNP, but not ANP, stimulated PLB phosphorylation, Ca(2+)i-handling, and contractility via cGKI. CONCLUSION: These results indicate that the loss of cGKI in cardiac myocytes compromises the hypertrophic program to pathological stimulation, rendering the heart more susceptible to dysfunction. In particular, cGKI mediates stimulatory effects of CNP on myocyte Ca(2+)i handling and contractility.

Mena/VASP and αII-Spectrin complexes regulate cytoplasmic actin networks in cardiomyocytes and protect from conduction abnormalities and dilated cardiomyopathy.

BACKGROUND: In the heart, cytoplasmic actin networks are thought to have important roles in mechanical support, myofibrillogenesis, and ion channel function. However, subcellular localization of cytoplasmic actin isoforms and proteins involved in the modulation of the cytoplasmic actin networks are elusive. Mena and VASP are important regulators of actin dynamics. Due to the lethal phenotype of mice with combined deficiency in Mena and VASP, however, distinct cardiac roles of the proteins remain speculative. In the present study, we analyzed the physiological functions of Mena and VASP in the heart and also investigated the role of the proteins in the organization of cytoplasmic actin networks. RESULTS: We generated a mouse model, which simultaneously lacks Mena and VASP in the heart. Mena/VASP double-deficiency induced dilated cardiomyopathy and conduction abnormalities. In wild-type mice, Mena and VASP specifically interacted with a distinct αII-Spectrin splice variant (SH3i), which is in cardiomyocytes exclusively localized at Z- and intercalated discs. At Z- and intercalated discs, Mena and ß-actin localized to the edges of the sarcomeres, where the thin filaments are anchored. In Mena/VASP double-deficient mice, ß-actin networks were disrupted and the integrity of Z- and intercalated discs was markedly impaired. CONCLUSIONS: Together, our data suggest that Mena, VASP, and αII-Spectrin assemble cardiac multi-protein complexes, which regulate cytoplasmic actin networks. Conversely, Mena/VASP deficiency results in disrupted ß-actin assembly, Z- and intercalated disc malformation, and induces dilated cardiomyopathy and conduction abnormalities.

Ablation of C-type natriuretic peptide/cGMP signaling in fibroblasts exacerbates adverse cardiac remodeling in mice.

Excessive activation of cardiac fibroblasts (CFs) in response to injury provokes cardiac fibrosis, stiffness, and failure. The local mediators counterregulating this response remain unclear. Exogenous C-type natriuretic peptide (CNP) exerts antifibrotic effects in preclinical models. To unravel the role of the endogenous hormone, we generated mice with fibroblast-restricted deletion (KO) of guanylyl cyclase-B (GC-B), the cGMP-synthesizing CNP receptor. CNP activated GC-B/cGMP signaling in human and murine CFs, preventing proliferative and promigratory effects of angiotensin II (Ang II) and TGF-ß. Fibroblast-specific GC-B-KO mice showed enhanced fibrosis in response to Ang II infusions. Moreover, after 2 weeks of mild pressure overload induced by transverse aortic constriction (TAC), such KO mice had augmented cardiac fibrosis and hypertrophy, together with systolic and diastolic contractile dysfunction. This was associated with increased expression of the profibrotic genes encoding collagen I, III, and periostin. Notably, such responses to Ang II and TAC were greater in female as compared with male KO mice. Enhanced Ang II-induced CNP expression in female hearts and augmented GC-B expression and activity in female CFs may contribute to this sex disparity. The results show that paracrine CNP signaling in CFs has antifibrotic and antihypertrophic effects. The CNP/GC-B/cGMP pathway might be a target for therapies combating pathological cardiac remodeling.

Identification of SPRED2 (sprouty-related protein with EVH1 domain 2) as a negative regulator of the hypothalamic-pituitary-adrenal axis.

