Anna R. Busija

Vinculin directly binds zonula occludens-1 and is essential for stabilizing connexin-43-containing gap junctions in cardiac myocytes

ABSTRACT Vinculin (Vcl) links actin filaments to integrin- and cadherin-based cellular junctions. Zonula occludens-1 (ZO-1, also known as TJP1) binds connexin-43 (Cx43, also known as GJA1), cadherin and actin. Vcl and ZO-1 anchor the actin cytoskeleton to the sarcolemma. Given that loss of Vcl from cardiomyocytes causes maldistribution of Cx43 and predisposes cardiomyocyte-specific Vcl-knockout mice with preserved heart function to arrhythmia and sudden death, we hypothesized that Vcl and ZO-1 interact and that loss of this interaction destabilizes gap junctions. We found that Vcl, Cx43 and ZO-1 colocalized at the intercalated disc. Loss of cardiomyocyte Vcl caused parallel loss of ZO-1 from intercalated dics. Vcl co-immunoprecipitated Cx43 and ZO-1, and directly bound ZO-1 in yeast two-hybrid studies. Excision of the Vcl gene in neonatal mouse cardiomyocytes caused a reduction in the amount of Vcl mRNA transcript and protein expression leading to (1) decreased protein expression of Cx43, ZO-1, talin, and &bgr;1D-integrin, (2) reduced PI3K activation, (3) increased activation of Akt, Erk1 and Erk2, and (4) cardiomyocyte necrosis. In summary, this is the first study showing a direct interaction between Vcl and ZO-1 and illustrates how Vcl plays a crucial role in stabilizing gap junctions and myocyte integrity.

Sarcolemmal cholesterol and caveolin-3 dependence of cardiac function, ischemic tolerance, and opioidergic cardioprotection

Cholesterol-rich caveolar microdomains and associated caveolins influence sarcolemmal ion channel and receptor function and protective stress signaling. However, the importance of membrane cholesterol content to cardiovascular function and myocardial responses to ischemia-reperfusion (I/R) and cardioprotective stimuli are unclear. We assessed the effects of graded cholesterol depletion with methyl-β-cyclodextrin (MβCD) and lifelong knockout (KO) or overexpression (OE) of caveolin-3 (Cav-3) on cardiac function, I/R tolerance, and opioid receptor (OR)-mediated protection. Langendorff-perfused hearts from young male C57Bl/6 mice were untreated or treated with 0.02-1.0 mM MβCD for 25 min to deplete membrane cholesterol and disrupt caveolae. Hearts were subjected to 25-min ischemia/45-min reperfusion, and the cardioprotective effects of morphine applied either acutely or chronically [sustained ligand-activated preconditioning (SLP)] were assessed. MβCD concentration dependently reduced normoxic contractile function and postischemic outcomes in association with graded (10-30%) reductions in sarcolemmal cholesterol. Cardioprotection with acute morphine was abolished with ≥20 μM MβCD, whereas SLP was more robust and only inhibited with ≥200 μM MβCD. Deletion of Cav-3 also reduced, whereas Cav-3 OE improved, myocardial I/R tolerance. Protection via SLP remained equally effective in Cav-3 KO mice and was additive with innate protection arising with Cav-3 OE. These data reveal the membrane cholesterol dependence of normoxic myocardial and coronary function, I/R tolerance, and OR-mediated cardioprotection in murine hearts (all declining with cholesterol depletion). In contrast, baseline function appears insensitive to Cav-3, whereas cardiac I/R tolerance parallels Cav-3 expression. Novel SLP appears unique, being less sensitive to cholesterol depletion than acute OR protection and arising independently of Cav-3 expression.

Neuron-Targeted Caveolin-1 Improves Molecular Signaling, Plasticity, and Behavior Dependent on the Hippocampus in Adult and Aged Mice.

