Simon Sedej

Nucleocytosolic Depletion of the Energy Metabolite Acetyl-Coenzyme A Stimulates Autophagy and Prolongs Lifespan

Summary Healthy aging depends on removal of damaged cellular material that is in part mediated by autophagy. The nutritional status of cells affects both aging and autophagy through as-yet-elusive metabolic circuitries. Here, we show that nucleocytosolic acetyl-coenzyme A (AcCoA) production is a metabolic repressor of autophagy during aging in yeast. Blocking the mitochondrial route to AcCoA by deletion of the CoA-transferase ACH1 caused cytosolic accumulation of the AcCoA precursor acetate. This led to hyperactivation of nucleocytosolic AcCoA-synthetase Acs2p, triggering histone acetylation, repression of autophagy genes, and an age-dependent defect in autophagic flux, culminating in a reduced lifespan. Inhibition of nutrient signaling failed to restore, while simultaneous knockdown of ACS2 reinstated, autophagy and survival of ach1 mutant. Brain-specific knockdown of Drosophila AcCoA synthetase was sufficient to enhance autophagic protein clearance and prolong lifespan. Since AcCoA integrates various nutrition pathways, our findings may explain diet-dependent lifespan and autophagy regulation.

Cardioprotection and lifespan extension by the natural polyamine spermidine

Aging is associated with an increased risk of cardiovascular disease and death. Here we show that oral supplementation of the natural polyamine spermidine extends the lifespan of mice and exerts cardioprotective effects, reducing cardiac hypertrophy and preserving diastolic function in old mice. Spermidine feeding enhanced cardiac autophagy, mitophagy and mitochondrial respiration, and it also improved the mechano-elastical properties of cardiomyocytes in vivo, coinciding with increased titin phosphorylation and suppressed subclinical inflammation. Spermidine feeding failed to provide cardioprotection in mice that lack the autophagy-related protein Atg5 in cardiomyocytes. In Dahl salt-sensitive rats that were fed a high-salt diet, a model for hypertension-induced congestive heart failure, spermidine feeding reduced systemic blood pressure, increased titin phosphorylation and prevented cardiac hypertrophy and a decline in diastolic function, thus delaying the progression to heart failure. In humans, high levels of dietary spermidine, as assessed from food questionnaires, correlated with reduced blood pressure and a lower incidence of cardiovascular disease. Our results suggest a new and feasible strategy for protection against cardiovascular disease.

Na+-dependent SR Ca2+ overload induces arrhythmogenic events in mouse cardiomyocytes with a human CPVT mutation

AIMSnMutations in the cardiac ryanodine receptor Ca(2+) release channel, RyR2, underlie catecholaminergic polymorphic ventricular tachycardia (CPVT), an inherited life-threatening arrhythmia. CPVT is triggered by spontaneous RyR2-mediated sarcoplasmic reticulum (SR) Ca(2+) release in response to SR Ca(2+) overload during beta-adrenergic stimulation. However, whether elevated SR Ca(2+) content--in the absence of protein kinase A activation--affects RyR2 function and arrhythmogenesis in CPVT remains elusive.nnnMETHODS AND RESULTSnIsolated murine ventricular myocytes harbouring a human RyR2 mutation (RyR2(R4496C+/-)) associated with CPVT were investigated in the absence and presence of 1 micromol/L JTV-519 (RyR2 stabilizer) followed by 100 micromol/L ouabain intervention to increase cytosolic [Na(+)] and SR Ca(2+) load. Changes in membrane potential and intracellular [Ca(2+)] were monitored with whole-cell patch-clamping and confocal Ca(2+) imaging, respectively. At baseline, action potentials (APs), Ca(2+) transients, fractional SR Ca(2+) release, and SR Ca(2+) load were comparable in wild-type (WT) and RyR2(R4496C+/-) myocytes. Ouabain evoked significant increases in diastolic [Ca(2+)], peak systolic [Ca(2+)], fractional SR Ca(2+) release, and SR Ca(2+) content that were quantitatively similar in WT and RyR2(R4496C+/-) myocytes. Ouabain also induced arrhythmogenic events, i.e. spontaneous Ca(2+) waves, delayed afterdepolarizations and spontaneous APs, in both groups. However, the ouabain-induced increase in the frequency of arrhythmogenic events was dramatically larger in RyR2(R4496C+/-) when compared with WT myocytes. JTV-519 greatly reduced the frequency of ouabain-induced arrhythmogenic events.nnnCONCLUSIONnThe elevation of SR Ca(2+) load--in the absence of beta-adrenergic stimulation--is sufficient to increase the propensity for triggered arrhythmias in RyR2(R4496C+/-) cardiomyocytes. Stabilization of RyR2 by JTV-519 effectively reduces these triggered arrhythmias.

cAMP increases Ca2+-dependent exocytosis through both PKA and Epac2 in mouse melanotrophs from pituitary tissue slices