Sprouty-related proteins with EVH1 (enabled/vasodilator-stimulated phosphoprotein homology 1) domain (SPREDs) are inhibitors of MAPK signaling. To elucidate SPRED2 in vivo function, we characterized body homeostasis in SPRED2(-/-) mice. They showed a doubled daily water uptake, induced by elevated serum osmolality, originating from increased blood salt load. Accordingly, serum aldosterone was doubled, accompanied by augmented adrenal aldosterone synthase (AS) expression. Surprisingly, serum vasopressin (AVP) was unaltered, and, as evidenced by halved angiotensin II (Ang II) levels, the renin angiotensin system (RAS) was down-regulated. Adrenocorticotropic hormone (ACTH) was significantly elevated in SPRED2(-/-) mice, together with its secretagogue corticotropin-releasing hormone (CRH) and its downstream target corticosterone. ERK phosphorylation in brains was augmented, and hypothalamic CRH mRNA levels were elevated, both contributing to the increased CRH release. Our data were supported by CRH promoter reporter assays in hypothalamic mHypoE-44 cells, revealing a SPRED-dependent inhibition of Ets (ERK/E-twenty-six)-dependent transcription. Furthermore, SPRED suppressed CRH production in these cells. In conclusion, our study suggests that SPRED2 deficiency leads to an increased MAPK signaling, which results in an augmented CRH promoter activity. The subsequent CRH overproduction causes an up-regulation of downstream hypothalamic-pituitary-adrenal (HPA) hormone secretion. This constitutes a possible trigger for the observed compulsive grooming in SPRED2(-/-) mice and may, together with hyperplasia of aldosterone-producing cells, contribute to the hyperaldosteronism and homeostatic imbalances.

Genotype- and Age-Dependent Differences in Ultrasound Vocalizations of SPRED2 Mutant Mice Revealed by Machine Deep Learning.

Vocalization is an important part of social communication, not only for humans but also for mice. Here, we show in a mouse model that functional deficiency of Sprouty-related EVH1 domain-containing 2 (SPRED2), a protein ubiquitously expressed in the brain, causes differences in social ultrasound vocalizations (USVs), using an uncomplicated and reliable experimental setting of a short meeting of two individuals. SPRED2 mutant mice show an OCD-like behaviour, accompanied by an increased release of stress hormones from the hypothalamic-pituitary-adrenal axis, both factors probably influencing USV usage. To determine genotype-related differences in USV usage, we analyzed call rate, subtype profile, and acoustic parameters (i.e., duration, bandwidth, and mean peak frequency) in young and old SPRED2-KO mice. We recorded USVs of interacting male and female mice, and analyzed the calls with the deep-learning DeepSqueak software, which was trained to recognize and categorize the emitted USVs. Our findings provide the first classification of SPRED2-KO vs. wild-type mouse USVs using neural networks and reveal significant differences in their development and use of calls. Our results show, first, that simple experimental settings in combination with deep learning are successful at identifying genotype-dependent USV usage and, second, that SPRED2 deficiency negatively affects the vocalization usage and social communication of mice.

The ß2-Subunit of Voltage-Gated Calcium Channels Regulates Cardiomyocyte Hypertrophy.

L-type voltage-gated calcium channels (LTCCs) regulate crucial physiological processes in the heart. They are composed of the Cavα1 pore-forming subunit and the accessory subunits Cavß, Cavα2δ, and Cavγ. Cavß is a cytosolic protein that regulates channel trafficking and activity, but it also exerts other LTCC-independent functions. Cardiac hypertrophy, a relevant risk factor for the development of congestive heart failure, depends on the activation of calcium-dependent pro-hypertrophic signaling cascades. Here, by using shRNA-mediated Cavß silencing, we demonstrate that Cavß2 downregulation enhances α1-adrenergic receptor agonist-induced cardiomyocyte hypertrophy. We report that a pool of Cavß2 is targeted to the nucleus in cardiomyocytes and that the expression of this nuclear fraction decreases during in vitro and in vivo induction of cardiac hypertrophy. Moreover, the overexpression of nucleus-targeted Cavß2 in cardiomyocytes inhibits in vitro-induced hypertrophy. Quantitative proteomic analyses showed that Cavß2 knockdown leads to changes in the expression of diverse myocyte proteins, including reduction of calpastatin, an endogenous inhibitor of the calcium-dependent protease calpain. Accordingly, Cavß2-downregulated cardiomyocytes had a 2-fold increase in calpain activity as compared to control cells. Furthermore, inhibition of calpain activity in Cavß2-downregulated cells abolished the enhanced α1-adrenergic receptor agonist-induced hypertrophy observed in these cells. Our findings indicate that in cardiomyocytes, a nuclear pool of Cavß2 participates in cellular functions that are independent of LTCC activity. They also indicate that a downregulation of nuclear Cavß2 during cardiomyocyte hypertrophy promotes the activation of calpain-dependent hypertrophic pathways.

