Valerie D. Myers

BAG3: a new player in the heart failure paradigm

BAG3 is a cellular protein that is expressed predominantly in skeletal and cardiac muscle but can also be found in the brain and in the peripheral nervous system. BAG3 functions in the cell include: serving as a co-chaperone with members of the heat-shock protein family of proteins to facilitate the removal of misfolded and degraded proteins, inhibiting apoptosis by interacting with Bcl2 and maintaining the structural integrity of the Z-disk in muscle by binding with CapZ. The importance of BAG3 in the homeostasis of myocytes and its role in the development of heart failure was evidenced by the finding that single allelic mutations in BAG3 were associated with familial dilated cardiomyopathy. Furthermore, significant decreases in the level of BAG3 have been found in end-stage failing human heart and in animal models of heart failure including mice with heart failure secondary to trans-aortic banding and in pigs after myocardial infarction. Thus, it becomes relevant to understand the cellular biology and molecular regulation of BAG3 expression in order to design new therapies for the treatment of patients with both hereditary and non-hereditary forms of dilated cardiomyopathy.

Decreased Levels of BAG3 in a Family With a Rare Variant and in Idiopathic Dilated Cardiomyopathy

The most common cause of dilated cardiomyopathy and heart failure (HF) is ischemic heart disease; however, in a third of all patients the cause remains undefined and patients are diagnosed as having idiopathic dilated cardiomyopathy (IDC). Recent studies suggest that many patients with IDC have a family history of HF and rare genetic variants in over 35 genes have been shown to be causative of disease. We employed whole‐exome sequencing to identify the causative variant in a large family with autosomal dominant transmission of dilated cardiomyopathy. Sequencing and subsequent informatics revealed a novel 10‐nucleotide deletion in the BCL2‐associated athanogene 3 (BAG3) gene (Ch10:del 121436332_12143641: del. 1266_1275 [NM 004281]) that segregated with all affected individuals. The deletion predicted a shift in the reading frame with the resultant deletion of 135 amino acids from the C‐terminal end of the protein. Consistent with genetic variants in genes encoding other sarcomeric proteins there was a considerable amount of genetic heterogeneity in the affected family members. Interestingly, we also found that the levels of BAG3 protein were significantly reduced in the hearts from unrelated patients with end‐stage HF undergoing cardiac transplantation when compared with non‐failing controls. Diminished levels of BAG3 protein may be associated with both familial and non‐familial forms of dilated cardiomyopathy. J. Cell. Physiol. 229: 1697–1702, 2014.

Arginine vasopressin enhances cell survival via a G protein-coupled receptor kinase 2/β-arrestin1/extracellular-regulated kinase 1/2-dependent pathway in H9c2 cells.

Circulating levels of arginine vasopressin (AVP) are elevated during hypovolemia and during cardiac stress. AVP activates arginine vasopressin type 1A (V1A)/Gαq–coupled receptors in the heart and vasculature and V2/Gαs–coupled receptors in the kidney. However, little is known regarding the signaling pathways that influence the effects of V1A receptor (V1AR) activation during cellular injury. Using hypoxia-reoxygenation (H/R) as a cell injury model, we evaluated cell survival and caspase 3/7 activity in H9c2 myoblasts after treatment with AVP. Pretreatment of H9c2 cells with AVP significantly reduced H/R-induced cell death and caspase 3/7 activity, effects that were blocked via both selective V1AR inhibition and mitogen-activated protein kinase (MEK1/2) inhibition. AVP increased extracellular-regulated kinase 1/2 (ERK1/2) phosphorylation in a concentration-dependent manner that was sensitive to MEK1/2 inhibition and V1AR inhibition, but not V1BR or V2R inhibition. Discrete elements of the V1A/Gαq-protein kinase C (PKC) and V1A/G protein–coupled receptor kinase (GRK)/β-arrestin signaling cascades were inhibited to dissect the pathways responsible for the protective effects of V1AR signaling: Gαq (overexpression of Gq-I-ires-green fluorescent protein), PKC (administration of Ro 31-82425; 2-[8-(aminomethyl)-6,7,8,9-tetrahydropyrido[1,2-a]indol-3-yl]-3-(1-methyl-1H-indol-3-yl)maleimide, HCl, bisindolylmaleimide X, HCl), GRK2 [C-terminal GRK2 peptide overexpression and small interfering RNA (siRNA) knockdown], GRK5 (siRNA knockdown), and β-arrestin1 (siRNA knockdown). These studies demonstrated that both Gαq/PKC- and GRK2/β-arrestin1–dependent V1AR signaling were capable of inducing ERK1/2 phosphorylation in response to AVP stimulation. However, AVP-mediated protection against H/R was elicited only via GRK2- and β-arrestin1–dependent signaling. These results suggest that activation of the V1AR in H9c2 cells mediates protective signaling via a GRK2/β−arrestin1/ERK1/2–dependent mechanism that leads to decreased caspase 3/7 activity and enhanced survival under conditions of ischemic stress.

