Testosterone induces cardiac hypertrophy through a mechanism that involves a concerted crosstalk between cytosolic and nuclear signaling pathways. Nuclear factor of activated T-cells (NFAT) is associated with the promotion of cardiac hypertrophy, glycogen synthase kinase-3β (GSK-3β) is considered to function as a negative regulator, mainly by modulating NFAT activity. However, the role played by calcineurin-NFAT and GSK-3β signaling in testosterone-induced cardiac hypertrophy has remained unknown. Here, we determined that testosterone stimulates cardiac myocyte hypertrophy through NFAT activation and GSK-3β inhibition. Testosterone increased the activity of NFAT-luciferase (NFAT-Luc) in a time- and dose-dependent manner, with the activity peaking after 24 h of stimulation with 100 nM testosterone. NFAT-Luc activity induced by testosterone was blocked by the calcineurin inhibitors FK506 and cyclosporine A and by 11R-VIVIT, a specific peptide inhibitor of NFAT. Conversely, testosterone inhibited GSK-3β activity as determined by increased GSK-3β phosphorylation at Ser9 and β-catenin protein accumulation, and also by reduction in β-catenin phosphorylation at residues Ser33, Ser37, and Thr41. GSK-3β inhibition with 1-azakenpaullone or a GSK-3β-targeting siRNA increased NFAT-Luc activity, whereas overexpression of a constitutively active GSK-3β mutant (GSK-3βS9A) inhibited NFAT-Luc activation mediated by testosterone. Testosterone-induced cardiac myocyte hypertrophy was established by increased cardiac myocyte size and [³H]-leucine incorporation (as a measurement of cellular protein synthesis). Calcineurin-NFAT inhibition abolished and GSK-3β inhibition promoted the hypertrophy stimulated by testosterone. GSK-3β activation by GSK-3βS9A blocked the increase of hypertrophic markers induced by testosterone. Moreover, inhibition of intracellular androgen receptor prevented testosterone-induced NFAT-Luc activation. Collectively, these results suggest that cardiac myocyte hypertrophy induced by testosterone involves a cooperative mechanism that links androgen signaling with the recruitment of NFAT through calcineurin activation and GSK-3β inhibition.

Inner mitochondrial membrane fusion and cristae shape depend on optic atrophy protein 1, OPA1. Mutations in OPA1 lead to autosomal dominant optic atrophy (ADOA), an important cause of inherited blindness. The Guanosin Triphosphatase (GTPase) and GTPase effector domains (GEDs) of OPA1 are essential for mitochondrial fusion; yet, their specific roles remain elusive. Intriguingly, patients carrying OPA1 GTPase mutations have a higher risk of developing more severe multisystemic symptoms in addition to optic atrophy, suggesting pathogenic contributions for the GTPase and GED domains, respectively. We studied OPA1 GTPase and GED mutations to understand their domain-specific contribution to protein function by analyzing patient-derived cells and gain-of-function paradigms. Mitochondria from OPA1 GTPase (c.870+5G>A and c.889C>T) and GED (c.2713C>T and c.2818+5G>A) mutants display distinct aberrant cristae ultrastructure. While all OPA1 mutants inhibited mitochondrial fusion, some GTPase mutants resulted in elongated mitochondria, suggesting fission inhibition. We show that the GED is dispensable for fusion and OPA1 oligomer formation but necessary for GTPase activity. Finally, splicing defect mutants displayed a posttranslational haploinsufficiency-like phenotype but retained domain-specific dysfunctions. Thus, OPA1 domain-specific mutants result in distinct impairments in mitochondrial dynamics, providing insight into OPA1 function and its contribution to ADOA pathogenesis and severity.

Testosterone is known to induce cardiac hypertrophy through androgen receptor (AR)-dependent and -independent pathways, but the molecular underpinnings of the androgen action remain poorly understood. Previous work has shown that Ca2+/calmodulin-dependent protein kinase II (CaMKII) and myocyte-enhancer factor 2 (MEF2) play key roles in promoting cardiac myocyte growth. In order to gain mechanistic insights into the action of androgens on the heart, we investigated how testosterone affects CaMKII and MEF2 in cardiac myocyte hypertrophy by performing studies on cultured rat cardiac myocytes and hearts obtained from adult male orchiectomized (ORX) rats. In cardiac myocytes, MEF2 activity was monitored using a luciferase reporter plasmid, and the effects of CaMKII and AR signaling pathways on MEF2C were examined by using siRNAs and pharmacological inhibitors targeting these two pathways. In the in vivo studies, ORX rats were randomly assigned to groups that were administered vehicle or testosterone (125 mg•kg-1•week-1) for 5 weeks, and plasma testosterone concentrations were determined using ELISA. Cardiac hypertrophy was evaluated by measuring well-characterized hypertrophy markers. Moreover, western blotting was used to assess CaMKII and phospholamban (PLN) phosphorylation, and MEF2C and AR protein levels in extracts of left-ventricle tissue from control and testosterone-treated ORX rats. Whereas testosterone treatment increased the phosphorylation levels of CaMKII (Thr286) and phospholambam (PLN) (Thr17) in cardiac myocytes in a time- and concentration-dependent manner, testosterone-induced MEF2 activity and cardiac myocyte hypertrophy were prevented upon inhibition of CaMKII, MEF2C, and AR signaling pathways. Notably, in the hypertrophied hearts obtained from testosterone-administered ORX rats, both CaMKII and PLN phosphorylation levels and AR and MEF2 protein levels were increased. Thus, this study presents the first evidence indicating that testosterone activates MEF2 through CaMKII and AR signaling. Our findings suggest that an orchestrated mechanism of action involving signal transduction and transcription pathways underlies testosterone-induced cardiac myocyte hypertrophy.

