STIM1 ablation impairs exercise-induced physiological cardiac hypertrophy and dysregulates autophagy in mouse hearts.

Cardiac stromal interaction molecule 1 (STIM1), a key mediator of store-operated Ca2+ entry (SOCE), is a known determinant of cardiomyocyte pathological growth in hypertrophic cardiomyopathy. We examined the role of STIM1 and SOCE in response to exercise-dependent physiological hypertrophy. Wild-type (WT) mice subjected to exercise training (WT-Ex) showed a significant increase in exercise capacity and heart weight compared with sedentary (WT-Sed) mice. Moreover, myocytes from WT-Ex hearts displayed an increase in length, but not width, compared with WT-Sed myocytes. Conversely, exercised cardiac-specific STIM1 knock-out mice (cSTIM1KO-Ex), although displaying significant increase in heart weight and cardiac dilation, evidenced no changes in myocyte size and displayed a decreased exercise capacity, impaired cardiac function, and premature death compared with sedentary cardiac-specific STIM1 knock-out mice (cSTIM1KO-Sed). Confocal Ca2+ imaging demonstrated enhanced SOCE in WT-Ex myocytes compared with WT-Sed myocytes with no measurable SOCE detected in cSTIM1KO myocytes. Exercise training induced a significant increase in cardiac phospho-Akt Ser473 in WT mice but not in cSTIM1KO mice. No differences were observed in phosphorylation of mammalian target of rapamycin (mTOR) and glycogen synthase kinase (GSK) in exercised versus sedentary cSTIM1KO mice hearts. cSTIM1KO-Sed mice showed increased basal MAPK phosphorylation compared with WT-Sed that was not altered by exercise training. Finally, histological analysis revealed exercise resulted in increased autophagy in cSTIM1KO but not in WT myocytes. Taken together, our results suggest that adaptive cardiac hypertrophy in response to exercise training involves STIM1-mediated SOCE. Our results demonstrate that STIM1 is involved in and essential for the myocyte longitudinal growth and mTOR activation in response to endurance exercise training.NEW & NOTEWORTHY Store-operated Ca2+ entry (SOCE) has been implicated in pathological cardiac hypertrophy; however, its role in physiological hypertrophy is unknown. Here we report that SOCE is also essential for physiological cardiac hypertrophy and functional adaptations in response to endurance exercise. These adaptations were associated with activation of AKT/mTOR pathway and curtailed cardiac autophagy and degeneration. Thus, SOCE is a common mechanism and an important bifurcation point for signaling paths involved in physiological and pathological hypertrophy.

Systemic γ-sarcoglycan AAV gene transfer results in dose-dependent correction of muscle deficits in the LGMD 2C/R5 mouse model.

Limb-girdle muscular dystrophy (LGMD) type 2C/R5 results from mutations in the γ-sarcoglycan (SGCG) gene and is characterized by muscle weakness and progressive wasting. Loss of functional γ-sarcoglycan protein in the dystrophin-associated protein complex destabilizes the sarcolemma, leading to eventual myofiber death. The SGCG knockout mouse (SGCG -/-) has clinical-pathological features that replicate the human disease, making it an ideal model for translational studies. We designed a self-complementary rAAVrh74 vector containing a codon-optimized human SGCG transgene driven by the muscle-specific MHCK7 promoter (SRP-9005) to investigate adeno-associated virus (AAV)-mediated SGCG gene transfer in SGCG -/- mice as proof of principle for LGMD 2C/R5. Gene transfer therapy resulted in widespread transgene expression in skeletal muscle and heart, improvements in muscle histopathology characterized by decreased central nuclei and fibrosis, and normalized fiber size. Histopathologic improvements were accompanied by functional improvements, including increased ambulation and force production and resistance to injury of the tibialis anterior and diaphragm muscles. This study demonstrates successful systemic delivery of the hSGCG transgene in SGCG -/- mice, with functional protein expression, reconstitution of the sarcoglycan complex, and corresponding physiological and functional improvements, which will help establish a minimal effective dose for translation of SRP-9005 gene transfer therapy in patients with LGMD 2C/R5.