β-Adrenergic Receptor-Mediated Cardiac Contractility is Inhibited via Vasopressin Type 1A-Receptor-Dependent Signaling

Background— Enhanced arginine vasopressin levels are associated with increased mortality during end-stage human heart failure, and cardiac arginine vasopressin type 1A receptor (V1AR) expression becomes increased. Additionally, mice with cardiac-restricted V1AR overexpression develop cardiomyopathy and decreased &bgr;-adrenergic receptor (&bgr;AR) responsiveness. This led us to hypothesize that V1AR signaling regulates &bgr;AR responsiveness and in doing so contributes to development of heart failure. Methods and Results— Transaortic constriction resulted in decreased cardiac function and &bgr;AR density and increased cardiac V1AR expression, effects reversed by a V1AR-selective antagonist. Molecularly, V1AR stimulation led to decreased &bgr;AR ligand affinity, as well as &bgr;AR-induced Ca2+ mobilization and cAMP generation in isolated adult cardiomyocytes, effects recapitulated via ex vivo Langendorff analysis. V1AR-mediated regulation of &bgr;AR responsiveness was demonstrated to occur in a previously unrecognized Gq protein–independent/G protein receptor kinase–dependent manner. Conclusions— This newly discovered relationship between cardiac V1AR and &bgr;AR may be informative for the treatment of patients with acute decompensated heart failure and elevated arginine vasopressin.

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

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 Ca(2+) channels in the sarcolemma and t-tubules. BAG3 co-immunoprecipitated with β1-adrenergic receptor, L-type Ca(2+) 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 [Ca(2+)]i transient amplitudes in LV myocytes stimulated with isoproterenol. L-type Ca(2+) current (ICa) and sarcoplasmic reticulum (SR) Ca(2+) content but not Na(+)/Ca(2+) exchange current (INaCa) or SR Ca(2+) uptake were reduced in isoproterenol-treated shBAG3 myocytes. Forskolin or dibutyryl 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 Ca(2+) 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 Ca(2+) channel provide novel insight into the role of BAG3 in cardiomyopathies and increased arrhythmia risks in heart failure.

Cardioprotection of Controlled and Cardiac-Specific Over-Expression of A2A-Adenosine Receptor in the Pressure Overload

Adenosine binds to three G protein-coupled receptors (R) located on the cardiomyocyte (A1-R, A2A-R and A3-R) and provides cardiac protection during both ischemic and load-induced stress. While the role of adenosine receptor-subtypes has been well defined in the setting of ischemia-reperfusion, far less is known regarding their roles in protecting the heart during other forms of cardiac stress. Because of its ability to increase cardiac contractility and heart rate, we hypothesized that enhanced signaling through A2A-R would protect the heart during the stress of transverse aortic constriction (TAC). Using a cardiac-specific and inducible promoter, we selectively over-expressed A2A-R in FVB mice. Echocardiograms were obtained at baseline, 2, 4, 8, 12, 14 weeks and hearts were harvested at 14 weeks, when WT mice developed a significant decrease in cardiac function, an increase in end systolic and diastolic dimensions, a higher heart weight to body weight ratio (HW/BW), and marked fibrosis when compared with sham-operated WT. More importantly, these changes were significantly attenuated by over expression of the A2A-R. Furthermore, WT mice also demonstrated marked increases in the hypertrophic genes β-myosin heavy chain (β-MHC), and atrial natriuretic factor (ANF) – changes that are mediated by activation of the transcription factor GATA-4. Levels of the mRNAs encoding β-MHC, ANP, and GATA-4 were significantly lower in myocardium from A2A-R TG mice after TAC when compared with WT and sham-operated controls. In addition, three inflammatory factors genes encoding cysteine dioxygenase, complement component 3, and serine peptidase inhibitor, member 3N, were enhanced in WT TAC mice, but their expression was suppressed in A2A-R TG mice. A2A-R over-expression is protective against pressure-induced heart failure secondary to TAC. These cardioprotective effects are associated with attenuation of GATA-4 expression and inflammatory factors. The A2A-R may provide a novel new target for pharmacologic therapy in patients with cardiovascular disease.