BACKGROUND Studies in vitro demonstrate that neuronal membrane/lipid rafts (MLRs) establish cell polarity by clustering progrowth receptors and tethering cytoskeletal machinery necessary for neuronal sprouting. However, the effect of MLR and MLR-associated proteins on neuronal aging is unknown. METHODS Here, we assessed the impact of neuron-targeted overexpression of an MLR scaffold protein, caveolin-1 (Cav-1) (via a synapsin promoter, SynCav1), in the hippocampus in vivo in adult (6-month-old) and aged (20-month-old) mice on biochemical, morphologic, and behavioral changes. RESULTS SynCav1 resulted in increased expression of Cav-1, MLRs, and MLR-localization of Cav-1 and tropomyosin-related kinase B receptor independent of age and time post gene transfer. Cav-1 overexpression in adult mice enhanced dendritic arborization within the apical dendrites of hippocampal cornu ammonis 1 and granule cell neurons, effects that were also observed in aged mice, albeit to a lesser extent, indicating preserved impact of Cav-1 on structural plasticity of hippocampal neurons with age. Cav-1 overexpression enhanced contextual fear memory in adult and aged mice demonstrating improved hippocampal function. CONCLUSIONS Neuron-targeted overexpression of Cav-1 in the adult and aged hippocampus enhances functional MLRs with corresponding roles in cell signaling and protein trafficking. The resultant structural alterations in hippocampal neurons in vivo are associated with improvements in hippocampal-dependent learning and memory. Our findings suggest Cav-1 as a novel therapeutic strategy in disorders involving impaired hippocampal function.

A new sense of protection: role of the Ca2+-sensing receptor in ischemic preconditioning

the phenomenon of ischemic preconditioning (IPC), a condition in which brief periods of ischemia increase the tolerance of the heart to subsequent ischemic injury, is of immense clinical interest. Many signaling pathways are implicated in the induction of IPC; however, the initiation of the response involves activation of G protein-coupled receptors (GPCRs) and their downstream effectors (e.g., a variety of prosurvival kinase cascades) that ultimately converge to inhibit the opening of the mitochondrial permeability transition pore and limit subsequent proapoptotic activity (2, 4).

Atorvastatin, but not pravastatin, inhibits cardiac Akt/mTOR signaling and disturbs mitochondrial ultrastructure in cardiac myocytes

Statins, which reduce LDL‐cholesterol by inhibition of 3‐hydroxy‐3‐methylglutaryl–coenzyme A reductase, are among the most widely prescribed drugs. Skeletal myopathy is a known statin‐induced adverse effect associated with mitochondrial changes. We hypothesized that similar effects would occur in cardiac myocytes in a lipophilicity‐dependent manner between 2 common statins: atorvastatin (lipophilic) and pravastatin (hydrophilic). Neonatal cardiac ventricular myocytes were treated with atorvastatin and pravastatin for 48 h. Both statins induced endoplasmic reticular (ER) stress, but only atorvastatin inhibited ERK1/2T202/Y204, AktSer473, and mammalian target of rapamycin signaling; reduced protein abundance of caveolin‐1, dystrophin, epidermal growth factor receptor, and insulin receptor‐β; decreased Ras homolog gene family member A activation; and induced apoptosis. In cardiomyocyte‐equivalent HL‐1 cells, atorvastatin, but not pravastatin, reduced mitochondrial oxygen consumption. When male mice underwent atorvastatin and pravastatin administration per os for up to 7 mo, only long‐term atorvastatin, but not pravastatin, induced elevated serum creatine kinase; swollen, misaligned, size‐variable, and disconnected cardiac mitochondria; alteration of ER structure; repression of mitochondria‐ and endoplasmic reticulum–related genes; and a 21% increase in mortality in cardiac‐specific vinculin‐knockout mice during the first 2 months of administration. To our knowledge, we are the first to demonstrate in vivo that long‐term atorvastatin administration alters cardiac ultrastructure, a finding with important clinical implications.——Godoy, J. C., Niesman, I. R., Busija, A.R., Kassan, A., Schilling, J. M., Schwarz, A., Alvarez, E. A., Dalton, N. D., Drummond, J. C., Roth, D.M., Kararigas, G., Patel, H. H., Zemljic‐Harpf, A. E. Atorvastatin, but not pravastatin, inhibits cardiac Akt/mTOR signaling and disturbs mitochondrial ultrastructure in cardiac myocytes. FASEB J. 33, 1209–1225 (2019). www.fasebj.org