In men, 70 of circulating testosterone binds with high affinity to plasma sex hormone binding globulin (SHBG), which determines its bioavailability in their target cells. In recent years, a growing body of evidence has shown that circulating SHBG not only is a passive carrier for steroid hormones but also actively regulates testosterone signaling through putative plasma membrane receptors and by local expression of androgen-binding proteins apparently to reach local elevated testosterone concentrations in specific androgen target tissues. Circulating SHBG levels are influenced by metabolic and hormonal factors, and they are reduced in obesity and insulin resistance, suggesting that SHBG may have a broader clinical utility in assessing the risk for cardiovascular diseases. Importantly, plasma SHBG levels are strongly correlated with testosterone concentrations, and in men, low testosterone levels are associated with an adverse cardiometabolic profile. Although obesity and insulin resistance are associated with an increased incidence of cardiovascular disease, whether they lead to abnormal expression of circulating SHBG or its interaction with androgen signaling remains to be elucidated. SHBG is produced mainly in the liver, but it can also be expressed in several tissues including the brain, fat tissue, and myocardium. Expression of SHBG is controlled by peroxisome proliferator-activated receptor γ (PPARγ) and AMP-activated protein kinase (AMPK). AMPK/PPAR interaction is critical to regulate hepatocyte nuclear factor-4 (HNF4), a prerequisite for SHBG upregulation. In cardiomyocytes, testosterone activates AMPK and PPARs. Therefore, the description of local expression of cardiac SHBG and its circulating levels may shed new light to explain physiological and adverse cardiometabolic roles of androgens in different tissues. According to emerging clinical evidence, here, we will discuss the potential mechanisms with cardioprotective effects and SHBG levels to be used as an early metabolic and cardiovascular biomarker in men.

Growth differentiation factor 11 (GDF11), a member of the transforming growth factor-β family, has been shown to act as a negative regulator in cardiac hypertrophy. Ca²⁺ signaling modulates cardiomyocyte growth; however, the role of Ca²⁺-dependent mechanisms in mediating the effects of GDF11 remains elusive. Here, we found that GDF11 induced intracellular Ca²⁺ increases in neonatal rat cardiomyocytes and that this response was blocked by chelating the intracellular Ca²⁺ with BAPTA-AM or by pretreatment with inhibitors of the inositol 1,4,5-trisphosphate (IP₃) pathway. Moreover, GDF11 increased the phosphorylation levels and luciferase activity of Smad2/3 in a concentration-dependent manner, and the inhibition of IP₃-dependent Ca²⁺ release abolished GDF11-induced Smad2/3 activity. To assess whether GDF11 exerted antihypertrophic effects by modulating Ca²⁺ signaling, cardiomyocytes were exposed to hypertrophic agents (100 nM testosterone or 50 μM phenylephrine) for 24 h. Both treatments increased cardiomyocyte size and [³H]-leucine incorporation, and these responses were significantly blunted by pretreatment with GDF11 over 24 h. Moreover, downregulation of Smad2 and Smad3 with siRNA was accompanied by inhibition of the antihypertrophic effects of GDF11. These results suggest that GDF11 modulates Ca²⁺ signaling and the Smad2/3 pathway to prevent cardiomyocyte hypertrophy.

Optic atrophy protein 1 (OPA1) mediates inner mitochondrial membrane (IMM) fusion and cristae organization. Mutations in OPA1 cause autosomal dominant optic atrophy (ADOA), a leading cause of blindness. Cells from ADOA patients show impaired mitochondrial fusion, cristae structure, bioenergetic function, and mitochondrial DNA (mtDNA) integrity. The mtDNA encodes electron transport chain subunits and is packaged into nucleoids spread within the mitochondrial population. Nucleoids interact with the IMM, and their distribution is tightly linked to mitochondrial fusion and cristae shaping. Yet, little is known about the physio-pathological relevance of nucleoid distribution. We studied the effect of OPA1 and ADOA-associated mutants on nucleoid distribution using high-resolution confocal microscopy. We applied a novel model incorporating the mitochondrial context, separating nucleoid distribution into the array in the mitochondrial population and intramitochondrial longitudinal distribution. Opa1-null cells showed decreased mtDNA levels and nucleoid abundance. Also, loss of Opa1 led to an altered distribution of nucleoids in the mitochondrial population, loss of cristae periodicity, and altered nucleoids to cristae proximity partly rescued by OPA1 isoform 1. Overexpression of WT OPA1 or ADOA-causing mutants c.870+5 G > A or c.2713 C > T in WT cells, showed perturbed nucleoid array in the mitochondria population associated with cristae disorganization, which was partly reproduced in Skeletal muscle-derived fibroblasts from ADOA patients harboring the same mutants. Opa1-null and cells overexpressing ADOA mutants accumulated mitochondria without nucleoids. Interestingly, intramitochondrial nucleoid distribution was only altered in Opa1-null cells. Altogether, our results highlight the relevance of OPA1 in nucleoid distribution in the mitochondrial landscape and at a single-organelle level and shed light on new components of ADOA etiology.