Vascular endothelial growth factor promotes atrial arrhythmias by inducing acute intercalated disk remodeling.

Atrial fibrillation (AF) is the most common arrhythmia and is associated with inflammation. AF patients have elevated levels of inflammatory cytokines known to promote vascular leak, such as vascular endothelial growth factor A (VEGF). However, the contribution of vascular leak and consequent cardiac edema to the genesis of atrial arrhythmias remains unknown. Previous work suggests that interstitial edema in the heart can acutely promote ventricular arrhythmias by disrupting ventricular myocyte intercalated disk (ID) nanodomains rich in cardiac sodium channels (NaV1.5) and slowing cardiac conduction. Interestingly, similar disruption of ID nanodomains has been identified in atrial samples from AF patients. Therefore, we tested the hypothesis that VEGF-induced vascular leak can acutely increase atrial arrhythmia susceptibility by disrupting ID nanodomains and slowing atrial conduction. Treatment of murine hearts with VEGF (30-60 min, at clinically relevant levels) prolonged the electrocardiographic P wave and increased susceptibility to burst pacing-induced atrial arrhythmias. Optical voltage mapping revealed slower atrial conduction following VEGF treatment (10 ± 0.4 cm/s vs. 21 ± 1 cm/s at baseline, p < 0.05). Transmission electron microscopy revealed increased intermembrane spacing at ID sites adjacent to gap junctions (GJs; 64 ± 9 nm versus 17 ± 1 nm in controls, p < 0.05), as well as sites next to mechanical junctions (MJs; 63 ± 4 nm versus 27 ± 2 nm in controls, p < 0.05) in VEGF-treated hearts relative to controls. Importantly, super-resolution microscopy and quantitative image analysis revealed reorganization of NaV1.5 away from dense clusters localized near GJs and MJs to a more diffuse distribution throughout the ID. Taken together, these data suggest that VEGF can acutely predispose otherwise normal hearts to atrial arrhythmias by dynamically disrupting NaV1.5-rich ID nanodomains and slowing atrial conduction. These data highlight inflammation-induced vascular leak as a potential factor in the development and progression of AF.

Muscarinic-dependent phosphorylation of the cardiac ryanodine receptor by protein kinase G is mediated by PI3K-AKT-nNOS signaling.

Post-translational modifications of proteins involved in calcium handling in myocytes, such as the cardiac ryanodine receptor (RyR2), critically regulate cardiac contractility. Recent studies have suggested that phosphorylation of RyR2 by protein kinase G (PKG) might contribute to the cardioprotective effects of cholinergic stimulation. However, the specific mechanisms underlying these effects remain unclear. Here, using murine ventricular myocytes, immunoblotting, proximity ligation as-says, and nitric oxide imaging, we report that phosphorylation of Ser-2808 in RyR2 induced by the muscarinic receptor agonist carbachol is mediated by a signaling axis comprising phosphoinositide 3-phosphate kinase, Akt Ser/Thr kinase, nitric oxide synthase 1, nitric oxide, soluble guanylate cyclase, cyclic GMP (cGMP), and PKG. We found that this signaling pathway is compartmentalized in myocytes, as it was distinct from atrial natriuretic peptide receptor-cGMP-PKG-RyR2 Ser-2808 signaling and independent of muscarinic-induced phosphorylation of Ser-239 in vasodilator-stimulated phosphoprotein. These results provide detailed insights into muscarinic-induced PKG signaling and the mediators that regulate cardiac RyR2 phosphorylation critical for cardiovascular function.

Chronic heart failure increases negative chronotropic effects of adenosine in canine sinoatrial cells via A1R stimulation and GIRK-mediated IKado.

