BAG3 regulates contractility and Ca2+ homeostasis in adult mouse ventricular myocytes

Abstract

Bcl2-associated athanogene 3 (BAG3) is a 575 amino acid anti-apoptotic protein that is constitutively expressed in the heart. BAG3 mutations, including mutations leading to loss of protein, are associated with familial cardiomyopathy. Furthermore, BAG3 levels have been found to be reduced in end-stage non-familial failing myocardium. In contrast to neonatal myocytes in which BAG3 is found in the cytoplasm and involved in protein quality control and apoptosis, in adult mouse left ventricular (LV) myocytes BAG3 co-localized with Na+-K+-ATPase and L-type Ca2+ channels in the sarcolemma and t-tubules. BAG3 co-immunoprecipitated with β1-adrenergic receptor, L-type Ca2+ channels and phospholemman. To simulate decreased BAG3 protein levels observed in human heart failure, we targeted BAG3 by shRNA (shBAG3) in adult LV myocytes. Reducing BAG3 by 55% resulted in reduced contraction and [Ca2+]i transient amplitudes in LV myocytes stimulated with isoproterenol. L-type Ca2+ current (ICa) and sarcoplasmic reticulum (SR) Ca2+ content but not Na+/Ca2+ exchange current (INaCa) or SR Ca2+ uptake were reduced in isoproterenol-treated shBAG3 myocytes. Forskolin or dibutyrl cAMP restored ICa amplitude in shBAG3 myocytes to that observed in WT myocytes, consistent with BAG3 having effects upstream and at the level of the receptor. Resting membrane potential and action potential amplitude were unaffected but APD50 and APD90 were prolonged in shBAG3 myocytes. Protein levels of Ca2+ entry molecules and other important excitation-contraction proteins were unchanged in myocytes with lower BAG3. Our findings that BAG3 is localized at the sarcolemma and t-tubules while modulating myocyte contraction and action potential duration through specific interaction with the β1-adrenergic receptor and L-type Ca2+ channel provide novel insight into the role of BAG3 in cardiomyopathies and increased arrhythmia risks in heart failure.

Bcl-2–associated athanogene 3 protects the heart from ischemia/reperfusion injury

“…Under normal conditions, confocal imaging demonstrated that BAG3 is found predominantly in the cytoplasm of neonatal NMVCs, consistent with our previous observations (9). However, when NMVCs were exposed to H/R, BAG3 was found predominantly in the perinuclear region and in the nucleus ( Figure 5A).…”

“…Ad-GFP, Ad-BAG3, or Ad-siBAG3 was constructed using the BD Adeno-X Expression System, 2PT3674-1, and BD Knockout RNAi Systems, PT3739 (BD Biosciences-Clontech) as previously described (9). Forty-eight hours after isolation, NMVCs were infected with adenovirus at a multiplicity of infection of 8.…”

“…Confocal microscopy was used to detect BAG3 localization in adult cardiomyocytes as described previously (9). Briefly, NMVCs were isolated and plated on laminin-coated 4-well chamber slides (Lab-Tek).…”

“…However, in adult myocytes, BAG3 localized at the sarcolemma and t-tubules, where it modulates myocyte contraction and action potential duration through specific interaction with the β1-adrenergic receptor and L-type Ca 2+ channel (9). Furthermore, overexpression of BAG3 using an adeno-associated virus serotype 9 (rAAV9-BAG3) restored left ventricular (LV) function in mice with diminished LV function secondary to a myocardial infarction (10).…”

BAG3 (Bcl-2–Associated Athanogene-3) Coding Variant in Mice Determines Susceptibility to Ischemic Limb Muscle Myopathy by Directing Autophagy

“…8 Second, evidence from our group and others supports a novel hypothesis for the etiology of tissue injury in limb ischemia in which the response of skeletal muscle cells to ischemia, and not solely tissue perfusion (e.g., collateral growth, arteriogenesis, or angiogenesis), is a critical determinant of whether skeletal muscle tissue survives an ischemic injury 16, 57 . Substantial evidence supports a role for BAG3 in the regulation of striated muscle function 20, 58–62 , and mutations in BAG3 have been linked to both myofibrillar myopathy and dilated cardiomyopathy in humans, providing a strong rationale for targeted investigation of the effects of BAG3 variation on ischemia-induced muscle necrosis. Finally, although narrowing the Lsq-1 locus (or related/overlapping loci) will be important, ultimately it will be necessary to tease out effects of individual target genes on specific phenotypes, whether limb muscle necrosis or collateral artery formation.…”