Cyclic AMP regulates Ca2+‐dependent exocytosis through a classical protein kinase A (PKA)‐dependent and an alternative cAMP–guanine nucleotide exchange factor (GEF)/Epac‐dependent pathway in many secretory cells. Although increased cAMP is believed to double secretory output in isolated pituitary cells, the direct target(s) for cAMP action and a detailed and high‐time resolved analysis of the effect of intracellular cAMP levels on the secretory activity in melanotrophs are still lacking. We investigated the effect of 200 μm cAMP on the kinetics of secretory vesicle depletion in mouse melanotrophs from fresh pituitary tissue slices. The whole‐cell patch‐clamp technique was used to depolarize melanotrophs and increase the cytosolic Ca2+ concentration ([Ca2+]i). Exogenous cAMP elicited an about twofold increase in cumulative membrane capacitance change and ∼34% increase of high‐voltage activated Ca2+ channel amplitude. cAMP‐dependent mechanisms did not affect [Ca2+]i, since the application of forskolin failed to change [Ca2+]i in melanotrophs, a phenomenon readily observed in anterior lobe. Depolarization‐induced secretion resulted in two distinct kinetic components: a linear and a threshold component, both stimulated by cAMP. The linear component (ATP‐independent) probably represented the exocytosis of the release‐ready vesicles, whereas the threshold component was assigned to the exocytosis of secretory vesicles that required ATP‐dependent reaction(s) and > 800 nm[Ca2+]i. The linear component was modulated by 8‐pCPT‐2Me‐cAMP (Epac agonist), while either H‐89 (PKA inhibitor) or Rp‐cAMPS (the competitive antagonist of cAMP binding to PKA) completely prevented the action of cAMP on the threshold component. In line with this, 6‐Phe‐cAMP, (PKA agonist), increased the threshold component. From our study, we suggest that the stimulation of cAMP production by application of oestrogen, as found in pregnant mice, increases the efficacy of the hormonal output through both PKA and cAMP–GEFII/Epac2‐dependent mechanisms.

JTV519 (K201) reduces sarcoplasmic reticulum Ca2+ leak and improves diastolic function in vitro in murine and human non-failing myocardium

BACKGROUND AND PURPOSE Ca2+ leak from the sarcoplasmic reticulum (SR) via ryanodine receptors (RyR2s) contributes to cardiomyocyte dysfunction. RyR2 Ca2+ leak has been related to RyR2 phosphorylation. In these conditions, JTV519 (K201), a 1,4‐benzothiazepine derivative and multi‐channel blocker, stabilizes RyR2s and decrease SR Ca2+ leak. We investigated whether JTV519 stabilizes RyR2s without increasing RyR2 phosphorylation in mice and in non‐failing human myocardium and explored underlying mechanisms.

Important Contribution of α-Neurexins to Ca2+-Triggered Exocytosis of Secretory Granules

α-Neurexins constitute a family of neuronal cell surface molecules that are essential for efficient neurotransmission, because mice lacking two or all three α-neurexin genes show a severe reduction of synaptic release. Although analyses of α-neurexin knock-outs and transgenic rescue animals suggested an involvement of voltage-dependent Ca2+channels, it remained unclear whether α-neurexins have a general role in Ca2+-dependent exocytosis and how they may affect Ca2+ channels. Here we show by membrane capacitance measurements from melanotrophs in acute pituitary gland slices that release from endocrine cells is diminished by >50% in adult α-neurexin double knock-out and newborn triple knock-out mice. There is a reduction of the cell volume in mutant melanotrophs; however, no ultrastructural changes in size or intracellular distribution of the secretory granules were observed. Recordings of Ca2+ currents from melanotrophs, transfected human embryonic kidney cells, and brainstem neurons reveal that α-neurexins do not affect the activation or inactivation properties of Ca2+ channels directly but may be responsible for coupling them to release-ready vesicles and metabotropic receptors. Our data support a general and essential role for α-neurexins in Ca2+-triggered exocytosis that is similarly important for secretion from neurons and endocrine cells.

Voltage-activated Ca2+ channels and their role in the endocrine function of the pituitary gland in newborn and adult mice

We have prepared fresh pituitary gland slices from adult and, for the first time, from newborn mice to assess modulation of secretory activity via voltage‐activated Ca2+ channels (VACCs). Currents through VACCs and membrane capacitance have been measured with the whole‐cell patch‐clamp technique. Melanotrophs in newborns were significantly larger than in adults. In both newborn and adult melanotrophs activation of VACCs triggered exocytosis. All pharmacologically isolated VACC types contributed equally to the secretory activity. However, the relative proportion of VACCs differed between newborns and adults. In newborn cells L‐type channels dominated and, in addition, an exclusive expression of a toxin‐resistant R‐type‐like current was found. The expression of L‐type VACCs was up‐regulated by the increased oestrogen levels observed in females, and was even more emphasized in the cells of pregnant females and oestrogen‐treated adult male mice. We suggest a general mechanism modulating endocrine secretion in the presence of oestrogen and particularly higher sensitivity to treatments with L‐type channel blockers during high oestrogen physiological states.