AIMS: Bradycardia contributes to tachy-brady arrhythmias or sinus arrest during heart failure (HF). Sinoatrial node (SAN) adenosine A1 receptors (ADO A1Rs) are upregulated in HF, and adenosine is known to exert negative chronotropic effects on the SAN. Here, we investigated the role of A1R signaling at physiologically relevant ADO concentrations on HF SAN pacemaker cells. MAIN METHODS: Dogs with tachypacing-induced chronic HF and normal controls (CTL) were studied. SAN tissue was collected for A1R and GIRK mRNA quantification. SAN cells were isolated for perforated patch clamp recordings and firing rate (bpm), slope of slow diastolic depolarization (SDD), and maximum diastolic potential (MDP) were measured. Action potentials (APs) and currents were recorded before and after addition of 1 and 10 µM ADO. To assess contributions of A1R and G protein-coupled Inward Rectifier Potassium Current (GIRK) to ADO effects, APs were measured after the addition of DPCPX (selective A1R antagonist) or TPQ (selective GIRK blocker). KEY FINDINGS: A1R and GIRK mRNA expression were significantly increased in HF. In addition, ADO induced greater rate slowing and membrane hyperpolarization in HF vs CTL (p < 0.05). DPCPX prevented ADO-induced rate slowing in CTL and HF cells. The ADO-induced inward rectifying current, IKado, was observed significantly more frequently in HF than in CTL. TPQ prevented ADO-induced rate slowing in HF. SIGNIFICANCE: An increase in A1R and GIRK expression enhances IKAdo, causing hyperpolarization, and subsequent negative chronotropic effects in canine chronic HF at relevant [ADO]. GIRK blockade may be a useful strategy to mitigate bradycardia in HF.

In Utero Particulate Matter Exposure Produces Heart Failure, Electrical Remodeling, and Epigenetic Changes at Adulthood.

BACKGROUND: Particulate matter (PM; PM2.5 [PM with diameters of <2.5 µm]) exposure during development is strongly associated with adverse cardiovascular outcomes at adulthood. In the present study, we tested the hypothesis that in utero PM2.5 exposure alone could alter cardiac structure and function at adulthood. METHODS AND RESULTS: Female FVB mice were exposed either to filtered air or PM2.5 at an average concentration of 73.61 µg/m3 for 6 h/day, 7 days/week throughout pregnancy. After birth, animals were analyzed at 12 weeks of age. Echocardiographic (n=9-10 mice/group) and pressure-volume loop analyses (n=5 mice/group) revealed reduced fractional shortening, increased left ventricular end-systolic and -diastolic diameters, reduced left ventricular posterior wall thickness, end-systolic elastance, contractile reserve (dP/dtmax/end-systolic volume), frequency-dependent acceleration of relaxation), and blunted contractile response to ß-adrenergic stimulation in PM2.5-exposed mice. Isolated cardiomyocyte (n=4-5 mice/group) function illustrated reduced peak shortening, ±dL/dT, and prolonged action potential duration at 90% repolarization. Histological left ventricular analyses (n=3 mice/group) showed increased collagen deposition in in utero PM2.5-exposed mice at adulthood. Cardiac interleukin (IL)-6, IL-1ß, collagen-1, matrix metalloproteinase (MMP) 9, and MMP13 gene expressions were increased at birth in in utero PM2.5-exposed mice (n=4 mice/group). In adult hearts (n=5 mice/group), gene expressions of sirtuin (Sirt) 1 and Sirt2 were decreased, DNA methyltransferase (Dnmt) 1, Dnmt3a, and Dnmt3b were increased, and protein expression (n=6 mice/group) of Ca2+-ATPase, phosphorylated phospholamban, and Na+/Ca2+ exchanger were decreased. CONCLUSIONS: In utero PM2.5 exposure triggers an acute inflammatory response, chronic matrix remodeling, and alterations in Ca2+ handling proteins, resulting in global adult cardiac dysfunction. These results also highlight the potential involvement of epigenetics in priming of adult cardiac disease.