Adeno-Associated Virus Serotype 9–Driven Expression of BAG3 Improves Left Ventricular Function in Murine Hearts With Left Ventricular Dysfunction Secondary to a Myocardial Infarction

Visual Abstract

Arginine vasopressin receptor signaling and functional outcomes in heart failure.

• Circulating AVP is elevated in HF patients, which is associated with increased morbidity and mortality.

-Adrenergic Receptor-Mediated Cardiac Contractility Is Inhibited via Vasopressin Type 1A-Receptor-Dependent Signaling

Background— Enhanced arginine vasopressin levels are associated with increased mortality during end-stage human heart failure, and cardiac arginine vasopressin type 1A receptor (V1AR) expression becomes increased. Additionally, mice with cardiac-restricted V1AR overexpression develop cardiomyopathy and decreased &bgr;-adrenergic receptor (&bgr;AR) responsiveness. This led us to hypothesize that V1AR signaling regulates &bgr;AR responsiveness and in doing so contributes to development of heart failure. Methods and Results— Transaortic constriction resulted in decreased cardiac function and &bgr;AR density and increased cardiac V1AR expression, effects reversed by a V1AR-selective antagonist. Molecularly, V1AR stimulation led to decreased &bgr;AR ligand affinity, as well as &bgr;AR-induced Ca2+ mobilization and cAMP generation in isolated adult cardiomyocytes, effects recapitulated via ex vivo Langendorff analysis. V1AR-mediated regulation of &bgr;AR responsiveness was demonstrated to occur in a previously unrecognized Gq protein–independent/G protein receptor kinase–dependent manner. Conclusions— This newly discovered relationship between cardiac V1AR and &bgr;AR may be informative for the treatment of patients with acute decompensated heart failure and elevated arginine vasopressin.

Association of Variants in BAG3 With Cardiomyopathy Outcomes in African American Individuals

Importance The prevalence of nonischemic dilated cardiomyopathy (DCM) is greater in individuals of African ancestry than in individuals of European ancestry. However, little is known about whether the difference in prevalence or outcomes is associated with functional genetic variants. Objective We hypothesized that Bcl2-associated anthanogene 3 (BAG3) genetic variants were associated with outcomes in individuals of African ancestry with DCM. Design This multicohort study of the BAG3 genotype in patients of African ancestry with dilated cardiomyopathy uses DNA obtained from African American individuals enrolled in 3 clinical studies: the Genetic Risk Assessment of African Americans With Heart Failure (GRAHF) study; the Intervention in Myocarditis and Acute Cardiomyopathy Trial-2 (IMAC-2) study; and the Genetic Risk Assessment of Cardiac Events (GRACE) study. Samples of DNA were also acquired from the left ventricular myocardium of patients of African ancestry who underwent heart transplant at the University of Colorado and University of Pittsburgh. Main Outcomes and Measures The primary end points were the prevalence of BAG3 mutations in African American individuals and event-free survival in participants harboring functional BAG3 mutations. Results Four BAG3 genetic variants were identified; these were expressed in 42 of 402 African American individuals (10.4%) with nonischemic heart failure and 9 of 107 African American individuals (8.4%) with ischemic heart failure but were not present in a reference population of European ancestry (P < .001). The variants included 2 nonsynonymous single-nucleotide variants; 1 three-nucleotide in-frame insertion; and 2 single-nucleotide variants that were linked in cis. The presence of BAG3 variants was associated with a nearly 2-fold (hazard ratio, 1.97 [95% CI, 1.19-3.24]; P = .01) increase in cardiac events in carriers compared with noncarriers. Transfection of transformed adult human ventricular myocytes with plasmids expressing the 4 variants demonstrated that each variant caused an increase in apoptosis and a decrease in autophagy when samples were subjected to the stress of hypoxia-reoxygenation. Conclusions and Relevance This study demonstrates that genetic variants in BAG3 found almost exclusively in individuals of African ancestry were not causative of disease but were associated with a negative outcome in patients with a dilated cardiomyopathy through modulation of the function of BAG3. The results emphasize the importance of biological differences in causing phenotypic variance across diverse patient populations, the need to include diverse populations in genetic cohorts, and the importance of determining the pathogenicity of genetic variants.