Cold-Induced Thermogenesis Depends on ATGL-Mediated Lipolysis in Cardiac Muscle, but Not Brown Adipose Tissue

Summary Fatty acids (FAs) activate and fuel UCP1-mediated non-shivering thermogenesis (NST) in brown adipose tissue (BAT). Release of FAs from intracellular fat stores by adipose triglyceride lipase (ATGL) is considered a key step in NST. Accordingly, the severe cold intolerance of global ATGL knockout (AKO) mice has been attributed to defective BAT lipolysis. Here we show that this conclusion is incorrect. We demonstrate that although the BAT-specific loss of ATGL impairs BAT lipolysis and alters BAT morphology, it does not compromise the β3-adrenergic thermogenic response or cold-induced NST. Instead, NST depends on nutrient supply or lipolysis in white adipose tissue during fasting, suggesting that circulating energy substrates are sufficient to fuel NST. Cold intolerance in AKO mice is not caused by BAT dysfunction as previously suspected but by severe cardiomyopathy. We conclude that functional NST requires adequate substrate supply and cardiac function, but does not depend on ATGL-mediated lipolysis in BAT.

In Situ Calibration of Nucleoplasmic versus Cytoplasmic Ca2+ Concentration in Adult Cardiomyocytes

Quantification of subcellularly resolved Ca²⁺ signals in cardiomyocytes is essential for understanding Ca²⁺ fluxes in excitation-contraction and excitation-transcription coupling. The properties of fluorescent indicators in intracellular compartments may differ, thus affecting the translation of Ca²⁺-dependent fluorescence changes into [Ca²⁺] changes. Therefore, we determined the in situ characteristics of a frequently used Ca²⁺ indicator, Fluo-4, and a ratiometric Ca²⁺ indicator, Asante Calcium Red, and evaluated their use for reporting and quantifying cytoplasmic and nucleoplasmic Ca²⁺ signals in isolated cardiomyocytes. Ca²⁺ calibration curves revealed significant differences in the apparent Ca²⁺ dissociation constants of Fluo-4 and Asante Calcium Red between cytoplasm and nucleoplasm. These parameters were used for transformation of fluorescence into nucleoplasmic and cytoplasmic [Ca²⁺]. Resting and diastolic [Ca²⁺] were always higher in the nucleoplasm. Systolic [Ca²⁺] was usually higher in the cytoplasm, but some cells (15%) exhibited higher systolic [Ca²⁺] in the nucleoplasm. Ca²⁺ store depletion or blockade of Ca²⁺ leak pathways eliminated the resting [Ca²⁺] gradient between nucleoplasm and cytoplasm, whereas inhibition of inositol 1,4,5-trisphosphate receptors by 2-APB reversed it. The results suggest the presence of significant nucleoplasmic-to-cytoplasmic [Ca²⁺] gradients in resting myocytes and during the cardiac cycle. Nucleoplasmic [Ca²⁺] in cardiomyocytes may be regulated via two mechanisms: diffusion from the cytoplasm and active Ca²⁺ release via inositol 1,4,5-trisphosphate receptors from perinuclear Ca²⁺ stores.

Intracellular Dyssynchrony of Diastolic Cytosolic [Ca2+] Decay in Ventricular Cardiomyocytes in Cardiac Remodeling and Human Heart Failure

Rationale: Synchronized release of Ca2+ into the cytosol during each cardiac cycle determines cardiomyocyte contraction. Objective: We investigated synchrony of cytosolic [Ca2+] decay during diastole and the impact of cardiac remodeling. Methods and Results: Local cytosolic [Ca2+] transients (1-µm intervals) were recorded in murine, porcine, and human ventricular single cardiomyocytes. We identified intracellular regions of slow (slowCaR) and fast (fastCaR) [Ca2+] decay based on the local time constants of decay (TAUlocal). The SD of TAUlocal as a measure of dyssynchrony was not related to the amplitude or the timing of local Ca2+ release. Stimulation of sarcoplasmic reticulum Ca2+ ATPase with forskolin or istaroxime accelerated and its inhibition with cyclopiazonic acid slowed TAUlocal significantly more in slowCaR, thus altering the relationship between SD of TAUlocal and global [Ca2+] decay (TAUglobal). Na+/Ca2+ exchanger inhibitor SEA0400 prolonged TAUlocal similarly in slowCaR and fastCaR. FastCaR were associated with increased mitochondrial density and were more sensitive to the mitochondrial Ca2+ uniporter blocker Ru360. Variation in TAUlocal was higher in pig and human cardiomyocytes and higher with increased stimulation frequency (2 Hz). TAUlocal correlated with local sarcomere relengthening. In mice with myocardial hypertrophy after transverse aortic constriction, in pigs with chronic myocardial ischemia, and in end-stage human heart failure, variation in TAUlocal was increased and related to cardiomyocyte hypertrophy and increased mitochondrial density. Conclusions: In cardiomyocytes, cytosolic [Ca2+] decay is regulated locally and related to local sarcomere relengthening. Dyssynchronous intracellular [Ca2+] decay in cardiac remodeling and end-stage heart failure suggests a novel mechanism of cellular contractile dysfunction.