Heart-Microcirculation Connection: Effects of ANP (Atrial Natriuretic Peptide) on Pericytes Participate in the Acute and Chronic Regulation of Arterial Blood Pressure.

Cardiac ANP (atrial natriuretic peptide) moderates arterial blood pressure. The mechanisms mediating its hypotensive effects are complex and involve inhibition of the renin-angiotensin-aldosterone system, increased natriuresis, endothelial permeability, and vasodilatation. The contribution of the direct vasodilating effects of ANP to blood pressure homeostasis is controversial because variable levels of the ANP receptor, GC-A (guanylyl cyclase-A), are expressed among vascular beds. Here, we show that ANP stimulates GC-A/cyclic GMP signaling in cultured microvascular pericytes and thereby the phosphorylation of the regulatory subunit of myosin phosphatase 1 by cGMP-dependent protein kinase I. Moreover, ANP prevents the calcium and contractile responses of pericytes to endothelin-1 as well as microvascular constrictions. In mice with conditional inactivation (knock-out) of GC-A in microcirculatory pericytes, such vasodilating effects of ANP on precapillary arterioles and capillaries were fully abolished. Concordantly, these mice have increased blood pressure despite preserved renal excretory function. Furthermore, acute intravascular volume expansion, which caused release of cardiac ANP, did not affect blood pressure of control mice but provoked hypertensive reactions in pericyte GC-A knock-out littermates. We conclude that GC-A/cGMP-dependent modulation of pericytes and microcirculatory tone contributes to the acute and chronic moderation of arterial blood pressure by ANP. Graphic Abstract A graphic abstract is available for this article.

C-type natriuretic peptide moderates titin-based cardiomyocyte stiffness.

Heart failure is often accompanied by titin-dependent myocardial stiffness. Phosphorylation of titin by cGMP-dependent protein kinase I (PKGI) increases cardiomyocyte distensibility. The upstream pathways stimulating PKGI-mediated titin phosphorylation are unclear. We studied whether C-type natriuretic peptide (CNP), via its guanylyl cyclase-B (GC-B) receptor and cGMP/PKGI signaling, modulates titin-based ventricular compliance. To dissect GC-B-mediated effects of endogenous CNP in cardiomyocytes, we generated mice with cardiomyocyte-restricted GC-B deletion (CM GC-B-KO mice). The impact on heart morphology and function, myocyte passive tension, and titin isoform expression and phosphorylation was studied at baseline and after increased afterload induced by transverse aortic constriction (TAC). Pressure overload increased left ventricular endothelial CNP expression, with an early peak after 3 days. Concomitantly, titin phosphorylation at Ser4080, the site phosphorylated by PKGI, was augmented. Notably, in CM GC-B-KO mice this titin response was abolished. TAC-induced hypertrophy and fibrosis were not different between genotypes. However, the KO mice presented mild systolic and diastolic dysfunction together with myocyte stiffness, which were not observed in control littermates. In vitro, recombinant PKGI rescued reduced titin-Ser4080 phosphorylation and reverted passive stiffness of GC-B-deficient cardiomyocytes. CNP-induced activation of GC-B/cGMP/PKGI signaling in cardiomyocytes provides a protecting regulatory circuit preventing titin-based myocyte stiffening during early phases of pressure overload.

Development and Characterization of an Inducible Rat Model of Chronic Thromboembolic Pulmonary Hypertension.

Chronic thromboembolic pulmonary hypertension (CTEPH) is an entity of PH that not only limits patients quality of life but also causes significant morbidity and mortality. The treatment of choice is pulmonary endarterectomy. However numerous patients do not qualify for pulmonary endarterectomy or present with residual vasculopathy post pulmonary endarterectomy and require specific vasodilator treatment. Currently, there is no available specific small animal model of CTEPH that could serve as tool to identify targetable molecular pathways and to test new treatment options. Thus, we generated and standardized a rat model that not only resembles functional and histological features of CTEPH but also emulates thrombi fibrosis. The pulmonary embolism protocol consisted of 3 sequential tail vein injections of fibrinogen/collagen-covered polystyrene microspheres combined with thrombin and administered to 10-week-old male Wistar rats. After the third embolism, rats developed characteristic features of CTEPH including elevated right ventricular systolic pressure, right ventricular cardiomyocyte hypertrophy, pulmonary artery remodeling, increased serum brain natriuretic peptide levels, thrombi fibrosis, and formation of pulmonary cellular-fibrotic lesions. The current animal model seems suitable for detailed study of CTEPH pathophysiology and permits preclinical testing of new pharmacological therapies against CTEPH.

Calcium/calmodulin-dependent protein kinase II contributes to cardiac arrhythmogenesis in heart failure.

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

2-Arachidonylglycerol acting on CB1 cannabinoid receptors mediates delayed cardioprotection induced by nitric oxide in rat isolated hearts.

Endocannabinoids have been implicated in protective effects in the heart and brain, but the mechanism of possible infarct-size-reducing effects remains controversial. Using a model of delayed preconditioning (PC), rats received the nitric oxide (NO) donor nitroglycerin (0.15 mg/h/kg) for 24 hours via transdermal application. Two days later, rat isolated perfused hearts were subjected to global, no-flow ischemia (20 min), and reperfusion (120 min). Cannabinoid receptor antagonists were given before no-flow throughout the protocol. Endocannabinoids were detected by liquid chromatography and mass spectrometry. NO-induced PC reduced the left ventricular infarct size from 40.9 +/- 3.9% to 27.5 +/- 3.8% (P < 0.05). Treatment with the specific CB1 cannabinoid receptor antagonist AM-251 (0.3 microM) prevented the protective effect of PC on infarct size (40.2 +/- 4.7%, P > 0.05 vs. controls). On the contrary, the specific CB2 receptor antagonist AM-630 (0.3 microM) did not alter infarct size (31.6 +/- 6.3%, P > 0.05 vs. PC alone). Recovery of left ventricular developed pressure and coronary flow was incomplete in control and NO-pretreated hearts and not consistently altered by cannabinoid receptor antagonists. PC increased the heart tissue content of the endocannabinoid 2-arachidonylglycerol (2-AG) from 4.6 +/- 1.0 nmol/g in controls to 12.0 +/- 2.1 nmol/g (P < 0.05). Tissue levels of the endocannabinoid arachidonylethanolamide (anandamide) remained unchanged (19.8 +/- 3.9 pmol/g vs. 19.5 +/- 4.8 pmol/g). 2-AG (1 microM) or its metabolically stable derivative noladinether (0.1 microM), given 30 minutes before ischemia/reperfusion in unpreconditioned hearts, mimicked the cardioprotective effects of PC and reduced infarct size. We conclude that delayed PC through transdermal nitroglycerin application increases the production of the endocannabinoid 2-AG which elicits protective effects against myocardial infarction via CB1 cannabinoid receptors which represents one new mechanism of NO-mediated PC.

Coronary vasodilator effects of endogenous cannabinoids in vasopressin-preconstricted unpaced rat isolated hearts.

The mechanisms by which cannabinoids alter coronary vascular tone and cardiac performance are controversial. We investigated the effects of various cannabinoids in spontaneously beating Langendorff-perfused rat hearts. Bolus injections of anandamide (0.1-1 micromol) caused no change in coronary flow (CF) or left ventricular systolic pressure (LVSP). In hearts preperfused with vasopressin to induce vasoconstrictor tone, anandamide or the selective CB1 receptor agonist ACEA (1-100 nmol) dose-dependently increased CF by up to 267% and LVSP by 20 mm Hg. The metabolically stable endocannabinoid derivatives, R-methanandamide and noladin ether, displayed similar effects. In contrast, Delta-THC (10-100 nmol), the major psychoactive ingredient of cannabis, strongly decreased CF and LVSP. The CB2 receptor agonist JWH-133 (10-100 nmol) elicited vasodilator and positive inotropic effects only at higher doses. The CB1 antagonists SR141716A and AM-251 as well as the potassium channel inhibitors tetraethylammonium and iberiotoxin blocked the anandamide-induced increases in CF and LVSP, whereas the CB2 antagonist SR144528 and the putative "CB3 antagonist" O-1918 did not have an inhibitory effect. Immunohistochemistry revealed the presence of cardiac CB1 but no CB2 receptors. Anandamide and 2-arachidonoylglycerol were detected in heart tissue. However, combined application of fatty acid amidohydrolase inhibitors and the transport inhibitor AM-404 to augment tissue levels of endocannabinoids was without effect on CF or LVSP. We conclude that in the rat isolated heart with reestablished vasoconstrictor tone, cannabinoids including anandamide elicit coronary vasodilation and a secondary increase in contractility via CB1 receptors and potassium channels.